DNA Methylation in the Diagnosis of Monogenic Diseases.

DNA methylation in the human genome is largely programmed and shaped by transcription factor binding and interaction between DNA methyltransferases and histone marks during gamete and embryo development. Normal methylation profiles can be modified at single or multiple loci, more frequently as consequences of genetic variants acting in cis or in trans, or in some cases stochastically or through interaction with environmental factors. For many developmental disorders, specific methylation patterns or signatures can be detected in blood DNA. The recent use of high-throughput assays investigating the whole genome has largely increased the number of diseases for which DNA methylation analysis provides information for their diagnosis. Here, we review the methylation abnormalities that have been associated with mono/oligogenic diseases, their relationship with genotype and phenotype and relevance for diagnosis, as well as the limitations in their use and interpretation of results

A restricted spectrum of missense KMT2D variants cause a multiple malformations disorder distinct from Kabuki syndrome

Purpose: To investigate if specific exon 38 or 39 KMT2D missense variants (MVs) cause a condition distinct from Kabuki syndrome type 1 (KS1). Methods: Multiple individuals, with MVs in exons 38 or 39 of KMT2D that encode a highly conserved region of 54 amino acids flanked by Val3527 and Lys3583, were identified and phenotyped. Functional tests were performed to study their pathogenicity and understand the disease mechanism. Results: The consistent clinical features of the affected individuals, from seven unrelated families, included choanal atresia, athelia or hypoplastic nipples, branchial sinus abnormalities, neck pits, lacrimal duct anomalies, hearing loss, external ear malformations, and thyroid abnormalities. None of the individuals had intellectual disability. The frequency of clinical features, objective software-based facial analysis metrics, and genome-wide peripheral blood DNA methylation patterns in these patients were significantly different from that of KS1. Circular dichroism spectroscopy indicated that these MVs perturb KMT2D secondary structure through an increased disordered to ɑ-helical transition. Conclusion: KMT2D MVs located in a specific region spanning exons 38 and 39 and affecting highly conserved residues cause a novel multiple malformations syndrome distinct from KS1. Unlike KMT2D haploinsufficiency in KS1, these MVs likely result in disease through a dominant negative mechanism.This article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site.16-17/10/Newlife - The Charity for Disabled Children FS/13/32/30069/BHF_/British Heart Foundation/United Kingdom 72160007/Chile's National Commission for Scientific and Technological Research MR/K011154/1/MRC_/Medical Research Council/United Kingdom WT_/Wellcome Trust/United Kingdompre-prin

Novel diagnostic DNA methylation episignatures expand and refine the epigenetic landscapes of Mendelian disorders.

Overlapping clinical phenotypes and an expanding breadth and complexity of genomic associations are a growing challenge in the diagnosis and clinical management of Mendelian disorders. The functional consequences and clinical impacts of genomic variation may involve unique, disorder-specific, genomic DNA methylation episignatures. In this study, we describe 19 novel episignature disorders and compare the findings alongside 38 previously established episignatures for a total of 57 episignatures associated with 65 genetic syndromes. We demonstrate increasing resolution and specificity ranging from protein complex, gene, sub-gene, protein domain, and even single nucleotide-level Mendelian episignatures. We show the power of multiclass modeling to develop highly accurate and disease-specific diagnostic classifiers. This study significantly expands the number and spectrum of disorders with detectable DNA methylation episignatures, improves the clinical diagnostic capabilities through the resolution of unsolved cases and the reclassification of variants of unknown clinical significance, and provides further insight into the molecular etiology of Mendelian conditions

A NGS-Targeted Autism/ID Panel Reveals Compound Heterozygous GNB5 Variants in a Novel Patient

Homozygous and compound heterozygous pathogenic variants in GNB5 have been recently associated with a spectrum of clinical presentations varying from a severe multisystem form of the disorder including intellectual disability, early infantile developmental and epileptic encephalopathy, retinal abnormalities and cardiac arrhythmias (IDDCA) to a milder form with language delay, attention-deficit/hyperactivity disorder, cognitive impairment, with or without cardiac arrhythmia (LADCI). Approximately twenty patients have been described so far; here we report a novel case of a 2.5-year-old female who is a compound heterozygote for a frameshift and a missense valiant in the GNB5 gene. Her clinical presentation is consistent with a moderate phenotype, corroborating the direct correlation between the type and pathogenic mechanism of the GNB5 genetic variant and the severity of related phenotype

