Journal articles: 'GeneMapper'

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Rinehart, Timothy A. "AFLP analysis using GeneMapper® software and an Excel® macro that aligns and converts output to binary." BioTechniques 37, no. 2 (2004): 186–88. http://dx.doi.org/10.2144/04372bm01.

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Schumm, James W., Heather M. Cunningham, Christopher A. Cave, Stephen Stafford, and David A. Leonard. "The BodeChecks solution: A high throughput analysis software combining GeneMapper® ID, FSS-i3, LIMS, and artificial intelligence." Forensic Science International: Genetics Supplement Series 1, no. 1 (2008): 125–27. http://dx.doi.org/10.1016/j.fsigss.2007.10.018.

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Padilla, J. A., F. J. Portilla, J. Salazar, et al. "Detección múltiple de SNPS relacionados con crecimiento y calidad de carne en porcino." Archivos de Zootecnia 59, no. 226 (2008): 233–44. http://dx.doi.org/10.21071/az.v59i226.4738.

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Se describe un método basado en análisis por primer extension para genotipar simultáneamente 7 SNPs relacionados con el crecimiento y la calidad de la carne en porcino. Las muestras de ADN genómico fueron obtenidas de 193 animales pertenecientes a varias razas (54 Ibérico, 63 Duroc, 47 Large White-Landrace x Large White) y a jabalí (29). A partir de las secuencias de los genes H-FABP, MC4R y LEPR, se diseñaron 4 parejas de cebadores con el fin de amplificar las regiones del genoma porcino que contienen esos 7 SNPs. La reacción de minisecuenciación primer extension (ABI PRISM SNaPshot MultiplexTM kit. Applied Biosystem) se realizó con cebadores diseñados en las regiones flanqueantes de cada uno de los SNPs de interés. Los genotipos fueron identificados por mini-secuenciación con ABIPrism 3130 y GeneMapper 3.7 (Applied Biosystems) pudiéndose así discriminar, en una sola reacción, los diferentes alelos de los 7 SNPs de cada animal. Gracias a esta técnica han sido detectados los polimorfismos T221C y C233T del gen LEPR (Genbank AF092422), que no eran reconocibles por enzimas de restricción. Este método permite identificar los animales con genotipo más deseables para la selección.

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Srivastava, Deepak K., Lauren S. Shoemaker, Craig E. Franks, and Michael D. Sussman. "Single Laboratory Validation of a Microsatellite Marker-Based Method in Tomato Variety Identification." Journal of AOAC INTERNATIONAL 94, no. 1 (2011): 251–58. http://dx.doi.org/10.1093/jaoac/94.1.251.

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Abstract The objective of this study was to develop a systematic and flexible method for assembling multiplex simple sequence repeat marker panels for high-throughput genome analysis in the tomato, Solanum lycopersicum, for varietal identification and to demonstrate the technical viability of these genetic markers for use in the enforcement of U.S. Department of Agriculture marketing order-based identity preservation programs. GeneMapper, a semiautomated software tool, was used for designing multiplex panels, allele identification, and polymorphism pattern evaluation of diverse tomato cultivars. Semiautomated genotyping was performed on a set of 12 microsatellite markers providing genome-wide coverage of the tomato chromosomes. Microsatellites were detected with fluorescently labeled primers grouped into five multiplex panels, and each primer pair was assessed in replicated trials for reliability of allele size estimates. Allele sizes for each locus were compared, and a database for 34 tomato varieties was developed. The microsatellite marker set identified distinct allelic peaks and unique genetic fingerprints for each of the studied tomato varieties. A "blind testing" exercise with UglyRipe™ and Vintage Ripe™ tomato varieties, using the above set of markers and database, further established the usefulness of these microsatellite markers for tomato commodity marketing order enforcement.

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Saamia, Vira, I. MADE WIRANATHA, Irfan Rofik, Setia betaria Aritonang, and Dwi Ana Oktaviani. "PEMERIKSAAN DNA SENTUHAN DARI BARANG BUKTI DENGAN PERBEDAAN JENIS BAHAN." Journal of Forensic Expert 1, no. 3 (2021): 1–7. http://dx.doi.org/10.54579/jfe.v1i3.2.

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Analysis of Touch DNA on forensic laboratory has been a favorite approach to identify a person. Every investigator demand the identity of whom the perpetrator that commit the crime, that leaved their DNA on the evidence. Many factors affect touch DNA, one of these is the substrate of the evidence. Common evidences that often examined in forensic lab are firearms, knife, swords, clothes, and switch bomb. To collect the cell on the evidence we use tapelift method using the duct tape. PrepFilerTM BTA Extraction Kit used to extract the DNA from the duct tape, followed by Quantifiler® Duo. For profiling the DNA we use GlobalFilerTM and fragment analyzed on ABI 3500 Genetic Analyzer followed by GeneMapper ID.X. V.1.4. Based on our analysis, DNA from fabric substrate has the higher percentage of success DNA profiling. The success DNA profiling rate of fabric and plastic substrate is 100%, and 0% for wood substrate. According to recent researches, smooth substrate, like plastic and glass, has higher percentage to get full profile than rough substrate, like woods. But on the fabric, they found has much higher percentage than smooth substrate. This can be due to the absorption ability of the fabric to obtain more cells

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Ilnitskaya, Elena, Marina Makarkina, Sergey Tokmakov, and Victoriya Kotlyar. "DNA-marker identification of Rpv3 and Rpv12 resistance loci in genotypes of table and seedless grape varieties." BIO Web of Conferences 25 (2020): 03004. http://dx.doi.org/10.1051/bioconf/20202503004.

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DNA markers are widely used in grapevine breeding to create forms with combined resistance genes. Downy mildew is one of the most common fungal diseases of the vine in the world. Growing grapevines with increased resistance allows to reduce the number of chemical treatments. The decrease in the use of pesticides is especially significant for viticulture of table varieties, since berries are directly consumed by humans for food. Currently, more than 20 resistance genes have been identified by molecular methods, and DNA markers for many genes have been developed. The genes Rpv3 (inherited from North American grape species) and Rpv12 (derived from V. amurensis) are among the most effective and have an additive effect. The study of 14 table grape varieties for the presence of the Rpv3 gene and 8 varieties for the presence of the Rpv12 gene was performed by using DNA-marker analysis. The analysis included varieties that could inherit these genes from the parent forms, according to their ancestry. The study was conducted using an automatic genetic analyzer ABI Prism 3130 and special software GeneMapper and PeakScanner, DNA-markers were taken from literature sources. According to the results of DNA-marker analysis, 9 varieties were identified, including 2 seedless varieties, with the Rpv3299-279 allele in the genotypes, which determines resistance to downy mildew, and 3 table varieties with the Rpv12 gene in the genotypes. One table grape genotype was identified with Rpv3 and Rpv12.

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Pombo, Ana Maria Lima, Pablo Abdon da Costa Francez, and R. S. Silva. "Risco de contaminação por DNA de alto peso molecular e por amplicons em Laboratório de Genética Forense no Brasil." Revista Brasileira de Criminalística 9, no. 2 (2020): 85–94. http://dx.doi.org/10.15260/rbc.v9i2.245.

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O DNA forense vem sendo usado na identificação criminal desde 1985, ele é considerado crucial para a identificação humana em situações como: Identificação de suspeitos em casos de crimes sexuais; Identificação de cadáveres; Investigação de vínculo genético em casos de gravidez resultante de estrupo, dentre outras aplicações. Na década de 70, o conceito de biossegurança vem sendo cada vez mais difundido e valorizado, na medida em que a responsabilidade do profissional envolvido em atividades laboratoriais tem se tornado maior, assim cuidados devem ser tomados com todas as evidências criminais envolvendo a análise do DNA, pois podem ocorrer contaminações, com isso é importante alertar sobre o uso correto de EPI´s no laboratório. O objetivo da pesquisa está em evidenciar a importância da técnica de PCR no que se refere a biossegurança em laboratórios de DNA forense. Foi realizada a aplicação da técnica de PCR no laboratório de DNA forense da POLITEC-AP, para investigar se há contaminações por DNA humano, utilizando 15 (Quinze) marcadores genéticos autossômicos (D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13St317, D7S820, D16S539, CSF1PO, Penta D, Vwa, D8s1179, TPOX, FGA e Amel) que serão coamplificados pelo método de PCR, utilizando o kit Powerplex16 HS (Promega). Os produtos de amplificação serão submetidos à eletroforese capilar em analisador genético ABI 3130 AvantMR (AppliedBiosystemsMR) e os perfis genéticos obtidos serão analisados, com o auxílio dos softwares ABI Prism 3100- Avant Data Collection v2.0,3100 e GeneMapper ID v3.2 após leitura das fluorescências.

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Jovanović, Jelena. "A Study on Effects of Immersion in River Water and Sea Water on DNA Placed on Guns." Kriminalističke teme 22, no. 3-4 (2022): 1–12. http://dx.doi.org/10.51235/kt.2022.22.3-4.1.

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To determine the quantity of DNA that can be obtained from that placed on guns after 30 minutes, 90 minutes and 270 minutes of immersion in water. Also, the aim of this study was to disclose how different types of water environments, such as freshwater and saltwater, affect the amount of DNA loss and degradation over the preset period of time. Firearms was swabbed with a double swabbing technique. DNA was isolated using a Qiagen procedure, quantified by real-time PCR, amplification was on thermal cycler and Capillary electrophoresis was performed on 3500 Genetic analyzer. The electropherograms obtained were analyzed with the GeneMapper ID-X software. Through the comparison of DNA amounts detected after isolation and DNA profiles/allele numbers detected on guns, it can be concluded that the influence of saltwater and freshwater are significantly different. The loss of DNA in saltwater from the outer firearms surface after 30 min is similar to the loss of DNA in freshwater after 90 minutes of immersion. Based on the results of this study, especially the results of real casework sample, it can be concluded as expected that saltwater has a more serious effects on the degradation and the loss DNA on guns than freshwater. Further, it point out that the guns found in freshwater after crime could be a valuable piece of forensic evidence, whilst the guns recovered from saltwater will have less value as forensic evidence if found after more than 30 minutes

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Gamar, Yasir A., Dan Kiambi, Mercy Kairichi, Martina Kyallo, and Mohamed H. Elgada. "Assessment of Genetic Diversity and Structure of Sudanese Sorghum Accessions using Simple Sequence Repeat (SSRs) Markers." Greener Journal of Plant Breeding and Crop Science 1, no. 1 (2013): 16–24. https://doi.org/10.15580/gjpbcs.2013.1.240913858.

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95 sorghum accessions (1,425 individuals) sampled represented most of crop- cultivated areas in Sudan. The genetic diversity and population structure was assessed using a panel of 39 SSRs marker, which covered the sorghum genome. Genotypic data was generated using the ABI 3730 genetic analyzer. The alleles were called and sized using GeneMapper software version 3.7. The molecular data analysis software’s PowerMarker v3.25, DARwin 5, and GenAIEx 6.5x were used to calculate the different diversity indices within and between populations. A total of 332 alleles were detected, with an average of 8.5 per marker pair. The gene diversity averaged at 0.6671. The Polymorphism Information Content (PIC) values averaged of 0.68 showing the highly polymorphic and discriminatory nature of the selected markers. The accessions showed lower mean of observed heterozygosity (Ho = 0.187) than the expected heterozygosity (He = 0.547). AMOVA calculated low variants among populations (1%), and moderate variants within individuals (20%). However, variants among individuals were relatively high within population (79%). The fixation indexes showed little genetic differentiation among populations (FST = 0.008, P = 0.012). However, in the total population high level of inbreeding (FIS = 0.802, P = 0.001) was exhibited with deviation from Hardy-Weinberg proportions (FIT = 0.804, P = 0.001). Neighbor joining rooted phylogeny tree based on genetic similarity coefficient revealed three distinct groups independent of their geographic origins clustering close to each other; groups also have sub-groups. The study estimated genetic diversity and structure of Sudanese sorghum accessions.

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Pérez Forero, Viviana Lucia, Victor Hugo Ochoa, Adriana María Gil Zapata, and Adriana Lucia Pico Romero. "Distribución del polimorfismos Gly185Cys del gen APOA5 en una población de profesores la Universidad de Santander UDES." Revista Facultad de Ciencias de la Salud UDES 4, no. 2.S1 (2017): 25. http://dx.doi.org/10.20320/rfcsudes.v4i2.s1.r12.

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Introducción: La genotipificación de los polimorfismos Gly185Cys del gen APOA5, permitirá evaluar si el gen se encuentra en equilibrio de Hardy Weinberg en la población estudiada. De esta manera se podrá validar posteriormente los posibles hallazgos de asociación de este polimorfismo, con la presencia de desarrollo de Síndrome Metabólico (SM) o de los criterios para el diagnóstico de esta patología, ampliando así el conocimiento sobre el posible riesgo de presentar esta enfermedad. Esta información será de utilidad para el programa de Estilos de Vida Saludable en el cual se podrán diseñar e implementar estrategias que disminuyan el efecto de los factores medioambientales desencadenantes de esta condición, disminuyendo así la tasa de morbilidad asociada con los criterios de diagnóstico de SM. Objetivo: Estimar las frecuencias alélicas, genotípicas y haplotípicas de los polimorfismos Gly185Cys del Gen APOA5, en una población de la Universidad de Santander UDES. Materiales y métodos: Se realizó un Estudio transversal con un total de 100 individuos. Se extrajo ADN con kit PrepFiler Forensic DNA® y amplificación por PCR multiplex. La identificación de las mutaciones de un solo nucleótido se realizó por medio de minisecuenciación empleando la metodología de SNaPshot. Los productos fueron analizados con el secuenciador ABI 310 empleando el software GeneMapper I.D.v 3.2. Los datos obtenidos fueron analizados con el software Arlequín® 3 y Stata. Resultados y conclusiones: Los análisis incluyeron el cálculo de las frecuencias alélicas, fenotípicas y haplotípicas de cada uno de los polimorfismos, así como el análisis del equilibrio de Hardy-Weinberg.

