Forensic parameters and population structure based on 21 autosomal STRs with the investigator 24plex QS in mestizos from the Mexico City population.

Allele frequencies and forensic parameters for 21 STR autosomal markers (CSF1PO, D10S1248, D12S391, D13S317,D16S539, D18S51, D19S433, D1S1656,D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, SE33, TH01, TPOX and vWA) were reported in 289 unrelated individuals from Mexico City, Mexico. In addition, an interpopulation analysis was performed including other world populations. In brief, the established population database of 21 autosomal STR markers in the present work is adequate for human identification purposes.

A miRNome analysis at the early postmortem interval.

The postmortem interval (PMI) is the time elapsing since the death of an individual until the body is examined. Different molecules have been analyzed to better estimate the PMI with variable results. The miRNAs draw attention in the forensic field to estimate the PMI as they can better support degradation. In the present work, we analyzed the miRNome at early PMI in rats' skeletal muscle using the Affymetrix GeneChip™ miRNA 4.0 microarrays. We found 156 dysregulated miRNAs in rats' skeletal muscle at 24 h of PMI, out of which 84 were downregulated, and 72 upregulated. The miRNA most significantly downregulated was miR-139-5p (FC = -160, p = 9.97 × 10-11), while the most upregulated was rno-miR-92b-5p (FC = 241.18, p = 2.39 × 10-6). Regarding the targets of these dysregulated miRNAs, the rno-miR-125b-5p and rno-miR-138-5p were the miRNAs with more mRNA targets. The mRNA targets that we found in the present study participate in several biological processes such as interleukin secretion regulation, translation regulation, cell growth, or low oxygen response. In addition, we found a downregulation of SIRT1 mRNA and an upregulation of TGFBR2 mRNA at 24 h of PMI. These results suggest there is an active participation of miRNAs at early PMI which could be further explored to identify potential biomarkers for PMI estimation.

Pathogenic variant profile in DNA damage response genes correlates with metastatic breast cancer progression-free survival in a Mexican-mestizo population.

Introduction: Metastatic breast cancer causes the most breast cancer-related deaths around the world, especially in countries where breast cancer is detected late into its development. Genetic testing for cancer susceptibility started with the BRCA 1 and 2 genes. Still, recent research has shown that variations in other members of the DNA damage response (DDR) are also associated with elevated cancer risk, opening new opportunities for enhanced genetic testing strategies. Methods: We sequenced BRCA1/2 and twelve other DDR genes from a Mexican-mestizo population of 40 metastatic breast cancer patients through semiconductor sequencing. Results: Overall, we found 22 variants -9 of them reported for the first time- and a strikingly high proportion of variations in ARID1A. The presence of at least one variant in the ARID1A, BRCA1, BRCA2, or FANCA genes was associated with worse progression-free survival and overall survival in our patient cohort. Discussion: Our results reflected the unique characteristics of the Mexican-mestizo population as the proportion of variants we found differed from that of other global populations. Based on these findings, we suggest routine screening for variants in ARID1A along with BRCA1/2 in breast cancer patients from the Mexican-mestizo population.

Species Delimitation of Scavenger Flies in the Valley of Mexico.

Identification of species involved in cadaveric decomposition, such as scavenger Diptera, is a fundamental step for the use of entomological evidence in court. Identification based on morphology is widely used in forensic cases; however, taxonomic knowledge of scavenger fauna is poor for many groups and for many countries, particularly Neotropical ones. A number of studies have documented the utility of a DNA barcoding strategy to assist in the identification of poorly known and diverse groups, particularly in cases involving immature states or fragmented organisms. To provide baseline knowledge of the diversity of scavenger Diptera in the Valley of Mexico, we generated a DNA barcode collection comprised of sequences of the cytochrome c oxidase subunit 1 (COI) gene for all families sampled at a nature reserve located in this region. We collected and identified specimens on the basis of morphology and a species delimitation analysis. Our analyses of 339 individuals delineated 42 species distributed across nine families of Diptera. The richest families were Calliphoridae (9 species), Sarcophagidae (7 species), and Phoridae (6 species). We found many of the species previously recorded for the Valley of Mexico, plus 18 new records for the region. Our study highlights the utility of DNA barcoding as a first-step strategy to assess species richness of poorly studied scavenger fly taxa.

