Abstract
Histone lysine methylation, mediated by mixed-lineage leukemia (MLL) proteins, is now known to be critical in the regulation of gene expression, genomic stability, cell cycle and nuclear architecture. Despite MLL proteins being postulated as essential for normal development, little is known about the specific functions of the different MLL lysine methyltransferases. Here we report heterozygous variants in the gene KMT2B (also known as MLL4) in 27 unrelated individuals with a complex progressive childhood-onset dystonia, often associated with a typical facial appearance and characteristic brain magnetic resonance imaging findings. Over time, the majority of affected individuals developed prominent cervical, cranial and laryngeal dystonia. Marked clinical benefit, including the restoration of independent ambulation in some cases, was observed following deep brain stimulation (DBS). These findings highlight a clinically recognizable and potentially treatable form of genetic dystonia, demonstrating the crucial role of KMT2B in the physiological control of voluntary movement.
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20 April 2017
Following publication of this article, the authors were asked to remove a clinical image and some video footage of one of the affected individuals. Although consent was obtained, in keeping with their ethical consent framework, the authors allow for withdrawal of consent and are carrying out the wishes of the research subjects under their consent process. This amendment has been made in the HTML and PDF versions of the article.
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Acknowledgements
We thank all our patients and their families for taking part in this study and encouraging international collaboration to seek out similar cases. Thanks are given to M. Ishida (GOS-ICH) for kindly providing the fetal cDNA, K. Tuschl (GOS-ICH) for kindly providing the human cDNA panel, L. Bassioni (Great Ormond Street Hospital, GOSH) for kindly selecting DaTSCAN images for the supplementary manuscript, M. Adams (National Hospital for Neurology and Neurosurgery, NHNN) for reviewing the imaging of the patients at NHNN and R. Meijer for helping with the sequencing analysis at the Department of Human Genetics (Nijmegen). We thank G. Moore (GOS-ICH) and P. Stanier (GOS-ICH) for proofreading the manuscript. Many thanks are given to A. Panahian-Jand (GOSH) for excellent administrative support. M.A.K. has a Wellcome Intermediate Clinical Fellowship (WT098524MA). E.M. and M.A.K. received funding from the Rosetrees Trust, the Great Ormond Street Hospital Children's Charity and the Gracious Heart Foundation. N.E.M. receives support from the UK Department of Health's NIHR Biomedical Research Centers funding streams. A. Papandreou has a joint Action Medical Research/British Paediatric Neurology Association Research Training Fellowship. J. Ng has an MRC Research Training Fellowship. A.N. has an Action Medical Research Training Fellowship. H.B.-P. has a DBS training travel grant from the Daniel Turnberg Trust Fund. H.H. is funded by the MRC and Wellcome Trust (Synaptopathies award). D.A. is supported by the Prusiner-Abramsky Award. H.P. has received grant support from the Dystonia Society (UK). K.J.P. has an Academy of Medical Sciences Clinical Starter Grant. B.P.-D. received funding from grants 20143130-La Marató de TV3 and PI15/00287-Ministerio Español de Economia y Competitividad. J.-P.L. has been supported by Guy's and St Thomas' Charity New Services and Innovation Grant G060708, the Dystonia Society (UK), grants 01/2011 and 07/2013 and an Action Medical Research, AMR-GN2097. This research was supported by the NIHR Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust, University College London and University of Cambridge and by funding from the NIHR for the BioResource for Rare Diseases (grant RG65966). This study makes use of data generated by the DECIPHER community. A full list of centers contributing to the generation of the data is available from http://decipher.sanger.ac.uk/ and via e-mail from decipher@sanger.ac.uk. Funding for the project was provided by the Wellcome Trust for UK10K (WT091310) and the DDD study. The DDD study presents independent research commissioned by the Health Innovation Challenge Fund (grant HICF-1009-003); see http://www.ddduk.org/access.html for full acknowledgment. This work was supported in part by the Intramural Research Program of the National Human Genome Research Institute and the Common Fund, NIH Office of the Director. This work was supported in part by the German Ministry of Research and Education (grants 01GS08160 and 01GS08167; German Mental Retardation Network) as part of the National Genome Research Network to A.R. and D.W., and by the Deutsche Forschungsgemeinschaft (AB393/2-2) to A.R. Brain expression data were provided by the UK Human Brain Expression Consortium (UKBEC), which comprises John A. Hardy, Mina Ryten, Michael Weale, Daniah Trabzuni, Adaikalavan Ramasamy, Colin Smith and Robert Walker, affiliated with the UCL Institute of Neurology (J.A.H., M.R. and D.T.), King's College London (M.R., M.W. and A.R.) and the University of Edinburgh (C.S. and R.W.).
