Early life stress unravels epistatic genetic associations of cortisol pathway genes with depression.

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis represents one of the most consistent pathophysiological findings in depressive disorders. Cortisol signaling is affected by proteins that mediate its cellular responses or alters its availability to mineralocorticoid and glucocorticoid receptors. In our study, we evaluated candidate genes that may influence the risk for depression and suicide due to its involvement in cortisol signaling. The aim of the study was to assess whether the genotypes of these genes are associated with the risk for depression, severity of depressive symptoms, suicidal ideation, and suicide attempts. And whether there is interaction between genes and early-life stress. In this study, 100 healthy controls and 140 individuals with depression were included. The subjects were clinically assessed using the 21-item GRID-Hamilton questionnaires (GRID-HAMD-21), Beck Scale for Suicidal Ideation (BSI), and the Childhood Trauma Questionnaire (CTQ). A robust multifactorial dimensionality reduction analysis was used to characterize the interactions between the genes HSD11B1, NR3C1, NR3C2, and MDR1 and early-life stress. It was found a significant association of the heterozygous genotype of the MDR1 gene rs1128503 polymorphism with reduced risk of at least one suicide attempt (OR: 0.08, p = 0.003*) and a reduction in the number of suicide attempts (ß = -0.79, p = 0.006*). Furthermore, it was found that the MDR1 rs1228503 and NR3C2 rs2070951 genes interact with early-life stress resulting in a strong association with depression (p = 0.001). Our findings suggest that polymorphisms in the MDR1 and NR3C2 genes and their interaction with childhood trauma may be important biomarkers for depression and suicidal behaviors.

Epigenetic control of adamantinomatous craniopharyngiomas.

INTRODUCTION: Studies addressing the methylation pattern in adamantinomatous craniopharyngioma (ACP) are lacking. OBJECTIVE: To identify methylation signatures in ACPs regarding clinical presentation and outcome. METHODS: Clinical and pathology data were collected from 35 ACP patients (54% male; 18.1 years [2-68]). CTNNB1 mutations and methylation profile (MethylationEPIC/Array-Illumina) were analyzed in tumoral DNA. Unsupervised machine learning analysis of this comprehensive methylome sample was achieved using hierarchical clustering and multi-dimensional scaling. Statistical associations between clusters and clinical features were achieved using Fisher's test and global biological process interpretations were aided by Gene Ontology enrichment analyses. RESULTS: Two clusters were revealed consistently by all unsupervised methods (ACP-1: n = 18; ACP-2: n = 17) with strong bootstrap statistical support. ACP-2 was enriched by CTNNB1 mutations (100% vs 56%, P = 0.0006), hypomethylated in CpG Island (CGI), non-CGI sites, and globally (P < 0.001), and associated with greater tumor size (24.1 vs 9.5cm3, P = 0.04). Enrichment analysis highlighted pathways on signaling transduction, transmembrane receptor, development of anatomical structures, cell-adhesion, cytoskeleton organization, and cytokine binding, and also cell-type specific biological processes as regulation of oligodendrocytes, keratinocyte, and epithelial cells differentiation. CONCLUSION: Two clusters of ACP patients were consistently revealed by unsupervised machine learning methods, being one of them significantly hypomethylated, enriched by CTNNB1 mutated ACPs, and associated with increased tumor size. Enrichment analysis reinforced pathways involved in tumor proliferation and in cell-specific tumoral microenvironment.

Nicotinamide Nucleotide Transhydrogenase Is Essential for Adrenal Steroidogenesis: Clinical and In Vitro Lessons.

