Caroline M. Gorvin

Whole-Exome Sequencing Studies of Nonhereditary (Sporadic) Parathyroid Adenomas

CONTEXT Genetic abnormalities, such as those of multiple endocrine neoplasia type 1 (MEN1) and Cyclin D1 (CCND1) genes, occur in <50% of nonhereditary (sporadic) parathyroid adenomas. OBJECTIVE To identify genetic abnormalities in nonhereditary parathyroid adenomas by whole-exome sequence analysis. DESIGN Whole-exome sequence analysis was performed on parathyroid adenomas and leukocyte DNA samples from 16 postmenopausal women without a family history of parathyroid tumors or MEN1 and in whom primary hyperparathyroidism due to single-gland disease was cured by surgery. Somatic variants confirmed in this discovery set were assessed in 24 other parathyroid adenomas. RESULTS Over 90% of targeted exons were captured and represented by more than 10 base reads. Analysis identified 212 somatic variants (median eight per tumor; range, 2-110), with the majority being heterozygous nonsynonymous single-nucleotide variants that predicted missense amino acid substitutions. Somatic MEN1 mutations occurred in six of 16 (∼35%) parathyroid adenomas, in association with loss of heterozygosity on chromosome 11. However, no other gene was mutated in more than one tumor. Mutations in several genes that may represent low-frequency driver mutations were identified, including a protection of telomeres 1 (POT1) mutation that resulted in exon skipping and disruption to the single-stranded DNA-binding domain, which may contribute to increased genomic instability and the observed high mutation rate in one tumor. CONCLUSIONS Parathyroid adenomas typically harbor few somatic variants, consistent with their low proliferation rates. MEN1 mutation represents the major driver in sporadic parathyroid tumorigenesis although multiple low-frequency driver mutations likely account for tumors not harboring somatic MEN1 mutations.

Mutant Prolactin Receptor and Familial Hyperprolactinemia

Hyperprolactinemia that is not associated with gestation or the puerperium is usually due to tumors in the anterior pituitary gland and occurs occasionally in hereditary multiple endocrine neoplasia syndromes. Here, we report data from three sisters with hyperprolactinemia, two of whom presented with oligomenorrhea and one with infertility. These symptoms were not associated with pituitary tumors or multiple endocrine neoplasia but were due to a heterozygous mutation in the prolactin receptor gene, PRLR, resulting in an amino acid change from histidine to arginine at codon 188 (His188Arg). This substitution disrupted the high-affinity ligand-binding interface of the prolactin receptor, resulting in a loss of downstream signaling by Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5). Thus, the familial hyperprolactinemia appears to be due to a germline, loss-of-function mutation in PRLR, resulting in prolactin insensitivity.

Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients

Receptor-mediated endocytosis, involving megalin and cubilin, mediates renal proximal-tubular reabsorption and is decreased in Dent disease because of mutations of the chloride/proton antiporter, chloride channel-5 (CLC-5), resulting in low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. To facilitate studies of receptor-mediated endocytosis and the role of CLC-5, we established conditionally immortalized proximal-tubular epithelial cell lines (ciPTECs) from three patients with CLC-5 mutations (30:insH, R637X, and del132-241) and a normal male. Confocal microscopy using the tight junction marker zona occludens-1 (ZO-1) and end-binding protein-1 (EB-1), which is specific for the plus end of microtubules demonstrated that the ciPTECs polarized. Receptor-mediated endocytic uptake of fluorescent albumin and transferrin in 30:insH and R637X ciPTECs was significantly decreased, compared with normal ciPTECs, and could be further reduced by competition with 10-fold excess of unlabeled albumin and transferrin, whereas in the del132-241 ciPTEC, receptor-mediated endocytic uptake was abolished. Investigation of endosomal acidification by live-cell imaging of pHluorin-VAMP2 (vesicle-associated membrane protein-2), a pH-sensitive-GFP construct, revealed that the endosomal pH in normal and 30:insH ciPTECs was similar, whereas in del132-241 and R637X ciPTECs, it was significantly more alkaline, indicating defective acidification in these ciPTECs. The addition of bafilomycin-A1, a V-ATPase inhibitor, raised the pH significantly in all ciPTECs, demonstrating that the differences in acidification were not due to alterations in the V-ATPase, but instead to abnormalities of CLC-5. Thus, our studies, which have established human Dent disease ciPTECs that will facilitate studies of mechanisms in renal reabsorption, demonstrate that Dent disease-causing CLC-5 mutations have differing effects on endosomal acidification and receptor-mediated endocytosis that may not be coupled.

