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ZFTA (C11orf95)—a gene of unknown function—partners with a variety of transcriptional coactivators in translocations that drive supratentorial ependymoma, a frequently lethal brain tumor. Understanding the function of ZFTA is key to developing therapies that inhibit these fusion proteins. Here, using a combination of transcriptomics, chromatin immunoprecipitation sequencing, and proteomics, we interrogated a series of deletion-mutant genes to identify a tripartite transformation mechanism of ZFTA-containing fusions, including: spontaneous nuclear translocation, extensive chromatin binding, and SWI/SNF, SAGA, and NuA4/Tip60 HAT chromatin modifier complex recruitment. Thereby, ZFTA tethers fusion proteins across the genome, modifying chromatin to an active state and enabling its partner transcriptional coactivators to promote promiscuous expression of a transforming transcriptome. Using mouse models, we validate further those elements of ZFTA-fusion proteins that are critical for transformation—including ZFTA zinc fingers and partner gene transactivation domains—thereby unmasking vulnerabilities for therapeutic targeting.Significance:Ependymomas are hard-to-treat brain tumors driven by translocations between ZFTA and a variety of transcriptional coactivators. We dissect the transforming mechanism of these fusion proteins and identify protein domains indispensable for tumorigenesis, thereby providing insights into the molecular basis of ependymoma tumorigenesis and vulnerabilities for therapeutic targeting.This article is highlighted in the In This Issue feature, p. 2113

ZFTA-RELAFUS1 domain interactomes

ZFTAFUS-sig

ZFTA (C11orf95)—a gene of unknown function—partners with a variety of transcriptional coactivators in translocations that drive supratentorial ependymoma, a frequently lethal brain tumor. Understanding the function of ZFTA is key to developing therapies that inhibit these fusion proteins. Here, using a combination of transcriptomics, chromatin immunoprecipitation sequencing, and proteomics, we interrogated a series of deletion-mutant genes to identify a tripartite transformation mechanism of ZFTA-containing fusions, including: spontaneous nuclear translocation, extensive chromatin binding, and SWI/SNF, SAGA, and NuA4/Tip60 HAT chromatin modifier complex recruitment. Thereby, ZFTA tethers fusion proteins across the genome, modifying chromatin to an active state and enabling its partner transcriptional coactivators to promote promiscuous expression of a transforming transcriptome. Using mouse models, we validate further those elements of ZFTA-fusion proteins that are critical for transformation—including ZFTA zinc fingers and partner gene transactivation domains—thereby unmasking vulnerabilities for therapeutic targeting. Significance: Ependymomas are hard-to-treat brain tumors driven by translocations between ZFTA and a variety of transcriptional coactivators. We dissect the transforming mechanism of these fusion proteins and identify protein domains indispensable for tumorigenesis, thereby providing insights into the molecular basis of ependymoma tumorigenesis and vulnerabilities for therapeutic targeting. This article is highlighted in the In This Issue feature, p. 2113

Background: DNA methylation profiles associated with childhood asthma might provide novel insights into disease pathogenesis. We did an epigenome-wide association study to assess methylation profiles associated with childhood asthma. Methods: We did a large-scale epigenome-wide association study (EWAS) within the Mechanisms of the Development of ALLergy (MeDALL) project. We examined epigenome-wide methylation using Illumina Infinium Human Methylation450 BeadChips (450K) in whole blood in 207 children with asthma and 610 controls at age 4–5 years, and 185 children with asthma and 546 controls at age 8 years using a cross-sectional case-control design. After identification of differentially methylated CpG sites in the discovery analysis, we did a validation study in children (4–16 years; 247 cases and 2949 controls) from six additional European cohorts and meta-analysed the results. We next investigated whether replicated CpG sites in cord blood predict later asthma in 1316 children. We subsequently investigated cell-type-specific methylation of the identified CpG sites in eosinophils and respiratory epithelial cells and their related gene-expression signatures. We studied cell-type specificity of the asthma association of the replicated CpG sites in 455 respiratory epithelial cell samples, collected by nasal brushing of 16-year-old children as well as in DNA isolated from blood eosinophils (16 with asthma, eight controls [age 2–56 years]) and compared this with whole-blood DNA samples of 74 individuals with asthma and 93 controls (age 1–79 years). Whole-blood transcriptional profiles associated with replicated CpG sites were annotated using RNA-seq data of subsets of peripheral blood mononuclear cells sorted by fluorescence-activated cell sorting. Findings: 27 methylated CpG sites were identified in the discovery analysis. 14 of these CpG sites were replicated and passed genome-wide significance (p

