REEV: review, evaluate and explain variants.

In the era of high throughput sequencing, special software is required for the clinical evaluation of genetic variants. We developed REEV (Review, Evaluate and Explain Variants), a user-friendly platform for clinicians and researchers in the field of rare disease genetics. Supporting data was aggregated from public data sources. We compared REEV with seven other tools for clinical variant evaluation. REEV (semi-)automatically fills individual ACMG criteria facilitating variant interpretation. REEV can store disease and phenotype data related to a case to use these for phenotype similarity measures. Users can create public permanent links for individual variants that can be saved as browser bookmarks and shared. REEV may help in the fast diagnostic assessment of genetic variants in a clinical as well as in a research context. REEV (https://reev.bihealth.org/) is free and open to all users and there is no login requirement.

Loss-of-function variants affecting the STAGA complex component SUPT7L cause a developmental disorder with generalized lipodystrophy.

Generalized lipodystrophy is a feature of various hereditary disorders, often leading to a progeroid appearance. In the present study we identified a missense and a frameshift variant in a compound heterozygous state in SUPT7L in a boy with intrauterine growth retardation, generalized lipodystrophy, and additional progeroid features. SUPT7L encodes a component of the transcriptional coactivator complex STAGA. By transcriptome sequencing, we showed the predicted missense variant to cause aberrant splicing, leading to exon truncation and thereby to a complete absence of SUPT7L in dermal fibroblasts. In addition, we found altered expression of genes encoding DNA repair pathway components. This pathway was further investigated and an increased rate of DNA damage was detected in proband-derived fibroblasts and genome-edited HeLa cells. Finally, we performed transient overexpression of wildtype SUPT7L in both cellular systems, which normalizes the number of DNA damage events. Our findings suggest SUPT7L as a novel disease gene and underline the link between genome instability and progeroid phenotypes.

Hi-C Identifies Complex Genomic Rearrangements and TAD-Shuffling in Developmental Diseases.

Genome-wide analysis methods, such as array comparative genomic hybridization (CGH) and whole-genome sequencing (WGS), have greatly advanced the identification of structural variants (SVs) in the human genome. However, even with standard high-throughput sequencing techniques, complex rearrangements with multiple breakpoints are often difficult to resolve, and predicting their effects on gene expression and phenotype remains a challenge. Here, we address these problems by using high-throughput chromosome conformation capture (Hi-C) generated from cultured cells of nine individuals with developmental disorders (DDs). Three individuals had previously been identified as harboring duplications at the SOX9 locus and six had been identified with translocations. Hi-C resolved the positions of the duplications and was instructive in interpreting their distinct pathogenic effects, including the formation of new topologically associating domains (neo-TADs). Hi-C was very sensitive in detecting translocations, and it revealed previously unrecognized complex rearrangements at the breakpoints. In several cases, we observed the formation of fused-TADs promoting ectopic enhancer-promoter interactions that were likely to be involved in the disease pathology. In summary, we show that Hi-C is a sensible method for the detection of complex SVs in a clinical setting. The results help interpret the possible pathogenic effects of the SVs in individuals with DDs.

Interpretable Clinical Genomics with a Likelihood Ratio Paradigm.

Human Phenotype Ontology (HPO)-based analysis has become standard for genomic diagnostics of rare diseases. Current algorithms use a variety of semantic and statistical approaches to prioritize the typically long lists of genes with candidate pathogenic variants. These algorithms do not provide robust estimates of the strength of the predictions beyond the placement in a ranked list, nor do they provide measures of how much any individual phenotypic observation has contributed to the prioritization result. However, given that the overall success rate of genomic diagnostics is only around 25%-50% or less in many cohorts, a good ranking cannot be taken to imply that the gene or disease at rank one is necessarily a good candidate. Here, we present an approach to genomic diagnostics that exploits the likelihood ratio (LR) framework to provide an estimate of (1) the posttest probability of candidate diagnoses, (2) the LR for each observed HPO phenotype, and (3) the predicted pathogenicity of observed genotypes. LIkelihood Ratio Interpretation of Clinical AbnormaLities (LIRICAL) placed the correct diagnosis within the first three ranks in 92.9% of 384 case reports comprising 262 Mendelian diseases, and the correct diagnosis had a mean posttest probability of 67.3%. Simulations show that LIRICAL is robust to many typically encountered forms of genomic and phenomic noise. In summary, LIRICAL provides accurate, clinically interpretable results for phenotype-driven genomic diagnostics.

