Differential Expression of miRNAs in Amyotrophic Lateral Sclerosis Patients.

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease that affects nerve cells in the brain and spinal cord, causing loss of muscle control, muscle atrophy and in later stages, death. Diagnosis has an average delay of 1 year after symptoms onset, which impairs early management. The identification of a specific disease biomarker could help decrease the diagnostic delay. MicroRNA (miRNA) expression levels have been proposed as ALS biomarkers, and altered function has been reported in ALS pathogenesis. The aim of this study was to assess the differential expression of plasma miRNAs in ALS patients and two control populations (healthy controls and ALS-mimic disorders). For that, 16 samples from each group were pooled, and then 1008 miRNAs were assessed through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). From these, ten candidate miRNAs were selected and validated in 35 ALS patients, 16 ALS-mimic disorders controls and 15 healthy controls. We also assessed the same miRNAs in two different time points of disease progression. Although we were unable to determine a miRNA signature to use as disease or condition marker, we found that miR-7-2-3p, miR-26a-1-3p, miR-224-5p and miR-206 are good study candidates to understand the pathophysiology of ALS.

Single Mutations in Cytochrome P450 Oxidoreductase Can Alter the Specificity of Human Cytochrome P450 1A2-Mediated Caffeine Metabolism.

A unique cytochrome P450 (CYP) oxidoreductase (CPR) sustains activities of human microsomal CYPs. Its function requires toggling between a closed conformation enabling electron transfers from NADPH to FAD and then FMN cofactors and open conformations forming complexes and transferring electrons to CYPs. We previously demonstrated that distinct features of the hinge region linking the FAD and FMN domain (FD) modulate conformer poses and their interactions with CYPs. Specific FD residues contribute in a CYP isoform-dependent manner to the recognition and electron transfer mechanisms that are additionally modulated by the structure of CYP-bound substrate. To obtain insights into the underlying mechanisms, we analyzed how hinge region and FD mutations influence CYP1A2-mediated caffeine metabolism. Activities, metabolite profiles, regiospecificity and coupling efficiencies were evaluated in regard to the structural features and molecular dynamics of complexes bearing alternate substrate poses at the CYP active site. Studies reveal that FD variants not only modulate CYP activities but surprisingly the regiospecificity of reactions. Computational approaches evidenced that the considered mutations are generally in close contact with residues at the FD-CYP interface, exhibiting induced fits during complexation and modified dynamics depending on caffeine presence and orientation. It was concluded that dynamic coupling between FD mutations, the complex interface and CYP active site exist consistently with the observed regiospecific alterations.

Performance of the ACMG-AMP criteria in a large familial renal glucosuria cohort with identified SLC5A2 sequence variants.

Familial Renal Glucosuria (FRG) is a co-dominantly inherited trait characterized by orthoglycaemic glucosuria. From 2003 to 2015 we have reported several cohorts validating SLC5A2 (16p11.2), encoding SGLT2 (Na+/glucose cotransporter family member 2), as the gene responsible for FRG. The aim of this work was to validate the variants identified in our extended FRG cohort of published, as well more recent unreported cases, according to the ACMG-AMP 2015 criteria. Forty-six variants were evaluated, including 16 novel alleles first described in this study. All are rare, ultra-rare or absent from population databases and most are missense changes. According to the ACMG-AMP standards, only 74% of the variants were classified as P/LP. The lack of descriptions of unrelated patients with similar variants or failing to test additional affected family members, averted a conclusion for pathogenicity in the alleles that scored VUS, highlighting the importance of both family testing and variant reporting. Finally, the cryo-EM structure of the hSGLT2-MAP17 complex in the empagliflozin-bound state improved the ACMG-AMP pathogenicity score by identifying critical/functional protein domains.

BRCA1 VUS: A functional analysis to differentiate pathogenic from benign variants identified in clinical diagnostic panels for breast cancer.