CONTEXT: Nicotinamide nucleotide transhydrogenase (NNT) acts as an antioxidant defense mechanism. NNT mutations cause familial glucocorticoid deficiency (FGD). How impaired oxidative stress disrupts adrenal steroidogenesis remains poorly understood. OBJECTIVE: To ascertain the role played by NNT in adrenal steroidogenesis. METHODS: The genotype-phenotype association of a novel pathogenic NNT variant was evaluated in a boy with FGD. Under basal and oxidative stress (OS) induced conditions, transient cell cultures of the patient's and controls' wild-type (WT) mononuclear blood cells were used to evaluate antioxidant mechanisms and mitochondrial parameters (reactive oxygen species [ROS] production, reduced glutathione [GSH], and mitochondrial mass). Using CRISPR/Cas9, a stable NNT gene knockdown model was built in H295R adrenocortical carcinoma cells to determine the role played by NNT in mitochondrial parameters and steroidogenesis. NNT immunohistochemistry was assessed in fetal and postnatal human adrenals. RESULTS: The homozygous NNT p.G866D variant segregated with the FGD phenotype. Under basal and OS conditions, p.G866D homozygous mononuclear blood cells exhibited increased ROS production, and decreased GSH levels and mitochondrial mass than WT NNT cells. In line H295R, NNT knocked down cells presented impaired NNT protein expression, increased ROS production, decreased the mitochondrial mass, as well as the size and the density of cholesterol lipid droplets. NNT knockdown affected steroidogenic enzyme expression, impairing cortisol and aldosterone secretion. In human adrenals, NNT is abundantly expressed in the transition fetal zone and in zona fasciculata. CONCLUSION: Together, these studies demonstrate the essential role of NNT in adrenal redox homeostasis and steroidogenesis.

The Association of Biochemical and Genetic Biomarkers in VEGF Pathway with Depression.

VEGF is an important neurotrophic and vascular factor involved in mental disorders. The objective of this study was to verify the effect of genetic polymorphisms in the VEGF pathway on the risk for depression, symptom intensity, and suicide attempts. To examine the association between the VEGF pathway and depression, we genotyped polymorphisms and measured the plasma concentrations of VEGF, KDR, and FLT1 proteins. The participants were 160 patients with depression and 114 healthy controls. The questionnaires that assessed the clinical profile of the patients were the MINI-International Neuropsychiatric Interview, GRID-HAMD21, CTQ, BSI, and the number of suicide attempts. Genotyping of participants was performed using the real-time PCR and protein measurements were performed using the enzyme-linked immunosorbent assay (ELISA). VEGF and its inhibitors were reduced in depression. Individuals with depression and displaying the homozygous AA of the rs699947 polymorphism had higher plasma concentrations of VEGF (p-value = 0.006) and were associated with a greater number of suicide attempts (p-value = 0.041). Individuals with depression that were homozygous for the G allele of the FLT1 polymorphism rs7993418 were associated with lower symptom severity (p-value = 0.040). Our results suggest that VEGF pathway polymorphisms are associated with the number of suicide attempts and the severity of depressive symptoms.

DNA methylation is a comprehensive marker for pediatric adrenocortical tumors.

Children diagnosed with pediatric adrenocortical tumors (pACT) have variable outcomes, and, to date, the disease lacks robust prognostic biomarkers. The prognostic potential of tumor methylation has been demonstrated in several cancers. We aimed to evaluate the pACT methylation profile and its association with disease presentation and survival. In this cross-sectional study, we accessed the DNA methylation (MethylationEPIC Array, Illumina) of 57 primary pACT from Southeastern Brazil and the respective patients' clinicopathological features. We also applied our analysis in an independent 48 pACT methylation dataset. Unsupervised learning whole-methylome analysis showed two groups with distinct methylation signatures: pACT-1 and pACT-2. Compared to pACT-2, pACT-1 tumors were enriched with higher methylation in CpG islands, mainly in gene promoter regions. The topmost hypermethylated gene in these samples was shown to be underexpressed. Patients in the pACT-1 group were older at diagnosis and were more likely to have carcinomas and nonlocalized/advanced and recurrent/metastatic disease. Univariate and bivariate regressions showed that pACT-1 methylation signature confers superior hazard ratio of disease progression and death than known prognostic features. The methylation groups had similar frequencies of germline mutations in the TP53 gene, including the regionally frequent p.R337H. Our analysis replication validated our findings and reproduced those recently described in pACT. We demonstrated the existence of different tumor methylation signatures associated with pACT presentation and clinical evolution, even in the context of germline TP53 mutations. Our data support tumor methylation profiling as a robust and independent prognostic biomarker for pACT and suggest a list of candidate genes for further validation.

Telomere length and Wnt/ß-catenin pathway in adamantinomatous craniopharyngiomas.

