María Eugenia Sáez

Specific polymorphisms in the RET proto-oncogene are over-represented in patients with Hirschsprung disease and may represent loci modifying phenotypic expression

Hirschsprung disease (HSCR) is a common genetic disorder presenting with functional intestinal obstruction secondary to enteric aganglionosis. HSCR can be familial or sporadic. Although five putative susceptibility genes have been identified, only germline mutations in the RET proto-oncogene account for a significant minority (up to 50%) of familial HSCR; 3% of sporadic HSCR in a population based series carry RETmutations. From 1998 to February 1999, we prospectively ascertained 64 cases of sporadic HSCR from the western Andalusia region. To determine if polymorphic sequence variants within RETcould act as low penetrance predisposing alleles, we examined allelic frequencies at seven polymorphic loci in this population based series. Whether allele frequencies differed from those in the control population were determined by either chi-squared analysis or Fisher’s exact test. For two sequence variants, A45A (c 135G→A) (exon 2) and L769L (c 2307T→G) (exon 13), the rarer polymorphic allele was over-represented among HSCR cases versus controls (p<0.0006). In contrast, two other polymorphisms, G691S (c 2071C→A) (exon 11) and S904S (c 2712C→G) (exon 15), were under-represented in the HSCR patients compared to controls (p=0.02). Polymorphisms in theRET proto-oncogene appear to predispose to HSCR in a complex, low penetrance fashion and may also modify phenotypic expression.

RET genotypes comprising specific haplotypes of polymorphic variants predispose to isolated Hirschsprung disease

BACKGROUND Hirschsprung disease (HSCR), which may be sporadic or familial, occurs in 1:5000 live births and presents with functional intestinal obstruction secondary to aganglionosis of the hindgut. Germline mutations of theRET proto-oncogene are believed to account for up to 50% of familial cases and up to 30% of isolated cases in most series. However, these series are highly selected for the most obvious and severe cases and large familial aggregations. Population based studies indicate that germline RETmutations account for no more than 3% of isolated HSCR cases. Recently, we and others have noted that specific polymorphic sequence variants, notably A45A (exon 2), are over-represented in isolated HSCR. PURPOSE In order to determine if it is the variant per se, a combination thereof, or another locus in linkage disequilibrium which predisposes to HSCR, we looked for association of RET haplotype(s) and disease in HSCR cases compared to region matched controls. METHODS Seven loci acrossRET were typed and haplotypes formed for HSCR cases, their unaffected parents, and region matched controls. Haplotype and genotype frequencies and distributions were compared among these groups using the transmission disequilibrium test and standard case-control statistic. RESULTS Twelve unique haplotypes, labelled A-L, were obtained. The distributions of haplotypes between cases and controls (χ11 2 =81.4, p<<0.0001) and between cases and non-transmitted parental haplotypes were significantly different (χ2 11=53.1, p<0.0001). Genotypes comprising pairs of haplotypes were formed for cases and controls. There were 38 different genotypes among cases and controls combined. Inspection of the genotypes in these two groups showed that the genotype distribution between cases and controls was distinct (χ37 2=93.8, p<<0.0001). For example, BB, BC, BD, and CD, all of which contain at least one allele with the polymorphic A45A, are prominently represented among HSCR cases, together accounting for >35% of the case genotypes, yet these four genotypes were not represented among the population matched normal controls. Conversely, AA, AG, DD, GG, and GJ, none of which contains A45A, are commonly represented in the controls, together accounting for 43% of the control genotypes, and yet they are never seen among the HSCR cases. CONCLUSIONS Our data suggest that genotypes comprising specific pairs of REThaplotypes are associated with predisposition to HSCR either in a simple autosomal recessive manner or in an additive, dose dependent fashion.

A method for detecting epistasis in genome-wide studies using case-control multi-locus association analysis.

BackgroundThe difficulty in elucidating the genetic basis of complex diseases roots in the many factors that can affect the development of a disease. Some of these genetic effects may interact in complex ways, proving undetectable by current single-locus methodology.ResultsWe have developed an analysis tool called Hypothesis Free Clinical Cloning (HFCC) to search for genome-wide epistasis in a case-control design. HFCC combines a relatively fast computing algorithm for genome-wide epistasis detection, with the flexibility to test a variety of different epistatic models in multi-locus combinations. HFCC has good power to detect multi-locus interactions simulated under a variety of genetic models and noise conditions. Most importantly, HFCC can accomplish exhaustive genome-wide epistasis search with large datasets as demonstrated with a 400,000 SNP set typed on a cohort of Parkinsons disease patients and controls.ConclusionWith the current availability of genetic studies with large numbers of individuals and genetic markers, HFCC can have a great impact in the identification of epistatic effects that escape the standard single-locus association analyses.

