Genetic analysis of childhood-onset dystonia-28 caused by a variant (c.5076G>A) in the KMT2B gene

Abstract

Objective

To investigate the clinical manifestations and genetic basis of childhood-onset dystonia-28 (DYT28) associated with a variant in the KMT2B gene.

Methods

A female patient, her mother, and her husband, who presented at Zhejiang Provincial People’s Hospital in November 2024, were enrolled in this study. Clinical data were collected, and trio-based whole-exome sequencing (WES) was performed using peripheral blood samples obtained from all three individuals. To further evaluate the effect of the identified variant on RNA splicing, mRNA sequencing (mRNA-seq) and Sanger sequencing were subsequently performed. This study was approved by the Medical Ethics Committee of Zhejiang Provincial People’s Hospital (Approval No. QT2026084).

Results

The patient presented with gait abnormalities beginning after 10 years of age, accompanied by impaired mathematical ability and language communication difficulties. Her mother exhibited similar clinical manifestations, including gait disturbance, language impairment, and a history of scoliosis. WES identified a heterozygous synonymous variant in exon 23 of the KMT2B gene, c.5076 G>A (p.Lys1692 =), in both the patient and her mother. mRNA-seq analysis demonstrated that the variant produced two aberrantly spliced transcripts: complete skipping of exon 23 and retention of an 18-bp sequence derived from the 5′ end of intron 23. According to the guidelines of the American College of Medical Genetics and Genomics (ACMG), the variant was classified as likely pathogenic based on the following evidence: PVS1_Strong, PP4 and PM2_Supporting.

Conclusion

The KMT2B c.5076 G>A (p.Lys1692 =) variant is likely the genetic cause of childhood-onset dystonia-28 in this patient. This study expands the mutational spectrum of KMT2B and provides additional evidence to support the genetic diagnosis and counseling of patients with KMT2B-related dystonia.

Introduction

Childhood-onset dystonia-28 (DYT28) is an autosomal dominant neurological disorder primarily caused by pathogenic variants in the KMT2B gene [1]. The disorder is typically characterized by progressive dystonia with onset during childhood, most commonly beginning in the lower limbs and resulting in gait abnormalities. As the disease progresses, dystonia gradually extends to the upper limbs, neck, and facial muscles. Affected individuals frequently exhibit varying degrees of motor developmental delay and/or cognitive impairment [2]. The KMT2B gene encodes a lysine methyltransferase involved in histone modification and transcriptional regulation. This protein plays an essential role in multiple cellular processes, including cell-cycle progression, cellular differentiation, and development. Pathogenic variants in KMT2B have been associated with a broad spectrum of neurodevelopmental disorders, particularly dystonia and other movement disorders [3]. To date, reported pathogenic variants in KMT2B include nonsense, frameshift, missense, and splice-site variants [4]. Although synonymous variants do not alter the encoded amino acid sequence, variants located at or near splice sites or within splicing regulatory regions may disrupt normal RNA splicing and result in aberrant transcripts [5].

In the present study, we performed genetic analyses in a patient presenting with gait abnormalities, impaired mathematical ability, and dysarthria, as well as in her mother and her husband. We identified a novel synonymous variant in the KMT2B gene, c.5076 G>A. Subsequent mRNA sequencing (mRNA-seq) demonstrated that this variant disrupts normal splicing, thereby suggesting a potential pathogenic mechanism. This study enhances clinical understanding of the association between KMT2B variants and DYT28, and provides additional evidence supporting the molecular diagnosis of this disorder.

Materials and Methods

Samples

A patient presenting with gait abnormalities, impaired mathematical ability, and dysarthria, who attended Zhejiang Provincial People’s Hospital on November 11, 2024, was enrolled in this study. Peripheral blood samples were collected from the patient, her mother, and her husband for WES analysis. This study was approved by the Medical Ethics Committee of Zhejiang Provincial People’s Hospital (Approval No. QT2026084), and written informed consent was obtained from all participants.

Methods

DNA extraction

Peripheral blood samples (2 mL each) were collected from the patient, her husband, and her mother in ethylenediaminetetraacetic-anticoagulated tubes. Genomic DNA was extracted using a tissue/blood genomic DNA extraction kit (Kaishuo Biotech, China) according to the manufacturer’s instructions. DNA concentration and purity were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). The extracted DNA samples were stored at −80°C until further analysis.

Whole exome sequencing (WES)

Genomic DNA obtained from the patient and her parents was fragmented, followed by end repair, adapter ligation, polymerase chain reaction amplification, and purification. DNA libraries were prepared using the Freyseq Clinical Exome Capture Kit and sequenced on the Illumina NextSeq 550DX platform. Sequencing targeted the exonic regions of 7099 clinically relevant genes and their flanking intronic regions (±20 bp). Raw sequencing data underwent quality-control processing and were aligned to the human reference genome hg19 using the Burrows-Wheeler Aligner (BWA). Variant annotation and filtering were performed using multiple public databases, including dbSNP, ExAC, Human Gene Mutation Database (HGMD), ClinVar, OMIM, and gnomAD, to identify candidate variants, including single nucleotide variants and insertions/deletions.

