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. 2018 Jan;26(1):107-116.
doi: 10.1038/s41431-017-0033-y. Epub 2017 Dec 4.

Molecular and cellular issues of KMT2A variants involved in Wiedemann-Steiner syndrome

Nicolas Lebrun  1   2   3 , Irina Giurgea  4 , Alice Goldenberg  5 , Anne Dieux  6 , Alexandra Afenjar  7 , Jamal Ghoumid  6 , Bertrand Diebold  8 , Léo Mietton  1   2   3 , Audrey Briand-Suleau  8 , Pierre Billuart  1   2   3 , Thierry Bienvenu  9   10   11   12
Affiliations

Molecular and cellular issues of KMT2A variants involved in Wiedemann-Steiner syndrome

Nicolas Lebrun et al. Eur J Hum Genet. 2018 Jan.

Abstract

Variants in KMT2A, encoding the histone methyltransferase KMT2A, are a growing cause of intellectual disability (ID). Up to now, the majority of KMT2A variants are non-sense and frameshift variants causing a typical form of Wiedemann-Steiner syndrome. We studied KMT2A gene in a cohort of 200 patients with unexplained syndromic and non-syndromic ID and identified four novel variants, one splice and three missense variants, possibly deleterious. We used primary cells from the patients and molecular approaches to determine the deleterious effects of those variants on KMT2A expression and function. For the putative splice variant c.11322-1G>A, we showed that it led to only one nucleotide deletion and loss of the C-terminal part of the protein. For two studied KMT2A missense variants, c.3460C>T (p.(Arg1154Trp)) and c.8558T>G (p.(Met2853Arg)), located at the cysteine-rich CXXC domain and the transactivation domain of the protein, respectively, we found altered KMT2A target genes expression in patient's fibroblasts compared to controls. Furthermore, we found a disturbed subcellular distribution of KMT2A for the c.3460C>T mutant. Taken together, our results demonstrated the deleterious impact of the splice variant and of the missense variants located at two different functional domains and suggested reduction of KMT2A function as the disease-causing mechanism.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
a Schematic of KMT2A protein structure (NP_001184033.1), with domains (and motifs) and the location of the identified de novo missense and splice variants. Domains were predicted by SMART program (http://smart.embl-heidelberg.de/). DNA binding AT hooks 1–3 (aa 169–180; aa 217–227; aa 301–309), CXXC domain (aa 1150–1198), zinc finger PHD-type 1–3 (aa 1431–1482; 1479–1533; 1566–1630), TAD domain (aa 2850–2858), bromodomain (aa 1703–1748), FYR N-terminal domain (aa 2021–2077), FYR-C terminal domain (aa 3666–3747), WDR5 interacting motif (aa 3765–3773), SET domain (aa 3832–3948), and post-SET domain (aa 3956–3972). b Sequence analysis of PCR products from the patient’s and parent’s (mother and father) DNA showing the different de novo variants. Asteriks indicate the position of the variants
Fig. 2
Fig. 2
Facial characteristics of the ID patients carrying a KMT2A variant. Patient 2 (P2) carries the c.8558T>G (p.(Met2853Arg)) variant, Patient 3 (P3) carries the c.3581G>A (p.(Cys1194Tyr)) variant and Patient 4 (P4) carries the c.11322–1G>A variant
Fig. 3
Fig. 3
a Sequence analysis of RT-PCR products from the patients (c.3460C>T (p.(Arg1154Trp)), c.8558T>G (p.(Met2853Arg)), c.11322–1G>A). In all cases, the two alleles were detected. b KMT2A mRNA expression in controls (WT) and mutated fibroblasts (c.3460C>T (p.(Arg1154Trp)), c.8558T>G (p.(Met2853Arg)), c.11322–1G>A). Y: KMT2A relative expression. Normalization factor was based on the GAPDH reference gene. Each chart represents one KMT2A relative quantification of a stimulated fibroblast culture. Errors bars represent SEM. c Western blot analysis of KMT2A protein of human control (WT1, WT2, WT3, and WT4; WT: all controls) and mutated fibroblasts (c.3460C>T (p.(Arg1154Trp)), c.8558T>G (p.(Met2853Arg)), c.11322–1G>A). GAPDH was used as loading control. Densitometry of western blotting was performed using Image J software. Data in arbitrary units (a.u.) represent the mean ± SEM of four separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001 between control and KMT2A mutated fibroblasts. d Concerning the patient bearing the potential splice variant, RT-PCR using primers in exon 32 and 35 on cDNA from the patient and controls only showed one distinct band. Sequencing of the RT products showed two transcripts, the wild-type transcript and the mutated transcript with a deletion of the first nucleotide G of exon 33. This variant results in a premature stop-codon and is predicted to produce a truncated protein deleted of its C-terminal SET domain (p.Lys3775Serfs*32). This data suggests a change in the acceptor site of intron 32 which uses the mutated nucleotide A and the first G of the exon 33 to reconstitute the acceptor site
Fig. 4
Fig. 4
Mapping nuclear targeting signals of wild-type (WT) and mutated KMT2A. Wild type or mutated KMT2A constructs (c.3460C>T (p.(Arg1154Trp)); c.8558T>G (p.(Met2853Arg))) were transiently transfected into COS7 cells and detected by staining with anti-MLL-1 (KMT2A) antibody. a Representative examples of typical patterns; uniform pattern and dot patterns (small dots or bigger patches absent within the nucleoli). b Distribution (% ± SEM) of the different nuclear patterns of cells expressing wild-type or mutated KMT2A constructs. Results were obtained by using data from more than 600 transfected cells of each construct in four independent experiments. The KMT2A c.3460C>T (p.(Arg1154Trp)) mutant abolishes significantly its capability to produce big dots (***χ 2 test with p < 0.0001)
Fig. 5
Fig. 5
Expression of target genes of KMT2A (CDKN2C, CDKN1B, SIX2, HOXA9 and MEIS1) in wild-type fibroblasts (WT) and in human fibroblasts bearing different KMT2A variants (patients, c.3460C>T (p.(Arg1154Trp)); c.8558T>G (p.(Met2853Arg)), c.11322–1G>A).**p < 0.001, ***p < 0.0005 between control and KMT2A mutated fibroblasts
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