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Purpose The study aimed to define the clinical, biochemical and genetic features of adult patients with osteopenia/osteoporosis and/or bone fragility and low serum alkaline phosphatase (sALP). Methods Twenty-two patients with at least two sALP values below the reference range were retrospectively enrolled after exclusion of secondary causes. Data about clinical features, mineral and bone markers, serum pyridoxal-5’-phosphate (PLP), urine phosphoethanolamine (PEA), lumbar and femur bone densitometry, and column X-ray were collected. Peripheral blood DNA of each participant was analyzed to detect ALPL gene anomalies. Results Pathogenic ALPL variants (pALPL) occurred in 23% and benign variants in 36% of patients (bALPL), while nine patients harbored wild-type alleles (wtALPL). Fragility fractures and dental anomalies were more frequent in patients harboring pALPL and bALPL than in wtALPL patients. Of note, wtALPL patients comprised women treated with tamoxifen for hormone-sensitive breast cancer. Mineral and bone markers were similar in the three groups. Mean urine PEA levels were significantly higher in patients harboring pALPL than those detected in patients harboring bALPL and wtALPL; by contrast, serum PLP levels were similar in the three groups. A 6-points score, considering clinical and biochemical features, was predictive of pALPL detection [P = 0.060, OR 1.92 (95% CI 0.972, 3.794)], and more significantly of pALPL or bALPL [P = 0.025, OR 14.33 (95% CI 1.401, 14.605)]. Conclusion In osteopenic/osteoporotic patients, single clinical or biochemical factors did not distinguish hypophosphatasemic patients harboring pALPL or bALPL from those harboring wtALPL. Occurrence of multiple clinical and biochemical features is predictive of ALPL anomalies, and, therefore, they should be carefully identified. Tamoxifen emerged as a hypophosphatasemic drug.

Objective: To provide Italian expert opinion-based practical recommendations to improve the cooperation between clinicians and geneticists in order to optimize diagnostic flow and care of X-linked hypophosphatemia (XLH). Methods: A panel of four geneticists and four clinicians from Italian reference centers for the diagnosis and management of XLH met virtually, first to highlight the critical issues in patient care and then to identify and share proposals to improve the diagnostic and care path of XLH. Results: Critical issues emerged regarding the transfer of adequate clinical information from clinicians to geneticists, standardization and clarity of genetic reporting, and adequate interactions between clinicians and geneticists during patients’ follow-up. The necessary requirements for an appropriate request for evaluation of genetic variants and the need for a clear and clinically useful genetic report were agreed upon. Specifically designed template forms to be adopted with appropriate adjustments were defined and are here proposed for both the clinician’s request and the geneticist’s report. Conclusions: The expert group strongly believes that collaboration between clinicians and geneticists should be encouraged in XLH, not only in the diagnostic phase but also during a patient’s follow-up, in order to manage patients more comprehensively and effectively.

A role for long non-coding RNAs (lncRNAs) in endocrine cancer pathogenesis is emerging. However, knowledge regarding their expression pattern, correlation with known genetic defects, and clinical implications in parathyroid tumors is still unclear. Here, we profiled 90 known lncRNAs in a first series of normal (PaN = 2), adenomatous (PAd = 12), and carcinomatous (PCa = 4) parathyroid glands and we confirmed deregulation of 11 lncRNAs using an independent cohort of patients (PaN = 4; PAd = 26; PCa = 9). Expression of lncRNAs was correlated with cytogenetic aberrations, status of genes multiple endocrine neoplasia 1 (MEN1) and cell division cycle 73 (CDC73), or clinical features. Globally, lncRNAs discriminate according to tissue histology. BC200 consistently identifies parathyroid cancers from adenomas and atypical adenomas. Loss-of-heterozygosity (LOH) at chromosomes 1, 11, 15, 21, and 22 significantly impacts expression of lncRNAs in PAds. Silencing of the key parathyroid gene MEN1 modulates the expression of six lncRNAs in primary PAds-derived cultures. Analogous levels of lncRNAs are measured in PAds with the mutation in the MEN1 gene compared with PAds with wild-type MEN1. Similarly, carcinomas with mutated CDC73 differ from PCas with wild-type protein in terms of expression of lncRNAs. PCas harboring CDC73 mutations overexpress BC200 compared to wild-type carcinomas. Overall, these findings shed light on deregulation of lncRNAs in human parathyroid tumors and propose that circuits between lncRNAs and the oncosuppressors MEN1 or CDC73 may have a role in parathyroid tumorigenesis as epigenetic modulators. © 2020 American Society for Bone and Mineral Research (ASBMR).