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Miskoska-Milevska, Elizabeta, Zoran Popovski, Blagica Dimitrievska, and Katerina Bandzo. "DNA microsatellite analysis for tomato genetic differentiation." Genetika 47, no. 3 (2015): 1123–30. http://dx.doi.org/10.2298/gensr1503123m.

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Commonly used method for determination of the genetic diversity among the populations is the test for genetic differentiation. DNA microsatellite markers are usually used to investigate the genetic structure of natural populations. The aim of this study was to evaluate the applicability of eight DNA microsatellite loci (LECH13, LE21085, LEMDDNa, LEEF1Aa, LELEUZIP, LE20592, TMS9 and LE2A11) in genetic differentiation of six morphologically different tomato varieties (var. grandifolium from subsp. cultum; var. cerasiforme - red and yellow, var. pruniforme and var. pyriforme from subsp. subspontaneum; and var. racemigerum from subsp. spontaneum). The fragment analyses was performed using Applied Biosystems DNA analyzer (ABI 3130) and GeneMapper? Software program. The data were analysed using the specific program Power Marker Software. The average number of detected alleles was 3,625. Also, the average PIC value for all 8 DNA microsatellites loci was 0,3571. The genetic differentiation test in the researched tomato subspecies showed minor differentiation for locus LELEUZIP (- 0,0009), modest differentiation for locus LECH13 (0,0896), locus LEMDDNa (0,0896) and locus LE21085 (0,0551) and major differentiation for locus LE2A11 (0,7633), locus LEEF1Aa (0,6167), locus TMS9 (0.4967) and locus LE20592 (0,4263). On the other hand, in the estimated tomato varieties, locus LE21085 (0,0297), locus LECH13 (0,0256) and locus LELEUZIP (0,0005) showed minor differentiation, locus LEMDDNa (0,1333) showed modest differentiation, while locus TMS9 (0,5929), locus LEEF1Aa (0,5006), locus LE2A11 (0,4013) and locus LE20592 (0,2606) showed major differentiation. The eight DNA microsatellite loci can be applicable solution for tomato genetic differentiation. The overall results suggest that these microsatellite loci could be used in further population genetic studies of tomatoes.

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Malchenkova, A. A., and E. N. Kosobokova. "Artifacts of analysis in cell line identification by short tandem repeat profiling." Cardiovascular Therapy and Prevention 23, no. 11 (2024): 4121. https://doi.org/10.15829/1728-8800-2024-4121.

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Aim. To study and describe the most common types of artifacts in detection of short tandem repeat (STR) amplicons by capillary electrophoresis and cause difficulties in interpreting the obtained STR profiles.Material and methods. Cell lines were obtained from the bioresource collection of cell lines of the Blokhin National Medical Research Center of Oncology. DNA was isolated according to the manufacturer’s instructions of the DNeasy Blood & Tissue (QIAGEN, Germany) and ExtractDNA Blood & Cells (Evrogen, Russia) kits. DNA concentration was measured using a Qubit 4.0 device (Thermo Fisher Scientific, USA) and a Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, USA). Multiplex PCR was performed using a COrDIS EXPERT26 reagent kit (Gordiz, Russia). Capillary electrophoresis of PCR products was performed on a 3500xL Genetic Analyzer (Applied Biosystems, USA). GeneMapper Software v6.0 (Thermo Fisher Scientific, USA) was used to process electrophoresis data.Results. The most well-known artifacts associated with the STR profiling and subsequent capillary electrophoretic separation of amplicons were studied. Cases of detection of these artifacts from personal practice are given. Recommendations for improving the electrophoresis pattern are given.Conclusion. The paper studies the artifacts of analysis in cell line STR profiling by capillary electrophoresis (STR-CE), which researchers encounter in laboratory practice. Common types of analysis artifacts that cause difficulties in interpreting the results obtained during STR profiling, as well as possible reasons for their occurrence, are described in detail and illustrated with examples from our own practice. Recommendations are given for reducing the number of non-specific fluorescent signals and their intensity.

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Rojas Pantoja, Rubén Darío, José René Jiménez Cardona, Daira Alicia del Pilar Cuarán Cuarán, Franco Alirio Vallejo Cabrera, Raul Dirceu Pazdiora, and Creuci Maria Caetano. "Genotipificación en introducciones de Capsicum chinense Jacq. mediante marcadores moleculares SSR fluorescentes." Magna Scientia UCEVA 3, no. 1 (2023): 79–87. http://dx.doi.org/10.54502/msuceva.v3n1a8.

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El objetivo del presente estudio fue utilizar marcadores SSR fluorescentes para seleccionar genotipos con amplia variabilidad genética, entre introducciones de C. chinense provenientes de México, Brasil y Colombia. En la genotipificación se empleó la plataforma Applied Biosystems 3730xI (Institute of Biotechnology, Cornell University) y la evaluación del tamaño de los alelos se realizó con el software GeneMapper 3.7 (Applied Biosystems). Los marcadores revelaron un total de 114 alelos con un promedio de 12 alelos por locus. El tamaño de los alelos osciló entre 91 y 341 pares de bases. El número de alelos por locus fue variable, de seis para Hpms 2-24 a 21 para Gpms -161. Las poblaciones estudiadas presentaron un índice de Shannon bajo. Las accesiones con mayor diversidad genética fue Brasil con I= 1.622, mientras las de Colombia fue la menor, con I= 0.995. Los valores medios de Ho fueron de 0.517 para Brasil, 0.317 para Colombia y 0.543 para México. Los valores medios de He fueron, en general, superiores a los observados. La tasa de He más baja se registró en accesiones colombianas (0.491), mientras la más alta en las mexicanas (0.719). El análisis de conglomerados mostró la conformación de tres grupos, diferenciados según el origen geográfico de los genotipos evaluados. Todos los cebadores mostraron bandas reproducibles, lo que demuestra su eficiencia para la cartografía genética y el etiquetado de genes en futuros estudios. El valor PIC refleja que la diversidad alélica y la frecuencia entre los genotipos fueron generalmente altas para los loci SSR probados.

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Ilnitskaya, E. T., M. V. Makarkina,, V. Sh Aiba, V. K. Kotlyar, and A. A. Krasilnikov. "Genotyping of Avasirkhva, an indigenous Abkhasian grapevine variety." Horticulture and viticulture, no. 1 (March 23, 2021): 5–10. http://dx.doi.org/10.31676/0235-2591-2021-1-5-10.

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Molecular genotyping of native varieties of Vitis vinifera L. from diﬀerent winegrowing areas is a current trend in the grapevine genetic diversity research. Abkhazia is among the world cradles of tamed grape, and its indigenous gene pool is of particular interest. Avasirkhva is a native Abkhasian grapevine variety mainly grown in the Gudauta District. Te research aimed to obtaining a genetic passport of Avasirkhva grapevine using microsatellite polymorphism data. Te study sampled grape plants from private farmsteads of the Gudauta District. Te plant phenotype corresponded to the variety’s ampelographic description. DNA was isolated from young shoot tip leaves with a CTAB-based protocol. Genotyping was performed with polymerase chain reaction (PCR) followed by capillary fragment separation. High-polymorphic SSR loci (VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG62, VrZAG79) recommended for grapevine varietal identiﬁcation were used as markers. Te amplicon size was estimated with an ABI Prism 3130 automated genetic analyser using the GeneMapper and PeakScanner soﬅware and Pinot Noir as a reference variety genotype. Four samples exhibited an identical microsatellite proﬁle. Te microsatellite assay-based genetic passport of the Avasirkhva variety is as follows: VVS2141-145 VVMD5234-242 VVMD7239- 249, VVMD25239-249, VVMD27184-190, VVMD28234-248, VVMD32248- 262, VrZAG62200-204, VrZAG79251-257. Te obtained passport is unique with respect to the known genotypes in the Vitis International Variety Catalogue (VIVC). A Principal Coordinate Analysis of microsatellite data was used to infer the genetic relationships between Avasirkhva and the Abkhasian varieties Kachich and Azhizhkvakva genotyped in our earlier studies, as well as nine native grapevines of Georgia, the nearest viticultural area. Te Avasirkhva genetic passport can be used in grapevine genotyping studies to clarify the varietal identity.

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Pardo Govea, Tatiana Carolina, Korana Tovar Stojcic, José Miguel Quintero Ferrer, et al. "Marcadores str’s autosómicos en población indígena añu zuliana de Venezuela." Anales de Antropología 54, no. 2 (2020): 107. http://dx.doi.org/10.22201/iia.24486221e.2020.2.69404.

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En el estado Zulia se encuentra 61.18% de la población aborigen de Venezuela. La etnia añú es el segundo grupo de indígenas más numeroso, después de los wayúu. Se estudiaron 29 individuos añú, a los cuales se les realizó extracción de adn por medio de un método que combina dos técnicas (Fenol/Sevag y Salting-Out), se procedió a la amplificación de las mismas a través de la pcr multiplex, se empleó el Kit Promega Powerplex®16HS, y se determinó las frecuencias alélicas de 15 marcadores STR´s autosómicos: D3S1358, TH01, D21S11, D18S51, PENTA E, D5S818, D13S317, D7S820, D16S539, CSF1P0, PENTA D, vWA, D8S1179, TPOX, FGA y AMELOGENINA. La genotipificación se realizó después de la comparación de fragmentos de STR con la escalera alélica y el tamaño del carril interno estándar utilizando el software GeneMapper® ID-X, Versión 1.2; las mayores frecuencias alélicas para cada marcador genético fueron las siguientes: el alelo 18 del locus D3S1358 (0.448), alelo 6 de TH01 (0.586), alelo 30 D21S11 (0.379), alelo 12 D18S51 (0.275), alelo 14 Penta E (0.258), alelo 11 D5S818 (0.603), alelo 12 D13S317 (0.327), alelo 11 D7S820 (0.586), alelo 12 D16S539 (0.310), alelo 10 CSF1PO (0.344), alelo 11 Penta D (0.327), alelo 16 vWA (0.620), alelo 14 D8S1179 (0.431), alelo 8 TPOX (0.551) y alelo 24 FGA (0.396). Según la matríz de distancia de Nei, la población añú no comparte su Acervo Genético con otros grupos indígenas zulianos ni del continente americano, lo cual indica que este grupo étnico tiene su propio origen ancestral con poco o ningún aporte genético externo.<div> </div>

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Maryna, Savelieva, and Rushkovsky Stanislav. "Analysis of frequencies of 21 autosomic microsatellite loci in populations of the cities of Kiev, Odessa, Kharkov, Dnipro and Western Ukraine." ScienceRise:Biological Science, no. 4(19) (October 31, 2019): 22–24. https://doi.org/10.15587/2519-8025.2019.182148.

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Autosomal STR-loci are currently the most used tool in forensic genetics. The allele frequencies of STR loci are used to calculate the probability of random coincidence of DNA profiles for identifying individuals and to calculate likelihood of kinship. The aim of the research was to investigate the polymorphism of 21 criminally significant STR-loci in mixed populations of Kiev, Odessa, Kharkov, Dnipro and Western Ukraine. Materials and methods. The reference sample consists of 1200 unrelated persons. Genomic DNA was isolated from buccal epithelial cells using a ChelexR100 ion exchange resin. The isolated DNA was typed using the polymerase chain reaction method at the 21 autosomal loci that make up the GlobalFiler™ Express PCR Amplification Kit. PCR products were electrophoretically fractionated using the SeqStudio™ Genetic Analyzer System. Allele sizes were analyzed using GeneMapper 6 software. The allele frequencies were compared between populations. Results. Population genetic data for 21 STR loci were included in the GlobalFiler™ Express system (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, vWA, TPOX, D18S51, D5S818, FGA, D2S4433, D19S4231, 1919 , D10S1248, D1S1656, D2S1338, D12S391). The expected and observed heterozygosity, matching probability, power or discrimination, power of exclusion, polymorphic information content were calculated. The correspondence of the observed distribution of genotypes of Hardy-Weinberg equilibrium was determined. Findings. High informativeness of the studied individualizing system of 21 autosomal STR loci was shown. SE33 locus was first analyzed, which turned out to be the most hypervariable. Differences in the frequencies of alleles of STR loci between populations were noted

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Pereira, Valéria Coelho Santa Rita, Fabrícia Lima Fontes-Dantas, Eduardo Ribeiro Paradela, et al. "Polymorphisms in the CIITA −168A/G (rs3087456) and CIITA +1614G/C (rs4774) may influence severity in multiple sclerosis patients." Arquivos de Neuro-Psiquiatria 77, no. 3 (2019): 166–73. http://dx.doi.org/10.1590/0004-282x20190026.

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ABSTRACT It is currently unknown how genetic factors may influence the clinical course of multiple sclerosis (MS). Objective: We examined the impact of CIITA polymorphisms −168A/G (rs3087456) and +1614G/C (rs4774) on the risk of disability progression, severity and on responses to first-line immunomodulator treatments. Methods: Genomic DNA was extracted from blood samples. We used ABI3730xl and GeneMapper v.4.0 software to identify genotype variations. All patients were followed up and clinically reassessed at three-month intervals. Disability progression was measured by the Expanded Disability Status Scale and disease severity by the Multiple Sclerosis Spasticity Scale (MSSS). Results: We included 37 men and 80 women. We found no evidence regarding the influence of the single nucleotide polymorphisms studied in the Expanded Disability Status Scale or therapeutic response of the evaluated drugs. We performed a logistic regression analysis with the MSSS and found that a less severe MS course was associated with wild type CIITA −168AA and CIITA +1614GG, as the chance of the patient progressing to MSSS2 and MSSS3 decreased in 61% and 75% with CIITA −168AA and 66% and 75% with CIITA +1614GG, respectively (p < 0.0001). Although less significant, the CIITA +1614 GC also pointed to a less severe MS course and the chance of the patient progressing to MSSS3 decreased 79% (p = 0.015). We also observed that the CIITA −168GG genotype was more frequent in MSSS2 and MSSS3 and had 40% lower odds ratio to becoming more severe MS. Conclusion: These data suggest that CIITA −168AA, CIITA +1614GG and CIITA +1614 GC polymorphisms may be associated with a better MS clinical course. This knowledge may be useful for a better understanding of MS and its therapeutic management.

