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OBJECTIVE: Current methods to determine chronological age from modern and ancient remains rely on both morphological and molecular approaches. However, low accuracy and the lack of standardized protocols make the development of alternative methods for the estimation of individual's age even more urgent for several research fields, such as biological anthropology, biodemography, forensics, evolutionary genetics, and ancient DNA studies. Therefore, the aim of this study is to identify genomic regions whose DNA methylation level correlates with age in modern teeth. METHODS: We used MALDI-TOF mass spectrometry to analyze DNA methylation levels of specific CpGs located in the ELOVL2, FHL2, and PENK genes. We considered methylation data from cementum, dentin and pulp of 21 modern teeth (from 17 to 77 years old) to construct a mathematical model able to exploit DNA methylation values to predict age of the individuals. RESULTS: The median difference between the real age and that estimated using DNA methylation values is 1.20 years (SD = 1.9) if DNA is recovered from both cementum and pulp of the same modern teeth, 2.25 years (SD = 2.5) if DNA is recovered from dental pulp, 2.45 years (SD = 3.3) if DNA is extracted from cementum and 7.07 years (SD = 7.0) when DNA is recovered from dentin only. DISCUSSION: We propose for the first time the evaluation of DNA methylation at ELOVL2, FHL2, and PENK genes as a powerful tool to predict age in modern teeth for anthropological applications. Future studies are needed to apply this method also to historical and relatively ancient human teeth.

Admixture-like plot displaying membership probabilities for NCWS and continental population clusters computed by DAPC. Probabilities belonging to the EAS, EUR, and AFR clusters are, respectively, displayed in green, red, and blue. (PDF 74Â kb)

Flowchart describing schematic representation of the implemented research approach detailing steps for NCWS sample selection and applied population genetics analytical workflow. (PDF 24Â kb)

Background: Although the pathogenesis of BCR-ABL1+ ALL is mainly related to the expression of BCR-ABL1, additional genetic lesions are supposed to be involved in its development and progression. Aim: In order to define the full repertoire of leukemia-related mutations, changes in expression profiles and alternative splicing (AS) events, the leukemia transcriptome of a BCR-ABL1+ ALL patient at diagnosis and relapse was sequenced using a Whole Transcriptome Sequencing (RNA-Seq) approach. Methods: Poly(A) RNA from blast cells was used to prepare cDNA libraries for Illumina/Solexa Genome Analyzer. Obtained sequence reads were mapped to the human genome reference sequence (UCSC hg18) to identify single nucleotide variants (SNVs). Reads that showed no match were mapped to a dataset of all possible splice junctions created in silico to identify AS events. The number of reads corresponding to RNA from known exons was also estimated and a normalized measure of gene expression level (RPKM) was computed. Results: RNA-seq analysis generated 13.9 and 15.8 million reads from de novo and relapsed ALL samples, respectively, detecting transcripts from 62% and 64% of human annotated genes. Low RPKM estimates (0.01

Next-Generation DNA sequencing data from the human intestinal microbiome provides new instruments to explore the correlation between changes in microbiome composition and the host physiologic status. Some studies show that newborns have a highly unstable microbiome composition, but few information are available in literature on the potential effect due to intrapartum antibiotic prophylaxis (IAP) administrated to the mothers positive to the infection by group B Streptococcus, which affects around 10% of pregnant woman. According to this, the aim of the project is to evaluate the main effects of the mentioned IAP on microbiome composition of newborns at seven days after birth, by a massive parallel sequencing of seven different regions of 16S gene. The analysis was performed with Ion PGM platform, taking advantage from “Ion 16s Metagenomics Sequencing Technology Access Program” (LifeTechnologies). Our first results show an evident difference in the microbial composition of treated newborns, resulting in a general decrease of microbial biodiversity and an over representation of Proteobacteria, suggesting that these infants are potentially more exposed to gastrointestinal diseases.

Environmental and cultural changes, such as those affecting dietary habits, are known to have substantially affected populations’ allele frequencies over human evolutionary history, triggering several adaptive processes. In some cases, especially since the Neolithic transition, they were established so rapidly to prevent adequate genetic adaptation to new nutritional/ecological conditions, thus potentially leading to various common chronic diseases. To detect genetic signatures of some of these evolutionary events and taking advantage from the 1000Genomes project dataset, we have analysed patterns of variation at a set of genes involved in nutritional and thermogenesis processes. Our preliminary results indicate PRDM16, a gene involved in the development of brown and beige adipocytes, as the most polymorphic locus within Europe. For instance, clusters and FST analyses pointed out completely different patterns between northern and southern European populations, suggesting the plausible action of climatic selective pressures on this gene. This study was conducted within the framework of the EPIC project (PRIN2012).

A string of biological and cultural changes strongly related to the occupation of new environmental contexts has characterized the evolution of the human lineage, especially during the widespread colonization of non-African continents by anatomically modern humans. Therefore, geographically and temporally defined variation of selective pressures acting on the genomes of both H. sapiens ancestors and modern populations triggered genetic adaptations in response to local environments. In particular, necessity to cope with unprecedented nutritional landscapes prompted a number of biological shifts in many human groups, mainly related to the introduction of considerable modifications of their diets and of new challenges to their metabolism [1,2], contributing to influence a variety of traits, among which differential disease susceptibility of human populations due to possible maladaptive processes [3]. In fact, the rate of dietary changes dramatically increased in the very recent evolutionary history of our species, as a consequence of introduction of agricultural and pastoralist practices, of consumption of industrial refined foods and of ever-increasing globalization [4], thus potentially converting some adaptive traits into maladaptive ones. In accordance to this view, increasing discordance between our anciently determined biology and contemporary nutritional or cultural patterns is supposed to have contributed to the spread of several complex diseases, which may represent by-products of past adaptive events due to loci that have become detrimental in modern societies [5]. To test this hypothesis, we have investigated the genetic legacy of natural selection on a panel of about one hundred genes representative of the main players driving functional pathways associated to food digestion and energy balance, or which turned out to be altered in metabolic and nutrition-related diseases. Sequence data for human populations living in different biomes were collected from public databases and used, in combination with unpublished data on individuals affected by certain nutrition-related diseases, to explore patterns of variation at more than 100,000 single nucleotide variants and/or indels. Population differentiation analyses based on the calculation of pairwise Fst indices among genetically homogeneous clusters of populations and on their comparison with genome-wide Fst distributions pointed out a few genes unusually differentiated with respect to average genomic patterns. Haplotype structure and potential deviations from a neutral model of evolution were then investigated at these candidate regions by applying several neutrality tests and by performing calibrated coalescent simulations, to disentangle the interplay between natural selection, demographic events and local mutation/recombination rates in shaping the observed patterns of variation. This enabled to elucidate implications of loci responsible for past adaptive events in the examined diseases, contributing to explain their underlying mechanisms and present-day epidemiological patterns, as well as some of the evolutionary processes that made our genome prone to the side effects of modern environmental stimuli.

The human mitochondrial DNA (mtDNA) phylogeny is an almost perfect molecular prototype for a non-recombining locus and knowledge on its variation has been extensively used in molecular anthropology and population genetics studies. Furthermore, its mode of inheritance, high mutation rate and high cellular copy number make this locus a primary choice also in the field of ancient DNA analysis, as well as in different contest such as biomedical research and diagnostic of mitochondrial disorders or human identification for forensic purposes. In the very last years, the study of mtDNA control region sequence and coding region single nucleotide polymorphisms have been gradually substituted by the investigation of variation at the whole mitochondrial genome, allowing analyses at a much higher resolution level and a deeper discrimination between haplogroups. In fact, high-throughput sequencing of whole mitochondrial genomes is now possible with lower costs with respect to those related to the traditional Sanger method thanks to Next Generation Sequencing (NGS) technologies. Among them, the Ion Personal Genome Machine (PGM, Life Technologies) is a new platform that uses an innovative method of sequencing. In fact, it does not use fluorescence or chemiluminescence, but measures the proton (H+) ions released during base incorporation. This new sequencing method allows to reduce times and costs of sequencing. The workflow, like other NGS, consists of four major steps: library construction, template preparation, sequencing and analysis. We develop a workflow for processing multiple samples in parallel through the preparation of barcoded libraries obtained by physical fragmentation of two long range PCR products with Bioruptor sonication system (Diagenode) at 200 base pairs reads. After equalization of single barcoded libraries in a pool the samples are subjected to an emulsion PCR reaction step. In this step, the library are attached to beads and amplified. During a step of enrichment only the beads that bind amplified template fragments are retrieved and these will be the template for the sequencing reaction. The sequencing technology is what really differentiates Ion Torrent platform; since optics are not required, the sequencing reaction is relatively fast and inexpensive. The obtained sequences are processed and aligned to the reference genome by means of the Ion Torrent suite and a dedicated bioinformatics tool was used to call single nucleotide and small insertion/deletion variants. We compared our results with those obtained by processing raw data via an ad hoc developed pipeline, as well as with those resulting from Sanger sequencing of the same samples to assess the reliability of the adopted protocol and sequencing technology.

