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.     

Computational analysis of candidate disease genes and variants for salt-sensitive hypertension in indigenous Southern Africans

Multiple factors underlie susceptibility to essential hypertension, including a significant genetic and ethnic component, and environmental effects. Blood pressure response of hypertensive individuals to salt is heterogeneous, but salt sensitivity appears more prevalent in people of indigenous African origin. The underlying genetics of salt-sensitive hypertension, however, are poorly understood. In this study, computational methods including text- and data-mining have been used to select and prioritize candidate aetiological genes for salt-sensitive hypertension. Additionally, we have compared allele frequencies and copy number variation for single nucleotide polymorphisms in candidate genes between indigenous Southern African and Caucasian populations, with the aim of identifying candidate genes with significant variability between the population groups: identifying genetic variability between population groups can exploit ethnic differences in disease prevalence to aid with prioritisation of good candidate genes. Our top-ranking candidate genes include parathyroid hormone precursor (PTH) and type-1 angiotensin II receptor (AGTR1). We propose that the candidate genes identified in this study warrant further investigation as potential aetiological genes for salt-sensitive hypertension.     

Association of assisted reproductive technology,germline de novo mutations and congenital heart defects in a prospective birth cohort study

Emerging evidence suggests that children conceived through assisted reproductive technology (ART) have a higher risk of congenital heart defects (CHDs) even when there is no family history. De novo mutation (DNM) is a well-known cause of sporadic congenital diseases; however, whether ART procedures increase the number of germline DNM (gDNM) has not yet been well studied. Here, we performed whole-genome sequencing of 1137 individuals from 160 families conceived through ART and 205 families conceived spontaneously. Children conceived via ART carried 4.59 more gDNMs than children conceived spontaneously, including 3.32 paternal and 1.26 maternal DNMs, after correcting for parental age at conception, cigarette smoking, alcohol drinking, and exercise behaviors. Paternal DNMs in offspring conceived via ART are characterized by C>T substitutions at CpG sites, which potentially affect protein-coding genes and are significantly associated with the increased risk of CHD. In addition, the accumulation of non-coding functional mutations was independently associated with CHD and 87.9% of the mutations were originated from the father. Among ART offspring, infertility of the father was associated with elevated paternal DNMs; usage of both recombinant and urinary follicle-stimulating hormone and high-dosage human chorionic gonadotropin trigger was associated with an increase of maternal DNMs. In sum, the increased gDNMs in offspring conceived by ART were primarily originated from fathers, indicating that ART itself may not be a major reason for the accumulation of gDNMs. Our findings emphasize the importance of evaluating the germline status of the fathers in families with the use of ART.Subject terms: Genomic analysis, Developmental biology     

Copy number variants in candidate genes are genetic modifiers of Hirschsprung disease

Hirschsprung disease (HSCR) is a neurocristopathy characterized by absence of intramural ganglion cells along variable lengths of the gastrointestinal tract. The HSCR phenotype is highly variable with respect to gender, length of aganglionosis, familiality and the presence of additional anomalies. By molecular genetic analysis, a minimum of 11 neuro-developmental genes (RET, GDNF, NRTN, SOX10, EDNRB, EDN3, ECE1, ZFHX1B, PHOX2B, KIAA1279, TCF4) are known to harbor rare, high-penetrance mutations that confer a large risk to the bearer. In addition, two other genes (RET, NRG1) harbor common, low-penetrance polymorphisms that contribute only partially to risk and can act as genetic modifiers. To broaden this search, we examined whether a set of 67 proven and candidate HSCR genes harbored additional modifier alleles. In this pilot study, we utilized a custom-designed array CGH with ～33,000 test probes at an average resolution of ～185 bp to detect gene-sized or smaller copy number variants (CNVs) within these 67 genes in 18 heterogeneous HSCR patients. Using stringent criteria, we identified CNVs at three loci (MAPK10, ZFHX1B, SOX2) that are novel, involve regulatory and coding sequences of neuro-developmental genes, and show association with HSCR in combination with other congenital anomalies. Additional CNVs are observed under relaxed criteria. Our research suggests a role for CNVs in HSCR and, importantly, emphasizes the role of variation in regulatory sequences. A much larger study will be necessary both for replication and for identifying the full spectrum of small CNV effects.     

