Manuel Lemos

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.

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.

Genetic background influences embryonic lethality and the occurrence of neural tube defects in Men1 null mice: relevance to genetic modifiers

Germline mutations of the multiple endocrine neoplasia type 1 (MEN1) gene cause parathyroid, pancreatic and pituitary tumours in man. MEN1 mutations also cause familial isolated primary hyperparathyroidism (FIHP) and the same MEN1 mutations, in different families, can cause either FIHP or MEN1. This suggests a role for genetic background and modifier genes in altering the expression of a mutation. We investigated the effects of genetic background on the phenotype of embryonic lethality that occurs in a mouse model for MEN1. Men1(+/-) mice were backcrossed to generate C57BL/6 and 129S6/SvEv incipient congenic strains, and used to obtain homozygous Men1(-/-) mice. No viable Men1(-/-) mice were obtained. The analysis of 411 live embryos obtained at 9.5-16.5 days post-coitum (dpc) revealed that significant deviations from the expected Mendelian 1:2:1 genotype ratio were first observed at 12.5 and 14.5 dpc in the 129S6/SvEv and C57BL/6 strains respectively (P<0.05). Moreover, live Men1(-/-) embryos were absent by 13.5 and 15.5 dpc in the 129S6/SvEv and C57BL/6 strains respectively thereby indicating an earlier lethality by 2 days in the 129S6/SvEv strain (P<0.01). Men1(-/-) embryos had macroscopic haemorrhages, and histology and optical projection tomography revealed them to have internal haemorrhages, myocardial hypotrophy, pericardial effusion, hepatic abnormalities and neural tube defects. The neural tube defects occurred exclusively in 129S6/SvEv embryos (21 vs 0%, P<0.01). Thus, our findings demonstrate the importance of genetic background in influencing the phenotypes of embryonic lethality and neural tube defects in Men1(-/-) mice, and implicate a role for genetic modifiers.

MEN1 gene replacement therapy reduces proliferation rates in a mouse model of pituitary adenomas

Multiple endocrine neoplasia type 1 (MEN1) is characterized by the combined occurrence of pituitary, pancreatic, and parathyroid tumors showing loss of heterozygosity in the putative tumor suppressor gene MEN1. This gene encodes the protein menin, the overexpression of which inhibits cell proliferation in vitro. In this study, we conducted a preclinical evaluation of MEN1 gene therapy in pituitary tumors of Men1(+/-) mice, using a recombinant nonreplicating adenoviral serotype 5 vector that contained the murine Men1 cDNA under control of a cytomegalovirus promoter (Men1.rAd5). Pituitary tumors in 55 Men1(+/-) female mice received a transauricular intratumoral injection of Men1.rAd5 or control treatments, followed by 5-bromo-2-deoxyuridine (BrdUrd) in drinking water for four weeks before magnetic resonance imaging (MRI) and immunohistochemical analysis. Immediate procedure-related and 4-week mortalities were similar in all groups, indicating that the adenoviral gene therapy was not associated with a higher mortality. Menin expression was higher in the Men1.rAd5-treated mice when compared with other groups. Daily proliferation rates assessed by BrdUrd incorporation were reduced significantly in Men1.rAd5-injected tumors relative to control-treated tumors. In contrast, apoptotic rates, immune T-cell response, and tumor volumes remained similar in all groups. Our findings establish that MEN1 gene replacement therapy can generate menin expression in pituitary tumors, and significantly reduce tumor cell proliferation.

A novel MEN1 intronic mutation associated with multiple endocrine neoplasia type 1.

Objective  To investigate a family with an unusual combination of multiple endocrine neoplasia (MEN1) and the McCune–Albright syndrome for MEN1 mutations and activating GNAS1 mutations at codons Arg201 and Gln227.