Several dietary shifts have occurred in the human lineage accompanying transitions to high-quality diets, the adoption of meat eating, food processing and cooking, and facilitating the evolution of energetically expensive brains. Adaptations to new nutritional resources have thus strongly contributed to shape the genome of human ancestors and of H. sapiens, influencing a variety of traits, being often related to crucial biological/cultural innovations, and introducing novel challenges to human metabolism. The relatively recent human occupation of a vast range of environments, in which populations encountered ever new and demanding nutritional landscapes, further boosted the rate of these dietary changes, producing dramatic and still on-going modifications in human diets. Selective forces associated to food availability have thus varied over a geographical scale, driving adaptations in response to local environmental/cultural pressures, the latter having played a key role in the evolution of human diets, mainly through plants and animals domestication. Accordingly, adaptive genetic variants have been plausibly selected in human groups exposed to novel dietary environments during the last 60,000 years, even though cultural/dietary changes are ever-accelerating since the Neolithic Transition, leading to possible maladaptation in contemporary populations. Exploring the genetic bases of nutrition-related adaptations will thus enable to shed light not only on some of the main mechanisms that have shaped the human genome, but also on those contributing to common metabolic diseases affecting present-day societies. To detect signatures of these adaptations, genetic variation at more than 40 nutrition-related genes selected according to their major role in functional pathways associated to processing and digestion of food, or associated with metabolic diseases, was investigated in 14 populations from the phase I of the 1000 Genomes project. For this purpose, information about several thousands of SNPs was retrieved from sequence data and used to assess derived allele frequency, differentiation among the examined groups and genetic structure at the investigated loci. Different neutrality tests were used to shortlist the best candidate genes potentially responsible for moderate to strong patterns of dietary adaptation in the studied populations. Further understanding of the mechanisms of action of these genes promises to give new insight into the recent history of human adaptation and its implication on the epidemiology of metabolic diseases.

Environmental changes, as well as evolution in cultural habits, are able to substantially affect populations’ allele frequencies: the well know case of selection on lactase persistence (LP) provides a good example for this assumption. Indeed, the process of dairy animal domestication seems to have positively selected different polymorphisms around the LCT gene, which are associated to LP[Enattah et al. 2002]. Several changes in dietary habits took place over human history since the introduction of meat consumption, the Neolithic transition, and the recent shift to an high caloric diet which led to a reversal of our dietary habits [Lindeberg 2012]. These new cultural habits have been established in such a recent time that it didn’t allow genomic adaptation to the new nutritional conditions. The mismatch between how our genome has adapted and the new dietary conditions is potentially at the basis of various common chronic diseases such as obesity, cardiovascular diseases, diabetes and cancer [Konner and Eaton 2010]. The interaction between the genome and the nutritional environment is obviously regulated not only by nutrition-related genes, but even by genes which modulate the perception of taste and thermoregulation, which rebalances the relation between temperature and the needed energy resources. According to these evidences, the aim of this study is to analyze, taking advantage from the 1000Genomes project phase I dataset, patterns of genetic variation at a set of genes, which have been selected for their involvement in nutritional processes, taste perception and thermogenesis, and to detect possible signs of selection at the investigated genomic regions. This in silico approach was used to detect the most interesting variants on the basis of their highest heterozygosity values among the examined European sub-populations. These results will subsequently drive experimental analyses of the most polymorphic genes on a Central-Southern Italian sample, collected according to a sampling strategy that takes into account the complex geographical and historical Italian background, as scheduled within the research activities of the EPIC project (PRIN2012).

Introduction. Mutations, chromosomal rearrangements and associated gene fusions resulting from inversions, interstitial deletion or translocations represent recurrent findings in leukemia. Nowadays high-throughput “Next Generation Sequencing” technologies and novel developed algorithmic methods allow a unified deep analysis of such alterations with a single base resolution. Taking advantage from these tools, we performed an RNA-Seq (whole transcriptome shotgun sequencing) approach using the Illumina/Solexa platform to define in a single procedure and at high sensitivity the full repertoire of leukemia-related mutations and fusion genes in 3 adult BCR-ABL1+ ALL cases treated with tyrosine kinase inhibitors. Methods. Patient median age was 55.3 years (range, 40-70); no additional chromosome abnormalities were detected except for one case with del(9)(p13p22). All the selected cases had previously been profiled by high resolution SNP (Affymetrix SNP6.0) and gene expression (Affymetrix Human Exon 1ST Array) arrays, as well as candidate gene re-sequencing (IKZF1, PAX5, JAK2, CDKN2A, CDKN2B, IDH1, IDH2), lacking missense point mutations. Two cases harbored the deletion of IKZF1, and in one case PAX5 and CDKN2A/B losses were also found. Poly(A) RNA from blast cells was used to prepare Illumina cDNA libraries according to the manufacturer’s recommendations. Sequencing by synthesis was performed on an Illumina Genome Analyzer IIx platform, with standard sequencing kits and nucleotide incorporation cycles, generating 75 base pairs (bp) paired end sequence reads. Results. A total of 57, 51 and 9 million reads were obtained from the 3 samples and high quality sequence reads were mapped to the reference sequence of the human genome (UCSC hg19) using the Maq software, finding out 58,205, 48,913 and 136,937 putative new single nucleotide variants (SNVs) in the CDS/EXON regions not reported in the dbSNP build 130. Of these, 874 distributed on 290 genes, affected both samples. Thereafter, we analyzed RNA-seq data by deFuse (McPherson A et al. PLoS Computational Biology May 2011), a novel computational method for fusion discovery between a gene and an intergenic region. This software uses clusters of discordant paired-end alignments to inform a split read alignment analysis for finding fusion boundaries. First of all, we evaluated the ability of deFuse to rediscovery the BCR-ABL1 gene fusion, then we focused on the most intriguing fusions such as the PBXIP1- PMVK (pre-B-cell leukemia homeobox interacting protein 1- phosphomevalonate kinase) gene fusion. All new gene fusions are under evaluation in a bigger ALL cohort. Conclusions. This study provided a comprehensive overview of a BCR-ABL1+ ALL transcriptome, identifying novel mutations and gene fusions involved in Ph+ ALL. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna- Ravenna, FIRB 2006, Ateneo RFO grants, PIO project, Programma di Ricerca Regione–Università 2007–2009.

Neandertal and Denisovan clades of archaic hominins experienced temperatures significantly lower than the present ones, occupying geographical regions involved in the Pleni-Wurm for a time interval long enough to adapt to this cold environment. Paleoanthropological evidences prove that Neandertals body proportions are in accordance with a minimization of the ratio between body surface area and volume to avoid heat loss. Their supposed massive muscular apparatus and hunter-gatherer subsistence strategies also suggest that their life-style was similar to H. sapiens populations living in circumpolar regions, being characterized by a diet rich in fat and proteins to satisfy high calorie needs. This may have favored the isolation from cold and the thermogenic functions in these archaic hominins. Moreover, since the advent of new subsistence strategies and the increase of environmental temperature during recent human evolution may have been too fast for completely retuning the basic processes of thermogenesis and thermoregulation in our species, even slight differences in their regulation between H. sapiens and Neandertals/Denisovans could be the key between our way to store and spend energy and that of our ancestors. Thermogenesis and thermoregulation consist in complex functional patterns centered on the dissipation of heat in the mitochondria by uncoupling oxidation and ATP synthesis and are mainly provided by adipocytes belonging to the Brown Adipose Tissue (BAT). In this preliminary work, we investigated the genetic similarities between 27 Neandertal, Denisovan and modern human genes involved in BAT metabolism, storage and neogenesis, or being found to be associated with fat accumulation and body mass index by GWAS studies. Archaic sequence data were retrieved from public databases, together with those from modern humans belonging to the “1000Genomes” project populations, and exploited to identify Neandertal/Denisovan specific variants and to explore whether they exhibit genetic similarities with respect to modern populations exposed to cold environments.

Western and eastern Eurasian populations of Neandertal and Denisovan sister clades of archaic hominins have basically experienced temperatures significantly lower than the present ones, also occupying geographical regions involved in the Pleni-Wurm for a time interval long enough to adapt to this cold environment. In fact, paleoanthropological evidences prove that Neandertal body proportions are in accordance with a minimization of the ratio between body surface area and volume in order to avoid heat loss, as regards both their voluminous thorax and their relatively short length of limbs that is found to be significantly lower even with respect to that of modern humans belonging to cold adapted populations. Moreover, Neandertal supposed massive muscular apparatus and hunter-gatherer subsistence strategies suggest that their life-style was similar to H. sapiens populations living in circumpolar regions, being mainly characterized by a diet rich in fat and proteins, thus able to satisfy a calorie needs ranging from 4,000 to 7,000 kcal/day. All these features, together with a high basal metabolic rate, may have favored the isolation from cold and the thermogenic functions in these archaic hominins. Furthermore, it can be hypothesized that the advent of new subsistence strategies and the increase of environmental temperature during recent human evolution may have been too fast for completely retuning the basic processes of thermogenesis and thermoregulation in our species, so that even slight differences in their regulation between H. sapiens and Neandertals/Denisovans could be the key between our way to store and spend energy and that of our ancestors. Thermogenesis and thermoregulation consist in complex functional patterns centered on the dissipation of heat in the mitochondria by uncoupling oxidation and ATP synthesis and are mainly provided by adipocytes belonging to the Brown Adipose Tissue (BAT), which contain a great amount of mitochondria and which differentiate from precursors of muscle cells. In this exploratory work, we investigated the genetic similarities/differences between 27 Neandertal, Denisovan and modern human genes involved in BAT metabolism, storage and neogenesis, or being found to be associated with fat accumulation and body mass index according to GWAS studies. Neandertal and Denisovan sequence data were thus retrieved from public databases, together with those from 1,122 modern humans belonging to 14 populations sequenced in the pilot phase of the “1000Genomes” project, and exploited to identify new Neandertal/Denisova-specific variants, as well as to explore whether the examined archaic samples exhibit genetic similarities, in the genomic regions under investigation, to individuals from modern human populations exposed to cold environments.

