Association Study of CACNA1D, KCNJ11, KCNQ1, and CACNA1E Single-Nucleotide Polymorphisms with Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is a complex chronic disease characterized by decreased insulin secretion and the development of insulin resistance. Previous genome-wide association studies demonstrated that single-nucleotide polymorphisms (SNPs) present in genes coding for ion channels involved in insulin secretion increase the risk of developing this disease. We determined the association of 16 SNPs found in CACNA1D, KCNQ1, KCNJ11, and CACNA1E genes and the increased probability of developing T2DM. In this work, we performed a case-control study in 301 Mexican adults, including 201 cases with diabetes and 100 controls without diabetes. Our findings indicate a moderate association between T2DM and the C allele, and the C/C genotype of rs312480 within CACNA1D. The CAG haplotype surprisingly showed a protective effect, whereas the CAC and CGG haplotypes have a strong association with T2DM. The C allele and C/C genotype of rs5219 were significantly associated with diabetes. Also, an association was observed between diabetes and the A allele and the A/A genotype of rs3753737 and rs175338 in CACNA1E. The TGG and CGA haplotypes were also found to be significantly associated. The findings of this study indicate that the SNPs examined could serve as a potential diagnostic tool and contribute to the susceptibility of the Mexican population to this disease.

Genomic sequencing revealed recombination event between clade 1 and clade 2 occurs in circulating varicella-zoster virus in China.

Varicella-zoster virus (VZV), a member of the Alphaherpesvirinae subfamily, causes varicella in primary infections and establishing a latent stage in sensory ganglia. Upon reactivation, VZV causes herpes zoster with severe neuralgia, especially in elderly patients. The mutation rate for VZV is comparatively lower than the other members of other alpha herpesviruses. Due to geographic isolation, different genotypes of VZV are circulating on separate continents. Here, we successfully isolated a VZV from the vesicular fluid of a youth zoster patient. Based on the single-nucleotide polymorphism profiles of different open reading frames that define the genotype, this newly isolated VZV primarily represents genotype clade 2 but also has characteristics of genotype clade 1. The next-generation sequencing provided a nearly full-length sequence, and further phylogenetic analysis revealed that this VZV isolate is distinct from clades 1 and 2. The Recombination Detection Program indicates that a possible recombinant event may occur between the VZV isolate and clade 1. In summary, we found that there is a circulating VZV isolate in China that may represent a recombinant between clade 1 and clade 2, providing new concerns that need to be considered in the future VZV vaccination program.

Discovering Genomic Regions Associated with Reproductive Traits and Frame Score in Mexican Simmental and Simbrah Cattle Using Individual SNP and Haplotype Markers.

Reproductive efficiency stands as a critical determinant of profitability within beef production systems. The incorporation of molecular markers can expedite advancements in reproductive performance. While the use of SNPs in association analysis is prevalent, approaches centered on haplotypes can offer a more comprehensive insight. The study used registered Simmental and Simbrah cattle genotyped with the GGP Bovine 150 k panel. Phenotypes included scrotal circumference (SC), heifer fertility (HF), stayability (STAY), and frame score (FS). After quality control, 105,129 autosomal SNPs from 967 animals were used. Haplotype blocks were defined based on linkage disequilibrium. Comparison between haplotypes and SNPs for reproductive traits and FS was conducted using Bayesian and frequentist models. 23, 13, 7, and 2 SNPs exhibited associations with FS, SC, HF, and STAY, respectively. In addition, seven, eight, seven, and one haplotypes displayed associations with FS, SC, HF, and STAY, respectively. Within these delineated genomic segments, potential candidate genes were associated.

