Detection of Genomic Variation by Selection of a 9 Mb DNA Region and High Throughput Sequencing

Detection of the rare polymorphisms and causative mutations of genetic diseases in a targeted genomic area has become a major goal in order to understand genomic and phenotypic variability. We have interrogated repeat-masked regions of 8.9 Mb on human chromosomes 21 (7.8 Mb) and 7 (1.1 Mb) from an individual from the International HapMap Project (NA12872). We have optimized a method of genomic selection for high throughput sequencing. Microarray-based selection and sequencing resulted in 260-fold enrichment, with 41% of reads mapping to the target region. 83% of SNPs in the targeted region had at least 4-fold sequence coverage and 54% at least 15-fold. When assaying HapMap SNPs in NA12872, our sequence genotypes are 91.3% concordant in regions with coverage≥4-fold, and 97.9% concordant in regions with coverage≥15-fold. About 81% of the SNPs recovered with both thresholds are listed in dbSNP. We observed that regions with low sequence coverage occur in close proximity to low-complexity DNA. Validation experiments using Sanger sequencing were performed for 46 SNPs with 15-20 fold coverage, with a confirmation rate of 96%, suggesting that DNA selection provides an accurate and cost-effective method for identifying rare genomic variants

Chromosome Conformation Capture Uncovers Potential Genome-Wide Interactions between Human Conserved Non-Coding Sequences

Comparative analyses of various mammalian genomes have identified numerous conserved non-coding (CNC) DNA elements that display striking conservation among species, suggesting that they have maintained specific functions throughout evolution. CNC function remains poorly understood, although recent studies have identified a role in gene regulation. We hypothesized that the identification of genomic loci that interact physically with CNCs would provide information on their functions. We have used circular chromosome conformation capture (4C) to characterize interactions of 10 CNCs from human chromosome 21 in K562 cells. The data provide evidence that CNCs are capable of interacting with loci that are enriched for CNCs. The number of trans interactions varies among CNCs; some show interactions with many loci, while others interact with few. Some of the tested CNCs are capable of driving the expression of a reporter gene in the mouse embryo, and associate with the oligodendrocyte genes OLIG1 and OLIG2. Our results underscore the power of chromosome conformation capture for the identification of targets of functional DNA elements and raise the possibility that CNCs exert their functions by physical association with defined genomic regions enriched in CNCs. These CNC-CNC interactions may in part explain their stringent conservation as a group of regulatory sequences

Gene expression variation and expression quantitative trait mapping of human chromosome 21 genes

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=7.46×10−8) 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=2.5×10−5) 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 individual

Galanin pathogenic mutations in temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is a common epilepsy syndrome with a complex etiology. Despite evidence for the participation of genetic factors, the genetic basis of TLE remains largely unknown. A role for the galanin neuropeptide in the regulation of epileptic seizures has been established in animal models more than two decades ago. However, until now there was no report of pathogenic mutations in GAL, the galanin-encoding gene, and therefore its role in human epilepsy was not established. Here, we studied a family with a pair of monozygotic twins affected by TLE and two unaffected siblings born to healthy parents. Exome sequencing revealed that both twins carried a novel de novo mutation (p.A39E) in the GAL gene. Functional analysis revealed that the p.A39E mutant showed antagonistic activity against galanin receptor 1 (GalR1)-mediated response, and decreased binding affinity and reduced agonist properties for GalR2. These findings suggest that the p.A39E mutant could impair galanin signaling in the hippocampus, leading to increased glutamatergic excitation and ultimately to TLE. In a cohort of 582 cases, we did not observe any pathogenic mutations indicating that mutations in GAL are a rare cause of TLE. The identification of a novel de novo mutation in a biologically-relevant candidate gene, coupled with functional evidence that the mutant protein disrupts galanin signaling, strongly supports GAL as the causal gene for the TLE in this family. Given the availability of galanin agonists which inhibit seizures, our findings could potentially have direct implications for the development of anti-epileptic treatmen

