Genetics of hereditary forms of primary hyperparathyroidism.

Primary hyperparathyroidism (PHPT), a relatively common disorder characterized by hypercalcemia with raised or inappropriately normal serum parathyroid hormone (PTH) concentrations, may occur as part of a hereditary syndromic disorder or as a non-syndromic disease. The associated syndromic disorders include multiple endocrine neoplasia types 1-5 (MEN1-5) and hyperparathyroidism with jaw tumor (HPT-JT) syndromes, and the non-syndromic forms include familial hypocalciuric hypercalcemia types 1-3 (FHH1-3), familial isolated hyperparathyroidism (FIHP), and neonatal severe hyperparathyroidism (NS-HPT). Such hereditary forms may occur in > 10% of patients with PHPT, and their recognition is important for implementation of gene-specific screening protocols and investigations for other associated tumors. Syndromic PHPT tends to be multifocal and multiglandular with most patients requiring parathyroidectomy with the aim of limiting end-organ damage associated with hypercalcemia, particularly osteoporosis, nephrolithiasis, and renal failure. Some patients with non-syndromic PHPT may have mutations of the MEN1 gene or the calcium-sensing receptor (CASR), whose loss of function mutations usually cause FHH1, a disorder associated with mild hypercalcemia and may follow a benign clinical course. Measurement of the urinary calcium-to-creatinine ratio clearance (UCCR) may help to distinguish patients with FHH from those with PHPT, as the majority of FHH patients have low urinary calcium excretion (UCCR < 0.01). Once genetic testing confirms a hereditary cause of PHPT, further genetic testing can be offered to the patients' relatives and subsequent screening can be carried out in these affected family members, which prevents inappropriate testing in normal individuals.

GNAQ/GNA11 Mosaicism Causes Aberrant Calcium Signaling Susceptible to Targeted Therapeutics.

Mosaic variants in genes GNAQ or GNA11 lead to a spectrum of vascular and pigmentary diseases including Sturge-Weber syndrome, in which progressive postnatal neurological deterioration led us to seek biologically targeted therapeutics. Using two cellular models, we find that disease-causing GNAQ/11 variants hyperactivate constitutive and G-protein coupled receptor ligand-induced intracellular calcium signaling in endothelial cells. We go on to show that the aberrant ligand-activated intracellular calcium signal is fueled by extracellular calcium influx through calcium-release-activated channels. Treatment with targeted small interfering RNAs designed to silence the variant allele preferentially corrects both the constitutive and ligand-activated calcium signaling, whereas treatment with a calcium-release-activated channel inhibitor rescues the ligand-activated signal. This work identifies hyperactivated calcium signaling as the primary biological abnormality in GNAQ/11 mosaicism and paves the way for clinical trials with genetic or small molecule therapies.

The role of DNA methylation in human pancreatic neuroendocrine tumours.

Pancreatic neuroendocrine tumours (PNETs) are the second most common pancreatic tumour. However, relatively little is known about their tumourigenic drivers, other than mutations involving the multiple endocrine neoplasia 1 (MEN1), ATRX chromatin remodeler, and death domain-associated protein genes, which are found in ~40% of sporadic PNETs. PNETs have a low mutational burden, thereby suggesting that other factors likely contribute to their development, including epigenetic regulators. One such epigenetic process, DNA methylation, silences gene transcription via 5'methylcytosine (5mC), and this is usually facilitated by DNA methyltransferase enzymes at CpG-rich areas around gene promoters. However, 5'hydroxymethylcytosine, which is the first epigenetic mark during cytosine demethylation, and opposes the function of 5mC, is associated with gene transcription, although the significance of this remains unknown, as it is indistinguishable from 5mC when conventional bisulfite conversion techniques are solely used. Advances in array-based technologies have facilitated the investigation of PNET methylomes and enabled PNETs to be clustered by methylome signatures, which has assisted in prognosis and discovery of new aberrantly regulated genes contributing to tumourigenesis. This review will discuss the biology of DNA methylation, its role in PNET development, and impact on prognostication and discovery of epigenome-targeted therapies.

A Mouse Model with a Frameshift Mutation in the Nuclear Factor I/X (NFIX) Gene Has Phenotypic Features of Marshall-Smith Syndrome.

