Microsatellite Regions Research Articles

Mithramycin analogues with unique mechanism of action in Ewing sarcoma

IntroductionChromosomal translocations involving ETS family proteins are common in Ewing Sarcoma Family of Tumors (ESFT) and form aberrant ETS‐fusion transcription factors. EWS‐FLI1 and EWS‐ERG are the most common transcription factors in ESFT and function by binding DNA at microsatellite regions, characterized by GGAA repeats, which are found at promoters of genes that drive ESFT growth. These fusion products also interact with the DNA damage response protein and transcriptional co‐regulator PARP‐1, which allows for the development and combination treatments with novel targeted therapeutics in ESFT. Mithramycin (MTM) has been identified as a potent inhibitor of EWS‐ETS, but this drug has a very narrow therapeutic window and its clinical use is marked by severe liver and hematological toxicities; likely the result of interference with the ubiquitously expressed Sp1 transcription factor as well as the pharmacokinetic and biodistribution characteristics of MTM. Here, we sought to identify analogues of MTM that selectively perturb the ETS transcription factor complex and afford efficacy in Ewing sarcoma as a monotherapy and potentially in combination with PARP inhibitors.MethodsQualitative interactions between drug‐DNA‐protein were assessed by electrophoretic mobility shift assays (EMSA). Time‐resolved fluorescence energy transfer (TR‐FRET) assays were used to quantitatively determine DNA bound ERG and Sp1 perturbation by MTM and analogues. Cellular thermal shift assays (CETSA) were used to semi‐quantitatively assess the physical interaction of compounds with EWS‐FLI1 and Sp1. DNA damage (c‐PARP, γ‐H2AX) and phosphorylation at the C‐terminal domain (CTD) of RNAPII was determined by western blot following drug treatments in ETS and non ETS expressing cell lines. mRNA expression of genes involved in DNA damage repair (DDR) was determined by RT‐PCR. Cell viability, by aerobic respiration, in single and combination treatments with a PARP inhibitor were assessed with resazurin.ResultsAnalogues with hydrophobic 3‐side chain modifications showed increased interaction with DNA bound ERG by TR‐FRET, CETSA, and luciferase reporter assays. Specifically, CETSA demonstrated that MTM analogues physically interact and stabilize EWS‐FLI1, but not with Sp1, which is a unique mechanism of action. MTM analogues enhanced DNA damage in Ewing Sarcoma cells and inhibited RNAP2 phosphorylation. As compared to MTM, treatment with analogues resulted in higher expression of DNA damage markers and down regulation of DDR genes specifically in cell lines containing EWS‐ETS translocations in a concentration dependent manner. Cell viability assays showed strong synergy when MTM analogues were combined with Olaparib, a PARP inhibitor. This effect was only seen in Ewing sarcoma cells and was abolished with EWS‐FLI1 silencing.ConclusionThese studies demonstrate that novel MTM analogues selectively perturb the EWS‐ETS transcription factor complex function. This interaction affects transcription of downstream genes and the DDR apparatus specifically in EWS‐ETS expressing cell lines. The recruitment of PARP to DNA damage sites induced by MTM analogues provides the opportunity for a rational and synergistic treatment combination with a PARP inhibitor.

Population structure of the New Zealand whelk, Cominella glandiformis (Gastropoda: Buccinidae), suggests sporadic dispersal of a direct developer

AbstractWe examined phylogeographic structure in the direct-developing New Zealand endemic intertidal mud whelk, Cominella glandiformis. Two hundred and ninety-six whelks from 12 sites were collected from sheltered shores around New Zealand’s four largest islands (North Island, South Island, Stewart Island and Chatham Island), encompassing the geographical range of this species. Despite being direct developers, gene flow among C. glandiformis populations may occur over short distances by adult floating, and over larger distances by rafting of egg masses. Primers were developed to amplify variable microsatellite regions at six loci. All loci were variable, with 8–34 alleles/loci. Observed and expected heterozygosities were high across all alleles, with minimal evidence of null alleles. The average number of alleles varied from 3.5 (Chatham Island) to 7.5 (Waitemata Harbour). Strong genetic structure was evident, with distinct ‘eastern’ and ‘western’ groups. Each group extended over a large geographic area, including regions of unsuitable habitat, but were linked by oceanic currents. We suggest that the intraspecific geographic genetic structure in C. glandiformis has arisen due a combination of ocean currents (promoting gene flow between geographically distant regions) and upwelling areas (limiting gene flow between certain regions).

