U.S. patent application number 11/389062 was filed with the patent office on 2006-07-27 for converting diploidy to haploidy for genetic diagnosis.
This patent application is currently assigned to The Johns Hopkins University. Invention is credited to Kenneth W. Kinzler, Nickolas Papadopoulos, Bert Vogelstein, Hai Yan.
Application Number | 20060166257 11/389062 |
Document ID | / |
Family ID | 46149886 |
Filed Date | 2006-07-27 |
United States Patent
Application |
20060166257 |
Kind Code |
A1 |
Vogelstein; Bert ; et
al. |
July 27, 2006 |
Converting diploidy to haploidy for genetic diagnosis
Abstract
Detection of mutations associated with hereditary diseases is
complicated by the diploid nature of mammalian cells. Mutations
present in one allele are often masked by the wild-type sequence of
the other allele. Individual alleles can be isolated from every
chromosome within somatic cell hybrids generated from a single
fusion. Nucleic acids from the hybrids can be analyzed for
mutations in an unambiguous manner. This approach was used to
detect two cancer-causing mutations that had previously defied
genetic diagnosis. One of the families studied, Warthin Family G,
was the first kindred with a hereditary colon cancer syndrome
described in the biomedical literature.
Inventors: |
Vogelstein; Bert;
(Baltimore, MD) ; Kinzler; Kenneth W.; (Bel Air,
MD) ; Papadopoulos; Nickolas; (Brookline, MA)
; Yan; Hai; (Chapel Hill, NC) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Assignee: |
The Johns Hopkins
University
Baltimore
MD
|
Family ID: |
46149886 |
Appl. No.: |
11/389062 |
Filed: |
March 27, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10210066 |
Aug 2, 2002 |
7045352 |
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11389062 |
Mar 27, 2006 |
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09504860 |
Feb 16, 2000 |
6475794 |
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10210066 |
Aug 2, 2002 |
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09461047 |
Dec 15, 1999 |
6399374 |
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09504860 |
Feb 16, 2000 |
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60158160 |
Oct 8, 1999 |
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Current U.S.
Class: |
435/6.13 ;
435/354; 435/455; 435/6.1 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12N 5/166 20130101; C12Q 2600/156 20130101 |
Class at
Publication: |
435/006 ;
435/455; 435/354 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 5/06 20060101 C12N005/06; C12N 15/87 20060101
C12N015/87 |
Goverment Interests
[0001] This invention was supported with U.S. government funds, NIH
grants CA43460, CA57345, CA62924, CA67409, CA72851. The government
therefore retains certain rights in the invention. This is a
divisional application of parent application Ser. No. 10/210,066,
filed Aug. 2, 2002, which is a Divisional Application of parent
application Ser. No. 09/504,860, filed Feb. 16, 2000, which is a
continuation-in-part of application Ser. No. 09/461,047 filed Dec.
15, 1999, which claims the benefit of provisional application Ser.
No. 60/170,260 filed Dec. 8, 1999.
Claims
1. A cell line comprising mouse embryonic fibroblasts which are
transformed by oncogenes, wherein said fibroblasts are defective in
a mismatch repair gene which causes microsatellite instability.
2. The cell line of claim 1 wherein the fibroblasts are deficient
in hypoxanthine phosphoribosyl transferase (HPRT).
3. The cell line of claim 1 wherein said oncogenes comprise
Ras.
4. The cell line of claim 1 wherein said oncogenes comprise
E1A.
5. The cell line of claim 1 wherein said oncogenes comprise Ras and
E1A.
6. A method of making a rodent cell recipient for fusion with
mammalian cells comprising: transforming rodent cell embryonic
fibrtoblasts with oncogenes, wherein said fibroblasts are defective
in a mismatch repair gene which causes microsatellite instability;
and selecting HPRT-deficient cells by growing in
2-amino-6-mercaptopurine.
7. The cell line of claim 6 wherein said oncogenes comprise
oncogene E1A.
8. The cell line of claim 6 wherein said oncogenes comprise
oncogene Ras.
9. The cell line of claim 6 wherein said oncogenes comprise
oncogene E1A and Ras.
10. The method of claim 6 wherein the step of detecting comprises
identifying a set comprising a plurality of distinctive markers on
the second and third non-rodent mammalian chromosomes.
11. The method of claim 10 wherein the distinctive markers comprise
microsatellite markers.
12. The method of claim 10 wherein the distinctive markers comprise
polymorphic markers.
