KMT2D regulates activation, localization, and integrin expression by T-cells.

Individuals with Kabuki syndrome present with immunodeficiency; however, how pathogenic variants in the gene encoding the histone-modifying enzyme lysine methyltransferase 2D (KMT2D) lead to immune alterations remain poorly understood. Following up on our prior report of KMT2D-altered integrin expression in B-cells, we performed targeted analyses of KMT2D's influence on integrin expression in T-cells throughout development (thymocytes through peripheral T-cells) in murine cells with constitutive- and conditional-targeted Kmt2d deletion. Using high-throughput RNA-sequencing and flow cytometry, we reveal decreased expression (both at the transcriptional and translational levels) of a cluster of leukocyte-specific integrins, which perturb aspects of T-cell activation, maturation, adhesion/localization, and effector function. H3K4me3 ChIP-PCR suggests that these evolutionary similar integrins are under direct control of KMT2D. KMT2D loss also alters multiple downstream programming/signaling pathways, including integrin-based localization, which can influence T-cell populations. We further demonstrated that KMT2D deficiency is associated with the accumulation of murine CD8+ single-positive (SP) thymocytes and shifts in both human and murine peripheral T-cell populations, including the reduction of the CD4+ recent thymic emigrant (RTE) population. Together, these data show that the targeted loss of Kmt2d in the T-cell lineage recapitulates several distinct features of Kabuki syndrome-associated immune deficiency and implicates epigenetic mechanisms in the regulation of integrin signaling.

[Clinical and genetic characteristics of four children with Kabuki syndrome due to de novo variants of KMT2D gene].

OBJECTIVE: To explore the clinical and genetic characteristics of four children with Kabuki syndrome (KS) due to variants of KMT2D gene. METHODS: Four children with KS diagnosed at the Children's Hospital of Shanxi Province between January 2020 and December 2022 were selected as the study subjects. Whole exome sequencing was carried out for the children and their family members. Candidate variants were verified by Sanger sequencing and pathogenicity analysis. RESULTS: The KS phenotype scores for the four children were 7, 8, 6, and 6, respectively. Child 2 also presented with a rare solitary kidney malformation. Genetic testing revealed that all children had harbored novel de novo variants of the KMT2D gene, including c.16472_16473del, c.858dup, c.11899C>T, and c.12844C>T, respectively. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), all of the variants were classified as pathogenic. CONCLUSION: For children showing phenotypes such as distinctive facial features, intellectual disability, developmental delay, cardiac abnormalities, and urinary system anomalies, KS should be considered. Early diagnosis and intervention can be achieved through genetic testing, especially in the presence of KMT2D gene mutations.

Autoimmune cytopenia and Kabuki syndrome in paediatrics: Insights in 11 patients.

Kabuki syndrome (KS) is now listed in the Human Inborn Errors of Immunity (IEI) Classification. It is a rare disease caused by KMT2D and KDM6A variants, dominated by intellectual disability and characteristic facial features. Recurrently, pathogenic variants are identified in those genes in patients examined for autoimmune cytopenia (AIC), but interpretation remains challenging. This study aims to describe the genetic diagnosis and the clinical management of patients with paediatric-onset AIC and KS. Among 11 patients with AIC and KS, all had chronic immune thrombocytopenic purpura, and seven had Evans syndrome. All had other associated immunopathological manifestations, mainly symptomatic hypogammaglobinaemia. They had a median of 8 (5-10) KS-associated manifestations. Pathogenic variants were detected in KMT2D gene without clustering, during the immunological work-up of AIC in three cases, and the clinical strategy to validate them is emphasized. Eight patients received second-line treatments, mainly rituximab and mycophenolate mofetil. With a median follow-up of 17 (2-31) years, 8/10 alive patients still needed treatment for AIC. First-line paediatricians should be able to recognize and confirm KS in children with ITP or multiple AIC, to provide early appropriate clinical management and specific long-term follow-up. The epigenetic immune dysregulation in KS opens exciting new perspectives.

Structural polymorphism of the nucleic acids in pentanucleotide repeats associated with the neurological disorder CANVAS.

