Using gene-network landscape to dissect genotype effects of TCF7L2 genetic variant on diabetes and cardiovascular risk

Vaquero AR, Ferreira NE, Omae SV, Rodrigues MV, Teixeira SK, Krieger JE, Pereira AC. Using gene-network landscape to dissect genotype effects of TCF7L2 genetic variant on diabetes and cardiovascular risk. Physiol Genomics 44: 903-914, 2012. First published August 7, 2012; doi:10.1152/physiolgenomics.00030.2012.-The single nucleotide polymorphism (SNP) within the TCF7L2 gene, rs7903146, is, to date, the most significant genetic marker associated with Type 2 diabetes mellitus (T2DM) risk. Nonetheless, its functional role in disease pathology is poorly understood. The aim of the present study was to investigate, in vascular smooth muscle cells from 92 patients undergoing aortocoronary bypass surgery, the contribution of this SNP in T2DM using expression levels and expression correlation comparison approaches, which were visually represented as gene interaction networks. Initially, the expression levels of 41 genes (seven TCF7L2 splice forms and 40 other T2DM relevant genes) were compared between rs7903146 wild-type (CC) and T2DM-risk (CT + TT) genotype groups. Next, we compared the expression correlation patterns of these 41 genes between groups to observe if the relationships between genes were different. Five TCF7L2 splice forms and nine genes showed significant expression differences between groups. RXR alpha gene was pinpointed as showing the most different expression correlation pattern with other genes. Therefore, T2DM risk alleles appear to be influencing TCF7L2 splice form's expression in vascular smooth muscle cells, and RXR alpha gene is pointed out as a treatment target candidate for risk reduction in individuals with high risk of developing T2DM, especially individuals harboring TCF7L2 risk genotypes.

Protective Effect of Human Endogenous Retrovirus K dUTPase Variants on Psoriasis Susceptibility

Previous genetic and functional studies have implicated the human endogenous retrovirus K (HERV-K) dUTPase located within the PSORS1 locus in the major histocompatibility complex region as a candidate psoriasis gene. Here, we describe a variant discovery and case-control association study of HERV-K dUTPase variants in 708 psoriasis cases and 349 healthy controls. Five common HERV-K dUTPase variants were found to be highly associated with psoriasis, with the strongest association occurring at the missense single-nucleotide polymorphism (SNP) rs3134774 (K158R, P=3.28 x 10(-15), odds ratio = 2.36 (95% confidence interval: 1.91-2.92)). After adjusting the association of the HERV-K dUTPase variants for the potential confounding effects of HLA alleles associated with psoriasis, the HERV-K SNPs rs9264082 and rs3134774 remained significantly associated. Haplotype analysis revealed that HERV-K haplotypes containing the non-risk alleles for rs3134774 and rs9264082 significantly reduced the risk of psoriasis. Functional testing showed higher antibody responses against recombinant HERV-K dUTPase in psoriasis patients compared with controls (P<0.05), as well as higher T-cell responses against a single HERV-K dUTPase peptide (P<0.05). Our data support an independent role for the HERV-K dUTPase on psoriasis susceptibility, and suggest the need for additional studies to clarify the role of this dUTPase in the pathogenesis of psoriasis. Journal of Investigative Dermatology (2012) 132, 1833-1840; doi:10.1038/jid.2012.69; published online 22 March 2012

Growth hormone pharmacogenetics: the interactive effect of a microsatellite in the IGF1 promoter region with the GHR-exon 3 and-202 A/C IGFBP3 variants on treatment outcomes of children with severe GH deficiency

