Case populations must match the respective disease model: Genotype diversity causes linkage disequilibrium mapping failure in monogenic disorders.

Traditional linkage analysis in large families is the most promising approach for mapping disease genes of monogenic heritable disorders when the number of informative meioses is sufficient. With rare diseases, however, the low availability of informative pedigrees poses a significant limitation. As an adjunct to family linkage methods, association studies based on the investigation of individual haplotypes from a number of unrelated patients (i.e. linkage disequilibrium analysis) have recently been employed in mapping hereditary disease loci. However, such haplotype analysis is hampered by a number of effects that influence statistical evaluation, e.g. i) population history and size, ii) allele and haplotype frequencies in the respective population(s), iii) heterogeneous mutation and natural selection processes, and iv) small sample sizes of patient groups. The purpose of the present study was to determine the utility and limitations of haplotype-based genetic mapping in estimating the location of the NYX gene, which has recently been identified as the causative gene for a rare inherited retinal disorder known as the complete type of X-linked congenital stationary night blindness (CSNB1). For this purpose we recapitulated haplotypes and tested for linkage disequilibrium in 20 unrelated male CSNB1 patients from three European populations and 44 healthy individuals. All subjects were genotyped for 17 polymorphic microsatellite loci covering the Xp11.4 region with an average marker density of approximately 0.29 cM. We found that a precise model to describe mutations at loci that erroneously break up linkage is highly required, and that the case population must match the respective disease model.

Complete form of X-linked congenital stationary night blindness: refined mapping and evidence of genetic homogeneity.

A number of distinct, partly non-overlapping genetic loci have been reported for the complete type of X-linked congenital stationary night blindness (CSNB1), suggesting genetic heterogeneity. In order to refine the localization of the CSNB1 gene and to demonstrate genetic homogeneity, linkage analysis was performed in two large CSNB1 families. Clinical features consistent with the diagnosis of CSNB1 were documented in five patients from a German seven-generation kindred by full ophthalmological examination including psychophysical and electroretinographical testing. Haplotype analysis in 30 members of the large German family was performed with 38 polymorphic markers predominantly covering the critical region. Linkage analyses defined a locus for CSNB1 with flanking markers DXS8042 and DXS228, refining the interval to 2.5 cM in Xp11.4. In addition, two-point linkage analysis was carried out using the MLINK computer program. In agreement with meiotic breakpoints, lod scores of 3.0 and greater were obtained for markers located to the proximal site of the former 5 cM CSNB consensus interval. A large Dutch CSNB1 family was re-evaluated with markers from the Xp11.4 region, and supports the CSNB1 minimal interval found in the German family. Together with previous results from three unrelated families from Sweden, Sardinia and Great Britain, our results provide evidence of genetic homogeneity in the disorder. Subsequent mutation analyses in CSNB1 patients revealed no pathogenic sequence alterations in DFFRX and CASK genes, but retain candidates for other diseases mapping to that region.

The complete form of X-linked congenital stationary night blindness is caused by mutations in a gene encoding a leucine-rich repeat protein.

X-linked congenital stationary night blindness (XLCSNB) is characterized by impaired scotopic vision with associated ocular symptoms such as myopia, hyperopia, nystagmus and reduced visual acuity. Genetic mapping in families with XLCSNB revealed two different loci on the proximal short arm of the X chromosome. These two genetic subtypes can be distinguished on the basis of electroretinogram (ERG) responses and psychophysical testing as a complete (CSNB1) and an incomplete (CSNB2) form. The CSNB1 locus has been mapped to a 5-cM linkage interval in Xp11.4 (refs 2,5-7). Here we construct and analyse a contig between the markers DXS993 and DXS228, leading to the identification of a new gene mutated in CSNB1 patients. It is partially deleted in 3 families and mutation analysis in a further 21 families detected another 13 different mutations. This gene, designated NYX, encodes a protein of 481 amino acids (nyctalopin) and is expressed at low levels in tissues including retina, brain, testis and muscle. The predicted polypeptide is a glycosylphosphatidylinositol (GPI)-anchored extracellular protein with 11 typical and 2 cysteine-rich, leucine-rich repeats (LRRs). This motif is important for protein-protein interactions and members of the LRR superfamily are involved in cell adhesion and axon guidance. Future functional analysis of nyctalopin might therefore give insight into the fine-regulation of cell-cell contacts in the retina.

Physical mapping and exclusion of GPR34 as the causative gene for congenital stationary night blindness type 1.

X-linked congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder characterized by impaired night vision, variably involving high myopia, nystagmus, decreased visual acuity, and strabismus. Linkage studies have identified two distinct loci for X-linked CSNB1 and CSNB2 on the short arm of chromosome X. The gene mutated in families displaying the "incomplete phenotype" of CSNB (i.e., CSNB2) has recently been identified. To identify novel candidate genes for the "complete form" of CSNB (i.e., CSNB1) we screened the physically vast region Xp11.3-Xp11.4 for cDNA sequences. This led us to identify and map the G protein coupled receptor (GPCR) gene GPR34 to Xp11.4 within 650 kb of the marker DXS993. Deletion screening via Southern blotting and direct sequencing of GPR34 revealed no mutations in 19 unrelated men with CSNB1, excluding a causal role in the disease. However, because of its expression in retinal and neural tissue and the involvement of GPCRs in transmembrane signal transduction, GPR34 remains a putative candidate gene for a number of ocular diseases which also map to the Xp11.4 region.