Molecular characterization of patients with colorectal and endometrial cancer andscreening of Lynch syndrome

El Cáncer Colorrectal (CCR) es la segunda causa de muerte por cáncer en Argentina, con una incidencia estimada de más de 13.000 nuevos casos por año. La patogénesis del CCR es compleja y diversa y está influenciada por múltiples factores, algunos de los cuales se encuentran relacionados a la dieta y estilo de vida y otros se relacionan a la predisposición genética.Entre el 3 y el 8% de los casos son producidos por mutaciones heredables en genes puntuales y constituyen los distintos síndromes de cáncer colorrectal hereditario. El más común de ellos es el Síndrome de Lynch o Cáncer Colorrectal Hereditario no Polipósico (CCHNP). Su patogénesis se relaciona con fallas en el sistema de reparación del ADN o MMR. Las alteraciones en MMR pueden deberse a mutaciones o silenciamiento por metilación de estos genes y llevan a la acumulación de mutaciones de nucleótido único y cambios en la longitud de secuencias repetitivas, fenómeno conocido como Inestabilidad de Microsatélites (MSI). La evaluación de la presencia o ausencia de MSI, estado de metilación y detección de deleciones o duplicaciones en los genes MMR en muestras tumorales es útil en la diferenciación de CCR esporádico de casos hereditarios. El objetivo del proyecto es evaluar las características moleculares de pacientes afectados de CCR o endometrio con sospecha clínica de Síndrome de Lynch. Para ello se pondrá en marcha la técnica molecular de MS-MLPA para genes de reparación del ADN (MMR) y se analizarán muestras tumorales con Inestabilidad de microsatélites (MSI) mediante las técnicas de MS-MLPA para genes de reparación del ADN. Los resultados de los estudios moleculares serán correlacionados con antecedentes clínicos y estudios anatomopatológicos y de inmunohistoquímica de pacientes con sospecha clínica de Síndrome de Lynch. Con esta información se protocolizará y generarán pautas diagnósticas para mejorar el tratamiento y asesoramiento genético de los pacientes con Síndrome de Lynch segun los análisis disponibles en Mendoza. Se incluirán al menos 40 pacientes con diagnóstico de cáncer de colon o endometrio y serán evaluados para MSI y análisis de perfil de metilación y deleciones/duplicaciones en genes de reparación del ADN. Los resultados permitirán realizar la pesquisa de Sindrome de Lynch y colaborar con el asesoramiento genético del paciente y su familia.Colorectal cancer (CRC) is the second cause of death from cancer in Argentina, with an estimated incidence of more than 13,000 new cases per year.The pathogenesis of CRC is complex and diverse and is influenced by multiple factors, some of which are related to diet and lifestyle and others are related to genetic predisposition.3 and 8% of the cases are produced by heritable mutations in point genes and constitute the different syndromes of hereditary colorectal cancer. The most common of these is Lynch Syndrome or Non-Polyposis Hereditary Colorectal Cancer (CCHNP). Its pathogenesis is related to failures in the DNA mismatch repair system (MMR).Alterations in MMR may be due to mutations or silencing by methylation of these genes and lead to the accumulation of single nucleotide mutations and changes in the length of repetitive sequences, a phenomenon known as Microsatellite Instability (MSI).The evaluation of the presence or absence of MSI, methylation status and detection of deletions or duplications in MMR genes in tumor samples is useful in the differentiation of sporadic CCR of hereditary cases.The objective of the project is to evaluate the molecular characteristics of patients affected by CRC or endometrium cancer with clinical suspicion of Lynch Syndrome. To this , the MS-MLPA molecular technique for DNA repair genes (MMR) will be implemented and tumor samples with microsatellite instability (MSI) will be analyzed by MS-MLPA techniques for DNA repair genes. The results of the molecular studies will be correlated with clinical antecedents and anatomopathological and immunohistochemical studies of patients with clinical suspicion of Lynch Syndrome. With this information, protocols will be generated to improve the treatment and genetic counseling of patients with Lynch Syndrome according to the analyzes available in Mendoza.At least 40 patients diagnosed with colon or endometrial cancer will be included and evaluated for MSI and methylation profile analysis and deletions / duplications in DNA repair genes. The results will allow us to carry out the Lynch Syndrome research and collaborate with the genetic counseling of the patient and his family