L’ipofosfatasia e una malattia multi-sistemica, causata da mutazioni inattivanti del gene ALPL codificante la fosfatasi alcalina, il cui quadro clinico puo esordire a diverse eta e progredire nel tempo. Il fenotipo clinico presenta in genere una severita inversamente correlata all’eta di insorgenza. Da alcuni anni e disponibile una terapia enzimatica sostitutiva che si e dimostrata efficace e sicura, in particolare nelle forme neonatali, infantili e giovanili.

Normocalcemic primary hyperparathyroidism (NPHPT) is diagnosed in the setting of elevated PTH concentrations with consistently normal albumin-adjusted and ionized serum calcium levels, in absence of secondary causes for elevated PTH concentrations. In order to confirm persistence of the hyperparathyroid state, PTH levels should be elevated on at least two occasions over a 3 to 6 months period. The prevalence of NPHPT depends on the population studied. Data from different studies are often not comparable; indeed, different criteria have been used to exclude secondary hyperparathyroidism. Notwithstanding such limits, the prevalence of NPHPT in studies including ionized calcium dosage was between 0.5% and 0.7%. Available data suggest that patients with NPHPT are likely to have more skeletal, kidney and metabolic complications compared to healthy subjects, but almost all studies suffer from possible misclassification of patients due to lack of ionized calcium dosage. The management of NPHPT is controversial in part due to lack of solid data about the natural history. However, surgical treatment is currently performed more frequently than in the past, although studies do not show, so far, a clear benefit from intervention.

Hereditary multiple osteochondromas (HMO) is a rare autosomal dominant skeletal disorder, caused by heterozygous variants in either EXT1 or EXT2, which encode proteins involved in the biogenesis of heparan sulphate. Pathogenesis and genotype-phenotype correlations remain poorly understood. We studied 114 HMO families (158 affected individuals) with causative EXT1 or EXT2 variants identified by Sanger sequencing, or multiplex ligation-dependent probe amplification and qPCR. Eighty-seven disease-causative variants (55 novel and 32 known) were identified including frameshift (42%), nonsense (32%), missense (11%), splicing (10%) variants and genomic rearrangements (5%). Informative clinical features were available for 42 EXT1 and 27 EXT2 subjects. Osteochondromas were more frequent in EXT1 as compared to EXT2 patients. Anatomical distribution of lesions showed significant differences based on causative gene. Microscopy analysis for selected EXT1 and EXT2 variants verified that EXT1 and EXT2 mutants failed to co-localize each other and loss Golgi localization by surrounding the nucleus and/or assuming a diffuse intracellular distribution. In a cell viability study, cells expressing EXT1 and EXT2 mutants proliferated more slowly than cells expressing wild-type proteins. This confirms the physiological relevance of EXT1 and EXT2 Golgi co-localization and the key role of these proteins in the cell cycle. Taken together, our data expand genotype-phenotype correlations, offer further insights in the pathogenesis of HMO and open the path to future therapies.

Brown tumors are osteolytic lesions associated with hyperparathyroidism (HPT). They may involve various skeletal segments, but rarely the cranio-facial bones. We report a case of a young boy with a swelling of the jaw secondary to a brown tumor presenting as the first manifestation of primary HPT (PHPT). He was found to have brown tumor located in the skull, as well. Different imaging technologies were employed for the diagnosis and follow-up after parathyroidectomy. We enclose a review of the literature on the employment of such imaging technologies in the differential diagnosis of osteolytic lesions. A multidisciplinary approach comprising clinical, laboratory and imaging findings is essential for the differential diagnosis of brown tumor in PHPT., Highlights • Brown tumors are associated with more severe hyperparathyroidism (HPT). • The case of a young patient with primary HPT and brown tumors is reported. • A multidisciplinary approach should be employed for the differential diagnosis. • We reviewed the main radiological characteristics of different types of osteolytic lesions.