ClearCNV: CNV calling from NGS panel data in the presence of ambiguity and noise.

MOTIVATION: While the identification of small variants in panel sequencing data can be considered a solved problem, the identification of larger, multi-exon copy number variants (CNVs) still poses a considerable challenge. Thus, CNV calling has not been established in all laboratories performing panel sequencing. At the same time, such laboratories have accumulated large datasets and thus have the need to identify CNVs on their data to close the diagnostic gap. RESULTS: In this article, we present our method clearCNV that addresses this need in two ways. First, it helps laboratories to properly assign datasets to enrichment kits. Based on homogeneous subsets of data, clearCNV identifies CNVs affecting the targeted regions. Using real-world datasets and validation, we show that our method is highly competitive with previous methods and preferable in terms of specificity. AVAILABILITY AND IMPLEMENTATION: The software is available for free under a permissible license at https://github.com/bihealth/clear-cnv. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Biallelic truncating variants in ATP9A cause a novel neurodevelopmental disorder involving postnatal microcephaly and failure to thrive.

BACKGROUND: Genes implicated in the Golgi and endosomal trafficking machinery are crucial for brain development, and mutations in them are particularly associated with postnatal microcephaly (POM). METHODS: Exome sequencing was performed in three affected individuals from two unrelated consanguineous families presenting with delayed neurodevelopment, intellectual disability of variable degree, POM and failure to thrive. Patient-derived fibroblasts were tested for functional effects of the variants. RESULTS: We detected homozygous truncating variants in ATP9A. While the variant in family A is predicted to result in an early premature termination codon, the variant in family B affects a canonical splice site. Both variants lead to a substantial reduction of ATP9A mRNA expression. It has been shown previously that ATP9A localises to early and recycling endosomes, whereas its depletion leads to altered gene expression of components from this compartment. Consistent with previous findings, we also observed overexpression of ARPC3 and SNX3, genes strongly interacting with ATP9A. CONCLUSION: In aggregate, our findings show that pathogenic variants in ATP9A cause a novel autosomal recessive neurodevelopmental disorder with POM. While the physiological function of endogenous ATP9A is still largely elusive, our results underline a crucial role of this gene in endosomal transport in brain tissue.

Transmission ratio distortion of mutations in the master regulator of centriole biogenesis PLK4.

The evolutionary conserved Polo-like kinase 4 (PLK4) is essential for centriole duplication, spindle assembly, and de novo centriole formation. In man, homozygous mutations in PLK4 lead to primary microcephaly, altered PLK4 expression is associated with aneuploidy in human embryos. Here, we report on a consanguineous four-generation family with 8 affected individuals compound heterozygous for a novel missense variant, c.881 T > G, and a deletion of the PLK4 gene. The clinical phenotype of the adult patients is mild compared to individuals with previously described PLK4 mutations. One individual was homozygous for the variant c.881G and phenotypically unaffected. The deletion was inherited by 14 of 16 offspring and thus exhibits transmission ratio distortion (TRD). Moreover, based on the already published families with PLK4 mutations, it could be shown that due to the preferential transmission of the mutant alleles, the number of affected offspring is significantly increased. It is assumed that reduced expression of PLK4 decreases the intrinsically high error rate of the first cell divisions after fertilization, increases the number of viable embryos and thus leads to preferential transmission of the deleted/mutated alleles.

Xq27.1 palindrome mediated interchromosomal insertion likely causes familial congenital bilateral laryngeal abductor paralysis (Plott syndrome).

Bilateral laryngeal abductor paralysis is a rare entity and the second most common cause of stridor in newborns. So far, no conclusive genetic or chromosomal aberration has been reported for X-linked isolated bilateral vocal cord paralysis, also referred to as Plott syndrome. Via whole genome sequencing (WGS), we identified a complex interchromosomal insertion in a large family with seven affected males. The 404 kb inserted fragment originates from chromosome 10q21.3, contains no genes and is inserted inversionally into the intergenic chromosomal region Xq27.1, 82 kb centromeric to the nearest gene SOX3. The patterns found at the breakpoint junctions resemble typical characteristics that arise in replication-based mechanisms with long-distance template switching. Non protein-coding insertions into the same genomic region have been described to result in different phenotypes, indicating that the phenotypic outcome likely depends on the introduction of regulatory elements. In conclusion, our data adds Plott syndrome as another entity, likely caused by the insertion of non-coding DNA into the intergenic chromosomal region Xq27.1. In this regard, we demonstrate the importance of WGS as a powerful diagnostic test in unsolved genetic diseases, as this genomic rearrangement has not been detected by current first-line diagnostic tests, i.e., exome sequencing and chromosomal microarray analysis.

