The study of gene expression in mammalian single cells via genomic technologies now provides the possibility to investigate the patterns of allelic gene expression. We used single-cell RNA sequencing to detect the allele-specific mRNA level in 203 single human primary fibroblasts over 133,633 unique heterozygous single-nucleotide variants (hetSNVs). We observed that at the snapshot of analyses, each cell contained mostly transcripts from one allele from the majority of genes; indeed, 76.4% of the hetSNVs displayed stochastic monoallelic expression in single cells. Remarkably, adjacent hetSNVs exhibited a haplotype-consistent allelic ratio; in contrast, distant sites located in two different genes were independent of the haplotype structure. Moreover, the allele-specific expression in single cells correlated with the abundance of the cellular transcript. We observed that genes expressing both alleles in the majority of the single cells at a given time point were rare and enriched with highly expressed genes. The relative abundance of each allele in a cell was controlled by some regulatory mechanisms given that we observed related single-cell allelic profiles according to genes. Overall, these results have direct implications in cellular phenotypic variability.

Cohen Syndrome (COH1) is a rare autosomal recessive disorder, principally identified by ocular, neural and muscular deficits. We identified three large consanguineous Pakistani families with intellectual disability and in some cases with autistic traits.

Recurrent airway obstruction (RAO) is a common and potentially debilitating lower airway disease in horses, which shares many similarities with human asthma. In susceptible horses RAO exacerbation is caused by environmental allergens and irritants present in hay dust. The objective of this study was the identification of genes and pathways involved in the pathology of RAO by global transcriptome analyses in stimulated peripheral blood mononuclear cells (PBMCs). We performed RNA-seq on PBMCs derived from 40 RAO affected and 45 control horses belonging to three cohorts of Warmblood horses: two half-sib families and one group of unrelated horses. PBMCs were stimulated with hay dust extract, lipopolysaccharides, a recombinant parasite antigen, or left unstimulated. The total dataset consisted of 561 individual samples. We detected significant differences in the expression profiles between RAO and control horses. Differential expression (DE) was most marked upon stimulation with hay dust extract. An important novel finding was a strong upregulation of CXCL13 together with many genes involved in cell cycle regulation in stimulated samples from RAO affected horses, in addition to changes in the expression of several HIF-1 transcription factor target genes. The RAO condition alters systemic changes observed as differential expression profiles of PBMCs. Those changes also depended on the cohort and stimulation of the samples and were dominated by genes involved in immune cell trafficking, development, and cell cycle regulation. Our findings indicate an important role of CXCL13, likely macrophage or Th17 derived, and the cell cycle regulator CDC20 in the immune response in RAO.

DNA methylation is essential in mammalian development. We have hypothesized that methylation differences induced by trisomy 21 (T21) contribute to the phenotypic characteristics and heterogeneity in Down syndrome (DS). In order to determine the methylation differences in T21 without interference of the interindividual genomic variation, we have used fetal skin fibroblasts from monozygotic (MZ) twins discordant for T21. We also used skin fibroblasts from MZ twins concordant for T21, normal MZ twins without T21, and unrelated normal and T21 individuals. Reduced Representation Bisulfite Sequencing (RRBS) revealed 35 differentially methylated promoter regions (DMRs) (Absolute methylation differences = 25%, FDR < 0.001) in MZ twins discordant for T21 that have also been observed in comparison between unrelated normal and T21 individuals. The identified DMRs are enriched for genes involved in embryonic organ morphogenesis (FDR = 1.60 e -03) and include genes of the HOXB and HOXD clusters. These DMRs are maintained in iPS cells generated from this twin pair and are correlated with the gene expression changes. We have also observed an increase in DNA methylation level in the T21 methylome compared to the normal euploid methylome. This observation is concordant with the up regulation of DNA methyltransferase enzymes (DNMT3B and DNMT3L) and down regulation of DNA demethylation enzymes (TET2 and TET3) observed in the iPSC of the T21 versus normal twin. Altogether, the results of this study highlight the epigenetic effects of the extra chromosome 21 in T21 on loci outside of this chromosome that are relevant to DS associated phenotypes.

