Biological and structural phenotypes associated with neurodevelopmental outcomes in congenital heart disease.

Neurodevelopmental disability (NDD) is recognised as one of the most common comorbidities in children with congenital heart disease (CHD) and is associated with altered brain structure and growth throughout the life course. Causes and contributors underpinning the CHD and NDD paradigm are not fully understood, and likely include innate patient factors, such as genetic and epigenetic factors, prenatal haemodynamic consequences as a result of the heart defect, and factors affecting the fetal-placental-maternal environment, such as placental pathology, maternal diet, psychological stress and autoimmune disease. Additional postnatal factors, including the type and complexity of disease and other clinical factors such as prematurity, peri-operative factors and socioeconomic factors are also expected to play a role in determining the final presentation of the NDD. Despite significant advances in knowledge and strategies to optimise outcomes, the extent to which adverse neurodevelopment can be modified remains unknown. Understanding biological and structural phenotypes associated with NDD in CHD are vital for understanding disease mechanisms, which in turn will advance the development of effective intervention strategies for those at risk. This review article summarises our current knowledge surrounding biological, structural, and genetic contributors to NDD in CHD and describes avenues for future research; highlighting the need for translational studies that bridge the gap between basic science and clinical practice.

ConanVarvar: a versatile tool for the detection of large syndromic copy number variation from whole-genome sequencing data.

BACKGROUND: A wide range of tools are available for the detection of copy number variants (CNVs) from whole-genome sequencing (WGS) data. However, none of them focus on clinically-relevant CNVs, such as those that are associated with known genetic syndromes. Such variants are often large in size, typically 1-5 Mb, but currently available CNV callers have been developed and benchmarked for the discovery of smaller variants. Thus, the ability of these programs to detect tens of real syndromic CNVs remains largely unknown. RESULTS: Here we present ConanVarvar, a tool which implements a complete workflow for the targeted analysis of large germline CNVs from WGS data. ConanVarvar comes with an intuitive R Shiny graphical user interface and annotates identified variants with information about 56 associated syndromic conditions. We benchmarked ConanVarvar and four other programs on a dataset containing real and simulated syndromic CNVs larger than 1 Mb. In comparison to other tools, ConanVarvar reports 10-30 times less false-positive variants without compromising sensitivity and is quicker to run, especially on large batches of samples. CONCLUSIONS: ConanVarvar is a useful instrument for primary analysis in disease sequencing studies, where large CNVs could be the cause of disease.

Using novel data linkage of congenital heart disease biobank data with administrative health data to identify cardiovascular outcomes to inform genomic analysis.

Introduction: Contemporary care of congenital heart disease (CHD) is largely standardised, however there is heterogeneity in post-surgical outcomes that may be explained by genetic variation. Data linkage between a CHD biobank and routinely collected administrative datasets is a novel method to identify outcomes to explore the impact of genetic variation. Objective: Use data linkage to identify and validate patient outcomes following surgical treatment for CHD. Methods: Data linkage between clinical and biobank data of children born from 2001-2014 that had a procedure for CHD in New South Wales, Australia, with hospital discharge data, education and death data. The children were grouped according to CHD lesion type and age at first cardiac surgery. Children in each 'lesion/age at surgery group' were classified into 'favourable' and 'unfavourable' cardiovascular outcome groups based on variables identified in linked administrative data including; total time in intensive care, total length of stay in hospital, and mechanical ventilation time up to 5 years following the date of the first cardiac surgery. A blind medical record audit of 200 randomly chosen children from 'favourable' and 'unfavourable' outcome groups was performed to validate the outcome groups. Results: Of the 1872 children in the dataset that linked to hospital or death data, 483 were identified with a 'favourable' cardiovascular outcome and 484 were identified as having a 'unfavourable' cardiovascular outcome. The medical record audit found concordant outcome groups for 182/192 records (95%) compared to the outcome groups categorized using the linked data. Conclusions: The linkage of a curated biobank dataset with routinely collected administrative data is a reliable method to identify outcomes to facilitate a large-scale study to examine genetic variance. These genetic hallmarks could be used to identify patients who are at risk of unfavourable cardiovascular outcomes, to inform strategies for prevention and changes in clinical care.

Insights into the genetic architecture underlying complex, critical congenital heart disease.

