IFNL4 Genotypes and Risk of Childhood Burkitt Lymphoma in East Africa.

Interferon lambda 4 (IFN-λ4) is a novel type-III interferon that can be expressed only by carriers of the genetic variant rs368234815-dG within the first exon of the IFNL4 gene. Genetic inability to produce IFN-λ4 (in carriers of the rs368234815-TT/TT genotype) has been associated with improved clearance of hepatitis C virus (HCV) infection. The IFN-λ4-expressing rs368234815-dG allele (IFNL4-dG) is most common (up to 78%) in West sub-Saharan Africa (SSA), compared to 35% of Europeans and 5% of individuals from East Asia. The negative selection of IFNL4-dG outside Africa suggests that its retention in African populations could provide survival benefits, most likely in children. To explore this hypothesis, we conducted a comprehensive association analysis between IFNL4 genotypes and the risk of childhood Burkitt lymphoma (BL), a lethal infection-associated cancer most common in SSA. We used genetic, epidemiologic, and clinical data for 4,038 children from the Epidemiology of Burkitt Lymphoma in East African Children and Minors (EMBLEM) and the Malawi Infections and Childhood Cancer case-control studies. Generalized linear mixed models fit with the logit link controlling for age, sex, country, P. falciparum infection status, population stratification, and relatedness found no significant association between BL risk and 3 coding genetic variants within IFNL4 (rs368234815, rs117648444, and rs142981501) and their combinations. Because BL occurs in children 6-9 years of age who survived early childhood infections, our results suggest that additional studies should explore the associations of IFNL4-dG allele in younger children. This comprehensive study represents an important baseline in defining the health effects of IFN-λ4 in African populations.

Genome-wide Association Study of Bladder Cancer Reveals New Biological and Translational Insights.

BACKGROUND: Genomic regions identified by genome-wide association studies (GWAS) for bladder cancer risk provide new insights into etiology. OBJECTIVE: To identify new susceptibility variants for bladder cancer in a meta-analysis of new and existing genome-wide genotype data. DESIGN, SETTING, AND PARTICIPANTS: Data from 32 studies that includes 13,790 bladder cancer cases and 343,502 controls of European ancestry were used for meta-analysis. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSES: Log-additive associations of genetic variants were assessed using logistic regression models. A fixed-effects model was used for meta-analysis of the results. Stratified analyses were conducted to evaluate effect modification by sex and smoking status. A polygenic risk score (PRS) was generated on the basis of known and novel susceptibility variants and tested for interaction with smoking. RESULTS AND LIMITATIONS: Multiple novel bladder cancer susceptibility loci (6p.22.3, 7q36.3, 8q21.13, 9p21.3, 10q22.1, 19q13.33) as well as improved signals in three known regions (4p16.3, 5p15.33, 11p15.5) were identified, bringing the number of independent markers at genome-wide significance (p < 5 × 10-8) to 24. The 4p16.3 (FGFR3/TACC3) locus was associated with a stronger risk for women than for men (p-interaction = 0.002). Bladder cancer risk was increased by interactions between smoking status and genetic variants at 8p22 (NAT2; multiplicative p value for interaction [pM-I] = 0.004), 8q21.13 (PAG1; pM-I = 0.01), and 9p21.3 (LOC107987026/MTAP/CDKN2A; pM-I = 0.02). The PRS based on the 24 independent GWAS markers (odds ratio per standard deviation increase 1.49, 95% confidence interval 1.44-1.53), which also showed comparable results in two prospective cohorts (UK Biobank, PLCO trial), revealed an approximately fourfold difference in the lifetime risk of bladder cancer according to the PRS (e.g., 1st vs 10th decile) for both smokers and nonsmokers. CONCLUSIONS: We report novel loci associated with risk of bladder cancer that provide clues to its biological underpinnings. Using 24 independent markers, we constructed a PRS to stratify lifetime risk. The PRS combined with smoking history, and other established risk factors, has the potential to inform future screening efforts for bladder cancer. PATIENT SUMMARY: We identified new genetic markers that provide biological insights into the genetic causes of bladder cancer. These genetic risk factors combined with lifestyle risk factors, such as smoking, may inform future preventive and screening strategies for bladder cancer.

