U.S. patent application number 10/731749 was filed with the patent office on 2004-06-24 for genetic polymorphism in a complement receptor.
Invention is credited to Kimberly, Robert P..
Application Number | 20040121393 10/731749 |
Document ID | / |
Family ID | 29715981 |
Filed Date | 2004-06-24 |
United States Patent
Application |
20040121393 |
Kind Code |
A1 |
Kimberly, Robert P. |
June 24, 2004 |
Genetic polymorphism in a complement receptor
Abstract
A method and a package for identifying single nucleotide
polymorphisms in complement receptor is useful in identifying
individual susceptibility to a disease. Complement receptors CR1
and CR2 are active in the immune response and autoimmune diseases.
The susceptibility and severity of autoimmune disease is determined
by genotyping or phenotyping an individual for complement
receptor.
Inventors: |
Kimberly, Robert P.;
(Birmingham, AL) |
Correspondence
Address: |
GIFFORD, KRASS, GROH, SPRINKLE
ANDERSON & CITKOWSKI, PC
280 N OLD WOODARD AVE
SUITE 400
BIRMINGHAM
MI
48009
US
|
Family ID: |
29715981 |
Appl. No.: |
10/731749 |
Filed: |
December 9, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10731749 |
Dec 9, 2003 |
|
|
|
09744346 |
Mar 23, 2001 |
|
|
|
6664052 |
|
|
|
|
09744346 |
Mar 23, 2001 |
|
|
|
PCT/US99/16264 |
Jul 23, 1999 |
|
|
|
60094096 |
Jul 24, 1998 |
|
|
|
60106643 |
Nov 2, 1998 |
|
|
|
Current U.S.
Class: |
435/6.18 ;
435/6.1; 435/7.2 |
Current CPC
Class: |
C07K 14/70596 20130101;
C12Q 1/6883 20130101; C12Q 2600/156 20130101; G01N 33/686 20130101;
A01K 2217/05 20130101 |
Class at
Publication: |
435/006 ;
435/007.2 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/567 |
Goverment Interests
[0002] The subject invention was made with government support from
the National Institutes of Health (NIH 1 P50 AR45231-01). The
government has certain rights in the invention.
Claims
1. The method of claim 5 wherein said complement receptor gene
encodes for a complement receptor single nucleotide polymorph which
is predominant in humans.
2. A method for correlating the ability of a cell to bind a
complement component, and cellular susceptibility to a disease,
said method comprising: identifying a complement receptor phenotype
of said cell; quantifying said complement component binding by said
cell; and comparing said complement component binding by said cell
to that of a second cell, said second cell having a second
complement receptor phenotype.
3. The method of claim 2 wherein identifying said complement
receptor phenotype utilizes antibody binding.
4. A method for correlating the ability of a cell to bind a
complement component and cellular susceptibility to a disease, said
method comprising: identifying a complement receptor genotype of
said cell; quantifying said complement component binding by said
cell expressing said complement receptor genotype; and comparing
said complement component binding by said cell and said complement
component binding by a second cell, said second cell expressing a
second complement receptor genotype.
5. The method of claim 4 wherein said complement receptor genotype
differs from said second complement receptor genotype by a point
mutation.
6. The method of claim 5 wherein said complement receptor genotype
and said second complement receptor genotype are for CR1.
7. The method of claim 5 wherein said complement receptor genotype
and said second complement receptor genotype are for CR2.
8. The method of claim 5 wherein said point mutation is a silent
mutation.
9. The method of claim 5 wherein said point mutation is a frame
shift mutation.
10. The method of claim 5 wherein said point mutation is a missense
mutation.
11. The method of claim 6 wherein said point mutation is a missense
mutation in nucleotide 5932.
12. The method of claim 11 wherein said missense mutation is within
a codon for an amino acid selected from the group consisting of
alanine and threonine.
13. The method of claim 4 wherein said cell is selected from the
group consisting of: erythrocyte, B lymphocyte, granulocyte,
monocyte, neutrophil and T cell.