A global analysis of Y-chromosomal haplotype diversity for 23 STR loci

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent.Fil: Corach, Daniel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Caputo, Mariela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marino, Miguel Eduardo. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Laboratorio de Analisis de ADN; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Purps, Josephine. Charité-Universitätsmedizin; AlemaniaFil: Siegert, Sabine. University of Cologne; AlemaniaFil: Willuweit, Sascha. Charité-Universitätsmedizin; AlemaniaFil: Nagy, Marion. Charité-Universitätsmedizin; AlemaniaFil: Alves, Cíntia. Universidad de Porto; PortugalFil: Salazar, Renato. Universidad de Porto; PortugalFil: Angustia, Sheila M. T.. Philippine National Police Crime Laboratory; FilipinasFil: Santos, Lorna H.. Philippine National Police Crime Laboratory; FilipinasFil: Anslinger, Katja. Universitat Genzentrum Der Ludwing-maximilians; AlemaniaFil: Bayer, Birgit. Universitat Genzentrum Der Ludwing-maximilians; AlemaniaFil: Ayub, Qasim. The Wellcome Trust Sanger Institute; Reino UnidoFil: Wei, Wei. The Wellcome Trust Sanger Institute; Reino UnidoFil: Xue, Yali. The Wellcome Trust Sanger Institute; Reino UnidoFil: Tyler Smith, Chris. The Wellcome Trust Sanger Institute; Reino UnidoFil: Baeta Bafalluy, Miriam. Universidad de Zaragoza; EspañaFil: Martínez Jarreta, Begoña. Universidad de Zaragoza; EspañaFil: Egyed, Balazs. Eotvos University, Budapest; ArgentinaFil: Balitzki, Beate. Universidad de Basilea; SuizaFil: Tschumi, Sibylle. Universidad de Basilea; SuizaFil: Ballard, David. King; Reino UnidoFil: Syndercombe Court, Denise. King; Reino UnidoFil: Barrantes, Xinia. Poder Judicial, Forensic Sciences Department; Costa RicaFil: Bäßler, Gerhard. Landeskriminalamt Baden-Württemberg; AlemaniaFil: Berger, Burkhard. Universidad de Innsbruck; AustriaFil: Niederstätter, Haral. Universidad de Innsbruck; AustriaFil: Parson, Walther. Universidad de Innsbruck; Austria. University Park; Estados UnidosFil: Davis, Carey. Department of Molecular and Medical Genetics; Estados Unidos. Institute of Applied Genetics; Estados UnidosFil: Furfuro, Sandra. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Laboratorio de Análisis de ADN; ArgentinaFil: Locarno, Laura. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Laboratorio de Análisis de ADN; Argentin

A global analysis of Y-chromosomal haplotype diversity for 23 STR loci

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent.Peer reviewe

Species identification in routine casework samples using the SPInDel kit

The identification of species in casework samples is of fundamental importance for forensic investigations. Laboratories are increasingly compelled to provide accurate and fast identifications in trace materials left on crime scenes, wildlife poaching, illegal trade of protected species, fraudulent food products cases, etc. However, the field of nonhuman forensic genetics is still working on the standardization of typing methods and practices. Here we describe the successful implementation of the Species Identification by Insertions/Deletions (SPInDel) method in routine casework analyses in 11 laboratories worldwide. The SPInDel was developed to detect human DNA, at the same time that identifies common animal species. The fragment size analysis of six mtDNA regions allows identification in suboptimal DNA samples, including mixtures, with no need for sequencing. The samples were collected from 2013 to 2018 and included hair, blood, meat, saliva, faeces, bones, etc. The SPInDel kit successfully identified >95% of the samples, being dog, human and pig the most frequently detected species. The six SPInDel loci were successfully amplified in mixtures and degraded samples (river water, sand, stains in clothes, etc.). Interestingly, several species that were not originally targeted by SPInDel primers were also identified (e.g., red fox, brown bear, fallow deer and red deer). In conclusion, the SPInDel kit was successfully used in crime scene investigations (often involving human DNA detection) and in cases of poaching, environmental contamination and food fraud. It is now becoming a useful tool for the routine analysis of nonhuman DNA samples within the high quality standards of forensic genetics.Fil: Pereira, Filipe. Universidad de Porto; PortugalFil: Alves, Cíntia. Universidad de Porto; PortugalFil: Couto, Cátia. Universidad de Porto; PortugalFil: López Díaz, Lourdes. Servicio de Criminalística de la Guardia Civil; EspañaFil: Parra, David. Servicio de Criminalística de la Guardia Civil; EspañaFil: Furfuro, Sandra. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Laboratorio de Análisis de ADN; ArgentinaFil: Aler, Mercedes. Instituto de Medicina Legal y Ciencias Forenses; EspañaFil: Burillo Borrego, Luís. Instituto de Medicina Legal y Ciencias Forenses de Valencia; EspañaFil: Olekšáková, Tereza. Institute of Criminalistics; República ChecaFil: Balsa, Filipa. Instituto Nacional de Medicina Legal e Ciências Forenses; PortugalFil: Sampaio, Lisa. Instituto Nacional de Medicina Legal e Ciências Forenses; PortugalFil: Porto, Maria João Anjos. Instituto Nacional de Medicina Legal e Ciências Forenses; PortugalFil: Costa, Heloisa Afonso. Serviço de Genética e Biologia Forenses; PortugalFil: Arévalo Voss, Cristina. Cuerpo Nacional de Policia; EspañaFil: Caputo, Mariela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; ArgentinaFil: Corach, Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; ArgentinaFil: García, Óscar. Basque Country Police-Ertzaintza; EspañaFil: Pedrosa Moro, Susana. Unidad de Laboratorio de Navarra de Servicios y Tecnologías; EspañaFil: Pereira, Rui. Universidad de Porto; PortugalFil: Amorim, António. Universidad de Porto; Portuga

