Michael R. Bowl

Characterization of GATA3 Mutations in the Hypoparathyroidism, Deafness, and Renal Dysplasia (HDR) Syndrome

The hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is an autosomal dominant disorder caused by mutations of the dual zinc finger transcription factor, GATA3. The C-terminal zinc finger (ZnF2) binds DNA, whereas the N-terminal finger (ZnF1) stabilizes this DNA binding and interacts with other zinc finger proteins, such as the Friends of GATA (FOG). We have investigated seven HDR probands and their families for GATA3 abnormalities and have identified two nonsense mutations (Glu-228 → Stop and Arg-367 → Stop); two intragenic deletions that result in frameshifts from codons 201 and 355 with premature terminations at codons 205 and 370, respectively; one acceptor splice site mutation that leads to a frameshift from codon 351 and a premature termination at codon 367; and two missense mutations (Cys-318 → Arg and Asn-320 → Lys). The functional effects of these mutations, together with a previously reported GATA3 ZnF1 mutation and seven other engineered ZnF1 mutations, were assessed by electrophoretic mobility shift, dissociation, yeast two-hybrid and glutathione S-transferase pull-down assays. Mutations involving GATA3 ZnF2 or adjacent basic amino acids resulted in a loss of DNA binding, but those of ZnF1 either lead to a loss of interaction with specific FOG2 ZnFs or altered DNA-binding affinity. These findings are consistent with the proposed three-dimensional model of ZnF1, which has separate DNA and protein binding surfaces. Thus, our results, which expand the spectrum of HDR-associated GATA3 mutations and report the first acceptor splice site mutation, help to elucidate the molecular mechanisms that alter the function of this zinc finger transcription factor and its role in causing this developmental anomaly.

An interstitial deletion-insertion involving chromosomes 2p25.3 and Xq27.1, near SOX3 , causes X-linked recessive hypoparathyroidism

X-linked recessive hypoparathyroidism, due to parathyroid agenesis, has been mapped to a 906-kb region on Xq27 that contains 3 genes (ATP11C, U7snRNA, and SOX3), and analyses have not revealed mutations. We therefore characterized this region by combined analysis of single nucleotide polymorphisms and sequence-tagged sites. This identified a 23- to 25-kb deletion, which did not contain genes. However, DNA fiber-FISH and pulsed-field gel electrophoresis revealed an approximately 340-kb insertion that replaced the deleted fragment. Use of flow-sorted X chromosome-specific libraries and DNA sequence analyses revealed that the telomeric and centromeric breakpoints on X were, respectively, approximately 67 kb downstream of SOX3 and within a repetitive sequence. Use of a monochromosomal somatic cell hybrid panel and metaphase-FISH mapping demonstrated that the insertion originated from 2p25 and contained a segment of the SNTG2 gene that lacked an open reading frame. However, the deletion-insertion [del(X)(q27.1) inv ins (X;2)(q27.1;p25.3)], which represents a novel abnormality causing hypoparathyroidism, could result in a position effect on SOX3 expression. Indeed, SOX3 expression was demonstrated, by in situ hybridization, in the developing parathyroid tissue of mouse embryos between 10.5 and 15.5 days post coitum. Thus, our results indicate a likely new role for SOX3 in the embryonic development of the parathyroid glands.

Identification of 70 calcium-sensing receptor mutations in hyper- and hypo-calcaemic patients: evidence for clustering of extracellular domain mutations at calcium-binding sites

The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that has an extracellular bilobed venus flytrap domain (VFTD) predicted to contain five calcium (Ca(2+))-binding sites. To elucidate the structure-function relationships of the VFTD, we investigated 294 unrelated probands with familial hypocalciuric hypercalcaemia (FHH), neonatal severe primary hyperparathyroidism (NSHPT) or autosomal dominant hypocalcaemic hypercalciuria (ADHH) for CaSR mutations and performed in vitro functional expression studies and three-dimensional modelling of mutations involving the VFTD. A total of 70 different CaSR mutations were identified: 35 in FHH, 10 in NSHPT and 25 in ADHH patients. Furthermore, a CaSR variant (Glu250Lys) was identified in FHH and ADHH probands and demonstrated to represent a functionally neutral polymorphism. NSHPT was associated with a large proportion of truncating CaSR mutations that occurred in the homozygous or compound heterozygous state. Thirty-four VFTD missense mutations were identified, and 18 mutations were located within 10 Å of one or more of the predicted Ca(2+)-binding sites, particularly at the VFTD cleft, which is the principal site of Ca(2+) binding. Mutations of residues 173 and 221, which are located at the entrance to the VFTD cleft binding site, were associated with both receptor activation (Leu173Phe and Pro221Leu) and inactivation (Leu173Pro and Pro221Gln), thereby highlighting the importance of these residues for entry and binding of Ca(2+) by the CaSR. Thus, these studies of disease-associated CaSR mutations have further elucidated the role of the VFTD cleft region in Ca(2+) binding and the function of the CaSR.