Genetic testing for susceptibility genes through next­generation sequencing (NGS) has become a widely used technique. Using this, a number of genetic variants have been identified, several of which are variants of unknown significance (VUS). These VUS can either be pathogenic or benign. However, since their biological effect remains unclear, functional assays are required to classify their functional nature. As the use of NGS becomes more mainstream as a diagnostic tool in clinical practice, the number of VUS is expected to increase. This necessitates their biological and functional classification. In the present study, a VUS was identified in the BRCA1 gene (NM_007294.3:c.1067A>G) in two women at risk for breast cancer, for which no functional data has been reported. Therefore, peripheral lymphocytes were isolated from the two women and also from two women without the VUS. DNA from all samples were sequenced by NGS of a breast cancer clinical panel. Since the BRCA1 gene is involved in DNA repair and apoptosis, the functional assays chromosomal aberrations, cytokinesis­blocked micronucleus, comet, γH2AX, caspase and TUNEL assays were then conducted on these lymphocytes after a genotoxic challenge by ionizing radiation or doxorubicin to assess the functional role of this VUS. The micronucleus and TUNEL assays revealed a lower degree of DNA induced­damage in the VUS group compared with those without the VUS. The other assays showed no significant differences between the groups. These results suggested that this BRCA1 VUS is likely benign, since the VUS carriers were apparently protected from deleterious chromosomal rearrangements, subsequent genomic instability and activation of apoptosis.

Genetic determinants and absence of breast cancer in Xavante Indians in Sangradouro Reserve, Brazil.

Genetic compositions of distinct human populations are different. How genomic variants influence many common and rare genetic diseases is always of great medical and anthropological interest, and understanding of genetic architectures of population groups in relation to diseases can advance our knowledge of medicine. Here, we have studied the genomic architecture of a group of Xavante Indians, an indigenous population in Brazil, and compared them with normal populations from the 1000 Genomes Projects. Principal component analysis (PCA) indicates that the Xavante Indians are genetically distinctive when compared to other ethnic groups. No incidence of breast cancer cases has ever been reported in the population, and polygenic risk analysis indicates extremely low breast cancer risk in this population when compared with germline TCGA (The Cancer Genome Atlas) breast cancer normal control samples. Low germinal mutation burden among this population is also observed. Our findings will help to deepen the understanding of breast cancer and might also provide new approaches to study the disease.

The Complex Dynamic of Phase I Drug Metabolism in the Early Stages of Doxorubicin Resistance in Breast Cancer Cells.

The altered activity of drug metabolism enzymes (DMEs) is a hallmark of chemotherapy resistance. Cytochrome P450s (CYPs), mainly CYP3A4, and several oxidoreductases are responsible for Phase I metabolism of doxorubicin (DOX), an anthracycline widely used in breast cancer (BC) treatment. This study aimed to investigate the role of Phase I DMEs involved in the first stages of acquisition of DOX-resistance in BC cells. For this purpose, the expression of 92 DME genes and specific CYP-complex enzymes activities were assessed in either sensitive (MCF-7 parental cells; MCF-7/DOXS) or DOX-resistant (MCF-7/DOXR) cells. The DMEs genes detected to be significantly differentially expressed in MCF-7/DOXR cells (12 CYPs and eight oxidoreductases) were indicated previously to be involved in tumor progression and/or chemotherapy response. The analysis of CYP-mediated activities suggests a putative enhanced CYP3A4-dependent metabolism in MCF-7/DOXR cells. A discrepancy was observed between CYP-enzyme activities and their corresponding levels of mRNA transcripts. This is indicative that the phenotype of DMEs is not linearly correlated with transcription induction responses, confirming the multifactorial complexity of this mechanism. Our results pinpoint the potential role of specific CYPs and oxidoreductases involved in the metabolism of drugs, retinoic and arachidonic acids, in the mechanisms of chemo-resistance to DOX and carcinogenesis of BC.

New "Omics" Approaches as Tools to Explore Mechanistic Nanotoxicology.

