Cullin 3 Exon 9 Deletion in Familial Hyperkalemic Hypertension Impairs Cullin3-Ring-E3 Ligase (CRL3) Dynamic Regulation and Cycling.

Cullin 3 (CUL3) is the scaffold of Cullin3 Ring E3-ligases (CRL3s), which use various BTB-adaptor proteins to ubiquitinate numerous substrates targeting their proteasomal degradation. CUL3 mutations, responsible for a severe form of familial hyperkalemia and hypertension (FHHt), all result in a deletion of exon 9 (amino-acids 403-459) (CUL3-∆9). Surprisingly, while CUL3-∆9 is hyperneddylated, a post-translational modification that typically activates CRL complexes, it is unable to ubiquitinate its substrates. In order to understand the mechanisms behind this loss-of function, we performed comparative label-free quantitative analyses of CUL3 and CUL3-∆9 interactome by mass spectrometry. It was observed that CUL3-∆9 interactions with COP9 and CAND1, both involved in CRL3 complexes' dynamic assembly, were disrupted. These defects result in a reduction in the dynamic cycling of the CRL3 complexes, making the CRL3-∆9 complex an inactive BTB-adaptor trap, as demonstrated by SILAC experiments. Collectively, the data indicated that the hyperneddylated CUL3-∆9 protein is inactive as a consequence of several structural changes disrupting its dynamic interactions with key regulatory partners.

TREM-1 orchestrates angiotensin II-induced monocyte trafficking and promotes experimental abdominal aortic aneurysm.

The triggering receptor expressed on myeloid cells 1 (TREM-1) drives inflammatory responses in several cardiovascular diseases but its role in abdominal aortic aneurysm (AAA) remains unknown. Our objective was to explore the role of TREM-1 in a mouse model of angiotensin II-induced (AngII-induced) AAA. TREM-1 expression was detected in mouse aortic aneurysm and colocalized with macrophages. Trem1 gene deletion (Apoe-/-Trem1-/-), as well as TREM-1 pharmacological blockade with LR-12 peptide, limited both AAA development and severity. Trem1 gene deletion attenuated the inflammatory response in the aorta, with a reduction of Il1b, Tnfa, Mmp2, and Mmp9 mRNA expression, and led to a decreased macrophage content due to a reduction of Ly6Chi classical monocyte trafficking. Conversely, antibody-mediated TREM-1 stimulation exacerbated Ly6Chi monocyte aorta infiltration after AngII infusion through CD62L upregulation and promoted proinflammatory signature in the aorta, resulting in worsening AAA severity. AngII infusion stimulated TREM-1 expression and activation on Ly6Chi monocytes through AngII receptor type I (AT1R). In human AAA, TREM-1 was detected and TREM1 mRNA expression correlated with SELL mRNA expression. Finally, circulating levels of sTREM-1 were increased in patients with AAA when compared with patients without AAA. In conclusion, TREM-1 is involved in AAA pathophysiology and may represent a promising therapeutic target in humans.

Mutation affecting the conserved acidic WNK1 motif causes inherited hyperkalemic hyperchloremic acidosis.

Gain-of-function mutations in with no lysine (K) 1 (WNK1) and WNK4 genes are responsible for familial hyperkalemic hypertension (FHHt), a rare, inherited disorder characterized by arterial hypertension and hyperkalemia with metabolic acidosis. More recently, FHHt-causing mutations in the Kelch-like 3-Cullin 3 (KLHL3-CUL3) E3 ubiquitin ligase complex have shed light on the importance of WNK's cellular degradation on renal ion transport. Using full exome sequencing for a 4-generation family and then targeted sequencing in other suspected cases, we have identified new missense variants in the WNK1 gene clustering in the short conserved acidic motif known to interact with the KLHL3-CUL3 ubiquitin complex. Affected subjects had an early onset of a hyperkalemic hyperchloremic phenotype, but normal blood pressure values"Functional experiments in Xenopus laevis oocytes and HEK293T cells demonstrated that these mutations strongly decrease the ubiquitination of the kidney-specific isoform KS-WNK1 by the KLHL3-CUL3 complex rather than the long ubiquitous catalytically active L-WNK1 isoform. A corresponding CRISPR/Cas9 engineered mouse model recapitulated both the clinical and biological phenotypes. Renal investigations showed increased activation of the Ste20 proline alanine-rich kinase-Na+-Cl- cotransporter (SPAK-NCC) phosphorylation cascade, associated with impaired ROMK apical expression in the distal part of the renal tubule. Together, these new WNK1 genetic variants highlight the importance of the KS-WNK1 isoform abundance on potassium homeostasis.

