U.S. patent application number 15/822994 was filed with the patent office on 2018-04-19 for methods of predicting predisposition to or risk of kidney disease.
The applicant listed for this patent is Beth Israel Deaconess Medical Center, Inc., Wake Forest University Health Sciences. Invention is credited to Barry I. Freedman, David J. Friedman, Giulio Genovese, Martin R. Pollak.
Application Number | 20180105880 15/822994 |
Document ID | / |
Family ID | 44834746 |
Filed Date | 2018-04-19 |
United States Patent
Application |
20180105880 |
Kind Code |
A1 |
Genovese; Giulio ; et
al. |
April 19, 2018 |
METHODS OF PREDICTING PREDISPOSITION TO OR RISK OF KIDNEY
DISEASE
Abstract
Methods are disclosed herein for detecting a genetic
predisposition to focal segmental glomerulosclerosis (FSGS) or
hypertensive end-stage kidney disease (ESKD) or both in a human
subject. The methods include detecting the presence of at least one
single nucleotide polymorphism (SNP) in an APOL1 gene, such as the
C-terminal exon of an APOL1 gene. In a further embodiment, methods
are disclosed for detecting resistance of a subject to a disease
associated with Trypanosoma infection. The methods include
detecting the presence of at least one single nucleotide
polymorphism (SNP) in an APOL1 gene, such as the C-terminal exon of
an APOL1 gene. Also disclosed are methods for treating a subject
infected with T. brucei (such as T. brucei brucei, T. b.
rhodesiense, or T. b. gambiense). The methods include administering
a therapeutically effective amount of an APOL1 protein including a
S342G substitution, an I384M substitution, and/or a deletion of
N388 and Y389 to the subject.
Inventors: |
Genovese; Giulio; (Boston,
MA) ; Friedman; David J.; (Boston, MA) ;
Pollak; Martin R.; (Boston, MA) ; Freedman; Barry
I.; (Winston-Salem, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Beth Israel Deaconess Medical Center, Inc.
Wake Forest University Health Sciences |
Boston
Winston-Salem |
MA
NC |
US
US |
|
|
Family ID: |
44834746 |
Appl. No.: |
15/822994 |
Filed: |
November 27, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13642054 |
Dec 12, 2012 |
9828637 |
|
|
PCT/US11/32924 |
Apr 18, 2011 |
|
|
|
15822994 |
|
|
|
|
61325343 |
Apr 18, 2010 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/172 20130101;
C12Q 1/6883 20130101; C12Q 2600/158 20130101; C12Q 2600/118
20130101; C12Q 2600/156 20130101 |
International
Class: |
C12Q 1/6883 20060101
C12Q001/6883 |
Claims
1. A method for identifying a genetic predisposition to renal
disease comprising: i) contacting a sample from a human subject
with a nucleic acid probe capable of hybridizing to a nucleic acid
molecule having the sequence of at least one apolipoprotein L1
(APOL1) gene risk allele or a complement thereof, or a nucleic acid
primer capable of amplifying the nucleic acid molecule or
complement thereof; and ii) detecting formation of a hybridization
complex between the nucleic acid probe and the nucleic acid
molecule or complement thereof or an amplification product
corresponding to the nucleic acid molecule or complement thereof,
wherein formation of the hybridization complex or the presence of
the at least one human APOL1 gene risk allele in the amplification
product indicates the human subject has an increased risk of
developing said renal disease, relative to a human subject that
lacks the APOL1 gene risk allele.
2. The method of claim 1, wherein the at least one human APOL1 gene
risk allele comprises at least one single nucleotide polymorphism
(SNP) and/or at least one inversion in a human APOL1 gene.
3. (canceled)
4. The method of claim 2, wherein the at least one SNP produces an
APOL1 polypeptide having a serine to glycine mutation at position
342 (S342G), an isoleucine to methionine mutation at position 384
(I384M), a deletion of amino acids N388 and Y389, or a combination
thereof.
5. The method of claim 4, wherein the at least one SNP produces an
APOL1 polypeptide having a S342G and an I384M mutation.
6. The method of claim 2, wherein the method comprises determining
the presence of the at least one SNP and/or the at least one
inversion on both chromosomes of the subject.
7. The method of claim 1, wherein the renal disease is focal
segmental glomerulosclerosis (FSGS) or hypertensive end-stage
kidney disease, or both.
8. The method of claim 7, wherein the method is for detecting a
genetic predisposition to focal segmental glomerulosclerosis, and
wherein the human subject is infected with human immunodeficiency
virus (HIV).
9. The method of claim 1, wherein the subject is of African or
Hispanic ancestry.
10. The method of claim 9, wherein the subject is an
African-American subject.
11. The method of claim 2, comprising detecting the presence of the
at least SNP and the at least one inversion in the human APOL1
gene.
12. The method of claim 2, wherein said inversion comprises
recombination between said human APOL1 gene and a human
apolipoprotein 4 (APOL4) gene.
13. The method of claim 2, wherein said inversion occurs in a
coding region of said APOL1 gene, a non-coding region of said APOL1
gene, or in both regions.
14. (canceled)
15. The method of claim 2, wherein detection of at least two SNPs
in said human subject indicates an increased likelihood said human
subject will develop said renal disease relative to a subject
having one or no APOL1 gene risk allele.
16. The method of claim 2, comprising determining whether said
human subject is homozygous or heterozygous for said at least one
SNP or said inversion, wherein a determination that the human
subject is homozygous for the at least one SNP or the at least one
inversion indicates an increased likelihood the human subject will
develop said renal disease relative to a human subject that is
heterozygous for the at least one SNP or the at least one
inversion.
17. (canceled)
18. The method of claim 2, wherein said inversion comprises
substitution of the 5' region of said APOL1 gene with the 5' region
of an APOL4 gene.
19-36. (canceled)
37. The method of claim 2, wherein the presence of said at least
one SNP or said inversion indicates said human subject has an
increased risk of renal damage following treatment with a
therapeutic.
38. The method of claim 37, wherein said therapeutic is selected
from a blood pressure medication, a steroid, and/or an
immunosuppressive agent.
39-54. (canceled)
55. The method of claim 2, wherein the presence of said at least
one SNP or said at least one inversion indicates said human subject
has an increased risk of kidney failure relative to a human subject
lacking said at least one SNP or said at least one inversion.
56. The method of claim 55, wherein said human subject has a
greater need for a kidney transplant relative to a human subject
lacking said at least one SNP or said at least one inversion.
57-88. (canceled)
89. The method of claim 2, wherein the sample is selected from the
group consisting or whole blood, serum, buccal cells, extracted
galls, biopsied or surgically removed tissue, tears, milk, a skin
scrape, a surface washing, urine, sputum, cerebrospinal fluid,
prostate fluid, pus, and a bone marrow aspirate.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of, and claims
priority to, U.S. patent application Ser. No. 13/642,054, filed
Dec. 12, 2012 (issued as U.S. Pat. No. 9,828,637 on Nov. 28, 2017),
which is a U.S. National Stage Application under 35 U.S.C. .sctn.
371 of International Application No. PCT/US2011/032924, filed Apr.
18, 2011, which claims the benefit of U.S. Provisional Application
No. 61/325,343, filed Apr. 18, 2010, the disclosure of each of
which is incorporated herein by reference in its entirety.
STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING
[0002] A Sequence Listing in ASCII text format, submitted under 37
C.F.R. .sctn. 1.821, entitled 9151-228TSDV_ST25.txt, 7,436 bytes in
size, generated on Nov. 27, 2017 and filed via EFS-Web, is provided
in lieu of a paper copy. This Sequence Listing is incorporated by
reference into the specification for its disclosures.
FIELD OF THE INVENTION
[0003] This disclosure relates to the field of individualized
medicine, particularly to the determination of risk of a subject to
develop renal disease, such as focal segmental glomerulosclerosis
(FSGS) or end-stage kidney disease (ESKD). The disclosure also
relates to the determination of resistance to disease associated
with Trypanosoma infection and methods for treating Trypanosoma
infection in a subject.
BACKGROUND OF THE INVENTION
[0004] The prevalence of chronic kidney disease (CKD) in the United
States is now estimated at 13%, and is associated with significant
morbidity and mortality (Coresh et al., Am J Kidney Dis 2003;
41(1):1-12). In particular, approximately 100,000 Americans develop
end-stage kidney disease (ESKD) each year. The cumulative life-time
risk for ESKD varies by race, and is approximately 7.5% for
African-Americans and 2.1% for European Americans (Kiberd et al., J
Am Soc Nephrol 2002; 13(6):1635-44). In particular,
African-Americans have a disproportionate risk for several forms of
CKD, among them diabetic nephropathy (Cowie et al., N Engl J Med
1989; 321(16):1074-9), hypertensive nephrosclerosis (Toto, Kidney
Int Suppl 2004(92):S102-4), lupus nephritis (Fernandez et al.,
Arthritis Rheum 2007; 57(4):576-84), focal segmental
glomerulosclerosis (Kitiyakara et al., Am J Kidney Dis 2004;
44(5):815-25) (FSGS), and HIV-associated nephropathy (a distinct
form of FSGS, also termed collapsing glomerulopathy).
[0005] FSGS is a clinical syndrome involving podocyte injury and
glomerular scarring, and includes genetic forms with Mendelian
inheritance, reactive forms associated with other illnesses
(including HIV-1 disease) or medications, and an idiopathic form,
which accounts for the majority of cases (Barisoni et al., Clin J
Am Soc Nephrol 2007; 2(3):529-42). African-Americans have a 4-fold
increased risk for sporadic FSGS (Kitiyakara et al., Semin Nephrol
2003; 23(2):172-82) and an 18-fold to 50-fold increased risk for
HIV-1-associated FSGS (Kopp et al., Kidney Int Suppl
2003(83):S43-9; Eggers et al., J Am Soc Nephrol 2004;
15(9):2477-85). Individuals of African ancestry also have increased
risk for FSGS in other geographic regions, suggesting that genetic
factors contribute to these disparities (Kitiyakara et al., Semin
Nephrol 2003; 23(2):172-82).
SUMMARY OF THE INVENTION
[0006] A first aspect of the invention features methods for
detecting a genetic predisposition to, or an increased risk of, the
development of a renal disease, such as focal segmental
glomerulosclerosis (FSGS) or hypertensive end-stage kidney disease
(ESKD), or both in a human subject. In one embodiment, the human
subject is of African (e.g., an African American) or Hispanic
ancestry (in preferred embodiments, the subject of Hispanic
ancestry also is of African ancestry). In another embodiment, the
human subject is of European ancestry. The methods include
detecting the presence of at least one APOL1 gene risk allele
(e.g., 2, 3 or 4 risk alleles; e.g., the risk allele is at least
one single nucleotide polymorphism (SNP) in an APOL1 gene, such as
the C-terminal exon of an APOL1 gene, or an inversion in an APOL1
gene (e.g., an inversion in a 5' region of an APOL1 gene, e.g., an
inversion in which the 5' region of an APOL1 gene is replaced with
a 5' region of an APOL4 gene). In other embodiments, the risk
allele is a G1, G2, del6, and/or a G3 allele. The presence of the
at least one SNP and/or the at least one inversion determines the
genetic predisposition to renal disease, such as focal segmental
glomerulosclerosis or hypertensive ESKD, or both. In other
embodiments, the inversion in the APOL1 gene replaces all or a
portion of up to three exons in APOL1 by sequence from APOL4 (e.g.,
the inversion may result in replacement of all or a portion of only
the first exon, and/or all or a portion of the first and/or second
exon, and/or all or a portion of the first, second, and/or all or a
portion of the third exon of the APOL1 gene). These three exons may
cover a range of 2000-2500 base pairs of genomic DNA (e.g., in a
range of from about 100 base pairs to about 3000 base pairs of
genomic DNA, such as a range from 1000 base pairs to about 2500
base pairs of genomic DNA), and may encode a maximum of about 420
base pairs of transcript (e.g., a range of from about 20 base pairs
to about 500 base pairs of transcript, such as from about 100 base
pairs to about 420 base pairs of transcript DNA). The actual coding
sequence replaced in the APOL1 protein may only code for about 1 to
about 30 amino acids, e.g., about 10 to about 20 amino acids, e.g.,
about 14 amino acids from APOL4. The substituted amino acids in the
APOL1 protein may all appear in the preprotein portion of the
hybrid APOL4/APOL1 protein and all or a portion of the replaced
amino acids may be cleaved depending upon the extent and actual
sequence of the inversion. In an embodiment, the inversion occurs
in a coding and/or non-coding region of the APOL1 gene and/or
results in a functional gene product.
[0007] In other embodiments, the method includes taking a sample
from the human subject to be tested. In still other embodiments,
the at least one SNP is a G at rs73885319; a G at rs60910145; a 6
base pair deletion at rs71785313; and/or a combination thereof. The
at least one SNP may produce an APOL1 polypeptide having a serine
to glycine mutation at position 342 (S342G), an isoleucine to
methionine mutation at position 384 (I384M), a deletion of amino
acids N388 and Y389, and/or a combination thereof (e.g., the at
least one SNP produces an APOL1 polypeptide having a S342G and an
I384M mutation). In yet other embodiments, the method includes
determining the presence of the at least one SNP and/or the at
least one inversion on both chromosomes of the subject. In another
embodiment, the subject is infected with human immunodeficiency
virus (HIV) and is at a greater risk of developing FSGS. In still
other embodiments, the subject is homozygous or heterozygous for
the at least one SNP and/or the inversion. In an embodiment, a
determination that the human subject is homozygous for the at least
one SNP and/or the at least one inversion indicates an increased
likelihood the human subject will develop renal disease relative to
a human subject that is heterozygous for the at least one SNP
and/or the at least one inversion.
[0008] In another embodiment, the presence of the at least one SNP
and/or the at least one inversion indicates the human subject has
an increased risk of renal disease following treatment with a
therapeutic. For example, a subject having one or more APOL1 gene
risk alleles may need to be offered a treatment regimen with
respect to blood pressure medications, steroids, and/or
immunosuppressive agents that is different from a subject lacking
any (or only having, e.g., one) APOL1 gene risk allele. In
particular, subjects having one or more APOL1 gene risk alleles are
more susceptible to renal damage and/or disease and the risk of
kidney damage increases in patients having one or more APOL1 gene
risk alleles that are treated with blood pressure medications,
steroids, and/or immunosuppressive agents. Thus, in patients having
one or more APOL1 gene risk alleles, the concentration of a given
blood pressure medication, steroid, and/or immunosuppressive agent
and/or the length of treatment may need to be decreased relative to
a patient lacking any (or having only one) APOL1 gene risk alleles.
Examples of therapeutics include blood pressure medications (e.g.,
a diuretic (e.g., chlorthalidone, chlorothiazide, furosemide,
hydrochlorothiazide, indapamide, metolazone, amiloride
hydrochloride, spironolactone, triamterene, bumetanide, or a
combination thereof), an alpha adrenergic antagonist (e.g.,
alfuzosin, doxazosin, prazosin, terazosin, or tamsulosin, or a
combination thereof), a central adrenergic inhibitor (e.g.,
clonidine, guanfacine, or methyldopa, or a combination thereof), an
angiotensin converting enzyme (ACE) inhibitor (e.g., benazepril,
captopril, enalapril, fosinopril, lisinopril, moexipril,
perindopril, quinapril, ramipril, or trandolapril, or combinations
thereof), an angiotensin II receptor blocker (e.g., candesartan,
eprosartan, irbesartan, losartan, olmesartan, telmisartan, or
valsartan, or combinations thereof), an alpha blocker (e.g.,
doxazosin, prazosin, or terazosin, or a combination thereof), a
beta blocker (e.g., acebutolol, atenolol, betaxolol, bisoprolol,
carteolol, metoprolol, nadolol, nebivolol, penbutolol, pindolol,
propranolol, solotol, or timolol, or a combination thereof), a
calcium channel blocker (e.g., amlodipine, bepridil, diltiazem,
felodipine, isradipine, nicardipine, nifedipine, nisoldipine, or
verapamil, or combination thereof), a vasodilator (e.g.,
hydralazine or minoxidil, or combination thereof), and a renin
inhibitor (e.g., aliskiren), or combinations thereof), a steroid
(e.g., a corticosteroid, such as cortisone, prednisone,
methylprednisolone, or prednisolone), or an anabolic steroid
(anatrofin, anaxvar, annadrol, bolasterone, decadiabolin,
decadurabolin, dehydropiandrosterone (DHEA), delatestryl,
dianiabol, dihydrolone, durabolin, dymethazine, enoltestovis,
equipose, gamma hydroxybutyrate, maxibolin, methatriol,
methyltestosterone, parabolin, primobolin, quinolone, therabolin,
trophobolene, and winstrol), or an immunosuppressive agent, such as
a glucocorticoid, a cytostatic, an antibody, or an
anti-immunophilin and/or mychophenolate mofetil (MMF), FK-506,
azathioprine, cyclophosphamide, methotrexate, dactinomycin,
antithymocyte globulin (ATGAM), an anti-CD20-antibody, a
muromonoab-CD3 antibody, basilizimab, daclizumab, cyclosporin,
tacrolimus, voclosporin, sirolimus, an interferon, infliximab,
etanercept, adalimumab, fingolimod, and/or myriocin).
[0009] In other embodiments, the presence of the at least one SNP
and/or the at least one inversion indicates the human subject has
an increased risk of kidney failure (and may have a greater need
for a kidney transplant) relative to a human subject lacking the at
least one SNP and/or the at least one inversion.
[0010] A second aspect of the invention features a method of
evaluating a human subject (e.g., a potential kidney donor) for
their suitability as a transplant donor by determining the presence
of at least one human APOL1 gene risk allele (e.g., at least one
(e.g., two, three, or four) single nucleotide polymorphism (SNP;
e.g., a G at rs73885319 (G1), a G at rs60910145 (G2), a 6 base pair
deletion at rs71785313 (del6), and/or a combination thereof) and/or
at least one inversion in a human APOL1 gene) in a cell, tissue, or
organ of the human subject, in which the presence of the at least
one APOL1 gene risk allele indicates the human subject is not
suitable as a transplant donor. In other embodiments, the at least
one SNP produces an APOL1 polypeptide having a serine to glycine
mutation at position 342 (S342G), an isoleucine to methionine
mutation at position 384 (I384M), a deletion of amino acids N388
and Y389, and/or a combination thereof (e.g., the at least one SNP
produces an APOL1 polypeptide having a S342G and an I384M
mutation). In other embodiments, the inversion is, e.g., a
substitution of the 5' region of the APOL1 gene with the 5' region
of another apolipoprotein gene (e.g., an APOL4 gene)). In yet other
embodiments, the method includes determining the presence of the at
least one SNP and/or the at least one inversion on both chromosomes
of the subject. The human subject may be of African or Hispanic
ancestry (e.g., an African American subject). In other embodiments,
the method includes detecting the presence of the at least one SNP
and the at least one inversion in said APOL1 gene; the inversion
includes recombination between the human APOL1 gene and another
apolipoprotein gene (e.g., a human APOL4 gene); the inversion
occurs in a coding and/or non-coding region of the APOL1 gene;
and/or the inversion results in a functional gene product. In still
other embodiments, detection of at least two SNPs in the human
subject further indicates the human subject is not suitable as a
transplant donor. The method may further include determining
whether the human subject is homozygous or heterozygous for the at
least one SNP and/or the at least one inversion (e.g., a
determination that the human subject is homozygous for the at least
one SNP and/or the at least one inversion indicates an increased
likelihood the human subject will develop renal disease relative to
a human subject that is heterozygous for the at least one SNP
and/or the at least one inversion).
[0011] A third aspect of the invention features methods for
detecting a disease associated with Trypanosoma spp. infection,
such as a disease associated with T. brucei infection, such as
African trypanosomiasis (sleeping sickness) in a subject (e.g., a
human subject) by detecting a resistance allele of an APOL1 gene.
In an embodiment, the resistance allele includes at least one
(e.g., two, three, or four) SNP in an APOL1 gene, such as the
C-terminal exon of an APOL1 gene, and/or at least one inversion in
an APOL1 gene (e.g., an inversion in a 5' region of an APOL1 gene,
e.g., an inversion in which the 5' region of an APOL1 gene is
replaced with a 5' region of an APOL4 gene). In other embodiments,
the resistance allele is a G1, G2, del6, and/or a G3 allele. The
presence of the SNP and/or the inversion determines resistance of
the subject to disease associated with Trypanosoma spp.
infection.
[0012] A fourth aspect of the invention features methods for
treating a subject infected with T. brucei (such as T. brucei
brucei, T. b. rhodesiense, or T. b. gambiense). The methods include
administering a therapeutically effective amount of an APOL1
protein (e.g., a human APOL1 protein) that includes at least one
resistance allele (e.g., a S342G substitution, an I384M
substitution, a deletion of N388 and Y389 and/or an inversion to
the subject. In some examples, the APOL1 protein is included in
human serum administered to the subject. In other examples, the
APOL1 protein is recombinant.
[0013] The foregoing and other features of the disclosure will
become more apparent from the following detailed description, which
proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIGS. 1A and 1B show a pair of plots showing logistic
regression adjusting for APOL1 alleles G1 and G2. Results of
association between 205 idiopathic biopsy-proven African-American
FSGS cases and 180 African-American controls. On the x-axis and
y-axis, genomic position and -log 10 of the p-values are shown.
Highlighted are also SNPs rs4821481 and rs3752462 whose combined
risk alleles define the E-1 haplotype (Kopp et al., Nature Genet.
40:1175-1184, 2008). (1A) Association of the studied variants with
FSGS using Fisher's exact test. (1B) Association of the studied
variants with FSGS after adjusting for allele G1 using logistic
regression.
[0015] FIG. 2 is a graph showing extended haplotype homozygosity
(EHH) values for the three APOL1 alleles computed after combining
Hapmap phase 2 genotype data with genotype data for alleles G1 and
G2 for Yoruba samples. This suggests an older age for allele G2,
although large uncertainty is introduced by the fact that only 8
haplotypes with the G2 allele were found in the Yoruba sample
set.
[0016] FIG. 3 shows a pair of maps showing average annual rate of
new cases of (Panel A) Trypanosoma brucei rhodesiense and (Panel B)
Trypanosoma brucei gambiense sleeping sickness reported between
1997 and 2006 in Africa.
[0017] FIG. 4 shows a series of panels showing trypanolytic
potential of ApoL1 variants on NHS-resistant (SRA+) and
NHS-sensitive (SRA-) T. b. rhodesiense ETat 1.2 (Edinburgh
Trypanozoon antigenic type 1, clone 2) clones. (Panel A) Titration
of trypanolytic activity in plasma samples after overnight
incubation (ctrl=control incubation in fetal calf serum without
plasma; horn, het=homozygous and heterozygous mutations,
respectively). (Panel B) ApoL1 content of various plasma samples
before and after affinity chromatography through SRA column
(NHS=normal human serum; WT=wild type apoL1; S=serine 342;
G=glycine 342; I=isoleucine 384; M=methionine 384; i=insertion of
N388/Y389; d=deletion of N388/Y389). (Panel C) Trypanolytic
activity of various recombinant ApoL1 variants after overnight
incubation (FCS=fetal calf serum). (Panel D) Kinetics of
trypanolysis by 20 .mu.g/ml recombinant ApoL1 variants in the
presence or absence of 25 .mu.M chloroquine (clq). (Panel E)
Phenotype of ETat1.2R trypanosomes incubated with various
recombinant ApoL1 (20 .mu.g/ml; 1 h 30 and 6h incubation, for G1
and G2 respectively; the arrows point to the swelling
lysosome).
[0018] FIG. 5 shows the mean age at dialysis initiation for
subjects by APOL1 risk allele status. Due to the proximity of the
alleles it is not expected for a diploid sample to have more then
two risk alleles, only six groups existed within this dataset;
Wt+Wt, Wt+G1, G1+G1, Wt+G2, G2+G2, G1+G2. Bar height is the mean
age. Error bars denote the standard error.
*significantly different from wild type (Wt+Wt)
[0019] FIG. 6 shows the mean age at dialysis for subjects with a G1
risk allele. Panel A shows the mean age at dialysis initiation by
G1 risk allele status in subjects with hypertension attributed ESRD
(H-ESKD). Panel B shows age by G1 risk allele status in subjects
with other ESRD causes, including HIV, inflammation, toxins, etc.
Horizontal bars denote mean age while vertical bars denote standard
error.
*significantly different from wild type (Wt+Wt)
[0020] FIG. 7. Panel A is a schematic showing the relationship of
the APOL1, APOL2, and APOL4 genes on chromosome 22. The G1 and G2
alleles are also shown. Panel B is a schematic showing the
inversion of a segment of DNA including the 5' end of APOL4, all of
APOL2, and the 5' end of APOL1.
[0021] FIG. 8 is a graph of HapMap gene expression data that
showing the coordinated regulation of APOL1 and APOL2.
[0022] FIG. 9 is a photograph of a gel showing the presence of a
APOL1-APOL4 hybrid gene following PCR amplification from 12 human
samples. Lane 1 shows a size ladder. Lanes 4 and 6 show the
inversion.
[0023] FIG. 10 shows the genomic sequence of APOL1 and APOL4
following G3 inversion (SEQ ID NO: 7).
[0024] FIG. 11. Panel A shows a potential transcript formed in
individuals with the G3 inversion and another SNP, rs9610445 (the C
allele), in which an essential splice site is eliminated. The donor
splice site sequence of APOL4 (SEQ ID NO: 8) and the acceptor
splice site of APOL1 (SEQ ID NO: 9) are shown. Panel B is a
potential transcript formed in individuals with the G3 inversion
and another SNP, rs6000181 T (minor) allele, in which a methionine
start site is eliminated.
SEQUENCE LISTING
[0025] The nucleic acid sequences and amino acid sequences listed
are shown using standard letter abbreviations for nucleotide bases,
and three letter code for amino acids, as defined in 37 C.F.R.
1.822. Only one strand of each nucleic acid sequence is shown, but
the complementary strand is understood as included by any reference
to the displayed strand. It should be noted that single nucleotide
polypmorphisms are identified in the leading strand, wherein the
risk nucleotide is listed first, and the protective nucleotide is
listed second. Due to the complementary nature of DNA, the single
nucleotide polymorphism is present in both DNA strands, and thus
can also be identified in the lagging strand.
[0026] SEQ ID NOs: 1-3 are nucleic acid sequences from the APOL1
gene, each include a single nucleotide polymorphism of
interest.
[0027] SEQ ID NOs: 4 and 5 are exemplary nucleic acid and amino
acid sequences of a human apolipoprotein L1, respectively.
[0028] SEQ ID NO: 7 is a genomic sequence of an APOL1 and APOL4
inversion.
DETAILED DESCRIPTION
I. Abbreviations and Terms
[0029] APOL1: apolipoprotein L1 gene or protein [0030] ESKD:
end-stage kidney disease [0031] FSGS: focal segmental
glomerulosclerosis [0032] HIV: human immunodeficiency virus [0033]
LD: linkage disequilibrium [0034] LOD: logarithm of the odds [0035]
MALD: mapping by admixture linkage disequilibrium [0036] NHS:
normal human serum [0037] OR: odds ratio [0038] ROC: receiver
operator characteristic [0039] SNP: single nucleotide polymorphism
[0040] SRA: serum resistance-associated gene or protein
[0041] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. It is further to be understood that all base
sizes or amino acid sizes, and all molecular weight or molecular
mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of this disclosure, suitable
methods and materials are described below. The term "comprises"
means "includes." All publications, patent applications, patents,
and other references mentioned herein are incorporated by reference
in their entirety. In case of conflict, the present specification,
including explanations of terms, will control. In addition, the
materials, methods, and examples are illustrative only and not
intended to be limiting.
[0042] Definitions of common terms in molecular biology may be
found in Benjamin Lewin, Genes V, published by Oxford University
Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The
Encyclopedia of Molecular Biology, published by Blackwell Science
Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.),
Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0043] Administration: To provide or give a subject an agent by any
effective route. Exemplary routes of administration include, but
are not limited to, oral, injection (such as subcutaneous,
intramuscular, intradermal, intraperitoneal, and intravenous),
sublingual, rectal, transdermal, intranasal, vaginal and inhalation
routes. Administration of extracorporeal treatment (e.g., dialysis)
is also included.
