We conducted a genome-wide association study (GWAS) on multiple sclerosis (MS) susceptibility in German cohorts with 4888 cases and 10,395 controls. In addition to associations within the major histocompatibility complex (MHC) region, 15 non-MHC loci reached genome-wide significance. Four of these loci are novel MS susceptibility loci. They map to the genes L3MBTL3, MAZ, ERG, and SHMT1. The lead variant at SHMT1 was replicated in an independent Sardinian cohort. Products of the genes L3MBTL3, MAZ, and ERG play important roles in immune cell regulation. SHMT1 encodes a serine hydroxymethyltransferase catalyzing the transfer of a carbon unit to the folate cycle. This reaction is required for regulation of methylation homeostasis, which is important for establishment and maintenance of epigenetic signatures. Our GWAS approach in a defined population with limited genetic substructure detected associations not found in larger, more heterogeneous cohorts, thus providing new clues regarding MS pathogenesis.

DNA sequencing identifies common and rare genetic variants for association studies, but studies typically focus on variants in nuclear DNA and ignore the mitochondrial genome. In fact, analyzing variants in mitochondrial DNA (mtDNA) sequences presents special problems, which we resolve here with a general solution for the analysis of mtDNA in next-generation sequencing studies. The new program package comprises 1) an algorithm designed to identify mtDNA variants (i.e., homoplasmies and heteroplasmies), incorporating sequencing error rates at each base in a likelihood calculation and allowing allele fractions at a variant site to differ across individuals; and 2) an estimation of mtDNA copy number in a cell directly from whole-genome sequencing data. We also apply the methods to DNA sequence from lymphocytes of ~2,000 SardiNIA Project participants. As expected, mothers and offspring share all homoplasmies but a lesser proportion of heteroplasmies. Both homoplasmies and heteroplasmies show 5-fold higher transition/transversion ratios than variants in nuclear DNA. Also, heteroplasmy increases with age, though on average only ~1 heteroplasmy reaches the 4% level between ages 20 and 90. In addition, we find that mtDNA copy number averages ~110 copies/lymphocyte and is ~54% heritable, implying substantial genetic regulation of the level of mtDNA. Copy numbers also decrease modestly but significantly with age, and females on average have significantly more copies than males. The mtDNA copy numbers are significantly associated with waist circumference (p-value = 0.0031) and waist-hip ratio (p-value = 2.4×10-5), but not with body mass index, indicating an association with central fat distribution. To our knowledge, this is the largest population analysis to date of mtDNA dynamics, revealing the age-imposed increase in heteroplasmy, the relatively high heritability of copy number, and the association of copy number with metabolic traits.

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.

Deep sequence-based imputation can enhance the discovery power of genome-wide association studies by assessing previously unexplored variation across the common- and low-frequency spectra. We applied a hybrid whole-genome sequencing (WGS) and deep imputation approach to examine the broader allelic architecture of 12 anthropometric traits associated with height, body mass, and fat distribution in up to 267,616 individuals. We report 106 genome-wide significant signals that have not been previously identified, including 9 low-frequency variants pointing to functional candidates. Of the 106 signals, 6 are in genomic regions that have not been implicated with related traits before, 28 are independent signals at previously reported regions, and 72 represent previously reported signals for a different anthropometric trait. 71% of signals reside within genes and fine mapping resolves 23 signals to one or two likely causal variants. We confirm genetic overlap between human monogenic and polygenic anthropometric traits and find signal enrichment in cis expression QTLs in relevant tissues. Our results highlight the potential of WGS strategies to enhance biologically relevant discoveries across the frequency spectrum.

Identifying the genes that influence levels of pro-inflammatory molecules can help to elucidate the mechanisms underlying this process. We first conducted a two-stage genome-wide association scan (GWAS) for the key inflammatory biomarkers Interleukin-6 (IL-6), the general measure of inflammation erythrocyte sedimentation rate (ESR), monocyte chemotactic protein-1 (MCP-1), and high-sensitivity C-reactive protein (hsCRP) in a large cohort of individuals from the founder population of Sardinia. By analysing 731,213 autosomal or X chromosome SNPs and an additional ∼1.9 million imputed variants in 4,694 individuals, we identified several SNPs associated with the selected quantitative trait loci (QTLs) and replicated all the top signals in an independent sample of 1,392 individuals from the same population. Next, to increase power to detect and resolve associations, we further genotyped the whole cohort (6,145 individuals) for 293,875 variants included on the ImmunoChip and MetaboChip custom arrays. Overall, our combined approach led to the identification of 9 genome-wide significant novel independent signals-5 of which were identified only with the custom arrays-and provided confirmatory evidence for an additional 7. Novel signals include: for IL-6, in the ABO gene (rs657152, p = 2.13×10(-29)); for ESR, at the HBB (rs4910472, p = 2.31×10(-11)) and UCN119B/SPPL3 (rs11829037, p = 8.91×10(-10)) loci; for MCP-1, near its receptor CCR2 (rs17141006, p = 7.53×10(-13)) and in CADM3 (rs3026968, p = 7.63×10(-13)); for hsCRP, within the CRP gene (rs3093077, p = 5.73×10(-21)), near DARC (rs3845624, p = 1.43×10(-10)), UNC119B/SPPL3 (rs11829037, p = 1.50×10(-14)), and ICOSLG/AIRE (rs113459440, p = 1.54×10(-08)) loci. Confirmatory evidence was found for IL-6 in the IL-6R gene (rs4129267); for ESR at CR1 (rs12567990) and TMEM57 (rs10903129); for MCP-1 at DARC (rs12075); and for hsCRP at CRP (rs1205), HNF1A (rs225918), and APOC-I (rs4420638). Our results improve the current knowledge of genetic variants underlying inflammation and provide novel clues for the understanding of the molecular mechanisms regulating this complex process.

