Estimation of Exon Dosage Using Real-Time Quantitative Polymerase Chain Reaction

Abstract

Hemophilia A is an X-linked bleeding disorder caused by a deficiency in blood coagulation factor VIII. Mutations in the F8 gene are responsible for this disease affecting 1 in 5,000 live male births. Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disease, also known as Osler-Weber-Rendu disease, associated with abnormal blood vessel formation. It is associated with alterations in at least two genes including Endoglin (ENG) on chromosome 9q34 and activin receptor-like kinase 1 (ACVRL1 or ALK1) on chromosome 12q13, which are involved in cell proliferation, differentiation, migration, and adhesion in the vascular system. Direct sequencing of the coding exons of the relevant genes in affected individuals is the primary screen for detecting a causative point mutation. However for most diseases, 10–15% of mutations involve intragenic deletions, or duplications of the whole gene or a subset of exons of the same gene. This necessitates an additional screen for exonic dosage estimation of all coding sequence mutation negative affected, or, carrier individuals. This is accomplished by using a real-time quantitative PCR assay for measuring the copy number of each exon of the relevant gene. This testing establishes the diagnosis in hemophilia A patients, as well as carrier status in at-risk females. Similarly, it can provide molecular confirmation for clinical diagnosis of HHT, as well as the identification of mutation carriers among presymptomatic individuals when the familial mutation is known.