Genomic and sequence variants of protein kinase A regulatory subunit type 1ß (PRKAR1B) in patients with adrenocortical disease and Cushing syndrome.

PURPOSE: Protein kinase A (PKA) subunit defects (in PRKAR1A and PRKACA) are known to contribute to adrenal tumor pathogenesis. We studied the PRKAR1B gene for any genetic changes in bilateral adrenocortical hyperplasia (BAH) and cortisol-producing adrenal adenomas (CPA). METHODS: Exome sequencing and PRKAR1B copy-number variant (CNV) analysis were performed in 74 patients with BAH and 21 with CPA. PKA activity was studied in tumors with defects; sequence variants were investigated in vitro. RESULTS: Three PRKAR1B germline variants (p.I40V, p.A67V, p.A300T) were identified among 74 patients with BAH. PRKAR1B copy-number gains (CNG) were found in 3 of 21 CPAs, one in a tumor carrying a somatic PRKACA "hotspot" pathogenic variant p.L206R. CPAs bearing PRKAR1B CNGs showed higher PRKAR1B messenger RNA (mRNA) levels and reduced PKA activity. Baseline PKA activity was also decreased for p.A67V and p.A300T in vitro, and mutant PRKAR1ß bound PRKACα in fluorescence resonance energy transfer (FRET) recordings of cotransfected HEK293 cells stronger than normal. CONCLUSION: PRKAR1B is yet another PKA subunit that may potentially contribute to adrenal tumor formation. Its involvement in adrenocortical disease may be different from that of other subunits, because PRKAR1B variants and PRKAR1B CNGs were associated with decreased (rather than increased) overall PKA activity in vitro.

PRKACB variants in skeletal disease or adrenocortical hyperplasia: effects on protein kinase A.

Genetic variants in components of the protein kinase A (PKA) enzyme have been associated with various defects and neoplasms in the context of Carney complex (CNC) and in isolated cases, such as in primary pigmented nodular adrenocortical disease (PPNAD), cortisol-producing adrenal adenomas (CPAs), and various cancers. PRKAR1A mutations have been found in subjects with impaired cAMP-dependent signaling and skeletal defects; bone tumors also develop in both humans and mice with PKA abnormalities. We studied the PRKACB gene in 148 subjects with PPNAD and related disorders, who did not have other PKA-related defects and identified two subjects with possibly pathogenic PRKACB gene variants and unusual bone and endocrine phenotypes. The first presented with bone and other abnormalities and carried a de novo c.858_860GAA (p.K286del) variant. The second subject carried the c.899C>T (p.T300M or p.T347M in another isoform) variant and had a PPNAD-like phenotype. Both variants are highly conserved in the PRKACB gene. In functional studies, the p.K286del variant affected PRKACB protein stability and led to increased PKA signaling. The p.T300M variant did not affect protein stability or response to cAMP and its pathogenicity remains uncertain. We conclude that PRKACB germline variants are uncommon but may be associated with phenotypes that resemble those of other PKA-related defects. However, detailed investigation of each variant is needed as PRKACB appears to be only rarely affected in these conditions, and variants such as p.T300M maybe proven to be clinically insignificant, whereas others (such as p.K286del) are clearly pathogenic and may be responsible for a novel syndrome, associated with endocrine and skeletal abnormalities.

Association of estrogen receptor gene variants (ESR1 and ESR2) with polycystic ovary syndrome in Tunisia.

SNV (single nucleotide variation) in estrogen receptor (ESR1 and ESR2) genes are susceptibility markers for complex diseases, such as cancer, metabolic disorders and women infertility. We explored six widely used SNVs in ESR1 (rs2234693, rs9340799, rs3798577, rs3020314) and ESR2 (rs1256049, rs4986938) in polycystic ovary syndrome (PCOS) in women from Tunisia (n = 254) compared to controls (n = 170). Genotyping was performed by RFLP-PCR or real-time PCR and analyzed in GoldenHelix statistical package. Logistic regression revealed association of rs2234693, rs3798577 and rs3020314 (ESR1) and rs1256049 (ESR2), the association of rs2234693 (C/T) being the strongest with P < 4.81 × 10-6, 2.88 × 10-5 after Bonferroni correction, OR 0.31, 95%CI (0.18-0.53)). Correlations were found with LH, LH/FSH or hyperandrogenism and even more significant with metabolic syndrome (rs9340799) and hyperglycemia (rs3798577). Among 14 haplotypes reconstructed in ESR1gene, four haplotypes (H1 to H4) were associated with PCOS the strongest being that of H1 (P < 0.002) supported by Bonferroni (P < 0.033) and permutation tests (P < 4 x10-4). In haplotype trend regression, concordant correlations were found with insulin resistance (P < 0.033) for H2 and with high blood pressure for H3 (P < 0.048). While these data revealed influential role on metabolic rather and hormonal features of PCOS, the association of rs2234693 was the strongest among all ethnic populations studied thus far giving a new insight on estrogen receptor gene variation in distant North African populations and their role in metabolic alteration of PCOS.

