KMT2A: Umbrella Gene for Multiple Diseases

KMT2A (Lysine methyltransferase 2A) is a member of the epigenetic machinery, encoding a lysine methyltransferase responsible for the transcriptional activation through lysine 4 of histone 3 (H3K4) methylation. KMT2A has a crucial role in gene expression, thus it is associated to pathological conditions when found mutated. KMT2A germinal mutations are associated to Wiedemann–Steiner syndrome and also in patients with initial clinical diagnosis of several other chromatinopathies (i.e., Coffin–Siris syndromes, Kabuki syndrome, Cornelia De Lange syndrome, Rubinstein–Taybi syndrome), sharing an overlapping phenotype. On the other hand, KMT2A somatic mutations have been reported in several tumors, mainly blood malignancies. Due to its evolutionary conservation, the role of KMT2A in embryonic development, hematopoiesis and neurodevelopment has been explored in different animal models, and in recent decades, epigenetic treatments for disorders linked to KMT2A dysfunction have been extensively investigated. To note, pharmaceutical compounds acting on tumors characterized by KMT2A mutations have been formulated, and even nutritional interventions for chromatinopathies have become the object of study due to the role of microbiota in epigenetic regulation.

KMTs catalyze the transfer of methyl groups from S-adenosylmethionine to the lysine residues on histone tails, particularly the histone H3 tail. Unlike other epigenetic enzymes such as acetyltransferases (HATs), KMTs are more specific and usually modify one or two lysines on a single histone [1]. Lysines can be monomethylated, bimethylated or trimethylated without changing the electric charge of the amino acid side chain. The effect on chromatin state, i.e., whether it activates transcription or represses it, depends on the methylation states and their positions ( Figure 1) [2][3][4][5][6][7][8][9][10][11][12][13][14][15]. KMTs are so called writers, enzymes that catalyze the addition of chemical groups to histone tails or to DNA; these modifications are not permanent but can be removed by erasers to reverse the influence on gene expression. Readers possess specialized domains able to recognize and interpret different chemical modifications. Writers, erasers and readers form the epigenetic machinery, and mutations in genes coding for this apparatus lead to ann altered chromatin conformation and an incorrect gene expression, resulting in a series of syndromes known as chromatinopathies, Mendelian genetic diseases, most of them with a dominant character [16][17][18]. Pathogenic mutations in KMTs and KDMs (Lysine demethylases) lead to haploinsufficiency in numerous developmental syndromes ( Figure 2) (Table 1) [10,19].
Genes 2022, 13, x FOR PEER REVIEW 2 of 17 on gene expression. Readers possess specialized domains able to recognize and interpret different chemical modifications. Writers, erasers and readers form the epigenetic machinery, and mutations in genes coding for this apparatus lead to ann altered chromatin conformation and an incorrect gene expression, resulting in a series of syndromes known as chromatinopathies, Mendelian genetic diseases, most of them with a dominant character [16][17][18]. Pathogenic mutations in KMTs and KDMs (Lysine demethylases) lead to haploinsufficiency in numerous developmental syndromes ( Figure  2) (Table 1) [10,19].  on gene expression. Readers possess specialized domains able to recognize and interpret different chemical modifications. Writers, erasers and readers form the epigenetic machinery, and mutations in genes coding for this apparatus lead to ann altered chromatin conformation and an incorrect gene expression, resulting in a series of syndromes known as chromatinopathies, Mendelian genetic diseases, most of them with a dominant character [16][17][18]. Pathogenic mutations in KMTs and KDMs (Lysine demethylases) lead to haploinsufficiency in numerous developmental syndromes ( Figure  2) (Table 1) [10,19].   Many species have a KMT2A ortholog, including fishes, birds, amphibians, and mammals; thus, its evolutionary conservation allowed a comprehensive study of KMT2A molecular functions through in vivo experiments on animal models (Drosophila melanogaster, Danio rerio, Mus musculus). KMT2A expression is mainly nuclear and ubiquitously present in 27 tissues, especially in ovary, lymph node, endometrium, thyroid and brain tissue [20]. KMT2A encodes a lysine methyltransferase (KMT) formed of 3969 amino acids, a transcriptional co-activator which plays a crucial role in hematopoiesis, in regulating gene expression at early developmental stages, and in the control of circadian gene expression. KMT2A is processed by the endopeptidase Taspase 1 in two fragments (MLL-C and MLL-N) which heterodimerize and regulate the transcription of specific genes, including HOX genes [21]. KMT2A protein has 18 domains, including the CXXC-type zinc finger, the extended PHD domain and the bromodomain. The SET domain has the methyltransferase activity (mono-, di-, tri-methylation) on lysine 4 of histone 3 (H3K4 me1/2/3), a post-transcriptional modification (PTM) responsible of epigenetic transcriptional activation and which efficiency can be increased when the protein is associated with another component of the MLL1/MLL complex ( Figure 3) [22]. genes, including HOX genes [21]. KMT2A protein has 18 domains, including the CXXCtype zinc finger, the extended PHD domain and the bromodomain. The SET domain has the methyltransferase activity (mono-, di-, tri-methylation) on lysine 4 of histone 3 (H3K4 me1/2/3), a post-transcriptional modification (PTM) responsible of epigenetic transcriptional activation and which efficiency can be increased when the protein is associated with another component of the MLL1/MLL complex ( Figure 3) [22]. As other members of KMTs family, KMT2A regulates gene transcription through chromatin opening or closure and its activity is antagonized by the lysine demethylases (KDMs) family.

