Identifizierung neuer Krankheitsgene als Ursache für Skelettfehlbildungen und syndromale geistige Behinderung

Die Stellung einer Diagnose, welche häufig nur durch Identifizierung der ursächlichen genetischen Veränderung möglich ist, hat für die genetische Beratung und klinische Betreuung von Patienten mit genetisch bedingten Erkrankungen einen hohen Stellenwert. Die molekulare Grundlage etwa der Hälfte aller bekannten monogenen Erkrankungen ist bisher unbekannt Seit Verfügbarkeit der Hochdurchsatzsequenzierung konnten die Diagnoserate und Anzahl an bekannten Krankheitsgenen deutlich erhöht werden. Im Rahmen dieser Arbeit wurden mittels Hochdurchsatzsequenzierung, vorwiegend Exomsequenzierung, Blutproben von Patienten mit Skelettfehlbildungssyndromen und syndromaler geistiger Behinderung untersucht. Über internationale Kooperationen konnten Kohorten von Patienten mit neuen Krankheitsgenen bzw. neuen Krankheitsentitäten gebildet werden. Der pathogene Effekt mehrerer der identifizierten Genveränderungen wurde in funktionellen In-vitro-Studien untersucht. Im Rahmen dieser Arbeit wurden die genetischen Ursachen für Catel-Manzke- und Fontaine-Progeroid-Syndrom untersucht. Als Ursache für Catel-Manzke-Syndrom haben wir biallelische pathogene Varianten in den Genen TGDS und KYNU identifiziert. Das TGDS-Protein spielt möglicherweise eine Rolle im Proteoglykanstoffwechsel. Das Enzym Kynureninase, das durch KYNU kodiert wird, katalysiert einen Schritt in der NAD+-Synthese. Trotz der Ähnlichkeit des resultierenden Phänotyps ist ein Zusammenhang zwischen dem TGDS- und KYNU-assoziierten Pathomechanismus bislang unklar. Als Ursache für Fontaine-Progeroid-Syndrom haben wir rekurrente De-novo-Varianten in SLC25A24, welches für einen mitochondrialen ATP-Mg/Pi-Transporter kodiert, identifiziert. In vitro wiesen wir eine mitochondriale Schwellung und Dysfunktion nach, welche bisher nicht als Ursache für Kraniosynostose-Syndrome beschrieben wurden und ihre pathogene Wirkung durch einen Einfluss der zellulären Differenzierung in den Schädelnähten entfalten könnten. Darüber hinaus wurden heterozygote pathogene Varianten in den Genen DLL1 und ACTB als Ursache für neue Formen der syndromalen geistigen Behinderung identifiziert. DLL1 kodiert für den Notch-Liganden Delta 1. Wenngleich zahlreiche Studien die Rolle des Notch-Signalweges in der Gehirnentwicklung untermauern, stellt diese Arbeit erstmals den Zusammenhang zu einem humanen primär entwicklungsneurologischen Phänotyp her. Mit der ACTB-assoziierten syndromalen Thrombozytopenie wird ein neuer Phänotyp beschrieben, der auf pathogenen Varianten in beta-Aktin beruht. Die Ergebnisse dieser Arbeit erweitern unser Verständnis der molekularen Ursachen unterschiedlicher genetisch bedingter Krankheitsbilder und geben neue Einblicke in verschiedene Mechanismen der Entwicklung des Skeletts und ZNS. Die gewonnenen Erkenntnisse können für die genetische Beratung und klinische Betreuung der betroffenen Familien genutzt werde und könnten die Grundlage für neue Therapieansätze darstellen.Finding a diagnosis is of great importance for genetic counseling and clinical care of patients with genetic diseases. Often, this is only possible through identifying the causative genetic alteration. But the molecular basis of around half of all known monogenic diseases is still unknown. When high-throughput sequencing becomes available, the diagnosis rate and the number of known disease genes increased significantly. Here, high-throughput sequencing, mainly exome sequencing, was used to study blood samples from patients with skeletal malformation syndromes and syndromic intellectual disability. Cohorts of patients with novel disease genes or novel disease entities were established through international collaborations. The pathogenic effect of several identified causative variants is further clarified in functional in vitro studies. In this work, the genetic causes of Catel-Manzke and Fontaine progeroid syndrome are investigated. We identified biallelic pathogenic variants in TGDS and KYNU as the cause of Catel-Manzke syndrome. The TGDS protein may play a role in proteoglycan metabolism. The enzyme kynureninase, encoded by KYNU, catalyzes a step in NAD+ synthesis. Despite the similarity of the resulting phenotype, a link between the TGDS- and KYNU-associated pathomechanisms remains unclear. We identified recurrent de-novo variants in SLC25A24 as the cause of Fontaine progeroid syndrome. SLC25A24 encodes a mitochondrial ATP-Mg/Pi transporter. In vitro, we detected mitochondrial swelling and dysfunction, which have not previously been described as a cause of craniosynostosis syndromes and may express their pathogenic effect through an influence of cellular differentiation in the cranial sutures. In addition, we identified heterozygous pathogenic variants in DLL1 and ACTB as the cause of novel forms of syndromic intellectual disability. DLL1 encodes the Notch ligand delta 1. Although numerous studies support a role for Notch signaling in brain development, this work is the first which links it to a human primary developmental neurological phenotype. ACTB-associated syndromic thrombocytopenia is a new phenotype described based on pathogenic variants in beta-actin. The results of this work expand our understanding of the molecular causes of different genetic phenotypes and provide new insights into different mechanisms of skeletal and central nervous system development. The findings can be used for genetic counseling and clinical management of affected families and can provide the basis for new therapeutic approaches

