BACKGROUND:Virtual panel analysis (VPA) of exome data is a common approach for the molecular diagnosis of congenital heart disease (CHD). However, differences in gene panel composition and patient inclusion criteria limit the evaluation of its diagnostic utility. This study aims to assess the diagnostic yield of VPA in a cohort of patients with CHD across 3 academic centers. METHODS:We collected clinical data including phenotypic features and family history, from 853 probands with CHD who underwent VPA analysis at the Center for Medical Genetics Ghent (525 probands; 471 genes), the University Medical Center Groningen (195 probands; 345 genes), and the University Medical Center Utrecht (133 probands; 55 genes). We evaluated the diagnostic yield by comparing the 3 centers with respect to panel composition and clinical presentation. RESULTS:The Center for Medical Genetics Ghent reported a higher diagnostic yield (9.9%) compared with the University Medical Center Groningen (7.2%) and the University Medical Center Utrecht (5.3%). In all centers, the diagnostic yield was higher in patients presenting with a syndromic constellation and did not differ significantly between the sporadic and familial cases. In 1.7% of the 536 nonsyndromic probands, a molecular cause was identified that typically is associated with syndromic CHD. Twelve genes showed likely pathogenic or pathogenic variants in multiple patients and contributed to 56.2% of the identified causes. CONCLUSIONS:We report an overall diagnostic yield of VPA for CHD of 8.6%, to which only a few genes contribute significantly, highlighting the complex origin of CHD. Since panel size, gene panel content, and local practices largely affect the diagnostic yield, we propose a (minimum) core gene panel for suspected isolated CHD, as well as a coordinated testing strategy for CHD to improve diagnosis and counseling and to catalyze collaborative efforts.

G protein-coupled receptors (GPCRs) control cell functions by responding to a myriad of extracellular signals, such as hormones and neurotransmitters. GPCRs comprise the most important drug targets. Cells express many different GPCRs each eliciting distinct and specific cell functions; however, GPCRs use only a few second messengers such as cyclic adenosine monophosphate (cAMP) to relay precise receptor stimuli. To control cell signaling specificity, we showed recently that cells organize subcellular cAMP signaling in so-called cAMP nanodomains, namely nanometer-sized signaling compartments of different local cAMP concentrations. GPCRs orchestrate high-concentration cAMP nanodomains to stimulate cell signaling, whereas phosphodiesterases (PDEs), enzymes that degrade cAMP, control low-concentration cAMP nanodomains to gate cAMP effector activation and downstream signaling. This precise cAMP signaling nanoarchitecture is essential for cell homeostasis. HTNB is a Mendelian hypertension form that results in dramatic increases in blood pressure leading to death by stroke at the age of 50 years when untreated. Increased peripheral vascular resistance is responsible. We present an HTNB patient with a novel, single-point mutation (L910P) in the catalytic PDE3A core. The localization indicates a direct influence on cAMP hydrolysis. We developed a FRET-based in vitro assay to determine the hydrolytic activities of the disease-causing PDE3 mutant to be 3-fold higher than PDE3A wt (V max (nM (cAMP)/s) were 143±7 and 45±3, respectively), substantially greater than earlier mutants we described. We used our recently developed nanoruler imaging technology for cAMP and downstream effectors and found that the increased cAMP turnover leads to pathologically large low-cAMP nanodomains in vicinity of PDE3A, thereby altering downstream cAMP signaling. Our data suggest that altered cAMP signaling, at the nanometer scale, causes the hypercontractile vascular smooth muscle phenotype and increased peripheral resistance in HTNB, since local protein kinase-A (PKA) activity is hypoactive in the PDE vicinity. We propose that disrupted cellular cAMP-signaling nanoarchitecture forms the molecular basis for HTNB. - 1 -