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Inokuchi, Shota, Natsuko Mizuno, and Kazumasa Sekiguchi. "Concordance study between GeneMapper^TM ID-X Software v1.6 and the earlier version: The impact of revision of peak height detection algorithm on forensic STR typing." Japanese Journal of Forensic Science and Technology 25, no. 2 (2020): 201–10. http://dx.doi.org/10.3408/jafst.780.

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Tlatelpa, Martín Aguilar, Guillermo Calderón Zavala, María Alejandra Gutiérrez Espinosa, et al. "Huella genética de variedades de fresa obtenidas en el Colegio de Postgraduados, México." Nova Scientia 11, no. 22 (2019): 186–206. http://dx.doi.org/10.21640/ns.v11i22.1794.

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En un programa de mejoramiento genético de fresa (Fragaria × ananassa) es importante contar con la metodología para evaluar la integridad genética de la planta en todas las etapas de incremento, desde diferentes criterios, como el fisiológico, morfológico y el molecular; para este propósito, una de las herramientas más apropiadas son los marcadores moleculares denominados Secuencias Simples Repetidas del inglés Simple Sequence Repeats (SSR), ya que permiten, por ejemplo, identificar poblaciones con una diversidad genética reducida, revelar genealogías, conocer el grado de parentesco entre individuos, proporcionar elementos sólidos en la defensa de la propiedad intelectual, evaluar la pureza del material vegetal, identificar el grado de variación somaclonal y evitar la mezcla de material vegetal en bancos de germoplasma. En este sentido, el presente estudio tuvo como objetivo obtener la huella genética de las variedades de fresa CP0201, CP0204, CP0615, CPLE7 desarrolladas en el Colegio de Postgraduados y como testigo la variedad Festival, desarrollada en Florida, USA, con el uso de nueve loci de microsatélites (SSR). El proceso incluyó la extracción de ADN a partir de tejido foliar de fresa, así como de la amplificación mediante PCR de punto final de los loci SSR agrupados en reacciones multiplex. Los productos de PCR fueron separados mediante electroforesis capilar y su tamaño en pares de bases se determinó con el programa GeneMapper® v. 4. A partir de las frecuencias alélicas se calcularon las matrices de distancia con los coeficientes de Jaccard y Dice. Se encontraron 63 alelos distintos, cada par de iniciadores amplificó entre 3 y 12 alelos. Los marcadores que presentaron mayor número de alelos fueron EMFn181 (11 alelos) y EMFv104 (12 alelos). Se generó la huella genética de cada variedad. Mediante los perfiles alélicos se encontraron diferencias entre las variedades CP0615, CPLE7 y Festival; CP0201 y CP0204 tuvieron una huella genética similar, ya que están emparentadas a través de su progenitor femenino; el índice de diversidad alélica dentro de las poblaciones varió de 3.96 a 5.93. Las variedades tuvieron un índice de uniformidad bajo debido al alto nivel de polimorfismo de los marcadores usados.

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Ilnitskaya, E. T., M. V. Makarkina, and S. V. Tokmakov. "DNA marker identification of Rpv3 downy mildew resistance gene in seedless grape varieties." Plant Biotechnology and Breeding 2, no. 3 (2020): 15–19. http://dx.doi.org/10.30901/2658-6266-2019-3-o4.

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Table grapes are a valuable dietary product. Seedless grapes are in high demand among consumers. For this reason, the breeding of seedless varieties is one of the popular trends in modern viticulture, along with the production of environmentally friendly products. Downy mildew (Plasmopara viticola (Berk. & M.A. Curtis) Berl. & De Toni) is one of the most common fungal diseases of the grapevine. Most downy mildew resistant grape accessions belong to North American species like Vitis aestivalis Michx., V. berlandieri Planch., V. cinerea (Engelm. ex A. Gray) Engelm. ex Millard, V. riparia Michx., V. rupestris Scheele, etc. The search for donors of resistance genes is an urgent task. Rpv3 is one of the most significant resistance genes from a number of North American grape varieties. The aim of this work is to identify the downy mildew resistance gene Rpv3 in seedless grape varieties by means of DNA-marker analysis. The grape varieties with rudimentary development of seed in berries and with North American species in the pedigree were chosen as the object of the study. The varieties “Dunavski lazur” and “Seyve Villard 12-375” with reference alleles were used as the positive control, while V. vinifera L. was used as the negative control. UDV305 and UDV737 DNA-markers were used in this study to identify the allelic type of the Rpv3 gene. The work was performed using the polymerase chain reaction. The reaction products were separated by capillary electrophoresis using the ABI Prism 3130 automatic genetic analyzer. Evaluation of the results was done using the GeneMapper and PeakScanner software. Functional alleles of the downy mildew resistance gene Rpv3 were revealed in grape varieties “Kishmish zaporozhskiy”, “Lady Patricia”, “Remaily seedless”, “Pamyati Smirnova” and “Shayan”. Rpv3299-279, one of the seven known haplotypes, was identified in all the varieties. The pedigree analysis of the studied varieties indicated that the parental forms – “Seyve Villard” and “Seibel” hybrids – are presumably the donors of the gene. Grape accessions with the identified Rpv3 gene can be used in seedless varieties breeding as donors of resistance to downy mildew.

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Karymsakov, T. "Characteristics of allele pool of cattle of simmental breed in Kazakhstan." Glavnyj zootehnik (Head of Animal Breeding), no. 8 (August 1, 2020): 31–36. http://dx.doi.org/10.33920/sel-03-2008-04.

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One of the key aspects of studying of new breeds, types and lines of farm animals is to evoluation the state of their allele pool by using DNA markers. At the same time, it should be noted that a significant contribution to the characteristic of allelic diversity of breeds is made by regional populations, the formation of which in most cases took place on the basis of local cattle, which are carriers of their own unique allele pool. It is known that studies on microsatellites make it possible to determine the genetic diversity within and between breeds, and the average number of alleles, observed and expected heterozygosity, allow us to calculate the genetic parameters of populations and evaluate their diversity. The purpose of the research was to study the features of the allele pool of a new intra-breed type Ertis of Red-and-White dairy cattle in the Simmental breed. The sampling of biological materials has been carried out according to generally accepted methods. Genotyping has been performed with a set of StockMarks Cattle at 12 loci. Identification of amplification products has been performed on the abi Prism 310 genetic analyzer (Applied Biosystems, USA) using capillary electrophoresis. Decoding of the obtained graphical results has been carried out in the program GeneMapper 4,0. The results of researches on the genetic structure of the studied population of cattle have been reflected in the article. It has been found that there are 65 alleles in 12 loci of the microsatellite, of which 7 alleles with a frequency of occurrence of ≤1 % have been identified. The most informative loci were TGLA227, TGLA122, ETH3, and TGLA53. The average level of expected heterozygosity was 0,68, with random inbreeding – 0,0488. Studies of the genetic profile of the new intra-breed type of dairy cattle Ertis allowed us to establish the wide variety of allele pool in it, and the small number of rare alleles allocated gives grounds for the possibility of determining this population as an independent structural unit in Simmental breed.

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Qu, K. Z., Y. Ren, J. Liu, A. Sferrozza, and R. A. Bender. "Detection of seven polymorphisms associated with colorectal cancer therapeutic response and toxicity." Journal of Clinical Oncology 24, no. 18_suppl (2006): 13160. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.13160.

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13160 Background: First-line therapy for metastatic colorectal cancer (CRC) is 5-fluorouracil/leucovorin in combination with oxaliplatin (ie, FOLFOX™) or irinotecan (ie, FOLFIRI). Optimizing this therapy for an individual patient, however, is complicated due to wide variability in drug response and potential for toxicity. Such variability may be due to functional genomic polymorphisms in the drug target gene or in the metabolizing or DNA repair enzyme genes. We report, herein, development and validation of 2 new assays that, together, detect 7 polymorphisms associated with CRC drug response and toxicity. Methods: The first assay (SNaPshot) simultaneously detects 4 polymorphisms by using primers targeting the ERCC1, XPD, GST-P1, and XRCC-1 genes, single nucleotide primer extension of PCR products, electrophoresis and fluorescent detection on an ABI PRISM® 3100 Genetic Analyzer, and data analysis using GeneMapper® software (Applied Biosystems, Inc.). The second assay (F-PCR) simultaneously detects 3 polymorphisms using paired primers targeting the TS and UGT1A1 genes. One primer per pair is labeled with a fluorescent tag, and electrophoresis, detection, and data analysis are performed as described for the SNaPshot assay. Results: Based on fragment size and color, the intra- and interassay precision for SnaPshot ranged from 0.04%-0.51% and 0.14%-0.59%, respectively, and for F-PCR from 0.02%-0.16% and 0.09%-0.29%, respectively. There was 100% concordance in the genotypes determined in 3 separate SNaPshot (n=44 samples) and F-PCR (n=32 samples) assays. Furthermore, there was 100% agreement between genotypes determined by the SNaPshot method and those determined by RFLP (n=10) and between genotypes determined by the F-PCR method and those determined by pyrosequencing (n=17). Conclusions: Our multiplex SNaPshot and F-PCR methods can accurately detect 7 polymorphisms associated with CRC drug response and toxicity. These multiplex PCRs should lead to improved precision and ease of interpretation relative to that of single PCR methods. These methods may help the clinician optimize CRC therapy for individual patients. No significant financial relationships to disclose.

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Nikitina, M. A., V. M. Alifirova, E. Yu Bragina, N. P. Babushkina, D. E. Gomboeva, and S. M. Nazarenko. "Environmental and genetic risk factors for Parkinson’s disease." Bulletin of Siberian Medicine 21, no. 4 (2023): 105–13. http://dx.doi.org/10.20538/1682-0363-2022-4-105-113.

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Aim. To analyze risk factors in the group of patients with Parkinson’s disease (PD) and compare them with the literature data.Materials and methods. The study included 439 patients with PD and 354 controls, comparable by gender and age. For each individual, a registration card was filled in containing demographic, epidemiological, clinical, and neuropsychological data. The severity of the disease was studied according to the MDS-UPDRS scale; the stage of PD was determined according to the Hoehn and Yahr scale. Cognitive functions were assessed by the MoCA test and MMSE. The length of the (CAG)n repeat region in the HTT gene was determined using fragment analysis on the ABI 3730 DNA analyzer. The obtained results were analyzed using GeneMapper Software v4.1 (Applied Biosystems, USA).Results. When comparing patients with PD and the control group, the odds ratio (OR) for PD in individuals with traumatic brain injury was 3.13 (95% confidence interval (CI): 2,27–4.34; p = 4.94 × 10–13), which showed the significance of this risk factor for PD. Consumption of coffee in the anamnesis distinguished the group of PD patients from the control group (OR = 0.41 (95% CI: 0.30–0.56); p < 0.0001), confirming its neuroprotective effect. Analysis of the variability in the length of the (CAG)n repeat regions in the HTT gene showed that patients whose genotype contained an allele with 17 repeats in combination with any allele other than an allele containing 18 repeats had a protective effect (OR = 0.50 (95% CI: 0.27–0.92); p = 0.025). All genotypes containing an allele with 18 repeats were predisposed to PD (OR = 2.57 (95% CI: 1.66–4.28); p = 0.007). The predisposing effect of the allele to PD, unrelated to the expansion of CAG repeats in the HTT gene, was revealed for the first time.Conclusion. Traumatic brain injury and the allele with 18 CAG repeats in the HTT gene are risk factors for PD. Coffee consumption can be attributed to protective factors in relation to PD.

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Zilberberg, Jenny, Kira Goldgirsh, Robert Korngold, and Thea M. Friedman. "Repertoire Analysis of Ex Vivo Polyclonally Expanded Regulatory T Cells." Blood 108, no. 11 (2006): 3222. http://dx.doi.org/10.1182/blood.v108.11.3222.3222.

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Abstract CD4+CD25+ regulatory T cells (Treg) are essential for the maintenance of self-tolerance and have also been implicated in the control of alloreactive immune responses. Several studies using murine models of graft-vs.-host disease (GVHD) have shown that addition of equivalent numbers of Treg to the donor T cell inoculum at time of hematopoietic stem cell transplantation can significantly reduce the incidence of GVHD. In addition, in an MHC-matched, minor histocompatibility disparate model, the infusion of Treg ten days post-transplantation was shown to ameliorate the progression of GVHD while permitting a graft-versus-leukemia effect. However, because Treg constitute <5% of peripheral CD4+ T cells in humans, the use of freshly isolated Treg to treat and/or prevent GVHD, as well as other diseases in the clinical situation, is limited. Therefore, much effort is now under way to expand Treg in order to have sufficient numbers for therapeutic use. There is little available information regarding the repertoire complexity of ex vivo, polyclonally expanded regulatory T cells. We hypothesize that like their CD4+CD25− T cell counterparts, the diversity of the Treg T cell receptor (TCR) repertoire will also be complex. To this end, CD4+CD25− and CD4+CD25+ T cells from B10.BR mice were purified using fluorescence activated cell sorting; both populations were polyclonally expanded using CD3/CD28 paramagnetic microbeads in combination with high levels (100 IU/ml) of hrIL-2. After achieving a greater than 50 fold expansion, RNA from 1–1.5×107 cells was isolated for RT-PCR. The complexity of the T cell repertoire of expanded CD4+CD25− and CD4+CD25+ was determined using TCR Vb CDR3-size spectratype analysis. The PCR products were run on a sequencing gel and analyzed by the GeneMapper Software from Applied Biosystems. This comparison revealed that the number of resolvable Vb families is more heterogeneous in the CD25− populations. Whether this reflected a lack of complexity in the regulatory repertoire warrants further investigation. However, for the resolvable Vb families there were no significant differences in the complexity indexes between these two groups.

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Aniskina, A. S., J. G. Payanidi, A. M. Stroganova, I. V. Manina, and K. I. Zhordania. "Detection of a status of microsatellite instability in tumors of patients with endometrioid adenocarcinoma of the ovaries and/or of uterine corpus." Russian Journal of Biotherapy 22, no. 3 (2023): 36–42. http://dx.doi.org/10.17650/1726-9784-2023-22-3-36-42.

