Abstract Methylenetetrahydrofolate reductase (MTHFR) gene is involved in homocysteine and folic acid metabolism. Tumour suppressor protein TP53 gene maintains cellular and genetic integrity. To date, no studies associated the MTHFR C677T rs1801133 and TP53 Pro72Arg rs1042522 with CRP levels and methotrexate (a folic acid antagonist) treatment outcomes in psoriatic arthritis (PsA) patients. The present study aimed to investigate whether the MTHFR rs1801133 and TP53 rs1042522 gene variants influences CRP levels and methotrexate treatment outcomes in South African Indian and Caucasian PsA patients. PsA patients (n=114) and healthy controls (n=100) were genotyped for the rs1801133 and rs1042522 using RFLP-PCR. (i) Results for rs1801133 genotyping: Caucasian patients had a higher frequency of the variant T-allele versus healthy Caucasian controls (40% versus 22%; OR=2.31, 95% CI=1.10-4.88, p=0.0379). Patients with the variant CT+TT genotypes had higher median CRP levels at baseline versus wildtype CC genotypes (11.70 (5.3-28.80) mg/mL versus 7.40 (5.00-15.05) mg/mL, p=0.0355). After 6 months of methotrexate treatment median CRP levels between genotypes reduced and remained similar. (ii) Results for rs1042522 genotyping: Indian patients had a higher frequency of the variant Arg-allele versus healthy Indian controls (42% versus 29%; OR=1.75, 95% CI=1.07-2.86, p=0.0275). In conclusion, patients with the MTHFR rs1801133 variant T-allele have elevated CRP levels, which can be ameliorated with methotrexate.

Abstract Neurofibromatosis type 1 (NF1) is a syndrome triggered by mutations in the NF1 gene, which alter the neurofibromin protein, a negative regulator of the RAS oncogenic pathway. Due to underreporting, the scarcity of studies on NF1 in Brazil and its importance in public health. This study aimed to assess the clinical and epidemiological characterisation of NF1 in a Reference Hospital in the country and DataSUS. The study analysed the electronic medical records of patients with NF1 and the DataSUS databases. The medical records showed a greater number of female, white and adult patients. There was a high frequency of clinical features adopted by the NIH consensus for the clinical diagnosis of the disease, such as CALMs, dermal neurofibromas and axillary/inguinal ephelides, bone and ophthalmological changes, in addition malignant and benign neoplasms and neurodevelopmental disorders. On the other hand, the data provided by DataSUS shows a disproportionate concentration of NF1 consultations between the country’s regions, with a low level of diagnoses of newborn with NF1 and a NF1 mortality rate of 3.06% in the population. There is therefore a need for new public policies on access to diagnosis, treatment and information about the disease for the Brazilian population.

Abstract Research has highlighted the significant role of methylated genes associated with DNA damage repair in pathogenesis of Lung adenocarcinoma (LUAD). However, the potential of DNA damage repair-related gene (DDRG) methylation as a prognostic biomarker remains underexplored. This study aimed to assess the prognostic value of methylated DDRGs in LUAD. Analysis of the TCGA-LUAD dataset revealed differentially expressed genes (DEGs) and differentially methylated genes (DE-MGs), from which methylated DE-DDRGs were identified. An independent prognostic risk model was constructed based on these methylated DE-DDRGs by integrating risk scores with clinical features. Additionally, the study examined responses to immunotherapy. Results indicated that CLU exhibited hypermethylation and elevated expression in LUAD tissues, while eight other genes (BUB1B, SHCBP1, RRM2, RPL39L, TRIP13, GAPDH, ENO1, and CENPM) showed high expression and hypomethylation. Among these, RRM2 and GAPDH were significantly linked to poorer overall survival. Furthermore, single-sample gene set enrichment analysis (ssGSEA) revealed that patients with LUAD in the high-risk group had lower immune scores and less immune cell infiltration. TIDE analysis suggested that patients in the low-risk group may exhibit greater sensitivity to immune checkpoint inhibitor therapy. In conclusion, RRM2 and GAPDH represent promising prognostic and immunotherapeutic biomarkers, offering new avenues for LUAD treatment strategies.

Abstract We report a viral metagenomic analysis of fecal samples from Bradypus crinitus (Pilosa: Bradypodidae), a recently described sloth species that occurs in the Atlantic Forest of Espírito Santo and Rio de Janeiro states, Southeast Brazil. Through Illumina sequencing, we generated a total of 2,065,344 raw reads, of which 945,386 reads (45.77%) passed the quality and size filter. The highest proportion of them was assigned to Eukarya, followed by Bacteria and only a small proportion to Virus. However, we identified 24 viral families using distinct taxonomic assignment tools, including phages and vertebrate viruses, such as retroviruses and papillomaviruses. Also, we identified four bacterial genus already associated with disease in sloths. Our study sheds light on the microbiome of a previously unexplored species, further contributing to the comprehension of metagenomic global diversity.

