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Positional cloning with microsatellite markers allowed further localization of the Darier disease gene to a 2-cM interval of chromosome 12, 12q23–24.1, between the polymorphic loci D12S234 and D12S129. A region this size is suitable for construction of a contig to identify the Darier disease gene. Use of a polymorphic intronic marker for nitric oxide synthetase 1 gene, which maps to the same chromosomal area as the Darier gene, allowed exclusion of that gene as the Darier disease gene.
The gene for Darier disease (DAR) has been localized to 12q23–24.1 by several different groups (Bashir et al, 1993, Craddock et al, 1993, Parfitt et al, 1994, Richard et al, 1994). We too previously reported a strong linkage of DAR to this same region of 12q in 10 affected families and have sublocalized DAR to a 5-cM region between microsatellite markers D12S84 and D12S354 (Ikeda et al, 1994). Subsequently, the area containing DAR was narrowed to a 3.8-cM interval between the microsatellite markers D12S105 and D12S129 (Carter et al, 1994). Studies of six new families affected with Darier disease (DD) using two-point lod score analysis of DAR in relation to polymorphic loci in 12q23–24.1 allowed us to further decrease the area containing the DAR gene to a 2-cM region. In addition, linkage studies with a polymorphism in the nitric oxide synthase gene (NOS1) (Twells et al, 1995) which also maps to 12q, excluded NOS1 as a candidate gene for DAR. Narrowing the gene’s location to a 1- to 2-cM region (1–2 million bp) will simplify the more detailed physically mapping of the region.
Affected individuals had characteristic lesions on the skin, and at least one member from each affected family had a histologically diagnostic skin biopsy. The six families are of Northern European ancestry: one from France (Bo), one from California (Sk), and four from Canada (Ca, Cb, Cc, and Cd).
DNA was extracted from peripheral blood leukocytes by standard techniques. Genotypes were determined by polyacrylamide gel electrophoresis of the products from polymerase chain reaction amplifications of genomic DNA at sites containing polymorphic simple sequence repeats (Gyapay et al, 1994, LeBlanc-Straceski et al, 1994). Two-point lod scores were calculated using LIPED or LINKAGE computer programs (Ott, 1992).
We studied six new DD-affected families using two-point lod score analysis of DAR in relation to polymorphic loci in the 12q23–24.1 region. These six families had positive lod scores for the marker D12S105 (Table I) similar to the 10 families that we originally studied (Ikeda et al, 1994). The Cd family had a single recombinant between DAR and D12S105, giving this family a negative lod score of −5.55 at θ = 0. This resulted in a combined two-point lod score of 26.42 at θ = 0.01 for all 16 families. The maximum lod score was 30.08 at θ = 0.0284 (SD = 0.0068).
Haplotype analysis of our families, using the microsatellite markers D12S105, D12S234, D12S129, and D12S354, has shown recombinants which further allow us to decrease the area containing DAR. In the Bo family, DAR is recombinant with the telomeric markers D12S129 and D12S354 (Fig 1a) in III-4, a 56-y-old unaffected female (examined by Y.S.). DAR also is recombinant with these same markers in an affected male in the previously reported Ba kindred (Ikeda et al, 1994) (data not shown). Figure 1b shows an affected male (III-3) in the Cd family, in whom DAR is recombinant with the centromeric markers D12S234 and D12S105. These results place DAR between D12S234 (cen) and D12S129 (tel).
Neuronal NOS1 has been localized by fluorescent in situ hybridization to the 12q24.2–24.31 region, and a trinucleotide repeat polymorphism has been described within an intronic region of the gene (Twells et al, 1995). We used this polymorphism to test whether NOS1 maps to the genetically delimited DAR region as a possible candidate gene for DAR. In using this polymorphism we have been able to localize NOS1 in relation to DAR and the microsatellite markers that flank DAR. As shown in Fig 2a, an affected male (III-7) in the As family (Ikeda et al, 1994) is recombinant between the more telomeric marker D12S79 and NOS1. Figure 2b shows the Be family (Ikeda et al, 1994), which contains an affected male (III-4) and an unaffected female (III-7); both have recombinants between NOS1 and D12S354. These recombinants place NOS1 between D12S354 (cen) and D12S79 (tel), telomeric to the D12S234–D12S129 interval containing DAR.
These findings further sublocalize DAR (Fig 3) and make physical mapping a more practical pursuit. In addition, our localization of DAR to 12q23–24.1 of an additional six families brings us to 37 as the total number of kindreds reported with this localization, suggesting that alterations in a single gene mapping to this region are responsible for DD. Haplotype analysis of our families with the NOS1 polymorphism allowed us to exclude NOS1 as a candidate gene for DD. Further studies will emphasize the construction of a contig for this region studies of new families and identification of epidermal-specific transcripts to identify the DD gene.
We thank Drs. Twells and Chamberlain for allowing us access to data on the NOS1 polymorphism before publication. We acknowledge the anonymous reviewer who kindly calculated the maximum lod score and its recombination fraction. Work supported in part by grant R01 AR 41700 from the USPHS.