University of Groningen
Paediatric cardiomyopathies
Herkert, Johanna Cornelia
DOI:
10.33612/diss.97534698
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Publication date:
2019
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Herkert, J. C. (2019). Paediatric cardiomyopathies: an evolving landscape of genetic aetiology and
diagnostic applications. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.97534698
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Chapter 10
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Summary
Since the early 1990s inherited gene variants have been implicated in cardiomyopathy, an insidious disease of the ventricular myocardium. Hypertrophic cardiomyopathy (HCM), characterized by unexplained cardiac hypertrophy and impaired relaxation, and dilated cardiomyopathy (DCM), defined as increased ventricular chamber volume with contractile impairment, were initially thought to be primarily diseases of sarcomeric proteins. However, recent advances in sequencing and array-based technologies have changed our understanding of the genetic basis of cardiomyopathies and dramatically increased the rate of detection of genomic variants. Particularly in paediatric-onset cardiomyopathy, which is even more heterogeneous than adult-onset cardiomyopathy, next-generation sequencing (NGS) has allowed identification of a still-increasing number of variants and genes related to inherited cardiomyopathies. This thesis focussed on the yield, optimal testing strategy and identification of variants in known and novel genes in children with cardiomyopathy, in particular those with HCM and DCM.
Chapter 1 provides a general introduction and explains the differences in the definition and
classification of cardiomyopathies between the American Heart Association (AHA) and the European Society of Cardiology (ESC), which are controversial in clinical practice. It also presents an introduction to current knowledge on incidence, presentation, causes, prognosis and therapies in paediatric HCM and DCM.
Pathogenic variants in cardiac myosin-binding protein C (MYBPC3) are found in approximately 15% of all HCM patients1 and account for approximately 40-50% of all HCM mutations, making
it the most frequently mutated gene in adult-onset HCM.2-4 It may not be surprising that biallelic
variants, either homozygous or compound heterozygous, have also been identified, and these individuals appear to develop severe early-onset disease. Chapter 3 describes four individuals with biallelic truncating variants in MYBPC3 and severe neonatal HCM leading to heart failure and death before age 13 weeks. All four children had septal defects. Three of them also presented with features with left ventricular noncompaction (LVNC). We then performed a literature study and ultimately identified 51 cases of homozygotes or compound heterozygotes: 21 cases with biallelic truncating variants (including our four patients), 13 cases with a missense variant plus a truncating variant and 17 cases with biallelic missense variants. Our analysis of the literature data indicates a more severe phenotype in patients with two functional null alleles: all of them presented with neonatal HCM and 71% died within the first year of life.In 62% (13/21), septal defects or a patent ductus arteriosus accompanied the cardiomyopathy. Chapter 2 describes the genetics and clinical utility of genetic testing in familial DCM in 2013. Many of these genes are now included in most diagnostic NGS panels targeting adult-onset cardiomyopathy. Forty of these 51 genes have been added to a ‘core list’ of 46 cardiomyopathy-related genes developed
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by a group of disease experts of the Dutch Society for Clinical Genetic Laboratory Diagnostics (VKGL).5 At least 26 of the genes listed in chapter 2 are also implicated in paediatric DCM,
and the taffazin (TAZ) gene (Barth syndrome) and SDHA gene (mitochondrial respiratory chain complex II deficiency) are exclusively related to paediatric-onset cardiomyopathy.
In adult-onset DCM, the most frequently mutated genes are TTN, LMNA, MYH7 and TNNT2.6
However, there are only a few large studies that report yield and the most common involved genes in children. In chapter 4, we report a yield of approximately 50% in a series of paediatric patients with and without extracardiac features. We show that whole exome sequencing (WES) with step-wise analysis of (i) well-known cardiomyopathy genes, (ii) copy number variants (CNVs), (iii) all genes assigned to the human phenotype ontology (HPO) term ‘cardiomyopathy’ and (iv), if appropriate, genes assigned to other HPO terms is a justified method for genetic testing in paediatric DCM. Consistent with previous and subsequent studies, we report a relatively high percentage of de novo variants in our DCM cohort (4/15, 26%). In chapter 5 we present GADO (GeneNetwork Assisted Diagnostic Optimization), a gene prioritization method that can be used without prior knowledge about gene function(s). To prioritize variants in genes, GADO uses gene co-regulation based on a high-quality dataset of 31,499 RNA-seq samples to link genes to HPO terms describing the patient’s phenotype. This resulted in the identification of several candidate genes that may have a role in the pathophysiology of cardiomyopathy and illustrates the importance of cross-talk between clinicians and bioinformaticians in providing an integrated approach to diagnose as many patients as possible.
