Pharmacogenetic variability of tuberculosis biomarkers in native and mestizo Peruvian populations.

In Peru, 29 292 people were diagnosed with tuberculosis in 2022. Although tuberculosis treatments are effective, 3.4%-13% are associated with significant adverse drug reactions, with drug-induced liver injury (DILI) considered the most predominant. Among the first-line antituberculosis drugs, isoniazid is the main drug responsible for the appearance of DILI. In liver, isoniazid (INH) is metabolized by N-acetyltransferase-2 (NAT2) and cytochrome P450 2E1 (CYP2E1). Limited information exists on genetic risk factors associated with the presence of DILI to antituberculosis drugs in Latin America, and even less is known about these factors in the native and mestizo Peruvian population. The aim of this study was to determine the prevalence of NAT2 and CYP2E1 genotypes in native and mestizo population. An analytical cross-sectional analysis was performed using genetic data from mestizo population in Lima and native participants from south of Peru. NAT2 metabolizer was determined as fast, intermediate and slow, and CYP2E1 genotypes were classified as c1/c1, c1/c2 and c2/c2, from molecular tests and bioinformatic analyses. Of the 472 participants, 36 and 6 NAT2 haplotypes were identified in the mestizo and native population, respectively. In mestizo population, the most frequent NAT2*5B and NAT2*7B haplotypes were associated with DILI risk; while in natives, NAT2*5G and NAT2*13A haplotypes were associated with decreased risk of DILI. For CYP2E1, c1/c1 and c1/c2 genotypes are the most frequent in natives and mestizos, respectively. The linkage disequilibrium of NAT2 single nucleotide polymorphisms (SNPs) was estimated, detecting a block between all SNPs natives. In addition, a block between rs1801280 and rs1799929 for NAT2 was detected in mestizos. Despite the limitations of a secondary study, it was possible to report associations between NAT2 and CYP2E alleles with Peruvian native and mestizo by prevalence ratios. The results of this study will help the development of new therapeutic strategies for a Tuberculosis efficient control between populations.

GSTT1/GSTM1 Genotype and Anti-Tuberculosis Drug-Induced Hepatotoxicity in Peruvian Patients.

In Peru, 24,581 people were diagnosed with tuberculosis (TB) in 2020. Although TB treatments are effective, 3.4-13% are associated with significant adverse drug reactions (ADRs), with drug-induced liver injury (DILI) considered the most predominant. Among the first-line antituberculosis drugs, isoniazid (INH) is the main drug responsible for the appearance of DILI. In the liver, INH is metabolized by the enzymes N-acetyltransferase-2 (NAT2), cytochrome P450 2E1 (CYP2E1), and glutathione S-transferase (GST) with two isoforms, GSTT1 and GSTM1. Based on previous studies, we hypothesized that interactions between the GSTT1 and GSTM1 null genotypes induce DILI in TB patients. In this cross-sectional study of 377 participants who completed their anti-TB treatment, we genotyped by revealing the presence or absence of 215- and 480-bp bands of GSTM1 and GSTT1, respectively. We found that the prevalence of the GSTM1 genotype was 52.79% and 47.21% for presence and null, respectively, and for GSTT1 it was 69.76% and 30.24% for presence and null, respectively. Neither genotype was prevalent in the patients who developed DILI (n = 16). We did not confirm our hypothesis; however, we found that the combination of GSTM1 present genotype, GSTT1 null genotype, fast NAT2 acetylators, and CYP2E1 c1/c1 genotype had a significant risk for the development of ADR (OR 11; p = 0.017; 95% CI: (0.54-186.35)). We propose that the presence of the GSTM1 present genotype, GSTT1 null genotype, fast NAT2 acetylators, and CYP2E1 c1/c1 genotype in the Peruvian population could be considered a risk factor for the development of ADR due to therapeutic drug intake.

NAT2 and CYP2E1 polymorphisms and antituberculosis drug-induced hepatotoxicity in Peruvian patients.