Congenital heart disease (CHD) has a multifactorial aetiology, raising the possibility of an underlying genetic burden, predisposing to disease but also variable expression, including variation in disease severity, and incomplete penetrance. Using whole genome sequencing (WGS), the findings of this study, indicate that complex, critical CHD is distinct from other types of disease due to increased genetic burden in common variation, specifically among established CHD genes. Additionally, these findings highlight associations with regulatory genes and environmental "stressors" in the final presentation of disease.

Common Genetic Variants Contribute to Risk of Transposition of the Great Arteries.

RATIONALE: Dextro-transposition of the great arteries (D-TGA) is a severe congenital heart defect which affects approximately 1 in 4,000 live births. While there are several reports of D-TGA patients with rare variants in individual genes, the majority of D-TGA cases remain genetically elusive. Familial recurrence patterns and the observation that most cases with D-TGA are sporadic suggest a polygenic inheritance for the disorder, yet this remains unexplored. OBJECTIVE: We sought to study the role of common single nucleotide polymorphisms (SNPs) in risk for D-TGA. METHODS AND RESULTS: We conducted a genome-wide association study in an international set of 1,237 patients with D-TGA and identified a genome-wide significant susceptibility locus on chromosome 3p14.3, which was subsequently replicated in an independent case-control set (rs56219800, meta-analysis P=8.6x10-10, OR=0.69 per C allele). SNP-based heritability analysis showed that 25% of variance in susceptibility to D-TGA may be explained by common variants. A genome-wide polygenic risk score derived from the discovery set was significantly associated to D-TGA in the replication set (P=4x10-5). The genome-wide significant locus (3p14.3) co-localizes with a putative regulatory element that interacts with the promoter of WNT5A, which encodes the Wnt Family Member 5A protein known for its role in cardiac development in mice. We show that this element drives reporter gene activity in the developing heart of mice and zebrafish and is bound by the developmental transcription factor TBX20. We further demonstrate that TBX20 attenuates Wnt5a expression levels in the developing mouse heart. CONCLUSIONS: This work provides support for a polygenic architecture in D-TGA and identifies a susceptibility locus on chromosome 3p14.3 near WNT5A. Genomic and functional data support a causal role of WNT5A at the locus.

Whole genome sequencing in transposition of the great arteries and associations with clinically relevant heart, brain and laterality genes.

BACKGROUND: The most common cyanotic congenital heart disease (CHD) requiring management as a neonate is transposition of great arteries (TGA). Clinically, up to 50% of TGA patients develop some form of neurodevelopmental disability (NDD), thought to have a significant genetic component. A "ciliopathy" and links with laterality disorders have been proposed. This first report of whole genome sequencing in TGA, sought to identify clinically relevant variants contributing to heart, brain and laterality defects. METHODS: Initial whole genome sequencing analyses on 100 TGA patients focussed on established disease genes related to CHD (n = 107), NDD (n = 659) and heterotaxy (n = 74). Single variant as well as copy number variant analyses were conducted. Variant pathogenicity was assessed using the American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines. RESULTS: Fifty-five putatively damaging variants were identified in established disease genes associated with CHD, NDD and heterotaxy; however, no clinically relevant variants could be attributed to disease. Notably, case-control analyses identified significantly more predicted-damaging, silent and total variants in TGA cases than healthy controls in established CHD genes (P < .001), NDD genes (P < .001) as well as across the three gene panels (P < .001). CONCLUSION: We present compelling evidence that the majority of TGA is not caused by monogenic rare variants and is most likely oligogenic and/or polygenic in nature, highlighting the complex genetic architecture and multifactorial influences on this CHD sub-type and its long-term sequelae. Assessment of variant burden in key heart, brain and/or laterality genes may be required to unravel the genetic contributions to TGA and related disabilities.

A new era of genetic testing in congenital heart disease: A review.

Genetic and genomic testing in pediatric CHD is becoming increasingly routine, and can have important psychosocial, clinical and reproductive implications. In this paper we highlight important challenges and considerations when providing genetics consults and testing in pediatric CHD and illustrate the role of a dedicated CHD genetics clinic. Key lessons include that a) a genetic diagnosis can have clinical utility that justifies testing early in life, b) adequate genetic counselling is crucial to ensure families are supported, understand the range of possible results, and are prepared for new or unexpected health information, and c) further integration of the clinical genetics and cardiology workflows will be required to effectively manage the burgeoning information arising from genetic testing. Our experience demonstrates that a dedicated CHD genetics clinic is a valuable addition to a multidisciplinary team providing care to children with CHD.

