A genome wide approach to identify genetic variants associated with left ventricular mass

Left ventricular mass (LVM) is an important clinical phenotype, whose assessment can predict adverse cardiovascular events and premature death in all genders, races, and ages. Increase in LVM defines left ventricular hypertrophy (LVH) with the thickening of the left ventricle of the heart. In community-based cohorts, the presence of left ventricular hypertrophy (LVH) and increased LVM predict the development of coronary heart disease, congestive heart failure, stroke, and cardiovascular disease. Thus this trait serves not only as measures of cardiac structure, but also as intermediate phenotype for clinical cardiovascular disease outcome. Several studies have indicated that LVM is influenced by genetic factors. Genome wide linkage and association studies in diverse population have been performed to identify genes influencing LVM, but much of the heritability remains unexplained, the identity of the underlying gene pathways and functional variants remain unknown, and the promise of genetically based risk prediction remains unfulfilled. The aim of the study was to investigate the association of common genetic variants with left ventricular mass using a genome wide approach in a large cohort of never treated mild-to-moderate essential hypertensive subjects. From the linear analysis, we selected 85 single nucleotide polymorphisms (SNPs), with a suggestive p-value of association with LVM ( 64 10-5). In particular, some SNPs lying in genes previously described as having a role in the pathogenesis of cardiac hypertrophy, such as ROCK1, IGF1, CACNA1D, FGFR1, TRAF5, SOX5, and KSR2. Each of them might play a putative role in determining the LVM phenotype as well as other pathophysiological pathways directly or indirectly linked to cardiac pathophysiology. To assess the risk alleles associated to the most interesting findings in relation to the phenotype studied, we performed a case-control analysis by dividing our sample in two subsets according to LVM values. Most of the SNPs associated with LVM in linear regression presented a significant association, showing that the carriers of the risk alleles have an odds ratio higher than 1 to have increased LVM, i.e. to be cases respect to controls. Nevertheless as for most of the complex traits, the observed odds ratios are modest (except for those biased by the absence of homozygous risk genotypes), so their relevance for a clinical use is uncertain. Thus, we defined a weighted genetic risk score using the effect size of the risk allele (beta value of the linear regression analysis) to account for the strength of the genetic association with each allele. The possibility to combine more variants in a global genetic risk score could be interesting and could add relevance to the results. In conclusion, our GWAS allowed us to pinpoint genes whose role in heart function and/or cardiac hypertrophy has been demonstrated in previously publications by different authors. Moreover, we highlighted the usefulness of an aggregate measure of risk of LVH to discriminate high-risk subjects. However, the results must be interpreted within the context of some potential limitations and perspectives. No SNPs reached a Bonferroni\u2019s significance level probably due to a limited sample size. However, the phenotypic homogeneity of our cohort and the absence of previous antihypertensive treatment are prerequisites for the identification of true genetic effects. A replication in independent cohorts is needed VII to further confirm the findings; however an independent cohort with similar criteria was not available for replication. Moreover, it often happens, as in our study, the significant SNPs map in non-coding regions, making it difficult to explain their causative role. These limitations should not reduce the relevance of the genes identified and confirmed by previously published papers. Future perspectives of this study should be the replication of the GWAS findings in independent cohorts and the assessment in independent samples of the prediction ability of wGRS to correctly classify true positives and true negatives according to their genetic background

Genome-Wide and Gene-Based Meta-Analyses Identify Novel Loci Influencing Blood Pressure Response to Hydrochlorothiazide

This study aimed to identify novel loci influencing the antihypertensive response to hydrochlorothiazide monotherapy. A genome-wide meta-analysis of blood pressure (BP) response to hydrochlorothiazide was performed in 1739 white hypertensives from 6 clinical trials within the International Consortium for Antihypertensive Pharmacogenomics Studies, making it the largest study to date of its kind. No signals reached genome-wide significance (P<5×10−8), and the suggestive regions (P<10−5) were cross-validated in 2 black cohorts treated with hydrochlorothiazide. In addition, a gene-based analysis was performed on candidate genes with previous evidence of involvement in diuretic response, in BP regulation, or in hypertension susceptibility. Using the genome-wide meta-analysis approach, with validation in blacks, we identified 2 suggestive regulatory regions linked to gap junction protein α1 gene (GJA1) and forkhead box A1 gene (FOXA1), relevant for cardiovascular and kidney function. With the gene-based approach, we identified hydroxy-delta-5-steroid dehydrogenase, 3 β- and steroid δ-isomerase 1 gene (HSD3B1) as significantly associated with BP response (P<2.28×10−4). HSD3B1 encodes the 3β-hydroxysteroid dehydrogenase enzyme and plays a crucial role in the biosynthesis of aldosterone and endogenous ouabain. By amassing all of the available pharmacogenomic studies of BP response to hydrochlorothiazide, and using 2 different analytic approaches, we identified 3 novel loci influencing BP response to hydrochlorothiazide. The gene-based analysis, never before applied to pharmacogenomics of antihypertensive drugs to our knowledge, provided a powerful strategy to identify a locus of interest, which was not identified in the genome-wide meta-analysis because of high allelic heterogeneity. These data pave the way for future investigations on new pathways and drug targets to enhance the current understanding of personalized antihypertensive treatment