[0044] African ancestry: An individual whose ancestors are from
Sub-Saharan Africa prior to the era of European expansion (prior to
about 1500). There are a number of programs that can be used to
analyze DNA to determine if an individual is of African ancestry,
such as STRUCTURE.TM. (available on the internet at
pritch.bsd.uchicago.edu/structure.html). In one example,
African-American individuals are those individuals who reside in
the United States and self-identify themselves as being of African
origin. In another example, African-Americans are individuals who
reside in the United States and self-identify as being of African
origin, and are of African ancestry as determined by a program that
analyzes DNA ancestry, such as STRUCTURE.TM..
[0045] Allele: A particular form of a genetic locus, distinguished
from other forms by its particular nucleotide sequence, or one of
the alternative polymorphisms found at a polymorphic site.
[0046] Allele frequency: A measure of the relative frequency of an
allele at a genetic locus in a population. Usually allele frequency
is expressed as a proportion or a percentage. In population
genetics, allele frequencies are used to depict the amount of
genetic diversity at the individual, population, or species level.
There are various databases in the public domain that contain SNPs
and a user may for example, determine the relative allele frequency
in some instances using such publicly available databases.
[0047] In the instant application the allele frequency for a risk
allele is greater than 5% in subjects of African ancestry. In a
further embodiment, the allele frequency for a risk allele is
greater than at least 10%, at least 15%, at least 20%, at least
25%, at least 30%, at least 35%, at least 40%, at least 45%, or at
least 50% in subjects of African ancestry. In some embodiments, an
unaffected population is used to calculate allele frequency. Risk
is elevated in individuals that carry the risk allele.
[0048] There are a number of diseases or disorders that are
associated with the identification of one or more SNPs or risk
alleles. In many cases, the diseases or disorders are autosomal
dominant mutations and any associated SNPs are observed only in
individuals who present with clinical manifestations of the
disease. In other circumstances, the occurrence of a
disease-associated SNP is so rare that no-known frequency can be
determined (for example, through the use of public domain SNP
databases or by comparison with the literature) and these
diseases/disorders are correctly defined as having an allele
frequency significantly lower than 1%.
[0049] Amplification: To increase the number of copies of a nucleic
acid molecule. The resulting amplification products are called
"amplicons." Amplification of a nucleic acid molecule (such as a
DNA or RNA molecule) refers to use of a technique that increases
the number of copies of a nucleic acid molecule in a sample. An
example of amplification is the polymerase chain reaction (PCR), in
which a sample is contacted with a pair of oligonucleotide primers
under conditions that allow for the hybridization of the primers to
a nucleic acid template in the sample. The primers are extended
under suitable conditions, dissociated from the template,
re-annealed, extended, and dissociated to amplify the number of
copies of the nucleic acid. This cycle can be repeated. The product
of amplification can be characterized by such techniques as
electrophoresis, restriction endonuclease cleavage patterns,
oligonucleotide hybridization or ligation, and/or nucleic acid
sequencing.
[0050] Other examples of in vitro amplification techniques include
quantitative real-time PCR; reverse transcriptase PCR (RT-PCR);
real-time PCR (rt PCR); real-time reverse transcriptase PCR (rt
RT-PCR); nested PCR; strand displacement amplification (see U.S.
Pat. No. 5,744,311); transcription-free isothermal amplification
(see U.S. Pat. No. 6,033,881); repair chain reaction amplification
(see PCT Publication No. WO 90/01069); ligase chain reaction
amplification (see European patent publication No. EP-A-320 308);
gap filling ligase chain reaction amplification (see U.S. Pat. No.
5,427,930); coupled ligase detection and PCR (see U.S. Pat. No.
6,027,889); and NASBA.TM. RNA transcription-free amplification (see
U.S. Pat. No. 6,025,134), amongst others.
[0051] APOL1: A gene encoding human apolipoprotein L, 1 (OMIM:
603743). This gene encodes a secreted high density lipoprotein
which binds to apolipoprotein A-I. Apolipoprotein A-I is a
relatively abundant plasma protein and is the major apoprotein of
HDL. Several different transcript variants encoding different
isoforms have been found for this gene.
[0052] Exemplary SNPs observed in APOL1 that are associated with a
predisposition to renal disease include a G at rs73885319 ("G1"); a
G at rs60910145 ("G2"); or a 6 base pair deletion at rs71785313
("del6") (incorporated by reference as present in dbSNP
(ncbi.nlm.nih.gov/SNP) on Apr. 18, 2010), as well as combinations
thereof.
[0053] Nucleic acid and protein sequences for human APOL1 are
publicly available. For example, GENBANK.RTM. Accession No.
NC_000022.10 (nucleotides 36649117.36663577) discloses an exemplary
human APOL1 genomic sequence (incorporated by reference as provided
by GENBANK.RTM. on Apr. 18, 2010). In other examples, GENBANK.RTM.
Accession Nos. AF305224.1, NM_003661.3, NM_145343.2,
NM_001136540.1, z82215, and BC127186.1 disclose exemplary human
APOL1 nucleic acid sequences, and GENBANK.RTM. Accession Nos.
CAQ09089, NP_003652, AAI43039.1, and AAI42721.1 disclose exemplary
human APOL1 protein sequences, all of which are incorporated by
reference as provided by GENBANK.RTM. on Apr. 18, 2010.
[0054] Caucasian: A human racial classification traditionally
distinguished by physical characteristics such as very light to
brown skin pigmentation and straight to wavy or curly hair, which
includes persons having origins in any of the original peoples of
Europe, North Africa, or the Middle East. Popularly, the word
"white" is used synonymously with "Caucasian" in North America.
Such persons retain substantial genetic similarity to natives or
inhabitants of Europe, North Africa, or the Middle East. In a
particular example, a Caucasian is at least 1/64 Caucasian.
[0055] Concordance: The presence of two or more loci or traits (or
combination thereof) derived from the same parental chromosome. The
opposite of concordance is discordance, that is, the inheritance of
only one (of two or more) parental alleles and/or traits associated
with a parental chromosome.
[0056] Correlation: A correlation between a phenotypic trait and
the presence or absence of a genetic marker (or haplotype or
genotype) can be observed by measuring the phenotypic trait and
comparing it to data showing the presence or absence of one or more
genetic markers. Some correlations are stronger than others,
meaning that in some instances subjects with FSGS will display a
particular genetic marker (e.g., 100% correlation). In other
examples the correlation will not be as strong, meaning that a
subject with FSGS will only display a particular genetic marker
90%, 85%, 70%, 60%, 55%, or 50% of the time. In some instances, a
haplotype which contains information relating to the presence or
absence of multiple markers can also be correlated to a genetic
predisposition such as the development of FSGS, or the type of
onset. Correlations can be described using various statistical
analyses known to the skilled artisan.
[0057] Decrease: Becoming less or smaller, as in number, amount,
size, or intensity. In one example, decreasing the risk of a
disease (such as FSGS or hypertensive ESKD) includes a decrease in
the likelihood of developing the disease by at least about 20%, for
example by at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In
another example, decreasing the risk of a disease includes a delay
in the development of the disease, for example a delay of at least
about six months, such as about one year, such as about two years,
about five years, or about ten years.
[0058] In one example, decreasing the signs and symptoms of FSGS
includes decreasing the effects of the disease such as podocyte
injury or glomerular scarring by a desired amount, for example by
at least 5%, at least 10%, at least 15%, at least 20%, at least
25%, at least 30%, at least 50%, at least 75%, or even at least
90%, as compared to a response in the absence of a therapeutic
composition.
[0059] In another, decreasing the signs and symptoms of Trypanosoma
infection, such as sleeping sickness, includes decreasing the
effects of the disease such as fever, headache, joint pain, lymph
node swelling, anemia, confusion, reduced coordination, or
disruption of the sleep cycle by a desired amount, for example by
at least 5%, at least 10%, at least 15%, at least 20%, at least
25%, at least 30%, at least 50%, at least 75%, or even at least
90%, as compared to a response in the absence of a therapeutic
composition.
[0060] DNA (deoxyribonucleic acid): DNA is a long chain polymer
which comprises the genetic material of most living organisms (some
viruses have genes comprising ribonucleic acid (RNA)). The
repeating units in DNA polymers are four different nucleotides,
each of which comprises one of the four bases, adenine, guanine,
cytosine and thymine bound to a deoxyribose sugar to which a
phosphate group is attached. Triplets of nucleotides (referred to
as codons) code for each amino acid in a polypeptide, or for a stop
signal (termination codon). The term codon is also used for the
corresponding (and complementary) sequences of three nucleotides in
the mRNA into which the DNA sequence is transcribed.
[0061] Unless otherwise specified, any reference to a DNA molecule
is intended to include the reverse complement of that DNA molecule.
Except where single-strandedness is required by the text herein,
DNA molecules, though written to depict only a single strand,
encompass both strands of a double-stranded DNA molecule. Thus, a
reference to the nucleic acid molecule that encodes a protein, or a
fragment thereof, encompasses both the sense strand and its reverse
complement. Thus, for instance, it is appropriate to generate
probes or primers from the reverse complement sequence of the
disclosed nucleic acid molecules.
[0062] Dominant Model: A genetic based model that tests the
association of having at least one risk allele (e.g. Dd or DD)
versus not having a risk allele at all (dd).
[0063] End-stage kidney disease (ESKD) or end-stage renal disease
(ESRD): A stage of kidney impairment that is irreversible and
cannot be controlled by conservative management alone. ESKD
requires dialysis or kidney transplantation to maintain life.
[0064] European ancestry: A type of ancestry shared by people who
derived from the fertile crescent of the Middle East some 50,000
years ago and spread to occupy Europe, the Middle East, parts of
Eurasia and South Asia. There are a number of programs that can be
used to analyze DNA to determine if an individual is of African
ancestry, such as STRUCTURE.TM. (available on the internet at
pritch.bsd.uchicago.edu/structure.html) and EURODNA.TM. and
ANCESTRYBYDNA.TM. (available through the DNA print website). In one
example, European-American individuals are those individuals who
reside in the United States and self-identify themselves as being
of European origin. In another example, European-Americans are
individuals who reside in the United States and self-identify as
being of European origin, and are of European ancestry as
determined by a program that analyzes DNA ancestry.
[0065] Focal segmental glomerulosclerosis (FSGS): A clinical
syndrome involving podocyte injury and glomerular scarring, and
includes genetic forms with Mendelian inheritance, reactive forms
associated with other illnesses (including HIV-1 disease) or
medications, and an idiopathic form, which accounts for the
majority of cases. The name refers to the appearance of the kidney
tissue on biopsy: focal--only some of the glomeruli are involved;
segmental--only part of an entire glomerulus is involved;
glomerulosclerosis--scarring of the glomerulus. FSGS presents as a
nephrotic syndrome (which is characterized by edema (associated
with weight gain), hypoalbuminemia (low serum albumin (a protein)
in the blood), hyperlipidemia and hypertension (high blood
pressure)). In adults it may also present as kidney failure and
proteinuria, without a full-blown nephrotic syndrome.
[0066] There are five mutually exclusive variants of FSGS that can
be distinguished by the pathologic findings seen on renal biopsy:
collapsing variant, glomerular tip lesion variant, cellular
variant, perihilar variant, and not otherwise specified (NOS)
variant. Determining the type of variant can have prognostic value
in individuals with primary FSGS (where no underlying cause is
determined). The collapsing variant is associated with higher rate
of progression to end-stage renal disease, whereas glomerular tip
lesion variant has low rate of progression to end-stage renal
disease in most patients. The cellular variant shows a similar
clinical presentation to collapsing and glomerular tip variant but
has intermediate outcomes between these two variants.
[0067] Genetic predisposition: Susceptibility of a subject to a
disease, such as renal disease, including FSGS and hypertensive end
stage renal disease. Detecting a genetic predisposition can
include, but does not necessarily include, detecting the presence
of the disease itself, such as but not limited to an early stage of
the disease process. Detecting a genetic predisposition also
includes detecting the risk of developing the disease, and
determining the susceptibility of that subject to developing the
disease or to having a poor prognosis for the disease. Thus, if a
subject has a genetic predisposition to a disease they do not
necessarily develop the disease but are at risk for developing the
disease.
[0068] Genomic target sequence: A sequence of nucleotides located
in a particular region in the human genome that corresponds to one
or more specific genetic abnormalities, such as a nucleotide
polymorphism, a deletion, an insertion, or amplification. The
target can be for instance a coding sequence; it can also be the
non-coding strand that corresponds to a coding sequence. The target
can also be a non-coding sequence, such as an intronic sequence. In
some examples, genomic target sequences are genomic sequences of
genes that encode apolipoprotein L1 (APOL1).
[0069] Gene: A segment of DNA that contains the coding sequence for
a protein, wherein the segment may include promoters, exons,
introns, and other untranslated regions that control
expression.
[0070] Genotype: An unphased 5' to 3' sequence of nucleotide
pair(s) found at a set of one or more polymorphic sites in a locus
on a pair of homologous chromosomes in an individual. "Genotyping"
is a process for determining a genotype of an individual.
[0071] Haplotype: A 5' to 3' sequence of nucleotides found at a set
of one or more polymorphic sites in a locus on a single chromosome
from a single individual. "Haplotype pair" is the two haplotypes
found for a locus in a single individual. With regard to a
population, haplotypes are the ordered, linear combination of
polymorphisms (e.g., single nucleotide polymorphisms (SNPs)) in the
sequence of each form of a gene (on individual chromosomes) that
exist in the population. "Haplotyping" is a process for determining
one or more haplotypes in an individual and includes use of family
pedigrees, molecular techniques and/or statistical inference.
"Haplotype data" is the information concerning one or more of the
following for a specific gene: a listing of the haplotype pairs in
an individual or in each individual in a population; a listing of
the different haplotypes in a population; frequency of each
haplotype in that or other populations, and any known associations
between one or more haplotypes and a trait.
[0072] Haplotype block: Sites of closely located SNPs which are
inherited in blocks. A haplotype block includes a group of SNP
locations that do not appear to recombine independently and that
can be grouped together. Regions corresponding to blocks have a few
common haplotypes which account for a large proportion of
chromosomes. Identification of haplotype blocks is a way of
examining the extent of linkage disequilibrium (LD) in the genome.
The "Hap-Map" project (see the internet at the Hap-Map website)
describes the mapping of haplotype blocks in the human genome.
[0073] There are programs available on the internet for the
identification of haplotype blocks, such as the program
HAPBLOCK.TM. which runs on both PC and Unix and is available from
the University of Southern California website on the internet. A
further program, which in addition to block identification also has
visualization and selection of "tagging" SNPs is
HAPLOBLOCKFINDER.TM., which runs interactively on the web or can be
downloaded for local machine use (Unix or PC). It can be accessed
at the program website available on the internet.
[0074] Hispanic Ancestry: A person of Mexican, Puerto Rican, Cuban,
Dominican, South or Central American, or other Spanish or
Portuguese culture or origin, regardless of race.
[0075] Hybridization: Oligonucleotides and their analogs hybridize
by hydrogen bonding, which includes Watson-Crick, Hoogsteen or
reversed Hoogsteen hydrogen bonding, between complementary bases.
Generally, nucleic acids consist of nitrogenous bases that are
either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or
purines (adenine (A) and guanine (G)). These nitrogenous bases form
hydrogen bonds between a pyrimidine and a purine, and the bonding
of the pyrimidine to the purine is referred to as "base pairing."
More specifically, A will hydrogen bond to T or U, and G will bond
to C. "Complementary" refers to the base pairing that occurs
between two distinct nucleic acid sequences or two distinct regions
of the same nucleic acid sequence. For example, an oligonucleotide
can be complementary to a specific genetic locus, so it
specifically hybridizes with a mutant allele (and not the reference
allele) or so that it specifically hybridizes with a reference
allele (and not the mutant allele).
[0076] "Specifically hybridizable" and "specifically complementary"
are terms that indicate a sufficient degree of complementarity such
that stable and specific binding occurs between the oligonucleotide
(or its analog) and the DNA or RNA target. The oligonucleotide or
oligonucleotide analog need not be 100% complementary to its target
sequence to be specifically hybridizable. An oligonucleotide or
analog is specifically hybridizable when binding of the
oligonucleotide or analog to the target DNA or RNA molecule
interferes with the normal function of the target DNA or RNA, and
there is a sufficient degree of complementarity to avoid
non-specific binding of the oligonucleotide or analog to non-target
sequences under conditions where specific binding is desired, for
example under physiological conditions in the case of in vivo
assays or systems. Such binding is referred to as specific
hybridization. In one example, an oligonucleotide is specifically
hybridizable to DNA or RNA nucleic acid sequences including an
allele of a gene, wherein it will not hybridize to nucleic acid
sequences containing a polymorphism.
[0077] Hybridization conditions resulting in particular degrees of
stringency will vary depending upon the nature of the hybridization
method of choice and the composition and length of the hybridizing
nucleic acid sequences. Generally, the temperature of hybridization
and the ionic strength (especially the Na.sup.+ concentration) of
the hybridization buffer will determine the stringency of
hybridization, though wash times also influence stringency.
Calculations regarding hybridization conditions required for
attaining particular degrees of stringency are discussed by
Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd
ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989, chapters 9 and 11.
[0078] The following is an exemplary set of hybridization
conditions and is not limiting: [0079] Very High Stringency
(detects sequences that share at least 90% identity) [0080]
Hybridization: 5.times.SSC at 65.degree. C. for 16 hours [0081]
Wash twice: 2.times.SSC at room temperature (RT) for 15 minutes
each [0082] Wash twice: 0.5.times.SSC at 65.degree. C. for 20
minutes each [0083] High Stringency (detects sequences that share
at least 80% identity) [0084] Hybridization: 5.times.-6.times.SSC
at 65.degree. C.-70.degree. C. for 16-20 hours [0085] Wash twice:
2.times.SSC at RT for 5-20 minutes each [0086] Wash twice:
1.times.SSC at 55.degree. C.-70.degree. C. for 30 minutes each
[0087] Low Stringency (detects sequences that share at least 50%
identity) [0088] Hybridization: 6.times.SSC at RT to 55.degree. C.
for 16-20 hours [0089] Wash at least twice: 2.times.-3.times.SSC at
RT to 55.degree. C. for 20-30 minutes each.
[0090] Hypertension: High blood pressure; transitory or sustained
elevation of systemic arterial blood pressure to a level likely to
induce cardiovascular damage or other adverse consequences.
Hypertension has been arbitrarily defined as a systolic blood
pressure above 140 mm Hg or a diastolic blood pressure above 90 mm
Hg. Consequences of uncontrolled hypertension include retinal
vascular damage (Keith-Wagener-Barker changes), cerebrovascular
disease and stroke, left ventricular hypertrophy and failure,
myocardial infarction, dissecting aneurysm, and renovascular
disease. An underlying disorder (such as renal disease, Cushing
syndrome, pheochromocytoma) is identified in fewer than 10% of all
cases of hypertension. The remainder, traditionally labeled
"essential" hypertension, probably arise from a variety of
disturbances in normal pressure-regulating mechanisms (which
involve baroreceptors, autonomic influences on the rate and force
of cardiac contraction and vascular tone, renal retention of salt
and water, formation of angiotensin II under the influence of renin
and angiotensin-converting enzyme, and other factors known and
unknown), and most are probably genetically conditioned.
[0091] Hypertensive nephropathy (or "hypertensive nephrosclerosis,"
or "hypertensive renal disease" or "hypertensive kidney disease"):
A medical condition referring to damage to the kidney due to
chronic high blood pressure. In the kidneys, as a result of benign
arterial hypertension, hyaline (pink, amorphous, homogeneous
material) accumulates in the wall of small arteries and arterioles,
producing the thickening of their walls and the narrowing of the
lumens--hyaline arteriolosclerosis. Consequent ischemia produces
tubular atrophy, interstitial fibrosis, glomerular alterations
(smaller glomeruli with different degrees of hyalinization--from
mild to sclerosis of glomeruli) and periglomerular fibrosis. In
advanced stages ("end-stage"), renal failure will occur.
[0092] Isolated: An "isolated" biological component (such as a
nucleic acid molecule, protein or organelle) has been substantially
separated or purified away from other biological components in the
cell of the organism in which the component naturally occurs, e.g.,
other chromosomal and extra-chromosomal DNA and RNA, proteins and
organelles. Nucleic acids and proteins that have been "isolated"
include nucleic acids and proteins purified by standard
purification methods. The term also embraces nucleic acids and
proteins prepared by recombinant expression in a host cell as well
as chemically synthesized nucleic acids.
[0093] Linkage: The association of two or more loci at positions on
the same chromosome, such that recombination between the two loci
is reduced to a proportion significantly less than 50%. The term
linkage can also be used in reference to the association between
one or more loci and a trait if an allele (or alleles) and the
trait, or absence thereof, are observed together in significantly
greater than 50% of occurrences. A linkage group is a set of loci,
in which all members are linked either directly or indirectly to
all other members of the set.
[0094] Linkage Disequilibrium: Co-occurrence of two genetic loci
(e.g., markers) at a frequency greater than expected for
independent loci based on the allele frequencies. Linkage
disequilibrium (LD) typically occurs when two loci are located
close together on the same chromosome. When alleles of two genetic
loci (such as a marker locus and a causal locus) are in strong LD,
the allele observed at one locus (such as a marker locus) is
predictive of the allele found at the other locus (for example, a
causal locus contributing to a phenotypic trait). The linkage
disequilibrium (LD) measure r.sup.2 (the squared correlation
coefficient) can be used to evaluate how SNPs are related on a
haplotype block. For each tag SNP, the r.sup.2 between that tag SNP
and each additional SNP in a genotyping set can be calculated. The
highest of these values is the maximum r.sup.2 value, m. In several
embodiments, a haplotype block can be identified by SNPs that have
an r.sup.2 value of greater than or equal to 0.75, greater than or
equal to 0.8, greater than or equal to about 0.85, greater than or
equal to 0.9, or greater than or equal to 0.95 from the tag SNP. A
low r.sup.2 value such as less than or equal to 0.3, less than or
equal to 0.2, less than or equal to 0.1, is generally considered to
be less predictive than a higher r.sup.2 value, which is considered
a stronger predictor of linkage disequilibrium, such as greater
than or equal to 0.75.
[0095] Locus: A location on a chromosome or DNA molecule
corresponding to a gene or a physical or phenotypic feature, where
physical features include polymorphic sites.
[0096] Mutation: Any change of a nucleic acid sequence as a source
of genetic variation. For example, mutations can occur within a
gene or chromosome, including specific changes in non-coding
regions of a chromosome, for instance changes in or near regulatory
regions of genes. Types of mutations include, but are not limited
to, base substitution point mutations (which are either transitions
or transversions), deletions, and insertions. Missense mutations
are those that introduce a different amino acid into the sequence
of the encoded protein; nonsense mutations are those that introduce
a new stop codon; and silent mutations are those that introduce the
same amino acid often with a base change in the third position of
the codon. In the case of insertions or deletions, mutations can be
in-frame (not changing the frame of the overall sequence) or frame
shift mutations, which may result in the misreading of a large
number of codons (and often leads to abnormal termination of the
encoded product due to the presence of a stop codon in the
alternative frame).
[0097] Non-coding: A change in nucleotide sequence that does not
result in the production of a codon that encodes for an amino acid
other than the wild-type human sequence, and therefore does not
result in the production of any alteration in polypeptide sequence.
In the instant application, the term "non-coding" refers to the
exclusion of non-synonymous SNPs or haplotypes. In addition, the
term "non-coding" also excludes promoter regions of a gene and is
therefore limited to intronic and exonic domains of the gene.
[0098] Odds Ratio: A calculation performed by analysis of a two by
two contingency table. In one example, the first column provides a
risk indicator in the absence of a disease (e.g., FSGS). The second
column provides the same risk indicator in the presence of the same
disease. The first row lists the risk indicator in the absence of a
risk factor (such as race) and the second row lists the same risk
indicator in the presence of the same risk factor (e.g., race). The
Odds Ratio (OR) is determined as the product of the two diagonal
entries in the contingency table divided by the product of the two
off-diagonal entries of the contingency table. An OR of 1 is
indicative of no association. Accordingly, very large or very small
ORs are indicative of a strong association between the factors
under investigation. The OR is independent of the ratio of cases or
controls in a study, group or subset.
[0099] Oligonucleotide: An oligonucleotide is a plurality of joined
nucleotides joined by native phosphodiester bonds, between about 6
and about 300 nucleotides in length. An oligonucleotide analog
refers to moieties that function similarly to oligonucleotides but
have non-naturally occurring portions. For example, oligonucleotide
analogs can contain non-naturally occurring portions, such as
altered sugar moieties or inter-sugar linkages, such as a
phosphorothioate oligodeoxynucleotide. Functional analogs of
naturally occurring polynucleotides can bind to RNA or DNA, and
include peptide nucleic acid (PNA) molecules.
[0100] In several examples, oligonucleotides and oligonucleotide
analogs can include linear sequences up to about 200 nucleotides in
length, for example a sequence (such as DNA or RNA) that is at
least 6 bases, for example at least 8, 10, 15, 20, 25, 30, 35, 40,
45, 50, 100, or even 200 bases long, or from about 6 to about 70
bases, for example about 10-25 bases, such as 12, 15, or 20
bases.
[0101] Phased: As applied to a sequence of nucleotide pairs for two
or more polymorphic sites in a locus, phased means the combination
of nucleotides present at those polymorphic sites on a copy of the
DNA for the locus.
[0102] Polymorphism: A variation in a gene sequence. The
polymorphisms can be those variations (DNA sequence differences)
which are generally found between individuals or different ethnic
groups and geographic locations which, while having a different
sequence, produce functionally equivalent gene products. Typically,
the term can also refer to variants in the sequence which can lead
to gene products that are not functionally equivalent.
Polymorphisms also encompass variations which can be classified as
alleles and/or mutations which can produce gene products which may
have an altered function. Polymorphisms also encompass variations
which can be classified as alleles and/or mutations which either
produce no gene product or an inactive gene product or an active
gene product produced at an abnormal rate or in an inappropriate
tissue or in response to an inappropriate stimulus. Alleles are the
alternate forms that occur at the polymorphism.
[0103] Polymorphisms can be referred to, for instance, by the
nucleotide position at which the variation exists, by the change in
amino acid sequence caused by the nucleotide variation, or by a
change in some other characteristic of the nucleic acid molecule or
protein that is linked to the variation.
[0104] In the instant application "polymorphism" refers a
traditional definition, in that the definition "polymorphism" means
that the minor allele frequency must be greater than at least
1%.
[0105] A "single nucleotide polymorphism (SNP)" is a single base
(nucleotide) polymorphism in a DNA sequence among individuals in a
population. Typically in the literature, a single nucleotide
polymorphism (SNP) may fall within coding sequences of genes,
non-coding regions of genes, or in the intergenic regions between
genes. SNPs within a coding sequence will not necessarily change
the amino acid sequence of the protein that is produced, due to
degeneracy of the genetic code. A SNP in which both forms lead to
the same polypeptide sequence is termed "synonymous" (sometimes
called a silent mutation). If a different polypeptide sequence is
produced they are "nonsynonymous." A nonsynonymous change may
either be missense or "nonsense," where a missense change results
in a different amino acid, while a nonsense change results in a
premature stop codon.
[0106] A tag SNP is a SNP that by itself or in combination with
additional tag SNPs indicates the presence of a specific haplotype,
or of one member of a group of haplotypes. The haplotype or
haplotypes can indicate a genetic factor is associated with risk
for disease, thus a tag SNP or combination of tag SNPs indicates
the presence or absence of risk factors for disease. A "tag SNP" is
a representative single nucleotide polymorphism (SNP) in a region
of the genome with high linkage disequilibrium (the non-random
association of alleles at two or more loci) that is associated with
a disease, such as renal disease, for example FSGS or ESKD. A tag
SNP can be used to identify other SNPs, such as those with a
specified r.sup.2 value from the tag SNP, which are associated with
a disease, such as FSGS or ESKD. Statistical methods to identify a
tag SNP are known (see Hoperin et al., Bioinformatics 21 (suppl):
i1954203, 2005, herein incorporated by reference).
[0107] Predictive power: A characteristic for a dichotomous test
(one that will return either a positive or negative result),
indicating increased risk with a positive result. Predictive power
is measured by sensitivity and specificity. In some examples, the
sensitivity of a test is the fraction of people who tested positive
for the presence of at least one APOL1 risk allele who will develop
renal disease, such as FSGS or hypertensive ESKD and the
specificity is the fraction of people who tested negative for the
presence of at least one APOL1 risk allele (e.g., absence of at
least one risk allele) who will not develop renal disease. A
measure of the predictive power of a test is the receiver operator
characteristic (ROC) C statistic. The ROC C statistic may be
defined as the probability (or fraction of the time) that an
individual with the condition has a risk score larger than of an
individual without the condition. For a test with no predictive
power, the C statistic will be 0.5; for a dichotomous test that can
invariably correctly identify positives and negatives (perfect
predictive power), the C statistic will be 1.
