Microsatellite Patterns Research Articles

Comparison of the Distribution Patterns of Microsatellites Across the Genomes of Reptiles

ABSTRACTMicrosatellites or simple sequence repeats (SSRs) are prevalent across various organisms' genomes. However, their distribution patterns and evolutionary dynamics in reptile genomes are rarely studied systematically. We herein conducted a comprehensive analysis of SSRs in the genomes of 36 reptile species. Our findings revealed that the total number of SSRs ranged from 1,840,965 to 7,664,452, accounting for 2.16%–8.19% of the genomes analyzed. The relative density ranged from 21,567.82 to 81,889.41 bp per megabase (Mbp). The abundance of different SSR categories followed the pattern of imperfect SSR (I‐SSR) > perfect SSR (P‐SSR) > compound SSR (C‐SSR). A significant positive correlation was observed between the number of SSRs and genome size (p = 0.0034), whereas SSR frequency (p = 0.013) or density (p = 0.0099) showed a negative correlation with genome size. Furthermore, no correlation was found between SSR length and genome size. Mononucleotide repeats were the most common P‐SSRs in crocodilians and turtles, whereas mononucleotides, trinucleotides, or tetranucleotides were the most common P‐SSRs in snakes, lizards, and tuatara. P‐SSRs of varying motif sizes showed nonrandom distribution across different genic regions, with AT‐rich repeats being predominant. The genomic SSR content of the squamate lineage ranked the highest in abundance and variability, whereas crocodilians and turtles showed a slowly evolving and reduced microsatellite landscape. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that genes harboring P‐SSRs in the coding DNA sequence regions were primarily involved in the regulation of transcription and translation processes. The SSR dataset generated in this study provides potential candidates for functional analysis and calls for broader‐scale analyses across the evolutionary spectrum.

Karyotype diversity of Polybia (gr. occidentalis) species complex (Hymenoptera: Vespidae): Taxonomic and evolutionary implications

AbstractPolybia is a common wasp genus found in most of South America. Polybia (gr. occidentalis) encompasses several species that are difficult to identify because of their similar morphologies. Our goal was to cytogenetically characterize three species belonging to Polybia (gr. occidentalis) and discuss the importance of cytogenetic data for integrative taxonomy. Polybia colonies were sampled from different regions of Brazil to perform classical and molecular cytogenetic analyses. Polybia paulista and Polybia sp. 1 showed 2n = 34 and 18S rDNA loci on two chromosomes, whereas Polybia sp. 2 showed 2n = 40 and 18S rDNA clusters on four chromosomes. The microsatellites GA(15), GAG(10), CAA(10), TTAGG(6), and TCAGG(6) showed similar distributions among the species, forming blocks in the euchromatic regions of the chromosomes, whereas CGG(10) did not yield any positive markings. In contrast, TAT(10) hybridized on the centromeric heterochromatin, showing differences in the number of marked chromosomes among the species. Therefore, it may potentially be a species‐specific cytotaxonomic marker in this group of wasps, but this feature needs further investigation. Fluorochromes evidenced that AT‐rich DAPI+ sequence distribution was coincident with heterochromatin, while the distribution of CG‐rich CMA3+ sequences was coincident with the 18S rDNA region. The present study revealed differences in diploid number, heterochromatin content, 18S rDNA sites, and microsatellite patterns between morphologically similar species, demonstrating the usefulness of cytotaxonomy in studying species complexes.

The Characteristics of Microsatellites and Development of SSR Markers in the Genome of Periplaneta americana

