Microsatellite Mutations Research Articles

Quantifying Microsatellite Mutation Rates from Intestinal Stem Cell Dynamics in Msh2-Deficient Murine Epithelium.

Microsatellite sequences have an enhanced susceptibility to mutation, and can act as sentinels indicating elevated mutation rates and increased risk of cancer. The probability of mutant fixation within the intestinal epithelium is dictated by a combination of stem cell dynamics and mutation rate. Here, we exploit this relationship to infer microsatellite mutation rates. First a sensitive, multiplexed, and quantitative method for detecting somatic changes in microsatellite length was developed that allowed the parallel detection of mutant [CA]n sequences from hundreds of low-input tissue samples at up to 14 loci. The method was applied to colonic crypts in Mus musculus, and enabled detection of mutant subclones down to 20% of the cellularity of the crypt (∼50 of 250 cells). By quantifying age-related increases in clone frequencies for multiple loci, microsatellite mutation rates in wild-type and Msh2-deficient epithelium were established. An average 388-fold increase in mutation per mitosis rate was observed in Msh2-deficient epithelium (2.4 × 10−2) compared to wild-type epithelium (6.2 × 10−5).

Microsatellite instability and mutations in BRAF and KRAS in small bowel adenocarcinoma.

e15037 Background: Small-bowel adenocarcinomas (SBAs) are rare cancers with a significantly lower incidence, later stage at diagnosis, and worse overall survival than other intestinal-derived cancers. Activating KRAS and/or BRAF mutations have been identified as predictors of resistance to anti-epidermal growth factor receptor (EGFR) The aim of this study was to perform a comprehensive immunohistochemical.analysis of KRAS, NRAS, V600E BRAF mutations and microsatellite instability using a cohort of surgically resected cases in our institution. Methods: A total of 17 patients (10 males and 7 females; mean age, 56.2 years old; range, 45-75 years old) received chemotherapy due to non-curative tumor resection, unresectable tumor or post-operative recurrence. Twelve patents received fluoropirimidine and oxaliplatin based first line chemotherapy. Molecular targeted agents were administered to 15 patients, for whom it was their first- or second-line therapy. Results: KRAS mutations were found in 7 cases (41%), out of which 5 (29%) were in exons 12/13. BRAFV600E mutation was observed in 1/17 pt. Microsatellite instability was identified in 3/17pt (MSI; 18%), mainly related to a loss of expression of MLH1 protein. Univariate analysis revealed a PS of 0 (P=0.0226) and treatment with platinum-based chemotherapy (P=0.0047) were significant factors for an improved prognosis. Among the 12 patients who received oxaliplatin-based chemotherapy as a first-line chemotherapy, a PS of 0 (P=0.0255) and treatment with anti-EGFR agents (P=0.0127) were significant positive prognostic factors. Toxicities due to the molecular targeted agents were not experienced. The median overall survival time was 14.3 months (range, 3-52 months), the median DFS was 14.2 months and the median OS was 32 months. Conclusions: To date, there is no standard chemotherapy regimen for advanced SBAs and little is known about their molecular characteristics. The results of the present study indicate that oxaliplatin based chemotherapy containing molecular targeted agents is a well-tolerated and effective treatment option for SBA. A better understanding of disease biology may help to identify therapeutic targets and advance precision medicine.

Intraspecific Variation in Microsatellite Mutation Profiles in Daphnia magna

Microsatellite loci (tandem repeats of short nucleotide motifs) are highly abundant in eukaryotic genomes and often used as genetic markers because they can exhibit variation both within and between populations. Although widely recognized for their mutability and utility, the mutation rates of microsatellites have only been empirically estimated in a few species, and have rarely been compared across genotypes and populations within a species. Here, we investigate the dynamics of microsatellite mutation over long- and short-time periods by quantifying the starting abundance and mutation rates for microsatellites for six different genotypes of Daphnia magna, an aquatic microcrustacean, collected from three populations (Finland, Germany, and Israel). Using whole-genome sequences of these six starting genotypes, descendent mutation accumulation (MA) lines, and large population controls (non-MA lines), we find each genotype exhibits a distinctive initial microsatellite profile which clusters according to the population-of-origin. During the period of MA, we observe motif-specific, highly variable, and rapid microsatellite mutation rates across genotypes of D. magna, the average of which is order of magnitude greater than the recently reported rate observed in a single genotype of the congener, Daphnia pulex. In our experiment, genotypes with more microsatellites starting out exhibit greater losses and those with fewer microsatellites starting out exhibit greater gains—a context-dependent mutation bias that has not been reported previously. We discuss how genotype-specific mutation rates and spectra, in conjunction with evolutionary forces, can shape both the differential accumulation of repeat content in the genome and the evolution of mutation rates.

