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The velo-cardio-facial syndrome (VCFS) is caused by hemizygous deletions on chromosome 22q11.2. The VCFS phenotype is complex and characterized by frequent occurrence of neuropsychiatric symptoms with up to 25–30% of cases suffering from psychotic disorders compared with only ~1% in the general population (odds ratio≈20–25). This makes the 22q11.2 deletion one of the most prominent risk factors for schizophrenia. However, its penetrance for neuropsychiatric phenotypes is incomplete suggesting that additional risk factors are required for disease development. These additional risk factors could lie anywhere on the genome, but by reducing the normal diploid to a haploid state, the 22q11.2 deletion could result in the unmasking of otherwise recessive alleles or functional variants on the non-deleted 22q11.2 allele. To test this hypothesis, we captured and sequenced the whole 22q11.2 non-deleted region in 88 VCFS patients with (n=40) and without (n=48) psychotic disorders to identify genetic variation that could increase the risk for schizophrenia. Single nucleotide variants (SNVs), small insertions/deletions (indels) and copy number variants were called and their distributions were compared between the two diagnostic groups using variant-, gene- and region-based association tests. None of these tests resulted in statistical evidence for the existence of a genetic variation in the non-deleted allele that would increase schizophrenia risk in VCFS patients. Power analysis showed that our study was able to achieve >80% statistical power to detect association of a risk variant with an odd ratio of ⩾22. However, it is certainly under-powered to detect risk variant of smaller effect sizes. Our study did not provide evidence that genetic variants of very large effect size located on the non-deleted 22q1.2 allele in VCFS patients increase the risk for developing psychotic disorders. Variants with smaller effects may be located in the remaining 22q11.2 allele and elsewhere in the genome. Therefore, whole exome or even genome sequencing for larger sample size would appear to be the next logical steps in the search for the genetic modifiers of the 22q11.2-deletion neuropsychiatric phenotype.

MicroRNAs (miRNAs) are regulatory noncoding RNAs that affect the production of a significant fraction of human mRNAs via post-transcriptional regulation. Interindividual variation of the miRNA expression levels is likely to influence the expression of miRNA target genes and may therefore contribute to phenotypic differences in humans, including susceptibility to common disorders. The extent to which miRNA levels are genetically controlled is largely unknown. In this report, we assayed the expression levels of miRNAs in primary fibroblasts from 180 European newborns of the GenCord project and performed association analysis to identify eQTLs (expression quantitative traits loci). We detected robust expression for 121 miRNAs out of 365 interrogated. We have identified significant cis- (10%) and trans- (11%) eQTLs. Furthermore, we detected one genomic locus (rs1522653) that influences the expression levels of five miRNAs, thus unraveling a novel mechanism for coregulation of miRNA expression.

Understanding how genetic variation affects distinct cellular phenotypes, such as gene expression levels, alternative splicing and DNA methylation levels, is essential for better understanding of complex diseases and traits. Furthermore, how inter-individual variation of DNA methylation is associated to gene expression is just starting to be studied. In this study, we use the GenCord cohort of 204 newborn Europeans' lymphoblastoid cell lines, T-cells and fibroblasts derived from umbilical cords. The samples were previously genotyped for 2.5 million SNPs, mRNA-sequenced, and assayed for methylation levels in 482,421 CpG sites. We observe that methylation sites associated to expression levels are enriched in enhancers, gene bodies and CpG island shores. We show that while the correlation between DNA methylation and gene expression can be positive or negative, it is very consistent across cell-types. However, this epigenetic association to gene expression appears more tissue-specific than the genetic effects on gene expression or DNA methylation (observed in both sharing estimations based on P-values and effect size correlations between cell-types). This predominance of genetic effects can also be reflected by the observation that allele specific expression differences between individuals dominate over tissue-specific effects. Additionally, we discover genetic effects on alternative splicing and interestingly, a large amount of DNA methylation correlating to alternative splicing, both in a tissue-specific manner. The locations of the SNPs and methylation sites involved in these associations highlight the participation of promoter proximal and distant regulatory regions on alternative splicing. Overall, our results provide high-resolution analyses showing how genome sequence variation has a broad effect on cellular phenotypes across cell-types, whereas epigenetic factors provide a secondary layer of variation that is more tissue-specific. Furthermore, the details of how this tissue-specificity may vary across inter-relations of molecular traits, and where these are occurring, can yield further insights into gene regulation and cellular biology as a whole.