Genome-wide meta-analysis of 241,258 adults accounting for smoking behaviour identifies novel loci for obesity traits

Few genome-wide association studies (GWAS) account for environmental exposures, like smoking, potentially impacting the overall trait variance when investigating the genetic contribution to obesity-related traits. Here, we use GWAS data from 51,080 current smokers and 190,178 nonsmokers (87% European descent) to identify loci influencing BMI and central adiposity, measured as waist circumference and waist-to-hip ratio both adjusted for BMI. We identify 23 novel genetic loci, and 9 loci with convincing evidence of gene-smoking interaction (GxSMK) on obesity-related traits. We show consistent direction of effect for all identified loci and significance for 18 novel and for 5 interaction loci in an independent study sample. These loci highlight novel biological functions, including response to oxidative stress, addictive behaviour, and regulatory functions emphasizing the importance of accounting for environment in genetic analyses. Our results suggest that tobacco smoking may alter the genetic susceptibility to overall adiposity and body fat distribution.Peer reviewe

Genome-wide meta-analysis of 241,258 adults accounting for smoking behaviour identifies novel loci for obesity traits

Few genome-wide association studies (GWAS) account for environmental exposures, like smoking, potentially impacting the overall trait variance when investigating the genetic contribution to obesity-related traits. Here, we use GWAS data from 51,080 current smokers and 190,178 nonsmokers (87% European descent) to identify loci influencing BMI and central adiposity, measured as waist circumference and waist-to-hip ratio both adjusted for BMI. We identify 23 novel genetic loci, and 9 loci with convincing evidence of gene-smoking interaction (GxSMK) on obesity-related traits. We show consistent direction of effect for all identified loci and significance for 18 novel and for 5 interaction loci in an independent study sample. These loci highlight novel biological functions, including response to oxidative stress, addictive behaviour, and regulatory functions emphasizing the importance of accounting for environment in genetic analyses. Our results suggest that tobacco smoking may alter the genetic susceptibility to overall adiposity and body fat distribution

Importanza del mappaggio genetico fine per la definizione farmacogenomica : esempio dell'ACE e risposta a LOSARTAN

RAZIONALE. L'allele D del gene ACE \ue8 associato a maggior rischio cardio-renale. I dati sono controversi per la risposta alla terapia antiipertensiva. Abbiamo eseguito uno studio farmacogenomico tra locus ACE e risposta a Losartan in ipertesi na\uefve (EH), mai trattati prima, per evitare ogni effetto carry-over. CASISTICA E METODI. Analisi della struttura di linkage disequilibrium (LD) di ACE per individuare SNPs tagging I/D, su 1013 soggetti etnicamente omogenei, caratterizzati anche per I/D. Gli SNP rs4341, rs4342, rs1987692 sono in LD assoluto con I/D, quindi tag. Farmacogenomica: 388 EH caucasici, genotipizzati con ILLUMINA 1MDuo. Imputazione con aplotipi CEU 1000Genomes. Fenotipo: caduta pressoria sistolica dopo 4 settimane di terapia con Losartan (\u394SBP), analizzata con regressione lineare, modello additivo, correggendo per et\ue0, sesso ed etnia. RISULTATI. I tre tag SNP sono significativamente associati a \u394SBP (p=3x10). Estendendo l'analisi a tutto il locus (400 kbp), abbiamo analizzato 110 SNP, notando un progressivo aumento di significativit\ue0 alla regione flanking-3\u2019 del gene (rs4461142, p=5.86x10 -2 ) a circa 2.000 bp dal codone di stop di ACE, associato a una caduta pressoria di 3.7 mmHg nei CC rispetto ai portatori dell\u2019allele T. Il polimorfismo \ue8 solo in debole LD con I/D (r =0.6). A conferma della specificit\ue0 per Losartan, ripetendo lo stesso espe- 2 rimento in 180 EH trattati con placebo e 660 con idroclorotiazide, -3 non \ue8 stato osservato nessun effetto attribuibile al genotipo (p=0.84 e p=0.97). CONCLUSIONI. Nonostante siano passati pi\uf9 di 20 anni dalla prima segnalazione, \ue8 probabile che il polimorfismo I/D non abbia un ruolo funzionale ma che sia solo un proxy di altri non ancora identificati. Questo studio di mappaggio fine punta alla regione flanking-3\u2019 che potrebbe essere connessa a un sito regolatore per il gene ACE, localizzata a circa 6.000 bp dallo pseudogene ACE3p

