Nicola Pirastu

A General Approach for Haplotype Phasing across the Full Spectrum of Relatedness

Many existing cohorts contain a range of relatedness between genotyped individuals, either by design or by chance. Haplotype estimation in such cohorts is a central step in many downstream analyses. Using genotypes from six cohorts from isolated populations and two cohorts from non-isolated populations, we have investigated the performance of different phasing methods designed for nominally ‘unrelated’ individuals. We find that SHAPEIT2 produces much lower switch error rates in all cohorts compared to other methods, including those designed specifically for isolated populations. In particular, when large amounts of IBD sharing is present, SHAPEIT2 infers close to perfect haplotypes. Based on these results we have developed a general strategy for phasing cohorts with any level of implicit or explicit relatedness between individuals. First SHAPEIT2 is run ignoring all explicit family information. We then apply a novel HMM method (duoHMM) to combine the SHAPEIT2 haplotypes with any family information to infer the inheritance pattern of each meiosis at all sites across each chromosome. This allows the correction of switch errors, detection of recombination events and genotyping errors. We show that the method detects numbers of recombination events that align very well with expectations based on genetic maps, and that it infers far fewer spurious recombination events than Merlin. The method can also detect genotyping errors and infer recombination events in otherwise uninformative families, such as trios and duos. The detected recombination events can be used in association scans for recombination phenotypes. The method provides a simple and unified approach to haplotype estimation, that will be of interest to researchers in the fields of human, animal and plant genetics.

Multiple Loci Are Associated with White Blood Cell Phenotypes

White blood cell (WBC) count is a common clinical measure from complete blood count assays, and it varies widely among healthy individuals. Total WBC count and its constituent subtypes have been shown to be moderately heritable, with the heritability estimates varying across cell types. We studied 19,509 subjects from seven cohorts in a discovery analysis, and 11,823 subjects from ten cohorts for replication analyses, to determine genetic factors influencing variability within the normal hematological range for total WBC count and five WBC subtype measures. Cohort specific data was supplied by the CHARGE, HeamGen, and INGI consortia, as well as independent collaborative studies. We identified and replicated ten associations with total WBC count and five WBC subtypes at seven different genomic loci (total WBC count—6p21 in the HLA region, 17q21 near ORMDL3, and CSF3; neutrophil count—17q21; basophil count- 3p21 near RPN1 and C3orf27; lymphocyte count—6p21, 19p13 at EPS15L1; monocyte count—2q31 at ITGA4, 3q21, 8q24 an intergenic region, 9q31 near EDG2), including three previously reported associations and seven novel associations. To investigate functional relationships among variants contributing to variability in the six WBC traits, we utilized gene expression- and pathways-based analyses. We implemented gene-clustering algorithms to evaluate functional connectivity among implicated loci and showed functional relationships across cell types. Gene expression data from whole blood was utilized to show that significant biological consequences can be extracted from our genome-wide analyses, with effect estimates for significant loci from the meta-analyses being highly corellated with the proximal gene expression. In addition, collaborative efforts between the groups contributing to this study and related studies conducted by the COGENT and RIKEN groups allowed for the examination of effect homogeneity for genome-wide significant associations across populations of diverse ancestral backgrounds.

Age- And Sex-Related Variations in Platelet Count in Italy: A Proposal of Reference Ranges Based on 40987 Subjects' Data

Background and Objectives Although several studies demonstrated that platelet count is higher in women, decreases with age, and is influenced by genetic background, most clinical laboratories still use the reference interval 150–400×109 platelets/L for all subjects. The present study was to identify age- and sex-specific reference intervals for platelet count. Methods We analysed electronic records of subjects enrolled in three population-based studies that investigated inhabitants of seven Italian areas including six geographic isolates. After exclusion of patients with malignancies, liver diseases, or inherited thrombocytopenias, which could affect platelet count, reference intervals were estimated from 40,987 subjects with the non parametric method computing the 2.5° and 97.5° percentiles. Results Platelet count was similar in men and women until the age of 14, but subsequently women had steadily more platelets than men. The number of platelets decreases quickly in childhood, stabilizes in adulthood, and further decreases in oldness. The final result of this phenomenon is that platelet count in old age was reduced by 35% in men and by 25% in women compared with early infancy. Based on these findings, we estimated reference intervals for platelet count ×109/L in children (176–452), adult men (141–362), adult women (156–405), old men (122–350) and, old women (140–379). Moreover, we calculated an “extended” reference interval that takes into account the differences in platelet count observed in different geographic areas. Conclusions The age-, sex-, and origin-related variability of platelet count is very wide, and the patient-adapted reference intervals we propose change the thresholds for diagnosing both thrombocytopenia and thrombocytosis in Italy.