The research was to analyze the number and pattern of microsatellites in Periplaneta americana’s genome, and also developed tetranucleotide SSR markers. We thoroughly scrutinized and dissected the inherent traits that govern the allocation of microsatellite sequences within the profound domain of P. americana’s genome, software MSDBv2 allowed for the utilization of 2.67 Gb. There were precisely 1,498,458 flawless microsatellite sequences, encompassed approximately 1.57%. The cumulative length of microsatellites was 45,076,707 bp, and the abundance of microsatellites was 16889.577 loci/Mb. Out of all the microsatellite repeat variations, the trinucleotide repeats accounted for 44.83% of the total, with a count of 671,830, which were the most abundant type. The tetranucleotide, mononucleotide, pentanucleotide, dinucleotide, and hexanucleotide repeats accounted for 29.01%, 13.62%, 8.37%, 3.70% and 0.47%, respectively. The numbers of different repeat copy categories in each repeat type were also quite different, such as the A in mononucleotide repeat type, the AT in dinucleotides, the AAT in trinucleotides, and AAAT in tetranucleotide were the most of each categories. 143 primers were designed. After undertaking the arduous task of enhancing the initial PCR conditions to perfection, we successfully determined and analyzed a gargantuan number of 38 different polymorphic tetranucleotide microsatellite markers with utmost precision, employed the assistance of two-color fluorescence markers and ingenious genotyping scaned for their comprehensive characterization. The genetic variation in P. americana population involved analyzing the diversity of microsatellite loci, which exhibited varying numbers of alleles per locus ranging from 4 to 21 among the 32 individuals studied. Among them, there were 24 microsatellite loci whose alleles were greater than 10, accounted for 63.16% of the total number of polymorphic microsatellite. The calculated degrees of genetic diversity varied between 0 and 1, the observed heterozygosities was between 0.219 and 1.0, with a mean of 0.6391. The expected heterozygosities was between 0.312 and 0.942, with a mean of 0.7663. The PIC was between 0.296 and 0.923, with a mean of 0.7294, and there were 36 microsatellite loci whose PIC was greater than 0.5, accounted for 94.74%. This study indicated that new development of microsatellite markers for P. americana was feasible. Furthermore, these new development microsatellite markers will provide adequate and reliable molecular genetics data for carrying out the research of molecular ecology and conservation genetics for P. americana.

Formation and identification of artificial gynogenetic mandarin fish (Siniperca chuatsi) induced by inactivated sperm of largemouth bass (Micropterus salmoides)

Gynogenesis is a special method of distant hybridization that has important application value in a variety of areas, such as rapid establishment of pure lines, restoration of endangered species and sexual control. Mandarin fish is an important aquaculture species in China that exhibits sexually dimorphic growth. However, there are few studies on the gynogenesis of mandarin fish at present, and there is no report on gynogenesis induced by cross-family heterologous sperm. In this study, we used the sperm of largemouth bass (Micropterus salmoides, LB, Family Centrarchidae, 2n = 46) with UV-inactivated genetic material to stimulate gynogenesis in mandarin fish (Siniperca chuatsi, MD, Family Serranidae, 2n = 48) to obtain surviving offspring. To determine whether the offspring obtained by the gynogenetic technique are gynogenetic fish, we analyzed the DNA content, chromosome number, karyotype, countable traits, 5S rDNA and mitochondrial DNA of the offspring. Based on the analysis of the above experimental results, it was found that the offspring was a diploid fish with 48 chromosomes, its karyotype was 6sm + 12st + 30 t, and the 5S rDNA gene included two fragments with sizes of 203 bp and 281 bp. These characteristics were consistent with MD. The DNA content, countable traits, and mitochondrial DNA sequence of the offspring were not significantly different from MD. Afterward, we identified the sex of the gynogenetic mandarin fish (GMD) through sex-specific markers. The results showed that all the mandarin fish obtained in this study are female. The microsatellite pattern showed that GMD contained bands from the male parent. In general, this is the first time that carnivore mandarin fish have obtained full female gynogenesis through gynogenesis. Additionally, this study provides a theoretical reference for the gynogenesis of fish in different subfamilies, and the acquisition of all-female mandarin fish has certain commercial value for production practices.

Are scattered microsatellites weak chromosomal markers? Guided mapping reveals new insights into Trachelyopterus (Siluriformes: Auchenipteridae) diversity.

