Farmacogenética y trastornos mentales / Pharmacogenetics and mental disorders

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Advancements in molecular marker technologies and their applications in diversity studies.

Crop improvement is a continuous effort, since some 10,000 years ago when primitive man made the transition from hunting and foraging to domestication and crop cultivation. Since then, man-made interventions have changed the entire scenario of crop evolution, by means of genetic alterations of plants and animals made to satisfy man's needs. The process of domestication has led to dramatic changes in their appearance, quality and productivity that have contributed substantially to global food security. The tremendous decline in cultivable land, freshwater, and increasing risk of biotic and abiotic stress demand immediate attention on crop improvement to cope with the higher demand of ∼ 40% of the food by 2020. Therefore, plant genetic variation plays a key role in plant breeding for its improvement. Most of the genetic variations useful for crop improvement have been deposited and maintained in seed gene banks across the world; they need to be brought into the mainstream of breeding lines. Recent advances and progress made in molecular markers have been substantial tools for deeper insights of genetics, and greatly complemented breeding strategies. Integration of the next-generation sequencing (NGS) technologies with precise phenotyping, association mapping, proteome and metabolome studies has increased the chances of finding candidate genes and their allelic variants controlling a trait of interest. Further, these functional markers (FMMs), genotype-by-sequencing and association mapping methodologies have opened new avenues for identification of novel genetic resources (lines) that can facilitate accelerated crop breeding programs for increased yield, high nutritional quality, and tolerance to a variety of abiotic and biotic stresses. The details of popular molecular markers, advancement in the technologies and strategies for crop diversity studies and their application in crop breeding programs are presented here.

Cryptosporidium genotyping in Europe: The current status and processes for a harmonised multi-locus genotyping scheme.

Due to the occurrence of genetic recombination, a reliable and discriminatory method to genotype Cryptosporidium isolates at the intra-species level requires the analysis of multiple loci, but a standardised scheme is not currently available. A workshop was held at the Robert Koch Institute, Berlin in 2016 that gathered 23 scientists with appropriate expertise (in either Cryptosporidium genotyping and/or surveillance, epidemiology or outbreaks) to discuss the processes for the development of a robust, standardised, multi-locus genotyping (MLG) scheme and propose an approach. The background evidence and main conclusions were outlined in a previously published report; the objectives of this further report are to describe 1) the current use of Cryptosporidium genotyping, 2) the elicitation and synthesis of the participants' opinions, and 3) the agreed processes and criteria for the development, evaluation and validation of a standardised MLG scheme for Cryptosporidium surveillance and outbreak investigations. Cryptosporidium was characterised to the species level in 7/12 (58%) participating European countries, mostly for human outbreak investigations. Further genotyping was mostly by sequencing the gp60 gene. A ranking exercise of performance and convenience criteria found that portability, biological robustness, typeability, and discriminatory power were considered by participants as the most important attributes in developing a multilocus scheme. The major barrier to implementation was lack of funding. A structured process for marker identification, evaluation, validation, implementation, and maintenance was proposed and outlined for application to Cryptosporidium, with prioritisation of Cryptosporidium parvum to support investigation of transmission in Europe.

Into the wild-WAMBAM goes to Canada.

The sixth Wild Animal Models Bi-Annual Meeting was held in July 2017 in Québec, with 42 participants. This report documents the evolution of questions asked and approaches used in evolutionary quantitative genetic studies of wild populations in recent decades, and how these questions and approaches were represented at the recent meeting. We explore how ideas from previous meetings in this series have developed to their present states, and consider how the format of the meetings may be particularly useful at fostering the rapid development and proliferation of ideas and approaches.

Factor IX Gene (F9) Genotyping Trends and Spectrum of Mutations Identified: A Reference Laboratory Experience.