Prioritizing cardiovascular disease-associated variants altering NKX2-5 and TBX5 binding through an integrative computational approach.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide and are heavily influenced by genetic factors. Genome-wide association studies have mapped >90% of CVD-associated variants within the noncoding genome, which can alter the function of regulatory proteins, such as transcription factors (TFs). However, due to the overwhelming number of single-nucleotide polymorphisms (SNPs) (>500,000) in genome-wide association studies, prioritizing variants for in vitro analysis remains challenging. In this work, we implemented a computational approach that considers support vector machine (SVM)-based TF binding site classification and cardiac expression quantitative trait loci (eQTL) analysis to identify and prioritize potential CVD-causing SNPs. We identified 1535 CVD-associated SNPs within TF footprints and putative cardiac enhancers plus 14,218 variants in linkage disequilibrium with genotype-dependent gene expression in cardiac tissues. Using ChIP-seq data from two cardiac TFs (NKX2-5 and TBX5) in human-induced pluripotent stem cell-derived cardiomyocytes, we trained a large-scale gapped k-mer SVM model to identify CVD-associated SNPs that altered NKX2-5 and TBX5 binding. The model was tested by scoring human heart TF genomic footprints within putative enhancers and measuring in vitro binding through electrophoretic mobility shift assay. Five variants predicted to alter NKX2-5 (rs59310144, rs6715570, and rs61872084) and TBX5 (rs7612445 and rs7790964) binding were prioritized for in vitro validation based on the magnitude of the predicted change in binding and are in cardiac tissue eQTLs. All five variants altered NKX2-5 and TBX5 DNA binding. We present a bioinformatic approach that considers tissue-specific eQTL analysis and SVM-based TF binding site classification to prioritize CVD-associated variants for in vitro analysis.

Amerindian ancestry proportion as a risk factor for inflammatory bowel diseases: results from a Latin American Andean cohort.

Background and aims: Latin American populations remain underrepresented in genetic studies of inflammatory bowel diseases (IBDs). Most genetic association studies of IBD rely on Caucasian, African, and Asian individuals. These associations have yet to be evaluated in detail in the Andean region of South America. We explored the contribution of IBD-reported genetic risk variants to a Chilean cohort and the ancestry contribution to IBD in this cohort. Methods: A total of 192 Chilean IBD patients were genotyped using Illumina's Global Screening Array. Genotype data were combined with similar information from 3,147 Chilean controls. The proportions of Aymara, African, European, and Mapuche ancestries were estimated using the software ADMIXTURE. We calculated the odds ratios (ORs) and 95% confidence intervals (CIs) for gender, age, and ancestry proportions. We also explored associations with previously reported IBD-risk variants independently and in conjunction with genetic ancestry. Results: The first and third quartiles of the proportion of Mapuche ancestry in IBD patients were 24.7 and 34.2%, respectively, and the corresponding OR was 2.30 (95%CI 1.52-3.48) for the lowest vs. the highest group. Only one variant (rs7210086) of the 180 reported IBD-risk SNPs was associated with IBD risk in the Chilean cohort (adjusted P = 0.01). This variant is related to myeloid cells. Conclusion: The type and proportion of Native American ancestry in Chileans seem to be associated with IBD risk. Variants associated with IBD risk in this Andean region were related to myeloid cells and the innate immune response.

Genome-wide association study for growth traits in Blanco Orejinegro and Romosinuano cattle.

Growth traits are economically important characteristics for the genetic improvement of local cattle breeds. Genome-wide association studies (GWAS) provide valuable information to enhance the understanding on the genetics of complex traits. The aim of this study was to perform a GWAS to identify genomic regions and genes associated to birth weight, weaning weight adjusted for 240 days, 16 months, and 24 months weight in Romosinuano (ROMO) and Blanco Orejinegro (BON) cattle. A single-step genomic-BLUP was implemented using 596 BON and 569 ROMO individuals that were genotyped with an Illumina BovineSNP50 BeadChip. There were 25 regions of interest identified on different chromosomes, with few of them simultaneously associated with two or more growth traits and some were common to both breeds. The gene mapping allowed to find 173 annotations on these regions, from which 49 represent potential candidate genes with known growth-related functions in cattle and other species. Among the regions that were associated with several growth traits, that at 24 - 27 MB of BTA14, has important candidate genes such as LYPLA1, XKR4, TMEM68 and PLAG1. Another region of interest at 0.40-0.77 Mb of BTA23 was identified in both breeds, containing KHDRBS2 as a potential candidate gene influencing body weight. Future studies targeting these regions could provide more knowledge to uncover the genetic architecture underlying growth traits in BON and ROMO cattle. The genomic regions and genes identified in this study could be used to improve the prediction of genetic merit for growth traits in these creole cattle breeds.

The importance of deep genotyping in crop breeding / La importancia de la genotipificación profunda en el mejoramiento genético

ABSTRACT One of the greatest challenges facing humanity is the development of sustainable strategies to ensure food availability in response to population growth and climate change. One approach that can contribute to increase food security is to close yield gaps and enhancing genetic gain; to such end, what is known as "molecular breeding" plays a fundamental role. Since a crop breeding program is mainly based on the quality of the germplasm, its detailed genetic characterization is mandatory to ensure the efficient use of genetic resources and accelerating development of superior varieties. Deep genotyping is an essential tool for a comprehensive characterization of the germplasm of interest and, fortunately, the technology is now accessible at a reasonable cost. What must be ensured is the correct interpretation of the genotypic information and on that basis develop efficient practical molecular crop breeding strategies that respond to the real needs of the breeding program.

