Towards positional cloning of the autosomal dominant doyne honeycomb retinal dystrophy (DHRD) gene localised to chromosome 2p16

Inherited retinal dystrophies are genetically diverse with a large number of gene defects implicated in a variety of clinical phenotypes. This thesis aims to investigate the molecular genetic basis of Doyne honeycomb retinal dystrophy (DHRD) which principally affects the central retina with the aim of discovering the underlying molecular pathology of this ocular disease. DHRD is an autosomal dominant disease that is characterised by the presence of drusen deposits in the macula of affected individuals leading to decreased visual acuity and eventually blindness. Mapping a disease to a chromosome is a prerequisite for the application of positional cloning strategies, leading to the characterisation of candidate gene(s) and its putative protein, addressing questions of its functional role and finally studying the pathophysiology of the disease phenotype with which it is associated. A similar strategy has been applied to DHRD that was initially mapped to the 2p16-21 region using the technique of linkage analysis thus placing the disease between the interval of D2S2316 and D2S378, of approximately 5 cM. Subsequent haplotype analysis, narrowed the critical region between the interval D2S2739 and D2S378 of approximately 4 cM. The highest lod score calculated (9.49 at [theta] = 0.06) was obtained with marker D2S378. Current genetic refinement has been achieved by haplotyping new members of the family that provided new recombination events placing the disease gene telomeric to D2S2352 and centromeric to D2S2251 in a 1 cM genetic interval. Additionally, two dominant drusen families were also mapped to the disease locus confirming the flanking markers previously published. These families did not refine the locus, but their haplotypes differed from the original DHRD haplotype, indicating the occurrence of 3 independent mutational events. Following linkage of DHRD, the construction of a YAC contig was initiated in order to map genes in the critical region. The previous disease interval was defined by markers D2S2739 and D2S378 was spanned by a YAC tiling path of approximately 3 Mb consisting of YAC clones from three different libraries. The contig physically ordered 17 STSs in 29 YAC clones. Following the construction of the contig, 2 ESTs and a gene (D2S1848E, D2S1981E, and [beta]-fodrin (SPTBN1)) were mapped to this interval. As SPTBN1 is expressed in rat retinal pigment epithelium (RPE) it was considered to be a good candidate gene to screen for DHRD. Thus immediate work concerned screening a partial region of SPTBN1 and mapping ESTs in the region in the hope of discovering the disease causative gene for DHRD. This work was later pre-empted due to the genetic refinement of the disease, excluding SPTBN1, D2S1848E and D2S1981E from the critical region. Meanwhile, PACs were isolated to reinforce the YAC contig and to provide non-chimaeric clones in a relatively narrow genetic region (between markers D2S2352 and D2S2251, of approximately 1 cM). Following the exclusion of obvious candidate genes a retinally expressed EST (WI-31133) was mapped to the refined genetic region. Subsequent work involved genomic characterisation of this gene in order to screen for mutations in the DHRD family and dominant drusen families in this region. The full-length coding sequence of this gene was capable of translating one exon which was screened but failed to reveal any single base pair mutations or polymorphism. Interestingly, a single variant (Arg345Trp) in the EFEMP1 has been demonstrated as the disease-causing gene for DHRD and Malattia (Stone et al., 1999). Although this change has been verified in the DHRD family, this gene lies external to our recent genetic refinement. It is proposed that this change is probably due to a polymorphism that was present in a common ancestor. Thus further work must be performed to reinforce that that EFEMP1 is the gene for DHRD. We are currently awaiting blood samples from a Japanese family that are affected with DHRD. It is of interest to screen their DNA with exon 10 of the EFFMP1 gene to demonstrate whether they possess the disease-causing (Arg345Trp) variant. If the affected individuals do not demonstrate this change, then the entire coding region of the EFFMP1 gene must be screened in search of a second mutation, thus reinforcing that is EFFMP1 the disease-causing gene for DHRD

Refined genetic and physical positioning of the gene for Doyne honeycomb retinal dystrophy (DHRD)

Doyne honeycomb retinal dystrophy (DHRD) is a late-onset autosomal dominant disorder that causes degeneration of the retina and can lead to blindness. We have previously assigned DHRD to a 5-cM region of chromosome 2p16 between marker loci D2S2739 and D2S378. Using sequence-tagged sites (STSs), expressed sequence tags (ESTs) and polymorphic markers within the DHRD region, we have identified 18 yeast artificial chromosomes (YACs) encompassing the DHRD locus? spanning approximately 3 Mb. The YAC contig was constructed by STS content mapping of these YACs and incorporates 13 STSs, including four genes and six polymorphic marker loci. We also report the genetic mapping of two families with a dominant drusen phenotype to the DHRD locus: and genetic refinement of the disease locus to a critical interval flanked by microsatellite marker loci D2S2352 and D2S2251, a distance of approximately 700 kb. These studies exclude a number of candidate genes and provide a resource for construction of a transcriptional map of the region, as a prerequisite to identification of the DHRD disease-causing gene and genes for other diseases mapping in the region, such as Malattia leventinese and Carney complex

RP1 protein truncating mutations predominate at the RP1 adRP locus

PURPOSE. Recent reports have shown that the autosomal dominant retinitis pigmentosa (adRP) phenotype linked to the pericentric region of chromosome 8 is associated with mutations in a gene designated RP1. Screening of the whole gene in a large cohort of patients has not been undertaken to date. To assess the involvement and character of RP1 mutations in adRP, the gene was screened in a panel of 266 unrelated patients of British origin and a Pakistani family linked to this locus. METHODS. Patients exhibiting the adRP phenotype were screened for mutations in the four exons of the RP1 gene by heteroduplex analysis and direct sequencing. Linkage of the Pakistani family was achieved using microsatellite markers. Polymerase chain reaction (PCR) products were separated by nondenaturing polyacrylamide gel electrophoresis. Alleles were assigned to individuals, which allowed calculation of LOD scores. Microsatellite marker haplotyping was used to determine ancestry of patients carrying the same mutation. RESULTS. In the 266 British patients and 1 Pakistani family analyzed, 21 loss-of-function mutations and 7 amino acid substitutions were identified, some of which may also be disease-causing. The mutations, many of which were deletion or insertion events, were clustered in the 5' end of exon 4. Most mutations resulted in a premature termination codon in the mRNA. Haplotype analysis of nine patients carrying an R677X mutation suggested that these patients are not ancestrally related. CONCLUSIONS. RP1 mutations account for 8% to 10% of the mutations in our cohort of British patients. The most common disease-causing mechanism is deduced to be one involving the presence of a truncated protein. Mutations in RP1 have now been described in adRP patients of four ethnically diverse populations. The different disease haplotype seen in the nine patients carrying the same mutation suggests that this mutation has arisen independently many times, possibly due to a mutation hot spot in this part of the gene