Anne Barlier

Heterogeneous Genetic Background of the Association of Pheochromocytoma/Paraganglioma and Pituitary Adenoma: Results From a Large Patient Cohort

Context: Pituitary adenomas and pheochromocytomas/paragangliomas (pheo/PGL) can occur in the same patient or in the same family. Coexistence of the two diseases could be due to either a common pathogenic mechanism or a coincidence. Objective: The objective of the investigation was to study the possible coexistence of pituitary adenoma and pheo/PGL. Design: Thirty-nine cases of sporadic or familial pheo/PGL and pituitary adenomas were investigated. Known pheo/PGL genes (SDHA-D, SDHAF2, RET, VHL, TMEM127, MAX, FH) and pituitary adenoma genes (MEN1, AIP, CDKN1B) were sequenced using next generation or Sanger sequencing. Loss of heterozygosity study and pathological studies were performed on the available tumor samples. Setting: The study was conducted at university hospitals. Patients: Thirty-nine patients with sporadic of familial pituitary adenoma and pheo/PGL participated in the study. Outcome: Outcomes included genetic screening and clinical characteristics. Results: Eleven germline mutations (five SDHB, one SDHC, one SDHD, two VHL, and two MEN1) and four variants of unknown significance (two SDHA, one SDHB, and one SDHAF2) were identified in the studied genes in our patient cohort. Tumor tissue analysis identified LOH at the SDHB locus in three pituitary adenomas and loss of heterozygosity at the MEN1 locus in two pheochromocytomas. All the pituitary adenomas of patients affected by SDHX alterations have a unique histological feature not previously described in this context. Conclusions: Mutations in the genes known to cause pheo/PGL can rarely be associated with pituitary adenomas, whereas mutation in a gene predisposing to pituitary adenomas (MEN1) can be associated with pheo/PGL. Our findings suggest that genetic testing should be considered in all patients or families with the constellation of pheo/PGL and a pituitary adenoma.

A Potential Inhibitory Role for the New Truncated Variant of Somatostatin Receptor 5, sst5TMD4, in Pituitary Adenomas Poorly Responsive to Somatostatin Analogs

CONTEXTnSomatostatin (SST) receptors, specially sst2 and sst5, provide a valuable target to inhibit excessive hormone release and cell growth in pituitary tumors by using SST analogs (SSAs). Unfortunately, an appreciable proportion of tumors fail to respond to SSA despite expressing high levels of one or more ssts. Recently we identified two novel truncated sst5 variants, sst5TMD5, and sst5TMD4, absent in normal pituitary but expressed in pituitary tumors.nnnOBJECTIVE AND DESIGNnWe aimed at exploring the potential role of sst5TMD5 and sst5TMD4 in the poor response of some tumors to SSA in vivo and in vitro. Specifically, 25 somatotropinomas showing different responses to octreotide in vivo and sst2 (BIM-23197)- and sst5(BIM-23268)-selective compounds in vitro were screened for sst5TMD5/sst5TMD4 expression by real-time PCR. Relationships between ssts expression and in vivo and in vitro secretory response of the corresponding pituitary samples were assessed.nnnRESULTSnsst5TMD5 was absent in all samples analyzed. sst5TMD4 was found in 85% of tumors, and its expression was positively correlated to that of sst5 (R(2) = 0.79, P < 0.001). Expression of sst5TMD4 was negatively correlated with the ability of octreotide to reduce GH levels in vivo and partially negatively correlated with inhibition of GH secretion by an sst5 selective agonist (BIM-23268) in vitro.nnnCONCLUSIONSnThese results indicate that sst5TMD4 is related to the reduced ability of octreotide at normalizing hormone secretion in poorly responsive tumors in vivo. Further studies will help to evaluate the potential use of sst5TMD4 expression in surgically removed pituitary adenomas as a predictor of the subsequent response of different pituitary tumors to SSA therapy.

A Novel Dysfunctional LHX4 Mutation with High Phenotypical Variability in Patients with Hypopituitarism