Disinfection By-Products in Drinking Water and Bladder Cancer: Evaluation of Risk Modification by Common Genetic Polymorphisms in Two Case-Control Studies.

BACKGROUND: By-products are formed when disinfectants react with organic matter in source water. The most common class of disinfection by-products, trihalomethanes (THMs), have been linked to bladder cancer. Several studies have shown exposure-response associations with THMs in drinking water and bladder cancer risk. Few epidemiologic studies have evaluated gene-environment interactions for total THMs (TTHMs) with known bladder cancer susceptibility variants. OBJECTIVES: In this study, we investigated the combined effect on bladder cancer risk contributed by TTHMs, bladder cancer susceptibility variants identified through genome-wide association studies, and variants in several candidate genes. METHODS: We analyzed data from two large case-control studies-the New England Bladder Cancer Study (n/n=989 cases/1,162 controls), a population-based study, and the Spanish Bladder Cancer Study (n/n=706 cases/772 controls), a hospital-based study. Because of differences in exposure distributions and metrics, we estimated effects of THMs and genetic variants within each study separately using adjusted logistic regression models to calculate odds ratios (ORs) and 95% confidence intervals (CI) with and without interaction terms, and then combined the results using meta-analysis. RESULTS: Of the 16 loci showing strong evidence of association with bladder cancer, rs907611 at 11p15.5 [leukocyte-specific protein 1 (LSP1 region)] showed the strongest associations in the highest exposure category in each study, with evidence of interaction in both studies and in meta-analysis. In the highest exposure category, we observed OR=1.66 (95% CI: 1.17, 2.34, p-trend=0.005) for those with the rs907611-GG genotype and p-interaction=0.02. No other genetic variants tested showed consistent evidence of interaction. DISCUSSION: We found novel suggestive evidence for a multiplicative interaction between a putative bladder carcinogen, TTHMs, and genotypes of rs907611. Given the ubiquitous exposure to THMs, further work is needed to replicate and extend this finding and to understand potential molecular mechanisms. https://doi.org/10.1289/EHP9895.

Intracellular Accumulation of IFN-λ4 Induces ER Stress and Results in Anti-Cirrhotic but Pro-HCV Effects.

IFNL3/IFNL4 polymorphisms are inversely associated with the risk of chronic hepatitis C virus (HCV) infection and cirrhosis, two major risk factors for developing hepatocellular carcinoma (HCC). To further explore these inverse associations and their molecular underpinnings, we analyzed IFNL3/IFNL4 polymorphisms represented by the IFNL4 genotype (presence of rs368234815-dG or rs12979860-T alleles) in HCV patients: 2969 from Japan and 2931 from Taiwan. IFNL4 genotype was associated with an increased risk of HCV-related HCC (OR=1.28, 95%CI=1.07-1.52, P=0.0058) in the general population of Japanese patients, but not in Taiwanese patients who achieved treatment-induced viral clearance. IFNL4 genotype was also associated with a decreased risk of cirrhosis (OR=0.66, 95%CI=0.46-0.93, P=0.018, in Taiwanese patients). We then engineered HepG2 cells to inducibly express IFN-λ4 in the presence or absence of interferon lambda receptor 1 (IFNLR1). Induction of IFN-λ4 resulted in its intracellular accumulation, mainly in lysosomes and late endosomes, and increased ER stress, leading to apoptosis and reduced proliferation. We identified the very-low-density lipoprotein receptor (VLDLR), which facilitates HCV entry into hepatocytes, as a transcript induced by IFN-λ4 but not IFN-λ3. Our results suggest that the molecular mechanisms underlying the anti-cirrhotic but pro-HCV associations observed for IFNL3/IFNL4 polymorphisms are, at least in part, contributed by intracellular accumulation of IFN-λ4 causing ER stress in hepatic cells.