Objectives: To evaluate how telomere length behaves in adamantinomtous craniopharyngioma (aCP) and if it contributes to the pathogenesis of aCPs with and without CTNNB1 mutations. Design: Retrospective cross-sectional study enrolling 42 aCP patients from 2 tertiary institutions. Methods: Clinicopathological features were retrieved from the patient's charts. Fresh frozen tumors were used for RNA and DNA analyses. Telomere length was evaluated by qPCR (T/S ratio). Somatic mutations in TERT promoter (TERTp) and CTNNB1 were detected by Sanger and/or whole-exome sequencing. We performed RNA-Seq to identify differentially expressed genes in aCPs presenting with shorter or longer telomere lengths. Results: Mutations in CTNNB1 were detected in 29 (69%) tumors. There was higher frequency of CTNNB1 mutations in aCPs from patients diagnosed under the age of 15 years (85% vs 15%; P = 0.04) and a trend to recurrent disease (76% vs 24%; P = 0.1). No mutation was detected in the TERTp region. The telomeres were shorter in CTNNB1-mutated aCPs (0.441, IQR: 0.297-0.597vs 0.607, IQR: 0.445-0.778; P = 0.04), but it was neither associated with clinicopathological features nor with recurrence. RNAseq identified a total of 387 differentially expressed genes, generating two clusters, being one enriched for short telomeres and CTNNB1-mutated aCPs. Conclusions: CTNNB1: mutations are more frequent in children and adolescents and appear to associate with progressive disease. CTNNB1-mutated aCPs have shorter telomeres, demonstrating a relationship between the Wnt/ß-catenin pathway and telomere biology in the pathogenesis of aCPs.

Vitamin D receptor hypermethylation as a biomarker for pediatric adrenocortical tumors.

Objective: Pediatric adrenocortical tumors (pACT) display complex genomic backgrounds, lacking robust prognostic markers and targeted therapeutic options. Vitamin D3 receptor (VDR) promoter hypermethylation and underexpression were reported in adrenocortical carcinomas from adult patients. In this study, we aimed to investigate VDR expression levels and methylation status in pACT and their clinical and prognostic significance. Design: Retrospective cross-sectional study enrolling pediatric patients with ACT from two tertiary referral institutions. Methods: We evaluated clinicopathological features, VDR mRNA (qPCR) and protein (immunohistochemistry) expression, and VDR-wide methylation of ACT samples from 108 pediatric patients. Fourteen pediatric and 32 fetal and postnatal normal adrenals were used as controls. Results: Unlike in pre- and post-natal normal adrenals, most pACT lacked nuclear VDR expression and had reduced mRNA levels, especially the carcinomas. Unsupervised analysis of VDR methylation data revealed two groups of pACT with distinct disease features and outcomes. Tumors with high VDR methylation presented lower mRNA levels, and the respective patients presented advanced disease and reduced disease-free and overall survival. Conclusions: VDR has a role in normal adrenocortical development and homeostasis, which is impaired during tumorigenesis. VDR hypermethylation and underexpression may be both predictive and prognostic biomarkers for pACT.

Arginase II polymorphisms modify the hypotensive responses to propofol by affecting nitric oxide bioavailability.

PURPOSE: Propofol anesthesia is usually accompanied by hypotensive responses, which are at least in part mediated by nitric oxide (NO). Arginase I (ARG1) and arginase II (ARG2) compete with NO synthases for their common substrate L-arginine, therefore influencing the NO formation. We examined here whether ARG1 and ARG2 genotypes and haplotypes affect the changes in blood pressure and NO bioavailability in response to propofol. METHODS: Venous blood samples were collected from 167 patients at baseline and after 10 min of anesthesia with propofol. Genotypes were determined by polymerase chain reaction. Nitrite concentrations were measured by using an ozone-based chemiluminescence assay, while NOx (nitrites + nitrates) levels were determined by using the Griess reaction. RESULTS: We found that patients carrying the AG + GG genotypes for the rs3742879 polymorphism in ARG2 gene and the ARG2 GC haplotype show lower increases in nitrite levels and lower decreases in blood pressure after propofol anesthesia. On the other hand, subjects carrying the variant genotypes for the rs10483801 polymorphism in ARG2 gene show more intense decreases in blood pressure (CA genotype) and/or higher increases in nitrite levels (CA and AA genotypes) in response to propofol. CONCLUSION: Our results suggest that ARG2 variants affect the hypotensive responses to propofol, possibly by modifying NO bioavailability. TRIAL REGISTRATION: NCT02442232.