Insulin-like growth factor type 1 (IGF1) is a mediator of growth hormone (GH) action, and therefore, IGF1 is a candidate gene for recombinant human GH (rhGH) pharmacogenetics. Lower serum IGF1 levels were found in adults homozygous for 19 cytosine-adenosine (CA) repeats in the IGF1 promoter. The aim of this study was to evaluate the influence of (CA)n IGF1 polymorphism, alone or in combination with GH receptor (GHR)-exon 3 and -202 A/C insulin-like growth factor binding protein-3 (IGFBP3) polymorphisms, on the growth response to rhGH therapy in GH-deficient (GHD) patients. Eighty-four severe GHD patients were genotyped for (CA) n IGF1, -202 A/C IGFBP3 and GHR-exon 3 polymorphisms. Multiple linear regressions were performed to estimate the effect of each genotype, after adjustment for other influential factors. We assessed the influence of genotypes on the first year growth velocity (1st y GV) (n = 84) and adult height standard deviation score (SDS) adjusted for target-height SDS (AH-TH SDS) after rhGH therapy (n = 37). Homozygosity for the IGF1 19CA repeat allele was negatively correlated with 1st y GV (P = 0.03) and AH-TH SDS (P = 0.002) in multiple linear regression analysis. In conjunction with clinical factors, IGF1 and IGFBP3 genotypes explain 29% of the 1st y GV variability, whereas IGF1 and GHR polymorphisms explain 59% of final height-target-height SDS variability. We conclude that homozygosity for IGF1 (CA) 19 allele is associated with less favorable short-and long-term growth outcomes after rhGH treatment in patients with severe GHD. Furthermore, this polymorphism exhibits a non-additive interaction with -202 A/C IGFBP3 genotype on the 1st y GV and with GHR-exon 3 genotype on adult height. The Pharmacogenomics Journal (2012) 12, 439-445; doi:10.1038/tpj.2011.13; published online 5 April 2011

Structural studies of Helicase NS3 variants from Hepatitis C virus genotype 3 in virological sustained responder and non-responder patients

Abstract Background About 130 million people are infected with the hepatitis C virus (HCV) worldwide, but effective treatment options are not yet available. One of the most promising targets for antiviral therapy is nonstructural protein 3 (NS3). To identify possible changes in the structure of NS3 associated with virological sustained response or non-response of patients, a model was constructed for each helicase NS3 protein coding sequence. From this, the goal was to verify the interaction between helicases variants and their ligands. Findings Evidence was found that the NS3 helicase portion of non-responder patients contained substitutions in its ATP and RNA binding sites. K210E substitution can cause an imbalance in the distribution of loads, leading to a decrease in the number of ligations between the essential amino acids required for the hydrolysis of ATP. W501R substitution causes an imbalance in the distribution of loads, leading and forcing the RNA to interact with the amino acid Thr269, but not preventing binding of ribavirin inhibitor. Conclusions Useful information is provided on the genetic profiling of the HCV genotype 3, specifically the coding region of the NS3 protein, improving our understanding of the viral genome and the regions of its protein catalytic site.

Mechanism of resistance to tyrosine kinase inhibitors in philadelphia-positive acute lymphblastic leukaemia (all): from genetic alterations to impaired RNA editing