BACKGROUND: In Peru, 32,970 people were diagnosed with tuberculosis (TB) in 2019. Although TB treatment is effective, 3.4%-13% is associated with significant adverse drug reactions (ADR), considering drug-induced liver injury (DILI) as the most prevalent. Among the first-line anti-TB drugs, isoniazid (INH) is primarily responsible for the occurrence of DILI. INH is metabolized in the liver by the enzymes N-acetyltransferase-2 (NAT2) and Cytochrome P450 2E1 (CYP2E1). Based on the previous studies, we hypothesized that the interactions between slow CYP2E1 genotype and NAT2 slow acetylators will induce DILI in TB patients. METHODS: In this cross-sectional study, all 377 participants completed their anti-TB treatment, and we genotyped SNPs: rs1041983, rs1801280, rs1799929, rs1799930, rs1208, and rs1799931 for NAT2 and rs3813867 and rs2031920 for CYP2E1. RESULTS: We found that rapid, intermediate, and slow NAT2 acetylator were 15%, 38%, and 47%, respectively, in the general population. Intermediate NAT2 acetylator is the least prevalent among patients with adverse reactions (p = 0.024). We did not confirm our hypothesis, however, we found that the combination of intermediate NAT2 acetylators and CYP2E1 c1/c1 genotype significantly protected (OR = 0.16; p = 0.049) against the development of DILI in our population. CONCLUSION: We propose that the presence of NAT2 intermediate and CYP2E1 c1/c1 genotype could help in therapeutic drug monitoring, and optimize its therapeutic benefits while minimizing its risk for side effects or toxicity.

Analysis of microbiome diversity in coprolites from Caral, Peru.

We analyzed human coprolites from the Sacred City of Caral, the oldest civilization in America (3000- and 1800-years BC). Our objective was to know the microbial diversity of the Caral Civilization through the use of a mobile ancient laboratory. DNA extraction conducted in a mobile laboratory placed near the collection site to reduce exposure of samples to contaminants and favor a rapid molecular processing. Using 16S rRNA and ITS 1 amplicon sequencing, we have elaborated the first list of the microbiomes of Caral, based on the bacterial and fungal community fingerprints detected in the coprolites recovered in six sectors of that ancient urban center. Among the most abundant sequences were those associated with Firmicutes for bacteria, Ascomycota and Basidiomycota for fungi. Bacillus was the most abundant bacterial genera in all samples analyzed, compromising up to 24.81% of the total bacterial abundance; while Aspergillus (11.43%) was the most abundant genera among fungal communities.

A mobile lab for ancient DNA extraction in Perug.

We report the use of a mobile laboratory set up to extract ancient DNA (aDNA) from 34 human coprolites (fossilized faeces) samples. Our approach enabled the rapid genetic characterization of 5,000 years old archeological samples. It is useful for the on-site screening of museums and freshly excavated samples for DNA. This approach is accessible to other investigators as the mobile laboratory was set up using commercially available instruments.

A Closer Look at ACE2 Signaling Pathway and Processing during COVID-19 Infection: Identifying Possible Targets.

Since the identification of its role as the functional receptor for SARS-CoV in 2003 and for SARS-CoV-2 in 2020, ACE2 has been studied in depth to understand COVID-19 susceptibility and severity. ACE2 is a widely expressed protein, and it plays a major regulatory role in the renin-angiotensin-aldosterone System (RAAS). The key to understanding susceptibility and severity may be found in ACE2 variants. Some variants have been shown to affect binding affinity with SARS-CoV-2. In this review, we discuss the role of ACE2 in COVID-19 infection, highlighting the importance of ACE2 isoforms (soluble and membrane-bound) and explore how ACE2 variants may influence an individual's susceptibility to SARS-CoV-2 infection and disease outcome.

Allelic and genotypic frequencies of NAT2, CYP2E1, and AADAC genes in a cohort of Peruvian tuberculosis patients.