Inter-individual differences in gene expression are likely to account for an important fraction of phenotypic differences, including susceptibility to common disorders. Recent studies have shown extensive variation in gene expression levels in humans and other organisms, and that a fraction of this variation is under genetic control. We investigated the patterns of gene expression variation in a 25 Mb region of human chromosome 21, which has been associated with many Down syndrome (DS) phenotypes. Taqman real-time PCR was used to measure expression variation of 41 genes in lymphoblastoid cells of 40 unrelated individuals. For 25 genes found to be differentially expressed, additional analysis was performed in 10 CEPH families to determine heritabilities and map loci harboring regulatory variation. Seventy-six percent of the differentially expressed genes had significant heritabilities, and genomewide linkage analysis led to the identification of significant eQTLs for nine genes. Most eQTLs were in trans, with the best result (P=7.46 x 10(-8)) obtained for TMEM1 on chromosome 12q24.33. A cis-eQTL identified for CCT8 was validated by performing an association study in 60 individuals from the HapMap project. SNP rs965951 located within CCT8 was found to be significantly associated with its expression levels (P=2.5 x 10(-5)) confirming cis-regulatory variation. The results of our study provide a representative view of expression variation of chromosome 21 genes, identify loci involved in their regulation and suggest that genes, for which expression differences are significantly larger than 1.5-fold in control samples, are unlikely to be involved in DS-phenotypes present in all affected individuals.

Trisomy 21 is the most frequent genetic cause of cognitive impairment. To assess the perturbations of gene expression in trisomy 21, and to eliminate the noise of genomic variability, we studied the transcriptome of fetal fibroblasts from a pair of monozygotic twins discordant for trisomy 21. Here we show that the differential expression between the twins is organized in domains along all chromosomes that are either upregulated or downregulated. These gene expression dysregulation domains (GEDDs) can be defined by the expression level of their gene content, and are well conserved in induced pluripotent stem cells derived from the twins' fibroblasts. Comparison of the transcriptome of the Ts65Dn mouse model of Down's syndrome and normal littermate mouse fibroblasts also showed GEDDs along the mouse chromosomes that were syntenic in human. The GEDDs correlate with the lamina-associated (LADs) and replication domains of mammalian cells. The overall position of LADs was not altered in trisomic cells; however, the H3K4me3 profile of the trisomic fibroblasts was modified and accurately followed the GEDD pattern. These results indicate that the nuclear compartments of trisomic cells undergo modifications of the chromatin environment influencing the overall transcriptome, and that GEDDs may therefore contribute to some trisomy 21 phenotypes.

Understanding the molecular basis of cardiomyocyte development is critical for understanding the pathogenesis of pre- and post-natal cardiac disease. MicroRNAs (miRNAs) are post-transcriptional modulators of gene expression that play an important role in many developmental processes. Here, we show that the miR-99a/let-7c cluster, mapping on human chromosome 21, is involved in the control of cardiomyogenesis by altering epigenetic factors. By perturbing miRNA expression in mouse embryonic stem cells, we find that let-7c promotes cardiomyogenesis by upregulating genes involved in mesoderm specification (T/Bra and Nodal) and cardiac differentiation (Mesp1, Nkx2.5 and Tbx5). The action of let-7c is restricted to the early phase of mesoderm formation at the expense of endoderm and its late activation redirects cells toward other mesodermal derivatives. The Polycomb complex group protein Ezh2 is a direct target of let-7c, which promotes cardiac differentiation by modifying the H3K27me3 marks from the promoters of crucial cardiac transcription factors (Nkx2.5, Mef2c, Tbx5). In contrast, miR-99a represses cardiac differentiation via the nucleosome-remodeling factor Smarca5, attenuating the Nodal/Smad2 signaling. We demonstrated that the identified targets are underexpressed in human Down syndrome fetal heart specimens. By perturbing the expression levels of these miRNAs in embryonic stem cells, we were able to demonstrate that these miRNAs control lineage- and stage-specific transcription factors, working in concert with chromatin modifiers to direct cardiomyogenesis.