The scattered distribution pattern of microsatellites is a challenging problem in fish cytogenetics. This type of array hinders the identification of useful patterns and the comparison between species, often resulting in over-limited interpretations that only label it as "scattered" or "widely distributed". However, several studies have shown that the distribution pattern of microsatellites is non-random. Thus, here we tested whether a scattered microsatellite could have distinct distribution patterns on homeologous chromosomes of closely related species. The clustered sites of 18S and 5S rDNA, U2 snRNA and H3/H4 histone genes were used as a guide to compare the (GATA)n microsatellite distribution pattern on the homeologous chromosomes of six Trachelyopterus species: T. coriaceus and Trachelyopterus aff. galeatus from the Araguaia River basin; T. striatulus, T. galeatus and T. porosus from the Amazonas River basin; and Trachelyopterus aff. coriaceus from the Paraguay River basin. Most species had similar patterns of the (GATA)n microsatellite in the histone genes and 5S rDNA carriers. However, we have found a chromosomal polymorphism of the (GATA)n sequence in the 18S rDNA carriers of Trachelyopterus galeatus, which is in Hard-Weinberg equilibrium and possibly originated through amplification events; and a chromosome polymorphism in Trachelyopterus aff. galeatus, which combined with an inversion polymorphism of the U2 snRNA in the same chromosome pair resulted in six possible cytotypes, which are in Hardy-Weinberg disequilibrium. Therefore, comparing the distribution pattern on homeologous chromosomes across the species, using gene clusters as a guide to identify it, seems to be an effective way to further the analysis of scattered microsatellites in fish cytogenetics.

Chromosomes of Asian Cyprinid Fishes: Genomic Differences in Conserved Karyotypes of 'Poropuntiinae' (Teleostei, Cyprinidae).

The representatives of cyprinid lineage 'Poropuntiinae' with 16 recognized genera and around 100 species form a significant part of Southeast Asian ichthyofauna. Cytogenetics are valuable when studying fish evolution, especially the dynamics of repetitive DNAs, such as ribosomal DNAs (5S and 18S) and microsatellites, that can vary between species. Here, karyotypes of seven 'poropuntiin' species, namely Cosmochilus harmandi, Cyclocheilichthys apogon, Hypsibarbus malcomi, H. wetmorei, Mystacoleucus chilopterus, M. ectypus, and Puntioplties proctozysron occurring in Thailand were examined using conventional and molecular cytogenetic protocols. Variable numbers of uni- and bi-armed chromosomes indicated widespread chromosome rearrangements with a stable diploid chromosome number (2n) of 50. Examination with fluorescence in situ hybridization using major and minor ribosomal probes showed that Cosmochilus harmandi, Cyclocheilichthys apogon, and Puntioplites proctozystron all had one chromosomal pair with 5S rDNA sites. However, more than two sites were found in Hypsibarbus malcolmi, H. wetmorei, Mystacoleucus chilopterus, and M. ectypus. The number of chromosomes with 18S rDNA sites varied amongst their karyotypes from one to three; additionally, comparative genomic hybridization and microsatellite patterns varied among species. Our results reinforce the trend of chromosomal evolution in cyprinifom fishes, with major chromosomal rearrangements, while conserving their 2n.

Evolutionary tracks of chromosomal diversification in Trichopsis (Anabantiformes, Osphronemidae) fishes: New insights from a molecular cytogenetic perspective

Abstract. Mingkwan B, Sassi FDMC, Muanglenm N, Pinmongkhonkul S, Pinthong K, Tongnunui S, Yeesin P, Tanomtong A, Liehr T, Cioffi MDB, Supiwong W. 2023. Evolutionary tracks of chromosomal diversification in Trichopsis (Anabantiformes, Osphronemidae) fishes: New insights from a molecular cytogenetic perspective. Biodiversitas 24: 1551-1559. This study investigated the chromosomes of three Trichopsisspecies (namely Trichopsis pumila, Trichopsis vittata, and Trichopsis schalleri) using conventional (Giemsa stain, Ag-NOR) and molecularcytogenetic techniques. Fluorescence in situ hybridization (FISH) with repetitive DNAs, including 5S and 18S rDNAs, and microsatellites as probes were also performed. Our results indicated a conserved diploid number of 46 (2n) for all analyzed species of both sexes, although varying in their karyotype structure. Furthermore, T. schalleri and T. vittata karyotypes were presented with only acrocentric chromosomes (46a), while T. pumila had a submetacentric pair in addition to acrocentric ones (2sm+44a). Positive Ag-NOR sites were differentially found in each species adjacent to the acrocentric chromosome's centromeric and/or interstitial sub-centromeric regions. Both 5S and 18S rDNAs vary between the analyzed species, but all were localized in multiple sites. However, the distribution pattern of microsatellites differed in each species, mostly scattering at peri-centromeric and telomeric regions. These findings show that Trichopsis species, while having a conserved diploid number, differ significantly in the distribution of repetitive sequences in their karyotypes, particularly for rDNAs. These species-specific patterns can be useful for characterizing and identifying further different species and examining the evolutionary mechanisms that drove the evolution of these fishes' karyotypes.