Genome-wide association study using diversity outcross mice identified candidate genes of pancreatic cancer

To understand the genetic causes of pancreatic cancer (PC), we conducted a genome-wide association study (GWAS) using the diversity outbred (DO) mice population to identify susceptibility genes underlying 7,12-dimethylbenzanthraene (DMBA) induced PC. The phenotype studied was the percent PC lesion area in the DO mice population. We genotyped 7851 SNP markers specifically designed for DO mice across the whole mouse genome. Four susceptibility genes with P values exceeding the genome-wide threshold for percent PC lesion area (P < 2.37 × 10⁻⁶) were identified, i.e., Epha4, Gpc5, Kcnj6, Arid1b. The most significant SNP of Gpc5 (UNC140360310) that is associated with PC lesion area in mice also significantly influences the Gpc5 expression, suggesting that this Gpc5 SNP exerts its role in PC through cis-regulating the gene expression of Gpc5. Together, our data supported that Gpc5 as a tumor suppressor gene involved in the etiology of PC.     

DNA microarray analysis for human congenital heart disease

Right ventricular hypertrophy and failure are prominent features in cyanotic congenital heart disease, tetralogy of Fallot (TF). Patients with TF require primary cardiac surgery at a very young age. To gain insight into the underlying molecular mechanisms of right ventricular hypertrophy and to identify gene(s) involved in TF, differential gene expression profile was assessed using expression-based microarray technology on right ventricular biopsies from young TF patients who underwent primary correction. By using quantitative immunohistochemistry, expression of vascular endothelial growth factor (VEGF), flk-1, and extracellular matrix (ECM) proteins (collagens and fibronectin) as well as vessel counts and myocyte cell size was evaluated in TF patients in relation to age-matched controls. Among 236 genes showing altered expression pattern in TF patients, VEGF (1.8-fold) and ECM markers were clearly upregulated (fibronectin, 2.4-fold; collagen Iα, 7.5-fold; and collagen III, 4.4-fold); flk-1 and most matrix metalloproteinases (MMPs) remained unchanged, except the levels of MMP-13 and-17 declined. Tissue inhibitors of metalloproteinases showed a downregulated pattern. Staining of VEGF in cardiomyocytes and of ECM proteins (fibronectin, collagen I and III) in interstitial as well as in perivascular area was increased (p<0.01) in TF patients. Morphometric analysis revealed enhanced vascular density (p<0.05) with unchanged wall thickness and enlarged myocyte cross-sectional areas (p<0.01) with linear correlation (p<0.01) with the age in TF-1 patients. We conclude that the upregulation of genes encoding VEGF and ECM proteins are the key events contributing to right ventricular hypertrophy and stunted angiogenesis in patients with TF.     

Genetic associations with coronary heart disease: Meta-analyses of 12 candidate genetic variants

Aims

The aim of this study was to evaluate the combined contribution of 12 genetic variants to the risk of coronary heart disease (CHD).

Methods

Through a comprehensive literature search for genetic variants involved in the CHD association study, we harvested a total of 10 genes (12 variants) for the current meta-analyses. These genes consisted of GPX1 (rs1050450), PPARD (rs2016520), ALOX15 (rs34210653), SELPLG (rs2228315), FCGR2A (rs1801274), CCL5 (rs2107538), CYP1A1 (rs4646903), TP53 (rs1042522), CX37 (rs1764391), and PECAM1 (rs668, rs12953, and rs1131012).

Results

A total of 45 studies among 23,314 cases and 28,430 controls were retrieved for the meta-analyses of 12 genetic variants. The results showed a significant association between the GPX1 rs1050450 polymorphism and CHD (odd ratio (OR) = 1.61, 95% confidence interval (CI) = 1.25–2.07, P = 0.0002). Other meta-analyses of the rest 11 variants suggested a lack of association with the risk of CHD.

Conclusion

Our results confirmed that GPX1 rs1050450 was associated with susceptibility to CHD in Chinese and Indian populations.     