Whole-exome sequencing studies of nonfunctioning pituitary adenomas.

CONTEXT The tumorigenic role of genetic abnormalities in sporadic pituitary nonfunctioning adenomas (NFAs), which usually originate from gonadotroph cells, is unknown. OBJECTIVE The objective of the study was to identify somatic genetic abnormalities in sporadic pituitary NFAs. DESIGN Whole-exome sequencing was performed using DNA from 7 pituitary NFAs and leukocyte samples obtained from the same patients. Somatic variants were confirmed by dideoxynucleotide sequencing, and candidate driver genes were assessed in an additional 24 pituitary NFAs. RESULTS Whole-exome sequencing achieved a high degree of coverage such that approximately 97% of targeted bases were represented by more than 10 base reads; 24 somatic variants were identified and confirmed in the discovery set of 7 pituitary NFAs (mean 3.5 variants/tumor; range 1-7). Approximately 80% of variants occurred as missense single nucleotide variants and the remainder were synonymous changes or small frameshift deletions. Each of the 24 mutations occurred in independent genes with no recurrent mutations. Mutations were not observed in genes previously associated with pituitary tumorigenesis, although somatic variants in putative driver genes including platelet-derived growth factor D (PDGFD), N-myc down-regulated gene family member 4 (NDRG4), and Zipper sterile-α-motif kinase (ZAK) were identified; however, DNA sequence analysis of these in the validation set of 24 pituitary NFAs did not reveal any mutations indicating that these genes are unlikely to contribute significantly in the etiology of sporadic pituitary NFAs. CONCLUSIONS Pituitary NFAs harbor few somatic mutations consistent with their low proliferation rates and benign nature, but mechanisms other than somatic mutation are likely involved in the etiology of sporadic pituitary NFAs.

Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype–phenotype correlations, codon bias and dominant-negative effects

The adaptor protein-2 sigma subunit (AP2σ2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2σ2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca2+o) homeostasis. To elucidate the role of AP2σ2 in Ca2+o regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2σ2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2σ2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype–phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2σ2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2σ2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2σ2 mutations to result in a more severe FHH phenotype with genotype–phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue.

Autosomal dominant hypercalciuria in a mouse model due to a mutation of the epithelial calcium channel, TRPV5.

Hypercalciuria is a major cause of nephrolithiasis, and is a common and complex disorder involving genetic and environmental factors. Identification of genetic factors for monogenic forms of hypercalciuria is hampered by the limited availability of large families, and to facilitate such studies, we screened for hypercalciuria in mice from an N-ethyl-N-nitrosourea mutagenesis programme. We identified a mouse with autosomal dominant hypercalciuria (HCALC1). Linkage studies mapped the Hcalc1 locus to a 11.94 Mb region on chromosome 6 containing the transient receptor potential cation channel, subfamily V, members 5 (Trpv5) and 6 (Trpv6) genes. DNA sequence analysis of coding regions, intron-exon boundaries and promoters of Trpv5 and Trpv6 identified a novel T to C transition in codon 682 of TRPV5, mutating a conserved serine to a proline (S682P). Compared to wild-type littermates, heterozygous (Trpv5 682P/+) and homozygous (Trpv5 682P/682P) mutant mice had hypercalciuria, polyuria, hyperphosphaturia and a more acidic urine, and ∼10% of males developed tubulointerstitial nephritis. Trpv5 682P/682P mice also had normal plasma parathyroid hormone but increased 1,25-dihydroxyvitamin D3 concentrations without increased bone resorption, consistent with a renal defect for the hypercalciuria. Expression of the S682P mutation in human embryonic kidney cells revealed that TRPV5-S682P-expressing cells had a lower baseline intracellular calcium concentration than wild-type TRPV5-expressing cells, suggesting an altered calcium permeability. Immunohistological studies revealed a selective decrease in TRPV5-expression from the renal distal convoluted tubules of Trpv5 682P/+ and Trpv5 682P/682P mice consistent with a trafficking defect. In addition, Trpv5682P/682P mice had a reduction in renal expression of the intracellular calcium-binding protein, calbindin-D28K, consistent with a specific defect in TRPV5-mediated renal calcium reabsorption. Thus, our findings indicate that the TRPV5 S682P mutant is functionally significant and study of HCALC1, a novel model for autosomal dominant hypercalciuria, may help further our understanding of renal calcium reabsorption and hypercalciuria.

