Assessment of genotyping markers in the molecular characterization of a population of clinical isolates of Fusarium in Colombia / Evaluación de marcadores de genotipificación en la caracterización molecular de una población de aislamientos clínicos de Fusarium en Colombia

Introduction: Fusarium is a very heterogeneous group of fungi, difficult to classify, with a wide range of living styles, acting as saprophytes, parasites of plants, or pathogens for humans and animals. Prevalence of clinical fusariosis and lack of effective treatments have increased the interest in the precise diagnosis, which implies a molecular characterization of Fusarium populations. Objective: We compared different genotyping markers in their assessment of the genetic variability and molecular identification of clinical isolates of Fusarium. Materials and methods: We evaluated the performance of the fingerprinting produced by two random primers: M13, which amplifies a minisatellite sequence, and (GACA)4, which corresponds to a simple repetitive DNA sequence. Using the Hunter Gaston Discriminatory Index (HGDI), an analysis of molecular variance (AMOVA), and a Mantel test, the resolution of these markers was compared to the reference sequencing-based and PCR genotyping methods. Results: The highest HGDI value was associated with the M13 marker followed by (GACA)4. AMOVA and the Mantel tests supported a strong correlation between the M13 classification and the reference method given by the partial sequencing of the transcription elongation factor 1-alpha (TEF1-α) and rDNA 28S. Conclusion: The strong correlation between the M13 classification and the sequencing-based reference together with its higher resolution demonstrates its adequacy for the characterization of Fusarium populations.

Introducción. Fusarium es un grupo heterogéneo de hongos, difícil de clasificar y con una amplia gama de estilos de vida, que actúa como saprófito, parásito de plantas o patógeno de humanos y animales. La prevalencia de la fusariosis clínica y la falta de tratamientos han incrementado el interés en su diagnóstico preciso, lo que conlleva la caracterización molecular de las poblaciones. Objetivo. Comparar marcadores de genotipificación en la evaluación de la variabilidad genética e identificación de aislamientos clínicos de Fusarium. Materiales y métodos. Se evaluó la huella genética producida por dos cebadores aleatorios: M13, que amplifica una secuencia minisatélite, y (GACA)4, que corresponde a una secuencia repetitiva de ADN. Utilizando el índice discriminatorio de Hunter Gaston (HGDI), el análisis de varianza molecular (AMOVA) y una prueba de Mantel, se comparó la resolución de estos marcadores con métodos de genotipificación basados en secuenciación y PCR. Resultados. El mayor HGDI se asoció con el marcador M13, seguido de (GACA)4. Las pruebas AMOVA y Mantel mostraron correlación entre las clasificaciones obtenidas con M13 y la referencia basada en la secuenciación parcial del factor de elongación de transcripción 1-alfa (TEF1-α) y el ADNr 28S. Conclusión. La fuerte correlación entre la clasificación obtenida con M13 y el método de referencia, así como su alta resolución, demuestran su idoneidad para la caracterización de poblaciones de Fusarium.

Association of single nucleotide polymorphisms in microRNAs with the risk of chronic spontaneous urticaria / 中华皮肤科杂志

Objective:To investigate the association between single nucleotide polymorphisms (SNPs) in microRNAs (miRNAs) and the risk of chronic spontaneous urticaria (CSU) .Methods:A case-control study was conducted. A total of 98 patients with CSU (CSU group) were collected from Department of Dermatology, Affiliated Hospital of Jining Medical University from January to June in 2019, and 148 health checkup examinees (control group) were collected at the same time, all of whom were of Han nationality from Shandong province. Genomic DNA was extracted from venous blood samples, and polymorphic sites rs2431697 (miRNA-146a) , rs57095329 (miRNA-146a) , rs3746444 (miRNA-499) , rs11614913 (miRNA-196a2) and rs895819 (miRNA-27a) were analyzed for SNP genotyping by multiplex PCR amplification and single-base extension. Chi-square test was used to analyze differences in the distribution of alleles, genotypes and genetic models between the two groups, and unconditional logistic regression to analyze the relationship between gene SNPs and the risk of CSU.Results:All samples were successfully genotyped by analysis of the 5 polymorphic sites. The alleles of the miRNA-196a2 SNP rs11614913 were T/C, and the absolute frequency of T allele was 110 (56.1%) in the CSU group and 131 (44.3%) in the control group; there was a significant difference in the T/C allele frequency distribution between the two groups ( χ2 = 6.64, P = 0.010) , and the T allele might be a risk factor for CSU ( OR=1.61, 95% CI: 1.12-2.32) . In addition, the absolute frequencies of CC, CT and TT genotypes of rs11614913 were 16 (16.3%) , 54 (55.1%) , 28 (28.6%) in the CSU group, and 48 (32.4%) , 69 (46.6%) , 31 (20.9%) in the control group respectively, and there was a significant difference in the genotype distribution between the two groups ( χ2 = 8.16, P = 0.017) ; the distribution of the dominant genetic model (TT + CT vs. CC) also significantly differed between the two groups ( χ2 = 7.95, P = 0.005) , which might increase the risk of CSU ( OR=2.46, 95% CI: 1.30-4.65) . Conclusion:The miRNA-196a2 SNPs may be associated with the risk of CSU in the Han population in Shandong, China, and the rs11614913 polymorphism may increase the risk of CSU.

