Integrative analysis of genomic variants reveals new associations of candidate haploinsufficient genes with congenital heart disease

Numerous genetic studies have established a role for rare genomic variants in Congenital Heart Disease (CHD) at the copy number variation (CNV) and de novo variant (DNV) level. To identify novel haploinsufficient CHD disease genes, we performed an integrative analysis of CNVs and DNVs identified in probands with CHD including cases with sporadic thoracic aortic aneurysm. We assembled CNV data from 7,958 cases and 14,082 controls and performed a gene-wise analysis of the burden of rare genomic deletions in cases versus controls. In addition, we performed variation rate testing for DNVs identified in 2,489 parent-offspring trios. Our analysis revealed 21 genes which were significantly affected by rare CNVs and/or DNVs in probands. Fourteen of these genes have previously been associated with CHD while the remaining genes (FEZ1, MYO16, ARID1B, NALCN, WAC, KDM5B and WHSC1) have only been associated in small cases series or show new associations with CHD. In addition, a systems level analysis revealed affected protein-protein interaction networks involved in Notch signaling pathway, heart morphogenesis, DNA repair and cilia/centrosome function. Taken together, this approach highlights the importance of re-analyzing existing datasets to strengthen disease association and identify novel disease genes and pathways.     

An Excess of Deleterious Variants in VEGF-A Pathway Genes in Down-Syndrome-Associated Atrioventricular Septal Defects

About half of people with trisomy 21 have a congenital heart defect (CHD), whereas the remainder have a structurally normal heart, demonstrating that trisomy 21 is a significant risk factor but is not causal for abnormal heart development. Atrioventricular septal defects (AVSD) are the most commonly occurring heart defects in Down syndrome (DS), and ∼65% of all AVSD is associated with DS. We used a candidate-gene approach among individuals with DS and complete AVSD (cases = 141) and DS with no CHD (controls = 141) to determine whether rare genetic variants in genes involved in atrioventricular valvuloseptal morphogenesis contribute to AVSD in this sensitized population. We found a significant excess (p < 0.0001) of variants predicted to be deleterious in cases compared to controls. At the most stringent level of filtering, we found potentially damaging variants in nearly 20% of cases but fewer than 3% of controls. The variants with the highest probability of being damaging in cases only were found in six genes: COL6A1, COL6A2, CRELD1, FBLN2, FRZB, and GATA5. Several of the case-specific variants were recurrent in unrelated individuals, occurring in 10% of cases studied. No variants with an equal probability of being damaging were found in controls, demonstrating a highly specific association with AVSD. Of note, all of these genes are in the VEGF-A pathway, even though the candidate genes analyzed in this study represented numerous biochemical and developmental pathways, suggesting that rare variants in the VEGF-A pathway might contribute to the genetic underpinnings of AVSD in humans.     

De Novo Loss-of-Function Mutations in CHD2 Cause a Fever-Sensitive Myoclonic Epileptic Encephalopathy Sharing Features with Dravet Syndrome

Dravet syndrome is a severe epilepsy syndrome characterized by infantile onset of therapy-resistant, fever-sensitive seizures followed by cognitive decline. Mutations in SCN1A explain about 75% of cases with Dravet syndrome; 90% of these mutations arise de novo. We studied a cohort of nine Dravet-syndrome-affected individuals without an SCN1A mutation (these included some atypical cases with onset at up to 2 years of age) by using whole-exome sequencing in proband-parent trios. In two individuals, we identified a de novo loss-of-function mutation in CHD2 (encoding chromodomain helicase DNA binding protein 2). A third CHD2 mutation was identified in an epileptic proband of a second (stage 2) cohort. All three individuals with a CHD2 mutation had intellectual disability and fever-sensitive generalized seizures, as well as prominent myoclonic seizures starting in the second year of life or later. To explore the functional relevance of CHD2 haploinsufficiency in an in vivo model system, we knocked down chd2 in zebrafish by using targeted morpholino antisense oligomers. chd2-knockdown larvae exhibited altered locomotor activity, and the epileptic nature of this seizure-like behavior was confirmed by field-potential recordings that revealed epileptiform discharges similar to seizures in affected persons. Both altered locomotor activity and epileptiform discharges were absent in appropriate control larvae. Our study provides evidence that de novo loss-of-function mutations in CHD2 are a cause of epileptic encephalopathy with generalized seizures.     

