Filomena Baorda

A rare S33C mutation of CTNNB1 encoding β-catenin in a parathyroid adenoma found in an Italian primary hyperparathyroid cohort

Primary hyperparathyroidism (PHPT) is one of the most common endocrine disorders with a prevalence of 21/1000 in women between 55 and 75 years of age, corresponding to a 3/1000 prevalence in the general population [1]. Sporadic (non-familial) PHPT accounts for 90–95% of all cases. While activation of the protooncogene cyclin D1 (CCDN1) and inactivation of the tumor suppressor menin gene (MEN1) contribute to parathyroid adenomatosis, and inactivation of the parafibromin gene (CDC73) contributes to parathyroid carcinogenesis, the molecular pathogenesis of these tumors is incompletely understood. Dysregulated Wnt signaling and activation of b-catenin is involved in several cancers and consequently there has been recent interest in whether this is implicated in parathyroid tumorigenesis. In the absence of Wnt, cytosolic b-catenin phosphorylation catalyzed by glycogen synthase kinase (GSK)-b on serine/threonine residues (S33, S37, T41) encoded by exon 3 of the CTNNB1 gene leads to the ubiquitination and to degradation mediated by the proteasome. Mutation of the serine/threonine residues causes stabilization of the b-catenin protein and localization to the nucleus where it activates gene transcription. Several studies have been reported examining CTNNB1 gene mutations and aberrant b-catenin localization in sporadic parathyroid adenomas. While studies from Sweden found the homozygous S37A mutation in *7% of 124 parathyroid adenomas and aberrant b-catenin localization in all cases [2, 3], other studies from Japan [4, 5], USA [6],

Calcium-sensing-related gene mutations in hypercalcaemic hypocalciuric patients as differential diagnosis from primary hyperparathyroidism: detection of two novel inactivating mutations in an Italian population

BACKGROUND Inactivating mutations of the calcium-sensing receptor (CaSR), of the G-protein subunit α11 (GNA11) and of the adaptor-related protein complex 2, sigma 1 subunit (AP2S1) genes are responsible for familial hypocalciuric hypercalcaemia (FHH). The aim of this study was to analyse prevalence and pathogenicity of CaSR, GNA11 and AP2S1 mutations in patients with an FHH phenotype and to compare them with a sample of patients with primary hyperparathyroidism (PHPT) in order to identify the most useful laboratory parameter for a differential diagnosis. METHODS Patients with an FHH phenotype were studied with polymerase chain reaction amplification and direct sequencing of the entire CaSR, GNA11 and AP2S1 coding sequences. Novel mutations were introduced in a Myc-tagged human wild-type (WT) CaSR cDNA-expressing vector, and functional assay was performed on human embryonic kidney cells evaluating expression and function of mutated proteins. RESULTS Among 16 FHH patients, none had an inactivating GNA11 or AP2S1 mutation while 3 (18.8%) carried a CaSR mutation and 10 (62.5%) at least one CaSR polymorphism. Within the latter group, 7 of 10 patients had more than one polymorphism (4.1 ± 2.1 per patient). Two novel CaSR mutations [c.2120A>T (E707V) and c.2320G>A (G774S)] were identified: the E707V mutation prevented CaSR expression (western blot), whereas the G774S mutation determined a reduced receptor sensitivity to calcium (IP3 assay). PHPT patients showed significantly (P < 0.001) higher serum calcium, parathyroid hormone, urinary calcium and calcium-creatinine clearance ratio (CCCR) and significantly lower serum phosphate than FHH ones. CONCLUSIONS FHH should be clearly differentiated by PHPT to avoid unnecessary surgery: CCCR could be a useful screening tool while genetic analysis should include the two novel CaSR mutations herein described. The role of multiple polymorphisms deserves further investigation in patients with an FHH phenotype.

Identification and Functional Characterization of Three NoLS (Nucleolar Localisation Signals) Mutations of the CDC73 Gene

Hyperparathyroidism Jaw-Tumour Syndrome (HPT-JT) is characterized by primary hyperparathyroidism (PHPT), maxillary/mandible ossifying fibromas and by parathyroid carcinoma in 15% of cases. Inactivating mutations of the tumour suppressor CDC73/HRPT2 gene have been found in HPT-JT patients and also as genetic determinants of sporadic parathyroid carcinoma/atypical adenomas and, rarely, typical adenomas, in familial PHPT. Here we report the genetic and molecular analysis of the CDC73/HRPT2 gene in three patients affected by PHPT due to atypical and typical parathyroid adenomas, in one case belonging to familial PHPT. Flag-tagged WT and mutant CDC73/HRPT2 proteins were transiently transfected in HEK293 cells and functional assays were performed in order to investigate the effect of the variants on the whole protein expression, nuclear localization and cell overgrowth induction. We identified four CDC73/HRPT2 gene mutations, three germline (c.679_680delAG, p.Val85_Val86del and p.Glu81_Pro84del), one somatic (p.Arg77Pro). In three cases the mutation was located within the Nucleolar Localisation Signals (NoLS). The three NoLS variants led to instability either of the corresponding mutated protein or mRNA or both. When transfected in HEK293 cells, NoLS mutated proteins mislocalized with a predeliction for cytoplasmic or nucleo-cytoplasmic localization and, finally, they resulted in overgrowth, consistent with a dominant negative interfering effect in the presence of the endogenous protein.

CASR gene activating mutations in two families with autosomal dominant hypocalcemia.

