Determination of Gene Dosage: Utilization of Endogenous and Exogenous Internal Standards

There are currently several screening methods for the detection of point mutations, such as single-stranded conformation polymorphism, heteroduplex analysis, denaturing gradient gel electrophoresis, and chemical cleavage. These are powerful tools for the identification of small sequence changes, but fail to detect heterozygous deletions or duplications of exons, genes or chromosomes. There are many genetic disorders where the primary defect is either owing to allelic deletions (Duchenne muscular dystrophy, spinal muscular atrophy, alpha thalassemia, growth hormone deficiency, familial hypercholesterolemia, and so on) or duplications (Charcot-Marie-Tooth, Klinefelter syndrome, Down syndrome, and so on). Furthermore, for the determination of the carrier state, for disorders such as Duchenne muscular dystrophy and spinal muscular atrophy, the accurate determination of heterozygous deletions is essential. This chapter will describe two methods for the determination of gene dosage, using Duchenne muscular dystrophy and spinal muscular atrophy as examples.