Anelia Horvath

A genome-wide scan identifies mutations in the gene encoding phosphodiesterase 11A4 ( PDE11A ) in individuals with adrenocortical hyperplasia

Phosphodiesterases (PDEs) regulate cyclic nucleotide levels. Increased cyclic AMP (cAMP) signaling has been associated with PRKAR1A or GNAS mutations and leads to adrenocortical tumors and Cushing syndrome. We investigated the genetic source of Cushing syndrome in individuals with adrenocortical hyperplasia that was not caused by known defects. We performed genome-wide SNP genotyping, including the adrenocortical tumor DNA. The region with the highest probability to harbor a susceptibility gene by loss of heterozygosity (LOH) and other analyses was 2q31–2q35. We identified mutations disrupting the expression of the PDE11A isoform-4 gene (PDE11A) in three kindreds. Tumor tissues showed 2q31–2q35 LOH, decreased protein expression and high cyclic nucleotide levels and cAMP-responsive element binding protein (CREB) phosphorylation. PDE11A codes for a dual-specificity PDE that is expressed in adrenal cortex and is partially inhibited by tadalafil and other PDE inhibitors; its germline inactivation is associated with adrenocortical hyperplasia, suggesting another means by which dysregulation of cAMP signaling causes endocrine tumors.

The role of germline AIP, MEN1, PRKAR1A, CDKN1B and CDKN2C mutations in causing pituitary adenomas in a large cohort of children, adolescents, and patients with genetic syndromes.

Stratakis CA, Tichomirowa MA, Boikos S, Azevedo MF, Lodish M, Martari M, Verma S, Daly AF, Raygada M, Keil MF, Papademetriou J, Drori‐Herishanu L, Horvath A, Tsang KM, Nesterova M, Franklin S, Vanbellinghen J‐F, Bours V, Salvatori R, Beckers A. The role of germline AIP, MEN1, PRKAR1A, CDKN1B and CDKN2C mutations in causing pituitary adenomas in a large cohort of children, adolescents, and patients with genetic syndromes.

Mutation in PDE8B, a Cyclic AMP–Specific Phosphodiesterase in Adrenal Hyperplasia

To the Editor: Cortisol-producing adrenocortical tumors have been associated with abnormalities of the cyclic AMP (cAMP)–signaling pathway. Examples are Cushings syndrome in infants with the McCun...

Mutations and Polymorphisms in the Gene Encoding Regulatory Subunit Type 1-alpha of Protein Kinase A (PRKAR1A): An Update

PRKAR1A encodes the regulatory subunit type 1‐alpha (RIα) of the cyclic adenosine monophosphate (cAMP)‐dependent protein kinase (PKA). Inactivating PRKAR1A mutations are known to be responsible for the multiple neoplasia and lentiginosis syndrome Carney complex (CNC). To date, at least 117 pathogenic variants in PRKAR1A have been identified (online database: http://prkar1a.nichd.nih.gov). The majority are subject to nonsense mediated mRNA decay (NMD), leading to RIα haploinsufficiency and, as a result, activated cAMP signaling. Recently, it became apparent that CNC may be caused not only by RIα haploinsufficiency, but also by the expression of altered RIα protein, as proven by analysis of expressed mutations in the gene, consisting of aminoacid substitutions and in‐frame genetic alterations. In addition, a new subgroup of mutations that potentially escape NMD and result in CNC through altered (rather than missing) protein has been analyzed—these are frame‐shifts in the 3′ end of the coding sequence that shift the stop codon downstream of the normal one. The mutation detection rate in CNC patients is recently estimated at above 60%; PRKAR1A mutation‐negative CNC patients are characterized by significant phenotypic heterogeneity. In this report, we present a comprehensive analysis of all presently known PRKAR1A sequence variations and discuss their molecular context and clinical phenotype. Hum Mutat 31:369–379, 2010. Published 2010 Wiley‐Liss, Inc.

RNA sequencing of cancer reveals novel splicing alterations

Breast cancer transcriptome acquires a myriad of regulation changes, and splicing is critical for the cell to “tailor-make” specific functional transcripts. We systematically revealed splicing signatures of the three most common types of breast tumors using RNA sequencing: TNBC, non-TNBC and HER2-positive breast cancer. We discovered subtype specific differentially spliced genes and splice isoforms not previously recognized in human transcriptome. Further, we showed that exon skip and intron retention are predominant splice events in breast cancer. In addition, we found that differential expression of primary transcripts and promoter switching are significantly deregulated in breast cancer compared to normal breast. We validated the presence of novel hybrid isoforms of critical molecules like CDK4, LARP1, ADD3, and PHLPP2. Our study provides the first comprehensive portrait of transcriptional and splicing signatures specific to breast cancer sub-types, as well as previously unknown transcripts that prompt the need for complete annotation of tissue and disease specific transcriptome.