G protein-coupled receptors (GPCRs) control cell functions by responding to a myriad of extracellular signals, such as hormones and neurotransmitters. GPCRs comprise the most important drug targets. Cells express many different GPCRs each eliciting distinct and specific cell functions; however, GPCRs use only a few second messengers such as cyclic adenosine monophosphate (cAMP) to relay precise receptor stimuli. To control cell signaling specificity, we showed recently that cells organize subcellular cAMP signaling in so-called cAMP nanodomains, namely nanometer-sized signaling compartments of different local cAMP concentrations. GPCRs orchestrate high-concentration cAMP nanodomains to stimulate cell signaling, whereas phosphodiesterases (PDEs), enzymes that degrade cAMP, control low-concentration cAMP nanodomains to gate cAMP effector activation and downstream signaling. This precise cAMP signaling nanoarchitecture is essential for cell homeostasis. HTNB is a Mendelian hypertension form that results in dramatic increases in blood pressure leading to death by stroke at the age of 50 years when untreated. Increased peripheral vascular resistance is responsible. We present an HTNB patient with a novel, single-point mutation (L910P) in the catalytic PDE3A core. The localization indicates a direct influence on cAMP hydrolysis. We developed a FRET-based in vitro assay to determine the hydrolytic activities of the disease-causing PDE3 mutant to be 3-fold higher than PDE3A wt (V max (nM (cAMP)/s) were 143±7 and 45±3, respectively), substantially greater than earlier mutants we described. We used our recently developed nanoruler imaging technology for cAMP and downstream effectors and found that the increased cAMP turnover leads to pathologically large low-cAMP nanodomains in vicinity of PDE3A, thereby altering downstream cAMP signaling. Our data suggest that altered cAMP signaling, at the nanometer scale, causes the hypercontractile vascular smooth muscle phenotype and increased peripheral resistance in HTNB, since local protein kinase-A (PKA) activity is hypoactive in the PDE vicinity. We propose that disrupted cellular cAMP-signaling nanoarchitecture forms the molecular basis for HTNB. - 1 -

Background: Splice prediction algorithms currently used in routine DNA diagnostics have limited sensitivity and specificity, therefore many potential splice variants are classified as variants of uncertain significance (VUSs). However, functional assessment of VUSs to test splicing is labour-intensive and time-consuming. We developed a decision tree to prioritise potential splice variants for functional studies and functionally verified the outcome of the decision tree. Materials and methods: We built the decision tree, SEPT-GD, by setting thresholds for the splice prediction programs implemented in Alamut. A set of 343 variants with known effects on splicing was used as control for sensitivity and specificity. We tested SEPT-GD using variants from a Dutch cardiomyopathy cohort of 2002 patients that were previously classified as VUS and predicted to have a splice effect according to diagnostic rules. We then selected 12 VUSs ranked by SEPT-GD to functionally verify the predicted effect on splicing using a minigene assay: 10 variants predicted to have a strong effect and 2 with a weak effect. RT-PCR was performed for nine variants. Variant classification was re-evaluated based on the functional test outcome. Results: Compared to similar individually tested algorithms, SEPT-GD shows higher sensitivity (91 %) and comparable specificity (88 %) for both consensus (dinucleotides at the start and end of the intron, GT at the 5 & PRIME; end and AG at the 3 & PRIME; end) and non-consensus splice-site variants (excluding middle of exon variants). Using clinical diagnostic criteria, 1295 unique variants in our cardiomyopathy cohort had originally been classified as VUSs, with 57 predicted by Alamut to have an effect on splicing. Using SEPT-GD, we prioritised 31 variants in 40 patients. In the minigene assay, all 12 variants showed results concordant with SEPT-GD predictions. RT-PCR confirmed the minigene results for two variants, TMEM43 c.1000 + 5G > T and TTN c.25922-6 T > G. Based on all outcomes, the SGCD c.4-1G > A and CSRP3 c.282-5_285del variants were reclassified as likely pathogenic. Conclusion: SEPT-GD outperforms the tools commonly used for RNA splicing prediction and improves prioritisation of variants in cardiomyopathy genes for functional splicing analysis in a diagnostic setting.