MeDALL (Mechanisms of the Development of ALLergy; EU FP7-CP-IP; Project No: 261357; 2010-2015) has proposed an innovative approach to develop early indicators for the prediction, diagnosis, prevention and targets for therapy. MeDALL has linked epidemiological, clinical and basic research using a stepwise, large-scale and integrative approach: MeDALL data of precisely phenotyped children followed in 14 birth cohorts spread across Europe were combined with systems biology (omics, IgE measurement using microarrays) and environmental data. Multimorbidity in the same child is more common than expected by chance alone, suggesting that these diseases share causal mechanisms irrespective of IgE sensitization. IgE sensitization should be considered differently in monosensitized and polysensitized individuals. Allergic multimorbidities and IgE polysensitization are often associated with the persistence or severity of allergic diseases. Environmental exposures are relevant for the development of allergy-related diseases. To complement the population-based studies in children, MeDALL included mechanistic experimental animal studies and in vitro studies in humans. The integration of multimorbidities and polysensitization has resulted in a new classification framework of allergic diseases that could help to improve the understanding of genetic and epigenetic mechanisms of allergy as well as to better manage allergic diseases. Ethics and gender were considered. MeDALL has deployed translational activities within the EU agenda.

Pharmacological inhibition of uncontrolled cell growth with small-molecule inhibitors is a potential strategy for treating glioblastoma multiforme (GBM), the most malignant primary brain cancer. We showed that the synthetic small-molecule KHS101 promoted tumor cell death in diverse GBM cell models, independent of their tumor subtype, and without affecting the viability of noncancerous brain cell lines. KHS101 exerted cytotoxic effects by disrupting the mitochondrial chaperone heat shock protein family D member 1 (HSPD1). In GBM cells, KHS101 promoted aggregation of proteins regulating mitochondrial integrity and energy metabolism. Mitochondrial bioenergetic capacity and glycolytic activity were selectively impaired in KHS101-treated GBM cells. In two intracranial patient-derived xenograft tumor models in mice, systemic administration of KHS101 reduced tumor growth and increased survival without discernible side effects. These findings suggest that targeting of HSPD1-dependent metabolic pathways might be an effective strategy for treating GBM.

Background: Long-term exposure to ambient air pollution can lead to adverse health effects in children; however, underlying biological mechanisms are not fully understood. Objectives: We evaluated the effect of air pollution exposure during different time periods on mRNA expression as well as circulating levels of inflammatory cytokines in children. Methods: We measured a panel of 10 inflammatory markers in peripheral blood samples from 670 8-y-old children in the Barn/Child, Allergy, Milieu, Stockholm, Epidemiology (BAMSE) birth cohort. Outdoor concentrations of nitrogen dioxide (NO2) and particulate matter (PM) with aerodynamic diameter