Genetic, structural, and functional characterization of POLE polymerase proofreading variants allows cancer risk prediction.

PURPOSE: Polymerase proofreading-associated polyposis is a dominantly inherited colorectal cancer syndrome caused by exonuclease domain missense variants in the DNA polymerases POLE and POLD1. Manifestations may also include malignancies at extracolonic sites. Cancer risks in this syndrome are not yet accurately quantified. METHODS: We sequenced POLE and POLD1 exonuclease domains in 354 individuals with early/familial colorectal cancer (CRC) or adenomatous polyposis. We assessed the pathogenicity of POLE variants with yeast fluctuation assays and structural modeling. We estimated the penetrance function for each cancer site in variant carriers with a previously published nonparametric method based on survival analysis approach, able to manage unknown genotypes. RESULTS: Pathogenic POLE exonuclease domain variants P286L, M294R, P324L, N363K, D368N, L424V, K425R, and P436S were found in ten families. The estimated cumulative risk of CRC at 30, 50, and 70 years was 11.1% (95% confidence interval [CI]: 4.2-17.5), 48.5% (33.2-60.3), and 74% (51.6-86.1). Cumulative risk of glioblastoma was 18.7% (3.2-25.8) at 70 years. Variants interfering with DNA binding (P286L and N363K) had a significantly higher mutagenic effect than variants disrupting ion metal coordination at the exonuclease site. CONCLUSION: The risk estimates derived from this study provide a rational basis on which to provide genetic counseling to POLE variant carriers.

Cullin 3 targets the tumor suppressor gene ARMC5 for ubiquitination and degradation.

ARMC5 (Armadillo repeat containing 5 gene) was identified as a new tumor suppressor gene responsible for hereditary adrenocortical tumors and meningiomas. ARMC5 is ubiquitously expressed and encodes a protein which contains a N-terminal Armadillo repeat domain and a C-terminal BTB (Bric-a-Brac, Tramtrack and Broad-complex) domain, both docking platforms for numerous proteins. At present, expression regulation and mechanisms of action of ARMC5 are almost unknown. In this study, we showed that ARMC5 interacts with CUL3 requiring its BTB domain. This interaction leads to ARMC5 ubiquitination and further degradation by the proteasome. ARMC5 alters cell cycle (G1/S phases and cyclin E accumulation) and this effect is blocked by CUL3. Moreover, missense mutants in the BTB domain of ARMC5, identified in patients with multiple adrenocortical tumors, are neither able to interact and be degraded by CUL3/proteasome nor alter cell cycle. These data show a new mechanism of regulation of the ARMC5 protein and open new perspectives in the understanding of its tumor suppressor activity.

Severe Arterial Hypertension from Cullin 3 Mutations Is Caused by Both Renal and Vascular Effects.

