Systematic large-scale application of ClinGen InSiGHT APC -specific ACMG/AMP variant classification criteria substantially alleviates the burden of variants of uncertain significance in ClinVar and LOVD databases.

Background: Pathogenic constitutional APC variants underlie familial adenomatous polyposis, the most common hereditary gastrointestinal polyposis syndrome. To improve variant classification and resolve the interpretative challenges of variants of uncertain significance (VUS), APC-specific ACMG/AMP variant classification criteria were developed by the ClinGen-InSiGHT Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel (VCEP). Methods: A streamlined algorithm using the APC -specific criteria was developed and applied to assess all APC variants in ClinVar and the InSiGHT international reference APC LOVD variant database. Results: A total of 10,228 unique APC variants were analysed. Among the ClinVar and LOVD variants with an initial classification of (Likely) Benign or (Likely) Pathogenic, 94% and 96% remained in their original categories, respectively. In contrast, 41% ClinVar and 61% LOVD VUS were reclassified into clinically actionable classes, the vast majority as (Likely) Benign. The total number of VUS was reduced by 37%. In 21 out of 36 (58%) promising APC variants that remained VUS despite evidence for pathogenicity, a data mining-driven work-up allowed their reclassification as (Likely) Pathogenic. Conclusions: The application of APC -specific criteria substantially reduced the number of VUS in ClinVar and LOVD. The study also demonstrated the feasibility of a systematic approach to variant classification in large datasets, which might serve as a generalisable model for other gene-/disease-specific variant interpretation initiatives. It also allowed for the prioritization of VUS that will benefit from in-depth evidence collection. This subset of APC variants was approved by the VCEP and made publicly available through ClinVar and LOVD for widespread clinical use.

Hematologically important mutations: Leukocyte adhesion deficiency (second update).

Leukocyte adhesion deficiency (LAD) is an immunodeficiency caused by defects in the adhesion of leukocytes (especially neutrophils) to the blood vessel wall. As a result, patients with LAD suffer from severe bacterial infections and impaired wound healing, accompanied by neutrophilia. In LAD-I, characterized directly after birth by delayed separation of the umbilical cord, mutations are found in ITGB2, the gene that encodes the ß subunit (CD18) of the ß2 integrins. In the rare LAD-II disease, the fucosylation of selectin ligands is disturbed, caused by mutations in SLC35C1, the gene that encodes a GDP-fucose transporter of the Golgi system. LAD-II patients lack the H and Lewis Lea and Leb blood group antigens. Finally, in LAD-III, the conformational activation of the hematopoietically expressed ß integrins is disturbed, leading to leukocyte and platelet dysfunction. This last syndrome is caused by mutations in FERMT3, encoding the kindlin-3 protein in all blood cells, involved in the regulation of ß integrin conformation. This article contains an update of the mutations that we consider to be relevant for the various forms of LAD.

Human Mutation special issue on "Variant Effect Prediction".

The journal Human Mutation has as its principal focus variants in the human genome, covering the entire spectrum from methods used to detect variants, to ways of answering the ultimate question: "What are the consequences of carrying a variant for the health of the individual?" This comprehensive collection of articles provides an excellent perspective of the advancements in variant effect prediction in recent years, as well as some caveats and cautions in this developing field. We believe that this resource will help to drive further evolution of the variant effect prediction process toward more robust understanding of genotype-phenotype relationships through reliable variant classification.

Stepwise ABC system for classification of any type of genetic variant.

The American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) system for variant classification is score based with five classes: benign, likely benign, variant of unknown significance (VUS), likely pathogenic, and pathogenic. Here, we present a variant classification model that can be an add-on or alternative to ACMG classification: A stepwise system that can classify any type of genetic variant (e.g., hypomorphic alleles, imprinted alleles, copy number variants, runs of homozygosity, enhancer variants, and variants related to traits). We call it the ABC system because classification is first functional (A), then clinical (B), and optionally a standard comment that fits the clinical question is selected (C). Both steps A and B have 1-5 grading when knowledge is sufficient, if not, class "zero" is assigned. Functional grading (A) only concerns biological consequences with the stages normal function (1), likely normal function (2), hypothetical functional effect (3), likely functional effect (4), and proven functional effect (5). Clinical grading (B) is genotype-phenotype focused with the stages "right type of gene" (1), risk factor (2), and pathogenic (3-5, depending on penetrance). Both grades are listed for each variant and combined to generate a joint class ranging from A to F. Importantly, the A-F classes are linked to standard comments, reflecting laboratory or national policy. In step A, the VUS class is split into class 0 (true unknown) and class 3 (hypothetical functional effect based on molecular predictions or de novo occurrence), providing a rationale for variant-of-interest reporting when the clinical picture could fit the finding. The system gives clinicians a better guide to variant significance.

