Exome-Wide Somatic Microsatellite Variation Is Altered in Cells with DNA Repair Deficiencies

Abstract

Microsatellites (MST), tandem repeats of 1–6 nucleotide motifs, are mutational hot-spots with a bias for insertions and deletions (INDELs) rather than single nucleotide polymorphisms (SNPs). The majority of MST instability studies are limited to a small number of loci, the Bethesda markers, which are only informative for a subset of colorectal cancers. In this paper we evaluate non-haplotype alleles present within next-gen sequencing data to evaluate somatic MST variation (SMV) within DNA repair proficient and DNA repair defective cell lines. We confirm that alleles present within next-gen data that do not contribute to the haplotype can be reliably quantified and utilized to evaluate the SMV without requiring comparisons of matched samples. We observed that SMV patterns found in DNA repair proficient cell lines without DNA repair defects, MCF10A, HEK293 and PD20 RV:D2, had consistent patterns among samples. Further, we were able to confirm that changes in SMV patterns in cell lines lacking functional BRCA2, FANCD2 and mismatch repair were consistent with the different pathways perturbed. Using this new exome sequencing analysis approach we show that DNA instability can be identified in a sample and that patterns of instability vary depending on the impaired DNA repair mechanism, and that genes harboring minor alleles are strongly associated with cancer pathways. The MST Minor Allele Caller used for this study is available at https://github.com/zalmanv/MST_minor_allele_caller.

Somatic microsatellite variability as a predictive marker for colorectal cancer and liver cancer progression

“…The unique repetitive genomic configuration of MSTs can lead to the development of complex DNA structures susceptible to polymerase slippage and DNA breaks [11, 15-17]. This results in a distinct mutational profile for MSTs with a bias for indels, as opposed to single nucleotide polymorphisms (SNPs) which are frequently observed in non-repetitive DNA [18]. Although MST expansion of tri-nucleotide (GCC and CAG) repeats have been regularly studied due to their connection to numerous neurological diseases, including Fragile X and Huntington's coria, recent work suggests that MSTs may also exhibit a contraction bias to which mono-nucleotide motifs are most susceptible [14, 19-23].…”

“…For example, cells with impaired MUTYH, MLH1 or MSH2/6 complexes (associated with familial colorectal cancer, Lynch or Muir-Torre syndrome), two of the three essential complexes required for removal and replacement of incorrect nucleotides, show a significant increase in MSI regardless of genomic localization [19, 27]. For these disorders, although the predominant mutated motif is composed of mono-nucleotides, other motifs, including di- and tetra-nucleotide MSTs, also show an increase in somatic mutations [18, 19, 27]. …”

“…To date, the only clinically approved test for MSI (Promega, Fitchburg WI) is based on five loci, that is Bethesda markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27). These MSI markers have been tested for a wide variety of cancers other than CRC, gastric and endometrial tumors, but their global applicability appears to be limited [17, 18, 22]. Expansion of analysis of genomic instability and/or microsatellite instability beyond these 5 loci may yield new markers with more general applicability.…”

“…Results from these publications revealed deficiencies in the current clinical assay in identifying MSI in gastric, cervical and even some colorectal tumors. Our group has recently developed a novel tool that identifies all the sequenced alleles for a given MST locus in a Next-Gen sequenced sample, and was subsequently used to quantify somatic microsatellite variation (SMV) in cell lines with known repair deficiencies [18]. In that study we demonstrated that it was possible to establish a baseline SMV profile in DNA repair proficient cell lines for comparison, and that the SMV profile of cell lines with DNA repair impairment changes in a pathway dependent manner.…”

“…Recent genomic studies have argued for an increased emphasis on global neoplastic MST changes to broaden of definition of MSI [18, 19, 27, 29, 30]. This work is the first to test somatic variability of MSTs and non-repetitive DNA sequences in colon and Hepatocellular carcinoma (LIHC) using Next-Gen sequencing.…”