Update on and future perspectives for the diagnosis of alpha-1 antitrypsin deficiency in Brazil / Atualização e perspectivas futuras para o diagnóstico da deficiência de alfa-1 antitripsina no Brasil

ABSTRACT Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disorder caused by a mutation in the SERPINA1 gene, which encodes the protease inhibitor alpha-1 antitrypsin (AAT). Severe AATD predisposes individuals to COPD and liver disease. Early diagnosis is essential for implementing preventive measures and limiting the disease burden. Although national and international guidelines for the diagnosis and management of AATD have been available for 20 years, more than 85% of cases go undiagnosed and therefore untreated. In Brazil, reasons for the underdiagnosis of AATD include a lack of awareness of the condition among physicians, a racially diverse population, serum AAT levels being assessed in a limited number of individuals, and lack of convenient diagnostic tools. The diagnosis of AATD is based on laboratory test results. The standard diagnostic approach involves the assessment of serum AAT levels, followed by phenotyping, genotyping, gene sequencing, or combinations of those, to detect the specific mutation. Over the past 10 years, new techniques have been developed, offering a rapid, minimally invasive, reliable alternative to traditional testing methods. One such test available in Brazil is the A1AT Genotyping Test, which simultaneously analyzes the 14 most prevalent AATD mutations, using DNA extracted from a buccal swab or dried blood spot. Such advances may contribute to overcoming the problem of underdiagnosis in Brazil and elsewhere, as well as being likely to increase the rate detection of AATD and therefore mitigate the harmful effects of delayed diagnosis.

RESUMO A deficiência de alfa-1 antitripsina (DAAT) é um distúrbio genético raro causado por uma mutação no gene SERPINA1, que codifica o inibidor de protease alfa-1 antitripsina (AAT). A DAAT predispõe os indivíduos a DPOC e doença hepática. O diagnóstico precoce é essencial para a implementação de medidas preventivas e para limitar a carga da doença. Embora diretrizes nacionais e internacionais para o diagnóstico e manejo da DAAT estejam disponíveis há 20 anos, mais de 85% dos casos não são diagnosticados e, portanto, não são tratados. No Brasil, os motivos para o subdiagnóstico da DAAT incluem o desconhecimento dos médicos sobre a condição, a diversidade racial da população, o fato de os níveis séricos de AAT serem avaliados em um número limitado de indivíduos e a falta de ferramentas diagnósticas convenientes. O diagnóstico da DAAT baseia-se em resultados de exames laboratoriais. A abordagem diagnóstica padrão envolve a avaliação dos níveis séricos de AAT, seguida de fenotipagem, genotipagem, sequenciamento gênico ou suas combinações para detecção da mutação específica. Nos últimos 10 anos, novas técnicas foram desenvolvidas, oferecendo uma alternativa rápida, minimamente invasiva e confiável aos métodos tradicionais de teste. Um desses testes disponíveis no Brasil é o teste de genotipagem A1AT, que analisa simultaneamente as 14 mutações mais prevalentes da DAAT usando DNA extraído de swab bucal ou de sangue em papel-filtro. Esses avanços podem contribuir para a superação do problema do subdiagnóstico no Brasil e em outros países, bem como podem aumentar a taxa de detecção da DAAT e, portanto, mitigar os malefícios do diagnóstico tardio.

Precision diagnosis of a full-term pregnancy with hydatidiform mole based on postnatal short tandem repeat DNA genotyping: a case report / 中华围产医学杂志

We describe a case of spontaneous conception following ovarian stimulation, in which a singleton pregnancy was revealed by ultrasound at 17 gestational weeks, with a multi-cystic "honeycomb" pattern in part of the placenta. With close monitoring, the patient delivered a healthy male neonate through cesarean section at 38 gestational weeks. The clinical findings, combined with ultrasound, laboratory, pathological, and immunohistochemistry examination, and short tandem repeat genotyping, confirmed a twin pregnancy consisting of a complete mole and coexisting fetus. No obvious abnormalities were found in the mother or the boy during a four-and-a-half-year's follow-up.

