TM6SF2 rs58542926 influences hepatic fibrosis progression in patients with non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is an increasingly common condition, strongly associated with the metabolic syndrome, that can lead to progressive hepatic fibrosis, cirrhosis and hepatic failure. Subtle inter-patient genetic variation and environmental factors combine to determine variation in disease progression. A common non-synonymous polymorphism in TM6SF2 (rs58542926 c.449 C>T, p.Glu167Lys) was recently associated with increased hepatic triglyceride content, but whether this variant promotes clinically relevant hepatic fibrosis is unknown. Here we confirm that TM6SF2 minor allele carriage is associated with NAFLD and is causally related to a previously reported chromosome 19 GWAS signal that was ascribed to the gene NCAN. Furthermore, using two histologically characterized cohorts encompassing steatosis, steatohepatitis, fibrosis and cirrhosis (combined n=1,074), we demonstrate a new association, independent of potential confounding factors (age, BMI, type 2 diabetes mellitus and PNPLA3 rs738409 genotype), with advanced hepatic fibrosis/cirrhosis. These findings establish new and important clinical relevance to TM6SF2 in NAFLD.

Patients with faecal peritonitis admitted to European intensive care units: an epidemiological survey of the GenOSept cohort.

INTRODUCTION Faecal peritonitis (FP) is a common cause of sepsis and admission to the intensive care unit (ICU). The Genetics of Sepsis and Septic Shock in Europe (GenOSept) project is investigating the influence of genetic variation on the host response and outcomes in a large cohort of patients with sepsis admitted to ICUs across Europe. Here we report an epidemiological survey of the subset of patients with FP. OBJECTIVES To define the clinical characteristics, outcomes and risk factors for mortality in patients with FP admitted to ICUs across Europe. METHODS Data was extracted from electronic case report forms. Phenotypic data was recorded using a detailed, quality-assured clinical database. The primary outcome measure was 6-month mortality. Patients were followed for 6 months. Kaplan-Meier analysis was used to determine mortality rates. Cox proportional hazards regression analysis was employed to identify independent risk factors for mortality. RESULTS Data for 977 FP patients admitted to 102 centres across 16 countries between 29 September 2005 and 5 January 2011 was extracted. The median age was 69.2 years (IQR 58.3-77.1), with a male preponderance (54.3%). The most common causes of FP were perforated diverticular disease (32.1%) and surgical anastomotic breakdown (31.1%). The ICU mortality rate at 28 days was 19.1%, increasing to 31.6% at 6 months. The cause of FP, pre-existing co-morbidities and time from estimated onset of symptoms to surgery did not impact on survival. The strongest independent risk factors associated with an increased rate of death at 6 months included age, higher APACHE II score, acute renal and cardiovascular dysfunction within 1 week of admission to ICU, hypothermia, lower haematocrit and bradycardia on day 1 of ICU stay. CONCLUSIONS In this large cohort of patients admitted to European ICUs with FP the 6 month mortality was 31.6%. The most consistent predictors of mortality across all time points were increased age, development of acute renal dysfunction during the first week of admission, lower haematocrit and hypothermia on day 1 of ICU admission.

Contemporary ecotypic divergence during a recent range expansion was facilitated by adaptive introgression

Although rapid phenotypic evolution during range expansion associated with colonization of contrasting habitats has been documented in several taxa, the evolutionary mechanisms that underlie such phenotypic divergence have less often been investigated. A strong candidate for rapid ecotype formation within an invaded range is the three-spine stickleback in the Lake Geneva region of central Europe. Since its introduction only about 140 years ago, it has undergone a significant expansion of its range and its niche, now forming phenotypically differentiated parapatric ecotypes that occupy either the pelagic zone of the large lake or small inlet streams, respectively. By comparing museum collections from different times with contemporary population samples, we here reconstruct the evolution of parapatric phenotypic divergence through time. Using genetic data from modern samples, we infer the underlying invasion history. We find that parapatric habitat-dependent phenotypic divergence between the lake and stream was already present in the first half of the twentieth century, but the magnitude of differentiation increased through time, particularly in antipredator defence traits. This suggests that divergent selection between the habitats occurred and was stable through much of the time since colonization. Recently, increased phenotypic differentiation in antipredator defence traits likely results from habitat-dependent selection on alleles that arrived through introgression from a distantly related lineage from outside the Lake Geneva region. This illustrates how hybridization can quickly promote phenotypic divergence in a system where adaptation from standing genetic variation was constrained.

