Genetic Alterations Precede DNA Methylation Changes in Juvenile Myelomonocytic Leukemia

Introduction Juvenile myelomonocytic leukemia (JMML) is a rare and highly aggressive myeloid malignancy of early childhood. Approximately 95% of patients have at least one mutation that leads to hyperactive RAS signaling. Both the presence of multiple mutations at diagnosis as well as altered DNA methylation are associated with a poor prognosis. Whether altered DNA methylation leads to the acquisition of additional genetic mutations or is a secondary event to genetic mutations is unknown. In this study, we analyzed a total of 33 newborn blood screening (NBS) cards from children who went on to develop JMML later in childhood. Using next generation sequencing to detect both genetic mutations and DNA methylation status we sought to determine the sequence of events that lead to the development of JMML. Patients and Methods We identified 33 patients that were born in the state of California from 1990 to 2017 who were confirmed to have JMML and were previously consented to participate in a JMML tissue bank study. Guthrie cards from these 33 patients as well as 12 healthy controls were obtained from the California Biobank Program. DNA samples (20 ng) were sequenced for mutations using a custom amplicon-based sequencing approach (Paragon Genomics, Hayward, CA) targeting 26 genes that are recurrently mutated in JMML. For DNA methylation profiling, 300ng of genomic DNA was processed for bisulfite conversion using the TrueMethyl oxBS Module (Tecan Genomics Inc., Redwood City, CA.) according to manufacturer's guidelines and next generetation sequencing (NGS) libraries were prepared by targeting 3000 CpG loci using custom probes for Targeted Methyl-Seq assay (Tecan Genomics Inc., Redwood City, CA). Sequence reads were trimmed using Cutadapt to remove adapters and methylation status called using Bismark. Samples were classified into one of three methylation subgroups: Low, Intermediate, or High using 1386 probes according to an international consensus definition. Results At diagnosis of JMML, somatic mutations were identified in 32 of 33 patients. Of the 32 patients with a somatic mutation present upon diagnosis, the same mutations were found in NBS cards in 13 (41%) patients using a mutant allele fraction (MAF) cut-off of 1%. Clonal mutations (MAF >15%) were found in 9 of 32 (28%) patients. Patients who had a somatic mutation detected at birth were significantly younger at diagnosis with a median age of 7.1 months (range 2.5-91.6 months) compared to patients who had no