The nuclear factor I/X (NFIX) gene encodes a ubiquitously expressed transcription factor whose mutations lead to two allelic disorders characterized by developmental, skeletal, and neural abnormalities, namely, Malan syndrome (MAL) and Marshall-Smith syndrome (MSS). NFIX mutations associated with MAL mainly cluster in exon 2 and are cleared by nonsense-mediated decay (NMD) leading to NFIX haploinsufficiency, whereas NFIX mutations associated with MSS are clustered in exons 6-10 and escape NMD and result in the production of dominant-negative mutant NFIX proteins. Thus, different NFIX mutations have distinct consequences on NFIX expression. To elucidate the in vivo effects of MSS-associated NFIX exon 7 mutations, we used CRISPR-Cas9 to generate mouse models with exon 7 deletions that comprised: a frameshift deletion of two nucleotides (Nfix Del2); in-frame deletion of 24 nucleotides (Nfix Del24); and deletion of 140 nucleotides (Nfix Del140). Nfix +/Del2, Nfix +/Del24, Nfix +/Del140, Nfix Del24/Del24, and Nfix Del140/Del140 mice were viable, normal, and fertile, with no skeletal abnormalities, but Nfix Del2/Del2 mice had significantly reduced viability (p < 0.002) and died at 2-3 weeks of age. Nfix Del2 was not cleared by NMD, and NfixDel2/Del2 mice, when compared to Nfix +/+ and Nfix +/Del2 mice, had: growth retardation; short stature with kyphosis; reduced skull length; marked porosity of the vertebrae with decreased vertebral and femoral bone mineral content; and reduced caudal vertebrae height and femur length. Plasma biochemistry analysis revealed Nfix Del2/Del2 mice to have increased total alkaline phosphatase activity but decreased C-terminal telopeptide and procollagen-type-1-N-terminal propeptide concentrations compared to Nfix +/+ and Nfix +/Del2 mice. Nfix Del2/Del2 mice were also found to have enlarged cerebral cortices and ventricular areas but smaller dentate gyrus compared to Nfix +/+ mice. Thus, Nfix Del2/Del2 mice provide a model for studying the in vivo effects of NFIX mutants that escape NMD and result in developmental abnormalities of the skeletal and neural tissues that are associated with MSS. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Preclinical Models of Neuroendocrine Neoplasia.

Neuroendocrine neoplasia (NENs) are a complex and heterogeneous group of cancers that can arise from neuroendocrine tissues throughout the body and differentiate them from other tumors. Their low incidence and high diversity make many of them orphan conditions characterized by a low incidence and few dedicated clinical trials. Study of the molecular and genetic nature of these diseases is limited in comparison to more common cancers and more dependent on preclinical models, including both in vitro models (such as cell lines and 3D models) and in vivo models (such as patient derived xenografts (PDXs) and genetically-engineered mouse models (GEMMs)). While preclinical models do not fully recapitulate the nature of these cancers in patients, they are useful tools in investigation of the basic biology and early-stage investigation for evaluation of treatments for these cancers. We review available preclinical models for each type of NEN and discuss their history as well as their current use and translation.

miR-3156-5p is downregulated in serum of MEN1 patients and regulates expression of MORF4L2.

Multiple endocrine neoplasia type 1 (MEN1), caused by mutations in the MEN1 gene encoding menin, is an autosomal dominant disorder characterised by the combined occurrence of parathyroid, pituitary and pancreatic neuroendocrine tumours (NETs). Development of these tumours is associated with wide variations in their severity, order and ages (from <5 to >80 years), requiring life-long screening. To improve tumour surveillance and quality of life, better circulating biomarkers, particularly for pancreatic NETs that are associated with higher mortality, are required. We, therefore, examined the expression of circulating miRNA in the serum of MEN1 patients. Initial profiling analysis followed by qRT-PCR validation studies identified miR-3156-5p to be significantly downregulated (-1.3 to 5.8-fold, P < 0.05-0.0005) in nine MEN1 patients, compared to matched unaffected relatives. MEN1 knock-down experiments in BON-1 human pancreatic NET cells resulted in reduced MEN1 (49%, P < 0.05), menin (54%, P < 0.05) and miR-3156-5p expression (20%, P < 0.005), compared to control-treated cells, suggesting that miR-3156-5p downregulation is a consequence of loss of MEN1 expression. In silico analysis identified mortality factor 4-like 2 (MOR4FL2) as a potential target of miR-3156-5p, and in vitro functional studies in BON-1 cells transfected with either miR-3156-5p mimic or inhibitors showed that the miR-3156-5p mimic significantly reduced MORF4L2 protein expression (46%, P < 0.005), while miR-3156-5p inhibitor significantly increased MORF4L2 expression (1.5-fold, P < 0.05), compared to control-treated cells, thereby confirming that miR-3156-5p regulates MORF4L2 expression. Thus, the inverse relationship between miR-3156-5p and MORF4L2 expression represents a potential serum biomarker that could facilitate the detection of NET occurrence in MEN1 patients.