Chapter 6 shows an example of the advantage of WES over conventional diagnostic procedures
in a child with congenital myopathy. Lack of centralized nuclei on muscle biopsy and limited genetic analysis, prevented an actual diagnosis until WES was performed several years after his death. This case also illustrates that use of HPO terms to describe the patient’s phenotype can be successfully applied to filter WES data. Chapter 7 describes the successful identification of a novel disease gene by combining two techniques, WES and homozygosity mapping, in two consanguineous families. We thereby identified homozygous truncating mutations in a new disease gene: alpha-kinase 3 (ALPK3). This gene encodes a nuclear kinase essential for early differentiation of cardiomyocytes. A third affected proband was identified upon cohort screening. Patients with biallelic mutations presented with severe cardiomyopathy leading to early lethality or biventricular dysfunction in childhood. We provided microscopic evidence of intercalated disc remodelling, as had been previously observed in Alpk3 knockout mice. In our follow-up study, chapter 8, we report novel damaging biallelic ALPK3 variants and expand the genetic and phenotypic spectrum of ALPK3-related cardiomyopathy. Nearly half of the individuals showed progression from DCM to ventricular hypertrophy, a phenomenon that has not been described before. We further show that the clinical presentation is more diverse and includes short stature, scoliosis, contractures, cleft palate or velopharyngeal insufficiency,
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and craniofacial dysmorphic features. We also report that (biallelic) missense variants lead to a phenotype similar to that seen for both the novel and previously published loss-of-function (LoF) variants. Since we noted that some of the heterozygous relatives described in chapter 7 also developed HCM at later age, we assessed the prevalence of ALPK3 variants in two unrelated adult-onset cardiomyopathy cohorts from the Netherlands and US. Here we showed that the burden of ALPK3 LoF variants was higher in these two cohorts than in the general population of gnomAD (p=1.6x10-5 and p=2.2x10-13, respectively). In chapter 9 we use exome sequencing and
histological and biochemical analyses to demonstrate that biallelic variants in SOD2 were the cause of an early-onset, rapidly progressive form of DCM in a neonate. Superoxide dismutase 2 (SOD2) is a mitochondrial matrix protein that scavenges oxygen radicals produced by oxidation-reduction and electron transport reactions occurring in mitochondria via conversion of superoxide anion (O2–•) into H
2O2. The SOD2 variant was shown to affect the catalytic activity
of the protein, leading to excess oxygen radical levels that can have strongly damaging effects in the neonatal heart. Lentiviral gene rescue completely restored superoxide dismutase activity. Together with previously reported evidence from Sod2 knockout mice, we provide compelling evidence for the role of SOD2 in cardiomyopathy that sheds new light on the mechanisms underlying myocardial dysfunction.
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References
1. Alfares AA, Kelly MA, McDermott G, et al. Results of clinical genetic testing of 2,912 probands with hypertrophic cardiomyopathy: expanded panels offer limited additional sensitivity. Genet Med 2015;17:880-888.
2. Behrens-Gawlik V, Mearini G, Gedicke-Hornung C, Richard P, Carrier L. MYBPC3 in hypertrophic cardiomyopathy: from mutation identification to RNA-based correction. Pflugers Arch 2014;466:215-223.
3. Schlossarek S, Mearini G, Carrier L. Cardiac myosin-binding protein C in hypertrophic cardiomyopathy: mechanisms and therapeutic opportunities. J Mol Cell Cardiol 2011;50:613-620.
4. Marian AJ, Braunwald E. Hypertrophic Cardiomyopathy: Genetics, Pathogenesis, Clinical Manifestations, Diagnosis, and Therapy. Circ Res 2017;121:749-770.
5. Weiss MM, Van der Zwaag B, Jongbloed JD, et al. Best practice guidelines for the use of next-generation sequencing applications in genome diagnostics: a national collaborative study of Dutch genome diagnostic laboratories. Hum Mutat 2013;34:1313-1321.
6. Pugh TJ, Kelly MA, Gowrisankar S, et al. The landscape of genetic variation in dilated cardiomyopathy as surveyed by clinical DNA sequencing. Genet Med 2014;16:601-608.