Dysregulation of miR-381-3p and miR-23b-3p in skeletal muscle could be a possible estimator of early post-mortem interval in rats.

BACKGROUND: The post-mortem interval (PMI) is the time elapsed since the dead of an individual until the body is found, which is relevant for forensic purposes. The miRNAs regulate the expression of some genes; and due to their small size, they can better support degradation, which makes them suitable for forensic analysis. In the present work, we evaluated the gene expression of miR-381-3p, miR-23b-3p, and miR-144-3p in skeletal muscle in a murine model at the early PMI. METHODS: We designed a rat model to evaluate the early PMI under controlled conditions. This model consisted in 25 rats divided into five groups of rats, that correspond to the 0, 3, 6, 12 and 24 hours of PMI. The 0 h-PMI was considered as the control group. Muscle samples were taken from each rat to analyze the expression of miR-381-3p, miR-23b-3p, and miR-144-3p by quantitative RT-PCR. The gene expression of each miRNA was expressed as Fold Change (FC) and compared among groups. To find the targets of these miRNAs and the pathways where they participate, we performed an in-silico analysis. From the gene targets of miR-381-3p identified in the silico analysis, the EPC1 gene was selected for gene expression analysis by quantitative RT-PCR in these samples. Also, to evaluate if miR-381-3p could predict the early PMI, a mixed effects model was calculated using its gene expression. RESULTS: An upregulation of miR-381-3p was found at 24 h-PMI compared with the control group of 0 h-PMI and (FC = 1.02 vs. FC = 1.96; p = 0.0079). This was the opposite for miR-23b-3p, which had a down-regulation at 24 h-PMI compared to 0 h-PMI (FC = 1.22 vs. FC = 0.13; p = 0.0079). Moreover, the gene expression of miR-381-3p increased throughout the first 24 h of PMI, contrary to miR-23b-3p. The targets of these two miRNAs, participate in biological pathways related to hypoxia, apoptosis, and RNA metabolism. The gene expression of EPC1 was found downregulated at 3 and 12 h of PMI, whereas it remained unchanged at 6 h and 24 h of PMI. Using a multivariate analysis, it was possible to predict the FC of miR-381-3p of all but 6 h-PMI analyzed PMIs. DISCUSSION: The present results suggest that miR-23b-3p and miR-381-3p participate at the early PMI, probably regulating the expression of some genes related to the autolysis process as EPC1 gene. Although the miR-381-3p gene expression is a potential estimator of PMI, further studies will be required to obtain better estimates.

Negative Regulation of Serine Threonine Kinase 11 (STK11) through miR-100 in Head and Neck Cancer.

BACKGROUND: Serine Threonine Kinase 11 (STK11), also known as LKB1, is a tumor suppressor gene that regulates several biological processes such as apoptosis, energetic metabolism, proliferation, invasion, and migration. During malignant progression, different types of cancer inhibit STK11 function by mutation or epigenetic inactivation. In Head and Neck Cancer, it is unclear what mechanism is involved in decreasing STK11 levels. Thus, the present work aims to determine whether STK11 expression might be regulated through epigenetic or post-translational mechanisms. METHODS: Expression levels and methylation status for STK11 were analyzed in 59 cases of head and neck cancer and 10 healthy tissue counterparts. Afterward, we sought to identify candidate miRNAs exerting post-transcriptional regulation of STK11. Then, we assessed a luciferase gene reporter assay to know if miRNAs directly target STK11 mRNA. The expression levels of the clinical significance of mir-100-3p, -5p, and STK11 in 495 HNC specimens obtained from the TCGA database were further analyzed. Finally, the Kaplan-Meier method was used to estimate the prognostic significance of the miRNAs for Overall Survival, and survival curves were compared through the log-rank test. RESULTS: STK11 was under-expressed, and its promoter region was demethylated or partially methylated. miR-17-5p, miR-106a-5p, miR-100-3p, and miR-100-5p could be negative regulators of STK11. Our experimental data suggested evidence that miR-100-3p and -5p were over-expressed in analyzed tumor patient samples. Luciferase gene reporter assay experiments showed that miR-100-3p targets and down-regulates STK11 mRNA directly. With respect to overall survival, STK11 expression level was significant for predicting clinical outcomes. CONCLUSION: This is, to our knowledge, the first report of miR-100-3p targeting STK11 in HNC. Together, these findings may support the importance of regulation of STK11 through post-transcriptional regulation in HNC and the possible contribution to the carcinogenesis process in this neoplasia.