Comprehensive Comparative Analysis Sheds Light on the Patterns of Microsatellite Distribution across Birds Based on the Chromosome-Level Genomes.

Microsatellites (SSRs) are widely distributed in the genomes of organisms and are an important genetic basis for genome evolution and phenotypic adaptation. Although the distribution patterns of microsatellites have been investigated in many phylogenetic lineages, they remain unclear within the morphologically and physiologically diverse avian clades. Here, based on high-quality chromosome-level genomes, we examined the microsatellite distribution patterns for 53 birds from 16 orders. The results demonstrated that each type of SSR had the same ratio between taxa. For example, the frequency of imperfect SSRs (I-SSRs) was 69.90-84.61%, while perfect SSRs (P-SSRs) were 14.86-28.13% and compound SSRs (C-SSRs) were 0.39-2.24%. Mononucleotide SSRs were dominant for perfect SSRs (32.66-76.48%) in most bird species (98.11%), and A(n) was the most abundant repeat motifs of P-SSRs in all birds (5.42-68.22%). Our study further confirmed that the abundance and diversity of microsatellites were less effected by evolutionary history but its length. The number of P-SSRs decreased with increasing repeat times, and longer P-SSRs motifs had a higher variability coefficient of the repeat copy number and lower diversity, indicating that longer motifs tended to have more stable preferences in avian genomes. We also found that P-SSRs were mainly distributed at the gene ends, and the functional annotation for these genes demonstrated that they were related to signal transduction and cellular process. In conclusion, our research provided avian SSR distribution patterns, which will help to explore the genetic basis for phenotypic diversity in birds.

Production of a diploid hybrid with fast growth performance derived from the distant hybridization of Hypophthalmichthys nobilis (female) × Megalobrama amblycephala (male)

Based on the distant hybridization derived from Hypophthalmichthys nobilis (bighead carp, BIC, 2n ​= ​48) (♀) ​× ​Megalobrama amblycephala (blunt snout bream, BSB, 2n ​= ​48) (♂), we obtained a novel hybrid (BB) in this study. We comparatively investigated the morphological traits, chromosome numbers, DNA contents, microsatellite patterns, and DNA variations in 5S rDNA and Hox genes. The chromosome and flow cytometer results indicated that BB was a diploid hybrid with 48 chromosomes. We confirmed successful hybridization with microsatellite DNA markers and analyzed the Hox gene family in the parental and hybrid lines. The sequences of 5S rDNA in BB mapped to those in BIC and BSB, indicating that the genetic materials of BB were derived from BIC and BSB. The breeding experiment showed that BB had the characteristics of herbivore and fast growth. This diploid hybrid is of great significance for fish genetic breeding and applied research.

Unique genetic structure of the human tapeworm Dibothriocephalus latus from the Alpine lakes region - a successful adaptation?