Process and pattern in cichlid radiations - inferences for understanding unusually high rates of evolutionary diversification

The cichlid fish radiations in the African Great Lakes differ from all other known cases of rapid speciation in vertebrates by their spectacular trophic diversity and richness of sympatric species, comparable to the most rapid angiosperm radiations. I review factors that may have facilitated these radiations and compare these with insights from recent work on plant radiations. Work to date suggests that it was a coincidence of ecological opportunity, intrinsic ecological versatility and genomic flexibility, rapidly evolving behavioral mate choice and large amounts of standing genetic variation that permitted these spectacular fish radiations. I propose that spatially orthogonal gradients in the fit of phenotypes to the environment facilitate speciation because they allow colonization of alternative fitness peaks during clinal speciation despite local disruptive selection. Such gradients are manifold in lakes because of the interaction of water depth as an omnipresent third spatial dimension with other fitness-relevant variables. I introduce a conceptual model of adaptive radiation that integrates these elements and discuss its applicability to, and predictions for, plant radiations.

Hybrid ‘superswarm’ leads to rapid divergence and establishment of populations during a biological invasion

Understanding the genetic background of invading species can be crucial information clarifying why they become invasive. Intraspecific genetic admixture among lineages separated in the native ranges may promote the rate and extent of an invasion by substantially increasing standing genetic variation. Here we examine the genetic relationships among threespine stickleback that recently colonized Switzerland. This invasion results from several distinct genetic lineages that colonized multiple locations and have since undergone range expansions, where they coexist and admix in parts of their range. Using 17 microsatellites genotyped for 634 individuals collected from 17 Swiss and two non-Swiss European sites, we reconstruct the invasion of stickleback and investigate the potential and extent of admixture and hybridization among the colonizing lineages from a population genetic perspective. Specifically we test for an increase in standing genetic variation in populations where multiple lineages coexist. We find strong evidence of massive hybridization early on, followed by what appears to be recent increased genetic isolation and the formation of several new genetically distinguishable populations, consistent with a hybrid ‘superswarm’. This massive hybridization and population formation event(s) occurred over approximately 140 years and likely fuelled the successful invasion of a diverse range of habitats. The implications are that multiple colonizations coupled with hybridization can lead to the formation of new stable genetic populations potentially kick-starting speciation and adaptive radiation over a very short time.

Evolutionary Genomics and Conservation of the Endangered Przewalski's Horse.

Przewalski's horses (PHs, Equus ferus ssp. przewalskii) were discovered in the Asian steppes in the 1870s and represent the last remaining true wild horses. PHs became extinct in the wild in the 1960s but survived in captivity, thanks to major conservation efforts. The current population is still endangered, with just 2,109 individuals, one-quarter of which are in Chinese and Mongolian reintroduction reserves [1]. These horses descend from a founding population of 12 wild-caught PHs and possibly up to four domesticated individuals [2-4]. With a stocky build, an erect mane, and stripped and short legs, they are phenotypically and behaviorally distinct from domesticated horses (DHs, Equus caballus). Here, we sequenced the complete genomes of 11 PHs, representing all founding lineages, and five historical specimens dated to 1878-1929 CE, including the Holotype. These were compared to the hitherto-most-extensive genome dataset characterized for horses, comprising 21 new genomes. We found that loci showing the most genetic differentiation with DHs were enriched in genes involved in metabolism, cardiac disorders, muscle contraction, reproduction, behavior, and signaling pathways. We also show that DH and PH populations split ∼45,000 years ago and have remained connected by gene-flow thereafter. Finally, we monitor the genomic impact of ∼110 years of captivity, revealing reduced heterozygosity, increased inbreeding, and variable introgression of domestic alleles, ranging from non-detectable to as much as 31.1%. This, together with the identification of ancestry informative markers and corrections to the International Studbook, establishes a framework for evaluating the persistence of genetic variation in future reintroduced populations.