Outlier-Based Identification of Copy Number Variations Using Targeted Resequencing in a Small Cohort of Patients with Tetralogy of Fallot

Copy number variations (CNVs) are one of the main sources of variability in the human genome. Many CNVs are associated with various diseases including cardiovascular disease. In addition to hybridization-based methods, next-generation sequencing (NGS) technologies are increasingly used for CNV discovery. However, respective computational methods applicable to NGS data are still limited. We developed a novel CNV calling method based on outlier detection applicable to small cohorts, which is of particular interest for the discovery of individual CNVs within families, de novo CNVs in trios and/or small cohorts of specific phenotypes like rare diseases. Approximately 7,000 rare diseases are currently known, which collectively affect ∼6% of the population. For our method, we applied the Dixon’s Q test to detect outliers and used a Hidden Markov Model for their assessment. The method can be used for data obtained by exome and targeted resequencing. We evaluated our outlier- based method in comparison to the CNV calling tool CoNIFER using eight HapMap exome samples and subsequently applied both methods to targeted resequencing data of patients with Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease. In both the HapMap samples and the TOF cases, our method is superior to CoNIFER, such that it identifies more true positive CNVs. Called CNVs in TOF cases were validated by qPCR and HapMap CNVs were confirmed with available array-CGH data. In the TOF patients, we found four copy number gains affecting three genes, of which two are important regulators of heart development (NOTCH1, ISL1) and one is located in a region associated with cardiac malformations (PRODH at 22q11). In summary, we present a novel CNV calling method based on outlier detection, which will be of particular interest for the analysis of de novo or individual CNVs in trios or cohorts up to 30 individuals, respectively.     

Genomic landscapes of Chinese sporadic autism spectrum disorders revealed by whole-genome sequencing

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with considerable clinical and genetic heterogeneity.In this study,we identified all classes of genomic variants from whole-genome sequencing (WGS) dataset of 32 Chinese trios with ASD,including de novo mutations,inherited variants,copy number variants (CNVs) and genomic structural variants.A higher mutation rate (Poisson test,P2.2×10~(-16)) in exonic (1.37×10~(-8)) and 3'-UTR regions (1.42×10~(-8)) was revealed in comparison with that of whole genome (1.05×10~(-8)).Using an integrated model,we identified 87 potentially risk genes (P0.01) from 4832 genes harboring various rare deleterious variants,including CHD8 and NRXN2,implying that the disorders may be in favor to multiple-hit.In particular,frequent rare inherited mutations of several microcephaly-associated genes (ASPM,WDR62,and ZNF335)were found in ASD.In chromosomal structure analyses,we found four de novo CNVs and one de novo chromosomal rearrangement event,including a de novo duplication of UBE3A-containing region at 15q11.2-q13.1,which causes Angelman syndrome and microcephaly,and a disrupted TNR due to de novo chromosomal translocation t (1;5) (q25.1;q33.2).Taken together,our results suggest that abnormalities of centrosomal function and chromatin remodeling of the microcephaly-associated genes may be implicated in pathogenesis of ASD.Adoption of WGS as a new yet efficient technique to illustrate the full genetic spectrum in complex disorders,such as ASD,could provide novel insights into pathogenesis,diagnosis and treatment.     

Limited Evidence for Parent-of-Origin Effects in Inflammatory Bowel Disease Associated Loci

Background

Genome-wide association studies of two main forms of inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), have identified 99 susceptibility loci, but these explain only ∼23% of the genetic risk. Part of the ‘hidden heritability’ could be in transmissible genetic effects in which mRNA expression in the offspring depends on the parental origin of the allele (genomic imprinting), since children whose mothers have CD are more often affected than children with affected fathers. We analyzed parent-of-origin (POO) effects in Dutch and Indian cohorts of IBD patients.

Methods

We selected 28 genetic loci associated with both CD and UC, and tested them for POO effects in 181 Dutch IBD case-parent trios. Three susceptibility variants in NOD2 were tested in 111 CD trios and a significant finding was re-evaluated in 598 German trios. The UC-associated gene, BTNL2, reportedly imprinted, was tested in 70 Dutch UC trios. Finally, we used 62 independent Indian UC trios to test POO effects of five established Indian UC risk loci.