The Bartter-Gitelman Spectrum: 50-Year Follow-up With Revision of Diagnosis After Whole-Genome Sequencing.

Bartter syndrome (BS) and Gitelman syndrome (GS) are renal tubular disorders affecting sodium, potassium, and chloride reabsorption. Clinical features include muscle cramps and weakness, in association with hypokalemia, hypochloremic metabolic alkalosis, and hyperreninemic hyperaldosteronism. Hypomagnesemia and hypocalciuria are typical of GS, while juxtaglomerular hyperplasia is characteristic of BS. GS is due to SLC12A3 variants, whereas BS is due to variants in SLC12A1, KCNJ1, CLCNKA, CLCNKB, BSND, MAGED2, or CASR. We had the opportunity to follow up one of the first reported cases of a salt-wasting tubulopathy, who based on clinical features was diagnosed with GS. The patient had presented at age 10 years with tetany precipitated by vomiting or diarrhea. She had hypokalemia, a hypochloremic metabolic alkalosis, hyponatremia, mild hypercalcemia, and normomagnesemia, and subsequently developed hypocalciuria and hypomagnesemia. A renal biopsy showed no evidence for juxtaglomerular hyperplasia. She developed chronic kidney failure at age 55 years, and ocular sclerochoroidal calcification, associated with BS and GS, at older than 65 years. Our aim was therefore to establish the genetic diagnosis in this patient using whole-genome sequencing (WGS). Leukocyte DNA was used for WGS analysis, and this revealed a homozygous c.226C > T (p.Arg76Ter) nonsense CLCNKB mutation, thereby establishing a diagnosis of BS type-3. WGS also identified 2 greater than 5-Mb regions of homozygosity that suggested likely mutational heterozygosity in her parents, who originated from a Greek island with fewer than 1500 inhabitants and may therefore have shared a common ancestor. Our results demonstrate the utility of WGS in establishing the correct diagnosis in renal tubular disorders with overlapping phenotypes.

Multiple endocrine neoplasia type 1 in children and adolescents: Clinical features and treatment outcomes.

BACKGROUND: Clinical manifestations and treatment outcomes in children and adolescents with multiple endocrine neoplasia type 1 are not well characterized. METHODS: We conducted a retrospective cohort study of 80 patients with multiple endocrine neoplasia type 1 who commenced tumor surveillance at ≤18 years of age. RESULTS: Fifty-six patients (70%) developed an endocrine tumor by age ≤18 years (median age = 14 years, range = 6-18 years). Primary hyperparathyroidism occurred in >80% of patients, with >70% undergoing parathyroidectomy, in which less-than-subtotal (<3-gland) resection resulted in decreased disease-free outcomes versus subtotal (3-3.5-gland) or total (4-gland) parathyroidectomy (median 27 months versus not reached; P = .005). Pancreaticoduodenal neuroendocrine tumors developed in ∼35% of patients, of whom >70% had nonfunctioning tumors, >35% had insulinomas, and <5% had gastrinomas, with ∼15% having metastases and >55% undergoing surgery. Pituitary tumors developed in >30% of patients, and ∼35% were macroprolactinomas. Tumor occurrence in male patients and female patients was not significantly different. Genetic analyses revealed 38 germline MEN1 mutations, of which 3 were novel. CONCLUSION: Seventy percent of children aged ≤18 years with multiple endocrine neoplasia type 1 develop endocrine tumors, which include parathyroid tumors for which less-than-subtotal parathyroidectomy should be avoided; pancreaticoduodenal neuroendocrine tumors that may metastasize; and pituitary macroprolactinomas.

