DNA methylation analysis in patients with neurodevelopmental disorders improves variant interpretation and reveals complexity.

Analysis of genomic DNA methylation by generating epigenetic signature profiles ("episignatures") is increasingly being implemented in genetic diagnosis. Here we report our experience using episignature analysis to resolve both uncomplicated and complex cases of neurodevelopmental disorder (NDD). We analysed 97 NDDs divided into: (i) a validation cohort of 59 patients with likely pathogenic/pathogenic variants characterized by a known episignature and (ii) a test cohort of 38 patients harbouring variants of unknown significance (VUS) or unidentified variants. The expected episignature was obtained in most cases with likely pathogenic/pathogenic variants (53/59; 90%), a revealing exception being the overlapping profile of two SMARCB1 pathogenic variants with ARID1A/B:c.6200, confirmed by the overlapping clinical features. In the test cohort, five cases showed the expected episignature, including: (i) novel pathogenic variants in ARID1B and BRWD3; (ii) a deletion in ATRX causing MRXFH1 X-linked mental retardation and (iii) confirmed the clinical diagnosis of Cornelia de Lange (CdL) syndrome in mutation negative CdL patients. Episignatures analysis of the in BAF complex components revealed novel functional protein interactions and common episignatures affecting homologous residues in highly conserved paralogous proteins (SMARCA2 M856V and SMARCA4 M866V). Finally, we also found sex-dependent episignatures in X-linked disorders. Implementation of episignature profiling is still in its early days but with increasing utilization come increasing awareness of the capacity of this methodology to help resolve the complex challenges of genetic diagnoses.

Skewed X-chromosome inactivation in unsolved neurodevelopmental disease cases can guide re-evaluation For X-linked genes.

Despite major advances in genome technology and analysis, >50% of patients with a neurodevelopmental disorder (NDD) remain undiagnosed after extensive evaluation. A point in case is our clinically heterogeneous cohort of NDD patients that remained undiagnosed after FRAXA testing, chromosomal microarray analysis and trio exome sequencing (ES). In this study, we explored the frequency of non-random X chromosome inactivation (XCI) in the mothers of male patients and affected females, the rationale being that skewed XCI might be masking previously discarded genetic variants found on the X chromosome. A multiplex fluorescent PCR-based assay was used to analyse the pattern of XCI after digestion with HhaI methylation-sensitive restriction enzyme. In families with skewed XCI, we re-evaluated trio-based ES and identified pathogenic variants and a deletion on the X chromosome. Linkage analysis and RT-PCR were used to further study the inactive X chromosome allele, and Xdrop long-DNA technology was used to define chromosome deletion boundaries. We found skewed XCI (>90%) in 16/186 (8.6%) mothers of NDD males and in 12/90 (13.3%) NDD females, far beyond the expected rate of XCI in the normal population (3.6%, OR = 4.10; OR = 2.51). By re-analyzing ES and clinical data, we solved 7/28 cases (25%) with skewed XCI, identifying variants in KDM5C, PDZD4, PHF6, TAF1, OTUD5 and ZMYM3, and a deletion in ATRX. We conclude that XCI profiling is a simple assay that targets a subgroup of patients that can benefit from re-evaluation of X-linked variants, thus improving the diagnostic yield in NDD patients and identifying new X-linked disorders.

Functional analysis of TLK2 variants and their proximal interactomes implicates impaired kinase activity and chromatin maintenance defects in their pathogenesis.

INTRODUCTION: The Tousled-like kinases 1 and 2 (TLK1 and TLK2) are involved in many fundamental processes, including DNA replication, cell cycle checkpoint recovery and chromatin remodelling. Mutations in TLK2 were recently associated with 'Mental Retardation Autosomal Dominant 57' (MRD57, MIM# 618050), a neurodevelopmental disorder characterised by a highly variable phenotype, including mild-to-moderate intellectual disability, behavioural abnormalities, facial dysmorphisms, microcephaly, epilepsy and skeletal anomalies. METHODS: We re-evaluate whole exome sequencing and array-CGH data from a large cohort of patients affected by neurodevelopmental disorders. Using spatial proteomics (BioID) and single-cell gel electrophoresis, we investigated the proximity interaction landscape of TLK2 and analysed the effects of p.(Asp551Gly) and a previously reported missense variant (c.1850C>T; p.(Ser617Leu)) on TLK2 interactions, localisation and activity. RESULTS: We identified three new unrelated MRD57 families. Two were sporadic and caused by a missense change (c.1652A>G; p.(Asp551Gly)) or a 39 kb deletion encompassing TLK2, and one was familial with three affected siblings who inherited a nonsense change from an affected mother (c.1423G>T; p.(Glu475Ter)). The clinical phenotypes were consistent with those of previously reported cases. The tested mutations strongly impaired TLK2 kinase activity. Proximal interactions between TLK2 and other factors implicated in neurological disorders, including CHD7, CHD8, BRD4 and NACC1, were identified. Finally, we demonstrated a more relaxed chromatin state in lymphoblastoid cells harbouring the p.(Asp551Gly) variant compared with control cells, conferring susceptibility to DNA damage. CONCLUSION: Our study identified novel TLK2 pathogenic variants, confirming and further expanding the MRD57-related phenotype. The molecular characterisation of missense variants increases our knowledge about TLK2 function and provides new insights into its role in neurodevelopmental disorders.

Expanding the clinical phenotype of the ultra-rare Skraban-Deardorff syndrome: Two novel individuals with WDR26 loss-of-function variants and a literature review.