Repeat-Associated Non-ATG Translation: Molecular Mechanisms and Contribution to Neurological Disease.

Microsatellite mutations involving the expansion of tri-, tetra-, penta-, or hexanucleotide repeats cause more than 40 different neurological disorders. Although, traditionally, the position of the repeat within or outside of an open reading frame has been used to focus research on disease mechanisms involving protein loss of function, protein gain of function, or RNA gain of function, the discoveries of bidirectional transcription and repeat-associated non-ATG (RAN) have blurred these distinctions. Here we review what is known about RAN proteins in disease, the mechanisms by which they are produced, and the novel therapeutic opportunities they provide.

Issue Highlights

Issue Highlights

Multiple interspecific hybridization and microsatellite mutations provide clonal diversity in the parthenogenetic rock lizard Darevskia armeniaca

BackgroundThe parthenogenetic Caucasian rock lizard Darevskia armeniaca, like most other parthenogenetic vertebrate species, originated through interspecific hybridization between the closely related sexual Darevskia mixta and Darevskia valentini. Darevskia armeniaca was shown to consist of one widespread allozyme clone and a few rare ones, but notwithstanding the origin of clonal diversity remains unclear. We conduct genomic analysis of D. armeniaca and its parental sexual species using microsatellite and SNP markers to identify the origin of parthenogenetic clonal lineages.ResultsFour microsatellite-containing loci were genotyped for 111 specimens of D. armeniaca, 17 D. valentini, and four D. mixta. For these species, a total of 47 alleles were isolated and sequenced. Analysis of the data revealed 13 genotypes or presumptive clones in parthenogenetic D. armeniaca, including one widespread clone, two apparently geographically restricted clones, and ten rare clones. Comparisons of genotype-specific markers in D. armeniaca with those of its parental species revealed three founder-events including a common and two rare clones. All other clones appeared to have originated via post-formation microsatellite mutations in the course of evolutionary history of D. armeniaca.ConclusionOur new approach to microsatellite genotyping reveals allele-specific microsatellite and SNP markers for each locus studied. Interspecies comparison of these markers identifies alleles inherited by parthenospecies from parental species, and provides new information on origin and evolution of clonal diversity in D. armeniaca. SNP analyses reveal at least three interspecific origins of D. armeniaca, and microsatellite mutations in these initial clones give rise to new clones. Thus, we first establish multiple origins of D. armeniaca. Our study identifies the most effective molecular markers for elucidating the origins of clonal diversity in other unisexual species that arose via interspecific hybridization.

Improved Microsatellite Instability Detection and Identification by Nuclease-Assisted Microsatellite Instability Enrichment Using HSP110 T17

To the Editor: Microsatellite instability (MSI)1 is characterized by the accumulation of mutations (insertions or deletions of several nucleotides) in microsatellites, caused by DNA mismatch repair system deficiency. Approximately 15% to 20% of colorectal cancers (CRCs) present with MSI, the origin of which can be sporadic or owing to genetic predisposition to Lynch syndrome/hereditary nonpolyposis CRC or constitutional mismatch repair deficiency syndrome. The MSI phenotype is associated with a better survival rate for patients with stage II/III CRC and is also a major predictive biomarker for the efficacy of immune checkpoint blockade therapy in metastatic CRC. The gold standard method for MSI detection includes standard PCR followed by fragment analysis using the Bethesda or the Pentaplex panel, each composed of 5 microsatellite markers. However, this approach can lack analytical sensitivity, thus limiting potential clinical applications. Recent attempts to improve the analytical sensitivity of MSI detection include a method based on mutation enrichment during E- ice -COLD-PCR. This method uses HSP110 T17 (HT17), which, compared with the Pentaplex panel, is an improved microsatellite biomarker for the diagnosis of MSI/MSI-High in CRC and also bears prognostic and predictive information …

HER2 status in patients with residual rectal cancer after preoperative chemoradiotherapy: the relationship with molecular results and clinicopathologic features.