Abundancia y diversidad genética de Quercus mulleri, especie microendémica amenazada de Oaxaca

Quercus mulleri es un encino microendémico de la Sierra Sur de Oaxaca y se encuentra dentro de la Lista Roja de Especies amenazadas de la UICN como “en peligro crítico”, sin embargo, debido a la falta de información actual sobre la especie, no se conoce el estado de conservación de sus poblaciones y, por lo tanto, no se ha podido asignar una categoría adecuada de riesgo. El estudio se realizó con el objetivo de analizar abundancia, distribución y diversidad genética de la especie, para proponer estrategias de conservación adecuadas. Los individuos localizados fueron georreferenciados, se les midió la altura y el diámetro a la altura del pecho para clasificarlos en cinco clases de tamaños; la diversidad genética se analizó empleando cinco regiones de microsatélites de la serie quru-GA. Se relocalizó la especie, encontrando que está restringida a una pequeña región de la Sierra Sur, donde se encuentra de manera fragmentada y aislada geográficamente. Sus poblaciones mostraron baja frecuencia de individuos por clase de tamaño (6.13 ± 5.6). Los valores de diversidad alélica, empleando cinco regiones de microsatélites de la serie quru-GA, fueron bajos (AT=22 y Ao=4.4) y los de diversidad genética fueron moderados (Ho=0.54), lo que sugiere que la población atravesó por un cuello de botella. Este trabajo representa el primer reporte de Q. mulleri después de más de 60 años de su última clasificación taxonómica, y los resultados indican que Q. mulleri es una especie vulnerable, dado que en su zona de distribución existe un proceso de pérdida de hábitat que, junto con la fragmentación de su población, ponen en riesgo la permanencia de la especie, por lo que se recomienda incluirla en la Norma Oficial Mexicana 059 como “Especie en Peligro de Extinción”.

Comprehensive analysis of indels in whole-genome microsatellite regions and microsatellite instability across 21 cancer types.

Microsatellites are repeats of 1- to 6-bp units, and approximately 10 million microsatellites have been identified across the human genome. Microsatellites are vulnerable to DNA mismatch errors and have thus been used to detect cancers with mismatch repair deficiency. To reveal the mutational landscape of microsatellite repeat regions at the genome level, we analyzed approximately 20.1 billion microsatellites in 2717 whole genomes of pan-cancer samples across 21 tissue types. First, we developed a new insertion and deletion caller (MIMcall) that takes into consideration the error patterns of different types of microsatellites. Among the 2717 pan-cancer samples, our analysis identified 31 samples, including colorectal, uterus, and stomach cancers, with a higher proportion of mutated microsatellite (≥0.03), which we defined as microsatellite instability (MSI) cancers of genome-wide level. Next, we found 20 highly mutated microsatellites that can be used to detect MSI cancers with high sensitivity. Third, we found that replication timing and DNA shape were significantly associated with mutation rates of microsatellites. Last, analysis of mutations in mismatch repair genes showed that somatic SNVs and short indels had larger functional impacts than germline mutations and structural variations. Our analysis provides a comprehensive picture of mutations in the microsatellite regions and reveals possible causes of mutations, as well as provides a useful marker set for MSI detection.

Accurately estimating the length distributions of genomic micro-satellites by tumor purity deconvolution

BackgroundGenomic micro-satellites are the genomic regions that consist of short and repetitive DNA motifs. Estimating the length distribution and state of a micro-satellite region is an important computational step in cancer sequencing data pipelines, which is suggested to facilitate the downstream analysis and clinical decision supporting. Although several state-of-the-art approaches have been proposed to identify micro-satellite instability (MSI) events, they are limited in dealing with regions longer than one read length. Moreover, based on our best knowledge, all of these approaches imply a hypothesis that the tumor purity of the sequenced samples is sufficiently high, which is inconsistent with the reality, leading the inferred length distribution to dilute the data signal and introducing the false positive errors.ResultsIn this article, we proposed a computational approach, named ELMSI, which detected MSI events based on the next generation sequencing technology. ELMSI can estimate the specific length distributions and states of micro-satellite regions from a mixed tumor sample paired with a control one. It first estimated the purity of the tumor sample based on the read counts of the filtered SNVs loci. Then, the algorithm identified the length distributions and the states of short micro-satellites by adding the Maximum Likelihood Estimation (MLE) step to the existing algorithm. After that, ELMSI continued to infer the length distributions of long micro-satellites by incorporating a simplified Expectation Maximization (EM) algorithm with central limit theorem, and then used statistical tests to output the states of these micro-satellites. Based on our experimental results, ELMSI was able to handle micro-satellites with lengths ranging from shorter than one read length to 10kbps.ConclusionsTo verify the reliability of our algorithm, we first compared the ability of classifying the shorter micro-satellites from the mixed samples with the existing algorithm MSIsensor. Meanwhile, we varied the number of micro-satellite regions, the read length and the sequencing coverage to separately test the performance of ELMSI on estimating the longer ones from the mixed samples. ELMSI performed well on mixed samples, and thus ELMSI was of great value for improving the recognition effect of micro-satellite regions and supporting clinical decision supporting. The source codes have been uploaded and maintained at https://github.com/YixuanWang1120/ELMSIfor academic use only.