Wiedemann-Steiner Syndrome
KMT2A germinal variants are associated to the Wiedemann-Steiner syndrome (WDSTS, OMIM #605130), a rare autosomal dominant disorder characterized by different features, mainly intellectual disability (ID), developmental delay (DD), pre-and postnatal growth deficiency, hypertrichosis, short stature, hypotonia, distinctive facial features (thick eyebrows, long eyelashes, narrow palpebral fissures, broad nasal tip, down slanting palpebral fissures), skeletal abnormalities (clinodactyly, brachydactyly, accelerated skeletal maturation), feeding problems and behavioral difficulties ( Figure 4A) ( Table 2) [23][24][25]. KMT2A variants are distributed throughout the gene, with a pathogenic mutation hotspot in exon 27, and most of them lead to KMT2A loss of function. WDSTS patients usually present de novo private mutations, and the diagnosis is based on clinical evaluation of signs and symptoms then confirmed by molecular analysis. Unfortunately, a specific treatment is not available, thus possible interventions aim at reducing the severity of symptoms. As other members of KMTs family, KMT2A regulates gene transcription through chromatin opening or closure and its activity is antagonized by the lysine demethylases (KDMs) family.

Wiedemann-Steiner Syndrome
KMT2A germinal variants are associated to the Wiedemann-Steiner syndrome (WD-STS, OMIM #605130), a rare autosomal dominant disorder characterized by different features, mainly intellectual disability (ID), developmental delay (DD), pre-and postnatal growth deficiency, hypertrichosis, short stature, hypotonia, distinctive facial features (thick eyebrows, long eyelashes, narrow palpebral fissures, broad nasal tip, down slanting palpebral fissures), skeletal abnormalities (clinodactyly, brachydactyly, accelerated skeletal maturation), feeding problems and behavioral difficulties ( Figure 4A) ( Table 2) [23][24][25]. KMT2A variants are distributed throughout the gene, with a pathogenic mutation hotspot in exon 27, and most of them lead to KMT2A loss of function. WDSTS patients usually present de novo private mutations, and the diagnosis is based on clinical evaluation of signs and symptoms then confirmed by molecular analysis. Unfortunately, a specific treatment is not available, thus possible interventions aim at reducing the severity of symptoms.

Other Chromatinopathies
Mutations in KMT2A have been also found in patients with a clinical presentation suggestive of other chromatinopathies but negative for alterations in the related knowncausative genes. Their clinical presentation shares with WDSTS some phenotypic features and it is caused by alterations of genes involved in the regulation and maintenance of chromatin state as KMT2A. Indeed, these syndromes are caused by mutations in genes of the epigenetic machinery and therefore are known as chromatinopathies [16,18].