Complete transcriptomic data at high resolution are available only for a few model organisms with medical importance. The gene structures of non-model organisms are mostly computationally predicted based on comparative genomics with other species. As a result, more than half of the horse gene models are known only by projection. Experimental data supporting these gene models are scarce. Moreover, most of the annotated equine genes are single-transcript genes. Utilizing RNA sequencing (RNA-seq) the experimental validation of predicted transcriptomes has become accessible at reasonable costs. To improve the horse genome annotation we performed RNA-seq on 561 samples of peripheral blood mononuclear cells (PBMCs) derived from 85 Warmblood horses. The mapped sequencing reads were used to build a new transcriptome assembly. The new assembly revealed many alternative isoforms associated to known genes or to those predicted by the Ensembl and/or Gnomon pipelines. We also identified 7,531 transcripts not associated with any horse gene annotated in public databases. Of these, 3,280 transcripts did not have a homologous match to any sequence deposited in the NCBI EST database suggesting horse specificity. The unknown transcripts were categorized as coding and noncoding based on predicted coding potential scores. Among them 230 transcripts had high coding potential score, at least 2 exons, and an open reading frame of at least 300 nt. We experimentally validated 9 new equine coding transcripts using RT-PCR and Sanger sequencing. Our results provide valuable detailed information on many transcripts yet to be annotated in the horse genome.

Next-generation sequencing (NGS) of exomes and genomes has accelerated the identification of genes involved in Mendelian phenotypes. However, many NGS studies fall short of identifying causal variants, with estimates for success rates as low as 25% for uncovering the pathological variant underlying disease etiology. An important reason for such failures is familial locus heterogeneity, where within a single pedigree causal variants in two or more genes underlie Mendelian trait etiology. As examples of intra- and inter-sibship familial locus heterogeneity, we present 10 consanguineous Pakistani families segregating hearing impairment due to homozygous variants in two different hearing impairment genes and a European-American pedigree in which hearing impairment is caused by four variants in three different genes. We have identified 41 additional pedigrees with syndromic and nonsyndromic hearing impairment for which a single previously reported hearing impairment gene has been identified but only segregates with the phenotype in a subset of affected pedigree members. We estimate that locus heterogeneity occurs in 15.3% (95% confidence interval: 11.9%, 19.9%) of the families in our collection. We demonstrate novel approaches to apply linkage analysis and homozygosity mapping (for autosomal recessive consanguineous pedigrees), which can be used to detect locus heterogeneity using either NGS or SNP array data. Results from linkage analysis and homozygosity mapping can also be used to group sibships or individuals most likely to be segregating the same causal variants and thereby increase the success rate of gene identification.

Ectodermal dysplasias (EDs) are a large heterogeneous group of inherited disorders exhibiting abnormalities in ectodermally derived appendages such as hair, nails, teeth and sweat glands. EDs associated with reticulated pigmentation phenotype are rare entities for which the genetic basis and pathophysiology are not well characterized. The present study describes a five generation consanguineous Pakistani family segregating an autosomal recessive form of a novel type of ectodermal dysplasia. The affected members present with sparse and woolly hair, severe nail dystrophy and reticulate skin pigmentation. After exclusion of known gene loci related with other skin disorders, genome-wide linkage analysis was performed using Illumina HumanOmniExpress beadchip SNP arrays. We linked this form of ED to human chromosome 18p11.32-p11.31 flanked by the SNPs rs9284390 (0.113Mb) and rs4797100 (3.14 Mb). A maximum two-point LOD score of 3.3 was obtained with several markers along the disease interval. The linkage interval of 3.03 Mb encompassed seventeen functional genes. However, sequence analysis of all these genes did not discover any potentially disease causing-variants. The identification of this novel locus provides additional information regarding the mapping of a rare form of ED. Further research, such as the use of whole-genome sequencing, would be expected to reveal any pathogenic mutation within the disease locus.

The HSA21 encoded Single-minded 2 (SIM2) transcription factor has key neurological functions and is a good candidate to be involved in the cognitive impairment of Down syndrome. We aimed to explore the functional capacity of SIM2 by mapping its DNA binding sites in mouse embryonic stem cells. ChIP-sequencing revealed 1229 high-confidence SIM2-binding sites. Analysis of the SIM2 target genes confirmed the importance of SIM2 in developmental and neuronal processes and indicated that SIM2 may be a master transcription regulator. Indeed, SIM2 DNA binding sites share sequence specificity and overlapping domains of occupancy with master transcription factors such as SOX2, OCT4 (Pou5f1), NANOG or KLF4. The association between SIM2 and these pioneer factors is supported by co-immunoprecipitation of SIM2 with SOX2, OCT4, NANOG or KLF4. Furthermore, the binding of SIM2 marks a particular sub-category of enhancers known as super-enhancers. These regions are characterized by typical DNA modifications and Mediator co-occupancy (MED1 and MED12). Altogether, we provide evidence that SIM2 binds a specific set of enhancer elements thus explaining how SIM2 can regulate its gene network in neuronal features.

