Comprehensive next-generation sequencing analyses of hypoparathyroidism: identification of novel GCM2 mutations

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.

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.

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.

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.

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.