A Strategy for Efficient Molecular Diagnosis of Genetically Heterogeneous Autosomal Recessive Diseases

Abstract

Purpose:: Genetic testing for many human Mendelian disorders can be time consuming and expensive because of extensive genetic heterogeneity. For example, the nine genes known to cause Leber congenital amaurosis (LCA) contain a total of 139 different exons. We hypothesized that the efficiency of genetic testing for autosomal recessive diseases could be dramatically increased by 1) using allele-specific assays to inexpensively identify known alleles, 2) using DNA sequencing of gene segments previously found to harbor disease-causing mutations to identify novel disease alleles, and 3) restricting the search for an individual's second recessive allele to the gene in which the first plausible disease-causing variant was identified.

Methods:: 416 patients with the clinical diagnosis of LCA were screened for disease-causing mutations with a combination of single strand conformational polymorphism analysis (SSCP), a multiplexed allele-specific ligation assay (SNPlex), and bidirectional DNA sequencing. Some exons were screened in parts so that a total of 170 different amplimers were evaluated.

Results:: 326 instances of 143 different disease-causing variations were identified in this cohort of 416 LCA patients. Only 92 of the 170 LCA amplimers were found to harbor a disease-causing variant in any individual. Although a multi-platform (SSCP and SNPlex) allele-specific assay can now rapidly and inexpensively detect all 143 of these alleles (as well as an additional 74 alleles that have been reported in the literature but were not observed in this cohort), 42% of the 100 most recently detected alleles were novel when they were discovered, and were detected with DNA sequencing.

Conclusions:: These data demonstrate that a tiered screening strategy can increase the efficiency of autosomal recessive mutation detection several fold. Multiplexed allele-specific assays can find known disease alleles very inexpensively while novel changes can be efficiently found with a sequencing strategy that takes advantage of the observed distribution of mutations within and among genes. Each novel mutation detected by DNA sequencing can be added to the allele specific tier of the assay shortly after its discovery, thereby steadily decreasing the average cost of the test and increasing its speed.

Keywords: mutations • genetics • retinal degenerations: hereditary