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BACKGROUND: Human adenoviruses (HAdVs) are an important cause of acute respiratory tract infections, conjunctivitis, hemorrhagic cystitis, and gastroenteritis. In addition to enteric serotypes 40 and 41, some serotypes belonging to subgroups A, B, and C have also been implicated to be etiological agents of gastroenteritis among infants and young children. The Vesikari Scoring System (VSS) is the severity scale that was originally developed to evaluate the effectiveness and efficacy of rotavirus vaccines on 20 points. The aim of this study was to evaluate and compare the diagnostic value of the VSS with HAdVs genome quantification in fecal samples collected from hospitalized children with acute gastroenteritis. METHODS: A total of 137 fecal specimens (69 male and 68 female) were tested for HAdVs. The samples were collected from under-five-year-old children with acute gastroenteritis in pediatric Hospital Regina Margherita of Turin in Italy. RESULTS: A total of 69 out of 137 (50.3%) samples were associated with HAdV genomic detection with a mean viral load of 1.08×1011±9.02×1011 genomes/mg fecal specimens. The samples were grouped on the basis of Mild VSS and Moderate VSS and the HAdV viral load was calculated in the two groups. No statistical differences were observed between two groups (P=0.6123 calculated by Mann-Whitney Test). CONCLUSIONS: Our results did not show a difference in mean viral load between the group with mild VVS and moderate VVS.
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BACKGROUND: MicroRNAs (miRNAs) are a class of short length double strand genome encoded RNAs that are produced to repress post-transcriptionally the expression of cellular mRNAs. 2578 unique mature miRNAs are currently annotated in the human genome and participate in the regulation of multiple events, such as cellular proliferation or apoptosis. The over-expression of miR-155 of cellular origin might play a key role in the life cycle of EBV. In this study 24 pediatric patients undergoing HSCT seropositive and seronegative to EBV were enrolled. Thirty-one peripheral blood samples were collected from these patients. The mir-155 expression profile has been evaluated by a stem-loop Real Time PCR in all these conditions. METHODS: Of 24 patients, 4 were seronegative to EBV and EBV negative to PCR (Group I), 10 were seropositive to EBV and EBV negative to PCR (Group II) and 10 were seropositive to EBV and EBV positive to PCR (Group III). RESULTS: Based on relative quantification, the mir-155 expression was compared among the groups. The comparison between HSCT patients without EBV infection seronegative to EBV (Group I) showed higher levels of mir-155 expression than patients seropositive to EBV (P=0.1419). The mir-155 expression levels in seronegative to EBV were not significantly different compared with the patients seropositive to EBV (P=0.6504). The mir-155 expression levels in seropositive to EBV without and with EBV infection (positive viral load), were not significantly (P=0.7667). Also, when we evaluated the mir-155 expression levels comparing all EBV negative patients with an active EBV infection, we did not observe a statistically significant difference (P=0.9782). CONCLUSIONS: Our results are controversial, showing a higher production of mir-155 levels during EBV infection.
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Infecções por Vírus Epstein-Barr , Transplante de Células-Tronco Hematopoéticas , MicroRNAs , Humanos , Criança , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , MicroRNAs/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: HPyV12 was found in organs of the digestive tract, in particular the liver but also in colon, rectum and feces. Until now, the prevalence of HPyV12 is not well characterized. METHODS: In this study, we investigate the presence of this novel polyomavirus DNA in stool specimens collected from under-five-year-old children with gastroenteritis compared to healthy infants. A total of 190 fecal specimens previously screened for rotavirus (RV) and adenovirus (ADV) and 80 fecal samples from healthy infants, were tested for HPyV12 DNA using a home-made real time PCR. All fecal specimens were tested for the presence of HPyV12 with specific primers and probes. RESULTS: None of 190 (0%) episodes of acute gastroenteritis was associated with HPyV12. We did not detect HPyV12 DNA in any of 80 control subjects, as well. CONCLUSIONS: Our study represents a pilot study aiming to clarify the current epidemiological pattern in pediatric Italian patients regarding the novel and rare HPyV12. Based on our negative data and the recent observations reported in literature, doubts remain on human tropism of the HPyV12 and epidemiology: these issues need further investigations.