In hemophilia B (HB), factor IX gene (F9) genotyping is used for molecular confirmation of affected individuals, for carrier testing, to facilitate the identification of those at risk for anaphylaxis/inhibitors (associated with large deletions), and to assist in assigning disease severity. Owing to test costs, optimal test utilization involves pre/post-test counseling and appropriate patient and test selection (e.g., mutation screening [F9MS] vs. known mutation [F9KM] testing). This article aims to review the trends and outcomes of F9-genotyping orders and describe the spectrum of variants identified in a sample of individuals in our reference laboratory. We performed a retrospective review of consecutive orders submitted to the Special Coagulation DNA Diagnostic Laboratory, Mayo Clinic, between 2012 and 2015. A total of 133 orders (38%) were identified for men: 118 (88%) were F9MS and 15 (12%) were F9KM. Thirteen orders (10%) were cancelled. A total of 209 orders were identified for women: 178 (85%) were F9MS and 31 (15%) were F9KM. Thirty-seven orders (18%) were cancelled and 30% of the tests performed yielded negative results. A total of 164 samples (47%) were received without clinical information. Seventeen previously unreported variants were identified. F9 genotyping provides useful information for HB management; however, 18% of our orders were cancelled and almost half were received without relevant clinical information, thus reaffirming the need for ongoing scrutiny of submitted orders. Optimal patient and test selection is important as is the accurate interpretation of variants identified. Most of the pathogenic variants identified were point mutations, with very few large deletions, consistent with the literature.

Common Genetic Variation and Susceptibility to Ovarian Cancer: Current Insights and Future Directions.

In this review, we summarize current progress in the genetic epidemiology of epithelial ovarian cancer (EOC), focusing exclusively on elucidating the role of common germline genetic variation in conferring susceptibility to EOC. We provide an overview of the more than 30 EOC risk loci identified to date by genome-wide association studies (GWAS) and describe the contribution of large-scale, cross-cancer type, custom genotyping projects, such as the OncoArray and the Collaborative Oncological Gene-Environment Study, to locus discovery and replication. We discuss the histotype-specific nature of these EOC risk loci, pleiotropy, or overlapping genetic effects between EOC and other hormone-related cancer types, and the application of findings to polygenic risk prediction for EOC. The second part of the article offers a concise review of primarily laboratory-based studies that have led to the identification of several putative EOC susceptibility genes using common variants at the known EOC risk loci as starting points. More global biological insights emerging from network- and pathway-based analyses of GWAS for EOC susceptibility are also highlighted. Finally, we delve into potential future directions, including the need to identify EOC risk loci in non-European populations and the next generation of GWAS functional studies that are likely to involve genome editing to establish the cell type-specific carcinogenic effects of EOC risk variants Cancer Epidemiol Biomarkers Prev; 27(4); 395-404. ©2018 AACRSee all articles in this CEBP Focus section, "Genome-Wide Association Studies in Cancer."

Liquid Biopsy: Approaches to Dynamic Genotyping in Cancer.

Malignant tumors release tumor cells and fragments of nucleic acids into the bloodstream. Liquid biopsies are non-invasive blood tests that detect circulating tumor cells (CTC) and circulating nucleic acids such as mRNA, microRNA, and cell-free circulating tumor DNA, also known as ctDNA. The presence of ctDNA or CTCs in the plasma has prognostic impact. Since ctDNA contains tumor-specific mutations, its detection in the blood or other body fluids can predict response to treatment and relapse. Moreover, repeated analysis and quantitation of ctDNA can inform about changes in clonal composition over time and thus allow dynamic treatment stratification. Today, the routine clinical use of liquid biopsy diagnostic tests is limited; however, in the near future, they might become commonly used sensitive and specific biomarkers to guide cancer treatment. This review will summarize recent findings on the use of ctDNA for monitoring response to therapy and dynamic genetic treatment stratification.

Future technologies for monitoring HIV drug resistance and cure.

PURPOSE OF REVIEW: Sensitive, scalable and affordable assays are critically needed for monitoring the success of interventions for preventing, treating and attempting to cure HIV infection. This review evaluates current and emerging technologies that are applicable for both surveillance of HIV drug resistance (HIVDR) and characterization of HIV reservoirs that persist despite antiretroviral therapy and are obstacles to curing HIV infection. RECENT FINDINGS: Next-generation sequencing (NGS) has the potential to be adapted into high-throughput, cost-efficient approaches for HIVDR surveillance and monitoring during continued scale-up of antiretroviral therapy and rollout of preexposure prophylaxis. Similarly, improvements in PCR and NGS are resulting in higher throughput single genome sequencing to detect intact proviruses and to characterize HIV integration sites and clonal expansions of infected cells. SUMMARY: Current population genotyping methods for resistance monitoring are high cost and low throughput. NGS, combined with simpler sample collection and storage matrices (e.g. dried blood spots), has considerable potential to broaden global surveillance and patient monitoring for HIVDR. Recent adaptions of NGS to identify integration sites of HIV in the human genome and to characterize the integrated HIV proviruses are likely to facilitate investigations of the impact of experimental 'curative' interventions on HIV reservoirs.