Objective The aim of this study was to analyze variants of the gene glial cells missing-2 (GCM2), encoding a parathyroid cell-specific transcription factor, in familial hypoparathyroidism and in familial isolated hyperparathyroidism (FIHP) without and with parathyroid carcinoma. Design We characterized 2 families with hypoparathyroidism and 19 with FIHP in which we examined the mechanism of action of GCM2 variants. Methods Leukocyte DNA of hypoparathyroid individuals was Sanger sequenced for CASR, PTH, GNA11 and GCM2 mutations. DNA of hyperparathyroid individuals underwent MEN1, CDKN1B, CDC73, CASR, RET and GCM2 sequencing. The actions of identified GCM2 variants were evaluated by in vitro functional analyses. Results A novel homozygous p.R67C GCM2 mutation which failed to stimulate transcriptional activity in a luciferase assay was identified in affected members of two hypoparathyroid families. Oligonucleotide pull-down assay and in silico structural modeling indicated that this mutant had lost the ability to bind the consensus GCM recognition sequence of DNA. Two novel (p.I383M and p.T386S) and one previously reported (p.Y394S) heterozygous GCM2 variants that lie within a C-terminal conserved inhibitory domain were identified in three affected individuals of the hyperparathyroid families. One family member, heterozygous for p.I138M, had parathyroid carcinoma (PC), and a heterozygous p.V382M variant was found in another patient affected by sporadic PC. These variants exerted significantly enhanced in vitrotranscriptional activity, including increased stimulation of the PTH promoter. Conclusions We provide evidence that two novel GCM2 R67C inactivating mutations with an inability to bind DNA are causative of hypoparathyroidism. Additionally, we provide evidence that two novel GCM2 variants increased transactivation of the PTH promoter in vitro and are associated with FIHP. Furthermore, our studies suggest that activating GCM2 variants may contribute to facilitating more aggressive parathyroid disease.

Background. Autoimmune polyglandular syndrome type 1 (APS-1) with or without reversible metaphyseal dysplasia is a rare genetic disorder due to inactivating variants of the autoimmune regulator, AIRE, gene. Clinical variability of APS-1 relates to pleiotropy, and the general dysfunction of self-tolerance to organ-specific antigens and autoimmune reactions towards peripheral tissues caused by the underlying molecular defect. Thus, early recognition of the syndrome is often delayed, mostly in cases with atypical presentation, and the molecular confirm through the genetic analysis of the AIRE gene might be of great benefit. Methods. Our methods were to investigate, with a multigene panel next generation sequencing approach, two clinical cases, both presenting with idiopathic hypoparathyroidism, also comprising the AIRE gene, as well as to comment our findings as part of a more extensive review of literature data. Results. In the first clinical case, two compound heterozygote pathogenic variants of the AIRE gene were identified, thus indicating an autosomal recessive inheritance of the disease. In the second case, only one AIRE gene variant was found and an atypical dominant negative form of APS-1 suggested, later confirmed by further medical ascertainments. Conclusions. APS-1 might present with variable and sometimes monosymptomatic presentations and, if not recognized, might associate with severe complications. In this context, next generation diagnostics focused on a set of genes causative of partially overlapping disorders may allow early diagnosis.

Stickler syndrome (SS) is a hereditary connective tissue disorder affecting bones, eyes, and hearing. Type 2 SS and the SS variant otospondylomegaepiphyseal dysplasia (OSMED) are caused by deleterious variants in COL11A1 and COL11A2, respectively. In both genes, available database information indicates a high rate of potentially deleterious intronic variants, but published evidence of their biological effect is usually insufficient for a definite clinical interpretation. We report four previously unpublished intronic variants in COL11A1 (c.2241 + 5G>T, c.2809 − 2A>G, c.3168 + 5G>C) and COL11A2 (c.4392 + 1G>A) identified in type 2 SS/OSMED individuals. The pathogenic effect of these variants was first predicted in silico and then investigated by an exon-trapping assay. We demonstrated that all variants can induce exon in-frame deletions, which lead to the synthesis of shorter collagen XI α1 or 2 chains. Lacking residues are located in the α-triple helical region, which has a crucial role in regulating collagen fibrillogenesis. In conclusion, this study suggests that these alternative COL11A1 and COL11A2 transcripts might result in aberrant triple helix collagen. Our approach may help to improve the diagnostic molecular pathway of COL11-related disorders.