Genetic structure and population dynamics of the silver pheasant (Lophuranycthemera) in southern China

In southern China, the silver pheasant (Lophura nycthemera) is a widespread species due to complex topography and sufficient habitats and food resources. We investigated the microsatellite and mitochondrial DNA control region sequence of 115 individuals from 7 locations in China to study population dynamics of the silver pheasant during the Pleistocene. The neutrality test and mismatch distribution analyses showed that population expansion began in the interglacial period (76,053 years ago), and the latest common ancestor appeared approximately 970,300 years ago. Most populations exhibited a high level of genetic diversity as well as gene flow. Results of phylogenetic trees and network and STRUCTURE analyses provided insights into the weak population structure of this species. The weak phylogeographic and complex historical expansion population of the silver pheasant during the interglacial period could probably be related to its complex topography and the sufficient amount of suitable habitats and food resources in southern China.

Rapid Detection of the Laurel Wilt Pathogen in Sapwood of Lauraceae Hosts

Laurel wilt (LW), caused by Raffaelea lauricola (RL), is a vascular fungal disease affecting species in the Lauraceae that has rapidly spread across the United States. This disease has caused significant tree losses in natural forests and Florida’s commercial avocado orchards. RL spreads through ambrosia beetle vectors and root grafts. Early detection and eradication are recommended to contain outbreaks. Therefore, rapid diagnosis is key for the timely implementation of mitigation strategies. Current LW diagnosis can take up to 10 days and involves pathogen isolation and the amplification of two microsatellite regions. To reduce diagnosis time, we optimized the standard PCR-based detection technique and assessed its potential use in the testing of woody samples. We further screened the microsatellite primers IFW and CHK on a higher number of fungal taxa as well as 11 host genotypes. Sensitivity was evaluated using RL-DNA at different concentrations in pure and mixed solutions. There was no cross-amplification in non-RL species. Both primers amplified all tested RL strains (100); however, the IFW primers were more sensitive than the CHK primers. Using the IFW primers, we detected RL in 89% of sapwood samples (76/85). This protocol provides a rapid and effective molecular-based approach that reduces the diagnostic time from 10 days to 24 h. This method can be an important tool for diagnostic laboratories. Altogether, our data and fungal collection, represent a robust foundation for future transferability of this protocol to more sensitive detection technologies (qPCR, LAMP) and for its application to samples from diverse origin (beetles, roots).

Allelic Loss Of 1Q32.1 The Kiss-1 Metastasis Suppressor Gene Locus in Iraqi Patients with Breast Cancer

Experimental cell lines provide evidence on Kiss-1 gene as a regulator of metastasis in many kinds of cancers, including breast cancer. However, there is scarcity in data for kiss1 gene expression profile in many malignancies, and to date, no effort has been made to explore kiss-1 gene-associated microsatellite markers in many types of tumors, including breast cancer. The present study aims to investigate the microsatellite markers associated with Kiss-1 gene and the current loss of Heterozygosity LOH in Iraqi patients with breast cancer; this was further correlated with immunohistochemical Kiss-1 gene expression. One hundred and three breast carcinoma samples were collected after mastectomy. Kiss-1 gene expression was analyzed using immunostaining. Three microsatellite marker sites: SHGC-76112, SHGC-76186 intragenic, and SHGC-33412 in chromosome 1q32.1 were selected to define allelotyping of kiss-1 gene in paired DNA samples `from tumor cells and control blood cells of each patient. The loss of Kiss-1 gene expression was significantly correlated with the clinical tumor stage (p-value andGLT; 0.01). Loss of heterozygosity (LOH) occurs in 60 (60%) samples. Seven of the examined samples show LOH in two markers; none of the samples show LOH in the three markers. Seven samples show LOH for both intragenic markers (SHGC-76112 and SHGC-76186). The highest LOH percentage was 28%, which occurred within the intragenic microsatellite marker (SHGC-76186), and the lowest was 14% within the flanking region microsatellite marker (SHGC-33412). Interestingly, a statistically significant relationship was found between the LOH and Loss of Kiss-1 gene expression (p-value = 0.0005). This study convinced us to consider the vital role of this gene in the progression of the carcinoma in Iraqi patients with breast cancer.