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Introduction. Multiple primary malignant neoplasms of female reproductive organs are a rare pathology. However, over the past decades, there has been an upsurge of interest in the study of this phenomenon in oncology. This is particularly the case for the diagnosis of synchronous endometrioid adenocarcinoma of the ovaries and uterine corpus, which histogenetically belong to the same germ layer and have similar histological structure. Until recently, clinicians relied only on morphological examination in these cases, but with the development of molecular genetic technologies, new diagnostic possibilities have emerged.Aim. Is the detection of the status of microsatellite instability in tumors of patients with endometrioid adenocarcinoma of the ovaries and/or uterine corpus.Materials and Methods. A pilot retrospective molecular genetic study (n = 48) was conducted to determine the status of microsatellite instability (MSI) in the tumors of the ovaries and/or uterine corpus: it involved 33 patients with solitary endometrioid ovarian cancer and 15 patients with synchronous endometrioid adenocarcinoma of the ovaries and uterine corpus. Microsatellite instability status was detected using PCR method with subsequent fragment analysis performed on ABI PRISM 3500 genetic analyzer (8 capillaries, Applied Biosystems). DNA was isolated from paraffin blocks of surgical specimens using DNAsorb B extraction kit (AmpliSens, Russia), according to the manufacturer’s manual. DNA concentration was estimated fluorometrically using Qubit 2.0 (Life Technologies, USA). The obtained data were analyzed using GeneMapper program (Thermo Fisher, USA). In case of polymorphism of two and more markers high-level microsatellite instability (MSI-H) was observed.Results. The incidence of MSI-H in solitary endometrioid ovarian cancer (n = 33) was 12,1 % (4 cases), while in synchronous ovarian and uterine corpus tumors (n = 15) MSI-H incidence made up 20 % (n = 3). Herewith, there have been only cases of a combination of endometrioid histotypes of ovarian and endometrial cancer with identical status of microsatellite instability. Thus, the incidence of MSI-H in synchronous endometrioid adenocarcinoma of the ovaries and uterine corpus (20 %) is comparable to that in solitary endometrial cancer.Conclusion. Our pilot study became a significant complement to the previously published materials, as it allowed to confirm the clonal origin of tumors in patients with endometrioid adenocarcinoma of the ovaries and uterine corpus, that can affect the stratification of treatment strategy for this category of patients.

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Ilnitskaya, E. T., V. Sh Ayba, M. V. Makarkina, S. V. Tokmakov та M. A. Avidzba. "DNA-fi ngerprinting of local Аbkhazian grape variety Kachich". Horticulture and viticulture, № 6 (20 грудня 2019): 9–13. http://dx.doi.org/10.31676/0235-2591-2019-6-9-13.

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The study of the indigenous gene pool of Vitis vinifera L. of diff erent zones of viticulture with the involvement of molecular genetic methods is an urgent due to the wide diversity of grape varieties. The territory of Abkhazia is a unique zone, one of the hotbeds of the emergence of cultural grapes. «Kachich» is the oldest Abkhazian grape variety, known for the quality of wines obtained from its harvest. The variety is more resistant to fungal diseases compared to popular European varieties cultivated in modern Abkhazia, and is particularly resistant to rot of berries. However, often the local population calls old bushes by the name «Kachich», which give grapes of good quality for making of red wines and not always these forms correspond to the morphological description of the variety. The aim of the research is DNA fi ngerprinting of the «Kachich» grape variety based on the analysis of polymorphism of microsatellite loci. The object of the study was the plants corresponding to the variety description, growing in the collection of the agricultural company “Wines and waters of Abkhazia” (Sukhum, Abkhazia). DNA was isolated from the young leaves of the top of the shoot of typical plants of the variety by the CTAB-method. Genotyping was carried out by polymerase chain reaction (PCR) with separation of reaction products by capillary electrophoresis. Highly polymorphic SSR-markers recommended for grapes varieties identifi cation were used for study (VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG62, VrZAG79). The size of amplifi ed fragments was estimated using the automatic genetic analyzer ABI Prism 3130 and special software GeneMapper and PeakScanner. To clarify the size of amplifi ed fragments, the DNA of reference varieties was used. According to the results of microsatellite analysis, the DNA-certifi cate of the genotype of the «Kachich» grape variety was compiled: VVS2153 155 VVMD5234 240 VVMD7239 249 VVMD25239 267 VVMD27186 193 VVMD28234 248 VVMD32262 272 VrZAG62194 196 VrZAG79236 236. The resulting DNA-profi le was checked for matches in the Vitis International Variety Catalogue VIVC. No matches of the obtained DNA-profi le with any other DNA-profi le presented in the Database were found. DNA-certifi cate of genotype «Kachich» can be successfully used for varieties identifi cation of grape plants if necessary to clarify their varietals affi liation.

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Batashova, Mariia, Liudmyla Kryvoruchko, Bohdana Makaova-Melamud, Volodymyr Tyshchenko, and Martin Spanoghe. "Application of SSR markers for assessment of genetic similarity and genotype identification in local winter wheat breeding program." Studia Biologica 18, no. 1 (2024): 83–98. http://dx.doi.org/10.30970/sbi.1801.762.

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Background. Simple sequence repeat (SSR) markers are widely used for genetic analysis in plant breeding, allowing for the investigation of genetic divergence and similarity of genotypes, identification of unique alleles and determination of levels of genetic diversity. Materials and Methods. Analysis of 42 wheat cultivars and lines from the breeding program of Poltava State Agrarian University was carried out using 11 SSR markers located on different chromosomes. A set of 11 microsatellite single locus primer pairs was used in this study (Xgwm 11, Xgwm 44, Xgwm 46, Xgwm 135, Xgwm 174, Xgwm 186, Xgwm 194, Xgwm 219, Xgwm 312, Xgwm 372, Xgwm 389). Amplification of 11 loci was performed using the Kapa2G FastHotStart PCR Kit (Kapa Biosystems, Boston, USA). The mixture for PCR amplification contained 1.5 x Kapa2G buffer, 0.5 mM dNTP mix, 0.5 μM of each primer (Sigma-Aldrich), 1 unit of Kapa2G FastHotStart DNA Polymerase and 11.8 ng of template DNA in a volume of 25 μl. Fragment lengths were determined using GeneMapper 4.0 software (Applied Biosystems). Dendrogram was constructed using UPGMA (unweighted pair-group method with arithmetic average) in DarWin 6.0 software (Perrier and Jacquemoud-Collet 2006) for clustering analysis. Results and Discussion. The number of alleles detected per locus varied from 5 (Xgwm 11, Xgwm 135, Xgwm 219) to 12 (Xgwm 174). A total of 80 alleles were identified for the 11 loci studied. Among these, 25 unique alleles were found, each of which was present in only one genotype. The polimorphism information content (PIC) values ranged from 0.48 to 0.87. The markers Xgwm 174 (PIC = 0.87), Xgwm 389 (PIC = 0.84) and Xgwm 372 (PIC = 0.83) were the most polymorphic in our study. We obtained a distribution of cultivars and lines by genetic similarity into five clusters. Conclusion. The use of SSR markers made it possible to identify rare alleles within the varieties presented. The study of the genetic similarity of the presented genotypes showed their relationship according to their origin. It was shown that unique alleles tended to occur in certain local breeding genotypes. This study has shown that genotypes representing the local Ukrainian breeding program often have the same allelic variants and at the same time some genotypes have unique allelic variants. The results obtained from the study of 42 winter wheat genotypes based on 11 SSR markers showed that molecular markers can be very useful in assessing genetic similarity and identifying genotypes in the local breeding program.

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Proff, C. "Work in “progress”—Applied Biosystems GeneMapperID™." International Congress Series 1288 (April 2006): 430–32. http://dx.doi.org/10.1016/j.ics.2005.10.022.

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Guyader-Joly, C., C. Gonzalez, D. Le Bourhis, B. Moulin, Y. Heyman, and P. Humblot. "135 PREGNANCY RATES AFTER SINGLE DIRECT TRANSFER OF BIOPSIED FROZEN-THAWED BOVINE EMBRYOS ACCORDING TO QUALITY." Reproduction, Fertility and Development 21, no. 1 (2009): 167. http://dx.doi.org/10.1071/rdv21n1ab135.

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In cattle, reliable methods for whole genome amplification (WGA) have been implemented for DNA pre-amplification and subsequent genotyping from embryo biopsy containing 5 cells or more. In France, these methods are now tested under field conditions. Several studies report pregnancy rates after direct transfer of biopsied frozen–thawed grade 1 embryos similar to that intact frozen ones. Even so, grade 2 and 3 embryos represent 25.5% of the transferable embryos (AETE data, 2007) and may limit the use of the above mentioned techniques if results are not satisfactory. The objective of this study was to investigate the impact of the embryo quality on the pregnancy rates after single direct transfer of biopsied frozen–thawed embryos. Embryos were collected on 12 donor cows after 15 sessions of superovulation treatment using FSH injected twice daily in decreasing doses over 4 days. Cows were inseminated at 12 and 24 hours after onset of estrus. Embryos were recovered 7 days post-insemination and evaluated according to IETS standards. Biopsies were performed on stage 4 to 7 grade 1 to 3 embryos using a microblade. Embryonic cells from the biopsy were dry deposited in microtubes and frozen before WGA (QIAGEN REPLI-g® Mini kit, Valencia, CA, USA) and multi-genotyping (GeneMapper software® – Applied Biosystems Europe). Each biopsied embryo was equilibrated for 10 min in 1.5 m Ethylene Glycol and then loaded into straw containing two columns of F1 medium separated by a central column of 1.5 m EG with the embryo. The freezing sequence was: –7°C directly; seeding; held for 10 min; 0.5°C min–1 until –35°C before plunging into liquid nitrogen. Embryos were thawed (straws 10 s in air and 20 s in water at 20°C) and directly transferred into synchronized recipient heifers. Pregnancy was diagnosed by ultrasonography at 35 and 90 days. Effect of embryonic stage and quality on pregnancy rates were analysed by log linear models (Proc CATMOD, SAS Institute Inc). A total of 58 embryos were micromanipulated. All grade 1 and 2 embryos were successfully biopsied and frozen and, 0.8% of grade 3 (2/25) were discarded due to their low quality after biopsy. No significant effect of embryo stage and quality on pregnancy rates was found after direct transfer (Table 1). These preliminary results suggest that high pregnancy rates can be achieved after direct transfer of biopsied frozen–thawed embryos of Grade 1, 2 and 3 allowing most of the embryos to be involved in the genotyping process. Table 1.Pregnancy rates following single direct transfer of biopsied frozen–thawed G1 to 3 bovine embryos This work has been performed through the programme TYPAGENAE (GENANIMAL 4-03) supported by FRT/ANR and Apis-Genes.

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Albano, Maria S., Linda Andrus, Dong H. Lee, Cladd E. Stevens, and Pablo Rubinstein. "Extra-Cellular DNA in Cord Blood (CB) Plasma: Use for Sample Identification [short Tandem Repeat (STR) Analysis] and for Infectious Disease Screening in CB Banks." Blood 106, no. 11 (2005): 1901. http://dx.doi.org/10.1182/blood.v106.11.1901.1901.

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Abstract Introduction: A commitment of CB banks is to test CB units and maternal blood for multiple genetic and infectious disease markers. As a consequence, several aliquots of blood, mononuclear cells and plasma are taken during processing for immediate and delayed testing. Whereas control of identity between aliquots containing cells can be performed by DNA assays, identity controls for plasma aliquots rely only on bar-coded label systems. We hypothesize that there is soluble extra-cellular DNA (EC-DNA) and that it can be used for identity control of plasma aliquots. The aims of our study were; to determine the concentration of EC-DNA in CB-plasma, to evaluate whether genetic identity testing by STR analysis is feasible and to investigate whether EC-DNA allows infectious disease screening by nucleic acid testing (NAT). Methods: CB (n=20) was collected, processed (Rubinstein et al, PNAS 1995) and EC-DNA from 200 μl of 0.45μm-filtered plasma was extracted both with the Qiagen and the High Pure Viral Nucleic Acid method (Roche). Presence of EC-DNA was evaluated by detection of HLA-DR and ALU gene sequences and by STR analysis (AmpFlSTR-Profiler Plus-Applied Biosystems). Purified HBV was added to CB and peripheral blood (PB) plasma, EC-DNA recovered as above and HBV-DNA assessed by real time PCR. Results: All 20 samples were positive for HLA-DR by PCR and for ALU sequence by real time PCR, confirming the presence of EC-DNA in CB-plasma in concentrations of 0.022 +/− 0.012 ng/μl at the time of CB collection, rising to 0.124 +/− 0.025 ng/μl 33 hrs later; p<0.016. Concentrations observed in CB-plasma were higher than those of adult donor plasma (PB-plasma 0.005+/−0.0065 ng/μl). STR analysis was validated on cellular DNA and, when applied for EC-DNA, showed good signal strengths and low backgrounds, allowing accurate automatic allele identification with no manual corrections (genemapper software, Applied Biosystems). An increase in STR-PCR product concentration was observed when we analyzed EC-DNA samples from CB after 33hs (7017.6 +/− 2014 RFU/μl at time of CB collection and 30290.9 +/− 3164 RFU/μl after 33 hs; RFU = relative fluorescence units). HBV was recovered from all spiked units, in correspondence with the viral concentrations added. Recovery of HBV was 96 +/− 21% with high and low viral concentrations. Three mothers with HBAg/HBcore positive had also HBV-DNA in PB-plasma. Six with anti-HBcore only and 70 with anti-HBs were negative. HBV-DNA was also negative in CB-plasma for all seventy nine respective newborns. Conclusions: there is infant EC-DNA in CB-plasma and viral nucleic acids can be obtained using the same extraction method. There is an increase of EC-DNA in CB over time that may reflect cell death. STR assay of EC-DNA can be a useful molecular tool for the identification of plasma aliquots used for infectious disease testing in CB banks.