13. A method of identifying a haplotype of a non-rodent mammalian
chromosome comprising: fusing cells of a non-rodent mammal to
rodent cell recipients to form non-rodent mammal-rodent cell
hybrids; selecting for fused cell hybrids by selecting for a first
marker contained on a rodent chromosome and for a second marker
contained on a first non-rodent mammalian chromosome, to form a
population of fused cell hybrids; detecting among the population of
fused cell hybrids a subset of hybrids which are haploid for a
second non-rodent mammalian chromosome which is not the same
chromosome as the first non-rodent mammalian chromosome and which
was not selected; analyzing said subset of hybrids to detect a
plurality of distinctive markers on the second non-rodent mammalian
chromosome.
14. The method of claim 13 wherein the marker set comprises
polymorphic markers.
15. The method of claim 13 wherein the marker set comprises
microsatellite markers.
16. A method of correlating a polymorphic marker with expression or
reduced expression of a gene in a non-rodent individual, comprising
the steps of: (a) fusing cells of the non-rodent individual to
rodent cell recipients to form non-rodent/rodent cell hybrids; (b)
selecting for fused cell hybrids by selecting for a first
selectable marker contained on a rodent chromosome and for a second
selectable marker contained on a first non-rodent individual
chromosome, to form a population of fused cell hybrids; (c)
detecting among the population of fused cell hybrids a subset of
hybrids which are haploid for a second non-rodent individual
chromosome which is not the same chromosome as the first non-rodent
individual chromosome and which was not selected; (d) analyzing
said subset of hybrids to detect a polymorphic marker on the second
non-rodent individual chromosome; (e) assaying for expression of a
gene on the second non-rodent individual chromosome; and (f)
identifying the polymorphic marker as correlated with expression of
the gene if the subset of hybrids comprises the polymorphic marker
and the gene is expressed in the hybrids or identifying the
polymorphic marker as correlated with reduced expression of the
gene if the subset of hybrids comprises the polymorphic marker and
expression of the gene is reduced in the hybrids.
17. The method of claim 16 wherein the polymorphic marker is a
single nucleotide polymorphism.
18. The method of claim 16 wherein the polymorphic marker is a
microsatellite marker.
19. The method of claim 16 wherein the polymorphic marker is a
plurality of polymorphic markers on the second non-rodent
individual chromosome.
20. The method of claim 16 wherein the polymorphic marker is a
mutation.
21. The method of claim 16 wherein an mRNA product of the gene is
analyzed in the subset of hybrids.
22. The method of claim 16 wherein a protein product of the gene is
analyzed in the subset of hybrids.
23. The method of claim 16 wherein the gene is analyzed in the
subset of hybrids.
24. The method of claim 16 wherein the non-rodent individual is a
human.
25. The method of claim 16 wherein the subset of hybrids is
analyzed to detect a plurality of polymorphic markers.
26. The method of claim 16 wherein the subset of hybrids is
analyzed to detect polymorphic markers in at least two different
genes.
27. A method of using a correlation between a polymorphic marker
and expression or reduced expression of a gene to select a medical
intervention for a non-rodent individual, comprising the steps of:
(a) assaying a biological sample obtained from the non-rodent
individual for a polymorphism which is correlated with expression
of a gene, wherein the correlation has been determined by a method
comprising the steps of: (1) fusing cells of the non-rodent
individual to rodent cell recipients to form non-rodent/rodent cell
hybrids; (2) selecting for fused cell hybrids by selecting for a
first selectable marker contained on a rodent chromosome and for a
second selectable marker contained on a first non-rodent individual
chromosome, to form a population of fused cell hybrids; (3)
detecting among the population of fused cell hybrids a subset of
hybrids which are haploid for a second non-rodent individual
chromosome which is not the same chromosome as the first non-rodent
individual chromosome and which was not selected; (4) analyzing
said subset of hybrids to detect a polymorphic marker on the second
non-rodent individual chromosome; (5) assaying for expression of a
gene on the second non-rodent individual chromosome; and (6)
identifying the polymorphic marker as correlated with expression of
the gene if the subset of hybrids comprises the polymorphic marker
and the gene is expressed in the hybrids or identifying the
polymorphic marker as correlated with reduced expression of the
gene if the subset of hybrids comprises the polymorphic marker and
expression of the gene is reduced in the hybrids; and (b) selecting
a medical intervention based on the presence or absence of the
polymorphic marker in the biological sample.
28. The method of claim 27 wherein the medical intervention is a
prophylactic intervention.
29. The method of claim 27 wherein the polymorphic marker
predisposes the non-rodent individual to a disorder.
30. The method of claim 27 wherein the polymorphic marker is
causally related to a disorder.
31. The method of claim 27 wherein the polymorphic marker is
associated with responsiveness to a drug and wherein the medical
intervention is administration of the drug.
32. The method of claim 27 wherein the polymorphic marker is
associated with resistance to a first drug useful for treating the
disorder and wherein the medical intervention is administration of
a second drug useful for treating the disorder.
