The molecular basis of X-linked spondyloepiphyseal dysplasia tarda

Abstract

The X-linked form of spondyloepiphyseal dysplasia tarda (SEDL), a radiologically distinct skeletal dysplasia affecting the vertebrae and epiphyses, is caused by mutations in the SEDL gene. To characterize the molecular basis for SEDL, we have identified the spectrum of SEDL mutations in 30 of 36 unrelated cases of X-linked SEDL ascertained from different ethnic populations. Twenty-one different disease-associated mutations now have been identified throughout the SEDL gene. These include nonsense mutations in exons 4 and 5, missense mutations in exons 4 and 6, small (2-7 bp) and large (>1 kb) deletions, insertions, and putative splicing errors, with one splicing error due to a complex deletion/insertion mutation. Eight different frameshift mutations lead to a premature termination of translation and account for >43% (13/30) of SEDL cases, with half of these (7/13) being due to dinucleotide deletions. Altogether, deletions account for 57% (17/30) of all known SEDL mutations. Four recurrent mutations (IVS3+5G-->A, 157-158delAT, 191-192delTG, and 271-275delCAAGA) account for 43% (13/30) of confirmed SEDL cases. The results of haplotype analyses and the diverse ethnic origins of patients support recurrent mutations. Two patients with large deletions of SEDL exons were found, one with childhood onset of painful complications, the other relatively free of additional symptoms. However, we could not establish a clear genotype/phenotype correlation and therefore conclude that the complete unaltered SEDL-gene product is essential for normal bone growth. Molecular diagnosis can now be offered for presymptomatic testing of this disorder. Appropriate lifestyle decisions and, eventually, perhaps, specific SEDL therapies may ameliorate the prognosis of premature osteoarthritis and the need for hip arthroplasty.

Figure 1

Schematic representation of the spectrum of mutations of the SEDL gene. Solid boxes represent the SEDL ORF, hatched boxes represent the UTR of the gene. Twenty-one different mutations of all types were found to have pathogenic consequences in 30 unrelated SEDL cases. Asterisks (*) indicate recurrent mutation events.

Figure 2

RT-PCR analysis of the SEDL gene in patient 27 (insertion/deletion of intron 5) and patient 2 (deletion of exon 3). Total RNA from blood lymphocytes was reverse transcribed with Superscript II reverse transcriptase (see Subjects, Material, and Methods section) and was PCR amplified with SEDL primers. Schematic diagrams indicate the positions of the RT-PCR primers used, the SEDL mutations tested, and the occurrence of the alternative splicing of exon 2. “gDNA” denotes normal genomic DNA used as a control; plus (+) and minus (−) signs denote presence and absence, respectively, of reverse transcriptase during the reverse transcription step of the RT-PCR; the asterisk (*) denotes presence of heteroduplexes; and pUC/HpaII and SPPI/EcoRI are molecular-weight markers. A, Primers spanning SEDL exons 1–4. These primers amplify a 451-bp SEDL-gene product, when exon 2 is spliced in, or a 309-bp product, when exon 2 is spliced out. These two primers also amplify genomic DNA of the chromosome 19 (SEDLP1) pseudogene (309-bp product). This product was present in lymphocyte RNA from patient 27. B, Primers spanning exons 3–6 of SEDL. When these primers were used, no SEDL PCR product was amplified from patient 27's lymphocyte RNA, indicating alternative 3′ processing of the SEDL RNA, as a consequence of the insertion/deletion mutation. Esterase D (ESD) RT-PCR is used as a control of RNA quality. C, RT-PCR amplification using primers spanning exons 1–6 from patient 2's lymphocyte RNA. This patient carries a deletion of exon 3. Exon 3 is missing from its spliced SEDL transcript, demonstrated as a shift of 112 bp (exon 3 size) of both SEDL exon 2⁺ and exon 2⁻ isoforms.

Figure 3

Sequence chromatograms of two common mutations. Restriction digestion of exon 3 and exon 4 PCR products with TaiI detects half of the recurrent mutations, accounting for >56% of all SEDL cases. A, IVS3+5G→A mutation, which abolishes a TaiI site (underlined) so that the 265-bp exon 3 PCR product is resistant to cleavage into 197-bp and 68-bp fragments. B, 157–158delAT mutation in exon 4, which creates a TaiI site cleaving the 289-bp product into 103-bp and 184-bp fragments. Carriers can be identified. A = affected, C = carriers, and N = normal control.