Diagnosis and management of Cornelia de Lange syndrome: first international consensus statement.

Cornelia de Lange syndrome (CdLS) is an archetypical genetic syndrome that is characterized by intellectual disability, well-defined facial features, upper limb anomalies and atypical growth, among numerous other signs and symptoms. It is caused by variants in any one of seven genes, all of which have a structural or regulatory function in the cohesin complex. Although recent advances in next-generation sequencing have improved molecular diagnostics, marked heterogeneity exists in clinical and molecular diagnostic approaches and care practices worldwide. Here, we outline a series of recommendations that document the consensus of a group of international experts on clinical diagnostic criteria, both for classic CdLS and non-classic CdLS phenotypes, molecular investigations, long-term management and care planning.

Mosaic divergent repeat interruptions in XDP influence repeat stability and disease onset.

While many genetic causes of movement disorders have been identified, modifiers of disease expression are largely unknown. X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disease caused by a SINE-VNTR-Alu(AGAGGG)n retrotransposon insertion in TAF1, with a polymorphic (AGAGGG)n repeat. Repeat length and variants in MSH3 and PMS2 explain ∼65% of the variance in age at onset (AAO) in XDP. However, additional genetic modifiers are conceivably at play in XDP, such as repeat interruptions. Long-read nanopore sequencing of PCR amplicons from XDP patients (n = 202) was performed to assess potential repeat interruption and instability. Repeat-primed PCR and Cas9-mediated targeted enrichment confirmed the presence of identified divergent repeat motifs. In addition to the canonical pure SINE-VNTR-Alu-5'-(AGAGGG)n, we observed a mosaic of divergent repeat motifs that polarized at the beginning of the tract, where the divergent repeat interruptions varied in motif length by having one, two, or three nucleotides fewer than the hexameric motif, distinct from interruptions in other disease-associated repeats, which match the lengths of the canonical motifs. All divergent configurations occurred mosaically and in two investigated brain regions (basal ganglia, cerebellum) and in blood-derived DNA from the same patient. The most common divergent interruption was AGG [5'-SINE-VNTR-Alu(AGAGGG)2AGG(AGAGGG)n], similar to the pure tract, followed by AGGG [5'-SINE-VNTR-Alu(AGAGGG)2AGGG(AGAGGG)n], at median frequencies of 0.425 (IQR: 0.42-0.43) and 0.128 (IQR: 0.12-0.13), respectively. The mosaic AGG motif was not associated with repeat number (estimate = -3.8342, P = 0.869). The mosaic pure tract frequency was associated with repeat number (estimate = 45.32, P = 0.0441) but not AAO (estimate = -41.486, P = 0.378). Importantly, the mosaic frequency of the AGGG negatively correlated with repeat number after adjusting for age at sampling (estimate = -161.09, P = 3.44 × 10-5). When including the XDP-relevant MSH3/PMS2 modifier single nucleotide polymorphisms into the model, the mosaic AGGG frequency was associated with AAO (estimate = 155.1063, P = 0.047); however, the association dissipated after including the repeat number (estimate = -92.46430, P = 0.079). We reveal novel mosaic divergent repeat interruptions affecting both motif length and sequence (DRILS) of the canonical motif polarized within the SINE-VNTR-Alu(AGAGGG)n repeat. Our study illustrates: (i) the importance of somatic mosaic genotypes; (ii) the biological plausibility of multiple modifiers (both germline and somatic) that can have additive effects on repeat instability; and (iii) that these variations may remain undetected without assessment of single molecules.

Bi-allelic variants of FILIP1 cause congenital myopathy, dysmorphism and neurological defects.

Filamin-A-interacting protein 1 (FILIP1) is a structural protein that is involved in neuronal and muscle function and integrity and interacts with FLNa and FLNc. Pathogenic variants in filamin-encoding genes have been linked to neurological disorders (FLNA) and muscle diseases characterized by myofibrillar perturbations (FLNC), but human diseases associated with FILIP1 variants have not yet been described. Here, we report on five patients from four unrelated consanguineous families with homozygous FILIP1 variants (two nonsense and two missense). Functional studies indicated altered stability of the FILIP1 protein carrying the p.[Pro1133Leu] variant. Patients exhibit a broad spectrum of neurological symptoms including brain malformations, neurodevelopmental delay, muscle weakness and pathology and dysmorphic features. Electron and immunofluorescence microscopy on the muscle biopsy derived from the patient harbouring the homozygous p.[Pro1133Leu] missense variant revealed core-like zones of myofibrillar disintegration, autophagic vacuoles and accumulation of FLNc. Proteomic studies on the fibroblasts derived from the same patient showed dysregulation of a variety of proteins including FLNc and alpha-B-crystallin, a finding (confirmed by immunofluorescence) which is in line with the manifestation of symptoms associated with the syndromic phenotype of FILIP1opathy. The combined findings of this study show that the loss of functional FILIP1 leads to a recessive disorder characterized by neurological and muscular manifestations as well as dysmorphic features accompanied by perturbed proteostasis and myopathology.