Description
BACKGROUND OF THE INVENTION
[0002] The problem with humans and other mammals, at least from a
genetic diagnostic perspective, is that they are diploid. Mutations
in one allele, such as those responsible for all dominantly
inherited syndromes, are always accompanied by the wild-type
sequence of the second allele. Though many powerful techniques for
genetic diagnosis have been developed over the past decade, all are
compromised by the presence of diploidy in the template. For
example, the presence of a wild-type band of the same
electrophoretic mobility as a mutant band can complicate
interpretation of sequencing ladders, especially when the mutant
band is of lower intensity. Deletions of a segment of DNA are even
more problematic, as in such cases only the wild-type allele is
amplified and analyzed by standard techniques. These issues present
difficulties for the diagnosis of monogenic diseases and are even
more problematic for multigenic diseases, where causative mutations
can occur in any of several different genes. Such multigenism is
the rule rather than the exception for common predisposition
syndromes, such as those associated with breast and colon cancer,
blindness, and hematologic, neurological, and cardiovascular
diseases. The sensitivity of genetic diagnostics for these diseases
is currently suboptimal, with 30% to 70% of cases refractory to
genetic analysis.
[0003] There is a need in the art for simply separating and
analyzing individual alleles from human and other mammalian
cells.
SUMMARY OF THE INVENTION
[0004] It is an object of the invention to provide a method for
detecting mutations in a gene of interest on a human or other
mammalian chromosome.
[0005] It is another object of the invention to provide a method
for making test cells suitable for sensitive genetic testing.
[0006] It is yet another object of the invention to provide a
population of fused cell hybrids which are useful for genetic
analysis.
[0007] These and other objects of the invention are provided by one
or more of the embodiments described below. In one embodiment a
method of detecting mutations in a gene of interest of a human or
other mammal is provided. Cells of a human or other mammal are
fused to rodent cell recipients to form human-rodent or other
mammal-rodent cell hybrids. Fused cell hybrids are selected by
selecting for a first marker contained on a rodent chromosome and
for a second marker contained on a first human or other mammalian
chromosome, forming a population of fused cell hybrids. A subset of
hybrids are detected among the population of fused cell hybrids.
The hybrids are haploid for a second human other mammalian
chromosome which is not the same chromosome as the first human or
other mammalian chromosome and which was not selected. The subset
of hybrids are tested to detect a gene, an mRNA product of said
gene, or a protein product of said gene. The gene resides on the
second human or other mammalian chromosome. Diminished amounts of
the mRNA or protein product or altered properties of the gene,
mRNA, or protein product indicate the presence of a mutation in the
gene in the human or other mammal.
[0008] According to another embodiment, a method is disclosed which
provides test cells for genetic testing. The test cells are haploid
for human or other mammalian genes. Cells of a human or other
mammal are fused to transformed, diploid, rodent cell recipients to
form human-rodent or other mammal-rodent cell hybrids. Fused cell
hybrids are selected by selecting for a marker on each of a first
hybrids. Fused cell hybrids are selected by selecting for a marker
on each of a first human or other mammalian chromosome and a rodent
chromosome, forming a population of cells which stably maintain one
or more human or other mammalian chromosomes in the absence of
selection for the human or other mammalian chromosomes. Cells which
are haploid for a second human or other mammalian chromosome which
is distinct from the first human or other mammlian chromosome are
detected among the population of cells; the second human or other
mammalian chromosome was not selected.
[0009] Also provided by the present invention is a population of
rodent-human or rodent-other mammalian hybrid cells wherein each
homolog of at least 2 human or other mammalian autosomes is present
in haploid form in at least one out of one hundred of the
cells.
[0010] The present invention thus provides the art with a method
which can be used to increase the sensitivity and effectiveness of
various diagnostic and analytic methods by providing hybrid cells
to analyze which are haploid for one or more genes of interest. The
human or other mammalian chromosome content of the hybrid cells is
stable and uniform.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1. Strategy for hybrid generation. The recipient mouse
cell line E2 was fused with human lymphocytes and clones were
subsequently selected with HAT plus geneticin, which kill unfused
E2 cells and lymphocytes, respectively. All clones contained a
human X chromosome responsible for growth in HAT. Clones were
genotyped to determine which human chromosomes were retained.
Chromosomes marked "A" and "B" represent the two homologs of a
representative human chromosome. The average proportion of clones
which retained neither, both, or either of the six chromosome
homologs analyzed is indicated (see text). Mutational analysis was
carried out on nucleic acids of clones which retained single
alleles of the genes to be tested.
[0012] FIG. 2. Allelic status and gene expression in hybrids. (FIG.
