X-连锁迟发性脊椎骨骺发育不良的基因诊断研究

目的 探讨应用连锁分析和基因测序方法对X-连锁迟发性脊椎骨骺发育不良(Xinked spondylepiphyseal dysplasia tarda,SEDL)进行基因诊断的可行性。方法 利用与SEDL基因毗邻的微卫星遗传标记DXSl6多态性对一个SEDL大家系的21名成员进行连锁分析,确定与该家系SEDL致病基因连锁的DXSl6等位基因型,对家系中的年轻女性和年幼男性进行基因诊断。为探讨连锁分析诊断的准确性,随后对SEDL基因编码外显子3—6进行PCR扩增产物双向直接测序以确定导致该家系发病的SEDL基因突变型,通过检测致病基因对待诊对象进行直接诊断。结果 21名家系成员的连锁分析结果显示与致病基因连锁的DXSl6是D2等位基因,6位待诊对象中IVt4、IVl9、IV21、V7各有一个DXSl6D2等位基因,表明她们是携带者;而IV23和V4不具有与致病基因连锁的DXSl6等位基因,表明她(他)们是正常人。对21名成员的DNA测序证实该家系的致病突变是首次发现的SEDL基因第2内含子剪接受体处IVS2-2A→C突变。待诊对象IVl4、IVl9、IV21、V7携带该突变,IV23和V4基因型正常。基因测序和连锁分析的诊断结果完全一致。结论 连锁分析用于SEDL基因诊断简便、快速、花费少、与基因测序方法一样准确,解决了长期以来SEDL症状前患者和潜在女性携带者无法诊断的难题,有重要的临床价值。

Gene diagnosis of X-linked spondyloepiphyseal dysplasia tarda by linkage analysis and DNA sequencing

OBJECTIVE: X linked spondyloepiphyseal dysplasia tarda (SEDL) is heritable osteochondrondysplasia characterized in affected males by disproportional short stature with short neck and trunk resulting from a growth defect of the vertebral bodies, accompanied by barrel chest and degenerative osteoarthropathy of hip joints. This progressive skeletal dysplasia is caused by the SEDL gene located approximately 100 kb centromeric of DXS16 at Xp22. The disorder usually manifests in late childhood without systemic complications, and generally female carriers of SEDL are asymptomatic. So the diagnosis of potential carriers and presymptomatic patients is almost impossible. This study aimed to establish methods of gene diagnosis for finding out potential carriers and presymptomatic patients. METHODS: The blood samples were collected from 21 individuals in a large Chinese pedigree with SEDL. Microsatellite marker DXS16 was selected for linkage analysis. In order to confirm the allele of DXS16 linked to the pathogenic SEDL gene, polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (PAGE) were used to examine the variability of the lengths of DXS16, and linkage analysis was performed for the diagnosis of potential carriers and presymptomatic patients. Then the pathogenic mutation of the SEDL gene in the family was identified by bi-directionally direct sequencing of PCR products amplified for each of the four coding exons as well as their exon/intron boundaries. The potential carriers and presymptomatic patients were also diagnosed in this way. RESULTS: Six young individuals (IV(14), IV(19), IV(21), IV(23), V(4), V(7))who wanted to know whether they were carriers or presymptomatic patients were diagnosed by linkage analysis. Four females of them (IV(14), IV(19), IV(21), V(7)) were determined being carriers because they carry the allele of DXS16 which links the pathogenic SEDL gene, and the other two (IV(23), V(4)) being normal individuals for their alleles of DXS16 linked with wild SEDL gene. DNA sequencing identified that the pathogenic mutation of SEDL gene in the family, which was a nucleotide substitution of the splice-acceptor site in intron 2, IVS2 -2 A-->C. This is a novel mutation in the SEDL gene. Four female individuals (IV(14), IV(19), IV(21), V(7)) carried the mutation; individuals IV(23) and V(4) carried the wild SEDL gene. The results of diagnosis of linkage analysis coincide completely with that of DNA sequencing. CONCLUSION: Linkage analysis is a simple, rapid and inexpensive gene diagnosis method for SEDL and its accuracy was the same as DNA sequencing. Each of linkage analysis and DNA sequencing can be used to diagnose SEDL, which is very helpful for finding potential carriers and presymptomatic patients.