Cell division cycle protein 73 homolog (CDC73) mutations in the hyperparathyroidism-jaw tumor syndrome (HPT-JT) and parathyroid tumors†

The hyperparathyroidism‐jaw tumor (HPT‐JT) syndrome is an autosomal dominant disorder characterized by the occurrence of parathyroid tumors in association with ossifying fibromas of the maxilla and/or mandible. The gene responsible for HPT‐JT, known as CDC73, was identified in 2002 and encodes a 531 amino acid protein known as parafibromin. Parafibromin is predominantly a nuclear protein that interacts directly with β‐catenin and also forms part of the RNA polymerase associated factor‐1 complex (Paf1C) that regulates transcription. Heterozygous germline CDC73 mutations are detected in the majority of patients with HPT‐JT, and the demonstration of loss of heterozygosity (LOH) at the CDC73 locus in tumors from affected individuals is consistent with a tumor suppressor role. Somatic CDC73 mutations are a frequent finding in nonfamilial (i.e., sporadic) parathyroid carcinomas and have also been reported in benign sporadic parathyroid tumors as well as sporadic renal and fibro‐osseous jaw tumors. To date, 111 independent CDC73 mutations have been identified (68 germline; 38 somatic; 5 undefined), and these occur throughout the coding region and splice sites of the CDC73 gene, with the majority (>80%) predicting premature truncation of the parafibromin protein. These CDC73 mutations, together with their clinical and biological relevance, are reviewed. Hum Mutat 31:295–307, 2010.

Parafibromin mutations in hereditary hyperparathyroidism syndromes and parathyroid tumours

Objective  To investigate two patients with the hyperparathyroidism‐jaw tumour (HPT‐JT) syndrome and three patients with familial isolated hyperparathyroidism (FIHP), together with 31 parathyroid tumours (2 HPT‐JT, 2 FIHP and 27 sporadic) for HRPT2 mutations. The HPT‐JT syndrome and FIHP are autosomal dominant disorders that may be caused by abnormalities of the HRPT2 gene, located on chromosome 1q31.2. HRPT2 encodes a 531 amino acid protein, parafibromin, which interacts with human homologues of the yeast Paf1 complex.

Parafibromin, a Component of the Human PAF Complex, Regulates Growth Factors and Is Required for Embryonic Development and Survival in Adult Mice

ABSTRACT Parafibromin, a transcription factor associated with the PAF complex, is encoded by the HRPT2 gene, mutations of which cause the hyperparathyroidism-jaw tumor syndrome (OMIM145001). To elucidate the function of parafibromin, we generated conventional and conditional Hrpt2 knockout mice and found that Hrpt2−/− mice were embryonic lethal by embryonic day 6.5 (E6.5). Controlled deletion of Hrpt2 after E8.5 resulted in apoptosis and growth retardation. Deletion of Hrpt2 in adult mice led to severe cachexia and death within 20 days. To explore the mechanism underlying the embryonic lethality and death of adult mice, mouse embryonic fibroblasts (MEFs) were cultured and Hrpt2 was deleted in vitro. Hrpt2−/− MEFs underwent apoptosis, while Hrpt2+/+ and Hrpt2+/− MEFs grew normally. To study the mechanism of this apoptosis, Hrpt2+/+ and Hrpt2−/− MEFs were used in cDNA microarray, semiquantitative reverse transcription-PCR, and chromatin immunoprecipitation assays to identify genes regulated by parafibromin. These revealed that Hrpt2 expression and the parafibromin/PAF complex directly regulate genes involved in cell growth and survival, including H19, Igf1, Igf2, Igfbp4, Hmga1, Hmga2, and Hmgcs2. Thus, our results show that expression of Hrpt2 and parafibromin is pivotal in mammalian development and survival in adults and that these functions are likely mediated by the transcriptional regulation of growth factors.