2A) Polymorphic markers from the indicated chromosomes were used to
determine the genotype of the indicated hybrids. "Donor" denotes
the human lymphocytes used for fusion with the mouse recipient
cells. (FIG. 2B) cDNA of E2 and four hybrids were used as templates
to amplify hMSH2, hMSH6, hMLH1, hTGF .beta.-RII, hPMS1, hPMS2, and
APC sequences. The results were concordant with the genotypes
observed in (FIG. 2A), in that hybrids 5-7 retained at least one
allele of each of the chromosomes containing the tested genes,
while hybrid 8 contained alleles of chromosomes 3, 5, and 7 but not
of chromosome 2 (containing the hMSH2, hPMS1, and hMSH6 genes).
[0013] FIG. 3. Mutational analysis of an HNPCC patient refractory
to standard genetic diagnosis. Nucleic acids from the indicated
hybrids were tested for retention of chromosomes 2 and 3 using
polymorphic markers (FIG. 3A) and for expression of hMSH2 and hMLH1
genes on chromosomes 2 and 3, respectively (FIG. 3B). Hybrids 1, 2,
3, and 6 contained allele A from chromosome 2 and did not express
hMSH2 transcripts, while hybrids 4 and 5 contained the B allele and
expressed hMSH2. hMLH1 expression served as a control for the
integrity of the cDNA. (FIG. 3C) Sequences representing the
indicated exons of hMSH2 were amplified from the indicated hybrids.
Exons 1-6 were not present in the hybrids containing allele A, but
exons 7-16 were present in hybrids containing either allele.
[0014] FIG. 4. Mutational analysis of Warthin family G. (FIG. 4A)
Sequence analysis of RT-PCR products from hMSH2 transcripts of
hybrid 1, containing the mutant allele of a Warthin family G
patient, illustrates a 24 bp insertion (underlined; antisense
primer used for sequencing). The wild-type sequence was found in
hybrid 3, containing the wt allele. RT-PCR analysis of transcripts
from lymphoid cells of the patient showed that the mutant
transcript was expressed at significantly lower levels than the
wild-type sequence. Sequence analysis of the genomic DNA of the
same hybrids (FIG. 4B) showed that the insertion was due to a A to
C mutation (antisense sequence, indicated in bold and underlined)
at the splice acceptor site of exon 4, resulting in the use of a
cryptic splice site 24 bp upstream. The signal of the mutant C is
not as strong as the wild-type A in the donor's DNA. Such
non-equivalence is not unusual in sequencing templates from diploid
cells, and can result in difficulties in interpretation of the
chromatograms. (FIG. 4C) Extracts from hybrids 1 and 5, carrying
the mutant allele of chromosome 2, were devoid of hMSH2 protein,
while extracts of hybrids 2 and 3, carrying the wt allele,
contained hMSH2 protein. Hybrid 4 did not contain either allele of
chromosome 2. Hybrids 1, 3, 4, and 5 each carried at least one
allele of chromosome 3 and all synthesized hMLH1 protein.
.alpha.-tubulin served as a protein loading control. Immunoblots
with antibodies to the indicated proteins are shown.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0015] We have devised a strategy for generating hybrids containing
any desired human or other mammal's chromosome using a single
fusion and selection condition. Importantly and unexpectedly, the
human or other mamalian chromosomes in these hybrids were stable,
and they expressed human or other mammalian genes at levels
sufficient for detailed analysis. The approach is based on the
principle that fusion between human or other mammal and rodent
cells creates hybrid cells that contain the full rodent genomic
complement but only a portion of the human or other mammalian
chromosomes. In the past, selection for retention of a specific
human or other mammalian chromosome (by complementation of an
auxotrophic rodent cell, for example) has allowed the isolation of
hybrids containing a desired chromosome (7, 8). Though such fusions
have proven useful for a variety of purposes (8, 9), their utility
has been limited by the availability of appropriate rodent
recipients for many chromosomes and by the inefficiencies and
variation of the fusion and selection conditions. For the analysis
of multigenic diseases, it would be necessary to perform a separate
fusion and selection for each chromosome.
[0016] The stability of the human or other mammalian chromosomes in
the hybrids of the present invention was surprising. Though the
human genetic constitution of radiation hybrids is relatively
stable, this stability has been presumed to be due to the
integration of small pieces of human DNA into rodent chromosomes
following irradiation of the donor cells. The human chromosomes in
whole cell fusions have been believed to be unstable unless
continuous selection pressure for individual chromosomes was
exerted. The reasons for the stability in our experiments is
unclear, but may be related to the diploid nature of the rodent
partner. Such diploidy reflects a chromosome stability that is
unusual among transformed rodent cells. Previous experiments have
indeed shown that chromosomally stable human cells retain all
chromosomes upon fusion with other chromosomally stable human
cells, unlike the situation when one of the two partners is
chromosomally unstable.