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Diarreia , Gastroenterite , Humanos , Lactente , Criança , Reação em Cadeia da Polimerase em Tempo Real , Projetos Piloto , Diarreia/diagnóstico , Diarreia/epidemiologia , Gastroenterite/epidemiologia , DNARESUMO
BACKGROUND: MXPyV, like MWPyV, was identified in stool samples from children suffering diarrhea in Mexico. In this study, we used a home-made real time PCR to investigate the presence of this novel viruses in stool specimen collected from under-five-year-old children with gastroenteritis. METHODS: A total of 192 fecal specimens previously screened for RV, ADV, NoV, HPeV and SaV, were tested for MWPyV with Taqman real time PCR. RESULTS: The most detected virus was NoV GII (33.8%), followed by RV (21.3%), SaV (10.9%), HPeV (8%), NoV GI (6.7%) and Adv (1%). Real time PCR detected MWPyV in 1/192 (0.5%) patients. CONCLUSIONS: We detected MWPyV in 0.5% of fecal specimens collected from pediatric patients suffering gastroenteritis which is smaller than the previously reported in literature (4.4% in Australia and 12% Mexico).
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Diarreia Infantil , Gastroenterite , Polyomavirus , Vírus , Humanos , Criança , Lactente , Diarreia , Gastroenterite/epidemiologia , Itália/epidemiologiaRESUMO
BACKGROUND: Gastroenteritis is a common disease in children, characterized by diarrhea, vomiting, abdominal pain, and fever. Human Cosavirus (HCoSV) and Saffold virus (SAFV) both have a worldwide distribution. Both viruses have been detected in the stools of patients with acute gastroenteritis in several countries. METHODS: In order to provide more insights into the epidemiology of enteric viruses that are not included usually in routine diagnostic tests, cases of childhood sporadic gastroenteritis of unknown etiology requiring hospital admission in Turin, Italy, during December 2014 to November 2015, were screened for HCoSV and SAFV. RESULTS: A total of 1 out of 164 (0.6%) episodes of acute gastroenteritis were associated with SAFV genomic detection. Among the 1 SAFV-positive cases, 1 were also positive for Adenovirus. The patient positive for SAFV do not present diarrheal episodes but vomiting. HCoSV was not detected in any of the samples. CONCLUSIONS: In conclusion, this study presents the current epidemiological data regarding the two viruses, HCoSV and SAFV, circulating in pediatric patients admitted to hospital with acute gastroenteritis in Turin, Italy.
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Gastroenterite , Picornaviridae , Vírus , Humanos , Criança , Pré-Escolar , Prevalência , Picornaviridae/genética , Gastroenterite/epidemiologia , Diarreia/epidemiologia , Itália/epidemiologia , Vômito/epidemiologia , Vômito/etiologiaRESUMO
INTRODUCTION: Most intractable diarrheas are treated with antibiotics, irrespective of the causative agent. This study aims to quantified Rotavirus with taqman real-time PCR in fecal samples of children with acute gastroenteritis in accordance with the program of reduction of drug-resistance and use of antibiotics. METHODS: A total of 190 fecal specimens were collected from under-five-year-old children with acute gastroenteritis in pediatric Hospital Regina Margherita of Turin in Italy in 2017. A total of 38 out of 67 (56.7%) episodes of acute gastroenteritis were associated with Rotavirus genomic detection with multiplex commercial kit. RESULTS: All fecal specimens were tested for the presence of RV and other GE viruses. The most commonly detected virus was norovirus (41%), astrovirus (15.8%), human bocavirus (8.9%), sapovirus (7.9%), human parechovirus (5.8%), rhinovirus (4.2%), and adenovirus (1%). In 66 out of 190 (34.7%) Rotavirus were detectable with the median viral load 7.2E+11±60E+11genomes/mg fecal specimens. DISCUSSION/CONCLUSIONS: Our results showed that RV was present in around 34.7% of children hospitalized for AGE, a rate similar to those reported in previous studies conducted elsewhere which were in the range of 33-75%. Our protocol are able to quantify the absolute number of viral particle/mg of feces. The clinical utility of quantitative molecular assays, such as Rotavirus viral load, will be markedly improved.