Sanger sequencing validation

Based on the WES findings, primers targeting the KMT2B c.5076 G>A variant were designed using Primer-BLAST. Sanger sequencing was subsequently performed, and ABI sequence files were generated. Sequence alignment and variant confirmation were conducted using SnapGene software against the reference to the corresponding GenBank sequence. The primer sequences were as follows: Forward primer: 5′-TCTTCCAGGATGACA-3′, Reverse primer: 5′-CGAGTCTATGTGGACT-3′.

mRNA-seq for splicing analysis

To investigate the effect of the KMT2B c.5076 G>A variant on mRNA splicing, total RNA was extracted from the peripheral blood of the patient. mRNA was enriched using oligo(dT) magnetic beads, followed by library construction for next-generation sequencing. Sequencing was performed on the Illumina NovaSeq 6000 platform. Raw sequencing data were processed using FastQC for quality assessment and aligned to the hg19 reference genome using BWA. The Integrative Genomics Viewer was used to identify abnormal splicing events near the variant site, with particular attention to exon skipping (SE), intron retention (RI), and other major splicing abnormalities.

Results

mRNA-seq analysis

WES was performed using genomic DNA extracted from peripheral blood samples obtained from the patient, her husband, and her mother. The average sequencing depth was 95.14 × , and 98.13% of the target regions achieved coverage greater than 20 × . The analysis identified a heterozygous c.5076 G>A variant in exon 23 of the KMT2B gene (NM_014727.3) in both the patient and her mother. To further investigate the functional impact of this variant, mRNA-seq was performed using RNA extracted from the peripheral blood of the patient. Compared with control samples, the KMT2B transcripts from the patient exhibited significant splicing abnormalities. Detailed analysis identified two major aberrant splicing events (Fig. 1). The first event was the complete skipping of exon 23 (Exon 23 skipping), which resulted in the complete exclusion of exon 23 (159 bp) from the mature mRNA transcript, and corresponded to the predicted protein alteration p.Arg1640_Lys1692del. The second event involved retention of 18 bp at the 5′ end of intron 23 (▽Intron23p_18bp retention): This abnormal splicing event was caused by activation of an aberrant 3′ splice site adjacent to exon 23, leading to retention of the first 18 nucleotides of intron 23 in the mature transcript. The resulting protein alteration corresponded to an in-frame insertion of six amino acids. Although the KMT2B c.5076 G>A variant is a synonymous substitution that does not alter the encoded amino acid sequence, mRNA-seq analysis clearly demonstrated the presence of abnormal transcript products associated with this variant. These findings indicate that mRNA-seq is a sensitive and effective approach for detecting aberrant transcripts caused by synonymous variants located near splice sites, highlighting its potential utility in clinical genetic diagnosis.

Fig. 1.

Results of mRNA-seq analysis. The patient's KMT2B gene has two abnormal spliced transcripts (the numbers between exons in the figure represent the number of reads supporting alternative splicing).

Genetic validation

Based on the findings of WES and mRNA-seq analyses, Sanger sequencing was performed for validation. Compared with control samples, the results confirmed that the KMT2B gene generated two distinct abnormal transcript products in the patient (Fig. 2). The splicing abnormalities caused by the c.5076 G>A variant are predicted to disrupt the fourth plant homeodomain (PHD) of the KMT2B gene, which may represent the primary molecular mechanism underlying the clinical phenotype of the patient.

Fig. 2.

Sanger sequencing verification diagram. There are two abnormal splicing variations in the transcript of the KMT2B gene in the patient.

Variant Pathogenicity Assessment

According to the ACMG guidelines for sequence variant interpretation, the KMT2B c.5076 G>A variant was classified as likely pathogenic (PVS1_Strong, PP4 and PM2_Supporting), based on the following evidence [6]: 1) This variant has not been previously reported in the HGMD; 2) PVS1_Strong: Although the variant is synonymous and located at the final nucleotide of exon 23, mRNA-seq functional analysis demonstrated that it causes aberrant splicing, including SE. This abnormal splicing event is predicted to disrupt the PHD4 functional domain and may result in loss of normal protein function through haploinsufficiency; 3) PP4: The patient’s clinical manifestations and family history were highly consistent with KMT2B-related disorders, and WES identified no other pathogenic or likely pathogenic variants; 4) PM2_Supporting: The variant is absent from population databases, including gnomAD v2.1.1.

Discussion

In this study, we report for the first time a case of a heterozygous KMT2B variant (c.5076 G>A, p.Lys1692 =) associated with DYT28. Through mRNA-seq analysis, we demonstrated that this synonymous variant produces two aberrantly spliced transcripts, namely exon 23 skipping and retention of 18 bp at the 5′ end of intron 23, thereby providing insight into its potential pathogenic mechanism.

