www.nature.com/scientificreports OPEN Five patients with disorders of calcium metabolism presented with GCM2 gene variants Alejandro García‑Castaño1,11*, Leire Madariaga2,11, Sara Gómez‑Conde3, Carmen Lourdes Rey Cordo4, María López‑Iglesias5, Yolanda Garcia‑Fernández6, Alicia Martín7, Pedro González7, Ignacio Goicolea7, Gustavo Pérez de Nanclares8, Ana Belén De la Hoz9, Aníbal Aguayo8, Idoia Martínez de LaPiscina1, Rosa Martínez1, Laura Saso3, Inés Urrutia1, Olaia Velasco3, Luis Castaño10 & Sonia Gaztambide10 The GCM2 gene encodes a transcription factor predominantly expressed in parathyroid cells that is known to be critical for development, proliferation and maintenance of the parathyroid cells. A cohort of 127 Spanish patients with a disorder of calcium metabolism were screened for mutations by Next‑ Generation Sequencing (NGS). A targeted panel for disorders of calcium and phosphorus metabolism was designed to include 65 genes associated with these disorders. We observed two variants of uncertain signifcance (p.(Ser487Phe) and p.Asn315Asp), one likely pathogenic (p.Val382Met) and one benign variant (p.Ala393_Gln395dup) in the GCM2 gene in the heterozygous state in fve families (two index cases had hypocalcemia and hypoparathyroidism, respectively, and three index cases had primary hyperparathyroidism). Our study shows the utility of NGS in unravelling the genetic origin of some disorders of the calcium and phosphorus metabolism, and confrms the GCM2 gene as an important element for the maintenance of calcium homeostasis. Importantly, a novel variant in the GCM2 gene (p.(Ser487Phe)) has been found in a patient with hypocalcemia. Calcium (Ca2+) is required for many physiological functions (muscle contraction, nerve conduction, hormone release, mineralization of bone, and blood coagulation). Ca2+ metabolism maintains a dynamic balance between intestinal absorption, exchange with the bone, and renal excretion. Parathyroid hormone (PTH), vitamin D and calcitonin regulate this balance by acting on their targets: intestine, bone and renal tubule. PTH is secreted by the parathyroid glands. Te GCM2 gene (glial cells missing transcription factor 2, MIM *603716) encodes a transcription factor predominantly expressed in parathyroid cells 1,2. Tis transcription factor is known to be critical for development of the parathyroid cells and plays a critical role in adult parathyroid cell proliferation and maintenance3,4. Tus, it has been demonstrated that a GCM2-defcient mouse lacked parathyroid glands 5. Te GCM2 gene was mapped to chromosome 6p24.22 and encodes the chorion-specifc transcription factor GCMb. Te full-length protein (506 amino acids) contains a N-terminal Zn-containing DNA binding domain (amino acids from 19 to 174), a nuclear localization sequence (amino acids from 176 to 191), a C-terminal conserved inhibitory domain (CCID, amino acids from 379 to 395), and two transcriptional activation domains in the C-terminal region: TAD1 (amino acids from 175 to 263) and TAD2 (amino acids from 428 to 506)2,6–8. Loss-of-function mutations in the GCM2 gene have recently been described in humans as a rare cause of autosomal dominant or recessive familial isolated type 2 hypoparathyroidism (MIM #618883)6. Patients usually 1Biocruces Bizkaia Health Research Institute, CIBERDEM, CIBERER, Plaza de Cruces, Barakaldo, 48903 Bizkaia, Spain. 2Biocruces Bizkaia Health Research Institute, CIBERDEM, CIBERER, Pediatric Nephrology Department, Hospital Universitario Cruces, University of the Basque Country (UPV/EHU), Bizkaia, Spain. 3Biocruces Bizkaia Health Research Institute, Bizkaia, Spain. 4Pediatric Endocrinology Department, Hospital Álvaro Cunqueiro, EOXI, Vigo, Spain. 5Endocrinology Department, Hospital General La Mancha Centro, Ciudad Real, Spain. 6Endocrinology and Nutrition Department, OSI Barrualde-Galdakao, Bizkaia, Spain. 7Biocruces Bizkaia Health Research Institute, Endocrinology and Nutrition Department, Hospital Universitario Cruces, Bizkaia, Spain. 8Biocruces Bizkaia Health Research Institute, CIBERDEM, CIBERER, Hospital Universitario Cruces, Bizkaia, Spain. 9Biocruces Bizkaia Health Research Institute, CIBERER, Bizkaia, Spain. 10Biocruces Bizkaia Health Research Institute, CIBERDEM, CIBERER, Endocrinology and Nutrition Department, Hospital Universitario Cruces, University of the Basque Country (UPV/EHU), Bizkaia, Spain. 