Evaluation of an eight marker-panel including long mononucleotide repeat markers to detect microsatellite instability in colorectal, gastric, and endometrial cancers.

BACKGROUND: Accurate determination of microsatellite instability (MSI) status is critical for optimal treatment in cancer patients. Conventional MSI markers can sometimes display subtle shifts that are difficult to interpret, especially in non-colorectal cases. We evaluated an experimental eight marker-panel including long mononucleotide repeat (LMR) markers for detection of MSI. METHODS: The eight marker-panel was comprised of five conventional markers (BAT-25, BAT-26, NR-21, NR-24, and NR-27) and three LMR markers (BAT-52, BAT-59 and BAT-62). MSI testing was performed against 300 specimens of colorectal, gastric, and endometrial cancers through PCR followed by capillary electrophoresis length analysis. RESULTS: The MSI testing with eight marker-panel showed 99.3% (295/297) concordance with IHC analysis excluding 3 MMR-focal deficient cases. The sensitivity of BAT-59 and BAT-62 was higher than or comparable to that of conventional markers in gastric and endometrial cancer. The mean shift size was larger in LMR markers compared to conventional markers for gastric and endometrial cancers. CONCLUSIONS: The MSI testing with eight maker-panel showed comparable performance with IHC analysis. The LMR markers, especially BAT-59 and BAT-62, showed high sensitivity and large shifts which can contribute to increased confidence in MSI classification, especially in gastric and endometrial cancers. Further study is needed with large number of samples for the validation of these LMR markers.

Loss of fragile WWOX gene leads to senescence escape and genome instability.

Induction of DNA damage response (DDR) to ensure accurate duplication of genetic information is crucial for maintaining genome integrity during DNA replication. Cellular senescence is a DDR mechanism that prevents the proliferation of cells with damaged DNA to avoid mitotic anomalies and inheritance of the damage over cell generations. Human WWOX gene resides within a common fragile site FRA16D that is preferentially prone to form breaks on metaphase chromosome upon replication stress. We report here that primary Wwox knockout (Wwox-/-) mouse embryonic fibroblasts (MEFs) and WWOX-knockdown human dermal fibroblasts failed to undergo replication-induced cellular senescence after multiple passages in vitro. Strikingly, by greater than 20 passages, accelerated cell cycle progression and increased apoptosis occurred in these late-passage Wwox-/- MEFs. These cells exhibited γH2AX upregulation and microsatellite instability, indicating massive accumulation of nuclear DNA lesions. Ultraviolet radiation-induced premature senescence was also blocked by WWOX knockdown in human HEK293T cells. Mechanistically, overproduction of cytosolic reactive oxygen species caused p16Ink4a promoter hypermethylation, aberrant p53/p21Cip1/Waf1 signaling axis and accelerated p27Kip1 protein degradation, thereby leading to the failure of senescence induction in Wwox-deficient cells after serial passage in culture. We determined that significantly reduced protein stability or loss-of-function A135P/V213G mutations in the DNA-binding domain of p53 caused defective induction of p21Cip1/Waf1 in late-passage Wwox-/- MEFs. Treatment of N-acetyl-L-cysteine prevented downregulation of cyclin-dependent kinase inhibitors and induced senescence in Wwox-/- MEFs. Our findings support an important role for fragile WWOX gene in inducing cellular senescence for maintaining genome integrity during DDR through alleviating oxidative stress.

Highly sensitive duplex MSI test and BAT40 germline polymorphism.

Tumors exhibiting DNA mismatch repair (MMR) deficiency and microsatellite instability (MSI) are responsive to immune checkpoint blockade. MSI is frequently diagnosed using five quasimonomorphic mononucleotide (pentaplex) markers; however, the assays have several technical limitations, including the lack of sensitivity of some of the markers. Although markers with increased sensitivity, such as CAT25 and BAT40, have been introduced, the majority of multiplex MSI tests have only been studied in Western populations and require further evaluation in an Asian cohort. This study tested the efficacy of BAT26, CAT25, and BAT40 mononucleotide MSI markers via triplex PCR on 300 samples from patients with advanced cancers from a Korean clinical population. The results were directly compared with those of a pentaplex MSI test and tumor mutation burden (TMB) status, and an additional 60 MSI-H cancers were used for further validation. Four (1.3%) out of 300 advanced tumors were MSI-high (MSI-H). In the pentaplex PCR assay, two colorectal cancers (0.7%) exhibited instability only with the BAT25 mononucleotide marker and were interpreted as MSI-low (MSI-L). In the triplex PCR assay, BAT40 was unstable in 64 cases (21%) and the results did not overlap with those of MSI-L from pentaplex. Given the high frequency of isolated BAT40 instability, we performed the same triplex PCR with DNA obtained from normal controls and found BAT40 polymorphisms in 37 cases (90%). Interestingly, the median TMB of the cases with BAT40 polymorphism was significantly higher (7.0 mt/Mb) than that of BAT40 wild-type cases (5.5 mt/Mb) (p = 0.003). The triplex PCR results from 60 additional MSI-H cancers correlated perfectly (100%) with those of pentaplex PCR, and the results were consistent for two (BAT26 and CAT25) markers. BAT40 germline polymorphism is common in the Korean population and is associated with higher TMB values. The simple duplex (BAT26 and CAT25) MSI test provided the same sensitivity and specificity as pentaplex PCR tests.