BACKGROUND: We determined the frequency of genetic polymorphisms in three anti-TB drug metabolic proteins previously reported: N-acetyltransferase 2 (NAT2), cytochrome P450 2E1 (CYP2E1), and arylacetamide deacetylase (AADAC) within a Peruvian population in a cohort of TB patients. METHODS: We genotyped SNPs rs1041983, rs1801280, rs1799929, rs1799930, rs1208, and rs1799931 for NAT2; rs3813867 and rs2031920 for CYP2E1; and rs1803155 for AADAC in 395 participants completed their antituberculosis treatment. RESULTS: Seventy-four percent of the participants are carriers of slow metabolizer genotypes: NAT2*5, NAT2*6, and NAT2*7, which increase the sensitivity of INH at low doses and increase the risk of drug-induced liver injuries. Sixty-four percent are homozygous for the wild-type CYP2E1*1A allele, which could increase the risk of hepatotoxicity. However, 16% had a NAT2 fast metabolizer phenotype which could increase the risk of acquiring resistance to INH, thereby increasing the risk of multidrug-resistant (MDR) or treatment failure. The frequency of rs1803155 (AADAC*2 allele) was higher (99.9%) in Peruvians than in European American, African American, Japanese, and Korean populations. CONCLUSIONS: This high prevalence of slow metabolizers for isoniazid in the Peruvian population should be further studied and considered to help individualize drug regimens, especially in countries with a great genetic diversity like Peru. These data will help the Peruvian National Tuberculosis Control Program develop new strategies for therapies.

CXCR4 Knockdown Via CRISPR/CAS9 in a Tumor-Associated Macrophage Model Decreases Human Breast Cancer Cell Migration.

Introduction Breast cancer is the leading cause of cancer-related deaths in women worldwide with the majority of deaths due to metastasis. The development of metastasis is closely related to the tumor microenvironment where tumor-associated macrophages (TAMs) are the main immune cell component playing a crucial role in tumor migration. Key players in tumor progression, metastasis and survival are the receptor CXCR4 and its ligand CXCL12. CXCR4 is expressed in multiple cell types including macrophages and breast cancer cells. Many studies have focus on the role of CXCR4 expressed in breast cancer cells. Methods In this study, we investigated the role of CXCR4 expressed in TAMs on breast cancer cell migration by reducing CXCR4 expression via CRISPR-CAS9 system in differentiated THP-1 cells (a TAMs model). Results According to wound healing migration assay, MCF7 cancer cells co-cultured with genetically edited dTHP-1 cells have a lower migration rate as compared to MCF7 cancer cells co-cultured with unedited and dTHP-1 cells. Conclusion The study demonstrates the role of CXCR4 on breast cancer cell migration through TAM-cancer cell crosstalk.

The genetic structure and adaptation of Andean highlanders and Amazonians are influenced by the interplay between geography and culture.

Western South America was one of the worldwide cradles of civilization. The well-known Inca Empire was the tip of the iceberg of an evolutionary process that started 11,000 to 14,000 years ago. Genetic data from 18 Peruvian populations reveal the following: 1) The between-population homogenization of the central southern Andes and its differentiation with respect to Amazonian populations of similar latitudes do not extend northward. Instead, longitudinal gene flow between the northern coast of Peru, Andes, and Amazonia accompanied cultural and socioeconomic interactions revealed by archeology. This pattern recapitulates the environmental and cultural differentiation between the fertile north, where altitudes are lower, and the arid south, where the Andes are higher, acting as a genetic barrier between the sharply different environments of the Andes and Amazonia. 2) The genetic homogenization between the populations of the arid Andes is not only due to migrations during the Inca Empire or the subsequent colonial period. It started at least during the earlier expansion of the Wari Empire (600 to 1,000 years before present). 3) This demographic history allowed for cases of positive natural selection in the high and arid Andes vs. the low Amazon tropical forest: in the Andes, a putative enhancer in HAND2-AS1 (heart and neural crest derivatives expressed 2 antisense RNA1, a noncoding gene related to cardiovascular function) and rs269868-C/Ser1067 in DUOX2 (dual oxidase 2, related to thyroid function and innate immunity) genes and, in the Amazon, the gene encoding for the CD45 protein, essential for antigen recognition by T and B lymphocytes in viral-host interaction.

Guillain-Barre syndrome outbreak in Peru: Association with polymorphisms in IL-17, ICAM1, and CD1.