The specific role of human epidermal growth factor receptor-2 (HER2) status in rectal cancers remains unclear. This study therefore aimed to explore clinicopathologic and molecular characteristics, and prognostic value of HER2-positivity in residual mid- and/or low-rectal cancers after preoperative chemoradiotherapy (CRT). Surgical specimens from 145 patients with residual rectal cancer after preoperative CRT between January 2006 and January 2011 were used to evaluate HER2 status. HER2 protein expression and gene amplification were determined using immunohistochemistry (IHC) and silver in situ hybridization (SISH) on whole tissue sections, respectively. Polymerase chain reaction was used to analyze molecular characteristics, including microsatellite instability (MSI) and mutations in KRAS exon 2 (codon 12 and 13) and BRAF V600E mutation. Of 139 eligible patients, 8 (5.8%) had HER2 overexpression (IHC 2+ and 3+) that was not associated with clinicopathologic characteristics and patient survival, except positive circumferential resection margin (CRM) (P = 0.012). SISH was performed on 24 patient samples with IHC 1+ (n = 16), 2+ (n = 6), and 3+ (n = 2). HER2 amplification was identified in 3 patients (2.2%); however, this was also associated with positive CRM (P = 0.009) but not survival (all P > 0.05). Moreover, HER2 overexpression and amplification had no relationship with KRAS or BRAF mutations, and MSI status (all P > 0.05). HER2 positivity was found in a minority of rectal cancer patients and was not significantly associated with clinicopathologic and molecular characteristics. Our findings can be helpful in understanding the clinicopathologic bases of HER2 status in rectal cancers.

Mutation burden and other molecular markers of prognosis in colorectal cancer treated with curative intent: results from the QUASAR 2 clinical trial and an Australian community-based series

SummaryBackgroundMolecular indicators of colorectal cancer prognosis have been assessed in several studies, but most analyses have been restricted to a handful of markers. We aimed to identify prognostic biomarkers for colorectal cancer by sequencing panels of multiple driver genes.MethodsIn stage II or III colorectal cancers from the QUASAR 2 open-label randomised phase 3 clinical trial and an Australian community-based series, we used targeted next-generation sequencing of 82 and 113 genes, respectively, including the main colorectal cancer drivers. We investigated molecular pathways of tumorigenesis, and analysed individual driver gene mutations, combinations of mutations, or global measures such as microsatellite instability (MSI) and mutation burden (total number of non-synonymous mutations and coding indels) for associations with relapse-free survival in univariable and multivariable models, principally Cox proportional hazards models.FindingsIn QUASAR 2 (511 tumours), TP53, KRAS, BRAF, and GNAS mutations were independently associated with shorter relapse-free survival (p<0·035 in all cases), and total somatic mutation burden with longer survival (hazard ratio [HR] 0·81 [95% CI 0·68–0·96]; p=0·014). MSI was not independently associated with survival (HR 1·12 [95% CI 0·57–2·19]; p=0·75). We successfully validated these associations in the Australian sample set (296 tumours). In a combined analysis of both the QUASAR 2 and the Australian sample sets, mutation burden was also associated with longer survival (HR 0·84 [95% CI 0·74–0·94]; p=0·004) after exclusion of MSI-positive and POLE mutant tumours. In an extended analysis of 1732 QUASAR 2 and Australian colorectal cancers for which KRAS, BRAF, and MSI status were available, KRAS and BRAF mutations were specifically associated with poor prognosis in MSI-negative cancers. MSI-positive cancers with KRAS or BRAF mutations had better prognosis than MSI-negative cancers that were wild-type for KRAS or BRAF. Mutations in the genes NF1 and NRAS from the MAPK pathway co-occurred, and mutations in the DNA damage-response genes TP53 and ATM were mutually exclusive. We compared a prognostic model based on the gold standard of clinicopathological variables and MSI with our new model incorporating clinicopathological variables, mutation burden, and driver mutations in KRAS, BRAF, and TP53. In both QUASAR 2 and the Australian cohort, our new model was significantly better (p=0·00004 and p=0·0057, respectively, based on a likelihood ratio test).InterpretationMultigene panels identified two previously unreported prognostic associations in colorectal cancer involving TP53 mutation and total mutation burden, and confirmed associations with KRAS and BRAF. Even a modest-sized gene panel can provide important information for use in clinical practice and outperform MSI-based prognostic models.FundingUK Technology Strategy Board, National Institute for Health Research Oxford Biomedical Research Centre, Cancer Australia Project, Cancer Council Victoria, Ludwig Institute for Cancer Research, Victorian Government.