RESUMEN Uno de los mayores desafíos que enfrenta la humanidad es el desarrollo de estrategias sostenibles para asegurar la disponibilidad de alimentos en respuesta al crecimiento de la población y el cambio climático. Un enfoque que puede contribuir a aumentar la seguridad alimentaria es cerrar las brechas de rendimiento y mejorar la ganancia genética; para tal fin, lo que se conoce como "mejoramiento molecular" juega un papel fundamental. Dado que un programa de mejoramiento de cultivos se basa principalmente en la calidad del germoplasma, su caracterización genética detallada es fundamental para garantizar el uso eficiente de los recursos genéticos y acelerar el desarrollo de variedades superiores. La genotipificación profunda es una herramienta esencial para una caracterización integral del germoplasma de interés y, afortunadamente, en la actualidad se puede acceder a la tecnología a un costo razonable. Lo que debe asegurarse es la interpretación correcta de la información genotípica y sobre esa base desarrollar estrategias eficientes y prácticas de mejoramiento molecular de cultivos que respondan a las necesidades reales del programa de mejoramiento.

Performance of the tetra-primer PCR technique compared to PCR-RFLP in the search for rs12979860 (C/T) and rs8099917 (T/G) single nucleotide polymorphisms (SNPs) in the IFNL4 gene / Desempenho da técnica tetra-primer PCR em relação a PCR-RFLP na pesquisa de polimorfismos de nucleotídeos únicos (SNPs) rs12979860 (C/T) e rs8099917 (T/G) no gene IFNL4

Single nucleotide polymorphisms (SNPs, rs12979860 e rs8099917) in the Interferon Lambda 4 gene (IFNL4, formerly IFNL3and/or IL28B) has been associated with failure in the innate immune response, sustained virological response in hepatitis C, and HTLV-1-associated myelopathy (HAM) development. To search for these polymorphisms several methodologies can be employed, such as sequencing, real-time or quantitative polymerase chain reaction (qPCR), restriction fragment length polymorphism analysis in PCR products (PCR-RFLP), and tetra-primer PCR. The present study compared the performance of the tetra-primer PCR in relation to the PCR-RFLP, both optimized in the Research HTLV Laboratory of the Center of Immunology of Instituto Adolfo Lutz in São Paulo. One hundred DNA samples obtained from patients of STD/Aids Reference Centre in São Paulo, previously analyzed for IL28B SNPs by PCR-RFLP were selected for analysis, after confirming that they represent all IL28B SNPs patterns described in the literature. The results obtained showed concordance between the PCR-RFLP and the tetra-primer PCR SNPs results, and because of the low cost, easy to perform, and minor employment of biological specimen and reagents, the tetra-primer PCR is of choice to be used in routine. (AU)

Polimorfismos de nucleotídeos únicos (single nucleotide polymorphisms, SNPs rs12979860 e rs8099917) no gene que codifica o Interferon Lambda 4 (IFNL4, antigamente IFNL3 e/ou IL28B) têm sido associados às falhas na resposta imune inata e resposta virológica sustentada na hepatite C, e a mielopatia associada ao HTLV-1 (HTLV-1-associated myelopathy, HAM). A pesquisa destes polimorfismos pode empregar diversas metodologias: sequenciamento, reação em cadeia da polimerase em tempo real ou quantitativa (quantitative polymerase chain reaction, qPCR), análise de fragmentos de restrição enzimática em produtos de PCR (restriction fragment length polymorphism in PCR products, PCR-RFLP) e a tetra-primer PCR. Este estudo comparou o desempenho da tetra-primer PCR em relação a PCR-RFLP, ambas otimizadas no Laboratório de Pesquisa em HTLV do Centro de Imunologia do Instituto Adolfo Lutz de São Paulo. Foram selecionadas 100 amostras de DNA obtidas de pacientes do Centro de Referência e Treinamento em DST/Aids de São Paulo cujos SNPs na IL28B foram anteriormente determinados por PCR-RFLP e representaram todos os perfis descritos em literatura. Os resultados obtidos mostraram concordância entre elas, e pelo fato da tetra-primer PCR ter menor custo, ser de fácil execução, empregar menos tempo, insumos e material biológico, é a técnica de escolha para uso em rotina. (AU)

ASGARD+: A New Modular Platform for Bacterial Antibiotic-Resistant Analysis.

