RESUMEN
BACKGROUND: Hymenoptera venom allergy (HVA) is of great concern because of the possibility of anaphylaxis, which may be fatal. Venom immunotherapy (VIT) is the only disease-modifying treatment in HVA and, although efficient, its mechanism remains partially unknown. Gene expression analysis may be helpful for establishing a proper model of tolerance induction during the build-up phase of VIT. The present study aimed to analyze how the start of VIT changes the expression of 15 selected genes. METHODS: Forty-five patients starting VIT with a wasp venom allergy were enrolled. The diagnosis was established based on anaphylaxis history (third or fourth grade on the Mueller scale) and positive soluble immunoglobulin E and/or skin tests. Two blood collections were performed in the patient group: before and after 3 months of VIT. One sample was taken in the control group. Gene expression analysis was performed using a reverse transcriptase-polymerase chain reaction with microfluidic cards and normalized to the 18S housekeeping gene. RESULTS: Commd8 was the only gene that changed expression significantly after the start of VIT (p = 0.012). Its expression decreased towards the levels observed in the healthy controls. Twelve out of 15 genes (commd8, cldn1, cngb3, fads1, hes6, hla-drb5, htr3b, prlr, slc16a4, snx33, socs3 and twist2) revealed a significantly different expression compared to the healthy controls. CONCLUSIONS: The present study shows that commd8 changes significantly its expression during initial phase of VIT. This gene might be a candidate for VIT biomarker in future studies.
Asunto(s)
Biomarcadores/metabolismo , Desensibilización Inmunológica/métodos , Himenópteros/inmunología , Hipersensibilidad Inmediata/terapia , Mordeduras y Picaduras de Insectos/terapia , Venenos de Avispas/uso terapéutico , Avispas/inmunología , Adolescente , Adulto , Anciano , Animales , Estudios de Casos y Controles , delta-5 Desaturasa de Ácido Graso , Femenino , Perfilación de la Expresión Génica , Humanos , Himenópteros/patogenicidad , Hipersensibilidad Inmediata/etiología , Hipersensibilidad Inmediata/metabolismo , Inmunoglobulina E/inmunología , Mordeduras y Picaduras de Insectos/complicaciones , Mordeduras y Picaduras de Insectos/metabolismo , Masculino , Persona de Mediana Edad , Pronóstico , Pruebas Cutáneas , Adulto JovenRESUMEN
MicroRNAs are small non-coding molecules playing a significant regulatory role in several allergic diseases. However their role in tolerance induction remains unclear. The aim of this study was to determine the expression of selected microRNAs during the first three months of wasp venom immunotherapy (VIT). 5 adult patients with a history of severe systemic reactions after stinging by wasps and confirmed sensitization were included. Venous blood samples were collected before VIT, 24 hours after completing its initial phase and after 3 months of the maintenance therapy. A control group was comprised of 5 healthy individuals with no history of allergy. In the blood samples expression of 96 microRNAs was determined with the use of microfluidic cards. In a statistical analysis the expression was compared between the study groups as well as between the pre- and post-VIT samples. Significant differences were found between the patients with wasp venom allergy and the healthy controls in the expression of miR-601 and miR-1201 upregulated in allergic patients at every time point (p = 0.04; p = 0.015, respectively). During VIT profile of microRNA was changing with lower expression of 6 microRNAs (including miR-182, miR-342, miR-375) and higher of 11 microRNAs (including let-7d, miR-34b, miR-143). To conclude, VIT has led to some changes in the expression of microRNA associated with Th2-type inflammation and tolerance induction.