BACKGROUND: Mutations in four genes, WNK lysine deficient protein kinase 1 and 4 (WNK1 and WNK4), kelch like family member 3 (KLHL3), or Cullin 3 (CUL3), can result in familial hyperkalemic hypertension (FHHt), a rare Mendelian form of human arterial hypertension. Although all mutations result in an increased abundance of WNK1 or WNK4, all FHHt-causing CUL3 mutations, resulting in the skipping of exon 9, lead to a more severe phenotype. METHODS: We created and compared two mouse models, one expressing the mutant Cul3 protein ubiquitously (pgk-Cul3∆9) and the other specifically in vascular smooth muscle cells (SM22-Cul3∆9). We conducted pharmacologic investigations on isolated aortas and generated stable and inducible HEK293 cell lines that overexpress the wild-type Cul3 or mutant Cul3 (Cul3∆9) protein. RESULTS: As expected, pgk-Cul3∆9 mice showed marked hypertension with significant hyperkalemia, hyperchloremia and low renin. BP increased significantly in SM22-Cul3∆9 mice, independent of any measurable effect on renal transport. Only pgk-Cul3∆9 mice displayed increased expression of the sodium chloride cotransporter and phosphorylation by the WNK-SPAK kinases. Both models showed altered reactivity of isolated aortas to phenylephrine and acetylcholine, as well as marked acute BP sensitivity to the calcium channel blocker amlodipine. Aortas from SM22-Cul3∆9 mice showed increased expression of RhoA, a key molecule involved in regulation of vascular tone, compared with aortas from control mice. We also observed increased RhoA abundance and t1/2 in Cul3∆9-expressing cells, caused by decreased ubiquitination. CONCLUSIONS: Mutations in Cul3 cause severe hypertension by affecting both renal and vascular function, the latter being associated with activation of RhoA.

Adrenal GIPR expression and chromosome 19q13 microduplications in GIP-dependent Cushing's syndrome.

GIP-dependent Cushing's syndrome is caused by ectopic expression of glucose-dependent insulinotropic polypeptide receptor (GIPR) in cortisol-producing adrenal adenomas or in bilateral macronodular adrenal hyperplasias. Molecular mechanisms leading to ectopic GIPR expression in adrenal tissue are not known. Here we performed molecular analyses on adrenocortical adenomas and bilateral macronodular adrenal hyperplasias obtained from 14 patients with GIP-dependent adrenal Cushing's syndrome and one patient with GIP-dependent aldosteronism. GIPR expression in all adenoma and hyperplasia samples occurred through transcriptional activation of a single allele of the GIPR gene. While no abnormality was detected in proximal GIPR promoter methylation, we identified somatic duplications in chromosome region 19q13.32 containing the GIPR locus in the adrenocortical lesions derived from 3 patients. In 2 adenoma samples, the duplicated 19q13.32 region was rearranged with other chromosome regions, whereas a single tissue sample with hyperplasia had a 19q duplication only. We demonstrated that juxtaposition with cis-acting regulatory sequences such as glucocorticoid response elements in the newly identified genomic environment drives abnormal expression of the translocated GIPR allele in adenoma cells. Altogether, our results provide insight into the molecular pathogenesis of GIP-dependent Cushing's syndrome, occurring through monoallelic transcriptional activation of GIPR driven in some adrenal lesions by structural variations.

Familial small-intestine carcinoids: Chromosomal alterations and germline inositol polyphosphate multikinase sequencing.

BACKGROUND: Familial small-intestine neuroendocrine tumors (SI-NETs) are an exceptional inherited entity. Underlying predisposing mechanisms are unelucidated, but inositol polyphosphate multikinase (IPMK) gene alterations might promote their tumorigenesis. METHODS: A retrospective-prospective nationwide cohort was constituted, by including patients with proven SI-NETs and at least one relative with the same disease. We performed constitutional and somatic IPMK sequencing, and somatic DNA comparative genomic hybridization (CGH). RESULTS: We included 17 patients from 8 families, who were characterized by high prevalence (57%) of multiple SI-NETs, and high frequency of distant metastases (82%) and carcinoid syndrome (65%). No IPMK mutation was found in constitutional or tumor DNA. CGH array revealed recurrent chromosome-18 deletions but no alteration in the IPMK region. CONCLUSION: We report here the first European series of patients with familial SI-NETs. Predisposing mechanisms may not involve the IPMK-encoding sequence or chromosomal region and might not differ from those of sporadic SI-NETs.