The NR3C1 gene expression is a potential surrogate biomarker for risk and diagnosis of posttraumatic stress disorder.

Posttraumatic Stress Disorder (PTSD) is an anxiety disorder which occurs after a traumatic event. The NR3C1 gene codes for the Glucocorticoid Receptor, which participate in the Hypothalamic-Pituitary-Adrenal (HPA) axis and is altered in PTSD patients. To evaluate whether the NR3C1 gene expression in peripheral blood could be useful as a diagnosis biomarker, a total of 32 PTSD patients and 59 healthy controls were analyzed with quantitative RT-PCR. Also, to assess if NR3C1 dysregulation is associated with hypocortisolism in PTSD patients, serum cortisol was quantified by ELISA in a subset of these samples. Significant NR3C1 over-expression was found in PTSD patients compared with controls, and this was higher in patients with acute PTSD. The Area Under the Curve (AUC) of NR3C1 gene expression was 0.797. The sensibility and specificity of NRC1 gene expression to diagnose PTSD was 62.5% and 89.8%, respectively. We also found that an up-regulation of NR3C1 increased the risk for being diagnosed with PTSD (OR= 12.8, 95%, CI 4-41.4). Finally, the NR3C1 gene expression was inversely related with serum cortisol in PTSD patients. The present results suggest that NR3C1 gene expression could be a promising biomarker for PTSD diagnosis and estimate the risk for disease development.

CDKN3 mRNA as a Biomarker for Survival and Therapeutic Target in Cervical Cancer.

The cyclin-dependent kinase inhibitor 3 (CDKN3) gene, involved in mitosis, is upregulated in cervical cancer (CC). We investigated CDKN3 mRNA as a survival biomarker and potential therapeutic target for CC. CDKN3 mRNA was measured in 134 CC and 25 controls by quantitative PCR. A 5-year survival study was conducted in 121 of these CC patients. Furthermore, CDKN3-specific siRNAs were used to investigate whether CDKN3 is involved in proliferation, migration, and invasion in CC-derived cell lines (SiHa, CaSki, HeLa). CDKN3 mRNA was on average 6.4-fold higher in tumors than in controls (p = 8 x 10-6, Mann-Whitney). A total of 68.2% of CC patients over expressing CDKN3 gene (fold change ≥ 17) died within two years of diagnosis, independent of the clinical stage and HPV type (Hazard Ratio = 5.0, 95% CI: 2.5-10, p = 3.3 x 10-6, Cox proportional-hazards regression). In contrast, only 19.2% of the patients with lower CDKN3 expression died in the same period. In vitro inactivation of CDKN3 decreased cell proliferation on average 67%, although it had no effect on cell migration and invasion. CDKN3 mRNA may be a good survival biomarker and potential therapeutic target in CC.

The distribution of high-risk human papillomaviruses is different in young and old patients with cervical cancer.

Despite numerous human papillomavirus (HPV) frequency studies in women with cervical cancer (CC), little is known of HPV frequency trends according to patient age. In this work, we compare the mean age and frequency distribution by age of CC patients positive for different HPVs. This study included 462 CC patients. HPVs were detected by PCR and typed using DNA sequencing. A total of 456 patients (98.7%) were positive for HPV: 418 (90.5%) had single and 38 (8.2%) had double HPV infections. HPV16 (46.5%), HPV18 (10.4%), HPV45 (6.7%), and HPV31 (4.1%) were the most frequent viral types in single-infected patients. The mean ages of single-infected patients with HPV16 (49.2±13.3), HPV18 (47.9±12.2), HPV45 (47.9±11.7), or HPV39 (42.6±8.9) were significantly lower than the mean ages of patients singly (53.9±12.7; p<0.001, t-test) or doubly (55.4±12.7; p<0.05, t-test) infected with the remaining HPVs. Three different trends were identified: one for HPV16, another for HPVs18/45/39, and a third for the rest of HPVs. The frequency trend of HPV16 shows two peaks. The first (63.2%) was found in the youngest women (≤35 years), followed by a decreasing trend until the age of 55-60 years (31.1%). The second peak arose at 61-65 years (52.5%), followed by a decreasing trend. The trend for HPVs18/45/39 declined from the youngest (19.3%) to the oldest (>70 years; 12.8%) women. In contrast, the trend for the remaining HPVs increased from the youngest (15.8%) to the oldest (46.2%) women. Unlike other life-style factors, low-risk sexual behavior was associated with late onset of CC independent of low-oncogenic HPV types (p<0.05, Wald chi-square statistic). The data indicate that most CCs in young women depend on the presence of high-oncogenic HPVs. In contrast, almost half of CCs in older patients had low-oncogenic HPVs, suggesting they could depend on the presence of other factors.