Asunto(s)
Mordeduras y Picaduras/inmunología , Expresión Génica/inmunología , Inmunoterapia/métodos , MicroARNs/genética , Venenos de Avispas/inmunología , Avispas/inmunología , Adulto , Animales , Desensibilización Inmunológica/métodos , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Masculino , Persona de Mediana Edad , Factores de Tiempo , Venenos de Avispas/administración & dosificación , Adulto JovenRESUMEN
BACKGROUND: Allergen-specific immunotherapy is the most effective method of treatment in allergy to wasp venom. However, its mechanism of action is still not fully understood. The aim of this study is to describe changes in microRNA (miRNA) expression in patients undergoing the buildup phase of venom immunotherapy. METHODS: The study group comprised 7 adult patients with a history of severe systemic reactions after stinging by a wasp. In all patients, sensitization to wasp venom had been confirmed by skin tests and serum IgE. The buildup phase of wasp venom immunotherapy (VIT) was conducted according to an ultrarush protocol. In blood samples collected before and 24 h after completing the VIT buildup phase, 740 miRNAs were assessed. RESULTS: Of the 740 miRNAs, 440 were detected in the study group, and in 5 expression was significantly changed after the buildup phase of VIT: miR-370, miR-539, miR-502-3p, miR-299, and miR-29c. Another 62 miRNAs changed 2-fold in some patients (nonsignificant), including increases in miR-143 (stimulating FOXp3 expression) and let-7d (reducing expression of IL-13, IL-6, and TLR4), and decreases in proinflammatory miR-301, miR-146b, miR-106, and miR-485. CONCLUSIONS: Several changes in miRNA expression have been found as a result of the buildup phase of wasp VIT, with lower expression of some miRNAs involved in allergic inflammation and higher expression of those possibly involved in tolerance induction. However, the role of the most significant changes is uncertain.
Asunto(s)
Desensibilización Inmunológica , Regulación de la Expresión Génica , Mordeduras y Picaduras de Insectos/genética , Mordeduras y Picaduras de Insectos/terapia , MicroARNs/genética , Venenos de Avispas/administración & dosificación , Avispas , Adulto , Anciano , Animales , Femenino , Perfilación de la Expresión Génica , Humanos , Tolerancia Inmunológica , Masculino , Persona de Mediana Edad , Proyectos PilotoRESUMEN
Degraded and low template DNA is often analyzed in forensic genetics laboratories. Reliable analysis of degraded and low template DNA is of great importance, because its results impact the quality and reliability of expert testimonies. Recently, a number of whole-genome amplification (WGA) methods have been proposed as preamplification tools improving quantity and quality of DNA. We chose, investigated, and compared 7 WGA methods to evaluate their ability to "recover" degraded and nondegraded DNA. These methods include degenerate oligonucleotide primed polymerase chain reaction, primer extension preamplification (PEP) polymerase chain reaction, GenomePlex WGA (Sigma), multiple displacement amplification, GenomiPhi Amplification Kit (Amersham Biosciences), restriction and circularization aided rolling circle amplification, and blunt-end ligation-mediated WGA. Recently, we have described the comparison of these methods' efficiency and reliability using SGMPlus kit. However, Y-chromosome profiling is also often used in analysis of both nondegraded and degraded DNA. This includes criminal cases and investigation of kinship in male linage. Here we demonstrate the impact of WGA methods on Y-chromosome loci (Yfiler) reactivation.The best results for nondegraded DNA were obtained with GenomiPhi kit and PEP method. In the case of degraded DNA (200 base pairs), the most complete profiles were obtained with GenomePlex kit and PEP method. None of the analyzed methods allowed full reactivation of degraded (200 base pairs) DNA in terms of Y-chromosome loci profiling.
Asunto(s)
Cromosomas Humanos Y , Genoma Humano , Técnicas de Amplificación de Ácido Nucleico/métodos , ADN/aislamiento & purificación , Degradación Necrótica del ADN , Dermatoglifia del ADN/métodos , Sitios Genéticos , Humanos , Masculino , Músculo Esquelético/químicaRESUMEN
The purpose of this paper is to formulate recommendations for the disclosure of biological traces in the laboratory and the handling of forensic evidence submitted for identification tests, recommended by the Polish Speaking Working Group of the International Society for Forensic Genetics. The paper organizes the knowledge of the most relevant stages of preliminary analysis of biological traces based on both literature sources and those resulting from years of research practice. Recommendations formulated in the course of multi-stage expert consultations contained in this study should be used in the development of laboratory procedures applied during the execution.
