Abnormal promoter DNA methylation in juvenile myelomonocytic leukemia is not caused by mutation in DNMT3A

To the editor:

Several recent publications reported the occurrence of mutations in the DNA methyltransferase 3A (DNMT3A) gene in acute myeloid leukemia (AML).^1-3  The majority of DNMT3A mutations identified affect residue R882 which is located within the methyltransferase domain.^1,2  A strong predilection for leukemias of monocytic or myelomonocytic lineage was observed.^2,3  This, and our previous findings linking altered DNA methylation patterns with poor prognosis in juvenile myelomonocytic leukemia (JMML),⁴  led us to hypothesize that somatic DNMT3A mutations might also occur in JMML.

We bidirectionally sequenced DNMT3A exon 23 (containing the hotspot codon 882) in granulocyte DNA from 113 JMML patients. All children were enrolled in the European Working Group of MDS in Childhood (EWOG-MDS) studies 98 or 2006, and informed consent had been obtained from patients' guardians. The patients' median age was 2.0 years. The hotspot mutation DNMT3A p.R882H was identified in JMML patient D101 (age at diagnosis 6.5 years, somatic NRAS^G12V mutation; Figure 1A), while all other samples exhibited wild-type sequence. Epstein-Barr virus-transformed B lymphocytes of D101 carried wild-type sequence, indicating somatic origin of the DNMT3A mutation.

Although DNMT3A is generally overexpressed in AML blasts versus healthy leukocytes,⁵  the expression does not correlate with occurrence of DNMT3A mutations.¹  Despite the finding that DNMT3A codon 882 mutations lead to reduced enzymatic activity,²  the genome-wide DNA methylation pattern of DNMT3A codon 882-mutant AML cells does not differ significantly from that of DNMT3A wild-type blasts.¹  We measured DNMT3A mRNA expression in mononuclear cells from 9 JMML patients by quantitative real-time PCR (RNA from patient D101 was unavailable). The abundance of the DNMT3A transcript in JMML cells varied considerably, with some cases expressing DNMT3A at higher levels than leukocytes from healthy subjects, and others at lower levels (range, 0.08-14.05 fold; Figure 1B). Unlike in AML where uniform blast populations are studied, our results may reflect the heterogeneous cellular composition of JMML samples. DNA methylation data were available for 6 of the 9 JMML samples studied,⁴  but there was no association of high DNMT3A expression with DNA hypermethylation (Figure 1C). Remarkably, leukemic cells of D101 carried hypermethylated BMP4, CALCA and CDKN2B promoters⁴  despite the presence of a DNMT3A loss-of-function mutation.

Several investigators highlighted the possible pathophysiologic role of the DNA-methylating enzyme DNMT3A in myeloid malignant disorders other than JMML.^1-3,6,7  We conclude that genetic or transcriptional aberrations of DNMT3A do not contribute to leukemogenesis in JMML. While the recurrent nature of DNMT3A alterations in AML, the strong preference to target codon 882 and the prognostic significance argue in favor of non-random pathogenetic relevance in AML, a DNA methylation profile that would distinguish DNMT3A-mutant AML cases has not yet been defined. By contrast, JMML cases with poor outcome exhibit promoter hypermethylation of several genes⁴  but for the most part lack DNMT3A alterations. The data presented here fit well with previous observations that DNMT3A mutations are much less prevalent in myeloid malignancies of children than in adults.^6,8  In summary, the functional implication of DNMT3A lesions in myeloid leukemogenesis requires further clarification.