Dibothriocephalus latus is the most frequent causative agent of fish-borne zoonosis (diphyllobothriosis) in Europe, where it is currently circulating mainly in the Alpine lakes region (ALR) and Russia. Three mitochondrial genes (cox1, cob and nad3) and 6 microsatellite loci were analysed to determine how is the recently detected triploidy/parthenogenesis in tapeworms from ALR displayed at the DNA level. A geographically distant population from the Krasnoyarsk Reservoir in Russia (RU-KR) was analysed as a comparative population. One or 2 alleles of each microsatellite locus was detected in plerocercoids from RU-KR, corresponding to the microsatellite pattern of a diploid organism. In contrast, 1–3 alleles were observed in tapeworms from ALR, in accordance with their triploidy. The high diversity of mitochondrial haplotypes in D. latus from RU-KR implied an original and relatively stable population, but the identical structure of mitochondrial genes of tapeworms from ALR was probably a consequence of a bottleneck typical of introduced populations. These results indicated that the diploid/sexually reproducing population from RU-KR was ancestral, located within the centre of the distribution of the species, and the triploid/parthenogenetically reproducing subalpine population was at the margin of the distribution. The current study revealed the allelic structure of the microsatellite loci in the triploid tapeworm for the first time.

Over time variation in microsatellite patterns in a natural wild potato population from northwest Argentina

AbstractThe common potato, Solanum tuberosum L. (2n = 4x = 48), has 100–200 related wild taxonomic species endemic to the Americas, which constitute invaluable sources of genetic diversity. They form a polyploid series with 2n = 2x – 6x (x = 12) and can reproduce asexually by tubers and stolons and/or sexually by seeds. Information is scarce on their preponderant mode of reproduction and its consequences on the genetic population structure over time. In previous work, a morphologically variable wild potato population from northwestern Argentina was sampled in two consecutive years. Two populations were ex situ regenerated from the sampled propagules (seeds in 2013 and tubers in 2014) for morphological and reproductive studies; these populations exhibited differences in reproductive behavior that could not be related to morphological phenotypes. To ascertain if the observed differences could be related to the captured genetic diversity in each year, a molecular analysis was carried out with seven microsatellite primer pairs located in seven chromosomes of the basic set. The captured genetic diversity, based on the proportion of individuals with a unique pattern of amplified fragments, varied significantly (χ2 α = .05) between sampling years. This could be due to changes in the preponderant mode of reproduction, differential genotype fitness resulting from genotype × environment interactions, or both. Wild potato accessions at germplasm banks are one‐time collection samples; thus, they may not be representative of the genetic diversity of the sampled population. To properly ex situ conserve the available genetic diversity, it is advisable to resample the sites whenever possible and to conform each accession with the successive samples.

Are Microsatellite Patterns Specific for Tumor Types? A Pilot Investigation

Microsatellite testing is an emerging field of molecular pathology, as microsatellite instability (MSI) appears to be a predictive biomarker for some cancers. Although multiple studies on microsatellites have been published, recent observations suggest that the microsatellites that define instability differ between tumor entities. This assumption is confirmed by the present study that compared different MSI assays validated for colorectal cancer. Whilst all assays deliver the same MSI/MSS status for colorectal cancers, they differ for tonsillar tumors, leading to the hypothesis that MSI patterns are tumor-type specific.

Karyomorphological delineation and linear differentiation of microsatellite patterns, and meiosis in giant Asian river frog (Limnonectes blyhii) from Thailand

The present investigation provides information on linear chromosomal differentiation and microsatellite patterns [d(CA)15, d(CGG)10, d(TA)15] in the genome of giant Asian river frog (Limnonectes blyhii). Five male and female samples each collected from southern Thailand, were used for the study. The metaphase chromosome preparations were prepared from the bone marrow using the standard protocol. Results show that both male and female have the diploid chromosome number 2n = 24, and the fundamental number NF = 48. The karyotypes compose of 4 large metacentric, 4 large submetacentric, 2 medium metacentric, 8 small metacentric and 6 small submetacentric chromosomes. The Ag-NOR, FISH as well as hybridization with the microsatellite d(CGG)10 confirm the location of NOR site in the subcentromeric region on chromosome pair 11. The in situ localization pattern of d(CA)15 microsatellite was positive on chromosome pairs 1 and 6, while microsatellite d(TA)15 show its location in the long arm of chromosome 5,8 and 10. The information provided has value as species specific marker for this species.

Distribution patterns of microsatellites and development of its marker in different genomic regions of forest musk deer genome based on high throughput sequencing.