Species identification in forensic samples using the SPInDel approach: A GHEP-ISFG inter-laboratory collaborative exercise

DNA is a powerful tool available for forensic investigations requiring identification of species. However, it is necessary to develop and validate methods able to produce results in degraded and or low quality DNA samples with the high standards obligatory in forensic research. Here, we describe a voluntary collaborative exercise to test the recently developed Species Identification by Insertions/Deletions (SPInDel) method. The SPInDel kit allows the identification of species by the generation of numeric profiles combining the lengths of six mitochondrial ribosomal RNA (rRNA) gene regions amplified in a single reaction followed by capillary electrophoresis. The exercise was organized during 2014 by a Working Commission of the Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG), created in 2013. The 24 participating laboratories from 10 countries were asked to identify the species in 11 DNA samples from previous GHEP-ISFG proficiency tests using a SPInDel primer mix and control samples of the 10 target species. A computer software was also provided to the participants to assist the analyses of the results. All samples were correctly identified by 22 of the 24 laboratories, including samples with low amounts of DNA (hair shafts) and mixtures of saliva and blood. Correct species identifications were obtained in 238 of the 241 (98.8%) reported SPInDel profiles. Two laboratories were responsible for the three cases of misclassifications. The SPInDel was efficient in the identification of species in mixtures considering that only a single laboratory failed to detect a mixture in one sample. This result suggests that SPInDel is a valid method for mixture analyses without the need for DNA sequencing, with the advantage of identifying more than one species in a single reaction. The low frequency of wrong (5.0%) and missing (2.1%) alleles did not interfere with the correct species identification, which demonstrated the advantage of using a method based on the analysis of multiple loci. Overall, the SPInDel method was easily implemented by laboratories using different genotyping platforms, the interpretation of results was straightforward and the SPInDel software was used without any problems. The results of this collaborative exercise indicate that the SPInDel method can be applied successfully in forensic casework investigations.Fil: Alves, Cíntia. Universidad de Porto; PortugalFil: Pereira, Rui. Universidad de Porto; PortugalFil: Prieto, Lourdes. Universidad de Santiago de Compostela; EspañaFil: Aler, Mercedes. Instituto de Medicina Legal y Ciencias Forenses de Valencia; EspañaFil: Amaral, Cesar R. L.. Universidade do Estado do Rio de Janeiro; BrasilFil: Arévalo, Cristina. Universidad de Alcalá; EspañaFil: Berardi, Gabriela. Fundación Favaloro; ArgentinaFil: Di Rocco, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Caputo, Mariela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Carmona, Cristian Hernandez. Poder Judicial. Departamento de Ciencias Forenses. Sección de Bioquímica; Costa RicaFil: Catelli, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Equipo Argentino de Antropología Forense; ArgentinaFil: Costa, Heloísa Afonso. Instituto Nacional de Medicina Legal e Ciências Forenses; PortugalFil: Coufalova, Pavla. Institute of Criminalistics Prague; República ChecaFil: Furfuro, Sandra Beatriz. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Laboratorio de Análisis de ADN; ArgentinaFil: García, Óscar. Polícia del País Vasco. Sección de Genética Forense; EspañaFil: Gaviria, Anibal. Cruz Roja Ecuatoriana; EcuadorFil: Goios, Ana. Universidad de Porto; PortugalFil: Gómez, Juan José Builes. Universidad de Antioquia; ColombiaFil: Hernández, Alexis. Instituto Nacional de Toxicología y Ciencias Forenses; EspañaFil: Betancor Hernández, Eva del Carmen. Instituto de Medicina Legal de Las Palmas. Laboratorio Genética Forense; EspañaFil: Miranda, Luís. Universidade de Aveiro; PortugalFil: Parra, David. Servicio de Criminalística de la Guardia Civil. Departamento de Química y Medio Ambiente; EspañaFil: Pedrosa, Susana. Unidad de Laboratorio de Navarra de Servicios y Tecnologías; EspañaFil: Porto, Maria João Anjos. Instituto Nacional de Medicina Legal e Ciências Forenses; PortugalFil: Rebelo, Maria de Lurdes. Instituto Nacional de Medicina Legal e Ciências Forenses; PortugalFil: Spirito, Matteo. Università Cattolica del Sacro Cuore; ItaliaFil: Torres, María del Carmen Villalobos. Universidad Autónoma de Nuevo León; MéxicoFil: Amorim, António. Universidad de Porto; PortugalFil: Pereira, Filipe. Universidad de Porto; Portuga