Other Chromatinopathies
Mutations in KMT2A have been also found in patients with a clinical presentation suggestive of other chromatinopathies but negative for alterations in the related knowncausative genes. Their clinical presentation shares with WDSTS some phenotypic features and it is caused by alterations of genes involved in the regulation and maintenance of chromatin state as KMT2A. Indeed, these syndromes are caused by mutations in genes of the epigenetic machinery and therefore are known as chromatinopathies [16,18].
Thanks to targeted sequencing and genome-wide DNA methylation analyses, in 2017, Sobreira and colleagues, investigating a cohort of 27 patients with a clinical diagnosis of Kabuki syndrome (KS1, OMIM #147920; KS2, OMIM #300867), found two patients positive for mutations in KMT2A (a de novo heterozygous missense mutation in pt#KS8 and a donor splice site mutation in pt#KS29) [31]. Kabuki syndrome is a congenital disease with a broad and variable spectrum, characterized by mild-to-moderate cognitive disability, post-natal growth deficit, characteristic facial features (long eyelid cracks with slight ectropion of lateral third of the lower eyelid), skeletal abnormalities and immunodeficiency ( Figure 4C) [32]. In about 60% of KS cases, the syndrome is caused by mutations in KMT2D (12q13.12, OMIM #602113; associated with KSS1), also known as MLL2, while in a few cases the causative mutation is carried by the KDM6A gene (Xp11.3, OMIM #300128; associated with KSS2). KMT2D is a methyltransferase that plays crucial roles in development, differentiation, metabolism, and tumor suppression [33]. Both patients analysed by Sobreira and colleagues presented hypotonia, persistent fetal fingerpads, eversion of the lower lateral lid and long palpebral fissure; patient #KS8 in addition showed seizures and recurrent infection and brachydactyly, while patient #KS29 presented ID and feeding difficulties ( Table 2) [31].

Effects of KMT2A Mutations in Animal Models
KMT2A is an evolutionary conserved gene, involved in several functional process of embryonic development, ranging from hematopoiesis to neurogenesis. Indeed, in 1995, Yu and colleagues showed that the complete disruption of KMT2A was embryonic lethal in mice, and heterozygous animals were anemic and affected by growth delay, hematopoietic anomalies and skeletal malformations [54]. Developmental defects were investigated in Drosophila melanogaster too, where mutations in KMT2A homolog (trx) led to a wide range of homeotic transformations [55]. Interestingly, KMT2A was demonstrated as having an important role in the maintenance of memory Th2 cell function [56] and in hematopoiesis, as its absence caused defects both in self-renewal of murine hematopoietic stem cells and in hematopoietic progenitor cell differentiation in zebrafish [57,58]. In addition, impairments in neural development were observed knocking down Kmt2a in zebrafish, and in murine models Mll1 was identified as a crucial component in memory formation, complex behaviors and synaptic plasticity [59][60][61][62][63].
Thus, KMT2A-depleted animal models recapitulate phenotypes described for patients with both germline and somatic mutations. KMT2A associated syndromes show clinical signs such as ID, behavioral problems, speech and growth delay and peculiar dysmorphisms, while the most frequent tumors enriched in KMT2A mutations are the

Effects of KMT2A Mutations in Animal Models
KMT2A is an evolutionary conserved gene, involved in several functional process of embryonic development, ranging from hematopoiesis to neurogenesis. Indeed, in 1995, Yu and colleagues showed that the complete disruption of KMT2A was embryonic lethal in mice, and heterozygous animals were anemic and affected by growth delay, hematopoietic anomalies and skeletal malformations [54]. Developmental defects were investigated in Drosophila melanogaster too, where mutations in KMT2A homolog (trx) led to a wide range of homeotic transformations [55]. Interestingly, KMT2A was demonstrated as having an important role in the maintenance of memory Th2 cell function [56] and in hematopoiesis, as its absence caused defects both in self-renewal of murine hematopoietic stem cells and in hematopoietic progenitor cell differentiation in zebrafish [57,58]. In addition, impairments in neural development were observed knocking down Kmt2a in zebrafish, and in murine models Mll1 was identified as a crucial component in memory formation, complex behaviors and synaptic plasticity [59][60][61][62][63].
Thus, KMT2A-depleted animal models recapitulate phenotypes described for patients with both germline and somatic mutations. KMT2A associated syndromes show clinical signs such as ID, behavioral problems, speech and growth delay and peculiar dysmorphisms, while the most frequent tumors enriched in KMT2A mutations are the hematological ones (e.g., B-cell lymphoma, T-cell lymphoblastic leukemia, acute myeloid leukemia), according to neurodevelopmental and hematopoietic defects found in the aforementioned in vivo models.