Development and application of a multiplex TaqMan® real-time qPCR assay for the simultaneous detection of Anaplasma marginale and Theileria annulata and molecular characterization of Anaplasma marginale from cattle in Western Cuba.

Anaplasmosis and theileriosis are considered the most important tick-borne diseases for livestock production worldwide, causing significant economic losses in tropical and subtropical regions. The present study was aimed to develop a multiplex TaqMan® qPCR assay to simultaneously detect Anaplasma marginale and Theileria annulata and to applied it to investigate naturally infected cattle in Cuba. The assay was highly specific, sensible, and efficient; it was more sensitive than a well-established nested PCR and detected 1 DNA copy of each target. Consistent repeatability and reproducibility within and between multiplex qPCR runs was shown. A total of 223 blood samples collected in western Cuba were analyzed for haemoparasites infection in cattle. The multiplex qPCR assay detected A. marginale in 213 samples (95.5%; CI: 95%; 91.9%–97.5%), but all samples were negative for T. annulata. Additionally, the genetic diversity of A. marginale was assessed using 16S rRNA, MSP1a and MSP4 nucleotide and protein sequences. The MSP1a tandem repeats ranged from three to five, and twelve different MSP1a tandem repeats of A. marginale were found, which presented genotypes C, E, and G in the 5ʹUTR microsatellite region. Phylogenetic analysis using the msp4 gene showed that Cuban strains were closely related to others previously reported in Mexico, Brazil and Asian countries. The multiplex qPCR described here proved to be a rapid, specific and cost-effective mean for the simultaneous detection of A. marginale and T. annulata. Further epidemiological studies using this assay will improve the surveillance of the associated diseases in regions where they are endemic.

Screening of LEP gene polymorphisms as a risk factor for obesity and type 2 diabetes in Iraqis.

The prevalence of obesity and diabetes changes dramatically with lifestyle and unequal risk among individuals have made scientists interested to understand how the environment interferes with genetic factors to make it so-called genetic predisposition. This study aimed to explore wherethe most variable region is in leptin gene and analyse microsatellite repeats with direct sequencing in Iraqis and compare our alleles with other populations as a risk for obesity and T2D predisposition. DNA was extracted from blood of 60 type 2 diabetics and 70 non diabetics individuals, LEP 5‛UTR, exon 2 and 3 were screened in 45 individuals (24 type 2 diabetes patients and 21 non- diabetics), LEP TTTC repeats region were amplified in all 130 participants from which 22 control samples were purified and sequenced, superimposed sequences were analyzed manually. Sequencing results showed G>A polymorphism (rs2167270) in 5‛UTR region. No polymorphisms detected in LEP exons 2 and 3. LEP microsatellites alleles were classified depending on sizes into class1 < (220bp) and class2 (> 220bp). Analysis of 22 control samples sequences of microsatellite region resulted in 6 type1 allele (unique sequence) and 5 type 3 allele (13 different isoforms) depending on TTTC arrangement separated by Ts bases. We concluded that LEP variations were in non- coding regions and no significant difference was observed in allele frequency between both groups, but there was a huge diversity in microsatellite repeat number and context among individuals. This may affects gene function thus prepare a predisposition for obesity and type 2 diabetes.

Noninvasive Detection of Microsatellite Instability and High Tumor Mutation Burden in Cancer Patients Treated with PD-1 Blockade.

Microsatellite instability (MSI) and high tumor mutation burden (TMB-High) are promising pan-tumor biomarkers used to select patients for treatment with immune checkpoint blockade; however, real-time sequencing of unresectable or metastatic solid tumors is often challenging. We report a noninvasive approach for detection of MSI and TMB-High in the circulation of patients. We developed an approach that utilized a hybrid-capture-based 98-kb pan-cancer gene panel, including targeted microsatellite regions. A multifactorial error correction method and a novel peak-finding algorithm were established to identify rare MSI frameshift alleles in cell-free DNA (cfDNA). Through analysis of cfDNA derived from a combination of healthy donors and patients with metastatic cancer, the error correction and peak-finding approaches produced a specificity of >99% (n = 163) and sensitivities of 78% (n = 23) and 67% (n = 15), respectively, for MSI and TMB-High. For patients treated with PD-1 blockade, we demonstrated that MSI and TMB-High in pretreatment plasma predicted progression-free survival (hazard ratios: 0.21 and 0.23, P = 0.001 and 0.003, respectively). In addition, we analyzed cfDNA from longitudinally collected plasma samples obtained during therapy to identify patients who achieved durable response to PD-1 blockade. These analyses demonstrate the feasibility of noninvasive pan-cancer screening and monitoring of patients who exhibit MSI or TMB-High and have a high likelihood of responding to immune checkpoint blockade.See related commentary by Wang and Ajani, p. 6887.