A G-protein Subunit-α11 Loss-of-Function Mutation, Thr54Met, Causes Familial Hypocalciuric Hypercalcemia Type 2 (FHH2).

Familial hypocalciuric hypercalcemia (FHH) is a genetically heterogeneous disorder with three variants, FHH1 to FHH3. FHH1 is caused by loss‐of‐function mutations of the calcium‐sensing receptor (CaSR), a G‐protein coupled receptor that predominantly signals via G‐protein subunit alpha‐11 (Gα11) to regulate calcium homeostasis. FHH2 is the result of loss‐of‐function mutations in Gα11, encoded by GNA11, and to date only two FHH2‐associated Gα11 missense mutations (Leu135Gln and Ile200del) have been reported. FHH3 is the result of loss‐of‐function mutations of the adaptor protein‐2 σ‐subunit (AP2σ), which plays a pivotal role in clathrin‐mediated endocytosis. We describe a 65‐year‐old woman who had hypercalcemia with normal circulating parathyroid hormone concentrations and hypocalciuria, features consistent with FHH, but she did not have CaSR and AP2σ mutations. Mutational analysis of the GNA11 gene was therefore undertaken, using leucocyte DNA, and this identified a novel heterozygous GNA11 mutation (c.161C>T; p.Thr54Met). The effect of the Gα11 variant was assessed by homology modeling of the related Gαq protein and by measuring the CaSR‐mediated intracellular calcium (Ca2+i) responses of HEK293 cells, stably expressing CaSR, to alterations in extracellular calcium (Ca2+o) using flow cytometry. Three‐dimensional modeling revealed the Thr54Met mutation to be located at the interface between the Gα11 helical and GTPase domains, and to likely impair GDP binding and interdomain interactions. Expression of wild‐type and the mutant Gα11 in HEK293 cells stably expressing CaSR demonstrate that the Ca2+i responses after stimulation with Ca2+o of the mutant Met54 Gα11 led to a rightward shift of the concentration‐response curve with a significantly (p < 0.01) increased mean half‐maximal concentration (EC50) value of 3.88 mM (95% confidence interval [CI] 3.76–4.01 mM), when compared with the wild‐type EC50 of 2.94 mM (95% CI 2.81–3.07 mM) consistent with a loss‐of‐function. Thus, our studies have identified a third Gα11 mutation (Thr54Met) causing FHH2 and reveal a critical role for the Gα11 interdomain interface in CaSR signaling and Ca2+o homeostasis.

Allosteric Modulation of the Calcium-sensing Receptor Rectifies Signaling Abnormalities Associated with G-protein α-11 Mutations Causing Hypercalcemic and Hypocalcemic Disorders