Disruption of the topologically associated domain at Xp21.2 is related to 46,XY gonadal dysgenesis.

BACKGROUND: Duplications at the Xp21.2 locus have previously been linked to 46,XY gonadal dysgenesis (GD), which is thought to result from gene dosage effects of NR0B1 (DAX1), but the exact disease mechanism remains unknown. METHODS: Patients with 46,XY GD were analysed by whole genome sequencing. Identified structural variants were confirmed by array CGH and analysed by high-throughput chromosome conformation capture (Hi-C). RESULTS: We identified two unrelated patients: one showing a complex rearrangement upstream of NR0B1 and a second harbouring a 1.2 Mb triplication, including NR0B1. Whole genome sequencing and Hi-C analysis revealed the rewiring of a topological-associated domain (TAD) boundary close to NR0B1 associated with neo-TAD formation and may cause enhancer hijacking and ectopic NR0B1 expression. Modelling of previous Xp21.2 structural variations associated with isolated GD support our hypothesis and predict similar neo-TAD formation as well as TAD fusion. CONCLUSION: Here we present a general mechanism how deletions, duplications or inversions at the NR0B1 locus can lead to partial or complete GD by disrupting the cognate TAD in the vicinity of NR0B1. This model not only allows better diagnosis of GD with copy number variations (CNVs) at Xp21.2, but also gives deeper insight on how spatiotemporal activation of developmental genes can be disrupted by reorganised TADs causing impairment of gonadal development.

Homozygous WASHC4 variant in two sisters causes a syndromic phenotype defined by dysmorphisms, intellectual disability, profound developmental disorder, and skeletal muscle involvement.

Recessive variants in WASHC4 are linked to intellectual disability complicated by poor language skills, short stature, and dysmorphic features. The protein encoded by WASHC4 is part of the Wiskott-Aldrich syndrome protein and SCAR homolog family, co-localizes with actin in cells, and promotes Arp2/3-dependent actin polymerization in vitro. Functional studies in a zebrafish model suggested that WASHC4 knockdown may also affect skeletal muscles by perturbing protein clearance. However, skeletal muscle involvement has not been reported so far in patients, and precise biochemical studies allowing a deeper understanding of the molecular etiology of the disease are still lacking. Here, we report two siblings with a homozygous WASHC4 variant expanding the clinical spectrum of the disease and provide a phenotypical comparison with cases reported in the literature. Proteomic profiling of fibroblasts of the WASHC4-deficient patient revealed dysregulation of proteins relevant for the maintenance of the neuromuscular axis. Immunostaining on a muscle biopsy derived from the same patient confirmed dysregulation of proteins relevant for proper muscle function, thus highlighting an affliction of muscle cells upon loss of functional WASHC4. The results of histological and coherent anti-Stokes Raman scattering microscopic studies support the concept of a functional role of the WASHC4 protein in humans by altering protein processing and clearance. The proteomic analysis confirmed key molecular players in vitro and highlighted, for the first time, the involvement of skeletal muscle in patients. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Novel Variants of SOX4 in Patients with Intellectual Disability.

SOX4 is a transcription factor with pleiotropic functions required for different developmental processes, such as corticogenesis. As with all SOX proteins, it contains a conserved high mobility group (HMG) and exerts its function via interaction with other transcription factors, such as POU3F2. Recently, pathogenic SOX4 variants have been identified in several patients who had clinical features overlapping with Coffin-Siris syndrome. In this study, we identified three novel variants in unrelated patients with intellectual disability, two of which were de novo (c.79G>T, p.Glu27*; c.182G>A p.Arg61Gln) and one inherited (c.355C>T, p.His119Tyr). All three variants affected the HMG box and were suspected to influence SOX4 function. We investigated the effects of these variants on transcriptional activation by co-expressing either wildtype (wt) or mutant SOX4 with its co-activator POU3F2 and measuring their activity in reporter assays. All variants abolished SOX4 activity. While our experiments provide further support for the pathogenicity of SOX4 loss-of-function (LOF) variants as a cause of syndromic intellectual disability (ID), our results also indicate incomplete penetrance associated with one variant. These findings will improve classification of novel, putatively pathogenic SOX4 variants.