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Le Bourhis, D., Y. Amigues, F. Charreaux, et al. "187 EMBRYO GENOTYPING FROM IN VIVO BIOPSIED BOVINE EMBRYOS AFTER WHOLE GENOME AMPLIFICATION." Reproduction, Fertility and Development 21, no. 1 (2009): 192. http://dx.doi.org/10.1071/rdv21n1ab187.

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Genomic tools are now available for most livestock species and used routinely for marker-assisted selection (MAS) in cattle. The detection of a large number of markers that are widespread over the genome is generally limited by the amount of genomic DNA available in an embryo biopsy of a small size not to be detrimental to embryonic survival. Amplification of DNA from such a biopsy is then necessary. In this study, the efficiency of embryo genotyping for 45 microsatellites (MS) following whole-genome amplification (WGA) was evaluated from samples of a variable number of cells isolated from cattle embryos. In a second part, this work aims to test the reliability of the MAS method for 45 MS and 13 single nucleotide polymorphisms (SNP) from bovine embryo biopsies under field conditions. In experiment 1, in vitro bovine morulae (n = 10) were produced, and 1, 5, and 10 embryonic cells were removed from each morula. Cells were dry frozen in tubes before further processing. Whole-genome amplification was performed using the commercial Qiagen REPLI-g® Mini Kit according to the manufacturer instructions (Qiagen, Valencia, CA, USA). WGA solution was then diluted, processed by PCR with 45 markers, and the resulting data were genotyped with GeneMapper software® (Applied Biosystems Europe). Accuracy and reliability of genotyping were assessed using different samples of cells from the same embryo. In experiment 2, after superovulation (10 cows), bovine embryos were in vivo-produced and collected at day 6 or day 7 of pregnancy. Only grade 1 embryos were washed and biopsied using a microblade. Biopsied embryos were either frozen or transferred back to synchronized recipients. Individual biopsies were transferred as dry samples to the laboratory. Genomic DNA was amplified using WGA, and embryos were genotyped. The results of experiment 1 clearly indicate that a conventional biopsy of 5 to 10 cells was sufficient for multi-markers detection after whole-genome amplification as 98% of the 45 markers were detected compared to 45% of marker detection using 1 cell (P < 0.01). In experiment 2, from 123 collected embryos, 79 were classified as grade I or II transferable embryos (64.2%) and 57 were biopsied (34 were classified as stage 4–5 and 23 as stage 5–6, according to the IETS criteria). Using the stereomicroscopic analysis, 44 biopsies had a number of cells ranging from 4 to 7 (5.6 ± 1.4) and 13 biopsies from 8 to 10 (8.4 ± 1.6). Overall, at least 95% of markers (MS + SNP) were detected in 49.1% of biopsies (28/57). The total detection rate for SNP was significantly higher than for MS; 70.2% (40/57) v. 31.6% (18/57), respectively, (chi-square, P < 0.01). The detection rate of the markers was not significantly affected by the embryo stage or the biopsy size. Our results confirm that genotyping a large number of markers from biopsy samples after whole-genome amplification is possible under field conditions. A larger number of biopsies is required to assess the reliability of this method that may allow the development of MAS from early embryo. This work has been performed through the programme TYPAGENAE (GENANIMAL 4-03) with the financial support of FRT/ANR and Apis-Genes.

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Ramasamy, Karthik, Shameem Mahmood, Laurence Pearce, et al. "Progression Free Survival (PFS) in Alemtuzumab Based RIC Allogeneic Transplantation for Myeloma Is Improved with Use of Pre-Emptive DLI (pDLI)." Blood 110, no. 11 (2007): 3034. http://dx.doi.org/10.1182/blood.v110.11.3034.3034.

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Abstract Allogeneic transplantation is the only potentially curative treatment option for myeloma (MM). While reduced intensity conditioning (RIC) regimens are associated with lower transplant related morbidity and mortality (TRM), this approach is offset by an increase in relapse rates in myeloma. Method & Materials: We conducted a prospective study on the use of pDLI in myeloma patients with declining donor T cell chimerism following alemtuzumab based RIC allogeneic transplants. 19 consecutive patients were recruited. Patients received 100mg of Alemtuzumab 1H in total over 5 days in vivo as part of the pre-transplant conditioning regimen. There were 8 females and 11 males with a median age of 54 years (range: 41–63). Median lines of prior therapy was 2 (1–4), and 17(84%) had at least one prior autograft. 15 patients were transplanted with related donors and 4 unrelated. The median followed up of the cohort was 25.5 months (4–82). Fractionated peripheral blood (PB) and unfractionated bone marrow (BM) chimerism was performed on day 30, 60, 100 and 3–6 monthly thereafter using promega powerplex 16 genetic identity system. The samples were electrophoresed on an ABI 3130 XL capillary sequencer and analysed using Genemapper 4.0 software. Metaphase cytogenetics of unfractionated BM overnight cultures and Y-FISH for sex mismatch transplants was performed at various time points. Escalating doses of pDLI was given to patients with falling donor T cell chimerism (<50%) after Day 100. The starting dose of DLI was 1 × 105 CD3 cells/kg. 6 patients received pDLI with a median number of pDLI infusions of 2.5 (1–4). Overall survival (OS) and PFS was calculated from date of transplant using Kaplan-Meier cumulative survival plot using SPSS 14.0 software in pDLI receiving (Cohort I) and non-pDLI receiving cohort (Cohort II). Results: 17 patients (84%) responded with CR rate at 100 days of 57%. One patient had primary graft failure and 1 patient had stable disease for a year post transplant. Median OS and PFS for all patients was 24 months (17.6–30.3) and 12 months (7.7–16.2) respectively. TRM at 2 years was 15.5%. In Cohort I, 13 patients did not receive pDLI. 1 patients (7.6%) experienced limited chronic graft versus host disease (cGVHD) with no cases of acute GVHD. In Cohort II, escalated doses of pDLI were administered to 6 patients at declining levels of donor CD3 chimerism in PB without evidence of morphological or cytogenetic relapse. 2/6(33.3%) patients developed cGVHD post DLI. Full donor chimerism was achieved in 4/6 patients, all of whom are long-term survivors. Median OS in Cohort I is 24 months (8.9–41.0) and 17 months (11.3–22.6) in Cohort II (p=NS). Median PFS in Cohort I is 25 months vs 9 months in Cohort II (p= 0.02). Cytogenetics was not helpful in determining requirement for DLI even in sex-mismatched patients. The presence of GvHD did not have a significant impact on OS. Conclusion: pDLI administration guided by PB CD3 chimerism significantly improves PFS in Alemtuzumab conditioned RIC transplants for myeloma. Monitoring CD3 chimerism and early application of pDLI improves survival with a 5-year survival rate of 20% in multiply treated high risk patients.

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Lekostaj, Jacqueline K., Juehua Gao, and Amy Chadburn. "Clonality Of Multiple Distinct Lymphoproliferative Disorders Occurring Within Individual HIV+ Patients." Blood 122, no. 21 (2013): 4329. http://dx.doi.org/10.1182/blood.v122.21.4329.4329.

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Abstract Introduction Lymphoma is usually considered a monoclonal process that can be present at one or more anatomic sites in a patient. In the setting of combined Epstein Barr virus (EBV) infection and immunosuppression, clonally distinct lymphoproliferative disorders (LPDs) may arise within a single individual. In the area of solid organ transplant, it has been previously described that multiple LPDs arising simultaneously or sequentially in the same patient, including those in the same anatomic site, can be of different genetic composition (Cancer 1995 75:2747). Furthermore, histologic morphology does not necessarily correlate with molecular clonality. HIV+ patients are another population who are immunosuppressed and often infected with EBV. However, whether multiple LPDs (reactive and/or neoplastic) occurring in an individual HIV+ patient are of identical or disparate clonal content, or whether they are clonally consistent with morphology, has not been fully studied. We previously reported data from 10 patients (Modern Path 2013 26(S2):340A) which suggested that recurrence or progression of lymphoma, regardless of site, is usually of the same clone. However, the vast majority of these cases involved specimens obtained fewer than 2 years apart, and half were only 6 months or less. Data on an additional 8 patients are now presented, with 5 cases representing greater than 2 years of follow-up. Materials/Methods DNA from touch preparations or formalin-fixed, paraffin-embedded tissue of 2-3 LPDs from 8 HIV+ patients (6 males/2 female; ages 32-62 yrs) was amplified using BIOMED-2 primer sets for heavy, kappa and lambda immunoglobulin loci (Invivoscribe). Tests performed were dependent on available material. Samples were assessed for quality using a specimen control size ladder primer set. PCR products were analyzed by capillary electrophoresis. The data was analyzed using GeneMapper software, and monoclonality was defined as a peak > 3 times the height of the third highest peak in the appropriate region. EBV was detected by in situ hybridization with an EBER probe. Results Clonal populations were seen in almost all specimens regardless of morphology. One reactive lymph node was polyclonal, but all lymphomas were monoclonal and three non-neoplastic –appearing lesions also contained clonal populations. Four patients had the same clone present in multiple locations at various times, ranging from 1 week to 2 years. In three cases with a longer time lapse between presentations (5-9 years), different clones were identified. Abbreviations (D)LBCL – (diffuse) large B cell lymphoma; LN – lymph node; MZL – marginal zone lymphoma; PEL – primary effusion lymphoma Conclusions The current data indicate that HIV-associated LPDs can contain clonal populations regardless of morphology. This may suggest either the presence of a benign reactive subpopulation, which could be transient, or of low-level visually-undetectable involvement by a neoplastic process. As was previously shown, the recurrence or redevelopment of lymphoma within 2 years of original diagnosis is usually of the same clone, and may therefore warrant a change in strategy for successful treatment. However, LPDs which appear more than 5 years from initial presentation generally represent a new disease and may not necessarily imply nonresponsiveness to a similar therapeutic regimen. Disclosures: No relevant conflicts of interest to declare.

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Noriega, Victor, Carolina Martinez-Laperche, Leyre Bento, et al. "The Genotype of the Donor for the (GT)n Polymorphism in the Promoter/Enhancer of the FOXP3 Gene Influences Graft Versus Host Disease without Affecting Graft Versus Leukemia Effect After Myeloablative HLA-Identical Allogeneic Stem Cell Transplantation." Blood 118, no. 21 (2011): 3051. http://dx.doi.org/10.1182/blood.v118.21.3051.3051.

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Abstract Abstract 3051 INTRODUCTION The FOXP3 gene encodes for a protein (Foxp3) involved in the development and functional activity of regulatory T cells (CD4+/CD25+/Foxp3+, Tregs) which exert regulatory and suppressive roles over the immune system. After allogeneic stem cell transplantation (allo-SCT), Tregs are known to mitigate graft versus host disease (GVHD) while maintaining a graft versus leukemia effect (GVL). Allele (GT)15 for the functional (GT)n polymorphism in the promoter/enhancer of the FOXP3 gene is associated with a higher expression of FOXP3 and production of a greater amount of Tregs. However, its impact in the allo-SCT setting has not been analyzed. OBJECTIVE To analyze the impact of the (GT)n polymorphism in the promoter/enhancer of the FOXP3 gene on the development of complications and ultimately on the success of conventional HLA-identical allo-SCT. MATERIALS AND METHODS The study includes 33 patients with hematological malignancies, treated with myeloablative HLA-identical peripheral blood allo-SCT (Table 1). Diagnosis, classification and grading of GVHD were made by clinical criteria and confirmed when necessary by pathological examination of histological samples from gut, skin, liver or lung, according to international consensus criteria. Donor and recipient genomic DNA was purified from EDTA anticoagulated peripheral blood before allo-SCT and using QIAamp Blood DNA extraction kit (Qiagen). Genotyping of the (GT)n microsatellite polymorphism in the FOXP3 gene was performed by a fluorescence-based short tandem repeat-polymerase chain reaction (STR-PCR) method (GeneAmp 7900; Applied Biosystems) and sized by capillary electrophoresis (POP7 - ABI PRISM 3130 xL Genetic Analyzer; Applied Biosystems) followed by fragment analysis (GeneMapper 4.0 Software; Applied Biosystems) as previously described [Bassuny WM, et al. Immunogenetics. 2003;55 :149–56]. RESULTS The median follow-up time for the cohort was 34 months (range 9.5–110). Allelic frequencies observed were similar to those previously reported (50.5% (GT)15, 41% (GT)16 and 7% (GT)17; no (GT)14 or (GT)18 alleles were found). Patients transplanted from donors harboring allele (GT)15 showed a lower incidence of grades II-IV acute GVHD (29% vs 67%; p =0.049). These patients also showed a trend to a lower incidence of severe (grades III-IV) GVHD (12% vs 33%; p =0.167) as well as chronic GVHD (75% vs 100%; p =0.143; Table 1, Figure 1). No statistically differences were found between patients transplanted from (GT)15 and non-(GT)15 donors in terms of relapse rate (38% vs 33%; p =0.825; Table 1) or cumulative incidence of relapse (CIR at 2 years 35.3% vs 37.5%, Figure 2). Finally, survival analysis did not show statistically significant differences between the two groups of patients in terms of median event (relapse) free survival (EFS, 15.6 months vs 4.5 months, p =0.686) or overall survival (OS, 29 months vs not reached, p =0.610). CONCLUSIONS Tregs are known to modulate the allotolerance-alloreactivity balance between donor and recipient in the allo-SCT setting, mitigating GVHD while preserving the anti-tumor effect (GVL) of the donor graft. In the present study, the presence of allele (GT)15 in the donor, which promotes a higher expression of FOXP3 and greater amount of Tregs, affected allo-SCT outcome by decreasing grades II-IV acute GVHD and chronic GVHD, without affecting GVL (no differences in CIR and OS). Analysis of this polymorphism can help in appropriate donor selection and, more importantly, drive a tailored management of patients submitted to allo-SCT. Disclosures: No relevant conflicts of interest to declare.

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Iacobucci, Ilaria, Annalisa Lonetti, Anna Ferrari, et al. "Different Isoforms of the B-Cell Mutator Activation-Induced Cytidine Deaminase (AID) Are Aberrantly Over-Expressed in BCR-ABL1-Positive Acute Lymphoblastic Leukemia (ALL) Patients and Promote Genetic Instability." Blood 112, no. 11 (2008): 1497. http://dx.doi.org/10.1182/blood.v112.11.1497.1497.

