Loss of dermatan-4-sulfotransferase 1 function results in adducted thumb-clubfoot syndrome

Abstract

Adducted thumb-clubfoot syndrome is an autosomal-recessive disorder characterized by typical facial appearance, wasted build, thin and translucent skin, congenital contractures of thumbs and feet, joint instability, facial clefting, and coagulopathy, as well as heart, kidney, or intestinal defects. We elucidated the molecular basis of the disease by using a SNP array-based genome-wide linkage approach that identified distinct homozygous nonsense and missense mutations in CHST14 in each of four consanguineous families with this disease. The CHST14 gene encodes N-acetylgalactosamine 4-O-sulfotransferase 1 (D4ST1), which catalyzes 4-O sulfation of N-acetylgalactosamine in the repeating iduronic acid-alpha1,3-N-acetylgalactosamine disaccharide sequence to form dermatan sulfate. Mass spectrometry of glycosaminoglycans from a patient's fibroblasts revealed absence of dermatan sulfate and excess of chondroitin sulfate, showing that 4-O sulfation by CHST14 is essential for dermatan sulfate formation in vivo. Our results indicate that adducted thumb-clubfoot syndrome is a disorder resulting from a defect specific to dermatan sulfate biosynthesis and emphasize roles for dermatan sulfate in human development and extracellular-matrix maintenance.

Figure 1

Biosynthesis of Dermatan Sulfate and Chondroitin Sulfate Chondroitin sulfate (CS), dermatan sulfate (DS), and also heparin sulfate share the synthesis of a tetrasaccharide linker region that attaches the glycosaminoglycan chains to a serine within the conserved attachment site of core proteins. The activity of a unique N-acetylgalactosaminyltransferase (GalNAcT-I) that transfers the first residue onto the tetrasaccharide linker starts a growing glycosaminoglycan chain to CS. This step is followed by the activities of specific enzymes that polymerize the glycosaminoglycan chain by the alternating addition of N-acetylgalactosamine (GalNAc) and glucoronic acid (GlcUA) moieties in CS. CS chains can be modified during elongation by a Golgi resident epimerase and a number of sulfotransferases. Epimerization of GlcUA to iduronic acid (IdoUA) by 5-hydroxyl epimerase (CS/DS5 epi) followed by sulfate addition to the C4 hydroxyl of the adjacent GalNAc residue by D4ST1 generates DS from CS and prevents back-epimerization of IdoUA to GlcUA.

Figure 2

Clinical Features of ATCS Left column: Austrian patient from family 1, homozygous for CHST14 mutation p.R213P at age 8 years; clinical findings were previously reported as case 2 by Janecke et al. Middle column: Turkish patient from family 3, homozygous for p.V49X at age 15 years; clinical findings were previously reported as case 2 by Dundar et al. Right column: 6-year-old sister of previous patient. Note the wasted build with weights below the 3rd centile and heights between the 25th to 50th centiles in all patients. The skin is translucent with readily visible venous pattern over the chest, abdomen, and extremities. Note long and tapering fingers, excessively wrinkled palms, and clubfeet (repaired in the Austrian patient).

Figure 3

Radiographs Showing Generalized, Mild to Moderate Osteopenia in All Examined ATCS Patients (A) Flat foot with plantar flexion of the talus. Rarefication of bone with reduced and coarse trabeculation, as well as thinning of the cortex, is shown. (B–F) Slender shafts of tubular bones with thin cortex and poorly trabeculated spongiosa. (A) and (B) show Austrian patient (family 1) homozygous for p.R213P at age 8 years; (C) and (D) show Turkish patient (family 3) homozygous for p.V49X at age 15 years; (E) and (F) show 6-year-old sister of previous patient).

Figure 4

Pedigrees and Linkage and Mutation Analyses of ATCS Families (A) The graph represents a parametric LOD score on the y axis in relation to genetic position on the x axis. Human chromosomes are concatenated from p-ter (left) to q-ter (right) on the x axis, and the genetic distance is given in cM. This scan identified a single region of extended homozygosity shared by all four affected individuals included in the genome scan, who are identified in (B), defining a critical interval of 3.76 Mb on chromosome 15q15. (B) The pedigrees of the four ATCS families included in this study are shown and the corresponding CHST14 mutations are shown on the right-hand side. A total of 15 subjects from families 1–3, identified by the & symbol, were included in the genome scan to map the disease. Family 1 originates from Austria and clinical findings were initially reported in 2001. The Turkish families 2 and 3 were first reported in 2001 and 1997, and detailed clinical findings of family 4 from Japan were reported in 2000.

Figure 5

Consequences of D4ST1 Mutations (A) Comparative analysis of D4ST1 orthologs and HNK-1 family members by ClustalW alignment shows a high degree of conservation of residues mutated in ATCS. (B) Analysis of epitope-tagged D4ST1 by SDS-PAGE. HEK293/T cells were transfected with pcDNA3.2/V5 (Invitrogen) expressing D4ST1 wild-type, p.[R135G; L137Q], p.R213P, pV49X, or p.Y293C. Cells were washed and dissolved in TPER. Samples that were or were not treated with N-glycanase prior to separation by SDS-PAGE are indicated by + and −, respectively. One percent of the cell extract (upper panel) and 2% and 5% of the concentrated medium (lower panel) was analyzed for each construct. The apparent molecular weight standards are indicated by arrows. Wild-type D4ST1 migrates as a major species with a Mr of 55 kD and less prominent species with a Mr of 44 kD that arises by proteolytic cleavage in the region between the transmembrane domain and the glycosylated asparagine located at position 110.

Figure 6

Light and Electron Microscopy of a Skin Biopsy from the Austrian ATCS Patient Homozygous for CHST14 p.R213P Normal light microscopy (LM) appearance of the skin using (A) H&E, (B) Elastica, and (C) PAS staining. (D) EM shows collagen fibrils, which are normally packed, round, and have a normal diameter and contour, as well as normal cross-sections of elastin fibers and collagen fibrils. Elastin contains normal microfibrillar material.

Figure 7

Defective Dermatan Sulfation and Increased CS Biosynthesis Cultured fibroblasts from a patient homozygous for D4ST1 mutation p.R213P detected by ³⁴S-sulfate metabolic labeling and capillary HPLC/MS analysis of CS-specific, DS-specific, and CS and DS-specific monosulfated (A) and nonsulfated (B) disaccharides. (A) Monosulfated disaccharides from cell extracts and media of patient (m/z 540) and normal fibroblasts (m/z 535) coeluted at 19 min. No DS-derived IdoUA-GalNAc4S disaccharide was detected by GRIL capillary HPLC-coupled MS disaccharide analysis of cell extracts from patient fibroblasts. In addition, GlcUA-GalNAc4S was greatly increased in glycosaminoglycans obtained from cell extracts and media of patient as compared with control fibroblasts. (B) Nonsulfated disaccharides from patient (m/z 460) and normal fibroblasts (m/z 455) eluted at 16 min after chondroitinase A, B, and ABC digestion of their glycosaminoglycans. The amount of GlcUA-GalNAc and IdoUA-GalNAc is increased in the cell extract and the medium from patient fibroblasts as compared to normal fibroblasts.