Asymptomatic Children with Multiple Endocrine Neoplasia Type 1 Mutations May Harbor Nonfunctioning Pancreatic Neuroendocrine Tumors

CONTEXT Multiple endocrine neoplasia type 1 (MEN1) is characterized by the occurrence of parathyroid, pituitary, and pancreatic tumors. MEN1, an autosomal dominant disorder, has a high degree of penetrance, such that more than 95% of patients develop clinical manifestations by the fifth decade, although this is lower at approximately 50% by age 20 yr. However, the lower penetrance in the younger group, which is based on detecting hormone-secreting tumors, may be an underestimate because patients may have nonfunctioning tumors and be asymptomatic. OBJECTIVE The aim of the study was to evaluate the occurrence of nonfunctioning pancreatic neuroendocrine tumors in asymptomatic children with MEN1. PATIENTS Twelve asymptomatic Northern European children, aged 6 to 16 yr, who were known to have MEN1 mutations were studied. RESULTS Two asymptomatic children, who were aged 12 and 14 yr, had normal plasma fasting gastrointestinal hormones and were found to have nonfunctioning pancreatic neuroendocrine tumors that were more than 2 cm in size. Surgery and immunostaining revealed that the tumors did not have significant expression of gastrointestinal hormones but did contain chromogranin A and synaptophysin, features consistent with those of nonfunctioning pancreatic neuroendocrine tumors. The tumors had a loss of menin expression. The 14 yr old also had primary hyperparathyroidism and a microprolactinoma, and the 12 yr old had a nonfunctioning pituitary microadenoma. Three other children had primary hyperparathyroidism and a microprolactinoma. CONCLUSION Nonfunctioning pancreatic neuroendocrine tumors may occur in asymptomatic children with MEN1 mutations, and screening for such enteropancreatic tumors in MEN1 children should be considered earlier than the age of 20 yr, as is currently recommended by the international guidelines.

Multiple endocrine neoplasia type 1 knockout mice develop parathyroid, pancreatic, pituitary and adrenal tumours with hypercalcaemia, hypophosphataemia and hypercorticosteronaemia

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized in man by parathyroid, pancreatic, pituitary and adrenal tumours. The MEN1 gene encodes a 610-amino acid protein (menin) which is a tumour suppressor. To investigate the in vivo role of menin, we developed a mouse model, by deleting Men1 exons 1 and 2 and investigated this for MEN1-associated tumours and serum abnormalities. Men1+/− mice were viable and fertile, and 220 Men1+/− and 94 Men1+/+ mice were studied between the ages of 3 and 21 months. Survival in Men1+/− mice was significantly lower than in Men1+/+ mice (<68% vs >85%, P<0.01). Men1+/− mice developed, by 9 months of age, parathyroid hyperplasia, pancreatic tumours which were mostly insulinomas, by 12 months of age, pituitary tumours which were mostly prolactinomas, and by 15 months parathyroid adenomas and adrenal cortical tumours. Loss of heterozygosity and menin expression was demonstrated in the tumours, consistent with a tumour suppressor role for the Men1 gene. Men1+/− mice with parathyroid neoplasms were hypercalcaemic and hypophosphataemic, with inappropriately normal serum parathyroid hormone concentrations. Pancreatic and pituitary tumours expressed chromogranin A (CgA), somatostatin receptor type 2 and vascular endothelial growth factor-A. Serum CgA concentrations in Men1+/− mice were not elevated. Adrenocortical tumours, which immunostained for 3-β-hydroxysteroid dehydrogenase, developed in seven Men1+/− mice, but resulted in hypercorticosteronaemia in one out of the four mice that were investigated. Thus, these Men1+/− mice are representative of MEN1 in man, and will help in investigating molecular mechanisms and treatments for endocrine tumours.

Parafibromin--functional insights.

Parafibromin is a predominantly nuclear protein with a tumour suppressor role in the development of hereditary and nonhereditary parathyroid carcinomas, and the hyperparathyroidism‐jaw tumour syndrome, which is associated with renal and uterine tumours. Parafibromin is a component of the highly conserved PAF1 complex, which regulates transcriptional events and histone modifications. The parafibromin/PAF1 complex regulates genes involved in cell growth and survival, and via these, parafibromin plays a pivotal role in embryonic development and survival of adults.

Functional characterization of calcium sensing receptor polymorphisms and absence of association with indices of calcium homeostasis and bone mineral density.

Objectives  Associations between calcium‐sensing receptor (CaSR) polymorphisms and serum calcium, PTH and bone mineral density (BMD) have been reported by six studies. However, three other studies have failed to detect such associations. We therefore further investigated three CaSR coding region polymorphisms (Ala986Ser, Arg990Gly and Gln1011Glu) for associations with indices of calcium homeostasis and BMD and for alterations in receptor function.