Mutational analysis of 12 patients with the phenotype of Ehlers–Danlos syndrome type VIB shows no linkage to the zinc transporter gene SLC39A13

The Ehlers–Danlos syndrome (EDS) encompasses a group of heterogeneous inherited connective tissue disorders in which patients are clinically characterized by fragile and hyperextensible skin and joint hypermobility. EDS has been divided into six distinct clinical subtypes [Beighton et al., 1998], one of which is the autosomal recessive kyphoscoliosis type (EDS VIA) (OMIM# 225400). EDS VIA is attributed to mutations in the lysyl hydroxylase 1 (LH1) gene (also known as PLOD1: procollagen-lysine, 2oxoglutarate 5-dioxygenase) [Yeowell and Walker, 2000], which codes for a post-translational modifying enzyme lysyl hydroxylase (LH) important in collagen biosynthesis [Yeowell and Pinnell, 1993; Steinmann et al., 2002]. Two other enzymes in the LH family include LH2 [Valtavaara et al., 1997], which exists in alternatively spliced forms, LH2 (short) and LH2 (long) [Yeowell and Walker, 1999], and LH3 [Passoja et al., 1998; Valtavaara et al., 1998]. A class of patients has been identified (EDS VIB) (OMIM# 229200) that has a similar clinical phenotype to EDS VIA but with normal LH activity. An earlier study on four EDS VIB patients showed no linkage to mutations in the genes for LH1, 2, and 3 [Walker et al., 2004a]. A second study on three LH2-deficient patients, with phenotypes ofmixed EDSVI and normal LHactivity, showed that their deficiency of LH2 was unrelated to mutations in either the coding or proximal promoter region of the LH2 gene [Walker et al., 2004b]. More recently, reports [Fukada et al., 2008; Giunta et al., 2008] have linked three families with EDS-like phenotypes to homozygous mutations in the zinc transporter gene (SLC39A13NM_152264.2). These included a homozygous 9 bp inframe deletion in exon 4 (c.483–491 del9) in two families [Giunta et al., 2008] and a homozygousG toA transition at nucleotide c.221 (c.221G>A) in exon 2 predicting the non-conservative amino acid substitution G74D in SLC39A13 in the third family [Fukada et al., 2008]. In addition to the typical EDS characteristics of hyperelastic, thin, bruisable skin, and hypermobile joints, the patients with the 9 bp deletion [Giunta et al., 2008] showed a generalized skeletal dysplasia and abnormalities of the hands, leading to the authors’ description of this disorder as the ‘‘spondylocheiro dysplastic form of EDS (SCD-EDS). ’’ Although the lysyl and prolyl collagen residues were underhydroxylated in these patients, their cultured skin fibroblasts showed normal enzyme activities. In vivo, this underhydroxylation could be attributed to an increased internal Zn2þ content which competed with Fe2þ, a cofactor required for lysyl and prolyl hydroxylation. In contrast, in the in vitro enzyme assay [Murad et al., 1985], addition of supplementary Fe2þ together with the other required cofactors resulted in normal LH and prolyl hydroxylase (PH) activities. Based on these results, we screened 12 cell lines in our cell repository from EDS VI-like patients, with normal LH activity measured in their cultured skin fibroblasts, for mutations in this gene. These included the fourEDSVIB cell lines previously reported in this journal [Walker et al., 2004a], the three EDSVI-like cell lines reported in a separate publication [Walker et al., 2004b], and five additional cell lines. The patients were selected on the basis of their general EDS VI-like clinical characteristics as described in Table I. Although not all of the clinical characteristics of this genetically