Glutathione-related genetic polymorphisms are associated with mercury retention and nephrotoxicity in gold-mining settings of a Colombian population.

Mercury (Hg) vapor can produce kidney injury, where the proximal tubule region of the nephron is the main target of the Hg-induced oxidative stress. Hg is eliminated from the body as a glutathione conjugate. Thus, single nucleotide polymorphisms (SNPs) in glutathione-related genes might modulate the negative impact of this metal on the kidneys. Glutathione-related SNPs were tested for association with levels of Hg and renal function biomarkers between occupationally exposed (n = 160) and non-exposed subjects (n = 121). SNPs were genotyped by TaqMan assays in genomic DNA samples. Total mercury concentration was measured in blood, urine and hair samples. Regression analyses were performed to estimate the effects of SNPs on quantitative traits. Alleles GCLM rs41303970-T and GSTP1 rs4147581-C were significantly overrepresented in the exposed compared with the non-exposed group (P < 0.01). We found significant associations for GCLM rs41303970-T with higher urinary clearance rate of Hg (ß = 0.062, P = 0.047), whereas GCLC rs1555903-C was associated with lower levels of estimated glomerular filtration rate in the non-exposed group (eGFR, ß = - 3.22, P = 0.008) and beta-2-microglobulin in the exposed group (ß-2MCG, ß = - 19.32, P = 0.02). A SNP-SNP interaction analysis showed significant epistasis between GSTA1 rs3957356-C and GSS rs3761144-G with higher urinary levels of Hg in the exposed (ß = 0.13, P = 0.04) but not in the non-exposed group. Our results suggest that SNPs in glutathione-related genes could modulate the pathogenesis of Hg nephrotoxicity in our study population by modulating glutathione concentrations in individuals occupationally exposed to this heavy metal.

Targeting natural splicing plasticity of APOBEC3B restricts its expression and mutagenic activity.

APOBEC3A (A3A) and APOBEC3B (A3B) enzymes drive APOBEC-mediated mutagenesis. Identification of factors affecting the activity of these enzymes could help modulate mutagenesis and associated clinical outcomes. Here, we show that canonical and alternatively spliced A3A and A3B isoforms produce corresponding mutagenic and non-mutagenic enzymes. Increased expression of the mutagenic A3B isoform predicted shorter progression-free survival in bladder cancer. We demonstrate that the production of mutagenic vs. non-mutagenic A3B protein isoforms was considerably affected by inclusion/skipping of exon 5 in A3B. Furthermore, exon 5 skipping, resulting in lower levels of mutagenic A3B enzyme, could be increased in vitro. Specifically, we showed the effects of treatment with an SF3B1 inhibitor affecting spliceosome interaction with a branch point site in intron 4, or with splice-switching oligonucleotides targeting exon 5 of A3B. Our results underscore the clinical role of A3B and implicate alternative splicing of A3B as a mechanism that could be targeted to restrict APOBEC-mediated mutagenesis.

Interferons and viruses induce a novel truncated ACE2 isoform and not the full-length SARS-CoV-2 receptor.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19, utilizes angiotensin-converting enzyme 2 (ACE2) for entry into target cells. ACE2 has been proposed as an interferon-stimulated gene (ISG). Thus, interferon-induced variability in ACE2 expression levels could be important for susceptibility to COVID-19 or its outcomes. Here, we report the discovery of a novel, transcriptionally independent truncated isoform of ACE2, which we designate as deltaACE2 (dACE2). We demonstrate that dACE2, but not ACE2, is an ISG. In The Cancer Genome Atlas, the expression of dACE2 was enriched in squamous tumors of the respiratory, gastrointestinal and urogenital tracts. In vitro, dACE2, which lacks 356 amino-terminal amino acids, was non-functional in binding the SARS-CoV-2 spike protein and as a carboxypeptidase. Our results suggest that the ISG-type induction of dACE2 in IFN-high conditions created by treatments, an inflammatory tumor microenvironment or viral co-infections is unlikely to increase the cellular entry of SARS-CoV-2 and promote infection.