[0108] Probes and primers: Isolated nucleic acids of at least ten
nucleotides capable of hybridizing to a target nucleic acid. A
detectable label or reporter molecule can be attached to a probe or
primer. Typical labels include radioactive isotopes, enzyme
substrates, co-factors, ligands, chemiluminescent or fluorescent
agents, haptens, and enzymes. Methods for labeling and guidance in
the choice of labels appropriate for various purposes are
discussed, for example in Sambrook et al. (In Molecular Cloning: A
Laboratory Manual, CSHL, New York, 1989) and Ausubel et al. (In
Current Protocols in Molecular Biology, John Wiley & Sons, New
York, 1998).
[0109] In a particular example, a probe or primer includes at least
one fluorophore, such as an acceptor fluorophore or donor
fluorophore. For example, a fluorophore can be attached at the 5'-
or 3'-end of the probe/primer. In specific examples, the
fluorophore is attached to the base at the 5'-end of the
probe/primer, the base at its 3'-end, the phosphate group at its
5'-end or a modified base, such as a T internal to the
probe/primer.
[0110] Probes are generally at least 15 nucleotides in length, such
as at least 15, at least 16, at least 17, at least 18, at least 19,
least 20, at least 21, at least 22, at least 23, at least 24, at
least 25, at least 26, at least 27, at least 28, at least 29, at
least 30, at least 31, at least 32, at least 33, at least 34, at
least 35, at least 36, at least 37, at least 38, at least 39, at
least 40, at least 41, at least 42, at least 43, at least 44, at
least 45, at least 46, at least 47, at least 48, at least 49, at
least 50 at least 51, at least 52, at least 53, at least 54, at
least 55, at least 56, at least 57, at least 58, at least 59, at
least 60, at least 61, at least 62, at least 63, at least 64, at
least 65, at least 66, at least 67, at least 68, at least 69, at
least 70, or more contiguous nucleotides complementary to the
target nucleic acid molecule, such as 20-70 nucleotides, 20-60
nucleotides, 20-50 nucleotides, 20-40 nucleotides, or 20-30
nucleotides.
[0111] Primers are short nucleic acid molecules, for instance DNA
oligonucleotides are 10 nucleotides or more in length, which can be
annealed to a complementary target nucleic acid molecule by nucleic
acid hybridization to form a hybrid between the primer and the
target nucleic acid strand. A primer can be extended along the
target nucleic acid molecule by a polymerase enzyme. Therefore,
primers can be used to amplify a target nucleic acid molecule.
[0112] The specificity of a primer increases with its length. Thus,
for example, a primer that includes 30 consecutive nucleotides will
anneal to a target sequence with a higher specificity than a
corresponding primer of only 15 nucleotides. Thus, to obtain
greater specificity, probes and primers can be selected that
include at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or
more consecutive nucleotides. In particular examples, a primer is
at least 15 nucleotides in length, such as at least 15 contiguous
nucleotides complementary to a target nucleic acid molecule.
Particular lengths of primers that can be used to practice the
methods of the present disclosure include primers having at least
15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, at least
25, at least 26, at least 27, at least 28, at least 29, at least
30, at least 31, at least 32, at least 33, at least 34, at least
35, at least 36, at least 37, at least 38, at least 39, at least
40, at least 45, at least 50, at least 55, at least 60, at least
65, at least 70, or more contiguous nucleotides complementary to
the target nucleic acid molecule to be amplified, such as a primer
of 15-70 nucleotides, 15-60 nucleotides, 15-50 nucleotides, or
15-30 nucleotides.
[0113] Primer pairs can be used for amplification of a nucleic acid
sequence, for example, by PCR, real-time PCR, or other nucleic-acid
amplification methods known in the art. An "upstream" or "forward"
primer is a primer 5' to a reference point on a nucleic acid
sequence. A "downstream" or "reverse" primer is a primer 3' to a
reference point on a nucleic acid sequence. In general, at least
one forward and one reverse primer are included in an amplification
reaction.
[0114] Nucleic acid probes and primers can be readily prepared
based on the nucleic acid molecules provided herein (such as
APOL1). It is also appropriate to generate probes and primers based
on fragments or portions of these disclosed nucleic acid molecules,
for instance regions that encompass the identified polymorphisms of
interest. PCR primer pairs can be derived from a known sequence by
using computer programs intended for that purpose such as Primer
(Version 0.5, .COPYRGT. 1991, Whitehead Institute for Biomedical
Research, Cambridge, Mass.) or PRIMER EXPRESS.RTM. Software
(Applied Biosystems, AB, Foster City, Calif.).
[0115] Recessive Model: A genetic based model that tests the
association of having two risk alleles (e.g. DD) versus having at
least one non-risk allele (e.g., Dd or dd). In some examples, it is
a genetic based model that tests the association of having two
copies of a specified allele versus having at least one copy of the
alternate (reference) allele.
[0116] Recombinant: A nucleic acid molecule, protein, cell, or
organism that results from the recombination of genes (e.g., a
sequence that is not naturally occurring or a sequence that is made
by an artificial combination of two otherwise separated segments of
sequence), regardless of whether naturally or artificially induced.
This artificial combination can be accomplished by chemical
synthesis or by the artificial manipulation of isolated segments of
nucleic acid molecules, such as by genetic engineering
techniques.
[0117] Reference Allele: A genotype that predominates in a natural
population of organisms that do not have a disease process, such as
renal disease, for example FSGS. In some examples, the reference
genotype differs from mutant forms. In other examples, the
reference allele is the alternative allele to a specified allele at
a specific locus.
[0118] Renal Disease (Nephropathy): A disorder that specifically
leads to damage of the kidneys. Renal diseases include but are not
limited to FSGS, hypertensive ESKD, nephropathy secondary to
systemic lupus erythematosus, diabetic nephropathy, hypertensive
nephropathy, IgA nephropathy, nephritis, and xanthine oxidase
deficiency.
[0119] Renal disease can be chronic or acute. Chronic renal
disease, or the type detected with the assays disclosed herein can
progress from stage 1 to stage 2, stage 3, stage 4 or stage 5. The
stages of chronic renal disease are:
[0120] Stage 1: Slightly diminished kidney function; Kidney damage
with normal or increased GFR (>90 mL/min/1.73 m2). Kidney damage
is defined as pathologic abnormalities or markers of damage,
including abnormalities in blood or urine test or imaging
studies.
[0121] Stage 2: Mild reduction in GFR (60-89 mL/min/1.73 m2) with
kidney damage. Kidney damage is defined as pathologic abnormalities
or markers of damage, including abnormalities in blood or urine
test or imaging studies.
[0122] Stage 3: Moderate reduction in GFR (30-59 mL/min/1.73
m2)
[0123] Stage 4: Severe reduction in GFR (15-29 mL/min/1.73 m2)
Stage 5: Established kidney failure (GFR<15 mL/min/1.73 m2, or
permanent renal replacement therapy (RRT)
[0124] The disclosed assays can be used to detect renal disease,
such as FSGS, at any of these stages, or prior to stage 1.
[0125] Risk Allele: A "risk" allele is an allele associated with a
particular type or form of disease. The risk allele identifies a
single nucleotide polymorphism that can be used to detect or
determine the risk for a disease, such as FSGS or hypertensive
ESKD.
[0126] Sample: A sample, such as a biological sample that includes
nucleic acid molecules, is a sample obtained from a subject. As
used herein, biological samples include all clinical samples useful
for detection of renal disease in subjects, including, but not
limited to, cells, tissues, and bodily fluids, such as: blood;
derivatives and fractions of blood, such as serum; extracted galls;
biopsied or surgically removed tissue, including tissues that are,
for example, unfixed, frozen, fixed in formalin and/or embedded in
paraffin; tears; milk; skin scrapes; surface washings; urine;
sputum; cerebrospinal fluid; prostate fluid; pus; or bone marrow
aspirates. In a particular example, a sample includes blood
obtained from a human subject, such as whole blood or serum. In
another particular example, a sample includes buccal cells, for
example collected using a swab or by an oral rinse.
[0127] Sequence identity/similarity: The identity/similarity
between two or more nucleic acid sequences, or two or more amino
acid sequences, is expressed in terms of the identity or similarity
between the sequences. Sequence identity can be measured in terms
of percentage identity; the higher the percentage, the more
identical the sequences are. Sequence similarity can be measured in
terms of percentage similarity (which takes into account
conservative amino acid substitutions); the higher the percentage,
the more similar the sequences are. Homologs or orthologs of
nucleic acid or amino acid sequences possess a relatively high
degree of sequence identity/similarity when aligned using standard
methods.
[0128] Methods of alignment of sequences for comparison are well
known in the art. Various programs and alignment algorithms are
described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981;
Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson &
Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins &
Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3,
1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et
al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson
et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol.
Biol. 215:403-10, 1990, presents a detailed consideration of
sequence alignment methods and homology calculations.
[0129] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul
et al., J. Mol. Biol. 215:403-10, 1990) is available from several
sources, including the National Center for Biological Information
(NCBI, National Library of Medicine, Building 38A, Room 8N805,
Bethesda, Md. 20894) and on the Internet, for use in connection
with the sequence analysis programs blastp, blastn, blastx, tblastn
and tblastx. Additional information can be found at the NCBI web
site.
[0130] BLASTN is used to compare nucleic acid sequences, while
BLASTP is used to compare amino acid sequences. To compare two
nucleic acid sequences, the options can be set as follows:--i is
set to a file containing the first nucleic acid sequence to be
compared (such as C:\seql.txt);--j is set to a file containing the
second nucleic acid sequence to be compared (such as
C:\seq2.txt);--p is set to blastn;--o is set to any desired file
name (such as C:\output.txt);--q is set to -1;--r is set to 2; and
all other options are left at their default setting. For example,
the following command can be used to generate an output file
containing a comparison between two sequences: C:\B12seq-i
c:\seql.txt-j c:\seq2.txt-p blastn-o c:\output.txt-q-1-r 2.
[0131] To compare two amino acid sequences, the options of B12seq
can be set as follows:--i is set to a file containing the first
amino acid sequence to be compared (such as C:\seql.txt);--j is set
to a file containing the second amino acid sequence to be compared
(such as C:\seq2.txt);--p is set to blastp;--o is set to any
desired file name (such as C:\output.txt); and all other options
are left at their default setting. For example, the following
command can be used to generate an output file containing a
comparison between two amino acid sequences: C:\B12seq
c:\seql.txt-j c:\seq2.txt-p blastp-o c:\output.txt. If the two
compared sequences share homology, then the designated output file
will present those regions of homology as aligned sequences. If the
two compared sequences do not share homology, then the designated
output file will not present aligned sequences.
[0132] Once aligned, the number of matches is determined by
counting the number of positions where an identical nucleotide or
amino acid residue is presented in both sequences. The percent
sequence identity is determined by dividing the number of matches
either by the length of the sequence set forth in the identified
sequence, or by an articulated length (such as 100 consecutive
nucleotides or amino acid residues from a sequence set forth in an
identified sequence), followed by multiplying the resulting value
by 100. For example, a nucleic acid sequence that has 1166 matches
when aligned with a test sequence having 1154 nucleotides is 75.0
percent identical to the test sequence (i.e., 1166/1554*100=75.0).
The percent sequence identity value is rounded to the nearest
tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down
to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up
to 75.2. The length value will always be an integer. In another
example, a target sequence containing a 20-nucleotide region that
aligns with 20 consecutive nucleotides from an identified sequence
as follows contains a region that shares 75 percent sequence
identity to that identified sequence (that is, 15/20*100=75).
[0133] One indication that two nucleic acid molecules are closely
related is that the two molecules hybridize to each other under
stringent conditions, as described above. Nucleic acid sequences
that do not show a high degree of identity may nevertheless encode
identical or similar (conserved) amino acid sequences, due to the
degeneracy of the genetic code. Changes in a nucleic acid sequence
can be made using this degeneracy to produce multiple nucleic acid
molecules that all encode substantially the same protein. Such
homologous nucleic acid sequences can, for example, possess at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%,
at least 98%, or at least 99% sequence identity (for example to a
known APOL1 gene sequence) determined by this method. An
alternative (and not necessarily cumulative) indication that two
nucleic acid sequences are substantially identical is that the
polypeptide which the first nucleic acid encodes is immunologically
cross reactive with the polypeptide encoded by the second nucleic
acid. One of skill in the art will appreciate that the particular
sequence identity ranges are provided for guidance only.
[0134] Subject: Living multi-cellular vertebrate organisms, a
category that includes human and non-human mammals (such as
laboratory or veterinary subjects).
[0135] Therapeutically effective amount: An amount of a therapeutic
agent that alone, or together with one or more additional
therapeutic agents, induces the desired response. In one example,
the desired response is decreasing the risk of developing FSGS or
decreasing the signs and symptoms of FSGS. In another example, the
desired response is ameliorating signs or symptoms associated with
a Trypanosoma brucei infection, such as sleeping sickness. For
example, a therapeutically effective amount of a human APOL1
protein comprising a S342G substitution, a I384M substitution
and/or a deletion removing amino acids N388 and Y389, can be used
to decrease symptoms associated with sleeping sickness, such as
fever, headache, joint pain, lymph node swelling, anemia,
confusion, reduced coordination, and disruption of the sleep
cycle.
[0136] Ideally, a therapeutically effective amount provides a
therapeutic effect without causing a substantial cytotoxic effect
in the subject. The preparations disclosed herein are administered
in therapeutically effective amounts. In general, a therapeutically
effective amount of a composition administered to a human subject
will vary depending upon a number of factors associated with that
subject, for example the overall health of the subject, the
condition to be treated, or the severity of the condition. A
therapeutically effective amount of a composition can be determined
by varying the dosage of the product and measuring the resulting
therapeutic response. The therapeutically effective amount can be
dependent on the source applied, the subject being treated, the
severity and type of the condition being treated, and the manner of
administration.
[0137] In one example, a desired response is to prevent the
development of FSGS. In another example, a desired response is to
delay the development or progression of FSGS, for example, by at
least about three months, at least about six months, at least about
one year, at least about two years, at least about five years, or
at least about ten years. In another example, a desired response is
to decrease the signs and symptoms of FSGS, such as the
neurological symptoms in the limbs or associated with speaking.
[0138] Treatment: With respect to disease, either term includes (1)
preventing the disease, e.g., causing the clinical symptoms of the
disease not to develop in an animal that may be exposed to or
predisposed to the disease but does not yet experience or display
symptoms of the disease, (2) inhibiting the disease, e.g.,
arresting the development of the disease or one or more of its
clinical symptoms, or (3) relieving the disease, e.g., causing
regression of the disease or one or more of its clinical symptoms.
For example, treatment can refer to relieving one or more symptoms
associated with Trypanosoma infection, such as sleeping sickness.
Treatment of a disease does not require a total absence of disease.
For example, a decrease of at least 25% or at least 50% of one or
more of the symptoms or undesired consequences of the disease can
be sufficient.
[0139] Trypanosoma brucei: A parasite that causes African
trypanosomiasis (sleeping sickness) in humans and nagana in animals
in Africa. The insect vector for T. brucei is the tsetse fly. There
are three sub-species of T. brucei: T. b. brucei, T. b. rhodesiense
and T. b. gambiense. A "disease associated with Trypanosoma
infection" includes those diseases that result from infection with
T. brucei, such as sleeping sickness. For example, infection by T.
brucei gambiense causes slow onset chronic trypanosomiasis in
humans while infection by T. brucei rhodesiense causes fast onset
acute trypanosomiasis in humans. T. brucei brucei infection causes
animal African trypanosomiasis.
II. Methods for Detecting a Genetic Predisposition to and/or an
Increased Risk of Developing Renal Disease
[0140] Methods for determining the genetic predisposition to, or an
increased risk of, development of a renal disease in a subject are
provided herein. Disclosed herein are methods for determining the
genetic predisposition to FSGS, as well as methods for determining
the genetic predisposition of a subject to hypertensive end-stage
kidney disease (EKSD). However, the methods disclosed herein also
can be used to detect any form of renal disease, such as, but not
limited to, FSGS or EKSD. The methods also can be used to determine
the risk of developing renal disease. The methods are also useful
in genetic confirmations of a diagnosis of renal disease, or to
determine a therapeutic regimen for a subject. The methods are
useful not only in determining risk, but for genetic confirmation
of suspected chronic renal disease, for example a subject who
presents with a reduced glomerular filtration rate (GFR) or other
laboratory evidence of renal impairment (such as elevated blood
urea nitrogen (BUN) or abnormal renal histology), of someone with
clinical presentation (symptoms) of renal disease, such as fatigue
and liquid retention. The methods disclosed herein can be used to
determine the genetic predisposition, detect, or determine the risk
of developing nephropathy secondary to systemic lupus erythematosus
and other nephropathies. The methods are also useful for
determining a therapeutic regimen in treating a subject of
interest, or determining if a subject will benefit from treatment
with a therapeutic regimen of interest. In patients having one or
more APOL1 gene risk alleles, treatment while the patient is
asymptomatic may be warranted.
[0141] In some embodiments, the method includes detecting the
presence of a genotype (e.g., the presence of at least one risk
allele), such as at least one single nucleotide polymorphism (SNP)
and/or at least one inversion in a subject (e.g., one or more of
the SNPs described herein, such as the G1 and/or G2 risk alleles,
or an inversion in the APOL1 gene, such as the G3 allele), such
that the presence of the at least one SNP and/or the at least one
inversion determines genetic predisposition to (and/or increased
risk of developing) renal disease in the subject. In other
embodiments, the method includes detecting the presence of a
genotype, such that both alleles of the genotype of the subject are
risk alleles that indicate a genetic predisposition to renal
disease in the subject. In other embodiments, the method includes
detecting the presence of a tag SNP. In further embodiments, the
method includes detecting the presence of a genotype, such that one
of the alleles of the genotype is a risk allele of a tag SNP. In
still other embodiments, the method includes determining whether
the subject is heterozygous for an APOL1 risk allele (e.g., the
subject has one risk and one non-risk allele at a given locus) or
whether the subject is homozygous for at least one APOL1 risk
allele (e.g., whether both alleles of the subject at a given locus
include an APOL1 risk allele). In an embodiment of the method,
determination that a subject is heterozygous for an APOL1 risk
allele at a given locus indicates the subject has an increased risk
of renal disease relative to a subject that is homozygous at that
locus for a non-risk APOL1 allele, and determination that a subject
is homozygous for an APOL1 risk allele at a given locus indicates
the subject has a substantially increased risk of renal disease
relative to a subject that is homozygous at that locus for a
non-risk APOL1 allele.
[0142] In some embodiments, the method uses two or more SNPs and/or
tag SNPs (alone or in combination with one or more inversions) to
identify the presence in the genome of a subject of one or two or
more risk haplotypes. In some embodiments, both of the haplotypes
identified as carried by the subject are copies of a risk
haplotype. In other embodiments, one of the haplotypes is a risk
haplotype.
[0143] In some embodiments, the method includes detecting the
presence of at least one SNP and at least one inversion in a gene
of interest, for example, APOL1.
[0144] In some embodiments, the methods disclosed herein can be
used to determine the genetic predisposition of a human subject to
renal disease, wherein the subject is of African ancestry, such as
an African-American subject (a subject who is of African ancestry
who resides in the United States) or an African-European subject (a
subject who is of African ancestry who resides in Europe) or a
subject of Hispanic ancestry. In additional embodiments, the
methods disclosed herein can be used to determine the genetic
predisposition of a human subject to renal disease, wherein the
subject is of European ancestry. The human subject can
self-identify themselves (such as on a questionnaire) as being of
European ancestry, such as identifying themselves as Caucasian.
There are a number of programs available to confirm European
ancestry, if such confirmation is desired. These include the
program STRUCTURE.TM. (available on the internet at
pritch.bsd.uchicago.edu/structure.html) and the program
EURASIANDNA.TM., version 1.0 and 2.0 (available from DNAPRINT.TM.).
In other embodiments, the subject can self-identify themselves
(such as on a questionnaire) as being of a specific ancestry.
However, there are a number of programs available to confirm
ancestry, if such confirmation is desired. These include the
program STRUCTURE.TM. (available on the internet at
pritch.bsd.uchicago.edu/structure.html). In several examples, the
subject is infected with a human immunodeficiency virus, such as
HIV-1 or HIV-2.
[0145] In some embodiments, the methods include obtaining a sample
including nucleic acids from a human subject of interest, and
analyzing the sample for the presence of at least one SNP and/or at
least one inversion, or a haplotype including at least one tag SNP
in these nucleic acids. In other embodiments, a sample is obtained
that contains nucleic acids from a human subject of interest, and
the sample is analyzed for the presence of a haplotype including at
least two tag SNPs in a non-coding region of a gene of interest.
The methods can include selecting a subject in need of detecting
the presence of the SNP, and obtaining a sample including nucleic
acids from this subject. For example, a subject can be selected who
is suspected to possess a genetic predisposition to renal disease,
such as FSGS or hypertensive ESKD. In another example, a subject
can be selected that is of African ancestry and/or is infected with
HIV. In a further example, a subject can be selected who has renal
disease, such as, but not limited to FSGS or hypertensive ESKD.
Thus, the subject's risk for progressing to another stage of renal
disease can be detected. The methods disclosed herein can also be
used to confirm the presence of renal disease in the subject. In
yet another example, a subject with renal disease is selected to
determine if a particular therapeutic regimen is appropriate for
the subject. A subject of interest (e.g., an asymptomatic subject)
can also be selected for preventative or prophylactic treatment
based on the presence of at least one risk allele.
[0146] Biological samples include all clinical samples useful for
detection of renal disease in subjects, such as cells, tissues, and
bodily fluids, for example blood; derivatives and fractions of
blood, such as serum; extracted galls; biopsied or surgically
removed tissue, including tissues that are, for example, unfixed,
frozen, fixed in formalin and/or embedded in paraffin; tears; milk;
skin scrapes; surface washings; urine; sputum; cerebrospinal fluid;
prostate fluid; pus; or bone marrow aspirates. In a particular
example, a sample includes blood obtained from a human subject,
such as whole blood or serum. In another particular example, a
sample includes buccal cells, for example collected using a swab or
by an oral rinse. In additional embodiments, the method includes
analyzing DNA sequence data previously obtained from the subject of
interest.
APOL1 SNPs and Inversions
[0147] In one example, a method for detecting genetic
predisposition to, or increased risk of developing, a renal
disease, such as FSGS or hypertensive ESKD, in a human subject is
performed by detecting the presence of at least one SNP and/or at
least one inversion in an APOL1 gene (e.g., an inversion that
replaces all or a portion of the first, second, and/or third exon
of the APOL1 gene with another apolipoprotein gene, such as the
APOL4 gene). In particular examples, specific SNPs of use in
identifying a genetic predisposition to renal disease (for example,
in a subject of African ancestry, such as an African-American
subject) include a G at rs73885319, a G at rs60910145, a 6 bp
deletion (-/TTATAA; SEQ ID NO:6) at rs71785313, and/or combinations
thereof. In some examples SNP rs73885319 results in a substitution
of glycine for serine at amino acid 342 of an APOL1 protein
(S342G). In other examples, SNP rs60910145 results in a
substitution of methionine for isoleucine at amino acid 384 of an
APOL1 protein (1384). In further examples, SNP rs71785313 results
in a deletion of amino acids 388 and 389 of an APOL1 protein. In
other examples, a specific inversion is the G3 inversion discussed
below).
[0148] The method can also include detecting one of more of the
APOL1 SNPs and/or inversions disclosed herein. Thus, the method can
include detecting at least one, at least two, or at least three
different SNPs and/or at least one, two, or three different
inversions. In some embodiments, the SNPs can be in any
combination, of at least two different SNPs. Detection of all of
the SNPs disclosed herein can also be used to detect a genetic
predisposition to renal disease, such as FSGS or hypertensive
ESKD.
[0149] In several embodiments, at least one SNP is detected in a
coding region of an APOL1 gene. Thus, the method can include
detecting at least one, at least two, or at least three different
SNPs in the coding region of an APOL1 gene, wherein at least one or
more SNPs in the coding region of the gene is a G at rs73885319, a
G at rs60910145, and/or a 6 bp deletion (-/TTATAA; SEQ ID NO: 6) at
rs71785313. In some examples, a G at rs73885319 includes an APOL1
nucleic acid having a G at nucleotide 1024 of SEQ ID NO: 4. In some
examples, a G at rs60910145 includes an APOL1 nucleic acid having a
G at nucleotide 1052 of SEQ ID NO: 4. In some examples, a 6 bp
deletion at rs71785313 includes an APOL1 nucleic acid having a
deletion of nucleotides 1064-1069 of SEQ ID NO: 4.
[0150] With regard to the SNPs, the SNPs can be identified by name.
The exact sequence of the SNP can be determined from the database
of SNPs available at the NCBI website (ncbi.nlm.nih.gov/SNP, Apr.
18, 2010). The "position" of the nucleotide of interest is the
location in the genome of the SNP, referring to the nucleotide
position from the p-terminus of the chromosome in the human genome,
see the NCBI SNP website, available on the internet. Sequence
information for each of the APOL1 SNPs listed above is provided in
table 1.
TABLE-US-00001 TABLE 1 APOL1 single nucleotide polymorphisms Risk
Reference SNP allele allele Flanking sequence rs73885319 G A
TCAAGCTCACGGATGTG GCCCCTGTA[G/A]GCT TCTTTCTTGTGCTGGAT GTAGT (SEQ ID
NO: 1) rs60910145 G T CAGGAGCTGGAGGAGAA GCTAAACAT[G/T]CTC
AACAATAATTATAAGAT TCTGC (SEQ ID NO: 2) rs71785313 del6 TTATAA
GAGAAGCTAAACATTCT (SEQ ID CAACAATAA[-/TTATA NO: 6)
A]GATTCTGCAGGCGGA CCAAGAACTG (SEQ ID NO: 3)
[0151] In Table 1, the "risk" allele identifies the SNP that can be
used to detect or determine the risk for renal disease, such as
FSGS or hypertensive ESKD. The "reference" allele is a different
allele not associated with renal disease, and thus is a "protective
allele" as this allele indicates that the subject does not have or
is not at risk for developing renal disease, such as FSGS or
hypertensive ESKD. In the sequences provided above, the notation
"[X/Y]" is used, wherein one of X or Y is the risk allele and one
of X or Y is the reference (protective) allele. For each sequence,
the allele associated with renal disease (the "risk" allele) is
listed. The allele that is associated with a decreased risk (or
absence) of renal disease is also listed (the "reference"
allele).
[0152] Another risk allele that can be used to detect or determine
the risk for renal disease, such as FSGS or hypertensive ESKD, is
an APOL1 gene chromosomal rearrangement that inverts a segment of
DNA including the 5' end of APOL4, all of APOL2, and the 5' end of
APOL1, which produces an APOL4/APOL1 hybrid gene. The inversion is
referred to herein as the "G3" risk allele.
[0153] The disclosed methods can include detecting at least one
risk allele (e.g., G1, G2, del6, and/or G3) on one or both
chromosomes, detecting the presence of a protective allele on one
or both chromosomes, or detecting the absence of the protective
allele on one or both chromosomes. In some embodiments, detecting
the presence of the risk allele indicates that the subject has a
genetic predisposition to renal disease, and detecting the absence
of the protective allele indicates that the subject has a genetic
predisposition to renal disease. Similarly, detecting the absence
of the risk allele indicates that the subject does not have a
genetic predisposition to renal disease, and detecting the presence
of the protective allele indicates that the subject does not have a
genetic predisposition to renal disease.
[0154] Thus, the disclosed methods can detect a low risk of
developing renal disease, or identify a subject that does not have
a genetic pre-disposition to developing renal disease. For example,
subjects that have at least one SNP associated with the reference
allele are not genetically pre-disposed to developing renal
disease, such as FSGS or hypertensive ESKD. These subjects do not
have renal disease and/or have a low risk for developing renal
disease.
