A fast linkage method for population GWAS cohorts with related individuals.

Linkage analysis, a class of methods for detecting co-segregation of genomic segments and traits in families, was used to map disease-causing genes for decades before genotyping arrays and dense SNP genotyping enabled genome-wide association studies in population samples. Population samples often contain related individuals, but the segregation of alleles within families is rarely used because traditional linkage methods are computationally inefficient for larger datasets. Here, we describe Population Linkage, a novel application of Haseman-Elston regression as a method of moments estimator of variance components and their standard errors. We achieve additional computational efficiency by using modern methods for detection of IBD segments and variance component estimation, efficient preprocessing of input data, and minimizing redundant numerical calculations. We also refined variance component models to account for the biases in population-scale methods for IBD segment detection. We ran Population Linkage on four blood lipid traits in over 70,000 individuals from the HUNT and SardiNIA studies, successfully detecting 25 known genetic signals. One notable linkage signal that appeared in both was for low-density lipoprotein (LDL) cholesterol levels in the region near the gene APOE (LOD = 29.3, variance explained = 4.1%). This is the region where the missense variants rs7412 and rs429358, which together make up the ε2, ε3, and ε4 alleles each account for 2.4% and 0.8% of variation in circulating LDL cholesterol. Our results show the potential for linkage analysis and other large-scale applications of method of moments variance components estimation.

GWAS of genetic factors affecting white blood cell morphological parameters in Sardinians uncovers influence of chromosome 11 innate immunity gene cluster on eosinophil morphology.

Few genome-wide association studies (GWAS) analyzing genetic regulation of morphological traits of white blood cells have been reported. We carried out a GWAS of 12 morphological traits in 869 individuals from the general population of Sardinia, Italy. These traits, included measures of cell volume, conductivity and light scatter in four white-cell populations (eosinophils, lymphocytes, monocytes, neutrophils). This analysis yielded seven statistically significant signals, four of which were novel (four novel, PRG2, P2RX3, two of CDK6). Five signals were replicated in the independent INTERVAL cohort of 11 822 individuals. The most interesting signal with large effect size on eosinophil scatter (P-value = 8.33 x 10-32, beta = -1.651, se = 0.1351) falls within the innate immunity cluster on chromosome 11, and is located in the PRG2 gene. Computational analyses revealed that a rare, Sardinian-specific PRG2:p.Ser148Pro mutation modifies PRG2 amino acid contacts and protein dynamics in a manner that could possibly explain the changes observed in eosinophil morphology. Our discoveries shed light on genetics of morphological traits. For the first time, we describe such large effect size on eosinophils morphology that is relatively frequent in Sardinian population.

Childhood body size directly increases type 1 diabetes risk based on a lifecourse Mendelian randomization approach.

The rising prevalence of childhood obesity has been postulated as an explanation for the increasing rate of individuals diagnosed with type 1 diabetes (T1D). In this study, we use Mendelian randomization (MR) to provide evidence that childhood body size has an effect on T1D risk (OR = 2.05 per change in body size category, 95% CI = 1.20 to 3.50, P = 0.008), which remains after accounting for body size at birth and during adulthood using multivariable MR (OR = 2.32, 95% CI = 1.21 to 4.42, P = 0.013). We validate this direct effect of childhood body size using data from a large-scale T1D meta-analysis based on n = 15,573 cases and n = 158,408 controls (OR = 1.94, 95% CI = 1.21 to 3.12, P = 0.006). We also provide evidence that childhood body size influences risk of asthma, eczema and hypothyroidism, although multivariable MR suggested that these effects are mediated by body size in later life. Our findings support a causal role for higher childhood body size on risk of being diagnosed with T1D, whereas its influence on the other immune-associated diseases is likely explained by a long-term effect of remaining overweight for many years over the lifecourse.

Predicting physiological aging rates from a range of quantitative traits using machine learning.