Influence of the Toll-Like Receptor 9 1635A/G Polymorphism on the CD4 Count, HIV Viral Load, and Clinical Progression

Objective:To analyze the influence of single-nucleotide polymorphisms (SNPs) in TLR2 (1892A/C and 2258G/A), TLR4 (896A/G and 1196C/T), and TLR9 (1635A/G) genes on CD4 count, HIV viral load, and clinical progression in a cohort of naive HIV-infected patients. Methods:TLR2, TLR4, and TLR9 SNPs were analyzed in 369 naive HIV-infected patients by real-time polymerase chain reaction and melting curve technology. TLR2 1892C/A and TLR9 1635A/G SNPs were also analyzed in a non-HIV-infected population. Multivariate multiple regression analysis and Cox regression analyses were performed to assess the potential association between the SNPs and the end points. Results:TLR2 and TLR4 SNPs were not associated with the end points of the study. Regarding TLR9 1635A/G SNP, patients with the AA genotype showed statistically lower CD4 count (P = 0.003) and higher HIV viral load (P = 0.0018) compared with AG+GG genotypes at cohort entry. The multivariate analysis showed a significant association between the 1635AA genotype and both end points. Cox regression analysis showed that HIV clinical progression to clinical stage C and death due to AIDS-related events under antiretroviral therapy was earlier in patients with the 1635AA genotype (P = 0.035, P = 0.017, respectively). Conclusions:TLR9 1635A/G SNP might have a role in HIV clinical disease progression.

The membrane-spanning 4-domains, subfamily A (MS4A) gene cluster contains a common variant associated with Alzheimer's disease.

BackgroundIn order to identify novel loci associated with Alzheimers disease (AD), we conducted a genome-wide association study (GWAS) in the Spanish population.MethodsWe genotyped 1,128 individuals using the Affymetrix Nsp I 250K chip. A sample of 327 sporadic AD patients and 801 controls with unknown cognitive status from the Spanish general population were included in our initial study. To increase the power of the study, we combined our results with those of four other public GWAS datasets by applying identical quality control filters and the same imputation methods, which were then analyzed with a global meta-GWAS. A replication sample with 2,200 sporadic AD patients and 2,301 controls was genotyped to confirm our GWAS findings.ResultsMeta-analysis of our data and independent replication datasets allowed us to confirm a novel genome-wide significant association of AD with the membrane-spanning 4-domains subfamily A (MS4A) gene cluster (rs1562990, P = 4.40E-11, odds ratio = 0.88, 95% confidence interval 0.85 to 0.91, n = 10,181 cases and 14,341 controls).ConclusionsOur results underscore the importance of international efforts combining GWAS datasets to isolate genetic loci for complex diseases.

Genetic Structure of the Spanish Population

BackgroundGenetic admixture is a common caveat for genetic association analysis. Therefore, it is important to characterize the genetic structure of the population under study to control for this kind of potential bias.ResultsIn this study we have sampled over 800 unrelated individuals from the population of Spain, and have genotyped them with a genome-wide coverage. We have carried out linkage disequilibrium, haplotype, population structure and copy-number variation (CNV) analyses, and have compared these estimates of the Spanish population with existing data from similar efforts.ConclusionsIn general, the Spanish population is similar to the Western and Northern Europeans, but has a more diverse haplotypic structure. Moreover, the Spanish population is also largely homogeneous within itself, although patterns of micro-structure may be able to predict locations of origin from distant regions. Finally, we also present the first characterization of a CNV map of the Spanish population. These results and original data are made available to the scientific community.

The CAPN10 Gene Is Associated with Insulin Resistance Phenotypes in the Spanish Population

Cardiovascular disease is the leading cause of morbidity and mortality in the industrialized world. Familial aggregation of cardiovascular risk factors is a frequent finding, but genetic factors affecting its presentation are still poorly understood. The calpain 10 gene (CAPN10) has been associated with type 2 diabetes (T2DM), a complex metabolic disorder with increased risk of cardiovascular disease. Moreover, the CAPN10 gene has been associated with the presence of metabolic syndrome (MS) in T2DM and in polycystic ovary syndrome (PCOS). In this work, we have analysed whether the polymorphisms UCSNP44, -43, -19 and -63 are related to several cardiovascular risk factors in the context of MS. Molecular analysis of CAPN10 gene was performed in 899 individuals randomly chosen from a cross-sectional population-based epidemiological survey. We have found that CAPN10 gene in our population is mainly associated with two indicators of the presence of insulin resistance: glucose levels two hours after a 75-g oral glucose tolerance test (OGTT) and HOMA values, although cholesterol levels and blood pressure values are also influenced by CAPN10 variants. In addition, the 1221/1121 haplogenotype is under-represented in individuals that fulfil the International Diabetes Federation (IDF) diagnostic criteria for MS. Our results suggest that CAPN10 gene is associated with insulin resistance phenotypes in the Spanish population.