Nurse empowerment through Pharmacogenetics.

OBJECTIVE: to verify the existence of elements that justify the use of pharmacogenetics by the Brazilian nurse. METHOD: this is a quantitative, cross-sectional, observational, descriptive study, whose final sample was 67 individuals. The participants were healthy at the time of the study and reported a history of previous use and the occurrence of adverse effects by drugs commonly used and metabolized by CYP2C9. We collected 4 mL of venous blood for subsequent DNA extraction by salting out method and genotyping of the CYP2C9*2 and CYP2C9*3 polymorphisms, using Polymerase Chain Reaction in real time using Taqman assays. RESULTS: the use of drugs metabolized by CYP2C9 was frequent (more than 75% of the individuals have already used between 2 or 4 of these drugs). Regarding adverse events, there were 19 perceived symptomatic occurrences associated with drugs metabolized by CYP2C9. The allele frequency of the polymorphism * 2 and * 3 in the population studied was 11.1% and 7.5%, respectively, and there was a coincidence between the presence of alleles of low enzyme activity and the occurrence of adverse effects. CONCLUSION: there are elements that justify the adoption of pharmacogenetics in the nursing care to reduce the occurrence of adverse reactions to drugs metabolized by CYP2C9.

Interaction Between NOS3 and HMOX1 on Antihypertensive Drug Responsiveness in Preeclampsia.

OBJECTIVE: We examined the interaction of polymorphisms in the genes heme oxygenase-1 (HMOX1) and nitric oxide synthase (NOS3) in patients with preeclampsia (PE) as well as the responsiveness to methyldopa and to total antihypertensive therapy. METHODS: The genes HMOX1 (rs2071746, A/T) and NOS3 (rs1799983, G/T) were genotyped using TaqMan allele discrimination assays (Applied Biosystems, Foster City, CA, USA ), and the levels of enzyme heme oxygenase-1 (HO-1) were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: We found interactions between genotypes of the HMOX-1 and NOS3 genes and responsiveness to methyldopa and that PE genotyped as AT presents lower levels of protein HO-1 compared with AA. CONCLUSION: We found interactions between the HMOX-1 and NOS3 genes and responsiveness to methyldopa and that the HMOX1 polymorphism affects the levels of enzyme HO-1 in responsiveness to methyldopa and to total antihypertensive therapy. These data suggest impact of the combination of these two polymorphisms on antihypertensive responsiveness in PE.

OBJETIVO: Examinamos a interação dos polimorfismos nos genes heme oxigenase-1 (HMOX1) eóxido nítrico sintase (NOS3) em pacientes com pré-eclâmpsia (PE)bem como as capacidades de resposta à metildopa e à terapia anti-hipertensiva. MéTODOS: Os polimorfismos nos genes HMOX1 (rs2071746, A/T) e NOS3 (rs1799983, G/T) foram genotipados usando TaqMan allele discrimination assays (Applied Biosystems, Foster City, CA, EUA), e os níveis da enzima heme oxigenase-1 (HO-1) foram medidos por enzyme-linked immunosorbent assay (ELISA). RESULTADOS: Foram encontradas interações entre os genótipos da HMOX-1 e NOS3 e responsividade à metildopa, e que pacientes genotipados como AT apresentam níveis mais baixos de proteína HO-1 em comparação com o genótipo AA. CONCLUSãO: Foram encontradas interações entre os genes HMOX-1 e NOS3 e responsividade à metildopa e que o polimorfismo localizado no gene HMOX1 afeta os níveis de enzima HO-1 na resposta à metildopa e à terapia anti-hipertensiva. Esses dados sugerem o impacto da combinação desses dois polimorfismos na resposta anti-hipertensiva na PE.

Clinical, Molecular, Functional, and Structural Characterization of CYP17A1 Mutations in Brazilian Patients with 17-Hydroxylase Deficiency.