Interferons and viruses induce a novel primate-specific isoform dACE2 and not the SARS-CoV-2 receptor ACE2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes COVID-19, utilizes angiotensin-converting enzyme 2 (ACE2) for entry into target cells. ACE2 has been proposed as an interferon-stimulated gene (ISG). Thus, interferon-induced variability in ACE2 expression levels could be important for susceptibility to COVID-19 or its outcomes. Here, we report the discovery of a novel, primate-specific isoform of ACE2, which we designate as deltaACE2 (dACE2). We demonstrate that dACE2, but not ACE2, is an ISG. In vitro, dACE2, which lacks 356 N-terminal amino acids, was non-functional in binding the SARS-CoV-2 spike protein and as a carboxypeptidase. Our results reconcile current knowledge on ACE2 expression and suggest that the ISG-type induction of dACE2 in IFN-high conditions created by treatments, inflammatory tumor microenvironment, or viral co-infections is unlikely to affect the cellular entry of SARS-CoV-2 and promote infection.

Laboratory genetic-based reference values for cholinesterase activity in a Colombian population: A step forward in personalized diagnostics / Valores de referencia basados en el contexto genético de la actividad enzimática de la colinesterasa en una población colombiana: un paso hacia el diagnóstico personalizado

Introduction: The determination of cholinesterase (ChE) activity has been commonly applied in the biomonitoring of exposure to organophosphate and carbamate pesticides. However, ChE activity is influenced by genetic factors. Integrating genotype and phenotype information in clinical laboratory tests would increase the accuracy of the reference values in well-defined populations. Objective: To establish genetic-based reference values for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity in a Colombian population. Materials and methods: A total of 397 healthy adults from Bucaramanga were included in the study. AChE and BChE activities were measured in blood samples by potentiometry and spectrophotometry, respectively. Genotyping for ACHE rs17880573 and BCHE rs1803274 was performed using the TaqMan allelic discrimination assay. The statistical analyses to obtain the reference values were performed with the MedCalc® software. Results: Allele frequencies were 10.58% for rs17880573 A and 8.82% for rs1803274 A. People with genotypes rs1803274 AA and AG showed a reduction of 20.69% and 10.92% respectively in mean BChE activity compared to people with genotype GG. No significant differences were identified in AChE activity between rs17880573 alleles or genotypes. In the overall sample, the corresponding reference values were as follows: for AChE activity, 0.62-0.98 D pH/h and for BChE activity, 4796.3-10321.1 U/L for people carrying the allele rs1803274A and 5768.2-11180.4 U/L for people carrying the genotype rs1803274 GG. Conclusion: We strongly recommend using these genetic-based reference values for ChE enzymes in our well-defined population in daily clinical practice.