STUDIES ON THE PATTERNS OF NUCLEOTIDE AND AMINO ACID SUBSTITUTION (ELECTROPHORESIS, MUTATION RATE, SELECTION, BASE, COMPOSITION)

Theoretical and empirical studies were conducted on the pattern of nucleotide and amino acid substitution in evolution, taking into account the effects of mutation at the nucleotide level and purifying selection at the amino acid level. A theoretical model for predicting the evolutionary change in electrophoretic mobility of a protein was also developed by using information on the pattern of amino acid substitution. The specific problems studied and the main results obtained are as follows: (1) Estimation of the pattern of nucleotide substitution in DNA nuclear genomes. The pattern of point mutations and nucleotide substitutions among the four different nucleotides are inferred from the evolutionary changes of pseudogenes and functional genes, respectively. Both patterns are non-random, the rate of change varying considerably with nucleotide pair, and that in both cases transitions occur somewhat more frequently than transversions. In protein evolution, substitution occurs more often between amino acids with similar physico-chemical properties than between dissimilar amino acids. (2) Estimation of the pattern of nucleotide substitution in RNA genomes. The majority of mutations in retroviruses accumulate at the reverse transcription stage. Selection at the amino acid level is very weak, and almost non-existent between synonymous codons. The pattern of mutation is very different from that in DNA genomes. Nevertheless, the pattern of purifying selection at the amino acid level is similar to that in DNA genomes, although selection intensity is much weaker. (3) Evaluation of the determinants of molecular evolutionary rates in protein-coding genes. Based on rates of nucleotide substitution for mammalian genes, the rate of amino acid substitution of a protein is determined by its amino acid composition. The content of glycine is shown to correlate strongly and negatively with the rate of substitution. Empirical formulae, called indices of mutability, are developed in order to predict the rate of molecular evolution of a protein from data on its amino acid sequence. (4) Studies on the evolutionary patterns of electrophoretic mobility of proteins. A theoretical model was constructed that predicts the electric charge of a protein at any given pH and its isoelectric point from data on its primary and quaternary structures. Using this model, the evolutionary change in electrophoretic mobilities of different proteins and the expected amount of electrophoretically hidden genetic variation were studied. In the absence of selection for the pI value, proteins will on the average evolve toward a mildly basic pI. (Abstract shortened with permission of author.) ^

Familial aggregation, candidate genes and genome scans: Analyzing the role of genetics in stroke

The genetic etiology of stroke likely reflects the influence of multiple loci with small effects, each modulating different pathophysiological processes. This research project utilized three analytical strategies to address the paucity of information related to the identification and characterization of genetic variation associated with stroke in the general population. ^ First, the general contribution of familial factors to stroke susceptibility was evaluated in a population-based sample of unrelated individuals. Increased risk of subclinical cerebral infarction was observed among individuals with a positive parental history of stroke. This association did not appear to be mediated by established stroke risk factors, specifically blood pressure levels or hypertension status. ^ The need to identify specific gene variation associated with stroke in the general population was addressed by evaluating seven candidate gene polymorphisms in a population-based sample of unrelated individuals. Three polymorphisms were significantly associated with increased subclinical cerebral infarction or incident clinical ischemic stroke risk. These relationships include the G-protein β3 subunit 825C/T polymorphism and clinical stroke in Whites, the lipoprotein lipase S/X447 polymorphism and subclinical and clinical stroke in men, and the angiotensin I-converting enzyme Ins/Del polymorphism and subclinical stroke in White men. These associations did not appear to be obfuscated by the stroke risk factors adjusted for in the analysis models specifically blood pressure levels or anti-hypertensive medication use. ^ The final research strategy considered, on a genome-wide scale, the idea that genetic variation may contribute to the occurrence of hypertension or stroke through a common etiologic pathway. Genomic regions were identified for which significant evidence of heterogeneity was observed among hypertensive sibpairs stratified by family history of stroke information. Regions identified on chromosome 15 in African Americans, and chromosome 13 in Whites and African Americans, suggest the presence of genes influencing hypertension and stroke susceptibility. ^ Insight into the role of genetics in stroke is useful for the potential early identification of individuals at increased risk for stroke and improved understanding of the etiology of the disease. The ultimate goal of these endeavors is to guide the development of therapeutic intervention and informed prevention to provide a lasting and positive impact on public health. ^