In the last years, "omics" approaches have been applied to study the toxicity of nanomaterials (NM) with the aim of obtaining insightful information on their biological effects. One of the most developed "omics" field, transcriptomics, expects to find unique profiles of differentially-expressed genes after exposure to NM that, besides providing evidence of their mechanistic mode of action, may also be used as biomarkers for biomonitoring purposes. Moreover, several NM have been associated with epigenetic alterations, i.e., changes in the regulation of gene expression caused by differential DNA methylation, histone tail modification and microRNA expression. Epigenomics research focusing on DNA methylation is increasingly common and the role of microRNAs is being better understood, either promoting or suppressing biological pathways. Moreover, the proteome is a highly dynamic system that changes constantly in response to a stimulus. Therefore, proteomics can identify changes in protein abundance and/or variability that lead to a better understanding of the underlying mechanisms of action of NM while discovering biomarkers. As to genomics, it is still not well developed in nanotoxicology. Nevertheless, the individual susceptibility to NM mediated by constitutive or acquired genomic variants represents an important component in understanding the variations in the biological response to NM exposure and, consequently, a key factor to evaluate possible adverse effects in exposed individuals. By elucidating the molecular changes that are involved NM toxicity, the new "omics" studies are expected to contribute to exclude or reduce the handling of hazardous NM in the workplace and support the implementation of regulation to protect human health.

The Central Role of Cytochrome P450 in Xenobiotic Metabolism-A Brief Review on a Fascinating Enzyme Family.

Human Cytochrome P450 (CYP) enzymes constitute a superfamily of membrane-bound hemoproteins that are responsible for the metabolism of a wide variety of clinically, physiologically, and toxicologically important compounds. These heme-thiolate monooxygenases play a pivotal role in the detoxification of xenobiotics, participating in the metabolism of many structurally diverge compounds. This short-review is intended to provide a summary on the major roles of CYPs in Phase I xenobiotic metabolism. The manuscript is focused on eight main topics that include the most relevant aspects of past and current CYP research. Initially, (I) a general overview of the main aspects of absorption, distribution, metabolism, and excretion (ADME) of xenobiotics are presented. This is followed by (II) a background overview on major achievements in the past of the CYP research field. (III) Classification and nomenclature of CYPs is briefly reviewed, followed by (IV) a summary description on CYP's location and function in mammals. Subsequently, (V) the physiological relevance of CYP as the cornerstone of Phase I xenobiotic metabolism is highlighted, followed by (VI) reviewing both genetic determinants and (VI) nongenetic factors in CYP function and activity. The last topic of the review (VIII) is focused on the current challenges of the CYP research field.

Male and female breast cancer: the two faces of the same genetic susceptibility coin.

BACKGROUND: Breast cancer (BC) is the most common cancer in women. In contrast, male BC is about 100 times less common than in women, being considered a rare disease. Male BC may be a distinctive subtype of BC and available data seems to indicate that male BC has a higher dependence on genetic variants than female BC. Nevertheless, the same prognostic and predictive markers are used to determine optimal management strategies for both male and female BC. Several studies have assessed the role of genetic polymorphisms (SNPs) in DNA repair genes in female BC susceptibility. However, data on male BC is scarce. Thus, the current study aimed to assess the role of SNPs in XRCC1, MUTYH and TP53 genes in a male cohort of BC, and, in addition, compare the male data with matched results previously genotyped in female BC patients. METHODS: The male BC cohort was genotyped through Real-Time PCR using TaqMan Assays for several SNPs previously analysed in Portuguese female BC patients. RESULTS: The results obtained indicate significant differences in BC susceptibility between males and females for the XRCC1 rs1799782, MUTYH rs3219489 and TP53 rs1042522 and rs8064946 variants. CONCLUSIONS: In males, XRCC1 and TP53 variants, when in heterozygosity, seem to be related with lower susceptibility for BC, contrasting with higher susceptibility for a MUTYH variant in females. These findings may help to explain the difference in incidence of BC between the two sexes.

Association Between miR-148a and DNA Methylation Profile in Individuals Exposed to Lead (Pb).

Experimental and epidemiologic studies have shown that lead (Pb) is able to induce epigenetic modifications, such as changes in DNA methylation profiles, in chromatin remodeling, as well as the expression of non-coding RNAs (ncRNAs). However, very little is known about the interactions between microRNAs (miRNAs) expression and DNA methylation status in individuals exposed to the metal. The aim of the present study was to investigate the impact of hsa-miR-148a expression on DNA methylation status, in 85 workers exposed to Pb. Blood and plasma lead levels (BLL and PLL, respectively) were determined by ICP-MS; expression of the miRNA-148a was quantified by RT-qPCR (TaqMan assay) and assessment of the global DNA methylation profile (by measurement of 5-methylcytosine; % 5-mC) was performed by ELISA. An inverse association was seen between miR-148a and % 5-mC DNA, as a function of BLL and PLL (ß = -3.7; p = 0.071 and ß = -4.1; p = 0.049, respectively) adjusted for age, BMI, smoking, and alcohol consumption. Taken together, our study provides further evidence concerning the interactions between DNA methylation profile and miR-148a, in individuals exposed to Pb.

