NADH dehydrogenase subunit 4 (ND4) is encoded by mitochondrial DNA and is an integral component of Complex I, one of the core enzymatic complexes critical for mitochondrial oxidative phosphorylation and regulation of the balance between NADH and NAD+. ND4 mutations have recently been described in adult acute myeloid leukemia (AML). In the current study, we investigated the frequency and prognostic impact of ND4 mutations in 289 pediatric leukemia patients (<= 18 years). Total cellular DNA was isolated from bone marrow or peripheral blood samples at diagnosis (n=289) and at complete remission (n=6) for children treated uniformly within multicenter treatment trials AML-Berlin-Frankfurt-Münster (BFM, n=180) and Dutch Childhood Oncology Group (DCOG, n=109).
ND4 mutations were detected by direct sequencing in 13 of 289 (4.5 %) pediatric AML patients. Mutations occurred throughout the ND4 sequence, and included missense mutations (n=10), deletions (n=2) and a nonsense mutation. The most commonly detected mutations were S86N (n=2), delA 11,032–11,038 (n=2), and F50L (n=2). All other mutations were detected in single cases. Four (30.8 %) ND4 mutations were heteroplasmic (i.e. both wild-type and mutated ND4 were detected) and 9 (69.2 %) were homoplasmic (i.e. only mutated ND4 was detected), which is similar to the distribution we previously observed for adult AML patients (37.9% and 62.1%, respectively). Of the 4 heteroplasmic mutations detected in the pediatric AML cohort, 3 are predicted to result in a truncated ND4 protein. The remaining heteroplasmic mutation, which results in an L72P substitution, is predicted to be damaging (PolyPhen2 score = 0.999). Thus all 4 heteroplasmic mutations are expected to interfere with ND4 protein function. In contrast, 3 of the 9 (33.3 %) homoplasmic mutations are within transmembrane regions and only 1 (11.1 %) is predicted to be damaging (S459Y, PolyPhen2 score = 0.906). The 11 predicted transmembrane domains (TMD) of ND4 may be important for mitochondrial proton transport. However, like in adult AML, the presence of ND4 mutations affecting or not affecting a TMD had no impact on pediatric AML patient outcome.
Non-tumoral DNA available through samples collected in routine follow-up examinations during complete remission allowed determination of mutation origin (e.g. somatic or germ-line) in 6 cases. Interestingly, the homoplasmic substitutions resulting in F50L, S86N and A131T were each defined to be germline mutations in both adult and pediatric AML samples. The heteroplasmic one base-pair deletion in a stretch of seven adenine residues (11,032–11,038) detected in two pediatric leukemia samples was determined to be somatic in the one case for whom a sample obtained during complete remission was available for analyses. Patient characteristics including age, FAB-subtype, WBC count, cytogenetic subgroup or presence of FLT3-ITD were similar regardless of ND4 mutation status.
In accordance with our earlier observations in adult AML, comparison of ND4mutated with ND4wildtype patients demonstrated no significant difference on overall survival (OS, P=.67). In the adult study, a survival advantage was observed for patients with somatic heteroplasmic ND4 mutations. No survival advantage was observed for children with heteroplasmic ND4 mutations, possibly due to limited numbers of ND4mutated patients treated in the BFM and DCOG study groups.
Gene expression profiles (GEP) for ND4mutated (n=11) and ND4wild-type (n=188) pediatric AML patients revealed no significant differences. However, 8 probe sets were found to be differentially regulated when GEP for heteroplasmic ND4mutated (n=4) and ND4wildtype (n=187) were compared. Two of these probe sets annotated the SETDB2 (CLLD8, KMT1F) gene, which encodes a histone H3 methyltransferase. Quantitative RT-PCR validated the lower SETDB2 expression as a characteristic of ND4mutated cases (P=.02). SETDB2 contributes to several important cellular functions, including heterochromatin formation, chromatin condensation and transcriptional repression.
In summary, ND4 mutations were not predictive for outcome in pediatric AML, but were significantly associated with decreased SETDB2 expression, providing a link between mitochondrial gene mutation and epigenetic control of gene expression.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.