title: Comprehensive next-generation sequencing analyses of hypoparathyroidism: identification of novel GCM2 mutations.
abstract: BACKGROUND
  In most patients with hypoparathyroidism (HP), the etiology is not defined clinically. Eight genes (AIRE, CASR, CLDN16, GATA3, GCM2, PTH, TBCE, and TRPM6) are known to be responsible genes associated with HP; however, no previous study has screened the eight responsible genes comprehensively in HP patients.
  
  
  OBJECTIVE
  This study was conducted to determine the genetic defect in HP patients. We also described clinical and molecular findings of two HP patients with novel GCM2 mutations.
  
  
  METHODS
  We enrolled 20 nonconsanguineous Japanese patients with child-onset permanent HP without 22q11 deletion. Mutations and genomic rearrangements involving the eight genes were screened by targeted next-generation sequencing (NGS). We also screened genetic rearrangements by array comparative genomic hybridization (aCGH) in the mutation-negative patients. A putative deletion, which was suspected by NGS, was additionally analyzed by droplet digital PCR (ddPCR) and junction PCR. Identified novel nucleotide-level GCM2 mutants were characterized in vitro.
  
  
  RESULTS
  We identified seven patients with a single gene disorder, including a CASR mutation, GATA3 mutations, and novel GCM2 mutations (R367Tfs*15, T370M, and the deletion encompassing exon 1). This submicroscopic deletion, which had been suspected by NGS, could not be detected by aCGH and was confirmed by ddPCR and junction PCR. Functional studies of R367Tfs*- and T370M-GCM2 demonstrated a reduction of target gene transactivation in both.
  
  
  CONCLUSIONS
  Using comprehensive NGS analyses, we identified the genetic defect in 35% of HP patients in our cohort and discovered novel GCM2 mutations including submicroscopic deletion that was undetectable by aCGH.
authors.affiliation: Department of Pediatrics (T.M., S.N., M.I., T.H.), Keio University School of Medicine, Tokyo 160-8582, Japan; Division of Pediatrics (K.N.), Department of Homeostatic Regulation and Development, Niigata University Graduate School of Medicine and Dental Sciences, Niigata 951-8510, Japan; Department of Pediatrics (M.N.), Tokyo Metropolitan Ohtsuka Hospital, Tokyo 170-8476, Japan; and Department of Pediatrics (G.S.), Tokyo Dental College Ichikawa General Hospital, Chiba 272-8513, Japan.
authors.forename: Toshikatsu
authors.initials: T
authors.lastname: Mitsui
authors.forename: Satoshi
authors.initials: S
authors.lastname: Narumi
authors.forename: Mikako
authors.initials: M
authors.lastname: Inokuchi
authors.forename: Keisuke
authors.initials: K
authors.lastname: Nagasaki
authors.forename: Mie
authors.initials: M
authors.lastname: Nakazawa
authors.forename: Goro
authors.initials: G
authors.lastname: Sasaki
authors.forename: Tomonobu
authors.initials: T
authors.lastname: Hasegawa
mesh_terms: D000293:Adolescent; D044466:Asian People / Q000235:genetics; D002648:Child; D002675:Child, Preschool; D055028:Comparative Genomic Hybridization; D004252:DNA Mutational Analysis; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007011:Hypoparathyroidism / Q000235:genetics*; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*
chemical_list: C119532:GCM2 protein, human; D009687:Nuclear Proteins; D014157:Transcription Factors