Mutations in Histone Lysine Methyltransferase Genes Are Associated with Autoimmune Cytopenias
Authors: Cole Daniel , Xiao Zhengrui , Gupta Varun , Anampa Mesias Jesus D. , Townsend Noelle , Mackie Gretchen , Sanghvi Ami , Thakur Rahul , Lachman Herbert , Murakhovskaya Irina 
Published: December 2023

Introduction
Epigenetic mechanisms are important in regulation of gene expression by modifying chromatin structure to facilitate DNA accessibility (Lau et al Nat Immunol. 2018). DNA methylation and histone modifications are critical for the pathogenesis of hematologic malignancies and autoimmune diseases (Surace et al Front Immunol. 2019) Alterations in enzymes involved in histone modification are associated with immunodeficiencies, including Kabuki syndrome (KS), a rare genetic disorder associated with autoimmune cytopenias, such as immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA) (Margot et al J Am Coll Med Genet 2020) caused by loss-of-function KMT2D and KDM6A mutations. Somatic mutations in the KMT2D gene have also been observed in patients with Cold Agglutinin Disease. (Małecka et al Br J Haematol. 2018).
Aims:
The aims of this study are: 1) to identify the frequency of mutations in histone methyltransferase genes (MT) KMT2D, KMT2A, KMT2C and KDM6A in patients with autoimmune cytopenias, and 2) to compare clinical, laboratory and immunological characteristics, and response to therapy in patients with or without mutation in MT and autoimmune cytopenias (AIC).
Methods
Data mining software from the electronic medical record was used to identify patients ≥ 18 years with a diagnosis of AIC, including ITP, warm and cold AIHA, autoimmune neutropenia, and Evans Syndrome (ES), who had Next Generation Sequencing (NGS) performed. Cytopenias due to malignancy, nutritional deficiency, medications, pregnancy, or infection were excluded. Patients were categorized into two groups based on the presence or absence of mutation in the MT genes (MT+, MT-) with variant allelic frequency (VAF) &gt; 2%. Demographic, laboratory, and clinical characteristics, treatment outcomes, and NGS sequencing data were collected and compared between groups. Next, KMT2 mutations seen in patients with AIC were cross-referenced against the 1000 Genome Project (1000GP) population database to evaluate the frequency of polymorphisms in the general population (Auton et al Nature 2015). CADD in silico prediction algorithm was used to score the potential deleterious effect of MT mutations (Kirchner Nat Genet. 2014). Chi-Square test was used for categorical variables. Statistical analysis was completed in R software.
Results
Among 495 patients who underwent NGS, 79 (16%) had a mutation in MT genes. These mutations were present in 36% of patients with AIC as compared to 13% in those without AIC (p=0.0005). Among 55 patients with AIC, mutations in the MT genes were present in 20 patients (MT+), 35 patients did not have these mutations (MT-). KMT2C mutations were seen in 10 (50%) patients, KMT2D mutations in 9 patients (45%, one patient had two KMT2D mutations), 1 patient (5%) had both KMT2C & KMT2D variants (Figure 1). The median VAF was 39.5% (IQR, 15.45 - 49.80). The MT+ group had a significantly higher proportion of Hispanic (60% vs 31%) and non-Hispanic White patients (25% vs 14%), p=0.038, Table 1. The median age at diagnosis for the MT+ cohort was younger compared to MT- (45.5 vs 58 years old, respectively), though not statistically significant.
AIC diagnoses in both groups were ITP (44%) wAIHA (22%) cAIHA (9%) Autoimmune Neutropenia (2%), and ES (24%). Compared to the MT- group, the MT+ group had lower IgG levels (p=0.0041) and absolute lymphocyte count (p=0.041). MT+ patients with AIHA were more likely to have positive Direct Coombs and complement involvement. No significant differences were found in median nadir hemoglobin and platelet counts, need for therapy, or median number of lines of therapy. Best overall response to therapy was similar in the two groups (93%), with more complete responses in MT- group (55% vs 33%) though not statistically significant. Next, we assessed KMT2 polymorphisms in the general population. Most of the polymorphisms were not present in the 1000GP database and none had a frequency &gt; 0.001. The average Phred score was 19.8; 12/20 patients had Phred score &gt; 20, suggesting that most mutations are in the top 1% of the deleterious variants.
Conclusion
We report for the first time that patients with autoimmune cytopenias have a high frequency of variants in MT genes. Median allelic frequency close to 50% suggests potential germline predisposition to immune dysregulation and autoimmunity however further studies are needed to better understand the impact of these observations.