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Background Since 2009, numerous tools have been developed to detect structural variants using short read technologies. Insertions >50 bp are one of the hardest type to discover and are drastically underrepresented in gold standard variant callsets. The advent of long read technologies has completely changed the situation. In 2019, two independent cross technologies studies have published the most complete variant callsets with sequence resolved insertions in human individuals. Among the reported insertions, only 17 to 28% could be discovered with short-read based tools. Results In this work, we performed an in-depth analysis of these unprecedented insertion callsets in order to investigate the causes of such failures. We have first established a precise classification of insertion variants according to four layers of characterization: the nature and size of the inserted sequence, the genomic context of the insertion site and the breakpoint junction complexity. Because these levels are intertwined, we then used simulations to characterize the impact of each complexity factor on the recall of several structural variant callers. We showed that most reported insertions exhibited characteristics that may interfere with their discovery: 63% were tandem repeat expansions, 38% contained homology larger than 10 bp within their breakpoint junctions and 70% were located in simple repeats. Consequently, the recall of short-read based variant callers was significantly lower for such insertions (6% for tandem repeats vs 56% for mobile element insertions). Simulations showed that the most impacting factor was the insertion type rather than the genomic context, with various difficulties being handled differently among the tested structural variant callers, and they highlighted the lack of sequence resolution for most insertion calls. Conclusions Our results explain the low recall by pointing out several difficulty factors among the observed insertion features and provide avenues for improving SV caller algorithms and their combinations.

Cells rely on a diverse repertoire of genes for maintaining homeostasis, but the transcriptional networks underlying their expression remain poorly understood. The MOF acetyltransferase-containing Non-Specific Lethal (NSL) complex is a broad transcription regulator. It is essential in Drosophila, and haploinsufficiency of the human KANSL1 subunit results in the Koolen-de Vries syndrome. Here, we perform a genome-wide RNAi screen and identify the BET protein BRD4 as an evolutionary conserved co-factor of the NSL complex. Using Drosophila and mouse embryonic stem cells, we characterise a recruitment hierarchy, where NSL-deposited histone acetylation enables BRD4 recruitment for transcription of constitutively active genes. Transcriptome analyses in Koolen-de Vries patient-derived fibroblasts reveals perturbations with a cellular homeostasis signature that are evoked by the NSL complex/BRD4 axis. We propose that BRD4 represents a conserved bridge between the NSL complex and transcription activation, and provide a new perspective in the understanding of their functions in healthy and diseased states. The MOF acetyltransferase-containing Non-Specific Lethal (NSL) complex is a broad transcription regulator and haploinsufficiency of its KANSL1 subunit results in the Koolen-de Vries syndrome in humans. Here, the authors identify the BET protein BRD4 as evolutionary conserved co-factor of the NSL complex and provide evidence that NSL-deposited histone acetylation induces BRD4 recruitment for transcription of constitutively active genes.

Mutations in chromatin-modifying complexes and metabolic enzymes commonly underlie complex human developmental syndromes affecting multiple organs. A major challenge is to determine how disease-causing genetic lesions cause deregulation of homeostasis in unique cell types. Here we show that neural-specific depletion of three members of the non-specific lethal (NSL) chromatin complex—Mof, Kansl2 or Kansl3—unexpectedly leads to severe vascular defects and brain haemorrhaging. Deregulation of the epigenetic landscape induced by the loss of the NSL complex in neural cells causes widespread metabolic defects, including an accumulation of free long-chain fatty acids (LCFAs). Free LCFAs induce a Toll-like receptor 4 (TLR4)–NFκB-dependent pro-inflammatory signalling cascade in neighbouring vascular pericytes that is rescued by TLR4 inhibition. Pericytes display functional changes in response to LCFA-induced activation that result in vascular breakdown. Our work establishes that neurovascular function is determined by the neural metabolic environment.Sheikh et al. show that deficiency in the lysine acetyltransferase MOF causes aberrant accumulation of fatty acids in neural cells, which in turn triggers inflammation in nearby pericytes, resulting in brain haemorrhaging.

