Haploinsufficiency of KMT2B, Encoding the Lysine-Specific Histone Methyltransferase 2B, Results in Early-Onset Generalized Dystonia

Abstract

Early-onset generalized dystonia represents the severest form of dystonia, a hyperkinetic movement disorder defined by involuntary twisting postures. Although frequently transmitted as a single-gene trait, the molecular basis of dystonia remains largely obscure. By whole-exome sequencing a parent-offspring trio in an Austrian kindred affected by non-familial early-onset generalized dystonia, we identified a dominant de novo frameshift mutation, c.6406delC (p.Leu2136Serfs^∗17), in KMT2B, encoding a lysine-specific methyltransferase involved in transcriptional regulation via post-translational modification of histones. Whole-exome-sequencing-based exploration of a further 30 German-Austrian individuals with early-onset generalized dystonia uncovered another three deleterious mutations in KMT2B-one de novo nonsense mutation (c.1633C>T [p.Arg545^∗]), one de novo essential splice-site mutation (c.7050-2A>G [p.Phe2321Serfs^∗93]), and one inherited nonsense mutation (c.2428C>T [p.Gln810^∗]) co-segregating with dystonia in a three-generation kindred. Each of the four mutations was predicted to mediate a loss-of-function effect by introducing a premature termination codon. Suggestive of haploinsufficiency, we found significantly decreased total mRNA levels of KMT2B in mutant fibroblasts. The phenotype of individuals with KMT2B loss-of-function mutations was dominated by childhood lower-limb-onset generalized dystonia, and the family harboring c.2428C>T (p.Gln810^∗) showed variable expressivity. In most cases, dystonic symptoms were accompanied by heterogeneous non-motor features. Independent support for pathogenicity of the mutations comes from the observation of high rates of dystonic presentations in KMT2B-involving microdeletion syndromes. Our findings thus establish generalized dystonia as the human phenotype associated with haploinsufficiency of KMT2B. Moreover, we provide evidence for a causative role of disordered histone modification, chromatin states, and transcriptional deregulation in dystonia pathogenesis.

Figure 1

KMT2B LoF Mutations in Individuals with Early-Onset Generalized Dystonia (A) Pedigree drawings and KMT2B LoF genotypes in families F1–F4. Filled black symbols represent individuals with early-onset generalized dystonia, filled gray symbols represent individuals with early-onset non-generalized dystonia, open symbols represent unaffected individuals, squares indicate males, and circles indicate females. Black asterisks indicate individuals subjected to WES. The mutation status is given below each individual. Mt indicates the mutant allele, WT indicates the wild-type allele, and N/A indicates an individual who has not been tested for the family mutation in KMT2B. Sanger-sequencing electropherograms confirm the de novo status of each LoF variant in families F1–F3 and co-segregation of the LoF variant in family F4. Mutated bases are boxed in black, and variants are indicated by an arrow. (B) Graphical view of KMT2B chromosomal position, transcript structure, and protein product, along with the distribution of four identified LoF variants. KMT2B maps to chromosomal region 19q13.12 and encodes a 37-exon canonical transcript (8,148 bp). The translated protein (2,715 amino acids) encompasses an AT-hook DNA-binding domain (in blue), a CXXC zinc-finger domain involved in protein-protein interactions (in green), three plant homodomain (PHD) fingers involved in protein-protein interactions (in yellow), two FY-rich domains (N-terminal, FYRN; C-terminal, FYRC) involved in protein heterodimerization (in violet), and a SET (su[var]3-9 enhancer-of-zeste trithorax) domain responsible for histone lysine methylation (in orange). The LoF variants, indicated by arrows, are shown below the protein. The structure of the KMT2B transcript and organization of the protein domains are not drawn to exact scale.

Figure 2

The KMT2B c.7050−2A>G Splice-Site Mutation Predictably Results in Protein Truncation (A) Gel electrophoresis of KMT2B cDNA fragments. Total RNA was extracted from whole blood of individual F3-II-3 and a healthy control individual (Ctrl) (QIAGEN) and reverse transcribed into cDNA (Invitrogen). The relevant cDNA fragment was amplified with specific primers in exon 28 and the exon 32-33 junction. Individual F3-II-3 and the control subject produce the expected cDNA fragment of 549 bp. In F3-II-3, the heterozygous splice acceptor AG>GG substitution in intron 29 leads to the accumulation of small amounts of a second cDNA species (473 bp). (B) Isolation and Sanger sequencing of the 549-bp cDNA fragment confirms a regular transcript structure with intact exon 28-29 and exon 29-30 boundaries. The 473-bp fragment represents a transcript that skips exon 29 and contains the terminal 14 nucleotides of intron 29. The mutant splice-acceptor allele is part of the misspliced transcript, as indicated by an arrow. (C) Scheme illustrating the complex effect of the KMT2B splicing mutation. The mutation at the conserved AG dinucleotide activates a cryptic splice acceptor in intron 29, resulting in a mixture of intronic sequence retention and exon skipping. The missplicing event is predicted to lead to a frameshift and introduction of a premature termination codon in the reading frame of exon 32 (p.Phe2321Serfs^∗93). Sites joined by splicing are indicated.

Figure 3

KMT2B Haploinsufficiency Causes Dystonia (A) Total KMT2B mRNA levels are reduced in dermal fibroblasts from individuals F1-II-5 and F3-II-3. Total RNA was extracted from fibroblasts of individuals F1-II-5 and F3-II-3, as well as two healthy control individuals (Ctrls) (QIAGEN). Integrity of the RNA was confirmed by the Agilent 2100 Bioanalyzer with the use of RNA 6000 Nano chips (RNA integrity number [RIN] = 10 for each sample). Reverse transcription was conducted with 1,000 ng total RNA as a template (Invitrogen). qRT-PCR was performed with TaqMan gene-expression assays (ThermoFisher) for KMT2B and an endogenous reference gene (GAPDH). Each sample was run in quadruplicate, and expression levels were determined by the ΔΔCT method. Error bars indicate the SEM of four independent experiments. (B) Published individuals with de novo KMT2B-involving 19q13.1 microdeletions manifest dystonia. A systematic search for original publications and individual clinical reports in PubMed and DECIPHER identified a total of four dystonia-affected individuals who harbor small interstitial de novo deletions of the 19q13.1 chromosomal region, including KMT2B. The dystonia-associated deletions (0.6–4.9 Mb in size) are depicted as violet horizontal bars in correlation to an ideogram of chromosome 19 and the relevant interval of 19q13.11–q13.12 (coordinates corresponding to UCSC Genome Browser build hg19). The solid green line extending through the diagram indicates the KMT2B locus (chr19: 36,208,921–36,229,781). Sources and individual case identifiers, along with the description of the associated dystonic manifestation, are given as provided. Sexes and ages at last examination (in years) of the individuals carrying the deletions are shown in parentheses. Abbreviations are as follows: M, male; F, female; N/A, not available.