TRα2-An Untuned Second Fiddle or Fine-Tuning Thyroid Hormone Action?

Thyroid hormones (THs) control a wide range of physiological functions essential for metabolism, growth, and differentiation. On a molecular level, TH action is exerted by nuclear receptors (TRs), which function as ligand-dependent transcription factors. Among several TR isoforms, the function of TRα2 remains poorly understood as it is a splice variant of TRα with an altered C-terminus that is unable to bind T3. This review highlights the molecular characteristics of TRα2, proposed mechanisms that regulate alternative splicing and indications pointing towards an antagonistic function of this TR isoform in vitro and in vivo. Moreover, remaining knowledge gaps and major challenges that complicate TRα2 characterization, as well as future strategies to fully uncover its physiological relevance, are discussed.

G Protein-Coupled Receptor 15 Expression Is Associated with Myocardial Infarction.

Beyond the influence of lifestyle-related risk factors for myocardial infarction (MI), the mechanisms of genetic predispositions for MI remain unclear. We sought to identify and characterize differentially expressed genes in early-onset MI in a translational approach. In an observational case−control study, transcriptomes from 112 early-onset MI individuals showed upregulated G protein-coupled receptor 15 (GPR15) expression in peripheral blood mononuclear cells compared to controls (fold change = 1.4, p = 1.87 × 10−7). GPR15 expression correlated with intima-media thickness (ß = 0.8498, p = 0.111), C-reactive protein (ß = 0.2238, p = 0.0052), ejection fraction (ß = −0.9991, p = 0.0281) and smoking (ß = 0.7259, p = 2.79 × 10−10). The relation between smoking and MI was diminished after the inclusion of GPR15 expression as mediator in mediation analysis (from 1.27 (p = 1.9 × 10−5) to 0.46 (p = 0.21)). The DNA methylation of two GPR15 sites was 1%/5% lower in early-onset MI individuals versus controls (p = 2.37 × 10−6/p = 0.0123), with site CpG3.98251219 significantly predicting risk for incident MI (hazard ratio = 0.992, p = 0.0177). The nucleotide polymorphism rs2230344 (C/T) within GPR15 was associated with early-onset MI (odds ratio = 3.61, p = 0.044). Experimental validation showed 6.3-fold increased Gpr15 expression in an ischemic mouse model (p < 0.05) and 4-fold increased Gpr15 expression in cardiomyocytes under ischemic stress (p < 0.001). After the induction of MI, Gpr15gfp/gfp mice showed lower survival (p = 0.042) and deregulated gene expression for response to hypoxia and signaling pathways. Using a translational approach, our data provide evidence that GPR15 is linked to cardiovascular diseases, mediating the adverse effects of smoking.

Transcriptional Alterations in X-Linked Dystonia-Parkinsonism Caused by the SVA Retrotransposon.

X-linked dystonia-parkinsonism (XDP) is a severe neurodegenerative disorder that manifests as adult-onset dystonia combined with parkinsonism. A SINE-VNTR-Alu (SVA) retrotransposon inserted in an intron of the TAF1 gene reduces its expression and alters splicing in XDP patient-derived cells. As a consequence, increased levels of the TAF1 intron retention transcript TAF1-32i can be found in XDP cells as compared to healthy controls. Here, we investigate the sequence of the deep intronic region included in this transcript and show that it is also present in cells from healthy individuals, albeit in lower amounts than in XDP cells, and that it undergoes degradation by nonsense-mediated mRNA decay. Furthermore, we investigate epigenetic marks (e.g., DNA methylation and histone modifications) present in this intronic region and the spanning sequence. Finally, we show that the SVA evinces regulatory potential, as demonstrated by its ability to repress the TAF1 promoter in vitro. Our results enable a better understanding of the disease mechanisms underlying XDP and transcriptional alterations caused by SVA retrotransposons.

Missense and truncating variants in CHD5 in a dominant neurodevelopmental disorder with intellectual disability, behavioral disturbances, and epilepsy.