11These authors contributed equally: Alejandro García-Castaño and Leire Madariaga. *email: [email protected] Scientifc Reports | (2021) 11:2968 | https://doi.org/10.1038/s41598-021-82661-y 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1. Pedigree of 5 families with variants in the GCM2 gene. Te novel variant is marked in bold. Index case are indicated by the arrows. Squares denote male family members, circles female family members, and solid symbols symptomatic subjects. develop seizures due to the hypocalcemia, in early life, with hyperphosphatemia, low to undetectable serum PTH levels and normal levels of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. On the other hand, gain-of- function mutations in the GCM2 gene cause autosomal dominant hyperparathyroidism type 4 (MIM #617343)9, a disorder characterized by hypercalcemia and elevated or inappropriate PTH secretion by parathyroid glands. In the present study, we report a novel variant of uncertain signifcance in the GCM2 gene in a family from Spain with severe hypocalcemia. Moreover, we report three previously described GCM2 gene variants, one likely pathogenic, one of uncertain signifcance and one benign, in four families from Spain presenting with diferent disorders of calcium metabolism. Materials and methods Ethics statement. Te study was approved by the Ethics Committee for Clinical Research of Euskadi (CEIC-E). Patients and their participating relatives provided written informed consent for the genetic study. Te research was carried out in accordance with the Declaration of Helsinki on human experimentation of the World Medical Association. Patients. A total of 65 genes whose mutations are a recognized cause of calcium and phosphorus metabo- lism disorders were tested by a Next-Generation Sequencing (NGS) panel in a cohort of 127 Spanish patients (50 had hypocalciuric hypercalcemia, 44 were diagnosed of primary hyperparathyroidism, 13 presented with hypocalcemia and/or hypoparathyroidism, 12 were diagnosed of pseudohypoparathyroidism and 8 had rickets). Clinical diagnoses were made by adult and pediatric endocrinologists. In all cases, the molecular analysis was done in the Molecular Genetic Laboratory at Biocruces Bizkaia Health Research Institute, Barakaldo, Spain. From the whole cohort, fve index cases with a suspected disorder of calcium metabolism (GS0198, CA0117, ME0292, ME0371 and CA0103) presented with GCM2 gene variants (Fig. 1). Index case GS0198 was a 7-month-old child who was referred for evaluation of irritability and refusal to eat. He had sufered three episodes of seizures accompanied by rapid and involuntary muscle contractions. Labora- tory results showed low serum Ca 2+ (less than 5 mg/dL, reference range 8.5–10.4) and 25-hydroxyvitamin D (7.49 ng/mL, reference range 9–47), and high serum phosphate (11.7 mg/dL, reference range 2.6–4.8), whereas serum intact PTH levels (54 pg/mL, reference range 10–65) were within the normal range. In addition, he pre- sented with moderate hypomagnesemia (1.39 mg/dL, reference range 1.7–2.5). While the mother had normal calcium and phosphate metabolism, proband’s father had small stature (1.57 cm) and body segment dispar- ity (short lower limbs). He had normal levels of serum Ca 2+ (9 mg/dL), phosphate (3.18 mg/dL), magnesium (1.94 mg/dL), intact PTH (34.38 pg/mL) and 25-hydroxyvitamin D (30.4 ng/mL) (Fig. 1a). Index case CA0117 was a 65-year-old male who had hypocalcemia and hypoparathyroidism. He had osteoar- thritis and sufered tingling of fngers and toes. Moreover, he was diagnosed with glaucoma. Laboratory results showed low serum Ca2+ (5.4 mg/dL) and serum intact PTH (5.8 pg/mL), whereas 25-hydroxyvitamin D (24 ng/ Scientifc Reports | (2021) 11:2968 | https://doi.org/10.1038/s41598-021-82661-y 2 Vol:.(1234567890) www.nature.com/scientificreports/ mL) and serum phosphate (4.3 mg/dL) were within the normal range. He was treated with calcium and vitamin D supplements. Regarding family history, there was no history of hypoparathyroidism or hypocalcemia (Fig. 1b). Index case ME0292 was a 67-year-old male presenting with elevated serum intact PTH levels, hypophos- phatemia, normocalcemia with vitamin D defciency, hypercalciuria, and nephrolithiasis. Laboratory evaluation showed normal serum calcium (10.2 mg/dL), high intact PTH (95.6 pg/mL), low serum phosphate (2 mg/dL), and 25-hydroxyvitamin D levels of 11 ng/mL. In addition, he exhibited high urinary calcium excretion (386 mg/24 h, reference range in adult male < 300 mg/24 h). Furthermore, index case ME0292 had a personal history of ankylos- ing spondylitis, prostate gland enlargement, hepatic steatosis, and hyperlipidemia. He was diagnosed of primary hyperparathyroidism and parathyroid glands surgery was performed. Parathyroid hyperplasia of two superior glands was verifed histologically and both were removed. However, afer the surgical intervention, he contin- ued with high intact PTH levels (113 pg/mL). He had a 61 year-old sister who had hyperparathyroidism (intact PTH 103 pg/mL) and nephrolithiasis as well (Fig. 1c). She had normal serum Ca2+ (9.4 mg/dL), normal serum phosphate (3.1 mg/dL) and exhibited slightly