It is widely thought that individuals age at different rates. A method that measures "physiological age" or physiological aging rate independent of chronological age could therefore help elucidate mechanisms of aging and inform an individual's risk of morbidity and mortality. Here we present machine learning frameworks for inferring individual physiological age from a broad range of biochemical and physiological traits including blood phenotypes (e.g., high-density lipoprotein), cardiovascular functions (e.g., pulse wave velocity) and psychological traits (e.g., neuroticism) as main groups in two population cohorts SardiNIA (~6,100 participants) and InCHIANTI (~1,400 participants). The inferred physiological age was highly correlated with chronological age (R2 > 0.8). We further defined an individual's physiological aging rate (PAR) as the ratio of the predicted physiological age to the chronological age. Notably, PAR was a significant predictor of survival, indicating an effect of aging rate on mortality. Our trait-based PAR was correlated with DNA methylation-based epigenetic aging score (r = 0.6), suggesting that both scores capture a common aging process. PAR was also substantially heritable (h2~0.3), and a subsequent genome-wide association study of PAR identified significant associations with two genetic loci, one of which is implicated in telomerase activity. Our findings support PAR as a proxy for an underlying whole-body aging mechanism. PAR may thus be useful to evaluate the efficacy of treatments that target aging-related deficits and controllable epidemiological factors.

Analysis of overlapping genetic association in type 1 and type 2 diabetes.

AIMS/HYPOTHESIS: Given the potential shared aetiology between type 1 and type 2 diabetes, we aimed to identify any genetic regions associated with both diseases. For associations where there is a shared signal and the allele that increases risk to one disease also increases risk to the other, inference about shared aetiology could be made, with the potential to develop therapeutic strategies to treat or prevent both diseases simultaneously. Alternatively, if a genetic signal co-localises with divergent effect directions, it could provide valuable biological insight into how the association affects the two diseases differently. METHODS: Using publicly available type 2 diabetes summary statistics from a genome-wide association study (GWAS) meta-analysis of European ancestry individuals (74,124 cases and 824,006 controls) and type 1 diabetes GWAS summary statistics from a meta-analysis of studies on individuals from the UK and Sardinia (7467 cases and 10,218 controls), we identified all regions of 0.5 Mb that contained variants associated with both diseases (false discovery rate <0.01). In each region, we performed forward stepwise logistic regression to identify independent association signals, then examined co-localisation of each type 1 diabetes signal with each type 2 diabetes signal using coloc. Any association with a co-localisation posterior probability of ≥0.9 was considered a genuine shared association with both diseases. RESULTS: Of the 81 association signals from 42 genetic regions that showed association with both type 1 and type 2 diabetes, four association signals co-localised between both diseases (posterior probability ≥0.9): (1) chromosome 16q23.1, near CTRB1/BCAR1, which has been previously identified; (2) chromosome 11p15.5, near the INS gene; (3) chromosome 4p16.3, near TMEM129 and (4) chromosome 1p31.3, near PGM1. In each of these regions, the effect of genetic variants on type 1 diabetes was in the opposite direction to the effect on type 2 diabetes. Use of additional datasets also supported the previously identified co-localisation on chromosome 9p24.2, near the GLIS3 gene, in this case with a concordant direction of effect. CONCLUSIONS/INTERPRETATION: Four of five association signals that co-localise between type 1 diabetes and type 2 diabetes are in opposite directions, suggesting a complex genetic relationship between the two diseases.

PRF1 mutation alters immune system activation, inflammation, and risk of autoimmunity.

BACKGROUND: Defective alleles within the PRF1 gene, encoding the pore-forming protein perforin, in combination with environmental factors, cause familial type 2 hemophagocytic lymphohistiocytosis (FHL2), a rare, severe autosomal recessive childhood disorder characterized by massive release of cytokines-cytokine storm. OBJECTIVE: The aim of this study was to determine the function of hypomorph PRF1:p.A91V g.72360387 G > A on multiple sclerosis (MS) and type 1 diabetes (T1D). METHODS: We cross-compare the association data for PRF1:p.A91V mutation derived from GWAS on adult MS and pediatric T1D in Sardinians. The novel association with T1D was replicated in metanalysis in 12,584 cases and 17,692 controls from Sardinia, the United Kingdom, and Scotland. To dissect this mutation function, we searched through the coincident association immunophenotypes in additional set of general population Sardinians. RESULTS: We report that PRF1:p.A91V, is associated with increase of lymphocyte levels, especially within the cytotoxic memory T-cells, at general population level with reduced interleukin 7 receptor expression on these cells. The minor allele increased risk of MS, in 2903 cases and 2880 controls from Sardinia p = 2.06 × 10-4, odds ratio OR = 1.29, replicating a previous finding, whereas it protects from T1D p = 1.04 × 10-5, OR = 0.82. CONCLUSION: Our results indicate opposing contributions of the cytotoxic T-cell compartment to MS and T1D pathogenesis.