A role for long non-coding RNAs (lncRNAs) in endocrine cancer pathogenesis is emerging. However, knowledge regarding their expression pattern, correlation with known genetic defects, and clinical implications in parathyroid tumors is still unclear. Here, we profiled 90 known lncRNAs in a first series of normal (PaN = 2), adenomatous (PAd = 12), and carcinomatous (PCa = 4) parathyroid glands and we confirmed deregulation of 11 lncRNAs using an independent cohort of patients (PaN = 4; PAd = 26; PCa = 9). Expression of lncRNAs was correlated with cytogenetic aberrations, status of genes multiple endocrine neoplasia 1 (MEN1) and cell division cycle 73 (CDC73), or clinical features. Globally, lncRNAs discriminate according to tissue histology. BC200 consistently identifies parathyroid cancers from adenomas and atypical adenomas. Loss-of-heterozygosity (LOH) at chromosomes 1, 11, 15, 21, and 22 significantly impacts expression of lncRNAs in PAds. Silencing of the key parathyroid gene MEN1 modulates the expression of six lncRNAs in primary PAds-derived cultures. Analogous levels of lncRNAs are measured in PAds with the mutation in the MEN1 gene compared with PAds with wild-type MEN1. Similarly, carcinomas with mutated CDC73 differ from PCas with wild-type protein in terms of expression of lncRNAs. PCas harboring CDC73 mutations overexpress BC200 compared to wild-type carcinomas. Overall, these findings shed light on deregulation of lncRNAs in human parathyroid tumors and propose that circuits between lncRNAs and the oncosuppressors MEN1 or CDC73 may have a role in parathyroid tumorigenesis as epigenetic modulators. © 2020 American Society for Bone and Mineral Research (ASBMR).

Objective. Atypical parathyroid adenoma is a rare neoplasm, showing atypical histological features intermediate between classic benign adenoma and the rarest parathyroid carcinoma, whose the clinical behaviour and outcome is not yet understood or predictable. Up to date only two cases of atypical adenoma were found associated to a MEN1 syndrome, and only one was proved to carry a pathogenic variant of the MEN1 gene. Design. We report the clinical, histologic, and molecular findings of a 44-year-old woman, presenting with a histologically proved atypical parathyroid adenoma with an apparent aggressive behaviour. Methods and Results. CDC73 gene was screened at germline and somatic levels with no results. Whole exome sequencing performed on DNA extracted from blood leukocytes and tumour tissue revealed a somatic MEN1 gene heterozygous variant, c.912+1G > A, of the splicing donor site of exon 6. On immunohistochemistry, downregulation of the menin protein expression in the neoplastic cells was also observed. Conclusions. We report the second case of a rare association of a somatic MEN1 gene mutation in a patient with atypical parathyroid adenoma. We suggest that MEN1 gene could be an underestimate genetic determinant of these rare histological entities, and we highlight the utility of a complete genetic screening protocol, by the use of next-generation sequencing technology in such undetermined clinical cases with no frank clinical presentation.

Stickler syndrome (SS) is a hereditary connective tissue disorder affecting bones, eyes, and hearing. Type 2 SS and the SS variant otospondylomegaepiphyseal dysplasia (OSMED) are caused by deleterious variants in COL11A1 and COL11A2, respectively. In both genes, available database information indicates a high rate of potentially deleterious intronic variants, but published evidence of their biological effect is usually insufficient for a definite clinical interpretation. We report four previously unpublished intronic variants in COL11A1 (c.2241 + 5G&gt, T, c.2809 &minus, 2A&gt, G, c.3168 + 5G&gt, C) and COL11A2 (c.4392 + 1G&gt, A) identified in type 2 SS/OSMED individuals. The pathogenic effect of these variants was first predicted in silico and then investigated by an exon-trapping assay. We demonstrated that all variants can induce exon in-frame deletions, which lead to the synthesis of shorter collagen XI &alpha, 1 or 2 chains. Lacking residues are located in the &alpha, triple helical region, which has a crucial role in regulating collagen fibrillogenesis. In conclusion, this study suggests that these alternative COL11A1 and COL11A2 transcripts might result in aberrant triple helix collagen. Our approach may help to improve the diagnostic molecular pathway of COL11-related disorders.