A multiplicity of study designs such as gene candidate analysis, genome wide search (GWS) and, recently, whole genome association studies have been employed for the identification of the genetic components of essential hypertension (EH). Several genome-wide linkage studies of EH and blood pressure-related phenotypes demonstrate that there is no single locus with a major effect while several genomic regions likely to contain EH-susceptibility loci were validated by multiple studies.

Genetic studies of complex traits have mainly identified associations with noncoding variants. To further determine the contribution of regulatory variation, we combined whole-genome and transcriptome data for 624 individuals from Sardinia to identify common and rare variants that influence gene expression and splicing. We identified 21,183 expression quantitative trait loci (eQTLs) and 6,768 splicing quantitative trait loci (sQTLs), including 619 new QTLs. We identified high-frequency QTLs and found evidence of selection near genes involved in malarial resistance and increased multiple sclerosis risk, reflecting the epidemiological history of Sardinia. Using family relationships, we identified 809 segregating expression outliers (median z score of 2.97), averaging 13.3 genes per individual. Outlier genes were enriched for proximal rare variants, providing a new approach to study large-effect regulatory variants and their relevance to traits. Our results provide insight into the effects of regulatory variants and their relationship to population history and individual genetic risk.

Cone rod-dystrophies (CRDs) are pigmentary retinopathies mainly involving cones. CRDs typically present with decreased visual acuity and loss of sensitivity in the central visual field, reflecting the primary dysfunction of cones associated with night blindness and concentric visual field loss due to rod dysfunction. We describe the phenotype, natural history, and molecular analysis results of an early onset form of CRD.

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.

Gut Microbiota (GM) dysbiosis associates with Atherosclerotic Cardiovascular Diseases (ACVD), but whether this also holds true in subjects without clinically manifest ACVD represents a challenge of personalized prevention. We connected exposure to diet (self-reported by food diaries) and markers of Subclinical Carotid Atherosclerosis (SCA) with individual taxonomic and functional GM profiles (from fecal metagenomic DNA) of 345 subjects without previous clinically manifest ACVD. Subjects without SCA reported consuming higher amounts of cereals, starchy vegetables, milky products, yoghurts and bakery products versus those with SCA (who reported to consume more mechanically separated meats). The variety of dietary sources significantly overlapped with the separations in GM composition between subjects without SCA and those with SCA (RV coefficient between nutrients quantities and microbial relative abundances at genus level = 0.65, p-value = 0.047). Additionally, specific bacterial species (Faecalibacterium prausnitzii in the absence of SCA and Escherichia coli in the presence of SCA) are directly related to over-representation of metagenomic pathways linked to different dietary sources (sulfur oxidation and starch degradation in absence of SCA, and metabolism of amino acids, syntheses of palmitate, choline, carnitines and Trimethylamine n-oxide in presence of SCA). These findings might contribute to hypothesize future strategies of personalized dietary intervention for primary CVD prevention setting.

EGFR is overexpressed in the majority of clear cell renal cell carcinomas (CCRCCs). Although EGFR deregulation was found to be of great significance in CCRCC biology, the EGFR overexpression is not associated with EGFR-targeted therapy responsiveness. Moreover, the prognostic role of EGFR expression remains controversial. In the present study, we evaluated the role played by EGFR overexpression in CCRCC and its prognostic significance associated with different immunohistochemical localization patterns. In our study, the Total Score (TS) related to membranous-cytoplasmic EGFR expression showed a significant correlation with grade, pathologic stage (pT), and Stage, Size, Grade, and Necrosis (SSIGN) score, and a negative correlation with nuclear EGFR expression. No significant correlations were shown between nuclear EGFR and clinic-pathological features. Additionally, a correlation between SGLT1 expression levels and pT was described. Multivariate analysis identifies pT and SSIGN score as independent prognostic factors for CCRCC. A significantly increased survival rate was found in the case of positive expression of nuclear EGFR and SGLT1. Based on our findings, SGLT1 and nuclear EGFR overexpression defines a subgroup of CCRCC patients with good prognosis. Membranous-cytoplasmic EGFR expression was shown to be a poor prognostic factor and could define a CCRCC subgroup with poor prognosis that should be responsive to anti-EGFR therapies.