14. The use of a single nucleotide polymorphism in a complement
receptor genotype to identify individual susceptibility to a
disease.
15. The use of claim 14 wherein said disease is selected from the
group consisting of: cancer, viral infection, bacterial infection,
systemic lupus erythematosus, systemic vasculitis, hemolytic
anemia, AIDS, rheumatoid arthritis, systemic sclerosis,
glomerulonephritides, Sjogren's syndrome and lepromatous
leprosy.
16. The use of claim 14 wherein the single nucleotide polymorphism
is at a codon 1969.
Description
RELATED APPLICATIONS
[0001] This patent application is a divisional application of U.S.
patent application Ser. No. 09/744,346 filed Mar. 23, 2001. Ser.
No. 09/744,346 is a U.S. National Phase under 35 U.S.C. 371 of
International Application Serial No. PCT/US99/16264 filed Jul. 23,
1999. Serial No. PCT/US99/16264 is a non-provisional of U.S.
Provisional Application Serial No. 60/094,096 filed Jul. 24, 1998,
and U.S. Provisional Application Serial No. 60/106,643 filed Nov.
2, 1998.
FIELD OF THE INVENTION
[0003] The present invention relates generally to compounds and
methods for identifying polymorphism in a cellular receptor, and
more particularly, to compounds and methods for identifying and
typing single nucleotide polymorphisms that code for a complement
receptor and applying these polymorphisms to delineation of disease
susceptibility and severity.
BACKGROUND OF THE INVENTION
[0004] The complement system involves a group of proteins that play
a role in host defense against infection. Complement is active in
immune defenses, especially antibody mediated events by way of the
"classical pathway." Complement response to an invasion by a
foreign particle can also embody an antibody independent mechanism
which is known as the "alternative pathway."
[0005] As part of the body's natural defenses, the complement
system operates as a biological cascade in which one component
activates successive components. Activation is usually by way of a
proteolytic cleavage event. The complement system functions to
promote the inflammatory response, to modify the membranes of
infectious organisms, and also to identify pathogenic material for
removal.
[0006] The inflammatory response is triggered by way of the
cascade, in which low molecular weight peptides are cleaved from
complement proteins. The resulting anaphylatoxins and chemotactic
factors attract leukocytes and modify vascular permeability.
[0007] The attachment of complement proteins, C3b/C4b to microbial
membranes or immune complexes, a process commonly known as
opsonization, facilitates the binding of opsonized material to cell
receptors. By modifying the membranes of a microbe, complement also
participates directly in microbial destruction.
[0008] Immune complex opsonization enables the process in which
specific receptors on a cell bind complement components. In some
cases this binding process leads to phagocytosis of the complement
bound antigen particles. The primary opsonins of the complement
system are C3b and C4b which are activated by cleavage of fragments
C3 and C4. C3b and C4b are capable of covalently binding to foreign
antigen particles. The resulting C3b/C4b coded complexes are
ligands for the C3b/C4b receptor on human peripheral blood cells.
The C3b/C4b receptor is also known as complement receptor 1
(CR1).
[0009] Receptors for complement proteins function to bind
complement coated antigens. CR1 is present on most peripheral blood
cells, including erythrocytes, B lymphocytes, granulocytes,
monocytes, neutrophils and some T cells. Erythrocyte CR1 upon
binding the C3b/C4b immune complex conveys the immune complex to
the liver or spleen where the ligand is transferred to hepatic
macrophages for internalization and disposal. As a result, CR1
plays a role in neutralizing complement activated immune complexes
within the body. Thus, defects in this receptor are associated with
impaired phagocytosis and can lead to enhanced destructive
processes such as joint destruction in rheumatoid arthritis, and
impaired host defense against infection.
[0010] The role of complement receptor 2 (CR2) is currently not
fully understood. Nonetheless, CR2 binds activated complement
components and is operative in response to foreign particles.