The enzyme that hydrolyzes lactose, the main carbohydrate in milk, is lactase-phlorizin hydrolase (LCT), which is encoded by the gene LCT located on chromosome 2. Normally, in all mammals, the ability to digest lactose rapidly declines after weaning, but in some human populations this ability persists in adulthood, a condition called lactase persistence (LP) (Swallow, 2003). Studies of the prevalence of the LP phenotype in worldwide populations have shown that the frequency of this trait is highly variable in different ethnic groups and appears to be positively correlated with the importance of the milk in the diet (Ingram et al., 2009). Several polymorphisms in the proximity of the LCT gene have been proved to be associated with LP phenotype. The first and the most important polymorphism was identified by Enattah et al. (2002), being located 13,910 base pairs 5’ of the start codon of LCT (-13,910*C/T) and being completely associated with LP in individuals of Finnish origin. In 2004, Bersaglieri et al. typed 101 single-nucleotide polymorphisms (SNPs) covering 3.2 Mb around the lactase gene. They demonstrated the presence of a common haplotype that extends largely undisrupted for >1 Mb in populations of northern European origins, confirming that this region of the human genome was recently subjected to a strong positive selection (Bersaglieri et al. 2004). According to these evidences, the aim of the project is to analyze the frequency spectrum of a panel of 51 SNPs selected on the basis of their highest heterozigosity values among those observed in the European subsample analyzed in Bersaglieri’s work. The sample is composed by 380 Italian healthy subjects distributed in six different macro-areas: North- Eastern, North-Western, Central-Eastern, Central-Western and Southern Italy, as well as Sicily and Sardinia. The project aims to be the first work that analyzes a so large numbers of SNPs potentially related to LP phenotype, in a wide and very wellcharacterized sample of Italian subjects. Genotyping on each study participant will be performed using the iPLEX Gold technology (Jurinke et al. 2002) and the MassARRAY DNA analysis with MALDI-TOF mass spectrometry (Sequenom, Inc., San Diego, CA). Preliminary results show a high variability in Italy with a long region of linkage disequilibrium around the - 13910*C/T variant in the North Italian subsamples.

Sicily, due to its strategic geographical position, located in the heart of Mediterranean Basin, has received the passage of various human groups both in pre-historical and historical times, acting as a bridge in different major migratory events. In addition to the Upper Paleolithic and Mesolithic cultures, several migrations have affected the genetic composition of the island after the Neolithic transition (Greeks, Phoenicians, Romans, Arabs and Normans), thus contributing to complicate the genetic landscape of the largest Mediterranean island. Made up of different superimposed historical genetic layers, Sicily offers a unique opportunity to test hypotheses connected with the peopling of Mediterranean shores. Both the presence (Romano et al. 2003, Di Gaetano et al. 2008) and absence (Rickards et al. 1998) of an internal genetic differentiation along the east-west axis has been previously described in Sicily, and several hypotheses, connected with the aforementioned dynamics of peopling, have been proposed to explain the heterogeneity observed. Nevertheless the extent of each independent historical contributions to the current genetic composition of Sicilian population is still highly debated. The present study aims to contribute to this debate using new samples and deeper analyzing the Y chromosome genetic markers. More precisely, a total of 24 Y-SNPs and 17 Y-STRs have been typed in more than 300 individuals from 8 different provinces, 5 in Sicily (Agrigento, Catania, Ragusa-Siracusa, Enna, Trapani) and 3 in Southern Italy (Matera, Lecce, Cosenza). Y-chromosome preliminary results reveal a substantial lack of genetic structure within the island, coupled with high levels of within-population genetic variability. These findings well agree with Sicily's historic role as major migration crossroads. Comparing our results with other Mediterranean populations (Southern Europe, Northern Africa and the Levant) we observe a marked geography-related global structure within the Mediterranean Basin. Two well separated clusters have been identified, suggesting a shared genetic background between north-western Mediterranean populations (Iberian Peninsula and Northern Italy) on one side, and south-eastern ones (Sicily and Southern Italy, Balkan Peninsula, North Africa and Levant) on the other side. The completion of Y-chromosome analysis, as well as the future study of mtDNA variation, will consent to explore more in detail specific issues related to the peopling of Sicily and to clarify its role in Mediterranean genetic context.

Introduction: Little is known about alterations of cyclin dependent kinase inhibitors p15INK4B, p16INK4A and p14ARF due to single nucleotide polymorphisms (SNPs) located within the CDKN2A/B genes and/or neighbouring loci. In order to investigate the potential involvement of such common DNA sequence variants in leukemia susceptibility, an association study was performed. Methods: 23 SNPs spanning the MTAP, CDKN2A/B and CDKN2BAS loci, as well as relative intergenic regions were genotyped in a case-control cohort made up of 149 leukemia patients, including Philadelphia positive (Ph+) ALL and acute myeloid leukemia (AML) samples, and 183 healthy controls. 6 SNPs were selected on the basis of their previous association with several diseases, such as coronary artery disease (rs2891168, rs518394, rs564398, rs10757278), type 2 diabetes mellitus (rs564398), frailty (rs2811712). The remaining 17 SNPs were selected to deepen the SNPs coverage for the examined region. Genotyping was performed using iPLEX Gold technology and MassARRAY high-throughput DNA analysis with Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Sequenom, Inc., San Diego, CA). Results: A total of 17 SNPs, spanning the 9p genomic interval that encompasses the MTAP, CDKN2A/B and CDKN2BAS loci, were successfully genotyped and used for investigating their potential associations with the leukemia phenotypes. Five SNPs (rs1012713, rs10965179, rs34011899, rs3731232, rs3218010) with MAF 20% missing call rates, were instead excluded from the association analysis and potential population stratification affecting the control sample was ruled out as its genotypes distribution satisfies the Hardy-Weinberg equilibrium criterion. Among the 17 SNPs, rs564398, mapping to the CDKN2BAS locus (exon 2) that encodes for ANRIL antisense non-coding RNA, showed a statistically significant correlation with the ALL phenotype, with a risk pattern that was compatible with an overdominant model of disease susceptibility and a OR of 2 (95% CI, 1.20 to 3.33; p= 7.1 x 10-3). Conclusions: Since a co-ordinated regulation of ANRIL and p14/ARF, p16/CDKN2A, p15/CDKN2B transcription has been already observed in both physiologic and pathologic conditions, we hypothesized that rs564398 association reflects a condition of high linkage disequilibrium between such polymorphism and a causative variant that is able to alter CDKN2A/B expression profiles by changing ANRIL dosage, thus leading to abnormal proliferative boosts and consequent increased ALL susceptibility. Recently, the rs564398 has been demonstrated by Cunnington M.S. et al. (2010) to alter ANRIL expression leading in turn to a deregulation of CDKN2A/B genes. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, Ateneo RFO grants, Project of integrated program (PIO), PRIN 2008, Programma di Ricerca Regione – Università 2007 – 2009.

Background: Genetic heterogeneity in drug-metabolizing enzyme and transporter genes affects specific drug-related phenotypes in cancer. In order to investigate a relationship between genetic variation and response in AML, we comprehensively assessed the allele frequencies of 1936 genetic variations of 225 absorption, distribution, metabolism and excretion genes in 94 CD33-positive AML patients younger than 65 years, using the new Affymetrix drug-metabolizing enzyme and transport (DMET Plus) genotyping platform. Patients and methods: All patients were enrolled in a phase III multicenter clinical trial combining low dose of Gemtuzumab-ozogamicin (GO) with FLAI regimen (Fludarabine, Cytarabine, Idarubicin) as Induction chemotherapy (ClinicalTrials.gov NCT00909168). Cytogenetics, multidrug-resistance phenotype, FLT3 and NPM mutation status, as well as WT1 quantitative expression analyses were performed at diagnosis. Furthermore, high-resolution single nucleotide polymorphism (SNP) array and DMET genotyping analyses (Affymetrix) were also performed. Results: Of the 94 patients, genotype results were evaluable for 91 cases. The median call rate was 99.48 (range, 96.32-100). Firstly, we tested the association among SNPs and response to the induction cycle (FLAI + GO) comparing the genotyping profile of 80 patients in complete (85%) and partial (3%) remission with that of patients (12%) with no response. A highly significant difference (p < 0.001) in the allele frequency of 2 variants, in complete linkage disequilibrium, in the alcohol dehydrogenase enzyme (ADH1A) was found. ADH1A metabolizes the conversion of ethanol to acetaldehyde which is thereafter converted to acetate by ALDH1, which is well known as stem cell marker. These variants were not associated with high risk AML, FLT3 and NPM1 mutations, but strongly influenced response to the induction phase also in a multivariate analysis. Secondly, SNPs were stratified according to liver toxicity and a significant difference in the allele frequency in CYP2E1, involved in the alcohol metabolism was found to be associated with a grade I/II liver toxicity. Conclusions: Genetic variations in ADH1A and CYP2E1 genes were for the first time identified and associated with response and toxicity in AML patients treated with a combination of GO and FLAI regimen. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione – Università 2007–2009.

