Genomic structure and identification of novel mutations in Usherin, the gene responsible for Usher syndrome type IIa

Usher syndrome type IIa (USHIIa) is an autosomal recessive disorder charact erized by moderate to severe sensorineural hearing loss and progressive ret initis pigmentosa, This disorder maps to human chromosome 1q41. Recently, m utations in USHIIa patients were identified in a novel gene isolated from t his chromosomal region. The USH2A gene encodes a protein with a predicted m olecular weight of 171.5 kD and possesses laminin epidermal growth factor a s well as fibronectin type III domains. These domains are observed in other protein components of the basal lamina and extracellular matrixes; they ma y also be observed in cell-adhesion molecules. The intron/exon organization of the gene whose protein we name "Usherin" was determined by direct seque ncing of PCR products and cloned genomic DNA with cDNA-specific primers. Th e gene is encoded by 21 exons and spans a minimum of 105 kb. A mutation sea rch of 57 independent USHIIa probands was performed with a combination of d irect sequencing and heteroduplex analysis of PCR-amplified exons. Fifteen new mutations were found. Of 114 independent USH2A alleles, 58 harbored pro bable pathologic mutations. Ten cases of USHIIa were true homozygotes and 1 0 were compound heterozygotes; 18 heterozygotes with only one identifiable mutation were observed. Sixty-five percent (38/58) of cases had at least on e mutation, and 51% (58/114) of the total number of possible mutations were identified. The allele 2299delG (previously reported as 2314delG) was the most frequent mutant allele observed (16%; 31/192). Three new missense muta tions (C319Y, N346H, and C419F) were discovered; all were restricted to the previously unreported laminin domain VI region of Usherin. The possible si gnificance of this domain, known to be necessary for laminin network assemb ly, is discussed in the context of domain VI mutations from other proteins.