Mycobacterium tuberculosis drug-resistance testing: challenges, recent developments and perspectives.

Drug-resistance testing, or antimicrobial susceptibility testing (AST), is mandatory for Mycobacterium tuberculosis in cases of failure on standard therapy. We reviewed the different methods and techniques of phenotypic and genotypic approaches. Although multiresistant and extensively drug-resistant (MDR/XDR) tuberculosis is present worldwide, AST for M. tuberculosis (AST-MTB) is still mainly performed according to the resources available rather than the drug-resistance rates. Phenotypic methods, i.e. culture-based AST, are commonly used in high-income countries to confirm susceptibility of new cases of tuberculosis. They are also used to detect resistance in tuberculosis cases with risk factors, in combination with genotypic tests. In low-income countries, genotypic methods screening hot-spot mutations known to confer resistance were found to be easier to perform because they avoid the culture and biosafety constraint. Given that genotypic tests can rapidly detect the prominent mechanisms of resistance, such as the rpoB mutation for rifampicin resistance, we are facing new challenges with the observation of false-resistance (mutations not conferring resistance) and false-susceptibility (mutations different from the common mechanism) results. Phenotypic and genotypic approaches are therefore complementary for obtaining a high sensitivity and specificity for detecting drug resistances and susceptibilities to accurately predict MDR/XDR cure and to gather relevant data for resistance surveillance. Although AST-MTB was established in the 1960s, there is no consensus reference method for MIC determination against which the numerous AST-MTB techniques can be compared. This information is necessary for assessing in vitro activity and setting breakpoints for future anti-tuberculosis agents.

Capturing Darwin's dream.

Evolutionary biologists from Darwin forward have dreamed of having data that would elucidate our understanding of evolutionary history and the diversity of life. Sequence capture is a relatively old DNA technology, but its use is growing rapidly due to advances in (i) massively parallel DNA sequencing approaches and instruments, (ii) massively parallel bait construction, (iii) methods to identify target regions and (iv) sample preparation. We give a little historical context to these developments, summarize some of the important advances reported in this special issue and point to further advances that can be made to help fulfill Darwin's dream.

CRISPR-Based Typing and Next-Generation Tracking Technologies.

Bacteria occur ubiquitously in nature and are broadly relevant throughout the food supply chain, with diverse and variable tolerance levels depending on their origin, biological role, and impact on the quality and safety of the product as well as on the health of the consumer. With increasing knowledge of and accessibility to the microbial composition of our environments, food supply, and host-associated microbiota, our understanding of and appreciation for the ratio of beneficial to undesirable bacteria are rapidly evolving. Therefore, there is a need for tools and technologies that allow definite, accurate, and high-resolution identification and typing of various groups of bacteria that include beneficial microbes such as starter cultures and probiotics, innocuous commensals, and undesirable pathogens and spoilage organisms. During the transition from the current molecular biology-based PFGE (pulsed-field gel electrophoresis) gold standard to the increasingly accessible omics-level whole-genome sequencing (WGS) N-gen standard, high-resolution technologies such as CRISPR-based genotyping constitute practical and powerful alternatives that provide valuable insights into genome microevolution and evolutionary trajectories. Indeed, several studies have shown potential for CRISPR-based typing of industrial starter cultures, health-promoting probiotic strains, animal commensal species, and problematic pathogens. Emerging CRISPR-based typing methods open new avenues for high-resolution typing of a broad range of bacteria and constitute a practical means for rapid tracking of a diversity of food-associated microbes.