Asunto(s)
Genética Forense , Humanos , Polonia , Genética Forense/normas , Genética Forense/métodos , Genética Forense/legislación & jurisprudencia , Sociedades Científicas/normas , Dermatoglifia del ADN/normas , Revelación/normas , Revelación/legislación & jurisprudenciaRESUMEN
Degraded DNA is often analyzed in forensic genetics laboratories. Reliable analysis of degraded DNA is of great importance, since its results impact the quality and reliability of expert testimonies. Recently, a number of whole genome amplification (WGA) methods have been proposed as preamplification tools. They work on the premise of being able to generate microgram quantities of DNA from as little as the quantity of DNA from a single cell. We chose, investigated, and compared seven WGA methods to evaluate their ability to "recover" degraded and nondegraded DNA: degenerate oligonucleotide-primed PCR, primer extension preamplification PCR, GenomePlex™ WGA commercial kit (Sigma), multiple displacement amplification, GenomiPhi™ Amplification kit (Amersham Biosciences), restriction and circularization-aided rolling circle amplification, and blunt-end ligation-mediated WGA. The efficiency and reliability of those methods were analyzed and compared using SGMPlus, YFiler, mtDNA, and Y-chromosome SNP typing. The best results for nondegraded DNA were obtained with GenomiPhi and PEP methods. In the case of degraded DNA (200 bp), the best results were obtained with GenomePlex which successfully amplified also severely degraded DNA (100 bp), thus enabling correct typing of mtDNA and Y-SNP loci. WGA may be very useful in analysis of low copy number DNA or degraded DNA in forensic genetics, especially after introduction of some improvements (sample pooling and replicate DNA typing).
Asunto(s)
Degradación Necrótica del ADN , Genoma Humano , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Alelos , Cromosomas Humanos Y , Dermatoglifia del ADN , Cartilla de ADN , ADN Mitocondrial/genética , Genética Forense , Humanos , Masculino , Repeticiones de Microsatélite , Polimorfismo de Nucleótido Simple , Reproducibilidad de los Resultados , Análisis de Secuencia de ADNRESUMEN
There is a necessity for deceased identification as a result of many accidents and sometimes bones are the only accessible source of DNA. So far, a universal method that allows for extraction of DNA from materials at different stages of degradation does not exist. The aims of this study were: the comparison of three methods of DNA extraction from bones with different degree of degradation and an evaluation of the usefulness of these methods in forensic genetics. The efficiency of DNA extraction, the degree of extract contamination by polymerase chain reaction (PCR) inhibitors and the possibility of determining the STR loci profile were especially being compared. Nuclear DNA from bones at different states of degradation was isolated using three methods: classical, organic phenol-chloroform extraction, DNA extraction from crystal aggregates and extraction by total demineralisation. Total demineralisation is the best method for most cases of DNA extraction from bones, although it does not provide pure DNA. DNA extraction from aggregates removes inhibitors much better and is also a good method of choice when identity determination of exhumed remains is necessary. In the case of not buried bones (remains found outside) total demineralisation or phenol-chloroform protocols are more efficient for successful DNA extraction.
Asunto(s)
Huesos/química , ADN/aislamiento & purificación , Antropología Forense/métodos , Genética Forense/métodos , Técnica de Descalcificación , Femenino , Humanos , Masculino , Reacción en Cadena de la Polimerasa Multiplex , Reacción en Cadena de la Polimerasa , Factores de TiempoRESUMEN
Background: Subcutaneous immunotherapy (SCIT) is widely used in the treatment of allergic rhinitis (AR). This study aimed to determine the expression of 48 miRNAs in patients with AR undergoing grass pollen SCIT and investigate relations with clinical outcomes. Methodology: Expression of selected miRNAs was determined using RT-PCR in the full blood of 16 patients with AR and seven healthy controls. Results: miR-136, miR-208 and miR-190 were upregulated in the AR group. After 6 months of SCIT, significant downregulation of some proinflammatory miRNAs and upregulation of several miRNAs regulating Th1/Th2 balance were found. No differences were found between good and poor responders. Conclusion: miRNAs may play a regulatory role in SCIT, leading to tolerance induction.