ASGARD+ (Accelerated Sequential Genome-analysis and Antibiotic Resistance Detection) is a command-line platform for automatic identification of antibiotic-resistance genes in bacterial genomes, providing an easy-to-use interface to process big batches of sequence files from whole genome sequencing, with minimal configuration. It also provides a CPU-optimization algorithm that reduces the processing time. This tool consists of two main protocols. The first one, ASGARD, is based on the identification and annotation of antimicrobial resistance elements directly from the short reads using different public databases. SAGA, enables the alignment, indexing, and mapping of whole-genome samples against a reference genome for the detection and call of variants, as well as the visualization of the results through the construction of a tree of SNPs. The application of both protocols is performed using just one short command and one configuration file based on JSON syntax, which modulates each pipeline step, allowing the user to do as many interventions as needed on the different software tools that are adapted to the pipeline. The modular ASGARD+ allows researchers with little experience in bioinformatic analysis and command-line use to quickly explore bacterial genomes in depth, optimizing analysis times and obtaining accurate results. © 2023 Wiley Periodicals LLC. Basic Protocol 1: ASGARD+ installation Basic Protocol 2: Configuration files general setup Basic Protocol 3: ASGARD execution Support Protocol: Results visualization with Phandango Basic Protocol 4: SAGA execution Alternative Protocol 1: Container installation Alternative Protocol 2: Run ASGARD and SAGA in container.

Polyploid SNP Genotyping Using the MassARRAY System.

Molecular marker discovery and genotyping are major challenges in polyploid breeding programs incorporating molecular biology tools. In this context, this work describes a method for single nucleotide polymorphism (SNP) genotyping in polyploid crops using matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry, the MassARRAY System.

Genome-wide association study for resistance in bread wheat (Triticum aestivum L.) to stripe rust (Puccinia striiformis f. sp. tritici) races in Argentina.

BACKGROUND: Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most devastating diseases of the wheat crop. It causes significant reductions in both grain yield and grain quality. In recent years, new and more virulent races have overcome many of the known resistance genes in Argentinian germplasm. In order to identify loci conferring resistance to the local races of Pst for effective utilization in future breeding programs, a genome-wide association study (GWAS) was performed using a collection of 245 bread wheat lines genotyped with 90 K SNPs. RESULTS: To search for adult plant resistance (APR) the panel was evaluated for disease severity (DS) and area under disease progress curve (AUDPC) in field trials during two years under natural infection conditions. To look for seedling or all-stage resistance (ASR) the panel was evaluated to determine infection type (IT) under greenhouse conditions against two prevalent races in Argentina. The phenotypic data showed that the panel possessed enough genetic variability for searching for sources of resistance to Pst. Significant correlations between years were observed for Pst response in the field and high heritability values were found for DS (H2 = 0.89) and AUDPC (H2 = 0.93). Based on GWAS, eight markers associated with Pst resistance (FDR < 0.01) were identified, of these, five were associated with ASR (on chromosomes 1B, 2A, 3A and 5B) and three with APR (on chromosomes 3B and 7A). These markers explained between 2% and 32.62% of the phenotypic variation. Five of the markers corresponded with previously reported Yr genes/QTL, while the other three (QYr.Bce.1B.sd.1, QYr.Bce.3A.sd and QYr.Bce.3B.APR.2) might be novel resistance loci. CONCLUSION: Our results revealed high genetic variation for resistance to Argentinian stripe rust races in the germplasm used here. It constitutes a very promising step towards the improvement of Pst resistance of bread wheat in Argentina. Also, the identification of new resistance loci would represent a substantial advance for diversifying the current set of resistance genes and to advance in the improvement of the durable resistance to the disease.

Inflammasome genetic variants are associated with tuberculosis, HIV-1 infection, and TB/HIV-immune reconstitution inflammatory syndrome outcomes.