BACKGROUND: Prenatal exposure to air pollution is considered to be associated with adverse effects on child health. This may partly be mediated by mechanisms related to DNA methylation. OBJECTIVES: We investigated associations between exposure to air pollution, using nitrogen dioxide (NO2) as marker, and epigenome-wide cord blood DNA methylation. METHODS: We meta-analyzed the associations between NO2 exposure at residential addresses during pregnancy and cord blood DNA methylation (Illumina 450K) in four European and North American studies (n = 1,508) with subsequent look-up analyses in children ages 4 (n = 733) and 8 (n = 786) years. Additionally, we applied a literature-based candidate approach for antioxidant and anti-inflammatory genes. To assess influence of exposure at the transcriptomics level, we related mRNA expression in blood cells to NO2 exposure in 4- (n = 111) and 16-year-olds (n = 239). RESULTS: We found epigenome-wide significant associations [false discovery rate (FDR) p < 0.05] between maternal NO2 exposure during pregnancy and DNA methylation in newborns for 3 CpG sites in mitochondria-related genes: cg12283362 (LONP1), cg24172570 (3.8 kbp upstream of HIBADH), and cg08973675 (SLC25A28). The associations with cg08973675 methylation were also significant in the older children. Further analysis of antioxidant and anti-inflammatory genes revealed differentially methylated CpGs in CAT and TPO in newborns (FDR p < 0.05). NO2 exposure at the time of biosampling in childhood had a significant impact on CAT and TPO expression. CONCLUSIONS: NO2 exposure during pregnancy was associated with differential offspring DNA methylation in mitochondria-related genes. Exposure to NO2 was also linked to differential methylation as well as expression of genes involved in antioxidant defense pathways. Citation: Gruzieva O, Xu CJ, Breton CV, Annesi-Maesano I, Antó JM, Auffray C, Ballereau S, Bellander T, Bousquet J, Bustamante M, Charles MA, de Kluizenaar Y, den Dekker HT, Duijts L, Felix JF, Gehring U, Guxens M, Jaddoe VV, Jankipersadsing SA, Merid SK, Kere J, Kumar A, Lemonnier N, Lepeule J, Nystad W, Page CM, Panasevich S, Postma D, Slama R, Sunyer J, Söderhäll C, Yao J, London SJ, Pershagen G, Koppelman GH, Melén E. 2017. Epigenome-wide meta-analysis of methylation in children related to prenatal NO2 air pollution exposure. Environ Health Perspect 125:104-110; http://dx.doi.org/10.1289/EHP36.

BACKGROUND: Dysregulated, stem cell-like self-renewal has been implicated in glioma treatment resistance and tumour recurrence. Drugs that eliminate tumour cells possessing this malignant characteristic are urgently needed. It remains, however, an experimental challenge to link heterogeneous glioma genotypes to drug response at scale. To this end, we successfully derived patient-specific induced pluripotent stem cell (iPSC) models from both low- (LGG) and high-grade gliomas (HGG) and developed an initial drug discovery application. METHODS: Brain tumour tissue, acquired at surgery, was reprogrammed. Derived iPSC models were characterised using pluripotency markers, tri-germinal layer differentiation, gene expression, karyology and deep whole genome sequencing (WGS, iPSC versus parental tumour). Glioma iPSC differentiation in 2-dimensional (adherent, optically clear 96-well imaging plates) and 3-dimensional (organoid) culture was carried out. Gene expression of neural induction and neuronal differentiation was analysed using mRNA-seq. Neural cancer stem cells from each glioma iPSC line were orthotopically implanted in vivo. RESULTS: Reprogrammed cells were confirmed as fully-reprogramed/stable iPSCs, with mutational variants (SNPs, CNVs) preserved as compared to the parental tumours. Glioma iPSC maturation and quantification of TUJ-1 expression indicated a ‘differentiation block’ in the HGG iPSC models. This phenotype was concordant in HGG iPSC-derived tumour organoids which displayed SOX2/MKI67-positive neural rosettes. Consistently, mice developed xenograft tumours with GBM histopathological characteristics. Expression profiling during neuronal differentiation (from iPSC to neural stem cells to neurons) has revealed candidate genes that may be responsible for the phenotypic differences between HGG and control/LGG iPSC models. CONCLUSIONS: Our adherent, organoid and in vivo iPSC models may uncover genetic mutations and regulatory networks underlying glioma stem cell self-renewal capability, and provide a basis for industrial-scale drug discovery. Here we have successfully implemented the first stages towards this development (in a 96-well assay format). Ultimately, our patient-derived iPSC-based approach may enable personalised precision medicine strategies against glioma.