Short tandem repeats are inherently unstable during DNA replication depending on repeat length, and the expansion of the repeat length in the human genome is responsible for repeat expansion disorders. Pentanucleotide AAGGG and ACAGG repeat expansions in intron 2 of the gene encoding replication factor C subunit 1 (RFC1) cause cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and other phenotypes of late-onset cerebellar ataxia. Herein, we reveal the structural polymorphism of the RFC1 repeats associated with CANVAS in vitro. Single-stranded AAGGG repeat DNA formed a hybrid-type G-quadruplex, whereas its RNA formed a parallel-type G-quadruplex with three layers. The RNA of the ACAGG repeat formed hairpin structure comprising C-G and G-C base pairs with A:A and GA:AG mismatched repeats. Furthermore, both pathogenic repeat RNAs formed more rigid structures than those of the nonpathogenic repeat RNAs. These findings provide novel insights into the structural polymorphism of the RFC1 repeats, which may be closely related to the disease mechanism of CANVAS.

Macular dystrophy in Kabuki syndrome due to de novo KMT2D variants: refining the phenotype with multimodal imaging and follow-up over 10 years: insight into pathophysiology.

BACKGROUND: Kabuki Syndrome is a rare and genetically heterogenous condition with both ophthalmic and systemic complications and typical facial features. We detail the macular phenotype in two unrelated patients with Kabuki syndrome due to de novo nonsense variants in KMT2D, one novel. A follow-up of 10 years is reported. Pathogenicity of both de novo nonsense variants is analyzed. METHODS: Four eyes of two young patients were studied by full clinical examination, kinetic perimetry, short wavelength autofluorescence, full field (ff) ERGs, and spectral-domain optical coherence tomography (SD-OCT). One patient had adaptive optic (AO) imaging. Whole exome sequencing was performed in both patients. RESULTS: Both patients had de novo nonsense variants in KMTD2. One patient had c.14843C>G; p. (Ser4948ter) novel variant and the second c.11119C>T; p. (Arg3707ter). Both had a stable Snellen visual acuity of 0.2-0.3. The retinal multimodal imaging demonstrated abnormalities at the fovea in both eyes: hyperreflectivity to blue light and a well-delimited gap-disruption of ellipsoid and interdigitation layer on OCT. The dark area on AO imaging is presumed to be absent for, or with structural change to photoreceptors. The ff ERGs and kinetic visual fields were normal. The foveal findings remained stable over several years. CONCLUSION: Kabuki syndrome-related maculopathy is a distinct loss of photoreceptors at the fovea as shown by multimodal imaging including, for the first time, AO imaging. This report adds to the literature of only one case with maculopathy with two additional macular dystrophies in patients with Kabuki syndrome. Although underestimated, these cases further raise awareness of the potential impact of retinal manifestations of Kabuki syndrome not only among ophthalmologists but also other healthcare professionals involved in the care of patients with this multisystem disorder.

Craniosynostosis in molecularly diagnosed Kabuki syndrome: Prevalence and clinical implications.

Kabuki syndrome (KS) is characterized by growth impairment, psychomotor delay, congenital heart disease, and distinctive facial features. KMT2D and KDM6A have been identified as the causative genes of KS. Craniosynostosis (CS) has been reported in individuals with KS; however, its prevalence and clinical implications remain unclear. In this retrospective study, we investigated the occurrence of CS in individuals with genetically diagnosed KS and examined its clinical significance. Among 42 individuals with genetically diagnosed KS, 21 (50%) exhibited CS, with 10 individuals requiring cranioplasty. No significant differences were observed based on sex, causative gene, and molecular consequence among individuals with KS who exhibited CS. Both individuals who underwent evaluation with three-dimensional computed tomography (3DCT) and those who required surgery tended to exhibit cranial dysmorphology. Notably, in several individuals, CS was diagnosed before KS, suggesting that CS could be one of the clinical features by which clinicians can diagnose KS. This study highlights that CS is one of the noteworthy complications in KS, emphasizing the importance of monitoring cranial deformities in the health management of individuals with KS. The findings suggest that in individuals where CS is a concern, conducting 3DCT evaluations for CS and digital impressions are crucial.

