DETERMINATION OF GENE DOSAGE BY A QUANTITATIVE ADAPTATION OF THE POLYMERASE CHAIN-REACTION (GD-PCR) - RAPID DETECTION OF DELETIONS AND DUPLICATIONS OF GENE-SEQUENCES

Screening methods based on the polymerase chain reaction (PCR), such a s denaturing gradient gel electrophoresis, single-stranded conformatio nal polymorphism, and heteroduplex analysis, are powerful tools for th e detection of point mutations as well as small deletions and insertio ns, but are unable to detect heterozygous deletions or duplications of exons, genes, or chromosomes. We now report a PCR-based approach, des ignated gene dosage-PCR (gd-PCR), that allows rapid screening for hete rozygous deletions and duplications of genes or exons. Gene dosage-PCR is a quantitative method in which two in vitro synthesized DNA intern al standards are coamplified with the genomic DNA sample, one correspo nding to the gene of interest (test sequence) and the other to a refer ence (disomic) gene (reference sequence). Both internal standards are designed to be amplified with the same primer pairs and with efficienc ies similar to those of their genomic DNA counterparts, yielding PCR p roducts slightly smaller than those derived from genomic DNA. Amplific ation of approximately equimolar amounts of the two internal standards and genomic DNA, in the presence of [P-32]dCTP, results in four radio labeled PCR products; after electrophoresis and quantification of the products, gene dosage is easily calculated. For validation, genomic DN A from 56 subjects, 28 with cytogenetically documented Down syndrome ( trisomy 21) and 28 controls that were disomic for chromosome 21, was a ssayed. Using the P-amyloid precursor protein gene (APP: chromosome 21 q21) as the test sequence, control subjects had an adjusted mean gene dose of 2.00 +/- 0.29, while subjects with Down syndrome had a mean ge ne dose of 3.05 +/- 0.27. There was a clear separation of all of the s amples between the two groups. We also successfully used gd-PCR to det ect allelic deletions by screening pertinent regions of the insulin re ceptor gene (IR; chromosome 19p13.2-p13.3) in three unrelated patients with genetic syndromes of extreme insulin resistance-known heterozygo tes for deletions of either exon 3, exon 14, or exons 17-22. Gene dosa ge-PCR is a versatile, rapid, and sensitive method for screening for d uplications and deletions of exons, genes, or chromosomes, with broad application in both clinical and research settings. (C) 1994 Academic Press, Inc.