TRIM50 regulates Beclin 1 proautophagic activity

Autophagy is a catabolic process needed for maintaining cell viability and homeostasis in response to numerous stress conditions. Emerging evidence indicates that the ubiquitin system has a major role in this process. TRIMs, an E3 ligase protein family, contribute to selective autophagy acting as receptors and regulators of the autophagy proteins recognizing endogenous or exogenous targets through intermediary autophagic tags, such as ubiquitin. Here we report that TRIM50 fosters the initiation phase of starvation-induced autophagy and associates with Beclin1, a central component of autophagy initiation complex. We show that TRIM50, via the RING domain, ubiquitinates Beclin 1 in a K63-dependent manner enhancing its binding with ULK1 and autophagy activity. Finally, we found that the Lys-372 residue of TRIM50, critical for its own acetylation, is necessary for its E3 ligase activity that governs Beclin1 ubiquitination. Our study expands the roles of TRIMs in regulating selective autophagy, revealing an acetylation-ubiquitination dependent control for autophagy modulation

Schilbach-Rott syndrome associated with 9q22.32q22.33 duplication, involving the PTCH1 gene

Schilbach-Rott syndrome (SRS, OMIM4220) is a disorder of unknown aetiology that is characterised by hypotelorism, epichantal folds, cleft palate, dysmorphic face, hypospadia in males and mild mental retardation in some patients. To date, 5 families and 17 patients have exhibited this phenotype, and recurrence in two of these families suggests an autosomal dominant inheritance. SRS overlaps with a mild form of holoprosencephaly (HPE), but array-CGH analysis and sequencing of some HPE-related genes (SEPT9, SHH and TWIST) did not reveal any variants in at least one family. Herein, we investigated by array-CGH analysis a 11-year-old female patient and her father, both exhibiting the typical SRS phenotype, disclosing in the daughter-father couple the same microduplication of chromosome 9q22.32q22.33 [arr[hg19]9q22.32(98,049,611_98,049,636)x3,9q22.33 (99,301,483_99,301,508)x3], involving eight genes, including PTCH1. The duplication segregated with the disease, since it was not found in the healthy paternal grandparents of the proband. The gain-of-function variants of the PTCH1 gene are responsible for a mild form of HPE. This is the first genetic variant found in SRS. This finding reinforces the hypothesis that SRS belongs to the HPE clinical spectrum and suggests to perform array-CGH in patients with SRS phenotype and, if negative, to consider a potential benefit from sequencing of HPE-related genes

Dissecting KMT2D missense mutations in Kabuki syndrome patients

Kabuki syndrome is a rare autosomal dominant condition characterized by facial features, various organs malformations, postnatal growth deficiency and intellectual disability. The discovery of frequent germline mutations in the histone methyltransferase KMT2D and the demethylase KDM6A revealed a causative role for histone modifiers in this disease. However, the role of missense mutations has remained unexplored. Here, we expanded the mutation spectrum of KMT2D and KDM6A in KS by identifying 37 new KMT2D sequence variants. Moreover, we functionally dissected 14 KMT2D missense variants, by investigating their impact on the protein enzymatic activity and the binding to members of the WRAD complex. We demonstrate impaired H3K4 methyltransferase activity in 9 of the 14 mutant alleles and show that this reduced activity is due in part to disruption of protein complex formation. These findings have relevant implications for diagnostic and counseling purposes in this disease

Clinical and Neurobehavioral Features of Three Novel Kabuki Syndrome Patients with Mosaic KMT2D Mutations and a Review of Literature

Kabuki syndrome (KS) is a rare disorder characterized by multiple congenital anomalies and variable intellectual disability caused by mutations in KMT2D/MLL2 and KDM6A/UTX, two interacting chromatin modifier responsible respectively for 56-75% and 5-8% of the cases. To date, three KS patients with mosaic KMT2D deletions in blood lymphocytes have been described. We report on three additional subjects displaying KMT2D gene mosaics including one in which a single nucleotide change results in a new frameshift mutation (p.L1199HfsX7), and two with already-known nonsense mutations (p.R4484X and p.R5021X). Consistent with previously published cases, mosaic KMT2D mutations may result in mild KS facial dysmorphisms and clinical and neurobehavioral features, suggesting that these characteristics could represent the handles for genetic testing of individuals with slight KS-like traits