[0017] The diploid, rodent recipient cells of the present invention
provide useful reagents for the facile creation of cells with
functionally haploid human genomes. Nucleic acids or proteins from
these hybrids can be used as reagents for any standard mutational
assay. As mutational assays are constantly being improved and
automated (1), the value of the hybrid-generated materials
correspondingly increases. It may soon become possible, in fact, to
examine the sequence of entire genes (promoters and introns in
addition to exons). Nucleic acid templates generated from single
alleles are clearly superior for such analyses, as the homogeneous
nature of the templates dramatically enhances the signal to noise
ratio of virtually any diagnostic assay. We therefore envision that
this approach can be productively applied to a wide variety of
research and clinical problems.
[0018] Genes of interest are typically those which have been found
to be involved in inherited diseases. These include genes involved
in colon cancer, breast cancer, Li-Fraumeni disease, cystic
fibrosis, neurofibromatosis type 2, von Hippel-Lindau disease, as
well as others. The identified genes include APC, merlin, CF, VHL,
hMSH2, p53, hPMS2, hMLH1, BRAC1, as well as others. Mutations which
can be identified at the protein level include those in sequences
that regulate transcription or translation, nonsense mutations,
splice site alterations, translocations, deletions, and insertions,
or any other changes that result in substantial reduction of the
full-length protein. Other subtler mutations can be detected at the
nucleic acid level, such as by sequencing of RT-PCR products.
[0019] Cells of the human which may be used in fusions are any
which can be readily fused to rodent cells. Peripheral blood
lymphocytes (PBL) which are readily available clinical specimens
are good fusion partners, with or without prior mitogenetic
stimulation, whether used fresh or stored for over one year at
-80.degree. C. Since inherited mutations are the subject of the
present method, any cells of the human body can be used, since all
such cells contain essentially the same genetic complement. Cells
of other mammals which can be used include in particular those of
cats, dogs, cows, sheep, goats, horses, chimpanzees, baboons, and
hogs. More generically, the cells of the other mammals can be
selected from the ruminants, primates, carnivora, lagomorpha, and
perissodactyla. Typically the other mammalian cell fusion partner
is not a rodent cell.
[0020] Rodent cell recipients for fusion are preferably diploid,
more preferably oncogene-transformed, and even more preferably have
microsatellite instability due to a defect in a mismatch repair
gene. Selection of particular clones which grow robustly, are
stably diploid, and fuse at a high rate is well within the skill of
the ordinary artisan. The rodent cells may be, for example, from
mice, rats, guinea pigs, or hamsters.
[0021] Fusion of cells according to the present invention can be
accomplished according to any means known in the art. Known
techniques for inducing fusion include polyethylene glycol-mediated
fusion, Sendai virus-mediated fusion, and electro-fusion. Cells can
desirably be mixed at a ratio of between 10:1 and 1:10 human to
rodent. Clones of fused cells generally become visible after about
two to three weeks of growth.
[0022] Fused hybrid cells can be selected using any markers which
result in a positively selectable phenotype. These include
antibiotic resistance genes, toxic metabolite resistance genes,
prototrophic markers, etc. The surprising advantage of the present
invention is that a single marker on a single human or other
mammalian chromosome can be used in the selection, and that stable
hybrids containing more than just the single, selected human or
other mammalian chromosome result. Thus markers on other
chromosomes can be analyzed even when the chromosomes on which the
markers reside were not selected.
[0023] Fused hybrid cells can be analyzed to determine that they do
in fact carry a human or other mammalian (non-rodent) chromosome
which carries a gene of interest. Hybrid cells which have either of
the two relevant human or other mammalian chromosomes can be
distinguished from each other as well as from hybrids which contain
both of the two human or other mammalian chromosomes. See FIG. 1.
While any means known in the art for identifying the human or other
mammalian chromosomes can be used, a facile analysis can be
performed by assessing microsatellite markers on the human or other
mammalian chromosome. Other linked polymorphic markers can be used
to identify a desired human or other mammalian chromosome in the
hybrids.
[0024] Once hybrid cells are isolated which contain one copy of a
human or other mammalian gene of interest from a human or other
mammal who is being tested, mutation analysis can be performed on
the hybrid cells. The genes can be tested directly for mutations,
or alternatively the mRNA or protein products of the genes can be
tested. Mutations that result in reduced expression of the
full-length gene product should be detectable by Western blotting
using appropriate antibodies. Tests which rely on the function of
the protein encoded by the gene of interest and enzyme assays can
also be performed to detect mutations. Other immunological
techniques can also be employed, as are known in the art.