Developmental eye defects often severely reduce vision. Despite extensive efforts, for a substantial fraction of these cases the molecular causes are unknown. Recessive eye disorders are frequent in consanguineous populations and such large families with multiple affected individuals provide an opportunity to identify recessive causative genes. We studied a Pakistani consanguineous family with three affected individuals with congenital vision loss and progressive eye degeneration. The family was analyzed by exome sequencing of one affected individual and genotyping of all family members. We have identified a non-synonymous homozygous variant (NM_001128918.2: c.1708C > G: p.Arg570Gly) in the MARK3 gene as the likely cause of the phenotype. Given that MARK3 is highly conserved in flies (I: 55%; S: 67%) we knocked down the MARK3 homologue, par-1, in the eye during development. This leads to a significant reduction in eye size, a severe loss of photoreceptors and loss of vision based on electroretinogram (ERG) recordings. Expression of the par-1 p.Arg792Gly mutation (equivalent to the MARK3 variant found in patients) in developing fly eyes also induces loss of eye tissue and reduces the ERG signals. The data in flies and human indicate that the MARK3 variant corresponds to a loss of function. We conclude that the identified mutation in MARK3 establishes a new gene-disease link, since it likely causes structural abnormalities during eye development and visual impairment in humans, and that the function of MARK3/par-1 is evolutionarily conserved in eye development.

Tissue cross-talk is emerging as a determinant way to coordinate the different organs implicated in glucose homeostasis. Among the inter-organ communication factors, muscle-secreted myokines can modulate the function and survival of pancreatic beta-cells. Using primary human myotubes from soleus, vastus lateralis and triceps brachii muscles, we report here that the impact of myokines on beta-cells depends on fiber types and their metabolic status. We show that Type I and type II primary myotubes present specific mRNA and myokine signatures as well as a different sensitivity to TNF-alpha induced insulin resistance. Finally, we show that angiogenin and osteoprotegerin are triceps specific myokines with beta-cell protective actions against proinflammatory cytokines. These results suggest that type I and type II muscles could impact insulin secretion and beta-cell mass differentially in type 2 diabetes through specific myokines secretion.

Eukaryotic cells make many types of primary and processed RNAs that are found either in specific subcellular compartments or throughout the cells. A complete catalogue of these RNAs is not yet available and their characteristic subcellular localizations are also poorly understood. Because RNA represents the direct output of the genetic information encoded by genomes and a significant proportion of a cell's regulatory capabilities are focused on its synthesis, processing, transport, modification and translation, the generation of such a catalogue is crucial for understanding genome function. Here we report evidence that three-quarters of the human genome is capable of being transcribed, as well as observations about the range and levels of expression, localization, processing fates, regulatory regions and modifications of almost all currently annotated and thousands of previously unannotated RNAs. These observations, taken together, prompt a redefinition of the concept of a gene.

Inherited deafness is clinically and genetically heterogeneous. We recently mapped DFNB86, a locus associated with nonsyndromic deafness, to chromosome 16p. In this study, whole-exome sequencing was performed with genomic DNA from affected individuals from three large consanguineous families in which markers linked to DFNB86 segregate with profound deafness. Analyses of these data revealed homozygous mutation c.208G>T (p.Asp70Tyr) or c.878G>C (p.Arg293Pro) in TBC1D24 as the underlying cause of deafness in the three families. Sanger sequence analysis of TBC1D24 in an additional large family in which deafness segregates with DFNB86 identified the c.208G>T (p.Asp70Tyr) substitution. These mutations affect TBC1D24 amino acid residues that are conserved in orthologs ranging from fruit fly to human. Neither variant was observed in databases of single-nucleotide variants or in 634 chromosomes from ethnically matched control subjects. TBC1D24 in the mouse inner ear was immunolocalized predominantly to spiral ganglion neurons, indicating that DFNB86 deafness might be an auditory neuropathy spectrum disorder. Previously, six recessive mutations in TBC1D24 were reported to cause seizures (hearing loss was not reported) ranging in severity from epilepsy with otherwise normal development to epileptic encephalopathy resulting in childhood death. Two of our four families in which deafness segregates with mutant alleles of TBC1D24 were available for neurological examination. Cosegregation of epilepsy and deafness was not observed in these two families. Although the causal relationship between genotype and phenotype is not presently understood, our findings, combined with published data, indicate that recessive alleles of TBC1D24 can cause either epilepsy or nonsyndromic deafness.