Optimization of HLA-B*27 ALLELE Genotyping by PCR-SSP

OBJECTIVES: HLA-B27 is strongly associated with ankylosing spondylitis (AS) and its presence helps to confirm AS diagnosis. Due to the high HLA polymorphism and the differentiated contribution of alleles and molecules encoded by them, HLA-B*27 allele identification is relevant in the clinical follow-up, diagnosis, and treatment of this spondyloarthropathy. Inexpensive genotyping techniques with high specificity and sensitivity are of great interest in histocompatibility laboratories. This work aimed to optimize HLA-B*27 genotyping by Polymerase Chain Reaction Sequence-specific Primer (PCR-SSP), which is an accessible and inexpensive technique. METHODS: The PCR-SSP was standardized using 26 HLA-B*27 positive and 3 HLA-B*27 negative samples previously defined by Polymerase Chain Reaction Sequence-specific Oligonucleotide Probes (PCR-SSOP) (medium resolution, One Lambda®) and primers described by Duangchanchot et al. (2009). For validating the technique, 397 samples were genotyped using PCR-SSP as well as PCR-SSOP. RESULTS: The PCR-SSP technique was standardized for identifying the alleles HLA-B*27:02, HLA-B*27:CAFRW (05/13/16/17/28/37/38/39/42), HLA-B*27:CAFRZ (08/26/40), HLA-B*27:09 and HLA-B*27:12, which were found in 90 positive samples (22.67%). There was 100% agreement between the two techniques for heterozygous samples; however, two homozygous samples could not be detected by PCR-SSP. CONCLUSION: The HLA-B*27 genotyping using PCR-SSP, an easy-to-use, specific, and affordable technique, was optimized for heterozygous samples. This technique may contribute to AS diagnosis.

Nontuberculous mycobacteria in patients with suspected tuberculosis and the genetic diversity of Mycobacterium avium in the extreme south of Brazil / Micobactérias não tuberculosas em pacientes com suspeita de tuberculose e a diversidade genética de Mycobacterium avium no extremo sul do Brasil

ABSTRACT Objective: Nontuberculous mycobacteria (NTM) are a heterogeneous group of bacteria that are widely distributed in nature and associated with opportunistic infections in humans. The aims of this study were to identify NTM in patients with suspected tuberculosis who presented positive cultures and to evaluate the genetic diversity of strains identified as Mycobacterium avium. Methods: We studied pulmonary and extrapulmonary samples obtained from 1,248 patients. The samples that tested positive on culture and negative for the M. tuberculosis complex by molecular identification techniques were evaluated by detection of the hsp65 and rpoB genes and sequencing of conserved fragments of these genes. All strains identified as M. avium were genotyped using the eight-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat method. Results: We found that NTM accounted for 25 (7.5%) of the 332 mycobacteria isolated. Of those 25, 18 (72%) were M. avium, 5 (20%) were M. abscessus, 1 (4%) was M. gastri, and 1 (4%) was M. kansasii. The 18 M. avium strains showed high diversity, only two strains being genetically related. Conclusions: These results highlight the need to consider the investigation of NTM in patients with suspected active tuberculosis who present with positive cultures, as well as to evaluate the genetic diversity of M. avium strains.

RESUMO Objetivo: As micobactérias não tuberculosas (MNT) são um grupo heterogêneo de bactérias amplamente distribuídas na natureza e relacionadas com infecções oportunistas em seres humanos. Os objetivos deste estudo foram identificar MNT em pacientes com suspeita de tuberculose e culturas positivas e avaliar a diversidade genética de cepas identificadas como Mycobacterium avium. Métodos: Foram estudadas amostras pulmonares e extrapulmonares provenientes de 1.248 pacientes. As amostras que apresentaram resultado positivo em cultura e negativo para o complexo M. tuberculosis na identificação molecular foram avaliadas por meio da detecção dos genes hsp65 e rpoB e de sequenciamento de fragmentos conservados desses genes. Todas as cepas identificadas como M. avium foram genotipadas pelo método mycobacterial interspersed repetitive unit-variable-number tandem-repeat com oito loci. Resultados: Das 332 micobactérias isoladas, 25 (7,5%) eram MNT. Dessas 25, 18 (72%) eram M. avium, 5 (20%) eram M. abscessus, 1 (4%) era M. gastri e 1 (4%) era M. kansasii. As 18 cepas de M. avium apresentaram alta diversidade, e apenas duas eram geneticamente relacionadas. Conclusões: Esses resultados mostram a necessidade de considerar a investigação de MNT em pacientes com suspeita de tuberculose ativa e culturas positivas e de avaliar a diversidade genética de cepas de M. avium.