Background: Tuberculosis (TB) and AIDS are the leading causes of infectious diseases death worldwide. Here, we investigated the relationship between from single nucleotide polymorphisms (SNPs) of the NLRP3, CARD8, AIM2, CASP-1, IFI16, and IL-1ß inflammasome genes, as well as the profiles of secreted proinflammatory cytokines (e.g., IL-1ß, IL-18, IL-33, and IL-6) with the TB clinical profiles, TB-HIV coinfection, and IRIS onset. Methods: The individuals were divided into four groups: TB-HIV group (n=88; 11 of them with IRIS), HIV-1 group (n=20), TB group (n=24) and healthy volunteers (HC) group (n=10), and were followed up at INI/FIOCRUZ and HGNI (Rio de Janeiro/Brazil) from 2006 to 2016. Real-time PCR was used to determine the genotypes of the Single Nucleotide Polymorphism (SNPs), and ELISA was used to measure the plasma cytokine levels. Unconditional logistic regression models were used to perform risk estimations. Results: A higher risk for extrapulmonary TB was associated with the TT genotype (aOR=6.76; P=0.026) in the NLRP3 rs4612666 Single Nucleotide Polymorphism (SNP) and the C-C-T-G-C haplotype (aOR=4.99; P= 0.017) in the NLRP3 variants. This same Single Nucleotide Polymorphism (SNP) was associated with lower risk against extrapulmonary TB when the carrier allele C (aOR=0.15; P=0.021) was present. Among those with HIV-1 infections, a higher risk for TB onset was associated with the GA genotype (aOR=5.5; P=0.044) in the IL1-ß rs1143634 Single Nucleotide Polymorphism (SNP). In contrast, lower risk against TB onset was associated with the A-G haplotype (aOR=0.17; P= 0.026) in the CARD8 variants. Higher IL-6 and IL-33 levels were observed in individuals with TB. A higher risk for IRIS onset was associated with CD8 counts ≤ 500 cells/mm3 (aOR=12.32; P=0.010), the presence of extrapulmonary TB (aOR=6.6; P=0.038), and the CT genotype (aOR=61.06; P=0.026) or carrier allele T (aOR=61.06; P=0.026) in the AIM2 rs2276405 Single Nucleotide Polymorphism (SNP), whereas lower risk against IRIS onset was associated with the AT genotype (aOR=0.02; P=0.033) or carrier allele T (aOR=0.02; P=0.029) in the CARD8 rs2043211 Single Nucleotide Polymorphism (SNP) and the T-G haplotype (aOR=0.07; P= 0.033) in the CARD8 variants. No other significant associations were observed. Conclusions: Our results depict the involvement of genetic polymorphisms of crucial innate immunity genes and proinflammatory cytokines in the clinical outcomes related to TB-HIV coinfection.

Genome-Environment Association Analysis for Bio-Climatic Variables in Common Bean (Phaseolus vulgaris L.) from Brazil.

Abiotic stress is a limiting factor for common bean (Phaseolus vulgaris L.) production globally. The study of the genotypic, phenotypic, and bio-climatic variables in a broad set of accessions may assist the identification of genomic regions involved in the climatic adaptation of the common bean. We conducted a genotyping-by-sequencing analysis using 28,823 SNPs on 110 georeferenced common bean accessions from Brazil to discover associations between SNPs and bio-climatic indexes. The population structure analysis clustered the accessions into two groups corresponding to the Andean and Mesoamerican gene pools. Of the 19 bioclimatic variables, 17 exhibited a significant association with SNPs on chromosomes Pv01, Pv02, Pv03, Pv04, Pv06, Pv09, Pv10, and Pv11 of common bean. Ten candidate genes were associated with specific bio-climatic variables related to temperature and precipitation. The candidate genes associated with this significant Pv09 region encode a Platz transcription factor family protein previously reported to be an essential regulator of drought stress. The SNP markers and candidate genes associated with the bio-climatic variables should be validated in segregating populations for water stress, which could further be used for marker-assisted selection. As a result, bean breeding programs may be able to provide advances in obtaining drought-tolerant cultivars.

Interleukin-1ß-31 (rs1143627) genetic variant and the risk of age-related macular degeneration in the Brazilian population.

