[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.