[0155] In subjects of African ancestry, the methods include
detecting the presence at least one SNP (e.g., G1, G2, and/or del6)
in the last (3') exon of the APOL1 gene or at least one inversion
in an APOL1 gene (e.g., G3). An exemplary nucleic acid sequence for
human apolipoprotein L1 is:
TABLE-US-00002 (SEQ ID NO: 4) atggagggag ctgctttgct gagagtctct
gtcctctgca tctggatgag tgcacttttc cttggtgtgg gagtgagggc agaggaagct
ggagcgaggg tgcaacaaaa cgttccaagt gggacagata ctggagatcc tcaaagtaag
cccctcggtg actgggctgc tggcaccatg gacccagaga gcagtatctt tattgaggat
gccattaagt atttcaagga aaaagtgagc acacagaatc tgctactcct gctgactgat
aatgaggcct ggaacggatt cgtggctgct gctgaactgc ccaggaatga ggcagatgag
ctccgtaaag ctctggacaa ccttgcaaga caaatgatca tgaaagacaa aaactggcac
gataaaggcc agcagtacag aaactggttt ctgaaagagt ttcctcggtt gaaaagtgag
cttgaggata acataagaag gctccgtgcc cttgcagatg gggttcagaa ggtccacaaa
ggcaccacca tcgccaatgt ggtgtctggc tctctcagca tttcctctgg catcctgacc
ctcgtcggca tgggtctggc acccttcaca gagggaggca gccttgtact cttggaacct
gggatggagt tgggaatcac agccgctttg accgggatta ccagcagtac catggactac
ggaaagaagt ggtggacaca agcccaagcc cacgacctgg tcatcaaaag ccttgacaaa
ttgaaggagg tgagggagtt tttgggtgag aacatatcca actttctttc cttagctggc
aatacttacc aactcacacg aggcattggg aaggacatcc gtgccctcag acgagccaga
gccaatcttc agtcagtacc gcatgcctca gcctcacgcc cccgggtcac tgagccaatc
tcagctgaaa gcggtgaaca ggtggagagg gttaatgaac ccagcatcct ggaaatgagc
agaggagtca agctcacgga tgtggcccct gtaagcttct ttcttgtgct ggatgtagtc
tacctcgtgt acgaatcaaa gcacttacat gagggggcaa agtcagagac agctgaggag
ctgaagaagg tggctcagga gctggaggag aagctaaaca ttctcaacaa taattataag
attctgcagg cggaccaaga actgtga
[0156] An exemplary amino acid sequence for human apolipoprotein L1
is:
TABLE-US-00003 (SEQ ID NO: 5) megaallrvs vlciwmsalf lgvgvraeea
garvqqnvps gtdtgdpqsk plgdwaagtm dpessified aikyfkekvs tqnllllltd
neawngfvaa aelprneade lrkaldnlar qmimkdknwh dkgqqyrnwf lkefprlkse
lednirrlra ladgvqkvhk gttianvvsg slsissgilt lvgmglapft eggslvllep
gmelgitaal tgitsstmdy gkkwwtqaqa hdlviksldk lkevreflge nisnflslag
ntyqltrgig kdiralrrar anlqsvphas asrprvtepi saesgeqver vnepsilems
rgvkltdvap vsfflvldvv ylvyeskhlh egaksetaee lkkvaqelee klnilnnnyk
ilqadqel
[0157] Methods are also disclosed for detection of a genetic
predisposition to renal disease, such as FSGS or hypertensive ESKD,
or both in a human subject of European ancestry. The assay can be
used for early diagnosis, for example before the development of
renal insufficiency or renal failure, or for confirming the
diagnosis of renal disease. The presence of at least one SNP or at
least one inversion in an APOL1 gene that encodes apolipoprotein L1
determines the genetic predisposition to FSGS or hypertensive ESKD
or both in the human subject of European ancestry. In one
embodiment, the method includes detecting at least one of a G at
rs73885319, a G at rs60910145, and/or a 6 bp deletion (-/TTATAA;
SEQ ID NO: 6) at rs71785313 and/or at least one inversion. In a
further example, the method includes detecting the absence of at
least one of a G at rs73885319, a G at rs60910145, and/or a 6 bp
deletion (-/TTATAA; SEQ ID NO: 6) at rs71785313 and/or at least one
inversion. In some examples, a G at rs73885319 includes an APOL1
nucleic acid having a G at nucleotide 1024 of SEQ ID NO: 4. In some
examples, a G at rs60910145 includes an APOL1 nucleic acid having a
G at nucleotide 1052 of SEQ ID NO: 4. In some examples, a 6 bp
deletion at rs71785313 includes an APOL1 nucleic acid having a
deletion of nucleotides 1064-1069 of SEQ ID NO: 4.
[0158] In a further embodiment, the frequency of the risk allele in
subjects of African ancestry is at least 5%, at least 10%, at least
15%, at least 20%, at least 25%, at least 30%, at least 35%, at
least 40% or at least 50%. In several instances, the SNP and/or
inversion is in a coding region of an APOL1 gene. In several
embodiments, the SNP used to identify the frequency of the risk
allele in subjects of African ancestry is set forth in Table 1 and
also may include at least one inversion. In one embodiment, the
subject of African ancestry is African-American.
[0159] In other embodiments, the risk of renal disease in a subject
of African or Hispanic ancestry increases if the subject has at
least one risk allele (e.g., at least one of a G1, G2, del6, and/or
G3 allele). Subjects of African or Hispanic ancestry that have at
least two (or more) of APOL1 gene risk alleles exhibit a
significantly increased risk of developing renal disease. The
methods of the invention may further include assaying the subject
for the presence of a wild type allele (relative to an APOL1 gene
risk allele) as a means for determining whether the subject has a
moderate or increased risk of renal disease. For example, a subject
that is heterozygous at a given locus for one or more of the APOL1
gene risk alleles may have a greater risk of renal disease relative
to a subject lacking any APOL1 gene risk alleles. A subject that is
homozygous at a given locus for one or more APOL1 gene risk alleles
may have a risk of renal disease that is greater than that of a
subject that is heterozygous for an APOL1 gene risk allele at that
locus and a subject that lacks any risk alleles in an APOL1 gene.
The presence of two or more (e.g., three, four, or more) risk
alleles at different loci further increases the likelihood of renal
disease in a subject.
[0160] In other embodiments, a subject having one or more (e.g.,
two, three, or four or more) APOL1 gene risk alleles (e.g., at
least one SNP, e.g., G1, G2, and/or del6, and/or at least one
inversion, such as the G3, risk allele; the subject may also be
heterozygous or homozygous for one or more of these risk alleles)
may be offered a treatment regimen that is different from that of a
subject having no or only one APOL1 gene risk alleles. For example,
a subject having one or more APOL1 gene risk alleles may be treated
with a medication or therapy to reduce or prevent renal disease
while the subject is asymptomatic (e.g., the subject may be
subjected to a change in diet, an increase in exercise, a reduction
in the intake of NSAIDs, a regimen of blood pressure medication(s)
(see list below) that do not produce a renal toxicity profile,
hemodialysis, peritoneal dialysis, or transplantation). The
treatment of such a patient may begin at a time point that is
earlier than that for a subject having no or only one APOL1 gene
risk allele; the amount of medication that is prescribed to such a
patient may be increased or decreased in order to avoid further
harm to the kidneys; or the type(s) of medication(s) may be
adjusted, relative to a subject having no or only one APOL1 risk
allele.
[0161] In other embodiments, a subject having one or more APOL1
gene risk alleles may be offered a treatment regimen with respect
to blood pressure medications, steroids, and/or immunosuppressive
agents, that is different from a subject lacking any (or only
having, e.g., one) APOL1 gene risk allele. In particular, subjects
having one or more APOL1 gene risk alleles are more susceptible to
renal damage and/or disease and the risk of kidney damage increases
in patients having one or more APOL1 gene risk alleles that are
treated with blood pressure medications, steroids, and/or
immunosuppressive agents that exhibit renal toxic side effects.
Thus, in patients having one or more APOL1 gene risk alleles, the
concentration of a given blood pressure medication, steroid, and/or
immunosuppressive agent and/or the length of treatment may be
decreased relative to a patient lacking any (or having only one)
APOL1 gene risk alleles to avoid damage to the patient's kidneys.
The change in therapeutic regimen in patients having one or more
APOL1 gene risk alleles may occur while the patients are
asymptomatic.
[0162] Examples of therapeutics include blood pressure medications
(e.g., a diuretic (e.g., chlorthalidone, chlorothiazide,
furosemide, hydrochlorothiazide, indapamide, metolazone, amiloride
hydrochloride, spironolactone, triamterene, bumetanide, or a
combination thereof), an alpha adrenergic antagonist (e.g.,
alfuzosin, doxazosin, prazosin, terazosin, or tamsulosin, or a
combination thereof), a central adrenergic inhibitor (e.g.,
clonidine, guanfacine, or methyldopa, or a combination thereof), an
angiotensin converting enzyme (ACE) inhibitor (e.g., benazepril,
captopril, enalapril, fosinopril, lisinopril, moexipril,
perindopril, quinapril, ramipril, or trandolapril, or combinations
thereof), an angiotensin II receptor blocker (e.g., candesartan,
eprosartan, irbesartan, losartan, olmesartan, telmisartan, or
valsartan, or combinations thereof), an alpha blocker (e.g.,
doxazosin, prazosin, or terazosin, or a combination thereof), a
beta blocker (e.g., acebutolol, atenolol, betaxolol, bisoprolol,
carteolol, metoprolol, nadolol, nebivolol, penbutolol, pindolol,
propranolol, solotol, or timolol, or a combination thereof), a
calcium channel blocker (e.g., amlodipine, bepridil, diltiazem,
felodipine, isradipine, nicardipine, nifedipine, nisoldipine, or
verapamil, or combination thereof), a vasodilator (e.g.,
hydralazine or minoxidil, or combination thereof), and a renin
inhibitor (e.g., aliskiren), or combinations thereof), a steroid
(e.g., a corticosteroid, such as cortisone, prednisone,
methylprednisolone, or prednisolone), or an anabolic steroid
(anatrofin, anaxvar, annadrol, bolasterone, decadiabolin,
decadurabolin, dehydropiandrosterone (DHEA), delatestryl,
dianiabol, dihydrolone, durabolin, dymethazine, enoltestovis,
equipose, gamma hydroxybutyrate, maxibolin, methatriol,
methyltestosterone, parabolin, primobolin, quinolone, therabolin,
trophobolene, and winstrol), or an immunosuppressive agent, such as
a glucocorticoid, a cytostatic, an antibody, or an
anti-immunophilin and/or mychophenolate mofetil (MMF), FK-506,
azathioprine, cyclophosphamide, methotrexate, dactinomycin,
antithymocyte globulin (ATGAM), an anti-CD20-antibody, a
muromonoab-CD3 antibody, basilizimab, daclizumab, cyclosporin,
tacrolimus, voclosporin, sirolimus, an interferon, infliximab,
etanercept, adalimumab, fingolimod, and/or myriocin).
[0163] Subjects having African ancestry (including some subjects of
Hispanic ancestry) exhibit a 35-45% increased risk of renal disease
when that subject is determined to have at least one APOL1 gene
risk allele (e.g., G1, G2, del6, and or G3). The risk of FSGS
increases by 10-fold in these subjects. Surprisingly, the risk of
HIV-associated nephropathy increases by 50-fold in subjects having
at least one risk allele. In addition, the risk of ESKD increases
by 7-8 fold in subjects having at least one risk allele. These risk
factors are not seen in non-African patients lacking one or more of
these risk alleles.
[0164] In a typical population of subjects of African ancestry, at
least 10-15% of the population is at high risk of renal disease due
to the presence of one or more risk alleles. Thirty percent of
these subjects are at slight or increased risk, while 55% are at
low risk of renal disease. Those subjects having two risk alleles
are at the greatest risk of renal disease. The rate of renal
disease in subjects of non-African ancestry is essentially the same
for subjects of African ancestry with 0 or, in some instances, 1
risk allele. Thus, the presence of APOL1 risk alleles account for
most of the large increase in renal disease risk in black compared
to white individuals.
[0165] Kidney Transplantation
[0166] A subject in need of kidney transplantation can also be
genotyped for the presence of at least one risk allele in the APOL1
gene disclosed herein. It is known that individuals of African
ancestry, including those individuals of Hispanic ancestry and, in
particular, African-Americans, have an elevated risk for carrying
one or two copies of at least one risk allele the APOL1 gene, which
increases their risk of developing idiopathic kidney disease. Thus,
in one embodiment, a kidney recipient can be genotyped to determine
if the recipient carries one or two copies of at least one of the
disclosed risk alleles the APOL1 gene. Additionally, a kidney
selected for transplantation can undergo genotyping prior to
surgery to establish the genotype status of the organ.
[0167] In some embodiments, if the recipient is negative for risk
alleles in the APOL1 gene and the donor kidney is positive for risk
alleles in the APOL1 gene, then the recipient is given pre- and/or
post-transplantation treatment regimens that reduce the risk of the
donated kidney undergoing subsequent kidney failure. Additionally,
it may be necessary to treat a subject who is to receive a kidney
that is positive for one or more risk alleles in the APOL1 gene
differently from a subject who is to receive a kidney that does not
possess an APOL1 risk allele. Therapeutic treatment and regimens
can therefore be developed after genotyping of a subject or an
organ for APOL1 genotype. These treatment regimens may include
decreasing the dosage of, or the length of treatment with, one or
more therapeutics in those individuals having at least one (e.g.,
two or more) risk alleles. These therapeutics include blood
pressure medications, steroids, and immunosuppressive agents (see
list above).
[0168] In other embodiments, the determination that a potential
transplantation donor has one or more risk alleles in the APOL1
gene (e.g., at least one risk allele at a given locus on one or
both chromosomes) indicates that an organ (e.g., a kidney) of the
donor is not suitable or has a lower suitability for transplant
into a recipient relative to a potential transplant donor that
lacks one or more risk alleles in the APOL1 gene (e.g., at least
one risk allele at a given locus on one or both chromosomes).
III. Methods for Identifying Resistance to Infection by
Trypanosoma
[0169] APOL1 is a trypanolytic factor of human serum (Vanhamme et
al., Nature 422:83-87, 2003; Perez-Morga et al., Science
309:469-472, 2005). The APOL1 variants disclosed herein exhibit the
ability to kill Trypanosoma brucei, the parasite responsible for
sleeping sickness disease. Therefore, the disclosed APOL1 variants
can be used to detect resistance of a subject (for example, a
mammal, such as a human subject) to a disease associated with
Trypanosoma infection.
[0170] Trypanosoma brucei is a heterotrophic species from the
Trypanosoma genus. It exists in two forms: an insect vector, and
once inside the bloodstream, a mammalian host. T. brucei exists as
its insect vector in the tsetse fly, a large, biting fly
originating in Africa. Once the tsetse fly bites a mammal, the
microbe enters the bloodstream where it transforms into the
mammalian host form, and is then capable of mutating and invading
the central nervous system, (CNS). Once inside the CNS, it has the
ability to inflict African trypanosomiasis, (sleeping
sickness).
[0171] There are three sub-species of T. brucei: T. b. brucei, T.
b. gambiense, and T. b. rhodesiense. T. b. gambiense causes slow
onset chronic trypanosomiasis in humans. It is most common in
central and western Africa, where humans are thought to be the
primary reservoir. T. brucei rhodesiense causes fast onset acute
trypanosomiasis in humans and is most common in southern and
eastern Africa, where game animals and livestock are thought to be
the primary reservoir. T. brucei brucei causes animal African
trypanosomiasis. T. b. brucei is generally not human infective due
to its susceptibility to lysis by human apolipoprotein L1. T. b.
gambiense parasites can further be divided into two types, type 1,
which is homogeneous and clearly distinct from T. b. rhodesiense,
and type 2, which is heterogeneous and shares characteristics with
T. b. rhodesiense.
[0172] In one example, a method for detecting resistance to a
disease associated with Trypanosoma (such as sleeping sickness) in
a human subject is performed by detecting the presence of at least
one SNP or at least one inversion in an APOL1 gene (e.g., G1, G2,
del6, and/or G3). In particular examples, specific SNPs of use in
identifying resistance to a disease associated with Trypanosoma
(for example, in a subject of African ancestry) include a G at
rs73885319, a G at rs60910145, a 6 bp deletion (-/TTATAA; SEQ ID
NO: 6) at rs71785313, and combinations thereof. In some examples
SNP rs73885319 results in a substitution of glycine for serine at
amino acid 342 of an APOL1 protein (S342G). In other examples, SNP
rs60910145 results in a substitution of methionine for isoleucine
at amino acid 384 of an APOL1 protein (1384). In further examples,
SNP rs71785313 results in a deletion of amino acids 388 and 389 of
an APOL1 protein.
[0173] The method can also include detecting one of more of the
APOL1 SNPs or inversions disclosed herein. Thus, the method can
include detecting at least one, at least two, or at least three
different SNPs (such as 1, 2, or 3 SNPs or inversions). In some
embodiments, the SNPs and/or inversion can be in any combination
(e.g., a combination of at least two different SNPs alone or in
combination with an inversion). Detection of one or more (e. g.,
all) of the SNPs and/or the inversions disclosed herein can also be
used to detect resistance to a disease associated with Trypanosoma
infection (e.g., G1, G2, del6, and/or G3).
[0174] In several embodiments, at least one SNP and/or at least one
inversion is detected in a coding region of an APOL1 gene. Thus,
the method can include detecting at least one, at least two, or at
least three different SNPs and/or inversions in the coding region
of an APOL1 gene, wherein at least one or more SNPs in the coding
region of the gene is a G at rs73885319, a G at rs60910145, or a 6
bp deletion (-/TTATAA; SEQ ID NO: 6) at rs71785313, and/or one of
the inversions is G3. Sequence information for each of the APOL1
SNPs listed above is provided in Table 2.
TABLE-US-00004 TABLE 2 APOL1 single nucleotide polymorphisms
Resistance Reference Flanking SNP allele allele sequence rs73885319
G A TCAAGCTCACGGATG TGGCCCCTGTA [G/A]GCTTCTTTCT TGTGCTGGATGTAGT
(SEQ ID NO: 1) rs60910145 G T CAGGAGCTGGAGGAG AAGCTAAACAT
[G/T]CTCAACAATA ATTATAAGATTCTGC (SEQ ID NO: 2) rs71785313 del6
TTATAA GAGAAGCTAAACATT (SEQ ID CTCAACAATAA[-/ NO: 6)
TTATAA]GATTCTGC AGGCGGACCAAGAAC TG (SEQ ID NO: 3)
[0175] In Table 2, the "resistance" allele identifies the SNP that
can be used to detect or determine resistance to a disease
associated with Trypanosoma infection, such as sleeping sickness.
The "reference" allele is a different allele not associated with
disease resistance. In the sequences provided above, the notation
"[X/Y]" is used, wherein one of X or Y is the resistance allele and
one of X or Y is the reference allele. For each sequence, the
allele associated with resistance to disease associated with
Trypanosoma infection (the "resistance" allele) is listed. The
allele that is not associated with a resistance to disease is also
listed (the "reference" allele).
[0176] The disclosed methods can include detecting the resistance
allele on one or both chromosomes, detecting the presence of a
reference allele on one or both chromosomes, or detecting the
absence of the resistance allele on one or both chromosomes. In
some embodiments, detecting the presence of the resistance allele
indicates that the subject has a resistance to disease associated
with Trypanosoma infection, and detecting the absence of the
reference allele indicates that the subject has a resistance to
disease associated with Trypanosoma infection. In particular
examples, detecting the presence of the resistance allele indicates
that the subject has a resistance to disease associated with T. b.
rhodesiense infection (such as disease associated with infection
with type 1 T. b. rhodesiense or type 2 T. b. rhodesiense).
Similarly, detecting the absence of the resistance allele indicates
that the subject does not have a resistance to disease associated
with Trypanosoma infection (such as disease associated with
infection with type 1 T. b. rhodesiense or type 2 T. b.
rhodesiense), and detecting the presence of the reference allele
indicates that the subject does not have a resistance to disease
associated with Trypanosoma infection.
[0177] Thus, the disclosed methods can detect resistance to disease
associated with Trypanosoma infection, such as decreased risk of
developing Trypanosoma-associated disease, or identify a subject
that does not have a resistance to disease associated with
Trypanosoma infection. For example, subjects that have at least one
APOL1 SNP associated with the resistance allele are genetically
pre-disposed to resistance to disease associated with Trypanosoma
infection. In particular examples, the subject is of African or
Hispanic ancestry. In further examples, the subject is
African-American.
[0178] Methods are also disclosed for detection of a resistance to
disease associated with Trypanosoma infection in a human subject of
European ancestry. The presence of at least one SNP in an APOL1
gene that encodes apolipoprotein L1 determines the genetic
predisposition to resistance to disease associated with Trypanosoma
infection in the human subject of European ancestry. In one
embodiment, the method includes detecting at least one of a G at
rs73885319, a G at rs60910145, and/or a 6 bp deletion (-/TTATAA;
SEQ ID NO: 6) at rs71785313, and/or at least one inversion in the
APOL1 gene (e.g., G3). In a further example, the method includes
detecting the absence of at least one of a G at rs73885319, a G at
rs60910145, and/or a 6 bp deletion (-/TTATAA; SEQ ID NO: 6) at
rs71785313 and/or at least at least one inversion in the APOL1 gene
(e.g., G3).
IV. Methods and Compositions for Treating Disease Associated with
Trypanosoma Infection
[0179] It has been discovered that human plasma from individuals
expressing variant APOL1 proteins (for example, S342G/I384M and/or
del N388/Y389 and/or the G3 inversion) lyses Trypanosoma brucei
parasites (such as SRA- or SRA+T. brucei) in vitro. Therefore,
disclosed herein are methods for treating a subject infected with
Trypanosoma brucei (such as T. b. brucei, T. b. rhodesiense, or T.
b. gambiense) utilizing the variant APOL1 proteins described
herein. In some embodiments, the method includes administering to a
subject a therapeutically effective amount of a variant APOL1
protein, such as an APOL1 protein with a S342G substitution, an
I384M substitution and/or a deletion removing amino acids N388 and
Y389 and/or an APOL1 with a G3 inversion. For example, a
therapeutically effective amount of a human APOL1 protein including
1, 2, 3 or all 4 of these mutations can be used. For example, a
therapeutically effective amount of a human APOL1 protein including
a S342G substitution, an I384M substitution and/or a deletion
removing amino acids N388 and Y389 and/or an APOL1 with a G3
inversion, can be used to decrease symptoms associated with
sleeping sickness, such as fever, headache, joint pain, lymph node
swelling, anemia, confusion, reduced coordination, and disruption
of the sleep cycle. In particular examples, the subject is infected
with T. b. rhodesiense (for example, type 1 T. b. rhodesiense or
type 2 T. b. rhodesiense).
[0180] A subject infected with T. brucei is identified by standard
diagnostic methods. In some examples, diagnosis includes
demonstrating presence of trypanosomes in the subject, for example
by microscopic examination of chancre fluid, lymph node aspirates,
blood, bone marrow, or, in the late stages of infection,
cerebrospinal fluid. In some examples, a wet preparation is
examined for motile trypanosomes and a smear is fixed, stained with
Giemsa (or Field), and examined. In other examples, a serological
test is used to detect presence of anti-trypanosome antibodies.
Particular serological tests include agglutination tests, such as
micro-CATT, wb-CATT, and wb-LATEX (e.g., Truc et al., Bull. World
Health Org. 80:882-886, 2002). In further examples, a diagnosis is
based on clinical symptoms, including non-specific symptoms (such
as fever, fatigue, headache, arthralgia, and pruritus), enlarged
cervical lymph nodes in the posterior cervical triangle
(Winterbottom's sign), and neuropsychiatric symptoms and signs
(such as mental disturbance, disturbance of the sleep-wake cycle,
rigidity and tremor, dysarthria, and ataxia).
[0181] Disclosed herein are methods of treating a subject infected
with T. brucei which include administering to the subject a
therapeutically effective amount of a variant APOL1 protein, such
as an APOL1 protein including a S342G substitution, an I384M
substitution and/or a deletion removing amino acids N388 and Y389,
and/or an APOL1 with a G3 inversion. In some embodiments, the
method includes administering a therapeutically effective amount of
human serum or HDL particles including at least one APOL1 variant
protein (such as an APOL1 protein including a S342G substitution,
an I384M substitution and/or a deletion removing amino acids N388
and Y389, and/or an APOL1 protein with a G3 inversion).
[0182] Appropriate human donors for obtaining human serum or HDL
particles containing APOL1 variant protein can be identified
utilizing the genotyping methods described herein. In some
examples, a donor is an individual with an APOL1 gene having at
least one of a G at rs73885319, a G at rs60910145, and/or a 6 base
pair deletion at rs71785313 and/or an APOL1 gene with a G3
inversion.
[0183] In some examples, a therapeutically effective amount of
human serum includes at least a 10-fold dilution of serum from a
donor with an APOL1 protein including a S342G substitution, an
I384M substitution and/or a deletion removing amino acids N388 and
Y389 and/or an APOL1 protein with a G3 inversion (such as at least
a 100-fold, 1000-fold, 10,000-fold, 100,000-fold or more dilution).
It will be appreciated that these dosages are examples only, and an
appropriate dose can be determined by one of ordinary skill in the
art using only routine experimentation.
[0184] In other embodiments, the method includes administering a
therapeutically effective amount of a recombinant APOL1 protein,
including at least one APOL1 variant (such as an APOL1 protein
including a S342G substitution, an I384M substitution and/or a
deletion removing amino acids N388 and Y389 and/or an APOL1 protein
with a G3 inversion.). In some examples, a therapeutically
effective amount of recombinant APOL1 variant protein includes
about 0.1 mg/kg to about 1000 mg/kg (such as about 1 mg/kg to 1000
mg/kg, about 10 mg/kg to 500 mg/kg, about 10 mg/kg to 100 mg/kg,
about 50 mg/kg to 500 mg/kg, or about 100 mg/kg to 1000 mg/kg).
Administration can be accomplished by single or multiple doses. The
dose required will vary from subject to subject depending on the
species, age, weight and general condition of the subject, the
particular therapeutic agent being used and its mode of
administration. It will be appreciated that these dosages are
examples only, and an appropriate dose can be determined by one of
ordinary skill in the art using only routine experimentation.
[0185] The preparation of recombinant proteins is well known to
those skilled in the art. See, e.g., Sambrook et al. (In Molecular
Cloning: A Laboratory Manual, CSHL, New York, 1989); Ausubel et al.
(In Current Protocols in Molecular Biology, John Wiley & Sons,
New York, 1998); and The Recombinant Protein Handbook, GE
Lifesciences, Code 18-1142-75.
[0186] Also disclosed herein are pharmaceutical compositions that
include a variant APOL1 protein (such as APOL1 protein including a
S342G variant, an I384M variant, and/or a del N388/Y389 variant,
and/or an APOL1 protein with a G3 inversion, or a combination
thereof), such as a recombinant APOL1 protein. In some embodiments,
the composition includes a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers are determined in part by the
particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of pharmaceutical
compositions of the present disclosure. See, e.g., Remington: The
Science and Practice of Pharmacy, The University of the Sciences in
Philadelphia, Editor, Lippincott, Williams, & Wilkins,
Philadelphia, Pa., 21.sup.st Edition (2005).
[0187] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives may also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like.
[0188] Formulations for topical administration may include
ointments, lotions, creams, gels, drops, suppositories, sprays,
liquids and powders. Conventional pharmaceutical carriers, aqueous,
powder or oily bases, thickeners and the like may be necessary or
desirable.
[0189] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets, or tablets. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids or binders may be desirable.
V. Molecular Methods
[0190] Generally, the methods disclosed herein involve an
assessment of nucleic acid sequence. Molecular techniques of use in
all of these methods are disclosed below.
[0191] Preparation of Nucleic Acids for Analysis:
[0192] Nucleic acid molecules can be prepared for analysis using
any technique known to those skilled in the art. Generally, such
techniques result in the production of a nucleic acid molecule
sufficiently pure to determine the presence or absence of one or
more variations at one or more locations in the nucleic acid
molecule. Such techniques are described for example, in Sambrook,
et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor
Laboratory, New York) (1989), and Ausubel, et al., Current
Protocols in Molecular Biology (John Wiley and Sons, New York)
(1997), incorporated herein by reference.
[0193] When the nucleic acid of interest is present in a cell, it
can be necessary to first prepare an extract of the cell and then
perform further steps, such as differential precipitation, column
chromatography, extraction with organic solvents and the like, in
order to obtain a sufficiently pure preparation of nucleic acid.
Extracts can be prepared using standard techniques in the art, for
example, by chemical or mechanical lysis of the cell. Extracts then
can be further treated, for example, by filtration and/or
centrifugation and/or with chaotropic salts such as guanidinium
isothiocyanate or urea or with organic solvents such as phenol
and/or chloroform to denature any contaminating and potentially
interfering proteins. When chaotropic salts are used, it can be
desirable to remove the salts from the nucleic acid-containing
sample. This can be accomplished using standard techniques in the
art such as precipitation, filtration, size exclusion
chromatography and the like. In some examples, nucleic acids can be
isolated using commercially available kits (e.g., Qiagen, Valencia,
Calif.; Life Technologies/Invitrogen, Carlsbad, Calif.; Epicentre,
Madison, Wis.).