New locus underlying auriculocondylar syndrome (ARCND): 430 kb duplication involving TWIST1 regulatory elements.

BACKGROUND: Auriculocondylar syndrome (ARCND) is a rare genetic disease that affects structures derived from the first and second pharyngeal arches, mainly resulting in micrognathia and auricular malformations. To date, pathogenic variants have been identified in three genes involved in the EDN1-DLX5/6 pathway (PLCB4, GNAI3 and EDN1) and some cases remain unsolved. Here we studied a large unsolved four-generation family. METHODS: We performed linkage analysis, resequencing and Capture-C to investigate the causative variant of this family. To test the pathogenicity of the CNV found, we modelled the disease in patient craniofacial progenitor cells, including induced pluripotent cell (iPSC)-derived neural crest and mesenchymal cells. RESULTS: This study highlights a fourth locus causative of ARCND, represented by a tandem duplication of 430 kb in a candidate region on chromosome 7 defined by linkage analysis. This duplication segregates with the disease in the family (LOD score=2.88) and includes HDAC9, which is located over 200 kb telomeric to the top candidate gene TWIST1. Notably, Capture-C analysis revealed multiple cis interactions between the TWIST1 promoter and possible regulatory elements within the duplicated region. Modelling of the disease revealed an increased expression of HDAC9 and its neighbouring gene, TWIST1, in neural crest cells. We also identified decreased migration of iPSC-derived neural crest cells together with dysregulation of osteogenic differentiation in iPSC-affected mesenchymal stem cells. CONCLUSION: Our findings support the hypothesis that the 430 kb duplication is causative of the ARCND phenotype in this family and that deregulation of TWIST1 expression during craniofacial development can contribute to the phenotype.

NR2F1 database: 112 variants and 84 patients support refining the clinical synopsis of Bosch-Boonstra-Schaaf optic atrophy syndrome.

Pathogenic variants of the nuclear receptor subfamily 2 group F member 1 gene (NR2F1) are responsible for Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS), an autosomal dominant disorder characterized by optic atrophy associated with developmental delay and intellectual disability, but with a clinical presentation which appears to be multifaceted. We created the first public locus-specific database dedicated to NR2F1. All variants and clinical cases reported in the literature, as well as new unpublished cases, were integrated into the database using standard nomenclature to describe both molecular and phenotypic anomalies. We subsequently pursued a comprehensive approach based on computed representation and analysis suggesting a refinement of the BBSOAS clinical description with respect to neurological features and the inclusion of additional signs of hypotonia and feeding difficulties. This database is fully accessible for both clinician and molecular biologists and should prove useful in further refining the clinical synopsis of NR2F1 as new data is recorded.

The Role of the European Society of Human Genetics in Delivering Genomic Education.

The European Society of Human Genetics (ESHG) was founded in 1967 as a professional organisation for members working in genetics in clinical practice, research and education. The Society seeks the integration of scientific research and its implementation into clinical practice and the education of specialists and the public in all areas of medical and human genetics. The Society works to do this through many approaches, including educational sessions at the annual conference; training courses in general and specialist areas of genetics; an online resource of educational materials (EuroGEMS); and a mentorship scheme. The ESHG Education Committee is implementing new approaches to expand the reach of its educational activities and portfolio. With changes in technology, appreciation of the utility of genomics in healthcare and the public's and patients' increased awareness of the role of genomics, this review will summarise how the ESHG is adapting to deliver innovative educational activity.

The LOVD3 platform: efficient genome-wide sharing of genetic variants.