CONTEXTnLHX4 is a LIM homeodomain transcription factor involved in pituitary ontogenesis. Only a few heterozygous LHX4 mutations have been reported to be responsible for congenital pituitary hormone deficiency.nnnSUBJECTS AND METHODSnA total of 136 patients with congenital hypopituitarism associated with malformations of brain structures, pituitary stalk, or posterior pituitary gland was screened for LHX4 mutations.nnnRESULTSnThree novel allelic variants that cause predicted changes in the protein sequence of LHX4 (2.3%) were found (p.Thr99fs, p.Thr90Met, and p.Gly370Ser). On the basis of functional studies, p.Thr99fs mutation was responsible for the patients phenotype, whereas p.Thr90Met and p.Gly370Ser were likely polymorphisms. Patients bearing the heterozygous p.Thr99fs mutation had variable phenotypes: two brothers presented somato-lactotroph and thyrotroph deficiencies, with pituitary hypoplasia and poorly developed sella turcica; the youngest brother (propositus) also had corpus callosum hypoplasia and ectopic neurohypophysis; their father only had somatotroph deficiency and delayed puberty with pituitary hyperplasia. Functional studies showed that the mutation induced a complete loss of transcriptional activity on POU1F1 promoter and a lack of DNA binding. Cotransfection of p.Thr99fs mutant and wild-type LHX4 failed to evidence any dominant negative effect, suggesting a mechanism of haploinsufficiency. We also identified prolactin and GH promoters as potential target genes of LHX4 and found that the p.Thr99fs mutant was also unable to transactivate these promoters.nnnCONCLUSIONSnThe present report describes three new exonic LHX4 allelic variants with at least one being responsible for congenital hypopituitarism. It also extends the phenotypical heterogeneity associated with LHX4 mutations, which includes variable anterior pituitary hormone deficits, as well as pituitary and extrapituitary abnormalities.

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

CONTEXTnHyperparathyroidism-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.nnnOBJECTIVEnThe 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 dÉtude des Tumeurs Endocrines.nnnMETHODSnPatients genomic DNA was screened by PCR-based sequencing for point mutations affecting HRPT2 and real-time quantitative PCR analysis for gross deletions.nnnRESULTSnWe 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.nnnCONCLUSIONnGross 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.

PROKR2 Variants in Multiple Hypopituitarism with Pituitary Stalk Interruption

CONTEXTnPituitary stalk interruption represents a frequent feature of congenital hypopituitarism, but only rare cases have been assigned to a known genetic cause.nnnOBJECTIVEnUsing a candidate gene approach, we tested several genes as potential causes of hypopituitarism with pituitary stalk interruption. We hypothesized that ectopic posterior pituitary may be a consequence of defective neuronal axon projections along the pituitary stalk or defective angiogenesis of hypophyseal portal circulation. Considering the role of the prokineticin 2 pathway in angiogenesis and neuronal migration, we screened PROK2 and PROKR2 genes.nnnDESIGNnPROK2 and PROKR2 and all genes previously known to be involved in hypopituitarism with pituitary stalk interruption (LHX4, HESX1, OTX2, and SOX3) were screened in 72 index cases with pituitary stalk interruption syndrome from the GENHYPOPIT database. In vitro studies were performed to assess the functional consequences of allelic variants.nnnRESULTSnWe identified two heterozygous PROKR2 mutations (p.Leu173Arg and p.Arg85His) previously reported in isolated hypogonadotroph hypogonadism and a novel PROKR2 variant (p.Ala51Thr) that, in contrast with both other mutations, did not impair receptor signaling activity. Three allelic variants of HESX1 were identified: the heterozygous p.Phe156Ser and the homozygous p.Arg109X mutations were functionally deleterious, whereas p.Ser67Thr was found as a rare allelic variant in association with p.Arg85His PROKR2 mutation in the same patient.nnnCONCLUSIONSnWe report PROKR2 variants in congenital hypopituitarism with pituitary stalk interruption, suggesting a potential role of the prokineticin pathway in pituitary development.

Phenotypic Homogeneity and Genotypic Variability in a Large Series of Congenital Isolated ACTH-Deficiency Patients with TPIT Gene Mutations

CONTEXTnCongenital isolated ACTH deficiency (IAD) is a rare disease characterized by low plasma ACTH and cortisol levels and preservation of all other pituitary hormones. This condition was poorly defined before we identified TPIT, a T-box transcription factor with a specific role in differentiation of the corticotroph lineage in mice and humans, as its principal molecular cause.nnnOBJECTIVEnWe have enlarged our series of IAD patients to better characterize the phenotype and the genotype of this rare disease.nnnDESIGNnEach exon of the TPIT gene was amplified and sequenced in IAD patients without any identified cause. A functional analysis of each new TPIT mutation was performed.nnnRESULTSnWe described the largest series of 91 IAD patients and identified three distinct groups: neonatal onset complete or partial IAD or late onset IAD. We did not identify any TPIT mutation in patients with partial or late-onset IAD. However, we found a TPIT mutation in 65% of patients with neonatal-onset complete IAD. These patients are homozygous or compound heterozygous for TPIT mutations, and their parents are healthy heterozygous carriers. We identified nine new mutations: four missense, one one-nucleotide deletion, three splice-site mutations, and one large deletion. TPIT mutations lead to loss of function by different mechanisms, such as non-sense-mediated mRNA decay, abnormal mRNA splicing, loss of TPIT DNA binding or protein-protein interaction defects.nnnCONCLUSIONnTPIT mutations are responsible for two thirds of neonatal-onset complete IAD but can not be detected in partial or late-onset IAD.