[0194] In some instances, messenger RNA can be extracted from
cells. Techniques and material for this purpose are known to those
skilled in the art and can involve the use of oligo dT attached to
a solid support such as a bead or plastic surface. In some
embodiments, the mRNA can be reverse transcribed into cDNA using,
for example, a reverse transcriptase enzyme. Suitable enzymes are
commercially available from, for example, Life
Technologies/Invitrogen (Carlsbad, Calif.). Optionally, cDNA
prepared from mRNA can also be amplified.
[0195] Amplification of Nucleic Acid Molecules:
[0196] Optionally, the nucleic acid samples obtained from the
subject are amplified prior to detection. Target nucleic acids are
amplified to obtain amplification products, including a SNP or
sequences from a haplotype block including a tag SNP, can be
amplified from the sample prior to detection. Typically, DNA
sequences are amplified, although in some instances RNA sequences
can be amplified or converted into cDNA, such as by using RT
PCR.
[0197] Any nucleic acid amplification method can be used. An
example of in vitro amplification is the polymerase chain reaction
(PCR), in which a biological sample obtained from a subject is
contacted with a pair of oligonucleotide primers, under conditions
that allow for hybridization of the primers to a nucleic acid
molecule in the sample. The primers are extended under suitable
conditions, dissociated from the template, and then re-annealed,
extended, and dissociated to amplify the number of copies of the
nucleic acid molecule. Other examples of in vitro amplification
techniques include quantitative real-time PCR, strand displacement
amplification (see U.S. Pat. No. 5,744,311); transcription-free
isothermal amplification (see U.S. Pat. No. 6,033,881); repair
chain reaction amplification (see PCT Publication No. WO 90/01069);
ligase chain reaction amplification (see EP-A-320 308); gap filling
ligase chain reaction amplification (see U.S. Pat. No. 5,427,930);
coupled ligase detection and PCR (see U.S. Pat. No. 6,027,889); and
NASBA.TM. RNA transcription-free amplification (see U.S. Pat. No.
6,025,134).
[0198] In specific examples, the target sequences to be amplified
from the subject include at least one APOL1 SNP, one or more
different haplotype blocks including a tag SNP, or a nucleotide
sequence of interest including the tag SNP. In certain embodiments,
target sequences containing one or more of SEQ ID NOs: 1-3, or a
subset thereof, are amplified. In an embodiment, a single SNP with
exceptionally high predictive value is amplified, or a nucleic acid
encoding the SNP is amplified.
[0199] A pair of primers can be utilized in the amplification
reaction. One or both of the primers can be labeled, for example
with a detectable radiolabel, fluorophore, or biotin molecule. The
pair of primers includes an upstream primer (which binds 5' to the
downstream primer) and a downstream primer (which binds 3' to the
upstream primer). The pair of primers used in the amplification
reactions are selective primers which permit amplification of a
size related marker locus. Primers can be selected to amplify a
nucleic acid including a SNP, a haplotype block including a tag
SNP, or a nucleic acid including a tag SNP. Numerous primers can be
designed by those of skill in the art simply by determining the
sequence of the desired target region of APOL1, for example, using
well known computer assisted algorithms that select primers within
desired parameters suitable for annealing and amplification.
[0200] If desired, an additional pair of primers can be included in
the amplification reaction as an internal control. For example,
these primers can be used to amplify a "housekeeping" nucleic acid
molecule, and serve to provide confirmation of appropriate
amplification. In another example, a target nucleic acid molecule
including primer hybridization sites can be constructed and
included in the amplification reactor. One of skill in the art will
readily be able to identify primer pairs to serve as internal
control primers.
[0201] Primer Design Strategy:
[0202] Increased use of polymerase chain reaction (PCR) methods has
stimulated the development of many programs to aid in the design or
selection of oligonucleotides used as primers for PCR. Four
examples of such programs that are freely available via the
Internet are: PRIMER.TM. by Mark Daly and Steve Lincoln of the
Whitehead Institute (UNIX, VMS, DOS, and Macintosh),
Oligonucleotide Selection Program by Phil Green and LaDeana Hiller
of Washington University in St. Louis (UNIX, VMS, DOS, and
Macintosh), PGEN.TM. by Yoshi (DOS only), and Amplify by Bill
Engels of the University of Wisconsin (Macintosh only). Generally
these programs help in the design of PCR primers by searching for
bits of known repeated-sequence elements and then optimizing the
T.sub.m by analyzing the length and GC content of a putative
primer. Commercial software is also available and primer selection
procedures are rapidly being included in most general sequence
analysis packages.
[0203] Designing oligonucleotides for use as either sequencing or
PCR primers requires selection of an appropriate sequence that
specifically recognizes the target APOL1, and then testing the
sequence to eliminate the possibility that the oligonucleotide will
have a stable secondary structure. Inverted repeats in the sequence
can be identified using a repeat-identification or RNA-folding
programs. If a possible stem structure is observed, the sequence of
the primer can be shifted a few nucleotides in either direction to
minimize the predicted secondary structure. When the amplified
sequence is intended for subsequence cloning, the sequence of the
oligonucleotide can also be compared with the sequences of both
strands of the appropriate vector and insert DNA. A sequencing
primer only has a single match to the target DNA. It is also
advisable to exclude primers that have only a single mismatch with
an undesired target DNA sequence. For PCR primers used to amplify
genomic DNA, the primer sequence can be compared to the sequences
in the GENBANK.TM. database to determine if any significant matches
occur. If the oligonucleotide sequence is present in any known DNA
sequence or, more importantly, in any known repetitive elements,
the primer sequence should be changed.
[0204] Detection of Alleles:
[0205] The nucleic acids obtained from the sample can be genotyped
to identify the particular allele present for a marker locus. A
sample of sufficient quantity to permit direct detection of marker
alleles from the sample can be obtained from the subject.
Alternatively, a smaller sample is obtained from the subject and
the nucleic acids are amplified prior to detection. Any APOL1
nucleic acid that is informative for a SNP or inversion or
chromosome haplotype can be detected. Generally, the target nucleic
acid corresponds to a tag SNP described above (SEQ ID NOs: 1-3).
Any method of detecting a nucleic acid molecule can be used, such
as hybridization and/or sequencing assays.
[0206] Hybridization is the binding of complementary strands of
DNA, DNA/RNA, or RNA. Hybridization can occur when primers or
probes bind to target sequences such as target sequences within
genomic DNA. Probes and primers that are useful generally include
nucleic acid sequences that hybridize (for example under high
stringency conditions) with a nucleic acid sequence including a SNP
or inversion of interest, but do not hybridize to a reference
allele, or that hybridize to the reference allele, but do not
hybridize to the SNP or inversion. Physical methods of detecting
hybridization or binding of complementary strands of nucleic acid
molecules, include but are not limited to, such methods as DNase I
or chemical footprinting, gel shift and affinity cleavage assays,
Southern and Northern blotting, dot blotting and light absorption
detection procedures. The binding between a nucleic acid primer or
probe and its target nucleic acid is frequently characterized by
the temperature (T.sub.m) at which 50% of the nucleic acid probe is
melted from its target. A higher (T.sub.m) means a stronger or more
stable complex relative to a complex with a lower (T.sub.m).
[0207] Generally, complementary nucleic acids form a stable duplex
or triplex when the strands bind, (hybridize), to each other by
forming Watson-Crick, Hoogsteen or reverse Hoogsteen base pairs.
Stable binding occurs when an oligonucleotide molecule remains
detectably bound to a target nucleic acid sequence under the
required conditions.
[0208] Complementarity is the degree to which bases in one nucleic
acid strand base pair with the bases in a second nucleic acid
strand. Complementarity is conveniently described by percentage,
that is, the proportion of nucleotides that form base pairs between
two strands or within a specific region or domain of two strands.
For example, if 10 nucleotides of a 15-nucleotide oligonucleotide
form base pairs with a targeted region of a DNA molecule, that
oligonucleotide is said to have 66.67% complementarity to the
region of DNA targeted.
[0209] In the present disclosure, "sufficient complementarity"
means that a sufficient number of base pairs exist between an
oligonucleotide molecule and a target nucleic acid sequence (such
as a tag SNP) to achieve detectable and specific binding. When
expressed or measured by percentage of base pairs formed, the
percentage complementarity that fulfills this goal can range from
as little as about 50% complementarity to full (100%)
complementary. In general, sufficient complementarity is at least
about 50%, for example at least about 75% complementarity, at least
about 90% complementarity, at least about 95% complementarity, at
least about 98% complementarity, or even at least about 100%
complementarity. The qualitative and quantitative considerations
involved in establishing binding conditions that allow one skilled
in the art to design appropriate oligonucleotides for use under the
desired conditions is provided by Beltz et al. Methods Enzymol
100:266-285, 1983, and by Sambrook et al. (ed.), Molecular Cloning:
A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989.
[0210] Hybridization conditions resulting in particular degrees of
stringency will vary depending upon the nature of the hybridization
method and the composition and length of the hybridizing nucleic
acid sequences. Generally, the temperature of hybridization and the
ionic strength (such as the Na.sup.+ concentration) of the
hybridization buffer will determine the stringency of
hybridization. Calculations regarding hybridization conditions for
attaining particular degrees of stringency are discussed in
Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual,
second edition, Cold Spring Harbor Laboratory, Plainview, N.Y.
(Chapters 9 and 11). Exemplary hybridization conditions are
provided above.
[0211] Methods for labeling nucleic acid molecules so they can be
detected are well known. Examples of such labels include
non-radiolabels and radiolabels. Non-radiolabels include, but are
not limited to an enzyme, chemiluminescent compound, fluorescent
compound (such as FITC, Cy3, and Cy5), metal complex, hapten,
enzyme, colorimetric agent, a dye, or combinations thereof.
Radiolabels include, but are not limited to, .sup.125I, .sup.32P
and .sup.35S. For example, radioactive and fluorescent labeling
methods, as well as other methods known in the art, are suitable
for use with the present disclosure. In one example, primers used
to amplify the subject's nucleic acids are labeled (such as with
biotin, a radiolabel, or a fluorophore). In another example,
amplified target nucleic acid samples are end-labeled to form
labeled amplified material. For example, amplified nucleic acid
molecules can be labeled by including labeled nucleotides in the
amplification reactions.
[0212] Nucleic acid molecules corresponding to one or more APOL1
SNPs and/or inversions, or haplotypes blocks, including a tag SNP,
can also be detected by hybridization procedures using a labeled
nucleic acid probe, such as a probe that detects only one
alternative allele at a marker locus. Most commonly, the target
nucleic acid (or amplified target nucleic acid) is separated based
on size or charge and transferred to a solid support. The solid
support (such as membrane made of nylon or nitrocellulose) is
contacted with a labeled nucleic acid probe, which hybridizes to it
complementary target under suitable hybridization conditions to
form a hybridization complex.
[0213] Hybridization conditions for a given combination of array
and target material can be optimized routinely in an empirical
manner close to the T.sub.m of the expected duplexes, thereby
maximizing the discriminating power of the method. For example, the
hybridization conditions can be selected to permit discrimination
between matched and mismatched oligonucleotides. Hybridization
conditions can be chosen to correspond to those known to be
suitable in standard procedures for hybridization to filters (and
optionally for hybridization to arrays). In particular, temperature
is controlled to substantially eliminate formation of duplexes
between sequences other than an exactly complementary allele of the
selected marker. A variety of known hybridization solvents can be
employed, the choice being dependent on considerations known to one
of skill in the art (see U.S. Pat. No. 5,981,185).
[0214] Once the target nucleic acid molecules have been hybridized
with the labeled probes, the presence of the hybridization complex
can be analyzed, for example by detecting the complexes.
[0215] Methods for detecting hybridized nucleic acid complexes are
well known in the art. In one example, detection includes detecting
one or more labels present on the oligonucleotides, the target
(e.g., amplified) sequences, or both. Detection can include
treating the hybridized complex with a buffer and/or a conjugating
solution to effect conjugation or coupling of the hybridized
complex with the detection label, and treating the conjugated,
hybridized complex with a detection reagent. In one example, the
conjugating solution includes streptavidin alkaline phosphatase,
avidin alkaline phosphatase, or horseradish peroxidase. Specific,
non-limiting examples of conjugating solutions include streptavidin
alkaline phosphatase, avidin alkaline phosphatase, or horseradish
peroxidase. The conjugated, hybridized complex can be treated with
a detection reagent. In one example, the detection reagent includes
enzyme-labeled fluorescence reagents or calorimetric reagents. In
one specific non-limiting example, the detection reagent is
enzyme-labeled fluorescence reagent (ELF) from Molecular Probes,
Inc. (Eugene, Oreg.). The hybridized complex can then be placed on
a detection device, such as an ultraviolet (UV) transilluminator
(manufactured by UVP, Inc. of Upland, Calif.). The signal is
developed and the increased signal intensity can be recorded with a
recording device, such as a charge coupled device (CCD) camera. In
particular examples, these steps are not performed when radiolabels
are used. In particular examples, the method further includes
quantification, for instance by determining the amount of
hybridization.
[0216] Allele Specific PCR:
[0217] Allele-specific PCR differentiates between target regions
differing in the presence of absence of a variation or
polymorphism. PCR amplification primers are chosen based upon their
complementarity an APOL1 sequence, such as nucleic acid sequence in
a SNP or inversion, haplotype block including a tag SNP, a
specified region of an allele including a tag SNP, or to the tag
SNP itself. The primers bind only to certain alleles of the target
sequence. This method is described by Gibbs, Nucleic Acid Res.
17:12427 2448, 1989, herein incorporated by reference.
[0218] Allele Specific Oligonucleotide Screening Methods:
[0219] Further screening methods employ the allele-specific
oligonucleotide (ASO) screening methods (e.g. see Saiki et al.,
Nature 324:163-166, 1986). Oligonucleotides with one or more base
pair mismatches are generated for any particular allele or
haplotype block. ASO screening methods detect mismatches between
one allele (or haplotype block) in the target genomic or PCR
amplified DNA and the other allele (or haplotype block), showing
decreased binding of the oligonucleotide relative to the second
allele (e.g., the other allele) oligonucleotide. Oligonucleotide
probes can be designed that under low stringency will bind to both
polymorphic forms of the allele, but which at high stringency, only
bind to the allele to which they correspond. Alternatively,
stringency conditions can be devised in which an essentially binary
response is obtained. For example, an ASO corresponding to a
variant form of the target gene will hybridize to that allele
(haplotype block), and not to the reference allele (haplotype
block).
[0220] Ligase Mediated Allele Detection Method:
[0221] Ligase can also be used to detect point mutations, such as
the SNPs disclosed herein, in a ligation amplification reaction
(e.g. as described in Wu et al., Genomics 4:560-569, 1989). The
ligation amplification reaction (LAR) utilizes amplification of
specific DNA sequence using sequential rounds of template dependent
ligation (e.g., as described in Wu, supra, and Barany, Proc. Nat.
Acad. Sci. 88:189-193, 1990).
[0222] Denaturing Gradient Gel Electrophoresis:
[0223] Amplification products generated using the polymerase chain
reaction can be analyzed by the use of denaturing gradient gel
electrophoresis. Different SNPs or alleles (haplotype blocks) can
be identified based on the different sequence-dependent melting
properties and electrophoretic migration of DNA in solution. DNA
molecules melt in segments, termed melting domains, under
conditions of increased temperature or denaturation. Each melting
domain melts cooperatively at a distinct, base-specific melting
temperature (T.sub.m). Melting domains are at least 20 base pairs
in length, and can be up to several hundred base pairs in
length.
[0224] Differentiation between SNPs or alleles (haplotype blocks)
based on sequence specific melting domain differences can be
assessed using polyacrylamide gel electrophoresis, as described in
Chapter 7 of Erlich, ed., PCR Technology, Principles and
Applications for DNA Amplification, W. H. Freeman and Co., New York
(1992).
[0225] Generally, a target region to be analyzed by denaturing
gradient gel electrophoresis is amplified using PCR primers
flanking the target region. The amplified PCR product is applied to
a polyacrylamide gel with a linear denaturing gradient as described
in Myers et al., Meth. Enzymol. 155:501-527, 1986, and Myers et
al., in Genomic Analysis, A Practical Approach, K. Davies Ed. IRL
Press Limited, Oxford, pp. 95 139, 1988. The electrophoresis system
is maintained at a temperature slightly below the T.sub.m of the
melting domains of the target sequences.
[0226] In an alternative method of denaturing gradient gel
electrophoresis, the target sequences can be initially attached to
a stretch of GC nucleotides, termed a GC clamp, as described in
Chapter 7 of Erlich, supra. In one example, at least 80% of the
nucleotides in the GC clamp are either guanine or cytosine. In
another example, the GC clamp is at least 30 bases long. This
method is particularly suited to target sequences with a high
T.sub.m.
[0227] Generally, the target region is amplified by polymerase
chain reaction. One of the oligonucleotide PCR primers carries at
its 5' end, the GC clamp region, at least 30 bases of the GC rich
sequence, which is incorporated into the 5' end of the target
region during amplification. The resulting amplified target region
is run on an electrophoresis gel under denaturing gradient
conditions. DNA fragments differing by a single base change will
migrate through the gel to different positions, which can be
visualized by ethidium bromide staining.
[0228] Temperature Gradient Gel Electrophoresis:
[0229] Temperature gradient gel electrophoresis (TGGE) is based on
the same underlying principles as denaturing gradient gel
electrophoresis, except the denaturing gradient is produced by
differences in temperature instead of differences in the
concentration of a chemical denaturant. Standard TGGE utilizes an
electrophoresis apparatus with a temperature gradient running along
the electrophoresis path. As samples migrate through a gel with a
uniform concentration of a chemical denaturant, they encounter
increasing temperatures. An alternative method of TGGE, temporal
temperature gradient gel electrophoresis (TTGE or tTGGE) uses a
steadily increasing temperature of the entire electrophoresis gel
to achieve the same result. As the samples migrate through the gel
the temperature of the entire gel increases, leading the samples to
encounter increasing temperature as they migrate through the gel.
Preparation of samples, including PCR amplification with
incorporation of a GC clamp, and visualization of products are the
same as for denaturing gradient gel electrophoresis.
[0230] Single-Strand Conformation Polymorphism Analysis:
[0231] Target sequences, such as alleles or haplotype blocks can be
differentiated using single-strand conformation polymorphism
analysis, which identifies base differences by alteration in
electrophoretic migration of single stranded PCR products, for
example as described in Orita et al., Proc. Nat. Acad. Sci.
85:2766-2770, 1989. Amplified PCR products can be generated as
described above, and heated or otherwise denatured, to form single
stranded amplification products. Single-stranded nucleic acids can
refold or form secondary structures which are partially dependent
on the base sequence. Thus, electrophoretic mobility of
single-stranded amplification products can detect base-sequence
difference between alleles or haplotype blocks.
[0232] Chemical or Enzymatic Cleavage of Mismatches:
[0233] Differences between target sequences, such as alleles or
haplotype blocks, can also be detected by differential chemical
cleavage of mismatched base pairs, for example as described in
Grompe et al., Am. J Hum. Genet. 48:212-222, 1991. In another
method, differences between target sequences, such as alleles or
haplotype blocks, can be detected by enzymatic cleavage of
mismatched base pairs, as described in Nelson et al., Nature
Genetics 4:11-18, 1993. Briefly, genetic material from an animal
and an affected family member can be used to generate mismatch free
heterohybrid DNA duplexes. As used herein, "heterohybrid" means a
DNA duplex strand comprising one strand of DNA from one animal, and
a second DNA strand from another animal, usually an animal
differing in the phenotype for the trait of interest. Positive
selection for heterohybrids free of mismatches allows determination
of small insertions, deletions or other polymorphisms.
[0234] Non-Gel Systems:
[0235] Other possible techniques include non-gel systems such as
TaqMan.TM. (Perkin Elmer). In this system oligonucleotide PCR
primers are designed that flank the mutation in question and allow
PCR amplification of the region. A third oligonucleotide probe is
then designed to hybridize to the region containing the base
subject to change between different alleles of the gene. This probe
is labeled with fluorescent dyes at both the 5' and 3' ends. These
dyes are chosen such that while in this proximity to each other the
fluorescence of one of them is quenched by the other and cannot be
detected. Extension by Taq DNA polymerase from the PCR primer
positioned 5' on the template relative to the probe leads to the
cleavage of the dye attached to the 5' end of the annealed probe
through the 5' nuclease activity of the Taq DNA polymerase. This
removes the quenching effect allowing detection of the fluorescence
from the dye at the 3' end of the probe. The discrimination between
different DNA sequences arises through the fact that if the
hybridization of the probe to the template molecule is not complete
(there is a mismatch of some form) the cleavage of the dye does not
take place. Thus only if the nucleotide sequence of the
oligonucleotide probe is completely complimentary to the template
molecule to which it is bound will quenching be removed. A reaction
mix can contain two different probe sequences each designed against
different alleles that might be present thus allowing the detection
of both alleles in one reaction.
[0236] Non-PCR Based Allele Detection:
[0237] The identification of a DNA sequence can be made without an
amplification step, based on polymorphisms including restriction
fragment length polymorphisms in a subject and a control, such as a
family member. Hybridization probes are generally oligonucleotides
which bind through complementary base pairing to all or part of a
target nucleic acid. Probes typically bind target sequences lacking
complete complementarity with the probe sequence depending on the
stringency of the hybridization conditions. The probes can be
labeled directly or indirectly, such that by assaying for the
presence or absence of the probe, one can detect the presence or
absence of the target sequence. Direct labeling methods include
radioisotope labeling, such as with .sup.32P or .sup.35S. Indirect
labeling methods include fluorescent tags, biotin complexes which
can be bound to avidin or streptavidin, or peptide or protein tags.
Visual detection methods include photoluminescents, Texas red,
rhodamine and its derivatives, red leuco dye and
3,3',5,5'-tetramethylbenzidine (TMB), fluorescein, and its
derivatives, dansyl, umbelliferone and the like or with horse
radish peroxidase, alkaline phosphatase and the like.
[0238] Hybridization probes include any nucleotide sequence capable
of hybridizing to a nucleic acid sequence wherein a polymorphism is
present that is associated with FSGS or hypertensive ESKD, such as
an APOL1 SNP and/or inversion, or a tag SNP, and thus defining a
genetic marker, including a restriction fragment length
polymorphism, a hypervariable region, repetitive element, or a
variable number tandem repeat. Hybridization probes can be any gene
or a suitable analog. Further suitable hybridization probes include
exon fragments or portions of cDNAs or genes known to map to the
relevant region of the chromosome.
[0239] Exemplary tandem repeat hybridization probes for use in the
methods disclosed are those that recognize a small number of
fragments at a specific locus at high stringency hybridization
conditions, or that recognize a larger number of fragments at that
locus when the stringency conditions are lowered.
[0240] The disclosure is illustrated by the following non-limiting
Examples.
EXAMPLES
Example 1
APOL1 Variants Associated with Focal Segmental
Glomerulosclerosis
Methods
[0241] FSGS Genotype Experiment:
[0242] Variants for the initial FSGS genotype study were selected
by accessing 1000 Genomes Project (1000genomes.org/) data using the
Integrative Genomics Viewer (broadinstitute.org/igv). All variants
in the region 34,930kb-35,060kb (NCBI 36) with estimated minor
allele frequency greater than 15% in Yoruba and minor allele
frequency less than 10% in Europeans were selected, together with
some additional ones with biological relevance, and sent for
genotyping using Sequenom technology (sequenom.com). A small amount
of SNPs were dropped due to Multiple eXTEND hits for scanned primer
triplets and some failed genotyping (<20%). Overall three plexes
were used for the FSGS analysis. Association of genotype data and
association controlling for allelles G1 and G2 were performed with
plink (pngu.mgh.harvard.edu/.about.purcell/plink; Purcell et al.,
Am. J. Hum. Genet. 81:559-575, 2007) using Fisher's exact test and
logistic regression.
[0243] Bounds on Causal Variant(s):
[0244] Due to the high frequency differentiation between frequency
of alleles G1 and G2 in cases and controls, some formal arguments
can be made to discard other variants as causal. Define with
A.sub.1 the combined allele G1 and G2 and with A.sub.2 the wild
type allele. Define with B.sub.1 the risk version of the combined
causal alleles and with B.sub.2 the non-risk version. Assume that
in controls the frequency of haplotype A.sub.1B.sub.1 is x.sub.11,
A.sub.1B.sub.2 is x.sub.12, A.sub.2B.sub.1 is x.sub.21, and
A.sub.2B.sub.2 is x.sub.22. Define the frequency in controls of
allele A.sub.1 as p.sub.1=x.sub.11+x.sub.12, and B.sub.1 as
q.sub.1=x.sub.11+x.sub.21. Say p'.sub.1 and q'.sub.1 for the
frequencies of A.sub.1 and B.sub.1 in cases with
p'.sub.1>p.sub.1 and q'.sub.1>q.sub.1. Think of
x.sub.11/q.sub.1 as the fraction of haplotypes containing B.sub.1
which also contain A.sub.1 and x.sub.12/(1-q.sub.1) as the fraction
of haplotypes containing B.sub.2 which also contain A.sub.1. We can
then write:
p.sub.1=(x.sub.11/q.sub.1)q.sub.1+x.sub.12/(1-q.sub.1)(1-q.sub.1),
p'.sub.1=(x.sub.11/q.sub.1)q'.sub.1+x.sub.12/(1-q.sub.1)(1-q'.sub.1).
[0245] By subtracting one equation from the other:
p'.sub.1-p.sub.1=x.sub.11/q.sub.1(q'.sub.1-q.sub.1)+x.sub.12/(1-q.sub.1)-
(1-q'.sub.1-1+q.sub.1),
p'.sub.1-p.sub.1=(x.sub.11/q.sub.1-x.sub.12/(1-q.sub.1)(q'-.sub.1+q.sub.-
1).
[0246] From this equation, since q'.sub.1<1 and
x.sub.11<p.sub.1, we get the inequality:
p'-p.sub.1<p.sub.1/q.sub.1(1-q.sub.1),
q.sub.1<p.sub.1/p'.sub.1
In the NIH FSGS cohort, p.sub.1=33% and p'.sub.1=72%, from which we
get the bound q.sub.1<46%. The rationale behind this argument is
that if the frequency of the causal allele is too high in controls,
then even if it was 100% in cases, this difference would not be
able to explain the disparity observed for alleles in APOL1.
Continuing from the previous equation:
p'.sub.1-p.sub.1=(x.sub.11-x.sub.11q.sub.1-x.sub.12q.sub.1)/(q.sub.1(1-q-
.sub.1))(q'.sub.1-q.sub.1).
Dividing both sides by \sqrt(p.sub.1(1-p.sub.1)) we get:
(p'.sub.1-p.sub.1)/\sqrt(p.sub.1(1-p.sub.1))=(x.sub.11-p.sub.1q.sub.1)/(-
\sqrt(p.sub.1(1-p.sub.1)q.sub.1(1-q.sub.1))(q'.sub.1-q.sub.1)/\sqrt(q.sub.-
1(1-q.sub.1)).
Define r as the correlation coefficient between the combined allele
G1 and G2 and the combined causal alleles. By the definition of
correlation coefficient, the previous equation can be written
as:
(p'.sub.1-p.sub.1)/\sqrt(p.sub.1(1-p.sub.1))=r(q'.sub.1-q.sub.1)/\sqrt(q-
.sub.1(1-q.sub.1)),
r=(p'.sub.1-p.sub.1)/\sqrt(p.sub.1(1-p.sub.1))\sqrt(q.sub.1(1-q.sub.1))(-
q'.sub.1-q.sub.1)).
Given that q'.sub.1-q.sub.1<1-q.sub.1,
r>(p'.sub.1-p.sub.1)/\sqrt(p.sub.1(1-p.sub.1))\sqrt(q.sub.1/(1-q.sub.-
1)),
r.sup.2>(p'.sub.1-p.sub.1).sup.2/(p.sub.1(1-p.sub.1))q.sub.1/(1-q.sub-
.1).
In the NIH FSGS cohort, p.sub.1=33% and p'.sub.1=72%. If we assume
that q.sub.1>30%, we get an estimate r.sup.2>29%, that could
be only explained by a short distance between the two variants.