Abstract 561: Circulating DNA clean up and enrichment from liquid biological samples for rare biomarker detection

Abstract A major roadblock to using circulating cell free DNA (ccfDNA) for cancer biomarker detection is the difficulty of obtaining a pure sample. Contaminating genomic DNA (gDNA) can overwhelm already rare events within a CCF sample and prevent sensitive detection of these key mutations. Unfortunately, liquid biological samples often are not collected and processed in an ideal manner, resulting in decreased quality and purity. Many studies report difficulties in detecting mutations in ccfDNA which may originate from variable levels of gDNA background based how samples are processed. To this end, we present an effective method for purifying relevant small nucleic acids from larger genomic species after DNA purification from plasma. Using a simple magnetic bead based technique, we demonstrate efficient removal of gDNA even in highly contaminated samples. This method can be used to enrich liquid biological samples for improved detection of rare genetic events such as microsatellite instability and mutations in key oncogenes. Using plasma from three individuals with colorectal cancer tumors harboring known mismatch repair gene mutations, we compare detection of somatic mutations in ccfDNA before and after this gDNA clean up method. Microsatellite instability profiles were obtained from ccfDNA and compared to normal and tumor profiles to determine if evidence of mismatch repair abnormalities could be detected. Targeted amplicon sequencing was then performed to detect tumor associated mutations. DNA isolated from matched adjacent normal and tumor tissues were used as controls in these experiments to confirm mutations were of tumor not germline mutations. Effectively removing gDNA contamination from ccfDNA samples can improve biomarker detection consistency from liquid biological samples. We demonstrate a magnetic bead-based method for ccfDNA clean-up, which can be used prior to microsatellite instability detection by fragment analysis or rare variant detection by next generation sequencing. Citation Format: Samantha R. Lewis, Charles Cowles, Kathryn Oostdik, Curtis Knox, Brad A. Hook. Circulating DNA clean up and enrichment from liquid biological samples for rare biomarker detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 561.

Population declines, genetic bottlenecks and potential hybridization in sea snakes on Australia's Timor Sea reefs

Population bottlenecks can result in loss of genetic variation, increased extinction risk, and hybridization with related sympatric species. Many challenges are associated with empirical detection of population declines, thus conservation biologists often use molecular approaches as surrogates. This study explored whether declines in abundances of viviparous sea snakes on Australia's Timor Sea reefs could have been foreshadowed using genetic surveys. Ashmore Reef (the largest Timor Sea reef) once hosted large breeding populations of sea snakes. Abundances have declined precipitously since 1994 and Ashmore Reef has been devoid of snakes since 2012. Moreover, high rates of hybridization between two sympatric species have been documented on Timor Sea reefs, possibly associated with sea snake declines. I analysed mitochondrial DNA and 11 nuclear microsatellites for >250 sea snakes from three species, Aipysurus laevis, Aipysurus fuscus and Emdocephalus annulatus, sampled on four Timor Sea reefs in 2002 and 2010. While there was strong spatial genetic structure among reefs, there was little temporal genetic divergence for A. laevis at Ashmore Reef, despite the massive declines in abundance during that temporal window. Positive Tajima's D and Fu's FS values at Ashmore Reef indicated demographic contraction for: A. laevis in 2002 and 2010; E. annulatus (2002); but not A. fuscus (2002). Microsatellites showed inbreeding depression (positive Fis values) and non-random mating (heterozygote deficit) for all three species at Ashmore Reef, consistent with population declines. Bottleneck tests were equivocal, with significant heterozygous excesses at Ashmore Reef, but non-significant M-ratios or mode-shifts in allele frequencies, with the significance of tests differing markedly with microsatellite mutation models. Thus genetic analyses alone would not have been sufficient to provide managers with unequivocal evidence of population declines. There was little evidence for hybridization between A. laevis and A. fuscus, despite previous research suggesting that the Endangered A. fuscus was at risk of reverse speciation secondary to the highly porous reproductive barriers between these species.