Interaction Modes of Microsomal Cytochrome P450s with Its Reductase and the Role of Substrate Binding.

The activity of microsomal cytochromes P450 (CYP) is strictly dependent on the supply of electrons provided by NADPH cytochrome P450 oxidoreductase (CPR). The variant nature of the isoform-specific proximal interface of microsomal CYPs implies that the interacting interface between the two proteins is degenerated. Recently, we demonstrated that specific CPR mutations in the FMN-domain (FD) may induce a gain in activity for a specific CYP isoform. In the current report, we confirm the CYP isoform dependence of CPR's degenerated binding by demonstrating that the effect of four of the formerly studied FD mutants are indeed exclusive of a specific CYP isoform, as verified by cytochrome c inhibition studies. Moreover, the nature of CYP's substrate seems to have a modulating role in the CPR:CYP interaction. In silico molecular dynamics simulations of the FD evidence that mutations induces very subtle structural alterations, influencing the characteristics of residues formerly implicated in the CPR:CYP interaction or in positioning of the FMN moiety. CPR seems therefore to be able to form effective interaction complexes with its structural diverse partners via a combination of specific structural features of the FD, which are functional in a CYP isoform dependent manner, and dependent on the substrate bound.

Micronuclei Formation upon Radioiodine Therapy for Well-Differentiated Thyroid Cancer: The Influence of DNA Repair Genes Variants.

Radioiodine therapy with 131I remains the mainstay of standard treatment for well-differentiated thyroid cancer (DTC). Prognosis is good but concern exists that 131I-emitted ionizing radiation may induce double-strand breaks in extra-thyroidal tissues, increasing the risk of secondary malignancies. We, therefore, sought to evaluate the induction and 2-year persistence of micronuclei (MN) in lymphocytes from 26 131I-treated DTC patients and the potential impact of nine homologous recombination (HR), non-homologous end-joining (NHEJ), and mismatch repair (MMR) polymorphisms on MN levels. MN frequency was determined by the cytokinesis-blocked micronucleus assay while genotyping was performed through pre-designed TaqMan® Assays or conventional PCR-restriction fragment length polymorphism (RFLP). MN levels increased significantly one month after therapy and remained persistently higher than baseline for 2 years. A marked reduction in lymphocyte proliferation capacity was also apparent 2 years after therapy. MLH1 rs1799977 was associated with MN frequency (absolute or net variation) one month after therapy, in two independent groups. Significant associations were also observed for MSH3 rs26279, MSH4 rs5745325, NBN rs1805794, and tumor histotype. Overall, our results suggest that 131I therapy may pose a long-term challenge to cells other than thyrocytes and that the individual genetic profile may influence 131I sensitivity, hence its risk-benefit ratio. Further studies are warranted to confirm the potential utility of these single nucleotide polymorphisms (SNPs) as radiogenomic biomarkers in the personalization of radioiodine therapy.

Copy number variations and constitutional chromothripsis (Review).

Both copy number variations (CNVs) and chromothripsis are phenomena that involve complex genomic rearrangements. Chromothripsis results in CNVs and other structural changes. CNVs are frequently observed in the human genome. Studies on CNVs have been increasing exponentially; the Database of Genomic Variants shows an increase in the number of data published on structural variations added to the database in the last 15 years. CNVs may be a result of replicative and non-replicative mechanisms, and are hypothesized to serve important roles in human health and disease. Chromothripsis is a phenomena of chromosomal rearrangement following chromosomal breaks at multiple locations and involves impaired DNA repair. In 2011, Stephens et al coined the term chromothripsis for this type of fragmenting event. Several proposed mechanisms have been suggested to underlie chromothripsis, such as p53 inactivation, micronuclei formation, abortive apoptosis and telomere fusions in telomere crisis. Chromothripsis gives rise to normal or abnormal phenotypes. In this review, constitutional chromothripsis, which may coexist with multiple de novo CNVs are described and discussed. This reviews aims to summarize recent advances in our understanding of CNVs and chromothripsis, and describe the effects of these phenomena on human health and birth defects.