Located in the critical 1p36 microdeletion region, the chromodomain helicase DNA-binding protein 5 (CHD5) gene encodes a subunit of the nucleosome remodeling and deacetylation (NuRD) complex required for neuronal development. Pathogenic variants in six of nine chromodomain (CHD) genes cause autosomal dominant neurodevelopmental disorders, while CHD5-related disorders are still unknown. Thanks to GeneMatcher and international collaborations, we assembled a cohort of 16 unrelated individuals harboring heterozygous CHD5 variants, all identified by exome sequencing. Twelve patients had de novo CHD5 variants, including ten missense and two splice site variants. Three familial cases had nonsense or missense variants segregating with speech delay, learning disabilities, and/or craniosynostosis. One patient carried a frameshift variant of unknown inheritance due to unavailability of the father. The most common clinical features included language deficits (81%), behavioral symptoms (69%), intellectual disability (64%), epilepsy (62%), and motor delay (56%). Epilepsy types were variable, with West syndrome observed in three patients, generalized tonic-clonic seizures in two, and other subtypes observed in one individual each. Our findings suggest that, in line with other CHD-related disorders, heterozygous CHD5 variants are associated with a variable neurodevelopmental syndrome that includes intellectual disability with speech delay, epilepsy, and behavioral problems as main features.

ANKRD11 variants: KBG syndrome and beyond.

Mutations affecting the transcriptional regulator Ankyrin Repeat Domain 11 (ANKRD11) are mainly associated with the multisystem developmental disorder known as KBG syndrome, but have also been identified in individuals with Cornelia de Lange syndrome (CdLS) and other developmental disorders caused by variants affecting different chromatin regulators. The extensive functional overlap of these proteins results in shared phenotypical features, which complicate the assessment of the clinical diagnosis. Additionally, re-evaluation of individuals at a later age occasionally reveals that the initial phenotype has evolved toward clinical features more reminiscent of a developmental disorder different from the one that was initially diagnosed. For this reason, variants in ANKRD11 can be ascribed to a broader class of disorders that fall within the category of the so-called chromatinopathies. In this work, we report on the clinical characterization of 23 individuals with variants in ANKRD11. The subjects present primarily with developmental delay, intellectual disability and dysmorphic features, and all but two received an initial clinical diagnosis of either KBG syndrome or CdLS. The number and the severity of the clinical signs are overlapping but variable and result in a broad spectrum of phenotypes, which could be partially accounted for by the presence of additional molecular diagnoses and distinct pathogenic mechanisms.

Linking Penetrance and Transcription in DYT-THAP1: Insights From a Human iPSC-Derived Cortical Model.

BACKGROUND: The THAP1 gene encodes a transcription factor, and pathogenic variants cause a form of autosomal dominant, isolated dystonia (DYT-THAP1) with reduced penetrance. Factors underlying both reduced penetrance and the disease mechanism of DYT-THAP1 are largely unknown. METHODS: We performed transcriptome analysis on 29 cortical neuronal precursors derived from human-induced pluripotent stem cell lines generated from manifesting and nonmanifesting THAP1 mutation carriers and control individuals. RESULTS: Whole transcriptome analysis showed a penetrance-linked signature with expressional changes more pronounced in the group of manifesting (MMCs) than in nonmanifesting mutation carriers (NMCs) when compared to controls. A direct comparison of the transcriptomes in MMCs versus NMCs showed significant upregulation of the DRD4 gene in MMCs. A gene set enrichment analysis demonstrated alterations in various neurotransmitter release cycle pathways, extracellular matrix organization, and deoxyribonucleic acid methylation between MMCs and NMCs. When specifically considering transcription factors, the expression of YY1 and SIX2 differed in MMCs versus NMCs. Further, THAP1 was upregulated in the group of MMCs. CONCLUSIONS: To our knowledge, this is the first report systematically analyzing reduced penetrance in DYT-THAP1 in a human model using transcriptomes. Our findings indicate that transcriptional alterations during cortical development influence DYT-THAP1 pathogenesis and penetrance. We reinforce previously linked pathways including dopamine and eukaryotic translation initiation factor 2 alpha signaling in the pathogenesis of dystonia including DYT-THAP1 and suggest extracellular matrix organization and deoxyribonucleic acid methylation as mediators of disease protection. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Syndromic neurodevelopmental disorder associated with de novo variants in DDX23.