[0025] If an immunological method is used to detect the protein
product of the gene of interest in the hybrids, it is desirable
that antibodies be used that do not cross-react with rodent
proteins. Alternatively, the rodent genes which are homologous to
the gene of interest can be inactivated by mutation to simplify the
analysis of protein products. Such mutations can be achieved by
targeted mutagenesis methods, as is well known in the art.
[0026] Functional tests can also be used to assess the normalcy of
each allelic product. For example, if one inserted an expression
construct comprising a .beta.-galactosidase gene downstream from a
p53 transcriptional activation site, into a rodent-human hybrid
cell that contained human chromosome 17 but no endogenous p53, then
one could detect mutations of the p53 on the human chromosome 17 by
staining clones with X-gal. Other enzymatic or functional assays
can be designed specifically tailored to the gene of interest.
[0027] Any method of detecting mutations at the DNA or RNA level as
are known in the art may be employed. These include without
limitation, sequencing, allele-specific PCR, allele-specific
hybridization, microarrays, DGGE, and automated sequencing.
[0028] It is a possibility that expression of the gene of interest
might be inhibited in the hybrid cell environment. In order for the
loss of expression of a gene of interest in the hybrid cells to be
meaningfully interpreted as indicating a mutation in the human or
other mammal, one must confirm that the gene of interest, when
wild-type, is expressed in rodent-human or other mammal hybrid
cells. This confirmation need not be done for each patient, but can
be done once when the assay is being established.
[0029] When the assay of the present invention indicates that a
mutation exists in the gene of interest, other family members can
be tested to ascertain whether they too carry the mutation.
Alternatively, the other family members can be tested to see if
they carry the same chromosome as the affected family member. This
can be determined by testing for a haplotype, i.e., a set of
distinctive markers which are found on the chromosome carrying the
mutation in the affected family member. Determination of a
haplotype is a by-product of performing the assay of the invention
on the first family member. When the hybrid cells are tested to
confirm the presence of the relevant chromosome in the hybrid, for
example by use of microsatellite markers, a distinctive marker set
will be identified, which can then be used as a haplotype.
[0030] Mixed populations of hybrid cells made by the fusion process
of the present invention may contain hybrid cells which are haploid
for a number of different human or other mammalian chromosomes.
Typically each homolog of at least 2, at least 5, at least 10, at
least 15, at least 20, or even 22 human or other mammalian
autosomes will be present in the population in a haploid condition
in at least one out of one hundred, seventy-five, fifty, thirty or
twenty-eight of the cells. Thus a high proportion of the cells
contain multiple human or other mammalian chromosomes, and a
relatively small number of cells must be tested to find cells
harboring a single copy of a non-selected chromosome.
[0031] Populations of cells resulting from a single hybrid are
uniform and homogeneous due to the high stability of the human or
other mammalian chromosomes in the hybrid cells of the invention.
Thus at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the
cells in the population resulting from a single hybrid cell contain
the same complement of human or other mammalian chromosomes.
[0032] The following examples provide experimental details which
demonstrate one of many ways to carry out the invention. The
invention is not limited to the particular methods of cells
employed in the examples. The claims and the specification as a
whole provide the measure of the invention.
EXAMPLES
Example 1
[0033] An outline of the approach is presented in FIG. 1. The
rodent fusion partner was a line derived from mouse embryonic
fibroblasts transformed with ras and adenovirus E1A oncogenes.
HPRT-deficient subclones of this line were generated, and one
subclone (E2) was chosen for further experimentation based on its
robust growth characteristics, maintenance of diploidy, and fusion
efficiency (10). Human lymphocytes cells were mixed with E2 cells
at an optimum ratio and electrofused, and hybrids selected in
geneticin (to kill unfused human cells) and HAT (to kill unfused E2
cells) (11). Colonies appearing after two weeks of growth were
expanded and RNA and DNA prepared for analysis. From a single
fusion experiment, an average of 36 hybrid clones were obtained
(range of 17 to 80 in five different individuals).
[0034] All hybrids contained the human X chromosome, as this
chromosome contains the HPRT gene allowing growth in HAT. To
determine whether other human chromosomes were present in the
hybrids, polymorphic microsatellite markers (12) were used as
probes in PCR-based assays (FIG. 2A). We focused on the chromosome
arms (2p, 2q, 3p, 5q, 7q, and 16q) known to contain colorectal
cancer (CRC) predisposition genes. One copy of each of these
chromosome arms was present in a significant fraction of the hybrid
clones. For example, of 476 hybrids derived from 14 individuals and
examined for chromosome 3, 136 hybrids contained neither donor
chromosome, 211 hybrids contained both donor chromosomes, 60
hybrids contained one parent's chromosome, and 69 hybrids contained
the other parent's chromosome. Similar retention frequencies were
found for all six chromosome arms analyzed. Testing of markers from
both arms of a single chromosome showed that whole chromosomes,
rather than chromosome fragments, were generally retained in the
hybrids. This result was confirmed with fluorescence in situ
hybridization (FISH) on metaphase spreads from the hybrids, which
indicated the presence of 11.+-.3 human chromosomes in each hybrid
cell. Calculations based on the genotypic data indicated that the
analysis of 25 hybrids would ensure a 95% probability of
identifying at least one hybrid containing the maternal allele and
one hybrid containing the paternal allele of a single chromosome
under study. Moreover, it would require only 45 hybrids to
similarly ensure that each allele of all 22 autosomes was present
and separated from its homolog in at least one hybrid (13).