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.

Complete lung agenesis caused by complex genomic rearrangements with neo-TAD formation at the SHH locus.

During human organogenesis, lung development is a timely and tightly regulated developmental process under the control of a large number of signaling molecules. Understanding how genetic variants can disturb normal lung development causing different lung malformations is a major goal for dissecting molecular mechanisms during embryogenesis. Here, through exome sequencing (ES), array CGH, genome sequencing (GS) and Hi-C, we aimed at elucidating the molecular basis of bilateral isolated lung agenesis in three fetuses born to a non-consanguineous family. We detected a complex genomic rearrangement containing duplicated, triplicated and deleted fragments involving the SHH locus in fetuses presenting complete agenesis of both lungs and near-complete agenesis of the trachea, diagnosed by ultrasound screening and confirmed at autopsy following termination. The rearrangement did not include SHH itself, but several regulatory elements for lung development, such as MACS1, a major SHH lung enhancer, and the neighboring genes MNX1 and NOM1. The rearrangement incorporated parts of two topologically associating domains (TADs) including their boundaries. Hi-C of cells from one of the affected fetuses showed the formation of two novel TADs each containing SHH enhancers and the MNX1 and NOM1 genes. Hi-C together with GS indicate that the new 3D conformation is likely causative for this condition by an inappropriate activation of MNX1 included in the neo-TADs by MACS1 enhancer, further highlighting the importance of the 3D chromatin conformation in human disease.

Genome sequencing in families with congenital limb malformations.

The extensive clinical and genetic heterogeneity of congenital limb malformation calls for comprehensive genome-wide analysis of genetic variation. Genome sequencing (GS) has the potential to identify all genetic variants. Here we aim to determine the diagnostic potential of GS as a comprehensive one-test-for-all strategy in a cohort of undiagnosed patients with congenital limb malformations. We collected 69 cases (64 trios, 1 duo, 5 singletons) with congenital limb malformations with no molecular diagnosis after standard clinical genetic testing and performed genome sequencing. We also developed a framework to identify potential noncoding pathogenic variants. We identified likely pathogenic/disease-associated variants in 12 cases (17.4%) including four in known disease genes, and one repeat expansion in HOXD13. In three unrelated cases with ectrodactyly, we identified likely pathogenic variants in UBA2, establishing it as a novel disease gene. In addition, we found two complex structural variants (3%). We also identified likely causative variants in three novel high confidence candidate genes. We were not able to identify any noncoding variants. GS is a powerful strategy to identify all types of genomic variants associated with congenital limb malformation, including repeat expansions and complex structural variants missed by standard diagnostic approaches. In this cohort, no causative noncoding SNVs could be identified.

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 CDK19G28R and CDK19Y32H. 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.

GLI3 variants causing isolated polysyndactyly are not restricted to the protein's C-terminal third.

Loss of function variants of GLI3 are associated with a variety of forms of polysyndactyly: Pallister-Hall syndrome (PHS), Greig-Cephalopolysyndactyly syndrome (GCPS), and isolated polysyndactyly (IPD). Variants affecting the N-terminal and C-terminal thirds of the GLI3 protein have been associated with GCPS, those within the central third with PHS. Cases of IPD have been attributed to variants affecting the C-terminal third of the GLI3 protein. In this study, we further investigate these genotype-phenotype correlations. Sequencing of GLI3 was performed in patients with clinical findings suggestive of a GLI3-associated syndrome. Additionally, we searched the literature for reported cases of either manifestation with mutations in the GLI3 gene. Here, we report 48 novel cases from 16 families with polysyndactyly in whom we found causative variants in GLI3 and a review on 314 previously reported GLI3 variants. No differences in location of variants causing either GCPS or IPD were found. Review of published data confirmed the association of PHS and variants affecting the GLI3 protein's central third. We conclude that the observed manifestations of GLI3 variants as GCPS or IPD display different phenotypic severities of the same disorder and propose a binary division of GLI3-associated disorders in either PHS or GCPS/polysyndactyly.

