Whole-Genome Linkage Scan Combined With Exome Sequencing Identifies Novel Candidate Genes for Carotid Intima-Media Thickness

Carotid intima-media thickness (cIMT) is an established heritable marker for subclinical atherosclerosis. In this study, we aim to identify rare variants with large effects driving differences in cIMT by performing genome-wide linkage analysis of individuals in the extremes of cIMT trait distribution (>90th percentile) in a large family-based study from a genetically isolated population in the Netherlands. Linked regions were subsequently explored by fine-mapping using exome sequencing. We observed significant evidence of linkage on chromosomes 2p16.3 [rs1017418, heterogeneity LOD (HLOD) = 3.35], 19q13.43 (rs3499, HLOD = 9.09), 20p13 (rs1434789, HLOD = 4.10), and 21q22.12 (rs2834949, HLOD = 3.59). Fine-mapping using exome sequencing data identified a non-coding variant (rs62165235) in PNPT1 gene under the linkage peak at chromosome 2 that is likely to have a regulatory function. The variant was associated with quantitative cIMT in the family-based study population (effect = 0.27, p-value = 0.013). Furthermore, we identified several genes under the linkage peak at chromosome 21 highly expressed in tissues relevant for atherosclerosis. To conclude, our linkage analysis identified four genomic regions significantly linked to cIMT. Further analyses are needed to demonstrate involvement of identified candidate genes in development of atherosclerosis

Targeted Genomic Sequencing of TSC1 and TSC2 Reveals Causal Variants in Individuals for Whom Previous Genetic Testing for Tuberous Sclerosis Complex Was Normal

Tuberous sclerosis complex (TSC) is caused by inactivating variants in TSC1 and TSC2. Somatic mosaicism, as well as the size and complexity of the TSC1 and TSC2 loci, makes variant identification challenging. Indeed, in some individuals with a clinical diagnosis of TSC, diagnostic testing fails to identify an inactivating variant. To improve TSC1 and TSC2 variant detection, we screened the TSC1 and TSC2 genomic regions using targeted HaloPlex custom capture and next-generation sequencing (NGS) in genomic DNA isolated from peripheral blood of individuals with definite, possible or suspected TSC in whom no disease-associated variant had been identified by previous diagnostic genetic testing. We obtained &gt;95% target region coverage at a read depth of 20 and &gt;50% coverage at a read depth of 300 and identified inactivating TSC1 or TSC2 variants in 83/155 individuals (54%); 65/113 (58%) with clinically definite TSC and 18/42 (43%) with possible or suspected TSC. These included 19 individuals with deep intronic variants and 54 likely cases of mosaicism (variant allele frequency 1-28%; median 7%). In 13 cases (8%), we identified a variant of uncertain significance (VUS). Targeted genomic NGS of TSC1 and TSC2 increases the yield of inactivating variants found in individuals with suspected TSC.</p

Next-generation sequencing-based genome diagnostics across clinical genetics centers: Implementation choices and their effects

Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found 'no causal variant', thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of 'greediness' to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care

Characterization of the ferret TRB locus guided by V, D, J, and C gene expression analysis

The domestic ferret, Mustela putorius furo, is an important mammalian animal model to study human respiratory infection. However, insufficient genomic annotation hampers detailed studies of ferret T cell responses. In this study, we analyzed the published T cell receptor beta (TRB) locus and performed high-throughput sequencing (HTS) of peripheral blood of four healthy adult ferrets to identify expressed V, D, J, and C genes. The HTS data is used as a guide to manually curate the expressed V, D, J, and C genes. The ferret locus appears to be most similar to that of the dog. Like other mammalian TRB loci, the ferret TRB locus contains a library of variable genes located upstream of two D-J-C gene clusters, followed by a (in the ferret non-functional) V gene with an inverted transcriptional orientation. All TRB genes (expressed or not) reported here have been approved by the IMGT/WHO-IUIS nomenclature committee

Characterization of the ferret TRB locus guided by V, D, J, and C gene expression analysis

The domestic ferret, Mustela putorius furo, is an important mammalian animal model to study human respiratory infection. However, insufficient genomic annotation hampers detailed studies of ferret T cell responses. In this study, we analyzed the published T cell receptor beta (TRB) locus and performed high-throughput sequencing (HTS) of peripheral blood of four healthy adult ferrets to identify expressed V, D, J, and C genes. The HTS data is used as a guide to manually curate the expressed V, D, J, and C genes. The ferret locus appears to be most similar to that of the dog. Like other mammalian TRB loci, the ferret TRB locus contains a library of variable genes located upstream of two D-J-C gene clusters, followed by a (in the ferret non-functional) V gene with an inverted transcriptional orientation. All TRB genes (expressed or not) reported here have been approved by the IMGT/WHO-IUIS nomenclature committee

Regulation of the cohesin-loading factor NIPBL: Role of the lncRNA NIPBL-AS1 and identification of a distal enhancer element