Loss-of-function genomic variants highlight potential therapeutic targets for cardiovascular disease.

Pharmaceutical drugs targeting dyslipidemia and cardiovascular disease (CVD) may increase the risk of fatty liver disease and other metabolic disorders. To identify potential novel CVD drug targets without these adverse effects, we perform genome-wide analyses of participants in the HUNT Study in Norway (n = 69,479) to search for protein-altering variants with beneficial impact on quantitative blood traits related to cardiovascular disease, but without detrimental impact on liver function. We identify 76 (11 previously unreported) presumed causal protein-altering variants associated with one or more CVD- or liver-related blood traits. Nine of the variants are predicted to result in loss-of-function of the protein. This includes ZNF529:p.K405X, which is associated with decreased low-density-lipoprotein (LDL) cholesterol (P = 1.3 × 10-8) without being associated with liver enzymes or non-fasting blood glucose. Silencing of ZNF529 in human hepatoma cells results in upregulation of LDL receptor and increased LDL uptake in the cells. This suggests that inhibition of ZNF529 or its gene product should be prioritized as a novel candidate drug target for treating dyslipidemia and associated CVD.

Complex genetic signatures in immune cells underlie autoimmunity and inform therapy.

We report on the influence of ~22 million variants on 731 immune cell traits in a cohort of 3,757 Sardinians. We detected 122 significant (P < 1.28 × 10-11) independent association signals for 459 cell traits at 70 loci (53 of them novel) identifying several molecules and mechanisms involved in cell regulation. Furthermore, 53 signals at 36 loci overlapped with previously reported disease-associated signals, predominantly for autoimmune disorders, highlighting intermediate phenotypes in pathogenesis. Collectively, our findings illustrate complex genetic regulation of immune cells with highly selective effects on autoimmune disease risk at the cell-subtype level. These results identify drug-targetable pathways informing the design of more specific treatments for autoimmune diseases.

Author Correction: Complex genetic signatures in immune cells underlie autoimmunity and inform therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Sequencing and imputation in GWAS: Cost-effective strategies to increase power and genomic coverage across diverse populations.

A key aim for current genome-wide association studies (GWAS) is to interrogate the full spectrum of genetic variation underlying human traits, including rare variants, across populations. Deep whole-genome sequencing is the gold standard to fully capture genetic variation, but remains prohibitively expensive for large sample sizes. Array genotyping interrogates a sparser set of variants, which can be used as a scaffold for genotype imputation to capture a wider set of variants. However, imputation quality depends crucially on reference panel size and genetic distance from the target population. Here, we consider sequencing a subset of GWAS participants and imputing the rest using a reference panel that includes both sequenced GWAS participants and an external reference panel. We investigate how imputation quality and GWAS power are affected by the number of participants sequenced for admixed populations (African and Latino Americans) and European population isolates (Sardinians and Finns), and identify powerful, cost-effective GWAS designs given current sequencing and array costs. For populations that are well-represented in existing reference panels, we find that array genotyping alone is cost-effective and well-powered to detect common- and rare-variant associations. For poorly represented populations, sequencing a subset of participants is often most cost-effective, and can substantially increase imputation quality and GWAS power.

Evidence of Polygenic Adaptation in Sardinia at Height-Associated Loci Ascertained from the Biobank Japan.

Adult height is one of the earliest putative examples of polygenic adaptation in humans. However, this conclusion was recently challenged because residual uncorrected stratification from large-scale consortium studies was considered responsible for the previously noted genetic difference. It thus remains an open question whether height loci exhibit signals of polygenic adaptation in any human population. We re-examined this question, focusing on one of the shortest European populations, the Sardinians, in addition to mainland European populations. We utilized height-associated loci from the Biobank Japan (BBJ) dataset to further alleviate concerns of biased ascertainment of GWAS loci and showed that the Sardinians remain significantly shorter than expected under neutrality (∼0.22 standard deviation shorter than Utah residents with ancestry from northern and western Europe [CEU] on the basis of polygenic height scores, p = 3.89 × 10-4). We also found the trajectory of polygenic height scores between the Sardinian and the British populations diverged over at least the last 10,000 years (p = 0.0082), consistent with a signature of polygenic adaptation driven primarily by the Sardinian population. Although the polygenic score-based analysis showed a much subtler signature in mainland European populations, we found a clear and robust adaptive signature in the UK population by using a haplotype-based statistic, the trait singleton density score (tSDS), driven by the height-increasing alleles (p = 9.1 × 10-4). In summary, by ascertaining height loci in a distant East Asian population, we further supported the evidence of polygenic adaptation at height-associated loci among the Sardinians. In mainland Europeans, the adaptive signature was detected in haplotype-based analysis but not in polygenic score-based analysis.