Neonatal Kabuki syndrome caused by KMT2D mutation: A case report.

BACKGROUND: Kabuki syndrome (KS) is an autosomal dominant inherited syndrome that involves multiple organs and systems. Gene mutation is the main cause of KS. The reported mutations in X-linked histone H3 lysine 4 methylase (KMT2D) and KDM6A genes are 2 relatively clear pathogenic pathways. In this paper, we report a case of KS with neonatal hypoglycemia and special features caused by KMT2D gene mutation confirmed by whole exome sequencing, it enriched the clinical phenotype spectrum and gene mutation spectrum of KS, which helps to improve the understanding of the disease. CASE REPORT: Through whole exome sequencing, we performed gene diagnosis of a newborn child with special facial features and multiple malformations, which revealed heterozygous mutation of NM_003482.3:c.755dupA(p.His252Glnfs*21) in KMT2D gene. It is consistent with the pathogenesis of KS, an autosomal dominat genetic disease caused by KMT2D gene mutation. This pathogenic mutation has not been prebiously reported. DISCUSSION: KS has strong clinical characteristics and biological heterogeneity. Genetic diagnosis can help identify mutant gene types. However, the relationship between genotype and phenotype has not been fully clarified. The molecular etiological mechanism still needs to be further explored and elucidated.

Normal and pathogenic variation of RFC1 repeat expansions: implications for clinical diagnosis.

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) is an autosomal recessive neurodegenerative disease, usually caused by biallelic AAGGG repeat expansions in RFC1. In this study, we leveraged whole genome sequencing data from nearly 10 000 individuals recruited within the Genomics England sequencing project to investigate the normal and pathogenic variation of the RFC1 repeat. We identified three novel repeat motifs, AGGGC (n = 6 from five families), AAGGC (n = 2 from one family) and AGAGG (n = 1), associated with CANVAS in the homozygous or compound heterozygous state with the common pathogenic AAGGG expansion. While AAAAG, AAAGGG and AAGAG expansions appear to be benign, we revealed a pathogenic role for large AAAGG repeat configuration expansions (n = 5). Long-read sequencing was used to characterize the entire repeat sequence, and six patients exhibited a pure AGGGC expansion, while the other patients presented complex motifs with AAGGG or AAAGG interruptions. All pathogenic motifs appeared to have arisen from a common haplotype and were predicted to form highly stable G quadruplexes, which have previously been demonstrated to affect gene transcription in other conditions. The assessment of these novel configurations is warranted in CANVAS patients with negative or inconclusive genetic testing. Particular attention should be paid to carriers of compound AAGGG/AAAGG expansions when the AAAGG motif is very large (>500 repeats) or the AAGGG motif is interrupted. Accurate sizing and full sequencing of the satellite repeat with long-read sequencing is recommended in clinically selected cases to enable accurate molecular diagnosis and counsel patients and their families.

Frequency and Phenotype of RFC1 Repeat Expansions in Bilateral Vestibulopathy.

BACKGROUND AND OBJECTIVES: Bilateral vestibulopathy (BVP) is a chronic debilitating neurologic disorder with no monogenic cause established so far despite familiar presentations. We hypothesized that replication factor complex subunit 1 (RFC1) repeat expansions might present a recurrent monogenic cause of BVP. METHODS: The study involved RFC1 screening and in-depth neurologic, vestibulo-oculomotor, and disease evolution phenotyping of 168 consecutive patients with idiopathic at least "probable BVP" from a tertiary referral center for balance disorders, with127 of them meeting current diagnostic criteria of BVP (Bárány Society Classification). RESULTS: Biallelic AAGGG repeat expansions in RFC1 were identified in 10/127 patients (8%) with BVP and 1/41 with probable BVP. Heterozygous expansions in 10/127 patients were enriched compared with those in reference populations. RFC1-related BVP manifested at a median age of 60 years (range 34-72 years) and co-occurred predominantly with mild polyneuropathy (10/11). Additional cerebellar involvement (7/11) was subtle and limited to oculomotor signs in early stages, below recognition of classic cerebellar ataxia, neuropathy, and vestibular areflexia syndrome. Clear dysarthria, appendicular ataxia, or cerebellar atrophy developed 6-8 years after onset. Dysarthria, absent patellar reflexes, and downbeat nystagmus best discriminated RFC1-positive BVP from RFC1-negative BVP, but neither sensory symptoms nor fine motor problems. Video head impulse gains of patients with RFC1-positive BVP were lower relative to those of patients with RFC1-negative BVP and decreased until 10 years disease duration, indicating a potential progression and outcome marker for RFC1-disease. DISCUSSION: This study identifies RFC1 as the first-and frequent-monogenic cause of BVP. It characterizes RFC1-related BVP as part of the multisystemic evolution of RFC1 spectrum disease, with implications for designing natural history studies and future treatment trials. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that RFC1 repeat expansions cause BVP.