Detection of pro-apoptotic Bax∆2 proteins in the human cerebellum.

Bax∆2 is a pro-apoptotic protein originally discovered in colon cancer patients with high microsatellite instability. Unlike most pro-apoptotic Bax family members, Bax∆2 mediates cell death through a non-mitochondrial caspase 8-dependent pathway. In the scope of analyzing the distribution of Bax∆2 expression in human tissues, we examined a panel of human brain samples. Here, we report four cerebellar cases in which the subjects had no neurological disorder or disease documented. We found Bax∆2 positive cells scattered in all areas of the cerebellum, but most strikingly concentrated in Purkinje cell bodies and dendrites. Two out the four subjects tested had strong Bax∆2-positive staining in nearly all Purkinje cells; one was mainly negative; and one had various levels of positive staining within the same sample. Further genetic analysis of the Purkinje cell layer, collected by microdissection from two subjects, showed that the samples contained G7 and G9 Bax microsatellite mutations. Both subjects were young and had no diseases reported at the time of death. As the distribution of Bax∆2 is consistent with that known for Baxα, but in a less ubiquitous manner, these results may imply a potential function of Bax∆2 in Purkinje cells.

Lynch Syndrome: Genomics Update and Imaging Review.

Lynch syndrome is the most common hereditary cancer syndrome, the most common cause of heritable colorectal cancer, and the only known heritable cause of endometrial cancer. Other cancers associated with Lynch syndrome include cancers of the ovary, stomach, urothelial tract, and small bowel, and less frequently, cancers of the brain, biliary tract, pancreas, and prostate. The oncogenic tendency of Lynch syndrome stems from a set of genomic alterations of mismatch repair proteins. Defunct mismatch repair proteins cause unusually high instability of regions of the genome called microsatellites. Over time, the accumulation of mutations in microsatellites and elsewhere in the genome can affect the production of important cellular proteins, spurring tumorigenesis. Universal testing of colorectal tumors for microsatellite instability (MSI) is now recommended to (a) prevent cases of Lynch syndrome being missed owing to the use of clinical criteria alone, (b) reduce morbidity and mortality among the relatives of affected individuals, and (c) guide management decisions. Organ-specific cancer risks and associated screening paradigms vary according to the sex of the affected individual and the type of germline DNA alteration causing the MSI. Furthermore, Lynch syndrome-associated cancers have different pathologic, radiologic, and clinical features compared with their sporadic counterparts. Most notably, Lynch syndrome-associated tumors tend to be more indolent than non-Lynch syndrome-associated neoplasms and thus may respond differently to traditional chemotherapy regimens. The high MSI in cases of colorectal cancer reflects a difference in the biologic features of the tumor, possibly with a unique susceptibility to immunotherapy. ©RSNA, 2018.

MLH1\u201393\xa0G/a polymorphism is associated with MLH1 promoter methylation and protein loss in dysplastic sessile serrated adenomas with BRAFV600E mutation