Fine-scale haplotype mapping of MUT, AACS, SLC6A15 and PRKCA genes indicates association with insulin resistance of metabolic syndrome and relationship with branched chain amino acid metabolism or regulation.

Branched chain amino acids (BCAA) are essential elements of the human diet, which display increased plasma levels in obesity and regained particular interest as potential biomarkers for development of diabetes. To define determinants of insulin resistance (IR) we investigated 73 genes involved in BCAA metabolism or regulation by fine-scale haplotype mapping in two European populations with metabolic syndrome. French and Romanians (n = 465) were genotyped for SNPs (Affymetrix) and enriched by imputation (BEAGLE 4.1) at 1000 genome project density. Initial association hits detected by sliding window were refined (HAPLOVIEW 3.1 and PHASE 2.1) and correlated to homeostasis model assessment (HOMAIR) index, in vivo insulin sensitivity (SI) and BCAA plasma levels (ANOVA). Four genomic regions were associated with IR located downstream of MUT, AACS, SLC6A15 and PRKCA genes (P between 9.3 and 3.7 x 10-5). Inferred haplotypes up to 13 SNPs length were associated with IR (e.g. MUT gene with P < 4.9 x 10-5; Bonferroni 1.3 x 10-3) and synergistic to HOMAIR. SNPs in the same regions were also associated with one order of magnitude lower P values (e.g. rs20167284 in the MUT gene with P < 1.27 x 10-4) and replicated in Mediterranean samples (n = 832). In French, influential haplotypes (OR > 2.0) were correlated with in vivo insulin sensitivity (1/SI) except for SLC6A15 gene. Association of these genes with BCAA levels was variable, but influential haplotypes confirmed implication of MUT from BCAA metabolism as well as a role of regulatory genes (AACS and PRKCA) and suggested potential changes in transcriptional activity. These data drive attention towards new regulatory regions involved in IR in relation with BCAA and show the ability of haplotypes in phased DNA to detect signals complimentary to SNPs, which may be useful in designing genetic markers for clinical applications in ethnic populations.

Branched-Chain Amino Acid Database Integrated in MEDIPAD Software as a Tool for Nutritional Investigation of Mediterranean Populations.

Branched-chained amino acids (BCAA) are essential dietary components for humans and can act as potential biomarkers for diabetes development. To efficiently estimate dietary intake, we developed a BCAA database for 1331 food items found in the French Centre d'Information sur la Qualité des Aliments (CIQUAL) food table by compiling BCAA content from international tables, published measurements, or by food similarity as well as by calculating 267 items from Greek, Turkish, Romanian, and Moroccan mixed dishes. The database embedded in MEDIPAD software capable of registering 24 h of dietary recalls (24HDR) with clinical and genetic data was evaluated based on archived 24HDR of the Saint Pierre Institute (France) from 2957 subjects, which indicated a BCAA content up to 4.2 g/100 g of food and differences among normal weight and obese subjects across BCAA quartiles. We also evaluated the database of 119 interviews of Romanians, Turkish and Albanians in Greece (27⁻65 years) during the MEDIGENE program, which indicated mean BCAA intake of 13.84 and 12.91 g/day in males and females, respectively, comparable to other studies. The MEDIPAD is user-friendly, multilingual, and secure software and with the BCAA database is suitable for conducting nutritional assessment in the Mediterranean area with particular facilities for food administration.

Diversity of Y-chromosomal and mtDNA Markers Included in Mediscope Chip within Two Albanian Subpopulations from Croatia and Kosovo: Preliminary Data.

The aim of this preliminary study is to analyze genetic specificity of Kosovo Albanians comparing with neighboring populations using new genetic tool - MEDISCOPE gene chip, to investigate the feasibility of this approach. We collected 37 DNA samples (9 Croats, 17 Albanians from Croatia and 11 Albanians from Kosovo) from unrelated males born in Croatia and Kosovo. Additionally, samples were expanded with female individuals and mtDNA analysis included a total of 61 samples (15 Croats, 23 Albanians from Croatia and 23 Albanians from Kosovo). This pilot study suggests that the usage of the MEDISCOPE chip could be recognized as an efficient tool within recognition of the population genetic specificity even within extremely small sample size.