Results
[0247] More than 50 genetic variants spanning the region including
APOL1, without bias towards either gene were selected for fine
mapping. Because the kidney disease risk allele(s) should have a
high frequency in African-Americans, as suggested by previous
studies (Kopp et al., Nature Genet. 40:1175-1184, 2008; Kao et al.,
Nature Genet. 40:1185-1192, 2008), causal alleles should be present
in the sequence data of Africans available from the 1000 Genomes
Project (available on the web at 1000genomes.org). The data was
searched for variants that were highly polymorphic in Yoruba that
were rare or absent in Europeans, as disease-causing variants are
expected to have this property. In addition, a single 6 bp deletion
(rs71785313) in the coding region of APOL1 also identified by the
1000 Genomes Project that was observed in three of the Yoruba
samples was studied. Many of these variants have not been genotyped
by the HapMap project.
[0248] An association analysis was performed with each of these
variants and disease, using DNA from 205 African-Americans with
biopsy proven FSGS and no family history of FSGS and 180
African-American controls. Association between disease and each
variant showed that the strongest associations were all clustered
in a 10 kb region centered on the last exon of APOL1 (Table 3).
These findings are summarized in FIG. 1A. The strongest association
was obtained for the haplotype termed "G1" consisting of the two
derived alleles for rs73885319 (S342G) and rs60910145 (I384M), in
the last exon of APOL1. These two alleles were found to be in
perfect linkage disequilibrium (LD) (r.sup.2=1). The G1 compound
allele (342G:384M) had a frequency of 52% in the combined set of
FSGS cases and 18% in controls (Table 4).
[0249] When logistic regression controlling for rs73885319 was
performed, a second strong signal was detected for a 6 bp deletion
termed "G2" recently entered into dbSNP as rs71785313 (-/TTATAA;
SEQ ID NO: 6) that removes amino acids N388 and Y389 (Table 5). Due
to the extremely close proximity of rs73885319, rs60910145, and
rs71785313, the two alleles G1 and G2 are mutually exclusive, as
recombination between them is very unlikely. FIG. 1B highlights
variants which still showed statistically significant associations.
These results are in accordance with recent studies (Freedman et
al., Kidney Int 75:736, 2009; Nelson et al., Hum. Mol. Genet.
19:1805-1815, 2010; Behar et al., Hum. Mol. Genet. 19:1816-1827,
2010), which also identified multiple different independent signals
of association. Allele G2 had a frequency of 23% in the combined
set of cases and 15% in the controls (Table 4).
[0250] Among the FSGS cases, all proven by kidney biopsy, 53
individuals were recruited through the Brigham and Women's Hospital
(BWH) from medical centers in the northeastern United States, and
152 individuals were recruited in the US National Institutes of
Health (NIH) FSGS Genetic Study from 22 academic medical centers in
the United States (MacKenzie et al., J. Am. Soc. Nephrol. 18:2987,
2007; Orloff et al., Physiol. Genom. 21:212, 2005). As controls,
DNA from 180 individuals from the NIH Blood Bank and the National
Cancer Institute-Frederick normal donor programs were used.
[0251] Odds ratios for disease were computed using the NIH samples,
as these samples were the best matched geographically. Table 6
shows the count for each one of the six possible compound genotypes
observable in each cohort of cases and controls. By combining the
two risk alleles G1 and G2, a .chi..sup.2 squared test showed no
association with FSGS between samples with no risk alleles and one
risk alleles (p=0.81). This supports a completely recessive model.
A second analysis comparing samples with one or no risk alleles and
samples with two risk alleles provided an odds ratio for FSGS of
10.5 (CI 6.0-18.4).
[0252] When comparing the number of samples with two risk alleles
among the BWH cases and the NIH cases, as shown in Table 3,
significant statistical differences were observed among frequencies
of alleles G1 and G2 using a Fisher's exact test (p=0.04). This
disparity cannot be explained by a difference in the amount of
African ancestry, as presence of risk alleles implies African
ancestry at the relevant locus, but may simply reflect a difference
in the frequency of allele G1 in the north eastern United
States.
Example 2
Replication in Hypertension-Attributed EKSD
Methods
[0253] Selection criteria for controls and hypertension-attributed
ESKD cases are described in detail in Freedman et al. (Kidney Int
75:736, 2009). Briefly, self-reported African-Americans from North
Carolina, South Carolina, Georgia, Virginia, or Tennessee were
recruited. Hypertension-attributed ESKD cases were diagnosed with
hypertension prior to initiation of renal replacement therapy, and
demonstrated hypertensive target end-organ damage (retinopathy or
left ventricular hypertrophy) and low grade or absence of
proteinuria. Only a minority of cases had quantified urinary
protein excretion. Patients with diabetic (type 1 and 2) ESKD were
excluded, as were known cases of cystic kidney disease, hereditary
nephritis, and urologic causes of ESKD.
[0254] Geographically similar controls all denied a history of
kidney disease and diabetes, or first-degree relatives with these
diseases. Most controls did not have direct measurements of
arterial blood pressure or renal function indices. Consequently,
some controls may have had occult kidney disease, which would
underestimate the effect size between cases and controls.
Results
[0255] Association of APOL1 variants and renal disease was tested
in a much larger cohort of 1030 African-American cases with
putative hypertensive ESKD and 1025 geographically matched
African-American controls from Wake Forest University. In this
cohort 36 variants were investigated that were chosen based on
strongest signals of positive selection in a broader region, nearby
coding variants together with rs73885319 (G1) and rs71785313 (G2).
The strongest association found was again for rs73885319
(p=1.1.times.10.sup.-39, Table 7). Upon controlling for rs73885319,
the strongest association was again for rs71785313
(p=8.8.times.10.sup.-18, Table 8). Frequencies for these alleles
are shown in Table 3.
[0256] With this larger population the mode of inheritance of G1
and G2 was explored. Cases and controls were partitioned into the
six possible genotypes. One risk allele was associated with only a
small increase in renal disease risk (odds ratio 1.26, CI
1.01-1.56, p=0.047). Two risk alleles versus zero risk alleles
yielded an odds ratio of 7.3 (CI 5.6-9.5). Two risk alleles versus
one risk allele gave an odds ratio of 5.8 (CI 4.5-7.5). Overall, a
recessive model best explains these findings and is in agreement
with the analysis of FSGS samples.
Example 3
Evidence of Natural Selection
Methods
[0257] Test for Genetic Divergence in African Populations:
[0258] To test for statistically significant differentiation of
allele frequency in between two populations we assume that the
difference in frequencies for a given polymorphism has mean 0 and
variance cp(1-p), where p is the ancestral frequency and
c=2xF.sub.ST (Ayodo et al., Am. J. Hum. Genet. 81:234-242. 2007).
Given the small size of the samples in the two populations
analyzed, it is also important to model sampling noise, which has
variance p(1-p)(1/N.sub.1+N.sub.2), where N.sub.1 and N.sub.2 are
the total count for the alleles for the two populations. Therefore,
to test for differentiation of frequency at a given allele, we
model the difference as a normal random variable with mean 0 and
variance p(1-p)(c+1/N.sub.1+1/N.sub.2) and we compute for each
allele a .chi..sup.2 statistic with 1 df.
[0259] Estimation of the Age of the Selected Allele:
[0260] Because of the presence of a recombination hotspot in
between APOL1 and MYH9 (Frazer et al., Nature 449:851-861, 2007),
SNP rs11912763, the variant most correlated with G1 available in
Hapmap, has genetic distance of about 0.2 centimorgans from APOL1
cSNPs rs73885319 and rs60910145, despite a physical distance of
less than 25kb from APOL1. The derived allele for SNP rs11912763,
absent outside of Africa, has a prevalence of about p=73% in
haplotypes containing the G1 allele. If we assume that the G1
allele arose in a haplotype already containing the rs11912763
derived allele, then the prevalence of the derived allele for
rs11912763 in haplotypes containing the G1 allele could not have
decreased at a rate faster than the expected frequency of
recombination 1-c per generation. This leads to an estimate for the
number t of generations
(1-c).sup.t.ltoreq.p,
t.gtoreq.log(p)/log(1-c),
from which we obtain a lower bound of about 150 generations, using
the values for c and p as above. If we assume an average of 20
years per generation, this estimate suggests an age of at least
3,000 years for allele G1. Given the prevalence of about p=72% for
the rs2239786 derived allele in haplotypes containing the G2
allele, a similar estimate also holds for the age of allele G2.
Results
[0261] The chromosomal region where APOL1 resides has previously
been shown to be a candidate for positive selection in the Yoruba
population using the long-range haplotype method (LRH) (Frazer et
al., Nature 449:851-861, 2007), the integrated haplotype score
(iHS) (Voight et al., PLoS Biol. 4:446, 2006; Barreiro et al.,
Nature Genet. 40:340-345, 2008), the rMHH (Kimura et al., PLoS One
2:e286, 2007), and the composite of multiple signals (CMS)
(Grossman et al., Science 327:883, 2010). The G1 and G2 allele was
present in all the Yoruba Hapmap samples and the extended haplotype
homozygosity (EHH) (Sabeti et al., Nature 419:832-837, 2002) was
computed for each one of the three alleles after phasing the data
using Beagle (Browning and Browning, Am. J Hum. Genet. 84:210-223,
2009) (FIG. 2). The iHS score was not computed, as the proximity of
a recombination hotspot makes this particular computation
unstable.
[0262] The frequency of allele G1 was also compared in Yoruba
samples from Nigeria and Luhya samples from Kenya to verify
statistically significant differences in these two populations. The
Yoruba population from Nigeria (YRI) and the Luhya population from
Kenya (LWK), despite being respectively from West Africa and East
Africa, are very closely related genetically with F.sub.ST=0.0043.
To test for selection, a model of allele-frequency differentiation
between two populations was used (Ayodo et al., Am. J Hum. Genet.
81:234-242. 2007), which corrects for genetic drift. The results
showed that differentiation for rs73885319, whose frequencies are
38% in the Yoruba and 5% in the Luhya, is highly significant
(F.sub.ST=0.16 and p=3.53.times.10.sup.-9). Interestingly, variant
rs73885319 was the second most highly differentiated variant in
these two populations across the whole genome. The frequencies of
variant rs71785313, respectively 0.08 and 0.07, did not show any
significant differentiation. Results of this analysis for the
region in between 34,900kb and 35,100kb (NCBI 36) are shown in
Table 9.
[0263] By analyzing the pattern of linkage disequilibrium between
these SNPs, it appears likely that alleles G1 and G2 are at least
3,000 years old. The true age is likely older than this number, but
not by orders of magnitude, and it might coincide with the Bantu
expansion event, a series of migrations across sub-Saharan Africa
that is estimated to have taken place between 4,500 and 5,000 years
ago (Excoffier et al., Am. J. Phys. Anthropol. 30:151-194, 2005).
In particular, frequency differentiation of allele G1 between two
populations from West and East Africa points to natural selection
having acted after the Bantu expansion, either to raise the
frequency in Yoruba or to decrease the frequency in Luhya.
Example 4
APOL1 and Resistance Against Trypanosome
Methods
[0264] Expression of ApoL1 proteins:
[0265] Two independent systems were used for expression of
recombinant ApoL1 in Escherichia coli and in 293T cells. The
various ApoL1 mutants were generated by site-directed mutagenesis
and expressed in E. coli essentially as described in Lecordier et
al. (PLoS Pathog. 5:e1000685, 2009), except that the pStaby1.2
plasmid (Delphi Genetics, Gosselies, Belgium) was used. For
production of ApoL1 protein in 293T cells with and without the G1
and G2 risk mutations, an image clone containing the ApoL1 cDNA
lacking the G1 and G2 mutations (reference sequence BC141823) was
purchased from Open Biosystems (Huntsville, Ala.). This cDNA was
provided in the pCMV-SPORT6 expression vector. The G1 and G2
mutations were introduced by synthesis of cDNA minigene fragments
(Integrated DNA Technologies, Coralville, Iowa) containing the
corresponding mutations flanked with 5' AleI and 3' XbaI
restriction sequences. The minigene fragments were then cloned into
the parental vector replacing the sequence between the AleI and
XbaI restriction sites. The resultant vectors were used to
transfect 293T cells using Fugene (Promega, Madison, Wis.). The
transfection media was replaced with OPTI-MEM reduced serum media
without phenol red (Life Technologies/Invitrogen, Carlsbad, Calif.)
at 12 hours post transfection. At 72 hours post transfection the
supernatants were harvested and concentrated 100 fold using an
Amicon Ultracel-10K centrifugal filter unit (Millipore, Billerica,
Mass.). The media was exchanged by centrifugation within the
Ultracel filters with fresh Iscove's Modified Dulbecco's Medium for
compatibility with the trypanosome killing assay.
[0266] Trypanolytic Assays:
[0267] The evaluation of trypanolytic activity of the various ApoL1
mutants was performed as described in Lecordier et al. (PLoS
Pathog. 5:e1000685, 2009).
Results
[0268] ApoL1 is the trypanolytic factor of human serum (Vanhamme et
al., Nature 422:83-87, 2003; Perez-Morga et al., Science 309:469,
2005) and confers resistance to the Trypanosoma brucei brucei
parasite. T. b. brucei has evolved into two forms, Trypanosoma
brucei gambiense and Trypanosoma brucei rhodesiense (Gibson
Parasitol. Today 9:255-257, 1986; Gibson Trends Parasitol.
18:486-490, 2002) which have both acquired the ability to infect
humans. FIGS. 3A and B show the relative distribution of infections
by T. b. rhodesiense and T. b. gambiense. Since these parasites
exist only in sub-Saharan Africa, it is plausible that the APOL1
gene had undergone natural selective pressure to counteract the
trypanosome adaptations.
[0269] T. b. rhodesiense can grow in humans because of a serum
resistance-associated (SRA) protein that interacts with the
C-terminal helix of ApoL1 and inhibits its anti-trypanosomal
activity (Xong et al., Cell 6:839-846, 1998; Vanhamme et al.,
Nature 422:83-87, 2003). A recent study showed that mutations and
deletions engineered into this helix prevent SRA from binding to
ApoL1 (Lecordier et al., PLoS Pathog. 5:e1000685, 2009). The 6 bp
deletion rs71785313 defining the G2 allele is located exactly at
the SRA binding site in the ApoL1 C-terminal helix.
[0270] Analysis of the in vitro lytic potential of 77 human plasma
samples was conducted on T. b. brucei, T. b. rhodesiense, and T. b.
gambiense. While all samples efficiently lysed T. b. brucei, none
lysed T. b. gambiense and 46 lysed normal human serum
(NHS)-resistant T. b. rhodesiense clones. All T. b. rhodesiense
lytic samples belonged to G1, G2 or both genotypes. As measured by
titration upon serial dilution, the lytic potential of these
plasmas against NHS-resistant (SRA+) T. b. rhodesiense was higher
for G2 than for G1, whereas it was similar for both genotypes
against NHS-sensitive (SRA-) parasites (FIG. 4A). While lysis of T.
b. rhodesiense by G2 could be explained by the incapacity of this
mutant ApoL1 to bind SRA, this conclusion did not hold for G1
plasmas, where ApoL1 still efficiently bound to SRA (FIG. 4B).
[0271] These results were confirmed with recombinant ApoL1
proteins. The S342G/I384M (G1) and delN388/Y389 (G2) variants
killed both NHS-sensitive (SRA-) and NHS-resistant (SRA+) T. b.
rhodesiense parasites (FIG. 4C), but not T. b. gambiense. While G2
was more active than G1 on NHS-resistant T. b. rhodesiense, the
reverse was true on NHS-sensitive parasites. ApoL1 variants with
either S342G or I384M alone were less lytic against T. b.
rhodesiense than was the combination of the two mutations, whereas
the S342G/1384M/delN388/Y389 variant was not more active than
delN388/Y389 alone (FIG. 4C). As shown in FIGS. 4D and E, all
measured features of the T. b. rhodesiense lysis process (kinetics,
transient inhibition by chloroquine, typical swelling of the
lysosome) were similar to those observed on T. b. brucei with
either NHS or recombinant ApoL1 (Perez-Morga et al., Science
309:469-472, 2005). Therefore, deletion of N388/Y389 was necessary
and sufficient to prevent interaction with SRA and to allow ApoL1
to kill T. b. rhodesiense, whereas the combination of S342G and
I384M was required for maximal ability to kill T. b. rhodesiense
despite the binding of SRA. None of these mutations affected the
resistance of T. b. gambiense.
Example 5
Predictive Power of APOL1 SNPs
[0272] HIV negative individuals carrying one APOL1 risk allele at
rs73885319 and one APOL1 risk allele at rs71785313 have a predicted
4.3 fold increase in risk of FSGS over the (African American)
population average. 40% of these individuals have a predicted 1.6
fold increased, while the remaining 60% have a predicted 5.6 fold
predicted risk; the individuals receiving an exaggerated prediction
of risk represent 22 out of 1000 individuals tested. Similar
although smaller improvements in risk estimates occur for other
APOL1 risk strata.
[0273] The ROC C statistic was calculated for FSGS. For FSGS, the C
statistic for at least one APOL1 risk allele was 0.822. In HIV
positive individuals, the C statistic for FSGS for at least one
APOL1 risk allele was 0.865. This increase in the C statistic
represents a 3% reduction in residual ignorance of FSGS risk.
Example 6
Nucleic Acid-Based Analysis of Genetic Predisposition to Renal
Disease
[0274] The methods disclosed herein are used for evaluating if a
subject has or is at risk for developing renal disease. For
example, the methods can be used to determine if a subject is at
risk for FSGS, or is at risk for hypertensive ESKD. One skilled in
the art will appreciate that methods that deviate from these
specific methods can also be used to successfully determine if a
subject is at risk for renal disease.
[0275] In one example, a sample including nucleic acids can be
obtained from a subject who is suspected to have a genetic
predisposition to renal disease, such as FSGS or hypertensive ESKD.
The subject can have family members who have had FSGS or
hypertensive renal disease. In another example, a sample including
nucleic acids can be obtained from a subject that is of African
ancestry. In a further example, a sample including nucleic acids is
obtained from a subject with African (such as African-American)
ancestry who is infected with HIV.
[0276] In a further example, a sample including nucleic acids is
obtained from a subject who has renal disease, wherein it is of
interest to determine if the subject has hypertensive ESKD. For
example, a sample can be obtained from a subject who presents with
a reduced glomerular filtration rate (GFR) or other laboratory
evidence of renal impairment (such as elevated blood urea nitrogen
(BUN) or abnormal renal histology), or someone with the clinical
presentation (symptoms) of renal disease, such as fatigue and
liquid retention. Additional indicators of renal disease that can
suggest chronic renal failure include hyperkalemia, acidemia,
elevated serum creatinine levels and/or the uremic syndrome. A
renal biopsy can be obtained from the subject to determine if the
subject has FSGS or hypertensive nephrosclerosis.
[0277] In some particular embodiments of the method, the subject is
seropositive for the HIV virus, and the test is performed to
predict whether the subject is likely to develop renal disease,
such as chronic renal failure, such as renal failure caused by
FSGS. In other embodiments, the subject is someone who has clinical
and laboratory evidence of early renal disease and the genetic test
is performed to confirm the diagnosis of renal disease. For
example, the subject may be an African-American with clinical
evidence of early renal failure without a known etiology.
Alternatively, the subject may have had a renal biopsy performed
with inconclusive or ambiguous results. In these instances, the
genetic test is performed to arrive at a diagnosis of chronic renal
disease (or FSGS) with a higher degree of clinical certainty than
would otherwise be possible. The genetic test can be used in
association with other clinical signs and symptoms to assign a
diagnosis, and from the diagnosis greater prognostic certainty can
be provided to the subject. Alternatively, the genetic test can be
used to provide a more specific diagnosis or etiology for chronic
renal failure, as may be needed in research studies or for the
selection of an appropriate therapeutic regimen.
[0278] In some examples a sample including nucleic acids is
obtained from a subject with lupus nephritis or sickle cell anemia.
These subjects can be tested to determine their haplotype at the
time of diagnosis. In other examples a sample including nucleic
acids is obtained from a subject with diabetes mellitus (type 1 or
type 2), IgA nephropathy, and/or renal vasculitis.
[0279] The finding of a susceptibility haplotype can initiate
screening annually or biannually for protein, using
albumin/creatinine ratio, such as beginning at about age 12 or
about age 15. For example, subjects who are found to have a
condition that is associated with renal injury, including
prematurity, small birth weight, obesity, hypertension, systemic
lupus erythematosus, sickle cell anemia, diabetes mellitus, and
HIV-1 infection can be screened using the methods disclosed
herein.
[0280] To perform the method, a biological sample of the subject is
assayed. The sample can, for example, be a blood sample or a buccal
sample. Methods of isolating nucleic acid molecules from a
biological sample are routine, for example using a commercially
available kit to isolate DNA. Nucleic acid molecules isolated from
PBMCs or any other biological sample can be amplified (for example,
by PCR) using routine methods to form nucleic acid amplification
products.
[0281] It is determined if the individual has an APOL1 SNP (such as
a G at rs73885319, a G at rs60910145, and/or a 6 base pair deletion
at rs71785313) using standard methods, such as real-time PCR (for
example, a TAQMAN.RTM. assay), allele-specific PCR, or sequence
analysis. The presence of at least one APOL1 SNP indicates that the
subject is at risk for developing renal disease. For example, the
methods can be used to determine if a subject is at risk for FSGS,
or is at risk for hypertensive ESKD.
[0282] Thus, in some cases, it is determined if the individual has
an APOL1 SNP (such as a G at rs73885319, a G at rs60910145, and/or
a 6 base pair deletion at rs71785313) using standard methods, such
as real-time PCR (for example, a TAQMAN.RTM. assay),
allele-specific PCR, or sequence analysis. The presence of at least
one APOL1 SNP indicates that the subject is at risk for developing
renal disease. For example, the methods can be used to determine if
a subject is at risk for FSGS, or is at risk for hypertensive
ESKD.
[0283] In another embodiment, the methods can be used to identify
protective alleles in a subject that are associated with the
absence of renal disease. In this instance, the detection of
protective alleles in a biological sample may be indicative of a
lower risk for developing renal disease in the subject.
Example 7
Nucleic Acid-Based Analysis of Resistance to Trypanosoma
[0284] The methods disclosed herein are used for evaluating if a
subject has a resistance to disease associated with Trypanosoma
infection. For example, the methods can be used to determine if a
subject has resistance to African trypanosomiasis (sleeping
sickness) caused by T. brucei. One skilled in the art will
appreciate that methods that deviate from these specific methods
can also be used to successfully determine if a subject has a
resistance to disease associated with Trypanosoma infection.
[0285] In one example, a sample including nucleic acids can be
obtained from a subject who is suspected to be at risk for disease
associated with Trypanosoma infection. The subject can live in,
have traveled to, or plan to travel to an area where Trypanosoma
parasites are endemic, for example, sub-Saharan Africa.
[0286] To perform the method, a biological sample of the subject is
assayed. The sample can, for example, be a blood sample or a buccal
sample. Methods of isolating nucleic acid molecules from a
biological sample are routine, for example using a commercially
available kit to isolate DNA. Nucleic acid molecules isolated from
PBMCs or any other biological sample can be amplified (for example,
by PCR) using routine methods to form nucleic acid amplification
products.
[0287] It is determined if the individual has an APOL1 SNP (such as
a G at rs73885319, a G at rs60910145, and/or a 6 base pair deletion
at rs71785313) using standard methods, such as real-time PCR (for
example, a TAQMAN.RTM. assay), allele-specific PCR, or sequence
analysis. The presence of at least one APOL1 SNP indicates that the
subject has a resistance to disease associated with Trypanosoma
infection. For example, the methods can be used to determine if a
subject is has resistance to disease associated with infection with
T. brucei.
[0288] In another embodiment, the methods can be used to identify
APOL1 SNPs (such as an A at rs73885319, a T at rs60910145, and
absence of a 6 base pair deletion at rs71785313) in a subject that
are associated with decreased resistance or susceptibility to
disease associated with Trypanosoma infection. In this instance,
the detection of these SNPs in a biological sample may be
indicative of decreased resistance or increased susceptibility of
the subject to disease associated with Trypanosoma infection.
Example 8
Genetic Variation in APOL1 and Age at Hemodialysis Initiation in
African Americans
[0289] African Americans have a markedly higher incidence of
end-stage renal disease (ESRD) compared with other racial groups.
Two coding sequence risk alleles in the APOL1 gene, found only in
individuals of recent African ancestry, have been identified as
risk alleles for renal disease in African Americans. We tested
whether these risk alleles were also linked to age of initiation of
chronic hemodialysis.
[0290] Methods:
[0291] We performed a cross-sectional study of 407 non-diabetic
African-Americans with ESRD who participated in Accelerated
Mortality on Renal Replacement (ArMORR), a prospective cohort study
of incident chronic hemodialysis subjects from across the United
States. We examined age of initiation of chronic hemodialysis
according to APOL1 risk alleles (G1 and G2). Analysis of variance
was used to compare mean age at dialysis initiation, and
multivariate linear regression modeling was used to adjust for
potential confounders.
[0292] Results:
[0293] African American subjects carrying two copies of the G1 risk
allele initiated chronic hemodialysis at a mean age of 49.0.+-.14.9
years, significantly earlier than subjects with one copy of the G1
allele (55.9.+-.16.7 years: p=0.014) or those without any risk
allele (61.8.+-.17.1 years; p=6.2.times.10.sup.-7). The G1
relationships remained statistically significant in multivariate
analysis adjusting for socio-demographic and other potential
confounders. G2 risk allele was not linked to age of chronic
hemodialysis initiation; however, limited sample size in this
analysis precluded definitive conclusions.
[0294] Conclusion:
[0295] Genetic variations in the APOL1 gene identify African
Americans that initiate chronic hemodialysis at an earlier age.
Early interventions to prevent progression of kidney disease may
benefit this high-risk population.
[0296] Introduction
[0297] African Americans have a four-fold greater risk of end stage
renal disease (ESRD) compared with white Americans (Klag et al.,
JAMA 277:1293-1298, 1997; System, N.I.o.D.a.D.a.K.D. National
Institutes of Health, Editorial, 2010). In 2009, the mean age for
African Americans at the start of renal replacement treatment was
59.2 years, compared with 66.8 years in Caucasians (System, supra).
This may be due in part to an accelerated progression of renal
disease in African Americans (Hsu et al., J. Am. Soc. Nephrol.
14:2902-2907, 2003; Walker et al., JAMA 268:3085-3091, 1992; Derose
et al., Kidney Int. 76:329-637). Several studies have found that
the high prevalence of ESRD in African Americans cannot be fully
explained by socioeconomic differences or differences in access to
medical care (Klag et al., supra; Tarver-Carr et al., J. Am. Soc.
Nephrol. 13:2363-2370, 2002). Thus, it is thought that biologic
factors, such as genetic differences, contribute to this disparity.
Indeed, previous studies have demonstrated strong familial
aggregation of kidney disease in African Americans (Freedman et
al., J. Am. Soc. Nephrol. 8:1942-1945, 1997). Two recent studies
used genetic admixture mapping to identify a region of chromosome
22 that explained the increased kidney disease risk in African
Americans (Kao et al., Nat. Genet. 40:1185-1192, 2008; Genovese et
al., Science 329:841-845, 2010).
[0298] Genovese et al. identified sequence variants in
apolipoprotein L-1 (APOL1) as risk alleles for focal segmental
glomerulosclerosis (FSGS) and hypertension-attributed ESRD (H-ESKD)
in African Americans (Genovese et al., Science, supra; Genovese et
al., Kidney Int. 78:698-704, 2010). APOL1 is located adjacent to
the MYH9 gene on chromosome 22, a locus that has previously been
reported to explain the high risk of renal disease in African
Americans (Kao et al., supra). Interestingly, APOL1 risk proteins
have lytic activity against a subspecies of trypanosomes known to
cause African sleeping sickness. Carrier status may have provided a
selective evolutionary advantage and thus maintained these risk
alleles in the African population. The two risk alleles found to
confer an elevated risk for FSGS and H-ESRD includes "G1," a two
locus allele found in a 10-kb region in the last exon of APOL1, and
"G2," a six base pair deletion located in close proximity to the G1
risk allele (Genovese et al., Science 329:841-845, 2010). These
risk alleles in APOL1 are only found in individuals of African
descent with allele frequencies of 38% for G1 and 8% for G2 in the
African Yoruba population. These alleles appear to act in a
recessive manner, with a 7 to 10 fold increased risk of H-ESKD or
FSGS conferred by the presence of a risk-associated allele in both
copies of APOL1.