Gene variant databases are the backbone of DNA-based diagnostics. These databases, also called Locus-Specific DataBases (LSDBs), store information on variants in the human genome and the observed phenotypic consequences. The largest collection of public databases uses the free, open-source LOVD software platform. To cope with the current demand for online databases, we have entirely redesigned the LOVD software. LOVD3 is genome-centered and can be used to store summary variant data, as well as full case-level data with information on individuals, phenotypes, screenings, and variants. While built on a standard core, the software is highly flexible and allows personalization to cope with the largely different demands of gene/disease database curators. LOVD3 follows current standards and includes tools to check variant descriptions, generate HTML files of reference sequences, predict the consequences of exon deletions/duplications on the reading frame, and link to genomic views in the different genomes browsers. It includes APIs to collect and submit data. The software is used by about 100 databases, of which 56 public LOVD instances are registered on our website and together contain 1,000,000,000 variant observations in 1,500,000 individuals. 42 LOVD instances share data with the federated LOVD data network containing 3,000,000 unique variants in 23,000 genes. This network can be queried directly, quickly identifying LOVD instances containing relevant information on a searched variant.

A Case Series of Familial ARID1B Variants Illustrating Variable Expression and Suggestions to Update the ACMG Criteria.

ARID1B is one of the most frequently mutated genes in intellectual disability (~1%). Most variants are readily classified, since they are de novo and are predicted to lead to loss of function, and therefore classified as pathogenic according to the American College of Medical Genetics and Genomics (ACMG) guidelines for the interpretation of sequence variants. However, familial loss-of-function variants can also occur and can be challenging to interpret. Such variants may be pathogenic with variable expression, causing only a mild phenotype in a parent. Alternatively, since some regions of the ARID1B gene seem to be lacking pathogenic variants, loss-of-function variants in those regions may not lead to ARID1B haploinsufficiency and may therefore be benign. We describe 12 families with potential loss-of-function variants, which were either familial or with unknown inheritance and were in regions where pathogenic variants have not been described or are otherwise challenging to interpret. We performed detailed clinical and DNA methylation studies, which allowed us to confidently classify most variants. In five families we observed transmission of pathogenic variants, confirming their highly variable expression. Our findings provide further evidence for an alternative translational start site and we suggest updates for the ACMG guidelines for the interpretation of sequence variants to incorporate DNA methylation studies and facial analyses.

ACO2 clinicobiological dataset with extensive phenotype ontology annotation.

Pathogenic variants of the aconitase 2 gene (ACO2) are responsible for a broad clinical spectrum involving optic nerve degeneration, ranging from isolated optic neuropathy with recessive or dominant inheritance, to complex neurodegenerative syndromes with recessive transmission. We created the first public locus-specific database (LSDB) dedicated to ACO2 within the "Global Variome shared LOVD" using exclusively the Human Phenotype Ontology (HPO), a standard vocabulary for describing phenotypic abnormalities. All the variants and clinical cases listed in the literature were incorporated into the database, from which we produced a dataset. We followed a rational and comprehensive approach based on the HPO thesaurus, demonstrating that ACO2 patients should not be classified separately between isolated and syndromic cases. Our data highlight that certain syndromic patients do not have optic neuropathy and provide support for the classification of the recurrent pathogenic variants c.220C>G and c.336C>G as likely pathogenic. Overall, our data records demonstrate that the clinical spectrum of ACO2 should be considered as a continuum of symptoms and refines the classification of some common variants.

Mutalyzer 2: next generation HGVS nomenclature checker.

MOTIVATION: Unambiguous variant descriptions are of utmost importance in clinical genetic diagnostics, scientific literature and genetic databases. The Human Genome Variation Society (HGVS) publishes a comprehensive set of guidelines on how variants should be correctly and unambiguously described. We present the implementation of the Mutalyzer 2 tool suite, designed to automatically apply the HGVS guidelines so users do not have to deal with the HGVS intricacies explicitly to check and correct their variant descriptions. RESULTS: Mutalyzer is profusely used by the community, having processed over 133 million descriptions since its launch. Over a five year period, Mutalyzer reported a correct input in ∼50% of cases. In 41% of the cases either a syntactic or semantic error was identified and for ∼7% of cases, Mutalyzer was able to automatically correct the description. AVAILABILITY AND IMPLEMENTATION: Mutalyzer is an Open Source project under the GNU Affero General Public License. The source code is available on GitHub (https://github.com/mutalyzer/mutalyzer) and a running instance is available at: https://mutalyzer.nl.

Newborn screening of duchenne muscular dystrophy specifically targeting deletions amenable to exon-skipping therapy.