Succinate Dehydrogenase (SDH) D Subunit (SDHD) Inactivation in a Growth-Hormone-Producing Pituitary Tumor: A New Association for SDH?

BACKGROUND Mutations in the subunits B, C, and D of succinate dehydrogenase (SDH) mitochondrial complex II have been associated with the development of paragangliomas (PGL), gastrointestinal stromal tumors, papillary thyroid and renal carcinoma (SDHB), and testicular seminoma (SDHD). AIM Our aim was to examine the possible causative link between SDHD inactivation and somatotropinoma. PATIENTS AND METHODS A 37-yr-old male presented with acromegaly and hypertension. Other family members were found with PGL. Elevated plasma and urinary levels of catecholamines led to the identification of multiple PGL in the proband in the neck, thorax, and abdomen. Adrenalectomy was performed for bilateral pheochromocytomas (PHEO). A GH-secreting macroadenoma was also found and partially removed via transsphenoidal surgery (TTS). Genetic analysis revealed a novel SDHD mutation (c.298_301delACTC), leading to a frame shift and a premature stop codon at position 133 of the protein. Loss of heterozygosity for the SDHD genetic locus was shown in the GH-secreting adenoma. Down-regulation of SDHD protein in the GH-secreting adenoma by immunoblotting and immunohistochemistry was found. A literature search identified other cases of multiple PGL and/or PHEO in association with pituitary tumors. CONCLUSION We describe the first kindred with a germline SDHD pathogenic mutation, inherited PGL, and acromegaly due to a GH-producing pituitary adenoma. SDHD loss of heterozygosity, down-regulation of protein in the GH-secreting adenoma, and decreased SDH enzymatic activity supports SDHDs involvement in the pituitary tumor formation in this patient. Older cases of multiple PGL and PHEO and pituitary tumors in the literature support a possible association between SDH defects and pituitary tumorigenesis.

Detection of somatic β-catenin mutations in primary pigmented nodular adrenocortical disease (PPNAD)

Background  Primary pigmented nodular adrenocortical disease (PPNAD) leads to Cushing syndrome (CS) and is often associated with Carney complex (CNC). Genetic alterations of the type 1‐α regulatory subunit of cAMP‐dependent protein kinase A (PRKAR1A) and phosphodiesterase 11A4 (PDE11A) genes have been found in PPNAD. Recent studies have demonstrated that β‐catenin mutations are frequent in adrenocortical adenomas and carcinomas and that the Wnt‐signalling pathway is involved in PPNAD tumorigenesis. We hypothesized that adrenocortical adenomas that form in the context of PPNAD may harbour β‐catenin mutations.

Phosphodiesterase 11A (PDE11A) and genetic predisposition to adrenocortical tumors.

Purpose: We have reported previously nonsense inactivating mutations of the phosphodiesterase 11A (PDE11A) gene in patients with micronodular adrenocortical hyperplasia and Cushing syndrome. The aim of this study is to investigate the presence of somatic or germ-line PDE11A mutations in various types of adrenocortical tumors: ACTH-independent macronodular adrenocortical hyperplasia (AIMAH), adrenocortical adenoma (ACA), and adrenocortical cancer (ACC). Experimental Design:PDE11A was sequenced in 117 adrenocortical tumors and 192 controls subjects; immunohistochemistry for PDE11A and tumor cyclic AMP levels were studied in a subgroup of adrenocortical tumors. Results: One PDE11A inactivating mutation (R307X) was found in one ACA, 22 germ-line missense variants (18.8%) were found in adrenocortical tumors, and only 11 missense variants (5.7%) were found in controls. By comparing the common mutations, a higher frequency of mutations in adrenocortical tumors than in age/sex-matched controls were observed [16% versus 10% in ACC, 19% versus 10% in ACA, and 24% versus 9% in AIMAH; odds ratio (OR), 3.53; P = 0.05]. Somatic DNA from adrenocortical tumors with missense variants showed a wild-type allelic loss. A significant difference between ACC and controls was observed for a polymorphism in exon 6 (E421E; OR, 2.1; P = 0.03) and three associated polymorphisms located in intron 10-exon 11-intron 11 (OR, 0.5; P = 0.01). In AIMAH/ACA, cyclic AMP levels were higher than in normal adrenals and decreased PDE11A immunostaining was present in adrenocortical tumors with PDE11A variants. Conclusions: The present investigation of a large cohort of adrenocortical tumors suggests that PDE11A sequence defects predispose to a variety of lesions (beyond micronodular adrenocortical hyperplasia) and may contribute to the development of these tumors in the general population.

