Defective initiation of glycosaminoglycan synthesis due to B3GALT6 mutations causes a pleiotropic Ehlers-Danlos-syndrome-like connective tissue disorder

Abstract

Proteoglycans are important components of cell plasma membranes and extracellular matrices of connective tissues. They consist of glycosaminoglycan chains attached to a core protein via a tetrasaccharide linkage, whereby the addition of the third residue is catalyzed by galactosyltransferase II (β3GalT6), encoded by B3GALT6. Homozygosity mapping and candidate gene sequence analysis in three independent families, presenting a severe autosomal-recessive connective tissue disorder characterized by skin fragility, delayed wound healing, joint hyperlaxity and contractures, muscle hypotonia, intellectual disability, and a spondyloepimetaphyseal dysplasia with bone fragility and severe kyphoscoliosis, identified biallelic B3GALT6 mutations, including homozygous missense mutations in family 1 (c.619G>C [p.Asp207His]) and family 3 (c.649G>A [p.Gly217Ser]) and compound heterozygous mutations in family 2 (c.323_344del [p.Ala108Glyfs(∗)163], c.619G>C [p.Asp207His]). The phenotype overlaps with several recessive Ehlers-Danlos variants and spondyloepimetaphyseal dysplasia with joint hyperlaxity. Affected individuals' fibroblasts exhibited a large decrease in ability to prime glycosaminoglycan synthesis together with impaired glycanation of the small chondroitin/dermatan sulfate proteoglycan decorin, confirming β3GalT6 loss of function. Dermal electron microcopy disclosed abnormalities in collagen fibril organization, in line with the important regulatory role of decorin in this process. A strong reduction in heparan sulfate level was also observed, indicating that β3GalT6 deficiency alters synthesis of both main types of glycosaminoglycans. In vitro wound healing assay revealed a significant delay in fibroblasts from two index individuals, pointing to a role for glycosaminoglycan defect in impaired wound repair in vivo. Our study emphasizes a crucial role for β3GalT6 in multiple major developmental and pathophysiological processes.

Figure 1

Schematic Illustration of Glycosaminoglycan Synthesis After synthesis in the endoplasmic reticulum (ER), the core protein is transported to the Golgi apparatus and simultaneously undergoes further modifications. First, a tetrasaccharide linker region is synthesized that originates from the addition of a xylose (Xyl) unit onto a serine residue of the core protein. Subsequent addition of two galactose (Gal) and one glucuronic acid (GlcA) residues completes this process. Depending on the glycosaminoglycan (GAG) chain type, disacharide units consisting of (N-acetylgalactosamine (GalNAc)-GlcA)_n or (N-acetylglucosamine (GlcNAc)-GlcA)_n are polymerized to form chondroitin sulfate (CS)/dermatan sulfate (DS)-PGs and heparan sulfate (HS)-PGs, respectively. The GAG chain is then further modified by epimerization and sulfation. Genes and the encoded enzymes are indicated in the figure. Disorders resulting from deficiency of the enzymes are indicated in black boxes. The deficient enzyme (β3GalT6) and the related disorder described in the text are depicted in red.

Figure 2

Clinical and Radiological Characteristics (A–D) P1 at age 7 years, illustrating (A) a hypotonic appearance, bruises on the lower limbs, and broad, flat feet; (B) skin hyperextensibility; and marked hyperlaxity of the finger joints (D) and broad distal phalanges (C). (E–H) P2 at age 26 years, illustrating (E) disproportionate short stature with short trunk, severe kyphoscoliosis and pectus deformity, and an elongated face with prominent chin; (F) thin and hyperextensible skin; (G) slender, tapered fingers with broad distal phalanges and finger contractures; and (H) broad, flat, and deformed feet and severe hallux valgus. (I–L) P3 at age 9 years, illustrating (I) severe hypotonia and low muscle mass, short trunk with pectus deformity and kyphoscoliosis, severely deformed lower limbs with contractures and club feet, and mild facial dysmorphism with low-set ears and prognathism; (J) small, yellowish teeth; (K, L) excessive wrinkling of the palms of hand and feet; and (K) spatulate fingers with broad distal phalanges. (M–P) P5 at age 20 months, illustrating (M) generalized hypotonia, low muscle mass, and pectus deformity; (M, N) a mild progeroid aspect of the face with shallow orbits, blue sclerae, proptosis, short nose, hypoplastic alae nasi, low nasal bridge, sparse hair, and bitemporal narrowing; (O) increased palmar wrinkling and broad distal phalanges; and (P) severe foot deformities. (Q–T) Radiographs of P2 at age 27 years, illustrating (Q) severe kyphosis, osteoporotic, wedged thoracic vertebral bodies; (R) severe prognathism; (S) flared iliac wings with increased trabecular patterning, coxa valga, and mild degenerative acetabular changes; and (T) osteoporosis of the femur and tibia with increased trabecular patterning. (U–X) Radiographs of P5 at age 2 years 3 months, illustrating (U) hypoplastic iliac bodies and poorly formed acetabulae; (U, V) osteoporotic aspect of the femur and tibia, diaphyseal narrowing, broadened metaphyses with abnormal trabecular patterning and epiphyseal changes; (W) severe kyphoscoliosis with ovoid vertebrae; and (X) mild bowing of humerus, radius, and ulna.