Forest musk deer (Moschus berezovskii, FMD) is an endangered artiodactyl species, male FMD produce musk. We have sequenced the whole genome of FMD, completed the genomic assembly and annotation, and performed bioinformatic analyses. Our results showed that microsatellites (SSRs) displayed nonrandomly distribution in genomic regions, and SSR abundances were much higher in the intronic and intergenic regions compared to other genomic regions. Tri- and hexanucleotide perfect (P) SSRs predominated in coding regions (CDSs), whereas, tetra- and pentanucleotide P-SSRs were less abundant. Trifold P-SSRs had more GC-contents in the 5′-untranslated regions (5'UTRs) and CDSs than other genomic regions, whereas mononucleotide P-SSRs had the least GC-contents. The repeat copy numbers (RCN) of the same mono- to hexanucleotide P-SSRs had different distributions in different genomic regions. The RCN of trinucleotide P-SSRs had increased significantly in the CDSs compared to the transposable elements (TEs), intronic and intergenic regions. The analysis of coefficient of variability (CV) of P-SSRs showed that the RCN of mononucleotide P-SSRs had relative higher variation in different genomic regions, followed by the CV pattern of RCN: dinucleotide P-SSRs > trinucleotide P-SSRs > tetranucleotide P-SSRs > pentanucleotide P-SSRs > hexanucleotide P-SSRs. The CV variations of RCN of the same mono- to hexanucleotide P-SSRs were relative higher in the intron and intergenic regions, followed by that in the TEs, and the relative lower was in the 5'UTR, CDSs and 3'UTRs. 58 novel polymorphic SSR loci were detected based on genotyping DNA from 36 captive FMD and 22 SSR markers finally showed polymorphism, stability, and repetition.

Two cases of airport-associated falciparum malaria in Frankfurt am Main, Germany, October 2019.

Two cases of presumably airport-acquired falciparum malaria were diagnosed in Frankfurt in October 2019. They were associated with occupation at the airport, and Plasmodium falciparum parasites from their blood showed genetically identical microsatellite and allele patterns. Both had severe malaria. It took more than a week before the diagnosis was made. If symptoms are indicative and there is a plausible exposure, malaria should be considered even if patients have not travelled to an endemic area.

Genome-wide characterization and analysis of microsatellite sequences in camelid species

Microsatellites or simple sequence repeats (SSRs) are among the genetic markers most widely utilized in research. This includes applications in numerous fields such as genetic conservation, paternity testing, and molecular breeding. Though ordered draft genome assemblies of camels have been announced, including for the Arabian camel, systemic analysis of camel SSRs is still limited. The identification and development of informative and robust molecular SSR markers are essential for marker assisted breeding programs and paternity testing. Here we searched and compared perfect SSRs with 1–6 bp nucleotide motifs to characterize microsatellites for draft genome sequences of the Camelidae. We analyzed and compared the occurrence, relative abundance, relative density, and guanine-cytosine (GC) content in four taxonomically different camelid species: Camelus dromedarius, C. bactrianus, C. ferus, and Vicugna pacos. A total of 546762, 544494, 547974, and 437815 SSRs were mined, respectively. Mononucleotide SSRs were the most frequent in the four genomes, followed in descending order by di-, tetra-, tri-, penta-, and hexanucleotide SSRs. GC content was highest in dinucleotide SSRs and lowest in mononucleotide SSRs. Our results provide further evidence that SSRs are more abundant in noncoding regions than in coding regions. Similar distributions of microsatellites were found in all four species, which indicates that the pattern of microsatellites is conserved in family Camelidae.

GTn Repeat Microsatellite Instability in Uterine Fibroids.