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Abstract Background: BCR-ABL1-positive ALL is the most frequent and prognostically most unfavorable subtype of adult ALL. The main reason for the poor clinical outcome of BCR-ABL1-positive ALL is genetic instability. However, how normal B-cell precursor cells acquire the genetic changes that lead to transformation and progression has not been completely defined. Activation-induced cytidine deaminase (AID) produces immunediversity by inducing somatic hypermutations and class-switch recombinations in human immunoglobulin genes (Ig). Aim: Since at much lower frequency AID can also target non-Ig genes and may even act as a genome-wide mutator, we investigated whether AID was expressed in BCR-ABL1-positive ALL and in chronic myeloid leukemia (CML) at the time of progression to blast crisis. Patients and methods: We analyzed 61 adult de novo Ph+ ALL patients (pts) and 60 CML pts (chronic phase and myeloid/lymphoid blast crisis). AID cDNA, obtained from bone marrow or peripheral blood, was amplified with two pairs of oligonucleotides, the forward primer of each couple conjugated with a fluorescent dye (fluorescein) at its 5′ end. PCR products were then loaded on the ABI Prism 3730 DNA Analyzer for automated capillary gel electrophoresis and the results were plotted with the AbiPrism GeneMapper v3.5 software (Applied Biosystems). Results: On the 61 de novo adult BCR-ABL1-positive ALL pts, AID mRNA and protein were detected in 41 (67%). AID expression correlated with the BCR-ABL1 transcript levels and disappeared after treatment with tyrosine kinase inhibitors at the time of remission. Moreover, AID expression was also found in lymphoid blast crisis CML (60%), but not in myeloid lineage or in chronic phase CML. Different isoforms of AID were expressed. In 10/61 (16%) BCR-ABL1-positive ALL pts the full-length isoform (GeneBank accession number NM_020661) was identified, in 16/61 (26%) the co-expression of the wild-type isoform and of different AID splice variants was found and in 15/61 (25%) only the expression of splice variants was found. These can result from retention of intron 4 (Variant A), omission of exon 4 (Variant B) and 3 (Variant C), and from a deletion of 30 bp in the initial portion of exon 4 (Variant D). In the wild-type mRNA, codon 148 spans exons 3 and 4. In both variants A and B, mRNA splicing disrupts this codon and causes a frameshift, which results in a premature stop codon. If translated, the splice variants produce truncated proteins of 187 and 145 amino acids, respectively. However, the putative deaminase active site, encoded by exon 3, is preserved in both splice variants, but is lacking in the variant C. Since enforced expression of Pax5 induces endogenous AID gene expression, we analyzed the expression levels of Pax5 in all pts (ΔΔCt method, GAPDH as control gene). As expected, we found a very strong difference (p&lt;0.0001) between chronic phase CML and BCR-ABL1-positive ALL, but total Pax5-transcripts did not differ significantly when BCR-ABL1-positive ALL/AID+ and BCR-ABL1-positive ALL/AID− were compared. To investigate whether AID introduces DNA-single strand breaks in BCR-ABL1-positive ALL, we performed a genome wide analysis by 250K NspI single nucleotide polymorphism (SNP) array (Affymetrix Inc., USA). We identified a region of high level amplification and homozygous deletion in all patients. Patients who expressed wild-type AID had a higher number of alterations compared to AID-negative patients (median copy number alteration of 14, range 5–27, versus 4, range 1–6, respectively, p&lt;0.03). Recurring copy number abnormalities were identified in genes with an established role in leukemogenesis, such as IKZF1, CDKN2A, CDKN2B, PAX5, MELK, BTG1 and MDS1. AID consensus motifs (DGYW/WRCH) were mapped very close to the breakpoint cluster regions. Conclusions: Our findings show that BCR-ABL1-positive ALL cells aberrantly express different isoforms of AID that can act as a mutator outside the Ig gene loci in promoting genetic instability in leukemia cells.

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Azhipa, Olga Y., Scott D. Rowley, Michele L. Donato, Robert Korngold, and Thea M. Friedman. "T Cell Receptor Repertoire of Patients with Chronic Graft-Versus Host Disease." Blood 108, no. 11 (2006): 5174. http://dx.doi.org/10.1182/blood.v108.11.5174.5174.

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Abstract Chronic GVHD (cGVHD) is a major risk factor in patients receiving allogeneic hematopoietic cell transplantation (HCT), and is a complicated syndrome with a combination of autoimmune-like features and a range of multiorgan manifestations. Currently, efforts are being made to standardize the criteria for diagnosis and staging of cGVHD, but there is little understanding of the pathogenesis of the disease, associated biomarkers, and the immune perturbations that may result. Reconstitution of the T cell repertoire after allo-HCT often takes several months to a year, and may be significantly impaired or skewed in patients who develop cGVHD. We thus sought to assess the immune T cell status of cGVHD patients by TCR Vβ CDR3-size spectratype analysis. A cohort of 9 patients who underwent allo-HCT (PBMC n=7; BM n=2) were enrolled in the study. The underlying diseases in these patients were CML (n=1), AML (n=4), ALL (n=1), CLL (n=1), and MM (n=2). Patients received either reduced intensity or myeloablative conditioning before transplantation, and 8 of the 9 had a previous history of acute GVHD. Furthermore, the patients did not have evidence of infectious disease. PBMC was collected from each patient at one time point ranging from 2 wk to 3 yr from the time they were diagnosed with cGVHD. The onset of cGVHD ranged from 100 d to 3 yr post-HCT (median of 5 mo). Flow cytometric analysis was performed on peripheral blood lymphocytes from 7 of the 9 patients to analyze recovery of different subpopulations. PCR amplification of the CDR3 region of 21 TCR Vβ genes was used to analyze the diversity of the T cell repertoire. The PCR products were run on a sizing gel to separate the CDR3-lengths, and further analyzed by ABI GeneMapper software. Flow cytometric analysis revealed diverse percentages of CD4+ and CD8+ T cells among the 7 patients tested, which were correlated with the post-HCT period. Two patients who received HCT, 4 and 9 months before blood sampling, had only 3% and 4% CD4+ and 3% and 9% CD8+ T cells in their PBMC sample, respectively. On the other hand, the remaining 5 patients, who were all at later time points post-HCT, had CD4+ and CD8+ T cell percentages within normal range. One patient had a ratio close to the normal 2:1 CD4/CD8 ratio, two patients had a 1:1 ratio, and four had inverse CD4/CD8 ratios. Based on CDR3-size spectratype analysis, we determined the recipient TCR-Vβ complexity index within each resoluble family, which represented the percentage of the number of peaks found for each Vβ relative to that found in the average corresponding Vβ family of 10 healthy donors. We considered Vβ to be fully complex if the complexity index exceeded 85%. The results indicated that 41 to 88% of resolved Vβ in all 9 patients were fully complex, with the lower range corresponding to those patients sampled early post-HCT. Vβ 1, 2, 4, 6, 8, 12, and 13 families revealed the best recovery in all patients, even in patients after 4-mo post-HCT. Importantly, extensive skewing of the repertoire within most of the TCR Vβ families were found in all 9 recipients, suggesting that there were active heterogenous T cell responses in those patients with cGVHD. As to what these T cell responses were directed to remains to be seen, and could theoretically involve autoantigens, alloantigens, tumor antigens, or sub-detectable infectious agents. In any case, the presence of a wide-ranging T cell response in these patients may serve as an important new diagnostic indicator for cGVHD.

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Follit, Courtney A., Patricia A. R. Brunker, and Willy A. Flegel. "SNP Genotyping and LD Testing in ERMAP: Revealing Scianna Blood Group Diversity in NIH Blood Donors." Blood 118, no. 21 (2011): 2322. http://dx.doi.org/10.1182/blood.v118.21.2322.2322.

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Abstract Abstract 2322 Background: The Scianna blood group system has been implicated in cases of hemolytic disease of the fetus and newborn and the detection of antibodies to rare antigens in this system have impacted on transfusion management in some patients. Recently, it has been discovered that the Scianna blood group antigens are expressed by the erythrocyte membrane-associated protein (ERMAP), a 475 amino acid red cell adhesion protein consisting of 12 exons with the transcription region spanning exons 3–12. Rare variants in exons 4 and 12 have been reported in patients who have made antibodies to Scianna antigens or have a serological null phenotype for the Scianna system. ERMAP is a member of the butyrophilin-like family, featuring an extracellular immunoglobulin variable and intracellular B30.2 domains. Although one ERMAP variant is detected in one commercial molecular assay (Sc1/Sc2), most reported variants in this gene are rare, and therefore remain largely unrecognized during transfusion planning. ERMAP polymorphisms remain unreported on a large scale, contributing to the uncertainty concerning their clinical significance. To fill this void, we characterized seventeen single nucleotide polymorphisms (SNPs) in exons 3, 4, and 12 of ERMAP in 905 repeat blood donors. Methods: The DNA of consenting, repeat NIH blood donors were genotyped for seventeen variants in the ERMAP gene. DNA was isolated from whole blood using the Qiagen's MagAttract EZ1 kit. Following polymerase chain reaction amplification, the samples were genotyped by ligation detection reaction (LDR). LDR utilizes a thermostable ligase to generate single stranded DNA fragments of engineered length with allele-specific fluorescent labels, allowing for rapid, multiplexed genotyping. Ligated products were resolved by capillary electrophoresis (3730 DNA analyzer and GeneMapper software (Life Technologies)). Results: Eleven of the seventeen variants (G35S, R81Q, nt307Δ2, Q296Q, R332X, R392H, L399L, L409L, S442P, L452P, and L452L) were monomorphic in this cohort (N=905). Overall, the 54c>t and 76c>t transitions in exon 3 had minor allele frequencies (MAF) of 0.21 and 0.23, respectively, and appeared in all self-identified ethnic groups (except Native American donors (n=2)) with maxima observed in donors of self-identified Hispanic ethnicity (n=16; MAF=0.41 and 0.44, respectively). These SNPs showed significant linkage disequilibrium (r2=0.86 [95%CI 0.85–0.88]). African-American donors (n=57) had the highest frequency of variant 11c>t (MAF 0.07) and variant 755c>t (MAF 0.018), which was absent or extremely rare in other ethnic groups. The Caucasian donor population was the only group to display variations 788g>a and 1094g>a (MAF 0.003 and 0.0008 respectively). Conclusions: This is the largest sample of blood donors to be comprehensively genotyped for Scianna variants to date. We observed population-specific polymorphism of these rare variants according to the donor's self-identified ethnicity, which is under further study. Determining the diversity in the Scianna blood group system may help explain otherwise unclear transfusion reactions, particularly if these variants impact on Scianna antigen surface density (especially the predicted leader sequence variants in exon 3) or other ERMAP functions (via variants in the intracellular domain encoded by exon 12). High throughput donor genotyping will allow evaluation of the clinical importance in alloimmunization for variants like the 11c>t, 54c>t, and 76c>t SNPs that lie in the predicted leader sequence and polymorphisms 755c>t, 788g>a, and 1094g>a that lie within the intracellular B30.2 domain of the ERMAP protein. Awareness of the frequencies of these variations can therefore be a clinically useful aid in the investigation of donors implicated in transfusion reactions. Disclosures: No relevant conflicts of interest to declare.

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Risinskaya, Natalya V., Olga A. Gavrilina, Julia A. Chabaeva, et al. "Loss of Heterozygosity in the Short Tandem Repeat (STR) Loci Found in Tumor DNA of De Novo Diagnosed ALL Patients As a Factor Predicting Poor Outcome." Blood 134, Supplement_1 (2019): 5204. http://dx.doi.org/10.1182/blood-2019-126668.

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Introduction: Loss of heterozygosity (LOH) was described for many malignancies including leukemia. Using chromosomal microarray analysis (CMA) we have shown preliminary (Risinskaya et al. HemaSphere, 2019, V.3, No S1, P.782) that LOH in STR loci in tumor cells with the normal karyotype represents the loss of extended chromosomal areas and could be associated with uniparental disomy of the entire chromosomal arm or the entire chromosome. Uniparental disomy results not only in the elimination of the wild type allele, but also may lead to the duplication of the mutant allele of certain candidate tumor gene(s). Retrospective analysis of archival samples have shown that in most cases STR loci absent at the relapse of disease were already lost at the onset of disease. We noted LOH at STR loci is especially frequent for relapsed ALL patients and could be used as prognostic factor for possible poor clinical outcome. The aim: To identify LOH in the blast cells of the patients with ALL at the onset of disease and to analyze therapy outcome relative to the patients without LOH. We expect LOH in the de novo ALL to be an unfavorable factor, and those patients possibly should be treated like high-risk group. Patients and Methods: This study includes a comparative analysis of the STR profiles of the DNA of tumor and normal cells from bone marrow samples on a cohort of 32 patients with de novo diagnosed Ph-negative ALL undergoing treatment according to the "RALL-2016" regimen at the National Research Center for Hematology (Moscow, Russia). Inclusion criteria: de novo diagnosed Ph-negative acute lymphoblastic leukemia (ALL) patients, 18-55 years old, intermediate risk group without MLL translocation t(4;11)(q21;q23), treatment by RALL-2016 protocol. Exclusion criteria: other diagnosis, adult patients older than 55 years, MLL translocation t(4;11)(q21;q23), pretreatment or treatment by other protocol. The presence of blast cells in bone marrow samples was confirmed morphologically. DNA was isolated from patient bone marrow samples taken at different disease stages. Control DNA samples were taken from blood of patients in complete remission and/or from buccal epithelium (buccal swab). STR-profiles were assessed by PCR with COrDIS Plus multiplex kit for amplification of 19 polymorphic STR-markers and amelogenin loci (Gordiz Ltd, Russia). The fragment analysis was performed on ABI3130 Genetic Analyzer. The data processing was accomplished using GeneMapper v.4-0 software. A multivariate survival analysis was used to assess the independent impact of the LOH as risk factor against the background of the abnormal tumor karyotype as standard factor for this cohort of patients. We have chosen Failure Free Survival (FFS) as primary endpoint for short-time follow-up and small patient sample size. Death from any reasons, relapse and second leukemia were chosen as failure events, survival time interval starts from begin of treatment. SAS V9/4 was used for statistical analysis of the data. Results: Of the 32 patients six were found with LOH in the certain STR loci (19%). Of these six, two were resistant to therapy and died from disease progression. One patient with LOH is currently in relapse and one patient with multiple LOH has a second leukemia (Fig.1a). Only two from six patients are "Failure Free" now, whereas only three patients from 26 in group without LOH died (two from infectious complications and one from GvHD after BMT). P value < 0,05 (Fig.1b). Conclusions: We have found statistically significant association of clinical failures with the LOH in STR loci measured at the onset of ALL. We assume that LOH is an unfavorable prognostic factor for de novo ALL patients and could be used for risk stratification and choice of adequate therapy implying allogeneic hematopoietic stem cells transplantation or modern innovative therapy approaches. Disclosures No relevant conflicts of interest to declare.