De novo variants in the WDR26 gene leading to haploinsufficiency have recently been associated with Skraban-Deardorff syndrome. This condition is an ultra-rare autosomal dominant neurodevelopmental disorder characterized by a broad range of clinical signs, including intellectual disability (ID), developmental delay (DD), seizures, abnormal facial features, feeding difficulties, and minor skeletal anomalies. Currently, 18 cases have been reported in the literature and for only 15 of them a clinical description is available. Here, we describe a child with Skraban-Deardorff syndrome associated with the WDR26 pathogenic de novo variant NM_025160.6:c.69dupC, p.(Gly24ArgfsTer48), and an adult associated with the pathogenic de novo variant c.1076G > A, p.(Trp359Ter). The adult patient was a 29-year-old female with detailed information on clinical history and pharmacological treatments since birth, providing an opportunity to map disease progression and patient management. By comparing our cases with published reports of Skraban-Deardorff syndrome, we provide a genetic and clinical summary of this ultrarare condition, describe the clinical management from childhood to adult age, and further expand on the clinical phenotype.

Is Focal Cortical Dysplasia/Epilepsy Caused by Somatic MTOR Mutations Always a Unilateral Disorder?

OBJECTIVE: To alert about the wide margin of unpredictability that distribution of somatic MTOR mosaicism may have in the brain and the risk for independent epileptogenesis arising from the seemingly healthy contralateral hemisphere after complete removal of epileptogenic focal cortical dysplasia (FCD). METHODS: Clinical, EEG, MRI, histopathology, and molecular genetics in 2 patients (1 and 2) treated with focal resections and subsequent complete hemispherectomy for epileptogenic FCD due to somatic MTOR mutations. Autoptic brain study of bilateral asymmetric hemispheric dysplasia and identification of alternative allele fraction (AAF) rates for AKT1 (patient 3). RESULTS: The strongly hyperactivating p.Ser2215Phe (patient 1) and p.Leu1460Pro (patient 2) MTOR mutations were at low-level AAF in the dysplastic tissue. After repeated resections and eventual complete hemispherectomy, both patients manifested intractable seizures arising from the contralateral, seemingly healthy hemisphere. In patient 3, the p.Glu17Lys AKT1 mutation exhibited random distribution and AAF rates in different tissues with double levels in the more severely dysplastic cerebral hemisphere. CONCLUSIONS: Our understanding of the distribution of somatic mutations in the brain in relation to the type of malformation and its hypothesized time of origin may be faulty. Large studies may reveal that the risk of a first surgery being disappointing might be related more to the specific somatic mammalian target of rapamycin mutation identified than to completeness of resection and that the advantages of repeated resections after a first unsuccessful operation should be weighed against the risk of the contralateral hemisphere becoming in turn epileptogenic.

Expanding Phenotype of Schimke Immuno-Osseous Dysplasia: Congenital Anomalies of the Kidneys and of the Urinary Tract and Alteration of NK Cells.

Schimke immuno-osseous dysplasia (SIOD) is a rare multisystemic disorder with a variable clinical expressivity caused by biallelic variants in SMARCAL1. A phenotype-genotype correlation has been attempted and variable expressivity of biallelic SMARCAL1 variants may be associated with environmental and genetic disturbances of gene expression. We describe two siblings born from consanguineous parents with a diagnosis of SIOD revealed by whole exome sequencing (WES). Results: A homozygous missense variant in the SMARCAL1 gene (c.1682G>A; p.Arg561His) was identified in both patients. Despite carrying the same variant, the two patients showed substantial renal and immunological phenotypic differences. We describe features not previously associated with SIOD-both patients had congenital anomalies of the kidneys and of the urinary tract and one of them succumbed to a classical type congenital mesoblastic nephroma. We performed an extensive characterization of the immunophenotype showing combined immunodeficiency characterized by a profound lymphopenia, lack of thymic output, defective IL-7Rα expression, and disturbed B plasma cells differentiation and immunoglobulin production in addition to an altered NK-cell phenotype and function. Conclusions: Overall, our results contribute to extending the phenotypic spectrum of features associated with SMARCAL1 mutations and to better characterizing the underlying immunologic disorder with critical implications for therapeutic and management strategies.

Accurate Detection of Hot-Spot MTOR Somatic Mutations in Archival Surgical Specimens of Focal Cortical Dysplasia by Molecular Inversion Probes.

BACKGROUND: Formalin-fixed, paraffin-embedded brain specimens are a potentially rich resource to identify somatic variants, but their DNA is characterised by low yield and extensive degradation, and matched peripheral samples are usually unavailable for analysis. METHODS: We designed single-molecule molecular inversion probes to target 18 MTOR somatic mutational hot-spots in unmatched, histologically proven focal cortical dysplasias from formalin-fixed, paraffin-embedded tissues of 50 patients. RESULTS: We achieved adequate DNA and sequencing quality in 28 focal cortical dysplasias, mostly extracted within 2 years from fixation, showing a statistically significant effect of time from fixation as a major determinant for successful genetic analysis. We identified and validated seven encompassing hot-spot residues (found in 14% of all patients and in 25% of those sequenced and analysed). The allele fraction had a range of 2-5% and variants were absent in available neighbouring non-focal cortical dysplasia specimens. We computed an alternate allele threshold for calling true variants, based on an experiment-wise mismatch count distribution, well predicting call reliability. CONCLUSIONS: Single-molecule molecular inversion probes are experimentally simple, cost effective and scalable, accurately detecting clinically relevant somatic variants in challenging brain formalin-fixed, paraffin-embedded tissues.