The Ph chromosome is the most frequent cytogenetic aberration associated with adult ALL and it represents the single most significant adverse prognostic marker. Despite imatinib has led to significant improvements in the treatment of patients with Ph+ ALL, in the majority of cases resistance developed quickly and disease progressed. Some mechanisms of resistance have been widely described but the full knowledge of contributing factors, driving both the disease and resistance, remains to be defined. The observation of rapid development of lymphoblastic leukemia in mice expressing altered Ikaros (Ik) isoforms represented the background of this study. Ikaros is a zinc finger transcription factor required for normal hemopoietic differentiation and proliferation, particularly in the lymphoid lineages. By means of alternative splicing, Ikaros encodes several proteins that differ in their abilities to bind to a consensus DNA-binding site. Shorter, DNA nonbinding isoforms exert a dominant negative effect, inhibiting the ability of longer heterodimer partners to bind DNA. The differential expression pattern of Ik isoforms in Ph+ ALL patients was analyzed in order to determine if molecular abnormalities involving the Ik gene could associate with resistance to imatinib and dasatinib. Bone marrow and peripheral blood samples from 46 adult patients (median age 55 yrs, 18-76) with Ph+ ALL at diagnosis and during treatment with imatinib (16 pts) or dasatinib (30 pts) were collected. We set up a fast, high-throughput method based on capillary electrophoresis technology to detect and quantify splice variants. 41% Ph+ ALL patients expressed high levels of the non DNA-binding dominant negative Ik6 isoform lacking critical N-terminal zinc-fingers which display abnormal subcellular compartmentalization pattern. Nuclear extracts from patients expressed Ik6 failed to bind DNA in mobility shift assay using a DNA probe containing an Ikaros-specific DNA binding sequence. In 59% Ph+ ALL patients there was the coexistence in the same PCR sample and at the same time of many splice variants corresponded to Ik1, Ik2, Ik4, Ik4A, Ik5A, Ik6, Ik6 and Ik8 isoforms. In these patients aberrant full-length Ikaros isoforms in Ph+ ALL characterized by a 60-bp insertion immediately downstream of exon 3 and a recurring 30-bp in-frame deletion at the end of exon 7 involving most frequently the Ik2, Ik4 isoforms were also identified. Both the insertion and deletion were due to the selection of alternative splice donor and acceptor sites. The molecular monitoring of minimal residual disease showed for the first time in vivo that the Ik6 expression strongly correlated with the BCR-ABL transcript levels suggesting that this alteration could depend on the Bcr-Abl activity. Patient-derived leukaemia cells expressed dominant-negative Ik6 at diagnosis and at the time of relapse, but never during remission. In order to mechanistically demonstrated whether in vitro the overexpression of Ik6 impairs the response to tyrosine kinase inhibitors (TKIs) and contributes to resistance, an imatinib-sensitive Ik6-negative Ph+ ALL cell line (SUP-B15) was transfected with the complete Ik6 DNA coding sequence. The expression of Ik6 strongly increased proliferation and inhibited apoptosis in TKI sensitive cells establishing a previously unknown link between specific molecular defects that involve the Ikaros gene and the resistance to TKIs in Ph+ ALL patients. Amplification and genomic sequence analysis of the exon splice junction regions showed the presence of 2 single nucleotide polymorphisms (SNPs): rs10251980 [A/G] in the exon2/3 splice junction and of rs10262731 [A/G] in the exon 7/8 splice junction in 50% and 36% of patients, respectively. A variant of the rs11329346 [-/C], in 16% of patients was also found. Other two different single nucleotide substitutions not recognized as SNP were observed. Some mutations were predicted by computational analyses (RESCUE approach) to alter cis-splicing elements. In conclusion, these findings demonstrated that the post-transcriptional regulation of alternative splicing of Ikaros gene is defective in the majority of Ph+ ALL patients treated with TKIs. The overexpression of Ik6 blocking B-cell differentiation could contribute to resistance opening a time frame, during which leukaemia cells acquire secondary transforming events that confer definitive resistance to imatinib and dasatinib.

TNFRSF13B Genetic variability an anthropological - evolutionary approach to Biomedical Research

In the recent years TNFRSF13B coding variants have been implicated by clinical genetics studies in Common Variable Immunodeficiency (CVID), the most common clinically relevant primary immunodeficiency in individuals of European ancestry, but their functional effects in relation to the development of the disease have not been entirely established. To examine the potential contribution of such variants to CVID, the more comprehensive perspective of an evolutionary approach was applied in this study, underling the belief that evolutionary genetics methods can play a role in dissecting the origin, causes and diffusion of human diseases, representing a powerful tool also in human health research. For this purpose, TNFRSF13B coding region was sequenced in 451 healthy individuals belonging to 26 worldwide populations, in addition to 96 control, 77 CVID and 38 Selective IgA Deficiency (IgAD) individuals from Italy, leading to the first achievement of a global picture of TNFRSF13B nucleotide diversity and haplotype structure and making suggestion of its evolutionary history possible. A slow rate of evolution, within our species and when compared to the chimpanzee, low levels of genetic diversity geographical structure and the absence of recent population specific selective pressures were observed for the examined genomic region, suggesting that geographical distribution of its variability is more plausibly related to its involvement also in innate immunity rather than in adaptive immunity only. This, together with the extremely subtle disease/healthy samples differences observed, suggests that CVID might be more likely related to still unknown environmental and genetic factors, rather than to the nature of TNFRSF13B variants only.

Analisi di “Copy Number Variants” ed identificazione di nuovi geni candidati per l’Autismo e Ritardo Mentale

