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.

Description

[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.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 p...

AI Technical Summary

Benefits of technology

[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 β-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.

Problems solved by technology

The problem with humans and other mammals, at least from a genetic diagnostic perspective, is that they are diploid.

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.

Images