Background: In acute myeloid leukemia (AML) the presence or absence of cytogenetic abnormalities allows the identification of favorable, intermediate and unfavorable subgroups. However, besides these specific subgroups, little it is known about the genetic variations influencing specific drug-related phenotypes. Aims: To perform an exploratory pharmacogenomic study that relates genetic variations in multidrug enzymes and transporters genes with the efficacy and toxicity to treatment. Patients and methods: We analyzed 94 CD33-positive AML patients younger than 65 previously untreated and enrolled in a phase III multicenter clinical trial combining low dose of Gemtuzumab-ozogamicin (GO) with FLAI regimen (Fludarabine, Cytarabine, Idarubicin) as Induction chemotherapy (eudract: 2007-005248-26; ClinicalTrials.gov NCT00909168). The induction regimen (GO-FLAI) included fludarabine (25 mg/sqm) and Ara-C (2 g/sqm) on days 1-5, idarubicin (10 mg/sqm) on days 1, 3, and 5 and GO (3 mg/sqm) on day 6. Hematopoietic stem cell transplant was planned for all high risk AML patients in first complete remission (CR) after consolidation with intermediate doses of Ara-C and idarubicin. Cytogenetics, multidrug-resistance phenotype, FLT3 and NPM mutation status, as well as WT1 quantitative expression analyses were performed at diagnosis in all patients. Furthermore, high-resolution single nucleotide polymorphism (SNP) array analysis (Affymetrix, Inc. Santa Clara, CA, USA) was also performed. The allele frequencies of 1936 genetic variations of 225 absorption, distribution, metabolism and excretion were assessed using the new Affymetrix drug-metabolizing enzyme and transport (DMET Plus) genotyping platform (Affymetrix). All statistical analyses were performed using the R package 2.11.1. Results: Of the 94 patients, genotype results were evaluable for 91 cases. The median call rate was 99.48 (range, 96.32-100). Three samples were run in duplicate and results with “passed call rate” were compared across all the polymorphic sites, showing a repeatability of 99.99%. In an initial screening procedure, we tested the association among SNPs and response to the induction cycle (FLAI + Gemtuzumab-Ozogamicin). Therefore, the genotyping profile of 80 patients in complete (85%) and partial (3%) remission was compared to that of patients (12%) with no response. We found a highly significant difference (p < 0.001) in the allele frequency of 2 variants, in complete linkage disequilibrium, in the alcohol dehydrogenase enzyme (ADH1A). These variants were not associated with high risk AML, FLT3 and NPM1 mutations, but strongly influenced response to the induction phase also in a multivariate analysis. Since genetic polymorphisms may influence the toxicity of chemotherapy drugs, we stratified SNPs according to liver toxicity and a significant difference in the allele frequency of a member of the cytochrome P450 family which is involved in the alcohol metabolism (CYP2E1) was found to be associated with a grade I/II liver toxicity. Conclusions: A pharmacogenomic panel made up of 1 gene (ADH1A) associated with clinical outcome and 1 gene (CYP2E1) associated with toxicity was for the first time identified in AML patients younger than 65 years treated with a combination of GO and FLAI regimen. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, Ateneo RFO grants, Project of integrated program, PRIN 2008, Programma di Ricerca Regione – Università 2007 – 2009.

Nowadays high throughput “Next Generation Sequencing” technologies are revolutionizing genomics and transcriptomics by providing a cost-efficient and single base resolution tool for a unified deep analysis of tumor complexity. Taking advantage from this tool, we used the Illumina/Solexa platform to define in a single procedure and at high sensitivity the full repertoire of leukemia-related mutations in 3 adult BCRABL1+ ALL cases treated with tyrosine kinase inhibitors. Patient median age was 55.3 years (range, 40-70); no additional chromosome abnormalities were detected except for one case. All the selected cases had previously been profiled by high resolution SNP (Affymetrix SNP6.0) and gene expression (Affymetrix Human Exon 1ST Array) arrays, as well as candidate gene re-sequencing (IKZF1, PAX5, JAK2, CDKN2A, CDKN2B, IDH1, IDH2), lacking missense point mutations. Two cases harbored the deletion of IKZF1, and in one case PAX5 and CDKN2A/B losses were also found. Poly(A) RNA from blast cells was used to prepare Illumina cDNA libraries according to the manufacturer’s recommendations. Sequencing by synthesis was performed on an Illumina Genome Analyzer IIx platform, with standard sequencing kits and nucleotide incorporation cycles, generating 75 base pairs (bp) paired end sequence reads. A total of 57, 51 and 9 million reads were obtained from the 3 samples and high quality sequence reads were mapped to the reference sequence of the human genome (UCSC hg19) using the Maq software, finding out 58,205, 48,913 and 136,937 putative new single nucleotide variants (SNVs) in the CDS/EXON regions not reported in the dbSNP build 130. Of these, 874 distributed on 290 genes, affected both samples. A functional analysis was carried out on common mutated genes using the GeneGo software (www.genego.com) and in the list of the most significant GeneGo Pathway Maps we found the “Development Hedgehog Signaling” (p value = 1.400e-4), including genes such as casein kinase 1, alpha 1-like (CSNK1A1L), catenin (cadherin-associated protein) beta 1 (CTNNB1) and heat shock protein HSP 90-beta (HSP90AB1). In conclusion, this study provided, for the first time, a comprehensive overview of a BCR-ABL1+ ALL transcriptome, identifying novel mutations potentially involved in ALL. Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione–Università 2007–2009.

Background: Inter-individual variations in genes encoding drug metabolizing enzymes and transporters have been demonstrated to influence the response to therapy. However so far, how these genetic variations interact to produce specific drug related phenotypes in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) has not yet been investigated. Aim: In order to investigate potential genetic structure and related pharmacogenetic profiles, around 2000 variants in more than 200 genes involved in drug absorption, distribution, metabolism and elimination were genotyped and studied with a population genetics approach in 45 Ph+ ALL patients. Methods: The Drug Metabolizing Enzymes and Transporters (DMET™, Affymetrix) platform, covering more than 90% of the most biologically relevant drug absorption, distribution, metabolism and excretion (ADME) markers was used for successfully genotyping 1931 variants in Ph+ ALL patients treated with the tyrosine kinase inhibitor Dasatinib. A model-based clustering method for inferring population structure using genotype data was applied by means of the Structure software assuming a model in which there are K populations - each one of them being characterized by a set of allele frequencies at each locus - to which individuals are probabilistically assigned according to their genotypes. Distribution of the genetic variance observed among the identified leukemia sub-groups was investigated with a locus by locus Analysis of the Molecular Variance (AMOVA) by means of the Arlequin 3.01 package, exploiting information on genotypes allelic content and frequencies. Results: Three different sub-groups (G1, G2, G3), made up of 2, 12 and 31 patients respectively, were identified in the examined ALL sample, according to their different patterns of allele frequency. A statistical support for this finding was provided by AMOVA results which pointed out a substantial level of genetic differentiation among G1 and the other two sub-groups (Fst = 0.099, p0.3) and significant Fst values; whereas a total of 50 loci, located on the NAT2, VKORC1, CYP4F2, CYP2B6, UGT2B7 and CYP2D6 genes, showed moderate to high (>0.08) significant Fst values in the G2/G3 comparison. Conclusions: Differences of allele frequencies observed among the identified ALL sub-groups prove that an evident genetic structure is detectable in our sample by genotyping loci involved in drug metabolism. Supported by: Fondazione GIMEMA Onlus, European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009.

Introduction. Little is known about alterations of cyclin dependent kinase inhibitors p15INK4B, p16INK4A and p14ARF due to single nucleotide polymorphisms (SNPs) located within the CDKN2A/B genes and/or neighbouring loci. In order to investigate the potential involvement of such common DNA sequence variants in leukemia susceptibility, an association study was performed. Methods. 23 SNPs spanning the MTAP, CDKN2A/Band CDKN2BAS loci, as well as relative intergenic regions were genotyped in a case-control cohort made up of 149 leukemia patients, including Philadelphia positive (Ph+) acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) samples, and 183 healthy controls. 6 SNPs were selected on the basis of their previous association with several diseases, such as coronary artery disease (rs2891168, rs518394, rs564398, rs10757278), type 2 diabetes mellitus (rs564398), frailty (rs2811712). The remaining 17 SNPs were selected to deepen the SNPs coverage for the examined region. Genotyping was performed using iPLEX Gold technology and MassARRAY high-throughput DNA analysis with Matrix-assisted laser desorption/ionization timeof-flight (MALDI-TOF) mass spectrometry (Sequenom, Inc., San Diego, CA). Results. A total of 17 SNPs, spanning the 9p genomic interval that encompasses the MTAP, CDKN2A/B and CDKN2BAS loci, were successfully genotyped and used for investigating their potential associations with the leukemia phenotypes. Five SNPs (rs1012713, rs10965179, rs34011899, rs3731232, rs3218010) with MAF 20% missing call rates, were instead excluded from the association analysis and potential population stratification affecting the control sample was ruled out as its genotypes distribution satisfies the Hardy-Weinberg equilibrium criterion. Among the 17 SNPs, rs564398, mapping to the CDKN2BAS locus that encodes for ANRIL antisense non-coding RNA, showed a statistically significant correlation with the ALL phenotype, with a risk pattern that was compatible with an overdominant model of disease susceptibility and a OR of 2 (95% CI, 1.20 to 3.33; P=7.1¥103). Conclusions. Since a co-ordinated regulation of ANRIL and p14/ARF, p16/CDKN2A, p15/CDKN2B transcription has been already observed in both physiologic and pathologic conditions, we hypothesized that rs564398 association reflects a condition of high linkage disequilibrium between such polymorphism and a causative variant that is able to alter CDKN2A/B expression profiles by changing ANRIL dosage, thus leading to abnormal proliferative boosts and consequent increased ALL susceptibility. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione - Università 2007-2009.