Background: Subcutaneous immunotherapy is widely used in the treatment of allergic rhinitis (AR). MicroRNAs (miRNAs) are small molecules controlling gene expression. Their role in the process of immunotherapy is not yet well understood. This study aimed to investigate the expression of 48 miRNAs in patients with AR undergoing grass pollen immunotherapy and relations between miRNAs and clinical outcomes. Methodology: The expression of selected miRNAs was determined in the blood of 16 patients with AR and seven healthy people. Results: Three miRNAs were found to be overproduced in allergic patients. During immunotherapy, the production of several proinflammatory miRNAs was reduced while those responsible for allergen tolerance were produced in larger amounts. Conclusion: miRNAs may play an important role in immunotherapy, leading to better tolerance of allergens.
Asunto(s)
MicroARNs , Rinitis Alérgica Estacional , Rinitis Alérgica , Inmunoterapia Sublingual , Alérgenos/genética , Alérgenos/uso terapéutico , Desensibilización Inmunológica , Humanos , Factores Inmunológicos/uso terapéutico , Inyecciones Subcutáneas , MicroARNs/genética , MicroARNs/uso terapéutico , Poaceae/genética , Polen/genética , Rinitis Alérgica/genética , Rinitis Alérgica/terapia , Rinitis Alérgica Estacional/terapiaRESUMEN
Numeric sex chromosome abnormalities are commonly associated with an increased cancer risk. Here, we report a 14-year-old boy with a rare mosaic 45, X/48, XYYY karyotype presenting with subtle dysmorphic features and relative height deficiency, requiring growth hormone therapy. As only 12 postnatal cases have been described so far with very limited follow-up data, to assess the proband's long-term prognosis, including cancer risk, we performed high-throughput single-cell RNA sequencing (scRNA-seq) analysis. Although comprehensive cytogenetic analysis showed seemingly near perfect balance between 45, X and 48, XYYY cell populations, scRNA-seq revealed widespread differences in genotype distribution among immune cell fractions, specifically in monocytes, B- and T-cells. These results were confirmed at DNA level by digital-droplet PCR on flow-sorted immune cell types. Furthermore, deregulation of predominantly autosomal genes was observed, including TCL1A overexpression in 45, X B-lymphocytes and other known genes associated with hematological malignancies. Together with the standard hematological results, showing increased fractions of monocytes and CD4+/CD8+T lymphocytes ratio, long-term personalized hemato-oncological surveillance was recommended in the reported patient.
Asunto(s)
Neoplasias , Masculino , Humanos , Adolescente , Cariotipificación , Cariotipo , Medición de Riesgo , Análisis de Secuencia de ARNRESUMEN
The available literature on traces characterised by a suboptimal amount of DNA, as well as expert research practice, show the complex nature of LT-DNA traces: from their detection and collection, through genetic analysis, up to the interpretation of final results. The aims of this paper are to systematise the current state of knowledge on handling LT-DNA traces and develop examination guidelines, as recommended by the Polish Speaking Working Group of the International Society for Forensic Genetics (ISFG-PL). The proposed guidelines should be followed by all Polish laboratories conducting forensic genetic analyses for the purpose of judicial proceedings.
Asunto(s)
Dermatoglifia del ADN , Genética Forense , ADN/genética , Humanos , Laboratorios , PoloniaRESUMEN
Y chromosome typing has been performed in forensic genetic practice for more than 20 years. The latest recommendations of the DNA Commission of the International Society of Forensic Genetics (ISFG) concerning the application of Y-chromosomal markers in forensic genetics were published in 2006. The aim of this report is to recapitulate, systematise and supplement existing recommendations on the forensic analysis of polymorphism of the Y chromosome with standards already implemented in practice, new capabilities linked to the development of research techniques as well as current solutions used in statistical analysis. The recommendations have been adapted specifically to aspects related to the preparation of expert opinions in the field of forensic genetics in Poland. The Polish Speaking Working Group of the ISFG believes that the presented guidelines should become a standard implemented by all Polish laboratories performing Y chromosome typing for forensic purposes.