Genotyping of Dombrock and Lutheran blood group systems in blood donors from the southwestern region of the state of Paraná, Southern Brazil

Abstract Background Lutheran and Dombrock are two blood group systems with low immunogenic antigens; they can cause mild-to-moderate transfusion reactions. For both, immunophenotyping is not performed in the pretransfusion routine in Brazil. In addition, the distribution of their antigenic frequencies is an important marker of ethnicity. Thus, the goal of this study was to carry out the genotyping of the LU*01, LU*02, DO*01 and DO*02 alleles of the Lutheran and Dombrock blood group systems in blood donors from the southwestern region of the state of Paraná, Southern Brazil. Method Genotyping was performed for 251 blood donors by specific allele-polymerase chain reaction. The genotype and allele frequencies were obtained through direct counting and compared with other Brazilian populations using the chi-square test with Yates correction. Results The distribution of genotype frequencies for LU were 0.4% for LU*01/LU*01, 6.8% for LU*01/LU*02 and 92.8% for LU*02/LU*02 and for DO, they were 19.9% for DO*01/DO*01, 44.6% for DO*01/DO*02 and 35.5% for DO*02/DO*02. The allele and genotype frequencies of LU and DO were similar to those expected for Caucasians, but the DO*01/DO*01 genotype frequency was different to other Brazilian populations. The rare LU*01/LU*01 genotype was found in a loyal blood donor. Conclusion The genotyping techniques allowed the evaluation of the LU*01, LU*02, DO*01 and DO*02 alleles in blood donors registered in the Hemotherapy Center of the southwestern region of Paraná, Southern Brazil, and contributed to a genotyped blood donor database.

Retinal dystrophies and variants in PRPH2 / Distrofias retinianas e variantes em PRPH2

ABSTRACT - This report presents three patients diagnosed with macular dystrophies with variants in PRPH2. Peripherin-2, the protein of this gene, is important in the morphogenesis and stabilization of the photoreceptor outer segment. Peripherin-2 deficiencies cause cellular apoptosis. Moreover, pathogenic variants in PRPH2 are associated with various diseases, such as pattern, butterfly-shaped pattern, central areolar, adult-onset vitelliform macular, and cone-rod dystrophies as well as retinitis pigmentosa, retinitis punctata albescens, Leber congenital amaurosis, fundus flavimaculatus, and Stargardt disease.

RESUMO - Este relato apresenta três pacientes com diagnóstico de distrofias maculares com mutações no PRPH2. Periferina 2, a proteína deste gene, é importante na morfogênese e estabilização do segmento externo dos fotorreceptores. Deficiências de periferina 2 causam apoptose celular. Além disso, variantes patogênicas no PRPH2 estão relacionadas a diferentes doenças, como distrofia padrão, distrofia padrão em asa de borboleta, distrofia central areolar, distrofia viteliforme do adulto, retinose pigmentar, distrofia de cones e bastonetes, retinite punctata albscens, amaurose congênita de Leber, fundus flavimaculatus e doença de Stargardt.

Polymorphism of Serotonin Transporter SLC6A4 (5-HTTLPR) Gene in Cheilitis Angularis Patients

Abstract Objective: To determine the relationship between the Serotonin transporter SLC6A4 (5-HTTLPR) gene polymorphism in cheilitis angularis patients. Material and Methods: We conducted a descriptive analysis of 100 DNA samples extracted from the blood serum of 50 patients with cheilitis angularis and 50 patients without cheilitis angularis. Analysis of the Serotonin transporter SLC6A4 (5-HTTLPR) gene polymorphism was observed by carrying out PCR method followed by electrophoresis for the analysis, without the usage of restriction enzyme. The Chi-square test was used for statistical analysis Results: In the cheilitis angularis group there were 24 samples with SS genotype, 23 samples with LS genotype, and 3 samples with LL genotype. Whereas in the non-cheilitis angularis group, there were 5 samples with SS genotype, 18 samples with LS genotype, and 27 samples with LL genotype. In the cheilitis angularis group found 71 S alleles and 29 L alleles, and in the non-cheilitis angularis group 28 S alleles and 72 L alleles were found. A statistically significant difference was found between the groups (p<0.001) Conclusion: There were significant differences in the distribution of the Serotonin transporter SLC6A4 (5-HTTLPR) gene polymorphism between patients with and without cheilitis angularis.

Molecular epidemiological characteristics of genital Chlamydia trachomatis / 中华皮肤科杂志

Chlamydia trachomatis (Ct) is the causative agent of bacterial sexually transmitted diseases (STDs) worldwide.The incidence of Ct infection has exceeded that of Neisseria gonorrhoeae,and becomes the highest in STDs in many countries.Ct infection can lead to urethritis,epididymitis,prostatitis and infertility in males,and cervicitis,endometritis,pelvic inflammatory disease,infertility in females,and neonatal conjunctivitis.Additionally,urogenital Ct infection is always ignored due to its concealed symptoms,leading to a long clinical course,recurrence or repeated infections.Furthermore,Ct infection can increase the risk of human immunodeficiency virus and human papilloma virus infections.Therefore,how to prevent and control the transmission of Ct has become one of the global public health issues.Currently,a growing body of researches have focused on the molecular epidemiological characteristics of Ct,which are aiming to identify the mutant strains,elaborate transmission dynamics,investigate the distribution of Ct serotypes in different populations,so as to provide molecular epidemiological evidence for the prevention and control of Ct infection.This review summarizes the epidemic status and research methods for molecular epidemiological characteristics of Ct,as well as application of Ct serotyping in clinical practice,providing references for the prevention,control and research of Ct infection.