Inactivation of PITX2 Transcription Factor Induced Apoptosis of Gonadotroph Tumoral Cells

Nonfunctioning pituitary adenomas (NFPA; gonadotroph derived), even not inducing hormonal hypersecretion, cause significant morbidity by compression neighboring structures. No effective and specific medical methods are available so far for treating these tumors. The pituitary homeobox 2 (PITX2) gene is a member of the bicoid-like homeobox transcription factor family, which is involved in the Wnt/Dvl/β-catenin pathway. PITX2 is overexpressed in NFPA. PITX2 mutations are known to be responsible for Axenfield Rieger syndrome, a genetic disorder in which pituitary abnormalities have been detected. The R91P mutant identified in Axenfeld Rieger syndrome is a dominant-negative factor, which is able to block the expression of several pituitary genes activated by PITX2. To better understand the role of Pitx2 on gonadotroph tumorigenesis and to explore new approach for inhibiting tumoral growth, the R91P mutant was transferred via a lentiviral vector in tumoral gonadotroph cells of two kinds: the αT3-1 cell line and human adenoma cells. R91P mutant and small interfering RNA directed against Pitx2 both decreased the viability of αT3-1 cells via an apoptotic mechanism involving the activation of executioner caspase. Similar effects of the R91P mutant were observed on human gonadotroph cells in primary culture. Therefore, Pitx2 overexpression may play an antiapoptotic role during NFPA tumorigenesis.

Genetic causes of combined pituitary hormone deficiencies in humans

Congenital hypopituitarism is a rare disease, usually induced by mutations of genes coding for transcription factors involved in pituitary development. PROP1 mutations represent the first cause of identified congenital hypopituitarism. Current techniques only identify 10-20% of congenital hypopituitarism etiologies, suggesting that new techniques are needed to improve this ratio. This should lead to a better management and follow-up of patients presenting with combined pituitary hormone deficiencies.

Case Report of GNAS Epigenetic Defect Revealed by a Congenital Hypothyroidism

Pseudohypoparathyroidism (PHP) is a group of disorders characterized by end-organ resistance to the parathyroid hormone (PTH). PHP type 1A includes multihormone resistance syndrome, Albright’s hereditary osteodystrophy, and obesity and is caused by mutations in GNAS exon 1 through 13. PHP type 1B (PHP1B), caused by epigenetic changes in the GNAS locus, was initially described as an isolated resistance to PTH. Epigenetic changes in GNAS have also been reported in patients who display mild Albright’s hereditary osteodystrophy or mild thyroid-stimulating hormone (TSH) resistance without mutation of GNAS. Here we report a case of PHP caused by epigenetic changes in GNAS in a patient with congenital hypothyroidism. The patient was referred for a positive newborn screening for hypothyroidism (TSH 50 mIU/L). She exhibited severe clinical features of congenital hypothyroidism. The thyroid was in place, and etiologic explorations were negative. TSH was normalized under L-thyroxin, and the symptoms disappeared, except for a macroglossia. In childhood, PHP was suspected in addition to elevated PTH, obesity, brachydactyly, and a rounded face. Sequencing, methylation analysis, and large deletion research were performed in GNAS. No genetic mutations were found. Methylation analysis revealed a broad epigenetic defect without deletion in GNAS consistent with sporadic PHP1B. The multilocus methylation analysis were negative. This finding expands the known onsets of PHP1B and emphasizes the need for a new PHP classification system. This case report has important consequences for the etiologic diagnosis of congenital hypothyroidism because it adds a new cause of the disease.

Detection of genetic hypopituitarism in an adult population of idiopathic pituitary insufficiency patients with growth hormone deficiency

Idiopathic pituitary insufficiency (IPI) is diagnosed in 10% of all hypopituitary patients. There are several known and unknown aetiologies within the IPI group. The aim of this study was to investigate an adult IPI population for genetic cause according a screening schedule. From files of 373 GH deficient (GHD) patients on GH replacement 50 cases with IPI were identified. Of the 39 patients that approved to the study, 25 patients were selected for genetic investigation according to phenotype and 14 patients were not further tested, as sporadic isolated GHD (nxa0=xa09) and GHD with diabetes insipidus (nxa0=xa05) have low probability for a known genetic cause. Genotyping of all coding exons of HESX1, LHX4, PROP1, POU1F1 and GH1 genes were performed according to a diagnostic algorithm based on clinical, hormonal and neuroradiological phenotype. Among the 25 patients, an overall rate of 8% of mutations was found, and a 50% rate in familial cases. Among two sibling pairs, one pair that presented with complete anterior pituitary insufficiency, had a compound heterozygous PROP1 gene mutation (codons 117 and 120: exon 3 p Phe 117 Ile (c349 T>A) and p Arg 120 Cys (c358 C>T)) with a phenotype of very late onset ACTH-insufficiency. In the other sibling pair and in the sporadic cases no mutation was identified. This study suggests that currently known genetic causes are rare in sporadic adult IPI patients, and that systematic genetic screening is not needed in adult-onset sporadic cases of IPI. Conversely, familial cases are highly suspect for genetic causes.