Corrigendum: The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners.

[This corrects the article DOI: 10.3389/fphar.2020.00299.].

ABC Efflux Transporters and the Circuitry of miRNAs: Kinetics of Expression in Cancer Drug Resistance.

Cancer drug resistance (CDR) is a major problem in therapeutic failure. Over 90% of patients with metastatic cancer present CDR. Several mechanisms underlie CDR, including the increased expression of efflux ABC transporters and epigenetic phenomena. Nevertheless, a topic that is not usually addressed is the mechanism underlying the loss of CDR once the challenge to these cells is withdrawn. A KCR cell line (doxorubicin-resistant, expressing ABCB1) was used to induce loss of resistance by withdrawing doxorubicin in culture medium. ABCB1 activity was analysed by fluorescence microscopy and flow cytometry through substrate (DiOC2) retention assays. The expression of 1008 microRNAs was assessed before and after doxorubicin withdrawal. After 16 weeks of doxorubicin withdrawal, a decrease of ABCB1 activity and expression occurred. Moreover, we determined a signature of 23 microRNAs, 13 underexpressed and 10 overexpressed, as a tool to assess loss of resistance. Through pathway enrichment analysis, "Pathways in cancer", "Proteoglycans in cancer" and "ECM-receptor interaction" were identified as relevant in the loss of CDR. Taken together, the data reinforce the assumption that ABCB1 plays a major role in the kinetics of CDR, and their levels of expression are in the dependence of the circuitry of cell miRNAs.

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners.

NADPH cytochrome P450 oxidoreductase (CPR) is the obligatory electron supplier that sustains the activity of microsomal cytochrome P450 (CYP) enzymes. The variant nature of the isoform-specific proximal interface of microsomal CYPs indicates that CPR is capable of multiple degenerated interactions with CYPs for electron transfer, through different binding mechanisms, and which are still not well-understood. Recently, we showed that CPR dynamics allows formation of open conformations that can be sampled by its structurally diverse redox partners in a CYP-isoform dependent manner. To further investigate the role of the CPR FMN-domain in effective binding of CPR to its diverse acceptors and to clarify the underlying molecular mechanisms, five different CPR-FMN-domain random mutant libraries were created. These libraries were screened for mutants with increased activity when combined with specific CYP-isoforms. Seven CPR-FMN-domain mutants were identified, supporting a gain in activity for CYP1A2 (P117H, G144C, A229T), 2A6 (P117L/L125V, G175D, H183Y), or 3A4 (N151D). Effects were evaluated using extended enzyme kinetic analysis, cytochrome b 5 competition, ionic strength effect on CYP activity, and structural analysis. Mutated residues were located either in or adjacent to several acidic amino acid stretches - formerly indicated to be involved in CPR:CYP interactions - or close to two tyrosine residues suggested to be involved in FMN binding. Several of the identified positions co-localize with mutations found in naturally occurring CPR variants that were previously shown to cause CYP-isoform-dependent effects. The mutations do not seem to significantly alter the geometry of the FMN-domain but are likely to cause very subtle alterations leading to improved interaction with a specific CYP. Overall, these data suggest that CYPs interact with CPR using an isoform specific combination of several binding motifs of the FMN-domain.

Thyroid Cancer: The Quest for Genetic Susceptibility Involving DNA Repair Genes.