[0299] Given the association between APOL1 risk alleles and
non-diabetic renal disease in African Americans, we tested whether
African Americans with ESRD and who are homozygous for APOL1 risk
alleles progress to ESRD at an earlier age than those who do not
have these risk alleles. The tests were performed in a cohort of
non-diabetic African American subjects initiating chronic
hemodialysis in the United States.
[0300] Results
[0301] Subject characteristics, including demographic information,
income, vascular access, cause of ESRD and laboratory values are
summarized in Table 10. The mean age of hemodialysis initiation
among all subjects was 55.2.+-.17.1 years.
[0302] When subjects were stratified into six unique groups
according to the number of G1 or G2 risk alleles, only subjects
with two G1 risk alleles had significantly lower mean age at
hemodialysis initiation compared to subjects without these APOL1
risk alleles (Wt+Wt=61.8.+-.17.0 vs. Wt+G1=55.9.+-.16.7 [p=0.152];
vs. G1+G1=49.0.+-.14.9 [p=3.0.times.10.sup.-6]; vs.
G1+G2-49.3.+-.17.0 [p=1.83.times.10.sup.-4], FIG. 5). In contrast,
subjects with one or two G2 risk alleles, but no G1 risk alleles,
did not begin hemodialysis at an earlier age compared with subjects
who lacked the G1 or G2 risk alleles (Wt+G2=58.1.+-.16.3 [p=0.96],
G2+G2=48.9.+-.15.3 [p=0.09]). However, the number of individuals
with the G2+G2 risk alleles was small. We therefore conducted
subsequent analyses only with G1 risk alleles. Decreased power from
exclusion of subjects with G2 risk alleles did not dramatically
change the results of the analysis. Including all subjects,
regardless of G2 risk, mean age at hemodialysis was significantly
lower among those with two G1 risk alleles compared to those
without G1 risk alleles (p=1.0.times.10.sup.-6).
[0303] Table 11 shows subject characteristics according to whether
subjects had zero, one, or two copies of the G1 risk allele. The
p-values in Table 11 represent comparisons between the genotypes.
G1 homozygotes, heterozygotes, and subjects without the G1 risk
allele had a similar proportion of male subjects and similar income
distribution, and similar locations of hemodialysis initiation.
Subjects with and without G1 alleles had similar systolic and
diastolic blood pressures, parathyroid hormone levels, calcium
levels, hemoglobin concentration, and albumin concentrations.
Subjects with G1 risk alleles tended to have higher serum
creatinine levels (p=1.0.times.10.sup.-6), perhaps due to their
age. To investigate possible confounders between G1 risk allele and
age at hemodialysis initiation, we also investigated factors
independently correlated with mean age at hemodialysis initiation;
BMI (r=-0.224, p=1.2.times.10.sup.-4); serum creatinine (r=-0.439,
p=1.5.times.10.sup.-14); systolic blood pressure (r=-0.188,
p=1.3.times.10.sup.-3); diastolic blood pressure (r=-0.490,
p=9.2.times.10.sup.-19). We estimated eGFR levels using the MDRD
formula and found that subjects with G1 risk alleles had lower eGFR
levels at ESRD initiation (p=8.1.times.10.sup.-5).
[0304] In consonance with other U.S. renal study populations, in
nearly three-fourths of our subjects ESRD was caused by
hypertension. Other causes of ESRD in our population, including
HIV, inflammation, toxins, etc. were grouped together as `other.`
In stratified analyses by cause of ESRD, the mean age at initiation
of hemodialysis remained younger in H-ESRD subjects with G1 risk
alleles but not in subjects with other reported causes of ESRD,
FIG. 6.
[0305] Subjects with ESRD due to causes other than hypertension
initiated chronic hemodialysis at an earlier mean age than subjects
with H-ESRD (50.6.+-.18.0 years vs. 58.1.+-.16.3 years,
p=7.8.times.10.sup.4). In multivariate regression models, the
presence of G1 risk alleles remained significantly associated with
early hemodialysis initiation after adjustment for demographic and
socioeconomic variables and cause of ESRD among non-diabetics
(Table 12). The average values in Table 12 represent the predicted
values estimated from multivariate regression equations controlling
for socio-demographic and clinical characteristics. In a similar
model, when we did not stratify by cause of ESRD (hypertension vs
other), we found that the G1 allele remained associated with age of
hemodialysis initiation.
[0306] Discussion
[0307] We aimed to determine if the G1 or G2 sequence risk alleles
of the APOL1 gene (which are associated with an increased risk of
renal disease in African Americans) are associated with initiating
chronic hemodialysis at a younger age--a marker of the severity of
progressive CKD. We found that African Americans with two copies of
the G1 risk allele initiated chronic hemodialysis approximately ten
years earlier than those without this allele. Subjects with one
copy of the G1 allele initiated hemodialysis an average of six
years earlier. These estimates were unchanged after adjustment for
a variety of socio-demographic factors. The presence of the G2
allele may be associated with the initiation of hemodialysis at a
younger age.
[0308] APOL1 risk alleles are associated with FSGS and H-ESKD
(Genovese et al., Kidney Int., supra). This study shows that these
risk alleles are also associated with age at first start of chronic
hemodialysis, a measure we used as a surrogate for age of
developing ESRD. Our data support the conceptual model that APOL1
risk alleles either trigger onset of renal disease at an earlier
age, or once initiated, alter the rate of progression.
[0309] Several factors may influence increased risk for ESRD in
African Americans. Milder forms of kidney disease, CKD stages are
less prevalent in African Americans while stage IV CKD and ESRD are
much more common (System, supra; Hsu et al., supra; Volkova et al.,
J. Am. Soc. Nephrol. 19:356-364, 2008). Consistent with this, in
the ArMORR study, the overall mean age at hemodialysis initiation
for Caucasians is 65.5.+-.14.9 years, compared to 57.8.+-.15.4
years in African Americans (p<1.0.times.10.sup.-4) (Shurraw et
al., Am. J Kidney Dis. 55:875-884, 2010). This is consistent with
an accelerated progression of ESRD in these subjects. This could
also be explained by an alternative pathogenesis for ESRD in some
African Americans, leading to early clinical onset of ESRD,
potentially mediated by mutations in the APOL1 gene. Several
factors have been associated with the rate of progression to ESRD
in African Americans, including, e.g., proteinuria,
25-hydroxyvitamin D levels, and late referral to specialty care.
One or more of these risk factors may mediate the relationship
between APOL1 risk alleles and ESRD in this population (Melamed et
al., J. Am. Soc. Nephrol. 20:2631-2639, 2009). In this study we
found that body mass index and income were unlikely to confound the
association between APOL1 genetic risk alleles and earlier age at
hemodialysis initiation.
[0310] Subjects with two copies of the G2 risk allele may initiate
hemodialysis at a younger age, while subjects with one copy of the
G2 risk allele appear to initiate hemodialysis at approximately the
same age as subjects without any risk alleles.
[0311] As there was less power to detect effects of the rarer G2
risk allele, it is less clear to what extent this characteristic
affects age at hemodialysis initiation. Post-hoc power analysis
revealed a sample size of 34 per G2 risk allele group is required
to obtain a power of 0.8, and 45 to obtain a power of 0.9. Based on
our current sample sizes it is likely that homozygous G2 risk
allele groups were underpowered to find any significant differences
between age of hemodialysis initiation. Also, just one copy of the
G1 allele was associated with a younger age at hemodialysis
initiation.
[0312] In conclusion, genetic variation in APOL1 is associated with
earlier onset of ESRD in African Americans without diabetes
mellitus as the etiology of end stage renal failure, and thus APOL1
genetic screening can be used to identify patients at risk so that
preventative interventions can be initiated much earlier than is
currently practiced.
[0313] Methods
Subjects
[0314] Subjects were African Americans with non-diabetic ESRD
enrolled in Accelerated Mortality on Renal Replacement (ArMORR), a
prospective cohort study of 10,044 subjects who initiated chronic
hemodialysis at any of 1056 US hemodialysis centers operated by
Fresenius Medical Care, North America between June 2004 and August
2005.[15] The study was approved by the Institutional Review Board
(IRB) of the Massachusetts General Hospital (MGH), which waived the
need for informed consent of this repository. Blood samples from
407 non-diabetic African Americans were available for DNA
extraction.
Data Collection
[0315] Data were collected prospectively by care-givers and
included demographics, body mass index, co-morbidities,
hemodialysis access (catheter, graft, or fistula), and reported
cause of ESRD. Laboratory tests on blood samples collected within
14 days of hemodialysis initiation were performed by a central
laboratory (Spectra East, Rockland, N.J.) and included albumin,
creatinine, calcium, phosphate, and hemoglobin, measured using
standard multisample automated analyzers. Intact parathyroid
hormone (PTH) was measured using Nichols Bio-intact assay of
full-length 1-84 PTH. Subject's eGFR levels were estimated using
the Modification of Diet in Renal Disease (MDRD) formula (Levey et
al., Ann. Intern. Med. 130:461-470, 1999).
[0316] Each subject received chronic hemodialysis at an outpatient
Fresenius Medical Center North American facility. Because age of
initiation of chronic hemodialysis may differ by region and income,
median household income of African Americans living in the zip code
for each facility was determined from U.S. Census data for the year
2000 and used as an estimate of socioeconomic status. (Kinchen et
al., Ann. Intern. Med 137:479-486, 2002.) We divided subjects into
three equally sized groups (high, medium, and low socioeconomic
status) based on median household income of African Americans in
the zip code in which they initiated dialysis. We also assigned
subjects to one of three geographic regions based on their
hemodialysis facility's zip code and U.S. Census Regions (U.S.
Census, available from:
http://factfinder.census.gov/servlet/DTTable?_bm=y&-context=dt&-ds_name=D-
EC_2000_SF3_U&-CONTEXT=dt&-mt_name=DEC_2000_SF3_U_P151B&-tree_id=403&-redo-
Log=false&-all_geo_types=N&-geo_id=01000US&-geo_id=02000US1&-geo_id=02000U-
S2&-geo_id=02000US3&-geo_id=02000US4&-search_results=01000US&-format=&-_la-
ng=en, retrieved 2002). These regions included Northeast
(Connecticut, Massachusetts, Maine, New Hampshire, New Jersey,
Pennsylvania, Puerto Rico, Rhode Island), Midwest/West (Arizona,
Colorado, Illinois, Kansas, New Mexico, Missouri, Minnesota,
Montana, Ohio, Wisconsin), and South (Alabama, Arkansas, Washington
D.C., Delaware, Florida, Georgia, Kentucky, Louisiana, Maryland,
Mississippi, North Carolina, Oklahoma, South Carolina, Tennessee,
Texas, Virginia, and West Virginia). Puerto Rico and the Island
Areas are not part of any census region or census division. For
this reason, Puerto Rico was assigned to the North East region
based on Standard Federal Regions where Puerto Rico is grouped in
Region III with New York and New Jersey (Federal Regions, available
from:
http://www.atsdr.cdc.gov/WebMaps/helpcontent/MapOptionsAdvance.asp#region-
s, retrieved 2010).
Genotyping
[0317] Genomic DNA was extracted from whole blood stored in PaxGene
tubes using a protocol adapted from PreAnalytix using a
QiagenAutoPure extraction robot (Harvard Partners Center for
Genetics and Genomics, Cambridge, Mass., USA). In all samples, DNA
quality was assessed with 260/280 OD ratios. The patients' DNA
samples were diluted in water to 10 nanograms per microliter, and
300 nanograms of each DNA sample were sent to Polymorphic DNA
Technologies in Alameda, Calif., which provided assay design,
oligonucleotide primers, PCR amplification, DNA sequencing and data
analysis. Polymorphic DNA Technologies uses Sanger dideoxy DNA
sequencing and employs automated high throughput capillary
electrophoresis DNA sequencing instruments and is less prone to
error rates, especially when two alleles, such as G1 and G2, are
juxtaposed close to each other.
[0318] We considered both G1 and G2 risk alleles and classified the
subjects by APOL1 risk allele status into groups depending on the
number of G1 or G2 alleles present. This created six unique groups
(WT+WT, G1+WT, G1+G1, G2+WT, G2+G2, G1+G2). Due to mutual
exclusivity of G1 and G2, no subjects had more than two risk
alleles in total (FIG. 5). We then considered G1 and G2 risk
alleles separately and compared the age of hemodialysis initiation
in subjects with zero, one, or two copies of each allele.
Statistical Analysis
[0319] Analysis of variance (ANOVA) with Sidak post-hoc tests was
used to compare mean ages at hemodialysis initiation and other
relevant continuous variables across genotypic groups. Pearson
correlation coefficients were used to examine the associations
between continuous variables. Chi-squared tests were used for
categorical variables. Multivariate linear regression modeling was
used to obtain the average predicted age of hemodialysis initiation
for G1 risk alleles, excluding G2 risk alleles, after adjustment
for socioeconomic, demographic and clinical factors. All
statistical analyses were performed using SAS version 9.2 software
(Cary, N.C.) and STATA version 11 (College Station, Tex.).
Two-tailed p-values of <0.05 were considered significant.
Example 9
An APOL1 Gene Inversion as a Risk/Resistance Allele
[0320] As discussed above, the G1 and G2 variants are located in
the C-terminal end of the APOL1 gene product. APOL1, APOL2, and
APOL4 are located in close proximity to one another on chromosome
22 (see FIG. 7A, not to scale).
[0321] The tail to tail (5' to 5') arrangement of APOL1 and APOL2
suggests coordinated regulation, a prediction that has been
confirmed with HapMap gene expression data (see FIG. 8).
[0322] Using bioinformatics, we discovered another APOL1 gene
risk/resistance allele that is a chromosomal rearrangement. The
chromosomal rearrangement inversion is predicted to invert a
segment of DNA including the 5' end of APOL4, all of APOL2, and the
5' end of APOL1 (see FIG. 7B). Individuals with the rearrangement
inversion on a given chromosome have several important changes:
[0323] a) The reference APOL4 gene is replaced by an APOL1/APOL4
hybrid gene;
[0324] b) The reference APOL1 gene is replaced by an APOL4/APOL1
hybrid gene;
[0325] c) APOL4 expression is driven by the reference APOL1
promoter, and APOL1 is driven by the reference APOL4 promoter;
and
[0326] d) APOL1 and APOL2 coordinated expression may now be
unlinked.
[0327] We validated the existence of the inversion at the
APOL1/APOL4 junction using PCR in human samples (see FIG. 9).
[0328] The inversion in the APOL1 gene may result in replacement of
up to three exons in APOL1 by sequence from APOL4 (e.g., the
inversion may result in replacement of all or a portion of only the
first exon, all or a portion of the first and/or second exon,
and/or all or a portion of the first, second, or third exon of the
APOL1 gene). These three exons may cover a range of 2000-2500 base
pairs of genomic DNA (e.g., in a range of from about 100 base pairs
to about 3000 base pairs of genomic DNA, such as a range from 1000
base pairs to about 2500 base pairs of genomic DNA), and may encode
a maximum of about 420 base pairs of transcript (e.g., a range of
from about 20 base pairs to about 500 base pairs of transcript,
such as from about 100 base pairs to about 420 base pairs of
transcript DNA). The actual coding sequence replaced in the APOL1
protein may only code for 1 to about 30 amino acids, e.g., about 10
to about 20 amino acids, e.g., about 14 amino acids from APOL4.
These substituted amino acids may all appear in the preprotein
portion of the hybrid APOL4/APOL1 protein and all or a portion of
the replaced amino acids may be cleaved depending upon the extent
and actual sequence of the inversion.
[0329] As shown in FIG. 10, the sequence in the inverted chromosome
is reference APOL1 at the 5' end and reference APOL4 at the 3' end.
The breakpoints on hg18 are at approximately chr22:34,981,580 to
chr22:34,981,980 at the APOL1 end, and chr22:34,927,460 to
chr22:34,927,060 at the APOL4 end.
[0330] The functional consequences of the inversion are predicted
to be one or more of the following:
[0331] a) the native APOL1 promoter is eliminated, and replaced by
a promoter that either expresses APOL1 at much lower levels or may
not express APOL1 at all;
[0332] b) in individuals with the inversion and another SNP,
rs9610445 (the C allele), an essential splice site is eliminated,
causing an alteration in the transcript with potential functional
consequences (see FIG. 11A);
[0333] c) in individuals with the inversion and another SNP,
rs6000181 T (minor) allele, a methionine start site is eliminated,
potentially altering translation of APOL1 (see FIG. 11B).
[0334] d) APOL1 and APOL2 expression are no longer coordinated;
and/or
[0335] e) The N-termini of ApoL1 and ApoL4 proteins are exchanged.
Under normal conditions, this may have no effect, as APOL1 has a
signal peptide that is cleaved prior to export from the cell,
effectively removing the amino acids contributed by APOL4. However,
in the setting of dramatic APOL1 upregulation that we have observed
when cells are exposed to inflammatory factors, the APOL4-encoded
region may not be efficiently cleaved and could affect molecular
function.
[0336] Despite the unusually large odds ratio for renal disease
associated with 2 APOL1 renal risk variants (G1 and G2), some
individuals with 2 risk variants do not develop disease, while some
with 0 or 1 variant do develop disease. The functional properties
of the inversion may be a "G3" that will improve predictive value
of APOL1 testing. Thus, the identification of the G1, G2, and/or G3
risk alleles in a human subject is predictive of a genetic
predisposition to and/or increased risk of the development of renal
disease in the human subject.
[0337] In addition, for the reasons given herein, G3 may also be a
resistance allele that can be detected alone or in combination with
G1 and/or G3 in a human subject to determine a resistance to a
disease associated with infection by a Trypanosoma spp. in the
human subject.
Other Embodiments
[0338] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure that come
within known or customary practice within the art to which the
invention pertains and may be applied to the essential features
hereinbefore set forth.
[0339] All publications and patent applications mentioned in this
specification, including the priority application, U.S. Application
Ser. No. 61/325,343, are herein incorporated by reference to the
same extent as if each independent publication or patent
application was specifically and individually indicated to be
incorporated by reference in their entirety.
[0340] In view of the many possible embodiments to which the
principles of the disclosure may be applied, it should be
recognized that the illustrated embodiments are only examples and
should not be taken as limiting the scope of the invention. Rather,
the scope of the invention is defined by the following claims. We
therefore claim as our invention all that comes within the scope
and spirit of these claims.
TABLE-US-00005 TABLE 3 Association of variants on chromosome 22
with FSGS using Fisher's exact test Frequency Frequency derived
derived Position Derived allele in allele in Ancestral Variant
(NCBI 36) allele cases controls allele P-value rs11089781 34886714
A 0.32 0.21 G 0.001341 rs7364143 34932129 T 0.56 0.4 G 9.904e-06
rs7289037 34938336 A 0.53 0.33 G 5.118e-08 rs8136528 34941252 T
0.52 0.34 C 4.977e-07 rs4821469 34946391 C 0.7 0.51 T 9.835e-08
rs73885303 34953617 T 0 0 C 0.4759 rs10854687 34954365 A 0.51 0.3 C
4.117e-09 rs9622362 34986390 A 0.04 0.09 C 0.01488 rs9622363
34986501 G 0.82 0.51 A 6.112e-20 ns41297245 34987686 A 0.01 0.05 G
0.000927 rs2239785 34991276 A 0.14 0.35 G 3.081e-12 rs136175
34991512 A 0.97 0.93 G 0.01712 rs73403889 34991637 A 0 0 G 1
rs16996616 34991837 A 0.03 0.08 G 0.00933 rs73885319 34991852 G
0.53 0.19 A 1.07e-23 rs60910145 34991980 G 0.52 0.18 T 2.591e-23
rs71785313 34991997 D 0.23 0.15 T 0.008882 rs58384577 34993159 C
0.49 0.19 T 9.782e-18 rs60295735 34997100 A 0.5 0.2 G 9.128e-17
rs56277602 34998706 G 0.5 0.76 T 2.359e-13 rs73885325 35000629 T
0.49 0.25 A 6.903e-12 SNP 2 bp after 35005098 A 0.44 0.22 G
1.482e-09 rs136196 rs73405714 35005359 G 0.44 0.22 A 9.451e-10
rs11703176 35008422 A 0.48 0.31 C 3.782e-06 rs58168942 35012680 A
0.4 0.19 G 2.652e-10 rs5756130 35014277 T 0.06 0.1 C 0.08136
rs6000226 35014513 T 0.32 0.26 C 0.09085 rs11912763 35014668 A 0.41
0.19 G 9.573e-12 rs11549907 35014926 T 0.06 0.08 C 0.2976 rs6000229
35016105 T 0.2 0.36 C 5.378e-06 rs6000223 35017908 0 0 0 A 1
rs73405726 35018652 C 0.47 0.29 G 3.09e-07 rs73405727 35020433 T
0.42 0.19 C 2.243e-11 rs2239786 35021873 C 0.32 0.29 G 0.4288
rs16996648 35022698 C 0.47 0.29 T 3.09e-07 rs56339459 35023558 T
0.42 0.19 C 8.107e-12 rs4821481 35025888 T 0.17 0.36 C 4.398e-09
rs6000235 35026033 T 0.78 0.53 C 7.393e-13 rs3752462 35040129 T
0.87 0.72 C 1.82e-07 rs2239784 35044581 T 0.72 0.63 C 0.005961
rs8141189 35044656 T 0.57 0.7 A 0.0002506 rs7285770 35045413 A 0.41
0.25 G 5.869e-06 rs55816447 35047283 T 0.42 0.25 C 7.291e-07
rs55670830 35049098 G 0.61 0.76 A 2.948e-05 rs16996668 35049131 G
0.58 0.75 C 2.92e-06 rs12160045 35049306 G 0.55 0.68 A 0.0004368
rs11912139 35052407 T 0.41 0.25 C 1.34e-06 rs11912881 35053384 A
0.41 0.25 T 8.857e-06 rs16996672 35055916 T 0.45 0.27 C 1.611e-07
rs16996674 35056598 T 0.41 0.25 C 3.157e-06 rs16996677 35057229 A
0.45 0.26 G 6.587e-08
TABLE-US-00006 TABLE 4 Number and frequencies of APOL1 genotypes
and alleles in FSGS and hypertension-attributed ESKD cases and
controls Hypertension- FSGS Cases and Controls attributed ESKD BWH
NIH Total NIH WFU WFU Cases Cases Cases Controls Cases Controls
Genotype WT + WT 3 26 29 77 239 409 WT + G1 6 21 27 41 173 250 WT +
G2 0 9 9 36 124 155 G1 + G1 25 35 60 9 219 41 G1 + G2 15 38 53 8
203 50 G2 + G2 3 11 14 5 44 18 Total 52 140 192 176 1002 923 Allele
G1 Freq. 0.68 0.47 0.52 0.18 0.41 0.21 G2 Freq. 0.19 0.25 0.23 0.15
0.21 0.13