Knock-In of the Recurrent R368X Mutation of PRKAR1A that Represses cAMP-Dependent Protein Kinase A Activation: A Model of Type 1 Acrodysostosis.

In humans, activating mutations in the PRKAR1A gene cause acrodysostosis 1 (ACRDYS1). These mutations result in a reduction in PKA activation caused by an impaired ability of cAMP to dissociate mutant PRKAR1A from catalytic PKA subunits. Two striking features of this rare developmental disease are renal resistance to PTH and chondrodysplasia resulting from the constitutive inhibition of PTHR1/Gsa/AC/cAMP/PKA signaling. We developed a knock-in of the recurrent ACRDYS1 R368X PRKAR1A mutation in the mouse. No litters were obtained from [R368X]/[+] females (thus no homozygous [R368X]/[R368X] mice). In [R368X]/[+] mice, Western blot analysis confirmed mutant allele heterozygous expression. Growth retardation, peripheral acrodysostosis (including brachydactyly affecting all digits), and facial dysostosis were shown in [R368X]/[+] mice by weight curves and skeletal measurements (µCT scan) as a function of time. [R368X]/[+] male and female mice were similarly affected. Unexpected, however, whole-mount skeletal preparations revealed a striking delay in mineralization in newborn mutant mice, accompanied by a decrease in the height of terminal hypertrophic chondrocyte layer, an increase in the height of columnar proliferative prehypertrophic chondrocyte layer, and changes in the number and spatial arrangement of proliferating cell nuclear antigen (PCNA)-positive chondrocytes. Plasma PTH and basal urinary cAMP were significantly higher in [R368X]/[+] compared to WT mice. PTH injection increased urinary cAMP similarly in [R368X]/[+] and WT mice. PRKACA expression was regulated in a tissue (kidney not bone and liver) manner. This model, the first describing the germline expression of a PRKAR1A mutation causing dominant repression of cAMP-dependent PKA, reproduced the main features of ACRDYS1 in humans. It should help decipher the specificity of the cAMP/PKA signaling pathway, crucial for numerous stimuli. In addition, our results indicate that PRKAR1A, by tempering intracellular cAMP levels, is a molecular switch at the crossroads of signaling pathways regulating chondrocyte proliferation and differentiation. © 2016 American Society for Bone and Mineral Research.

DNA Methylation Is an Independent Prognostic Marker of Survival in Adrenocortical Cancer.

CONTEXT: Adrenocortical cancer (ACC) is an aggressive tumor with a heterogeneous outcome. Prognostic stratification is difficult even based on tumor stage and Ki67. Recently integrated genomics studies have demonstrated that CpG islands hypermethylation is correlated with poor survival. OBJECTIVE: The goal of this study was to confirm the prognostic value of CpG islands methylation on an independent cohort. DESIGN: Methylation was measured by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). SETTING: MS-MLPA was performed in a training cohort of 50 patients with ACC to identify the best set of probes correlating with disease-free survival (DFS) and overall survival (OS). These outcomes were validated in an independent cohort from 21 ENSAT centers. PATIENTS: The validation cohort included 203 patients (64% women, median age 50 years, 80% localized tumors). MAIN OUTCOME MEASURES: DFS and OS. RESULTS: In the training cohort, mean methylation of 4 genes (PAX5, GSTP1, PYCARD, PAX6) was the strongest methylation marker. In the validation cohort, methylation was a significant prognostic factor of DFS (P < 0.0001) and OS (P < 0.0001). Methylation, Ki67, and ENSAT stage were combined in multivariate models. For DFS, methylation (P = 0.0005) and stage (P < 0.0001) but not Ki67 (P = 0.19) remained highly significant. For OS, methylation (P = 0.0006), stage (P < 0.0001), and Ki67 (P = 0.024) were independent prognostic factors. CONCLUSIONS: Tumor DNA methylation emerges as an independent prognostic factor in ACC. MS-MLPA is readily compatible with clinical routine and should enhance our ability for prognostication and precision medicine.