17-Hydroxylase-deficiency (17OHD) is a rare form of congenital adrenal hyperplasia. The aim of the work was to study clinical, biochemical, and the follow up of 17OHD patients and evaluate the function and structure of CYP17A1 mutations. Brazilian patients (three 46, XX and four 46, XY; 17±1.9 years) with combined 17-hydroxylase/17,20-lyase deficiency were evaluated. CYP17A1 gene was sequenced. Functional analysis was performed transfecting COS7 cells, which were exposed to progesterone or 17α-hydroxypregnolone substrates. Hormones were determined by RIA or LC-MS/MS. Three-dimensional structural modeling was performed by Modeller software. All patients presented prepubertal female external genitalia, primary amenorrhea, hypergonadotrophic hypogonadism, hypokalemic hypertension, decreased cortisol, and increased ACTH and corticosterone levels. Five patients presented previously described mutations: p.W406R/p.W406R, IVS2-2A>C/p.P428L, and p.P428L/p.P428L. Two patients presented the compound heterozygous p.G478S/p.I223Nfs*10 mutations, whose CYP17A1 activity and the three dimensional structural modeling are originally studied in this paper. CYP17A1 activity of p.G478S was 13 and 58% against progesterone and 17-hydroxypregnenolone, respectively. The p.I223Nfs*10 caused a truncated inactive protein. Three-dimensional p.G478S structural modeling showed different internal hydrophobic interaction with W313 and created an additional chain side contact with L476 residue. Due to the rarity of 17OHD, the long term follow up (15.3±3.1 years) of our patients will help endocrinologists on the management of patients with 17OHD. The mutation p.G478S/pI223Nfs*10 led to severe 17OHD and impaired CYP17A1 structure and function. The integration of in silico and in vitro analysis showed how the amino acid changes affected the CYP17A1 activity and contributed to clarify the molecular interactions of CYP17A1.

USP8 Mutations and Cell Cycle Regulation in Corticotroph Adenomas.

Corticotroph adenomas frequently harbor somatic USP8 mutations. These adenomas also commonly exhibit underexpression of P27, a cell cycle regulator. The present study aimed to determine the influence of USP8 mutations on clinical features of Cushing's disease and to elucidate the relationship between USP8 mutations and P27 underexpression in these tumors. Retrospective study with 32 patients with Cushing's disease was followed at the Ribeirao Preto Medical School University Hospital. We evaluated the patients' clinical data, the USP8 mutation status and the gene expression of cell cycle regulators P27/CDKN1B, CCNE1, CCND1, CDK2, CDK4, and CDK6 in tumor tissue in addition to the protein expression of P27/CDKN1B. We observed somatic mutations in the exon 14 of USP8 in 31.3% of the patients. Larger tumor size was observed in patients harboring USP8 mutations (p=0.04), with similar rates of remission, age of presentation, salivary cortisol at 23:00 h and after 1 mg dexamethasone, ACTH levels, and early postoperative plasma cortisol. We observed no differences regarding the gene or protein expression of the cell cycle regulators according to USP8 mutation status. In this Brazilian series, the observed frequency of USP8 somatic mutations was similar to that reported in European ancestry populations. Although it was reasonable that USP8 mutations could contribute to cell cycle dysregulation and P27 underexpression in corticotroph adenomas, our data did not confirm this hypothesis. It is possible that increased deubiquitinase activity observed in mutated USP8 might influence other pathways related to cell growth and proliferation.

Association of 11ß-hydroxysteroid dehydrogenase type1 (HSD11b1) gene polymorphisms with outcome of antidepressant therapy and suicide attempts.