Somatic mutations of GNA11 and GNAQ in CTNNB1-mutant aldosterone-producing adenomas presenting in puberty, pregnancy or menopause.

Most aldosterone-producing adenomas (APAs) have gain-of-function somatic mutations of ion channels or transporters. However, their frequency in aldosterone-producing cell clusters of normal adrenal gland suggests a requirement for codriver mutations in APAs. Here we identified gain-of-function mutations in both CTNNB1 and GNA11 by whole-exome sequencing of 3/41 APAs. Further sequencing of known CTNNB1-mutant APAs led to a total of 16 of 27 (59%) with a somatic p.Gln209His, p.Gln209Pro or p.Gln209Leu mutation of GNA11 or GNAQ. Solitary GNA11 mutations were found in hyperplastic zona glomerulosa adjacent to double-mutant APAs. Nine of ten patients in our UK/Irish cohort presented in puberty, pregnancy or menopause. Among multiple transcripts upregulated more than tenfold in double-mutant APAs was LHCGR, the receptor for luteinizing or pregnancy hormone (human chorionic gonadotropin). Transfections of adrenocortical cells demonstrated additive effects of GNA11 and CTNNB1 mutations on aldosterone secretion and expression of genes upregulated in double-mutant APAs. In adrenal cortex, GNA11/Q mutations appear clinically silent without a codriver mutation of CTNNB1.

The bromodomain inhibitor JQ1+ reduces calcium-sensing receptor activity in pituitary cell lines.

Corticotrophinomas represent 10% of all surgically removed pituitary adenomas, however, current treatment options are often not effective, and there is a need for improved pharmacological treatments. Recently, JQ1+, a bromodomain inhibitor that promotes gene transcription by binding acetylated histone residues and recruiting transcriptional machinery, has been shown to reduce proliferation in a murine corticotroph cell line, AtT20. RNA-Seq analysis of AtT20 cells following treatment with JQ1+ identified the calcium-sensing receptor (CaSR) gene as significantly downregulated, which was subsequently confirmed using real-time PCR and Western blot analysis. CaSR is a G protein-coupled receptor that plays a central role in calcium homeostasis but can elicit non-calcitropic effects in multiple tissues, including the anterior pituitary where it helps regulate hormone secretion. However, in AtT20 cells, CaSR activates a tumour-specific cAMP pathway that promotes ACTH and PTHrP hypersecretion. We hypothesised that the Casr promoter may harbour binding sites for BET proteins, and using chromatin immunoprecipitation (ChIP)-sequencing demonstrated that the BET protein Brd3 binds to the promoter of the Casr gene. Assessment of CaSR signalling showed that JQ1+ significantly reduced Ca2+e-mediated increases in intracellular calcium (Ca2+i) mobilisation and cAMP signalling. However, the CaSR-negative allosteric modulator, NPS-2143, was unable to reduce AtT20 cell proliferation, indicating that reducing CaSR expression rather than activity is likely required to reduce pituitary cell proliferation. Thus, these studies demonstrate that reducing CaSR expression may be a viable option in the treatment of pituitary tumours. Moreover, current strategies to reduce CaSR activity, rather than protein expression for cancer treatments, may be ineffective.

Multiple Endocrine Neoplasia Type 1 (MEN1) 5'UTR Deletion, in MEN1 Family, Decreases Menin Expression.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the occurrence of parathyroid, pancreatic and pituitary tumors, and is due to mutations in the coding region of the MEN1 gene, which encodes menin. We investigated a family with identical twins that had MEN1, with different MEN1 tumors. DNA sequence analysis of the MEN1 coding region had not identified any abnormalities and we hypothesized that deletions and mutations involving the untranslated regions may be involved. Informed consent and venous blood samples were obtained from five family members. Sanger DNA sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses were performed using leukocyte DNA. This revealed a heterozygous 596bp deletion (Δ596bp) between nucleotides -1087 and -492 upstream of the translation start site, located within the MEN1 5' untranslated region (UTR), and includes the core promoter and multiple cis-regulatory regions. To investigate the effects of this 5'UTR deletion on MEN1 promoter activity, we generated luciferase reporter constructs, containing either wild-type 842bp or mutant 246bp MEN1 promoter, and transfected them into human embryonic kidney HEK293 and pancreatic neuroendocrine tumor BON-1 cells. This revealed the Δ596bp mutation to result in significant reductions by 37-fold (p < 0.0001) and 16-fold (p < 0.0001) in luciferase expression in HEK293 and BON-1 cells, respectively, compared to wild-type. The effects of this 5'UTR deletion on MEN1 transcription and translation were assessed using qRT-PCR and Western blot analyses, respectively, of mRNA and protein lysates obtained from Epstein-Barr-virus transformed lymphoblastoid cells derived from affected and unaffected individuals. This demonstrated the Δ596bp mutation to result in significant reductions of 84% (p < 0.05) and 88% (p < 0.05) in MEN1 mRNA and menin protein, respectively, compared to unaffected individuals. Thus, our results report the first germline MEN1 5'UTR mutation and highlight the importance of investigating UTRs in MEN1 patients who do not have coding region mutations. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Multiple Endocrine Neoplasia Type 1: Latest Insights.

