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

Delineating the molecular and phenotypic spectrum of the SETD1B-related syndrome

Purpose: Pathogenic variants in SETD1B have been associated with a syndromic neurodevelopmental disorder including intellectual disability, language delay, and seizures. To date, clinical features have been described for 11 patients with (likely) pathogenic SETD1B sequence variants. This study aims to further delineate the spectrum of the SETD1B-related syndrome based on characterizing an expanded patient cohort. Methods: We perform an in-depth clinical characterization of a cohort of 36 unpublished individuals with SETD1B sequence variants, describing their molecular and phenotypic spectrum. Selected variants were functionally tested using in vitro and genome-wide methylation assays. Results: Our data present evidence for a loss-of-function mechanism of SETD1B variants, resulting in a core clinical phenotype of global developmental delay, language delay including regression, intellectual disability, autism and other behavioral issues, and variable epilepsy phenotypes. Developmental delay appeared to precede seizure onset, suggesting SETD1B dysfunction impacts physiological neurodevelopment even in the absence of epileptic activity. Males are significantly overrepresented and more severely affected, and we speculate that sex-linked traits could affect susceptibility to penetrance and the clinical spectrum of SETD1B variants. Conclusion: Insights from this extensive cohort will facilitate the counseling regarding the molecular and phenotypic landscape of newly diagnosed patients with the SETD1B-related syndrome

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Diagnostic Value of a Protocolized In-Depth Evaluation of Pediatric Bone Marrow Failure: A Multi-Center Prospective Cohort Study

Background: Severe multilineage cytopenia in childhood caused by bone marrow failure (BMF) often represents a serious condition requiring specific management. Patients are at risk for invasive infections and bleeding complications. Previous studies report low rates of identifiable causes of pediatric BMF, rendering most patients with a descriptive diagnosis such as aplastic anemia (AA). Methods: We conducted a multi-center prospective cohort study in which an extensive diagnostic approach for pediatric patients with suspected BMF was implemented. After exclusion of malignant and transient causes of BMF, patients entered thorough diagnostic evaluation including bone marrow analysis, whole exome sequencing (WES) including copy number variation (CNV) analysis and/or single nucleotide polymorphisms (SNP) array analysis. In addition, functional and immunological evaluation were performed. Here we report the outcomes of the first 50 patients (2017-2021) evaluated by this approach. Results: In 20 patients (40%) a causative diagnosis was made. In this group, 18 diagnoses were established by genetic analysis, including 14 mutations and 4 chromosomal deletions. The 2 remaining patients had short telomeres while no causative genetic defect was found. Of the remaining 30 patients (60%), 21 were diagnosed with severe aplastic anemia (SAA) based on peripheral multi-lineage cytopenia and hypoplastic bone marrow, and 9 were classified as unexplained cytopenia without bone marrow hypoplasia. In total 28 patients had undergone hematopoietic stem cell transplantation (HSCT) of which 22 patients with an unknown cause and 6 patients with an identified cause for BMF. Conclusion: We conclude that a standardized in-depth diagnostic protocol as presented here, can increase the frequency of identifiable causes within the heterogeneous group of pediatric BMF. We underline the importance of full genetic analysis complemented by functional tests of all patients as genetic causes are not limited to patients with typical (syndromal) clinical characteristics beyond cytopenia. In addition, it is of importance to apply genome wide genetic analysis, since defects in novel genes are frequently discovered in this group. Identification of a causal abnormality consequently has implications for the choice of treatment and in some cases prevention of invasive therapies

PRRT2-related phenotypes in patients with a 16p11.2 deletion

We studied the presence of benign infantile epilepsy (BIE), paroxysmal kinesigenic dyskinesia (PKD), and PKD with infantile convulsions (PKD/IC) in patients with a 16p11.2 deletion including PRRT2 or with a PRRT2 loss-of-function sequence variant. Index patients were recruited from seven Dutch university hospitals. The presence of BIE, PKD and PKD/IC was retrospectively evaluated using questionnaires and medical records. We included 33 patients with a 16p11.2 deletion: three (9%) had BIE, none had PKD or PKD/IC. Twelve patients had a PRRT2 sequence variant: BIE was present in four (p = 0.069), PKD in six (p < 0.001) and PKD/IC in two (p = 0.067). Most patients with a deletion had undergone genetic testing because of developmental problems (87%), whereas all patients with a sequence variant were tested because of a movement disorder (55%) or epilepsy (45%). BIE, PKD and PKD/IC clearly showed incomplete penetrance in patients with 16p11.2 deletions, but were found in all and 95% of patients with a PRRT2 sequence variant in our study and a large literature cohort, respectively. Deletions and sequence variants have the same underlying loss-of-function disease mechanism. Thus, differences in ascertainment have led to overestimating the frequency of BIE, PKD and PKD/IC in patients with a PRRT2 sequence variant. This has important implications for counseling if genome-wide sequencing shows such variants in patients not presenting the PRRT2-related phenotypes