Asunto(s)
Cromosomas Humanos Y , Dermatoglifia del ADN/normas , Genética Forense/normas , Polimorfismo Genético , Mapeo Cromosómico/normas , Testimonio de Experto/normas , Guías como Asunto , Humanos , Polonia , Sociedades Científicas/normasRESUMEN
The trisomy of the 21 chromosomes is one of the most important chromosomal anomalies and is responsible for Down syndrome. It persists in Poland in 1/700 birth. The risk of childbirth with Down syndrome grows with age, especially in women after 35 years old. In this study we performed QF-PCR method with specific polymorphic sequence--D21S11 for detection of trisomy of the 21 chromosome. DNA for PCR was isolated from amniotic fluid, blood of healthy and with Down syndrome patients. PCR was performed with ProfilerPlus Kit, which permits for amplification of 9 STR loci (also D21S11) and gender marker--amelogenin gene. We detected incorrect amplification of STR sequence for D21S11 in one amniotic fluid, which was confirmed by kariotyping as trisomy of the 21 chromosome of fetus.
Asunto(s)
Aneuploidia , Síndrome de Down/diagnóstico , Diagnóstico Prenatal/métodos , Estudios de Casos y Controles , Femenino , Fluorescencia , Marcadores Genéticos , Humanos , Reacción en Cadena de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
Although mitochondrial DNA (mtDNA) testing has been used in forensic genetics only since the mid-1990s, forensic DNA laboratories have been recently increasing the range of mtDNA sequencing, employing new analytical approaches and methods of data analysis. Therefore, it seems fitting to gather and systematize existing recommendations in the field of mtDNA analysis for forensic purposes, and formulate a set of interpretative guidelines which are especially relevant in view of recent developments in the forensic casework. The starting point is the recommendations of the International Society for Forensic Genetics (ISFG) which, in the opinion of the Polish Speaking Working Group of the ISFG (ISFG- PL), should be followed by all Polish laboratories conducting forensic testing.
Asunto(s)
Dermatoglifia del ADN/normas , ADN Mitocondrial/genética , Genética Forense/normas , Análisis de Secuencia de ADN/normas , Genética Forense/métodos , Humanos , Polonia , Alineación de Secuencia/normas , Sociedades CientíficasRESUMEN
Establishing the cellular or tissue-type origin of human biological traces found at crimes scenes is forensically relevant, as it allows evaluating the crime relevance of such traces and enables reconstructing the sequence of crime events. Messenger RNA and micro RNA markers are useful for forensic tissue identification, but provide challenges for linking RNA-identified cell/tissue types with DNA-identified trace donors, especially in mixed traces. DNA methylation markers overcome this problem, but provide technical challenges due to the DNA treatment required by most analysis methods. Here we introduce a novel type of DNA markers for forensic tissue identification analysed without prior DNA treatment, namely copy number variation (CNV). Using genome-wide CNV screening followed-up by targeted qPCR confirmation, and using qPCR analysis of additional CNV-like candidate DNA markers, in samples of several individuals from all commonly encountered forensically-relevant tissue types, we identified DNA markers specific for blood and semen, respectively. Preliminary forensic validation testing demonstrates that the developed qPCR assays are highly sensitive - delivering positive results down to picogram level of input DNA, specific, and can cope well with degraded DNA, providing suitable prerequisites for forensic applications. Moreover, we exemplified that using the CNV qPCR products as input material for subsequent forensic STR analysis delivered full STR profiles, opening-up new avenues of using the same DNA aliquot for both forensic purposes, tissue and individual identification. Provided additional forensic validation studies, we envision the application of these novel DNA markers for forensic tissue identification in future forensic casework. Such CNV markers are particularly useful for tissue identification in old/cold cases, where aged/old DNA extracts are available that contain no RNA and are not suitable for DNA methylation analysis due to limited DNA quantity and quality.