KMT2B is widely expressed in human tissues and is particularly abundant in the brain, with the highest expression observed in the cerebellum [2]. Previous studies have shown that pathogenic variants in KMT2B can cause early-onset generalized dystonia with autosomal dominant inheritance. Dystonia is the predominant clinical manifestation of KMT2B-related disorders, with highly variable manifestations that may arise from infancy to late adulthood [2]. The disorder typically progresses over time, initially affecting in the lower limbs, and subsequently spreading to other body regions, including the upper limbs, neck, and cranial muscles. Additional neurological manifestations may include myoclonus, spasticity, and intellectual disability [7]. The clinical course of DYT28 is generally characterized by onset of dystonia during the first decade of life, although later onset may occur; initial involvement of the lower limbs presenting as toe walking, gait abnormalities, and impaired balance; progression to cervical, laryngeal, or cranial dystonia, manifesting as torticollis, dysarthria, dysphonia, and difficulties with swallowing and chewing and eventual progression to generalized dystonia within 2–10 years after disease onset [8]. In the present study, patient developed gait abnormalities after 10 years of age and had exhibited impaired mathematical ability since childhood, accompanied by dysarthria. Her mother also developed gait abnormalities at approximately 10 years of age over time, with progressive worsening, accompanied by lower limb pain during walking, scoliosis, and impaired mathematical ability, but without muscle atrophy. Although the age at onset in both individuals was slightly later than that typically reported for DYT28, their clinical manifestations were broadly consistent with KMT2B-related dystonia and were accompanied by mild intellectual impairment. However, because dystonia is clinically heterogeneous and relatively nonspecific, molecular analyses, including WES and mRNA-seq, were essential for establishing the genotype–phenotype correlation in these cases.

The KMT2B gene, located on chromosome 19q13.12, encodes a 2,715-amino-acid protein that functions as a lysine methyltransferase responsible for histone H3 lysine 4 (H3K4) methylation and plays a crucial role in the regulation of gene expression during normal development [1], [2]. The KMT2B protein contains multiple functional domains involved in chromatin targeting, histone modification recognition of histone modifications, and catalytic activity, including the AT-hook domain, CXXC zinc finger domain, four PHD fingers, bromodomain, FY-rich domain, and SET domain [9]. In the present study, WES identified a heterozygous c.5076 G>A variant in exon 23 of KMT2B in both the patient and her mother. Subsequent mRNA-seq analysis revealed that this variant caused two abnormal splicing events: exon 23 skipping and insertion of 18 bp derived from intron 23. These alterations generated two aberrant transcripts: NM_014727.3:c.4918_5076del/p.Arg1640_Lys1692del and NM_014727.3:c.5076_5077insGTGGGCCAGAACTGTGGG/p.Lys1692_Glu1693insValGlyGlnAsnCysGly. To date, a broad spectrum of pathogenic KMT2B variants associated with DYT28 has been reported, including missense, nonsense, splice-site variants, small insertions/deletions causing frameshifts, as well as larger structural variations such as translocations and deletions [4]. The c.5076 G>A variant identified in this study has not been reported in HGMD, gnomAD v2.1.1, or other publicly available databases. Although this variant is synonymous and does not alter the encoded amino acid sequence [5], mRNA-seq analysis demonstrated that it disrupts normal splicing, thereby providing strong functional evidence supporting its pathogenicity. Notably, several likely pathogenic variants affecting exon 23 of KMT2B have previously been reported in patients with dystonia. Loss of exon 23 is predicted to disrupt the PHD4 domain, thereby impairing normal KMT2B function [2], [9], [10], [11]. Based on the currently available evidence, the c.5076 G>A variant was therefore classified as likely pathogenic according to ACMG criteria [6]. The c.5076 G>A variant is predicted to disrupt the PHD4 domain (amino acids 1639–1686) of the KMT2B protein. The PHD domain plays a critical role in recognizing specific histone modifications and mediating interactions with downstream regulatory complexes [9], [12]. Disruption of this domain may therefore impair chromatin regulation, potentially affecting embryonic development and neuronal differentiation, ultimately contributing to the pathogenesis of DYT28.

Nevertheless, several limitations of this study should be acknowledged. Although mRNA-seq identified two aberrant transcripts, additional in vitro and in vivo functional studies were not performed to directly evaluate the structural and functional consequences of these transcript alterations at the protein level. Furthermore, the precise molecular mechanisms by which disruption of KMT2B functional domains leads to DYT28 remain incompletely understood. Elucidating these mechanisms represents an important direction for future research.

In summary, we identified a novel synonymous variant in KMT2B (c.5076 G>A) associated with dystonia. Functional analysis using mRNA-seq demonstrated that this variant disrupts normal RNA splicing, resulting in exon 23 skipping and partial RI, which likely constitute the underlying pathogenic mechanism. These findings expand the mutational spectrum of KMT2B and provide additional evidence supporting the diagnosis and genetic counseling of patients with DYT28.

Ethical approval

This study was reviewed and approved by the Medical Ethics Committee of Zhejiang Provincial People's Hospital (Approval Number: QT2026084).

Funding

This research was funded by the "Sharp soldier Leading Goose + X" Research and Development Program of Zhejiang Province (2024C03004) and Natural Science Foundation of Zhejiang Province (LY22H160039).

Consent

Written informed consent was obtained from the patients.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.