Multiple endocrine neoplasia type 1 (MEN1), a rare tumor syndrome that is inherited in an autosomal dominant pattern, is continuing to raise great interest for endocrinology, gastroenterology, surgery, radiology, genetics, and molecular biology specialists. There have been 2 major clinical practice guidance papers published in the past 2 decades, with the most recent published 8 years ago. Since then, several new insights on the basic biology and clinical features of MEN1 have appeared in the literature, and those data are discussed in this review. The genetic and molecular interactions of the MEN1-encoded protein menin with transcription factors and chromatin-modifying proteins in cell signaling pathways mediated by transforming growth factor ß/bone morphogenetic protein, a few nuclear receptors, Wnt/ß-catenin, and Hedgehog, and preclinical studies in mouse models have facilitated the understanding of the pathogenesis of MEN1-associated tumors and potential pharmacological interventions. The advancements in genetic diagnosis have offered a chance to recognize MEN1-related conditions in germline MEN1 mutation-negative patients. There is rapidly accumulating knowledge about clinical presentation in children, adolescents, and pregnancy that is translatable into the management of these very fragile patients. The discoveries about the genetic and molecular signatures of sporadic neuroendocrine tumors support the development of clinical trials with novel targeted therapies, along with advancements in diagnostic tools and surgical approaches. Finally, quality of life studies in patients affected by MEN1 and related conditions represent an effort necessary to develop a pharmacoeconomic interpretation of the problem. Because advances are being made both broadly and in focused areas, this timely review presents and discusses those studies collectively.

Multiple Endocrine Neoplasia Type 1 (MEN1) Phenocopy Due to a Cell Cycle Division 73 (CDC73) Variant.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the combined occurrence of parathyroid tumors, pituitary adenomas, and pancreatic neuroendocrine neoplasms (PNENs). MEN1 is caused by germline MEN1 mutations in > 75% of patients, and the remaining 25% of patients may have mutations in unidentified genes or represent phenocopies with mutations in genes such as cell cycle division 73 (CDC73), the calcium sensing receptor (CASR), and cyclin-dependent kinase inhibitor 1B (CDKN1B), which are associated with the hyperparathyroidism-jaw tumor syndrome, familial hypocalciuric hypercalcemia type 1, and MEN4, respectively. Here, we report a heterozygous c.1138C>T (p.Leu380Phe) CDC73 germline variant in a clinically diagnosed MEN1 patient, based on combined occurrence of primary hyperparathyroidism, acromegaly, and a PNEN. Characterization of the PNEN confirmed it was a neuroendocrine neoplasm as it immuno-stained positively for chromogranin and glucagon. The rare variant p.Leu380Phe occurred in a highly conserved residue, and further analysis using RNA-Scope indicated that it was associated with a significant reduction in CDC73 expression in the PNEN. Previously, CDC73 mutations have been reported to be associated with tumors of the parathyroids, kidneys, uterus, and exocrine pancreas. Thus, our report of a patient with PNEN and somatotrophinoma who had a CDC73 variant, provides further evidence that CDC73 variants may result in a MEN1 phenocopy.

Whole genome sequence analysis identifies a PAX2 mutation to establish a correct diagnosis for a syndromic form of hyperuricemia.