Context Paget disease of bone (PDB) is a metabolic bone disease whose genetic cause remains unknown in up to 50% of familial patients. Objective Our aim was to investigate the underlying genetic defect in a large pedigree with a severe, early onset, autosomal dominant form of PDB across 3 generations. Methods Whole exome sequencing was performed in affected and unaffected family members, and then mutation screening was replicated in a sample of PDB patients with early-onset, polyostotic PDB. Results We identified a frameshift D107Rfs*3 mutation in PFN1 (encoding for profilin 1, a highly conserved regulator of actin-polymerization and cell motility) causing the truncation of the C-terminal part of the protein. The mutation was also detected in a 17-year-old asymptomatic family member who upon biochemical and radiological analyses was indeed found to be affected. Sequencing of the entire PFN1 coding region in unrelated PDB patients identified the same mutation in 1 patient. All mutation carriers had a reduced response to bisphosphonates, requiring multiple zoledronate infusions to control bone pain and achieve biochemical remission over a long term. In vitro osteoclastogenesis in peripheral blood mononuclear cells (PBMCs) from mutation carriers showed a higher number of osteoclasts with PDB-like features. A similar phenotype was observed upon PFN1 silencing in murine bone marrow-derived monocytes, suggesting that the frameshift PFN1 mutation confers a loss of function in profilin 1 activity that induces PDB-like features in the osteoclasts, likely due to enhanced cell motility and actin ring formation. Conclusions Our findings indicate that PFN1 mutation causes an early onset, polyostotic PDB-like disorder.

Parathyroid carcinoma (PC) is a rare disease with an indolent behavior due to the low malignant potential. The etiology is unknown. Somatic mutations of CDC73 gene, the same gene involved in the hyperparathyroidism-jaw tumor syndrome, can be identified in up to 70% of patients with PC and in one-third of cases the mutations are germline. Therefore, in patients who carry germline CDC73 gene mutations, its finding permits to identify the carriers among relatives and sometimes to early detect a parathyroid lesion in such subjects. The diagnosis of PC is commonly made after surgery, however there are some clinical/biochemical features that should raise the suspicion of PC, namely markedly elevated serum calcium and PTH levels, a large parathyroid lesion with suspected ultrasonographic features of malignancy, the damages of kidney and bones. The best chance of cure is the complete surgical resection with the en-bloc excision at the first operation, however several recurrences are often observed during the follow-up. Since PC is an indolent tumor with long-lasting survival and the death is due to complications of untreatable hypercalcemia, multiple surgical interventions with debulking of tumoral tissues along with medical treatment for reducing hypercalcemia are often needed. Patients with PC should be followed up along their lifetime.

Parathyroid tumors deregulate microRNAs belonging to the two clusters on the chromosome 19, the C19MC and miR-371-373 clusters. Here, we report that the embryonic miR-372 is aberrantly expressed in half of parathyroid adenomas (PAds) in most of atypical adenomas and carcinomas (n = 15). Throughin situhybridization, we identified that miR-372-positive parathyroid tumor cells were scattered throughout the tumor parenchyma. In PAd-derived cells, ectopic miR-372 inhibited the expression of its targetsCDKN1A/p21 and LATS2 at both mRNA and protein levels. Although the viability of parathyroid cells was not affected by miR-372 overexpression, the miRNA blunted camptothecin-induced apoptosis in primary PAd-derived cultures. miR-372 overexpression in parathyroid tumor cells increased parathormone (PTH) mRNA levels, and it positively correlatedin vivowith circulating PTH levels. Conversely, the parathyroid-specific genesTBX1andGCM2were not affected by miR-372 mimic transfection. Finally, miR-372 dampened the Wnt pathway in parathyroid tumor cells through DKK1 upregulation. In conclusion, miR-372 is a novel mechanism exploited by a subset of parathyroid tumor cells to partially decrease sensitivity to apoptosis, to increase PTH synthesis and to deregulate Wnt signaling.

