Loss of chondroitin 6-O-sulfotransferase-1 function results in severe human chondrodysplasia with progressive spinal involvement

Abstract

We studied two large consanguineous families from Oman with a distinct form of spondyloepiphyseal dysplasia (SED Omani type). By using a genome-wide linkage approach, we were able to map the underlying gene to a 4.5-centimorgan interval on chromosome 10q23. We sequenced candidate genes from the region and identified a missense mutation in the chondroitin 6-O-sulfotransferase (C6ST-1) gene (CHST3) changing an arginine into a glutamine (R304Q) in the well conserved 3'-phosphoadenosine 5'-phosphosulfate binding site. C6ST-1 catalyzes the modifying step of chondroitin sulfate (CS) synthesis by transferring sulfate to the C-6 position of the N-acetylgalactosamine of chondroitin. From the crystal structures of other sulfotransferases, it could be inferred that Arg-304 is essential for the structure of the cosubstrate binding site. We used recombinant C6ST-1 to show that the identified missense mutation completely abolishes C6ST-1 activity. Disaccharide composition analysis of CS chains by anion-exchange HPLC shows that both Delta HexA-GalNAc(6S) and Delta HexA(2S)-GalNAc(6S) were significantly reduced in the patient's cells and that Delta HexA-GalNAc(4S,6S), undetectable in controls, was elevated. Analysis of the patient's urine shows marked undersulfation of CS, in particular reduction in 6-O-sulfated disaccharide and an increase in the nonsulfated unit. Our results indicate that the mutation in CHST3 described here causes a specific but generalized defect of CS chain sulfation resulting in chondrodysplasia with major involvement of the spine.

Fig. 1.

Haplotype analysis for family A and B at chromosome 10q23–24 are shown for the individuals where DNA was available. Eight microsatellite markers are shown in black, and the position of the CHST3 gene is shown in red. The order is from centromere to q-terminal using physical distances derived from National Center for Biotechnology Information (NCBI) assembly build 33. Circles denote females, squares denote males. Filled symbols indicate diagnosis of SED. Haplotypes are presented as gray and black bars. All affected people are homozygous for the affected (black) haplotype. The fragment from an ancestral founder haplotype remaining in both families is drawn as a dark yellow box. Position 911 from CHST3 gene, carrying the G → A mutation, is presented as trace sequence picture cutout under the haplotype from each individual. Patterns of gel electrophoresis after SmaI restriction enzyme digest from PCR fragments covering the SmaI site-destroying mutation are shown for family B.

Fig. 2.

CHST3 gene structure. Black filled boxes represent coding sequence. White boxes represent untranslated regions. The arrow denotes the missense mutation 911G → A resulting in an R304Q exchange. A multiple sequence alignment is shown below demonstrating the high conservation around R304. The conserved sequence is boxed.

Fig. 3.

Disaccharide composition analysis of CS chains produced by fibroblasts from a patient (4493) and a control subject. The GAG fraction prepared from fibroblasts of age-matched control 1 (A) or affected individual (B), respectively, was digested with chondroitinase ABC. Each digest was labeled with 2-aminobenzamide and analyzed by anion-exchange HPLC on an amine-bound silica PA-03 column (2). The elution positions of authentic 2-aminobenzamide-labeled disaccharide standards derived from CS are indicated by numbered arrows in A. 1, ΔHexA-GalNAc; 2, ΔHexA-GalNAc(6S); 3, ΔHexA-GalNAc(4S); 4, ΔHexA(2S)-GalNAc(6S); 5, ΔHexA-GalNAc(4S,6S).

Fig. 4.

Schematic representation of the PAPS-binding region of human C6ST-1 (model) (Upper) and human hydroxysteroid sulfotransferase (HST) (PDB ID code 1EFH) (Lower). Arg-142 (Lys-44) supposed to be involved in catalysis is shown in dark green; important residues in contact with PAPS [Phe-147 (Trp-49), Arg-301 (Arg-121), and Ser-309 (Ser-129)] are shown in light green, Arg-304 (Arg-124) (shown in blue) is converted to Gln (shown in light blue) in the mutant C6ST enzyme; residues surrounding Arg-304 (Arg-124) are shown in yellow [Asn-302 (Asp-122), Pro-303 (Pro-123), (Trp-254), (Phe-258), (Phe-266), and (Asp-267)]; PEPS is shown in red; secondary structure is shown in gray. Residue numbering for HST is given in parentheses.