Background: In the molecular genetic diagnostics of Mendelian disorders, solutions are needed for the major challenge of dealing with the large number of variants of uncertain significance (VUSs) identified using next-generation sequencing (NGS). Recently, promising approaches using constraint metrics to calculate case excess scores (CE), etiological fractions (EF), and gnomAD-derived constraint scores have been reported that estimate the likelihood of rare variants in specific genes or regions that are pathogenic. Our objective is to study the usability of these constraint data into variant interpretation in a diagnostic setting, using our cardiomyopathy cohort. Methods and Results: Patients (N = 2002) referred for clinical genetic diagnostics underwent NGS testing of 55–61 genes associated with cardiomyopathies. Previously classified likely pathogenic (LP) and pathogenic (P) variants were used to validate the use of data from CE, EF, and gnomAD constraint analyses for (re)classification of associated variant types in specific cardiomyopathy subtype-related genes. The classifications corroborated in 94% (354/378) of cases. Next, we reclassified 23 unique VUSs to LP, increasing the diagnostic yield by 1.2%. In addition, 106 unique VUSs (5.3% of patients) were prioritized for co-segregation or functional analyses. Conclusions: Our analysis confirms that the use of constraint metrics data can improve variant interpretation, and we, therefore, recommend using constraint scores on other cohorts and disorders and its inclusion in variant interpretation protocols.

Vici syndrome (OMIM 242840) is a very rare autosomal recessive multisystem disorder first described in 1988. In 2013, bi-allelic loss-of-function mutations in EPG5 were reported to cause Vici syndrome. Five principal diagnostic features of Vici syndrome have been proposed: agenesis of the corpus callosum, cataracts, cardiomyopathy, hypopigmentation, and combined immunodeficiency. We identified 15 patients carrying a homozygous founder missense variant in EPG5 who all exhibit a less severe clinical phenotype than classic Vici syndrome. All 15 show typical brain abnormalities on MRI. The homozygous founder variant in EPG5 they carry results in a shorter in-frame transcript and truncated, but likely still residual, EPG5 protein. We speculate that the residual EPG5 protein explains their attenuated phenotype, which is consistent with two previous observations that low expression of EPG5 can lead to an attenuated Vici syndrome phenotype. We propose renaming this condition EPG5-related neurodevelopmental disorder to emphasize the clinical variability of patients with bi-allelic mutations in EPG5.

Uncombable hair syndrome is a hair shaft condition in which the hair is frizzy, light in color (silver to light brown), and cannot be combed flat. Autosomal dominant (with complete or incomplete penetrance), autosomal recessive, and sporadic cases have been reported. In 2016 causative mutations in three genes were identified for uncombable hair syndrome, all with an autosomal recessive inheritance pattern: PADI3 , TGM3 , and TCHH . In many cases, however, there is still no molecular diagnosis. Here, we describe a case of autosomal recessive uncombable hair syndrome resulting from maternal uniparental disomy of chromosome 1.

Mendelian syndromes give great insight into pathogenesis and have implicated salt handling. Hypertension with brachydactyly (HTNB) is unique in that a direct increase in peripheral vascular resistance is produced by activating mutations in phosphodiesterase 3A (PDE3A). A 50 mm Hg blood-pressure elevation by age 50 years causes stroke in untreated persons. Here, we report mutations in the PDE3 catalytic domain found in two new HTNB families. Since structural predictions indicated increased PDE3A cleavage activity, we used Förster resonance energy transfer (FRET) to measure the PDE3-specific cAMP degradation in cytosolic fractions from transfected cells. The newly discovered catalytic-domain PDE3A mutants, R862C and L910P, both showed a shorter transient emission-ratio change upon cAMP addition than PDE3A wildtype, indicating faster cAMP turnover. The V max of all experiments were extracted from FRET data and quantitated and analyzed statistically. The mean values V max (nM (cAMP)/s) were 143±7 for L910P, 63±3 for R862C and 73±4 for T445N, and 45±3 for WT respectively, (p<0.0001, <0.001, <0.05). We found that all disease-relevant mutations led to a clearly hyperactive PDE3A. The L910P catalytic-domain mutation resulted in the most dramatic increase in cAMP hydrolysis. That mutation also exhibited the most stepwise resistance to milrinone. Recently, we showed that cAMP signaling is organized in nanometer-sized cellular compartments. We suggest that the gain-of-function PDE3 mutations result in unphysiologically larger cAMP nanodomains and thereby dysregulate crucially important cAMP nanoarchitecture. Spatiotemporal cAMP signaling and precise PDE-controlled effector activation, could usher in a site-specific nanomolecular pharmacology.