Invasions of gladiolus rust in North America are caused by a widely-distributed clone of Uromycestransversalis.

Uromyces transversalis, the causal agent of Gladiolus rust, is an invasive plant pathogen in the United States and is regulated as a quarantine pathogen in Europe. The aim of this research was to: (i) determine the origin of introductions of U. transversalis to the United States, (ii) track the movement of genotypes, and (iii) understand the worldwide genetic diversity of the species. To develop molecular markers for genotyping, whole genome sequencing was performed on three isolates collected in the United States. Genomes were assembled de novo and searched for microsatellite regions. Primers were developed and tested on ten isolates from the United States resulting in the identification of 24 polymorphic markers. Among 92 isolates collected from Costa Rica, Mexico, New Zealand, Australia, and the United States there were polymorphisms within isolates with no genotypic diversity detected among isolates; however, missing data among the New Zealand and Australia isolates due to either poor amplification of degraded DNA or null alleles as a result of genetic differences made it difficult to generate conclusions about these populations. The microsatellite loci and flanking regions showed high diversity and two divergent genomes within dikaryotic individuals, yet no diversity among individuals, suggesting that the invasive U. transversalis populations from North America are strictly clonal.

Anti-PD-1 therapy as monotherapy and the development of biomarkers for patient selection in gastroesophageal cancers

Abstract Gastroesophageal cancers, eg cancers of the stomach and esophagus, are a leading cause of cancer related death worldwide. Recent evidence suggests that these cancers are complex and heterogeneous diseases with emerging subtypes shown to influence response to treatment and survival. Immunotherapy is an advancing field and immune checkpoint inhibitors have become standard treatment options in numerous tumor types and is emerging therapy in gastroesophageal malignancies. Several immune checkpoint inhibitors have been examined in gastric and esophageal cancers, both as single agents and in combination with cytotoxic therapy. Tumors with deficiency in mismatch repair have demonstrated considerable activity with single agent immunotherapy. These tumors, typically characterized by loss or inactivation of DNA repair proteins MLH1, MSH2, MSH6, and PMS2, accumulate hundreds to thousands of mutations in the microsatellite regions of DNA during replication, mutations that would normally be repaired if the mismatch repair system were intact. These tumors are noted to have high lymphocytic infiltrates, and are primed for an anti-tumor immune response by host immunity, with high single agent immunotherapy activity of ∼50% response rates. However across several studies, particularly without patient selection or by selection based on PD-L1 expression alone, the response rates to anti-PD-1 or anti-PD-L1 therapy in gastric and esophageal cancer is marginal, ranging from 5-15%. The most compelling and transformative aspect of immunotherapy is that the few patients who do respond have durable responses, often greater than 1 year, generally with minimal side effects. Our challenge is to better select patients who will benefit from single agent immunotherapy. We will discuss the emerging data examining immunotherapy in upper gastrointestinal malignancies across first, second, and third line studies and the development of biomarkers to improve patient selection.

The Expression of CNS-Specific PPARGC1A Transcripts Is Regulated by Hypoxia and a Variable GT Repeat Polymorphism

PPARGC1A encodes a transcriptional co-activator also termed peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1-alpha (PGC-1α) which orchestrates multiple transcriptional programs. We have recently identified CNS-specific transcripts that are initiated far upstream of the reference gene (RG) promoter. The regulation of these isoforms may be relevant, as experimental and genetic studies implicated the PPARGC1A locus in neurodegenerative diseases. We therefore studied cis- and trans-regulatory elements activating the CNS promoter in comparison to the RG promoter in human neuronal cell lines. A naturally occurring variable guanidine thymidine (GT) repeat polymorphism within a microsatellite region in the proximal CNS promoter increases promoter activity in neuronal cell lines. Both the RG and the CNS promoters are activated by ESRRA, and the PGC-1α isoforms co-activate ESRRA on their own promoters suggesting an autoregulatory feedback loop. The proximal CNS, but not the RG, promoter is induced by FOXA2 and co-activated by PGC-1α resulting in robust activation. Furthermore, the CNS, but not the RG, promoter is targeted by the canonical hypoxia response involving HIF1A. Importantly, the transactivation by HIF1A is modulated by the size of the GT polymorphism. Increased expression of CNS-specific transcripts in response to hypoxia was observed in an established rat model, while RG transcripts encoding the full-length reference protein were not increased. These results suggest a role of the CNS region of the PPARGC1A locus in ischemia and warrant further studies in humans as the activity of the CNS promoter as well as its induction by hypoxia is subject to inter-individual variability due to the GT polymorphism.