Results

We identified POO effects for NOD2 (L1007fs; OR = 21.0, P-value = 0.013) for CD; these results could not be replicated in an independent cohort (OR = 0.97, P-value = 0.95). A POO effect in IBD was observed for IL12B (OR = 3.2, P-value = 0.019) and PRDM1 (OR = 5.6, P-value = 0.04). In the Indian trios the IL10 locus showed a POO effect (OR = 0.2, P-value = 0.03).

Conclusions

Little is known about the effect of genomic imprinting in complex diseases such as IBD. We present limited evidence for POO effects for the tested IBD loci. POO effects explain part of the hidden heritability for complex genetic diseases but need to be investigated further.     

Gene-Network Analysis Identifies Susceptibility Genes Related to Glycobiology in Autism

The recent identification of copy-number variation in the human genome has opened up new avenues for the discovery of positional candidate genes underlying complex genetic disorders, especially in the field of psychiatric disease. One major challenge that remains is pinpointing the susceptibility genes in the multitude of disease-associated loci. This challenge may be tackled by reconstruction of functional gene-networks from the genes residing in these loci. We applied this approach to autism spectrum disorder (ASD), and identified the copy-number changes in the DNA of 105 ASD patients and 267 healthy individuals with Illumina Humanhap300 Beadchips. Subsequently, we used a human reconstructed gene-network, Prioritizer, to rank candidate genes in the segmental gains and losses in our autism cohort. This analysis highlighted several candidate genes already known to be mutated in cognitive and neuropsychiatric disorders, including RAI1, BRD1, and LARGE. In addition, the LARGE gene was part of a sub-network of seven genes functioning in glycobiology, present in seven copy-number changes specifically identified in autism patients with limited co-morbidity. Three of these seven copy-number changes were de novo in the patients. In autism patients with a complex phenotype and healthy controls no such sub-network was identified. An independent systematic analysis of 13 published autism susceptibility loci supports the involvement of genes related to glycobiology as we also identified the same or similar genes from those loci. Our findings suggest that the occurrence of genomic gains and losses of genes associated with glycobiology are important contributors to the development of ASD.     

Four new loci associations discovered by pathway-based and network analyses of the genome-wide variability profile of Hirschsprung’s disease

Finding gene associations in rare diseases is frequently hampered by the reduced numbers of patients accessible. Conventional gene-based association tests rely on the availability of large cohorts, which constitutes a serious limitation for its application in this scenario. To overcome this problem we have used here a combined strategy in which a pathway-based analysis (PBA) has been initially conducted to prioritize candidate genes in a Spanish cohort of 53 trios of short-segment Hirschsprung’s disease. Candidate genes have been further validated in an independent population of 106 trios. The study revealed a strong association of 11 gene ontology (GO) modules related to signal transduction and its regulation, enteric nervous system (ENS) formation and other HSCR-related processes. Among the preselected candidates, a total of 4 loci, RASGEF1A, IQGAP2, DLC1 and CHRNA7, related to signal transduction and migration processes, were found to be significantly associated to HSCR. Network analysis also confirms their involvement in the network of already known disease genes. This approach, based on the study of functionally-related gene sets, requires of lower sample sizes and opens new opportunities for the study of rare diseases.     

Prospective Associations of Coronary Heart Disease Loci in African Americans Using the MetaboChip: The PAGE Study

Background

Coronary heart disease (CHD) is a leading cause of morbidity and mortality in African Americans. However, there is a paucity of studies assessing genetic determinants of CHD in African Americans. We examined the association of published variants in CHD loci with incident CHD, attempted to fine map these loci, and characterize novel variants influencing CHD risk in African Americans.

Methods and Results

Up to 8,201 African Americans (including 546 first CHD events) were genotyped using the MetaboChip array in the Atherosclerosis Risk in Communities (ARIC) study and Women''s Health Initiative (WHI). We tested associations using Cox proportional hazard models in sex- and study-stratified analyses and combined results using meta-analysis. Among 44 validated CHD loci available in the array, we replicated and fine-mapped the SORT1 locus, and showed same direction of effects as reported in studies of individuals of European ancestry for SNPs in 22 additional published loci. We also identified a SNP achieving array wide significance (MYC: rs2070583, allele frequency 0.02, P = 8.1×10⁻⁸), but the association did not replicate in an additional 8,059 African Americans (577 events) from the WHI, HealthABC and GeneSTAR studies, and in a meta-analysis of 5 cohort studies of European ancestry (24,024 individuals including 1,570 cases of MI and 2,406 cases of CHD) from the CHARGE Consortium.