Impact of gene dosage on gene expression, biological processes and survival in cervical cancer: a genome-wide follow-up study.

We investigated the role of tumor copy number (CN)-altered genome (CN-AG) in the carcinogenesis of cervical cancer (CC), especially its effect on gene expression, biological processes, and patient survival. Fifty-nine human papillomavirus 16 (HPV16)-positive CCs were investigated with microarrays-31 for mapping CN-AG and 55 for global gene expression, with 27 CCs in common. Five-year survival was investigated in 55 patients. Deletions and amplifications >2.5 Mb were defined as CN alterations. The %CN-AG varied from 0 to 32.2% (mean = 8.1±8.9). Tumors were classified as low (mean = 0.5±0.6, n = 11), medium (mean = 5.4±2.4, n = 10), or high (mean = 19.2±6.6, n = 10) CN. The highest %CN-AG was found in 3q, which contributed an average of 55% of all CN alterations. Genome-wide, only 5.3% of CN-altered genes were deregulated directly by gene dosage. In contrast, the rate in fully duplicated 3q was twice as high. Amplification of 3q explained 23.2% of deregulated genes in whole tumors (r2 = 0.232, p = 0.006; analysis of variance), including genes located in 3q and other chromosomes. A total of 862 genes were deregulated exclusively in high-CN tumors, but only 22.9% were CN altered. This suggests that the remaining genes are not deregulated directly by gene dosage, but by mechanisms induced in trans by CN-altered genes. Anaphase-promoting complex/cyclosome (APC/C)-dependent proteasome proteolysis, glycolysis, and apoptosis were upregulated, whereas cell adhesion and angiogenesis were downregulated exclusively in high-CN tumors. The high %CN-AG and upregulated gene expression profile of APC/C-dependent proteasome proteolysis were associated with poor patient survival (p<0.05, log-rank test). Along with glycolysis, they were linearly associated with FIGO stage (r>0.38, p<0.01, Spearman test). Therefore, inhibition of APC/C-dependent proteasome proteolysis and glycolysis could be useful for CC treatment. However, whether they are indispensable for tumor growth remains to be demonstrated.

Mitosis is a source of potential markers for screening and survival and therapeutic targets in cervical cancer.

The effect of preventive human papillomavirus (HPV) vaccination on the reduction of the cervical cancer (CC) burden will not be known for 30 years. Therefore, it's still necessary to improve the procedures for CC screening and treatment. The objective of this study was to identify and characterize cellular targets that could be considered potential markers for screening or therapeutic targets. A pyramidal strategy was used. Initially the expression of 8,638 genes was compared between 43 HPV16-positive CCs and 12 healthy cervical epitheliums using microarrays. A total of 997 genes were deregulated, and 21 genes that showed the greatest deregulation were validated using qRT-PCR. The 6 most upregulated genes (CCNB2, CDC20, PRC1, SYCP2, NUSAP1, CDKN3) belong to the mitosis pathway. They were further explored in 29 low-grade cervical intraepithelial neoplasias (CIN1) and 21 high-grade CIN (CIN2/3) to investigate whether they could differentiate CC and CIN2/3 (CIN2+) from CIN1 and controls. CCNB2, PRC1, and SYCP2 were mostly associated with CC and CDC20, NUSAP1, and CDKN3 were also associated with CIN2/3. The sensitivity and specificity of CDKN3 and NUSAP1 to detect CIN2+ was approximately 90%. The proteins encoded by all 6 genes were shown upregulated in CC by immunohistochemistry. The association of these markers with survival was investigated in 42 CC patients followed up for at least 42 months. Only CDKN3 was associated with poor survival and it was independent from clinical stage (HR = 5.9, 95%CI = 1.4-23.8, p = 0.01). CDKN3 and NUSAP1 may be potential targets for the development of screening methods. Nevertheless, further studies with larger samples are needed to define the optimal sensitivity and specificity. Inhibition of mitosis is a well-known strategy to combat cancers. Therefore, CDKN3 may be not only a screening and survival marker but a potential therapeutic target in CC. However, whether it's indispensable for tumor growth remains to be demonstrated.