[0011] Autoimmune diseases are often characterized by abnormal
deposits of complement fixing immune complexes. Such deposits may
result from excessive immune complex formation, inappropriate
antibody production illustratively including IgG, IgM and IgA,
complement deficiencies or receptor anomalies. Receptors that are
incapable of binding and/or releasing immune complex or that are
underexpressed on the cell surface would result in incomplete
removal of immune complexes thereby resulting in deposition.
[0012] Such abnormal deposits are associated with a variety of
diseases including several types of human glomerulonephritis. In
particular, systemic lupus erythematosus (SLE) is associated with
glomerular deposits of early complement cascade components such as
C1q, C4 and C2. Deposition of these components is suggestive of the
classical pathway of complement activation in this disease. Other
types of glomerulonephritis such as IgA neuropathy, bacterial
infections and membranoproliferative glomerulonephritis are
typically associated with glomerular deposits of C3 and properdin,
thus suggesting alternative pathway dysfunction in these diseases.
The CR1 receptor in humans and primates is found on most types of
peripheral blood leukocytes and erythrocytes, however not on
platelets. Due to the large fraction of erythrocytes in blood, a
great majority of all CR1 in peripheral blood is found on
erythrocytes. Perhaps greater than 90% of all CR1 is found on
erythrocytes. The characterization of CR1 has previously failed to
make a definitive correlation between CR1 structure and
immunological disease severity.
[0013] CR1 is a single polypeptide chain that exhibits a size
polymorphism derived from four codominant inherited alleles. The
alleles are: type A (220 kD), type B (250 kD), type C (190 kD) and
type D (280 kD). These four alleles result in ten phenotypes of CR1
within the human population which have varying numbers of C3b
binding sites. Fluctuations in erythrocyte CR1 expression
associated with SLE progression suggests that genetic
predisposition as to size polymorphism is not a controlling factor
in SLE (Please fill in reference prop #110, now 1). While SLE is an
important autoimmune disease, fluctuating levels of CR1 are also
associated with diseases including hemolytic anemias, AIDS,
rheumatoid arthritis, Sjogren's syndrome and lepromatous
leprosy.
[0014] CR1, in addition to having the four codominant size
polymorph alleles, also exhibits Knops blood group polymorphism and
cis-acting Hind III restriction fragment length polymorphism
(RFLP). Little is known about the role of the Knops antigen in
receptor function and therefore its role in autoimmune diseases,
such as SLE. RFLP correlates with quantitative expression of CR1 on
erythrocytes. RFLP has a 6.9 kilobase polymorph which correlates
with low copy number expression of CR1 on erythrocytes. The other
RFLP is 7.4 kilobases in size and correlates with high numeric
expression. There is a controversy as to whether or not the 6.9
kilobase RFLP is found with increased frequency in SLE patients and
their relatives. Similarly, controversy persists regarding the role
of size polymorphism in genetic predisposition to SLE, autoimmune
and pathogenic diseases.
[0015] The present invention is based on the discovery of a novel
form of polymorphism in CR1. This novel polymorphism is exploited
in a testing methodology which allows for early identification of
individuals susceptible to diseases associated with CR1 function,
offering the possibility of early and aggressive treatment in those
patients.
SUMMARY OF THE INVENTION
[0016] The present invention is a system and method for correlating
the ability of binding properties of human complement receptor (CR)
and cellular susceptibility to a disease by identifying a CR
genotype of a cell and quantifying complement protein binding by
the cell expressing the CR genotype. Thereafter, the complement
protein binding by the cell and complement protein binding by a
second cell expressing a second Fc.gamma.RI genotype is
compared.
[0017] The present invention uses a single nucleotide polymorphism
or combinations thereof within a CR genotype to identify individual
susceptibility to a disease.
[0018] The methods of the present invention also extend to
correlating the ability of a cell to bind complement protein and
cellular susceptibility to a disease through identifying a Cr
phenotype of a given cell and quantifying complement protein
binding by said cell. Thereafter, complement protein binding by the
cell is compared to a second cell having a second CR phenotype. In
particular, single nucleotide polymorphisms are responsible for
genotypical and phenotypical differences in CR herein.