Cost effectiveness analysis of plasma genotyping versus tumor genotyping in detection of advanced non-small-cell lung cancer with epidermal growth factor receptor and T790M mutation under the Brazilian private healthcare system perspective / Custo-efetividade do uso da biópsia líquida versus biópsia tecidual para detecção de câncer de pulmão de não pequenas células avançado com receptor do fator de crescimento epidérmico e mutação T790M sob a perspectiva do sistema suplementar de saúde do Brasil

Objective: Comparing the costs and effectiveness of plasma genotyping versus tumor genotyping for detecting the T790M mutation in advanced non-small cell lung cancer (NSCLC) with a mutation in the epidermal growth factor receptor (EGFR) and that progressed after use of an EGFR tyrosine kinase inhibitor (EGFR-TKI), from the perspective of the private healthcare system in Brazil. Methods: Patients with a post-EGFR-TKI T790M mutation are eligible for a second-line treatment with a third-generation EGFR-TKI (osimertinib). In order to estimate the costs associated with the diagnosis method for the T790M mutation, a decision tree model has been used. Resource use was estimated by a team of experts, and the direct costs were estimated based on official databases. Results: Plasma genotyping provided a R$391 reduction per patient, due to the reduced cost with complications; it prevented 40.96% of the patients from undergoing an invasive procedure and 31.91% of the patients from having any kind of complication. Conclusion: Data found support a new paradigm for treating the resistance to EGFR-TKIs, with plasma genotyping as the first diagnostic choice, what can help to define the treatment and to reduce the costs of Brazilian private healthcare system.

Objetivo: Comparar os custos e efetividade da biópsia líquida versus biópsia tecidual para detecção da mutação T790M no câncer de pulmão de não pequenas células (CPNPC) avançado com mutação no receptor do fator de crescimento epidérmico (EGFR) e que progrediram após o uso de um inibidor do sítio da tirosina cinase associada ao EGFR (EGFR-TKI), sob a perspectiva do sistema suplementar de saúde do Brasil. Métodos: Pacientes com mutação EGFR-T790M pós-EGFR-TKI são elegíveis ao tratamento de segunda linha com um EGFR-TKI de terceira geração (osimertinibe). Para a estimativa dos custos relacionados ao método de diagnóstico de mutação T790M, foi elaborado um modelo de árvore de decisão. A utilização de recursos foi estimada por painel de especialistas e os custos diretos foram estimados utilizando-se bases de dados oficiais. Resultados: A biópsia líquida proporcionou redução de R$ 391 por paciente, devido a uma redução no custo com complicações; evitou que 40,96% dos pacientes passassem por um procedimento invasivo e que 31,95% dos pacientes tivessem algum tipo de complicação. Conclusão: Os dados observados embasam um novo paradigma para o manejo da resistência aos EGFR-TKIs, com genotipagem pelo plasma como primeira opção diagnóstica, o que pode auxiliar na melhor definição do tratamento e reduzir custos ao sistema de saúde suplementar brasileiro.

Comparison of DNA extraction using proteinase K and extraction kit: analysis of the quality of the genetic material / Comparação da extração de DNA utilizando proteinase K e kit de extração: análise da qualidade do material genético

ABSTRACT INTRODUCTION: Deoxyribonucleic acid (DNA) is the raw material for genetic studies, and therefore laboratory techniques are developed to obtain it with appropriate concentration and integrity. OBJECTIVE: To compare two methods of DNA extraction regarding sample concentration and integrity. METHODS: DNA was extracted from the tail end (2 mm long) of mice, stored at -20ºC. The proteinase K method (PKM) and the Kappa Express Extract® kit were used for extraction. The concentrations and ratios 260/280 and 260/230 were determined by spectrophotometry. DNA integrity was checked on 2% agarose gel with ethidium bromide. For the final test of the extracted samples, a multiplex polymerase chain reaction (PCR) was performed, with primers for the Large gene. RESULTS: Samples extracted by the PKM presented mean concentration value of 59.4 ± 18.5 ng/µl (260/280 = 1.74 ± 0.04 and 260/230 + 1.85 ± 0.14) and the samples extracted by the commercial kit presented mean concentration value of 178.8 ± 42.0 ng/µl (260/280 = 1.09 ± 0.04 and 260/230 = 0.62 ± 0.66). PCR amplified the Large gene in the DNA extracted, regardless of the methodology used. CONCLUSION: Both methodologies studied can be used, and the PKM is cheap but a time-consuming method, while the commercial kit is more expensive, however DNA extraction is faster.