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E.M., K.J.C., J.M.E.N., J.R.C., F.L.R. and M.A.K. conceived and designed experiments. J.R., N.E.M., A. Papandreou, J. Ng, H.B.-P., M.A.W., D.A., A. Barnicoat, H.B., S. Bhate, N.D., N.F., N.G., A.H., H.H., J.A.H., Z.I., M.K., P.L., D.L., S. McKee, S. Misra, S.S.M., V.N., J. Nicolai, M.N., H.P., K.J.P., G.B.P., P.P., M.S.R., P.R., R.S., M. Sinnema, M. Smith, P.T., S.M.W., D.W., B.T.W., G.W., the UK10K Consortium, the DDD study, the NIHRBR-RD Consortium, L.J.C., B.P.-D., J.-P.L., A.R., W.A.G., C.T., K.P.B., N.W.W., E.-J.K., P.G., R.C.D., F.L.R. and M.A.K. ascertained patients and contributed clinical information, photographs, videos and neuroimaging studies. M.A.K. performed phenotypic characterization of all patients. W.K.C. and M.A.K. reviewed patient neuroimaging. A. Papandreou and M.A.K. edited patient videos. A. Boys, C.W. and D.M. undertook chromosomal microarray analysis. E.M., K.J.C., D.G., N.E.M., S.W., A. Pittman, the UK10K Consortium, the DDD Study, the NIHRBR-RD Consortium, A.R., W.A.G., C.T., E.-J.K. and M.A.K. carried out whole-exome and whole-genome sequencing analysis. E.M. and A.N. performed variant validation by direct Sanger sequencing. K.J.C. performed enrichment analysis (and corresponding statistical analysis). S.P. and S.J.H.H. analyzed CSF neurotransmitters. A.P.J. and M.T. undertook comparative homology modeling. J.M.E.N. and J.R.C. undertook the histone methylation assays (and corresponding statistical analysis) and cloning of the Set1 point substitution in Dictyostelium. S. Barral generated dopaminergic neurons, collected RNA and cDNA samples, and undertook qRT–PCR experiments. E.M. maintained fibroblast cultures, collected RNA, cDNA and protein samples, and performed fibroblast immunoblotting analysis (and corresponding statistical analysis) and CSF immunoblotting (and corresponding statistical analysis). J. Ng carried out CSF immunoblotting analysis. P.G. and F.L.R. contributed critical suggestions for experimental work. E.M. and M.A.K. wrote the manuscript. K.J.C., J.R., J.M.E.N., D.G., A.P.J., N.E.M., A.R., W.A.G., C.T., E.-J.K., W.K.C., M.T., J.R.C. and F.L.R. contributed written sections for the manuscript. M.A.K. oversaw the overall project. All authors critically reviewed manuscript.
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H.P. has unrestricted support for educational activity from Medtronic.
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A list of members and affiliations appears in the Supplementary Note.
A list of members and affiliations appears in the Supplementary Note.
A list of members and affiliations appears in the Supplementary Note.
Integrated supplementary information
Supplementary Figure 1 Schematic representation of the cohorts screened for KMT2B variants.
Thirty-four patients with childhood-onset dystonia were identified in the GOS-ICH Dystonia Cohort for molecular genetic investigations. Chromosomal microarray, whole-exome sequencing and whole-genome sequencing were undertaken. Collaborations with other centers facilitated identification of further individuals with KMT2B variants.
Supplementary Figure 2 Sanger confirmation and segregation of the identified KMT2B variants.
Sequence chromatograms of patients and their parents (where available) are shown. For patients 11–19, 21, 23–25 and 26b, the sequence chromatograms show the de novo status of the respective KMT2B variant. For patients 22, 26a and 27, the sequencing chromatograms show that the respective KMT2B variant is maternally inherited.
Supplementary Figure 3 Effect of age on clinical presentation.
Age of clinical presentation for patients with KMT2B dystonia. Twenty-seven patients with variants in KMT2B were identified. Seven patients had missense variants (black squares), and the remainder had microdeletions (Del), frameshift mutations (Fs), nonsense and splice-site mutations (black circles). The age of clinical presentation for those with missense variants (n = 7; mean = 6.4 ± 2.07 (s.d.) years) was significantly higher than for those with mutations predicted to truncate or elongate the protein (n = 19; mean = 4.1 ± 2.18 (s.d.) years). Data were analyzed by two-tailed unpaired t test: *P = 0.0223 (t = 2.443, d.f. = 24). No significant difference in variances between the groups was detected by F test. Individual data points are plotted with the center bar showing the mean and error bars showing s.d.