Transcription factors active in embryonic parathyroid cells can be maintained in adult parathyroids and be involved in tumorigenesis. TBX1, the candidate gene of 22q11.2-DiGeorge syndrome, which includes congenital hypoparathyroidism, is involved in parathyroid embryogenesis. The study aimed to investigate expression, function, and regulation of the parathyroid embryonic transcription factor TBX1 in human parathyroid adult normal and tumor tissues. TBX1 transcripts were detected in normal parathyroids and were deregulated in parathyroid tumors. Using immunohistochemistry, TBX1 protein was detected, mainly at the nuclear level, in a consistent proportion of cells in normal adult parathyroids, whereas TBX1 immunoreactivity was absent in fetal parathyroids. TBX1-expressing cells were markedly reduced in about a half of adenomas (PAds) and two-thirds of carcinomas and the proportion of TBX1-expressing cells negatively correlated with the serum albumin-corrected calcium levels in the analyzed tumors. Moreover, a subset of TBX1-expressing tumor cells coexpressed PTH. TBX1 silencing in HEK293 cells, expressing endogenous TBX1, increased the proportion of cells in the G0/G1 phase of cell cycle; concomitantly, CDKN1A/p21 and CDKN2A/p16 transcripts increased and ID1 mRNA levels decreased. TBX1 silencing exerted similar effects in PAd-derived cells, suggesting cell cycle arrest. Moreover, in PAd-derived cells GCM2 and PTH mRNA levels were unaffected by TBX1 deficiency, whereas it was associated with reduction of WNT5A, an antagonist of canonical WNT/β-catenin pathway. WNT/β-catenin activation by lithium chloride inhibited TBX1 expression levels both in HEK293 and PAd-derived cells. In conclusion, TBX1 is expressed in adult parathyroid cells and deregulated in parathyroid tumors, where TBX1 deficiency may potentially contribute to the low proliferative nature of parathyroid tumors.

Background The occurrence of parathyroid carcinoma in multiple endocrine neoplasia type I (MENI) is rare and the 15 cases of malignant parathyroid tumor reported so far have been associated with MENI in individuals and not with multiple members within a family. Methods We report on a 61-year-old male, operated for a 7.3 cm parathyroid carcinoma infiltrating the esophagus. In his brother, a 4.6 cm parathyroid carcinoma was diagnosed histologically, while in the daughter, neck ultrasonography revealed 2 extrathyroidal nodules, yet to be excised. Results Screening of the MEN1 gene identified a known germline heterozygous missense mutation (c.1252G>A; p.D418N) in exon 9, in all affected subjects. Conclusions The occurrence of parathyroid carcinoma in more than one affected member of a single MEN1 family represents the first reported familial case. This suggests that additional constitutional genetic mutations may contribute to the variation in malignant potential and clinical behavior of parathyroid tumors in MEN1.

TNXB-related classical-like Ehlers-Danlos syndrome (TNXB-clEDS) is an ultrarare type of Ehlers-Danlos syndrome due to biallelic null variants in TNXB, encoding tenascin-X. Less than 30 individuals have been reported to date, mostly of Dutch origin and showing a phenotype resembling classical Ehlers-Danlos syndrome without atrophic scarring. TNXB-clEDS is likely underdiagnosed due to the complex structure of the TNXB locus, a fact that complicates diagnostic molecular testing. Here, we report two unrelated Italian women with TNXB-clEDS due to compound heterozygosity for null alleles in TNXB. Both presented soft and hyperextensible skin, generalized joint hypermobility and related musculoskeletal complications, and chronic constipation. In addition, individual 1 showed progressive finger contractures and shortened metatarsals, while individual 2 manifested recurrent subconjunctival hemorrhages and an event of spontaneous rupture of the brachial vein. Molecular testing found the two previously unreported c.8278C &gt, T p.(Gln2760*) and the c.(2358 + 1_2359 &minus, 1)_(2779 + 1_2780 &minus, 1)del variants in Individual 1, and the novel c.1150dupG p.(Glu384Glyfs*57) and the recurrent c.11435_11524+30del variants in Individual 2. mRNA analysis confirmed that the c.(2358 + 1_2359 &minus, 1)del variant causes a frameshift leading to a predicted truncated protein [p.(Thr787Glyfs*40)]. This study refines the phenotype recently delineated in association with biallelic null alleles in TNXB, and adds three novel variants to its mutational repertoire. Unusual digital anomalies seem confirmed as possibly peculiar of TNXB-clEDS, while vascular fragility could be more than a chance association also in this Ehlers-Danlos syndrome type.

The Von Hippel-Lindau (VHL) syndrome has been rarely associated with renal oncocytomas, and tumors usually show HIF1α chronic stabilization. By contrast, oncocytomas mainly associated with respiratory chain (RC) defects due to severe mitochondrial DNA (mtDNA) mutations are incapable of stabilizing HIF1α, since oxygen consumption by the RC is dramatically diminished and prolylhydroxylase activity is increased by α-ketoglutarate accumulation following Krebs cycle slowdown. Here, we investigate the cooccurrence of a pseudohypoxic condition with oncocytic transformation in a case of VHL-associated renal oncocytoma. While HIF1α was abundant in nuclei concordantly with defects in VHL, negative staining of its targets carbonic anhydrase IX (CAIX) and glucose transporter GLUT1, usually overexpressed in VHL-associated neoplasms, suggested HIF1α to be present in its inactive (hydroxylated) form. MtDNA sequencing and immunohistochemistry analyses revealed a MT-CO1 stop-gain mutation and cytochrome c oxidase loss. We suggest that a mitochondrial respiration impairment may lead to hyperhydroxylation of the transcription factor, which we confirmed by specific staining of hydroxylated HIF1α. Such inactive form hence accumulated in the VHL-deficient tumor, where it may contribute to the benign nature of the neoplasm. We propose that the protumorigenic role of HIF1α in VHL cancers may be blunted through drugs inhibiting mitochondrial respiratory complexes, such as metformin.

