Identification of new leukemia associated gene mutations by whole genome sequencing of DNA from two siblings with congenital neutropenia and secondary AML

We have performed a whole genome sequencing (WGS) of DNA samples from a family with two affected siblings suffering from CN. Both children harbored ELANE gene mutations and developed AML. Father of the children demonstrates somatic mosaicism for the ELANE mutation and has a mild neutropenia. For WGS we used Complete Genomics technology (Complete Genomics, USA). More than 90% of genomes were sequenced at high quality with minimum coverage of at least 20-fold. As an example, 3437845 single nucleotide variants (SNVs) were identified in DNA isolated from leukemia blasts of sequenced CN patient. The set of filters were used to identify candidate mutations in the leukemic cells from affected siblings. At the end, 14 candidate SNVs with predicted damaging effects on the protein function were used for further analysis. All candidate SNVs were validated by Sanger sequencing. We detected nine inherited candidate SNVs presented in the two affected children but not in healthy siblings. SNV in the ELANE gene (dbsnp.129:rs57246956) was inherited from the father. Novel SNVs inherited from the father were as follows: in the TCTE1, FAM135A, M6PR, C20orf144 and PTPN23 genes. Only three SNVs were inherited from the healthy mother (in BLOC1S1, DUS3L and KIAA1543 genes). All SNVs were heterozygous. We also found 5 sporadic SNVs presented in leukemia sample of sequenced CN patient. These are heterozygous mutations in the CSF3R, ACAP2, GRM1, LASS3, and RUNX1 gene. Interestingly, both affected patients had a somatic mutation in the RUNX1 gene at the same nucleotide position (c.415C>G, p.R139G in sick brother and c.415C>T, p.R139* in sick sister). Our study also establishes WGS as an unbiased method for discovering leukemia-associated mutations.