| X-linked spondyloepiphyseal dysplasia tarda (SEDL) is a genetically heterogeneous disorder caused by growth defect of vertebrae and epiphyses. The clinical features of this progressive skeletal dysplasia are disproportionate short stature with short neck and barrel-shaped chest which does not appear until late childhood. SEDL often involves the lumbar spine and the major proximal weight-bearing joints. The distinctive radiological changes in affected males are platyspondyly with hump-shaped central and posterior portions, narrow disc spaces, and degeneration of the hips. SEDL is lack of systemic complications and generally female carriers do not show obvious symptoms.SEDL was first described in 1939 by Jacobsen in a five-generation American family. In 1988, the SEDL gene was first mapped to the distal part of the short arm of the X chromosome. The localization was subsequently refined to an interval spanning approximately 2 Mb on Xp22.12-p22.31. The gene was ultimately identified in 1999. To explore the molecular pathogenesis of SEDL, different disease-associated mutations including nonsense mutation, missense mutations, small and large deletions, insertions, and some splicing errors have been identified in different ethnic populations.At present there have been only a few case reports about SEDL in Chinese. Because the disorder usually manifests in late childhood and appears absence of systemic complications, and generally female carriers of SEDL are not symptomatic, the diagnosis of potential carriers and presymptomatic patients can not be made. This study aimed to establish methods of gene diagnosis for potential carriers andpresymptomatic patients ; to identify the mutation of SEDL gene in a large Chinese family with X-linked spondyloepiphyseal dysplasia tarda ; to explore the effect of the mutation of the splice-acceptor site in intronl of SEDL gene on mRNA processing and to elucidate the molecular base of SEDL. Subjects and Methods21 participants are from a five-generational Chinese pedigree with 13 affected males and 11 obligate female carriers. The diagnosis of X-linked SEDL was established by clinical and radiological means as well as by the pattern of X-linked recessive inheritance.The blood samples were collected from 21 individuals in the large pedigree with SEDL from Chinese. Microsatellite marker DXS16 was selected for linkage analysis. In order to confirm the allele of DXS16 linked 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 the diagnosis of potential carriers and presymptomatic patients was performed by linkage analysis. Then the pathogenic mutation of the SEDL gene in the family was identified by bi-directionally direct sequencing of PCR products amplified each of the four coding exons as well as their exon/intron boundaries. The potential carriers and presymptomatic patients were also diagnosed by this way. Total RNA was isolated from EDTA blood samples of the patients ^ female carriers and normal controls, and RT-PCR was performed on total RNA. The sequence of cDNA were analyzed by bi-directionally direct sequencing of PCR products. The results of sequencing were compared against normal sequences to find the variations. Polyacrylamide gel electrophoresis (PAGE) was used to confirm them. ResultsSix young individuals (IY14, IVw, IV2i, IV23,V4, V7) who want to know whether they are carriers or presymptomatic patients were diagnosed by linkage analysis. Four females of them (IVu, IVw, IV2i, V7) were determined being carriers because they carry the allele of DXS16 which links the pathogenic SEDL gene , and the other two (IV23, V4) being normal individuals for their alleles of DXS16 link with wild SEDL gene . Direct sequencing of genomic DNA of the proband and V3 revealed an A→C transversion at the second base of the intron 2 acceptor splice site. No other sequence changes were detected in the SEDL ORF and flanking intronic sequences.Subsequently,... |
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