BACKGROUND: Guillain-Barre Syndrome (GBS) is considered a complex disorder with significant environmental effect and genetic susceptibility. Genetic polymorphisms in CD1E, CD1A, IL-17, and/or ICAM1 had been proposed as susceptibility genetic variants for GBS mainly in Caucasian population. This study explores the association between selected polymorphisms in these genes and GBS susceptibility in confirmed GBS cases reported in mestizo population from northern Peru during the most recent GBS outbreak of May 2018. METHODS: A total of nine nonrelated cases and 11 controls were sequenced for the polymorphic regions of CD1A, CD1E, IL-17, and ICAM1. RESULTS: We found a significant protective association between heterozygous GA genotype in ICAM1 (241Gly/Arg) and GBS (p < .047). IL-17 was monomorphic in both controls and patients. No significant differences were found in the frequency of SNPs in CD1A and CD1E between the group with GBS patients and healthy controls. CONCLUSION: ICAM1 polymorphisms might be considered as potential genetic markers of GBS susceptibility. Further studies with larger sample size will be required to validate these findings.

Genetics and genomics in Peru: Clinical and research perspective.

Peruvians currently preserve in their DNA the history of 2.5 million years of human evolution and 150,000 years of migration from Africa to Peru or the Americas. The development of Genetics and Genomics in the clinical and academic field is shown in this review.

A rapid identification technique for drug-resistant Mycobacterium tuberculosis isolates using mismatch specific cleavage enzyme.

The emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains is a major health problem for high Tuberculosis (TB) incidence countries. Therefore, it is of interest to identify antibiotic resistant bacteria by mismatch detection using DNA hybridization. We generated PCR products for five genes (rpoB, inhA, katG, gyrA and rrs) associated with drug resistance TB from MDR and XDR Mycobacterium tuberculosis (MTB) DNA samples. These were hybridized to PCR products from MTB H37Rv (pansusceptible laboratory strain) to generate DNA hetero-duplex products, which was digested by Detection Enzyme (GeneArt Genomic Cleavage Detection Kit) and visualized by agarose gel electrophoresis. Results show different bands with sizes of 400 bp and 288 bp (rpoB), 280 bp (inhA), 310 bp (katG), 461 bp (gyrA) and 427 bp (rrs) suggesting mutations in DNA heteroduplex for each gene. Detection Enzyme specifically cleaves DNA hetero-duplex with mismatch. The technique helps in the improved detection of MDR (mutations in rpoB, inhA and katG) and XDR (mutations in rpoB, inhA katG, gyrA and rrs) MTB strains. Moreover, the technique is customized without expensive specialized equipment to detect mutations. It is also fast, efficient and easy to implement in standard molecular biology laboratories.

Evolutionary genomic dynamics of Peruvians before, during, and after the Inca Empire.

Native Americans from the Amazon, Andes, and coastal geographic regions of South America have a rich cultural heritage but are genetically understudied, therefore leading to gaps in our knowledge of their genomic architecture and demographic history. In this study, we sequence 150 genomes to high coverage combined with an additional 130 genotype array samples from Native American and mestizo populations in Peru. The majority of our samples possess greater than 90% Native American ancestry, which makes this the most extensive Native American sequencing project to date. Demographic modeling reveals that the peopling of Peru began ∼12,000 y ago, consistent with the hypothesis of the rapid peopling of the Americas and Peruvian archeological data. We find that the Native American populations possess distinct ancestral divisions, whereas the mestizo groups were admixtures of multiple Native American communities that occurred before and during the Inca Empire and Spanish rule. In addition, the mestizo communities also show Spanish introgression largely following Peruvian Independence, nearly 300 y after Spain conquered Peru. Further, we estimate migration events between Peruvian populations from all three geographic regions with the majority of between-region migration moving from the high Andes to the low-altitude Amazon and coast. As such, we present a detailed model of the evolutionary dynamics which impacted the genomes of modern-day Peruvians and a Native American ancestry dataset that will serve as a beneficial resource to addressing the underrepresentation of Native American ancestry in sequencing studies.