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Gómez-Seguí, Inés, Esperanza Such, Jose Cervera, et al. "Gene Microdeletions in Adult and Pediatric Acute Lymphoblastic Leukemia,." Blood 118, no. 21 (2011): 3539. http://dx.doi.org/10.1182/blood.v118.21.3539.3539.

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Abstract Abstract 3539 Background: Microdeletions of genes involved in B lymphopoiesis and cell-cycle regulation, such as CDKN2A/B, PAX5, IKZF1, ETV6, RB1, BTG1 and EBF1 have been reported as a frequent event in pediatric acute lymphoblastic leukemia (ALL). Whether these findings are found in adulthood and the possible differences with childhood ALL, as well as its prognostic implication, are still unknown. Aims: To assess the differences between two cohorts of children and adults diagnosed with ALL on the frequency of deletions in these genes and their relationship with clinical data and prognosis. Methods: We studied 70 children and 83 adults diagnosed with ALL with available DNA sample at diagnosis. In children, median age was 4y. (1 – 14), median leukocytes 10.3×109/L (0.7 – 675) and the cytogenetic risk distribution was 42(39%), 30(27%) and 12(11%) for favourable [t(12;21) and hyperdiploidy], intermediate (normal karyotype and miscellaneous) and high risk [t(9;22), t(4;11), hypodiploid and complex karyotype], respectively. In adults, median age was 38y. (15 – 85), median leukocytes 16.8×109/L (1 – 371) and 29(35%) patients belonged to the high risk cytogenetic group. We performed Multiplex Ligation Probe Amplification (MLPA) using SALSA kit P335-A1 (MRC-Holland). PCR products were separated on an ABIPRISM 310 DNA Analyzer and analyzed using GeneMapper v3.2 (Applied Biosystems). Results: Frequency of deletions in the studied genes was similar in children and adults, except for IKZF1 deletions that were more frequent in adults (P<.001) (Table 1). In children, ETV6 deletions occurred more frequently in patients with t(12;21) (67% of patients with deletion vs. 17% without, P <.001); CDKN2A/B deletions were found in patients assigned to the intermediate cytogenetic risk group (59% of patients with deletion vs. 23% without, P =.028); and the three cases with RB1 deletions were found in patients with hypodiploidy (P <.001). In adults, ETV6 and CDKN2A/B deletions occurred more frequently in women (67% vs. 39%, P =.022 and 77% vs. 42%, P =.021, for patients with and without deletions, respectively); PAX5 and IKZF1 deletions appeared more frequently in patients with >30×109/L leukocytes (60% vs. 27%, P =.032 and 52% vs. 21%, P =.007, for patients with and without deletions, respectively); besides, PAX5 deletions occurred in patients who belonged to the standard cytogenetic risk group (55% vs. 6% for patients with and without deletions, P <.001). In the pediatric cohort, the leukocytes >30×109/L and the cytogenetic risk group were the variables that reached statistical significance for both overall survival (OS) and relapse free survival (RFS) and also age >10y. for OS, but in the multivariate analyses, just the cytogenetic risk classification remained significant [HR: 4 (CI 95%: 1.6 – 10), P =. 004 for OS and HR: 3.5 (CI 95%: 1.7 – 7.2), P =. 001 for RFS]. In the adult cohort, multivariate analysis for OS including all significant variables in the univariate analysis (age >60y, karyotype, CDKN2A/B and ETV6 deletions) showed as independent variables: age >60y. [HR: 4.3 (CI 95%: 2.1 – 8.6), P<. 001] and CDKN2A/B deletions [HR: 2.6 (CI 95%: 1.4 – 5.3), P=. 004]. Similarly, taking into account karyotype, CDKN2A/B and ETV6 deletions for the RFS multivariate analyses, just ETV6 deletions arose as an independent factor [HR: 3.8 (CI 95%: 1.5 – 9.4), P=. 004]. In fact, having CDKN2A/B and/or ETV6 deletions conferred a worse prognosis to patients in both standard risk cytogenetic group (3y. RFS: 45% vs. 70% for patients with and without deletions, respectively; P =.049) and high risk cytogenetic group (3y. RFS: 14% vs. 66% for patients with and without deletions, respectively; P =.025). Conclusions: This study shows the high incidence of deletions in genes of cell-cycle and B-lymphopoiesis in adult and pediatric ALL. However, the biological and prognostic implications of these deletions seem to differ between both patient groups: while cytogenetics was the strongest variable for risk assessment in children, gene microdeletions in CDKN2A/B and ETV6 added a prognostic value to karyotype in our adult cohort. Fundings: AP-194/10, R06/0020/0031, BES2008–008053, CM10/00321, CM09/00038, and CA08/00141. Disclosures: No relevant conflicts of interest to declare.

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Thol, Felicitas, Frederik Damm, Katharina Wagner, et al. "Next Generation Sequencing for Minimal Residual Disease Monitoring in AML Patients with FLT3-ITD,." Blood 118, no. 21 (2011): 3548. http://dx.doi.org/10.1182/blood.v118.21.3548.3548.

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Abstract Abstract 3548 Minimal Residual Disease (MRD) monitoring has become an important tool for risk and treatment stratification in hematological malignancies. MRD monitoring in FLT3 mutated patients has been difficult in the past as FLT3-ITDs vary from patient to patient and individual primer/probe sets would be required to assess MRD over time. In the present study we evaluated next-generation sequencing (NGS) as a new tool for MRD monitoring in patients with FLT3-ITD and NPM1 mutations. Five pediatric and 5 adult AML patients with FLT3-ITD and 10 adult patients with NPM1 mutations were analyzed by NGS with a target coverage of 10,000 reads per amplicon. Pediatric samples were collected at diagnosis, day 22 and after consolidation chemotherapy while adult samples were collected at different time points (average 4 timepoints per patient). Samples were sequenced unidirectionally on eight-lane PicoTiterPlates on a GS FLX sequencing system. In total, 2,563,550 sequencing reads were generated, corresponding to a total of 1,176,171 high-quality sequencing reads. NPM1 mutations were analyzed by quantitative RT-PCR using the MutaQuant kit from Ipsogen (Ispogen, Marseille, France). Allelic ratios of FLT3-ITDs were determined by fragment analysis on a DNA sequencer using GeneMapper software 4.0. First, the sensitivity of NGS to detect mutated alleles was evaluated by sequencing serial dilutions of a patient sample that had 46.3 percent mutated FLT3-ITD alleles at diagnosis. With a target coverage of 10,000 sequences and an allelic ratio of 46.3 percent the theoretical detection sensitivity was at most 1 in 4630 sequences. In fact, the allelic ratio in the sequenced samples linearly decreased in the tested dilutions down to the 5×10-4 dilution (Pearson correlation R2=.996). Samples from healthy volunteers were tested negative for both FLT3-ITD and NPM1 mutations (n=3). Allelic ratios from three diagnostic specimens of FLT3-ITD mutated patients were highly reproducible when determined in two independent NGS runs. As proof of principle we analyzed NPM1 mutated patients by NGS and quantitative RT-PCR in parallel. The mean allelic ratio of NPM1 mutants at diagnosis was 0.37 (range 0.29–0.46). An allelic ratio of 0.37 and 0.4 was measured in peripheral blood of two patients, and thus was similar to ratios in bone marrow. Concordant results between NGS and qRT-PCR were found in 38 samples (95%), whereas in two samples one method did not detect the mutation while the other did (NGS and RT-PCR were negative once each). We analyzed relapse samples in four patients. The NPM1 mutation was detected consistently by both methods in three patients at allelic ratios of 0.013, 0.19, and 0.32, while one patient had lost the mutation at relapse. One patient had an atypical NPM1 mutation for which no RT-PCR kit was available. NGS allowed quantification of the allelic ratio in this patient, which was 0.37 at diagnosis, 0.06 after one cycle of induction therapy, and 0 after the second cycle of induction therapy. In FLT3-ITD mutated patients we could determine insertion site, insertion length, number of individual clones, and allelic ratio from NGS data. The mean allelic ratio in diagnostic samples was 0.27 as measured by NGS and 0.4 as measured by fragment analysis. Three follow up samples were negative by fragment analysis, while a small clone could still be detected with NGS in these samples (allelic ratio 0.0004 to 0.001). All other samples were concordant between fragment analysis and NGS. NGS was used to determine MRD status in 5 patients with childhood AML harboring mutated FLT3. A reduction of 2–3 orders of magnitude was achieved during induction chemotherapy. During consolidation a further decrease or disappearance of mutated alleles was achieved in 3 patients, who remained in remission. However, allelic burden increased in 2 patients after first consolidation treatment (HAM) by 9- and 735-fold compared to the allelic ratio after induction therapy, and they relapsed 74 and 303 days later. Thus, accurate determination of the FLT3-ITD allelic ratio by NGS may become useful to identify patients before overt relapse. In summary, we show that NGS can be used for minimal residual disease assessment in FLT3-ITD mutated AML patients. The sensitivity of the method is scalable depending on the read depth, however, an adequate sensitivity level for efficient MRD detection still needs to be determined. Disclosures: No relevant conflicts of interest to declare.

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Valencia, Ana, Jose Cervera, Esperanza Such, et al. "A Methylation Profile of Tumor Suppressor Genes Predicts a Poorer Survival in Patients with Refractory Anemia with Ringed Sideroblasts." Blood 114, no. 22 (2009): 2780. http://dx.doi.org/10.1182/blood.v114.22.2780.2780.

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Abstract Abstract 2780 Poster Board II-756 Patients with refractory anemia with ring sideroblasts (RARS) are considered to have good prognosis and anemia is usually managed with best supportive care and erythroid stimulating agents. Nowadays, no specific molecular marker related to outcome and disease progression has been identified. Several genes involved in cell cycle and apoptosis that may become inactivated by aberrant hypermethylation have been identified in patients with myelodysplastic syndromes (MDS) but the significance of a methylation profile (studying several genes at the same time) in RARS is unknown, mainly because the number of patients with RARS in published reports is rather low. To assess the implication of aberrant methylation in RARS, we studied the methylation status of 25 sequences of a set of tumor suppressor genes in 40 patients (median age 70 yr, 25 male gender) with RARS according to FAB criteria [WHO classification, RARS in 22 patients (55%); refractory citopenia with multilineage dysplasia and ring sideroblasts, 18 (45%)] by means of methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. The MS-MLPA procedure (SALSA MLPA kit ME001, MRC-Holland, Amsterdam, The Netherlands) has been developed for detecting in a semi-quantitative manner changes in the methylation status of 25 tumor suppressor genes in a single experiment. Briefly, approximately 50 ng of DNA were denatured and hybridized with MLPA probes. Subsequently, the probes were ligated in half of every sample, whereas for the rest of the sample, ligation was combined with an endonuclease HhaI digestion resulting in ligation of the methylated sequences only. PCR was performed as described by the manufacturer, and then separated by capillary gel electrophoresis and quantified using the Genemapper software (ABI 310, Applied Biosystems, Foster City, CA). Quantification of the methylation status was done by dividing the peak area with the combined areas of the control probes lacking the target sequence of the HhaI. Finally, the relative peak area of each target probe from the digested sample was compared with those obtained from the undigested sample. Aberrant methylation was scored when the calculated methylation percentage was >10%. To validate the MS-MLPA method the methylation status of CDKN2B was also analyzed by methylation-specific PCR (MSP). Actuarial curves of OS were built by Kaplan-Meier method and differences between curves compared with log-rank tests. Small numbers precluded the use of multivariate methodology. The MS-MLPA analysis detected methylation of at least one gene in 17 patients (42.5%). The genes aberrantly methylated were CDKN2B (n = 8, 20%), RASSF1 (n = 7, 17%), RARB (n = 3, 7.5%), CDH13 (n = 3, 7.5%) and FHIT (n = 2; 5%). Of the 17 patients, five (12%) presented methylation in two genes (RASSF1-FHIT in 2, RASSF1-RARB in 1, RASSF1-CDH13 in 1, and CDKN2B-CDH13 in 1, who was the only patient who progressed to AML). The presence of aberrant gene methylation was not significantly associated with any clinical or biological characteristic or WHO morphological subtype. Patients with aberrant gene methylation had a significantly shorter overall survival (OS) than patients without methylated genes (median OS, 72 mo vs 114 mo, respectively; P = 0.03). Patients with hemoglobin level below 10 g/dL and platelet count below 150 ×109/L also had a significantly poorer OS (P= 0.01 and P= 0.02, respectively). As the majority of probes used in the MS-MPLA method detect methylation in only one CpG pair, the results of CDKN2B methylation were validated by MSP, which yielded the same methylation results than with MS-MPLA methodology. The 8 patients with methylated CDKN2B show a trend for a shorter survival than the remaining 32 with unmethylated CDKN2B (median 72 mo vs 114 mo, P = 0.08). The results of this study indicate that aberrant methylation of several tumor suppressor genes is observed in a substantial proportion of patients with RARS. As occurs in patients with high-risk MDS, our results suggest that aberrant gene methylation confers a poor prognosis in RARS, but these data and their potential independent value require confirmation in larger series that employ multivariate methods. Finally, these findings provide a strong rationale for the use of hypomethylating agents (e.g., azacitidine or decitabine) in patients with RARS. This work has been partially supported by ISCIII grants RD06/0020/0031 and CA08/00141. Disclosures: No relevant conflicts of interest to declare.