Familial cerebral cavernous malformation (FCCM) is an autosomal dominant vascular disorder caused by heterozygous deleterious variants in KRIT1, CCM2 or PDCD10. In a previous study, we presented the clinical and molecular findings in 140 FCCM individuals. In the present work, we report supporting information on (a) applied diagnostic workflow; (b) clinical significance of molecular findings according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology recommendations; (c) standardization of molecular and clinical data according to the Human Phenotype Ontology; (d) preliminary genotype-phenotype correlations on a subgroup of patients by considering sex, age at diagnosis, neurological symptoms, and number and anatomical site(s) of vascular anomalies; (e) datasets submitted to the Leiden Open Variation Database. An overview of the changes of our diagnostic approach before and after the transition to next-generation sequencing is also reported. This work presents the full procedure that we apply for molecular testing, data interpretation and storing in public databases in FCCM.

The Hippo pathway is involved in human tumorigenesis and tissue repair. Here, we investigated the Hippo coactivator Yes-associated protein 1 (YAP1) and the kinase large tumor suppressor 1/2 (LATS1/2) in tumors of the parathyroid glands, which are almost invariably associated with primary hyperparathyroidism. Compared with normal parathyroid glands, parathyroid adenomas (PAds) and carcinomas show variably but reduced nuclear YAP1 expression. The kinase LATS1/2, which phosphorylates YAP1 thus promoting its degradation, was also variably reduced in PAds. Further, YAP1 silencing reduces the expression of the key parathyroid oncosuppressor multiple endocrine neoplasia type 1(MEN1), while MEN1 silencing increases YAP1 expression. Treatment of patient-derived PAds-primary cell cultures and Human embryonic kidney 293A (HEK293A) cells expressing the calcium-sensing receptor (CASR) with the CASR agonist R568 induces YAP1 nuclear accumulation. This effect was prevented by the incubation of the cells with RhoA/Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitors Y27632 and H1152. Lastly, CASR activation increased the expression of the YAP1 gene targets CYR61, CTGF, and WNT5A, and this effect was blunted by YAP1 silencing. Concluding, here we provide preliminary evidence of the involvement of the Hippo pathway in human tumor parathyroid cells and of the existence of a CASR-ROCK-YAP1 axis. We propose a tumor suppressor role for YAP1 and LATS1/2 in parathyroid tumors.

Several studies reported somatic mutations of many genes (MEN1, CTNNB1, CDKIs and others) in parathyroid adenoma, although with different prevalence. Recently, activating mutations of the EZH2 and ZFX oncogenes were identified in benign parathyroid adenoma by whole exome sequencing. The same mutations had been found in blood and ovary malignant tumours. On one hand, this result raised the hypothesis that these oncogenes may play a role in the onset of parathyroid tumour, but it would also suggest they may be involved in malignant, rather benign, parathyroid neoplasm. Our aim was to verify the occurrence of selected mutations of the EZH2 and ZFX genes in an Italian cohort of 23 sporadic parathyroid carcinomas, 12 atypical and 45 typical adenomas. DNA was extracted from paraffin-embedded tissues, PCR amplified and directly sequenced. No mutations were detected in the coding sequence and boundaries of both genes in any of the samples. Two polymorphisms of the EZH2 gene were identified with different prevalence: the rs2072407 variant was present in the 30 % of the samples, in keeping with the overall frequency in larger populations, while the rs78589034 variant, located close to the 5' end of the exon 16, was detected in only one proband with familial isolated hyperparathyroidism; we investigated the possible outcome on the splicing process. EZH2 and ZFX genes do not seem to have an impact on the onset of most parathyroid tumours, both benign and malignant, though further studies on larger cohorts of different ethnicity are needed.