Asunto(s)
Análisis Químico de la Sangre , Variaciones en el Número de Copia de ADN , Marcadores Genéticos , Semen/química , Moco del Cuello Uterino/química , Dermatoglifia del ADN , ADN Mitocondrial/genética , Femenino , Genética Forense/métodos , Humanos , Masculino , Repeticiones de Microsatélite , Reacción en Cadena de la Polimerasa , Saliva , Piel/químicaAsunto(s)
Dermatoglifia del ADN/métodos , Patologia Forense/métodos , Rol del Médico , Violación/diagnóstico , Manejo de Especímenes/métodos , ADN/genética , ADN/aislamiento & purificación , Medicina Legal/métodos , Examen Ginecologíco/métodos , Conocimientos, Actitudes y Práctica en Salud , Humanos , Patología Molecular/métodos , Polonia , Competencia Profesional , Rol ProfesionalAsunto(s)
Dermatoglifia del ADN/métodos , Frecuencia de los Genes/genética , Repeticiones de Microsatélite/genética , Polimorfismo Genético , Población Blanca/genética , Alelos , Manchas de Sangre , Femenino , Genes Dominantes/genética , Marcadores Genéticos/genética , Genética de Población , Humanos , Masculino , Polonia , Reacción en Cadena de la Polimerasa/métodos , Valores de ReferenciaRESUMEN
Recently mRNA profiling has been widely proposed as a universal tool for biological fluids identification. Here, we describe a test for vaginal fluid identification that combines detection of five markers: vaginal mRNAs and Lactobacilli in end-point PCR reaction. The test detects the following transcripts: HBD1 (Human beta-defensin 1), MUC4 (Mucin 4), MMP11 (Matrix metalloproteinase 11), housekeeping gene G6PDH (glucose 6-phosphate dehydrogenase), and the 16S-23S rRNA intergenic spacer regions of L. crispatus and L. gasseri/L. johnsonii. Simultaneous analysis of five vaginal markers and a housekeeping gene ensures high specificity and reliability in the detection of vaginal material, which could not be obtained using detection of a single marker.
Asunto(s)
Biomarcadores/metabolismo , Sangre , Líquidos Corporales/química , Menstruación , ARN Mensajero/genética , Análisis de Secuencia de ARN/métodos , Vagina , Femenino , Genética Forense , Humanos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reproducibilidad de los ResultadosRESUMEN
Mastocytosis is an uncommon disease classified as a myeloproliferative neoplasm, however, its symptoms are broad and place patients at crossroads between dermatology, hematology and allergology. Patients with mastocytosis often suffer from symptoms resulting from the activation and release of mediators from the mast cells, such as generalized itching, redness, headache, abdominal cramps, diarrhea, bone pain or arthritis, hypotension and shock. The possible severe, fatal or near fatal reactions caused by food hypersensitivity are reasons for the research focused on marker identification. The aim of the study was to analyse the gene expression differences in mastocytosis patients with and without food and drug hypersensitivity and insect venom allergy (IVA). A total of 57 Caucasian patients with mastocytosis were studied [median age 41.8; range 18-77 years; 15 (26.3 %) males and 42 (73.7 %) females]. Quantitative RT-PCRs of 11 genes plus ribosomal 18S RNA were run. Symptoms of food hypersensitivity were found in 12 patients (21 %), including 3 patients (13 %) with cutaneous mastocytosis (CM), and 9 (28 %) with indolent systemic mastocytosis (ISM). IVA was confirmed in 13 patients (22.8 %) including 6 patients (10.5 %) with CM, and 7 patients (12.3 %) with ISM. Drug hypersensitivity was diagnosed in 10 patients (17.5 %). Significant differences in the gene expression were found for TRAF4 (p = 0.008) in the comparison of the mastocytosis patients with and without concomitant food hypersensitivity. Furthermore significant differences were found in gene expression for B3GAT1 (p = 0.003) in patients with IVA compared to patients without insect sting anaphylaxis in the medical history. The expression of studied genes did not differ according to the presence of drug hypersensitivity. The TRAF4 expression was higher in mastocytosis patients with food hypersensitivity in their medical history, the B3GAT1 expression was lower in mastocytosis patients with IVA in history.
