Genetic landscape of pediatric acute liver failure of indeterminate origin.

BACKGROUND AND AIMS: Pediatric acute liver failure (PALF) is a life-threatening condition. In Europe, the main causes are viral infections (12%-16%) and inherited metabolic diseases (14%-28%). Yet, in up to 50% of cases the underlying etiology remains elusive, challenging clinical management, including liver transplantation. We systematically studied indeterminate PALF cases referred for genetic evaluation by whole-exome sequencing (WES), and analyzed phenotypic and biochemical markers, and the diagnostic yield of WES in this condition. APPROACH AND RESULTS: With this international, multicenter observational study, patients (0-18 y) with indeterminate PALF were analyzed by WES. Data on the clinical and biochemical phenotype were retrieved and systematically analyzed. RESULTS: In total, 260 indeterminate PALF patients from 19 countries were recruited between 2011 and 2022, of whom 59 had recurrent PALF. WES established a genetic diagnosis in 37% of cases (97/260). Diagnostic yield was highest in children with PALF in the first year of life (41%), and in children with recurrent acute liver failure (64%). Thirty-six distinct disease genes were identified. Defects in NBAS (n=20), MPV17 (n=8), and DGUOK (n=7) were the most frequent findings. When categorizing, the most frequent were mitochondrial diseases (45%), disorders of vesicular trafficking (28%), and cytosolic aminoacyl-tRNA synthetase deficiencies (10%). One-third of patients had a fatal outcome. Fifty-six patients received liver transplantation. CONCLUSIONS: This study elucidates a large contribution of genetic causes in PALF of indeterminate origin with an increasing spectrum of disease entities. The high proportion of diagnosed cases and potential treatment implications argue for exome or in future rapid genome sequencing in PALF diagnostics.

Heterozygous BRCA1 and BRCA2 and Mismatch Repair Gene Pathogenic Variants in Children and Adolescents With Cancer.

BACKGROUND: Genetic predisposition is has been identified as a cause of cancer, yet little is known about the role of adult cancer predisposition syndromes in childhood cancer. We examined the extent to which heterozygous pathogenic germline variants in BRCA1, BRCA2, PALB2, ATM, CHEK2, MSH2, MSH6, MLH1, and PMS2 contribute to cancer risk in children and adolescents. METHODS: We conducted a meta-analysis of 11 studies that incorporated comprehensive germline testing for children and adolescents with cancer. ClinVar pathogenic or likely pathogenic variants (PVs) in genes of interest were compared with 2 control groups. Results were validated in a cohort of mainly European patients and controls. We employed the Proxy External Controls Association Test to account for different pipelines. RESULTS: Among 3975 children and adolescents with cancer, statistically significant associations with cancer risk were observed for PVs in BRCA1 and 2 (26 PVs vs 63 PVs among 27 501 controls, odds ratio = 2.78, 95% confidence interval = 1.69 to 4.45; P < .001) and mismatch repair genes (19 PVs vs 14 PVs among 27 501 controls, odds ratio = 7.33, 95% confidence interval = 3.64 to 14.82; P <.001). Associations were seen in brain and other solid tumors but not in hematologic neoplasms. We confirmed similar findings in 1664 pediatric cancer patients primarily of European descent. CONCLUSION: These data suggest that heterozygous PVs in BRCA1 and 2 and mismatch repair genes contribute with reduced penetrance to cancer risk in children and adolescents. No changes to predictive genetic testing and surveillance recommendations are required.

Uncovering the Contribution of Moderate-Penetrance Susceptibility Genes to Breast Cancer by Whole-Exome Sequencing and Targeted Enrichment Sequencing of Candidate Genes in Women of European Ancestry.

Rare variants in at least 10 genes, including BRCA1, BRCA2, PALB2, ATM, and CHEK2, are associated with increased risk of breast cancer; however, these variants, in combination with common variants identified through genome-wide association studies, explain only a fraction of the familial aggregation of the disease. To identify further susceptibility genes, we performed a two-stage whole-exome sequencing study. In the discovery stage, samples from 1528 breast cancer cases enriched for breast cancer susceptibility and 3733 geographically matched unaffected controls were sequenced. Using five different filtering and gene prioritization strategies, 198 genes were selected for further validation. These genes, and a panel of 32 known or suspected breast cancer susceptibility genes, were assessed in a validation set of 6211 cases and 6019 controls for their association with risk of breast cancer overall, and by estrogen receptor (ER) disease subtypes, using gene burden tests applied to loss-of-function and rare missense variants. Twenty genes showed nominal evidence of association (p-value < 0.05) with either overall or subtype-specific breast cancer. Our study had the statistical power to detect susceptibility genes with effect sizes similar to ATM, CHEK2, and PALB2, however, it was underpowered to identify genes in which susceptibility variants are rarer or confer smaller effect sizes. Larger sample sizes would be required in order to identify such genes.

