Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) deficiency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) of PTH through in vitro functional study. Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbutyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified. Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%-38%, and 51%-96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. In this study, two novel RVs of PTH (Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

Parathyroid hormone (PTH) gene mutations represent a rare cause of familial isolated hypoparathyroidism (FIH). These defects can cause hypoparathyroidism with increased or decreased serum levels of PTH through 1) impaired PTH synthesis; 2) induction of parathyroid cell apoptosis; or 3) secretion of bioinactive PTH molecules. Eight pathogenic mutations of this gene have been described previously. Through describing 2 novel mutations of the PTH gene, we aim to extend the molecular basis for FIH and further refine the proposed mechanisms by which PTH mutations cause hypoparathyroidism. Proband case reports were compiled with extended family analysis. The probands in both kindreds presented before age 10 days with hypocalcemia and elevated phosphate levels. Proband A had low PTH levels, whereas these levels were elevated in Proband B. Proband B was initially diagnosed with pseudohypoparathyroidism. Methylation analysis was performed of CpG dinucleotides within 3 GNAS differentially methylated regions; whole-genome sequencing; and PTH infusion with analysis of nephrogenous 3',5'-cyclic adenosine 5'-monophosphate. Proband A had a novel heterozygous sequence change in exon 2 of the PTH gene, c.46_47delinsAA (p.Ala16Lys), and proband B had a novel homozygous nucleotide transition in PTH exon 3 (c.128G > A; p.G43E) that led to replacement of glycine by glutamic acid at position 12 of PTH 1-84. PTH 1-34 infusion demonstrated that renal responsiveness to PTH was intact and not antagonized by circulating bioinactive PTH. PTH gene mutations are uncommon causes of hypoparathyroidism, but can be misdiagnosed as disorders of gland development or receptor function if PTH levels are decreased or elevated, respectively. Genetic testing should be considered early in the diagnostic approach to these presentations.

Familial isolated hypoparathyroidism (FIH) is a genetically heterogeneous disorder due to mutations of the calcium-sensing receptor (CASR), glial cells missing-2 (GCM2), guanine nucleotide binding protein α11 (GNA11), or parathyroid hormone (PTH) genes. Thus far, only four cases with homozygous and two cases with heterozygous mutations in the PTH gene have been reported. To clinically describe an FIH family and identify and characterize the causal gene mutation. Genomic DNA of the family members was subjected to CASR, GCM2, GNA11, and PTH gene mutational analysis. Functional assays were performed on the variant identified. Six subjects of a three-generation FIH family with three affected individuals having severe hypocalcemia and inappropriately low serum PTH. No mutations were detected in the CASR, GCM2, and GNA11 genes. A heterozygous variant that segregated with the disease was identified in PTH gene exon 2 (c.41T>A; p.M14K). This missense variant, in the hydrophobic core of the signal sequence, was predicted in silico to impair cleavage of preproPTH to proPTH. Functional assays in HEK293 cells demonstrated much greater retention intracellularly but impaired secretion into the medium of the M14K mutant relative to wild type. The addition of the pharmacological chaperone, 4-phenylbutyric acid, led to a reduction of cellular retention and increased accumulation in the cell medium of the M14K mutant. We report a heterozygous PTH mutation in an FIH family and demonstrate accumulation of the mutant intracellularly and its impaired secretion. An accurate genetic diagnosis in such hypoparathyroid patients is critical for appropriate treatment and genetic counseling.