Evidence of Inbreeding Depression on Human Height

Stature is a classical and highly heritable complex trait, with 80%–90% of variation explained by genetic factors. In recent years, genome-wide association studies (GWAS) have successfully identified many common additive variants influencing human height; however, little attention has been given to the potential role of recessive genetic effects. Here, we investigated genome-wide recessive effects by an analysis of inbreeding depression on adult height in over 35,000 people from 21 different population samples. We found a highly significant inverse association between height and genome-wide homozygosity, equivalent to a height reduction of up to 3 cm in the offspring of first cousins compared with the offspring of unrelated individuals, an effect which remained after controlling for the effects of socio-economic status, an important confounder (χ2 = 83.89, df = 1; p = 5.2×10−20). There was, however, a high degree of heterogeneity among populations: whereas the direction of the effect was consistent across most population samples, the effect size differed significantly among populations. It is likely that this reflects true biological heterogeneity: whether or not an effect can be observed will depend on both the variance in homozygosity in the population and the chance inheritance of individual recessive genotypes. These results predict that multiple, rare, recessive variants influence human height. Although this exploratory work focuses on height alone, the methodology developed is generally applicable to heritable quantitative traits (QT), paving the way for an investigation into inbreeding effects, and therefore genetic architecture, on a range of QT of biomedical importance.

Hearing function and thresholds: a genome-wide association study in European isolated populations identifies new loci and pathways

Background Hearing is a complex trait, but until now only a few genes are known to contribute to variability of this process. In order to discover genes and pathways that underlie auditory function, a genome-wide association study was carried out within the International Consortium G-EAR. Methods Meta-analysis of genome-wide association studys data from six isolated populations of European ancestry for an overall number of 3417 individuals. Results Eight suggestive significant loci (p<10−7) were detected with a series of genes expressed within the inner ear such as: DCLK1, PTPRD, GRM8, CMIP. Additional biological candidates marked by a single nucleotide polymorphism (SNP) with a suggestive association (p<10−6) were identified, as well as loci encompassing ‘gene desert regions’—genes of unknown function or genes whose function has not be linked to hearing so far. Some of these new loci map to already known hereditary hearing loss loci whose genes still need to be identified. Data have also been used to construct a highly significant ‘in silico’ pathway for hearing function characterised by a network of 49 genes, 34 of which are certainly expressed in the ear. Conclusion These results provide new insights into the molecular basis of hearing function and may suggest new targets for hearing impairment treatment and prevention.

Association analysis of bitter receptor genes in five isolated populations identifies a significant correlation between TAS2R43 variants and coffee liking.

Coffee, one of the most popular beverages in the world, contains many different physiologically active compounds with a potential impact on people’s health. Despite the recent attention given to the genetic basis of its consumption, very little has been done in understanding genes influencing coffee preference among different individuals. Given its markedly bitter taste, we decided to verify if bitter receptor genes (TAS2Rs) variants affect coffee liking. In this light, 4066 people from different parts of Europe and Central Asia filled in a field questionnaire on coffee liking. They have been consequently recruited and included in the study. Eighty-eight SNPs covering the 25 TAS2R genes were selected from the available imputed ones and used to run association analysis for coffee liking. A significant association was detected with three SNP: one synonymous and two functional variants (W35S and H212R) on the TAS2R43 gene. Both variants have been shown to greatly reduce in vitro protein activity. Surprisingly the wild type allele, which corresponds to the functional form of the protein, is associated to higher liking of coffee. Since the hTAS2R43 receptor is sensible to caffeine, we verified if the detected variants produced differences in caffeine bitter perception on a subsample of people coming from the FVG cohort. We found a significant association between differences in caffeine perception and the H212R variant but not with the W35S, which suggests that the effect of the TAS2R43 gene on coffee liking is mediated by caffeine and in particular by the H212R variant. No other significant association was found with other TAS2R genes. In conclusion, the present study opens new perspectives in the understanding of coffee liking. Further studies are needed to clarify the role of the TAS2R43 gene in coffee hedonics and to identify which other genes and pathways are involved in its genetics.

High Differentiation among Eight Villages in a Secluded Area of Sardinia Revealed by Genome-Wide High Density SNPs Analysis

To better design association studies for complex traits in isolated populations its important to understand how history and isolation moulded the genetic features of different communities. Population isolates should not “a priori” be considered homogeneous, even if the communities are not distant and part of a small region. We studied a particular area of Sardinia called Ogliastra, characterized by the presence of several distinct villages that display different history, immigration events and population size. Cultural and geographic isolation characterized the history of these communities. We determined LD parameters in 8 villages and defined population structure through high density SNPs (about 360 K) on 360 unrelated people (45 selected samples from each village). These isolates showed differences in LD values and LD map length. Five of these villages show high LD values probably due to their reduced population size and extreme isolation. High genetic differentiation among villages was detected. Moreover population structure analysis revealed a high correlation between genetic and geographic distances. Our study indicates that history, geography and biodemography have influenced the genetic features of Ogliastra communities producing differences in LD and population structure. All these data demonstrate that we can consider each village an isolate with specific characteristics. We suggest that, in order to optimize the study design of complex traits, a thorough characterization of genetic features is useful to identify the presence of sub-populations and stratification within genetic isolates.