Multi-omics profiling identifies a deregulated FUS-MAP1B axis in ALS/FTD-associated UBQLN2 mutants.

Ubiquilin-2 (UBQLN2) is a ubiquitin-binding protein that shuttles ubiquitinated proteins to proteasomal and autophagic degradation. UBQLN2 mutations are genetically linked to the neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). However, it remains elusive how UBQLN2 mutations cause ALS/FTD. Here, we systematically examined proteomic and transcriptomic changes in patient-derived lymphoblasts and CRISPR/Cas9-engineered HeLa cells carrying ALS/FTD UBQLN2 mutations. This analysis revealed a strong up-regulation of the microtubule-associated protein 1B (MAP1B) which was also observed in UBQLN2 knockout cells and primary rodent neurons depleted of UBQLN2, suggesting that a UBQLN2 loss-of-function mechanism is responsible for the elevated MAP1B levels. Consistent with MAP1B's role in microtubule binding, we detected an increase in total and acetylated tubulin. Furthermore, we uncovered that UBQLN2 mutations result in decreased phosphorylation of MAP1B and of the ALS/FTD-linked fused in sarcoma (FUS) protein at S439 which is critical for regulating FUS-RNA binding and MAP1B protein abundance. Together, our findings point to a deregulated UBQLN2-FUS-MAP1B axis that may link protein homeostasis, RNA metabolism, and cytoskeleton dynamics, three molecular pathomechanisms of ALS/FTD.

Clonal hematopoiesis as a pitfall in germline variant interpretation in the context of Mendelian disorders.

Clonal hematopoiesis because of somatic mutations in hematopoietic stem/progenitor cells is an age-related phenomenon and commonly observed when sequencing blood DNA in elderly individuals. Several genes that are implicated in clonal hematopoiesis are also associated with Mendelian disorders when mutated in the germline, potentially leading to variant misinterpretation. We performed a literature search to identify genes associated with age-related clonal hematopoiesis followed by an OMIM query to identify the subset of genes in which germline variants are associated with Mendelian disorders. We retrospectively screened for diagnostic cases in which the presence of age-related clonal hematopoiesis confounded exome sequencing data interpretation. We found 58 genes in which somatic mutations are implicated in clonal hematopoiesis, while germline variants in the same genes are associated with Mendelian (mostly neurodevelopmental) disorders. Using five selected cases of individuals with suspected monogenic disorders, we illustrate how clonal hematopoiesis in either variant databases or exome sequencing datasets poses a pitfall, potentially leading to variant misclassification and erroneous conclusions regarding gene-disease associations.

Cell cycle defects underlie childhood-onset cardiomyopathy associated with Noonan syndrome.

Childhood-onset myocardial hypertrophy and cardiomyopathic changes are associated with significant morbidity and mortality in early life, particularly in patients with Noonan syndrome, a multisystemic genetic disorder caused by autosomal dominant mutations in genes of the Ras-MAPK pathway. Although the cardiomyopathy associated with Noonan syndrome (NS-CM) shares certain cardiac features with the hypertrophic cardiomyopathy caused by mutations in sarcomeric proteins (HCM), such as pathological myocardial remodeling, ventricular dysfunction, and increased risk for malignant arrhythmias, the clinical course of NS-CM significantly differs from HCM. This suggests a distinct pathophysiology that remains to be elucidated. Here, through analysis of sarcomeric myosin conformational states, histopathology, and gene expression in left ventricular myocardial tissue from NS-CM, HCM, and normal hearts complemented with disease modeling in cardiomyocytes differentiated from patient-derived PTPN11 N308S/+ induced pluripotent stem cells, we demonstrate distinct disease phenotypes between NS-CM and HCM and uncover cell cycle defects as a potential driver of NS-CM.

Sequential Defects in Cardiac Lineage Commitment and Maturation Cause Hypoplastic Left Heart Syndrome.

BACKGROUND: Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role. METHODS: To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent-offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls. RESULTS: Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. CONCLUSIONS: Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

Mitochondrial DNA mutation analysis from exome sequencing-A more holistic approach in diagnostics of suspected mitochondrial disease.