Background: Age-related macular degeneration (AMD) is a multifactorial disease and one of the main causes of blindness in people over 50 years old. The etiology and pathophysiology of AMD are not well understood. The aim of this study was to investigate whether the rs1143627 variant allele of IL1B, which encodes Interleukin (IL)-1ß, a key cytokine, mediates immune and inflammatory responses.Methods: A case-control study was conducted with 397 AMD patients and 402 controls in Brazil. IL1B genotyping was carried out with TaqMan® genotyping assay. Differences in IL1B allele frequencies and genotypes were evaluated between patients and controls and between wet and dry subgroups of AMD. Relationships between allele presence/genotype and disease risk are reported as odds ratios (ORs) with 95% confidence intervals (CIs).Results: Genotype proportions for the rs1143627 variant allele of IL1B were similar between AMD patients and controls (p = .21), with 84.38% of AMD patients and 79.60% of the controls carrying the variant allele. We observed a trend toward the variant allele being associated with AMD risk (OR = 1.38, 95% CI 0.95-2.03, p = .08), as well as a trend toward the variant allele being associated with increased risk for wet AMD in particular (OR = 1.23, 95% CI 0.96-1.56, p = .08).Conclusions: The rs1443627 variant was not associated with AMD risk in this Brazilian population sample. Larger studies are warranted to determine whether the trends observed in this study reflect a relationship between this variant and risk of AMD, especially wet AMD.

Bovine TMEM95 gene: Polymorphisms detecting in five Chinese indigenous cattle breeds and their association with growth traits

BACKGROUND: Transmembrane protein 95 (TMEM95) plays a role in male fertility. Previous studies showed that genes with a significant impact on reproductive traits can also affect the growth traits of livestock. Thus, we speculated that the genetic variation of TMEM95 gene may have effects on growth traits of cattle. RESULTS: Two SNPs were genotyped. The rs136174626 and rs41904693 were in the intron 4 and 30 -untranslated region, respectively. The linkage disequilibrium analysis illustrated that these two loci were not linked. The rs136174626 was associated with six growth traits of Nanyang cattle, four traits of Luxi cattle, and three traits of Ji'an cattle. For rs41904693 locus, the GG individuals had greater body height and abdominal girth in Ji' an cattle than TT and TG individuals. In Jinnan cattle, GG and TT individuals had greater body height, height at hip cross, body length, and heart girth than TG individuals. The potential splice site prediction results suggest that the rs136174626 may influence the splicing efficiency of TMEM95, and the miRNA binding site prediction results showed that the rs41904693 may influence the expression of TMEM95 by affecting the binding efficiency of Bta-miR-1584 and TMEM95 30 -UTR. CONCLUSIONS: The findings of the study suggested that the two SNPs in TMEM95 could be a reliable basis for molecular breeding in cattle.

M. bovis BCG Moreau N-Terminal Loss Leads to a Less Stable Dodecin With Lower Flavin Binding Capacity.

Tuberculosis still remains a concerning health problem worldwide. Its etiologic agent, Mycobacterium tuberculosis, continues to be the focus of research to unravel new prophylactic and therapeutic strategies against this disease. The only vaccine in use against tuberculosis is based on the in vitro attenuated strain, M. bovis BCG. Dodecin is a dodecameric complex important for flavin homeostasis in Archea and Eubacteria, and the M. tuberculosis protein is described as thermo- and halostable. M. bovis BCG Moreau, the Brazilian vaccine strain, has a single nucleotide polymorphism in the dodecin start codon, leading to a predicted loss of seven amino acids at the protein N-terminal end. In this work we aimed to characterize the effect of this mutation in the BCG Moreau protein features. Our recombinant protein assays show that the predicted BCG homolog is less thermostable than M.tb's but maintains its dodecamerization ability, although with a lower riboflavin-binding capacity. These data are corroborated by structural analysis after comparative modeling, showing that the predicted BCG dodecin complex has a lower interaction energy among its monomers and also a distinct electrostatic surface near the flavin binding pocket. However, western blotting assays with the native proteins were unable to detect significant differences between the BCG Moreau and M.tb orthologs, indicating that other factors may be modulating protein structure/function in the bacterial context.

Diseño y validación de un ensayo de minisecuenciación múltiple para detectar polimorfismos asociados con Síndrome Metabólico / Design and validation of a multiple minisequencing assay to detect polymorphisms associated with Metabolic Syndrome