This study aimed to elucidate the observed variable phenotypic expressivity associated with NRXN1 (Neurexin 1) haploinsufficiency by analyses of the largest cohort of patients with NRXN1 exonic deletions described to date and by comprehensively reviewing all comparable copy number variants in all disease cohorts that have been published in the peer reviewed literature (30 separate papers in all). Assessment of the clinical details in 25 previously undescribed individuals with NRXN1 exonic deletions demonstrated recurrent phenotypic features consisting of moderate to severe intellectual disability (91%), severe language delay (81%), autism spectrum disorder (65%), seizures (43%), and hypotonia (38%). These showed considerable overlap with previously reported NRXN1-deletion associated phenotypes in terms of both spectrum and frequency. However, we did not find evidence for an association between deletions involving the -isoform of neurexin-1 and increased head size, as was recently published in four cases with a deletion involving the C-terminus of NRXN1. We identified additional rare copy number variants in 20% of cases. This study supports a pathogenic role for heterozygous exonic deletions of NRXN1 in neurodevelopmental disorders. The additional rare copy number variants identified may act as possible phenotypic modifiers as suggested in a recent digenic model of neurodevelopmental disorders. (c) 2013 Wiley Periodicals, Inc.

Variations occur throughout our genome. These variations can cause genes to be expressed (switched on) in slightly different ways among individuals. Moreover, the same gene can also be expressed in different ways in different cells within an individual. A third level of variation is supplied by epigenetic markers: these are molecules that bind to the DNA at specific points and can have profound effects on the expression of nearby genes. One such epigenetic marker is the addition of a methyl group to a cytosine base, a process that is known as DNA methylation. DNA methylation usually happens when a cytosine base is next to a guanine base, forming a CpG site. In mammals, most CpG sites have methyl groups attached, although regions with a lot of CpG sites (called CpG islands) are mostly unmethylated. Initial studies suggested that methylation prevented particular genes from being expressed, but more recent work has indicated that methylation can be associated with both reduced and increased expression of genes. Moreover, it is not clear if this association is active (i.e., changes in methylation drive changes in gene expression) or passive (DNA methylation is the result of gene regulation). Now, Gutierrez-Arcelus et al. have carried out a large-scale study to clarify the relationships between three different types of gene-related variations among individuals. They extracted fibroblasts, T-cells and lymphoblastoid cells from the umbilical cords of 204 babies, and analysed them for variations in DNA sequence, gene expression and DNA methylation. Their results show that the associations between the three are more complex than was previously thought. Gutierrez-Arcelus et al. show that the mechanisms that control the association between the variations in DNA methylation and gene expression in individuals are likely to be different to those that are responsible for the establishment of methylation patterns during the process of cell differentiation. They also find that the association between DNA methylation and gene expression can be either active or passive, and can depend on the context in which they occur in our genome. Finally, where the two copies or alleles of a gene are not equally expressed in a given cell, the difference in expression is primarily regulated by DNA sequence variation, with DNA methylation having little or no role on its own. Equally complex interactions and effects are expected in further studies of genetic and epigenetic variation.

Association studies have revealed expression quantitative trait loci (eQTLs) for a large number of genes. However, the causative variants that regulate gene expression levels are generally unknown. We hypothesized that copy-number variation of sequence repeats contribute to the expression variation of some genes. Our laboratory has previously identified that the rare expansion of a repeat c.-174CGGGGCGGGGCG in the promoter region of the CSTB gene causes a silencing of the gene, resulting in progressive myoclonus epilepsy. Here, we genotyped the repeat length and quantified CSTB expression by quantitative real-time polymerase chain reaction in 173 lymphoblastoid cell lines (LCLs) and fibroblast samples from the GenCord collection. The majority of alleles contain either two or three copies of this repeat. Independent analysis revealed that the c.-174CGGGGCGGGGCG repeat length is strongly associated with CSTB expression (P = 3.14 × 10(-11)) in LCLs only. Examination of both genotyped and imputed single-nucleotide polymorphisms (SNPs) within 2 Mb of CSTB revealed that the dodecamer repeat represents the strongest cis-eQTL for CSTB in LCLs. We conclude that the common two or three copy variation is likely the causative cis-eQTL for CSTB expression variation. More broadly, we propose that polymorphic tandem repeats may represent the causative variation of a fraction of cis-eQTLs in the genome.