Diagnostics for suspected mitochondrial disease (MD) can be challenging and necessitate invasive procedures like muscle biopsy. This is due to the extremely broad genetic and phenotypic spectrum, disease genes on both nuclear and mitochondrial DNA (mtDNA), and the tissue specificity of mtDNA variants. Exome sequencing (ES) has revolutionized the diagnostics for MD. However, the nuclear and mtDNA are investigated with separate tests, increasing costs and duration of diagnostics. The full potential of ES is often not exploited as the additional analysis of "off-target reads" deriving from the mtDNA can be used to analyze both genomes. We performed mtDNA analysis by ES of 2111 cases in a clinical setting. We further assessed the recall rate and precision as well as the estimation of heteroplasmy by ES data by comparison with targeted mtDNA next generation sequencing in 49 cases. ES identified known pathogenic mtDNA point mutations in 38 individuals, increasing the diagnostic yield by nearly 2%. Analysis of mtDNA variants by ES had a high recall rate (96.2 ± 5.6%) and an excellent precision (99.5 ± 2.2%) when compared to the gold standard of targeted mtDNA next generation sequencing. ES estimated heteroplasmy levels with an average difference of 6.6 ± 3.8%, sufficient for clinical decision making. Taken together, the mtDNA analysis from ES is of sufficient quality for clinical diagnostics. We therefore propose ES, investigating both nuclear and mtDNA, as first line test in individuals with suspected MD. One should be aware, that a negative result does not exclude MD and necessitates further test (in additional tissues).

Bi-allelic ADPRHL2 Mutations Cause Neurodegeneration with Developmental Delay, Ataxia, and Axonal Neuropathy.

ADP-ribosylation is a reversible posttranslational modification used to regulate protein function. ADP-ribosyltransferases transfer ADP-ribose from NAD+ to the target protein, and ADP-ribosylhydrolases, such as ADPRHL2, reverse the reaction. We used exome sequencing to identify five different bi-allelic pathogenic ADPRHL2 variants in 12 individuals from 8 families affected by a neurodegenerative disorder manifesting in childhood or adolescence with key clinical features including developmental delay or regression, seizures, ataxia, and axonal (sensori-)motor neuropathy. ADPRHL2 was virtually absent in available affected individuals' fibroblasts, and cell viability was reduced upon hydrogen peroxide exposure, although it was rescued by expression of wild-type ADPRHL2 mRNA as well as treatment with a PARP1 inhibitor. Our findings suggest impaired protein ribosylation as another pathway that, if disturbed, causes neurodegenerative diseases.

Mutations in PPCS, Encoding Phosphopantothenoylcysteine Synthetase, Cause Autosomal-Recessive Dilated Cardiomyopathy.

Coenzyme A (CoA) is an essential metabolic cofactor used by around 4% of cellular enzymes. Its role is to carry and transfer acetyl and acyl groups to other molecules. Cells can synthesize CoA de novo from vitamin B5 (pantothenate) through five consecutive enzymatic steps. Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the pathway during which phosphopantothenate reacts with ATP and cysteine to form phosphopantothenoylcysteine. Inborn errors of CoA biosynthesis have been implicated in neurodegeneration with brain iron accumulation (NBIA), a group of rare neurological disorders characterized by accumulation of iron in the basal ganglia and progressive neurodegeneration. Exome sequencing in five individuals from two unrelated families presenting with dilated cardiomyopathy revealed biallelic mutations in PPCS, linking CoA synthesis with a cardiac phenotype. Studies in yeast and fruit flies confirmed the pathogenicity of identified mutations. Biochemical analysis revealed a decrease in CoA levels in fibroblasts of all affected individuals. CoA biosynthesis can occur with pantethine as a source independent from PPCS, suggesting pantethine as targeted treatment for the affected individuals still alive.

Molecular diversity of combined and complex dystonia: insights from diagnostic exome sequencing.

Combined and complex dystonias are heterogeneous movement disorders combining dystonia with other motor and/or systemic signs. Although we are beginning to understand the diverse molecular causes of these disease entities, clinical pattern recognition and conventional genetic workup achieve an etiological diagnosis only in a minority of cases. Our goal was to provide a window into the variable genetic origins and distinct clinical patterns of combined/complex dystonia more broadly. Between August 2016 and January 2017, we applied whole-exome sequencing to a cohort of nine patients with varied combined and/or complex dystonic presentations, being on a diagnostic odyssey. Bioinformatics analyses, co-segregation studies, and sequence-interpretation algorithms were employed to detect causative mutations. Comprehensive clinical review was undertaken to define the phenotypic spectra and optimal management strategies. On average, we observed a delay in diagnosis of 23 years before whole-exome analysis enabled determination of each patient's genetic defect. Whereas mutations in ACTB, ATP1A3, ADCY5, and SGCE were associated with particular phenotypic clues, trait manifestations arising from mutations in PINK1, MRE11A, KMT2B, ATM, and SLC6A1 were different from those previously reported in association with these genes. Apart from improving counseling for our entire cohort, genetic findings had actionable consequences on preventative measures and therapeutic interventions for five patients. Our investigation confirms unique genetic diagnoses, highlights key clinical features and phenotypic expansions, and suggests whole-exome sequencing as a first-tier diagnostic for combined/complex dystonia. These results might stimulate independent teams to extend the scope of agnostic genetic screening to this particular phenotypic group that remains poorly characterized through existing studies.