Using an integrative approach in which genetic variation, gene expression, and clinical phenotypes are assessed in relevant tissues may help functionally characterize the contribution of genetics to disease susceptibility. We sought to identify genetic variation influencing skeletal muscle gene expression (expression quantitative trait loci [eQTLs]) as well as expression associated with measures of insulin sensitivity. We investigated associations of 3,799,401 genetic variants in expression of >7,000 genes from three cohorts (n = 104). We identified 287 genes with cis-acting eQTLs (false discovery rate [FDR] <5%; P < 1.96 × 10(-5)) and 49 expression-insulin sensitivity phenotype associations (i.e., fasting insulin, homeostasis model assessment-insulin resistance, and BMI) (FDR <5%; P = 1.34 × 10(-4)). One of these associations, fasting insulin/phosphofructokinase (PFKM), overlaps with an eQTL. Furthermore, the expression of PFKM, a rate-limiting enzyme in glycolysis, was nominally associated with glucose uptake in skeletal muscle (P = 0.026; n = 42) and overexpressed (Bonferroni-corrected P = 0.03) in skeletal muscle of patients with T2D (n = 102) compared with normoglycemic controls (n = 87). The PFKM eQTL (rs4547172; P = 7.69 × 10(-6)) was nominally associated with glucose uptake, glucose oxidation rate, intramuscular triglyceride content, and metabolic flexibility (P = 0.016-0.048; n = 178). We explored eQTL results using published data from genome-wide association studies (DIAGRAM and MAGIC), and a proxy for the PFKM eQTL (rs11168327; r(2) = 0.75) was nominally associated with T2D (DIAGRAM P = 2.7 × 10(-3)). Taken together, our analysis highlights PFKM as a potential regulator of skeletal muscle insulin sensitivity.

Hypophosphatemic rickets (HR) comprises a rare group of inherited diseases. Very recently, mutations in the dentin matrix protein 1 (DMP1) gene were identified in patients with an extremely rare autosomal recessive form of HR (ARHR). To date, very few cases of these mutations were reported.

Gene expression is a heritable cellular phenotype that defines the function of a cell and can lead to diseases in case of misregulation. In order to detect genetic variations affecting gene expression, we performed association analysis of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) with gene expression measured in 869 lymphoblastoid cell lines of the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort in cis and in trans. We discovered that 3,534 genes (false discovery rate (FDR) = 5%) are affected by an expression quantitative trait locus (eQTL) in cis and 48 genes are affected in trans. We observed that CNVs are more likely to be eQTLs than SNPs. In addition, we found that variants associated to complex traits and diseases are enriched for trans-eQTLs and that trans-eQTLs are enriched for cis-eQTLs. As a variant affecting both a gene in cis and in trans suggests that the cis gene is functionally linked to the trans gene expression, we looked specifically for trans effects of cis-eQTLs. We discovered that 26 cis-eQTLs are associated to 92 genes in trans with the cis-eQTLs of the transcriptions factors BATF3 and HMX2 affecting the most genes. We then explored if the variation of the level of expression of the cis genes were causally affecting the level of expression of the trans genes and discovered several causal relationships between variation in the level of expression of the cis gene and variation of the level of expression of the trans gene. This analysis shows that a large sample size allows the discovery of secondary effects of human variations on gene expression that can be used to construct short directed gene regulatory networks.

Down syndrome (DS) is due to increased copy number of human chromosome 21. The contribution of different genetic regions has been tested using mouse models. As shown previously, the Abcg1-U2af1 genetic region contributes to cognitive defects in working and short-term recognition memory in Down syndrome mouse models. Here we analyzed the impact of monosomy of the same genetic interval, using a new mouse model, named Ms2Yah. We used several cognitive paradigms and did not detect defects in the object recognition or the Morris water maze tests. However, surprisingly, Ms2Yah mice displayed increased associative memory in a pure contextual fear-conditioning test and decreased social novelty interaction along with a larger long-term potentiation recorded in the CA1 area following stimulation of Schaffer collaterals. Whole-genome expression studies carried out on hippocampus showed that the transcription of only a small number of genes is affected, mainly from the genetic interval (Cbs, Rsph1, Wdr4), with a few additional ones, including the postsynaptic Gabrr2, Gabbr1, Grid2p, Park2, and Dlg1 and the components of the Ubiquitin-mediated proteolysis (Anapc1, Rnf7, Huwe1, Park2). The Abcg1-U2af1 region is undeniably encompassing dosage-sensitive genes or elements whose change in copy number directly affects learning and memory, synaptic function, and autistic related behavior.