Conclusions

Our findings suggest that some CHD loci previously identified in individuals of European ancestry may be relevant to incident CHD in African Americans.     

Inflammatory Cytokines Induce NOTCH Signaling in Nucleus Pulposus Cells: IMPLICATIONS IN INTERVERTEBRAL DISC DEGENERATION*

The objective of the study was to investigate how inflammatory cytokines, IL-1β, and TNF-α control NOTCH signaling activity in nucleus pulposus (NP) cells. An increase in expression of selective NOTCH receptors (NOTCH1 and -2), ligand (JAGGED2), and target genes (HES1, HEY1, and HEY2) was observed in NP cells following cytokine treatment. A concomitant increase in NOTCH signaling as evidenced by induction in activity of target gene HES1 and HEY1 promoters and reporter 12xCSL was seen. Moreover, treatment increased activity of a 2-kb NOTCH2 promoter. Treatment of cells with NF-κB and MAPK inhibitors abolished the inductive effect of cytokines on NOTCH2 promoter and its expression. Gain and loss-of-function studies confirmed the inductive effect of p65 on NOTCH2 promoter activity. In contrast, p50 blocked the cytokine induction of promoter activity. Supporting promoter studies, lentiviral delivery of sh-p65, and sh-IKKβ significantly decreased cytokine dependent change in NOTCH2 expression. Interestingly, MAPK signaling showed an isoform-specific control of NOTCH2 promoter; p38α/β2/δ, ERK1, and ERK2 contributed to cytokine dependent induction, whereas p38γ played no role. Analysis of human NP tissues showed that NOTCH1 and -2 and HEY2 expression correlated with each other. Moreover, expression of NOTCH2 and IL-1β as well as the number of cells immunopositive for NOTCH2 significantly increased in histologically degenerate discs compared with non-degenerate discs. Taken together, these results explain the observed dysregulated expression of NOTCH genes in degenerative disc disease. Thus, controlling IL-1β and TNF-α activities during disc disease may restore NOTCH signaling and nucleus pulposus cell function.     

Genotypes with the apolipoprotein ε4 allele are predictors of coronary heart disease mortality in a longitudinal study of elderly Finnish men

Earlier we reported that allelic variation in the gene coding for apolipoprotein (apoE) is a significant predictor of variation in the risk of coronary heart disease (CHD) death in a longitudinal study of elderly Finnish men. Here we address the question: which of the apoE genotypes confers the risk information in these men, and whether such information persists after other CHD risk factors are considered? We followed two cohorts of elderly Finnish men aged 65 to 84 years, one in Eastern (n = 281) and the other in the Southwestern (n = 344) Finland for 5 years during which 26 (9.3%) of the men from the Eastern cohort and 40 (11.6%) of the men in the Southwestern cohort died from CHD. Baseline high density lipoprotein (HDL) cholesterol and (HDL cholesterol)² in the Eastern cohort and age, and total and HDL cholesterol and smoking status in the Southwestern cohort were significant predictors of CHD death (P < 0.05). The apoE genotypes were significant predictors in the Southwestern cohort atP = 0.02 and in the Eastern cohort atP = 0.18. In multivariable models, information about apoE genotypes improved the prediction atP = 0.10 level of statistical significance in both cohorts. When genotypes were considered separately, the ε2/4 combined with the ε4/4 in the Eastern cohort (odds ratio = 7.69, 95% CI = 1.67-35.52) and the ε3/4 in the Southwestern cohort (odds ratio = 2.44, 95% CI = 1.16–5.10) had sigificanctly greater odds of CHD death compared to the common ε3/3 genotype. We conclude that apoE genotypes confer risk information about CHD death in two cohorts of elderly Finnish men in a longitudinal study, and this information persists after adjustment for other CHD risk factors. Because different genotypes were predictors in these two cohorts, we further conclude that the utility of a particular genotype as a predictor of CHD death in other populations may depend on the distribution of risk factor profiles at baseline, geographically defined environmental exposures, the CHD mortality history, and the evolutionary history of background genotypes in the population considered.     