The hypothalamic-pituitary-adrenal axis has been implicated in the pathophysiology of depressive disorders. HSD11B1 encodes 11ß-hydroxysteroid dehydrogenase type1 enzyme, responsible for converting cortisone to cortisol. Genetic polymorphisms in HSD11B1 may impact in depression outcome and risk of suicide. This study aimed to assess whether HSD11B1 genotypes and haplotypes are associated with depression risk, severity of symptoms and suicidal attempts, considering early-life stress as an environmental factor. Here, 142 depressive patients and 103 healthy controls were included. Patients were enrolled from the Affective Disorders ambulatory and day hospital units, both within the University General Hospital of Ribeirao Preto. All subjects were clinically assessed applying the Mini-PLUS International Neuropsychiatric Interview, followed by the 21-item GRID-Hamilton Depression Scale, Childhood Trauma Questionnaire and Beck Scale for Suicidal Ideation (BSI). All subjects underwent antecubital vein puncture to obtain blood for DNA extraction. Genotyping of rs11119328 and rs11811440 were performed using allele-specific oligonucleotide polymerase chain reaction. We found a significant association of rs11119328 variant genotypes with increased risk for at least one suicide attempt (OR: 7.10, p = 0.049) and an association of variant genotypes of rs11811440 with euthymic mood under optimized pharmacological treatment (OR: 0.05, P = 0.014). These tests included correction for confounding factors. The association of genetic markers with depression risk, GRID-HAM-D21 and BSI scores and the number of suicidal attempts were nonsignificant. Haplotypes combining both markers were not associated with the studied phenotypes. We conclude that HSD11B1 polymorphisms may be relevant biomarkers for detecting subjects genetically vulnerable to poorer antidepressant response and higher risk of suicide attempts.

Nurse empowerment through Pharmacogenetics / Empoderamento do enfermeiro por meio da Farmacogenética / Empoderamiento del enfermero a través de la farmacogenética

Objective: to verify the existence of elements that justify the use of pharmacogenetics by the Brazilian nurse. Method: this is a quantitative, cross-sectional, observational, descriptive study, whose final sample was 67 individuals. The participants were healthy at the time of the study and reported a history of previous use and the occurrence of adverse effects by drugs commonly used and metabolized by CYP2C9. We collected 4 mL of venous blood for subsequent DNA extraction by salting out method and genotyping of the CYP2C9*2 and CYP2C9*3 polymorphisms, using Polymerase Chain Reaction in real time using Taqman assays. Results: the use of drugs metabolized by CYP2C9 was frequent (more than 75% of the individuals have already used between 2 or 4 of these drugs). Regarding adverse events, there were 19 perceived symptomatic occurrences associated with drugs metabolized by CYP2C9. The allele frequency of the polymorphism * 2 and * 3 in the population studied was 11.1% and 7.5%, respectively, and there was a coincidence between the presence of alleles of low enzyme activity and the occurrence of adverse effects. Conclusion: there are elements that justify the adoption of pharmacogenetics in the nursing care to reduce the occurrence of adverse reactions to drugs metabolized by CYP2C9.

Objetivo: verificar a existência de elementos que justifiquem o uso da farmacogenética pelo enfermeiro brasileiro. Método: trata-se de um estudo quantitativo, do tipo transversal, observacional descritivo, cuja amostra final foi de 67 indivíduos. Os participantes estavam saudáveis no momento do estudo e reportaram histórico de uso prévio e ocorrência de efeitos adversos por fármacos comumente utilizados e metabolizados pela CYP2C9. Coletamos 4 mL de sangue venoso para posterior extração de DNA por método salting out e genotipagem dos polimorfismos CYP2C9*2 e CYP2C9*3 através de Polymerase Chain Reaction em tempo real, utilizando ensaios Taqman. Resultados: o uso de fármacos metabolizados pela CYP2C9 foi frequente (mais de 75% dos sujeitos já utilizaram entre 2 ou 4 desses fármacos). A respeito dos eventos adversos, houve 19 ocorrências sintomáticas percebidas, associadas a fármacos metabolizados pela CYP2C9. A frequência alélica do polimorfismo *2 e *3 na população estudada foi de 11,1% e 7,5%, respectivamente, com coincidência entre a presença dos alelos de baixa atividade enzimática e ocorrência de efeitos adversos. Conclusão: existem elementos que justificam a adoção da farmacogenética no cuidado do enfermeiro com objetivo de redução da ocorrência de reações adversas a fármacos metabolizados pela CYP2C9.