X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3

By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2–DNAAF4–HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins

Gene Expression From the Aneuploid Chromosome in a Trisomy Mouse Model of Down Syndrome

Trisomy 21 is the prototype of human aneuploidies. Since its discovery in 1959, the hypothesis has been that overexpression of the ∼230 human chromosome 21 (Hsa21) genes result in the complex phenotype. However, the level of overexpression of Hsa21 genes in trisomic individuals is presently unknown. We have used Taqman real-time quantitative PCR to accurately measure expression of the mouse orthologs of Hsa21 in the partial trisomy mouse model Ts65Dn. The transcript levels of 78 protein-coding genes present in three copies in Ts65Dn and 21 control genes were compared between Ts65Dn and normal mouse littermates. The mean overexpression of the aneuploid genes is very close to the expected 1.5-fold in all six tissues studied. However, only approximately a third of the genes (37%) are expressed at the theoretical value of 1.5-fold. On average, 45% of the genes are expressed at significantly lower than 1.5-fold, and 9% are not significantly different from 1.0. Interestingly, 18% of the aneuploid genes were expressed at levels significantly greater than 1.5-fold. These data provide candidate genes that might be involved in the phenotypes of Down syndrome, and reveal a complex regulation of gene expression that is not only related to gene copy number

An apparently dominant bipolar affective disorder (BPAD) locus on chromosome 20p11.2-q11.2 in a large Turkish pedigree

Bipolar affective disorder (BPAD), also known as manic-depressive illness, is a common complex, polygenic disorder characterised by recurrent cyclic episodes of mania and depression. Family, twin, and adoption studies strongly suggest a genetic predisposition/susceptibility to BPAD, but no genes have yet been identified. We studied a large Turkish pedigree, with an apparently autosomal dominant BPAD, which contained 13 affected individuals. The age of onset ranged from 15-40 with a mean of 25 years. The phenotypes consisted of recurrent manic and major depressive episodes, including suicidal attempts; there was usually full remission with lithium treatment. A genome-wide linkage analysis using a dominant mode of inheritance showed strong evidence for a BPAD susceptibility locus on chromosome 20p11.2-q11.2. The highest 2-point lod score of 4.34 at theta = 0 was obtained with markers D20S604, D20S470, D20S836 and D20S838 using a dominant model with full penetrance. Haplotype analysis enabled the mapping of the BPAD locus in this family between markers D20S186 and D20S109, to a region of approximately 42 cM

Cardiomyogenesis is controlled by the miR-99a/let-7c cluster and epigenetic modifications

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

A DNA resequencing array for pathogenic mutation detection in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a heterogeneous autosomal dominant cardiac disorder with a prevalence of 1 in 500. Over 450 different pathogenic mutations in at least 16 genes have been identified so far. The large allelic and genetic heterogeneity of HCM requires high-throughput, rapid, and affordable mutation detection technologies to efficiently integrate molecular screening into clinical practice. We developed a custom DNA resequencing array that contains both strands of all coding exons (160), splice-site junctions, and 5'UTR regions of 12 genes that have been clearly implicated in HCM (MYH7, MYBPC3, TNNT2, TPM1, TNNI3, MYL3, MYL2, CSRP3, PLN, ACTC, TNNC1, and PRKAG2). We analyzed a first series of 38 unrelated patients with HCM (17 familial, 21 sporadic). A total of 953,306 bp across the 38 patients were sequenced with a mean nucleotide call rate of 96.92% (range: 93-99.9%). Pathogenic mutations (single nucleotide substitutions) in MYH7, MYBPC3, TNNI3, and MYL3 (six known and six novel) were identified in 60% (10/17) of familial HCM and 10% of sporadic cases (2/21). The high-throughput HCM resequencing array is the most rapid and cost-effective tool for molecular testing of HCM to date; it thus has considerable potential in diagnostic and predictive testing, and prognostic stratification