Lijiang Ma

De novo mutations in histone-modifying genes in congenital heart disease.

Congenital heart disease (CHD) is the most frequent birth defect, affecting 0.8% of live births. Many cases occur sporadically and impair reproductive fitness, suggesting a role for de novo mutations. Here we compare the incidence of de novo mutations in 362 severe CHD cases and 264 controls by analysing exome sequencing of parent–offspring trios. CHD cases show a significant excess of protein-altering de novo mutations in genes expressed in the developing heart, with an odds ratio of 7.5 for damaging (premature termination, frameshift, splice site) mutations. Similar odds ratios are seen across the main classes of severe CHD. We find a marked excess of de novo mutations in genes involved in the production, removal or reading of histone 3 lysine 4 (H3K4) methylation, or ubiquitination of H2BK120, which is required for H3K4 methylation. There are also two de novo mutations in SMAD2, which regulates H3K27 methylation in the embryonic left–right organizer. The combination of both activating (H3K4 methylation) and inactivating (H3K27 methylation) chromatin marks characterizes ‘poised’ promoters and enhancers, which regulate expression of key developmental genes. These findings implicate de novo point mutations in several hundreds of genes that collectively contribute to approximately 10% of severe CHD.

Whole Exome Sequencing to Identify a Novel Gene (Caveolin-1) Associated with Human Pulmonary Arterial Hypertension

Background— Heritable and idiopathic pulmonary arterial hypertension (PAH) are phenotypically identical and associated with mutations in several genes related to transforming growth factor (TGF) beta signaling, including bone morphogenetic protein receptor type 2, activin receptor-like kinase 1, endoglin, and mothers against decapentaplegic 9. Approximately 25% of heritable cases lack identifiable mutations in any of these genes. Methods and Results— We used whole exome sequencing to study a 3-generation family with multiple affected family members with PAH, but no identifiable TGF beta mutation. We identified a frameshift mutation in caveolin-1 (CAV1), which encodes a membrane protein of caveolae abundant in the endothelium and other cells of the lung. An independent de novo frameshift mutation was identified in a child with idiopathic PAH. Western blot analysis demonstrated a reduction in caveolin-1 protein, while lung tissue immunostaining studies demonstrated a reduction in normal caveolin-1 density within the endothelial cell layer of small arteries. Conclusions— Our study represents successful elucidation of a dominant Mendelian disorder using whole exome sequencing. Mutations in CAV1 are associated in rare cases with PAH. This may have important implications for pulmonary vascular biology, as well as PAH-directed therapeutic development.

De novo mutations in congenital heart disease with neurodevelopmental and other congenital anomalies

Putting both heart and brain at risk For reasons that are unclear, newborns with congenital heart disease (CHD) have a high risk of neurodevelopmental disabilities. Homsy et al. performed exome sequence analysis of 1200 CHD patients and their parents to identify spontaneously arising (de novo) mutations. Patients with both CHD and neurodevelopmental disorders had a much higher burden of damaging de novo mutations, particularly in genes with likely roles in both heart and brain development. Thus, clinical genotyping of patients with CHD may help to identify those at greatest risk of neurodevelopmental disabilities, allowing surveillance and early intervention. Science, this issue p. 1262 Genotyping of children with congenital heart disease may identify those at high risk of neurodevelopmental disorders. Congenital heart disease (CHD) patients have an increased prevalence of extracardiac congenital anomalies (CAs) and risk of neurodevelopmental disabilities (NDDs). Exome sequencing of 1213 CHD parent-offspring trios identified an excess of protein-damaging de novo mutations, especially in genes highly expressed in the developing heart and brain. These mutations accounted for 20% of patients with CHD, NDD, and CA but only 2% of patients with isolated CHD. Mutations altered genes involved in morphogenesis, chromatin modification, and transcriptional regulation, including multiple mutations in RBFOX2, a regulator of mRNA splicing. Genes mutated in other cohorts examined for NDD were enriched in CHD cases, particularly those with coexisting NDD. These findings reveal shared genetic contributions to CHD, NDD, and CA and provide opportunities for improved prognostic assessment and early therapeutic intervention in CHD patients.