Background: Type I collagen is a triple helix structure with two α1 and one α2 chains. Coordinated biosynthesis of α1 and α2 subunits is very important for tissue morphogenesis, growth, and repair. In contrast, abnormal deposition in response to proinflammatory cytokines is associated with organ dysfunction. In humans, COL1A2 contains two microsatellite loci: one located at the 5’-flanking region is composed of poly CA and poly CG; the other located in the 1st intron is constituted of poly GT. Expression of COL1A2 has been noted in gastric cancer and was positively correlated with degree of invasion and metastases. But no genetic study taking into account polymorphism of COL1A2 in uterine fibroids has been undertaken. Methods: In this study, repeated dinucleotide GT n of intron 1 COL1A2 was highlighted in 55 patients with uterine fibroids (UF). Clinical and pathological data were obtained from patient’s records, and other parameters were recorded. Mutation Surveyor version 5.0.1, DnaSP version 5.10, MEGA version 7.0.26, and Arlequin version 3.5.1.3 were used to determine genetics parameters. To estimate genetic variation according to epidemiological parameters, index of genetic differentiation (Fst) and genetic structure (AMOVA) were determined with Arlequin version. Results: Based on reference microsatellite pattern (GT) 14 CT(GT) 3 CT(GT) 3, 15 haplotypes were found. Among the 15 haplotypes, 12 have mutation at position 2284C > G and 7 at position 2292C > G. Insertions of repeated dinucleotide GTn were found on three haplotypes against eight haplotypes in which they are deletions. Intron 1 of COL1A2 gene exhibits high genetic diversity in uterine fibroids with 35.34% polymorphic sites, 95.74% of which were parsimoniously variable and an average number of nucleotide difference of 10.442, which reflects an important genetic variability. According to epidemiological parameters, our results showed, for the first time, a genetic structuring of uterine fibroids according to ethnicity, marital status, use of contraception, diet, and physical activity, beyond confirming the involvement dinucleotide length polymorphism GTn in occurrence of uterine fibroids in Senegalese women. Conclusion: Results obtained open up avenues for understanding the mechanisms involved in the racial variation in the prevalence of uterine fibroids as well as the predisposing factors.

Abstract 3490: Molecular profiling of MSI-high colorectal cancer (CRC) in Indian population

Abstract Background: CRC is the 3rd most commonly diagnosed malignancy and 4th leading cause of cancer related deaths worldwide. Over two decades of extensive research from the West has revealed three major tumorigenesis pathways responsible for CRC viz. a) Chromosomal Instability (CIN) arising from aberrant activation of Wnt signalling, b) Microsatellite Instability (MSI) caused by inactivation of MisMatch Repair (MMR) genes and c) CpG Island Methylator Phenotype resulting in promoter DNA methylation induced inactivation of tumour suppressor genes. Previous results from our laboratory showed a significant proportion of Early Onset Sporadic Rectal Cancer (EOSRC) to be driven neither by aberrant Wnt signalling nor MSI. We also identified a significant proportion of MSI+ familial CRC not exhibiting loss either of the four major MMR genes indicating involvement of addition MMR or non-MMR genes. We therefore endeavoured to determine status of MSI in sporadic CRC samples from the Indian population. We are performing analysis of the status of other molecular features including CIMP, BRAF/KRAS/p53 mutation status, Wnt, etc. to obtain a wholesome molecular profile of sporadic CRC from the Indian population. Methodology: The microsatellite pattern in CRC tumours was determined using the five microsatellite marker panel as per the Bethesda guidelines. Expression status of MMR proteins (MLH1, MSH2, MSH6 & PMS2) was evaluated using immunohistochemistry. Mutations in BRAF (V600E) and KRAS (Codon 12 and 13) were identified by Sanger sequencing. CIMP status is being determined using the MethyLight technology as well as methylation specific PCR. Results: A comprehensive screen of close to 200 CRC samples revealed a significantly higher proportion of sporadic CRC tumors (41%; 50% of colonic and 31% of rectal cancer samples) exhibiting MSI as compared to 14% reported in The Cancer Genome Atlas (TCGA) data. Among MSI-high tumours, only 27% and 15% showed loss of MLH1 or MSH2 respectively, a significantly lower frequency compared to Caucasian data. Instability in the BAT25 microsatellite was significantly high in tumors harboring MLH1 loss. More importantly, a significant proportion of MSI+ CRC tumors were devoid of loss of either of the four major MMR genes. In addition, the BRAF V600E mutation, a known cause of MLH1 silencing, is absent in all but one MSI+ tumors. Work on determining status of CIMP and Wnt signalling is currently underway. Conclusions: Significantly higher proportion of sporadic CRCs from Indian population exhibit MSI but the underlying mechanism appears to be novel and distinct from those identified from the Caucasian population. Deep sequencing based studies are currently underway to determine molecular aberrations driving MSI in the Indian sporadic CRC. Note: This abstract was not presented at the meeting. Citation Format: Ashmala Naz, Vijaya Tourani, Satish Rao, Murali Dharan Bashyam. Molecular profiling of MSI-high colorectal cancer (CRC) in Indian population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3490.