Characterizing microsatellite polymorphisms using assembly-based and mapping-based tools.

Microsatellite polymorphism has always been a challenge for genome assembly and sequence alignment due to sequencing errors, short read lengths, and high incidence of polymerase slippage in microsatellite regions. Despite the information they carry being very valuable, microsatellite variations have not gained enough attention to be a routine step in genome sequence analysis pipelines. After the completion of the 1000 Genomes Project, which aimed to establish the most detailed genetic variation catalog for humans, the consortium released only two microsatellite prediction sets generated by two tools. Many other large research efforts have failed to shed light on microsatellite variations. We evaluated the performance of three different local assembly methods on three different experimental settings, focusing on genotype-based performance, coverage impact, and preprocessing including flanking regions. All these experiments supported our initial expectations on assembly. We also demonstrate that overlap-layout-consensus (OLC)-basedassembly methods show higher sensitivity to microsatellite variant calling when compared to a de Bruijn graph-based approach. We conclude that assembly with OLC is the better method for genotyping microsatellites. Our pipeline is available at https://github.com/gulfemd/STRAssembly.

Transcriptome characterization and development of functional polymorphic SSR marker resource for Himalayan endangered species, Taxus contorta (Griff)

Taxus contorta is an important medicinal plant currently listed as endangered in IUCN Red Data List. It produces an anticancer drug, paclitaxel which is well known in the industrial sector. Due to habitat destruction and overexploitation, it is at the verge of extinction. Genomic and transcriptomic data for this species is scarce which has hampered its genomic studies. Moreover, large scale polymorphic informative codominant marker resource is also scarce which hinders its population and landscape genetic analysis. Here, we generated a reference transcriptome for this species which would facilitate the understanding of the functional elements and promote genomic research in this species. Also, a robust polymorphic SSR marker resource was characterized which can be used in conservation of this species. More than 100 million paired end raw reads were obtained through Illumina sequencing. A total of 129,869 unigenes with mean sequence length of 1244 nt were obtained from 209,860 de novo assembled transcripts. Of these, 35,752 transcripts were assigned 5971 unique GO terms. Around 40,386 transcripts were found to have 2163 unique Pfam Ids. Pathway analysis against KEGG database yielded 3721 unique enzyme numbers. Screening of the transcripts for microsatellite regions yielded 7041 SSRs. Among the 100 SSRs selected for characterization on 30 genotypes, 37 polymorphic markers showed a total of 214 alleles with mean of 5.78 alleles per locus. Mean effective number of alleles (Ne) was found to be 3.64 and average PIC value of 0.64 was observed. Observed heterozygosity (0.57) was found to be lower than expected (0.69). This effective polymorphic SSR marker resource will act as valuable tool for deciphering its genetic diversity.

Leptin Gene Polymorphism in Goats Fed with Diet at Different Energy Level: Effects on Feed Intake, Milk Traits, Milk Fatty Acids Composition, and Metabolic State.

Simple SummaryIn this study it has been highlighted the role of a leptin polymorphism on goat milk yield and quality and of its interaction with the energy content of the diet. Energy did not interfere with genotype effect. The studied leptin polymorphism increased healthy fatty acids in milk, thus representing a potential tool to improve functional characteristics of goat milk.The study investigated the effects of a polymorphism at the LEP gene intron 1 microsatellite region and its interaction with diet energy level on feed intake, milk traits, milk fatty acid composition, and metabolic state in goats. Sixteen Girgentana lactating goats at mid-lactation, selected on the basis of their genotype (8 goats homozygous 266 bp/266 bp, L genotype; 8 goats heterozygous 266 bp/264 bp, H genotype), were fed ad libitum according to a change-over design, with two diets at different energy levels reached with different hay inclusion: low energy diet (LE)—100% of hay; and high energy diet (HE)—65% of hay. No differences in milk yield and composition or in dry matter intake were found between leptin genotypes or between diets. Leptin genotype had no effect on plasma metabolite concentrations. The differences between diets were recorded for plasma β-hydroxybutyric acid (BHBA) concentrations with higher (p = 0.01) values for the HE compared to the LE diet (0.44 vs. 0.24 mmol/L, respectively). Nonesterified fatty acid (NEFA) values seem to indicate a positive energy balance in goats. No interaction genotype per diet was evident for most of the studied parameters. Fatty acid composition was strongly influenced by LEP genotype: L goats, compared to H goats, showed higher levels of monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), and 14:1/14:0 desaturation index; lower levels of saturated fatty acids (SFA); and a more favorable atherogenic index. These results seem to suggest an improvement of health characteristics of milk with the L genotype.