Hereditary hyperuricemia may occur as part of a syndromic disorder or as an isolated nonsyndromic disease, and over 20 causative genes have been identified. Here, we report the use of whole genome sequencing (WGS) to establish a diagnosis in a family in which individuals were affected with gout, hyperuricemia associated with reduced fractional excretion of uric acid, chronic kidney disease (CKD), and secondary hyperparathyroidism, that are consistent with familial juvenile hyperuricemic nephropathy (FJHN). However, single gene testing had not detected mutations in the uromodulin (UMOD) or renin (REN) genes, which cause approximately 30-90% of FJHN. WGS was therefore undertaken, and this identified a heterozygous c.226G>C (p.Gly76Arg) missense variant in the paired box gene 2 (PAX2) gene, which co-segregated with renal tubulopathy in the family. PAX2 mutations are associated with renal coloboma syndrome (RCS), which is characterized by abnormalities in renal structure and function, and anomalies of the optic nerve. Ophthalmological examination in two adult brothers affected with hyperuricemia, gout, and CKD revealed the presence of optic disc pits, consistent with optic nerve coloboma, thereby revising the diagnosis from FJHN to RCS. Thus, our results demonstrate the utility of WGS analysis in establishing the correct diagnosis in disorders with multiple etiologies.

Preclinical drug studies in MEN1-related neuroendocrine neoplasms (MEN1-NENs).

Neuroendocrine neoplasms (NENs) occur usually as sporadic tumours; however, rarely, they may arise in the context of a hereditary syndrome, such as multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant disorder characterised by the combined development of pancreatic NENs (pNENs) together with parathyroid and anterior pituitary tumours. The therapeutic decision for sporadic pNENs patients is multi-disciplinary and complex: based on the grade and stage of the tumor, various options (and their combinations) are considered, such as surgical excision (either curative or for debulking aims), biological drugs (somatostatin analogues), targeted therapies (mTOR inhibitors or tyrosine kinases (TK)/receptors inhibitors), peptide receptor radioligand therapy (PRRT), chemotherapy, and liver-directed therapies. However, treatment of MEN1-related NENs' patients is even more challenging, as these tumours are usually multifocal with co-existing foci of heterogeneous biology and malignant potential, rendering them more resistant to the conventional therapies used in their sporadic counterparts, and therefore associated with a poorer prognosis. Moreover, clinical data using standard therapeutic options in MEN1-related NENs are scarce. Recent preclinical studies have identified potentially new targeted therapeutic options for treating MEN1-associated NENs, such as epigenetic modulators, Wnt pathway-targeting ß-catenin antagonists, Ras signalling modulators, Akt/mTOR signalling modulators, novel somatostatin receptors analogues, anti-angiogenic drugs, as well as MEN1 gene replacement therapy. The present review aims to summarize these novel therapeutic opportunities for NENs developing in the context of MEN1 syndrome, with an emphasis on pancreatic NENs, as they are the most frequent ones studied in MEN1-NENs models to date; moreover, due to the recent shifting nomenclature of 'pituitary adenomas' to 'pituitary neuroendocrine neoplasms', relevant data on MEN1-pituitary tumours, when appropriate, are briefly described.

Clinical MEN-1 Among a Large Cohort of Patients With Acromegaly.

CONTEXT: Clinical multiple endocrine neoplasia type 1 (MEN-1) is diagnosed by the presence of at least 2 MEN-1-associated tumors. Many patients with acromegaly and clinical MEN-1 yield negative testing for MEN1 mutations. While cases of acromegaly and primary hyperparathyroidism (PHP) with negative genetic testing have been reported, its prevalence among patients with acromegaly is undetermined, and the clinical presentation has not been well characterized. OBJECTIVES: The main goals of this study are: (1) To determine the prevalence of clinical MEN-1 with PHP in patients with acromegaly and characterize their clinical features; and (2) to evaluate the genetic basis for the coexistence of acromegaly and PHP. DESIGN: Retrospective record review and genetic analysis. SETTING: Clinical Research Centers. PARTICIPANTS: 414 patients with acromegaly. INTERVENTIONS: Clinical evaluation and DNA sequencing for MEN1, CDKN1A, CDKN1B, CDKN2B, CDKN2C, and AIP genes. MAIN OUTCOME MEASUREMENTS: Clinical and genetic analysis. RESULTS: Among patients with acromegaly, clinical MEN-1, as defined by the presence of at least one other MEN-1-associated tumor, was present in 6.6%. PHP occurred in 6.1%; more than half had parathyroid hyperplasia. DNA sequencing was unrevealing for genetic mutations, except for 1 case of a CDC73 mutation. Acromegaly was diagnosed at an older age with a higher prevalence of malignancies (specifically breast and thyroid) in patients with coexisting PHP than those with isolated acromegaly. CONCLUSIONS: A distinct phenotype is described in patients with clinical MEN-1 and negative genetic testing for mutations previously associated with this syndrome. Further studies are needed to identify other genes that may explain the association between PHP and acromegaly.