Human genetic diversity in Europe has been extensively studied using uniparentally inherited sequences (mitochondrial DNA (mtDNA) and the Y chromosome), which reveal very different patterns indicating sex-specific demographic histories. The X chromosome, haploid in males and inherited twice as often from mothers as from fathers, could provide insights into past female behaviours, but has not been extensively investigated. Here, we use HapMap single-nucleotide polymorphism data to identify genome-wide segments of the X chromosome in which recombination is historically absent and mutations are likely to be the only source of genetic variation, referring to these as phylogeographically informative haplotypes on autosomes and X chromosome (PHAXs). Three such sequences on the X chromosome spanning a total of ~49 kb were resequenced in 240 males from Europe, the Middle East and Africa at an average coverage of 181 ×. These PHAXs were confirmed to be essentially non-recombining across European samples. All three loci show highly homogeneous patterns across Europe and are highly differentiated from the African sample. Star-like structures of European-specific haplotypes in median-joining networks indicate past population expansions. Bayesian skyline plots and time-to-most-recent-common-ancestor estimates suggest expansions pre-dating the Neolithic transition, a finding that is more compatible with data on mtDNA than the Y chromosome, and with the female bias of X-chromosomal inheritance. This study demonstrates the potential of the use of X-chromosomal haplotype blocks, and the utility of the accurate ascertainment of rare variants for inferring human demographic history.

BACKGROUND AND AIMS: Shorter telomeres have been associated with increased risk of malignancy, including colorectal cancer (CRC). Telomere length is heritable and may be an intermediate phenotype linked to genetic susceptibility to CRC.METHODS: In a large sample, the study investigated whether candidate single nucleotide polymorphisms (SNP) in 'telomere biology' genes were associated with telomere length in leucocytes. SNP associated with an increased risk of CRC were searched for separately.RESULTS: Carriers of the common allele at SNP rs10936599, near the telomerase RNA component (TERC) locus, had significantly longer telomeres. It was independently found that the same rs10936599 allele was associated with increased risk of both CRC and colorectal adenomas. Neither telomere length nor CRC risk was associated with variation near telomerase reverse transcriptase or other telomere biology genes. In silico analysis showed that SNP rs2293607 was strongly correlated with rs10936599, mapped within TERC transcripts, had a predicted effect on messenger RNA folding and lay at a reported transcription factor binding site. TERC mRNA were expressed, differing only at the alleles of rs2293607, in CRC cell line HCT116. The long-telomere/CRC-risk allele was associated with higher levels of TERC mRNA and the formation of longer telomeres.CONCLUSIONS: Common genetic variation at TERC is associated with both longer telomeres and an increased risk of CRC, a potential mechanism being reduced levels of cell senescence or death. This finding is somewhat paradoxical, given retrospective studies reporting that CRC cases have shorter telomeres than controls. One possibility is that that association actually results from poorer survival in patients with longer telomeres.

OBJECTIVE: Colorectal cancer (CRC) has a substantial heritable component. Common genetic variation has been shown to contribute to CRC risk. A study was conducted in a large multi-population study to assess the feasibility of CRC risk prediction using common genetic variant data combined with other risk factors. A risk prediction model was built and applied to the Scottish population using available data. DESIGN: Nine populations of European descent were studied to develop and validate CRC risk prediction models. Binary logistic regression was used to assess the combined effect of age, gender, family history (FH) and genotypes at 10 susceptibility loci that individually only modestly influence CRC risk. Risk models were generated from case-control data incorporating genotypes alone (n=39,266) and in combination with gender, age and FH (n=11,324). Model discriminatory performance was assessed using 10-fold internal cross-validation and externally using 4187 independent samples. The 10-year absolute risk was estimated by modelling genotype and FH with age- and gender-specific population risks. RESULTS: The median number of risk alleles was greater in cases than controls (10 vs 9, p5% predicted 10-year absolute risk. CONCLUSION: Genotype data provide additional information that complements age, gender and FH as risk factors, but individualised genetic risk prediction is not currently feasible. Nonetheless, the modelling exercise suggests public health potential since it is possible to stratify the population into CRC risk categories, thereby informing targeted prevention and surveillance.