Abstract LB-215: CMSI: A Bayesian model for estimating clonal micro-satellites instability from NGS data

Abstract Genomic micro-satellites are the genomic regions consisting of short and repetitive DNA motifs. Micro-satellite region usually exposes intrinsic polymorphism in terms of the numbers of repetitive motifs, which is often described as a probability distribution of the numbers of repeats. In cancer genomics, for any micro-satellite region, it is considered as a micro-satellite instability (MSI) event, if the probability distribution sampled from tumor tissue is significantly different from the distribution sampled from the corresponding normal tissue. Since recent studies have emphasized the importance of micro-satellite instability events in cancer diagnosis and treatments, a series of computational approaches have been developed to detect MSI events from the sequencing data. However, the existing methods suffer an accuracy loss when clonal micro-satellites exist, which are recently observed in some TCGA/ICGC samples. For a clonal micro-satellite, different sub-clones may carry different distributions, while the observed “distribution” from the sequencing data is actually a convolution of the sub-clonal ones. In this case, a sub-clonal distribution may present a true MSI event, but the convolutional one dilutes the data signal and misleads the detection algorithm to report a micro-satellite stability (MSS) event, which introduces type-I error. In addition, a comprehensive understanding of the micro-satellite distribution of each sub-clone is also quite informative for downstream analyses. Thus, to overcome the potential weakness of existing approaches and further improve the computational model, here, we proposed a probabilistic framework, named CMSI, to identify the MSI events under tumor heterogeneous structure. Similar to other approaches, CMSI works on the next generation sequencing data. The proposed framework follows the assumption that the probability density function of the numbers of repeats of a micro-satellite region usually follows a normal distribution. Then, when clonal micro-satellite exists, the convolution distribution observed from the sequencing data should obey a Gaussian mixture distribution. CMSI establishes a variational Bayesian mixture model for the Gaussian distribution calculated from the sequencing reads. This mixture model clusters the reads by the numbers of repeats they bring or infer, and further provide a probabilistic assignment to each read by maximizing the global posterior distribution. By solving this computational model by an EM algorithm, CMSI estimates the number of sub-clones, the proportion of each sub-clone and the parameters of each distribution. Finally, each sub-clonal distribution is examined by statistical test by weighting the clonal proportion, and CMSI outputs the MSI events of sub-clones. To verify the performance of the proposed framework, we conducted several experiments on both simulation datasets and real datasets, where CMSI effectively identified an acceptable percentage of the preset MSI events. Note: This abstract was not presented at the meeting. Citation Format: Yixuan Wang, Xuanping Zhang, Yi Huang, Tao Liu, Xiao Xiao, Jiayin Wang. CMSI: A Bayesian model for estimating clonal micro-satellites instability from NGS data [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 LB-215.

Abstract 571: The shared mutation and neoantigen landscape of MMR-deficient colorectal cancers suggests immunoediting during tumor evolution