Epigenetic Strategies for Pharmacological Approaches
Targeting the regulators of lysine methylation is an emerging strategy for therapeutic approaches, given the role of chromatin post translational modification in regulating gene expression, and considering that lysine methylation has a pivotal role in this process. Indeed, mutations in one of the components of the epigenetic machinery affect the normal pattern of covalent histone modifications, leading to an incorrect gene expression pattern that may consequently result in tumor evolution. In addition, given the very high specificity of each methyltransferase to its target, the development of drugs directed to those enzymes would have the advantage to minimize the off-target effects [64].
As described above, KMT2A alterations have been reported in several blood cancers such as mixed-lineage, acute lymphoblastic and acute myeloid leukemia [65]. Acute leukemia with rearrangements of the KMT2A gene (KMT2Ar) is associated with a higher risk of relapse and is more resistant to standard therapies. KMT2A exerts its function by forming a core-complex with other proteins [66]; for this reason, the inhibition of KMT2A with its interaction partners, both histone and non-histone proteins, is a promising pharmacological strategy when KMT2A rearrangements are drivers of pathology, such as in leukemia. For example, recent studies have shown that the use of peptidomimetics disrupting the interaction between KMT2A and WDR5 (a member of the above-mentioned core-complex) in murine cell line reduces the expression of target genes responsible for KMT2A-mediated leukemogenesis and inhibits the growth of leukemia cells [67,68].
Similarly, it was demonstrated that the small molecule EPZ-5676 has a modest clinical activity reducing the proliferation of MLL-rearranged cells and inducing apoptosis by targeting the enzymatic core of DOT1L, a H3K79 methyltransferase recruited to fusion partners of KMT2A in disease-linked translocations and required for leukemogenesis [69][70][71][72]. Advances in treating MLL-rearranged leukemia were also achieved by using small molecules to block the KMT2A binding site on Menin, a protein encoded by MEN1 and required for oncogenic transformation, leading to the inhibition of the aberrant leukemogenic transcription program [73][74][75][76][77].
Another pharmacological efficient approach in cancer treatment might be the targeting of pathways deregulated in tumorigenesis. Indeed, the inhibition of glycogen synthase kinase 3 (GSK3) can induce the growth arrest of leukemia cells in KMT2Ar leukemia [78], while targeting the DNA damage response (DDR) pathway can lead to specific synthetic lethality in leukemic cells with MLL-rearrangements [79].
Besides the leukemia treatment, the KMT inhibitors are considered potential drugs for other cancers. In particular, Tazemetostat has been approved in January 2020 for the treatment of a rare tumor, epithelioid sarcoma, and then for follicular lymphoma, sustaining the role of the lysine methylation pathways as potential effective targets for treating various diseases [80].
On the contrary, in genetic disorders related to KMT2A, the altered histone methylation status is mainly attributed to loss of functions mutations or missense mutations involving this gene. For this reason, a possible pharmacological approach could counteract the lack of KMT2A activity.
Altered epigenetic control of gene expression may cause psychosis and other psychiatric diseases, it was demonstrated that the atypical antipsychotic clozapine can induce the methylation of GABAergic gene promoters through Mll1 recruitment in a mouse model of schizophrenia [81,82]. Moreover, a study comparing clozapine-responder and non-responder twins demonstrated that clozapine increases DNA methylation of the MECP2 promoter, leading to its downregulation, and consequently enhancing the expression of genes that are regulated by MeCP2 protein [83]. Similarly, the antidepressant phenelzine and its analogue bizine enhance H3K4me2 status in H460, A549 and MDA-MB-231 cancer cell lines by inhibiting the activity of the histone demethylase LSD1 [84]. Furthermore, tranylcypromine (TCP), another antidepressant, has been demonstrated to specifically inhibit LSD1, and its administration in combination with all-trans-retinoic-acid (ATRA) induces the differentiation of acute promyelocytic leukemia (APL) and acute myeloid leukemia (AML) blasts [85]. Moreover, a phase I/II trial (ClinicalTrials.gov: NCT02261779) have demonstrated that TCP-ATRA combined therapy can be used to treat refractory or relapsed AML patients, even if the required high dosage and the prolonged treatment may cause the onset of several side-effects [86]. For this reason, a selective LSD1 inhibitor, ORY-1001, has been developed using TCP structure. Sub-nanomolar doses of this molecule reduce the proliferation of MLL-translocated leukemic cell lines, both in vitro and in vivo, and display synergistic action with the common anti-leukemic drugs, opening the possibility of a targeted and personalized therapy [87]. A phase I/IIa clinical trial has already evaluated the tolerability, pharmacokinetics and pharmacodynamics of ORY-1001 in relapsed or acute refractory leukemia (EUDRACT no.2013-002447-29) [88].
Interestingly, epigenetic interventions could be either pharmaceutical or nutritional. It is well known that dynamic crosstalk between gut microbiota and the host is present and that it can be modulated by diet. Krautkramer and colleagues reported that microbiota regulates histone methylation and acetylation in different tissues as a diet-dependent process [89] and, notably, a microbiota-dependent epigenetic signature was reported in specific diseases, e.g., inflammatory bowel disease [90]. Indeed, the microbial community within the intestine can produce metabolites such as short-chain fatty acids (SCFAs) with a known role of histone deacetylase (HDAC) inhibitors. These compounds or diets able to increase them were recently used as possible therapeutic approach for several diseases, including drugresistant epilepsy [91,92], cancer [93], neurodegenerative disease [94], heart failure [95], and diabetes mellitus [96], and their effect was even studied in experimental models of chromatinopathies, i.e., Kabuki syndrome [97] and Rubinstein-Taybi syndrome [98]. Furthermore, bacteria synthetize essential vitamins, fundamental for immune systems, such as B12, but also folate, required for DNA, histone and protein methylation [99,100]. Intriguingly, in a kdm5-deficient Drosophila model, not only an increase in gut H3K4me3 but also the disruption of intestinal barrier together with aberrant immune activation and anomalies in social behavior were observed. All these changes correlated with alterations in gut microbiota composition, which were rescued by probiotic administration [101].
Thus, considering the latest developments on epigenetic intervention, a deepening understanding of microbiota composition of patients with KMT2A mutations could help new therapeutic approaches investigation among the epigenetic treatments.