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Human endogenous retroviruses (HERVs) are relics of ancestral infections and represent 8% of the human genome. They are no longer infectious, but their activation has been associated with several disorders, including neuropsychiatric conditions. Enhanced expression of HERV-K and HERV-H envelope genes has been found in the blood of autism spectrum disorder (ASD) patients, but no information is available on syncytin 1 (SYN1), SYN2, and multiple sclerosis-associated retrovirus (MSRV), which are thought to be implicated in brain development and immune responses. HERV activation is regulated by TRIM28 and SETDB1, which are part of the epigenetic mechanisms that organize the chromatin architecture in response to external stimuli and are involved in neural cell differentiation and brain inflammation. We assessed, through a PCR realtime Taqman amplification assay, the transcription levels of pol genes of HERV-H, -K, and -W families, of env genes of SYN1, SYN2, and MSRV, as well as of TRIM28 and SETDB1 in the blood of 33 ASD children (28 males, median 3.8 years, 25-75% interquartile range 3.0-6.0 y) and healthy controls (HC). Significantly higher expressions of TRIM28 and SETDB1, as well as of all the HERV genes tested, except for HERV-W-pol, were found in ASD, as compared with HC. Positive correlations were observed between the mRNA levels of TRIM28 or SETDB1 and every HERV gene in ASD patients, but not in HC. Overexpression of TRIM28/SETDB1 and several HERVs in children with ASD and the positive correlations between their transcriptional levels suggest that these may be main players in pathogenetic mechanisms leading to ASD.
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Transtorno do Espectro Autista , Retrovirus Endógenos , Esclerose Múltipla , Transtorno do Espectro Autista/genética , Criança , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Produtos do Gene env/metabolismo , Genoma Humano , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Masculino , Esclerose Múltipla/patologia , Fatores de Transcrição/genética , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismoRESUMO
BACKGROUND: The etiopathogenesis of MF remains obscure. CD27 is a member of the tumor necrosis factor receptor superfamily (TNFRS) that regulates lymphocyte function. Expression of CD27 protein and mRNA has been reported in B-cell lymphomas and adult T-cell leukemia/lymphoma. In this study, we examined the expression of CD27 in the skin of MF patients by real time PCR. The amount of CD27 was measured in MF patients and healthy controls. METHODS: A total of 98 skin biopsies were analyzed: 12 obtained from healthy donors and 86 obtained cryostatic sections OCT-embedded affected by MF. Relative quantification of mRNA CD27 expression was achieved by means of TaqMan (Thermo Fisher Scientific, Waltham, MA, USA) amplification and normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RESULTS: Housekeeping gene was detectable in all skin samples and there is no difference between healthy control and MF P value =0.1564. CD27 mRNA sequences were found in 3 of 12 (25%) of skin obtained from healthy donors and in 59 of 86 (68%) of skin obtained from cryostatic sections OCT-embedded affected by MF. The χ2 statistic with Yates correction is 6.8413 and the P value is 0.0089. When we compared the CD27 expression in MF and controls the RQ analysis showed a value of 9.12±14.13. A RQ of 9.12 means that this gene is 9.12 times more expressed in MF skin samples then in the healthy skin samples. No difference was observed in the MF clustered by stages. CONCLUSIONS: Our findings indicates that CD27 can be used as diagnostic/prognostic markers, and whether anti-CD27 antibodies can be used in therapy.