MutationDistiller: user-driven identification of pathogenic DNA variants

MutationDistiller is a freely available online tool for user-driven analyses of Whole Exome Sequencing data. It offers a user-friendly interface aimed at clinicians and researchers, who are not necessarily bioinformaticians. MutationDistiller combines Mutation- Taster’s pathogenicity predictions with a phenotypebased approach. Phenotypic information is not limited to symptoms included in the Human Phenotype Ontology (HPO), but may also comprise clinical diagnoses and the suspected mode of inheritance. The search can be restricted to lists of candidate genes (e.g. virtual gene panels) and by tissue-specific gene expression. The inclusion of GeneOntology (GO) and metabolic pathways facilitates the discovery of hitherto unknown disease genes. In a novel approach, we trained MutationDistiller’s HPO-based prioritization on authentic genotype–phenotype sets obtained from ClinVar and found it to match or outcompete current prioritization tools in terms of accuracy. In the output, the program provides a list of potential disease mutations ordered by the likelihood of the affected genes to cause the phenotype. MutationDistiller provides links to gene-related information from various resources. It has been extensively tested by clinicians and their suggestions have been valued in many iterative cycles of revisions. The tool, a comprehensive documentation and examples are freely available at https://www.mutationdistiller.org

VarFish: comprehensive DNA variant analysis for diagnostics and research

VarFish is a user-friendly web application for the quality control, filtering, prioritization, analysis, and user-based annotation of DNA variant data with a focus on rare disease genetics. It is capable of processing variant call files with single or multiple samples. The variants are automatically annotated with population frequencies, molecular impact, and presence in databases such as ClinVar. Further, it provides support for pathogenicity scores including CADD, MutationTaster, and phenotypic similarity scores. Users can filter variants based on these annotations and presumed inheritance pattern and sort the results by these scores. Variants passing the filter are listed with their annotations and many useful link-outs to genome browsers, other gene/variant data portals, and external tools for variant assessment. VarFish allows users to create their own annotations including support for variant assessment following ACMG-AMP guidelines. In close collaboration with medical practitioners, VarFish was designed for variant analysis and prioritization in diagnostic and research settings as described in the software's extensive manual. The user interface has been optimized for supporting these protocols. Users can install VarFish on their own in-house servers where it provides additional lab notebook features for collaborative analysis and allows re-analysis of cases, e.g. after update of genotype or phenotype databases

TGDS pathogenic variants cause Catel-Manzke syndrome without hyperphalangy

Catel-Manzke syndrome, also known as micrognathia-digital-syndrome, is a rare autosomal recessive disorder characterized by the combination of the two cardinal features Pierre-Robin sequence and bilateral hyperphalangy leading to ulnar clinodactyly (ulnar curvature of the phalanges) and radial deviation (radial angulation at the metacarpophalangeal joint) of the index finge