Microsatellite instability detection from plasma of colorectal cancer patients.

515 Background: MSI, detected in 15% of colorectal cancer (CRC) patients, is a hypermutable phenotype caused by deficiency in DNA mismatch repair system. Currently, MSI detection is limited to tissue samples. Detection of MSI from blood samples has been explored but confounded by low sensitivity due to the trace amount of ctDNA present. In this study, we developed a NGS read-count based algorithm-bMSISEA (blood MSI signature enriched analysis) to detect MSI from blood samples. Methods: Matched tumor and adjacent normal tissues were obtained from 50 MSI-H CRC patients to establish a microsatellite pattern for MSI-H tumors. White blood cells (WBCs) from 100 CRC patients were used to establish a microsatellite pattern for MSS. Plasma samples were obtained from 75 CRC patients with known MSI status assessed by IHC (38 MSI-H and 37 MSS) to validate the algorithm. Results: To establish features for MSI-H and MSS tumors, 51 microsatellite loci covered by ColonCore panel with mononucleotide repeats longer than 10bp were scanned using paired tumor and adjacent normal tissues and 8 loci with significant differential read length distribution were identified. MSI signature enrichment analysis was performed using WBCs to establish a microsatellite signature for MSS tumors. For each locus, the numbers of total reads supporting either MSI-H (denoted as K in the equation) or MSS (N-K, where N refers to the total number of reads supporting either MSI-H or MSS at a specific locus) were counted. The MSI signature enrichment analysis was performed using hypergeometric test. The log-transformed p values were used to calculate z scores. The final microsatellite status was reflected by MS score, which was the sum of z scores of all loci. A MS score of 15 was derived to differentiate MSS and MSI-H tumors. Both simulation data and data generated using clinical samples revealed more than 98% sensitivity and 100% specificity with ctDNA percentage greater than 1%. This method is still reliable if the ctDNA percentage drops to 0.4%, yielding sensitivity of 93.3% and specificity of 100%. Conclusions: We developed a sensitive algorithm which can reliably detect MSI status with ctDNA fraction greater than 0.4% with sensitivity of 93.3% and specificity of 100%.

Karyotypic Divergence of Two Co-Occurring Species of Andean Climbing Catfishes (Siluriformes: Loricarioidei: Astroblepidae).

The monotypic Astroblepidae fish family includes 81 species distributed along Central and Northern Andes in South America and Panamá in Central America; most aspects regarding its biology, taxonomy, and chromosomal features remain largely unknown. This study reports the karyotype of two sympatric Astroblepus species from the Colombian Andes, aiming to provide novel information on karyotype organization and reveal possible chromosomal rearrangements occurred on these species, through mapping of different repetitive DNA classes, including microsatellites and ribosomal DNA multigene families. The results showed differences in the chromosome number and karyotypic formula: Astroblepus grixalvii had 2n = 52 (28m+8sm +12st +4a) with the metacentric pair number 1 as the largest chromosome pair, whereas Astroblepus homodon had 2n = 54 (30m+8sm +8st +8a) and less evident chromosome size differences. Microsatellite probes marked the tips of all chromosomes in both species except the short arms of acrocentric pair numbers 24 and 25 in A. homodon. Each ribosomal probe marked different chromosome pairs in both species. Microsatellite patterns suggest that the 2n increase probably involved a centric fission event that occurred during the evolutionary history of these species. This is the first karyotype description of an Astroblepus species and it contributes to the theoretical framework about the karyoevolutionary trends within Loricarioidei.