Introduction. The BCR-ABL1+ ALL is the most frequent and prognostically unfavorable subtype of ALL in adults. In order to define the full repertoire of leukemia-related mutations, changes in expression profiles and alternative splicing (AS) events, the leukemia transcriptome of a BCR-ABL1+ ALL patient at diagnosis and relapse was sequenced using a Whole Transcriptome Sequencing (RNA-Seq) approach. The selected cases had previously been profiled by high-resolution SNP and gene expression arrays and candidate gene re-sequencing. Methods. Poly(A) RNA from blast cells was used to prepare cDNA libraries for Illumina/Solexa Genome Analyzer. Obtained sequence reads were mapped to the human genome reference sequence (UCSC hg18) to identify single nucleotide variants (SNVs). Reads that showed no match were mapped to a dataset of all possible splice junctions created in silico to identify AS events. The number of reads corresponding to RNA from known exons was also estimated and a normalized measure of gene expression level (RPKM) was computed. Results. RNA-seq generated 13.9 and 15.8 million reads from de novo and relapsed ALL samples, most of which successfully mapped to the reference sequence of the human genome. With the exclusion of the T315I BCR-ABL mutation, 7 novel missense mutations were detected after applying stringent criteria to reduce the SNV discovery false positive rate: 4 were exclusively found in the primary ALL sample and affected genes involved in metabolic processes (DPEP1, ZC3H12D, TMEM46) or transport (MVP); 3 relapse-related mutations affected genes involved in cell cycle regulation (CDC2L1) and catalytic activity (CTSZ, CXorf21). Differences in mutational patterns suggest that the leukemia clone from which relapsed cells have been developed was not the predominant one at diagnosis and that relapse specific variants were mutations probably acquired during progression. Moreover, 4,334 and 3,651 primary and relapse isoforms with at least one AS event were identified. An average of 1.5 and 1.3 AS per isoform was estimated. Finally, a detailed gene expression profile was obtained indicating that more than 60% of annotated human genes were transcribed in leukemia cells in both diagnosis and relapse phases. Approximately 23% of genes were up-regulated at relapse compared to diagnosis, and most of them affected cell cycle progression (AURORA A, SURVIVIN, PLK1, CDK1, Cyclin A, Cyclin B), suggesting that the loss of cell cycle control may play a role in disease progression. Conversely, only 9% of active genes in both samples were down-regulated at relapse compared to diagnosis. Conclusions. Discovery of novel missense mutations, as well as exhaustive alternative splicing and gene expression profiles were achieved for the first time for a BCR-ABL1+ positive ALL demonstrating that RNA-Seq is a suitable approach for identifying a wide spectrum of genetic alterations. Supported by AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna,FIRB 2006, PRIN 2008, European LeukemiaNet, GIMEMA ONLUS, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione - Università 2007-2009.

In the recent years TNFRSF13B coding variants have been implicated by clinical genetics studies in Common Variable Immunodeficiency (CVID), the most common clinically relevant primary immunodeficiency in individuals of European ancestry, but their functional effects in relation to the development of the disease have not been entirely established. To examine the potential contribution of such variants to CVID, the more comprehensive perspective of an evolutionary approach was applied in this study, underling the belief that evolutionary genetics methods can play a role in dissecting the origin, causes and diffusion of human diseases, representing a powerful tool also in human health research. For this purpose, TNFRSF13B coding region was sequenced in 451 healthy individuals belonging to 26 worldwide populations, in addition to 96 control, 77 CVID and 38 Selective IgA Deficiency (IgAD) individuals from Italy, leading to the first achievement of a global picture of TNFRSF13B nucleotide diversity and haplotype structure and making suggestion of its evolutionary history possible. A slow rate of evolution, within our species and when compared to the chimpanzee, low levels of genetic diversity geographical structure and the absence of recent population specific selective pressures were observed for the examined genomic region, suggesting that geographical distribution of its variability is more plausibly related to its involvement also in innate immunity rather than in adaptive immunity only. This, together with the extremely subtle disease/healthy samples differences observed, suggests that CVID might be more likely related to still unknown environmental and genetic factors, rather than to the nature of TNFRSF13B variants only.

Additional file 1 : Figure S1. Procrustes analysis projecting genomic information summarized by first and second principal components onto geographic coordinates of Italian population samples. Figure S2. Decay of the length of chromosome chunks inherited by Italian population clusters from possible pairs of parental groups calculated with the GLOBETROTTER pipeline. Figure S3. PCA projecting variation of 559 ancient samples onto the genetic space defined by 239 individuals belonging to 40 modern Euro-Mediterranean populations. Figure S4. Outgroup f3 biplot comparing shared genetic drift between the N_ITA and S_ITA population clusters and, in turn, all ancient population groups included in the “modern + aDNA dataset”. Figure S5. Representation of the Insulin secretion pathway and of its components subjected to positive selection in the Italian population. Figure S6. Representation of the Mucin type O-glycan biosynthesis pathway and of its components subjected to positive selection in the S_ITA cluster. Figure S7. Representation of the Basal cell carcinoma pathway and of its components subjected to positive selection in the S_ITA cluster. Table S1. Admixture proportions inferred for N_ITA and S_ITA population clusters with the GLOBETROTTER method. Table S2. Admixture dates inferred for N_ITA and S_ITA population clusters with the GLOBETROTTER method. Table S3. Gene networks showing significant signatures of positive selection according to signet analysis performed on the obtained genome-wide distribution of DIND scores. Table S4. Gene networks showing significant signatures of positive selection according to signet analysis performed on the obtained genome-wide distribution of nSL scores. Table S5. Gene networks showing significant signatures of balancing selection according to signet analysis performed on the obtained genome-wide distribution of BALLET scores. Supplementary Results.

Additional file 1 : Figure S1. Procrustes analysis projecting genomic information summarized by first and second principal components onto geographic coordinates of Italian population samples. Figure S2. Decay of the length of chromosome chunks inherited by Italian population clusters from possible pairs of parental groups calculated with the GLOBETROTTER pipeline. Figure S3. PCA projecting variation of 559 ancient samples onto the genetic space defined by 239 individuals belonging to 40 modern Euro-Mediterranean populations. Figure S4. Outgroup f3 biplot comparing shared genetic drift between the N_ITA and S_ITA population clusters and, in turn, all ancient population groups included in the “modern + aDNA dataset”. Figure S5. Representation of the Insulin secretion pathway and of its components subjected to positive selection in the Italian population. Figure S6. Representation of the Mucin type O-glycan biosynthesis pathway and of its components subjected to positive selection in the S_ITA cluster. Figure S7. Representation of the Basal cell carcinoma pathway and of its components subjected to positive selection in the S_ITA cluster. Table S1. Admixture proportions inferred for N_ITA and S_ITA population clusters with the GLOBETROTTER method. Table S2. Admixture dates inferred for N_ITA and S_ITA population clusters with the GLOBETROTTER method. Table S3. Gene networks showing significant signatures of positive selection according to signet analysis performed on the obtained genome-wide distribution of DIND scores. Table S4. Gene networks showing significant signatures of positive selection according to signet analysis performed on the obtained genome-wide distribution of nSL scores. Table S5. Gene networks showing significant signatures of balancing selection according to signet analysis performed on the obtained genome-wide distribution of BALLET scores. Supplementary Results.

reserved 19 no Objectives: The Yaghnobis are an ethno-linguistic minority historically settled along the Yaghnob River in the Upper-Zarafshan Valley in Tajikistan. They speak a language of Old Sogdian origin, which is the only present-day witness of the Lingua Franca used along the Silk Road in Late Antiquity. The aim of this study was to reconstruct the genetic history of this community in order to shed light on its isolation and genetic ancestry within the Euro-Asiatic context. Materials and Methods: A total of 100 DNA samples were collected in the Yaghnob and Matcha Valleys during several expeditions and their mitochondrial, Y-chromosome and autosomal genome-wide variation were compared with that from a large set of modern and ancient Euro-Asiatic samples. Results: Findings from uniparental markers highlighted the long-term isolation of the Yaghnobis. Mitochondrial DNA ancestry traced an ancient link with Middle Eastern populations, whereas Y-chromosome legacy showed more tight relationships with Central Asians. Admixture, outgroup-f3, and D-statistics computed on autosomal variation corroborated Y-chromosome evidence, pointing respectively to low Anatolian Neolithic and high Steppe ancestry proportions in Yaghnobis, and to their closer affinity with Tajiks than to Iranians. Discussion: Although the Yaghnobis do not show evident signs of recent admixture, they could be considered a modern proxy for the source of gene flow for many Central Asian and Middle Eastern groups. Accordingly, they seem to retain a peculiar genomic ancestry probably ascribable to an ancient gene pool originally wide spread across a vast area and subsequently reshuffled by distinct demographic events occurred in Middle East and Central Asia. mixed Cilli, Elisabetta*; Sarno, Stefania; Gnecchi Ruscone, Guido Alberto; Serventi, Patrizia; De Fanti, Sara; Delaini, Paolo; Ognibene, Paolo; Basello, Gian Pietro; Ravegnini, Gloria; Angelini, Sabrina; Ferri, Gianmarco; Gentilini, Davide; Di Blasio, Anna Maria; Pelotti, Susi; Pettener, Davide; Sazzini, Marco; Panaino, Antonio; Luiselli, Donata; Gruppioni, Giorgio Cilli, Elisabetta*; Sarno, Stefania; Gnecchi Ruscone, Guido Alberto; Serventi, Patrizia; De Fanti, Sara; Delaini, Paolo; Ognibene, Paolo; Basello, Gian Pietro; Ravegnini, Gloria; Angelini, Sabrina; Ferri, Gianmarco; Gentilini, Davide; Di Blasio, Anna Maria; Pelotti, Susi; Pettener, Davide; Sazzini, Marco; Panaino, Antonio; Luiselli, Donata; Gruppioni, Giorgio

