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

The invention provides a method for detecting mutations in a gene of interest on a human or other mammalian chromosome by fusing cells of a human or other mammal with rodent cells to form hybrids. These hybrids are selected for a marker on each of a first human or other mammalian chromosome and a rodent chromosome. The resulting population of fused cell hybrids contains a subset of cells that are haploid for a second human or other mammalian chromosome. The presence of a mutation in the gene of interest is detected by testing the hybrids for the presence of the gene, its mRNA or protein product. The invention also provides test cells that are haploid for human or other mammalian genes, which can be used for genetic testing. The human or other mammalian chromosome content of the hybrid cells is stable and uniform. The technical effect of the invention is to increase the sensitivity and effectiveness of various diagnostic and analytic methods by providing stable and uniform hybrid cells for analysis.

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.

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