The genetic architecture of membranous nephropathy and its potential to improve non-invasive diagnosis.

Membranous Nephropathy (MN) is a rare autoimmune cause of kidney failure. Here we report a genome-wide association study (GWAS) for primary MN in 3,782 cases and 9,038 controls of East Asian and European ancestries. We discover two previously unreported loci, NFKB1 (rs230540, OR = 1.25, P = 3.4 × 10-12) and IRF4 (rs9405192, OR = 1.29, P = 1.4 × 10-14), fine-map the PLA2R1 locus (rs17831251, OR = 2.25, P = 4.7 × 10-103) and report ancestry-specific effects of three classical HLA alleles: DRB1*1501 in East Asians (OR = 3.81, P = 2.0 × 10-49), DQA1*0501 in Europeans (OR = 2.88, P = 5.7 × 10-93), and DRB1*0301 in both ethnicities (OR = 3.50, P = 9.2 × 10-23 and OR = 3.39, P = 5.2 × 10-82, respectively). GWAS loci explain 32% of disease risk in East Asians and 25% in Europeans, and correctly re-classify 20-37% of the cases in validation cohorts that are antibody-negative by the serum anti-PLA2R ELISA diagnostic test. Our findings highlight an unusual genetic architecture of MN, with four loci and their interactions accounting for nearly one-third of the disease risk.

Genetic history from the Middle Neolithic to present on the Mediterranean island of Sardinia.

The island of Sardinia has been of particular interest to geneticists for decades. The current model for Sardinia's genetic history describes the island as harboring a founder population that was established largely from the Neolithic peoples of southern Europe and remained isolated from later Bronze Age expansions on the mainland. To evaluate this model, we generate genome-wide ancient DNA data for 70 individuals from 21 Sardinian archaeological sites spanning the Middle Neolithic through the Medieval period. The earliest individuals show a strong affinity to western Mediterranean Neolithic populations, followed by an extended period of genetic continuity on the island through the Nuragic period (second millennium BCE). Beginning with individuals from Phoenician/Punic sites (first millennium BCE), we observe spatially-varying signals of admixture with sources principally from the eastern and northern Mediterranean. Overall, our analysis sheds light on the genetic history of Sardinia, revealing how relationships to mainland populations shifted over time.

Meta-MultiSKAT: Multiple phenotype meta-analysis for region-based association test.

The power of genetic association analyses can be increased by jointly meta-analyzing multiple correlated phenotypes. Here, we develop a meta-analysis framework, Meta-MultiSKAT, that uses summary statistics to test for association between multiple continuous phenotypes and variants in a region of interest. Our approach models the heterogeneity of effects between studies through a kernel matrix and performs a variance component test for association. Using a genotype kernel, our approach can test for rare-variants and the combined effects of both common and rare-variants. To achieve robust power, within Meta-MultiSKAT, we developed fast and accurate omnibus tests combining different models of genetic effects, functional genomic annotations, multiple correlated phenotypes, and heterogeneity across studies. In addition, Meta-MultiSKAT accommodates situations where studies do not share exactly the same set of phenotypes or have differing correlation patterns among the phenotypes. Simulation studies confirm that Meta-MultiSKAT can maintain the type-I error rate at the exome-wide level of 2.5 × 10-6 . Further simulations under different models of association show that Meta-MultiSKAT can improve the power of detection from 23% to 38% on average over single phenotype-based meta-analysis approaches. We demonstrate the utility and improved power of Meta-MultiSKAT in the meta-analyses of four white blood cell subtype traits from the Michigan Genomics Initiative (MGI) and SardiNIA studies.

Parent-of-origin effects on quantitative phenotypes in a large Hutterite pedigree.