Abstract Acute lymphoblastic leukemia (ALL) stands out as the most prevalent neoplasm during childhood, characterized by the rapid production of abnormal lymphoid cells. Chemotherapy administered to these patients may induce a substantial imbalance in the oral microbiota. A prospective pediatric study encompassing a control group (without ALL) and ALL patients at two treatment stages (pre-induction and consolidation) was conducted. Clinical and laboratory data were meticulously collected. Moreover, DNA from saliva samples was extracted for 16S rRNA sequencing. Clinical data revealed a heightened incidence of oral mucositis during the consolidation phase. Analysis of alpha biodiversity (observed taxa) exhibited a significant reduction in bacterial richness among patients in the consolidation phase. Network analysis identified key taxa during this phase, namely Neisseria flavescens, Prevotella melaninogenica and Porphyromonas. The findings underscore the substantial impact of ALL treatment on the oral microbiota composition, indicating diminished bacterial diversity and an elevated prevalence of oral mucositis.

Abstract Here we analyze the Yaravirus brasiliense, an amoeba-infecting 80-nm-sized virus with a 45-kbp dsDNA, using structural molecular modeling. Almost all of its 74 genes were previously identified as ORFans. Considering its unprecedented genetic content, we analyzed Yaravirus genome to understand its genetic organization, its proteome, and how it interacts with its host. We reported possible functions for all Yaravirus proteins. Our results suggest the first ever report of a fragment proteome, in which the proteins are separated in modules and joined together at a protein level. Given the structural resemblance between some Yaravirus proteins and proteins related to tricarboxylic acid cycle (TCA), glyoxylate cycle, and the respiratory complexes, our work also allows us to hypothesize that these viral proteins could be modulating cell metabolism by upregulation. The presence of these TCA cycle-related enzymes specifically could be trying to overcome the cycle’s control points, since they are strategic proteins that maintain malate and oxaloacetate levels. Therefore, we propose that Yaravirus proteins are redirecting energy and resources towards viral production, and avoiding TCA cycle control points, “unlocking” the cycle. Altogether, our data helped understand a previously almost completely unknown virus, and a little bit more of the incredible diversity of viruses.

Abstract Sperm-associated antigen 9 (SPAG9) is a member of cancer-testis antigen, having characteristics of a scaffold protein, which is involved in the c-Jun N-terminal kinase JNK signaling pathway, suggesting its key involvement in different physiological processes, such as survival, apoptosis, tumorigenesis, and cell proliferation. We identified two families (A and B) having multisystem features like coarse facial features, albinism, cataracts, skeletal abnormalities, and developmental delay. Whole genome sequencing (WGS) in families A and B revealed a homozygous frameshift variant (c.903del; p.Phe301Leufs*2) in the SPAG9 gene. Sanger sequencing of both families revealed perfect segregation of the identified variant in all family members. 3D protein modeling revealed substantial changes in the protein’s secondary structure. Furthermore, RT-qPCR revealed a substantial reduction of SPAG9 gene expression at the mRNA level in the affected individuals of both families, thus supporting the pathogenic nature of the identified variant. For the first time in the literature, biallelic SPAG9 gene variation was linked to multisystem-exhibiting features like coarse facial features, albinism, cataracts, skeletal abnormalities, and developmental delay. Thus, this data supports the notion that SPAG9 plays an important role in a multisystemic disorder in humans.

Abstract Rare heterozygous variants in IRF6 (interferon regulatory factor-6) gene cause van der Woude syndrome 1 (VWS1) or Popliteal Pterygium syndrome, two forms of syndromic cleft lip/palate (CLP) that present with a variety of congenital malformations due to impairment ectodermal homeostasis. These malformations include, in addition to CLP, lip pits, pterygia, and intraoral and eyelid fibrous bands. Amniotic band sequence (ABS) is a rare condition of unknown genetic etiology that involves a range of congenital anomalies caused by the entanglement of fibrous bands, which disrupt fetal body parts. However, ABS co-occurs with CLP and other malformations that cannot be explained by this mechanism. Therefore, investigating the genetic relationship between ABS and CLP may provide clues regardind the genes involved in these conditions. Here, we report a case of a girl diagnosed with VWS1, autism, intellectual disability, and congenital right limb anomalies compatible with ABS. Molecular analysis revealed a novel, rare heterozygous missense variant in IRF6 (NM_006147.3:c.970T>C) located in exon 7, inherited from her father. This variant results in the replacement of serine by proline at position 324 of the IRF6 protein with potentially deleterious effects. This report expands the mutational landscape of IRF6 and provides further support for a possible link between the genetics of CLP and ABS.

