title: Functional validation of novel compound heterozygous variants in B3GAT3 resulting in severe osteopenia and fractures: expanding the disease phenotype.
abstract: 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.
  
  
  METHODS
  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.
authors.affiliation: Institute for Human Genetics and Molecular Biology, Martin Luther University Halle-Wittenberg, Magdeburger Str. 2, 06112, Halle (Saale), Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Job
authors.affiliation: Department of Pathobiochemistry, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya, Aichi, 468-8503, Japan.
authors.forename: Shuji
authors.initials: S
authors.lastname: Mizumoto
authors.affiliation: University of North Carolina School of Medicine, Division of Pediatric Genetics and Metabolism, Department of Pediatrics, Raleigh, NC, USA.
authors.forename: Laurie
authors.initials: L
authors.lastname: Smith
authors.affiliation: University of North Carolina School of Medicine, Division of Pediatric Genetics and Metabolism, Department of Pediatrics, Raleigh, NC, USA.
authors.forename: Natario
authors.initials: N
authors.lastname: Couser
authors.affiliation: Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, 45229, USA.
authors.forename: Ashley
authors.initials: A
authors.lastname: Brazil
authors.affiliation: Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, 45229, USA.
authors.forename: Howard
authors.initials: H
authors.lastname: Saal
authors.affiliation: Department of Pathology, Children's Mercy Hospitals and Clinics, Kansas City, MO, USA.
authors.forename: Melanie
authors.initials: M
authors.lastname: Patterson
authors.affiliation: Department of Pathology, Children's Mercy Hospitals and Clinics, Kansas City, MO, USA.
authors.forename: Margaret I
authors.initials: MI
authors.lastname: Gibson
authors.affiliation: Department of Pediatrics, Children's Mercy Hospitals and Clinics, Kansas City, MO, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Soden
authors.affiliation: Department of Medical Informatics, Children's Mercy Hospitals and Clinics, Kansas City, MO, USA.
authors.forename: Neil
authors.initials: N
authors.lastname: Miller
authors.affiliation: Department of Pathology, Children's Mercy Hospitals and Clinics, Kansas City, MO, USA.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Thiffault
authors.affiliation: Department of Pathology, Children's Mercy Hospitals and Clinics, Kansas City, MO, USA.
authors.forename: Carol
authors.initials: C
authors.lastname: Saunders
authors.affiliation: Department of Pathobiochemistry, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya, Aichi, 468-8503, Japan.
authors.forename: Shuhei
authors.initials: S
authors.lastname: Yamada
authors.affiliation: Institute for Human Genetics and Molecular Biology, Martin Luther University Halle-Wittenberg, Magdeburger Str. 2, 06112, Halle (Saale), Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Hoffmann
authors.affiliation: Department of Pathobiochemistry, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya, Aichi, 468-8503, Japan. k-sugar@sci.hokudai.ac.jp.|The Laboratory of Proteoglycan Signaling and Therapeutics, Graduate School of Life Science, Faculty of Advanced Life Science, Hokkaido University, Sapporo, 001-0021, Japan. k-sugar@sci.hokudai.ac.jp.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Sugahara
authors.affiliation: Department of Pediatrics, Children's Mercy Hospitals and Clinics, Kansas City, MO, USA. egfarrow@cmh.edu.|Center for Pediatric Genomic Medicine, Children's Mercy Hospitals and Clinics, 2420 Pershing, Suite 100, Kansas City, MO, USA. egfarrow@cmh.edu.
authors.forename: Emily
authors.identifier: http://orcid.org/0000-0002-8864-7073
authors.initials: E
authors.lastname: Farrow
mesh_terms: D001851:Bone Diseases, Metabolic / Q000235:genetics* / Q000473:pathology; D002675:Child, Preschool; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D050723:Fractures, Bone / Q000235:genetics*; D014644:Genetic Variation; D005838:Genotype; D014453:Glucuronosyltransferase / Q000235:genetics* / Q000378:metabolism; D006579:Heterozygote; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D020125:Mutation, Missense; D010375:Pedigree; D010641:Phenotype; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D004247:DNA; C000594393:B3GAT3 protein, human; D014453:Glucuronosyltransferase
keywords: B3GAT3; Glucuronyltransferase-I; Glycosaminoglycan; Linkeropathy; Proteoglycan