"Why and how did this happen?": development and evaluation of an information resource for parents of children with CHD.

BACKGROUND: The causes of CHD are complex and often unknown, leading parents to ask how and why this has happened. Genetic counselling has been shown to benefit these parents by providing information and support; however, most parents currently do not receive this service. This study aimed to develop a brochure to determine whether an information resource could improve parents' knowledge about CHD causation and inheritance and increase psychosocial functioning. METHODS: In development, the resource was assessed against several readability scales and piloted. Parents of children attending preadmission clinic for surgery were included. Assessments occurred pre- and post-receiving the information resource using a purpose-designed knowledge measure and validated psychological measures. RESULTS: Participant's (n = 52) knowledge scores increased significantly from the pre-questionnaire ( ${\overline x}\, = \,5/10$ , sd = 2.086) to post-questionnaire ( $\overline x\, = \,7.88/10$ , sd = 2.094, p < 0.001), with all aware that CHD can be caused by genetic factors after reading the brochure. Perceived personal control also increased from pre- ( $\overline x\, = \,11.856/18$ , sd = 4.339) to post-brochure ( $\overline x\, = \,14.644/18$ , sd = 3.733, p < 0.001), and many reported reduced feelings of guilt. No negative emotional response to the brochure was reported. The information provided was considered relevant (88%), reassuring (86%), and 88% would recommend the brochure to other parents. However, some wanted more emotional support and assistance in what to tell their child. CONCLUSIONS: Use of the information resource significantly enhanced parents' knowledge of CHD causation and increased their psychosocial functioning. It is a valuable resource in the absence of genetic counselling; however, it should not replace formal genetic counselling when required.

Functional genomics and gene-environment interaction highlight the complexity of congenital heart disease caused by Notch pathway variants.

Congenital heart disease (CHD) is the most common birth defect and brings with it significant mortality and morbidity. The application of exome and genome sequencing has greatly improved the rate of genetic diagnosis for CHD but the cause in the majority of cases remains uncertain. It is clear that genetics, as well as environmental influences, play roles in the aetiology of CHD. Here we address both these aspects of causation with respect to the Notch signalling pathway. In our CHD cohort, variants in core Notch pathway genes account for 20% of those that cause disease, a rate that did not increase with the inclusion of genes of the broader Notch pathway and its regulators. This is reinforced by case-control burden analysis where variants in Notch pathway genes are enriched in CHD patients. This enrichment is due to variation in NOTCH1. Functional analysis of some novel missense NOTCH1 and DLL4 variants in cultured cells demonstrate reduced signalling activity, allowing variant reclassification. Although loss-of-function variants in DLL4 are known to cause Adams-Oliver syndrome, this is the first report of a hypomorphic DLL4 allele as a cause of isolated CHD. Finally, we demonstrate a gene-environment interaction in mouse embryos between Notch1 heterozygosity and low oxygen- or anti-arrhythmic drug-induced gestational hypoxia, resulting in an increased incidence of heart defects. This implies that exposure to environmental insults such as hypoxia could explain variable expressivity and penetrance of observed CHD in families carrying Notch pathway variants.

Management of People With a Fontan Circulation: a Cardiac Society of Australia and New Zealand Position statement.

The Fontan circulation describes the circulatory state resulting from an operation in congenital heart disease where systemic venous return is directed to the lungs without an intervening active pumping chamber. As survival increases, so too does recognition of the potential health challenges. This document aims to allow clinicians, people with a Fontan circulation, and their families to benefit from consensus agreement about management of the person with a Fontan circulation. The document was crafted with input from a multidisciplinary group of health care providers as well as individuals with a Fontan circulation and families. It is hoped that the shared common vision of long-term wellbeing will continue to drive improvements in care and quality of life in this patient population and eventually translate into improved survival. KEYPOINTS.

'Big issues' in neurodevelopment for children and adults with congenital heart disease.