Broadening the phenotypic and molecular spectrum of FINCA syndrome: Biallelic NHLRC2 variants in 15 novel individuals

FINCA syndrome [MIM: 618278] is an autosomal recessive multisystem disorder characterized by fibrosis, neurodegeneration and cerebral angiomatosis. To date, 13 patients from nine families with biallelic NHLRC2 variants have been published. In all of them, the recurrent missense variant p.(Asp148Tyr) was detected on at least one allele. Common manifestations included lung or muscle fibrosis, respiratory distress, developmental delay, neuromuscular symptoms and seizures often followed by early death due to rapid disease progression.Here, we present 15 individuals from 12 families with an overlapping phenotype associated with nine novel NHLRC2 variants identified by exome analysis. All patients described here presented with moderate to severe global developmental delay and variable disease progression. Seizures, truncal hypotonia and movement disorders were frequently observed. Notably, we also present the first eight cases in which the recurrent p.(Asp148Tyr) variant was not detected in either homozygous or compound heterozygous state.We cloned and expressed all novel and most previously published non-truncating variants in HEK293-cells. From the results of these functional studies, we propose a potential genotype-phenotype correlation, with a greater reduction in protein expression being associated with a more severe phenotype.Taken together, our findings broaden the known phenotypic and molecular spectrum and emphasize that NHLRC2-related disease should be considered in patients presenting with intellectual disability, movement disorders, neuroregression and epilepsy with or without pulmonary involvement

CDK19-related disorder results from both loss-of-function and gain-of-function de novo missense variants

Purpose To expand the recent description of a new neurodevelopmental syndrome related to alterations in CDK19. Methods Individuals were identified through international collaboration. Functional studies included autophosphorylation assays for CDK19 Gly28Arg and Tyr32His variants and in vivo zebrafish assays of the CDK19(G28R) and CDK19(Y32H). Results We describe 11 unrelated individuals (age range: 9 months to 14 years) with de novo missense variants mapped to the kinase domain of CDK19, including two recurrent changes at residues Tyr32 and Gly28. In vitro autophosphorylation and substrate phosphorylation assays revealed that kinase activity of protein was lower for p.Gly28Arg and higher for p.Tyr32His substitutions compared with that of the wild-type protein. Injection of CDK19 messenger RNA (mRNA) with either the Tyr32His or the Gly28Arg variants using in vivo zebrafish model significantly increased fraction of embryos with morphological abnormalities. Overall, the phenotype of the now 14 individuals with CDK19-related disorder includes universal developmental delay and facial dysmorphism, hypotonia (79%), seizures (64%), ophthalmologic anomalies (64%), and autism/autistic traits (56%). Conclusion CDK19 de novo missense variants are responsible for a novel neurodevelopmental disorder. Both kinase assay and zebrafish experiments showed that the pathogenetic mechanism may be more diverse than previously thought.Peer reviewe

PEDIA: prioritization of exome data by image analysis.

PURPOSE: Phenotype information is crucial for the interpretation of genomic variants. So far it has only been accessible for bioinformatics workflows after encoding into clinical terms by expert dysmorphologists. METHODS: Here, we introduce an approach driven by artificial intelligence that uses portrait photographs for the interpretation of clinical exome data. We measured the value added by computer-assisted image analysis to the diagnostic yield on a cohort consisting of 679 individuals with 105 different monogenic disorders. For each case in the cohort we compiled frontal photos, clinical features, and the disease-causing variants, and simulated multiple exomes of different ethnic backgrounds. RESULTS: The additional use of similarity scores from computer-assisted analysis of frontal photos improved the top 1 accuracy rate by more than 20-89% and the top 10 accuracy rate by more than 5-99% for the disease-causing gene. CONCLUSION: Image analysis by deep-learning algorithms can be used to quantify the phenotypic similarity (PP4 criterion of the American College of Medical Genetics and Genomics guidelines) and to advance the performance of bioinformatics pipelines for exome analysis

