Abstract
Stickler syndrome (hereditary arthro-ophthalmopathy) is a dominantly inherited disorder of connective tissue that affects the eyes, ears, joints and skeleton. It is the most common autosomal dominant connective tissue dysplasia, the commonest cause of inherited retinal detachment and an important cause of premature osteoarthritis. The diagnosis and classification of Stickler syndrome is made challenging by clinical variability and genetic (locus) heterogeneity. Half to two-thirds of affected families show linkage to the gene encoding type II procollagen, with all reported mutations resulting in a premature stop codon. Defects in type XI collagen are also associated with Stickler syndrome, with mutations in both the α1(XI) and α2(XI) chains having been characterized. We have developed a novel molecular test that will facilitate screening of families with Stickler syndrome due to COL2A1 nonsense mutations. This test employs an RNA-based approach, due to the large size of the gene with 52 exons, and messenger RNA extracted from transformed lymphoblasts is used as source material for the protein truncation test (PTT). Five overlapping primer sets were designed to cover the entire coding region of the COL2A1 gene. Resulting PCR products were then transcribed and translated in vitro, with the presence of a smaller protein product indicating a stop codon mutation in the corresponding PCR product. RNA-based approaches such as this can be compromised by low levels of mutant mRNA, due to the phenomenon of nonsense-mediated mRNA decay triggered by premature stop codons. To overcome this problem the protein synthesis inhibitor cycloheximide was added to cells prior to mRNA extraction and PTT. To develop this test we selected a candidate family with seven affected living members spanning two generations. Employment of our methodology led to the detection of a protein truncating mutation in all affected individuals and subsequent mutation analysis confirmed its predicted protein truncating nature. This screening test should become a powerful tool in the diagnosis of this group of Stickler patients, who are at risk of the disastrous ocular and other complications of this disorder such as premature osteoarthritis. It would enable these individuals to undergo regular medical surveillance with access to preventative or ameliorative treatment and accurate counseling.
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Savarirayan, R., Freddi, S. & Bateman, J. Novel Protein Truncation Test to detect COL2AI Nonsense Mutations in Stickler syndrome. Genet Med 1, 44 (1999). https://doi.org/10.1097/00125817-199901000-00020
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DOI: https://doi.org/10.1097/00125817-199901000-00020