Analysis of CRX Mutations in LCA Patients

Abstract

Abstract: : Purpose: Leber congenital amaurosis (LCA) is a clinically and genetically heterogeneous disorder presenting in infancy with profound vision loss and a markedly attenuated ERG. Six causative genes, GUCY2D, RPE-65, CRX, AIPL1, CRB1 and RPGRIP1 have been identified. As recently published, the human cone-rod homeobox (CRX) gene consists of six exons spanning 25kb on chromosome 19q13. It encodes photoreceptor-specific transcripts. 5'-RACE and primer extension studies combined with EST database search revealed that the CRX 5'-UTR is coded by two more exons, 1a and 1b, upstream of exon 2 harboring the initiation codon ATG, and the major transcription start site is located at 5' of exon 1b. Mutations in CRX account for ~ 6% of LCA and the mutations identified were heterozygous, except for R90W. We sequenced the recently identified additional upstream exon and promoter sequence in patients with previously identified heterozygous CRX mutations. We also tested for the presence of mutations in other LCA genes. Methods: Mutation screening was performed using direct sequencing in eleven probands of worldwide distribution with a previously identified heterozygous mutation in CRX. Primer sequences were obtained from the public databases. Results: Four heterozygous missense mutations were identified in the probands and also in the unaffected first degree relatives. Two of these changes were non-conservative (G122D and T273M) and were not detected in 100 controls. Frame-shift mutations resulting from insertions (G248ins23bps) and deletions (L237del1bp, A177del1bp,Y191del1bp, T251del1bp) were not detected in the normal unaffected relatives, except P9ins1bp. Since these mutations disrupt one or both of the activation domains, they are expected to cause a marked reduction in CRX's transactivational activity. S448X was detected in GUCY2D in a proband with CRX A158T. Conclusions: CRX mutations impair CRX-mediated transcriptional activity; they alter function of genes involved in normal retinal structure and survival. Sequencing of upstream promoter and enhancer regions continues in an effort to identify sequence variants and to study their functional significance.

Keywords: genetics • retina • photoreceptors