Identification of a KDM6A somatic mutation responsible for Kabuki syndrome by excluding a conflicting KMT2D germline variant through episignature analysis.

Kabuki syndrome (KS) is a congenital disorder caused by mutations in either KMT2D on chromosome 12 or KDM6A on chromosome X, encoding a lysine methyltransferase and a lysine demethylase, respectively. A 9-year-4-month-old male patient with a normal karyotype presented with KS and autism spectrum disorder. Genetic testing for KS was conducted by Sanger sequencing and episignature analysis using DNA methylation array data. The patient had a mosaic stop-gain variant in KDM6A and a heterozygous missense variant (rs201078160) in KMT2D. The KDM6A variant is expected to be deleterious. The KMT2D variant pathogenicity has been inconsistently reported in the ClinVar database. Using biobanking resources, we identified two heterozygous individuals possessing the rs201078160 variant. In a subsequent episignature analysis, the KS patient showed the KS episignature, but two control individuals with the rs201078160 variant did not. Our results indicate that the mosaic stop-gained variant in KDM6A, but not the rs201078160 variant in KMT2D, is responsible for the KS phenotype in the patient. This study further demonstrated the utility of DNA methylation information in diagnosing rare genetic diseases and emphasized the importance of a reference dataset containing both genotype and DNA methylation information.

Cerebellar ataxia-neuropathy-vestibular areflexia-syndrome.

CANVAS including its clinical components of cerebellar ataxia, sensory neuropathy and vestibular areflexia is presented in this review. An intronic biallelic pentanucleotide expansion in RFC1 is the genetic cause of CANVAS. Several patients diagnosed with isolated "idiopathic" neurological or otological conditions might have a CANVAS spectrum disorder. The number of CANVAS patients may well increase considerably in the near future, making it important to consider the diagnostic set-up and infrastructure for counselling, treatment and follow-up in the Danish healthcare system.

Characterizing the molecular impact of KMT2D variants on the epigenetic and transcriptional landscapes in Kabuki syndrome.

Kabuki syndrome (KS) is a rare, multisystem disorder with a variable clinical phenotype. The majority of KS is caused by dominant loss-of-function mutations in KMT2D (lysine methyltransferase 2D). KMT2D mediates chromatin accessibility by adding methyl groups to lysine residue 4 of histone 3, which plays a critical role in cell differentiation and homeostasis. The molecular underpinnings of KS remain elusive partly because of a lack of histone modification data from human samples. Consequently, we profiled and characterized alterations in histone modification and gene transcription in peripheral blood mononuclear cells (PBMCs) from 33 patients with KMT2D mutations and 36 unaffected healthy controls. Our analysis identified unique enhancer signatures in H3K4me1 and H3K4me2 in KS compared with controls. Reduced enhancer signals were present for promoter-distal sites of immune-related genes for which co-binding of PBMC-specific transcription factors was predicted; 31% of super-enhancers of normal blood cells overlapped with disrupted enhancers in KS, supporting an association of reduced enhancer activity of immune-related genes with immune deficiency phenotypes. In contrast, increased enhancer signals were observed for promoter-proximal regions of metabolic genes enriched with EGR1 and E2F2 motifs, whose transcriptional levels were significantly increased in KS. Additionally, we identified ~100 de novo enhancers in genes, such as in MYO1F and AGAP2. Together, our results underscore the effect of KMT2D haploinsufficiency on dysregulation of enhancer states and gene transcription and provide a framework for the identification of therapeutic targets and biomarkers in preparation for clinical trial readiness.