Duchenne Muscular Dystrophy (DMD) is a lethal progressive muscle-wasting disease. New treatment strategies relying on DMD gene exon-skipping therapy have recently been approved and about 30% of patients could be amenable to exon 51, 53 or 45 skipping. We evaluated the spectrum of deletions reported in DMD registries, and designed a method to screen newborns and identify DMD deletions amenable to exon 51, 53 and 45 skipping. We developed a multiplex qPCR assay identifying hemi(homo)-zygotic deletions of the flanking exons of these therapeutic targets in DMD exons (i.e. exons 44, 46, 50, 52 and 54). We conducted an evaluation of our new method in 51 male patients with a DMD phenotype, 50 female carriers of a DMD deletion and 19 controls. Studies were performed on dried blood spots with patient's consent. We analyzed qPCR amplification curves of controls, carriers, and DMD patients to discern the presence or the absence of the target exons. Analysis of the exons flanking the exon-skipping targets permitted the identification of patients that could benefit from exon-skipping. All samples were correctly genotyped, with either presence or absence of amplification of the target exon. This proof-of-concept study demonstrates that this new assay is a highly sensitive method to identify DMD patients carrying deletions that are rescuable by exon-skipping treatment. The method is easily scalable to population-based screening. This targeted screening approach could address the new management paradigm in DMD, and could help to optimize the beneficial therapeutic effect of DMD therapies by permitting pre-symptomatic care.

Integrating Whole-Genome Sequencing in Clinical Genetics: A Novel Disruptive Structural Rearrangement Identified in the Dystrophin Gene (DMD).

While in most patients the identification of genetic alterations causing dystrophinopathies is a relatively straightforward task, a significant number require genomic and transcriptomic approaches that go beyond a routine diagnostic set-up. In this work, we present a Becker Muscular Dystrophy patient with elevated creatinine kinase levels, progressive muscle weakness, mild intellectual disability and a muscle biopsy showing dystrophic features and irregular dystrophin labelling. Routine molecular techniques (Southern-blot analysis, multiplex PCR, MLPA and genomic DNA sequencing) failed to detect a defect in the DMD gene. Muscle DMD transcript analysis (RT-PCR and cDNA-MLPA) showed the absence of exons 75 to 79, seen to be present at the genomic level. These results prompted the application of low-coverage linked-read whole-genome sequencing (WGS), revealing a possible rearrangement involving DMD intron 74 and a region located upstream of the PRDX4 gene. Breakpoint PCR and Sanger sequencing confirmed the presence of a ~8 Mb genomic inversion. Aberrant DMD transcripts were subsequently identified, some of which contained segments from the region upstream of PRDX4. Besides expanding the mutational spectrum of the disorder, this study reinforces the importance of transcript analysis in the diagnosis of dystrophinopathies and shows how WGS has a legitimate role in clinical laboratory genetics.

Verifying nomenclature of DNA variants in submitted manuscripts: Guidance for journals.

Documenting variation in our genomes is important for research and clinical care. Accuracy in the description of DNA variants is therefore essential. To address this issue, the Human Variome Project convened a committee to evaluate the feasibility of requiring authors to verify that all variants submitted for publication complied with a widely accepted standard for description. After a pilot study of two journals, the committee agreed that requiring authors to verify that variants complied with Human Genome Variation Society nomenclature is a reasonable step toward standardizing the worldwide inventory of human variation.

Using Personal Genomic Data within Primary Care: A Bioinformatics Approach to Pharmacogenomics.

One application of personalized medicine is the tailoring of medication to the individual, so that the medication will have the highest chance of success. In order to individualize medication, one must have a complete inventory of all current pharmaceutical compounds (a detailed formulary) combined with pharmacogenetic datasets, the genetic makeup of the patient, their (medical) family history and other health-related data. For healthcare professionals to make the best use of this information, it must be visualized in a way that makes the most medically relevant data accessible for their decision-making. Similarly, to enable bioinformatics analysis of these data, it must be prepared and provided through an interface for controlled computational analysis. Due to the high degree of personal information gathered for such initiatives, privacy-sensitive implementation choices and ethical standards are paramount. The Personal Genetic Locker project provides an approach to enable the use of personal genomic data in primary care. In this paper, we provide a description of the Personal Genetic Locker project and show its utility through a use case based on open standards, which is illustrated by the 4MedBox system.

