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Converting diploidy to haploidy for genetic diagnosisRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidConverting diploidy to haploidy for genetic diagnosis description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060166257, Converting diploidy to haploidy for genetic diagnosis. Brief Patent Description - Full Patent Description - Patent Application Claims
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. Continue reading about Converting diploidy to haploidy for genetic diagnosis... 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