Abstract
Purpose::
To map, identify and functionally characterize the genetic mutation underlying autosomal dominant childhood cataract segregating in a 6-generation Caucasian-American family.
Methods::
Following ethical approval and informed consent, genomic DNA was prepared from blood leucocytes, and genotyping performed using STR and SNP markers. Pedigree and haplotype data were managed using Cyrillic, and Lod scores calculated using MLINK. PCR amplicons were cycle sequenced using dye-terminator chemistry and capillary electrophoresis. RNA transcripts were quantified by qRT-PCR in real-time with SYBR-Green. Site-specific in vitro mutagenesis was performed by mutagenic primer-directed plasmid replication. Cultured cells were transfected with CMV-driven expression plasmids, and recombinant FLAG-tagged proteins detected by immuno-blot and immuno-fluorescence microscopy techniques.
Results::
After exclusion of multiple loci for autosomal dominant cataract on chromosomes 1-3,10-13, 15-17, 19, 21 and 22, we obtained significant evidence of linkage (Z = >3.0, θ = 0) to STR markers on chromosome 20. Fine mapping with SNP markers and haplotype analysis indicated that the cataract locus was confined to a ~4Mb physical interval harboring ~100 positional candidate genes. Mutation profiling detected a heterozygous A>T transversion that co-segregated with the disease but was not detected in 192 controls. The A>T coding change was detected at the RNA level, consistent with a missense mutation, and was predicted to result in the non-conservative substitution of a phylogenetically conserved amino acid at the protein level. Transfection studies revealed that the mutant protein displayed an abnormal sub-cellular localization and impaired transport properties.
Conclusions::
Our data identify the first causative mutation in a gene not previously associated with inherited cataract, and suggest that a deleterious gain-of-function mechanism leads to loss of lens transparency.
Keywords: genetics • gene mapping • candidate gene analysis