Amplified genes may be overexpressed, unchanged, or downregulated in cervical cancer cell lines.

Several copy number-altered regions (CNAs) have been identified in the genome of cervical cancer, notably, amplifications of 3q and 5p. However, the contribution of copy-number alterations to cervical carcinogenesis is unresolved because genome-wide there exists a lack of correlation between copy-number alterations and gene expression. In this study, we investigated whether CNAs in the cell lines CaLo, CaSki, HeLa, and SiHa were associated with changes in gene expression. On average, 19.2% of the cell-line genomes had CNAs. However, only 2.4% comprised minimal recurrent regions (MRRs) common to all the cell lines. Whereas 3q had limited common gains (13%), 5p was entirely duplicated recurrently. Genome-wide, only 15.6% of genes located in CNAs changed gene expression; in contrast, the rate in MRRs was up to 3 times this. Chr 5p was confirmed entirely amplified by FISH; however, maximum 33.5% of the explored genes in 5p were deregulated. In 3q, this rate was 13.4%. Even in 3q26, which had 5 MRRs and 38.7% recurrently gained SNPs, the rate was only 15.1%. Interestingly, up to 19% of deregulated genes in 5p and 73% in 3q26 were downregulated, suggesting additional factors were involved in gene repression. The deregulated genes in 3q and 5p occurred in clusters, suggesting local chromatin factors may also influence gene expression. In regions amplified discontinuously, downregulated genes increased steadily as the number of amplified SNPs increased (p<0.01, Spearman's correlation). Therefore, partial gene amplification may function in silencing gene expression. Additional genes in 1q, 3q and 5p could be involved in cervical carcinogenesis, specifically in apoptosis. These include PARP1 in 1q, TNFSF10 and ECT2 in 3q and CLPTM1L, AHRR, PDCD6, and DAP in 5p. Overall, gene expression and copy-number profiles reveal factors other than gene dosage, like epigenetic or chromatin domains, may influence gene expression within the entirely amplified genome segments.

The Amerindian mtDNA haplogroup B2 enhances the risk of HPV for cervical cancer: de-regulation of mitochondrial genes may be involved.

Although human papillomavirus (HPV) infection is the main causal factor for cervical cancer (CC), there are data suggesting that genetic factors could modulate the risk for CC. Sibling studies suggest that maternally inherited factors could be involved in CC. To assess whether mitochondrial DNA (mtDNA) polymorphisms are associated to CC, HPV infection and HPV types, a case-control study was performed in the Mexican population. Polymorphism of mtDNA D-loop was investigated in 187 CC patients and 270 healthy controls. HPV was detected and typed in cervical scrapes. The expression of 29 mitochondrial genes was analyzed in a subset of 45 tumor biopsies using the expression microarray ST1.0. The Amerindian haplogroup B2 increased the risk for CC (odds ratio (OR)=1.6; 95% confidence interval (CI): 1.05-2.58) and enhanced 36% (OR=208; 95% CI: 25.2-1735.5) the risk conferred by the HPV alone (OR=152.9; 95% CI: 65.4-357.5). In cases, the distribution of HPV types was similar in all haplogroups but one (D1), in which is remarkable the absence of HPV18, a very low frequency of HPV16 and high frequencies of HPV45, HPV31 and other HPV types. Two mtDNA genes (mitochondrial aspartic acid tRNA (MT-TD), mitochondrial lysine tRNA (MT-TK)) could be involved in the increased risk conferred by the haplogroup B2, as they were upregulated exclusively in B2 tumors (P<0.01, t-test). Although the association of mtDNA with CC and HPV infection is clear, other studies with higher sample size will be needed to elucidate the role of mtDNA in cervical carcinogenesis.