The evidence on traffic-related air pollution exposure (TRAP) and incident childhood asthma is inconsistent, and may depend on genetic factors.We aimed to identify mechanisms of childhood asthma using genome-wide SNP data and individual TRAP exposure, and to evaluate the effect of susceptibility SNPs and TRAP on DNA-methylation and gene expression.We used LUR models to estimate individual outdoor NO2 levels at the birth address and performed a genome-wide interaction study for doctor's diagnosis of asthma up to 8 years in three European birth cohorts with replication in two North American cohorts (n=3,322 subjects). The top GWIS and replicated SNPs were assessed for methQTL effects in peripheral blood cells and eQTL effects in human lung specimens. Short- and long-term TRAP associations with methylation patterns and TRAP-induced differential gene expression in blood cells were also assessed.The novel loci MAGI1, B4GALT5, MOCOS and DLG2, and the previously lung disease linked locus ADCY2 showed strong evidence for interaction with TRAP (genome-wide significance or replication). The top replication SNP rs686237 was identified as an eQTL for B4GALT5 (p=1.18x10-17) and affected TRAP-induced gene expression (p=0.03). Differential methylation following TRAP exposure was seen for DLG2, ADCY2, MAGI1 and MOCOS. Identified genes belong to the guanylate kinase, sphingolipid and calcium signaling pathways, suggesting involvement in asthma pathogenesis.Our results indicate that gene-environment interactions are important for asthma development and that functional genomics analyses in conjunction with environmental exposures may give valuable insights about pathophysiologic mechanisms.

Allergic diseases (asthma, rhinitis and atopic dermatitis) are complex. They are associated with allergen-specific IgE and non-allergic mechanisms that may coexist in the same patient. In addition, these diseases tend to cluster and patients present concomitant or consecutive diseases (multimorbidity). IgE sensitization should be considered as a quantitative trait. Important clinical and immunological differences exist between mono or polysensitized subjects. Multimorbidities of allergic diseases share common causal mechanisms that are only partly IgE-mediated. Persistence of allergic diseases over time is associated with multimorbidity and/or IgE polysensitization. The importance of the family history of allergy may decrease with age. This review puts forward the hypothesis that allergic multimorbidities and IgE polysensitization are associated and related to the persistence or re-occurrence of foetal Type 2 signalling. Asthma, rhinitis and atopic dermatitis are manifestations of a common systemic immune imbalance (mesodermal origin) with specific patterns of remodelling (ectodermal or endodermal origin). This paper proposes a new classification of IgE-mediated allergic diseases that allows the definition of novel phenotypes in order to (i) better understand genetic and epigenetic mechanisms, (ii) better stratify allergic preschool children for prognosis, and (iii) propose novel strategies of treatment and prevention.

SummarySurnames are cultural markers of shared ancestry within human populations. The Y chromosome, like many surnames, is paternally inherited, so men sharing surnames might be expected to share similar Y chromosomes as a signature of coancestry. Such a relationship could be used to connect branches of family trees [1], to validate population genetic studies based on isonymy [2], and to predict surname from crime-scene samples in forensics [3]. However, the link may be weak or absent due to multiple independent founders for many names, adoptions, name changes and nonpaternities, and mutation of Y haplotypes. Here, rather than focusing on a single name [4], we take a general approach by seeking evidence for a link in a sample of 150 randomly ascertained pairs of males who each share a British surname. We show that sharing a surname significantly elevates the probability of sharing a Y-chromosomal haplotype and that this probability increases as surname frequency decreases. Within our sample, we estimate that up to 24% of pairs share recent ancestry and that a large surname-based forensic database might contribute to the intelligence-led investigation of up to ∼70 rapes and murders per year in the UK. This approach would be applicable to any society that uses patrilineal surnames of reasonable time-depth.

Asthma, rhinitis, and eczema are complex diseases with multiple genetic and environmental factors interlinked through IgE-associated and non–IgE-associated mechanisms. Mechanisms of the Development of ALLergy (MeDALL; EU FP7-CP-IP; project no: 261357; 2010-2015) studied the complex links of allergic diseases at the clinical and mechanistic levels by linking epidemiologic, clinical, and mechanistic research, including invivo and invitro models. MeDALL integrated 14 European birth cohorts, including 44,010 participants and 160 cohort follow-ups between pregnancy and age 20years. Thirteen thousand children were prospectively followed after puberty by using a newly standardized MeDALL Core Questionnaire. Amicroarray developed for allergen molecules with increased IgE sensitivity was obtained for 3,292 children. Estimates of air pollution exposure from previous studies were available for 10,000 children. Omics data included those from historical genome-wide association studies (23,000 children) and DNA methylation (2,173), targeted multiplex biomarker (1,427), andtranscriptomic (723) studies. Using classical epidemiology and machine-learning methods in 16,147 children aged 4years and 11,080 children aged 8years, MeDALL showed the multimorbidity of eczema, rhinitis, and asthma and estimated that only 38% of multimorbidity was attributable to IgE sensitization. MeDALL has proposed a new vision of multimorbidity independent of IgE sensitization, and has shown that monosensitization and polysensitization represent 2 distinct phenotypes. The translational component of MeDALL is shown by the identification of a novel allergic phenotype characterized by polysensitization and multimorbidity, which is associated with the frequency, persistence, and severity of allergic symptoms. The results of MeDALL will help integrate personalized, predictive, preventative, and participatory approaches in allergic diseases.

