Functional validation of novel compound heterozygous variants in B3GAT3 resulting in severe osteopenia and fractures: expanding the disease phenotype

Abstract

BackgroundA new disease class of syndromes, described as linkeropathies, which are derived from defects in the glycosaminoglycan-linker region as well as glycosaminoglycan-side chains of proteoglycans is increasingly being recognized as a cause of human disease. Proteoglycans are an essential component of the extracellular matrix. Defects in the enzymatic process of proteoglycan synthesis broadly occur due to the incorrect addition of side chains. Previously, homozygous missense variants within the B3GAT3 gene encoding beta 1,3 glucuronyltransferase 3(GlcAT-I) responsible for the biosynthesis of glycosaminoglycans have been described in 7 individuals.Case presentationIn this study, a 4-year-old patient with a severe phenotype of osteoporosis, hypotonia, joint laxity, fractures, scoliosis, biscuspid aortic valve and myopia was referred for next generation sequencing after extensive negative clinical testing. Whole exome sequencing was performed on the proband and his unaffected parents to identify the molecular basis of his disease. Sequencing revealed compound heterozygous variants in B3GAT3: c.1A > G (p.Met1?) and c.671 T > A (p.L224Q). Clinical and in vitro functional studies were then completed to verify the pathogenicity of the genotype and further characterize the functional basis of the patient’s disease demonstrating the patient had a decrease both in the protein level of B3GAT3 and in the glucuronyltransferase activity when compared to control samples. Independent in vitro assessment of each variant confirmed the B3GAT3: c.1A > G (p.Met1?) variant is functionally null and the c.671 T > A (p.L224Q) missense variant has significantly reduced glucuronyltransferase activity (~3% of control).ConclusionsThis is the first report of a patient with compound heterozygosity for a null variant in trans with a missense in B3GAT3 resulting in a severe phenotype, expanding both the genotypic and phenotypic spectrum of B3GAT3-related disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12881-016-0344-9) contains supplementary material, which is available to authorized users.

B3GAT3-related disorder with craniosynostosis and bone fragility due to a unique mutation

“…Besides the observation of a presumed well-separated phenotype, a phenotypic continuum could be considered in light of the description of a 6-year-old boy 17 who presented hipjoint dislocation due to severe ligamentous laxity, osteoporosis, kyphoscoliosis, multiple fractures, and cardiac abnormalities (patent foramen ovale, bicuspid aortic valve, mild aortic root enlargement, and ascending aorta dilation). The patient had a compound mutation in B3GAT3 (c.1A > G, p.Met1?…”

Pseudodiastrophic dysplasia expands the known phenotypic spectrum of defects in proteoglycan biosynthesis

“…The impact of the B3GAT3 variants (Arg169Trp and Arg225*) on the function of B3GAT3/GlcAT-I in the biosynthesis of disaccharide units was investigated using cell-based ELISA (In-Cell ELISA). As described previously,22 CS and DS chains on the cell surface and extracellular matrix were compared between fibroblasts from patient 1A and control subjects. Briefly, fibroblasts were cultured in 96-well plates (5000 cells/well) for 1 day.…”

CSGALNACT1‐congenital disorder of glycosylation: A mild skeletal dysplasia with advanced bone age

“…Cell‐based ELISA was carried out as described (Job et al, ) to determine the relative number of CS/DS chains on core proteins. Briefly, fibroblasts from the patients and a control subject were cultured on 96‐well plates (5,000 cells/well) for a day, washed with PBS, and treated with chondroitinase ABC at 37°C for 30 min.…”

“…P2 at the age of 5 years showing marked disproportionate stature, macrocephaly, pectus excavatum, and skin laxity. (c) P3 at the age of 10 years with camptodactyly, mild skeletal anomalies and facial gestalt similar to P2 with short and downslanting palpebral fissures, midface hypoplasia, flat nasal bridge, prominent nasal tip, and dysplastic external ears 2.8 | Determination of the CS/DS chain number on core proteins by cell-based enzyme-linked immunosorbent assay (ELISA) Cell-based ELISA was carried out as described (Job et al, 2016) to determine the relative number of CS/DS chains on core proteins. Briefly, fibroblasts from the patients and a control subject were cultured on 96-well plates (5,000 cells/well) for a day, washed with PBS, and treated with chondroitinase ABC at 37°C for 30 min.…”