I disturbi dello spettro autistico (DSA) ed il ritardo mentale (RM) sono caratterizzati da un’eziologia genetica complessa ed eterogenea. Grazie ai recenti sviluppi nella ricerca genomica, è stato possibile dimostrare il ruolo di numerose copy number variants (CNVs) nella patogenesi di questi disturbi, anche se nella maggior parte dei casi l’eziologia rimane ancora sconosciuta. Questo lavoro riguarda l’identificazione e la caratterizzazione dei CNVs in famiglie con DSA e RM. E’ stata studiata una microdelezione in 7q31 che coinvolge i geni IMMP2L e DOCK4, trasmessa dalla madre con dislessia a due figli con autismo ed una figlia con dislessia. Nella stessa famiglia segrega una seconda microdelezione in 2q14 che inattiva il gene CNTNAP5 ed è trasmessa dal padre (con tratti autistici) ai due figli con autismo. Abbiamo quindi ipotizzato che i geni DOCK4 e CNTNAP5 potessero essere implicati, rispettivamente, nella suscettibilità a dislessia e DSA. Lo screening di numerosi individui affetti ha supportato la nostra ipotesi, con l’identificazione di una nuova microdelezione di DOCK4 che segrega con la dislessia, e 3 nuove varianti missenso in CNTNAP5 in individui con autismo. Dall’analisi genomica comparativa su array (aCGH) di individui con RM, è stata identificata una delezione nella regione 7q31.32, che coinvolge il gene CADPS2, in due fratelli con RM e tratti autistici, probabilmente ereditata dalla madre. Lo screening di mutazione di questo gene in individui con autismo o RM, ha portato all’identificazione di 3 varianti non sinonime, assenti nei controlli, ed ereditate per via materna. Poiché CADPS2 risiede in una regione genomica che contiene loci soggetti ad imprinting, abbiamo ipotizzato che il gene CADPS2 possa essere anch’esso caratterizzato da imprinting, con espressione monoallelica materna. Lo studio di espressione di CADPS2 in cellule del sangue ha avvalorato questa ipotesi, implicando perciò CADPS2 come un nuovo gene di suscettibilità per il RM e DSA.

Investigating the role of Copy Number Variants in Specific Language Impairment and identification of new candidate genes

Specific language impairment (SLI) is a complex neurodevelopmental disorder defined as an unexpected failure to develop normal language abilities for no obvious reason. Copy number variants (CNVs) are an important source of variation in the susceptibility to neuropsychiatric disorders. Therefore, a CNV study within SLI families was performed to investigate the role of structural variants in SLI. Among the identified CNVs, we focused on CNVs on chromosome 15q11-q13, recurrently observed in neuropsychiatric conditions, and a homozygous exonic microdeletion in ZNF277. Since this microdeletion falls within the AUTS1 locus, a region linked to autism spectrum disorders (ASD), we investigated a potential role of ZNF277 in SLI and ASD. Frequency data and expression analysis of the ZNF277 microdeletion suggested that this variant may contribute to the risk of language impairments in a complex manner, that is independent of the autism risk previously described in this region. Moreover, we identified an affected individual with a dihydropyrimidine dehydrogenase (DPD) deficiency, caused by compound heterozygosity of two deleterious variants in the gene DPYD. Since DPYD represents a good candidate gene for both SLI and ASD, we investigated its involvement in the susceptibility to these two disorders, focusing on the splicing variant rs3918290, the most common mutation in the DPD deficiency. We observed a higher frequency of rs3918290 in SLI cases (1.2%), compared to controls (~0.6%), while no difference was observed in a large ASD cohort. DPYD mutation screening in 4 SLI and 7 ASD families carrying the splicing variant identified six known missense changes and a novel variant in the promoter region. These data suggest that the combined effect of the mutations identified in affected individuals may lead to an altered DPD activity and that rare variants in DPYD might contribute to a minority of cases, in conjunction with other genetic or non-genetic factors.

Analysis of Copy Number Variants identifies new candidate genes for Autism Spectrum Disorder and Intellectual Disability

Autism spectrum disorder (ASD) and Intellectual Disability (ID) are complex neuropsychiatric disorders characterized by extensive clinical and genetic heterogeneity and with overlapping risk factors. The aim of my project was to further investigate the role of Copy Numbers Variants (CNVs), identified through genome-wide studies performed by the Autism Geome Project (AGP) and the CHERISH consortium in large cohorts of ASD and ID cases, respectively. Specifically, I focused on four rare genic CNVs, selected on the basis of their impact on interesting ASD/ID candidate genes: a) a compound heterozygous deletion involving CTNNA3, predicted to cause the lack of functional protein; b) a 15q13.3 duplication containing CHRNA7; c) a 2q31.1 microdeletion encompassing KLHL23, SSB and METTL5; d) Lastly, I investigated the putative imprinting regulation of the CADPS2 gene, disrupted by a maternal deletion in two siblings with ASD and ID. This study provides further evidence for the role of CTNNA3, CHRNA7, KLHL23 and CADPS2 as ASD and/or ID susceptibility genes, and highlights that rare genetic variation contributes to disease risk in different ways: some rare mutations, such as those impacting CTNNA3, act in a recessive mode of inheritance, while other CNVs, such as those occurring in the 15q13.3 region, are implicated in multiple developmental and/or neurological disorders possibly interacting with other susceptibility variants elsewhere in the genome. On the other hand, the discovery of a tissue-specific monoallelic expression for the CADPS2 gene, implicates the involvement of epigenetic regulatory mechanisms as risk factors conferring susceptibility to ASD/ID.