The incidence of thyroid cancer (TC), particularly well-differentiated forms (DTC), has been rising and remains the highest among endocrine malignancies. Although ionizing radiation (IR) is well established on DTC aetiology, other environmental and genetic factors may also be involved. DNA repair single nucleotide polymorphisms (SNPs) could be among the former, helping in explaining the high incidence. To further clarify the role of DNA repair SNPs in DTC susceptibility, we analyzed 36 SNPs in 27 DNA repair genes in a population of 106 DTCs and corresponding controls with the aim of interpreting joint data from previously studied isolated SNPs in DNA repair genes. Significant associations with DTC susceptibility were observed for XRCC3 rs861539, XPC rs2228001, CCNH rs2230641, MSH6 rs1042821 and ERCC5 rs2227869 and for a haplotype block on chromosome 5q. From 595 SNP-SNP combinations tested and 114 showing relevance, 15 significant SNP combinations (p < 0.01) were detected on paired SNP analysis, most of which involving CCNH rs2230641 and mismatch repair variants. Overall, a gene-dosage effect between the number of risk genotypes and DTC predisposition was observed. In spite of the volume of data presented, new studies are sought to provide an interpretability of the role of SNPs in DNA repair genes and their combinations in DTC susceptibility.

The Role of Caspase Genes Polymorphisms in Genetic Susceptibility to Philadelphia-Negative Myeloproliferative Neoplasms in a Portuguese Population.

Our main aim was to evaluate the role of caspases' genes SNPs in Philadelphia-chromosome negative chronic myeloproliferative neoplasms (PN-MPNs) susceptibility. A case-control study in 133 Caucasian Portuguese PN-MPNs patients and 281 matched controls was carried out, studying SNPs in apoptosis related caspases: rs1045485 and rs1035142 (CASP8), rs1052576, rs2308950, rs1132312 and rs1052571 (CASP9), rs2227309 and rs2227310 (CASP7) and rs13006529 (CASP10). After stratification by pathology diagnosis for essential thrombocythemia (ET), female gender or JAK2 positive, there is a significant increased risk for those carrying at least one variant allele for CASP9 (C653T) polymorphism (OR 2.300 CI 95% [1.180-4.484], P = 0.014). However, when considered individually, none of the studied caspases polymorphisms was associated with PN-MPNs risk. Our results do not reveal a significant involvement of caspase genes polymorphisms on the individual susceptibility towards PN-MPNs as a whole. However, for essential thrombocythemia (ET), female gender or JAK2 positive, there is a significant increased risk to those carrying at least one variant allele for CASP9. Although larger studies are required to confirm these results and to provide conclusive evidence of association between these and other caspases variants and PN-MPNs susceptibility, these new data may contribute to a best knowledge of the pathophysiology of these disorders and, in the future, to a more rational and efficient choice of therapeutic strategies to be adopted in PN-MPNs treatment.

Regulation of ABCB1 activity by microRNA-200c and microRNA-203a in breast cancer cells: the quest for microRNAs' involvement in cancer drug resistance.

Aim: ABCB1 is a major player in cancer drug resistance. The purpose of this study was to functionally assess the regulation of ABCB1 activity in a doxorubicin-resistant breast cancer cell line by miR-200c and miR-203. Methods: Human breast carcinoma cell lines MCF-7 (Doxorubicin-sensitive and not expressing ABCB1) and KCR (Doxorubicin-resistant and expressing ABCB1) were used to evaluate the expression levels of miR-200c and miR-203 by Real-time quantitative PCR (RT-qPCR). The effects of transient ectopic expression of miRNA-200c and miR-203 on the expression of ABCB1 in KCR and MCF-7 cells was verified by RT-qPCR and Western Blot. The extrusion activity of the ABCB1 pump was analyzed by fluorescence microscopy and flow cytometry through fluorescence substrate retention assays (DiOC2) in the presence and absence of the ABCB1 inhibitor verapamil. Results: RT-qPCR results indicated a 100,000-fold increase in ABCB1 mRNA expression levels in KCR cells compared to MCF-7 cells, and is inversely correlated with the expression of miR-203 and miR-200c. The insertion of miR-200c and miR-203 led to a higher retention of DiOC2 within KCR cells, and slightly reduced the protein levels of ABCB1 in KCR cells, although the high initial expression of ABCB1 masked the reduction in protein levels. The increased intracellular accumulation of the fluorescent due DiOC2 in the presence of the ABCB1 inhibitor verapamil correlated with the inhibition caused by miR-203 and miR-200c in transfected cells. Conclusion: The present study demonstrates that miR-200c and miR-203 exert a negative modulating effect on the activity of ABCB1 associated with doxorubicin resistance.