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.

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.

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.

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.

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.

MiRNAs, a small family of non-coding RNA, are now emerging as regulators of stem cell pluripotency, differentiation, and autophagy, thus controlling stem cell behavior. Stem cells are undifferentiated elements capable to acquire specific phenotype under different kind of stimuli, being a main tool for regenerative medicine. Within this context, we have previously shown that stem cells isolated from Wharton jelly multipotent stem cells (WJ-MSCs) exhibit gender differences in the expression of the stemness related gene OCT4 and the epigenetic modulator gene DNA-Methyltransferase (DNMT1). Here, we further analyze this gender difference, evaluating adipogenic and osteogenic differentiation potential, autophagic process, and expression of miR-145, miR-148a, and miR-185 in WJ-MSCs derived from males and females. These miRNAs were selected since they are involved in OCT4 and DNMT1 gene expression, and in stem cell differentiation. Our results indicate a difference in the regulatory circuit involving miR-148a/DNMT1/OCT4 autophagy in male WJ-MSCs as compared to female cells. Moreover, no difference was detected in the expression of the two-differentiation regulating miRNA (miR-145 and miR-185). Taken together, our results highlight a different behavior of WJ-MSCs from males and females, disclosing the chance to better understand cellular processes as autophagy and stemness, usable for future clinical applications.

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.

Thyroid dysfunction is an important public health problem, which affects 10% of the general population and increases the risk of cardiovascular morbidity and mortality. Many aspects of thyroid hormone regulation have only partly been elucidated, including its transport, metabolism, and genetic determinants. Here we report a large meta-analysis of genome-wide association studies for thyroid function and dysfunction, testing 8 million genetic variants in up to 72,167 individuals. One-hundred-and-nine independent genetic variants are associated with these traits. A genetic risk score, calculated to assess their combined effects on clinical end points, shows significant associations with increased risk of both overt (Graves' disease) and subclinical thyroid disease, as well as clinical complications. By functional follow-up on selected signals, we identify a novel thyroid hormone transporter (SLC17A4) and a metabolizing enzyme (AADAT). Together, these results provide new knowledge about thyroid hormone physiology and disease, opening new possibilities for therapeutic targets.

FOXL2 belongs to the evolutionarily conserved forkhead box (FOX) superfamily and is a master transcription factor in a spectrum of developmental pathways, including ovarian and eyelid development and bone, cartilage and uterine maturation. To analyse its action, we searched for proteins that interact with FOXL2. We found that FOXL2 interacts with specific C-terminal propeptides of several fibrillary collagens. Because these propeptides can participate in feedback regulation of collagen biosynthesis, we inferred that FOXL2 could thereby affect the transcription of the cognate collagen genes. Focusing on COL1A2, we found that FOXL2 indeed affects collagen synthesis, by binding to a DNA response element located about 65Kb upstream of this gene. According to our hypothesis we found that in Foxl2(-/-) mouse ovaries, Col1a2 was elevated from birth to adulthood. The extracellular matrix (ECM) compartmentalizes the ovary during folliculogenesis, (with type I, type III and type IV collagens as primary components), and ECM composition changes during the reproductive lifespan. In Foxl2(-/-) mouse ovaries, in addition to up-regulation of Col1a2, Col3a1, Col4a1 and fibronectin were also upregulated, while laminin expression was reduced. Thus, by regulating levels of extracellular matrix components, FOXL2 may contribute to both ovarian histogenesis and the fibrosis attendant on depletion of the follicle reserve during reproductive aging and menopause.

Transposons and γ-retroviruses have been efficiently used as insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells and that hamper the identification of early cancer-driving events among bystander and progression-related events. We developed an insertional mutagenesis platform based on lentiviral vectors (LVVs) by which we could efficiently induce hepatocellular carcinoma (HCC) in three different mouse models. By virtue of the LVV's replication-deficient nature and broad genome-wide integration pattern, LVV-based insertional mutagenesis allowed identification of four previously unknown liver cancer-associated genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. The newly identified genes are likely to play a role in human cancer because they are upregulated, amplified and/or deleted in human HCCs and can predict clinical outcomes of patients.

Idiopathic pulmonary fibrosis (IPF) is a complex disease in which a multitude of proteins and networks are disrupted. Interrogation of the transcriptome through RNA sequencing (RNA-Seq) enables the determination of genes whose differential expression is most significant in IPF, as well as the detection of alternative splicing events which are not easily observed with traditional microarray experiments. We sequenced messenger RNA from 8 IPF lung samples and 7 healthy controls on an Illumina HiSeq 2000, and found evidence for substantial differential gene expression and differential splicing. 873 genes were differentially expressed in IPF (FDR<5%), and 440 unique genes had significant differential splicing events in at least one exonic region (FDR<5%). We used qPCR to validate the differential exon usage in the second and third most significant exonic regions, in the genes COL6A3 (RNA-Seq adjusted pval = 7.18e-10) and POSTN (RNA-Seq adjusted pval = 2.06e-09), which encode the extracellular matrix proteins collagen alpha-3(VI) and periostin. The increased gene-level expression of periostin has been associated with IPF and its clinical progression, but its differential splicing has not been studied in the context of this disease. Our results suggest that alternative splicing of these and other genes may be involved in the pathogenesis of IPF. We have developed an interactive web application which allows users to explore the results of our RNA-Seq experiment, as well as those of two previously published microarray experiments, and we hope that this will serve as a resource for future investigations of gene regulation in IPF.