Objetivo: verificar la existencia de elementos que justifiquen el uso de la farmacogenética por parte del enfermero brasileño. Método: se trata de un estudio cuantitativo, transversal, observacional, descriptivo, cuya muestra final fue de 67 individuos. Los participantes estaban sanos en el momento del estudio e informaron un historial de uso previo y la aparición de efectos adversos por fármacos comúnmente utilizados y metabolizados por el CYP2C9. Recolectamos 4 ml de sangre venosa para la posterior extracción de ADN mediante el método de salazón y genotipificación de los polimorfismos CYP2C9 * 2 y CYP2C9 * 3 a través de la reacción en cadena de la polimerasa en tiempo real utilizando ensayos Taqman. Resultados: el uso de drogas metabolizadas por el CYP2C9 fue frecuente (más del 75% de las personas ya han usado entre 2 o 4 de estas drogas). Con respecto a los eventos adversos, hubo 19 casos sintomáticos percibidos asociados con medicamentos metabolizados por el CYP2C9. La frecuencia alélica del polimorfismo * 2 y * 3 en la población estudiada fue de 11.1% y 7.5%, respectivamente, y hubo una coincidencia entre la presencia de alelos de baja actividad enzimática y la aparición de efectos adversos. Conclusión: existen elementos que justifican la adopción de la farmacogenética en el cuidado del enfermero para reducir la aparición de reacciones adversas a los medicamentos metabolizados por el CYP2C9.

DDAH1 and DDAH2 polymorphisms associate with asymmetrical dimethylarginine plasma levels in erectile dysfunction patients but not in healthy controls.

Erectile Dysfunction (ED) is one of the main complaints of aging male. A reduced production of Nitric Oxide (NO) may be involved in ED pathogenesis. NO is synthesized from l-Arginine, and asymmetrical dimethylarginine inhibits all NO synthases. DDAH1 and DDAH2 are genes that encode enzymes responsible for metabolizing ADMA. We aimed to assess whether: 1) ADMA and nitrite levels associated with ED risk and with symptoms intensity; and whether 2) DDAH1 and DDAH2 gene polymorphisms associate with changes in biochemical data, and with ED risk and symptoms intensity. In this study were included 98 healthy controls and 130 ED patients. ADMA levels were measured by ELISA and nitrite levels by Chemiluminescence. DDAH1 and DDAH2 polymorphisms were assessed by Taqman assays. We found that ED had increased nitrite levels and lower ADMA levels than Control group (P < 0.05). We found a significant correlation of ADMA with Nitrite levels only in ED (B = -0.57, P < 0.001). Genotypes and haplotypes of DDAH1 were associated with ADMA levels in ED (P < 0.05), while haplotypes of DDAH2 were associated with levels of nitrite in ED (P < 0.05). Erectile dysfunction patients show an association between DDAH1 and DDAH2 polymorphisms with ADMA levels, which in turn are negatively correlated with nitrite levels. This is not evident on healthy controls.

Circulating HO-1 levels are not associated with plasma sFLT-1 and GTn HMOX1 polymorphism in preeclampsia.

OBJECTIVES: We aimed to assess the plasma HO-1 level and its interrelationship with the plasma sFLT-1 level in preeclamptic and healthy pregnant women with different variants of microsatellite polymorphism (GTn) located in the promoter region of the HMOX-1 gene. METHODS: HO-1 and sFLT-1 were measured by ELISA. HMOX1 genotyping was performed using fragment analysis. RESULTS: We found similar and higher levels of plasma HO-1 and sFLT-1, respectively, in preeclampsia. Similar genotypes and alleles frequencies were found in both groups and the absence of modulation of HO-1 levels by genotypes were observed. CONCLUSION: The plasma HO-1 levels are not increased in preeclampsia women and neither related to sFLT-1 levels and GTn polymorphism.

Relationship between asymmetric dimethylarginine, nitrite and genetic polymorphisms: Impact on erectile dysfunction therapy.

Sildenafil is the most used treatment of erectile dysfunction, however a large part of patients do not respond to therapy. This drug enhances nitric oxide (NO) signaling, and therefore factors that alter NO production may impact this drug responsiveness. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of all NO synthases, and is metabolized by Dimethylarginine Dimethilaminohydrolase (DDAH) 1 and 2. Here we aimed to assess the relationship between plasma levels of ADMA and nitrite (marker of nitric oxide production) with Sildenafil responsiveness. We also studied genetic polymorphisms in DDAH1 and DDAH2 genes and their relation with biochemical and clinical data. Were included here 140 patients, divided in Clinical Erectile Dysfunction (CED) or Post-Prostatectomy Erectile Dysfunction (PPED) groups. Erectile function was evaluated before and after Sildenafil on-demand treatment using the International Index for Erectile Function Questionnaire. We have found that nitrite was associated with worse response to Sildenafil (r = - 0.25, P = 0.040). rs1554597 and rs18582 DDAH1 polymorphisms were associated with changes in ADMA levels in CED (B = - 0.23, P = 0.002; B = - 0.15, P = 0.017 for both variant genotypes, respectively). Finally, DDAH2 polymorphisms were associated with altered responsiveness to Sildenafil in PPED (B = +0.19, P = 0.027).