Final Remarks
Epigenetic modifications are fundamental for many biological processes; indeed, alterations of genes with this activity can lead to neurodevelopmental disorders or tumorigenesis, when germinal or somatic mutations respectively occur [102,103]. This is the case of KMT2A, a lysin methyltransferase-coding gene, whose variants are associated with a chromatinopathy (WDSTS) at germinal level or can be found in both blood cancers and solid tumors in regard to malignancies.
Interestingly, due to exome-and genome-wide analyses, patients described above with a defined initial chromatinopathy diagnosis but lacking the molecular one were found to be carriers of pathogenetic variants in the KMT2A gene and could have obtained a clinical re-evaluation. In detail, nearly the totality of patients previously diagnosed with CdLS, CSS, KS and RSTS showed features common to WDSTS, such as ID (11/12), speech delay (7/10), peculiar dysmorphisms affecting eyes (12/12) (i.e., thick eyebrows, synophrys, long eyelashes, ptosis and downslanting/narrow palpebral fissure) and nose (12/12) (i.e., depressed nasal bridge and broad nasal tip), while about half of them shared with WDSTS feeding problems (5/10), hirsutism (6/10) and hypotonia (6/10). Oddly, almost all of these patients displayed features less frequently present in WDSTS, such as dysmorphisms affecting mouth (7/12) (i.e., high arched palate and thin upper vermilion) and anomalies of hands/feet (11/12) (i.e., clinodactyly, brachydactyly, persistent fetal fingerpads and broad halluces). Indeed, mutations in different genes involved in the regulation and maintenance of chromatin state can lead to a clinical overlapping phenotype, suggesting a common affected pathway during embryonic development and the evaluation of an expanded set of genes when investigating the molecular causes for a correct diagnosis of these syndromes.
In addition, somatic mutations in KMT2A have been reported in different tumors, as well as alterations in all KMT2 family genes [104] and in other genes associated to chromatinopathies. Curiously, we observed that germline mutations described in the literature are more frequently nonsense than missense, in contrast to somatic ones. This could be explained by the consequent loss of function mechanism characterizing most of chromatinopathies due to a defective protein production, which strongly impacts on embryonic development.
To conclude, since molecular defects in KMT2A also characterize some types of tumors, and research in the field of epigenetic drugs for malignancies is rapidly evolving [101], a therapeutic approach targeting KMT2A interaction or its pathway could be considered also for chromatinopathies, modulating epigenetic dysfunction with pharmaceutical products or diet-based interventions.

Conflicts of Interest:
The authors declare no conflict of interest.