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

BACKGROUND: A 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 PRESENTATION: In 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). CONCLUSIONS: This 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.

Mutation in erythroid specific transcription factor KLF1 causes Hereditary Spherocytosis in the Nan hemolytic anemia mouse model.

KLF1 regulates definitive erythropoiesis of red blood cells by facilitating transcription through high affinity binding to CACCC elements within its erythroid specific target genes including those encoding erythrocyte membrane skeleton (EMS) proteins. Deficiencies of EMS proteins in humans lead to the hemolytic anemia Hereditary Spherocytosis (HS) which includes a subpopulation with no known genetic defect. Here we report that a mutation, E339D, in the second zinc finger domain of KLF1 is responsible for HS in the mouse model Nan. The causative nature of this mutation was verified with an allelic test cross between Nan/+ and heterozygous Klf1(+/-) knockout mice. Homology modeling predicted Nan KLF1 binds CACCC elements more tightly, suggesting that Nan KLF1 is a competitive inhibitor of wild-type KLF1. This is the first association of a KLF1 mutation with a disease state in adult mammals and also presents the possibility of being another causative gene for HS in humans.