Introducción. La determinación de la actividad enzimática de la colinesterasa se utiliza comúnmente en la vigilancia biológica de la exposición a pesticidas organofosforados y carbamatos. Sin embargo, los factores genéticos afectan la actividad de la colinesterasa, por lo que la integración de la información sobre genotipos y fenotipos en las pruebas de laboratorio clínico, incrementaría la precisión de los valores de referencia. Objetivo. Establecer los valores de referencia basados en el contexto genético para la actividad enzimática de la acetilcolinesterasa (AChE) y la butirilcolinesterasa (BChE), en una población colombiana. Materiales y métodos. Se incluyeron 397 adultos sanos. La actividad de la acetilcolinesterasa y la de la butirilcolinesterasa, se determinaron en muestras de sangre por potenciometría y espectrofotometría, respectivamente. Los genotipos de los ACHE rs17880573 y BCHE rs1803274 se obtuvieron mediante el ensayo TaqMan y los valores de referencia se estimaron con el programa MedCalc®. Resultados. La frecuencia alélica fue de 10,58 % para rs17880573 A y de 8,82 % para rs1803274 A. Las personas con los genotipos rs1803274 AA y AG, mostraron una reducción en el promedio de la actividad de la butirilcolinesterasa de 20,69 % y de 10,92 %, respectivamente, comparados con aquellas con el genotipo GG. No se encontraron diferencias significativas en la actividad de la acetilcolinesterasa con respecto a los alelos y genotipos del rs17880573. Los valores de referencia determinados para esta población fueron de 0,62-0,98 D pH/h para acetilcolinesterasa y de 4796,3-10321,1 U/L para butirilcolinesterasa, en las personas portadoras del alelo rs1803274 A, y de 5768,2-11180,4 U/L, en las portadoras del genotipo rs1803274 GG. Conclusión. Se recomienda el uso de estos valores de referencia basados en el contexto genético para las colinesterasas, en la práctica clínica diaria en esta población.

Actividad enzimática colinesterasa en muestras de sangre humana: efecto de las condiciones de almacenamiento / Cholinesterase enzyme activity in human blood samples: effects of the store conditions

Introducción: La determinación de la actividad enzimática colinesterasa (ChE) es el principal biomarcador de efecto de la exposición a los plaguicidas organofosforados y carbamatos. Por lo tanto, la estabilidad de la actividad de las ChEs en muestras de sangre es un parámetro pre-analítico importante que necesita ser considerado en términos de la seguridad diagnóstica. Objetivo: Determinar el efecto del tiempo y de la temperatura de almacenamiento sobre la actividad de las ChEs en muestras de sangre humana. Metodología: Muestras de sangre entera y suspensiones de eritrocitos (eritrocitos + solución salina 0.9% proporción 1:1) fueron almacenados a -20°C, 4°C y 25°C. Las determinaciones enzimáticas se realizaron una hora después de la toma de la muestra y se repitieron entre el día 1 hasta el día 90. La actividad enzimática ChE total y Acetil-ChE se determinaron respectivamente mediante el método colorimétrico de Limperos & Ranta y mediante el método potenciométrico de Michel. Resultados: La máxima estabilidad de la actividad ChE total se observó a -20°C hasta por 60 días, además, dicha estabilidad perduró hasta por el tiempo máximo del estudio a 4°C para la Acetil-ChE. Una considerable disminución de la actividad Acetil-ChE se observó después de los días 7 a 25°C y 4 a -20°C. Conclusión: Considerando la seguridad diagnóstica, nosotros recomendamos almacenar las muestras de sangre entera a -20°C por un tiempo máximo de 30 días para la determinación de la actividad ChE total y la suspensión de eritrocitos en 0.9% de NaCl a 4°C por 14 días máximo para la determinación de la Acetil-ChE.

Introduction: Determination of cholinesterase (ChE) enzyme activity is the main biomarker of exposure to pesticides organophosphorus and carbamate. Therefore, the enzyme stability of ChEsin blood samples is an important pre-analytical factor to take into account in the diagnosis. Objective: To determine the effect of storage time and temperature on ChEs enzyme activity in human blood samples. Methodology: Whole-blood samples and erythrocyte suspensions (erythrocyte + 0.9% saline solution; ratio 1:1) were stored at -20°C, 4°C and 25°C. Enzyme activity measurements were performed at one hour after the blood samples have been obtained and then were repeated between days 1 and 90. Total ChE and Acetyl-ChE activities were determined using the Limperos & Ranta colorimetric method and the potentiometric method of Michel respectively. Results: The maximum stability of the total ChE enzyme activity was achieved at -20°C for 60 days and, in the case of Acetyl-ChE, at 4°C for the time the study was conducted. A decrease of Acetyl-ChE activity was shown after 7 days at 25°C and 4 days at -20°C. Conclusion: In terms of diagnosis, we recommend that in order to measure the total ChE activity the wholeblood samples should be stored at -20°C for 30 days, whereas to measure the Acetyl-ChE activity the erythrocyte suspensions in 0.9% NaCl at 4°C for 14 days.