Supplementary Figure 4 Additional clinical features in KMT2B dystonia.
(a,b) Examples of handwriting from patient 11, age 7 years, showing deterioration over a 4-week period, with reduced legibility and increased letter size due to worsening upper-limb dystonia. (c) Patient 3, age 8 years, with evidence of scalp cutis aplasia.
Supplementary Figure 5 Effect of age on neuroradiological findings.
(a) Age of neuroimaging for patients with KMT2B dystonia. Twenty-two of 27 patients had neuroimaging, specifically T2/T2*, diffusion and susceptibility weighted sequences of sufficient quality for assessment of hypointensity in the globus pallidus (Supplementary Table 7). The mean age of 17 patients with an abnormal scan (n = 21; mean = 11.7 ± 4.31 (s.d.) years; black circles) was significantly lower than that of the 5 patients with normal scans (n = 7; 19.0 ± 11.12 (s.d.) years; black squares). Data were analyzed by two-tailed unpaired t test: *P = 0.0167 (t = 2.559, d.f. = 26). A significant difference in variances between the groups was detected by F test (**P = 0.0011). Individual data points are plotted with the center bar showing the mean and error bars showing s.d. (b,c) Serial magnetic resonance imaging (MRI) in patient 22. MRI with diffusion imaging data sets with b value of zero in patient 22 at 13 years, 1 month (b) and 17 years (c) showing reduction in the hypointense appearance of the globus pallidus over time. (d) Normal DaTSCAN imaging on patient 14, age 18 years.
Supplementary Figure 6 Uncut agarose gel of PCR analysis for expression of KMT2B.
The top row of this uncut agarose gel (relating to Fig. 5a) shows KMT2B-specific PCR bands of human fetal and adult cDNA samples amplified by the intron-spanning primer pair KMT2B-ex29/30-F and KMT2B-ex29/30-R (Supplementary Table 9). The bottom row displays the GAPDH-specific PCR bands for the same cDNA samples amplified by the intron-spanning primer pair GAPDH-F and GAPDH-R (Supplementary Table 9). The last sample in both rows represents a negative control in which no DNA was included in the PCR reaction.
Supplementary Figure 7 Histone methylation assays.
(a) Example immunoblots of methylated and pan histone H3 extracted from control and patient-derived fibroblasts (relating to Fig. 5d). (b) Example immunoblots of H3K4me3, pan histone H3 and GFP in Dictyostelium cell lysates (relating to Fig. 5e). (c) Quantification of anti-GFP immunoblotting, showing expression of GFP-DdSet1(I1447T) as a percentage of GFP-DdSet1 expression. Individual data points are plotted with the center bar showing the mean and error bars showing s.d. Three independently transformed GFP-DdSet1(I1447T) cell lines were tested. n = 3 technical replicates prepared and blotted on different days (normalized expression level (by anti-GFP): set1– GFP-DdSet1(I1447T) 1 = 34.39 ± 19.52 (s.d.), set1– GFP-DdSet1(I1447T) 2 = 68.32 ± 9.191 (s.d.), set1– GFP-DdSet1(I1447T) 3 = 52 ± 5.575 (s.d.)). Dilutions of GFP-DdSet1-containing lysates were tested to ensure that quantification by densitometry was conducted in the linear range of assay sensitivity.
Supplementary Figure 8 Conservation of the amino acids altered by the identified KMT2B missense variants.
(a–f) The KMT2B amino acid sequences from seven different species and the human paralog KMT2A (another member of the MLL protein family) are aligned. (a) Additionally, the amino acid sequence of the human PHF6 protein, used to model the PHD-like domain, is shown. Gly1652 (in red) is highly conserved in all listed amino acid sequences, while the Phe1662 residue (in red) is either conserved or tolerates replacement by the similar amino acid tyrosine (Supplementary Fig. 12). (b) Arg1705 (in red) is conserved to zebrafish and in human KMT2A. (c) Arg1762 is fully conserved throughout the species. (d) Leu1781 (in red) is conserved in all listed mammalian homologs of KMT2B. (e) Arg2517 (in red) is conserved to zebrafish and in human KMT2A. (f) Additionally, the amino acid sequences of SET domain–containing proteins (human SETD1A and SETD1B, yeast Set1 and Dictyostelium discoideum Set1) are shown. Ile2674 (in red) is highly conserved in all listed amino acid sequences. Human KMT2B (NP_055542.1); dog KMT2B (XP_003432729.3); cow KMT2B (XP_003587289.1); mouse Kmt2B (NP_083550.2); zebrafish kmt2ba (XP_689347.5); zebrafish kmt2bb (XP_002664717.2); fruit fly trx (NP_599109.1); human KMT2A (NP_001184033.1); human PHF6 (NP_115834.1); yeast Set1 (NP_011987.1); Dicty Set1 (XP_636258.1); human SETD1A (NP_055527.1); human SETD1B (NP_055863.1).