A Genomic Signature for Genotyping Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), has a vast diversity of genotypes including Beijing, CAS, EAI, Haarlem, LAM, X, Ural, T, AFRI1 and AFRI2. However, genotyping can be expensive, time consuming and in some cases, results may vary depending on methodology used. Here, we proposed a new set of 10 SNPs using a total of 249 MTB genomes, and selected by first the inclusion/ exclusion (IE) criteria using spoligotyping and phylogenies, followed by the selection of the nonsynonymous SNPs present in the most conserved cluster of orthologous groups (COG) of each genotype of MTB. Genotype assignment of the new set of 10 SNPs was validated using an additional of 34 MTB genomes and results showed 100% correlation with their known genotypes. Our set of 10 SNPs have not been previously reported and cover the MTB genotypes that are prevalent worldwide. This set of SNPs could be used for molecular epidemiology with drug resistant markers.

[Comparative genomic analysis of peruvian strains of Mycobacterium tuberculosis]. / Análisis genómico comparativo de cepas peruanas de Mycobacterium tuberculosis.

OBJECTIVES: To comparatively analyze three genomic sequences of Mycobacterium tuberculosis(MTB), including sensitive (INS-SEN), multi-drug-resistant (INS-MDR), and extremely drug-resistant (INS-XDR) strains, collected in Lima, Peru. MATERIALS AND METHODS: Specific single nucleotide polymorphisms (SNPs) were identified in the INS SEN, INS-MDR, and INS-XDR strains according to the inclusion/exclusion criteria. The three MTB genomes were compared and a molecular phylogeny was constructed with 27 MTB strains from other studies available from the Genbank database. RESULTS: The specific SNPs in each genome were organized in clusters of orthologous groups (COGs). The genomic analysis allowed for the identification of a set of SNPs associated mainly with virulence determinants (family of mce proteins, polyketides, phiRv1, transposase, and methyltransferases, and other related to vitamin synthesis). A close correlation between the INS-MDR and INS-XDR strains was observed, with only a 6.1% difference in SNPs; however, the INS-SEN strain had 50.2% and 50.3% different SNPs from the MDR and XDR strains, respectively. The molecular phylogeny grouped the Peruvian strains within the LAM lineage and closely to the F11 and KZN strains from South Africa. CONCLUSIONS: High similarity (99.9%) was noted between the INS-SEN strain and the F11 South African strain with broadglobal scope, while the analysis of the INS-MDR and INS-XDR strains showed a likely expansion of the KZN family, a South African strain with high virulence and pathogenicity.

High-resolution melting analysis for molecular detection of multidrug resistance tuberculosis in Peruvian isolates.

BACKGROUND: The emergence of multidrug-resistant strains is a major health problem especially for countries with high TB incidence such as Peru. In this study, we evaluated High Resolution Melting (HRM) assay in Peruvian isolates for the detection of mutations within rpoB, katG genes and promoter region inhA to determine isoniazid and rifampicin resistance in Mycobacterium tuberculosis (Mtb). METHODS: DNA samples extracted from a total of 167 clinical isolates of Mtb, 89 drug-sensitive and 78 multidrug-resistant, were blindly analyzed by HRM analysis and verified by DNA sequencing. RESULTS: The HRM analysis generated patterns that were specific to distinguish between sensitive and resistance isolates. The sensitivity and specificity of the HRM assays in comparison with drug susceptibility testing (DST) for detection of rifampicin resistance were 98.7 % and 97.5 %, and for isoniazid resistance were 98.7 % and 100 %. CONCLUSION: This study suggests that HRM Analysis could help with rapid diagnosis of MDR-TB cases in Peru.

Análisis genómico comparativo de cepas peruanas de Mycobacterium tuberculosis / Comparative genomic analysis of peruvian strains of Mycobacterium tuberculosis