none 15 si Background: The ability to digest lactose after weaning, namely, lactase persistence (LP), is encoded by polymorphisms in the MCM6 gene and varies widely in frequency among different human populations. Although, evolution of LP-related genetic variants was investigated in many groups of Sub-Saharan African, Middle Eastern, and European ancestry, only few studies have focused on populations from North Africa and no data are especially available from the Tunisian one. For this reason, there is an urgent need to investigate the frequency patterns at these loci in Tunisia since this adaptive trait is implicated in health. Methods: Forty SNPs covering the LCT/MCM6 genes and including the two functional variants − 13,910 C > T and − 22,018 G > A were genotyped in 117 Tunisian individuals using the Sequenom Mass Array technology. The observed nucleotide and haplotype patterns of variation were then compared with those of several African, European, and Mediterranean human groups for which comparable data were publicly available. Admixture analysis on a 5 Mb genomic region surrounding the LCT/MCM6 loci was also performed by extracting genotypes from a previously generated genome-wide dataset in order to deepen the reconstruction of the evolutionary history of these loci. Results: We found that lactase non-persistence (LNP)-related alleles and haplotypes were predominantly present in the examined population. A clear differentiation between Tunisian, African, and North European/North Italian samples was found, while the Tunisian population showed more genetic affinity to Central and South Italian groups. Conclusions: Our study provided a first report of LP-associated alleles and haplotypes in the Tunisian population. We highlighted a gradient followed by LP diffusion from Europe to North Africa. Based on the rich historic background of Tunisia, we suggest that this adaptive trait was introduced in that geographic region by a relatively recent gene flow. open Ben Halima, Yosra; Kefi, Rym; Sazzini, Marco; Giuliani, Cristina; De Fanti, Sara; Nouali, Chokri; Nagara, Majdi; Mengozzi, Giacomo; Elouej, Sahar; Abid, Abdelmajid; Jamoussi, Henda; Chouchane, Lotfi; Romeo, Giovanni; Abdelhak, Sonia; Luiselli, Donata Ben Halima, Yosra; Kefi, Rym; Sazzini, Marco; Giuliani, Cristina; De Fanti, Sara; Nouali, Chokri; Nagara, Majdi; Mengozzi, Giacomo; Elouej, Sahar; Abid, Abdelmajid; Jamoussi, Henda; Chouchane, Lotfi; Romeo, Giovanni; Abdelhak, Sonia; Luiselli, Donata

none 10 no Published online: 08 Nov 2016 Investigation of human mitochondrial DNA variation patterns and phylogeny has been extensively used in Anthropological and Population Genetics studies and sequencing the whole mitochondrial genome is progressively becoming the gold standard. Among the currently available massive parallel sequencing technologies, Ion Torrent™ semiconductor sequencing represents a promising approach for such studies. Nevertheless, an experimental protocol conceived to enable the achievement of both as high as possible yield and of the most homogeneous sequence coverage through the whole mitochondrial genome is still not available. The present work was thus aimed at improving the overall performance of whole mitochondrial genomes Ion Torrent™ sequencing, with special focus on the capability to obtain robust coverage and highly reliable variants calling. For this purpose, a series of cost-effective modifications in standard laboratory workflows was fine-tuned to optimize them for medium- and large-scale population studies. A total of 54 human samples were thus subjected to sequencing of the whole mitochondrial genome with the Ion Personal Genome Machine™ System in four distinct experiments and using Ion 314 chips. Seven of the selected samples were also characterized by means of conventional Sanger sequencing for the sake of comparison. Obtained results demonstrated that the implemented optimizations had definitely improved sequencing outputs in terms of both variants calling efficiency and coverage uniformity, enabling to setup an effective and accurate protocol for whole mitochondrial genome sequencing and a considerable reduction in experimental time consumption and sequencing costs. none De Fanti, Sara; Vianello, Dario; Giuliani, Cristina; Quagliariello, Andrea; Cherubini, Anna; Sevini, Federica; Iaquilano, Nicoletta; Franceschi, Claudio; Sazzini, Marco; Luiselli, Donata De Fanti, Sara; Vianello, Dario; Giuliani, Cristina; Quagliariello, Andrea; Cherubini, Anna; Sevini, Federica; Iaquilano, Nicoletta; Franceschi, Claudio; Sazzini, Marco; Luiselli, Donata

none 15 no Background: Non-celiac wheat sensitivity is an emerging wheat-related syndrome showing peak prevalence in Western populations. Recent studies hypothesize that new gliadin alleles introduced in the human diet by replacement of ancient wheat with modern varieties can prompt immune responses mediated by the CXCR3-chemokine axis potentially underlying such pathogenic inflammation. This cultural shift may also explain disease epidemiology, having turned European-specific adaptive alleles previously targeted by natural selection into disadvantageous ones. Methods: To explore this evolutionary scenario, we performed ultra-deep sequencing of genes pivotal in the CXCR3-inflammatory pathway on individuals diagnosed for non-celiac wheat sensitivity and we applied anthropological evolutionary genetics methods to sequence data from worldwide populations to investigate the genetic legacy of natural selection on these loci. Results: Our results indicate that balancing selection has maintained two divergent CXCL10/CXCL11 haplotypes in Europeans, one responsible for boosting inflammatory reactions and another for encoding moderate chemokine expression. Conclusions: This led to considerably higher occurrence of the former haplotype in Western people than in Africans and East Asians, suggesting that they might be more prone to side effects related to the consumption of modern wheat varieties. Accordingly, this study contributed to shed new light on some of the mechanisms potentially involved in the disease etiology and on the evolutionary bases of its present-day epidemiological patterns. Moreover, overrepresentation of disease homozygotes for the dis-adaptive haplotype plausibly accounts for their even more enhanced CXCR3-axis expression and for their further increase in disease risk, representing a promising finding to be validated by larger follow-up studies. open Sazzini, Marco; De Fanti, Sara; Cherubini, Anna; Quagliariello, Andrea; Profiti, Giuseppe; Martelli, Pier Luigi; Casadio, Rita; Ricci, Chiara; Campieri, Massimo; Lanzini, Alberto; Volta, Umberto; Caio, Giacomo; Franceschi, Claudio; Spisni, Enzo; Luiselli, Donata Sazzini, Marco; De Fanti, Sara; Cherubini, Anna; Quagliariello, Andrea; Profiti, Giuseppe; Martelli, Pier Luigi; Casadio, Rita; Ricci, Chiara; Campieri, Massimo; Lanzini, Alberto; Volta, Umberto; Caio, Giacomo; Franceschi, Claudio; Spisni, Enzo; Luiselli, Donata

Since prehistoric times, Italy has represented a bridge between peoples, genes and cultures. Its peculiar geographical position explains why: it is located in the center of the Mediterranean Sea, flanked by the Balkans and the Hellenic Peninsula to the east, Iberia to the west and surrounded by North Africa to the south and central Europe to the north. This makes Italy of extraordinary interest for the study of some different aspects of human diversity. Here we overview current knowledge regarding the relationships between the structure of the genetic variation of Italian populations and the geographical, ecological and cultural factors that have characterized their evolutionary history. Human presence in Italian territory is deeply rooted in the past. Lithic artifacts produced by the genus Homo and remains of Homo sapiens are among the earliest to have been found on the continent, as shown by the lithic industry of Pirro Nord (between 1.3 and 1.6 Mya) and the dental remains of the "Grotta del Cavallo" (between 45 and 43 Kya). Genetic and genomic studies relating to existing and extinct human groups have shed light on the migrations from Europe, Africa and Asia that created the ancient layers of the genetic structure of today's Italian populations, especially before the Iron Age. The important role of isolation (genetic and cultural) in shaping genetic structure is clearly visible in the patterns of intra- and inter-population diversity observed among Italian ethno-linguistic minorities that settled on the peninsula and on the major islands until the 19th century. Finally, selective pressures have likely driven the distribution of originally adaptive variants and haplotypes that now confer protection or susceptibility to major diseases such as diabetes and cardiovascular disease (in northern Italy) and tuberculosis and leprosy (in the south). What emerges is a picture where the combined effects of migration, isolation and natural selection generated by the interplay of geography, environment and culture have shaped a complex pattern of human diversity that is unique in Europe and which goes hand in hand with today's rich animal and plant biodiversity. In a nutshell, scientific evidence and cultural heritage paint Italy as a place with extremely diverse environments where distant peoples have met since the deep past, bringing and sharing genes and ideas.