Supplementary Figure 9 H3K4 methylation profiles.
(a–e) The publically available ENCODE (Encyclopedia of DNA Elements) data (Nucleic Acids Res. 41, D56–D63, 2013) on the UCSC genome browser (https://genome.ucsc.edu/) were interrogated for the histone H3 lysine 4 trimethylation (H3K4me3) pattern in different cell lines. The layered H3K4me3 lane includes data from seven cell lines (GM12878, H1-hESC, HSMM, HUVEC, K562, NHEK and NHLF). (a) The H3K4 methylation profile from THAP1 showed a very strong, sharp methylation peak around exon 1 in all investigated cell lines. (b) A similar but slightly less sharp H3K4 methylation peak around exon 1 of TOR1A was observed. (c) The identified weaker H3K4 methylation in GNAL did not coincide with exon 1 and was located in a large intron. (d,e) For the two investigated markers of the dopaminergic system, D2R and TH, no H3K4 methylation peak was seen. NH-A, cell line from brain tissue containing astrocytes; NHDF-Ad, adult dermal fibroblast cell line based on skin tissue; NHEK, cell line of epidermal keratinocytes of skin tissue.
Supplementary Figure 10 Box plots of THAP1 mRNA expression levels in ten adult brain regions.
Expression levels are based on exon array experiments and are plotted on a log2 scale (y axis). As previously described, this data set was generated using Affymetrix Exon 1.0 ST arrays and brain tissue originating from 134 control individuals, collected by the Medical Research Council (MRC) Sudden Death Brain and Tissue Bank, Edinburgh, UK, and the Sun Health Research Institute (SHRI), an affiliate of Sun Health Corporation, USA (J. Neurochem. 119, 275–282, 2011). This plot shows that THAP1 is ubiquitously expressed across all ten brain regions analyzed; as for KMT2B, expression is higher in the cerebellum than in any other region. Putamen (PUTM), frontal cortex (FCTX), temporal cortex (TCTX), occipital cortex (OCTX), hippocampus (HIPP), substantia nigra (SNIG), medulla (specifically inferior olivary nucleus, MEDU), intralobular white matter (WHMT), thalamus (THAL), and cerebellar cortex (CRBL). N indicates the number of brain samples analyzed to generate the results for each brain region. Ranges extend from the box to 1.5 times the interquartile range.
Supplementary Figure 11 Functional investigation of the downstream effects of mutations in KMT2B.
(a–c) Investigation of downstream effects in fibroblast cell culture samples (patients, n = 4; controls, n = 2). (a) Quantitative RT–PCR of THAP1 and TOR1A indicates that patients have a reduction of THAP1 and, to a lesser extent, of TOR1A transcripts in comparison to controls (THAP1: controls = 1.04 ± 0.40 (s.d.); patients = 0.55 ± 0.05 (s.d.); TOR1A: controls = 1.00 ± 0.05 (s.d.); patients = 0.79 ± 0.06 (s.d.)). Data were analyzed by two-tailed unpaired t test (THAP1: *P = 0.0498 (t = 2.780, d.f. = 4), TOR1A: *P = 0.0140 (t = 4.170, d.f. = 4)). While a significant difference in variances between the groups was detected by F test (**P = 0.0066) for THAP1, no significant difference in variances between the groups was detected for TOR1A. (b) Immunoblotting studies (for every fibroblast line, n = 3 protein samples were collected and analyzed) indicate a significant reduction in THAP1 levels for patients when compared to controls (controls = 1.57 ± 0.25 (s.d.); patient 2 = 0.83 ± 0.06 (s.d.); patient 13 = 0.77 ± 0.17 (s.d.); patient 14 = 0.53 ± 0.04 (s.d.); patient 16 = 0.54 ± 0.06 (s.d.)). Data were analyzed by one-way ANOVA: ***P < 0.0001 (F = 28.46, d.f. = 4). (c) Immunoblotting studies indicate a statistically reduced level of torsin-1A in patient 14 when compared to controls (controls = 1.10 ± 0.14 (s.d.); patient 14 = 0.64 ± 0.05 (s.d.)) but not for the other patients (patient 2 = 1.09 ± 0.27 (s.d.); patient 13 = 1.39 ± 0.18 (s.d.); patient 16 = 1.13 ± 0.29 (s.d.)). Data were analyzed by one-way ANOVA: **P = 0.0066 (F = 5.800, d.f. = 4). (d) CSF immunoblotting studies on patients 2 and 16 (n = 2 patient CSF samples) show markedly reduced levels of D2R and increased levels of TH when compared to control CSF (n = 4 control CSF samples) (D2R: controls = 1.09 ± 0.21 (s.d.); patients = 0.64 ± 0.02 (s.d.); TH: controls = 0.52 ± 0.08 (s.d.); patients = 0.90 ± 0.01 (s.d.)). Data were analyzed by two-tailed unpaired t test: D2R: *P = 0.0471 (t = 2.835, d.f. = 4); TH: **P = 0.0036 (t = 6.147, d.f. = 4). No significant difference in variances between the groups was detected by F test. CSF immunoblotting was undertaken once owing to limited volume of CSF. Individual data points are plotted with the center bar showing the mean and error bars showing s.d.