It is established that neurodevelopmental disability (NDD) is common in neonates undergoing complex surgery for congenital heart disease (CHD); however, the trajectory of disability over the lifetime of individuals with CHD is unknown. Several 'big issues' remain undetermined and further research is needed in order to optimise patient care and service delivery, to assess the efficacy of intervention strategies and to promote best outcomes in individuals of all ages with CHD. This review article discusses 'gaps' in our knowledge of NDD in CHD and proposes future directions.

Identification of clinically actionable variants from genome sequencing of families with congenital heart disease.

PURPOSE: Congenital heart disease (CHD) affects up to 1% of live births. However, a genetic diagnosis is not made in most cases. The purpose of this study was to assess the outcomes of genome sequencing (GS) of a heterogeneous cohort of CHD patients. METHODS: Ninety-seven families with probands born with CHD requiring surgical correction were recruited for genome sequencing. At minimum, a proband-parents trio was sequenced per family. GS data were analyzed via a two-tiered method: application of a high-confidence gene screen (hcCHD), and comprehensive analysis. Identified variants were assessed for pathogenicity using the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines. RESULTS: Clinically relevant genetic variants in known and emerging CHD genes were identified. The hcCHD screen identified a clinically actionable variant in 22% of families. Subsequent comprehensive analysis identified a clinically actionable variant in an additional 9% of families in genes with recent disease associations. Overall, this two-tiered approach provided a clinically relevant variant for 31% of families. CONCLUSIONS: Interrogating GS data using our two-tiered method allowed identification of variants with high clinical utility in a third of our heterogeneous cohort. However, association of emerging genes with CHD etiology, and development of novel technologies for variant assessment and interpretation, will increase diagnostic yield during future reassessment of our GS data.

Genetic burden and associations with adverse neurodevelopment in neonates with congenital heart disease.

BACKGROUND: Up to 20% of children with congenital heart disease (CHD) undergoing cardiac surgery develop neurodevelopmental disabilities (NDD), with some studies reporting persistent impairment. Recent large-scale studies have demonstrated shared genetic mechanisms contributing to CHD and NDD. In this study, a targeted approach was applied to assess direct clinical applicability of this information. METHODS: A gene panel comprising 148 known CHD and/or NDD genes was used to sequence 15 patients with CHD + NDD, 15 patients with CHD, and 15 healthy controls. The number and types of variants between the 3 groups were compared using Poisson log-linear regression, and the SNP-set (Sequence) Kernel Association Test-Optimized was used to conduct single-gene and gene-pathway burden analyses. RESULTS: A significant increase in rare (minor allele frequency < 0.01) and novel variants was identified between the CHD + NDD cohort and controls, P < .001 and P = .001, respectively. There was also a significant increase in rare variants in the CHD cohort compared with controls (P = .04). Rare variant burden analyses implicated pathways associated with "neurotransmitters," "axon guidance," and those incorporating "RASopathy" genes in the development of NDD in CHD patients. CONCLUSIONS: These findings suggest that an increase in novel and rare variants in known CHD and/or NDD genes is associated with the development of NDD in patients with CHD. Furthermore, burden analyses point toward rare variant burden specifically in pathways related to brain development and function as contributors to NDD. Although promising variants and pathways were identified, further research, utilizing whole-genome approaches, is required prior to demonstrating clinical utility in this patient group.

A Screening Approach to Identify Clinically Actionable Variants Causing Congenital Heart Disease in Exome Data.

BACKGROUND: Congenital heart disease (CHD)-structural abnormalities of the heart that arise during embryonic development-is the most common inborn malformation, affecting ≤1% of the population. However, currently, only a minority of cases can be explained by genetic abnormalities. The goal of this study was to identify disease-causal genetic variants in 30 families affected by CHD. METHODS: Whole-exome sequencing was performed with the DNA of multiple family members. We utilized a 2-tiered whole-exome variant screening and interpretation procedure. First, we manually curated a high-confidence list of 90 genes known to cause CHD in humans, identified predicted damaging variants in genes on this list, and rated their pathogenicity using American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines. RESULTS: In 3 families (10%), we found pathogenic variants in known CHD genes TBX5, TFAP2B, and PTPN11, explaining the cardiac lesions. Second, exomes were comprehensively analyzed to identify additional predicted damaging variants that segregate with disease in CHD candidate genes. In 10 additional families (33%), likely disease-causal variants were uncovered in PBX1, CNOT1, ZFP36L2, TEK, USP34, UPF2, KDM5A, KMT2C, TIE1, TEAD2, and FLT4. CONCLUSIONS: The pathogenesis of CHD could be explained using our high-confidence CHD gene list for variant filtering in a subset of cases. Furthermore, our unbiased screening procedure of family exomes implicates additional genes and variants in the pathogenesis of CHD, which suggest themselves for functional validation. This 2-tiered approach provides a means of (1) identifying clinically actionable variants and (2) identifying additional disease-causal genes, both of which are essential for improving the molecular diagnosis of CHD.