Asunto(s)
Hipersensibilidad a los Alimentos , Glucuronosiltransferasa/metabolismo , Mordeduras y Picaduras de Insectos/inmunología , Mastocitosis Sistémica/inmunología , Mastocitosis/inmunología , Factor 4 Asociado a Receptor de TNF/metabolismo , Adolescente , Adulto , Anciano , Alérgenos , Anafilaxia/inmunología , Hipersensibilidad a las Drogas , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Mastocitosis/metabolismo , Mastocitosis Sistémica/metabolismo , Persona de Mediana Edad , Ponzoñas , Adulto JovenRESUMEN
The role of adjuvant chemotherapy in stage T2-T3N0 colon cancer (CC) is controversial and there are currently no reliable factors allowing for individual selection of patients with high risk of relapse for such therapy. We searched for microRNA-based signature with prognostic significance in this group. We assessed by qRT-PCR expression of 754 microRNAs (miRNAs) in tumour samples from 85 stage pT2-3N0 CC patients treated with surgery alone. MiRNA expression was compared between two groups of patients: 40 and 45 patients who did and did not develop distant metastases after resection, respectively. Additionally, miRNA expression was compared between CC and normal colon mucosa samples and between the mismatch repair (MMR) competent and deficient tumours. Low expression of miR-1300 and miR-939 was significantly correlated with shorter distant metastasis-free survival (DMFS) in Cox univariate analysis (p.adjusted = 0.049). The expression signature of five miRNAs (miR-1296, miR-135b, miR-539, miR-572 and miR-185) was found to be prognostic [p = 1.28E-07, HR 8.4 (95 % CI: 3.81-18.52)] for DMFS and cross-validated in a leave-one-out analysis, with the sensitivity and specificity of 74 and 78 %, respectively. The expression of miR-592 was significantly associated with the MMR status (p.adjusted <0.01). The expression of several novel miRNAs were found to be tumour specific, e.g. miR-888, miR-523, miR-18b, miR-302a, miR-423-5p, miR-582-3p (p < 0.05). We developed a miRNA expression signature that may be predictive for the risk of distant relapse in early stage CC and confirmed previously reported association between miR-592 expression and MMR status.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias del Colon/genética , MicroARNs/genética , Metástasis de la Neoplasia/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/biosíntesis , Neoplasias del Colon/patología , Reparación de la Incompatibilidad de ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , MicroARNs/biosíntesis , Persona de Mediana Edad , Metástasis de la Neoplasia/patología , Valor Predictivo de las Pruebas , PronósticoRESUMEN
INTRODUCTION: The identification of unknown victims of high temperatures (fire, terrorist attack, and other disasters) is one of the most difficult tasks faced by forensic geneticists. The main aim of this study was to investigate the availability of DNA isolated from various human tissue samples exposed to high temperatures of 1001000°C for 5 and 10 minutes. MATERIAL AND METHODS: Samples of varying thickness of thigh muscle, liver, heart, adipose tissue, bone, teeth, hair and nails of 52 fresh cadavers and 59 healthy teeth of 29 volunteers were used. The study was performed using the following commercially available STR (Short Tandem Repeats) and miniSTR kits: AmpFlSTR®SGM Plus® and AmpFlSTR®MiniFilerTM. Hyper variable region I (HVI) of human mitochondrial DNA (mtDNA) was sequenced with BigDye Terminator Cycle Sequencing Kit 1.1. The PEP (Primer-Extension Preamplification) method was used for the whole human genome amplification. RESULTS: It was possible to obtain complete DNA profiles (AmpFlSTR®SGM Plus®, AmpFlSTR®MiniFilerTM Applied Biosystems, USA and mtDNA HVI region) for tissue samples of heart, liver and thigh muscle, exposed up to 900°C for 5 min. However, under the applied conditions, limited usefulness of hair, nails and teeth for identification purposes was shown. CONCLUSIONS: DNA stability in tissues subjected to incineration depends on many factors, like tissue type and its thickness, temperature and time of exposure. In the cases of human remains exposed to high temperatures, samples of soft tissues of the highest weight (thickness) provide the best chance of successful identification through the genetic analysis. In some cases of negative results, even if using mtDNA typing, application of the whole genome amplification (WGA) technique could provide the expected results for highly degraded DNA templates.