Sonic Hedgehog mutations are not a common cause of congenital hypopituitarism in the absence of complex midline cerebral defects.

CONTEXT AND OBJECTIVE: Sonic Hedgehog (SHH) and GLI2, an obligatory mediator of SHH signal transduction, are holoprosencephaly (HPE)-associated genes essential in pituitary formation. GLI2 variants have been found in patients with congenital hypopituitarism without complex midline cerebral defects (MCD). However, data on the occurrence of SHH mutations in these patients are limited. We screened for SHH and GLI2 mutations or copy number variations (CNV) in patients with congenital hypopituitarism without MCD or with variable degrees of MCD. PATIENTS AND METHODS: Detailed data on clinical, laboratory and neuroimaging findings of 115 patients presenting with congenital hypopituitarism without MCD, septo-optic dysplasia or HPE were analysed. The SHH and GLI2 genes were directly sequenced, and the presence of gene CNV was analysed by multiplex ligation-dependent probe amplification (MLPA). RESULTS: Anterior pituitary deficiency was found in 74% and 53% of patients with SOD or HPE, respectively. Diabetes insipidus was common in patients with HPE (47%) but infrequent in patients with congenital hypopituitarism or SOD (7% and 8%, respectively). A single heterozygous nonsense SHH mutation (p.Tyr175Ter) was found in a patient presenting with hypopituitarism and alobar HPE. No other SHH mutations or CNV were found. Nine GLI2 variations (8 missense and 1 frameshift) including a homozygous and a compound heterozygous variation were found in patients with congenital hypopituitarism or SOD, but not in HPE patients. No GLI2 CNV were found. CONCLUSION: SHH mutations or copy number variations are not a common cause of congenital hypopituitarism in patients without complex midline cerebral defects. GLI2 variants are found in some patients with congenital hypopituitarism without complex midline cerebral defects or septo-optic dysplasia. However, functional analyses of these variants are needed to strengthen genotype-phenotype relationship.

A rational, non-radioactive strategy for the molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

CONTEXT: Molecular diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) has not been straightforward. OBJECTIVE: To conduct a comprehensive genetic analysis by Multiplex Ligation dependent Probe Amplification (MLPA) and evaluate its reliability for the molecular CAH-21OHD diagnosis. PATIENTS AND METHODS: We studied 99 patients from 90 families with salt-wasting (SW; n=32), simple-virilizing (SV; n=29), and non-classical (NC; n=29) CAH-21OHD. Molecular analysis was sequentially performed by detecting the most frequent point mutations by allele-specific oligonucleotide polymerase chain reaction (ASO-PCR), large rearrangements by MLPA, and rare mutations by direct sequencing. Parental segregation was evaluated. RESULTS: ASO-PCR detected microconversions in 164 alleles (91.1%). MLPA identified CYP21A1P large conversions to CYP21A2 in 7 of the remaining 16 (43.7%), 30-kb deletions including the 3'-end of CYP21A1P, C4B, and the 5'-end of CYP21A2 in 3 of the 16 (18.7%), and a complete CYP21A2 deletion in one (6.3%). Five alleles (2.7%) required direct sequencing; three mutations located in the CYP21A2 gene and two derived from CYP21A1P were found. No parental segregation was observed in patients with the c.329_336del and/or the CL6 cluster mutations. These cases were not diagnosed by ASO-PCR, but MLPA detected deletions in the promoter region of the CYP21A2 gene, explaining the genotype/phenotype dissociation. CONCLUSION: Using the proposed algorithm, all alleles were elucidated. False-positive results in MLPA occurred when mutations or polymorphisms were located close to the probe-binding regions. These difficulties were overcome by the association of MLPA with ASO-PCR and paternal segregation. Using these approaches, we can successfully use MLPA in a cost-effective laboratory routine for the molecular diagnosis of CAH-21OHD.