The impact of the parental origin of associated alleles in GWAS has been largely ignored. Yet sequence variants could affect traits differently depending on whether they are inherited from the mother or the father, as in imprinted regions, where identical inherited DNA sequences can have different effects based on the parental origin. To explore parent-of-origin effects (POEs), we studied 21 quantitative phenotypes in a large Hutterite pedigree to identify variants with single parent (maternal-only or paternal-only) effects, and then variants with opposite parental effects. Here we show that POEs, which can be opposite in direction, are relatively common in humans, have potentially important clinical effects, and will be missed in traditional GWAS. We identified POEs with 11 phenotypes, most of which are risk factors for cardiovascular disease. Many of the loci identified are characteristic of imprinted regions and are associated with the expression of nearby genes.

Relative impact of indels versus SNPs on complex disease.

It is unclear whether insertions and deletions (indels) are more likely to influence complex traits than abundant single-nucleotide polymorphisms (SNPs). We sought to understand which category of variation is more likely to impact health. Using the SardiNIA study as an exemplar, we characterized 478,876 common indels and 8,246,244 common SNPs in up to 5,949 well-phenotyped individuals from an isolated valley in Sardinia. We assessed association between 120 traits, resulting in 89 nonoverlapping-associated loci.We evaluated whether indels were enriched among credible sets of potential causal variants. These credible sets included 1,319 SNPs and 88 indels. We did not find indels to be significantly enriched. Indels were the most likely causal variant in seven loci, including one locus associated with monocyte count where an indel with causality and mechanism previously demonstrated (rs200748895:TGCTG/T) had a 0.999 posterior probability. Overall, our results show a very modest and nonsignificant enrichment for common indels in associated loci.

Genome Analyses of >200,000 Individuals Identify 58 Loci for Chronic Inflammation and Highlight Pathways that Link Inflammation and Complex Disorders.

C-reactive protein (CRP) is a sensitive biomarker of chronic low-grade inflammation and is associated with multiple complex diseases. The genetic determinants of chronic inflammation remain largely unknown, and the causal role of CRP in several clinical outcomes is debated. We performed two genome-wide association studies (GWASs), on HapMap and 1000 Genomes imputed data, of circulating amounts of CRP by using data from 88 studies comprising 204,402 European individuals. Additionally, we performed in silico functional analyses and Mendelian randomization analyses with several clinical outcomes. The GWAS meta-analyses of CRP revealed 58 distinct genetic loci (p < 5 × 10-8). After adjustment for body mass index in the regression analysis, the associations at all except three loci remained. The lead variants at the distinct loci explained up to 7.0% of the variance in circulating amounts of CRP. We identified 66 gene sets that were organized in two substantially correlated clusters, one mainly composed of immune pathways and the other characterized by metabolic pathways in the liver. Mendelian randomization analyses revealed a causal protective effect of CRP on schizophrenia and a risk-increasing effect on bipolar disorder. Our findings provide further insights into the biology of inflammation and could lead to interventions for treating inflammation and its clinical consequences.

Genomic history of the Sardinian population.

The population of the Mediterranean island of Sardinia has made important contributions to genome-wide association studies of complex disease traits and, based on ancient DNA studies of mainland Europe, Sardinia is hypothesized to be a unique refuge for early Neolithic ancestry. To provide new insights on the genetic history of this flagship population, we analyzed 3,514 whole-genome sequenced individuals from Sardinia. Sardinian samples show elevated levels of shared ancestry with Basque individuals, especially samples from the more historically isolated regions of Sardinia. Our analysis also uniquely illuminates how levels of genetic similarity with mainland ancient DNA samples varies subtly across the island. Together, our results indicate that within-island substructure and sex-biased processes have substantially impacted the genetic history of Sardinia. These results give new insight into the demography of ancestral Sardinians and help further the understanding of sharing of disease risk alleles between Sardinia and mainland populations.

XCAVATOR: accurate detection and genotyping of copy number variants from second and third generation whole-genome sequencing experiments.

BACKGROUND: We developed a novel software package, XCAVATOR, for the identification of genomic regions involved in copy number variants/alterations (CNVs/CNAs) from short and long reads whole-genome sequencing experiments. RESULTS: By using simulated and real datasets we showed that our tool, based on read count approach, is capable to predict the boundaries and the absolute number of DNA copies CNVs/CNAs with high resolutions. To demonstrate the power of our software we applied it to the analysis Illumina and Pacific Bioscencies data and we compared its performance to other ten state of the art tools. CONCLUSION: All the analyses we performed demonstrate that XCAVATOR is capable to detect germline and somatic CNVs/CNAs outperforming all the other tools we compared. XCAVATOR is freely available at http://sourceforge.net/projects/xcavator/ .