Targeted sequencing

Targeted sequencing of 10 known disease genes (ACD, ANKRD26, CEBPA, DDX41, ETV6, GATA2, RUNX1, SRP72, TERC, and TERT) was performed for the index case of each family using the accredited testing facility at the West Midlands Regional Genetics Laboratories (WMRGL) in Birmingham, UK [https://www.geneadviser.com/genetictest/familial_mds_familial_aml_sequencing_birmingham]. Variants were considered pathogenic if they had been previously described to be associated with familial MDS/AML or new variants (SNVs or indels) meeting the following criteria: (1) minor allele frequency in healthy population (ExAC and gnomAD databases) <0.00001, (2) variant allele frequency >30%, (3) predicted to be pathogenic with two out of four tools for functional annotation (PolyPhen2, MutationTaster, SIFT and Provean), (4) sufficient evidence of pathogenicity and not meeting any criteria for a benign assertion following ACMG guidelines⁷⁴, and (5) where practical, the segregation with affected individuals.

A targeted myeloid panel of 33 genes frequently mutated in MDS/AML (ASXL1 (exon 12), BCOR (all), CALR (exon 9), CBL (exons 7 + 8 + 9), CEBPA (all), CSF3R (exons 14–17), DNMT3A (all), ETV6 (all), EZH2 (all), FLT3 (exons 14 + 15 + 20), GATA2 (all), GNAS (exons 8 + 9), IDH1 (exon 4), IDH2 (exon 4), IKZF1 (all), JAK2 (exons 12 + 14), KIT (exons 2, 8–11, 13 + 17), KRAS (exons 2 + 3), MPL (exon 10), NPM1 (exon 12), NRAS (exons 2 + 3), PDGFRA (exons 12, 14, 18), PHF6 (all), PTPN11 (exons 3 + 13), RUNX1 (all), SETBP1 (exon 4), SF3B1 (exons 12 – 16), SRSF2 (exon 1), TET2 (all), TP53 (all), U2AF1 (exons 2 + 6), WT1 (exons 7 + 9) and ZRSR2 (all)) was employed to determine the acquired variants in the five patients with germline DHX34 variants. An in-house True SeqCustom Amplicon (TSCA) design (Illumina, San Diego, California, USA) was used for target enrichment. Pooled library targets were sequenced the MiSeq sequencing platform. Minimum read depth threshold was 150 reads and lower limit of sensitivity was 5–10% variant allele frequency (VAF).