The NBN gene has been included in breast cancer (BC) multigene panels based on early studies suggesting an increased BC risk for carriers, though not confirmed by recent research. To evaluate the impact of NBN analysis, we assessed the results of NBN sequencing in 116 BRCA-negative BC patients and reviewed the literature. Three patients (2.6%) carried potentially relevant variants: two, apparently unrelated, carried the frameshift variant c.156_157delTT and another one the c.628G&gt, T variant. The latter was subsequently found in 4/1390 (0.3%) BC cases and 8/1580 (0.5%) controls in an independent sample, which, together with in silico predictions, provided evidence against its pathogenicity. Conversely, the rare c.156_157delTT variant was absent in the case-control set, moreover, a 50% reduction of NBN expression was demonstrated in one carrier. However, in one family it failed to co-segregate with BC, while the other carrier was found to harbor also a probably pathogenic TP53 variant that may explain her phenotype. Therefore, the c.156_157delTT, although functionally deleterious, was not supported as a cancer-predisposing defect. Pathogenic/likely pathogenic NBN variants were detected by multigene panels in 31/12314 (0.25%) patients included in 15 studies. The risk of misinterpretation of such findings is substantial and supports the exclusion of NBN from multigene panels.

Native American genetic ancestry has been remarkably implicated with increased risk of diverse health issues in several Mexican populations, especially in relation to the dramatic changes in environmental, dietary, and cultural settings they have recently undergone. In particular, the effects of these ecological transitions and Westernization of lifestyles have been investigated so far predominantly on Mestizo individuals. Nevertheless, indigenous groups, rather than admixed Mexicans, have plausibly retained the highest proportions of genetic components shaped by natural selection in response to the ancient milieu experienced by Mexican ancestors during their pre-Columbian evolutionary history. These formerly adaptive variants have the potential to represent the genetic determinants of some biological traits that are peculiar to Mexican people, as well as a reservoir of loci with possible biomedical relevance. To test such a hypothesis, we used genome-wide genotype data to infer the unique adaptive evolution of Native Mexican groups selected as reasonable descendants of the main pre-Columbian Mexican civilizations. A combination of haplotype-based and gene-network analyses enabled us to detect genomic signatures ascribable to polygenic adaptive traits plausibly evolved by the main genetic clusters of Mexican indigenous populations to cope with local environmental and/or cultural conditions. Some of these adaptations were found to play a role in modulating the susceptibility/resistance of these groups to certain pathological conditions, thus providing new evidence that diverse selective pressures have contributed to shape the current biological and disease-risk patterns of present-day Native and Mestizo Mexican populations.

Although Tibetans and Sherpa present several physiological adjustments evolved to cope with selective pressures imposed by the high-altitude environment, especially hypobaric hypoxia, few selective sweeps at a limited number of hypoxia related genes were confirmed by multiple genomic studies. Nevertheless, variants at these loci were found to be associated only with downregulation of the erythropoietic cascade, which represents an indirect aspect of the considered adaptive phenotype. Accordingly, the genetic basis of Tibetan/Sherpa adaptive traits remains to be fully elucidated, in part due to limitations of selection scans implemented so far and mostly relying on the hard sweep model. In order to overcome this issue, we used whole-genome sequence data and several selection statistics as input for gene network analyses aimed at testing for the occurrence of polygenic adaptation in these high-altitude Himalayan populations. Being able to detect also subtle genomic signatures ascribable to weak positive selection at multiple genes of the same functional subnetwork, this approach allowed us to infer adaptive evolution at loci individually showing small effect sizes, but belonging to highly interconnected biological pathways overall involved in angiogenetic processes. Therefore, these findings pinpointed a series of selective events neglected so far, which likely contributed to the augmented tissue blood perfusion observed in Tibetans and Sherpa, thus uncovering the genetic determinants of a key biological mechanism that underlies their adaptation to high altitude.

Background Aneuploidy occurs in more than 20% of acute myeloid leukemia (AML) cases and correlates with an adverse prognosis. Methods To understand the molecular bases of aneuploid acute myeloid leukemia (A‐AML), this study examined the genomic profile in 42 A‐AML cases and 35 euploid acute myeloid leukemia (E‐AML) cases. Results A‐AML was characterized by increased genomic complexity based on exonic variants (an average of 26 somatic mutations per sample vs 15 for E‐AML). The integration of exome, copy number, and gene expression data revealed alterations in genes involved in DNA repair (eg, SLX4IP, RINT1, HINT1, and ATR) and the cell cycle (eg, MCM2, MCM4, MCM5, MCM7, MCM8, MCM10, UBE2C, USP37, CK2, CK3, CK4, BUB1B, NUSAP1, and E2F) in A‐AML, which was associated with a 3‐gene signature defined by PLK1 and CDC20 upregulation and RAD50 downregulation and with structural or functional silencing of the p53 transcriptional program. Moreover, A‐AML was enriched for alterations in the protein ubiquitination and degradation pathway (eg, increased levels of UHRF1 and UBE2C and decreased UBA3 expression), response to reactive oxygen species, energy metabolism, and biosynthetic processes, which may help in facing the unbalanced protein load. E‐AML was associated with BCOR/BCORL1 mutations and HOX gene overexpression. Conclusions These findings indicate that aneuploidy‐related and leukemia‐specific alterations cooperate to tolerate an abnormal chromosome number in AML, and they point to the mitotic and protein degradation machineries as potential therapeutic targets., Aneuploid acute myeloid leukemia (A‐AML) is associated with genomic and transcriptional alterations in the cell cycle and protein degradation pathways. The upregulation of PLK1 and CDC20 and the downregulation of RAD50 and of a p53‐related signature are hallmarks of A‐AML.

Natural epigenetic diversity has been suggested as a key mechanism in microevolutionary processes due to its capability to create phenotypic variability within individuals and populations. It constitutes an important reservoir of variation potentially useful for rapid adaptation in response to environmental stimuli. The analysis of population epigenetic structure represents a possible tool to study human adaptation and to identify external factors that are able to naturally shape human DNA methylation variability. The aim of this study is to investigate the dynamics that create epigenetic diversity between and within different human groups. To this end, we first used publicly available epigenome-wide data to explore population-specific DNA methylation changes that occur at macro-geographic scales. Results from this analysis suggest that nutrients, UVA exposure and pathogens load might represent the main environmental factors able to shape DNA methylation profiles. Then, we evaluated DNA methylation of candidate genes (KRTCAP3, MAD1L1, and BRSK2), emerged from the previous analysis, in individuals belonging to different populations from Morocco, Nigeria, Philippines, China, and Italy, but living in the same Italian city. DNA methylation of the BRSK2 gene is significantly different between Moroccans and Nigerians (pairwise t-test: CpG 6 P-value = 5.2*10 (-) (3); CpG 9 P-value = 2.6*10 (-) (3); CpG 10 P-value = 3.1*10 (-) (3); CpG 11 P-value = 2.8*10 (-) (3)). Comprehensively, these results suggest that DNA methylation diversity is a source of variability in human groups at macro and microgeographical scales and that population demographic and adaptive histories, as well as the individual ancestry, actually influence DNA methylation profiles.

The pathogenesis of idiopathic and heritable forms of pulmonary arterial hypertension is still not completely understood, even though several causative genes have been proposed, so that a third of patients remains genetically unresolved. Here we applied a multistep approach to extend identification of the genetic bases of such a disease by searching for novel candidate genes/pathways. Twenty-eight patients belonging to 18 families were screened for BMPR2 mutations and BMPR2-negative samples were tested for 12 additional candidate genes by means of a specific massive parallel sequencing-based assay. Finally, whole exome sequencing was performed on four patients showing no mutations at known disease genes, as well as on their unaffected parents. In addition to EIF2AK4, which has been already suggested to be associated with pulmonary veno-occlusive disease, we identified the novel candidate genes ATP13A3, CD248, EFCAB4B, involved in lung vascular remodeling that represent reliable drivers contributing to the disease according to their biological functions/inheritance patterns. Therefore, our results suggest that combining gene panel and whole exome sequencing provides new insights useful for the genetic diagnosis of familial and idiopathic pulmonary arterial hypertension, as well as for the identification of biological pathways that will be potentially targeted by new therapeutic strategies.

Extensive European and African admixture coupled with loss of Amerindian lineages makes the reconstruction of pre-Columbian history of Native Americans based on present-day genomes extremely challenging. Still open questions remain about the dispersals that occurred throughout the continent after the initial peopling from the Beringia, especially concerning the number and dynamics of diffusions into South America. Indeed, if environmental and historical factors contributed to shape distinct gene pools in the Andes and Amazonia, the origins of this East-West genetic structure and the extension of further interactions between populations residing along this divide are still not well understood. To this end, we generated new high-resolution genome-wide data for 229 individuals representative of one Central and ten South Amerindian ethnic groups from Mexico, Peru, Bolivia, and Argentina. Low levels of European and African admixture in the sampled individuals allowed the application of fine-scale haplotype-based methods and demographic modeling approaches. These analyses revealed highly specific Native American genetic ancestries and great intragroup homogeneity, along with limited traces of gene flow mainly from the Andes into Peruvian Amazonians. Substantial amount of genetic drift differentially experienced by the considered populations underlined distinct patterns of recent inbreeding or prolonged isolation. Overall, our results support the hypothesis that all non-Andean South Americans are compatible with descending from a common lineage, while we found low support for common Mesoamerican ancestors of both Andeans and other South American groups. These findings suggest extensive back-migrations into Central America from non-Andean sources or conceal distinct peopling events into the Southern Continent.