Resumen Introducción: Es importante identificar los polimorfismos de interés clínico en patologías complejas como el Síndrome Metabólico. Por esto, las metodologías para su evaluación deben estar diseñadas y validadas correctamente, esto permite optimizar recursos y tiempo en la genotipificación y detección correcta de los alelos presentes en los individuos. Objetivo: Diseñar y validar una PCR múltiple, seguida de detección por minisecuenciación, para la genotipificación de ocho polimorfismos de nucleótido simple ubicados en el gen del Receptor Beta 3-Adrenérgico (rs4994 y rs4998), gen de la Apolipoproteina A5 (rs3135506 y rs2075291), gen de la Adiponectina (rs1501299 y rs2241766) y gen del Receptor Activador de la Proliferación de los Peroxisomas tipo gamma (rs1801282 y rs1800571), asociados con el síndrome metabólico. Materiales y métodos: Se diseñaron 24 cebadores para la amplificación y detección de ocho polimorfismos de nucleótido sencillo ubicados en cuatro genes candidatos a estar asociados con el síndrome metabólico, usando el software Primer3®. Dieciséis fueron diseñados para amplificar los polimorfismos y ocho para detectarlos por minisecuenciación. Las estructuras secundarias entre los cebadores se verificaron con el software Autodimer. Los polimorfismos se amplificaron simultáneamente y los fragmentos amplificados se acoplaron a las sondas diseñadas para detectar por minisecuenciación el alelo presente, por medio de bases marcadas con fluorocromos. Finalmente, los alelos fueron detectados por electroforesis capilar en un analizador genético ABI 310 y se interpretaron con el software GeneMapper®. La validación del multiplex se realizó genotipando 20 muestras de individuos, cada uno de ellos autorizó este procedimiento por medio del consentimiento informado. Resultados: Se obtuvieron los perfiles genéticos de los 20 controles genotipados, a partir de la amplificación múltiple, seguida de minisecuenciación, diseñada y validada para detectar los ocho polimorfismos. Conclusión: Se diseñó y validó un ensayo para la detección simultánea de los polimorfismos, ubicados en cuatro genes asociados con el Síndrome metabólico. Los cuales pueden ser empleados como referencia para futuros estudios poblacionales.

Abstract Introduction: It is important to identify the polymorphisms of clinical interest in complex pathologies such as Metabolic Syndrome. Therefore, the methodologies for its evaluation must be designed and validated correctly, this permits optimization of resources and time in genotyping and correct detection of the alleles present in individuals. Objective: To design and validate a multiplex PCR, followed by detection by minisequencing, for the genotyping of eight single nucleotide polymorphisms located in the Beta 3-Adrenergic Receptor gene (rs4994 and rs4998), Apolipoprotein A5 gene (rs3135506 and rs2075291), Adiponectin gene (rs1501299 and rs2241766) and gamma-type Peroxisome Proliferation Activating Receptor gene (rs1801282 and rs1800571), associated with metabolic syndrome. Materials and methods: Twenty-four primers were designed for the amplification and detection of eight single nucleotide polymorphisms located in four candidate genes to be associated with the metabolic syndrome, using the Primer3® software. Sixteen were designed to amplify the polymorphisms and eight to detect them by minisequencing. The secondary structures between the primers were verified with Autodimer software. The polymorphisms were simultaneously amplified, and the amplified fragments were coupled to probes designed to minisequence the present allele using fluorochrome-labeled bases. Finally, the alleles were detected by capillary electrophoresis using an ABI 310 genetic analyzer and analyzed with the GeneMapper® software. The validation of the multiplex was performed by genotyping 20 individual samples, each of them authorized this procedure through informed consent. Results: The genetic profiles of the 20 genotyped controls were obtained, from multiple amplification, followed by minisequencing, designed and validated to detect the eight polymorphisms. Conclusion: An essay was designed and validated for the simultaneous detection of polymorphisms, located in four genes associated with metabolic syndrome, and can used as a reference for future population studies.

Variants in HSD11B1 gene modulate susceptibility to diabetes kidney disease and to insulin resistance in type 1 diabetes.

BACKGROUND AND AIM: 11ß-Hydroxysteroid dehydrogenase 1 has been implicated in insulin resistance (IR) in the setting of metabolic disorders, and single nucleotide polymorphisms (SNPs) in its encoding gene (HSD11B1) have been associated with type 2 diabetes and metabolic syndrome. In type 1 diabetes (T1D), IR has been related to the development of chronic complications. We investigated the association of HSD11B1 SNPs with microvascular complications and with IR in a Brazilian cohort of T1D individuals. MATERIALS AND METHODS: Five SNPs were genotyped in 466 T1D individuals (57% women; median of 37 years old, diabetes duration of 25 years and HbA1c of 8.4%). RESULTS: The minor allele T of rs11799643 was nominally associated with diabetic retinopathy (OR = 0.52; confidence interval [CI] 95% = 0.28-0.96; P = .036). The minor allele C of rs17389016 was nominally associated with overt diabetic kidney disease (DKD) (OR = 1.90; CI 95% = 1.07-3.37; P = .028). A follow-up study revealed that 29% of the individuals lost ≥5 mL min-1 × 1.73 m2 per year of the estimated glomerular filtration rate (eGFR). In these individuals (eGFR decliners), C allele of rs17389016 was more frequent than in non-decliners (OR = 2.10; CI 95% = 1.14-3.89; P = .018). Finally, minor allele T of rs846906 associated with higher prevalence of arterial hypertension, higher body mass index and waist circumference, thus conferring risk to a lower estimated glucose disposal rate, a surrogate marker of insulin sensitivity (OR = 1.23; CI 95% = 1.06-1.42; P = .004). CONCLUSION: SNPs in the HSD11B1 gene may confer susceptibility to DKD and to IR in T1D individuals.

