Yang Wei, Hu Zhou-jun, Yu Xiao-fen, Li Qi-huan, Zhang Ai-ju, Deng Xi, Zhang Ai-ying, Gao Chun-sheng, Liu Yang, Ao Yang, Lo Wilson H-Y, Zhang Xue
Department of Medical Genetics & National Key Laboratory of Medical Molecular Biology, Peking Union Medical College, Beijing 100005, China.
Zhonghua Yi Xue Za Zhi. 2006 Mar 14;86(10):652-8.
OBJECTIVE: To identify the disease-causing genetic alteration of split-hand/split-foot malformation (SHFM) in a Chinese family. METHODS: Three of the 5 affected individuals from a four-generation Chinese SHFM family were examined physically and radiologically. Peripheral blood samples were collected from Digital photographs of the malformed hands and feet were taken. Peripheral blood samples were collected from 2 affected individuals, and lymphocytes were isolated to undergo high resolution G-banding. Genomic DNA was extracted from the whole blood samples of 4 available family members, including the 3 affected individuals. All 16 exons and their flanking intronic sequences of the TP63 gene were amplified using polymerase chain reaction (PCR) and sequenced directly. Microsatellite markers from the five SHFM loci were analyzed in the available family members by PCR, polyacrylamide gel electrophoresis and silver staining. For semi-quantitative determination of the allele copy number, the polymorphic PCR-amplified fragments representing genetic markers from the SHFM3 locus at chromosome 10q24.3 were sequenced in the affected individuals using normal individuals with identical genotypes as controls. RESULTS: All 3 existing affected individuals showed absence of 3 radial fingers, 2 affected individuals had a deep central cleft and central ray deficiency in the feet, and 1 affected individual had a fibular monodactyli, all limb malformations being bilateral and consistent with the phenotype of typical SHFM. G-banding showed normal karyotypes in the 3 affected individuals and no visible cytogenetic abnormality was found. Moreover, no mutation was identified in the TP63 gene. While no haplotype sharing was observed in the markers from loci SHFM1, SHFM4 and SHFM5, potential haplotype sharing was detected in the markers from two loci, SHFM2 and SHFM3, indicating possible causative mutation at SHFM2 or SHFM3. Furthermore, obviously biased silver density toward the allele fragments shared by the 3 affected individuals was observed in the markers from the SHFM3 locus. Comparative sequencing showed roughly one-fold increase of fluorescent signal of the shared fragments in the affected individuals. These results suggested a large-scale DNA duplication within the SHFM3 locus. CONCLUSION: A large-scale DNA duplication within the SHFM3 locus at chromosome 10q24.3 has been identified as the pathogenic genetic change in Chinese patients with SHFM.
目的：鉴定一个中国家庭中导致裂手/裂足畸形（SHFM）的致病基因改变。 方法：对一个四代中国SHFM家庭的5名受累个体中的3名进行体格检查和放射学检查。拍摄畸形手和足的数码照片。采集2名受累个体的外周血样本，分离淋巴细胞进行高分辨率G显带分析。从4名可获得的家庭成员（包括3名受累个体）的全血样本中提取基因组DNA。使用聚合酶链反应（PCR）扩增TP63基因的所有16个外显子及其侧翼内含子序列，并直接进行测序。通过PCR、聚丙烯酰胺凝胶电泳和银染分析可获得的家庭成员中来自5个SHFM位点的微卫星标记。为了半定量测定等位基因拷贝数，以基因型相同的正常个体为对照，对受累个体中代表10q24.3染色体上SHFM3位点遗传标记的多态性PCR扩增片段进行测序。 结果：所有3名现有的受累个体均表现为缺3根桡侧手指，2名受累个体足部有深部中央裂和中央射线缺如，1名受累个体有腓侧单指，所有肢体畸形均为双侧性，与典型SHFM的表型一致。G显带显示3名受累个体的核型正常，未发现可见的细胞遗传学异常。此外，在TP63基因中未鉴定到突变。虽然在SHFM1、SHFM4和SHFM5位点的标记中未观察到单倍型共享，但在SHFM2和SHFM3两个位点的标记中检测到潜在的单倍型共享，表明SHFM2或SHFM3可能存在致病突变。此外，在SHFM3位点的标记中观察到明显偏向于3名受累个体共享的等位基因片段的银密度。比较测序显示受累个体中共享片段的荧光信号大约增加了一倍。这些结果提示SHFM3位点存在大规模DNA重复。 结论：已确定10q24.3染色体上SHFM3位点的大规模DNA重复是中国SHFM患者的致病基因改变。
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