Valor predictivo y utilidad clínica del genotipado HLA-DQ en el diagnóstico de la enfermedad celiaca en mayores de 50 años / Predictive value and clinical utility of HLA-DQ typing in diagnosis of celiac disease in patients over 50

Introducción. Existe un alto número de casos no diagnosticados de enfermedad celiaca, especialmente en individuos de edad avanzada. El objetivo del presente trabajo es determinar el valor predictivo del genotipado de HLA-DQ en el diagnóstico de la enfermedad celiaca en mayores de 50 años y analizar una posible relación entre la gradación de riesgo atribuida a los alelos y el diagnóstico en edad avanzada. Materiales y métodos. Seiscientos treinta y cinco pacientes fueron estudiados durante 2013 con clínica sugestiva de enfermedad celiaca. El diagnóstico fue confirmado mediante estudios serológicos y biopsia intestinal. El genotipado del HLA-DQ se realizó mediante una técnica PCR-SSOP. Resultados. Un 10,7% de los pacientes estudiados eran mayores de 50 años con un ratio hombre mujer 1:3. La frecuencia de los alelos considerados de riesgo para la enfermedad celiaca (HLA-DQ2.5 y/o HLA-DQ8) fue del 87,5% (56 pacientes), siendo celiacos 13 de ellos. Veintiún pacientes portaban solo un alelo del DQ2.5 (DQA1*05 o DQB1*02) sin confirmarse el diagnóstico de celiaquía. Un paciente celiaco confirmado no expresaba ni DQ2 ni DQ8. El valor predictivo positivo del genotipado de HLA-DQ para el diagnóstico de EC en la población mayor de 50 años fue del 29,27% y el valor predictivo negativo del 93%. Conclusiones. Debido al alto valor predictivo negativo, la determinación del HLA-DQ es un marcador útil en el diagnóstico de la EC en individuos de edad avanzada con clínica asociada. No se encontró relación entre los alelos considerados de menor riesgo y la aparición tardía de la enfermedad (AU)

Introduction. Celiac disease is significantly undiagnosed, especially in older individuals. The aim of this study is to determine the predictive value of HLA-DQ typing in the diagnosis of celiac disease in patients over 50 and analyze the possible relationship between the gradation of risk attributed to the alleles and diagnosis in elderly. Materials and methods. 635 patients were studied during 2013 with suggestive symptoms of celiac disease. The diagnosis was confirmed by serologic studies and small bowel biopsy. The HLA-DQ genotyping was performed using a PCR-SSOP technique. Results. 68 of out 635 patients studied (10.7%) were older than 50 years with a male to female ratio of 1:3. The frequency of the alleles that has been associated with risk of CD (HLA DQ2.5 and/or HLA DQ8) was 87.5% (56 patients), being celiac 13 of them. 21 patients carry the half allele of HLA-DQ2.5 (DQA1*05 or DQB1*02) associated with low risk of celiac disease being none of them celiac. One patient with celiac disease did not carry DQ2.5 or DQ8 heterodimers but was positive for serological and histological analysis. The positive predictive value of the use of HLA testing in the population older than 50 years is 29.27% and the negative predictive value is 93%. Conclusions. Due to the high negative predictive value, the determination of HLA-DQ is a useful marker in the diagnosis of CD, in individuals over 50 with associated clinical. There was no relationship between low risk alleles and late onset of disease (AU)

Association of PSMA4 polymorphisms with lung cancer susceptibility and response to cisplatin-based chemotherapy in a Chinese Han population