RESUMO INTRODUÇÃO: O ácido desoxirribonucleico (DNA) é a matéria-prima para os estudos genéticos, por isso são desenvolvidas técnicas laboratoriais para sua obtenção, com concentração e integridades adequadas. OBJETIVO: Comparar dois métodos de extração de DNA em relação à concentração e à integridade da amostra. MÉTODOS: O DNA foi extraído da extremidade das caudas (2 mm de comprimento) de camundongos, as quais foram estocadas a -20ºC. Utilizou-se a metodologia de extração com proteinase K (MPK) e o kit Kappa Express Extract®. As concentrações e as relações 260/280 e 260/230 foram determinadas por espectrofotometria. A integridade do DNA foi verificada em gel de agarose a 2%, com brometo de etídeo. Para o teste final das amostras extraídas, realizou-se reação em cadeia da polimerase (PCR) multiplex, com primers para o gene Large. RESULTADOS: As amostras extraídas pela MPK apresentaram concentração média de 59,4 ± 18,5 ng/µl (260/280 = 1,74 ± 0,04 e 260/230 + 1,85 ± 0,14) e as extraídas pelo kit comercial, concentração média de 178,8 ± 42 ng/µl (260/280 = 1,09 ± 0,04 e 260/230 = 0,62 ± 0,66). A PCR amplificou o gene Large no DNA extraído, independente da metodologia utilizada. CONCLUSÃO: As metodologias estudadas podem ser utilizadas, sendo a MPK uma metodologia barata, porém demorada, enquanto o kit comercial é mais oneroso, contudo a extração do DNA é célere.

Prevalência de genótipos predisponentes para doença celíaca, incluindo HLA-DQ2. 2, em crianças brasileiras / Prevalence of celiac disease predisposing genotypes, including hla-dq2. 2 variant, in brazilian children

ABSTRACT BACKGROUND: Celiac disease is an autoimmune enteropathy triggered by the ingestion of gluten in genetically susceptible individuals. Almost all celiac patients carry immune recognition genes coding for HLA-DQ2.5 and DQ8 heterodimers. Over the last few years, great importance has been given to HLA-DQ2.2 as probable predisposing variant, although controversies still exist regarding its relevance. OBJECTIVE: The aim of our study was to determine the possible existence of an association between HLA-DQ2.2 and celiac disease in Brazilian children by analyzing the prevalence of the predisposing variants for celiac disease in a representative group of children of a population in which this determination is still missing. METHODS: HLA-DQ typing was performed in samples from a group of celiac (n=100) and non-celiac children (n=110). All samples were tested for the presence of the following variants: DQA1*05-DQB1*02 (DQ2.5), DQA1*03-DQB1*03:02 (DQ8) and DQA1*02:01-DQB1*02:02 (DQ2.2). Fisher`s exact test was used for statistical analysis. RESULTS: In the group of 100 celiac children, 78 (78%) were positive for DQ2, 13 (13 %) were DQ2/DQ8 and 6 (6%) were DQ8 positives. The HLA-DQ pattern in the 110 non-celiac children was as follows: positive for DQ2 in 33 (29.9%) samples, in 2 (1.8 %) was positive for DQ2/DQ8 and in 15 (13.6%) was positive for DQ8. We found significant differences between the distribution of some but not all of the analyzed alleles when comparing celiac and non-celiac children. CONCLUSION: The genotyping of celiac disease HLA-DQ predisposing alleles showed similarities with HLA-DQ patterns found in both European and non-European populations, which may be a reflection of the miscegenation, which gave origin to the current Brazilian population. No significant association was found between DQ2.2 variant and celiac disease in the studied population.

RESUMO CONTEXTO: A doença celíaca é uma enteropatia autoimune, desencadeada pela ingestão do glúten em indivíduos geneticamente predispostos. Quase todos os pacientes celíacos possuem genes que codificam os heterodímeros HLA-DQ2.5 e DQ8. Nos últimos anos, mesmo com algumas controvérsias a respeito, tem se dado grande importância ao HLA-DQ2.2 como outra provável variante predisponente para doença celíaca. OBJETIVO: O objetivo do nosso trabalho foi determinar a provável associação entre HLA-DQ2.2 e a doença celíaca em crianças brasileiras, mediante a análise da prevalência das variantes predisponentes para doença celíaca em um grupo representativo desta população que ainda carece de dita informação. MÉTODOS: A genotipagem das variantes HLA-DQ foi realizada em populações de crianças celíacas (n=100) e não celíacas (n=110). A presença das seguintes variantes foi testada em todas as amostras: DQA1*05-DQB1*02 (DQ2.5), DQA1*03-DQB1*03:02 (DQ8) e DQA1*02:01-DQB1*02:02 (DQ2.2). A análise estatística foi realizada utilizando o teste exato de Fisher. RESULTADOS: No grupo de 100 crianças celíacas, 78 (78%) foram positivas para DQ2, 13 (13%) para DQ2/DQ8 e 6 (6%) foram DQ8 positivas. O padrão de variantes predisponentes no grupo de 110 crianças não celíacas foi: 33 (29.9%) amostras positivas para DQ2, 2 (1.8%) DQ2/DQ8 positivas e 15 (13.6%) DQ8 positivas. Quando as prevalências de ambos grupos foram compradas, foram achadas diferenças significativas entre algumas, mas não todas as variantes predisponentes. CONCLUSÃO: A genotipagem das variantes HLA-DQ predisponentes para doença celíaca mostrou um padrão similar ao achado em populações europeias e não-europeias, o qual pode ser resultado da miscigenação que deu origem à população brasileira atual. Nosso trabalho não mostrou associação significativa entre a variante DQ2.2 e a doença celíaca na população estudada.