Large Deletions of the PRKAR1A Gene in Carney Complex

Purpose: Since the identification of PRKAR1A mutations in Carney complex, substitutions and small insertions/deletions have been found in ∼70% of the patients. To date, no germ-line PRKAR1A deletion and/or insertion exceeded a few base pairs (up to 15). Although a few families map to chromosome 2, it is possible that current sequencing techniques do not detect larger gene changes in PRKAR1A–mutation-negative individuals with Carney complex. Experimental Design: To screen for gross alterations of the PRKAR1A gene, we applied Southern hybridization analysis on 36 unrelated Carney complex patients who did not have small intragenic mutations or large aberrations in PRKAR1A, including the probands from two kindreds mapping to chromosome 2. Results: We found large PRKAR1A deletions in the germ-line of two patients with Carney complex, both sporadic cases; no changes were identified in the remaining patients, including the two chromosome-2-mapping families. In the first patient, the deletion is expected to lead to decreased PRKAR1A mRNA levels but no other effects on the protein; the molecular phenotype is predicted to be PRKAR1A haploinsufficiency, consistent with the majority of PRKAR1A mutations causing Carney complex. In the second patient, the deletion led to in-frame elimination of exon 3 and the expression of a shorter protein, lacking the primary site for interaction with the catalytic protein kinase A subunit. In vitro transfection studies of the mutant PRKAR1A showed impaired ability to bind cyclic AMP and activation of the protein kinase A enzyme. The patient bearing this mutation had a more-severe-than-average Carney complex phenotype that included the relatively rare psammomatous melanotic schwannoma. Conclusions: Large PRKAR1A deletions may be responsible for Carney complex in patients that do not have PRKAR1A gene defects identifiable by sequencing. Preliminary data indicate that these patients may have a different phenotype especially if their defect results in an expressed, abnormal version of the PRKAR1A protein.

Ochratoxin A concentrations in food and feed from a region with Balkan Endemic Nephropathy.

Balkan Endemic Nephropathy (BEN), a chronic renal disease of unknown aetiology, is found in geographically close areas of Bulgaria, Romania, Serbia, Croatia, Bosnia and Herzegovina, Slovenia, and the former Yugoslav Republic of Macedonia. Ochratoxin A (OTA), a secondary metabolite of Aspergillus and Penicillium species and a natural contaminant of food and feed, is a putative cause of BEN. Some studies have found a geographic covariation between OTA content in food/feed and BEN manifestation; others have not. In May 2000, using a competitive direct ELISA assay for OTA (detection limit 1 μg kg-1), we investigated OTA contamination in 165 samples of home-produced food (beans, potatoes, corn, wheat, flour) and feed from households in villages from the BEN region (Vratza district) of north-western Bulgaria. Samples were collected from: (a) BEN villages (n = 8), and therein from BEN households (20), and BEN-free households (16) (within-village controls, WVC households); and (b) BEN-free villages (7) and therein BEN-free households (22) (between-village controls, BVC). BEN households consistently had a higher proportion of OTA-positive samples than WVC households, but similar (for some foods) or lower (for other foods) proportions to BVC households. The proportion of OTA-positive samples was also higher in BVC than in WVC households. Furthermore, BEN households had a similar proportion of OTA-positive samples to the pooled, WVC and BVC, group of households. OTA-exposure estimates, derived from our OTA-concentration findings and the reported average per capita monthly consumption of basic foods in rural Bulgaria, showed the highest OTA intake in BEN households (1.21 μg day-1), versus 1.03 μg day-1 in BVC and 0.71 μ g day-1 in WVC households. These OTA intakes are higher than those in the EU, and are close to the upper limits acceptable to several food-safety organizations. The results indicate that OTA may not alone cause BEN; only synergistically with other environmental toxicants and/or predisposing genotypes may do so.