Outcomes of Japanese patients with non-alcoholic fatty liver disease according to genetic background and lifestyle-related diseases.

INTRODUCTION AND OBJECTIVES: Genetic background may be involved in the mechanisms of liver injury and the development of non-alcoholic fatty liver disease (NAFLD). However, its contributions to the long-term outcome of NAFLD have been unclear. METHODS: We enrolled 314 Japanese patients with biopsy-confirmed NAFLD from 2000 to 2018 (161 men [51.3%]; median age, 53 [14-84] years; 114 with advanced fibrosis [37.5%]) in the patients without hepatocellular carcinoma at diagnosis. Genomic DNA was extracted from peripheral blood and single nucleotide polymorphisms (SNPs) were analyzed. Associations of mortality with patatin-like phospholipase 3 (PNPLA3) and aldehyde dehydrogenase 2 (ALDH2) were analyzed. Finally, a subgroup analysis according to lifestyle-related disease was performed. RESULTS: During the median 7 years of follow-up, 20 patients (6.4%) died (13 liver-related [4.1%] and 7 non-liver-related deaths [2.2%]). Patients with ALDH2 (non-GG genotype) who had reduced alcohol metabolism tended to have a poor prognosis (p = 0.06). Patients carrying both risk SNPs of PNPLA3 (GG) and ALDH2 (non-GG) had a significantly poor prognosis (p = 0.01). In the subgroup analysis, patients with PNPLA3 (GG) who were non-diabetics (p = 0.06) or non-dyslipidemic (p = 0.03), with ALDH2 (non-GG) who were non-dyslipidemic (p = 0.01) or hypertensive (p = 0.03), also had a poor prognosis. The Cox analysis revealed that ALDH2 (non-GG) was associated with a poor prognosis (Hazard ratio: 4.568, 95% Confidence Interval: 1.294-16.131, p = 0.02) similar to the liver function tests. CONCLUSIONS: Genetic background may affect NAFLD prognosis and ALDH2 SNP could predict the outcome.

Serum mir-195-5p exhibits clinical significance in the diagnosis of essential hypertension with type 2 diabetes

OBJECTIVES: Diagnosis and management of essential hypertension (EH) or type 2 diabetes mellitus (T2DM) by combining comprehensive treatment and classificatory diagnosis have been continuously improved. However, understanding the pathogenesis of EH patients with concomitant T2DM and subsequent treatment remain the major challenges owing to the lack of non-invasive biomarkers and information regarding the underlying mechanisms. METHODS: Herein, we collected 200 serum samples from EH and/or T2DM patients and healthy donors (N). Gene-expression profiling was conducted to identify candidate microRNAs with clinical significance. Then, a larger cohort of the aforementioned patients and 50 N were used to identify the correlation between the tumor suppressor miR-195-5p and EH and/or T2DM. The dual-luciferase reporter assay was used to explore the target genes of miR-195-5p. The suppressive effects of miR-195-5p on the 3′-UTR of the dopamine receptor D1 (DRD1) transcript in EH patients with concomitant T2DM were verified as well. RESULTS: Compared with that in other groups, serum miR-195-5p was highly downregulated in EH patients with concomitant T2DM. miR-195-5p overexpression efficiently suppressed DRD1 expression by binding to the two 3′-UTRs. Additionally, two single nucleotide polymorphisms, including 231T-A and 233C-G, in the miR-195-5p binding sites of the DRD1 3′-UTR were further identified. Collectively, we identified the potential clinical significance of DRD1 regulation by miR-195-5p in EH patients with concomitant T2DM. CONCLUSIONS: Our data suggested that miR-195-5p circulating in the peripheral blood served as a novel biomarker and therapeutic target for EH and T2DM, which could eventually help address major challenges during the diagnosis and treatment of EH and T2DM.