[0019] The present invention further includes a commercial
packaging including reagents for identifying single nucleotide
polymorphisms in the CR genotype or phenotype of an individual as a
test to identify individual susceptibility to a disease. The
reagents further include instructions for the use thereof.
BRIEF DESCRIPTION OF THE DRAWING
[0020] FIG. 1 is a bar graph showing the expression of CR1 on
erythrocytes from CR1 genotype normal donors using fluorescence
measurements associated with anti-CR1 mAb.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention relates to compounds and methods for
identifying single nucleotide polymorphisms in the complement
receptors CR1 and CR2. The single nucleotide allelic differences in
a complement receptor are determined by direct DNA sequencing. It
is appreciated that other techniques known to the art may also be
employed, these illustratively include the use of allele specific
oligonucleotides as hybridization probes and/or as primers for DNA
amplification, and immunological binding of antibodies that
distinguish between different polymorphic forms of the complement
receptor expressed on erythrocytes.
[0022] The present invention is based on the finding that single
nucleotide allelic polymorphism in a gene encoding a complement
receptor results in functionally distinct gene products. The
expression of a polymorphic complement receptor in an individual
may have important consequences for the physiological activity of
the receptor by itself, or in combination with other single
nucleotide polymorphs. This in turn can effect functioning of the
cell types that carry the receptor. Because of the important role
of complement in the immune response, the present invention has
utility as a diagnostic to identify high risk patients that warrant
early and aggressive treatment. As a diagnostic for infectious
disease, the present invention has utility in predicting
susceptibility to specific microbes and thereby guiding the use of
therapeutics. As a diagnostic for autoimmune disease, the present
invention has utility in diseases illustratively including SLE,
systemic vasculitis, glomerulonephritides, Sjogren's syndrome and
IgA nephropathy. Identification of the appropriate allelic forms
further allows for gene therapy transduction of host cells to
correct hereditary limitations in the host's complement receptor
genes through delivery of a translatable complement receptor gene
to a defective host cell. Generally, a predominant "normal" gene of
the total human population or a derivative thereof would be
delivered.
[0023] The present invention provides a method for identifying the
complement receptor single nucleotide allelic pattern in human
patients which involves testing DNA from individual patients for
the presence of different allelic variants. The present invention
also encompasses the identification analysis of new single
nucleotide allelic forms of complement receptor genes, the analysis
being achieved using methods well known in the art, such as direct
DNA sequencing; single strand conformational polymorphism analysis
(SSCP); "HOT" cleavage; denaturing gradient gel electrophoresis
(DVGE) and combinations thereof.
[0024] Once a new polymorphism has been identified, immunological
and/or molecular biological tests are used to genotype patients for
the presence or absence of a given single nucleotide polymorphism.
For example, monoclonal antibodies specific to the protein encoded
by a newly identified single nucleotide allele are prepared by well
known methods. Such methods are described in U.S. Application
60/094,096, filed on Jul. 24, 1998. These antibodies can be used
for genotyping the patient population as described above.
Alternatively, allele specific oligonucleotides may be designed for
use as probes and/or as primers in hybridization or PCR based
detection methods, respectively.
[0025] Through the establishment of statistically significant
correlations between the different single nucleotide polymorphic
allelic forms of a complement receptor and various physiological or
clinical manifestations of variable complement receptor function,
the role of naturally occurring point mutations in clearing immune
complex from the bloodstream is identified in homozygous genotype
donors and in stable transfectants. These correlations are utilized
to provide diagnostic utilities of the present invention. In
practicing the present invention, preferably the correlations
sought are those between particular complement receptor single
nucleotide allelic polymorphs and the risk for developing any of
the illustratively aforementioned diseases.
[0026] The term "allele" or "allelic form" is intended to mean an
alternative version of a gene encoding the same functional protein
but containing differences in nucleotide sequence relative to
another version of the same gene.
[0027] The term "allelic polymorphism" or "allelic variant" is
intended to mean a variation in the nucleotide sequence within a
gene, wherein different individuals in a general population express
different variants of the gene.