Background Omphalocele is a congenital birth defect characterised by the presence of internal organs located outside of the ventral abdominal wall. The purpose of this study was to identify the underlying genetic mechanisms of a large autosomal dominant Caucasian family with omphalocele. Methods and findings A genetic linkage study was conducted in a large family with an autosomal dominant transmission of an omphalocele using a genome-wide single nucleotide polymorphism (SNP) array. The analysis revealed significant evidence of linkage (non-parametric NPL = 6.93, p=0.0001; parametric logarithm of odds (LOD) = 2.70 under a fully penetrant dominant model) at chromosome band 1p31.3. Haplotype analysis narrowed the locus to a 2.74 Mb region between markers rs2886770 (63014807 bp) and rs1343981 (65757349 bp). Molecular characterisation of this interval using array comparative genomic hybridisation followed by quantitative microsphere hybridisation analysis revealed a 710 kb duplication located at 63.5–64.2 Mb. All affected individuals who had an omphalocele and shared the haplotype were positive for this duplicated region, while the duplication was absent from all normal individuals of this family. Multipoint linkage analysis using the duplication as a marker yielded a maximum LOD score of 3.2 at 1p31.3 under a dominant model. The 710 kb duplication at 1p31.3 band contains seven known genes including FOXD3 , ALG6 , ITGB3BP , KIAA1799 , DLEU2L , PGM1 , and the proximal portion of ROR1 . Importantly, this duplication is absent from the database of genomic variants. Conclusions The present study suggests that development of an omphalocele in this family is controlled by overexpression of one or more genes in the duplicated region. To the authors9 knowledge, this is the first reported association of an inherited omphalocele condition with a chromosomal rearrangement.

Nature 508, 345–350 (2014); doi:10.1038/nature13200 Owing to a labelling error in the input files, one of the two replicate data sets used for Fig. 5d and e and Supplementary Fig. 6d of this Article was incorrect. We have now repeated the analysis with a correct, independent replicate experiment. This confirms our previous conclusion that there are no detectable differences in nuclear lamina interactions between the normal and trisomy 21 twin cells.

The third author, Reedik Magi, should be noted as affiliated with: Estonian Biocentre, Tartu, Estonia, and the Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.

Using principal component (PC) analysis, we studied the genetic constitution of 3,112 individuals from Europe as portrayed by more than 270,000 single nucleotide polymorphisms (SNPs) genotyped with the Illumina Infinium platform. In cohorts where the sample size was >100, one hundred randomly chosen samples were used for analysis to minimize the sample size effect, resulting in a total of 1,564 samples. This analysis revealed that the genetic structure of the European population correlates closely with geography. The first two PCs highlight the genetic diversity corresponding to the northwest to southeast gradient and position the populations according to their approximate geographic origin. The resulting genetic map forms a triangular structure with a) Finland, b) the Baltic region, Poland and Western Russia, and c) Italy as its vertexes, and with d) Central- and Western Europe in its centre. Inter- and intra- population genetic differences were quantified by the inflation factor lambda (lambda) (ranging from 1.00 to 4.21), fixation index (F(st)) (ranging from 0.000 to 0.023), and by the number of markers exhibiting significant allele frequency differences in pair-wise population comparisons. The estimated lambda was used to assess the real diminishing impact to association statistics when two distinct populations are merged directly in an analysis. When the PC analysis was confined to the 1,019 Estonian individuals (0.1\% of the Estonian population), a fine structure emerged that correlated with the geography of individual counties. With at least two cohorts available from several countries, genetic substructures were investigated in Czech, Finnish, German, Estonian and Italian populations. Together with previously published data, our results allow the creation of a comprehensive European genetic map that will greatly facilitate inter-population genetic studies including genome wide association studies (GWAS).

Animal microRNAs (miRNAs) regulate gene expression through base pairing to their targets within the 3' untranslated region (UTR) of protein-coding genes. Single-nucleotide polymorphisms (SNPs) located within such target sites can affect miRNA regulation. We mapped annotated SNPs onto a collection of experimentally supported human miRNA targets. Of the 143 experimentally supported human target sites, 9 contain 12 SNPs. We further experimentally investigated one of these target sites for hsa-miR-155, within the 3' UTR of the human AGTR1 gene that contains SNP rs5186. Using reporter silencing assays, we show that hsa-miR-155 down-regulates the expression of only the 1166A, and not the 1166C, allele of rs5186. Remarkably, the 1166C allele has been associated with hypertension in many studies. Thus, the 1166C allele may be functionally associated with hypertension by abrogating regulation by hsa-miR-155, thereby elevating AGTR1 levels. Since hsa-miR-155 is on chromosome 21, we hypothesize that the observed lower blood pressure in trisomy 21 is partially caused by the overexpression of hsa-miR-155 leading to allele-specific underexpression of AGTR1. Indeed, we have shown in fibroblasts from monozygotic twins discordant for trisomy 21 that levels of AGTR1 protein are lower in trisomy 21.