We report a 19-month-old patient with cardiomyopathy as the first presenting feature of primary COQ10 deficiency-6. This case expands the phenotypic spectrum of this disorder. Furthermore, it shows that genetic testing for primary COQ10 deficiency should be considered in patients with pediatric-onset cardiomyopathy as it can guide treatment options.

Background Cardiotoxicity presenting as cardiomyopathy is a common side effect in cancer treatment especially with anthracyclines. The role of genetic predisposition is still being investigated. Case summary Four unrelated patients with a familial burden for cardiac disease, who developed cardiomyopathy after anthracycline treatment are presented. Case 1 received chemotherapy for breast cancer and developed a dilated left ventricle just after treatment. Her father had died unexpectedly while being screened for heart transplant. Case 2 was known with a family history of sudden cardiac death prior to her breast cancer diagnosis. She received anthracycline-containing chemotherapy treatment twice in 5 years due to recurrence of breast cancer. During that period, two brothers developed a cardiomyopathy. Eighteen years later, a genetic predisposition for cardiomyopathy was ascertained and at screening an asymptomatic non-ischaemic cardiomyopathy was established. Case 3 was diagnosed with a dilated cardiomyopathy 1 year after chemotherapy treatment for breast cancer. Her mother had developed a dilated cardiomyopathy several years before. Case 4 received chemotherapy treatment for Non-Hodgkin's lymphoma and developed dilated cardiomyopathy 1 year later. His brother died from congestive heart failure which he developed after chemotherapy for Non-Hodgkin's lymphoma and a grandmother had died suddenly during child delivery. In all four cases, genetic screening showed (likely) pathogenic variants in cardiomyopathy-associated genes. Discussion Current guidelines recommend cardiac evaluation in cancer patients receiving chemotherapy based on the presence of cardiovascular risk factors at the start of treatment. This series emphasizes the importance of including a thorough family history in this process.

Background: Next-generation sequencing (NGS) is increasingly used for clinical evaluation of cardiomyopathy patients as it allows for simultaneous screening of multiple cardiomyopathy-associated genes. Adding copy number variant (CNV) analysis of NGS data is not routine yet and may contribute to the diagnostic yield. Objectives: Determine the diagnostic yield of our targeted NGS gene panel in routine clinical diagnostics of Dutch cardiomyopathy patients and explore the impact of exon CNVs on diagnostic yield. Methods: Patients (N = 2002) referred for clinical genetic analysis underwent diagnostic testing of 55-61 genes associated with cardiomyopathies. Samples were analyzed and evaluated for single nucleotide variants (SNVs), indels and CNVs. CNVs identified in the NGS data and suspected of being pathogenic based on type, size and location were confirmed by additional molecular tests. Results: A (likely) pathogenic (L)P variant was detected in 22.7% of patients, including 3 with CNVs and 25 where a variant was identified in a gene currently not associated with the patient's cardiomyopathy subtype. Only 15 out of 2002 patients (0.8%) were found to carry two (L)P variants. Conclusion: The yield of routine clinical diagnostics of cardiomyopathies was relatively low when compared to literature. This is likely due to the fact that our study reports the outcome of patients in daily routine diagnostics, therefore also including patients not fully fulfilling (subtype specific) cardiomyopathy criteria. This may also explain why (L)P variants were identified in genes not associated with the reported subtype. The added value of CNV analysis was shown to be limited but not negligible. (C) 2021 The Authors. Published by Elsevier B.V.