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Leucemia-Linfoma de Células T do Adulto , Micose Fungoide , Neoplasias Cutâneas , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Adulto , Humanos , Micose Fungoide/genética , RNA Mensageiro/genética , Pele/metabolismo , Neoplasias Cutâneas/genéticaRESUMO
BACKGROUND: HERVs expression seems impaired in several diseases, ranging from autoimmune to neoplastic disorders. However, various stimuli may re-activate HERVs transcription. Henoch-Schönlein purpura is the most common cause of vasculitis in children. The role of microbial antigens in the pathogenesis of HSP remains elusive. Toll-like receptors (TLRs) are the first line of defense for the host to initiate an immune and inflammatory response. We aimed to investigate HERV, K and W expression in peripheral mononuclear cells of children with HSP and relation with TLRs activation. METHODS: The study enrolled 63 children affected by HSP. We used RT-PCR to detect GAPDH in the peripheral blood mononuclear cells samples to normalize the data. Subsequently, the viral pol gene HERVs and TLRs transcripts in the same samples were assessed. RESULTS: HERV K and W was expressed in all samples analyzed but the level of expression was much lower in the HSP that in HC P=0.0006 and P=0.0004 for HERV-K and -W respectively. TLR2 was hyper-expressed in 39 vs. 63 (62%) of the HSP, TLR3 in 23 vs. 63 (42%), TLR4 in 42 vs. 63 (66%) and TLR9 in 32 vs. 63 (52%). The different expression was statistically significant only for TLR4 (P=0.0276) no for TLR2,3 and 9 (P=0.1251; 0.3964 and 0.1882 respectively). Spearman's test demonstrated no correlation between the low expression of HERV-K and HERV-W and high expression of TLRs. CONCLUSIONS: The results of the present study demonstrated that mRNA expression of HERV-K and -W and TLRs did not directly correlated.
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Retrovirus Endógenos , Vasculite por IgA , Receptores Toll-Like , Criança , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Humanos , Vasculite por IgA/genética , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 3 Toll-Like , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9 , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
BACKGROUND: Human Parechovirus (HPeVs), along with human enteroviruses is associated with gastrointestinal and respiratory infections. The aim of this study was to assess the performance characteristics of two nucleic acid extraction for HPeV-RNA quantitation, the RNAlev Extraction Kit associated with Maxwell automated nucleic acid extractor (Promega, Milan, Italy) and RNAzol manually protocol. METHODS: A total of 137 fecal specimens previously routinely screened for Rotavirus and Adenovirus were tested for HPeV virus. RESULTS: Methods 1 and 2 detected HPeV-RNA in 11 and 10 samples, respectively, with a 96.2% concordance. In particular, 124/137 (90.5%) were concordantly negative by both methods; 8/137 (5.8%) concordantly positive. For the 8 specimens that were positive by both tests, the population mean (SD) was 320 (601) copies/mg with method 1 and 1216 (2338) copies/mg with method 2. By referring to the 8 specimens that were concordantly positive, the correlation value between the two methods was not statistically significant (r=0.381 and P=0.3599). Bland-Altman analysis showed that differences between the two methods were within ±1000 copies/mg of the averaged results for all of the tested specimens. CONCLUSIONS: Method 2, being a semi-automated method, provides benefits over a manual method, in terms of turnaround time, less errors and reliability.
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Enterovirus , Parechovirus , Infecções por Picornaviridae , Criança , Enterovirus/genética , Humanos , Parechovirus/genética , Infecções por Picornaviridae/diagnóstico , Infecções por Picornaviridae/epidemiologia , RNA Viral/genética , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Human endogenous retroviruses (HERVs), remnants of ancestral infections, represent 8% of the human genome. HERVs are co-opted for important physiological functions during embryogenesis; however, little is known about their expression in human gametes. We evaluated the transcriptional levels of several retroviral sequences in human spermatozoa. METHODS AND RESULTS: We assessed, through a Real-Time PCR assay, the transcription levels of the pol genes of HERV-H, -K and -W families and of env genes of syncytin (Syn)1 and Syn2 in the spermatozoa from 8 normospermic subjects. The entity and distribution of their expressions were compared to values found in white blood cells (WBCs) from 16 healthy volunteers. The level of HERV transcripts was significantly lower in spermatozoa than in WBCs for HERV-H-pol, HERV-K-pol, HERV-W-pol, and Syn2.In contrast, the level of expression of Syn1 in the sperm was similar to that found in WBCs and it was significantly higher than the mRNA concentrations of other HERV genes in spermatozoa. CONCLUSIONS: Our findings show, for the first time, the presence of several retroviral mRNAs in the sperm, although in low amounts. The higher concentration of Syn1 suggests that it could play a key role in the fusion process between gametes during fertilization and, perhaps, be involved in embryo development. Further studies could clarify whether aberrant HERV expressions, in particular of Syn1, negatively affect fertilization and embryo growth and whether sperm manipulation procedures, such as cryopreservation, may potentially influence HERV transcription in the human male gamete.