Epigenetic modifications such as DNA methylation play a key role in gene regulation and disease susceptibility. However, little is known about the genome-wide frequency, localization, and function of methylation variation and how it is regulated by genetic and environmental factors. We utilized the Multiple Tissue Human Expression Resource (MuTHER) and generated Illumina 450K adipose methylome data from 648 twins. We found that individual CpGs had low variance and that variability was suppressed in promoters. We noted that DNA methylation variation was highly heritable (h(2)median = 0.34) and that shared environmental effects correlated with metabolic phenotype-associated CpGs. Analysis of methylation quantitative-trait loci (metQTL) revealed that 28% of CpGs were associated with nearby SNPs, and when overlapping them with adipose expression quantitative-trait loci (eQTL) from the same individuals, we found that 6% of the loci played a role in regulating both gene expression and DNA methylation. These associations were bidirectional, but there were pronounced negative associations for promoter CpGs. Integration of metQTL with adipose reference epigenomes and disease associations revealed significant enrichment of metQTL overlapping metabolic-trait or disease loci in enhancers (the strongest effects were for high-density lipoprotein cholesterol and body mass index [BMI]). We followed up with the BMI SNP rs713586, a cg01884057 metQTL that overlaps an enhancer upstream of ADCY3, and used bisulphite sequencing to refine this region. Our results showed widespread population invariability yet sequence dependence on adipose DNA methylation but that incorporating maps of regulatory elements aid in linking CpG variation to gene regulation and disease risk in a tissue-dependent manner.

Transcript co-expression is regulated by a combination of shared genetic and environmental factors. Here, we estimate the proportion of co-expression that is due to shared genetic variance. To do so, we estimated the genetic correlations between each pairwise combination of 2469 transcripts that are highly heritable and expressed in whole blood in 1748 unrelated individuals of European ancestry. We identify 556 pairs with a significant genetic correlation of which 77% are located on different chromosomes, and report 934 expression quantitative trait loci, identified in an independent cohort, with significant effects on both transcripts in a genetically correlated pair. We show significant enrichment for transcription factor control and physical proximity through chromatin interactions as possible mechanisms of shared genetic control. Finally, we construct networks of interconnected transcripts and identify their underlying biological functions. Using genetic correlations to investigate transcriptional co-regulation provides valuable insight into the nature of the underlying genetic architecture of gene regulation.Covariance of gene expression pairs is due to a combination of shared genetic and environmental factors. Here the authors estimate the genetic correlation between highly heritable pairs and identify transcription factor control and chromatin interactions as possible mechanisms of correlation.

The importance of natural gene expression variation for human behavior is undisputed, but its impact on circadian physiology remains mostly unexplored. Using umbilical cord fibroblasts, we have determined by genome-wide association how common genetic variation impacts upon cellular circadian function. Gene set enrichment points to differences in protein catabolism as one major source of clock variation in humans. The two most significant alleles regulated expression of COPS7B, a subunit of the COP9 signalosome. We further show that the signalosome complex is imported into the nucleus in timed fashion to stabilize the essential circadian protein BMAL1, a novel mechanism to oppose its proteasome-mediated degradation. Thus, circadian clock properties depend in part upon a genetically-encoded competition between stabilizing and destabilizing forces, and genetic alterations in these mechanisms provide one explanation for human chronotype.

There is increasing evidence that heritable variation in gene expression underlies genetic variation in susceptibility to disease. Therefore, a comprehensive understanding of the similarity between relatives for transcript variation is warranted--in particular, dissection of phenotypic variation into additive and non-additive genetic factors and shared environmental effects. We conducted a gene expression study in blood samples of 862 individuals from 312 nuclear families containing MZ or DZ twin pairs using both pedigree and genotype information. From a pedigree analysis we show that the vast majority of genetic variation across 17,994 probes is additive, although non-additive genetic variation is identified for 960 transcripts. For 180 of the 960 transcripts with non-additive genetic variation, we identify expression quantitative trait loci (eQTL) with dominance effects in a sample of 339 unrelated individuals and replicate 31% of these associations in an independent sample of 139 unrelated individuals. Over-dominance was detected and replicated for a trans association between rs12313805 and ETV6, located 4MB apart on chromosome 12. Surprisingly, only 17 probes exhibit significant levels of common environmental effects, suggesting that environmental and lifestyle factors common to a family do not affect expression variation for most transcripts, at least those measured in blood. Consistent with the genetic architecture of common diseases, gene expression is predominantly additive, but a minority of transcripts display non-additive effects.