In vitro study of new DUOX2 mutants

Context: Some cases of congenital hypothyroidism (CH) are associated with a gland of normal size. Objective: To explore the cause of organification defect in one child with CH and a eutopic thyroid gland, genetic analyses of TPO, DUOX2, and DUOXA2 genes were performed. Patient: One child with CH, a eutopic thyroid gland, and a partial organification defect was shown after 123I scintigraphy and perchlorate test. Methods: In the child with the organification defect, TPO, DUOX2, and DUOXA2 genes were analyzed. The functional activity of the DUOX2 mutants was studied after expression in eukaryotic cells. Results: No TPO or DUOXA2 gene mutations were identified. Direct sequencing of the DUOX2 gene revealed a compound heterozygous genotype for S911L and C1052Y substitutions. S911L and C1052Y caused a partial defect in H2O2 production after transient expression in HeLa cells. Conclusions: We performed a genetic analysis in one child with CH and a eutopic thyroid gland. Two new mutations in DUOX2 gene responsible for the partial deficit in the organification process were identified. A new case of congenital hypothyroidism with a normally located thyroid gland and two new mutations in DUOX2 protein partially impairing H2O2 generation is described

Ipotiroidismo congenito causato da una mutazione omozigote del gene della tireoperossidasi

L\u2019ipotiroidismo congenito con gozzo dovuto a difetti di organificazione dello iodio \ue8 ereditato come tratto recessivo ed \ue8 spesso dovuto a mutazioni del gene della tireoperossidasi (TPO). Il gene umano della TPO contiene 17 esoni ed \ue8 localizzato sul cromosoma 2p25. Il gene codifica per un enzima contenente un gruppo eme di 933 aminoacidi molto simile alla mieloperossidasi umana. Ad oggi sono state descritte pi\uf9 di 50 mutazioni del gene della TPO che determinano una alterata attivit\ue0 funzionale. Lo scopo del nostro studio \ue8 stato quello di eseguire l\u2019analisi del gene della TPO in una paziente affetta da ipotiroidismo congenito trattato tardivamente associati a gozzo nodulare. La paziente presentava un alterato sviluppo psicosomatico ed era in terapia con L9T4 dall\u2019et\ue0 di circa 3 anni. All\u2019esame ecografico era presente una tiroide di dimensioni superiori alla norma con nodulo destro di 5 cm. Gli esami di laboratorio mostravano valori di FT3, FT4 e TG nella norma, TSH inferiore alla norma ed assenza di anticorpi anti tiroide in terapia con L9 T4. A completamento dell\u2019iter diagnostico \ue8 stato effettuato test al perclorato dopo somministrazione di TSH ricombinante che si rivelava positivo con dismissione del radioiodio del 91% a 1 ora dalla somministrazione di perclorato di potassio. Il DNA genomico \ue8 stato estratto dal sangue della paziente e di 50 soggetti normali di controllo utilizzando un kit commerciale. Tutti i 17 esoni che compongono il gene della TPO sono stati amplificati per PCR, sequenziati con BigDye Terminator Kit e analizzati su 3130xl genetic analyzer. Nel sangue della paziente \ue8 stata identificata una nuova mutazione puntiforme omozigote a livello dell\u2019esone 10 del gene della TPO (ACG/ATG) che determina la sostituzione in posizione 561 dell\u2019aminoacido treonina con l\u2019aminoacido metionina (T561M). Erano inoltre presenti varianti alleliche gi\ue0 descritte in letteratura. La mutazione non \ue8 stata identificata nel DNA da sangue dei 50 soggetti normali. In conclusione, abbiamo identificato una nuova mutazione omozigote del gene della TPO in una paziente affetta da ipotiroidismo congenito trattato tardivamente. La mutazione \ue8 responsabile del difetto di organificazione dello iodio che ha determinato l\u2019ipotiroidismo