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Regulação da Expressão Gênica , Produtos do Gene env/genética , Proteínas da Gravidez/genética , Espermatozoides/metabolismo , Adolescente , Adulto , Humanos , Masculino , Adulto JovemRESUMO
Three newly discovered viruses have been recently described in diarrheal patients: Cosavirus (CosV) and Salivirus (SalV), 2 picornaviruses, and bufavirus (BuV), a parvovirus. The detection rate and the role of these viruses remain to be established in acute gastroenteritis (AGE) in diarrheal Italian infants. From November 2016 to November 2017, stool samples were collected from 160 children <5 years old suffering from AGE and attending the Children's Hospital in Turin, Italy. During the study period, 1 (0.5%) sample was positive for 1 of the 3 investigated viruses: 0 (0%) CosV, 1 (0.5%) SalV, and 0 (0%) BuV, whereas 42 (26.0%) children were infected with rotavirus and 2 (1%) with adenovirus. No mixed infections involving the 3 viruses were found. Although these viruses are suspected to be responsible for AGE in children, our data showed that this association was uncertain. Therefore, further studies with large cohorts of healthy and diarrheal children will be needed to evaluate their clinical role in AGE.
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Gastroenterite , Picornaviridae , Criança , Pré-Escolar , Diarreia/epidemiologia , Fezes , Gastroenterite/epidemiologia , Humanos , Lactente , Itália/epidemiologia , Picornaviridae/genéticaRESUMO
Chronic hepatitis C virus (HCV) infection is associated with several hepatic and extrahepatic complications, including cancers and autoimmune disorders, whose frequency is reduced but not abolished after drug-induced viral clearance. The causes of these complications and of their persistence are ill-defined. Human endogenous retroviruses (HERVs) are remnants of ancestral infections and constitute 8% of the human genome. Most HERV elements are inactive, but some are transcribed. HERV overexpression is associated with many cancers and autoimmune diseases with a putative pathogenetic role. Several viral infections trigger HERV activation, but there are no studies on HCV-infected subjects. We assessed, through a PCR real-time amplification assay, the transcription levels of the pol genes of HERV-H, -K, and -W, and of their repressor TRIM28 in white blood cells (WBCs) of vertically infected children, both before and after therapy with direct-acting antivirals (DAAs). The results documented significantly higher expressions of HERV-H-pol and HERV-K-pol, not of HERV-W-pol, in HCV-infected subjects as compared to age-matched controls. HERV RNA levels remained unchanged after DAA-driven viral clearance. No significant variations in transcription levels of TRIM28 were observed in infected subjects. Our findings demonstrate HERV-H-pol and HERV-K-pol overexpression in subjects with chronic HCV infection, without variations after a positive response to DAAs; this might justify their predisposition to cancers and autoimmune disorders that persist after a DAA-induced resolution of viremia.
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Retrovirus Endógenos/genética , Regulação Viral da Expressão Gênica , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/virologia , Adolescente , Antivirais/uso terapêutico , Doenças Autoimunes/metabolismo , Criança , Pré-Escolar , Feminino , Genoma Humano , Genótipo , Humanos , Lactente , Leucócitos/virologia , Masculino , RNA Viral/genética , Proteína 28 com Motivo Tripartido/metabolismo , Proteínas Virais/metabolismoRESUMO
The human endogenous retroviruses (HERVs) are endogenous retroviruses that are inserted into the germ cell DNA of humans over 30 million years ago. Using real-time RT-PCR we describe HERV modulation by commensal microbes in the human gut. Infants, exclusively or predominant breast milk feeding, less than 12 weeks of age, during bacteria gut colonization, were assessed for eligibility. Our data demonstrate that the colonization with commensal microbes, in particular, Bifidobacterium spp., of the gut causes modulation of HERVs.