Germline loss- and gain-of-function mutations of G-protein α-11 (Gα11), which couples the calcium-sensing receptor (CaSR) to intracellular calcium (Ca2+i) signaling, lead to familial hypocalciuric hypercalcemia type 2 (FHH2) and autosomal dominant hypocalcemia type 2 (ADH2), respectively, whereas somatic Gα11 mutations mediate uveal melanoma development by constitutively up-regulating MAPK signaling. Cinacalcet and NPS-2143 are allosteric CaSR activators and inactivators, respectively, that ameliorate signaling disturbances associated with CaSR mutations, but their potential to modulate abnormalities of the downstream Gα11 protein is unknown. This study investigated whether cinacalcet and NPS-2143 may rectify Ca2+i alterations associated with FHH2- and ADH2-causing Gα11 mutations, and evaluated the influence of germline gain-of-function Gα11 mutations on MAPK signaling by measuring ERK phosphorylation, and assessed the effect of NPS-2143 on a uveal melanoma Gα11 mutant. WT and mutant Gα11 proteins causing FHH2, ADH2 or uveal melanoma were transfected in CaSR-expressing HEK293 cells, and Ca2+i and ERK phosphorylation responses measured by flow-cytometry and Alphascreen immunoassay following exposure to extracellular Ca2+ (Ca2+o) and allosteric modulators. Cinacalcet and NPS-2143 rectified the Ca2+i responses of FHH2- and ADH2-associated Gα11 loss- and gain-of-function mutations, respectively. ADH2-causing Gα11 mutations were demonstrated not to be constitutively activating and induced ERK phosphorylation following Ca2+o stimulation only. The increased ERK phosphorylation associated with ADH2 and uveal melanoma mutants was rectified by NPS-2143. These findings demonstrate that CaSR-targeted compounds can rectify signaling disturbances caused by germline and somatic Gα11 mutations, which respectively lead to calcium disorders and tumorigenesis; and that ADH2-causing Gα11 mutations induce non-constitutive alterations in MAPK signaling.

Cinacalcet for Symptomatic Hypercalcemia Caused by AP2S1 Mutations

The authors show that cinacalcet, which mediates allosteric modulation of the calcium-sensing receptor, corrects loss of function of AP2S1 mutations that cause familial hypocalciuric hypercalcemia type 3 and ameliorates symptomatic hypercalcemia.

Identification of a G-Protein Subunit-α11 Gain-of-Function Mutation, Val340Met, in a Family With Autosomal Dominant Hypocalcemia Type 2 (ADH2).

Autosomal dominant hypocalcemia (ADH) is characterized by hypocalcemia, inappropriately low serum parathyroid hormone concentrations and hypercalciuria. ADH is genetically heterogeneous with ADH type 1 (ADH1), the predominant form, being caused by germline gain‐of‐function mutations of the G‐protein coupled calcium‐sensing receptor (CaSR), and ADH2 caused by germline gain‐of‐function mutations of G‐protein subunit α‐11 (Gα11). To date Gα11 mutations causing ADH2 have been reported in only five probands. We investigated a multigenerational nonconsanguineous family, from Iran, with ADH and keratoconus which are not known to be associated, for causative mutations by whole‐exome sequencing in two individuals with hypoparathyroidism, of whom one also had keratoconus, followed by cosegregation analysis of variants. This identified a novel heterozygous germline Val340Met Gα11 mutation in both individuals, and this was also present in the other two relatives with hypocalcemia that were tested. Three‐dimensional modeling revealed the Val340Met mutation to likely alter the conformation of the C‐terminal α5 helix, which may affect G‐protein coupled receptor binding and G‐protein activation. In vitro functional expression of wild‐type (Val340) and mutant (Met340) Gα11 proteins in HEK293 cells stably expressing the CaSR, demonstrated that the intracellular calcium responses following stimulation with extracellular calcium, of the mutant Met340 Gα11 led to a leftward shift of the concentration‐response curve with a significantly (p < 0.0001) reduced mean half‐maximal concentration (EC50) value of 2.44 mM (95% CI, 2.31 to 2.77 mM) when compared to the wild‐type EC50 of 3.14 mM (95% CI, 3.03 to 3.26 mM), consistent with a gain‐of‐function mutation. A novel His403Gln variant in transforming growth factor, beta‐induced (TGFBI), that may be causing keratoconus was also identified, indicating likely digenic inheritance of keratoconus and ADH2 in this family. In conclusion, our identification of a novel germline gain‐of‐function Gα11 mutation, Val340Met, causing ADH2 demonstrates the importance of the Gα11 C‐terminal region for G‐protein function and CaSR signal transduction.