Constitutional Chromothripsis Rearrangements Involve Clustered Double-Stranded DNA Breaks and Nonhomologous Repair Mechanisms

Chromothripsis represents a novel phenomenon in the structural variation landscape of cancer genomes. Here, we analyze the genomes of ten patients with congenital disease who were preselected to carry complex chromosomal rearrangements with more than two breakpoints. The rearrangements displayed unanticipated complexity resembling chromothripsis. We find that eight of them contain hallmarks of multiple clustered double-stranded DNA breaks (DSBs) on one or more chromosomes. In addition, nucleotide resolution analysis of 98 breakpoint junctions indicates that break repair involves nonhomologous or microhomology-mediated end joining. We observed that these eight rearrangements are balanced or contain sporadic deletions ranging in size between a few hundred base pairs and several megabases. The two remaining complex rearrangements did not display signs of DSBs and contain duplications, indicative of rearrangement processes involving template switching. Our work provides detailed insight into the characteristics of chromothripsis and supports a role for clustered DSBs driving some constitutional chromothripsis rearrangements

An activating mutation in the kinase homology domain of the natriuretic peptide receptor-2 causes extremely tall stature without skeletal deformities

Background: C-type natriuretic peptide (CNP)/natriuretic peptide receptor 2 (NPR2) signaling is essential for long bone growth. Enhanced CNP production caused by chromosomal translocations results in tall stature, a Marfanoid phenotype, and skeletal abnormalities.Asimilar phenotype was described in a family with an activating NPR2 mutation within the guanylyl cyclase domain. Case: Here we describe an extremely tall male without skeletal deformities, with a novel NPR2 mutation (p.Arg655Cys) located in the kinase homology domain. Objectives: The objective of the study was to investigate the functional and structural effects of the NPR2 mutation. Methods: Guanylyl cyclase activities of wild-type vs mutant NPR2 were analyzed in transfected human embryonic kidney 293 cells and in skin fibroblasts. The former were also used to study possible interactions between both isoforms. Homology modeling was performed to understand the molecular impact of the mutation. Results: CNP-stimulated cGMP production by the mutant NPR2 was markedly increased in patient skin fibroblasts and transfected human embryonic kidney 293 cells. The stimulatory effects of ATP on CNP-dependent guanylyl cyclase activity were augmented, suggesting that this novel mutation enhances both the responsiveness of NPR2 to CNP and its allosteric modulation/stabilization by ATP. Coimmunoprecipitation showed that wild-type and mutant NPR2 can form stable heterodimers, suggesting a dominant-positive effect. In accordance with augmented endogenous receptor activity, plasma N-terminal pro-CNP (a marker of CNP production in tissues) was reduced in the proband. Conclusions:Wereport the first activating mutation within the kinase homology domain of NPR2, resulting in extremely tall stature. Our observations emphasize the important role of this domain in the regulation of guanylyl cyclase activity and bone growth in response to CNP

AHDC1 missense mutations in Xia-Gibbs syndrome

Xia-Gibbs syndrome (XGS; MIM: 615829) is a phenotypically heterogeneous neurodevelopmental disorder (NDD) caused by newly arising mutations in the AT-Hook DNA-Binding Motif-Containing 1 (AHDC1) gene that are predicted to lead to truncated AHDC1 protein synthesis. More than 270 individuals have been diagnosed with XGS worldwide. Despite the absence of an independent assay for AHDC1 protein function to corroborate potential functional consequences of rare variant genetic findings, there are also reports of individuals with XGS-like trait manifestations who have de novo missense AHDC1 mutations and who have been provided a molecular diagnosis of the disorder. To investigate a potential contribution of missense mutations to XGS, we mapped the missense mutations from 10 such individuals to the AHDC1 conserved protein domain structure and detailed the observed phenotypes. Five newly identified individuals were ascertained from a local XGS Registry, and an additional five were taken from external reports or databases, including one publication. Where clinical data were available, individuals with missense mutations all displayed phenotypes consistent with those observed in individuals with AHDC1 truncating mutations, including delayed motor milestones, intellectual disability (ID), hypotonia, and speech delay. A subset of the 10 reported missense mutations cluster in two regions of the AHDC1 protein with known conserved domains, likely representing functional motifs. Variants outside the clustered regions score lower for computational prediction of their likely damaging effects. Overall, de novo missense variants in AHDC1 are likely diagnostic of XGS when in silico analysis of their position relative to conserved regions is considered together with disease trait manifestations.Among >270 individuals diagnosed with Xia-Gibbs syndrome (XGS; MIM: 615829), almost all have de novo “truncating” mutations in AHDC1. There are now also 10 reports of XGS arising from de novo missense AHDC1 mutations. We mapped these to the AHDC1 protein domain structure and identified two critical, sensitive regions