Expanding the clinical and molecular spectrum of ATP6V1A related metabolic cutis laxa.

Several inborn errors of metabolism show cutis laxa as a highly recognizable feature. One group of these metabolic cutis laxa conditions is autosomal recessive cutis laxa type 2 caused by defects in v-ATPase components or the mitochondrial proline cycle. Besides cutis laxa, muscular hypotonia and cardiac abnormalities are hallmarks of autosomal recessive cutis laxa type 2D (ARCL2D) due to pathogenic variants in ATP6V1A encoding subunit A of the v-ATPase. Here, we report on three affected individuals from two families with ARCL2D in whom we performed whole exome and Sanger sequencing. We performed functional studies in fibroblasts from one individual, summarized all known probands' clinical, molecular, and biochemical features and compared them, also to other metabolic forms of cutis laxa. We identified novel missense and the first nonsense variant strongly affecting ATP6V1A expression. All six ARCL2D affected individuals show equally severe cutis laxa and dysmorphism at birth. While for one no information was available, two died in infancy and three are now adolescents with mild or absent intellectual disability. Muscular weakness, ptosis, contractures, and elevated muscle enzymes indicated a persistent myopathy. In cellular studies, a fragmented Golgi compartment, a delayed Brefeldin A-induced retrograde transport and glycosylation abnormalities were present in fibroblasts from two individuals. This is the second and confirmatory report on pathogenic variants in ATP6V1A as the cause of this extremely rare condition and the first to describe a nonsense allele. Our data highlight the tremendous clinical variability of ATP6V1A related phenotypes even within the same family.

Variants in the SK2 channel gene (KCNN2) lead to dominant neurodevelopmental movement disorders.

KCNN2 encodes the small conductance calcium-activated potassium channel 2 (SK2). Rodent models with spontaneous Kcnn2 mutations show abnormal gait and locomotor activity, tremor and memory deficits, but human disorders related to KCNN2 variants are largely unknown. Using exome sequencing, we identified a de novo KCNN2 frameshift deletion in a patient with learning disabilities, cerebellar ataxia and white matter abnormalities on brain MRI. This discovery prompted us to collect data from nine additional patients with de novo KCNN2 variants (one nonsense, one splice site, six missense variants and one in-frame deletion) and one family with a missense variant inherited from the affected mother. We investigated the functional impact of six selected variants on SK2 channel function using the patch-clamp technique. All variants tested but one, which was reclassified to uncertain significance, led to a loss-of-function of SK2 channels. Patients with KCNN2 variants had motor and language developmental delay, intellectual disability often associated with early-onset movement disorders comprising cerebellar ataxia and/or extrapyramidal symptoms. Altogether, our findings provide evidence that heterozygous variants, likely causing a haploinsufficiency of the KCNN2 gene, lead to novel autosomal dominant neurodevelopmental movement disorders mirroring phenotypes previously described in rodents.

Variable pulmonary manifestations in Chitayat syndrome: Six additional affected individuals.

Hand hyperphalangism leading to shortened index fingers with ulnar deviation, hallux valgus, mild facial dysmorphism and respiratory compromise requiring assisted ventilation are the key features of Chitayat syndrome. This condition results from the recurrent heterozygous missense variant NM_006494.2:c.266A>G; p.(Tyr89Cys) in ERF on chromosome 19q13.2, encoding the ETS2 repressor factor (ERF) protein. The pathomechanism of Chitayat syndrome is unknown. To date, seven individuals with Chitayat syndrome and the recurrent pathogenic ERF variant have been reported in the literature. Here, we describe six additional individuals, among them only one presenting with a history of assisted ventilation, and the remaining presenting with variable pulmonary phenotypes, including one individual without any obvious pulmonary manifestations. Our findings widen the phenotype spectrum caused by the recurrent pathogenic variant in ERF, underline Chitayat syndrome as a cause of isolated skeletal malformations and therefore contribute to the improvement of diagnostic strategies in individuals with hand hyperphalangism.

Pathogenic variants in USP7 cause a neurodevelopmental disorder with speech delays, altered behavior, and neurologic anomalies.