Detecting the signature of natural selection with microsatellites

Natural selection is one of the major factors in the evolution of all organisms. Detecting the signature of natural selection has been a central theme in evolutionary genetics. With the availability of microsatellite data, it is of interest to study how natural selection can be detected with microsatellites. ^ The overall aim of this research is to detect signatures of natural selection with data on genetic variation at microsatellite loci. The null hypothesis to be tested is the neutral mutation theory of molecular evolution, which states that different alleles at a locus have equivalent effects on fitness. Currently used tests of this hypothesis based on data on genetic polymorphism in natural populations presume that mutations at the loci follow the infinite allele/site models (IAM, ISM), in the sense that at each site at most only one mutation event is recorded, and each mutation leads to an allele not seen before in the population. Microsatellite loci, which are abundant in the genome, do not obey these mutation models, since the new alleles at such loci can be created either by contraction or expansion of tandem repeat sizes of core motifs. Since the current genome map is mainly composed of microsatellite loci and this class of loci is still most commonly studied in the context of human genome diversity, this research explores how the current test procedures for testing the neutral mutation hypothesis should be modified to take into account a generalized model of forward-backward stepwise mutations. In addition, recent literature also suggested that past demographic history of populations, presence of population substructure, and varying rates of mutations across loci all have confounding effects for detecting signatures of natural selection. ^ The effects of the stepwise mutation model and other confounding factors on detecting signature of natural selection are the main results of the research. ^

Modifier genes and susceptibility to colorectal cancer in individuals with Lynch syndrome

Lynch syndrome, is caused by inherited germ-line mutations in the DNA mismatch repair genes resulting in cancers at an early age, predominantly colorectal (CRC) and endometrial cancers. Though the median age at onset for CRC is about 45 years, disease penetrance varies suggesting that cancer susceptibility may be modified by environmental or other low-penetrance genes. Genetic variation due to polymorphisms in genes encoding metabolic enzymes can influence carcinogenesis by alterations in the expression and activity level of the enzymes. Variation in MTHFR, an important folate metabolizing enzyme can affect DNA methylation and DNA synthesis and variation in xenobiotic-metabolizing enzymes can affect the metabolism and clearance of carcinogens, thus modifying cancer risk. ^ This study examined a retrospective cohort of 257 individuals with Lynch syndrome, for polymorphisms in genes encoding xenobiotic-metabolizing enzymes-- CYP1A1 (I462V and MspI), EPHX1 (H139R and Y113H), GSTP1 (I105V and A114V), GSTM1 and GSTT1 (deletions) and folate metabolizing enzyme--MTHFR (C677T and A1298C). In addition, a series of 786 cases of sporadic CRC were genotyped for CYP1A1 I462V and EPHX1 Y113H to assess gene-gene interaction and gene-environment interaction with smoking in a case-only analysis. ^ Prominent findings of this study were that the presence of an MTHFR C677T variant allele was associated with a 4 year later age at onset for CRC on average and a reduced age-associated risk for developing CRC (Hazard ratio: 0.55; 95% confidence interval: 0.36–0.85) compared to the absence of any variant allele in individuals with Lynch syndrome. Similarly, Lynch syndrome individuals heterozygous for CYP1A1 I462V A>G polymorphism developed CRC an average of 4 years earlier and were at a 78% increased age-associated risk (Hazard ratio for AG relative to AA: 1.78; 95% confidence interval: 1.16-2.74) than those with the homozygous wild-type genotype. Therefore these two polymorphisms may be additional susceptibility factors for CRC in Lynch syndrome. In the case-only analysis, evidence of gene-gene interaction was seen between CYP1A1 I462V and EPHX1 Y113H and between EPHX1 Y113H and smoking suggesting that genetic and environmental factors may interact to increase sporadic CRC risk. Implications of these findings are the ability to identify subsets of high-risk individuals for targeted prevention and intervention. ^

Diabetes genes and risk of prostate cancer in the Atherosclerosis Risk in Communities study

