Clonal diversity of recurrently mutated genes in myelodysplastic syndromes

Abstract

Recent studies suggest that most cases of myelodysplastic syndrome (MDS) are clonally heterogeneous, with a founding clone and multiple subclones. It is not known whether specific gene mutations typically occur in founding clones or subclones. We screened a panel of 94 candidate genes in a cohort of 157 patients with MDS or secondary acute myeloid leukemia (sAML). This included 150 cases with samples obtained at MDS diagnosis and 15 cases with samples obtained at sAML transformation (8 were also analyzed at the MDS stage). We performed whole-genome sequencing (WGS) to define the clonal architecture in eight sAML genomes and identified the range of variant allele frequencies (VAFs) for founding clone mutations. At least one mutation or cytogenetic abnormality was detected in 83% of the 150 MDS patients and 17 genes were significantly mutated (false discovery rate ≤0.05). Individual genes and patient samples displayed a wide range of VAFs for recurrently mutated genes, indicating that no single gene is exclusively mutated in the founding clone. The VAFs of recurrently mutated genes did not fully recapitulate the clonal architecture defined by WGS, suggesting that comprehensive sequencing may be required to accurately assess the clonal status of recurrently mutated genes in MDS.

Figure 1

Clonality of sAML samples. VAF vs read depth at the site of all validated Tier 1–3 somatic mutations in the diploid regions of eight MDS-derived sAML genomes. Above each scatterplot, the histograms depict clustered VAFs using a Bayesian approach that performs outlier detection and removal, and determines the optimal number of clusters. Founding clones (yellow) are defined by the region with the maximum density peak. Points falling outside this region are in subclones (blue). A heterozygous mutation present in every bone marrow cell would have a VAF = 50% (depicted by the vertical dashed lines in the lower plots), implying that 100% of cells are clonal in a sample.

Figure 2

Candidate gene sequencing. (a) The number of validated somatic mutations per patient in 94 candidate genes in 150 paired MDS/ normal samples. (b) The predicted translational consequences of all mutations in 150 MDS samples. (c) Mutation spectrum in recurrently mutated genes (≥3 cases) in 150 MDS (solid fill) or seven sAML (hatched fill) samples. Left, genes with predominantly missense substitutions; Right, genes with predominantly truncating mutations (frameshift, nonsense and splice site).

Figure 3

Recurrently mutated genes in MDS and AML. (a) The distribution of mutations in 100 MDS samples with at least one mutation in 27 genes. Each column represents an individual patient sample and each row represents a gene with a mutation. Mutations are indicated by colored cells and gene groups/families are indicated by different colors at the left. The International Prognostic Scoring System-Revised cytogenetic score and cases with chromosome 5 and/or 7 deletions are indicated at the bottom. Normal karyotype (classified as ‘Good’ cytogenetic risk within the International Prognostic Scoring System-Revised) is indicated by a white box. (b) The frequency of genes with non-silent mutations in ≥5/150 MDS or ≥5/200 AML cases (*P<0.05).

Figure 4

Comparison of VAFs from whole-genome and candidate gene sequencing. (a) The VAFs for all validated SNVs in 157 MDS or sAML samples. Green horizontal bar, median VAF (±2 s.d.) of founding clone from WGS data (Figure 1). (b) VAFs for genes with ≥3 validated somatic SNVs. The vertical boxed regions show the range and mean (horizontal line) of VAFs for each gene. Dotted lines, boundary of founding clone (as in panel a). (c) Comparison of VAFs from capture validation of Tier 1–3 SNVs (restricted to mutations on autosomes without copy number alteration or uniparental disomy) vs only Tier 1 (open) or candidate genes (green) in eight sAML genomes. The founding clones (yellow) and subclones (blue) were identified by clustering, as described in the text. For each founding clone, the mean (±s.d.) of VAFs is shown. All data were generated simultaneously in a single liquid hybridization and deep sequencing experiment.