Down syndrome (DS) is characterized by extensive phenotypic variability, with most traits occurring in only a fraction of affected individuals. Substantial gene-expression variation is present among unaffected individuals, and this variation has a strong genetic component. Since DS is caused by genomic-dosage imbalance, we hypothesize that gene-expression variation of human chromosome 21 (HSA21) genes in individuals with DS has an impact on the phenotypic variability among affected individuals. We studied gene-expression variation in 14 lymphoblastoid and 17 fibroblast cell lines from individuals with DS and an equal number of controls. Gene expression was assayed using quantitative real-time polymerase chain reaction on 100 and 106 HSA21 genes and 23 and 26 non-HSA21 genes in lymphoblastoid and fibroblast cell lines, respectively. Surprisingly, only 39% and 62% of HSA21 genes in lymphoblastoid and fibroblast cells, respectively, showed a statistically significant difference between DS and normal samples, although the average up-regulation of HSA21 genes was close to the expected 1.5-fold in both cell types. Gene-expression variation in DS and normal samples was evaluated using the Kolmogorov-Smirnov test. According to the degree of overlap in expression levels, we classified all genes into 3 groups: (A) nonoverlapping, (B) partially overlapping, and (C) extensively overlapping expression distributions between normal and DS samples. We hypothesize that, in each cell type, group A genes are the most dosage sensitive and are most likely involved in the constant DS traits, group B genes might be involved in variable DS traits, and group C genes are not dosage sensitive and are least likely to participate in DS pathological phenotypes. This study provides the first extensive data set on HSA21 gene-expression variation in DS and underscores its role in modulating the outcome of gene-dosage imbalance.

Natural variation in DNA sequence contributes to individual differences in quantitative traits. While multiple studies have shown genetic control over gene expression variation, few additional cellular traits have been investigated. Here, we investigated the natural variation of NADPH oxidase-dependent hydrogen peroxide (H(2)O(2) release), which is the joint effect of reactive oxygen species (ROS) production, superoxide metabolism and degradation, and is related to a number of human disorders. We assessed the normal variation of H(2)O(2) release in lymphoblastoid cell lines (LCL) in a family-based 3-generation cohort (CEPH-HapMap), and in 3 population-based cohorts (KORA, GenCord, HapMap). Substantial individual variation was observed, 45% of which were associated with heritability in the CEPH-HapMap cohort. We identified 2 genome-wide significant loci of Hsa12 and Hsa15 in genome-wide linkage analysis. Next, we performed genome-wide association study (GWAS) for the combined KORA-GenCord cohorts (n = 279) using enhanced marker resolution by imputation (>1.4 million SNPs). We found 5 significant associations (p

The elucidation of the largely unknown transcriptome of small RNAs is crucial for the understanding of genome and cellular function. We report here the results of the analysis of small RNAs (< 50 nt) in the ENCODE regions of the human genome. Size-fractionated RNAs from four different cell lines (HepG2, HelaS3, GM06990, SK-N-SH) were mapped with the forward and reverse ENCODE high-density resolution tiling arrays. The top 1% of hybridization signals are termed SmRfrags (Small RNA fragments). Eight percent of SmRfrags overlap the GENCODE genes (CDS), given that the majority map to intergenic regions (34%), intronic regions (53%), and untranslated regions (UTRs) (5%). In addition, 9.6% and 16.8% of SmRfrags in the 5' UTR regions overlap significantly with His/Pol II/TAF250 binding sites and DNase I Hypersensitive sites, respectively (compared to the 5.3% and 9% expected). Interestingly, 17%-24% (depending on the cell line) of SmRfrags are sense-antisense strand pairs that show evidence of overlapping transcription. Only 3.4% and 7.2% of SmRfrags in intergenic regions overlap transcribed fragments (Txfrags) in HeLa and GM06990 cell lines, respectively. We hypothesized that a fraction of the identified SmRfrags corresponded to microRNAs. We tested by Northern blot a set of 15 high-likelihood predictions of microRNA candidates that overlap with smRfrags and validated three potential microRNAs ( approximately 20 nt length). Notably, most of the remaining candidates showed a larger hybridizing band ( approximately 100 nt) that could be a microRNA precursor. The small RNA transcriptome is emerging as an important and abundant component of the genome function.