Insights in the etiopathology of galactosyltransferase II (GalT-II) deficiency from transcriptome-wide expression profiling of skin fibroblasts of two sisters with compound heterozygosity for two novel B3GALT6 mutations

Abstract

Mutations in B3GALT6, encoding the galactosyltransferase II (GalT-II) involved in the synthesis of the glycosaminoglycan (GAG) linkage region of proteoglycans (PGs), have recently been associated with a spectrum of connective tissue disorders, including spondyloepimetaphyseal dysplasia with joint laxity type 1 (SEMDJL1) and Ehlers–Danlos-like syndrome. Here, we report on two sisters compound heterozygous for two novel B3GALT6 mutations that presented with severe short stature and progressive kyphoscoliosis, joint hypermobility and laxity, hyperextensible skin, platyspondyly, short ilia, and elbow malalignment. Microarray-based transcriptome analysis revealed the differential expression of several genes encoding extracellular matrix (ECM) structural components, including COMP, SPP1, COL5A1, and COL15A1, enzymes involved in GAG synthesis and in ECM remodeling, such as CSGALNACT1, CHPF, LOXL3, and STEAP4, signaling transduction molecules of the TGFβ/BMP pathway, i.e., GDF6, GDF15, and BMPER, and transcription factors of the HOX and LIM families implicated in skeletal and limb development. Immunofluorescence analyses confirmed the down-regulated expression of some of these genes, in particular of the cartilage oligomeric matrix protein and osteopontin, encoded by COMP and SPP1, respectively, and showed the predominant reduction and disassembly of the heparan sulfate specific GAGs, as well as of the PG perlecan and type III and V collagens. The key role of GalT-II in GAG synthesis and the crucial biological functions of PGs are consistent with the perturbation of many physiological functions that are critical for the correct architecture and homeostasis of various connective tissues, including skin, bone, cartilage, tendons, and ligaments, and generates the wide phenotypic spectrum of GalT-II-deficient patients.

COMP-assisted collagen secretion - a novel intracellular function required for fibrosis

“…A microarray-based transcriptome analysis of skin fibroblasts from two individuals with mutations in this gene has revealed the pronounced downregulation of COMP, which is accompanied by alterations in the secretion and matrix assembly of collagen I and collagen III, eventually leading to hyperextensible skin (Ritelli et al, 2015). These changes in the biomechanical properties of the skin of such individuals are reminiscent of the softer skin that we observed in COMP-deficient mice.…”

The Ehlers–Danlos syndromes, rare types

“…Following the identification of B3GALT6 as the causal gene for SEMD‐JL1, Vorster et al [] identified the same mutations and a novel p.(Thr79Ala) mutations, all located in or in the vicinity of the stem region, in eight prototype South African families. A few additional patients with an EDS/SEMDJL1 overlap phenotype have also been reported [Sellars et al, ; Ritelli et al, ; Alazami et al, ].…”

“…The molecular basis of these observations remains to be established. In two patients harboring a compound deletion and catalytic domain mutation [Ritelli et al, ] showed by micro‐array transcriptome and immunofluorescence analyses a reduced expression of cartilage oligomeric matrix protein ( COMP ) and osteopontin ( SPP1 ). Interestingly, these authors reported reduced expression and disassembly of HS GAG chains and of the HS‐matrix PG perlecan.…”

A B3GALT6 variant in patient originally described as Al‐Gazali syndrome and implicating the endoplasmic reticulum quality control in the mechanism of some β3GalT6‐pathy mutations

“…To investigate the pathogenic effects of the c.618C > G, p.(Cys206Trp) variant, as well as 12 other selected missense pathogenic variants spanning the β3GalT6 protein (NP_542172.2) (Table ): c.16C > T, p.(Arg6Trp); c.193A > G, p.(Ser65Gly); c.200C > T, p.(Pro67Leu); c.466G > A, p.(Asp156Asn); c.694C > T, p.(Arg232Cys); c.899G > C, p.(Cys300Ser); c.925 T > A, p.(Ser309Thr); c.235A > G, p.(Thr79Ala); c.476C > A, p.(Ser159Tyr); c.795A > C, p.(Glu265Asp); c.556 T > C, p.(Phe186Leu) and c.766C > T, p.(Arg256Trp) . These variants were generated by site‐directed mutagenesis (Pfu UltraHF cat#600384‐51) using cMyc‐tagged B3GALT6 ‐pCMV6 plasmid (RC223942; OriGene, Rockville, MD) as template.…”

“…To investigate the pathogenic effects of the c.618C > G, p. (Arg256Trp). 16 These variants were generated by site-directed mutagenesis (Pfu UltraHF cat#600384-51) using cMyc-tagged B3GALT6-pCMV6 plasmid (RC223942; OriGene, Rockville, MD) as template. All primers were listed in Table S1, Supporting Information and generated variants were systematically confirmed by Sanger sequencing.…”