In Search of the Forgotten Exon of the Human Crumbs Homolog 1 and Its Implication in LCA

Abstract

Abstract: : Purpose: To identify the gene responsible for Leber congenital amaurosis in a very large consanguineous family originating from Palestine. Methods: A genome wide search for homozygosity was undertaken in the nine affected patients belonging to one large consanguineous family. Three hundred and eighty two pairs of fluorescent oligonucleotides of the Genescan Linkage Mapping Set, version II (Perkin Elmer Cetus) were used. Amplified fragments were electrophoresed and analysed on an automatic sequencer (ABI377). Genes lying in the candidate region were screened by direct sequencing in affected individuals. Novel CRB1 exons were searched by the screening of the Human Genome Working Draft and confirmed by RT-PCR of retinal mRNA. Results & Conclusion: Homozygosity was found in several regions of the genome. Further analyses allowed to exclude these regions except one since homozygosity for the markers only reflected the lack of informativity in the parents. Conversely, we found evidence for homozygosity for markers of the 1q31 region. Recombination events in two affected patients allowed reducing the genetic interval containing the disease-causing gene between D1S2757 and D1S413. The CRB1 gene was found to map in this 4 cM interval. However, prior to this genome wide search for homozygosity, a screening of the six already known LCA-causing gene including CRB1 was performed but no mutation was found. The mapping of the disease-causing gene in this family prompted us to consider that the mutation might lie in a forgotten exon of the CRB1 gene. Subsequently, we screened the Human Genome Working Draft in order to identify novel CRB1 exons. This study revealed that a twelfth exon lies in the 3' end of the gene. The sequence of this exon revealed that the disease in this family is accounted for by a homozygous 10 bp deletion leading to apparition of a premature Stop codon. Further study of this exon in 125 unrelated LCA patients was therefore undertaken. The results of the whole study will be presented.

Keywords: 562 retinal degenerations: hereditary • 335 candidate gene analysis • 420 genetics