International audienceCohesin is crucial for genome stability, cell division, transcription and chromatin organization. Its functions critically depend on NIPBL, the cohesin-loader protein that is found to be mutated in > 60% of the cases of Cornelia de Lange syndrome (CdLS). Other mutations are described in the cohesin subunits SMC1A, RAD21, SMC3 and the HDAC8 protein. In 25-30% of CdLS cases no mutation in the known CdLS genes is detected. Until now, functional elements in the noncoding genome were not characterized in the molecular etiology of CdLS and therefore are excluded from mutation screening, although the impact of such mutations has now been recognized for a wide range of diseases. We have identified different elements of the noncoding genome involved in regulation of the NIPBL gene. NIPBLAS1 is a long non-coding RNA transcribed upstream and antisense to NIPBL. By knockdown and transcription blocking experiments, we could show that not the NIPBL-AS1 gene product, but its actual transcription is important to regulate NIPBL expression levels. This reveals a possibility to boost the transcriptional activity of the NIPBL gene by interfering with the NIPBL-AS1 lncRNA. Further, we have identified a novel distal enhancer regulating both NIPBL and NIPBLAS1. Deletion of the enhancer using CRISPR genome editing in HEK293T cells reduces expression of NIPBL, NIPBL-AS1 as well as genes found to be dysregulated in CdLS

Exome-Wide Meta-Analysis Identifies Rare 3′-UTR Variant in ERCC1/CD3EAP Associated with Symptoms of Sleep Apnea

Obstructive sleep apnea (OSA) is a common sleep breathing disorder associated with an increased risk of cardiovascular and cerebrovascular diseases and mortality. Although OSA is fairly heritable (~40%), there have been only few studies looking into the genetics of OSA. In the present study, we aimed to identify genetic variants associated with symptoms of sleep apnea by performing a whole-exome sequence meta-analysis of symptoms of sleep apnea in 1,475 individuals of European descent. We identified 17 rare genetic variants with at least suggestive evidence of significance. Replication in an independent dataset confirmed the association of a rare genetic variant (rs2229918; minor allele frequency = 0.3%) with symptoms of sleep apnea (p-valuemeta = 6.98 × 10−9, βmeta = 0.99). Rs2229918 overlaps with the 3′ untranslated regions of ERCC1 and CD3EAP genes on chromosome 19q13. Both genes are expressed in tissues in the neck area, such as the tongue, muscles, cartilage and the trachea. Further, CD3EAP is localized in the nucleus and mitochondria and involved in the tumor necrosis factor-alpha/nuclear factor kappa B signaling pathway. Our results and biological functions of CD3EAP/ERCC1 genes suggest that the 19q13 locus is interesting for further OSA research

<i>NIPBL-AS1</i> does not influence <i>NIPBL</i> transcription.

<p>A) Overview of the genomic position of <i>NIPBL</i> and <i>NIPBL-AS1</i> genes. Strand-specific read coverage of RNA-sequencing data (positive in green; negative in red) from HEK293T cells shows the transcription of <i>NIPBL-AS1</i> antisense to <i>NIPBL</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref001" target="_blank">1</a>]. CTCF binding sites in HEK293 cells (ENCODE hg18) are shown. Primers used in the transcript analysis are indicated as green bars. (B-C) Transcript levels of (B) <i>NIPBL-AS1</i> and (C) <i>NIPBL</i> after antisense oligonucleotide knockdown (ASO2, ASO3) of <i>NIPBL-AS1</i> in HEK293T cells. ASO C was used as control. Transcript levels were normalized against the control sample (ASO C) and the housekeeping <i>SNAPIN</i> using the ΔΔCt method (mean n = 3, error bars +/- s.d., p-values determined with t-Test).</p

Interactions of <i>NIPBL</i> and <i>NIPBL-AS1</i> with a potential distal enhancer.

<p>A) Long-range chromosomal interactions of the <i>NIPBL</i> and <i>NIPBL-AS1</i> promoter detected by chromosome conformation capture (3C-seq) in HEK293T cells using an ApoI digest. The positions of the different viewpoints used are marked in yellow. Three different viewpoints at the promoter (VP4, blue track) and the candidate enhancers regions R1 (VP5, green track) and R2 (R2—VP6, red track) were used. B) CTCF ChIP sequencing track from HEK293 cells (ENCODE). The orientations of the CTCF motifs as determined with JASPAR are shown below the track (red triangle–forward orientation, green triangle–reverse orientation). The CTCF sites involved in the promoter-enhancer interaction are indicated with yellow triangles above the track. C) DNAse clusters as well as histone modification profiles—H2A.z, H3K4me1, H3K4me2 and H3K4me3—of six different cell lines (G312878, K562, HeLa-S3, HEMEC, HSMM and HUVEC, available from ENCODE) are displayed as density graph. Black represents areas with the highest enrichment of the signals.</p