Figure 3

Identification and Analysis of B3GALT6 Mutations and Their Effect on B3GALT6 Expression (A) Pedigrees of families 1–3. Circles indicate females, squares indicate males. Symbols filled in black indicate affected family members with proven homozygous or compound heterozygous B3GALT6 mutations. Partially filled symbols indicate proven heterozygous carriers. Index persons are indicated by an arrow. Plus sign in the pedigree of family 1 indicates all members included in the homozygosity mapping. Electropherograms are included when available. (B) Representation of β3GalT6 protein structure with indication of the different domains and positions of the described mutations. Four conserved motifs (I–IV) are shown in orange and a predicted N-linked glycosylation site is highlighted in pink. (C) Clustal Omega protein sequence alignment showing part of the β3GalT6 galactosyltransferase domain including the substituted amino acid residues. The protein sequence is highly conserved across vertebrates, invertebrates, and plants, and Asp207 and Gly217 residues are evolutionary highly conserved (with the latter localized within a consensus motif). Asterisk (^∗) indicates a single, fully conserved residue; colon (:) indicates strong similar properties (>0.5 in the Gonnet PAM 250 matrix); and period (.) indicates weak similar properties (≤0.5 in the Gonnet PAM 250 matrix). Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence. At protein level, +1 corresponds to the methionine translator initiator. (D) qRT-PCR analysis of B3GALT6 in P2, P3, P5, and three control individuals shows reduced B3GALT6 expression in affected individuals’ samples. Total RNA was extracted with the RNeasy kit (QIAGEN), submitted to DNase Digestion (QIAGEN), and reverse-transcribed with the iScript cDNA Synthesis Kit (Bio-Rad Laboratories). All PCR reactions were carried out in duplicate. Relative expression was determined with the qbase^PLUS software (Biogazelle, v.2.4) with HPRT1 and YWHAZ as reference genes. Data are expressed as mean ± SEM; ^∗p < 0.05, ^∗∗p < 0.01 (Student’s t test).

Figure 4

Effect of B3GALT6 Mutations on β3GalT6 Activity and CS/DS and HS GAG Synthesis (A) B3GALT6 mutations affect priming of GAG synthesis. GAG synthesis rate was evaluated in cultured fibroblasts from affected individuals and control by radiolabeled sulfate incorporation from the exogenous xyloside 4-methylumbelliferyl-β-D-xylopyranoside (4-MUX, Sigma-Aldrich) as exogenous substrate as a function of time and concentration. Data are expressed as mean ± SEM; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 (Student’s t test). (B) Glycanation of decorin core protein is decreased in P2 and P5 fibroblast cultures compared to controls. Concentrated serum-free conditioned medium was collected at day 7, subjected to SDS-PAGE (4%–12% Bis-Tris gel, Life Technologies) and blotting, and immunolabeled with anti-human decorin antibody (Clone 115402, R&D Systems). The Precision Plus Protein All Blue Standard (Bio-Rad Laboratories) was used as molecular mass marker. Control samples show a broad band of decorin-PG around ∼75 kDa. In samples from affected individuals, this decorin-PG band is decreased in intensity, and a second band (∼50 kDa) is observed, corresponding to the decorin core protein devoid of its CS/DS chain. In P3 a third, low-molecular band (∼32 kDa), possibly a degradation product, is seen. (C) Confocal immunofluorescence microscopy of HS with 10E4 anti-HS monoclonal antibodies shows a clear pericellular staining of HS epitope in control human fibroblasts, in contrast to a reduced staining intensity for P2 and P5 and virtually no staining for P3 fibroblasts. (D) qRT-PCR analysis shows a strong reduction of DCN expression in all affected persons. A decreased expression of HSPG2 and LUM was observed in P3, whereas slight changes are noted for XYLT2 and B4GALT7 in some affected indivuals compared to controls. Experiments were performed in duplicate as described in Figure 3D. Data are expressed as mean ± SEM; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 (Student’s t test).

Figure 5

Effect of β3GalT6 Deficiency on Wound Closure and Collagen Fibril and Elastin Ultrastructure (A) Transmission electron microscopy shows a disturbed collagen organization and ECM architecture. A skin biopsy was taken from the upper anterior thigh of P5, fixed with 3% (w/v) glutaraldehyde in phosphate buffer (pH 7.4), and analyzed with a Zeiss EM900 equipped with a 1K SlowScan CCD camera (Tröndle). The observations were compared with control samples available from the skin biopsy bank of the EM-Lab at Dermatology, University Clinic Heidelberg. Collagen fibrils are loosely packed, with varying fibril diameters and occasional fibrils with very irregular contours (arrowhead). (B) Quantitative analysis of in vitro wound closure in dermal fibroblasts from β3GalT6-deficient persons shows a delayed wound closure for P2 and P3. Confluent cultures from control, P2, P3, and P5 were subjected to a wound healing assay. Wound closure was monitored 20 and 40 hr after wounding and recorded as number of migrated cells in the denuded area. Data are expressed as mean ± SEM; ^∗p < 0.05 (Student’s t test).