Glut1 deficiency syndrome and erythrocyte glucose uptake assay

The Glut1 deficiency syndrome (Glut1 DS) phenotype has expanded dramatically since first described in 1991. Hypoglycorrhachia and decreased erythrocyte 3‐OMG uptake are confirmatory laboratory biomarkers. The objective is to expand previous observations regarding the diagnostic value of the uptake assay.

De novo copy number variants are associated with congenital diaphragmatic hernia

Background Congenital diaphragmatic hernia (CDH) is a common birth defect with significant morbidity and mortality. Although the aetiology of CDH remains poorly understood, studies from animal models and patients with CDH suggest that genetic factors play an important role in the development of CDH. Chromosomal anomalies have been reported in CDH. Methods In this study, the authors investigated the frequency of chromosomal anomalies and copy number variants (CNVs) in 256 parent–child trios of CDH using clinical conventional cytogenetic and microarray analysis. The authors also selected a set of CDH related training genes to prioritise the genes in those segmental aneuploidies and identified the genes and gene sets that may contribute to the aetiology of CDH. Results The authors identified chromosomal anomalies in 16 patients (6.3%) of the series including three aneuploidies, two unbalanced translocation, and 11 patients with de novo CNVs ranging in size from 95 kb to 104.6 Mb. The authors prioritised the genes in the CNV segments and identified KCNA2, LMNA, CACNA1S, MYOG, HLX, LBR, AGT, GATA4, SOX7, HYLS1, FOXC1, FOXF2, PDGFA, FGF6, COL4A1, COL4A2, HOMER2, BNC1, BID, and TBX1 as genes that may be involved in diaphragm development. Gene enrichment analysis identified the most relevant gene ontology categories as those involved in tissue development (p=4.4×10−11) or regulation of multicellular organismal processes (p=2.8×10−10) and ‘receptor binding’ (p=8.7×10−14) and ‘DNA binding transcription factor activity’ (p=4.4×10−10). Conclusions The present findings support the role of chromosomal anomalies in CDH and provide a set of candidate genes including FOXC1, FOXF2, PDGFA, FGF6, COL4A1, COL4A2, SOX7, BNC1, BID, and TBX1 for further analysis in CDH.

Mutations in ZIC3 and ACVR2B are a common cause of heterotaxy and associated cardiovascular anomalies.

BACKGROUND Heterotaxy syndrome is caused by left-right asymmetry disturbances and is associated with abnormal lateralisation of the abdominal and thoracic organs. The heart is frequently involved and the severity of the abnormality usually determines the outcome. METHODS We performed a direct sequence analysis of the coding sequence of genes including Zinc Finger Protein of the Cerebellum 3, Left-Right Determination Factor 2, Activin A Receptor Type IIB, and Cryptic in 47 patients with laterality defects and congenital cardiac disease. RESULTS Of the 47 patients, 31 (66%) had atrioventricular septal defects, 34 (72%) had abnormal systemic venous return, 25 (53%) had transposed or malposed great arteries, and 20 (43%) had pulmonary venous abnormalities. We identified two novel genetic changes in Zinc Finger Protein of the Cerebellum 3, and these variants were not present in 100 ethnically matched control samples. One previously reported missense mutation in Activin A Receptor Type IIB was identified in two unrelated subjects. The genetic changes identified in this study are all located in conserved regions and are predicted to affect protein function in left-right axis formation and cardiovascular development. CONCLUSIONS Mutations in Zinc Finger Protein of the Cerebellum 3 and Activin A Receptor Type IIB were identified in 4 of the 47 patients with heterotaxy syndrome for a yield of approximately 8.5%. Our results expand the mutation spectrum of monogenic heterotaxy syndrome with associated cardiac anomalies and suggest that there are other causes of heterotaxy yet to be identified.