Aldosterone-Producing Adenoma With a Somatic KCNJ5 Mutation Revealing APC-Dependent Familial Adenomatous Polyposis.

CONTEXT: Recurrent somatic mutations in KCNJ5, CACNA1D, ATP1A1, and ATP2B3 have been identified in aldosterone-producing adenomas (APAs). The question as to whether they are responsible for both nodulation and aldosterone production is not solved. CASE DESCRIPTION: We describe the case of a young patient who was diagnosed with severe arterial hypertension due to primary aldosteronism at age 26 years, followed by hemorrhagic stroke 4 years later. Abdominal computed tomography showed bilateral macronodular adrenal hyperplasia. Identification of lateralized aldosterone secretion led to right adrenalectomy, followed by normalization of biochemical and hormonal parameters and amelioration of blood pressure. The resected adrenal showed three nodules, one of them expressing aldosterone synthase and harboring a somatic KNCJ5 mutation. A Weiss revisited index of 3 of the APA prompted us to perform a second 18F-2-fluoro-2-deoxy-D-glucose-positron emission tomography after surgery, which revealed abnormal rectal activity despite the absence of clinical symptoms. Gastrointestinal exploration showed multiple polyps with severe dysplasia, and the diagnosis of familial adenomatous polyposis was established in the presence of a germline heterozygous APC gene mutation. Sequencing of somatic DNA from the APA and a second adrenal nodule revealed biallelic APC inactivation due to loss of heterozygosity in both nodules. CONCLUSIONS: This case report underlines the need for establishing the frequency of germline APC variants in patients with primary aldosteronism and bilateral macronodular adrenal hyperplasia because their presence may predispose to APA development and severe hypertension well before the first familial adenomatous polyposis symptoms appear. From a mechanistic point of view, it supports a two-hit model for APA development, whereby the first hit drives increased cell proliferation whereas the second hit specifies the pattern of hormonal secretion.

Functional Characterization of PRKAR1A Mutations Reveals a Unique Molecular Mechanism Causing Acrodysostosis but Multiple Mechanisms Causing Carney Complex.

The main target of cAMP is PKA, the main regulatory subunit of which (PRKAR1A) presents mutations in two genetic disorders: acrodysostosis and Carney complex. In addition to the initial recurrent mutation (R368X) of the PRKAR1A gene, several missense and nonsense mutations have been observed recently in acrodysostosis with hormonal resistance. These mutations are located in one of the two cAMP-binding domains of the protein, and their functional characterization is presented here. Expression of each of the PRKAR1A mutants results in a reduction of forskolin-induced PKA activation (measured by a reporter assay) and an impaired ability of cAMP to dissociate PRKAR1A from the catalytic PKA subunits by BRET assay. Modeling studies and sensitivity to cAMP analogs specific for domain A (8-piperidinoadenosine 3',5'-cyclic monophosphate) or domain B (8-(6-aminohexyl)aminoadenosine-3',5'-cyclic monophosphate) indicate that the mutations impair cAMP binding locally in the domain containing the mutation. Interestingly, two of these mutations affect amino acids for which alternative amino acid substitutions have been reported to cause the Carney complex phenotype. To decipher the molecular mechanism through which homologous substitutions can produce such strikingly different clinical phenotypes, we studied these mutations using the same approaches. Interestingly, the Carney mutants also demonstrated resistance to cAMP, but they expressed additional functional defects, including accelerated PRKAR1A protein degradation. These data demonstrate that a cAMP binding defect is the common molecular mechanism for resistance of PKA activation in acrodysosotosis and that several distinct mechanisms lead to constitutive PKA activation in Carney complex.

IGF2 promotes growth of adrenocortical carcinoma cells, but its overexpression does not modify phenotypic and molecular features of adrenocortical carcinoma.

