Heterozygous Variants in the DNA-binding Domain of C-Myb May Affect Normal B/T Cell Development

Inborn errors of immunity (IEI) are serious immunological disorders characterized by aberrant development, proliferation, regulation, or function of immune cells.1 The introduction of next-generation sequencing has facilitated the discovery of new causes of IEI.2 Already >400 IEI-related genes have been identified and classified according to the affected component of the immune system.1 The MYB proto-oncogene (MYB) has previously been identified in both in vitro and in vivo models as a key player in transcription factor networks involved in stem cell and hematopoietic cell development but has not yet been related to IEI.3 While homozygous null variants of MYB in mice have been shown to be lethal, heterozygous, temporal and local null models of the DNA-binding domain exon in mice and human cell lines have shown that its product, the c-Myb transcription factor, is crucial for pro- and pre-B cell differentiation by controlling the expression of interleukin-7 receptor-α, and recombinase activating gene (Rag) and the initiation of survival signals.3,4 Moreover, c-Myb is vital for thymocyte development and regulates 2 distinct pathways in mature CD4+ and CD8+ cells; in CD4+ cells, it is involved in T helper 2 (Th2) development through GATA binding protein-3 (GATA3) regulation, while it regulates central memory stemness in CD8+ cells, through Tcf7 and Bcl2 upregulation and Zeb2 repression (Figure 1A).5,6Figure 1.: c-Myb and its downstream targets are differently expressed in 2 technical replicates of T cells carrying the p.Lys182Arg variant. Results were analyzed with 2-tailed Student t tests, *P < 0.05 **P < 0.01. (A), In activated mice CD8+ T cells, transcription factor c-Myb has been found to increase Tcf7 and Bcl2 expression, while repressing Zeb2 expression through competitive binding to the promoter.5 (B), Proportions of CD3+ subsets in the patient vs 3 healthy controls showing that the patient has reduced naive CD3+ fractions and increased TEMRA fractions. (C), Expression of levels of c-Myb, Tcf7, Bcl2, and Zeb2 in unstimulated T cells. 2^–ΔΔCt values were obtained using the mean ΔCt values of the healthy controls. Zeb2 expression is higher in patient samples. (D), Peak c-Myb, Tcf7, and Bcl2 expression and Zeb2 repression in T cells after stimulation. 2^–ΔΔCt values were obtained using the mean ΔCt values of unstimulated T cells. Patient cells show a trend towards decreased c-Myb, Tcf7, and Bcl2 expression and similar Zeb2 expression. (E–G), MFI of c-Myb, Tcf7, and Zeb2 in CD3+, CD4+, and CD8+ cells. Peak protein expression of both c-Myb and Tcf7 was lower in CD3+ and CD8+ cells but not in CD4+ cells. MFI = mean fluorescence intensity; TEMRA = T effector memory cells expressing CD45RA.Here, we describe 2 patients who presented with a combined immunodeficiency that progressed into severe bone marrow dysfunction with distinct de novo MYB heterozygous DNA-binding domain variants. Participants provided written informed consent for institutional review board-approved studies in the Netherlands (National PID study, METC: NL40331.078) and Canada (Care4Rare). Single nucleotide polymorphism (SNP)-array copy number variant (CNV) profiling and analysis of regions of homozygosity were performed on DNA isolated from peripheral blood according to standard procedures, using the Infinium Human CytoSNP-850K v1.0 BeadChip (Illumina) and the Nexus software v7 (BioDiscovery) with Human genome build February 2009 GRCh37/hg19. Peripheral blood mononuclear cells were isolated from heparinized blood according to standardized protocols and stored in liquid nitrogen. After thawing, T cells were isolated with CD3+ magnetic beads and MS Columns (Miltenyi Biotec) and seeded in 24-wells plates at a 1 × 10^6/mL density and cultured for 0, 24, 72, and 120 hours with CD3/CD28 Dynabeads (Thermo Fisher Scientific). RNA was isolated with the RNeasy Mini Kit (Qiagen) and complementary DNA was obtained using the RevertAid First Strand Kit (Thermo Fisher Scientific). Primers selected from earlier publications (Suppl. Table S1, Integrated DNA