The role of genetics in pulmonary arterial hypertension

Group 1 pulmonary hypertension or pulmonary arterial hypertension (PAH) is a rare disease characterized by proliferation and occlusion of small pulmonary arterioles, leading to progressive elevation of pulmonary artery pressure and pulmonary vascular resistance, and right ventricular failure. Historically, it has been associated with a high mortality rate, although, over the last decade, treatment has improved survival. PAH includes idiopathic PAH (IPAH), heritable PAH (HPAH), and PAH associated with certain medical conditions. The aetiology of PAH is heterogeneous, and genetics play an important role in some cases. Mutations in BMPR2, encoding bone morphogenetic protein receptor 2, a member of the transforming growth factor‐β superfamily of receptors, have been identified in 70% of cases of HPAH, and in 10–40% of cases of IPAH. Other genetic causes of PAH include mutations in the genes encoding activin receptor‐like type 1, endoglin, SMAD9, caveolin 1, and potassium two‐pore‐domain channel subfamily K member 3. Mutations in the gene encoding T‐box 4 have been identified in 10–30% of paediatric PAH patients, but rarely in adults with PAH. PAH in children is much more heterogeneous than in adults, and can be associated with several genetic syndromes, congenital heart disease, pulmonary disease, and vascular disease. In addition to rare mutations as a monogenic cause of HPAH, common variants in the gene encoding cerebellin 2 increase the risk of PAH by approximately two‐fold. A PAH panel of genes is available for clinical testing, and should be considered for use in clinical management, especially for patients with a family history of PAH. Copyright

Quantitative Analysis of Copy Number Variants Based on Real‐Time LightCycler PCR

Quantitative real‐time PCR is PCR visualized in real time by the use of fluorescent or intercalating dyes, which are employed to measure gene expression or gene quantification including contiguous gene deletions or duplications. A simple method is described here to quantify DNA copy number from human samples. Curr. Protoc. Hum. Genet. 80:7.21.1‐7.21.8.

De novo missense variants in PPP1CB are associated with intellectual disability and congenital heart disease

Intellectual disabilities are genetically heterogeneous and can be associated with congenital anomalies. Using whole-exome sequencing (WES), we identified five different de novo missense variants in the protein phosphatase-1 catalytic subunit beta (PPP1CB) gene in eight unrelated individuals who share an overlapping phenotype of dysmorphic features, macrocephaly, developmental delay or intellectual disability (ID), congenital heart disease, short stature, and skeletal and connective tissue abnormalities. Protein phosphatase-1 (PP1) is a serine/threonine-specific protein phosphatase involved in the dephosphorylation of a variety of proteins. The PPP1CB gene encodes a PP1 subunit that regulates the level of protein phosphorylation. All five altered amino acids we observed are highly conserved among the PP1 subunit family, and all are predicted to disrupt PP1 subunit binding and impair dephosphorylation. Our data suggest that our heterozygous de novo PPP1CB pathogenic variants are associated with syndromic intellectual disability.

Exome Sequencing in Children With Pulmonary Arterial Hypertension Demonstrates Differences Compared With Adults

Background: Pulmonary arterial hypertension (PAH) is a rare disease characterized by pulmonary arteriole remodeling, elevated arterial pressure and resistance, and subsequent heart failure. Compared with adult-onset disease, pediatric-onset PAH is more heterogeneous and often associated with worse prognosis. Although BMPR2 mutations underlie ≈70% of adult familial PAH (FPAH) cases, the genetic basis of PAH in children is less understood. Methods: We performed genetic analysis of 155 pediatric- and 257 adult-onset PAH patients, including both FPAH and sporadic, idiopathic PAH (IPAH). After screening for 2 common PAH risk genes, mutation-negative FPAH and all IPAH cases were evaluated by exome sequencing. Results: We observed similar frequencies of rare, deleterious BMPR2 mutations in pediatric- and adult-onset patients: ≈55% in FPAH and 10% in IPAH patients in both age groups. However, there was significant enrichment of TBX4 mutations in pediatric- compared with adult-onset patients (IPAH: 10/130 pediatric versus 0/178 adult-onset), and TBX4 carriers had younger mean age-of-onset compared with BMPR2 carriers. Mutations in other known PAH risk genes were infrequent in both age groups. Notably, among pediatric IPAH patients without mutations in known risk genes, exome sequencing revealed a 2-fold enrichment of de novo likely gene-damaging and predicted deleterious missense variants. Conclusions: Mutations in known PAH risk genes accounted for ≈70% to 80% of FPAH in both age groups, 21% of pediatric-onset IPAH, and 11% of adult-onset IPAH. Rare, predicted deleterious variants in TBX4 are enriched in pediatric patients and de novo variants in novel genes may explain ≈19% of pediatric-onset IPAH cases.