BackgroundSessile serrated adenomas with BRAF mutation progress rapidly to cancer following the development of dysplasia (SSAD). Approximately 75% of SSADs methylate the mismatch repair gene MLH1, develop mismatch repair deficiency and the resultant cancers have a good prognosis. The remaining SSADs and BRAF mutant traditional serrated adenomas (TSA) develop into microsatellite stable cancers with a poor prognosis. The reason for this dichotomy is unknown. In this study, we assessed the genotypic frequency of the MLH1–93 polymorphism rs1800734 in SSADs and TSAs to determine if the uncommon variant A allele predisposes to MLH1 promoter hypermethylation.MethodsWe performed genotyping for the MLH1–93 polymorphism, quantitative methylation specific PCR, and MLH1 immunohistochemistry on 124 SSAD, 128 TSA, 203 BRAF mutant CRCs and 147 control subjects with normal colonoscopy.ResultsThe minor A allele was significantly associated with a dose dependent increase in methylation at the MLH1 promoter in SSADs (p = 0.022). The AA genotype was only observed in SSADs with MLH1 loss. The A allele was also overrepresented in BRAF mutant cancers with MLH1 loss. Only one of the TSAs showed loss of MLH1 and the overall genotype distribution in TSAs did not differ from controls.ConclusionsThe MLH1–93 AA genotype is significantly associated with promoter hypermethylation and MLH1 loss in the context of SSADs. BRAF mutant microsatellite stable colorectal cancers with the AA genotype most likely arise in TSAs since the A allele does not predispose to methylation in this context.

High numbers of PDCD1 (PD-1)-positive T cells and B2M mutations in microsatellite-unstable colorectal cancer

ABSTRACT DNA mismatch repair (MMR)-deficient cancers accumulate high numbers of coding microsatellite mutations, which lead to the generation of highly immunogenic frameshift peptide (FSP) neoantigens. MMR-deficient cells can grow out to clinically manifest cancers either if they evade immune cell attack or if local T-cells get exhausted. Therefore, a subset of MSI cancer patients responds particularly well to treatment with immune checkpoint inhibitors. We analyzed whether immune evasion in MMR-deficient cancer mediated by loss of HLA class I or II antigens is related to local immune cell activation status. Microsatellites located in Beta2-microglobulin (B2M) and the HLA class II-regulatory genes RFX5 and CIITA were analyzed for mutations in MMR-deficient colorectal cancers (n = 53). The results were related to CD3-positive and PDCD1 (PD-1)-positive T-cell infiltration. PDCD1 (PD-1)-positive T-cell counts were significantly higher in B2M-mutant compared to B2M-wild type tumors (median: 22.2 cells per 0.25 mm2 vs. 2.0 cells per 0.25 mm2, Wilcoxon test p = 0.002). Increasing PDCD1 (PD-1)-positive T-cell infiltration was significantly related to an increased likelihood of B2M mutations (OR = 1.81). HLA class II antigen expression status was significantly associated with enhanced overall T-cell infiltration, but not related to PDCD1 (PD-1)-positive T-cells. These results suggest that immune evasion mediated by B2M mutation-induced loss of HLA class I antigen expression predominantly occurs in an environment of activated PDCD1 (PD-1)-positive T cell infiltration. If B2M mutations interfere with anti-PDCD1 (PD-1)/CD274 (PD-L1) therapy success, we predict that resistance towards anti-PDCD1 (PD-1) therapy may – counterintuitively – be particularly common in patients with MMR-deficient cancers that show high PDCD1 (PD-1)-positive T cell infiltration.

Multistep microsatellite mutation at D18S51 locus in a parentage testing case

Mutations of short tandem repeats (STRs) loci might cause allelic mismatchs between the child and the parents, which entangled the forensic inference in paternity testing. Most of the reported microsatellite mutations are confined to single-step mutations, and multistep mutations were rarely reported and only account for a very limited number of STR mutation events in previously studied cases. Here we reported a paternity case with a mismatch in locus D18S51. The genotypes of the alleged father, the mother and the child in D18S51 locus were 14/23, 15/16, and 15/20, respectively. Examination of 38 autosomal STR loci revealed no mismatches, and the paternity index is above 1.58E+15. These results suggested that the alleged father is the biological father of the child that a rare three-steps mutation had occurred in the paternal allele of D18S51.

The origin of multiple clones in the parthenogenetic lizard species Darevskia rostombekowi.

The all-female Caucasian rock lizard Darevskia rostombekowi and other unisexual species of this genus reproduce normally via true parthenogenesis. Typically, diploid parthenogenetic reptiles exhibit some amount of clonal diversity. However, allozyme data from D. rostombekowi have suggested that this species consists of a single clone. Herein, we test this hypothesis by evaluating variation at three variable microsatellite loci for 42 specimens of D. rostombekowi from four populations in Armenia. Analyses based on single nucleotide polymorphisms of each locus reveal five genotypes or presumptive clones in this species. All individuals are heterozygous at the loci. The major clone occurs in 24 individuals and involves three populations. Four rare clones involve one or several individuals from one or two populations. Most variation owes to parent-specific single nucleotide polymorphisms, which occur as heterozygotes. This result fails to reject the hypothesis of a single hybridization founder event that resulted in the initial formation of one major clone. The other clones appear to have originated via post-formation microsatellite mutations of the major clone.