Genetic background influences tumour development in heterozygous Men1 knockout mice.

Multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant disorder caused by MEN1 germline mutations, is characterised by parathyroid, pancreatic and pituitary tumours. MEN1 mutations also cause familial isolated primary hyperparathyroidism (FIHP), a milder condition causing hyperparathyroidism only. Identical mutations can cause either MEN1 or FIHP in different families, thereby implicating a role for genetic modifiers in altering phenotypic expression of tumours. We therefore investigated the effects of genetic background and potential for genetic modifiers on tumour development in adult Men1+/- mice, which develop tumours of the parathyroids, pancreatic islets, anterior pituitary, adrenal cortex and gonads, that had been backcrossed to generate C57BL/6 and 129S6/SvEv congenic strains. A total of 275 Men1+/- mice, aged 5-26 months were macroscopically studied, and this revealed that genetic background significantly influenced the development of pituitary, adrenal and ovarian tumours, which occurred in mice over 12 months of age and more frequently in C57BL/6 females, 129S6/SvEv males and 129S6/SvEv females, respectively. Moreover, pituitary and adrenal tumours developed earlier, in C57BL/6 males and 129S6/SvEv females, respectively, and pancreatic and testicular tumours developed earlier in 129S6/SvEv males. Furthermore, glucagon-positive staining pancreatic tumours occurred more frequently in 129S6/SvEv Men1+/- mice. Whole genome sequence analysis of 129S6/SvEv and C57BL/6 Men1+/- mice revealed >54,000 different variants in >300 genes. These included, Coq7, Dmpk, Ccne2, Kras, Wnt2b, Il3ra and Tnfrsf10a, and qRT-PCR analysis revealed that Kras was significantly higher in pituitaries of male 129S6/SvEv mice. Thus, our results demonstrate that Kras and other genes could represent possible genetic modifiers of Men1.

Activating Mutations of the G-protein Subunit α 11 Interdomain Interface Cause Autosomal Dominant Hypocalcemia Type 2.

CONTEXT: Autosomal dominant hypocalcemia types 1 and 2 (ADH1 and ADH2) are caused by germline gain-of-function mutations of the calcium-sensing receptor (CaSR) and its signaling partner, the G-protein subunit α 11 (Gα 11), respectively. More than 70 different gain-of-function CaSR mutations, but only 6 different gain-of-function Gα 11 mutations are reported to date. METHODS: We ascertained 2 additional ADH families and investigated them for CaSR and Gα 11 mutations. The effects of identified variants on CaSR signaling were evaluated by transiently transfecting wild-type (WT) and variant expression constructs into HEK293 cells stably expressing CaSR (HEK-CaSR), and measuring intracellular calcium (Ca2+i) and MAPK responses following stimulation with extracellular calcium (Ca2+e). RESULTS: CaSR variants were not found, but 2 novel heterozygous germline Gα 11 variants, p.Gly66Ser and p.Arg149His, were identified. Homology modeling of these revealed that the Gly66 and Arg149 residues are located at the interface between the Gα 11 helical and GTPase domains, which is involved in guanine nucleotide binding, and this is the site of 3 other reported ADH2 mutations. The Ca2+i and MAPK responses of cells expressing the variant Ser66 or His149 Gα 11 proteins were similar to WT cells at low Ca2+e, but significantly increased in a dose-dependent manner following Ca2+e stimulation, thereby indicating that the p.Gly66Ser and p.Arg149His variants represent pathogenic gain-of-function Gα 11 mutations. Treatment of Ser66- and His149-Gα 11 expressing cells with the CaSR negative allosteric modulator NPS 2143 normalized Ca2+i and MAPK responses. CONCLUSION: Two novel ADH2-causing mutations that highlight the Gα 11 interdomain interface as a hotspot for gain-of-function Gα 11 mutations have been identified.