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Retrovirus Endógenos/genética , Microbioma Gastrointestinal/fisiologia , Transcrição Gênica , Bactérias/classificação , Bactérias/isolamento & purificação , Aleitamento Materno , Retrovirus Endógenos/classificação , Fezes/microbiologia , Microbioma Gastrointestinal/genética , Produtos do Gene pol/sangue , Produtos do Gene pol/genética , Humanos , LactenteRESUMO
BACKGROUND: According to the latest update, 2578 unique mature miRNAs are currently annotated in the human genome and participate in the regulation of multiple events, such as cellular proliferation or apoptosis. A previous study analyzing global miRNA expression patterns in GH cells (high HERV-K versus low) showed that two miRNAs (miR-663 and miR-638) are differentially regulated and exhibit expression parallel to that of HERV-K. The aim of this study was to evaluate HERV-K and -W pol gene and mir-155 expression in SS patients and possible relationship between them. METHODS: The comparison between SS patients and healthy donor showed a significant difference in terms of mir-155 expression P=0.0003 as previously reported by our groups. RESULTS: We demonstrated that HERV-K and -W pol gene expression was significantly higher in SS patients vs. healthy donor as previously reported by our groups. Our correlation data suggest that miR-155 are not directly involved in regulating the HERVs. CONCLUSIONS: Furthermore, further studies including other cohorts of pathology with mir-155 and HERVs involvement such as inflammatory diseases are needed to investigate the role of mir-155 in the cross-activations of HERVs.
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Retrovirus Endógenos/genética , MicroRNAs/genética , Síndrome de Sézary/genética , Neoplasias Cutâneas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-IdadeRESUMO
BACKGROUND: A novel human protoparvovirus named Cutavirus has been discovered. We investigated the presence of Cutavirus in a sample of Cutaneous T-cell lymphomas by using PCR real time TaqMan® (Thermo Fisher Scientific, Waltham, MA, USA). METHODS: In total, 55 CTCL samples were analyzed using a TaqMan® Real time PCR on a 7500 ABI instrument. All of these shown internal control amplification. RESULTS: The presence of Cutavirus DNA corresponding was examined. CuV DNA sequences were not detected in any skin specimen. CONCLUSIONS: The role of Cutaviruses in cutaneous cancers remains to be investigated.
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DNA Viral/análise , Linfoma Cutâneo de Células T/virologia , Parvovirinae/isolamento & purificação , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Parvovirinae/genética , Parvovirinae/patogenicidade , Reação em Cadeia da Polimerase em Tempo RealRESUMO
McCune-Albright syndrome (MAS) is a rare congenital disorder characterized by the association of endocrine and nonendocrine anomalies caused by somatic activating variants of GNAS. The mosaic state of variants makes the clinical presentation extremely heterogeneous depending on involved tissues. Biological samples bearing a low level of mosaicism frequently lead to false-negative results with an underestimation of causative molecular alterations, and the analysis of biopsies is often needed to obtain a molecular diagnosis. To date, no reliable analytical method for the noninvasive testing of blood is available. This study was aimed at validating a novel and highly sensitive technique, the digital PCR (dPCR), to increase the detection rate of GNAS alterations in patients with a clinical suspicion of MAS and, in particular, in blood. We screened different tissues (blood, bone, cutis, ovary, and ovarian cyst) collected from 54 MAS patients by different technical approaches. Considering blood, Sanger was unable to detect mutations, the allele-specific PCR and the co-amplification at lower denaturation temperature had a 9.1% and 18.1% detection rate, respectively, whereas the dPCR reached a 37.8% detection rate. In conclusion, the dPCR resulted in a cost-effective, reliable, and rapid method allowing the selective amplification of low-frequency variants and able to improve GNAS mutant allele detection, especially in the blood.