BACKGROUND Autosomal dominant hypocalcemia (ADH) is an endocrine disorder caused by activating mutations of the calcium-sensing receptor (CASR) gene which plays a major role in maintaining calcium homeostasis. Biochemical features of ADH are hypocalcemia and hypercalciuria with inappropriately low levels of parathyroid hormone (PTH). We report on two four-generation families affected by ADH. AIM To identify mutations of CASR gene in subjects affected by familial idiopathic hypoparathyroidism. To perform functional assays of identified CASR variants by transient transfection on HEK293 cells. RESULTS We identified two CASR variants (Q681R and P221L): the Q681R variant was novel while the P221L had been previously published. Functional assays on the Q681R variant showed that it did not alter the whole expression nor the correct plasmamembrane localization, but enhanced the signaling function, increasing the sensitivity of the receptor as compared to the WT. CONCLUSIONS We report two activating CASR mutations in two families affected by ADH and the functional assays performed on the novel variant Q681R. Our work enlarged the spectrum of mutations of the CASR and contributed to a better elucidation of the protein function.

Large intragenic deletion of CDC73 (exons 4–10) in a three-generation hyperparathyroidism-jaw tumor (HPT-JT) syndrome family

BackgroundInactivating mutations of CDC73 cause Hyperparathyroidism-Jaw Tumour syndrome (HPT-JT), Familial Isolated Hyperparathyroidism (FIHP) and sporadic parathyroid carcinoma. We conducted CDC73 mutation analysis in an HPT-JT family and confirm carrier status of the proband’s daughter.MethodsThe proband had primary hyperparathyroidism (parathyroid carcinoma) and uterine leiomyomata. Her father and daughter had hyperparathyroidism (parathyroid adenoma) but no other manifestations of HPT-JT. CDC73 mutation analysis (sequencing of all 17 exons) and whole-genome copy number variation (CNV) analysis was done on leukocyte DNA of the three affecteds as well as the proband’s unaffected sister.ResultsA novel deletion of exons 4 to 10 of CDC73 was detected by CNV analysis in the three affecteds. A novel insertion in the 5’UTR (c.-4_-11insG) that co-segregated with the deletion was identified. By in vitro assay the 5’UTR insertion was shown to significantly impair the expression of the parafibromin protein. Screening for the mutated CDC73 confirmed carrier status in the proband’s daughter and the biochemistry and ultrasonography led to pre-emptive surgery and resolution of the hyperparathyroidism.ConclusionsA novel gross deletion mutation in CDC73 was identified in a three-generation HPT-JT family emphasizing the importance of including screening for large deletions in the molecular diagnostic protocol.

Large deletion at the CDC73 gene locus and search for predictive markers of the presence of a CDC73 genetic lesion

The Hyperparathyroidism with Jaw-Tumours syndrome is caused by mutations of the CDC73 gene: it has been suggested that early onset of the disease and high Ca2+ levels may predict the presence of a CDC73 mutation. We searched for large deletions at the CDC73 locus in patients with: HPT-JT (nr 2), atypical adenoma (nr 7) or sporadic parathyroid carcinoma (nr 11) with a specific MLPA and qRT-PCR assays applied on DNA extracted from whole blood. A Medline search in database for all the papers reporting a CDC73 gene mutation, clinical/histological diagnosis, age at onset, Ca2+, PTH levels for familial/sporadic cases was conducted with the aim to possibly identify biochemical/clinical markers predictive, in first diagnosis, of the presence of a CDC73 gene mutation. A novel genomic deletion of the first 10 exons of the CDC73 gene was found in a 3-generation HPT-JT family, confirmed by SNP array analysis. A classification tree built on the published data, showed the highest probability of having a CDC73 mutation in subjects with age at the onset < 41.5 years (44/47 subjects, 93.6%, had the mutation). Whereas the lowest probability was found in subjects with age at the onset ≥ 41.5 years and Ca2+ levels <13.96 mg/dL (7/20 subjects, 35.0%, had the mutation, odds ratio = 27.1, p < 0.001). We report a novel large genomic CDC73 gene deletion identified in an Italian HPT-JT family. Age at onset < 41.5 ys and Ca2+ > 13.96 mg/dL are predictive for the presence of a CDC73 genetic lesion.

Autosomal Dominant PTH Gene Signal Sequence Mutation in a Family With Familial Isolated Hypoparathyroidism

Context: 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 &agr;11 (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. Objective: To clinically describe an FIH family and identify and characterize the causal gene mutation. Design: 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. Participants: Six subjects of a three‐generation FIH family with three affected individuals having severe hypocalcemia and inappropriately low serum PTH. Results: 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. Conclusions: 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.

Erratum: Large intragenic deletion of CDC73 (exons 4-10) in a three-generation hyperparathyroidism-jaw tumor (HPT-JT) syndrome family [BMC Med Genet. 2017;18:83] DOI 10.1186/s12881-017-0445-0

Author details Medical Genetics, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy. Department of Otolaryngology Head and Neck Surgery, University of Toronto, Toronto, ON, Canada. Department of Medicine, University of Toronto, Toronto, ON, Canada. Division of Endocrinology, Women’s College Hospital, Toronto, ON, Canada. Department of Otolaryngology, Head & Neck Surgery, Sunnybrook Health Sciences Centre, Toronto, ON, Canada. Department of Anatomic Pathology, Sunnybrook Health Sciences Centre, Toronto, ON, Canada. Departments of Laboratory Medicine and Pathobiology, Medicine and Genetics, University of Toronto, Toronto, ON, Canada. Endocrinology, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy. Metabolic Disorders and Complications, McGill University Health Centre-Research Institute, Montreal, QC, Canada. Departments of Medicine, Physiology and Human Genetics, McGill University, Montreal, QC, Canada.