HER2+ breast cancer (BC) is an aggressive subtype genetically and biologically heterogeneous. We evaluate the predictive and prognostic role of HER2 protein/gene expression levels combined with clinico-pathologic features in 154 HER2+ BCs patients who received trastuzumab-based neoadjuvant chemotherapy (NACT). The tumoral pathological complete response (pCR) rate was 40.9%. High tumoral pCR show a scarce mortality rate vs subjects with a lower response. 93.7% of ypT0 were HER2 IHC3+ BC, 6.3% were HER2 IHC 2+/SISH+ and 86.7% of ypN0 were HER2 IHC3+, the remaining were HER2 IHC2+/SISH+. Better pCR rate correlate with a high percentage of infiltrating immune cells and right-sided tumors, that reduce distant metastasis and improve survival, but no incidence difference. HER2 IHC score and laterality emerge as strong predictors of tumoral pCR after NACT from machine learning analysis. HER2 IHC3+ and G3 are poor prognostic factors for HER2+ BC patients, and could be considered in the application of neoadjuvant therapy. Increasing TILs concentrations, lower lymph node ratio and lower residual tumor cellularity are associated with a better outcome. The immune microenvironment and scarce lymph node involvement have crucial role in clinical outcomes. The combination of all predictors might offer new options for NACT effectiveness prediction and stratification of HER2+ BC during clinical decision-making.

Through genome-wide association meta-analyses of up to 133,010 individuals of European ancestry without diabetes, including individuals newly genotyped using the Metabochip, we have increased the number of confirmed loci influencing glycemic traits to 53, of which 33 also increase type 2 diabetes risk (q < 0.05). Loci influencing fasting insulin concentration showed association with lipid levels and fat distribution, suggesting impact on insulin resistance. Gene-based analyses identified further biologically plausible loci, suggesting that additional loci beyond those reaching genome-wide significance are likely to represent real associations. This conclusion is supported by an excess of directionally consistent and nominally significant signals between discovery and follow-up studies. Functional analysis of these newly discovered loci will further improve our understanding of glycemic control.

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.

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.

We report sequencing-based whole-genome association analyses to evaluate the impact of rare and founder variants on stature in 6,307 individuals on the island of Sardinia. We identify two variants with large effects. One variant, which introduces a stop codon in the GHR gene, is relatively frequent in Sardinia (0.87% versus <0.01% elsewhere) and in the homozygous state causes Laron syndrome involving short stature. We find that this variant reduces height in heterozygotes by an average of 4.2 cm (-0.64 s.d.). The other variant, in the imprinted KCNQ1 gene (minor allele frequency (MAF) = 7.7% in Sardinia versus <1% elsewhere) reduces height by an average of 1.83 cm (-0.31 s.d.) when maternally inherited. Additionally, polygenic scores indicate that known height-decreasing alleles are at systematically higher frequencies in Sardinians than would be expected by genetic drift. The findings are consistent with selection for shorter stature in Sardinia and a suggestive human example of the proposed 'island effect' reducing the size of large mammals.

Next-Generation Sequencing methods have led to a great increase in phylogenetically useful markers within the male specific portion of the Y chromosome, but previous studies have limited themselves to the study of the X-degenerate regions.

Family samples, which can be enriched for rare causal variants by focusing on families with multiple extreme individuals and which facilitate detection of de novo mutation events, provide an attractive resource for next-generation sequencing studies. Here, we describe, implement, and evaluate a likelihood-based framework for analysis of next generation sequence data in family samples. Our framework is able to identify variant sites accurately and to assign individual genotypes, and can handle de novo mutation events, increasing the sensitivity and specificity of variant calling and de novo mutation detection. Through simulations we show explicit modeling of family relationships is especially useful for analyses of low-frequency variants and that genotype accuracy increases with the number of individuals sequenced per family. Compared with the standard approach of ignoring relatedness, our methods identify and accurately genotype more variants, and have high specificity for detecting de novo mutation events. The improvement in accuracy using our methods over the standard approach is particularly pronounced for low-frequency variants. Furthermore the family-aware calling framework dramatically reduces Mendelian inconsistencies and is beneficial for family-based analysis. We hope our framework and software will facilitate continuing efforts to identify genetic factors underlying human diseases.

Molecular and cellular changes are intrinsic to aging and age-related diseases. Prior cross-sectional studies have investigated the combined effects of age and genetics on gene expression and alternative splicing; however, there has been no long-term, longitudinal characterization of these molecular changes, especially in older age.

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.