Prostate cancer (PrCa) is a leading cause of morbidity and mortality, yet the etiology remains uncertain. Meta-analyses show that PrCa risk is reduced by 16% in men with type 2 diabetes (T2D), but the mechanism is unknown. Recent genome-wide association studies and meta-analyses have found single nucleotide polymorphisms (SNPs) that consistently predict T2D risk. We evaluated associations of incident PrCa with 14 T2D SNPs in the Atherosclerosis Risk in Communities (ARIC) study. From 1987-2000, there were 397 incident PrCa cases ascertained from state or local cancer registries among 6,642 men (1,560 blacks and 5,082 whites) aged 45-64 years at baseline. Genotypes were determined by TaqMan assay. Cox proportional hazards models were used to assess the association between PrCa and increasing number of T2D risk-raising alleles for individual SNPs and for genetic risk scores (GRS) comprised of the number of T2D risk-raising alleles across SNPs. Two-way gene-gene interactions were evaluated with likelihood ratio tests. Using additive genetic models, the T2D risk-raising allele was associated with significantly reduced risk of PrCa for IGF2BP2 rs4402960 (hazard ratio [HR]=0.79; P=0.07 among blacks only), SLC2A2 rs5400 (race-adjusted HR=0.85; P=0.05) and UCP2 rs660339 (race-adjusted HR=0.84; P=0.02), but significantly increased risk of PrCa for CAPN10 rs3792267 (race-adjusted HR=1.20; P=0.05). No other SNPs were associated with PrCa using an additive genetic model. However, at least one copy of the T2D risk-raising allele for TCF7L2 rs7903146 was associated with reduced PrCa risk using a dominant genetic model (race-adjusted HR=0.79; P=0.03). These results imply that the T2D-PrCa association may be partly due to shared genetic variation, but these results should be verified since multiple tests were performed. When the combined, additive effects of these SNPs were tested using a GRS, there was nearly a 10% reduction in risk of PrCa per T2D risk-raising allele (race-adjusted HR=0.92; P=0.02). SNPs in IGF2BP2, KCNJ11 and SLC2A2 were also involved in multiple synergistic gene-gene interactions on a multiplicative scale. In conclusion, it appears that the T2D-PrCa association may be due, in part, to common genetic variation. Further knowledge of T2D gene-PrCa mechanisms may improve understanding of PrCa etiology and may inform PrCa prevention and treatment.^

Functional data analysis approaches for genotype-phenotype association studies from next-generation sequencing

Next-generation DNA sequencing platforms can effectively detect the entire spectrum of genomic variation and is emerging to be a major tool for systematic exploration of the universe of variants and interactions in the entire genome. However, the data produced by next-generation sequencing technologies will suffer from three basic problems: sequence errors, assembly errors, and missing data. Current statistical methods for genetic analysis are well suited for detecting the association of common variants, but are less suitable to rare variants. This raises great challenge for sequence-based genetic studies of complex diseases.^ This research dissertation utilized genome continuum model as a general principle, and stochastic calculus and functional data analysis as tools for developing novel and powerful statistical methods for next generation of association studies of both qualitative and quantitative traits in the context of sequencing data, which finally lead to shifting the paradigm of association analysis from the current locus-by-locus analysis to collectively analyzing genome regions.^ In this project, the functional principal component (FPC) methods coupled with high-dimensional data reduction techniques will be used to develop novel and powerful methods for testing the associations of the entire spectrum of genetic variation within a segment of genome or a gene regardless of whether the variants are common or rare.^ The classical quantitative genetics suffer from high type I error rates and low power for rare variants. To overcome these limitations for resequencing data, this project used functional linear models with scalar response to develop statistics for identifying quantitative trait loci (QTLs) for both common and rare variants. To illustrate their applications, the functional linear models were applied to five quantitative traits in Framingham heart studies. ^ This project proposed a novel concept of gene-gene co-association in which a gene or a genomic region is taken as a unit of association analysis and used stochastic calculus to develop a unified framework for testing the association of multiple genes or genomic regions for both common and rare alleles. The proposed methods were applied to gene-gene co-association analysis of psoriasis in two independent GWAS datasets which led to discovery of networks significantly associated with psoriasis.^

Single nucleotide polymorphisms (SNPs) associated with TGF-beta pathway and their significance in systemic sclerosis - A multilevel analysis