RESUMEN Objetivos. Analizar comparativamente tres secuencias genómicas de Mycobacterium tuberculosis(MTB): INS-SEN,cepa sensible; INS-MDR, cepa multidrogorresistente e INS-XDR, cepa extensamente resistente, procedentes de la Ciudad de Lima, Perú. Materiales y métodos. Se identificaron los polimorfismos de un solo nucleótido (SNPs) específicos en las cepas INS-SEN, INS-MDR y INS-XDR mediante el criterio de inclusión/exclusión. Se compararon los tres genomas de MTB y se construyó una filogenia molecular con 27 cepas de MTB de otros estudios, disponibles de la base de datos Genbank. Los SNPs específicos en cada genoma fueron organizados en clústers de grupos ortólogos (COGs). Resultados. El análisis de genomas permitió identificar un conjunto de SNPs asociados a determinantes de virulencia (familia de proteínas mce, policetidos, phiRv1, transposasas, metiltransferasas y relacionados a síntesis de vitaminas) principalmente. Se observa una estrecha relación entre la cepa INS-MDR y INS-XDR, con solo un 6,1% de SNPs diferentes, sin embargo, la cepa INS-SEN presenta un 50,2 y 50,3% de SNPs diferentes a las cepas MDR y XDR, respectivamente. La filogenia molecular agrupó a las cepas peruanas dentro del linaje LAM y cercanamente a las cepas F11 y KZN de Sudáfrica. Conclusiones . Se evidenció una alta similitud (99,9%) de la cepa INS-SEN con la cepa sudafricana F11, de gran alcance mundial, mientras los análisis de las cepas INS-MDR e INS-XDR demuestran una probable expansión de la familia KZN, cepa de Sudáfrica con alta virulencia y patogenicidad.

ABSTRACT Objectives. To comparatively analyze three genomic sequences of Mycobacterium tuberculosis(MTB), including sensitive (INS-SEN), multi-drug-resistant (INS-MDR), and extremely drug-resistant (INS-XDR) strains, collected in Lima, Peru. Materials and Methods. Specific single nucleotide polymorphisms (SNPs) were identified in the INS SEN, INS-MDR, and INS-XDR strains according to the inclusion/exclusion criteria. The three MTB genomes were compared and a molecular phylogeny was constructed with 27 MTB strains from other studies available from the Genbank database. Results. The specific SNPs in each genome were organized in clusters of orthologous groups (COGs). The genomic analysis allowed for the identification of a set of SNPs associated mainly with virulence determinants (family of mce proteins, polyketides, phiRv1, transposase, and methyltransferases, and other related to vitamin synthesis). A close correlation between the INS-MDR and INS-XDR strains was observed, with only a 6.1% difference in SNPs; however, the INS-SEN strain had 50.2% and 50.3% different SNPs from the MDR and XDR strains, respectively. The molecular phylogeny grouped the Peruvian strains within the LAM lineage and closely to the F11 and KZN strains from South Africa. Conclusions. High similarity (99.9%) was noted between the INS-SEN strain and the F11 South African strain with broadglobal scope, while the analysis of the INS-MDR and INS-XDR strains showed a likely expansion of the KZN family, a South African strain with high virulence and pathogenicity.

Germline mutations of the DNA repair pathways in uterine serous carcinoma.

OBJECTIVE: Treatment options are limited for patients with uterine serous carcinoma (USC). Knowledge of USC's somatic mutation landscape is rapidly increasing, but its role in hereditary cancers remains unclear. We aim to evaluate the frequency and characteristics of germline mutations in genes commonly implicated in carcinogenesis, including those within homologous recombination (HR) and mismatch repair (MMR) pathways in patients with pure USC. METHODS: By using targeted capture exome sequencing, 43 genes were analyzed in a cohort of 7 consecutive patients with paired tumor and non-tumor USC samples in our institutional tumor repository. Mutations predicted to have damaging effects on protein function are validated by Sanger Sequencing. RESULTS: We found 21 germline mutations in 11 genes in our USC cohort. Five patients harbored 7 germline mutations (33.3%) within genes involved in the HR pathway, RAD51D being the most common. Four patients had 9 (42.8%) germline mutations in hereditary colon cancer genes, most commonly MLH. All patients (42.7%) who are platinum-sensitive had HR germline mutations (RAD50, NBN, ATM). Patients with HER2 overexpression (2/7, 28.6%) had germline HR mutations and were platinum-sensitive. Three patients in our cohort reported a personal history of breast cancer, one with HR germline mutation, and 2 in patients with germline mutations in HCC genes. In addition, 5 out of 7 patients had germline mutations in genes associated with growth factor signaling pathway. CONCLUSIONS: A significant proportion of our cohort harbor germline mutations in DNA repair genes. This may be associated with the high rate of breast cancer in our patients and their family, and suggests a targeted cohort for genetic counseling. If validated in a larger cohort, our findings may allow clinicians to expand therapeutic options to include targeted therapies and inclusion of USC patient in preventative and genetic counseling.