Length variations within the Merle retrotransposon of canine PMEL: correlating genotype with phenotype.

BACKGROUND: The antisense insertion of a canine short interspersed element (SINEC_Cf) in the pigmentation gene PMEL (or SILV) causes a coat pattern phenotype in dogs termed merle. Merle is a semi-dominant trait characterized by patches of full pigmentation on a diluted background. The oligo(dT) tract of the Merle retrotransposon is long and uninterrupted and is prone to dramatic truncation. Phenotypically wild-type individuals carrying shorter oligo(dT) lengths of the Merle allele have been previously described and termed cryptic merles. Two additional coat patterns, dilute merle (uniform, steely-grey coat) and harlequin merle (white background with black patches), also appear in breeds segregating the Merle allele. RESULTS: Sequencing of all PMEL exons in a dilute and a harlequin merle reveals that variation exists solely within the oligo(dT) tract of the SINEC_Cf insertion. In fragment analyses from 259 dogs heterozygous for Merle, we observed a spectrum of oligo(dT) lengths spanning 25 to 105 base pairs (bp), with ranges that correspond to the four varieties of the merle phenotype: cryptic (25-55 bp), dilute (66-74 bp), standard (78-86 bp), and harlequin (81-105 bp). Somatic contractions of the oligo(dT) were observed in 43% of standard and 51% of harlequin merle dogs. A small proportion (4.6%) of the study cohort inherited de novo contractions or expansions of the Merle allele that resulted in dilute or harlequin coat patterns, respectively. CONCLUSIONS: The phenotypic consequence of the Merle SINE insertion directly depends upon oligo(dT) length. In transcription, we propose that the use of an alternative splice site increases with oligo(dT) length, resulting in insufficient PMEL and a pigment dilution spectrum, from dark grey to complete hypopigmentation. We further propose that during replication, contractions and expansions increase in frequency with oligo(dT) length, causing coat variegation (somatic events in melanocytes) and the spontaneous appearance of varieties of the merle phenotype (germline events).

A mononucleotide repeat in PRRT2 is an important, frequent target of mismatch repair deficiency in cancer.

The DNA mismatch repair (MMR) system corrects DNA replication mismatches thereby contributing to the maintenance of genomic stability. MMR deficiency has been observed in prostate cancer but its impact on the genomic landscape of these tumours is not known. In order to identify MMR associated mutations in prostate cancer we have performed whole genome sequencing of the MMR deficient PC346C prostate cancer cell line. We detected a total of 1196 mutations in PC346C which was 1.5-fold higher compared to a MMR proficient prostate cancer sample (G089). Of all different mutation classes, frameshifts in mononucleotide repeat (MNR) sequences were significantly enriched in the PC346C sample. As a result, a selection of genes with frameshift mutations in MNR was further assessed regarding its mutational status in a comprehensive panel of prostate, ovarian, endometrial and colorectal cancer cell lines. We identified PRRT2 and DAB2IP to be frequently mutated in MMR deficient cell lines, colorectal and endometrial cancer patient samples. Further characterization of PRRT2 revealed an important role of this gene in cancer biology. Both normal prostate cell lines and a colorectal cancer cell line showed increased proliferation, migration and invasion when expressing the mutated form of PRRT2 (ΔPRRT2). The wild-type PRRT2 (PRRT2wt) had an inhibitory effect in proliferation, consistent with the low expression level of PRRT2 in cancer versus normal prostate samples.

Identification and characterization of a -1 reading frameshift in the heavy chain constant region of an IgG1 recombinant monoclonal antibody produced in CHO cells.

Frameshifts lead to complete alteration of the intended amino acid sequences, and therefore may affect the biological activities of protein therapeutics and pose potential immunogenicity risks. We report here the identification and characterization of a novel -1 frameshift variant in a recombinant IgG1 therapeutic monoclonal antibody (mAb) produced in Chinese hamster ovary cells during the cell line selection studies. The variant was initially observed as an atypical post-monomer fragment peak in size exclusion chromatography. Characterization of the fragment peak using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analyses determined that the fragment consisted of a normal light chain disulfide-linked to an aberrant 26 kDa fragment that could not be assigned to any HC fragment even after considering common modifications. Further analysis using LC-MS/MS peptide mapping revealed that the aberrant fragment contained the expected HC amino acid sequence (1-232) followed by a 20-mer novel sequence corresponding to expression of heavy chain DNA sequence in the -1 reading frame. Examination of the DNA sequence around the frameshift initiation site revealed that a mononucleotide repeat GGGGGG located in the IgG1 HC constant region was most likely the structural root cause of the frameshift. Rapid identification of the frameshift allowed us to avoid use of a problematic cell line containing the frameshift as the production cell line. The frameshift reported here may be observed in other mAb products and the hypothesis-driven analytical approaches employed here may be valuable for rapid identification and characterization of frameshift variants in other recombinant proteins.