Sex and Body Mass Index Specific Regulation of Blood Pressure by CYP19A1 Gene Variants

Sexual dimorphism in blood pressure (BP) regulation has been observed both in humans and experimental animals, and estrogens have been shown to contribute to this epidemiological observation. A key enzyme in determining estrogen levels is aromatase cytochrome P450. The aim of this study was to evaluate the role of the gene encoding aromatase, CYP19A1, as an independent risk factor for hypertension and its relationship with systolic and diastolic BP measures. We genotyped 2 polymorphisms within the CYP19A1 gene, IVS4 rs11575899 and 3′UTR rs10046, in 3448 individuals. In quantitative analysis, we observed significant associations between the 2 polymorphisms and BP values in women, being these associations dependent on BMI and independent of menopause status. The case-control analysis revealed that the most prominent associations were found for nonobese women in diastolic hypertension (DHT): the IVS4_22 and 3′UTR_11 are risk genotypes (OR=1.61, P=0.027 and OR=1.59, P=0.012, respectively), whereas IVS4_11 and 3′UTR_22 genotypes have a protective effect against DHT (OR=0.63, P=0.009, and OR=0.61, P=0.020, respectively). Haplotype analysis confirmed the above associations: among nonobese women the haplotype 21 is overrepresented in hypertensive women (OR=1.33, P=0.004, for DHT and OR=1.25, P=0.026, for systolic hypertension, SHT) and, conversely, the haplotype 12 protects against hypertension (OR=0.78, P=0.015 for DHT and OR=0.82, P=0.04 for SHT). Our study has shown that the CYP19A1 gene may be involved in the genetic regulation of BP in women. This effect is dependent on BMI and independent of menopause status, suggesting that this action is mainly driven by aromatase activity in fat tissue.

Estrogen receptor alpha gene variants are associated with Alzheimer's disease

The present research is aimed at assessing the role of 3 estrogen receptor alpha (ESR1) gene variants in late onset Alzheimers disease (AD) susceptibility. One thousand one hundred thirteen unrelated late onset sporadic AD patients, 1109 healthy controls and 121 neurologically healthy elderly controls were used to carry out case-control genetic association studies with ESR1 rs3844508, rs2234693, and ESR1 noncoding deletion 1 (ESR1-NCD1) polymorphisms. Thirty-five healthy male samples were used for molecular analyses. The rs2234693 polymorphism is associated with AD in our population (odds ratio [OR], 1.29; p = 0.008). The rs3844508 marker confers protection against AD in males (OR, 0.57; p = 0.001) and the deletion ESR1-NCD1 is a risk factor for AD in women (OR, 1.67; p < 0.001). Molecular analyses on ESR1-NCD1 indicate that this deletion confers a higher response to estradiol activity on ESR1 receptor and it is also associated with differential expression of ESR1 isoforms. Our results support the involvement of ESR1 gene in AD and point to the existence of sexual dimorphism for ESR1 markers. In addition, carriers of ESR1-NCD1 deletion could overrespond to estradiol action.

ATP5H/KCTD2 locus is associated with Alzheimer's disease risk

To identify loci associated with Alzheimer disease, we conducted a three-stage analysis using existing genome-wide association studies (GWAS) and genotyping in a new sample. In Stage I, all suggestive single-nucleotide polymorphisms (at P<0.001) in a previously reported GWAS of seven independent studies (8082 Alzheimer’s disease (AD) cases; 12 040 controls) were selected, and in Stage II these were examined in an in silico analysis within the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium GWAS (1367 cases and 12904 controls). Six novel signals reaching P<5 × 10−6 were genotyped in an independent Stage III sample (the Fundació ACE data set) of 2200 sporadic AD patients and 2301 controls. We identified a novel association with AD in the adenosine triphosphate (ATP) synthase, H+ transporting, mitochondrial F0 (ATP5H)/Potassium channel tetramerization domain-containing protein 2 (KCTD2) locus, which reached genome-wide significance in the combined discovery and genotyping sample (rs11870474, odds ratio (OR)=1.58, P=2.6 × 10−7 in discovery and OR=1.43, P=0.004 in Fundació ACE data set; combined OR=1.53, P=4.7 × 10−9). This ATP5H/KCTD2 locus has an important function in mitochondrial energy production and neuronal hyperpolarization during cellular stress conditions, such as hypoxia or glucose deprivation.