Comprehensive Evaluation of Protein Coding Mononucleotide Microsatellites in Microsatellite-Unstable Colorectal Cancer.

Approximately 15% of colorectal cancers exhibit microsatellite instability (MSI), which leads to accumulation of large numbers of small insertions and deletions (indels). Genes that provide growth advantage to cells via loss-of-function mutations in microsatellites are called MSI target genes. Several criteria to define these genes have been suggested, one of them being simple mutation frequency. Microsatellite mutation rate, however, depends on the length and nucleotide context of the microsatellite. Therefore, assessing the general impact of mismatch repair deficiency on the likelihood of mutation events is paramount when following this approach. To identify MSI target genes, we developed a statistical model for the somatic background indel mutation rate of microsatellites to assess mutation significance. Exome sequencing data of 24 MSI colorectal cancers revealed indels at 54 million mononucleotide microsatellites of three or more nucleotides in length. The top 105 microsatellites from 71 genes were further analyzed in 93 additional MSI colorectal cancers. Mutation significance and estimated clonality of mutations determined the most likely MSI target genes to be the aminoadipate-semialdehyde dehydrogenase AASDH and the solute transporter SLC9A8 Our findings offer a systematic profiling of the somatic background mutation rate in protein-coding mononucleotide microsatellites, allowing a full cataloging of the true targets of MSI in colorectal cancer. Cancer Res; 77(15); 4078-88. ©2017 AACR.

Molecular Population Genetics of the Northern Elephant Seal Mirounga angustirostris.

The northern elephant seal, Mirounga angustirostris, was heavily hunted and declared extinct in the 19th century. However, a colony remained on remote Guadalupe Island, Mexico and the species has since repopulated most of its historical distribution. Here, we present a comprehensive evaluation of genetic variation in the species. First, we assess the effect of the demographic bottleneck on microsatellite variability and compare it with that found in other pinnipeds, demonstrating levels of variation similar to that in species that continue to be threatened with extinction. Next, we use sequence data from these markers to demonstrate that some of the limited polymorphism predates the bottleneck. However, most contemporary variation appears to have arisen recently and persisted due to exponential growth. We also describe how we use the range in allele size of microsatellites to estimate ancestral effective population size before the bottleneck, demonstrating a large reduction in effective size. We then employ a classical method for bacteria to estimate the microsatellite mutation rate in the species, deriving an estimate that is extremely similar to that estimated for a similar set of loci in humans, indicating consistency of microsatellite mutation rates in mammals. Finally, we find slight significant structure between some geographically separated colonies, although its biological significance is unclear. This work demonstrates that genetic analysis can be useful for evaluating the population biology of the northern elephant seal, in spite of the bottleneck that removed most genetic variation from the species.

Microsatellite Mutation Rate in Atlantic Sturgeon (Acipenser oxyrinchus).

Understanding mutation rates can greatly extend the utility of population and conservation genetic analyses. Herein, we present an estimate of genome-wide microsatellite mutation rate in Atlantic sturgeon (Acipenser oxyrinchus) based on parent-offspring transmission patterns. We screened 307 individuals for parentage and mutation-rate analysis applying 43 variable markers. Out of 13228 allele transfers, 11 mutations were detected, producing a mutation rate of 8.3 × 10-4 per locus per generation (95% confidence interval: 1.48 × 10-3, 4.15 × 10-4). Single-step mutations predominated and there were trends toward mutations in loci with greater polymorphism and allele length. Two of the detected mutations were most probably cluster mutations, being identified in 12 and 28 sibs, respectively. Finally, we observed evidences of polyploidy based on the sporadic presence of 3 or 4 alleles per locus in the genotyped individuals, supporting previous reports of incomplete diploidization in Atlantic sturgeon.