Systemic sclerosis (SSc) or Scleroderma is a complex disease and its etiopathogenesis remains unelucidated. Fibrosis in multiple organs is a key feature of SSc and studies have shown that transforming growth factor-β (TGF-β) pathway has a crucial role in fibrotic responses. For a complex disease such as SSc, expression quantitative trait loci (eQTL) analysis is a powerful tool for identifying genetic variations that affect expression of genes involved in this disease. In this study, a multilevel model is described to perform a multivariate eQTL for identifying genetic variation (SNPs) specifically associated with the expression of three members of TGF-β pathway, CTGF, SPARC and COL3A1. The uniqueness of this model is that all three genes were included in one model, rather than one gene being examined at a time. A protein might contribute to multiple pathways and this approach allows the identification of important genetic variations linked to multiple genes belonging to the same pathway. In this study, 29 SNPs were identified and 16 of them located in known genes. Exploring the roles of these genes in TGF-β regulation will help elucidate the etiology of SSc, which will in turn help to better manage this complex disease. ^

Pathway analysis of genome-wide association study data in primary biliary cirrhosisa

Genome-wide association studies (GWAS) have successfully identified several genetic loci associated with inherited predisposition to primary biliary cirrhosis (PBC), the most common autoimmune disease of the liver. Pathway-based tests constitute a novel paradigm for GWAS analysis. By evaluating genetic variation across a biological pathway (gene set), these tests have the potential to determine the collective impact of variants with subtle effects that are individually too weak to be detected in traditional single variant GWAS analysis. To identify biological pathways associated with the risk of development of PBC, GWAS of PBC from Italy (449 cases and 940 controls) and Canada (530 cases and 398 controls) were independently analyzed. The linear combination test (LCT), a recently developed pathway-level statistical method was used for this analysis. For additional validation, pathways that were replicated at the P <0.05 level of significance in both GWAS on LCT analysis were also tested for association with PBC in each dataset using two complementary GWAS pathway approaches. The complementary approaches included a modification of the gene set enrichment analysis algorithm (i-GSEA4GWAS) and Fisher's exact test for pathway enrichment ratios. Twenty-five pathways were associated with PBC risk on LCT analysis in the Italian dataset at P<0.05, of which eight had an FDR<0.25. The top pathway in the Italian dataset was the TNF/stress related signaling pathway (p=7.38×10 -4, FDR=0.18). Twenty-six pathways were associated with PBC at the P<0.05 level using the LCT in the Canadian dataset with the regulation and function of ChREBP in liver pathway (p=5.68×10-4, FDR=0.285) emerging as the most significant pathway. Two pathways, phosphatidylinositol signaling system (Italian: p=0.016, FDR=0.436; Canadian: p=0.034, FDR=0.693) and hedgehog signaling (Italian: p=0.044, FDR=0.636; Canadian: p=0.041, FDR=0.693), were replicated at LCT P<0.05 in both datasets. Statistically significant association of both pathways with PBC genetic susceptibility was confirmed in the Italian dataset on i-GSEA4GWAS. Results for the phosphatidylinositol signaling system were also significant in both datasets on applying Fisher's exact test for pathway enrichment ratios. This study identified a combination of known and novel pathway-level associations with PBC risk. If functionally validated, the findings may yield fresh insights into the etiology of this complex autoimmune disease with possible preventive and therapeutic application.^

Sources of familial aggregation and co-aggregation of fasting blood glucose and nutritional factors among Mexican -Americans in Starr County, Texas

The purpose of this study was to determine the effects of nutrient intake, genetic factors and common household environmental factors on the aggregation of fasting blood glucose among Mexican-Americans in Starr County, Texas. This study was designed to determine: (a) the proportion of variation of fasting blood glucose concentration explained by unmeasured genetic and common household environmental effects; (b) the degree of familial aggregation of measures of nutrient intake; and (c) the extent to which the familial aggregation of fasting blood glucose is explained by nutrient intake and its aggregation. The method of path analysis was employed to determine these various effects.^ Genes play an important role in fasting blood glucose: Genetic variation was found to explain about 40% of the total variation in fasting blood glucose. Common household environmental effects, on the other hand, explained less than 3% of the variation in fasting blood glucose levels among individuals. Common household effects, however, did have significant effects on measures of nutrient intake, though it explained only about 10% of the total variance in nutrient intake. Finally, there was significant familial aggregation of nutrient intake measures, but their aggregation did not contribute significantly to the familial aggregation of fasting blood glucose. These results imply that similarities among relatives for fasting blood glucose are not due to similarities in nutrient intake among relatives. ^