Analysis of DICER1 in familial and sporadic cases of transposition of the great arteries.

OBJECTIVE: We previously identified a pathogenic germline DICER1 variant in a child with transposition of the great arteries who was a member of a family with DICER1 syndrome. In view of a report linking Dicer1 knockout in murine cardiomyocytes to cardiac outflow defects, we investigated the involvement of DICER1 in transposition of the great arteries. DESIGN: We used Fluidigm access array followed by next generation sequencing to screen for variants in the coding exons, their exon/intron boundaries and the 3' untranslated region of DICER1 in patient DNA. CASES: Germline DNA was collected from 129 patients with either sporadic or familial forms of transposition of the great arteries from two sites in Australia and Italy. RESULTS: Most cases (85%) did not have any germline DICER1 variants. In the remaining 15% of cases, we identified 16 previously reported variants (5 synonymous, 6 intronic, and 5 missense) and 2 novel variants (1 intronic and 1 missense). None of the identified variants were predicted to be pathogenic. CONCLUSIONS: Here, we report that neither likely pathogenic nor pathogenic variants in DICER1 appear to play a major role in transposition of the great arteries.

Advances in the Genetics of Congenital Heart Disease: A Clinician's Guide.

Our understanding of the genetics of congenital heart disease (CHD) is rapidly expanding; however, many questions, particularly those relating to sporadic forms of disease, remain unanswered. Massively parallel sequencing technology has made significant contributions to the field, both from a diagnostic perspective for patients and, importantly, also from the perspective of disease mechanism. The importance of de novo variation in sporadic disease is a recent highlight, and the genetic link between heart and brain development has been established. Furthermore, evidence of an underlying burden of genetic variation contributing to sporadic and familial forms of CHD has been identified. Although we are still unable to identify the cause of CHD for most patients, recent findings have provided us with a much clearer understanding of the types of variants and their individual contributions and collectively mark an important milestone in our understanding of both familial and sporadic forms of disease.

The promises and challenges of exome sequencing in familial, non-syndromic congenital heart disease.

BACKGROUND: Exome sequencing is an established strategy to identify causal variants in families with two or more members affected by congenital heart disease (CHD). This unbiased approach, in which both rare and common variants are identified, makes it suitable to research complex, heterogeneous diseases such as CHD. METHODS AND RESULTS: Exome sequencing was performed on two affected members of a three generation family with atrial septal defects (ASD), suggesting a dominant inheritance pattern. Variants were filtered using two bioinformatics pipelines and prioritised according to in silico prediction programs. Segregation studies and functional analyses were used to assess co-segregation with disease and effects on protein function, respectively. Following the data and in silico analyses, ten candidate variants were prioritised. Of these, SRPK2 (c.2044C>T[p.Arg682Trp]) and NOTCH1 (c.3835C>T[p.Arg1279Cys]), co-segregated with disease in the family; however, previous functional analyses on SRPK2 make this an unlikely candidate. Functional analyses in the variant (c.3835C>T[p.Arg1279Cys]) of the known CHD gene NOTCH1 demonstrated a non-significant decrease in signalling activity. CONCLUSION: This study demonstrates both the potential, as well as the challenges, of applying exome sequencing to complex diseases such as CHD. While in silico evidence and segregation analyses in the NOTCH1 p.Arg1279Cys variant are highly suggestive of pathogenicity, the minimal change in signalling capacity suggests that other variants may be required for CHD development. This study highlights the difficulties of applying exome sequencing in familial, non-syndromic CHD in the clinical environment and a cautionary note in the interpretation of apparently causal abnormalities in silico without supportive functional data.