Amplificación por PCR de tejidos de archivo: efecto de los fijadores / PCR amplification of stored tissues: Effect of tissue-fixatives

El diagnóstico a nivel de los laboratorios de Patología está aprovechando las ventajas que ofrecen las técnicas moleculares, entre ellas la alta sensibilidad, especificidad y la estabilidad y fácil manipulación del DNA. La utilidad de los datos obtenidos a partir del análisis de tejidos está directamente relacionada con la calidad de la muestra, la cual se puede ver afectada por las condiciones de manejo y almacenamiento que pueden contribuir a su deterioro o degradación. En este estudio se determinó la calidad y la integridad del DNA extraído de tejido cardíaco por análisis en electroforesis de agarosa y amplificación por Reacción en Cadena de la Polimerasa (PCR) de fragmentos de diferentes tamaños. Adicionalmente se analizó la presencia de DNA de Trypanosoma cruzi. Los mejores resultados se obtuvieron cuando el tejido se congeló sin la utilización de fijadores. De los fijadores el que mostró los mejores resultados fue la formalina tamponada antes de su uso y el almacenamiento a –20 ºC.

The diagnosis in Patology is taking advantages offered by molecular approach, including, high sensibility, specificity, stability and facility to manipulate DNA. The usefulness of data obtained from tissue analysis is related to specimen quality, wich may be affected by conditions of manipulation and storage that may contribute to its degradation. In this report the quality and integrity of DNA obtained fron cardiac tissues was measured for electrophoretic pattern and specific amlied polymerase chain reaction (PCR) products to different sizes. DNA to T. cruzi was also amplified. Bests results for PCR products were obtained with frozen tissues without fixatives. Buffered-Formaline and incubation at -20ºC was the best fixative.

Influencia del método de desparafinación y el tiempo de almacenamiento en la extracción de DNA a partir de tejidos de archivo / Influence of the metod of dewaxing and the time of storage in DNA's extraction from stored tissues

Los tejidos fijados en formalina y embebidos en parafina son un material de incalculable valor para estudios retrospectivos que requieran aplicar el análisis molecular a muestras clínicas de archivo. Sin embargo, la extracción del DNA a partir de este material consume mucho tiempo y los reactivos usados pueden contribuir a su fragmentación y contaminación, dificultando de esta forma su uso para pruebas moleculares. En este trabajo se analizaron cuatro métodos de desparafinación y su efecto sobre la cantidad y calidad del DNA. Todos los métodos mostraron una alta fragmentación del DNA, con amplificación de fragmentos de tamaños menores a 200 pb. El método de microondas fue el único que permitió amplificación de fragmentos de mayor tamaño, además de presentar la ventaja de su simplicidad, poco tiempo de ejecución y bajo costo. El tiempo de almacenamiento influyó, obteniéndose amplificación hasta 6 años y no en muestras almacenadas 10 años.

Formaline-fixed and paraffin wax embedded tissues are an invaluable source for retrospectives molecular analysis with clinical correlation. However, DNA extraction from this type of material demands a prolonged time and the DNA is often highly fragmented and contaminated by protein agents, making it inadequate for molecular purposes. Four methods to deparaffinization are tested and compared the quality and quantity to DNA obtained. All techniques lead to DNA degradation but only short sequences can be amplified, less than 200 bases in all methods. The most efficient method uses a microwave to melt the wax. This method was quick, straighforward, cheaper and effective to amplify fragments the 450 pb. The time of storage influenced, with amplification to specimens to 6 years and not in samples stored 10 years