ApoE genotyping and its clinical significance / 中华检验医学杂志

Apolipoprotein E has a dominant function in the metabolic process of triglyceride and cholesterol in multiple tissues of human .The allele of ApoE has three variants ε2,ε3,ε4 and six genotypesε2/ε2, ε3/ε3, ε4/ε4, ε2/ε3, ε2/ε4, ε3/ε4, due to polymorphisms of two common SNPs . These genotypes associated with kinds of diseases ,such as hyperlipidemia , ASCVD, and AD.In addition, they are also related to the treatment effect of statins .Thus, the detection of the ApoE genotypes is valuable and meaningful in the clinic.In this article, there is a simple discussion about the methodology of the ApoE genotypes, from the protein electrophoresis phenotyping to the rapid , precise and convenient genotyping methods, and its clinical significances.

Individual Identification of Cartilage by Direct Amplification in Mass Disasters / 法医学杂志

OBJECTIVES@#To explore the effectiveness of direct amplification for the STR analysis of cartilage, and to accelerate the effectiveness of disaster victim identification.@*METHODS@#Eighty-eight cartilage samples were directly amplified by PowerPle® 21 kit, and the results of genotyping were compared with that obtained by the magnetic beads method.@*RESULTS@#In 88 cartilage samples, the STR genotypes were successfully detected from 84 samples by direct amplification and magnetic beads method, and both the results of genotyping by two method were consistent.@*CONCLUSIONS@#Direct amplification with PowerPlex® 21 kit can be used for STR genotyping of cartilages. This method is operated easily and promptly, which has a potential application in the individual identification of mass disasters.

Mutation analysis in a pedigree with oculocutaneous albinism / 中华皮肤科杂志

Objective To investigate gene mutations in a pedigree with oculocutaneous albinism by using targeted next-generation sequencing technology.Methods Clinical data were collected from a pedigree with oculocutaneous albinism.Genomic DNA was extracted from peripheral blood cells of the proband and his parents.High-throughput sequencing technology was used for sequence analysis of coding regions in exons of 29 genes including TYR,OCA2,TYRP1 and SLC45A2 in the proband to find potential pathogenic gene mutations.Sanger sequencing was conducted to detect the corresponding genetic loci in the parents.Results Two heterozygous mutations were identified in the TYR gene of the proband,including a novel mutation c.534G ＞ C (p.Trp178Cys) and a known mutation c.1147G ＞ A (p.Asp383Asn).The detection of the TYR gene mutations in the parents of the proband showed that the c.534G ＞ C and c.1147G ＞ A mutations in the proband were inherited from his father and mother respectively.Conclusion A novel pathogenic mutation c.534G ＞ C in the TYR gene is identified in the pedigree with oculocutaneous albinism by using targeted next-generation sequencing technology.

Relationship between human papillomavirus genotypes in and clinical characteristics of adult plantar warts / 中华皮肤科杂志

Objective To investigate human papillomavirus (HPV) genotypes in adult patients with plantar warts,and to explore their relationship with clinical features of adult plantar warts.Methods PCR,gene sequencing and TA-cloning technique were performed to detect HPV types in 221 patients with plantar warts.Meanwhile,clinical data were recorded,including patients' age,disease duration,symptoms and number of warts.Results Of 221 specimens,HPV DNA was detected in 215,with the HPV-positive rate being 97％.Single HPV infections were detected in 206 specimens (96％,206/215),among which,HPV types included HPV-27 (44.7％,92/206),-2 (12.1％,25/206),-57 (7.3％,15/206),-7 (Alpha genus;0.5％,1/206),-65 (Gamma genus;0.5％,1/206),-1 (33.5％,69/206) and-63 (Mu genus;1.5％,3/206).There were no significant differences in age,disease duration,number of warts,incidence of pain and gender among patients with HPV-27,-2 or-57 infections.Compared with patients with HPV-27 infection,patients with HPV-1 infection were more related to age ≤ 30 years,disease duration ≤ 1 year,number of warts ≤ 2 and high incidence of pain.Conclusion HPV-27,-2,-57 and-1 are the main types in adult patients with plantar warts,and HPV types are correlated with clinical features of adult plantar warts.