De novo mutations in SLC25A24 cause a craniosynostosis syndrome with hypertrichosis, progeroid appearance, and mitochondrial dysfunction

Gorlin-Chaudhry-Moss syndrome (GCMS) is a dysmorphic syndrome characterized by coronal craniosynostosis and severe midface hypoplasia, body and facial hypertrichosis, microphthalmia, short stature, and short distal phalanges. Variable lipoatrophy and cutis laxa are the basis for a progeroid appearance. Using exome and genome sequencing, we identified the recurrent de novo mutations c.650G>A (p.Arg217His) and c.649C>T (p.Arg217Cys) in SLC25A24 in five unrelated girls diagnosed with GCMS. Two of the girls had pronounced neonatal progeroid features and were initially diagnosed with Wiedemann-Rautenstrauch syndrome. SLC25A24 encodes a mitochondrial inner membrane ATP-Mg/Pi carrier. In fibroblasts from affected individuals, the mutated SLC25A24 showed normal stability. In contrast to control cells, the probands' cells showed mitochondrial swelling, which was exacerbated upon treatment with hydrogen peroxide (H2O2). The same effect was observed after overexpression of the mutant cDNA. Under normal culture conditions, the mitochondrial membrane potential of the probands' fibroblasts was intact, whereas ATP content in the mitochondrial matrix was lower than that in control cells. However, upon H2O2 exposure, the membrane potential was significantly elevated in cells harboring the mutated SLC25A24. No reduction of mitochondrial DNA copy number was observed. These findings demonstrate that mitochondrial dysfunction with increased sensitivity to oxidative stress is due to the SLC25A24 mutations. Our results suggest that the SLC25A24 mutations induce a gain of pathological function and link mitochondrial ATP-Mg/Pi transport to the development of skeletal and connective tissue.N.E. is a participant in the Berlin Institute of Health Charité Clinician Scientist Program, funded by the Charité - Universitätsmedizin Berlin and the Berlin Institute of Health. S.M. was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) and the Max Planck Foundation, B.W. was supported by grants from the DFG SFB1002 project D02, and B.F.-Z. was supported by a grant from the DFG (FI 2240/1-1). U.K. received funding from FP7-EU grant agreement no. 602300 (SYBIL) and the DFG Research Unit FOR 2165 (249509554). Research reported in this publication was supported by National Institute of Neurological Disorders and Stroke of the National Institutes of Health under award number R01NS08372 to P.E.B

Biallelic truncating variants in MAPKAPK5 cause a new developmental disorder involving neurological, cardiac, and facial anomalies combined with synpolydactyly

[Purpose]: This study aimed to identify the genetic cause of a new multiple congenital anomalies syndrome observed in three individuals from two unrelated families.[Methods]: Clinical assessment was conducted prenatally and at different postnatal stages. Genetic studies included exome sequencing (ES) combined with single-nucleotide polymorphism (SNP) array based homozygosity mapping and trio ES. Dermal fibroblasts were used for functional assays.[Results]: A clinically recognizable syndrome characterized by severe developmental delay, variable brain anomalies, congenital heart defects, dysmorphic facial features, and a distinctive type of synpolydactyly with an additional hypoplastic digit between the fourth and fifth digits of hands and/or feet was identified. Additional features included eye abnormalities, hearing impairment, and electroencephalogram anomalies. ES detected different homozygous truncating variants in MAPKAPK5 in both families. Patient-derived cells showed no expression of MAPKAPK5 protein isoforms and reduced levels of the MAPKAPK5-interacting protein ERK3. F-actin recovery after latrunculin B treatment was found to be less efficient in patient-derived fibroblasts than in control cells, supporting a role of MAPKAPK5 in F-actin polymerization.[Conclusion]: Our data indicate that loss-of-function variants in MAPKAPK5 result in a severe developmental disorder and reveal a major role of this gene in human brain, heart, and limb development.We are grateful to patients and their parents for their participation in this study. The work at IIB was financially supported by the Spanish Ministry of Science, Innovation and Universities (PID2019-105620RB-I00/AEI/10.13039/501100011033 and SAF2016‐75434‐R (AEI/FEDER, UE)