The DEAD/DEAH box RNA helicases are a superfamily of proteins involved in the processing and transportation of RNA within the cell. A growing literature supports this family of proteins as contributing to various types of human disorders from neurodevelopmental disorders to syndromes with multiple congenital anomalies. This article presents a cohort of nine unrelated individuals with de novo missense alterations in DDX23 (Dead-Box Helicase 23). The gene is ubiquitously expressed and functions in RNA splicing, maintenance of genome stability, and the sensing of double-stranded RNA. Our cohort of patients, gathered through GeneMatcher, exhibited features including tone abnormalities, global developmental delay, facial dysmorphism, autism spectrum disorder, and seizures. Additionally, there were a variety of other findings in the skeletal, renal, ocular, and cardiac systems. The missense alterations all occurred within a highly conserved RecA-like domain of the protein, and are located within or proximal to the DEAD box sequence. The individuals presented in this article provide evidence of a syndrome related to alterations in DDX23 characterized predominantly by atypical neurodevelopment.

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.

Chromatinopathies: A focus on Cornelia de Lange syndrome.

In recent years, many genes have been associated with chromatinopathies classified as "Cornelia de Lange Syndrome-like." It is known that the phenotype of these patients becomes less recognizable, overlapping to features characteristic of other syndromes caused by genetic variants affecting different regulators of chromatin structure and function. Therefore, Cornelia de Lange syndrome diagnosis might be arduous due to the seldom discordance between unexpected molecular diagnosis and clinical evaluation. Here, we review the molecular features of Cornelia de Lange syndrome, supporting the hypothesis that "CdLS-like syndromes" are part of a larger "rare disease family" sharing multiple clinical features and common disrupted molecular pathways.

Heterozygous de novo variants in CSNK1G1 are associated with syndromic developmental delay and autism spectrum disorder.

The gamma-1 isoform of casein kinase 1, the protein encoded by CSNK1G1, is involved in the growth and morphogenesis of cells. This protein is expressed ubiquitously among many tissue types, including the brain, where it regulates the phosphorylation of N-methyl-D-aspartate receptors and plays a role in synaptic transmission. One prior individual with a de novo variant in CSNK1G presenting with severe developmental delay and early-onset epilepsy has been reported. Here we report an updated clinical history of this previously published case, as well as four additional individuals with de novo variants in CSNK1G1 identified via microarray-based comparative genomic hybridization, exome, or genome sequencing. All individuals (n = 5) had developmental delay. At least three individuals had diagnoses of autism spectrum disorder. All participants were noted to have dysmorphic facial features, although the reported findings varied widely and therefore may not clearly be recognizable. None of the participants had additional major malformations. Taken together, our data suggest that CSNK1G1 may be a cause of syndromic developmental delay and possibly autism spectrum disorder.

A hexanucleotide repeat modifies expressivity of X-linked dystonia parkinsonism.

OBJECTIVE: X-linked dystonia parkinsonism (XDP) is a neurodegenerative movement disorder caused by a single mutation: SINE-VNTR-Alu (SVA) retrotransposon insertion in TAF1. Recently, a (CCCTCT)n repeat within the SVA insertion has been reported as an age-at-onset (AAO) modifier in XDP. Here we investigate the role of this hexanucleotide repeat in modifying expressivity of XDP. METHODS: We genotyped the hexanucleotide repeat in 355 XDP patients and correlated the repeat number (RN) with AAO (n = 295), initial clinical manifestation (n = 294), site of dystonia onset (n = 238), disease severity (n = 28), and cognitive function (n = 15). Furthermore, we investigated i) repeat instability by segregation analysis and Southern blotting using postmortem brain samples from two affected individuals and ii) relative TAF1 expression in blood RNA from 31 XDP patients. RESULTS: RN showed significant inverse correlations with AAO and with TAF1 expression and a positive correlation with disease severity and cognitive dysfunction. Importantly, AAO (and not RN) was directly associated with whether dystonia or parkinsonism will manifest at onset. RN was lower in patients affected by mouth/tongue dystonia compared with blepharospasm. RN was unstable across germline transmissions with an overall tendency to increase in length and exhibited somatic mosaicism in brain. INTERPRETATION: The hexanucleotide repeat within the SVA insertion acts as a genetic modifier of disease expressivity in XDP. RN-dependent TAF1 repression and subsequent differences in TAF1 mRNA levels in patients may be potentiated in the brain through somatic variability leading to the neurological phenotype. ANN NEUROL 2019;85:812-822.

DNA Methylation as a Potential Molecular Mechanism in X-linked Dystonia-Parkinsonism.