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BACKGROUND/AIMS: Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth disorder predisposing to tumorigenesis caused by abnormal expression or function of imprinted genes of the chromosome 11p15.5 imprinting gene cluster. This real-time PCR-based assay determines the methylation status of a selected CpG island and has been proposed for use in high-throughput methylation analysis. METHODS: Here, we use quantitative analysis of methylated alleles (QAMA) for the detection of methylation status of the KCNQ10T1 gene, in a region immediately upstream of the transcription initiation site, and the CTCF binding site 6, located approximately 2 kb upstream of the SmaI site currently used for clinical laboratory testing. We assayed a series of controls and patients diagnosed with BWS at two different loci at 11p15.5 to assess the diagnostic yield of QAMA PCR for clinical laboratory testing. RESULTS: These results compare favorably with methylation-specific multiple ligation probe amplification (MS-MLPA) analysis at both differentially methylated region (DMR)1 and DMR2. There are several advantages of the QAMA PCR over MS-MLPA. The QAMA PCR is less labor-intensive and therefore more cost-effective and does not require dedicated analysis software. A second advantage is that the assay is amenable to high-throughput analysis. CONCLUSIONS: The small sample size reflects the rare nature of this epigenetic disorder, and the range of ages was quite wide, as was the degree of disease severity. Therefore, further validation with larger cohorts is warranted.
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Síndrome de Beckwith-Wiedemann/diagnóstico , Síndrome de Beckwith-Wiedemann/genética , Metilação de DNA , Reação em Cadeia da Polimerase Multiplex , Reação em Cadeia da Polimerase em Tempo Real , Alelos , Ilhas de CpG , Sondas de DNA/genética , Humanos , Técnicas de Diagnóstico MolecularRESUMO
OBJECTIVE: The human endogenous retroviruses (HERVs) are endogenous retroviruses that were inserted into the germ cell DNA of humans over 30 million years ago. Insertion of HERVs into the chromosomal DNA can influence a number of host genes in various modes during human evolution and their proviral long terminal repeats can participate in the transcriptional regulation of various cellular genes. Our aim was to evaluate the pol gene expression of HERV-K and HERV-H in mesenchymal stem cells (MSCs) in relation with the expression of stemness genes such as NANOG, OCT-4, and SOX-2. METHODS: MSCs were isolated from bone marrow of healthy donors and expanded until the 5th passage in α-MEM with 10% fetal bovine serum. HERV-K, HERV-H pol gene, NANOG, OCT-4, SOX-2, and GAPDH expression was quantified by real-time PCR in MSCs during the expansion. RESULTS: HERV-K and HERV-H expression was always higher at p1 compared to other passages and this difference reached a high statistical significance when passage p1 was compared with passage 3. In addition, NANOG, OCT-4, and SOX-2 expression at p1 was significantly higher than their expression at p3. Pearson's test demonstrated a strong correlation between the expression of HERV-K and HERV-H and the expression of NANOG, OCT-4, and SOX-2. CONCLUSIONS: Our findings showed that HERV-K and H were concurrently expressed with pluripotency biomarkers NANOG, OCT-4, and SOX-2.
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Retrovirus Endógenos/genética , Expressão Gênica , Genes pol , Células-Tronco Mesenquimais/virologia , Biomarcadores , Humanos , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genéticaRESUMO
OBJECTIVE: Transcription of human endogenous retrovirus (HERV) elements is usually suppressed by epigenetic factors such as DNA methylation and heterochromatin silencing by histone modifications. There is an association between maternal smoking during pregnancy and DNA methylation levels in placental tissue and in DNA from cord blood. STUDY DESIGN: We assessed the transcriptional activity of HERV-H, HERV-K, and HERV-W in umbilical cord blood from 47 term babies unexposed to tobacco smoke in utero and 23 term babies exposed to tobacco smoke in utero. RESULTS: In our population, the HERV-H, HERV-K, and HERV-W families were always transcriptionally active, and the levels of all HERVs (H, K, W) were significantly higher in unexposed than smoke-exposed babies. CONCLUSION: This study provides preliminary information about the transcriptional activity of HERV-H, HERV-K, and HERV-W families in human umbilical cord blood.