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Jann, Johann-Christoph, Daniel Nowak, Florian Nolte, et al. "Development of a DNA-Based Targeted Assay Suitable for Accurate Quantification of Chromosomal Deletions in Myelodysplastic Syndromes with Deletion (5q) and Other Clonal Diseases." Blood 124, no. 21 (2014): 1925. http://dx.doi.org/10.1182/blood.v124.21.1925.1925.

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Abstract Introduction The acquisition of large-scale chromosomal lesions is a frequent event in malignant disorders. One example is the recurrent deletion of chromosome 5q (del(5q)) in myelodysplastic syndromes (MDS). The detection and monitoring of such deletions are important elements in routine clinical diagnostics and cancer genomic studies. Currently established methods for their assessment are metaphase cytogenetics (MC), fluorescence in situ hybridization (FISH) and micro-array based techniques. However, each of these methods harbours specific disadvantages as they depend on (viable) cells, are expensive, labour-intensive or only semi-quantitative. One possible approach to interrogate chromosomal deletions constitutes the assessment of allelic loss at heterozygous short tandem repeat (STR) loci within deleted regions. Therefore, we aimed to establish a robust, quick and inexpensive PCR assay that measures allelic imbalance at such STR loci in order to reliably estimate frequencies of cells carrying del(5q) from only minute amounts of DNA. Methods Genomic DNA (gDNA) was isolated from bone marrow (BM) or blood cells of MDS and acute myeloid leukemia (AML) patients with cytogenetically confirmed del(5q). Based on NCBI UniSTS database, we designed 12 fluorochrome-labelled PCR amplicons with size ranges of 100-400 bp that surround STR loci between chromosomal bands 5q21 and 5q31. Using only 10 ng gDNA, all 12 PCR amplicons were amplified in a single optimized multiplex-PCR reaction. Subsequently, amplicon fragment analysis was carried out via capillary electrophoresis on an ABI 3130 Genetic Analyzer. Allele size quantification of informative heterozygous loci was performed using ABI Genemapper software. Furthermore, size calculations of individual alleles were corrected for PCR-stutter, which was estimated from corresponding loci in homozygous samples. Finally, using mesenchymal stromal cells as a germline control, the degree of skewing in the allelic ratios of all informative STR markers in the tumor sample relative to the corresponding allelic ratios in the control was averaged and subsequently translated into fractions of cells carrying the del(5q) lesion. Results Application of our novel assay for quantification of del(5q) burden in n=559 samples from n=67 patients revealed a high frequency of informative markers with an average of 7 heterozygous STR loci per patient. The data shows a strong inter-marker concordance with a standard deviation (SD) of 2.3% for del(5q) cell frequencies. Moreover, duplicate analysis of 328 samples revealed an average SD of 0.86%. Most importantly, paired analysis of the proportion of del(5q) cells estimated using interphase-FISH and our PCR assay was carried out for n=9 samples and resulted in strong correlation with r²=0.93. A serial dilution series with deleted and non-deleted gDNA also revealed highly concordant results with r²=0.96. When comparing matched germline and tumor STR profiles, no case of microsatellite instability was detectable in our MDS/AML cohort thus highlighting the suitability of STR based lesion quantification in this disease entity. Furthermore, we could use our large dataset to calculate amplification efficiencies for each locus in order to predict “surrogate” germline profiles eventually allowing us to calculate del(5q) frequencies in tumor samples that lack germline controls. Finally, our assay reliably confirmed molecular remission in BM from del(5q) MDS patients after Lenalidomide treatment, in agreement with concomitant MC analyses. Conclusion Using a multiplexed PCR assay for measurement of STRs in deleted chromosomal regions we present a highly adaptable tool for precise quantification of large scale lesions. We show a very good correlation with established methods exemplarily for del(5q) lesions. Even without available germline control our assay provides robust results. Requiring only minute amounts of gDNA, this assay is ideally suitable for copy number quantification in samples for which only residual archival gDNA and no cells for FISH analysis are available. Due to the small amplicon sizes it should also be useful for investigation of fragmented gDNA from formalin-fixed archival specimen. In summary, our newly developed assay offers a mean to obtain quantitative data for basically any large scale chromosomal deletion which contains STRs and should be easily applicable to other clonal diseases. Disclosures Nolte: Celgene Corp., Novartis Pharma: Honoraria, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Verger, Emmanuelle, Bruno Cassinat, Christine Dosquet, et al. "Impact of the Molecular Profile of Malignant Clones on the Response to Interferon Alpha (IFNa) Therapy in JAK2V617F-Negative Essential Thrombocythemia (ET)." Blood 124, no. 21 (2014): 407. http://dx.doi.org/10.1182/blood.v124.21.407.407.

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Abstract Background: The majority of ET patients (pts) without JAK2 or MPL mutations present somatic mutations in the calreticulin gene (CALR). However, a series of mutations in other genes involved in the epigenome, the splicing machinery or leukemic transformation have been described in ET, but contrary to MF, their impact has not been clearly assessed. We have previously shown that IFNa may considerably reduce the JAK2-mutated clones, and we also observed that TET2 mutated clones are resistant to IFNa therapy in JAK2+TET2+ pts with polycythemia vera. As little is known about the IFNa response of clones harboring mutations other than JAK2V617F in ET, we took advantage of a cohort of pts without JAK2 mutation but CALRmutated and treated with IFNa (according to international and local guidelines) to assess the dynamics of the different MPN clones during therapy. Aims: 1) To determine, using a Next Generation Sequencing (NGS) approach, the molecular pattern of mutations in genes previously shown to have a prognostic impact in MPNs in a series of CALR-mutated ET pts; 2) To study the evolution of these mutational patterns during IFNa therapy. Methods: JAK2 and MPL-negative ET pts followed in our department fulfilling the following criteria were included in the study: presence of a CALR mutation; availability of at least 3 sequential blood samples including one taken before IFNa; IFNa therapy for at least 3 months; informed consent for molecular analysis. Total DNA was extracted from blood samples (Qiagen blood DNA mini kits) for molecular analyses. Mutations in TET2, ASXL1, EZH2, SRSF2, IDH1, IDH2, SH2B3 and CSF3R were searched through a NGS approach on a MiSeq instrument using a TruSeq custom amplicon approach (Illumina). CALRgene mutation detection was done by direct Sanger sequencing of exon 9, and mutant allele burden (%CALR) was estimated by fragment analysis (GeneMapper software , Life technologies) with a sensitivity of about 1%. Both sequencing and fragment analysis were performed on a 3500xL DX Genetic Analyser (Life technologies). Molecular response was defined as complete (CMR) when CALR mutation was no longer detectable, partial (PMR) when %CALR was decreased by >50%, minor (mMR) when %CALR was decreased by 25 to 49%, and non responders if %CALR was reduced by less than 25%. Results: Among 238 ET pts without JAK2 or MPL mutations, we identified 52 pts (22%) treated with peg-IFNa-2a, of whom 24 fulfilled the inclusion criteria. Median age was 52 years (range 31-68), 67% were women, median follow up was 12 years (range : 1.5 – 29) and median IFN treatment duration was 30 months (range: 12 – 102). 23/24 (96%) pts achieved complete or partial hematological response to IFNa. CALR mutations (present in all patients, quantifiable in all but 1 because of 1bp difference between mutated and WT) were of type 1 in 10 (42%), type 2 in 8 (33%) and of neither type in 6 (25%) pts, respectively. In addition to CALR, a second mutation was found in 8 pts (33%) by NGS, affecting ASXL1 (n=2), TET2 (n=2), IDH2 (n=2), CSF3R (n=1) and SH2B3 (n=1) genes. Comparison of sequential samples showed that the %CALR decreased from a median of 43% (range: 8-57) to 26% (range: 0-49) in the last sample (p= 0.018). In details, %CALR decreased in 13 (57%) pts, including 1 CMR, 7 PMR and 5 mMR. Interestingly, molecular response to IFNa of CALR+clones appeared poorer in pts with additional mutations (vs. pts with CALR mutation alone): 50% were non responders (vs. 40%), %CALR even increased during follow up in 25% (vs. 7%), and the median decrease in %CALR was 32% (vs. 45%). Dynamics of clones harboring additional mutated genes showed that, in contrast to CALR, mutant allele burden did not vary in 6/8 pts. However, an IDH2 mutation decreased from 9% to 1% in 1 pt, and a TET2 mutation increased from 1% to 21% in another (while %CALR remained unchanged in both pts, at 44% and 19% respectively). Of note, after 12 years of median follow-up, no emergence of new mutation was observed in any patient. Conclusion: In this cohort of JAK2V617F-negative ET, 96% of pts achieved hematological response with IFNa therapy. However, molecular profiling suggests that the mutational pattern found in malignant clones modulates the molecular response to IFNa: 1) Existence of more than 1 mutation induces poorer molecular response; 2) Clones with CALR mutation alone are sensitive to IFNa; 3) Clones harboring mutations in genes other than CALR seem less responsive to IFNa therapy. Disclosures Off Label Use: Interferon alpha was used off-label in selected ET patients according to local and international guidelines (Barbui et al., J Clin Oncol. 2011 Feb 20;29(6):761-70).

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Risinskaya, Natalya, Julia Chabaeva, Elena Nikulina, et al. "Loss of Heterozygosity and Possible Loss of Allele Amplification in Homozygous Short Tandem Repeats Loci in Two ALL Patients with Central Nervous System and Bone Marrow Relapses." Blood 132, Supplement 1 (2018): 5135. http://dx.doi.org/10.1182/blood-2018-99-114613.

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Abstract Introduction: Loss of heterozygosity (LOH) was described for many forms of cancer including leukemia. LOH may contribute to oncogenesis through the deletion of tumor suppressor genes due to appropriate allelic loss. LOH in short tandem repeats loci (STR) has been reported for solid tumors and is widely used as malignant cells population marker. It should be noted that little is known yet about possible loss of one of certain homozygous alleles in tumour cells and STR analysis might be a promising approach to investigate this phenomenon. STR analysis is used as a routine test for chimerism monitoring after bone marrow transplantation. It could not also be excluded that recipient's chimerism estimation may be distorted in some cases due to LOH issues. The aim: To investigate possible allele loss in homozygous loci in tumour cells and to assess the contribution of LOH to the error in mixed chimerism estimation. Patients and Methods: This study includes analysis of STR DNA-profiles of two ALL patients after allogeneic stem cells transplantation. Patient 1 was 54 years old female with B-ALL, she underwent allogenic BM transplantation but after it she had four central nervous system (CNS) relapses and three bone marrow relapses. Patient 2 was 23 years old male with preT-ALL, two CNS relapses: first before transplantation and second after it. The DNAs were isolated from bone marrow samples of patients in remission before transplantation, from donors blood samples, from cerebrospinal fluid during neuro relapse and from bone marrow samples. The presence of blast cells in bone marrow samples and cerebrospinal fluid was confirmed morphologically and cytogenetically. STR-profiles were assessed by polymerase chain reaction with COrDIS Plus multiplex kit for amplification of 19 polymorphic STR-markers and amelogenin loci (Gordiz Ltd, Russia). The fragment analysis was performed on ABI3130 Genetic Analyzer. The data processing was accomplished using GeneMapper v.4-0 software. Results: The STR profile analysis for Patient 1 showed complete recipient chimerism in cerebrospinal fluid with LOH in 10 from 15 heterozygous loci (Fig.1a,b) comparative to control sample of DNA (bone marrow before transplantation). The same LOH pattern was detected in archival DNA sample from first spinal relapse. Mixed chimerism in bone marrow samples estimated by STR analysis was equal to blast cell percentage assessed by cytogenetics. Loss of non-informative allele in D16S539 (Fig.1b) didn't distort chimerism estimation. However decreased levels of mixed chimerism were calculated in two homozygous loci (Fig.1a,c). We suspected that this decrease could be caused by the loss of one allele amplification in homozygous locus. Therefore, we proposed the mathematical model f(x), where x - is donor percentage in chimera, f(x) - difference between real recipient percentage (100-x) and calculated for locus with loss of allele amplification ((100-x)/2:((100-x)/2+x))*100% (Fig.1c) to test this hypothesis. The highest difference in chimerism calculation (about 17%) is expected in about 40% of donor and 60% recipient percentage. For Patient 2 the STR analysis detected only one LOH locus in DNA samples from cerebrospinal fluid during CNS relapse before and after BMT. However this patient has eight homozygous STR loci and complete donor chimerism in bone marrow. To find possible "loss of allele amplification" we used the artificial chimera, the mix of Patient 2 and unrelated random healthy donor DNA (Fig.1d). We used control DNA of Patient 2 (bone marrow before BMT) to check chimerism level deviations in loci of interest. The decreased level of mixed chimerism was detected in two homozygous loci from cerebrospinal fluid DNA-sample. The decreased estimate for chimerism correlated with proposed math model. For the same STR loci in control DNA no chimerism deviations were detected in mixed artificial chimera (Fig.1e). Conclusions: The LOH loci pattern might be preserved during long period of disease for certain malignant clone. Mixed chimerism or artificial mixed chimera can be useful in finding of homozygous loci with loss of one allele amplification. We suppose homozygous loci are the preferable subject for investigating LOH mechanisms. Loci with "loss of allele amplification" might be the used as malignant cells population marker. This study was supported by the Russian Foundation for Basic Research (RFBR) grant 18-015-00399 a . Disclosures Risinskaya: Russian Foundation for Basic Research grant 18-015-00399 A: Research Funding. Chabaeva:Russian Foundation for Basic Research grant 18-015-00399 A: Research Funding. Kulikov:Russian Foundation for Basic Research grant 18-015-00399 A: Research Funding.

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Journal articles on the topic 'GeneMapper'

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