Abstract The immune system can recognize and attack cancer cells and their precursors, especially those with a high load of mutation-induced neoantigens. Such neoantigens are particularly abundant in DNA mismatch repair (MMR)-deficient cancers. MMR deficiency results in microsatellite instability (MSI), which leads to multiple insertion/deletion mutations at coding microsatellites and to neoantigen-inducing translational frameshifts. The significance of immune selection and immunoediting potentially shaping the neoantigen landscape during the progression from premalignant MMR-deficient lesions into cancers has not yet been analyzed. We hypothesized that the neoantigen landscape of MSI cancers may reflect the impact of immunoediting. We developed a novel tool for quantitative analysis of microsatellite mutations to explore the neoantigen landscape of MSI colorectal (CRC, n=139) cancers. Frameshift mutations were examined in 41 coding microsatellite (cMS) regions using our new algorithm. We predicted the resulting frameshift neoantigen sequences and used the publicly available prediction tool NetMHCpan 4.0 for prediction of MHC binding sequences. Immunological scores were generated to quantify the likelihood of defined cMS mutations to generate immunogenic neoantigens in different populations with defined HLA allele distributions. Across the 41 cMS analyzed, 77% of all mutations were in the reading frame of 1 nucleotide deletions (m1). The cMS mutation frequency and FSP epitope distribution across HLA genotypes (described by a general epitope likelihood score, GELS) showed a significant negative correlation (Pearson’s r=-0.42, p=0.0149). Some cMS presented with high mutation frequencies despite a high GELS (i.e. TGFBR2: pmut = 88%, GELS = 78.9%), suggesting mutation-induced driver effects, which may outweigh the increased immunogenicity. Our results show that MSI cancers share several highly immunogenic neoantigens. Importantly, a negative correlation between the antigenic strength of neoepitopes and their mutation frequency in MMR-deficient cancers points towards continuous immunoediting during their evolution. These findings will have substantial impact on the optimization of vaccines designed to potentially prevent or treat MSI-driven cancers. Citation Format: Matthias Kloor, Alexej Ballhausen, Moritz Przybilla, Michael Jendrusch, Elisabeth Pfaffendorf, Markus Draxlbauer, Florian Seidler, Sonja Krausert, Aysel Ahadova, Simon Kalteis, Daniel Heid, Johannes Gebert, Maria Bonsack, Sarah Schott, Hendrik Bläker, Toni Seppälä, Jukka-Pekka Mecklin, Sanne Ten Broeke, Maartje Nielsen, Julia Krzykalla, Axel Benner, Angelika Riemer, Magnus von Knebel Doeberitz. The shared mutation and neoantigen landscape of MMR-deficient colorectal cancers suggests immunoediting during tumor evolution [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 571.

Abstract 2882: Mithramycin impedes EWS-FLI1 driven transcription by preventing target promoter clearance

Abstract Background: Ewing sarcoma (ES) is a primary bone tumor of adolescence with poor prognosis. ES is characterized by the presence of EWS-FLI1 which acts as an oncogenic transcription factor to promote and maintain a tumorigenic phenotype. All ES cells express EWS-FLI1, however, other actionable mutations are exceedingly rare. We previously identified mithramycin (MMA) as a potent and specific EWS-FLI1 inhibitor. MMA effectively decreases ES tumor cell viability and tumor volume in vivo by reversing the EWS-FLI1 transcriptional signature. Structural and biochemical data suggest that sub-micromolar MMA increases the stability of the DNA binding domain of EWS-FLI1 at GGAA repeats. These data suggest that MMA may prevent EWS-FLI1 from successfully promoting Pol II transcriptional elongation. Methods: We used ChIP-qPCR to detect EWS-FLI1 binding at the promoter of well characterized targets. We used a Pol II processivity assay to measure the efficiency of transcriptional elongation at EWS-FLI1 targets. We use RT-qPCR, western blotting, and RNAseq to confirm that MMA sensitizes ES cells to elongation inhibitors. Finally, we use GROseq to identify the changes to Pol II dynamics at EWS-FLI1 targets following MMA treatment. Results: Our ChIP-qPCR data demonstrate an increased fraction of EWS-FLI1 DNA binding at the promoters of target genes with GGAA microsatellite regions. MMA significantly inhibits the processivity of Pol II at EWS-FLI1 targets when added both into a run-on reaction or following ES cell pretreatment. Combining MMA with elongation inhibitors induces strong synergy as measured by Bliss-Independence, renders multiple ES cell lines unviable by inhibiting the effects of EWS-FLI1 at the mRNA and protein level, and is effective in vivo. MMA biases GROseq reads toward the 5’ end of genes, a result consistent with a promoter trapping mechanism. We are currently correlating GROseq data to global EWS-FLI1 DNA binding using ChIPseq. Conclusions: We characterized a mechanism of MMA-induced EWS-FLI1 inhibition that involves increased stabilization of the fusion protein at target promoter regions. This mechanism is supported by changes to EWS-FLI1 DNA-binding, EWS-FLI1 target transcriptional processivity, and a reversal of the EWS-FLI1 transcriptional signature by combining MMA with downstream elongation inhibition. We hope that this work will inform future endeavors to develop a targeted therapy for ES that inhibits the main driver of disease, EWS-FLI1. Citation Format: Guillermo Flores, Susan Kitchen-Goosen, Brandon Oswald, Elissa Boguslawski, Marie Adams, Ian Beddows, Zachary Madaj, Patrick Grohar. Mithramycin impedes EWS-FLI1 driven transcription by preventing target promoter clearance [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 2882.