G-Quadruplex Forming DNA Sequence Context Is Enriched around Points of Somatic Mutations in a Subset of Multiple Myeloma Patients

Abstract

Multiple myeloma (MM) is the second most common hematological malignancy, which remains incurable despite recent advances in treatment strategies. Like other forms of cancer, MM is characterized by genomic instability, caused by defects in DNA repair. Along with mutations in DNA repair genes and genotoxic drugs used to treat MM, non-canonical secondary DNA structures (four-stranded G-quadruplex structures) can affect accumulation of somatic mutations and chromosomal abnormalities in the tumor cells of MM patients. Here, we tested the hypothesis that G-quadruplex structures may influence the distribution of somatic mutations in the tumor cells of MM patients. We sequenced exomes of normal and tumor cells of 11 MM patients and analyzed the data for the presence of G4 context around points of somatic mutations. To identify molecular mechanisms that could affect mutational profile of tumors, we also analyzed mutational signatures in tumor cells as well as germline mutations for the presence of specific SNPs in DNA repair genes or in genes regulating G-quadruplex unwinding. In several patients, we found that sites of somatic mutations are frequently located in regions with G4 context. This pattern correlated with specific germline variants found in these patients. We discuss the possible implications of these variants for mutation accumulation and specificity in MM and propose that the extent of G4 context enrichment around somatic mutation sites may be a novel metric characterizing mutational processes in tumors.

Keywords: G-quadruplex structures; Next Generation Sequencing (NGS); multiple myeloma; mutational signatures; somatic mutations.

Figure 1

Percentage of G4 strong context occurrence near mutation sites in different patients and in randomly sampled sequences. The random1 and random2 sets include 2000 randomly selected sequences from genomic intervals corresponding to the All Exon V6+UTR V6 enrichment panel (random2) or Truseq Exome panel (random1). The graph displays the percentage proportion along with the confidence interval for the proportion. The asterisk denotes a statistically significant difference between the proportions of G4 context occurrence around point of somatic mutations in patients and in randomly sampled sequences as determined by a z-test.

Figure 2

Mutation signatures observed in the analyzed tumors. (a) Visualization of SBS proportions in each of the analyzed tumors based on SigProfilerAssignment. (b) Visualization of small insertions and deletions (ID) among somatic mutations determined in different patients by SigProfilerAssignment. (c) t-SNE analysis based on SigProfilerAssignment SBS classification, percentage of SBS in each sample used, samples with G4 strong context enrichment are salmon, samples without G4 strong context enrichment are cyan. (d) k-means cluster analysis based on SigProfilerAssignment SBS classification was performed for illustration of similarity between samples; percentage of SBS in each sample used. Mutational signature associations: SBS1—aging, clock-like signature, spontaneous or enzymatic deamination of 5-methylcytosine to thymine; SBS5—aging, clock-like signature, may implicate NER [55]; SBS6—defective DNA mismatch repair, is very specific to MM with high genomic risk [56]; SBS7a—DNA damage due to exposure to ultraviolet light; SBS9—activity of activation-induced deaminase (AID) in non-coding regions, mutation pattern found in B-cell cancers that develop after the germinal center stage. This signature results from the off-target activity of AID (normally working during the germinal center phase of the hypermutation of immunoglobulin genes [57], MMR, and gap repair with participation of DNA polymerase eta); SBS10b—polymerase epsilon exonuclease (POLE-Exo) domain mutations [58]; SBS11—a mutation pattern similar to that of alkylating agents; SBS12—defective mismatch repair [59]; SBS15—defective DNA mismatch repair [60]; SBS17a and b—unidentified etiology, were found in MM [61]; SBS32—treatment with azathioprine prior to induce immunosuppression, the presence of transcription-coupled nucleotide excision repair activity on damaged DNA [62]; SBS38—indicating possible secondary harm caused by UV exposure; SBS40b—related to indicators of decreased kidney function; SBS84—activity of AID [62,63]; SBS87—thiopurine chemotherapy treatment; SBS88—explore to the colibactin from E. coli-carrying pks pathogenicity island, displays heightened activity during early childhood; SBS19, SBS37, SBS93, SBS94—unknown; SBS45, SBS47, SBS58—possible sequencing artefact. ID1, ID2—indicate DNA mismatch repair deficiency; ID5—possible clock-like signature; ID6—defective homologous recombination repair; ID13—UV exposure; ID23—aristolochic acid exposure; ID4, ID9, ID11, ID12, ID20—unknown.

Figure 3

Types of mutations in samples with G4 context enrichment around points of somatic mutations and without G4 context enrichment, classified by the type of context. Standard deviation of a proportion is shown as error bars.

Figure 4

Consequence of somatic mutations found in different groups of samples in respect to the G4 context.

Figure 5

Germline SNPs associated with MM according to publications, GWAS catalog, and Clinvar. SNPs affect such genes as XRCC5, ULK4, ADH1B, ELL2, NDUFA8, CCND1, SLC28A2, RFWD3, CTC1, TNFRSF13B, KLF2, ZBTB46, MYNN, LRRC34, SMARCD3, ICAM1, SAA4, DCLRE1B, CASP3, and MRTFA. CoMut were used for SNP visualization [64].