Purpose. Genetic factors play an important role in our predisposition to cancer. Genome-wide association studies have linked the chromosome 15q25.1 locus to lung cancer susceptibility and implicated proteasome subunit alpha type-4 (PSMA4) as a candidate gene. In this case–control study, pathologically confirmed lung cancer patients and controls from the Chinese Han population were investigated to determine the effect of variant genotypes within the PSMA4 locus on susceptibility to lung cancer and sensitivity to cisplatin-based chemotherapy. Methods. We identified validated tagged single nucleotide polymorphisms (tSNPs) with minor allele frequency >5 % in the HapMap Chinese Han Beijing population and genotyped seven SNPs within the PSMA4 locus. Their correlation with lung cancer risks and treatment response were examined using χ 2 test and haplotype analysis. Multivariate logistic regression analysis tested the association between the polymorphisms and chemotherapy response. Results. rs12901682 is associated with lung cancer risks (OR = 1.45, 95 % CI, 1.04–2.02; P = 0.029). Using SNPStats software, we found rs12901682 (OR = 6.30, 95 % CI, 1.31–30.26; P = 0.0073) associated with lung cancer risks in the recessive model. Haplotype analysis showed that “CAGAATC” conferred an increased risk of lung cancer (OR = 1.50, 95 % CI, 1.07–2.11; P = 0.019). After adjustment for age, this association was pronounced in the male gender (OR = 6.30, 95 % CI, 1.31–30.26; P = 0.0073). PSMA4 polymorphisms did not affect the tumor sensitivity to cisplatin combination chemotherapy. Conclusions. Our study suggests a potential association between PSMA4 variants and lung cancer risk in Chinese Han population (AU)

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Genotyping and interpretation of STR-DNA: Low-template, mixtures and database matches-Twenty years of research and development.

The introduction of Short Tandem Repeat (STR) DNA was a revolution within a revolution that transformed forensic DNA profiling into a tool that could be used, for the first time, to create National DNA databases. This transformation would not have been possible without the concurrent development of fluorescent automated sequencers, combined with the ability to multiplex several loci together. Use of the polymerase chain reaction (PCR) increased the sensitivity of the method to enable the analysis of a handful of cells. The first multiplexes were simple: 'the quad', introduced by the defunct UK Forensic Science Service (FSS) in 1994, rapidly followed by a more discriminating 'six-plex' (Second Generation Multiplex) in 1995 that was used to create the world's first national DNA database. The success of the database rapidly outgrew the functionality of the original system - by the year 2000 a new multiplex of ten-loci was introduced to reduce the chance of adventitious matches. The technology was adopted world-wide, albeit with different loci. The political requirement to introduce pan-European databases encouraged standardisation - the development of European Standard Set (ESS) of markers comprising twelve-loci is the latest iteration. Although development has been impressive, the methods used to interpret evidence have lagged behind. For example, the theory to interpret complex DNA profiles (low-level mixtures), had been developed fifteen years ago, but only in the past year or so, are the concepts starting to be widely adopted. A plethora of different models (some commercial and others non-commercial) have appeared. This has led to a confusing 'debate' about the 'best' to use. The different models available are described along with their advantages and disadvantages. A section discusses the development of national DNA databases, along with details of an associated controversy to estimate the strength of evidence of matches. Current methodology is limited to searches of complete profiles - another example where the interpretation of matches has not kept pace with development of theory. STRs have also transformed the area of Disaster Victim Identification (DVI) which frequently requires kinship analysis. However, genotyping efficiency is complicated by complex, degraded DNA profiles. Finally, there is now a detailed understanding of the causes of stochastic effects that cause DNA profiles to exhibit the phenomena of drop-out and drop-in, along with artefacts such as stutters. The phenomena discussed include: heterozygote balance; stutter; degradation; the effect of decreasing quantities of DNA; the dilution effect.

Laboratory diagnosis of melioidosis: past, present and future.

Melioidosis is an emerging, potentially fatal disease caused by Burkholderia pseudomallei, which requires prolonged antibiotic treatment to prevent disease relapse. However, difficulties in laboratory diagnosis of melioidosis may delay treatment and affect disease outcomes. Isolation of B. pseudomallei from clinical specimens has been improved with the use of selective media. However, even with positive cultures, identification of B. pseudomallei can be difficult in clinical microbiology laboratories, especially in non-endemic areas where clinical suspicion is low. Commercial identification systems may fail to distinguish between B. pseudomallei and closely related species such as Burkholderia thailandensis. Genotypic identification of suspected isolates can be achieved by sequencing of gene targets such as groEL which offer higher discriminative power than 16S rRNA. Specific PCR-based identification of B. pseudomallei has also been developed using B. pseudomallei-specific gene targets such as Type III secretion system and Tat-domain protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolutionary technique for pathogen identification, has been shown to be potentially useful for rapid identification of B. pseudomallei, although existing databases require optimization by adding reference spectra for B. pseudomallei. Despite these advances in bacterial identification, diagnostic problems encountered in culture-negative cases remain largely unresolved. Although various serological tests have been developed, they are generally unstandardized "in house" assays and have low sensitivities and specificities. Although specific PCR assays have been applied to direct clinical and environmental specimens, the sensitivities for diagnosis remain to be evaluated. Metabolomics is an uprising tool for studying infectious diseases and may offer a novel approach for exploring potential diagnostic biomarkers. The metabolomics profiles of B. pseudomallei culture supernatants can be potentially distinguished from those of related bacterial species including B. thailandensis . Further studies using bacterial cultures and direct patient samples are required to evaluate the potential of metabolomics for improving diagnosis of melioidosis.