Inflammatory lung diseases are highly complex in respect of pathogenesis and relationships between inflammation, clinical disease and response to treatment. Sophisticated large-scale analytical methods to quantify gene expression (transcriptomics), proteins (proteomics), lipids (lipidomics) and metabolites (metabolomics) in the lungs, blood and urine are now available to identify biomarkers that define disease in terms of combined clinical, physiological and patho-biological abnormalities. The aspiration is that these approaches will improve diagnosis, i.e. define pathological phenotypes, and facilitate the monitoring of disease and therapy, and also, unravel underlying molecular pathways. Biomarker studies can either select predefined biomarker(s) measured by specific methods or apply an "unbiased" approach involving detection platforms that are indiscriminate in focus. This article reviews the technologies presently available to study biomarkers of lung disease within the 'omics field. The contributions of the individual 'omics analytical platforms to the field of respiratory diseases are summarised, with the goal of providing background on their respective abilities to contribute to systems medicine-based studies of lung disease.

Network analysis is an essential component of systems biology approaches toward understanding the molecular and cellular interactions underlying biological systems functionalities and their perturbations in disease. Regulatory and signalling pathways involve DNA, RNA, proteins and metabolites as key elements to coordinate most aspects of cellular functioning. Cellular processes depend on the structure and dynamics of gene regulatory networks and can be studied by employing a network representation of molecular interactions. This chapter describes several types of biological networks, how combination of different analytic approaches can be used to study diseases, and provides a list of selected tools for network visualization and analysis. It also introduces protein–protein interaction networks, gene regulatory networks, signalling networks and metabolic networks to illustrate concepts underlying network representation of cellular processes and molecular interactions. It finally discusses how the level of accuracy in inferring functional relationships influences the choice of methods applied for the analysis of a particular biological network type.

This chapter introduces systems biology, its context, aims, concepts and strategies. It then describes approaches and methods used for collection of high-dimensional structural and functional genomics data, including epigenomics, transcriptomics, proteomics, metabolomics and lipidomics, and how recent technological advances in these fields have moved the bottleneck from data production to data analysis and bioinformatics. Finally, the most advanced mathematical and computational methods used for clustering, feature selection, prediction analysis, text mining and pathway analysis in functional genomics and systems biology are reviewed and discussed in the context of use cases.

12 páginas, 6 figuras.-- et al., Most studies of European genetic diversity have focused on large-scale variation and interpretations based on events in prehistory, but migrations and invasions in historical times could also have had profound effects on the genetic landscape. The Iberian Peninsula provides a suitable region for examination of the demographic impact of such recent events, because its complex recent history has involved the long-term residence of two very different populations with distinct geographical origins and their own particular cultural and religious characteristics—North African Muslims and Sephardic Jews. To address this issue, we analyzed Y chromosome haplotypes, which provide the necessary phylogeographic resolution, in 1140 males from the Iberian Peninsula and Balearic Islands. Admixture analysis based on binary and Y-STR haplotypes indicates a high mean proportion of ancestry from North African (10.6%) and Sephardic Jewish (19.8%) sources. Despite alternative possible sources for lineages ascribed a Sephardic Jewish origin, these proportions attest to a high level of religious conversion (whether voluntary or enforced), driven by historical episodes of social and religious intolerance, that ultimately led to the integration of descendants. In agreement with the historical record, analysis of haplotype sharing and diversity within specific haplogroups suggests that the Sephardic Jewish component is the more ancient. The geographical distribution of North African ancestry in the peninsula does not reflect the initial colonization and subsequent withdrawal and is likely to result from later enforced population movement—more marked in some regions than in others—plus the effects of genetic drift., M.A.J. was supported by a Wellcome Trust Senior Fellowship in Basic Biomedical Science (grant no. 057559), and S.M.A., E.B., P.L.B., S.J.B., and A.C.L. were supported by the Wellcome Trust.