Supplementary Figure 12 Comparative modeling of p.Phe1662Tyr indicates a tolerated substitution.
In fruit flies and zebrafish, the phenylalanine is replaced by a tyrosine at position 1662 of KMT2B. Based on the similar side chains for these amino acids, tyrosine can be accommodated instead of phenylalanine at position 1662 in KMT2B without disruption of the structure of the protein. Furthermore, the different prediction programs propose this substitution to be non-deleterious (PolyPhen-2, benign with a score of 0.005; SIFT, tolerated with a score of 1.000; PROVEAN, neutral with a score of 1.16).
Supplementary Figure 13 Exclusion of KMT2B imprinting.
(a,b) The chromosomal microarray of patients 2 and 10 harboring KMT2B microdeletions and their parents indicated that the inherited SNPs were only of maternal origin, implying that the paternal KMT2B allele was deleted. While in at least these two cases the KMT2B mutation occurs on the paternal allele, we have shown that KMT2B changes in patients 22, 26a and 27 are maternally inherited. This proves that KMT2B variants are not restricted to either the maternal or parental allele. (c–e) Sanger sequencing of cDNA samples from control fibroblasts, patient fibroblasts and generated dopaminergic neurons showed biallelic expression of the common KMT2B SNP rs231591 (c.7091A>G). (f) Although there are nine imprinted regions reported in the literature (http://igc.otago.ac.nz/home.html), none of them coincide with the chromosomal location of KMT2B (hg19: chromosome 19: 36,208,921–36,229,779).
Supplementary Figure 14 Evaluation of the differentiation efficiency of midbrain dopaminergic neurons.
Immunofluorescence of generated midbrain dopaminergic neurons at day 50 of differentiation using MAP2 (green) as a neuronal marker and TH (red) as a specific dopaminergic marker showed cells with colocalization (merge) of both markers indicating the successful generation of midbrain dopaminergic neurons. Nuclei were stained with DAPI (blue). Quantification of the two markers from three independent differentiations (n = 3; 600 cells per experiment) yielded 50.3 ± 17.2% of cells as MAP2 positive, of which 39.1 ± 11.0% were also TH positive.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–14, Supplementary Tables 1–11 and Supplementary Note. (PDF 4367 kb)
Supplementary Video 1
Lower-limb dystonia and gait disturbance in patients with KMT2B variants. (MPG 9702 kb)
Supplementary Video 2
Upper-limb dystonia in patients with KMT2B variants. (MPG 7260 kb)
Supplementary Video 3
Progression to generalized dystonia in patients with KMT2B variants. (MPG 9312 kb)
Supplementary Video 4
Cranial, cervical and laryngeal features in patients with KMT2B variants. (MPG 19576 kb)
Supplementary Video 5
Motor overflow secondary to dystonia in patients with KMT2B variants. (MPG 6358 kb)
Supplementary Video 6
Dystonic crisis in a patient with a KMT2B mutation. (MPG 3298 kb)
Supplementary Video 7
Myoclonus dystonia in a patient with a KMT2B mutation (MPG 2234 kb)
Supplementary Video 8
Response to deep brain stimulation in patient 9. (MPG 1946 kb)
Supplementary Video 9
Response to deep brain stimulation in patient 17. (MPG 3862 kb)
Supplementary Video 10
Response to deep brain stimulation in patient 21. (MPG 3402 kb)
Supplementary Video 11
Response to deep brain stimulation in patient 22. (MPG 10030 kb)
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Meyer, E., Carss, K., Rankin, J. et al. Mutations in the histone methyltransferase gene KMT2B cause complex early-onset dystonia. Nat Genet 49, 223–237 (2017). https://doi.org/10.1038/ng.3740
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DOI: https://doi.org/10.1038/ng.3740
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