Comparative analysis of microsatellites in chloroplast genomes of lower and higher plants.

Microsatellites, or simple sequence repeats (SSRs), contain repetitive DNA sequence where tandem repeats of one to six base pairs are present number of times. Chloroplast genome sequences have been  shown to possess extensive variations in the length, number and distribution of SSRs. However, a comparative analysis of chloroplast microsatellites is not available. Considering their potential importance in generating genomic diversity, we have systematically analysed the abundance and distribution of simple and compound microsatellites in 164 sequenced chloroplast genomes from wide range of plants. The key findings of these studies are (1) a large number of mononucleotide repeats as compared to SSR(2-6)(di-, tri-, tetra-, penta-, hexanucleotide repeats) are present in all chloroplast genomes investigated, (2) lower plants such as algae show wide variation in relative abundance, density and distribution of microsatellite repeats as compared to flowering plants, (3) longer SSRs are excluded from coding regions of most chloroplast genomes, (4) GC content has a weak influence on number, relative abundance and relative density of mononucleotide as well as SSR(2-6). However, GC content strongly showed negative correlation with relative density (R (2) = 0.5, P < 0.05) and relative abundance (R (2) = 0.6, P < 0.05) of cSSRs. In summary, our comparative studies of chloroplast genomes illustrate the variable distribution of microsatellites and revealed that chloroplast genome of smaller plants possesses relatively more genomic diversity compared to higher plants.

Mononucleotide repeats in the SMAD4 gene promoter in colon carcinoma tissue of Croatian patients.

This study was aimed at the analysis of mononucleotide repeats -462T(15) and -4T(12) in the SMAD4 gene promoter in sporadic colon adenocarcinoma tissue of Croatian patients. The analysis has included 60 pairs of samples of colon tumor and adjacent normal tissue. The number of thymidines in the tracts -462T(15) and -4T(12) of the SMAD4 gene promoter was determined by PCR with fluorescently labeled primers followed by the analysis of obtained DNA fragments by capillary electrophoresis. In the normal colon tissue two haplotypes were present: -462T(15)/-4T(12) in 51 patients (85%) and -462T(16)/-4T(12) in 9 patients (15%). Among the cases with haplotype -462T(15)/-4T(12) detected in normal colon tissue, in 5 cases (8%) malignant tissue displayed different haplotypes: 462T(10)/-4T(10), -462T(12)/-4T(12), 462T(13)/-4T(11), -462T(14)/-4T(10) and -462T(15)/-4T(11). Haplotype -462T(14)/-4T(10) was previously found to be associated with significantly decreased SMAD4 gene promoter activity in comparison to the wild type, while the other detected haplotypes remain to be functionally characterized. This study has shown that functionally relevant somatic alterations of the SMAD4 gene promoter are found in some colon cancer tumors. Although not as frequent in colon as in pancreatic cancer, they may be of significance for certain cases and their role in colon tumorigenesis should be investigated further.

The mitochondrial genome of the pinewood nematode (Bursaphelenchus xylophilus) lineage introduced in Europe.

The pinewood nematode (PWN) Bursaphelenchus xylophilus is the causative agent of pine wilt disease and the greatest biological threat to conifer forests worldwide. Here we describe the near-complete mitochondrial DNA (mtDNA) sequence (12,945 bp) of the PWN lineage recently introduced in Europe. The absence of polymorphisms across the mtDNA of three Portuguese isolates suggests that a single mitochondrial lineage was introduced in southwestern Europe. We also found that Portuguese isolates have an incomplete stop codon (TA) at COX3, while the reference mtDNA from a South Korean isolate has a complete stop codon (TAA). Moreover, two insertion/deletion polymorphisms change the ND4 protein in a stretch of seven amino acids, and a polymorphic mononucleotide repeat alters the predicted structure of the tyrosine tRNA in different geographical isolates. Overall, the new PWN mtDNA sequence provides a basis for studying the European dispersion of this important invasive species.

Non-LTR retrotransposons and microsatellites: Partners in genomic variation.

The human genome is laden with both non-LTR (long-terminal repeat) retrotransposons and microsatellite repeats. Both types of sequences are able to, either actively or passively, mutagenize the genomes of human individuals and are therefore poised to dynamically alter the human genomic landscape across generations. Non-LTR retrotransposons, such as L1 and Alu, are a major source of new microsatellites, which are born both concurrently and subsequently to L1 and Alu integration into the genome. Likewise, the mutation dynamics of microsatellite repeats have a direct impact on the fitness of their non-LTR retrotransposon parent owing to microsatellite expansion and contraction. This review explores the interactions and dynamics between non-LTR retrotransposons and microsatellites in the context of genomic variation and evolution.