Origin and timing of de novo variants implicated in type 2 von Willebrand disease

Very few studies have shown the real origin and timing of de novo variants (DNV) implicated in von Willebrand disease (VWD). We investigated four families with type 2 VWD. First, we conducted linkage analysis using single nucleotide variant genotyping to recognize the possible provenance of DNV. Second, we performed amplification refractory mutation system‐quantitative polymerase chain reaction to confirm the real origin of variant (~0% mutant cells) or presence of a genetic mosaic variant (0%–50% mutant cells) in three embryonic germ layer‐derived tissues and sperm cells. Then, three possible timings of DNV were categorized based on the relative likelihood of occurrence according to the number of cell divisions during embryogenesis. Two each with type 2B VWD (proband 1 p.Arg1308Cys, proband 4 p.Arg1306Trp) and type 2A VWD (proband 2 p.Leu1276Arg, proband 3 p.Ser1506Leu) were identified. Variant origins were identified for families 1, 2 and 3 and confirmed to originate from the mother, father and father, respectively. However, the father of family 4 was confirmed to have isolated germline mosaicism with 2.2% mutant sperm cells. Further investigation confirmed the paternal grandfather to be the origin of variant. Thus, we proposed that DNV originating from the two fathers most likely occurred at the single sperm cell, the one originating from the mother occurred at the zygote during the first few cellular divisions; alternatively, in family 4, the DNV most likely occurred at the early postzygotic development in the father. Our findings are essential for understanding genetic pathogenesis and providing accurate genetic counselling.     

Association analysis of candidate genes for neuropsychiatric disease: the perpetual campaign

Association studies have been proposed to identify the genetic determinants of complex neuropsychiatric traits. Although such studies of candidate genes offer great potential to identify genetic variants that contribute to the expression of psychiatric disease, no consistent associations have been identified. Studies to date have focused on candidate genes that are selected for analysis on the basis of incomplete information about gene function in the brain, therefore the majority of genes expressed in the brain have been ignored. Additionally, most genetic determinants of psychiatric disease will probably be of modest effect and therefore require association studies of large samples. As genomic technologies advance, massive genotyping of large samples should allow identification of alleles that contribute to psychopathology.     

Novel quantitative trait loci for central corneal thickness identified by candidate gene analysis of osteogenesis imperfecta genes

Osteogenesis imperfecta (OI) is a rare connective tissue disorder caused by mutations in the type I collagen genes, COL1A1 and COL1A2, and is characterised by low bone mass and bone fragility. In this study, we explored the relationship between type 1 collagen genes and the quantitative trait central corneal thickness (CCT). CCT was measured in a cohort of 28 Australian type I OI patients and mean CCT was found to be significantly lower compared to a normal population (P < 0.001). We then investigated CCT and corneal collagen fibril diameter and density in a mouse model of OI with a col1a2 mutation. Mean CCT was significantly lower in mutant mice (P = 0.002), as was corneal collagen fibril diameter (P = 0.034), whilst collagen fibril density was significantly greater in mutants (P = 0.034). Finally, we conducted a genetic study to determine whether common single nucleotide polymorphisms (SNPs) in COL1A1 and COL1A2 are associated with CCT variation in the normal human population. Polymorphism rs2696297 (P = 0.003) in COL1A1 and a three SNP haplotype in COL1A2 (P = 0.007) were all significantly associated with normal CCT variation. These data implicate type 1 collagen in the determination of CCT in both OI patients and normal individuals. This provides the first evidence of quantitative trait loci that influence CCT in a normal population and has potential implications for investigating genes involved in glaucoma pathogenesis, a common eye disease in which the severity and progression is influenced by CCT.     

Stringent analysis of gene function and protein-protein interactions using fluorescently tagged genes

In Drosophila collections of green fluorescent protein (GFP) trap lines have been used to probe the endogenous expression patterns of trapped genes or the subcellular localization of their protein products. Here, we describe a method, based on nonoverlapping, highly specific, shRNA transgenes directed against GFP, that extends the utility of these collections to loss-of-function studies. Furthermore, we used a MiMIC transposon to generate GFP traps in Drosophila cell lines with distinct subcellular localization patterns, which will permit high-throughput screens using fluorescently tagged proteins. Finally, we show that fluorescent traps, paired with recombinant nanobodies and mass spectrometry, allow the study of endogenous protein complexes in Drosophila.