Modern human genetic diversity is the result of demographic history, and selective effects that have acted to adapt different populations to their environments. Broad patterns of global diversity are well explained by geography, based on an out-of-Africa model of early human evolution. Genome-wide searches for signals of selection, plus studies of specific candidate loci and candidate phenotypes, have identified genes that show population differences due to adaptation to pathogens, climate, diet and possibly cognitive challenges. Some past adaptations are now maladaptive, and can lead to disease. However, the history of adaptation is complex, and adaptive explanations are often unsupported by hard evidence.

We have compared the accuracy, efficiency and robustness of three methods of genotyping single nucleotide polymorphisms on pooled DNAs. We conclude that (i) the frequencies of the two alleles in pools should be corrected with a factor for unequal allelic amplification, which should be estimated from the mean ratio of a set of heterozygotes (k); (ii) the repeatability of an assay is more important than pinpoint accuracy when estimating allele frequencies, and assays should therefore be optimised to increase the repeatability; and (iii) the size of a pool has a relatively small effect on the accuracy of allele frequency estimation. We therefore recommend that large pools are genotyped and replicated a minimum of four times. In addition, we describe statistical approaches to allow rigorous comparison of DNA pool results. Finally, we describe an extension to our ACeDB database that facilitates management and analysis of the data generated by association studies.

The male-specific region of the human Y chromosome (MSY) includes eight large inverted repeats (palindromes) in which arm-to-arm similarity exceeds 99.9%, due to gene conversion activity. Here, we studied one of these palindromes, P6, in order to illuminate the dynamics of the gene conversion process. We genotyped ten paralogous sequence variants (PSVs) within the arms of P6 in 378 Y chromosomes whose evolutionary relationships within the SNP-defined Y phylogeny are known. This allowed the identification of 146 historical gene conversion events involving individual PSVs, occurring at a rate of 2.9–8.4×10−4 events per generation. A consideration of the nature of nucleotide change and the ancestral state of each PSV showed that the conversion process was significantly biased towards the fixation of G or C nucleotides (GC-biased), and also towards the ancestral state. Determination of haplotypes by long-PCR allowed likely co-conversion of PSVs to be identified, and suggested that conversion tract lengths are large, with a mean of 2068 bp, and a maximum in excess of 9 kb. Despite the frequent formation of recombination intermediates implied by the rapid observed gene conversion activity, resolution via crossover is rare: only three inversions within P6 were detected in the sample. An analysis of chimpanzee and gorilla P6 orthologs showed that the ancestral state bias has existed in all three species, and comparison of human and chimpanzee sequences with the gorilla outgroup confirmed that GC bias of the conversion process has apparently been active in both the human and chimpanzee lineages., Author Summary The sex-determining role of the human Y chromosome makes it male-specific, and always present in only a single copy. This solo lifestyle has endowed it with some bizarre features, among which are eight large DNA units constituting about a quarter of the chromosome's length, and containing many genes important for sperm production. These units are called palindromes, since, taking into account the polarity of the DNA strands, the sequence is the same read from either end of the unit. We investigated the details of a process (gene conversion) that transfers sequence variants in one half of a palindrome into the other, thereby maintaining >99.9% similarity between the halves. We analysed patterns of sequence variants within one palindrome in a set of Y chromosomes whose evolutionary relationships are known. This allowed us to identify past gene conversion events, and to demonstrate a bias towards events that eliminate new variants, and retain old ones. Gene conversion has therefore acted during human evolution to retard sequence change in these regions. Analysis of the chimpanzee and gorilla versions of the palindrome shows that the dynamic processes we see in human Y chromosomes have a deep evolutionary history.

A report from HGM2001, the sixth annual International Human Genome Meeting organized by The Human Genome Organisation (HUGO), Edinburgh, UK, 19-22 April 2001.