Association between variants of the leptin receptor gene (LEPR) and overweight: a systematic review and an analysis of the CoLaus study

Background Three non-synonymous single nucleotide polymorphisms (Q223R, K109R and K656N) of the leptin receptor gene (LEPR) have been tested for association with obesity-related outcomes in multiple studies, showing inconclusive results. We performed a systematic review and meta-analysis on the association of the three LEPR variants with BMI. In addition, we analysed 15 SNPs within the LEPR gene in the CoLaus study, assessing the interaction of the variants with sex. Methodology/Principal Findings We searched electronic databases, including population-based studies that investigated the association between LEPR variants Q223R, K109R and K656N and obesity- related phenotypes in healthy, unrelated subjects. We furthermore performed meta-analyses of the genotype and allele frequencies in case-control studies. Results were stratified by SNP and by potential effect modifiers. CoLaus data were analysed by logistic and linear regressions and tested for interaction with sex. The meta-analysis of published data did not show an overall association between any of the tested LEPR variants and overweight. However, the choice of a BMI cut-off value to distinguish cases from controls was crucial to explain heterogeneity in Q223R. Differences in allele frequencies across ethnic groups are compatible with natural selection of derived alleles in Q223R and K109R and of the ancient allele in K656N in Asians. In CoLaus, the rs10128072, rs3790438 and rs3790437 variants showed interaction with sex for their association with overweight, waist circumference and fat mass in linear regressions. Conclusions Our systematic review and analysis of primary data from the CoLaus study did not show an overall association between LEPR SNPs and overweight. Most studies were underpowered to detect small effect sizes. A potential effect modification by sex, population stratification, as well as the role of natural selection should be addressed in future genetic association studies.

Genetic diversity of EBV-encoded LMP1 in the Swiss HIV Cohort Study and implication for NF-Κb activation

Epstein-Barr virus (EBV) is associated with several types of cancers including Hodgkin's lymphoma (HL) and nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane protein 1 (LMP1), a multifunctional oncoprotein, is a powerful activator of the transcription factor NF-κB, a property that is essential for EBV-transformed lymphoblastoid cell survival. Previous studies reported LMP1 sequence variations and induction of higher NF-κB activation levels compared to the prototype B95-8 LMP1 by some variants. Here we used biopsies of EBV-associated cancers and blood of individuals included in the Swiss HIV Cohort Study (SHCS) to analyze LMP1 genetic diversity and impact of sequence variations on LMP1-mediated NF-κB activation potential. We found that a number of variants mediate higher NF-κB activation levels when compared to B95-8 LMP1 and mapped three single polymorphisms responsible for this phenotype: F106Y, I124V and F144I. F106Y was present in all LMP1 isolated in this study and its effect was variant dependent, suggesting that it was modulated by other polymorphisms. The two polymorphisms I124V and F144I were present in distinct phylogenetic groups and were linked with other specific polymorphisms nearby, I152L and D150A/L151I, respectively. The two sets of polymorphisms, I124V/I152L and F144I/D150A/L151I, which were markers of increased NF-κB activation in vitro, were not associated with EBV-associated HL in the SHCS. Taken together these results highlighted the importance of single polymorphisms for the modulation of LMP1 signaling activity and demonstrated that several groups of LMP1 variants, through distinct mutational paths, mediated enhanced NF-κB activation levels compared to B95-8 LMP1.

Genome-wide association study identifies variants associated with progression of liver fibrosis from HCV infection

Polymorphisms in IL28B were shown to affect clearance of hepatitis C virus (HCV) infection in genome-wide association (GWA) studies. Only a fraction of patients with chronic HCV infection develop liver fibrosis, a process that might also be affected by genetic factors. We performed a 2-stage GWA study of liver fibrosis progression related to HCV infection.