OPA1: 516 unique variants and 831 patients registered in an updated centralized Variome database.

BACKGROUND: The dysfunction of OPA1, a dynamin GTPase involved in mitochondrial fusion, is responsible for a large spectrum of neurological disorders, each of which includes optic neuropathy. The database dedicated to OPA1 ( https://www.lovd.nl/OPA1 ), created in 2005, has now evolved towards a centralized and more reliable database using the Global Variome shared Leiden Open-source Variation Database (LOVD) installation. RESULTS: The updated OPA1 database, which registers all the patients from our center as well as those reported in the literature, now covers a total of 831 patients: 697 with isolated dominant optic atrophy (DOA), 47 with DOA "plus", and 83 with asymptomatic or unclassified DOA. It comprises 516 unique OPA1 variants, of which more than 80% (414) are considered pathogenic. Full clinical data for 118 patients are documented using the Human Phenotype Ontology, a standard vocabulary for referencing phenotypic abnormalities. Contributors may now make online submissions of phenotypes related to OPA1 mutations, giving clinical and molecular descriptions together with detailed ophthalmological and neurological data, according to an international thesaurus. CONCLUSIONS: The evolution of the OPA1 database towards the LOVD, using unified nomenclature, should ensure its interoperability with other databases and prove useful for molecular diagnoses based on gene-panel sequencing, large-scale mutation statistics, and genotype-phenotype correlations.

Dutch genome diagnostic laboratories accelerated and improved variant interpretation and increased accuracy by sharing data.

Each year diagnostic laboratories in the Netherlands profile thousands of individuals for heritable disease using next-generation sequencing (NGS). This requires pathogenicity classification of millions of DNA variants on the standard 5-tier scale. To reduce time spent on data interpretation and increase data quality and reliability, the nine Dutch labs decided to publicly share their classifications. Variant classifications of nearly 100,000 unique variants were catalogued and compared in a centralized MOLGENIS database. Variants classified by more than one center were labeled as "consensus" when classifications agreed, and shared internationally with LOVD and ClinVar. When classifications opposed (LB/B vs. LP/P), they were labeled "conflicting", while other nonconsensus observations were labeled "no consensus". We assessed our classifications using the InterVar software to compare to ACMG 2015 guidelines, showing 99.7% overall consistency with only 0.3% discrepancies. Differences in classifications between Dutch labs or between Dutch labs and ACMG were mainly present in genes with low penetrance or for late onset disorders and highlight limitations of the current 5-tier classification system. The data sharing boosted the quality of DNA diagnostics in Dutch labs, an initiative we hope will be followed internationally. Recently, a positive match with a case from outside our consortium resulted in a more definite disease diagnosis.

Templated Insertions: A Smoking Gun for Polymerase Theta-Mediated End Joining.

A recognized source of disease-causing genome alterations is erroneous repair of broken chromosomes, which can be executed by two distinct mechanisms: non-homologous end joining (NHEJ) and the recently discovered polymerase theta-mediated end joining (TMEJ) pathway. While TMEJ has previously been considered to act as an alternative mechanism backing up NHEJ, recent work points to a role for TMEJ in the repair of replication-associated DNA breaks that are excluded from repair through homologous recombination. Because of its mode of action, TMEJ is intrinsically mutagenic and sometimes leaves behind a recognizable genomic scar when joining chromosome break ends (i.e., 'templated insertions'). This review article focuses on the intriguing observation that this polymerase theta signature is frequently observed in disease alleles, arguing for a prominent role of this double-strand break repair pathway in genome diversification and disease-causing spontaneous mutagenesis in humans.

Phenotype predictions for exon deletions/duplications: A user guide for professionals and clinicians using Becker and Duchenne muscular dystrophy as examples.

Variations in the DMD gene that affect dystrophin production underlie both the severe Duchenne and the milder Becker muscular dystrophies (DMD and BMD, respectively). Depending on their location, deletions and duplications involving one or more exons of a gene can have a range of consequences. This overview, summarizing the important points to consider, was drafted in response to frequent questions we receive about deletions/duplications involving the dystrophin encoding DMD gene. Although directed at DMD, the observations made can be applied to many other genes. The overview is meant primarily for healthcare professionals involved with interpreting the results of genetic analyses in clinical practice.