The presence of aflatoxin B1-FAPY adduct and human papilloma virus in cervical smears from cancer patients in Mexico.

The carcinogenic biomarker aflatoxin B(1)-formamidopyrimidine 2,3-dihydro-2-(N-formyl)-2',5',6'-triamino-4'-4'-oxy-N-pyrimidyl-3-hydroxy-AFB(1) called AFB(1)-FAPY adduct, and Human Papilloma Virus (HPV) types 16 and 18 were quantified from DNA cervical scrapes from 40 women with cervical cancer (CC) and 14 healthy women as controls. The relationship between the AFB(1)-FAPY adduct and HPV types 16 and 18 was determined. Competitive inhibitory indirect ELISA was validated with 94% inhibition to quantify the AFB(1)-FAPY adducts in picograms per milligram of DNA (limit of detection = 0.1 pg/mg, and limit of quantification = 10 pg/mg), polymerase chain reaction and DNA sequencing to identify HPV types. The average concentration of AFB(1)-FAPY adducts/mg DNA in the CC cases was 1025 pg, 1420 pg with HPV16 and 630 pg sharing HPV18 (p = 0.03). In comparison, healthy controls had ≤ 2.6 pg/mg DNA, a statistically significant difference (p = 0.00006). The presence of AFB(1)-FAPY adduct increased six-fold the risk for CC between cases and controls, the odds ratio was 6.1 (95% CI = 1.4-25.4). There was a close relationship between the AFB(1)-FAPY adducts and HPV16 in CC samples.

HPV-16 and HLA-DRB1 alleles are associated with cervical carcinoma in Mexican Mestizo women.

The aim of this report was to investigate the contribution of HLA-DRB1/DQB1 alleles to the expression of cervical cancer (CC) and squamous intraepithelial lesion (SIL) in Mexican patients. A total of 257 women were included in the study: 61with low-grade squamous intraepithelial lesions (LSIL), 30 with high-grade (HSIL), 73 with CC and 93 healthy females. All were Mexican Mestizos. For HLA class II typing, PCR-SSOP methodology was used. HPV-16 viral DNA was detected by PCR with specific primers for E6-E7 region. HPV-16 was found in 52% of the patients with CC as well as in 19% of women with HSIL and in 12.5% of females with LSIL. HLA-DRB1∗04:03 (OR = 5.88) was found increased in patients with HSIL as compared with controls, although significance (p = 0.04) was lost after correction (pc =NS). HLA-DRB1∗04:03 seems to influence the risk for developing HSIL, disregarding the presence of HPV-16. HLA-DRB1∗01:01 (OR = 0.12; p = 0.01) may confer protection to the development of CC. An analysis performed stratifying by the presence of HPV-16 infection showed that the frequency of HLA-DRB1∗04:07 (OR = 2.71) was increased in CC patients infected with HPV-16, confirming that the HLA association is HPV dependent. These results shed light on the influence that this virus may have in the expression of CC in the susceptible host. Genetic background is, therefore, a crucial factor in understanding the etiopathogenesis of CC in HPV-positive patients.

A great diversity of Amerindian mitochondrial DNA ancestry is present in the Mexican mestizo population.

There are limited data on mitochondrial DNA (mtDNA) variation in the Mexican mestizo population. To examine the genetic diversity and matrilineal ancestry, the full mtDNA hypervariable regions I and II were sequenced in 270 unrelated mestizos from different regions of Mexico. A total of 202 different haplotypes were identified and the haplotype diversity was 0.9945. Amerindian haplotypes predominated in the sample with a proportion of 93.3%, followed by European (6.0%) and African haplotypes (0.7%). The frequency of the Amerindian haplogroups A2, B2, C1 and D1 was 51.1, 17.8, 18.5 and 5.9%, respectively. The frequency of Amerindian haplogroups was higher in the central region than in Mexico City, whereas it was the contrary for European haplogroups. This difference was accounted principally by the high frequency of B2 haplotypes in the central region. The minimum spanning network, the mismatch distribution and Tajima's D neutrality test suggest a population expansion for each Amerindian haplogroup, which could be initiated more recently for haplogroups A2 and D1. The present knowledge combined with other nuclear genetic markers will be essential in future association studies to correct for genetic substructure in mestizo populations.