PURPOSE: Haploinsufficiency of USP7, located at chromosome 16p13.2, has recently been reported in seven individuals with neurodevelopmental phenotypes, including developmental delay/intellectual disability (DD/ID), autism spectrum disorder (ASD), seizures, and hypogonadism. Further, USP7 was identified to critically incorporate into the MAGEL2-USP7-TRIM27 (MUST), such that pathogenic variants in USP7 lead to altered endosomal F-actin polymerization and dysregulated protein recycling. METHODS: We report 16 newly identified individuals with heterozygous USP7 variants, identified by genome or exome sequencing or by chromosome microarray analysis. Clinical features were evaluated by review of medical records. Additional clinical information was obtained on the seven previously reported individuals to fully elucidate the phenotypic expression associated with USP7 haploinsufficiency. RESULTS: The clinical manifestations of these 23 individuals suggest a syndrome characterized by DD/ID, hypotonia, eye anomalies,feeding difficulties, GERD, behavioral anomalies, and ASD, and more specific phenotypes of speech delays including a nonverbal phenotype and abnormal brain magnetic resonance image findings including white matter changes based on neuroradiologic examination. CONCLUSION: The consistency of clinical features among all individuals presented regardless of de novo USP7 variant type supports haploinsufficiency as a mechanism for pathogenesis and refines the clinical impact faced by affected individuals and caregivers.

Targeted panel sequencing in pediatric primary cardiomyopathy supports a critical role of TNNI3.

The underlying genetic mechanisms and early pathological events of children with primary cardiomyopathy (CMP) are insufficiently characterized. In this study, we aimed to characterize the mutational spectrum of primary CMP in a large cohort of patients ≤18 years referred to a tertiary center. Eighty unrelated index patients with pediatric primary CMP underwent genetic testing with a panel-based next-generation sequencing approach of 89 genes. At least one pathogenic or probably pathogenic variant was identified in 30/80 (38%) index patients. In all CMP subgroups, patients carried most frequently variants of interest in sarcomere genes suggesting them as a major contributor in pediatric primary CMP. In MYH7, MYBPC3, and TNNI3, we identified 18 pathogenic/probably pathogenic variants (MYH7 n = 7, MYBPC3 n = 6, TNNI3 n = 5, including one homozygous (TNNI3 c.24+2T>A) truncating variant. Protein and transcript level analysis on heart biopsies from individuals with homozygous mutation of TNNI3 revealed that the TNNI3 protein is absent and associated with upregulation of the fetal isoform TNNI1. The present study further supports the clinical importance of sarcomeric mutation-not only in adult-but also in pediatric primary CMP. TNNI3 is the third most important disease gene in this cohort and complete loss of TNNI3 leads to severe pediatric CMP.

HLA-MA: simple yet powerful matching of samples using HLA typing results.

SUMMARY: We propose the simple method HLA-MA for consistency checking in pipelines operating on human HTS data. The method is based on the HLA typing result of the state-of-the-art method OptiType. Provided that there is sufficient coverage of the HLA loci, comparing HLA types allows for simple, fast and robust matching of samples from whole genome, exome and RNA-seq data. Our approach uses information from small but genetically highly variable regions and thus complements approaches that rely on genome or exon-wide variant profiles. AVAILABILITY AND IMPLEMENTATION: The software is implemented In Python 3 and freely available under the MIT license at https://github.com/bihealth/hlama and via Bioconda. CONTACT: dieter.beule@bihealth.de. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Methods for the detection and assembly of novel sequence in high-throughput sequencing data.

MOTIVATION: Large insertions of novel sequence are an important type of structural variants. Previous studies used traditional de novo assemblers for assembling non-mapping high-throughput sequencing (HTS) or capillary reads and then tried to anchor them in the reference using paired read information. RESULTS: We present approaches for detecting insertion breakpoints and targeted assembly of large insertions from HTS paired data: BASIL and ANISE. On near identity repeats that are hard for assemblers, ANISE employs a repeat resolution step. This results in far better reconstructions than obtained by the compared methods. On simulated data, we found our insert assembler to be competitive with the de novo assemblers ABYSS and SGA while yielding already anchored inserted sequence as opposed to unanchored contigs as from ABYSS/SGA. On real-world data, we detected novel sequence in a human individual and thoroughly validated the assembled sequence. ANISE was found to be superior to the competing tool MindTheGap on both simulated and real-world data. AVAILABILITY AND IMPLEMENTATION: ANISE and BASIL are available for download at http://www.seqan.de/projects/herbarium under a permissive open source license.