Linkage analysis between microsatellite markers and cleft lip and/or palate in Japanese families

本研究采用参数化方法检测了候选基因BCL3及其附近19号染色体基因与日本家庭非综合征性唇裂伴或不伴腭裂（CL/P）的连锁关系。通过新泻大学口腔医院口腔颌面外科二科确定了9个多代CL/P家族。在获得知情同意后，从60个人中抽取血样，其中20人受到影响，并提取基因组dna。利用位于19q13.2的4个微卫星标记D19S178、BCL3、007/008和AC1/AC2进行pcr扩增，经8%聚丙烯酰胺凝胶电泳分离，银染色显示。此外，还分析了50例日本非亲属健康个体的4种标记的等位基因频率和杂合度值。使用linkage软件包中的MLINK程序进行两点连锁分析，并计算每个家庭的LOD分数，假设外显率降低的常染色体显性遗传模式。我们还测试了每个家庭中受影响个体的联系，包括表型信息。因此，在仅受影响的模型中，没有在四个位点检测到连锁的证据。假设受影响个体的外显率分别设置为0.8、0.6和0.3，则在D19S178、007/008和AC1/AC2位点两侧约1 cM内未检测到连锁的证据。另一方面，当外显率设置为0.999时，重组分数为0时，BCL3位点的最大LOD得分为0.206，说明候选基因的连锁不明确。

We examined linkage between candidate genes, BCL3 and nearby genes on chromosome 19, and non-syndromic cleft lip with or without cleft palate (CL/P) in Japanese families using parametric method. Nine multigenerational CL/P families were ascertained through the Second Department of Oral and Maxillofacial Surgery, Niigata University Dental Hospital. After informed consent was obtained, blood samples were drawn from 60 individuals, 20 of whom were affected, and genomic DNAs were extracted. PCR-amplified products using four microsatellite markers, D19S178, BCL3, 007/008 and AC1/AC2 located in 19q13.2, were separated by 8% polyacrylamide gel electrophoresis and visualized by silver staining. In addition, allele frequencies and heterozygosity values of four markers were analyzed for 50 healthy unrelative Japanese individuals. Two-point linkage analysis was carried out using the MLINK program of the LINKAGE package, and LOD scores were calculated for each family, assuming an autosomal dominant model of inheritance with reduced penetrance. We also tested for linkage including phenotypic information only for affected individuals in each family. Consequently, no evidence of linkage was detected at four loci in affected-only model. And assuming that penetrance for affected individuals was set alternatively at 0.8, 0.6, and 0.3, no evidence of linkage was detected within about 1 cM on both sides of D19S178, 007/008 and AC1/AC2 loci. On the other hand, the maximum LOD score for BCL3 locus was 0.206 at recombination fraction of 0 when penetrance was set at 0.999, this result indicated that linkage to candidate genes was not defined.