[0028] The term "allelic pattern" is intended to mean the identity
of each of the two copies of a particular gene in a patient i.e.,
homozygosity or heterozygosity.
[0029] The term "allelic pattern" is used herein interchangeably
with "genotype."
[0030] The term "genotyping" as used herein as being the process of
determining the allelic patterns of a human individual.
[0031] The term "point mutation" as used herein is intended to mean
a mutation involving a single nucleotide.
[0032] The term "silent mutation" as used herein is intended to
mean a change of a nucleotide within a gene sequence that does not
result in change in the coded amino acid sequence.
[0033] The term "missense mutation" as used herein is intended to
mean a change of a nucleotide within a gene sequence that results
in a change in the meaning of a codon, thereby changing the coded
amino acid.
[0034] The term "frame shift mutation" as used herein is intended
to mean a mutation involving the insertion or deletion of a single
nucleotide that results in the remaining downstream sequence being
transcribed or translated out of phase.
[0035] Examination of the DNA sequence encoding CR1 by direct
sequence analysis shows a single nucleotide variation at position
5932. The single nucleotide polymorphism at position 5932 results
in a missense codon, which translates to an alanine (A) to
threonine (T) substitution at amino acid residue 1969 within the
CR1 protein. Homozygous normal donors of each genotype at residue
1969 have been identified. FIG. 1 shows that 1969 A/A individuals
have higher expression numbers of CR1 than homozygous T/T
individuals. Correlations between CR1 expression on erythrocytes
and donor genotype were made using quantitative flow cytometry
following genotyping.
[0036] DNA was obtained from a donor and the presence of DNA
sequences corresponding to a particular CR1 single nucleotide
allelic polymorphism are determined. The DNA may be obtained from
any cell source or body fluid containing intact nucleic acid
bearing cells (expressing a complement receptor). DNA is extracted
from the cell source using any of the numerous methods that are
standard in the art. Once extracted, the DNA may be employed in the
present invention without further manipulation. Direct sequencing
is accomplished by chemical sequencing, using the Maxam-Gilbert
method. It is appreciated that alternate sequencing methods such as
enzymatic sequencing by way of methods such as the Sanger method
are also operative herein and are described in Application
60/094,096, filed Jul. 24, 1998.
[0037] Once an individual has been genotyped, erythrocytes are
exposed separately to anti-CR1 mAb species YZ1, E11, and 3D9.
Fluorescence intensity associated with standardized cell
populations correlates with copy number expression of CR1.
[0038] Standard analytical flow cytometry using one to four colors
was performed on a FACScan flow cytometer (Becton-Dickinson).
Sorting of cell lines is performed on a FACS-Vantage
(Becton-Dickinson).
[0039] By extension of the flow cytometry process, a study was
conducted to determine the distribution of CR1 1969 genotypes and
1969 allele frequencies. A control group consisted of healthy
individuals and rheumatoid arthritis patients. The results from
these groups were compared to the genotype distributions and allele
frequencies for SLE patients. An over representation of the 1969 T
allele in SLE patients is noted in Table 1.
1TABLE 1 Distribution of CR1 Alleles in SLE Patients Normal + RA
Comparison SLE patients (N = 70) (N = 30) Genotype 1969 A/A 45
(64%) 18 (60%) 1969 A/T 23 (33%) 9 (30%) 1969 T/T 2 (3%) 3 (10%)
Allele frequency 1969 A 0.81 0.75 1969 T 0.19 0.25
[0040] The correlation of various single nucleotide polymorphisms
in individuals including the 1969 naturally occurring A/T polymorph
in CR1 as well as other single nucleotide polymorphs in CR1 and CR2
are correlated to cis-acting Hind III RFLP and the severity or
susceptibility of individuals in SLE diathesis. In this way
relative importance of specific single nucleotide polymorphs in
disease progression is assessed.
[0041] Any patents, applications or publications mentioned in the
specification are indicative of the levels of those skilled in the
art to which the invention pertains. These patents and publications
are herein incorporated by reference to the same extent as if each
individual publication or patent was specifically and individually
indicated to be incorporated by reference.
* * * * *