Example 2
[0035] Two other features of the hybrids were essential for the
analyses described below. First, the human chromosome complements
of the hybrids were remarkably stable. Polymorphic marker analysis
in ten hybrids revealed identical patterns of retention after
growth for 90 (30 passages) generations after initial genotyping.
Second, those hybrids containing the relevant chromosome expressed
every human gene assessed, including all known colorectal cancer
susceptibility genes (the hMSH2 and hMSH6 genes on chromosome 2p,
the hPMS1 gene on chromosome 2q, the TGF-.beta. Receptor Type II
gene and hMLH1 gene on chromosome 3p, the APC gene on chromosome
5q, the hPMS2 gene on chromosome 7q, and the E-cadherin gene on
chromosome 16q; representative examples in FIG. 2B) (14).
Example 3
[0036] Having established the stability and expression patterns of
CRC-predisposition genes in these hybrids, we used this
"conversion" approach to investigate ten patients who had proven
refractory to standard genetic diagnostic techniques. Each of these
patients had a significant family history of colorectal cancer and
evidence of mismatch repair deficiency in their tumors, yet
sequencing of the entire coding sequence of each known MMR gene had
failed to reveal mutations. Indeed, these and similar studies have
prompted the speculation that other major HNPCC genes must exist.
(25-34) Hybrids were generated from lymphocytes of each patient,
and at least one hybrid containing the maternal allele and one
hybrid containing the paternal allele of each MMR gene was
isolated. Analysis of the nucleic acids from these hybrids revealed
specific mutations in all ten patients (Table 1). In every case, an
abnormality was found in a single allele of either hMSH2 or hMLH1.
The nature of the abnormalities revealed why they had not been
detected with the standard methods previously used for their
analysis. Three cases were due to large deletions, encompassing six
or seven exons. When corresponding nucleic acids from the cells of
such patients are evaluated by any PCR-based method, only the wild
type sequences from the unaffected parent would be amplified,
leading to the false impression of normalcy (for example, case #1
in FIG. 3). Though Southern blotting can reveal deletions of one or
a few exons in MMR, larger deletions are refractory to such
blotting methods. In three cases (#4, 6, and 9), no transcript was
generated from one allele, though the sequences of all exons and
intron-exon borders from this allele were normal. Presumably,
mutations deep within an intron or within the promoter of the gene
were responsible. The absence of transcripts from one specific
allele of these three patients was confirmed in at least three
other converted hybrids from each patient. In four other cases,
point mutations were found (Table 1). These mutations were not
detected in the original sequence analyses because the signals from
the mutant allele were not as robust as those from the wild type.
Such asymmetry can be caused by instability of mutant transcripts
due to nonsense mediated decay (36-38), or to nucleotide
preferences of the polymerases in specific sequence contexts, and
represents a common problem for both manual and automated
sequencing methods (39). The conversion approach eliminates these
problems because only one sequence can possibly be present at each
position. A good example of this was provided by Warthin G (17).
The mutation in this prototype kindred was an A to C transversion
at a splice site. The signal from the mutant "C" in the sequencing
ladder was not as intense as the wild type "A" (FIG. 4b). This
mutation led to the use of a cryptic splice site 24 bp upstream of
exon 4, and an under-represented transcript with a 24 base
insertion (FIG. 4a). To demonstrate that this mutation had an
effect at the protein level, we analyzed the hybrids by
immunoblotting with specific antibodies (19). The hybrids
containing the mutant allele did not make detectable levels of
human hMSH2 protein, though they did synthesize normal levels of a
control protein (FIG. 4C).
[0037] The results described above demonstrate that individual
alleles of human chromosomes can be readily isolated upon fusion to
mouse cells.