The study of the genetics of longevity has been mainly addressed by GWASs that considered subjects from different populations to reach higher statistical power. The "price to pay" is that population-specific evolutionary histories and trade-offs were neglected in the investigation of gene-environment interactions. We propose a new "diachronic" approach that considers processes occurred at both evolutionary and lifespan timescales. We focused on a well-characterized population in terms of evolutionary history (i.e. Italians) and we generated genome-wide data for 333 centenarians from the peninsula and 773 geographically-matched healthy individuals. Obtained results showed that: (i) centenarian genomes are enriched for an ancestral component likely shaped by pre-Neolithic migrations; (ii) centenarians born in Northern Italy unexpectedly clustered with controls from Central/Southern Italy suggesting that Neolithic and Bronze Age gene flow did not favor longevity in this population; (iii) local past adaptive events in response to pathogens and targeting arachidonic acid metabolism became favorable for longevity; (iv) lifelong changes in the frequency of several alleles revealed pleiotropy and trade-off mechanisms crucial for longevity. Therefore, we propose that demographic history and ancient/recent population dynamics need to be properly considered to identify genes involved in longevity, which can differ in different temporal/spatial settings.

Liddle syndrome is considered a rare Mendelian hypertension. We have previously described 3 reportedly unrelated families, native of an Italian area around the Strait of Messina, carrying the same mutation (βP617L) of the epithelial sodium channel. The aims of our study were (1) to evaluate whether a close genomic relationship exists between the 3 families through the analysis of mitochondrial DNA and Y chromosome; and (2) to quantify the genomic relatedness between the patients with Liddle syndrome belonging to the 3 families and assess the hypothesis of a mutation shared through identity by descent. HVRI (the hypervariable region I) of the mitochondrial DNA genome and the Y chromosome short tandem repeats profiles were analyzed in individuals of the 3 families. Genotyping 542 585 genome-wide single nucleotide polymorphisms was performed in all the patients with Liddle syndrome of the 3 families and some of their relatives. A panel of 780 healthy Italian adult samples typed for the same set of markers was used as controls. espite different lineages between the 3 families based on the analysis of mitochondrial DNA and Y chromosome, the 3 probands and their 6 affected relatives share the same ≈5 Mbp long haplotype which encompasses the mutant allele. Using an approach based on coalescent theory, we estimate that the 3 families inherited the mutant allele from a common ancestor ≈13 generations ago and that such an ancestor may have left ≈20 carriers alive today. The prevalence of Liddle syndrome in the region of origin of the 3 families may be much higher than that estimated worldwide.

Several studies highlighted the role of climate in shaping many human evolutionary processes. This occurred even in relatively recent times, having affected various human phenotypic traits, among which metabolic processes that orchestrate absorption and accumulation of substances to maintain energy homeostasis, that is critical for the survival of individuals in high energy-expenditure environments. To date, most researches have focalized on detection of climatic influence on SNPs' frequency in populations exposed to extreme environmental conditions or by comparing variation patterns between populations from different continents. In this study, we instead explored the genetic background of distinct western European human groups at loci involved in nutritional and thermoregulation processes, to test whether patterns of differential local adaptation to environmental conditions could be appreciated also at a lower geographical scale. Taking advantage from the 1000 Genomes Project data, genetic information for 21 genes involved in nutritional and thermoregulation processes was analysed for three western European populations. The applied Anthropological Genetics methods pointed to appreciable differentiation between the examined groups especially for the PRDM16 gene. Moreover, several neutrality tests suggested that balancing selection has acted on different regions of the gene in people from Great Britain, as well as that more recent positive selection could have also targeted some PRDM16 SNPs in Finn and Italian populations. These series of adaptive footprints are plausibly related to climate variability in both ancient and relatively recent times. Since this locus is involved in thermoregulation mechanisms and adipogenesis, local adaptations mediated by a pathway related to the brown adipose tissue activity could have evolved in response to changing cold temperature exposures of such populations.

The Mediterranean shores stretching between Sicily, Southern Italy and the Southern Balkans witnessed a long series of migration processes and cultural exchanges. Accordingly, present-day population diversity is composed by multiple genetic layers, which make the deciphering of different ancestral and historical contributes particularly challenging. We address this issue by genotyping 511 samples from 23 populations of Sicily, Southern Italy, Greece and Albania with the Illumina GenoChip Array, also including new samples from Albanian-and Greek-speaking ethno-linguistic minorities of Southern Italy. Our results reveal a shared Mediterranean genetic continuity, extending from Sicily to Cyprus, where Southern Italian populations appear genetically closer to Greek-speaking islands than to continental Greece. Besides a predominant Neolithic background, we identify traces of Post-Neolithic Levantine-and Caucasus-related ancestries, compatible with maritime Bronze-Age migrations. We argue that these results may have important implications in the cultural history of Europe, such as in the diffusion of some Indo-European languages. Instead, recent historical expansions from North-Eastern Europe account for the observed differentiation of present-day continental Southern Balkan groups. Patterns of IBD-sharing directly reconnect Albanian-speaking Arbereshe with a recent Balkan-source origin, while Greek-speaking communities of Southern Italy cluster with their Italian-speaking neighbours suggesting a long-term history of presence in Southern Italy., This study was supported by the Genographic Project 2.0 (Geno 2.0) Scientific Research Grant 4–13 and by the European Research Council ERC-2011-AdG 295733 grant (Langelin).

Observable patterns of cultural variation are consistently intertwined with demic movements, cultural diffusion, and adaptation to different ecological contexts [Cavalli-Sforza and Feldman (1981) Cultural Transmission and Evolution: A Quantitative Approach; Boyd and Richerson (1985) Culture and the Evolutionary Process]. The quantitative study of gene–culture coevolution has focused in particular on the mechanisms responsible for change in frequency and attributes of cultural traits, the spread of cultural information through demic and cultural diffusion, and detecting relationships between genetic and cultural lineages. Here, we make use of worldwide whole-genome sequences [Pagani et al. (2016) Nature 538:238–242] to assess the impact of processes involving population movement and replacement on cultural diversity, focusing on the variability observed in folktale traditions (n = 596) [Uther (2004) The Types of International Folktales: A Classification and Bibliography. Based on the System of Antti Aarne and Stith Thompson] in Eurasia. We find that a model of cultural diffusion predicted by isolation-by-distance alone is not sufficient to explain the observed patterns, especially at small spatial scales (up to ~4,000 km). We also provide an empirical approach to infer presence and impact of ethnolinguistic barriers preventing the unbiased transmission of both genetic and cultural information. After correcting for the effect of ethnolinguistic boundaries, we show that, of the alternative models that we propose, the one entailing cultural diffusion biased by linguistic differences is the most plausible. Additionally, we identify 15 tales that are more likely to be predominantly transmitted through population movement and replacement and locate putative focal areas for a set of tales that are spread worldwide., E.B. is supported by SimulPast Consolider Ingenio Project CSD2010-00034 funded by the Spanish Ministry of Economy, Industry, and Competitiveness. L.P. is supported by the European Union through European Regional Development Fund Projects 2014-2020.4.01.16-0030 and 2014-2020.4.01.15-0012.

While much research attention has focused on demographic processes that enabled human diffusion on the Tibetan plateau, little is known about more recent colonization of Southern Himalayas. In particular, the history of migrations, admixture and/or isolation of populations speaking Tibeto-Burman languages, which is supposed to be quite complex and to have reshaped patterns of genetic variation on both sides of the Himalayan arc, remains only partially elucidated. We thus described the genomic landscape of previously unsurveyed Tibeto-Burman (i.e. Sherpa and Tamang) and Indo-Aryan communities from remote Nepalese valleys. Exploration of their genomic relationships with South/East Asian populations provided evidence for Tibetan admixture with low-altitude East Asians and for Sherpa isolation. We also showed that the other Southern Himalayan Tibeto-Burmans derived East Asian ancestry not from the Tibetan/Sherpa lineage, but from low-altitude ancestors who migrated from China plausibly across Northern India/Myanmar, having experienced extensive admixture that reshuffled the ancestral Tibeto-Burman gene pool. These findings improved the understanding of the impact of gene flow/drift on the evolution of high-altitude Himalayan peoples and shed light on migration events that drove colonization of the southern Himalayan slopes, as well as on the role played by different Tibeto-Burman groups in such a complex demographic scenario.

The increased risk for autosomal recessive disorders is one of the most well-known medical implications of consanguinity. In the Sultanate of Oman, a country characterized by one of the highest rates of consanguineous marriages worldwide, prevalence of genetic hearing loss (GHL) is estimated to be 6/10 000. Families of GHL patients have higher consanguinity rates than the general Omani population, indicating a major role for recessive forms. Mutations in GJB2, the most commonly mutated GHL gene, have been sporadically described. We collected 97 DNA samples of GHL probands, affected/unaffected siblings and parents from 26 Omani consanguineous families. Analyzing a first family by whole-exome sequencing, we identified a novel homozygous frameshift duplication (c.1171_1177dupGCCATCT) in MYO15A, the gene linked to the deafness locus DFNB3. This duplication was then found in a total of 8/26 (28%) families, within a 849 kb founder haplotype. Reconstruction of haplotype structure at MYO15A surrounding genomic regions indicated that the founder haplotype branched out in the past two to three centuries from a haplotype present worldwide. The MYO15A duplication emerges as the major cause of GHL in Oman. These findings have major implications for the design of GHL diagnosis and prevention policies in Oman.