Individual Identification of Cartilage by Direct Amplification in Mass Disasters / 法医学杂志

Objective T o explore the effectiveness of direct am plification for the ST R analysis of carti-lage, and to accelerate the effectiveness of disaster victim identification. Methods E ighty-eight cartilage sam ples w ere directly am plified by Pow erPlex誖21 kit, and the results of genotyping w ere com pared w ith that obtained by the m agnetic beads m ethod. Results In 88 cartilage sam ples, the ST R genotypes w ere successfully detected from 84 sam ples by direct am plification and m agnetic beads m ethod, and both the results of genotyping by tw o m ethod w ere consistent. Conclusion D irect am plification w ith Pow er-Plex誖21 kit can be used for ST R genotyping of cartilages. T his m ethod is operated easily and prom ptly, w hich has a potential application in the individual identification of m ass disasters.

Evaluation of SYTO13 as fluorescent dye for high resolution melting based single nucleotide polymorphism genotyping / 中华检验医学杂志

Objective To evaluate the utility of fluorescent dye SYTO 13 for high -resolution melting ( HRM) detection in single nucleotide polymorphism ( SNP) genotyping and its clinical application . Methods This is a performance verification study .36 genotype defined samples were divided into three groups:SNP rs3125734 C>T (class Ⅰ SNP) ,rs255758 A>C (class ⅡSNP) and rs688C>T.These samples were used to evaluate SYTO 13′s SNP genotyping capability of class ⅠSNP, classⅡSNP, and two PCR products of different lengths (52 and 107 bp) covering the same SNP of rs688C>T.The commercial HRM dye of LCGreen Plus was used as the control .The genotyping capability is indicated by the Tm difference(ΔTm) between wild type and homozygous mutant genotypes .The Tm differences between wild genotype and homozygous mutant genotype were compared using the Independent Samples t test.Paired t test was used to evaluate genotyping capability of the two dyes .The clinical applicability is evaluated by synchronously performing PCR amplification and HRM analysis on thirty -five randomly selected DNA samples with known genotypes of the three SNPs .Results The SNPs of class Ⅰ and class Ⅱ can be genotyped directly and clearly with SYTO13 (ΔTmclas Ⅰ =0.36 ±0.05,tclas Ⅰ =14.827,Pclas Ⅰ =0.000;ΔTm clas Ⅱ =0.42 ±0.110,tclasⅡ =9.539,Pclas Ⅱ =0.000).The classⅠSNP genotyping results was better using SYTO13 (ΔTmSYTO13 =0.39 ±0.027), while the SNP genotyping for small amplicon did not discriminated clearly in this study .Long amplicons of class ⅠandⅡSNPs can be identified directly except for several samples which can be genotyped accurately after having performed reexamination .Conclusion SYTO13 can apply for HRM analysis of genotyping classⅠand ⅡSNPs with long amplicon and for clinical routine detection.

Genotyping of Treponema pallidum in patients with early syphilis in Guangxi province / 中华皮肤科杂志

Objective To investigate genotyps of Treponema pallidum (Tp) in several cities in Guangxi province. Methods A total of 300 patients with suspected early syphilis were enrolled from STD clinics in Guangxi between January 2012 and July 2016, and tissue fluid samples were collected from skin lesions. Silver staining was performed to detect Tp, and PCR to amplify the Tp polA gene for the diagnosis of early syphilis. Positive samples were subjected to PCR amplification of a 60-bp tandem repeat region within the arp gene, restriction fragment length polymorphism(RFLP)analysis of the tpr Ⅱgene after digestion with Mse Ⅰ enzyme and tp0548 genotyping. Results Finally, 215 patients were diagnosed with early syphilis, including 210(97.7%)patients positive for PCR and 105(48.8%)patients positive for silver staining, and the positive rate significantly differed between the two methods (χ2 = 103.01, P < 0.05). Among the PCR-positive samples, 190 could be genotyped by analysis of three target genes, and 17 genotypes were identified. The genotype 14d/f was predominant (45.3%, 86/190), followed by 15d/f (13.7%, 26/190), 16d/f(11.6%, 22/190), 17d/f(7.4%, 14/190), 13d/f(6.8%, 13/190), 10d/f(4.2%, 8/190), 18d/f(1.6%, 3/190), 16a/f(1.6%, 3/190), 5d/f(1.1%, 2/190), 7d/f(1.1%, 2/190), 12d/f(1.1%, 2/190), 16d/e(1.1%, 2/190), 14a/f(1.1%, 2/190), 9h/c(1.1%, 2/190), 15l/f(0.5%, 1/190), 25a/e(0.5%, 1/190), 15i/f(0.5%, 1/190). Conclusion Tp genotypes are diversified in patients with early syphilis in Guangxi, and the genotype 14 d/f is predominant.