Insulin-like growth factor 2 (IGF2) overexpression is an important molecular marker of adrenocortical carcinoma (ACC), which is a rare but devastating endocrine cancer. It is not clear whether IGF2 overexpression modifies the biology and growth of this cancer, thus more studies are required before IGF2 can be considered as a major therapeutic target. We compared the phenotypical, clinical, biological, and molecular characteristics of ACC with or without the overexpression of IGF2, to address these issues. We also carried out a similar analysis in an ACC cell line (H295R) in which IGF2 expression was knocked down with si- or shRNA. We found no significant differences in the clinical, biological and molecular (transcriptomic) traits between IGF2-high and IGF2-low ACC. The absence of IGF2 overexpression had little influence on the activation of tyrosine kinase pathways both in tumors and in H295 cells that express low levels of IGF2. In IGF2-low tumors, other growth factors (FGF9, PDGFA) are more expressed than in IGF2-high tumors, suggesting that they play a compensatory role in tumor progression. In addition, IGF2 knock-down in H295R cells substantially impaired growth (>50% inhibition), blocked cells in G1 phase, and promoted apoptosis (>2-fold). Finally, analysis of the 11p15 locus showed a paternal uniparental disomy in both IGF2-high and IGF2-low tumors, but low IGF2 expression could be explained in most IGF2-low ACC by an additional epigenetic modification at the 11p15 locus. Altogether, these observations confirm the active role of IGF2 in adrenocortical tumor growth, but also suggest that other growth promoting pathways may be involved in a subset of ACC with low IGF2 expression, which creates opportunities for the use of other targeted therapies.

Integrated genomic characterization of adrenocortical carcinoma.

Adrenocortical carcinomas (ACCs) are aggressive cancers originating in the cortex of the adrenal gland. Despite overall poor prognosis, ACC outcome is heterogeneous. We performed exome sequencing and SNP array analysis of 45 ACCs and identified recurrent alterations in known driver genes (CTNNB1, TP53, CDKN2A, RB1 and MEN1) and in genes not previously reported in ACC (ZNRF3, DAXX, TERT and MED12), which we validated in an independent cohort of 77 ACCs. ZNRF3, encoding a cell surface E3 ubiquitin ligase, was the most frequently altered gene (21%) and is a potential new tumor suppressor gene related to the ß-catenin pathway. Our integrated genomic analyses further identified two distinct molecular subgroups with opposite outcome. The C1A group of ACCs with poor outcome displayed numerous mutations and DNA methylation alterations, whereas the C1B group of ACCs with good prognosis displayed specific deregulation of two microRNA clusters. Thus, aggressive and indolent ACCs correspond to two distinct molecular entities driven by different oncogenic alterations.

Mass-array screening of frequent mutations in cancers reveals RB1 alterations in aggressive adrenocortical carcinomas.

CONTEXT: Adrenocortical carcinoma (ACC) is a rare disease with a poor overall outcome. Transcriptome analysis identified two groups of ACCs with different prognosis. In aggressive ACCs, somatic mutations of the tumor suppressor gene TP53 and the proto-oncogene ß-catenin are detected in 50% of cases. For the remaining aggressive ACCs and for the group with a better prognosis, molecular alterations are unknown. OBJECTIVE: To identify new molecular actors driving adrenal tumorigenesis. EXPERIMENTAL DESIGN: Analysis by mass array of 374 mutations among 32 common oncogenes or tumor suppressor genes was performed on the tumoral DNA of 26 ACCs, using Sequenom OncoCarta Panels. RESULTS: Four mutations were identified, two previously known ß-catenin mutations and one alteration in two other genes: JAK3 and retinoblastoma gene (RB1). The JAK3 alteration was found in leukocyte DNA and therefore considered as a polymorphism and not a somatic event. The full RB1 tumor suppressor gene was subsequently sequenced in a cohort of 49 ACCs (26 ACCs from the 'OncoCarta cohort' and 23 other ACCs): three somatic mutations were identified, all in the poor-outcome ACC group. By immunohistochemistry, a loss of the retinoblastoma protein (pRb) was found exclusively in aggressive ACCs in 27% of cases (seven out of 26), three of them with an inactivating RB1 mutation. Among the seven pRb-negative ACCs, five had an allele loss at the RB1 locus. CONCLUSIONS: Parallel analysis of somatic mutations among known cancer genes allowed us to identify RB1 as a new actor in aggressive ACCs. These results suggest a prognostic significance of pRb expression loss in ACCs.