Ipotiroidismo congenito causato da una nuova mutazione omozigote del gene della tireoglobulina

L\u2019ipotiroidismo congenito dovuto a deficit di tireoglobulina (TG) \ue8 una malattia autosomica recessiva con una prevalenza di 1:4000091:100000 nati vivi e caratterizzata da gozzo, bassi livelli sierici di TG, elevati livelli di TSH con bassi livelli di ormoni tiroidei e test al perclorato negativo. La TG umana, una proteina di 2768 aminoacidi, \ue8 codificata da un grosso gene con 48 esoni localizzato su cromosoma 8q24. Mutazioni del gene della TG sono associate a gozzo congenito con ipotiroidismo o eutiroidismo. Ad oggi sono state descritte e caratterizzate 50 mutazioni del gene della TG umana. Lo scopo del nostro studio \ue8 stato quello di eseguire l\u2019analisi del gene della TG in due sorelle nate da genitori consanguinei affette da ipotiroidismo congenito. Le due sorelle sono state identificate allo screening neonatale e trattate dopo la conferma di ipotiroidismo con L9Tiroxina. Alla et\ue0 di 7 anni, una sorella, dopo sospensione della L9tiroxina presentava un franco ipotiroidismo, TG indosabile; alla scintigrafia era presente una tiroide in sede con captazione del 12% dopo 24 ore; la ecografia dimostrava la presenza di una ghiandola in sede di normali dimensioni. La seconda sorella, nonostante il precoce trattamento con L9 tiroxina, era anche affetta da ritardo mentale. La ecografia confermava la presenza di una tiroide in sede di normali dimensioni, e la tireoglobulina era indosabile. Il DNA genomico \ue8 stato estratto dal sangue delle pazienti e da quello del padre, unico genitore in vita, utilizzando un kit commerciale. Tutti i 48 esoni che compongono il gene della TG sono stati amplificati per PCR, sequenziati con BigDye Terminator Kit e analizzati su 3130xl genetic analyzer. Nel sangue di entrambe le pazienti \ue8 stata identificata una mutazione puntiforme omozigote a livello dell\u2019esone 10 del gene della TG (CGA/TGA) che determina la formazione di un codone di stop in posizione 768 (R768X). Il risultato \ue8 quindi la presenza di una proteina precocemente troncata e quindi non funzionante. Erano inoltre presenti varianti alleliche gi\ue0 descritte in letteratura. In conclusione, abbiamo identificato due sorelle con ipotiroidismo congenito e tiroide in sede di normali dimensioni e TG indosabile. L\u2019analisi genetica ha dimostrato una alterazione della Tireoglobulina come causa dell\u2019ipotiroidismo

TET2 and CSMD1 genes affect SBP response to hydrochlorothiazide in never-treated essential hypertensives

Background: Thiazide diuretics have been recommended as a first-line antihypertensive treatment, although the choice of 'the right drug in the individual essential hypertensive patient' remains still empirical. Essential hypertension is a complex, polygenic disease derived from the interaction of patient's genetic background with the environment. Pharmacogenomics could be a useful tool to pinpoint gene variants involved in antihypertensive drug response, thus optimizing therapeutic advantages and minimizing side effects. Methods and results: We looked for variants associated with blood pressure response to hydrochlorothiazide over an 8-week follow-up by means of a genome-wide association analysis in two Italian cohorts of never-treated essential hypertensive patients: 343 samples from Sardinia and 142 from Milan. TET2 and CSMD1 as plausible candidate genes to affect SBP response to hydrochlorothiazide were identified. The specificity of our findings for hydrochlorothiazide was confirmed in an independent cohort of essential hypertensive patients treated with losartan. Our best findings were also tested for replication in four independent hypertensive samples of European Ancestry, such as GENetics of drug RESponsiveness in essential hypertension, Genetic Epidemiology of Responses to Antihypertensives, NORdic DILtiazem intervention, Pharmacogenomics Evaluation of Antihypertensive Responses, and Campania Salute Network-StayOnDiur. We validated a polymorphism in CSMD1 and UGGT2. Conclusion: This exploratory study reports two plausible loci associated with SBP response to hydrochlorothiazide: TET2, an aldosterone-responsive mediator of ENaC gene transcription; and CSMD1, previously described as associated with hypertension in a case-control study