Advances in plant genotyping: where the future will take us.

Genetic diversity between individuals can be tracked and monitored using a range of molecular markers. These markers can detect variation ranging in scale from a single base pair up to duplications and translocations of entire chromosomal regions. The genotyping of individuals allows the detection of this variation and it has been successfully applied in plant science for many years. The increasing amounts of sequence data able to be generated using next-generation sequencing (NGS) technologies have produced a vast expansion in the rate of discovery of polymorphisms, with single nucleotide polymorphisms (SNPs) predominating as the marker of choice. This increase in polymorphic marker resources through efficient discovery, coupled with the utility of SNPs, has enabled the shift to high-throughput genotyping assays and these methods are reviewed and discussed here, alongside the recent innovations allowing increased throughput.

Genotype-phenotype correlation--promiscuity in the era of next-generation sequencing.

Newly cost-effective next-generation sequencing has led to an explosion of discoveries of novel genetic mutations that reveal the rampant "promiscuity" of genotype-phenotype relationships. Such discoveries should ultimately revolutionize clinical care.

Genotyping Mycobacterium bovis from cattle in the Central Pampas of Argentina: temporal and regional trends

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB), a disease that affects approximately 5% of Argentinean cattle. Among the molecular methods for genotyping, the most convenient are spoligotyping and variable number of tandem repeats (VNTR). A total of 378 samples from bovines with visible lesions consistent with TB were collected at slaughterhouses in three provinces, yielding 265 M. bovis spoligotyped isolates, which were distributed into 35 spoligotypes. In addition, 197 isolates were also typed by the VNTR method and 54 combined VNTR types were detected. There were 24 clusters and 27 orphan types. When both typing methods were combined, 98 spoligotypes and VNTR types were observed with 27 clusters and 71 orphan types. By performing a meta-analysis with previous spoligotyping results, we identified regional and temporal trends in the population structure of M. bovis. For SB0140, the most predominant spoligotype in Argentina, the prevalence percentage remained high during different periods, varying from 25.5-57.8% (1994-2011). By contrast, the second and third most prevalent spoligotypes exhibited important fluctuations. This study shows that there has been an expansion in ancestral lineages as demonstrated by spoligotyping. However, exact tandem repeat typing suggests dynamic changes in the clonal population of this microorganism.

Genotyping Mycobacterium bovis from cattle in the Central Pampas of Argentina: temporal and regional trends.

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB), a disease that affects approximately 5% of Argentinean cattle. Among the molecular methods for genotyping, the most convenient are spoligotyping and variable number of tandem repeats (VNTR). A total of 378 samples from bovines with visible lesions consistent with TB were collected at slaughterhouses in three provinces, yielding 265 M. bovis spoligotyped isolates, which were distributed into 35 spoligotypes. In addition, 197 isolates were also typed by the VNTR method and 54 combined VNTR types were detected. There were 24 clusters and 27 orphan types. When both typing methods were combined, 98 spoligotypes and VNTR types were observed with 27 clusters and 71 orphan types. By performing a meta-analysis with previous spoligotyping results, we identified regional and temporal trends in the population structure of M. bovis. For SB0140, the most predominant spoligotype in Argentina, the prevalence percentage remained high during different periods, varying from 25.5-57.8% (1994-2011). By contrast, the second and third most prevalent spoligotypes exhibited important fluctuations. This study shows that there has been an expansion in ancestral lineages as demonstrated by spoligotyping. However, exact tandem repeat typing suggests dynamic changes in the clonal population of this microorganism.