Technologies) were used for real-time polymerase chain reaction (PCR) using the SYBR Select Master Mix (Thermo Fisher Scientific).7–9 Antibodies used for flow cytometry are listed in Suppl. Table S2. For intracellular staining of c-Myb, Tcf7, and Zeb2, cells were fixed and permeabilized (eBioscience, 00–5524). BDFortessa II (BD Biosciences) was used for flow cytometry acquisition. Samples were analyzed with FlowJo software (TreeStar). Telomere length was measured by a 2-panel assay by flow fish technology at RepeatDx (https://repeatdx.com). Results were analyzed using the 2-tailed Student t test in GraphPad Prism v9. A 22-year-old male of Dutch origin with a history of tetralogy of Fallot presented with recurrent lower tract infections at the age of 3 years. Laboratory test results showed reduced IgG and IgM and absent peripheral B cells. Fourteen B cell disorder-associated genes were analyzed and no disease-associated variants were identified. He was diagnosed with common variable immunodeficiency and treated with intravenous immunoglobulin replacement therapy (IgRT). At the age of 9 years, he developed a slowly progressive thrombocytopenia and at the age of 19 years, bone marrow examination showed normal cellularity with dysplastic features in all lineages. Furthermore, he developed oral aphthous lesions and leukoplakia. Immunological investigations showed monocytopenia, neutropenia, reduced natural killer (NK) cells and low fractions of naive CD8+ T cells with impaired proliferation upon antigen stimulation and a reduced V-beta repertoire (Table 1). Table 1 - Baseline Characteristics, CBC at Onset, Immunoglobulins at Onset, Immunophenotyping at Onset, and Functional Tests of Both MYB Cases Parameter Case 1 Case 2 Baseline characteristics Sex Male Male Age at presentation 3 y 3 y Current age 23 y 11 y MYB variant c.545A>G c.383A>G CID classification T+B–NK– T+B–NK+ CBC Hemoglobin (mmol/L) 8.1 8.3 Platelets (×10^9/L) 326 148 Leukocytes (×10^9/L) 4.6 2.1 Neutrophils (×10^9/L) 1.32 2.1 Lymphocytes (×10^9/L) 2.39 0.2 Igs IgM (g/L) <0.04 0.25 IgA (g/L) 0.55 <0.0667 IgG (g/L) 5.19 0.34 Immunophenotyping CD3+ (×10^9/L) 2.3 0.726 CD3+CD4+ (×10^9/L) 1.2 0.440 CD3+CD8+ (×10^9/L) 1.1 0.165 CD19+ (×10^9/L) 0 0.011 CD56+ (×10^9/L) 0.03 0.319 Functional tests TREC NA Absent v-beta repertoire Reduced Normal T cell proliferation Reduced for CD8+ Normal Lymphocytic telomeres G variant in MYB causing a p.(Lys182Arg) substitution within the DNA-binding domain of c-Myb. This variant had never been reported in genome aggregation database (gnomAD) and prediction software predicted the variant to be damaging. Hence the variant was classified as a variant of unknown significance and was submitted to the Matchmaker Exchange in order to identify other unrelated individuals with rare variants in the same gene and overlapping phenotypes.10 Other pathogenic variants in known genes associated with primary immunodeficiencies were not found and SNP-array analysis showed a normal array profile without indications for pathogenic CNVs in 22q11. T cell phenotyping showed decreased naive T cell fractions and increased TEMRA fractions in the patient compared with healthy controls and age-adjusted reference values (Figure 1B). Real-time reverse transcription PCR showed that c-Myb, Tcf7, and Bcl2 expression was comparable to that of healthy controls in unstimulated T cells, while Zeb2 expression was increased 11-fold (Figure 1C; P < 0.01). After 72 hours of CD3/CD28 stimulation, peak c-Myb and Bcl2 expression was reached, while Tcf7 expression and Zeb2 repression peaked after 120 hours (Suppl. Figure S1). There was a trend towards lower peak expression of c-Myb, Tcf7, and Bcl2 (P = 0.19, P = 0.19 and P = 0.21) in patient cells, while peak repression of Zeb2 was not significantly different (P = 0.42) (Figure 1D). Protein analyses using flow cytometry for c-Myb, Tcf7, and Zeb2 showed similar results (Suppl. Figure S2), and additionally indicated that expression of c-Myb and Tcf7 was especially hampered in the patient’s CD8+ T cells (P < 0.01 and P < 0.01) (Figure 1E–G). A telomere length assay demonstrated extremely short lymphocytic telomeres (