Estrogen-related receptor gamma and hearing function: evidence of a role in humans and mice

Since estrogen is thought to protect pre-menopausal women from age-related hearing loss, we investigated whether variation in estrogen-signalling genes is linked to hearing status in the 1958 British Birth Cohort. This analysis implicated the estrogen-related receptor gamma (ESRRG) gene in determining adult hearing function and was investigated further in a total of 6134 individuals in 3 independent cohorts: (i) the 1958 British Birth Cohort; (ii) a London ARHL case-control cohort; and (iii) a cohort from isolated populations of Italy and Silk Road countries. Evidence of an association between the minor allele of single nucleotide polymorphism (SNP) rs2818964 and hearing status was found in females, but not in males in 2 of these cohorts: p = 0.0058 (London ARHL) and p = 0.0065 (Carlantino, Italy). Furthermore, assessment of hearing in Esrrg knock-out mice revealed a mild 25-dB hearing loss at 5 weeks of age. At 12 weeks, average hearing thresholds in female mice(-/-) were 15 dB worse than in males(-/-). Together these data indicate ESRRG plays a role in maintenance of hearing in both humans and mice.

Food Preference Patterns in a UK Twin Cohort.

Food liking-disliking patterns may strongly influence food choices and health. Here we assess: (1) whether food preference patterns are genetic/environmentally driven; and (2) the relationship between metabolomics profiles and food preference patterns in a large population of twins. 2,107 individuals from TwinsUK completed an online food and lifestyle preference questionnaire. Principle components analysis was undertaken to identify patterns of food liking-disliking. Heritability estimates for each liking pattern were obtained by structural equation modeling. The correlation between blood metabolomics profiles (280 metabolites) and each food liking pattern was assessed in a subset of 1,491 individuals and replicated in an independent subset of monozygotic twin pairs discordant for the liking pattern (65 to 88 pairs). Results from both analyses were meta-analyzed. Four major food-liking patterns were identified (Fruit and Vegetable, Distinctive Tastes, Sweet and High Carbohydrate, and Meat) accounting for 26% of the total variance. All patterns were moderately heritable (Fruit and Vegetable, h(2)[95% CI]: 0.36 [0.28; 0.44]; Distinctive Tastes: 0.58 [0.52; 0.64]; Sweet and High Carbohydrate: 0.52 [0.45, 0.59] and Meat: 0.44 [0.35; 0.51]), indicating genetic factors influence food liking-disliking. Overall, we identified 14 significant metabolite associations (Bonferroni p < 4.5 × 10(-5)) with Distinctive Tastes (8 metabolites), Sweet and High Carbohydrate (3 metabolites), and Meat (3 metabolites). Food preferences follow patterns based on similar taste and nutrient characteristics and these groupings are strongly determined by genetics. Food preferences that are strongly genetically determined (h(2) ≥ 0.40), such as for meat and distinctive-tasting foods, may influence intakes more substantially, as demonstrated by the metabolomic associations identified here.

Patterns of linkage disequilibrium between SNPs in a sardinian population isolate and the selection of markers for association studies

Objective: In isolated populations, ‘background’ linkage disequilibrium (LD) has been shown to extend over large genetic distances. This and their reduced environmental and genetic heterogeneity has stimulated interest in their potential for association mapping. We compared LD unit map distances with pair-wise measurements of LD in a dense single nucleotide polymorphism (SNP) set. Methods: We genotyped 771 SNPs in an 8 Mb segment of chromosome 22 on 101 individuals from the isolated village of Talana, Sardinia, and compared with outbred European populations. Results: Heterozygosity was remarkably similar in both populations. In contrast, the extent of LD observed was quite different. The decay of LD with distance is slower in the isolate. The differences in LD map lengths suggest that useful LD extends up to three times farther in the Sardinian population; smaller differences are seen with pairwise LD metrics. While LD map length slightly decreases with average relatedness, cryptic relatedness does not explain the decrease in LD map length. Haplotypes, block boundaries, and patterns of LD are similar in both populations, suggesting a shared distribution of recombination hotspots. Conclusions: About 15% fewer haplotype tagging SNPs need to be genotyped in the isolate, and possibly 70% fewer if selecting SNPs evenly spaced on the metric LD map.