Determining Gene Dosage

Abstract

Numerous investigations (both clinical and basic) in today’s molecular biology laboratory require accurate quantification of DNA by the polymerase chain reaction (PCR). To name a few important examples, quantitative PCR has been used to quantify viral copy number, to perform gene expression studies, and to diagnose genetic diseases. In this issue of Clinical Chemistry , Poropat and Nicholson describe a quantitative PCR assay for the determination of gene dosage (i.e., the number of copies of a gene per somatic cell) of the PMP22 gene, a myelin gene involved in inherited neuropathies (1). The PMP22 gene is extremely sensitive to copy number; for example, it is duplicated in the autosomal dominant Charcot-Marie Tooth type I (CMT1A) disease and is deleted in the autosomal dominant hereditary neuropathy with liability to pressure palsies (HNPP). Because the expression of the disorders can be quite variable and CMT1A shows genetic heterogeneity, the DNA testing for these diseases establishes a secure diagnosis; it also permits genetic counseling and testing for high-risk family members. For molecular laboratories involved in genetic disorders, gene dosage determinations are clinically important and frequently performed. In addition to CMT1A and HNPP, there are many genetic disorders where the primary defect is due either to allelic deletions (e.g., Duchenne muscular dystrophy, spinal muscular atrophy, α-thalassemia, growth hormone deficiency, and familial hypercholesterolemia) or to duplications (e.g., Klinefelter syndrome and Down syndrome). Furthermore, for the determination of the carrier state of disorders such as Duchenne muscular dystrophy and spinal muscular atrophy, the accurate determination of heterozygous deletions is essential. Originally, gene dosage determinations were made from Southern blots. The protocol was laborious and required large amounts of DNA. Furthermore, optimal conditions were required; very high-quality blots were necessary, with consistent transfer and hybridization and low background. We have found that Southern autoradiographic bands >10 kb …