Higher risk of death among MEN1 patients with mutations in the JunD interacting domain: a Groupe d'etude des Tumeurs Endocrines (GTE) cohort study.

Multiple endocrine neoplasia syndrome type 1 (MEN1), which is secondary to mutation of the MEN1 gene, is a rare autosomal-dominant disease that predisposes mutation carriers to endocrine tumors. Although genotype-phenotype studies have so far failed to identify any statistical correlations, some families harbor recurrent tumor patterns. The function of MENIN is unclear, but has been described through the discovery of its interacting partners. Mutations in the interacting domains of MENIN functional partners have been shown to directly alter its regulation abilities. We report on a cohort of MEN1 patients from the Groupe d'étude des Tumeurs Endocrines. Patients with a molecular diagnosis and a clinical follow-up, totaling 262 families and 806 patients, were included. Associations between mutation type, location or interacting factors of the MENIN protein and death as well as the occurrence of MEN1-related tumors were tested using a frailty Cox model to adjust for potential heterogeneity across families. Accounting for the heterogeneity across families, the overall risk of death was significantly higher when mutations affected the JunD interacting domain (adjusted HR = 1.88: 95%-CI = 1.15-3.07). Patients had a higher risk of death from cancers of the MEN1 spectrum (HR = 2.34; 95%-CI = 1.23-4.43). This genotype-phenotype correlation study confirmed the lack of direct genotype-phenotype correlations. However, patients with mutations affecting the JunD interacting domain had a higher risk of death secondary to a MEN1 tumor and should thus be considered for surgical indications, genetic counseling and follow-up.

Frequent large germline HRPT2 deletions in a French National cohort of patients with primary hyperparathyroidism.

CONTEXT: Hyperparathyroidism-jaw tumor syndrome (HPT-JT) is an autosomal dominant syndrome with incomplete penetrance that can associate in a single patient parathyroid adenoma or carcinoma, fibro-osseous jaw tumor, cystic kidney lesion, and uterine tumor. Germline mutations of the HRPT2 gene (CDC73) coding for parafibromin are identified in approximately 50%-75% of HPT-JT cases and in approximately 14% of familial isolated hyperparathyroidism. A whole deletion of this gene has recently been reported in 1 sporadic case and in a family presenting with HPT-JT. OBJECTIVE: The objective of the study was to report molecular abnormalities of the HRPT2 gene in patients with primary hyperparathyroidism in a French National cohort from the Groupe d'Étude des Tumeurs Endocrines. METHODS: Patients' genomic DNA was screened by PCR-based sequencing for point mutations affecting HRPT2 and real-time quantitative PCR analysis for gross deletions. RESULTS: We report 20 index patients with a germinal HRPT2 abnormality. Median age at diagnosis of primary hyperparathyroidism was 23 years (range 14-65 years). Median serum total calcium level at diagnosis was 3.19 mmol/L (range 2.8-4.3 mmol/L). Thirteen different mutations were identified by routine sequencing, including 7 mutations never reported. Seven patients (35%) carried a gross deletion of this gene (3 complete and 4 partial deletions). No genotype-phenotype correlation could be identified. A gross deletion of the HRPT2 gene was identified in 7% of patients for whom a routine screening by direct sequencing came up as negative. CONCLUSION: Gross deletion analysis of the HRPT2 gene is indicated for all patients negative for mutation, presenting with HPT-JT or familial isolated hyperparathyroidism, parathyroid carcinoma, or in patients with apparently sporadic parathyroid adenoma diagnosed at a young age, having a severe hypercalcemia.