Keratin variants associate with progression of fibrosis during chronic hepatitis C infection

Keratins 8 and 18 (K8/K18) protect the liver from various forms of injury. Studies of liver explants from a large cohort of U.S. patients showed that K8/K18 mutations confer a risk to developing end-stage liver diseases, though which diseases are preferentially involved is unknown. We tested the hypothesis that K8/K18 variants are associated with chronic hepatitis C (CHC) and that their presence correlates with progression of fibrosis. Genomic DNA was isolated from peripheral blood of a well-characterized German cohort of 329 patients with CHC infection. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Our findings showed: (1) amino acid altering keratin heterozygous variants in 24 of 329 CHC patients (7.3%) and non-coding heterozygous variants in 26 patients (7.8%), and (2) 3 new exonic K8 variants (T26R/G55A/A359T); 6 novel non-coding variants and one K18 coding variant (K18 S230T; 2 patients). The most common variants were K8 R341H (10 patients), K8 G62C (6 patients) and K8 I63V (4 patients). A novel and exclusive association of an intronic KRT8 IVS7+10delC deletion in all 10 patients with K8 R341H was observed. Notably, there was a significant association of exonic, but not of intronic K8 variants with increased fibrosis. In conclusion, previously described and novel K8 variants are present in a German population and collectively associate with progression of fibrosis in CHC infection. The unique 100% segregation of the most common K8 variant, R341H, with an intronic deletion suggests that one of these two genetic changes might lead to the other.

No role of matrixmetalloproteinase-3 genetic promoter polymorphism 1171 as a risk factor for cirrhosis in alcoholic liver disease

BACKGROUND: As only a minority of alcoholics develop cirrhosis, polymorphic genes, whose products are involved in fibrosis development were suggested to confer individual susceptibility. We tested whether a functional promoter polymorphism in the gene encoding matrix metalloproteinase-3 (MMP-3; 1171 5A/6A) was associated liver cirrhosis in alcoholics. METHODS: Independent cohorts from the UK and Germany were studied. (i) UK cohort: 320 alcoholic cirrhotics and 183 heavy drinkers without liver damage and (ii) German cohort: 149 alcoholic cirrhotics, 220 alcoholic cirrhotics who underwent liver transplantation and 151 alcoholics without liver disease. Patients were genotyped for MMP-3 variants by restriction fragment length polymorphism, single strand confirmation polymorphism, and direct sequencing. In addition, MMP-3 transcript levels were correlated with MMP-3 genotype in normal liver tissues. RESULTS: Matrix metalloproteinase-3 genotype and allele distribution in all 1023 alcoholic patients were in Hardy-Weinberg equilibrium. No significant differences in MMP-3 genotype and allele frequencies were observed either between alcoholics with or without cirrhosis. There were no differences in hepatic mRNA transcription levels according to MMP-3 genotype. CONCLUSIONS: Matrix metalloproteinase-3 1171 promoter polymorphism plays no role in the genetic predisposition for liver cirrhosis in alcoholics. Stringently designed candidate gene association studies are required to exclude chance observations.

STrengthening the REporting of Genetic Association studies (STREGA)--an extension of the STROBE statement

Making sense of rapidly evolving evidence on genetic associations is crucial to making genuine advances in human genomics and the eventual integration of this information in the practice of medicine and public health. Assessment of the strengths and weaknesses of this evidence, and hence the ability to synthesize it, has been limited by inadequate reporting of results. The STrengthening the REporting of Genetic Association studies (STREGA) initiative builds on the STrengthening the Reporting of OBservational Studies in Epidemiology (STROBE) Statement and provides additions to 12 of the 22 items on the STROBE checklist. The additions concern population stratification, genotyping errors, modelling haplotype variation, Hardy-Weinberg equilibrium, replication, selection of participants, rationale for choice of genes and variants, treatment effects in studying quantitative traits, statistical methods, relatedness, reporting of descriptive and outcome data and the volume of data issues that are important to consider in genetic association studies. The STREGA recommendations do not prescribe or dictate how a genetic association study should be designed, but seek to enhance the transparency of its reporting, regardless of choices made during design, conduct or analysis.