Genome sequencing of the 5,300-year-old mummy of the Tyrolean Iceman, found in 1991 on a glacier near the border of Italy and Austria, has yielded new insights into his origin and relationship to modern European populations. A key finding of that study was an apparent recent common ancestry with individuals from Sardinia, based largely on the Y chromosome haplogroup and common autosomal SNP variation. Here, we compiled and analyzed genomic datasets from both modern and ancient Europeans, including genome sequence data from over 400 Sardinians and two ancient Thracians from Bulgaria, to investigate this result in greater detail and determine its implications for the genetic structure of Neolithic Europe. Using whole-genome sequencing data, we confirm that the Iceman is, indeed, most closely related to Sardinians. Furthermore, we show that this relationship extends to other individuals from cultural contexts associated with the spread of agriculture during the Neolithic transition, in contrast to individuals from a hunter-gatherer context. We hypothesize that this genetic affinity of ancient samples from different parts of Europe with Sardinians represents a common genetic component that was geographically widespread across Europe during the Neolithic, likely related to migrations and population expansions associated with the spread of agriculture.

We focused on an integrated view of genomic changes in Colorectal cancer (CRC) and distant normal colon tissue (NTC) to test the effectiveness of expression profiling on identification of molecular targets. We performed transcriptome on 16 primary coupled CRC and NTC tissues. We identified pathways and networks related to pathophysiology of CRC and selected potential therapeutic targets. CRC cells have multiple ways to reprogram its transcriptome: a functional enrichment analysis in 285 genes, 25% mutated, showed that they control the major cellular processes known to promote tumorigenesis. Among the genes showing alternative splicing, cell cycle related genes were upregulated (CCND1, CDC25B, MCM2, MCM3), while genes involved in fatty acid metabolism (ACAAA2, ACADS, ACAT1, ACOX, CPT1A, HMGCS2) were downregulated. Overall 148 genes showed differential splicing identifying 17 new isoforms. Most of them are involved in the pathogenesis of CRC, although the functions of these variants remain unknown. We identified 2 in-frame fusion events, KRT19-KRT18 and EEF1A1-HSP90AB1, encoding for chemical proteins in two CRC patients. We draw a functional interactome map involving integrated multiple genomic features in CRC. Finally, we underline that two functional cell programs are prevalently deregulated and absolutely crucial to determinate and sustain CRC phenotype.

Crisponi syndrome (CS)/cold-induced sweating syndrome type 1 (CISS1) is a very rare autosomal-recessive disorder characterized by a complex phenotype with high neonatal lethality, associated with the following main clinical features: hyperthermia and feeding difficulties in the neonatal period, scoliosis, and paradoxical sweating induced by cold since early childhood. CS/CISS1 can be caused by mutations in cytokine receptor-like factor 1 (CRLF1). However, the physiopathological role of CRLF1 is still poorly understood. A subset of CS/CISS1 cases remain yet genetically unexplained after CRLF1 sequencing. In five of them, exome sequencing and targeted Sanger sequencing identified four homozygous disease-causing mutations in kelch-like family member 7 (KLHL7), affecting the Kelch domains of the protein. KLHL7 encodes a BTB-Kelch-related protein involved in the ubiquitination of target proteins for proteasome-mediated degradation. Mono-allelic substitutions in other domains of KLHL7 have been reported in three families affected by a late-onset form of autosomal-dominant retinitis pigmentosa. Retinitis pigmentosa was also present in two surviving children reported here carrying bi-allelic KLHL7 mutations. KLHL7 mutations are thus associated with a more severe phenotype in recessive than in dominant cases. Although these data further support the pathogenic role of KLHL7 mutations in a CS/CISS1-like phenotype, they do not explain all their clinical manifestations and highlight the high phenotypic heterogeneity associated with mutations in KLHL7.

Methylation at 5-cytosine (5-mC) is a fundamental epigenetic DNA modification associated recently with cardiac disease. In contrast, the role of 5-hydroxymethylcytosine (5-hmC)-5-mC's oxidation product-in cardiac biology and disease is unknown. Here we assess the hydroxymethylome in embryonic, neonatal, adult and hypertrophic mouse cardiomyocytes, showing that dynamic modulation of hydroxymethylated DNA is associated with specific transcriptional networks during heart development and failure. DNA hydroxymethylation marks the body of highly expressed genes as well as distal regulatory regions with enhanced activity. Moreover, pathological hypertrophy is characterized by a shift towards a neonatal 5-hmC distribution pattern. We also show that the ten-eleven translocation 2 (TET2) enzyme regulates the expression of key cardiac genes, such as Myh7, through 5-hmC deposition on the gene body and at enhancers. Thus, we provide a genome-wide analysis of 5-hmC in the cardiomyocyte and suggest a role for this epigenetic modification in heart development and disease.