Insights into the genotype-phenotype relationship of ocular manifestations in Kabuki syndrome.

We aim to assess if genotype-phenotype correlations are present within ocular manifestations of Kabuki syndrome (KS) among a large multicenter cohort. We conducted a retrospective, medical record review including clinical history and comprehensive ophthalmological examinations of a total of 47 individuals with molecularly confirmed KS and ocular manifestations at Boston Children's Hospital and Cincinnati Children's Hospital Medical Center. We assessed information regarding ocular structural, functional, and adnexal elements as well as pertinent associated phenotypic features associated with KS. For both type 1 KS (KS1) and type 2 KS (KS2), we observed more severe eye pathology in nonsense variants towards the C-terminus of each gene, KMT2D and KDM6A, respectively. Furthermore, frameshift variants appeared to be not associated with structural ocular elements. Between both types of KS, ocular structural elements were more frequently identified in KS1 compared with KS2, which only involved the optic disc in our cohort. These results reinforce the need for a comprehensive ophthalmologic exam upon diagnosis of KS and regular follow-up exams. The specific genotype may allow risk stratification of the severity of the ophthalmologic manifestation. However, additional studies involving larger cohorts are needed to replicate our observations and conduct powered analyses to more formally risk-stratify based on genotype, highlighting the importance of multicenter collaborations in rare disease research.

Insights into the perinatal phenotype of Kabuki syndrome in infants identified by genome-wide sequencing.

Increasing use of unbiased genomic sequencing in critically ill infants can expand understanding of rare diseases such as Kabuki syndrome (KS). Infants diagnosed with KS through genome-wide sequencing performed during the initial hospitalization underwent retrospective review of medical records. Human phenotype ontology terms used in genomic analysis were aggregated and analyzed. Clinicians were surveyed regarding changes in management and other care changes. Fifteen infants met inclusion criteria. KS was not suspected prior to genomic sequencing. Variants were classified as Pathogenic (n = 10) or Likely Pathogenic (n = 5) by American College of Medical Genetics and Genomics Guidelines. Fourteen variants were de novo (KMT2D, n = 12, KDM6A, n = 2). One infant inherited a likely pathogenic variant in KMT2D from an affected father. Frequent findings involved cardiovascular (14/15) and renal (7/15) systems, with palatal defects also identified (6/15). Three infants had non-immune hydrops. No minor anomalies were universally documented; ear anomalies, micrognathia, redundant nuchal skin, and hypoplastic nails were common. Changes in management were reported in 14 infants. Early use of unbiased genome-wide sequencing enabled a molecular diagnosis prior to clinical recognition including infants with atypical or rarely reported features of KS while also expanding the phenotypic spectrum of this rare disorder.

DNA methylation signature classification of rare disorders using publicly available methylation data.

Disease-specific DNA methylation patterns (DNAm signatures) have been established for an increasing number of genetic disorders and represent a valuable tool for classification of genetic variants of uncertain significance (VUS). Sample size and batch effects are critical issues for establishing DNAm signatures, but their impact on the sensitivity and specificity of an already established DNAm signature has not previously been tested. Here, we assessed whether publicly available DNAm data can be employed to generate a binary machine learning classifier for VUS classification, and used variants in KMT2D, the gene associated with Kabuki syndrome, together with an existing DNAm signature as proof-of-concept. Using publicly available methylation data for training, a classifier for KMT2D variants was generated, and individuals with molecularly confirmed Kabuki syndrome and unaffected individuals could be correctly classified. The present study documents the clinical utility of a robust DNAm signature even for few affected individuals, and most importantly, underlines the importance of data sharing for improved diagnosis of rare genetic disorders.

Truncating Variants in RFC1 in Cerebellar Ataxia, Neuropathy, and Vestibular Areflexia Syndrome.