SKI pathogenic variations are associated with Shprintzen–Goldberg Syndrome (SGS), a rare systemic connective tissue disorder characterized by craniofacial, skeletal and cardiovascular features. So far, the clinical description, including intellectual disability, has been relatively homogeneous, and the known pathogenic variations were located in two different hotspots of the SKI gene. In the course of diagnosing Marfan syndrome and related disorders, we identified nine sporadic probands (aged 2–47 years) carrying three different likely pathogenic or pathogenic variants in the SKI gene affecting the same amino acid (Thr180). Seven of these molecular events were confirmed de novo. All probands displayed a milder morphological phenotype with a marfanoid habitus that did not initially lead to a clinical diagnosis of SGS. Only three of them had learning disorders, and none had intellectual disability. Six out of nine presented thoracic aortic aneurysm, which led to preventive surgery in the oldest case. This report extends the phenotypic spectrum of variants identified in the SKI gene. We describe a new mutational hotspot associated with a marfanoid syndrome with no intellectual disability. Cardiovascular involvement was confirmed in a significant number of cases, highlighting the importance of accurately diagnosing SGS and ensuring appropriate medical treatment and follow-up.

Silver-Russell syndrome (SRS) (mainly secondary to 11p15 molecular disruption) and Temple syndrome (TS) (secondary to 14q32.2 molecular disruption) are imprinting disorders with phenotypic (prenatal and postnatal growth retardation, early feeding difficulties) and molecular overlap. To describe the clinical overlap between SRS and TS and extensively study the molecular aspects of TS. We retrospectively collected data on 28 patients with disruption of the 14q32.2 imprinted region, identified in our center, and performed extensive molecular analysis. Seventeen (60.7%) patients showed loss of methylation of the MEG3/DLK1 intergenic differentially methylated region by epimutation. Eight (28.6%) patients had maternal uniparental disomy of chromosome 14 and three (10.7%) had a paternal deletion in 14q32.2. Most patients (72.7%) had a Netchine-Harbison SRS clinical scoring system ≥4/6, and consistent with a clinical diagnosis of SRS. The mean age at puberty onset was 7.2 years in girls and 9.6 years in boys; 37.5% had premature pubarche. The body mass index of all patients increased before pubarche and/or the onset of puberty. Multilocus analysis identified multiple methylation defects in 58.8% of patients. We identified four potentially damaging genetic variants in genes encoding proteins involved in the establishment or maintenance of DNA methylation. Most patients with 14q32.2 disruption fulfill the criteria for a clinical diagnosis of SRS. These clinical data suggest similar management of patients with TS and SRS, with special attention to their young age at the onset of puberty and early increase of body mass index.

PUF60 encodes a nucleic acid-binding protein, a component of multimeric complexes regulating RNA splicing and transcription. In 2013, patients with microdeletions of chromosome 8q24.3 including PUF60 were found to have developmental delay, microcephaly, craniofacial, renal and cardiac defects. Very similar phenotypes have been described in six patients with variants in PUF60, suggesting that it underlies the syndrome. We report 12 additional patients with PUF60 variants who were ascertained using exome sequencing: six through the Deciphering Developmental Disorders Study and six through similar projects. Detailed phenotypic analysis of all patients was undertaken. All 12 patients had de novo heterozygous PUF60 variants on exome analysis, each confirmed by Sanger sequencing: four frameshift variants resulting in premature stop codons, three missense variants that clustered within the RNA recognition motif of PUF60 and five essential splice-site (ESS) variant. Analysis of cDNA from a fibroblast cell line derived from one of the patients with an ESS variants revealed aberrant splicing. The consistent feature was developmental delay and most patients had short stature. The phenotypic variability was striking; however, we observed similarities including spinal segmentation anomalies, congenital heart disease, ocular colobomata, hand anomalies and (in two patients) unilateral renal agenesis/horseshoe kidney. Characteristic facial features included micrognathia, a thin upper lip and long philtrum, narrow almond-shaped palpebral fissures, synophrys, flared eyebrows and facial hypertrichosis. Heterozygote loss-of-function variants in PUF60 cause a phenotype comprising growth/developmental delay and craniofacial, cardiac, renal, ocular and spinal anomalies, adding to disorders of human development resulting from aberrant RNA processing/spliceosomal function.