BACKGROUND: X-linked dystonia-parkinsonism is a neurodegenerative movement disorder. The underlying molecular basis has still not been completely elucidated, but likely involves dysregulation of TAF1 expression. In X-linked dystonia-parkinsonism, 3 disease-specific single-nucleotide changes (DSCs) introduce (DSC12) or abolish (DSC2 and DSC3) CpG dinucleotides and consequently sites of putative DNA methylation. Because transcriptional regulation tightly correlates with specific epigenetic marks, we investigated the role of DNA methylation in the pathogenesis of X-linked dystonia-parkinsonism. METHODS: DNA methylation at DSC12, DSC3, and DSC2 was quantified by bisulfite pyrosequencing in DNA from peripheral blood leukocytes, fibroblasts, induced pluripotent stem cell-derived cortical neurons and brain tissue from X-linked dystonia-parkinsonism patients and age- and sex-matched healthy Filipino controls in a prospective study. RESULTS: Compared with controls, X-linked dystonia-parkinsonism patients showed striking differences in DNA methylation at the 3 investigated CpG sites. Using methylation-sensitive luciferase reporter gene assays and immunoprecipitation, we demonstrated (1) that lack of DNA methylation because of DSC2 and DSC3 affects gene promoter activity and (2) that methylation at all 3 investigated CpG sites alters DNA-protein interaction. Interestingly, DSC3 decreased promoter activity per se compared with wild type, and promoter activity further decreased when methylation was present. Moreover, we identified specific binding of proteins to the investigated DSCs that are associated with splicing and RNA and DNA binding. CONCLUSIONS: We identified altered DNA methylation in X-linked dystonia-parkinsonism patients as a possible additional mechanism modulating TAF1 expression and putative novel targets for future therapies using DNA methylation-modifying agents. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Regulation of the cohesin-loading factor NIPBL: Role of the lncRNA NIPBL-AS1 and identification of a distal enhancer element.

Cohesin is crucial for genome stability, cell division, transcription and chromatin organization. Its functions critically depend on NIPBL, the cohesin-loader protein that is found to be mutated in >60% of the cases of Cornelia de Lange syndrome (CdLS). Other mutations are described in the cohesin subunits SMC1A, RAD21, SMC3 and the HDAC8 protein. In 25-30% of CdLS cases no mutation in the known CdLS genes is detected. Until now, functional elements in the noncoding genome were not characterized in the molecular etiology of CdLS and therefore are excluded from mutation screening, although the impact of such mutations has now been recognized for a wide range of diseases. We have identified different elements of the noncoding genome involved in regulation of the NIPBL gene. NIPBL-AS1 is a long non-coding RNA transcribed upstream and antisense to NIPBL. By knockdown and transcription blocking experiments, we could show that not the NIPBL-AS1 gene product, but its actual transcription is important to regulate NIPBL expression levels. This reveals a possibility to boost the transcriptional activity of the NIPBL gene by interfering with the NIPBL-AS1 lncRNA. Further, we have identified a novel distal enhancer regulating both NIPBL and NIPBL-AS1. Deletion of the enhancer using CRISPR genome editing in HEK293T cells reduces expression of NIPBL, NIPBL-AS1 as well as genes found to be dysregulated in CdLS.

Evaluating Face2Gene as a Tool to Identify Cornelia de Lange Syndrome by Facial Phenotypes.

Characteristic or classic phenotype of Cornelia de Lange syndrome (CdLS) is associated with a recognisable facial pattern. However, the heterogeneity in causal genes and the presence of overlapping syndromes have made it increasingly difficult to diagnose only by clinical features. DeepGestalt technology, and its app Face2Gene, is having a growing impact on the diagnosis and management of genetic diseases by analysing the features of affected individuals. Here, we performed a phenotypic study on a cohort of 49 individuals harbouring causative variants in known CdLS genes in order to evaluate Face2Gene utility and sensitivity in the clinical diagnosis of CdLS. Based on the profile images of patients, a diagnosis of CdLS was within the top five predicted syndromes for 97.9% of our cases and even listed as first prediction for 83.7%. The age of patients did not seem to affect the prediction accuracy, whereas our results indicate a correlation between the clinical score and affected genes. Furthermore, each gene presents a different pattern recognition that may be used to develop new neural networks with the goal of separating different genetic subtypes in CdLS. Overall, we conclude that computer-assisted image analysis based on deep learning could support the clinical diagnosis of CdLS.