BACKGROUND AND OBJECTIVE: Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is an autosomal recessive neurodegenerative disease characterized by adult-onset and slowly progressive sensory neuropathy, cerebellar dysfunction, and vestibular impairment. In most cases, the disease is caused by biallelic (AAGGG)n repeat expansions in the second intron of the replication factor complex subunit 1 (RFC1). However, a small number of cases with typical CANVAS do not carry the common biallelic repeat expansion. The objective of this study was to expand the genotypic spectrum of CANVAS by identifying sequence variants in RFC1-coding region associated with this condition. METHODS: Fifteen individuals diagnosed with CANVAS and carrying only 1 heterozygous (AAGGG)n expansion in RFC1 underwent whole-genome sequencing or whole-exome sequencing to test for the presence of a second variant in RFC1 or other unrelated gene. To assess the effect of truncating variants on RFC1 expression, we tested the level of RFC1 transcript and protein on patients' derived cell lines. RESULTS: We identified 7 patients from 5 unrelated families with clinically defined CANVAS carrying a heterozygous (AAGGG)n expansion together with a second truncating variant in trans in RFC1, which included the following: c.1267C>T (p.Arg423Ter), c.1739_1740del (p.Lys580SerfsTer9), c.2191del (p.Gly731GlufsTer6), and c.2876del (p.Pro959GlnfsTer24). Patient fibroblasts containing the c.1267C>T (p.Arg423Ter) or c.2876del (p.Pro959GlnfsTer24) variants demonstrated nonsense-mediated mRNA decay and reduced RFC1 transcript and protein. DISCUSSION: Our report expands the genotype spectrum of RFC1 disease. Full RFC1 sequencing is recommended in cases affected by typical CANVAS and carrying monoallelic (AAGGG)n expansions. In addition, it sheds further light on the pathogenesis of RFC1 CANVAS because it supports the existence of a loss-of-function mechanism underlying this complex neurodegenerative condition.

New Cerebellar Ataxia, Neuropathy, Vestibular Areflexia Syndrome cases are caused by the presence of a nonsense variant in compound heterozygosity with the pathogenic repeat expansion in the RFC1 gene.

The biallelic pathogenic repeat (AAGGG)400-2000 intronic expansion in the RFC1 gene has been recently described as the cause of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and as a major cause of late-onset ataxia. Since then, many heterozygous carriers have been identified, with an estimated allele frequency of 0.7% to 4% in the healthy population. Here, we describe in two affected CANVAS sisters the presence of the nonsense c.724C > T p.(Arg242*) variant in compound heterozygosity with the pathogenic repeat expansion in the RFC1 gene. Further RNA analysis demonstrated a reduced expression of the p.Arg242* allele in patients confirming an efficient nonsense-mediated mRNA decay. We also highlight the importance of considering the sequencing of the RFC1 gene for the diagnosis, especially in patients with CANVAS diagnosis carriers of the AAGGG repeat expansion.

Four cases of audio-vestibular disorders related to immunisation with SARS-CoV-2 mRNA vaccines.

OBJECTIVE: To gain medical insight into the clinical course and safety of otolaryngologic disorders following immunisation with severe acute respiratory coronavirus (SARS-CoV-2) mRNA-based vaccines. DESIGN: Case description. STUDY SAMPLE: We report four cases of transient audio-vestibular symptoms, which occurred shortly after inoculation of two BNT162b2 (Pfizer-BioNTech®) and mRNA-1273 (Moderna®) vaccines. RESULTS: Hearing loss was unilateral in all cases and recovered at least partially: it was associated with persistent gait instability in two cases, after 1 and 7 months. Trigger mechanisms underpinning audio-vestibular impairment remain uncertain. Immune tolerance mechanisms with off-target innate activation of T-lymphocytes may be involved in vestibulocochlear nerve disorders, as for other cranial nerves involvement. CONCLUSIONS: The occurrence of audio-vestibular manifestations following mRNA-based vaccines needs ENT monitoring to support their causality in such rare vaccine-related adverse events. Audio-vestibular disorders appeared of transitory nature, including hearing loss, and should not deter further efforts in large-scale vaccination campaigns against SARS-CoV-2.