Deletions that include the gene TAB2 and TAB2 loss-of-function variants have previously been associated with congenital heart defects and cardiomyopathy. However, other features, including short stature, facial dysmorphisms, connective tissue abnormalities and a variable degree of developmental delay, have only been mentioned occasionally in literature and thus far not linked to TAB2 . In a large-scale, social media-based chromosome 6 study, we observed a shared phenotype in patients with a 6q25.1 deletion that includes TAB2 . To confirm if this phenotype is caused by haploinsufficiency of TAB2 and to delineate a TAB2 -related phenotype, we subsequently sequenced TAB2 in patients with matching phenotypes and recruited patients with pathogenic TAB2 variants detected by exome sequencing. This identified 11 patients with a deletion containing TAB2 (size 1.68–14.31 Mb) and 14 patients from six families with novel truncating TAB2 variants. Twenty (80%) patients had cardiac disease, often mitral valve defects and/or cardiomyopathy, 18 (72%) had short stature and 18 (72%) had hypermobility. Twenty patients (80%) had facial features suggestive for Noonan syndrome. No substantial phenotypic differences were noted between patients with deletions and those with intragenic variants. We then compared our patients to 45 patients from the literature. All literature patients had cardiac diseases, but syndromic features were reported infrequently. Our study shows that the phenotype in 6q25.1 deletions is caused by haploinsufficiency of TAB2 and that TAB2 is associated not just with cardiac disease, but also with a distinct phenotype, with features overlapping with Noonan syndrome. We propose the name “ TAB2 -related syndrome”.

Objective To describe the prevalence of pulmonary arterial hypertension (PAH)-associated gene mutations, and other genetic characteristics in a national cohort of children with PAH from the Dutch National registry and to explore genotype-phenotype associations and outcomes. Study design Children (n = 70) diagnosed with idiopathic PAH, heritable PAH, PAH associated with congenital heart disease with coincidental shunt (PAH-congenital heart disease group 3), PAH after closure of a cardiac shunt (PAH-congenital heart disease group 4), or PAH associated with other noncardiac conditions were enrolled. Targeted next-generation sequencing was performed on PAH-associated genes (BMPR2, ACVRL1 , EIF2AK4, CAV1 , ENG, KCNK3, SMAD9, and TBX4). Also, children were tested for specific genetic disorders in case of clinical suspicion. Additionally, children were tested for copy number variations. Results Nineteen children (27%) had a PAH-associated gene mutation/variant: BMPR2 n = 7, TBX4 n = 8, ACVRL1 n = 1, KCNK3 n = 1, and EIF2AK4 n = 2. Twelve children (17%) had a genetic disorder with an established association with PAH (including trisomy 21 and cobalamin C deficiency). In another 16 children (23%), genetic disorders without an established association with PAH were identified (including Noonan syndrome, Beals syndrome, and various copy number variations). Survival rates differed between groups and was most favorable in TBX4 variant carriers. Conclusions Children with PAH show a high prevalence of genetic disorders, not restricted to established PAH-associated genes. Genetic architecture could play a role in risk-stratified care management in pediatric PAH.

The etiology of nonimmune hydrops fetalis is extensive and includes genetic disorders. We describe a term-born female neonate with late onset extensive nonimmune hydrops, that is, polyhydramnios, edema, and congenital bilateral chylothorax. This newborn was successfully treated with repetitive thoracocentesis, total parenteral feeding, octreotide intravenously and finally surgical pleurodesis and corticosteroids. A genetic cause seemed plausible as the maternal history revealed a fatal nonimmune hydrops fetalis. A homozygous truncating variant in GDF2 (c.451C>T, p.(Arg151*)) was detected with exome sequencing. Genetic analysis of tissue obtained from the deceased fetal sibling revealed the same homozygous variant. The parents and two healthy siblings were heterozygous for the GDF2 variant. Skin and lung biopsies in the index patient, as well as the revised lung biopsy of the deceased fetal sibling, showed lymphatic dysplasia and lymphangiectasia. To the best of our knowledge, this is the first report of an association between a homozygous variant in GDF2 with lymphatic dysplasia, hydrothorax and nonimmune hydrops fetalis.

Previously, intragenic CAMTA1 copy number variants (CNVs) have been shown to cause non-progressive, congenital ataxia with or without intellectual disability (OMIM#614756). However, ataxia, intellectual disability, and dysmorphic features were all incompletely penetrant, even within families. Here, we describe four patients with de novo nonsense, frameshift or missense CAMTA1 variants. All four patients predominantly manifested features of ataxia and/or spasticity. Borderline intellectual disability and dysmorphic features were both present in one patient only, and other neurological and behavioural symptoms were variably present. Neurodevelopmental delay was found to be mild. Our findings indicate that also nonsense, frameshift and missense variants in CAMTA1 can cause a spastic ataxia syndrome as the main phenotype.