Exome sequencing (ES) has revolutionized diagnostic procedures in medical genetics, particularly for developmental diseases. The variety and complexity of the information produced has raised issues regarding its use in a clinical setting. Of particular interest are patients’ expectations regarding the information disclosed, the accompaniment provided, and the value patients place on these. To explore these issues in parents of children with developmental disorders and no diagnosis with known etiology, a multidisciplinary group of researchers from social and behavioral sciences and patient organizations conducted a mixed-methodology study (quantitative and qualitative) in two centers of expertise for rare diseases in France. The quantitative study aimed to determine the preferences of 513 parents regarding the disclosure of ES results. It showed that parents wished to have exhaustive information, including variants of unknown significance possibly linked to their child’s disorder and secondary findings. This desire for information could be a strategy to maximize the chances of obtaining a diagnosis. The qualitative study aimed to understand the expectations and reactions of 57 parents interviewed just after the return of ES results. In-depth analysis showed that parents had ambivalent feelings about the findings whatever the results returned. The contrasting results from these studies raise questions about the value of the information provided and parents’ high expectations regarding the results. The nature of parental expectations has emerged as an important topic in efforts to optimize accompaniment and support for families during the informed decision-making process and after disclosure of the results in an overall context of uncertainty.

Cohen syndrome (CS) is a rare genetic disorder due to mutations in VPS13B gene. Among various clinical and biological features, CS patients suffer from inconsistent neutropenia, which is associated with recurrent but minor infections. We demonstrate here that this neutropenia results from an exaggerate rate of neutrophil apoptosis. Besides this increased cell death, which occurs in the absence of any endoplasmic reticulum stress or defect in neutrophil elastase (ELANE) expression or localization, all neutrophil functions appeared to be normal. We showed a disorganization of the Golgi apparatus in CS neutrophils precursors, that correlates with an altered glycosylation of ICAM-1 in these cells, as evidenced by a migration shift of the protein. Furthermore, a striking decrease in the expression of SERPINB1 gene, which encodes a critical component of neutrophil survival, was detected in CS neutrophils. These abnormalities may account for the excessive apoptosis of neutrophils leading to neutropenia in CS.Key messagesCohen syndrome patients’ neutrophils display normal morphology and functions.Cohen syndrome patients’ neutrophils have an increased rate of spontaneous apoptosis compared to healthy donors’ neutrophils.No ER stress or defective ELA2 expression or glycosylation was observed in Cohen syndrome patients’ neutrophils.SerpinB1 expression is significantly decreased in Cohen syndrome neutrophils as well as in VPS13B-deficient cells.

Fragile-X syndrome (FXS) is a frequent genetic form of intellectual disability (ID). The main recurrent mutagenic mechanism causing FXS is the expansion of a CGG repeat sequence in the 5'-UTR of the FMR1 gene, therefore, routinely tested in ID patients. We report here three FMR1 intragenic pathogenic variants not affecting this sequence, identified using high-throughput sequencing (HTS): a previously reported hemizygous deletion encompassing the last exon of FMR1, too small to be detected by array-CGH and inducing decreased expression of a truncated form of FMRP protein, in three brothers with ID (family 1) and two splice variants in boys with sporadic ID: a de novo variant c.990+1G>A (family 2) and a maternally inherited c.420-8A>G variant (family 3). After clinical reevaluation, the five patients presented features consistent with FXS (mean Hagerman's scores=15). We conducted a systematic review of all rare non-synonymous variants previously reported in FMR1 in ID patients and showed that six of them are convincing pathogenic variants. This study suggests that intragenic FMR1 variants, although much less frequent than CGG expansions, are a significant mutational mechanism leading to FXS and demonstrates the interest of HTS approaches to detect them in ID patients with a negative standard work-up.