Pseudohypoparathyroidism type 1a due to a novel mutation in the GNAS gene

Dear Editor, Pseudohypoparathyroidism type 1a (PHP1a) (OMIM #103580) is characterized by hypocalcaemia and hyperphosphataemia due to parathyroid hormone (PTH) resistance, associated with features of Albright’s Hereditary Osteodystrophy (AHO) which include short stature, obesity, subcutaneous calcifications and brachydactyly. PHP1a is caused by heterozygous germline mutations of the alpha subunit of the stimulatory form of the GTP-binding protein (Gs-alpha), which is a downstream signalling protein of the PTH receptor and of other G protein-coupled hormone receptors. Gs-alpha is encoded by the GNAS gene (chromosome 20q13.3), which is a complex imprinted locus that also produces additional coding and noncoding transcripts through the use of alternative promoters and alternative splicing, in a tissue-specific manner. PHP1a results from maternally inherited loss-of-function mutations of Gs-alpha, but paternally inherited mutations usually result in pseudopseudohypoparathyroidism (PPHP), which is characterized by the presence of AHO without hormone resistance. Thus, PHP1a and PPHP, which are inherited as autosomal dominant disorders with parental imprinting, are frequently found in the same kindred. We identified a kindred with PHP1a/PPHP and investigated the patients for the underlying molecular abnormality. The index case was an 11-year-old Portuguese boy that presented with seizures due to hypocalcaemia. Serum concentration of calcium was 1 65 mmol/l (normal: 2 0–2 6 mmol/l), phosphate 3 6 mmol/l (normal: 0 9–1 5 mmol/l) and PTH 607 ng/l (normal: 9–55). Height and weight were on the 50th percentile and 90th percentile, respectively. He had subcutaneous and intracranial calcifications (Fig. 1a); brachydactyly, which was due to shortened metacarpals and metatarsals (Fig. 1b); and learning disability. He also had abnormal thyroid function tests with serum concentrations of TSH 4 8 mIU/l (normal: 0 3–3 0), free T4 6 4 pmol/l (normal: 7 7–23 2) and free T3 6 6 pmol/l (normal: 3 4–8 4). His mother had short stature (3rd percentile) and brachydactyly (Fig. 1c), but no biochemical abnormalities. Family history also included an older sibling with a similar phenotype, who had died due to an unknown cause, at age 7 years, and two younger unaffected siblings. Venous blood samples were obtained after informed consent, from the index case, the mother, two siblings and the maternal grandparents. Leucocyte DNA was extracted and used with appropriate PCR primers to amplify exons 1–13 of the GNAS gene, utilizing conditions previously described. Bidirectional sequencing of the PCR products was carried out by the use of the same primers, and an automated capillary sequencer (GenomeLab GeXP System, Beckman Coulter, Fullerton, CA, USA). A novel heterozygous 2-base pair (bp) deletion in exon 2 (c.188_189delTG) was found in the proband and his mother (Fig. 1d). The deletion of this dinucleotide sequence is predicted to cause a frameshift, with the incorporation of two missense amino acids, followed by a premature stop codon (TAA) in the new frame at codon 65. The mutation resulted in the loss of an NspI restriction endonuclease site and this was used to confirm its presence and to assess the other family members (Fig. 1e). The mutation was absent in the unaffected family members, including the maternal grandparents, thereby indicating that the mother (individual II-2 in Fig. 1e) either has a de novo mutation involving the paternal allele or that her father has undetected mosaicism. In addition, an analysis of the DNA from 55 unrelated normal individuals (110 alleles) confirmed the absence of this DNA sequence abnormality (data not shown). The different clinical presentation in the son (PHP1a) and mother (PPHP), who harbour the same mutation, can be explained by the characteristic mode of inheritance, which is autosomal dominant with parental imprinting of hormone resistance. The identification of the causative mutation in the index case may be useful for screening other family members in order to avoid late or misdiagnosis, as probably occurred with individual III-1 (Fig. 1e). Age of onset of the hormone resistance is quite variable among mutation-positive individuals and can be delayed for several years. This latency of PTH resistance in patients with PHP1a has been attributed to a gradual development of paternal Gs-alpha silencing in target tissues. Therefore, genetic screening of family members can be useful for presymptomatic diagnosis. There are over 340 reported kindreds with PHP1a/PPHP due to a GNAS mutation, and these are scattered across the 13 exons that encode Gs-alpha, with no known genotype–phenotype correlation. However, exons 2 and 3 are the least affected in these disorders. To date, only three other mutations of exon 2, one missense and two insertions, have been reported. Therefore, the 2-bp deletion in exon 2 identified by the present study is unusual and further expands the spectrum of known GNAS mutations associated with these complex disorders.