The Contribution of Mosaic Variants to Autism Spectrum Disorder

De novo mutation is highly implicated in autism spectrum disorder (ASD). However, the contribution of post-zygotic mutation to ASD is poorly characterized. We performed both exome sequencing of paired samples and analysis of de novo variants from whole-exome sequencing of 2,388 families. While we find little evidence for tissue-specific mosaic mutation, multi-tissue post-zygotic mutation (i.e. mosaicism) is frequent, with detectable mosaic variation comprising 5.4% of all de novo mutations. We identify three mosaic missense and likely-gene disrupting mutations in genes previously implicated in ASD (KMT2C, NCKAP1, and MYH10) in probands but none in siblings. We find a strong ascertainment bias for mosaic mutations in probands relative to their unaffected siblings (p = 0.003). We build a model of de novo variation incorporating mosaic variants and errors in classification of mosaic status and from this model we estimate that 33% of mosaic mutations in probands contribute to 5.1% of simplex ASD diagnoses (95% credible interval 1.3% to 8.9%). Our results indicate a contributory role for multi-tissue mosaic mutation in some individuals with an ASD diagnosis.     

Mutations in NOTCH1 Cause Adams-Oliver Syndrome

Notch signaling determines and reinforces cell fate in bilaterally symmetric multicellular eukaryotes. Despite the involvement of Notch in many key developmental systems, human mutations in Notch signaling components have mainly been described in disorders with vascular and bone effects. Here, we report five heterozygous NOTCH1 variants in unrelated individuals with Adams-Oliver syndrome (AOS), a rare disease with major features of aplasia cutis of the scalp and terminal transverse limb defects. Using whole-genome sequencing in a cohort of 11 families lacking mutations in the four genes with known roles in AOS pathology (ARHGAP31, RBPJ, DOCK6, and EOGT), we found a heterozygous de novo 85 kb deletion spanning the NOTCH1 5′ region and three coding variants (c.1285T>C [p.Cys429Arg], c.4487G>A [p.Cys1496Tyr], and c.5965G>A [p.Asp1989Asn]), two of which are de novo, in four unrelated probands. In a fifth family, we identified a heterozygous canonical splice-site variant (c.743−1 G>T) in an affected father and daughter. These variants were not present in 5,077 in-house control genomes or in public databases. In keeping with the prominent developmental role described for Notch1 in mouse vasculature, we observed cardiac and multiple vascular defects in four of the five families. We propose that the limb and scalp defects might also be due to a vasculopathy in NOTCH1-related AOS. Our results suggest that mutations in NOTCH1 are the most common cause of AOS and add to a growing list of human diseases that have a vascular and/or bony component and are caused by alterations in the Notch signaling pathway.     

Squamousness: Next-generation sequencing reveals shared molecular features across squamous tumor types

In order to gain a better understanding of the underlying biology of squamous cell carcinoma (SCC), we tested the hypothesis that SCC originating from different organs may possess common molecular alterations. SCC samples (N = 361) were examined using clinical-grade targeted next-generation sequencing (NGS). The most frequent SCC tumor types were head and neck, lung, cutaneous, gastrointestinal and gynecologic cancers. The most common gene alterations were TP53 (64.5% of patients), PIK3CA (28.5%), CDKN2A (24.4%), SOX2 (17.7%), and CCND1 (15.8%). By comparing NGS results of our SCC cohort to a non-SCC cohort (N = 277), we found that CDKN2A, SOX2, NOTCH1, TP53, PIK3CA, CCND1, and FBXW7 were significantly more frequently altered, unlike KRAS, which was less frequently altered in SCC specimens (all P < 0.05; multivariable analysis). Therefore, we identified “squamousness” gene signatures (TP53, PIK3CA, CCND1, CDKN2A, SOX2, NOTCH 1, and FBXW7 aberrations, and absence of KRAS alterations) that were significantly more frequent in SCC versus non-SCC histologies. A multivariable co-alteration analysis established 2 SCC subgroups: (i) patients in whom TP53 and cyclin pathway (CDKN2A and CCND1) alterations strongly correlated but in whom PIK3CA aberrations were less frequent; and (ii) patients with PIK3CA alterations in whom TP53 mutations were less frequent (all P ≤ 0 .001, multivariable analysis). In conclusion, we identified a set of 8 genes altered with significantly different frequencies when SCC and non-SCC were compared, suggesting the existence of patterns for “squamousness.” Targeting the PI3K-AKT-mTOR and/or cyclin pathway components in SCC may be warranted.