[0038] HNPCC provides a cogent demonstration of the power of the
conversion approach because it is a common genetic disease that has
been widely studied. In the last three years, for example,
extensive analyses of the major MMR genes have been performed in
303 HNPCC kindreds from nine cohorts distributed throughout the
world (25-34). Based on the fraction of such patients with
characteristic microsatellite instability in their cancers (30-34),
it can be estimated that 239 (78%) of the kindreds had germ-line
mutations of mismatch repair genes. Yet MMR gene mutations were
identified in only 127 (42%) of these 239 kindreds (25-34). Our
cohort was similar, in that it was derived from a total of 25
kindreds, 22 of whom had tumors with microsatellite instability and
presumptive MMR gene mutations. Of these 22, our initial analyses
revealed mutations in only 12 (54%) (ref. 14 and unpublished data).
Mutations of the other ten patients were only revealed upon
conversion analysis, which thereby increased the sensitivity from
54% to 100%. The conclusion that virtually all cases of HNPCC
associated with MSI are due to germline mutations of known MMR
genes is consistent with recent immunohistochemical data
demonstrating the absence of either MSH2 or MLH1 protein staining
in the cancers from the great majority of HNPCC patients (40, 41).
A corollary of these results is that the search for new human MMR
genes should not based on the premise that a large fraction of
HNPCC cases will prove attributable to such unknown genes.
[0039] The system described above can be applied to other genetic
diseases in a straight forward manner. It should be emphasized that
this approach is not a substitute for the many powerful methods
currently available to search for specific mutations. Rather,
conversion can be used to maximize the sensitivity of existing
techniques. Converted nucleic acids provide the preferred
substrates for such methods because of the higher signal to noise
attainable and the inability of the wild type allele to mask or
confound detection of the mutant allele. As DNA-based mutational
assays are improved in the future, and progressive incorporate
microarrays and other automatable features (42-44), the value of
conversion-generated nucleic acids will correspondingly increase,
significantly enhancing the effectiveness of genetic tests for
hereditary disease.
Methods
Cell Culture
[0040] Mouse embryonic fibroblasts were derived from MSH2-deficient
mice (46) and transformed with adenovirus E1A and RAS oncogenes.
HPRT-deficient subclones were selected by growing the fibroblasts
in 10 .mu.M 2-amino-6-mercaptopurine. Clones were maintained in
Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% FCS
and 10 .mu.M 2-amino-6-mercaptopurine.
Cell Fusion and the Generation of Hybrids
[0041] The patients were from kindreds with HNPCC as defined by the
Amsterdam criteria (44); in no case was linkage analysis feasible
due to the lack of a sufficient number of affected individuals.
Microsatellite instability (MSI) in the cancers from these patients
was determined through the markers recommended in ref. 45.
3.times.10.sup.6 E2 cells and 12.times.10.sup.6 lymphocytes cells
were mixed, washed, and centrifuged twice in fusion medium (0.25 M
D-sorbitol, 0.1 mM calcium acetate, 0.5 mM magnesium acetate, 0.1%
Bovine Serum Albumin (BSA), pH 7) and resuspended in 640 .mu.l
fusion medium. The solution was pipetted into a cuvette (BTX
cuvette electrode 470; BTX, San Diego). Cells were fused using a
BTX ElectroCell Manipulator, model ECM200. The settings that
yielded the greatest number of hybrids were: 30V (AC) for 22
seconds, followed by three 300V (DC) pulses of 15 .mu.sec each. The
cells from one fusion were plated into three 48-well plates
(Costar) in DMEM supplemented with 10% FCS. After 24 hours, the
medium was replaced by DMEM supplemented with 10% FCS, 0.5 mg/ml
geneticin and 1.times.HAT (Life Technologies, Gaithersburg, Md.).
The medium was changed after a week. Hybrid clones became visible
two weeks after fusion and were expanded for another week prior to
genotyping. From a single fusion, an average of 23.+-.15 hybrid
clones were obtained. The lymphocytes used for the experiments
described here were derived from Epstein-Barr Virus infection of
peripheral blood leukocytes, but it was found that freshly drawn
lymphocytes could also be successfully fused and analyzed using
identical methods.
Genotyping
[0042] Genotyping was performed as described (12). PCR products
were separated on 6% denaturing gels and visualized by
autoradiography. The microsatellite markers used were D2S1788 and
D2S1360, D2S1384, D3S2406, D7S1824, and D16S3095, from chromosome
2p, 2q, 3p, 5q, 7q and 16q, respectively. Fluorescence in situ
hybridization was performed as described previously (21).
PCR and Sequencing
[0043] Polyadenylated RNA was purified and RT-PCR performed as
described previously. Sequencing was performed using ABI Big Dye
terminators and an ABI 377 automated sequencer. All primers used
for amplification and sequencing will be made available through an
internet site.
Statistical Analysis
[0044] the number of hybrids containing none, both or a single
allele of each chromosome tested were consistent with a multinomial
distribution. Monte Carlo simulations were used to estimate the
number of hybrids required to generate mono-allelic hybrids
containing specific numbers of each chromosomes.
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