Samples for which either the G1 or G2 assay failed are not
reported
TABLE-US-00007 TABLE 5 Association of different variants on
chromosome 22 with FSGS after controlling for variant rs73885319
(which co-segregates with rs60910145) using logistic regression
Position Variant (NCBI 36) P-value rs11089781 34886714 0.925
rs7364143 34932129 0.9919 rs7289037 34938336 0.2779 rs8136528
34941252 0.5498 rs4821469 34946391 0.05454 rs73885303 34953617
0.9993 rs10854687 34954365 0.3387 rs9622362 34986390 0.8758
rs9622363 34986501 0.0001509 rs41297245 34987686 0.1298 rs2239785
34991276 0.00566 rs136175 34991512 0.2976 rs73403889 34991637
0.9993 rs16996616 34991837 0.5397 rs73885319 34991852 NA rs60910145
34991980 0.6051 rs71785313 34991997 4.377e-07 rs58384577 34993159
0.1982 rs60295735 34997100 0.6759 rs56277602 34998706 0.3886
rs73885325 35000629 0.3589 SNP 2bp after 35005098 0.1207 rs136196
rs73405714 35005359 0.1746 rs11703176 35008422 0.02567 rs58168942
35012680 0.2972 rs5756130 35014277 0.2318 rs6000226 35014513
0.0001059 rs11912763 35014668 0.4741 rs11549907 35014926 0.9798
rs6000229 35016105 0.1074 rs6000223 35017908 NA rs73405726 35018652
0.1368 rs73405727 35020433 0.5361 rs2239786 35021873 0.0005036
rs16996648 35022698 0.1368 rs56339459 35023558 0.3864 rs4821481
35025888 0.003736 rs6000235 35026033 0.002684 rs3752462 35040129
0.003073 rs2239784 35044581 0.743 rs8141189 35044656 0.3762
rs7285770 35045413 0.3736 rs55816447 35047283 0.3485 rs55670830
35049098 0.2393 rs16996668 35049131 0.4054 rs12160045 35049306
0.1751 rs11912139 35052407 0.289 rs11912881 35053384 0.3462
rs16996672 35055916 0.6268 rs16996674 35056598 0.3884 rs16996677
35057229 0.7119
TABLE-US-00008 TABLE 6 Association of different variants on
chromosome 22 with FSGS after controlling for variants rs73885319
and rs71785313 using logistic regression Position (NCBI Variant 36)
P-value rs11089781 34886714 0.2439 rs7364143 34932129 0.1461
rs7289037 34938336 0.8734 rs8136528 34941252 0.3631 rs4821469
34946391 0.3533 rs73885303 34953617 0.9993 rs10854687 34954365
0.4109 rs9622362 34986390 0.13 rs9622363 34986501 0.8217 rs41297245
34987686 0.5326 rs2239785 34991276 0.9337 rs136175 34991512 0.8123
rs73403889 34991637 0.9993 rs16996616 34991837 0.3839 rs73885319
34991852 NA rs60910145 34991980 0.7417 rs71785313 34991997 NA
rs58384577 34993159 0.3419 rs60295735 34997100 0.9987 rs56277602
34998706 0.5788 rs73885325 35000629 0.5589 SNP 2bp after 35005098
0.285 rs136196 rs73405714 35005359 0.3297 rs11703176 35008422
0.1264 rs58168942 35012680 0.4001 rs5756130 35014277 0.5038
rs6000226 35014513 0.1419 rs11912763 35014668 0.529 rs11549907
35014926 0.4356 rs6000229 35016105 0.9112 rs6000223 35017908 NA
rs73405726 35018652 0.3053 rs73405727 35020433 0.5282 rs2239786
35021873 0.1924 rs16996648 35022698 0.3063 rs56339459 35023558
0.4316 rs4821481 35025888 0.2728 rs6000235 35026033 0.4815
rs3752462 35040129 0.1644 rs2239784 35044581 0.1031 rs8141189
35044656 0.5344 rs7285770 35045413 0.5092 rs55816447 35047283
0.4334 rs55670830 35049098 0.3437 rs16996668 35049131 0.5467
rs12160045 35049306 0.45 rs11912139 35052407 0.3368 rs11912881
35053384 0.4146 rs16996672 35055916 0.8562 rs16996674 35056598
0.4494 rs16996677 35057229 0.954
TABLE-US-00009 TABLE 7 Association of variants on chromosome 22
with hypertensive ESKD using basic association test Frequency
Frequency derived derived Position Derived allele allele Ancestral
Variant (NCBI 36) allele in cases in controls allele P-value
rs5999985 34452302 A 0.05 0.04 G 0.3545 rs41283201 34452326 A 0.07
0.06 T 0.2416 rs2157258 34672336 C 0.31 0.33 T 0.2191 rs16996299
34778586 T 0.41 0.34 C 3.215e-06 rs6000152 34868999 A 0.08 0.07 G
0.3209 rs7284379 34881360 T 0.28 0.19 C 8.155e-11 rs11089781
34886714 A 0.29 0.21 G 1.327e-10 rs132653 34886769 T 0.44 0.48 G
0.01523 rs6000173 34917169 T 0.74 0.66 G 2.658e-08 rs61730819
34917292 T 0.08 0.11 C 0.000395 rs2016703 34948899 T 0.42 0.25 C
1.448e-29 rs1001293 34960895 T 0.45 0.31 C 3.059e-20 rs9622363
34986501 G 0.72 0.53 A 6.125e-34 rs136168 34990788 A 0.44 0.54 G
2.395e-10 rs16996616 34991837 A 0.05 0.08 G 0.000549 rs73885319
34991852 G 0.41 0.21 A 1.097e-39 rs71785313 34991997 D 0.21 0.13 I
7.276e-10 rs7078 35007860 G 0.1 0.15 A 3.42e-05 rs12107 35007928 A
0.07 0.11 G 1.731e-05 rs16996639 35008348 A 0.1 0.08 G 0.0599
rs11089787 35008399 G 0.48 0.38 C 1.642e-10 rs735853 35009159 G
0.07 0.11 C 1.144e-06 rs58168942 35012680 A 0.34 0.2 G 7.018e-24
rs5756129 35014038 C 0.15 0.21 T 6.297e-07 rs11912763 35014668 A
0.34 0.19 G 8.035e-24 rs56020676 35020066 C 0.4 0.26 T 1.324e-19
rs73885341 35021424 A 0.4 0.27 G 5.333e-17 rs4821480 35025193 T
0.26 0.4 G 6.622e-21 rs2032487 35025374 T 0.25 0.38 C 3.941e-20
rs4821481 35025888 T 0.26 0.4 C 8.434e-21 rs2413396 35038030 C 0.72
0.58 T 1.114e-16 rs5750250 35038429 G 0.68 0.5 A 2.757e-26
rs3752462 35040129 T 0.81 0.73 C 5.73e-09 rs11912881 35053384 A
0.34 0.25 T 5.993e-11 rs16996674 35056598 T 0.34 0.23 C 6.825e-13
rs16996677 35057229 A 0.36 0.27 G 1.709e-10
TABLE-US-00010 TABLE 8 Association of different variants on
chromosome 22 with hypertensive ESKD after controlling for variant
rs73885319 using logistic regression Position Variant (NCBI 36)
P-value rs5999985 34452302 0.2828 rs41283201 34452326 0.3638
rs2157258 34672336 0.02322 rs16996299 34778586 0.0567 rs6000152
34868999 0.2081 rs7284379 34881360 0.004519 rs11089781 34886714
0.006513 rs132653 34886769 0.5453 rs6000173 34917169 0.0008435
rs61730819 34917292 0.1326 rs2016708 34948899 9.595e-06 rs1001293
34960895 0.006298 rs9622363 34986501 6.124e-08 rs136168 34990788
0.01352 rs16996616 34991837 0.5301 rs73885319 34991852 NA
rs71785313 34991997 8.798e-18 rs7078 35007860 0.1648 rs12107
35007928 0.05038 rs16996639 35008348 0.002146 rs11089787 35008399
0.156 rs735853 35009159 0.0159 rs58168942 35012680 0.9944 rs5756129
35014038 0.06101 rs11912763 35014668 0.3702 rs56020676 35020066
0.5497 rs73885341 35021424 0.9711 rs4821480 35025193 6.763e-06
rs2032487 35025374 2.137e-05 rs4821481 35025888 8.516e-06 rs2413396
35038030 0.0006269 rs5750250 35038429 4.145e-08 rs3752462 35040129
0.02872 rs11912881 35053384 0.8152 rs16996674 35056598 0.47
rs16996677 35057229 0.9985
TABLE-US-00011 TABLE 9 Frequency differentiation analysis of
variants near APOL1 for two African populations, Yoruba from
Nigeria and Luhya from Kenya Frequency Frequency Non reference non-
Position Reference reference allele in reference Variant (NCBI 36)
allele allele YRI allel in LWK F.sub.ST P-value rs12185880 34900774
C G 0.86 0.76 0.02 0.0453 rs132681 34904713 A G 0.07 0.05 0 0.5108
rs132683 34905610 G A 0.67 0.58 0.01 0.1986 rs132686 34906596 A G 1
0.97 0.02 0.0815 rs132688 34906886 G A 1 0.97 0.01 0.1726 rs6000164
34907077 C T 0.89 0.8 0.02 0.0869 rs132689 34907092 G A 0.9 0.95
0.01 0.1459 rs5995235 34907888 C T 0.67 0.62 0.01 0.3792 rs6000167
34908800 G A 0.45 0.52 0.01 0.3370 rs132692 34909112 T C 0.03 0.16
0.05 0.0012 rs132693 34909507 A G 0.01 0.02 0 0.6093 rs2239831
34913030 T C 0.13 0.21 0.01 0.1274 rs916338 34914376 T C 0.01 0.02
0 0.6093 rs132697 34914659 A G 0.01 0.07 0.02 0.0319 rs8136064
34914892 T G 0.98 0.91 0.03 0.0201 rs1053982 34915510 T C 0.25 0.41
0.03 0.0133 rs5756091 34915667 T G 0.24 0.41 0.03 0.0087 rs5756093
34915917 G A 1 1 NaN NaN rs6000172 34917148 G A 0.24 0.37 0.02
0.0413 rs6000174 34917225 A G 0.24 0.37 0.02 0.0407 rs2227167
34917432 A G 0.24 0.37 0.02 0.0378 rs2269596 34920892 C T 0.23 0.3
0.01 0.2156 rs2007468 34921326 A G 0.12 0.14 0 0.6150 rs2007706
34922316 C T 0.01 0.1 0.04 0.0059 rs132717 34926598 C T 0.23 0.44
0.05 0.0015 rs132734 34927823 G A 0.23 0.43 0.05 0.0012 rs132735
34927827 G T 0.52 0.67 0.02 0.0307 rs5995251 34930704 A T 0.61 0.36
0.06 0.0003 rs6000190 34930787 A G 0.61 0.36 0.06 0.0003 rs5995252
34931145 C T 0.6 0.36 0.06 0.0004 rs7364143 34932129 G T 0.53 0.79
0.08 5.315e-05 rs5995255 34932725 G T 0.58 0.38 0.04 0.0038
rs6000197 34933240 G A 0.58 0.37 0.04 0.0029 rs132744 34934551 T C
0.48 0.27 0.05 0.0015 rs132745 34935277 C T 0.15 0.32 0.04 0.0040
rs132746 34935337 C T 0.15 0.32 0.04 0.0030 rs8142325 34935923 A T
0.54 0.83 0.09 8.998e-06 rs132749 34936575 C T 0.81 0.67 0.03
0.0262 rs9610448 34938151 A G 0.22 0.31 0.01 0.1381 rs132750
34938295 C T 0.03 0.04 0 0.4781 rs7289037 34938336 G A 0.51 0.81
0.1 6.039e-06 rs11704479 34939580 G A 1 1 NaN NaN rs4820222
34939685 C T 0.22 0.32 0.02 0.0975 rs6000199 34939878 G A 0.92 0.84
0.02 0.0661 rs8140384 34940517 C T 0.21 0.22 0 0.7640 rs8136528
34941252 C T 0.52 0.79 0.09 1.990e-05 rs5995259 34941809 G A 0.82
0.76 0.01 0.3005 rs1315 34946081 A C 0.9 0.9 0 0.9650 rs4821467
34946146 G A 0.51 0.79 0.09 1.390e-05 rs4821469 34946391 T C 0.34
0.45 0.02 0.0877 rs763086 34949003 G A 0.34 0.49 0.03 0.0273
rs11703398 34950907 A G 0.83 0.7 0.03 0.0223 rs2006259 34951559 A C
0.35 0.49 0.02 0.0366 rs132757 34951655 T C 0 0.01 0.01 0.4369
rs9619597 34952768 G T 1 1 NaN NaN rs129607 34952852 T C 0.39 0.64
0.06 0.0003 rs132760 34953677 T C 0 0 NaN NaN rs7285167 34953866 G
A 0.54 0.86 0.12 3.653e-07 rs11089784 34956223 C T 0.9 0.9 0 0.8770
rs11703957 34956901 A G 0.79 0.72 0.01 0.2647 rs2010467 34958853 T
C 0.54 0.24 0.1 6.779e-06 rs2010659 34959579 A C 0.86 0.87 0 0.8853
rs9610462 34960296 C A 0.86 0.87 0 0.8162 rs1001294 34960936 C T
0.86 0.86 0 0.9329 rs2157249 34960985 T C 0.86 0.86 0 0.9545
rs2157250 34961637 G A 0.05 0.03 0 0.4757 rs136142 34962971 C T
0.65 0.44 0.05 0.0024 rs1557534 34963171 G A 0.97 0.94 0.01 0.3752
rs136145 34965913 A G 0.3 0.48 0.04 0.0063 rs4821472 34977906 T C
0.97 0.91 0.02 0.0473 rs5995271 34978039 G T 0.93 0.84 0.02 0.0496
rs5756115 34978498 A G 1 0.96 0.02 0.0804 rs9610467 34979520 G A
0.91 0.84 0.01 0.1329 rs7284919 34982110 T C 0.93 0.83 0.03 0.0229
rs136148 34982877 C T 0.1 0.16 0.01 0.1986 rs4820224 34983221 G A
0.99 0.97 0.01 0.4534 rs2413395 34984662 G A 0.93 0.92 0 0.7441
rs136159 34986969 T C 0 0.04 0.02 0.0390 rs129423 34987275 T C 0
0.03 0.02 0.0551 rs136161 34987378 G C 0.8 0.65 0.03 0.0143
rs713929 34987542 A G 0 0.03 0.02 0.0561 rs713753 34988480 C T 0.89
0.72 0.04 0.0026 rs4419330 34988801 T C 0.89 0.89 0 0.8258
rs2239785 34991276 G A 0.73 0.56 0.03 0.0088 rs136174 34991432 C A
0 0.07 0.03 0.0137 rs136175 34991512 G A 0 0.07 0.03 0.0137
rs136176 34991592 G A 0 0.04 0.03 0.0270 rs136177 34991788 G A 0.03
0.13 0.04 0.0063 rs16996616 34991837 G A 0.94 0.87 0.02 0.0974
rs73885319 34991852 G A 0.38 0.05 0.16 3.533e-09 rs71785313
34991997 D I 0.08 0.07 0 0.8949 rs2012928 34993948 G A 0.83 0.67
0.03 0.0086 rs136183 34996271 T C 0.34 0.55 0.05 0.0022 rs4821475
34999041 C T 0.37 0.46 0.01 0.1825 rs9306308 34999716 T A 0.98 0.86
0.05 0.0015 rs136187 35002222 A C 0.5 0.62 0.02 0.0783 rs136196
35005096 A G 0.31 0.37 0.01 0.4163 rs2481 35007346 G A 0.91 0.77
0.04 0.0063 rs735854 35009004 T C 0.93 0.78 0.05 0.0014 rs5756129
35014038 T C 0.79 0.7 0.01 0.1210 rs5756130 35014277 C T 0.82 0.83
0 0.8596 rs2269529 35014300 T C 0.97 0.87 0.04 0.0039 rs2269530
35014304 C A 0.97 0.88 0.03 0.0094 rs11912763 35014668 G A 0.67
0.94 0.12 4.030e-07 rs1476009 35016002 A G 0.04 0.02 0 0.5721
rs6000229 35016105 T C 0.28 0.34 0.01 0.3473 rs6000233 35017908 T C
0.64 0.47 0.03 0.0098 rs710181 35021553 A C 0.02 0.02 0 0.9960
rs75725 35021637 T C 0.93 0.94 0 0.6467 rs2239786 35021873 G C 0.71
0.53 0.03 0.0085 rs875726 35021915 G A 0.81 0.82 0 0.8952
rs16996648 35022698 T C 0.6 0.88 0.1 3.222e-06 rs9610486 35023388 G
A 0.81 0.82 0 0.7884 rs5756133 35023926 T A 0.91 0.95 0.01 0.2300
rs2187776 35025119 C T 0.31 0.46 0.02 0.0276 rs4821481 35025888 C T
0.73 0.64 0.01 0.1505 rs2239787 35028938 C A 1 0.99 0 0.9095
rs9619601 35030121 A G 0.96 0.97 0 0.9326 rs8137674 35032048 A G
0.99 0.94 0.02 0.0888 rs8138016 35032095 G A 0.94 0.96 0 0.5345
rs17806543 35034780 C A 1 0.99 0.01 0.4376 rs2239781 35034987 C T
0.98 0.89 0.04 0.0052 rs2239782 35035050 G A 0.95 0.87 0.02 0.0403
rs1557529 35035475 A G 0.52 0.39 0.02 0.0577 rs1557530 35035568 G A
0.79 0.8 0 0.8724 rs2187777 35036688 C T 1 0.99 0.01 0.2717
rs2157252 35036825 C A 0.77 0.79 0 0.7517 rs2157254 35037146 G C
0.77 0.79 0 0.7517 rs2157256 35037607 A G 0.71 0.69 0 0.7725
rs2413396 35038030 C T 0.67 0.54 0.02 0.0633 rs5750250 35038429 G A
0.64 0.54 0.01 0.1494 rs3830104 35038570 T C 0.97 0.99 0.01 0.2224
rs4820229 35038699 A G 0.77 0.79 0 0.7517 rs4820230 35039485 G A
0.71 0.7 0 0.8089 rs3752462 35040129 T C 0.77 0.79 0 0.7954
rs4820232 35040487 A G 0.75 0.75 0 0.9919 rs8141971 35041308 A G
0.78 0.79 0 0.8734 rs5756152 35042418 A G 0.42 0.33 0.01 0.2106
rs9610489 35043477 T C 0.91 0.73 0.05 0.0010 rs2239784 35044581 C T
0.3 0.48 0.04 0.0050 rs1005570 35045220 A G 0.5 0.43 0.01 0.2882
rs2071731 35048804 G A 0.79 0.74 0.01 0.4006 rs12159211 35049109 G
A 0.99 0.99 0 0.5521 rs5756154 35050370 C T 0.7 0.72 0 0.7700
rs5756156 35050725 C T 0.75 0.77 0 0.6751 rs8136069 35052436 C A
0.79 0.74 0.01 0.4006 rs8136336 35052480 G A 0.04 0.02 0.01 0.3053
rs16996672 35055916 C T 0.63 0.78 0.03 0.0151 rs16996677 35057229 G
A 0.63 0.79 0.03 0.0113 rs11704382 35058098 C A 1 1 NaN NaN
ns4820234 35059020 A G 0.27 0.29 0 0.7013 rs2413398 35060893 T G
0.73 0.74 0 0.8116 rs1557540 35062483 C T 0.26 0.28 0 0.7824
rs713839 35063884 A G 0.73 0.74 0 0.8269 rs739096 35071686 G C 0.97
0.94 0.01 0.2819 rs6000244 35071832 C T 0.94 0.85 0.02 0.0357
rs739097 35076025 G A 0.35 0.36 0 0.9082 rs5756164 35078939 A G
0.05 0.09 0.01 0.2460 rs11089788 35081047 C A 0.56 0.63 0.01 0.3345
rs136206 35085444 A G 0.67 0.61 0.01 0.3496 rs16996693 35086202 A C
0.99 0.99 0 0.7813 rs9306310 35088204 G A 0.99 0.94 0.02 0.0367
rs136211 35088493 A G 0.45 0.38 0.01 0.3437 rs16996704 35094734 A G
0.46 0.52 0.01 0.3706 rs933224 35095949 T C 0.42 0.41 0 0.9430
rs1883273 35099631 G A 0.59 0.54 0 0.5139 rs6000262 35099984 A G
0.59 0.55 0 0.5608
TABLE-US-00012 TABLE 10 Subject Characteristics (n = 407) Mean .+-.
SD Range Percent (n) (Min-Max) Age at Dialysis Initiation 55.2 .+-.
17.1 18.9-94.7 Sex Male 52.3% (213) Female 47.7% (194) Median
Income Tertile Three 31.9% (130) $31,924-$107,479 Tertile Two 33.2%
(135) $21,076-$31,611 Tertile One 32.4% (132) $6,878-$20,985
Unknown 2.5% (10) Census Region Northeast 11.3% (46) Midwest/West
17.0% (69) South 69.8% (284) Unknown 2.0% (8) Access Catheter 58.5%
(238) Graft 11.1% (45) Fistula 23.3% (95) Unknown 7.1% (29)
Location of Dialysis Initiation Inpatient 83.5% (340) Outpatient
16.5% (67) Cause of ESRD Hypertension 72.7% (296) Other 27.0% (110)
Unknown 0.3% (1) Body Mass Index 27.2 .+-. 7.8 13.8-67.5 Systolic
Blood Pressure, mmHg 145.0 .+-. 22.4 90.0-219.0 Diastolic Blood
Pressure, mmHg 79.4 .+-. 14.1 49.0-137.0 Albumin, g/dl 3.4 .+-. 0.6
1.3-4.7 Creatinine, mg/dl 8.1 .+-. 3.6 2.1-22.1 eGFR, mL/min/1.73
m.sup.2 9.9 .+-. 5.6 3.0-45.4 PTH, pg/ml 387.9 .+-. 325.8
4.6-2,353.4 Calcium, mg/dl 8.4 .+-. 1.0 4.3-12.7 Hemoglobin, g/dl
9.9 .+-. 1.3 5.9-14.9 eGFR = Estimated Glomerular Filtration
Rate
TABLE-US-00013 TABLE 11 Subject Characteristics by G1 Risk Allele
Status Wild Type Heterozygous Homozygous (n = 104) (n = 101) (n =
85) p-value Age at Dialysis Initiation* 61.8 .+-. 17.1.sup.a,b 55.9
.+-. 16.7.sup.a 49.0 .+-. 14.9.sup.b 1.0 .times. 10.sup.-6 Sex
0.9314 Male 51.0% (53) 53.5% (54) 52.9% (45) Female 49.0% (51)
46.5% (47) 47.1% (40) Median Income 0.6869 Third tertile 26.9% (28)
34.7% (35) 35.3% (30) Second tertile 29.8% (31) 33.7% (34) 32.9%
(28) First tertile 37.5% (39) 30.7% (31) 31.8% (27) Unknown 5.8%
(6) 1.0% (1) 0.0% (0) Census Region 0.7810 Northeast 11.5% (12)
13.9% (14) 9.4% (8) Midwest/West 20.2% (21) 15.8% (16) 17.7% (15)
South 64.4% (67) 69.3% (70) 72.9% (62) Unknown 3.9% (4) 1.0% (1)
0.0% (0) Access 0.8344 Catheter 57.7% (60) 63.4% (64) 58.8% (50)
Graft 15.4% (16) 10.9% (11) 12.9% (11) Fistula 19.2% (20) 20.8%
(21) 23.5% (20) Unknown 7.7% (8) 5.0% (5) 4.7% (4) Location of
Dialysis Initiation 0.4177 Inpatient 78.9% (82) 85.2% (86) 84.7%
(72) Outpatient 21.2% (22) 14.9% (15) 15.3% (13) Cause of ESRD
0.9573 Hypertension 73.1% (76) 71.3% (72) 71.8% (61) Other 26.9%
(28) 28.7% (29) 27.1% (23) Unknown 0.0% (0) 0.0% (0) 1.2% (1) Body
Mass Index 25.9 .+-. 6.3 26.9 .+-. 7.5 27.6 .+-. 7.9 0.2838
Systolic Blood Pressure, mmHg 146.6 .+-. 22.3 145.4 .+-. 23.5 141.3
.+-. 20.6 0.2514 Diastolic Blood Pressure, mmHg 77.8 .+-. 12.9 79.3
.+-. 15.2 80.6 .+-. 14.7 0.3898 Albumin, g/dl 3.5 .+-. 0.6 3.4 .+-.
0.6 3.5 .+-. 0.6 0.6550 Creatinine, mg/dl* 6.8 .+-. 2.8.sup.a,b 7.7
.+-. 3.4.sup.a 9.4 .+-. 3.8.sup.b 1.0 .times. 10.sup.-6 eGFR,
mL/min/1.73 m.sup.2 11.6 .+-. 6.6.sup.a,b 10.4 .+-. 5.5.sup.a 8.0
.+-. 3.5.sup.b 8.1 .times. 10.sup.-5 PTH, pg/ml 332.2 .+-. 318.7
3337.2 .+-. 249.7 444.9 .+-. 382.1 0.0585 Calcium, mg/dl 8.5 .+-.
0.9 8.4 .+-. 1.0 8.3 .+-. 1.0 0.5680 Hemoglobin, g/dl 9.9 .+-. 1.3
9.8 .+-. 1.4 10.1 .+-. 1.2 0.3316 *Values with the same letter
differ significantly from each other based on post-hoc tests eGFR =
Estimated Glomerular Filtration Rate
TABLE-US-00014 TABLE 12 Average Predicted Age at Dialysis
Initiation from Linear Regression Models by G1 Risk Allele Wild
Type Heterozygous.sup.c Homozygous.sup.c Age 61.8 55.9 49.0 -- p =
0.011 p = 2.1 .times. 10.sup.-7 Age + Hypertensive ESRD 61.8 55.9
49.1 -- p = 0.011 p = 1.710.sup.-7 Age + Hypertensive ESRD + Male
Sex 61.8 55.9 49.1 -- p = 0.012 p = 1.710.sup.-7 Age + Hypertensive
ESRD + Male Sex + Third 62.1 55.8 49.1 Tertile Income.sup.a + First
Tertile Income.sup.a -- p = 8.0 .times. 10.sup.-3 p = 1.210.sup.-7
Age + Hypertensive ESRD + Male Sex + Third 62.1 55.8 49.1 Tertile
Income.sup.a + First Tertile Income.sup.a + -- p = 0.012 p = 2.1
.times. 10.sup.-7 Inpatient Dialysis Age + Hypertensive ESRD + Male
Sex + Third 62.1 55.9 49.1 Tertile Income.sup.a + First tertile
Income.sup.a + -- p = 0.013 p = 1.7 .times. 10.sup.-7 Inpatient
Dialysis + Northeast Region.sup.b + South Region.sup.b P-values
represent significance of G1 risk allele coefficients controlling
for all other variables in the model .sup.aReference group is
Medium Income .sup.bReference group is Midwest/West .sup.cReference
group is Wild Type
Sequence CWU 1
1
9152DNAHomo sapiens 1tcaagctcac ggatgtggcc cctgtargct tctttcttgt
gctggatgta gt 52252DNAHomo sapiens 2caggagctgg aggagaagct
aaacatkctc aacaataatt ataagattct gc 52357DNAHomo
sapiensmisc_feature(27)..(32)This region may or may not be present
3gagaagctaa acattctcaa caataattat aagattctgc aggcggacca agaactg
5741197DNAHomo sapiens 4atggagggag ctgctttgct gagagtctct gtcctctgca
tctggatgag tgcacttttc 60cttggtgtgg gagtgagggc agaggaagct ggagcgaggg
tgcaacaaaa cgttccaagt 120gggacagata ctggagatcc tcaaagtaag
cccctcggtg actgggctgc tggcaccatg 180gacccagaga gcagtatctt
tattgaggat gccattaagt atttcaagga aaaagtgagc 240acacagaatc
tgctactcct gctgactgat aatgaggcct ggaacggatt cgtggctgct
300gctgaactgc ccaggaatga ggcagatgag ctccgtaaag ctctggacaa
ccttgcaaga 360caaatgatca tgaaagacaa aaactggcac gataaaggcc
agcagtacag aaactggttt 420ctgaaagagt ttcctcggtt gaaaagtgag
cttgaggata acataagaag gctccgtgcc 480cttgcagatg gggttcagaa
ggtccacaaa ggcaccacca tcgccaatgt ggtgtctggc 540tctctcagca
tttcctctgg catcctgacc ctcgtcggca tgggtctggc acccttcaca
600gagggaggca gccttgtact cttggaacct gggatggagt tgggaatcac
agccgctttg 660accgggatta ccagcagtac catggactac ggaaagaagt
ggtggacaca agcccaagcc 720cacgacctgg tcatcaaaag ccttgacaaa
ttgaaggagg tgagggagtt tttgggtgag 780aacatatcca actttctttc
cttagctggc aatacttacc aactcacacg aggcattggg 840aaggacatcc
gtgccctcag acgagccaga gccaatcttc agtcagtacc gcatgcctca
900gcctcacgcc cccgggtcac tgagccaatc tcagctgaaa gcggtgaaca
ggtggagagg 960gttaatgaac ccagcatcct ggaaatgagc agaggagtca
agctcacgga tgtggcccct 1020gtaagcttct ttcttgtgct ggatgtagtc
tacctcgtgt acgaatcaaa gcacttacat 1080gagggggcaa agtcagagac
agctgaggag ctgaagaagg tggctcagga gctggaggag 1140aagctaaaca
ttctcaacaa taattataag attctgcagg cggaccaaga actgtga 11975398PRTHomo
sapiens 5Met Glu Gly Ala Ala Leu Leu Arg Val Ser Val Leu Cys Ile
Trp Met 1 5 10 15 Ser Ala Leu Phe Leu Gly Val Gly Val Arg Ala Glu
Glu Ala Gly Ala 20 25 30 Arg Val Gln Gln Asn Val Pro Ser Gly Thr
Asp Thr Gly Asp Pro Gln 35 40 45 Ser Lys Pro Leu Gly Asp Trp Ala
Ala Gly Thr Met Asp Pro Glu Ser 50 55 60 Ser Ile Phe Ile Glu Asp
Ala Ile Lys Tyr Phe Lys Glu Lys Val Ser 65 70 75 80 Thr Gln Asn Leu
Leu Leu Leu Leu Thr Asp Asn Glu Ala Trp Asn Gly 85 90 95 Phe Val
Ala Ala Ala Glu Leu Pro Arg Asn Glu Ala Asp Glu Leu Arg 100 105 110
Lys Ala Leu Asp Asn Leu Ala Arg Gln Met Ile Met Lys Asp Lys Asn 115
120 125 Trp His Asp Lys Gly Gln Gln Tyr Arg Asn Trp Phe Leu Lys Glu
Phe 130 135 140 Pro Arg Leu Lys Ser Glu Leu Glu Asp Asn Ile Arg Arg
Leu Arg Ala 145 150 155 160 Leu Ala Asp Gly Val Gln Lys Val His Lys
Gly Thr Thr Ile Ala Asn 165 170 175 Val Val Ser Gly Ser Leu Ser Ile
Ser Ser Gly Ile Leu Thr Leu Val 180 185 190 Gly Met Gly Leu Ala Pro
Phe Thr Glu Gly Gly Ser Leu Val Leu Leu 195 200 205 Glu Pro Gly Met
Glu Leu Gly Ile Thr Ala Ala Leu Thr Gly Ile Thr 210 215 220 Ser Ser
Thr Met Asp Tyr Gly Lys Lys Trp Trp Thr Gln Ala Gln Ala 225 230 235
240 His Asp Leu Val Ile Lys Ser Leu Asp Lys Leu Lys Glu Val Arg Glu
245 250 255 Phe Leu Gly Glu Asn Ile Ser Asn Phe Leu Ser Leu Ala Gly
Asn Thr 260 265 270 Tyr Gln Leu Thr Arg Gly Ile Gly Lys Asp Ile Arg
Ala Leu Arg Arg 275 280 285 Ala Arg Ala Asn Leu Gln Ser Val Pro His
Ala Ser Ala Ser Arg Pro 290 295 300 Arg Val Thr Glu Pro Ile Ser Ala
Glu Ser Gly Glu Gln Val Glu Arg 305 310 315 320 Val Asn Glu Pro Ser
Ile Leu Glu Met Ser Arg Gly Val Lys Leu Thr 325 330 335 Asp Val Ala
Pro Val Ser Phe Phe Leu Val Leu Asp Val Val Tyr Leu 340 345 350 Val
Tyr Glu Ser Lys His Leu His Glu Gly Ala Lys Ser Glu Thr Ala 355 360
365 Glu Glu Leu Lys Lys Val Ala Gln Glu Leu Glu Glu Lys Leu Asn Ile
370 375 380 Leu Asn Asn Asn Tyr Lys Ile Leu Gln Ala Asp Gln Glu Leu
385 390 395 66DNAHomo sapiens 6ttataa 6 7599DNAHomo sapiens
7agacgcccct ctgcatactc ccctggtgaa ctgctgccca ggactgggtc ccccttttac
60ccttgctgca tggagtcccc agaagacaaa catctgtgtg tctgaaccct gagacaaagg
120caggaaaggg aaagagggag gcgagtggct tttgaggagg gggctttagt
atgagagctg 180gaggatggaa ccccatcagg gggcccggga accactgagc
tgttaaaata aagtctgcaa 240acaaagacca gctgctggaa gtgggtgtgc
cagggagtgc gcagagacac acggtgagaa 300aagaacaatg gtaatgcttg
gagccgcccc taactgggat gggcctgaag tggtattgtt 360attatttata
gtatcattat tagtcatttt catcttattt gtaccctccc tctatctctc
420tctccacctt ttcctaacat tctatcacca gttttatgtc tcccattagc
aactttgtag 480ctgtaaacaa tttacttaca actttcttat accctcagtt
gtacccagta tttcttaact 540tccctcttta aaaaatgaca atattaatcc
tccttctcct ttgttcagtg cttcacatc 599816DNAHomo sapiens 8agcgtcgggt
gagtcc 16911DNAHomo sapiens 9aacaggatga g 11
* * * * *
References