Abstract Tumor necrosis factor-alpha (TNF-α), is partly attributed to pathogenesis of end-stage renal disease (ESRD). Inconsistency of reported associations between TNF-α G-308A polymorphism (rs1800629) and ESRD prompted a meta-analysis to obtain more precise estimates. Eleven case-control studies from 11 articles were included. Pooled odds ratios (OR) and 95% confidence intervals (95% CIs) were estimated to evaluate the association. Subgroup analysis was based on ethnicity (Caucasian and Asian). Multiple comparisons were Bonferroni-corrected. Trial sequential analysis (TSA) was implemented to ascertain the reliability of results. Sensitivity analyses and publication bias tests were performed on significant results. There were no significant association (pa >0.05) in the overall and ethnic subgroup. Indians, three significant pool ORs (pa < 0.01-0.03) showed increased susceptibility to ESRD in homozygous (OR, 6.57; 95% CI, 1.45 to 29.75; pa = 0.01), recessive (OR, 6.75; 95% CI, 1.44 to 31.56; pa = 0.02), and codominant (OR, 2.06; 95% CI, 1.08 to 3.94; pa = 0.03) models. TSA indicated the robustness of such association in the Indian population. The main outcomes were robust without evidence of publication bias. This study showed associations between TNF-α G-308A and ESRD are confined to Indians, which are susceptible to ESRD up to approximately 7 times.

Abstract Centromochlus heckelii has the lowest diploid chromosome number (2n = 46) and the only described heteromorphic sex chromosome system in Auchenipteridae. This study presents a population of C. heckelii from the Central Amazon basin with subtle variations in the karyotype composition and a variant W chromosome with distinct morphology and increased C-positive heterochromatin content. In this population, the W chromosome is subtelocentric, whereas the only previous study on C. heckelii reported a metacentric W chromosome. Constitutive heterochromatin (CH) and accumulation of microsatellite motifs have significantly contributed to this W chromosome enlargement. Notably, this population exhibits numerous interstitial telomeric sites (ITSs). Some of these ITSs might represent genuine chromosomal fusion points due to the reduced 2n; however, additional mechanisms, such as chromosomal inversions, translocations, transpositions, or association with satellite DNA, are likely responsible for this unusual pattern. The 18S rDNA sites were found in both the Z and W chromosomes of all individuals. However, two individuals exhibited an additional 18S rDNA site in a single homologous of the chromosome pair 20, characterizing an intrapopulation polymorphism. The 5S rDNA sites were found in two chromosome pairs, distinguishing this population from other Centromochlinae species and further supporting it as one of the most efficient cytotaxonomic markers within the subfamily.

Abstract The genome of Corynebacterium pseudotuberculosis, etiologic agent of Caseous Lymphadenitis (CLA), was sequenced to comprehend its genetics, pathogenicity, and virulence mechanisms due to its economic importance. A focus was placed on the G/U mismatch-specific DNA glycosylase (Mug), an enzyme vital for base excision repair in DNA that can play an important role in uracil repair, since the high G+C content of C. pseudotuberculosis makes it prone to deamination events, accentuating the potential significance of Mug. Through in silico and in vitro analyses, the Corynebacterium pseudotuberculosis Mug protein (CpMug) was characterized to confirm its DNA glycosylase activity and lesion affinity. The mug gene was identified in both pathogenic and non-pathogenic Corynebacterium species, lacking a discernible ancestry pattern. Bioinformatics analyses revealed the preservation of essential uracil DNA glycosylase catalytic residues in CpMug. The 3D structure of CpMug was constructed, and molecular docking analysis demonstrated its interaction with DNA containing uracil and other lesions. Comparative analyses revealed a higher affinity of CpMug’s catalytic residues for uracil over other DNA lesions and enzymatic assays with purified CpMug affirmed its uracil DNA glycosylase activity, while it exhibited no activity on 8-oxoguanine, tetrahydrofuran, or thymine glycol, consistent with computational simulations.

Abstract Single nucleotide polymorphisms (SNPs) in the HLA DR/DQ region have the greatest impact on susceptibility to type 1 diabetes mellitus (T1DM). Non-HLA SNPs interact with the HLA, influencing the risk for T1DM. The aim of this study was to develop a genetic risk score (GRS) based on HLA DR/DQ and non-HLA SNPs to discriminate patients with T1DM. The sample comprised 466 patients with T1DM and 469 controls. The rs689/INS, rs2476601/PTPN22, rs231775/CTLA-4, rs2304256/TYK2, rs2292239/ERBB3, and HLA DR/DQ SNPs were genotyped using real-time PCR. The unweighted GRS (uGRS) was calculated by summing the risk alleles of each SNP and the weighted GRS (wGRS) by multiplying the risk alleles by their odds ratios. The uGRS was higher in T1DM patients than in non-diabetic controls (0.34 ± 0.14 vs. 0.26 ± 0.13, P <0.0001), being positively correlated with HbA1c levels (P <0.0001). wGRSs exhibited higher AUCs than uGRSs. The wGRS containing only HLA DR/DQ SNPs showed an AUC of 0.75 (95% CI 0.72 - 0.78). The wGRS containing both HLA DR/DQ and non-HLA SNPs, adjusted for race, demonstrated the best discriminative power [AUC 0.91 (95% CI 0.89 - 0.93)]. The race adjusted-wGRS, including all SNPs, seems to be a useful genetic tool for assessing individual’s predisposition to T1DM.

Abstract The contemporary genetic diversity of the cheetah (Acinonyx jubatus) has been the focus of several studies, which have revealed very low levels of variation. Different hypotheses have been proposed to explain this pattern of low diversity, and require additional scrutiny. Here, we used published microsatellite data and coalescence-based analytical methods to explore the historical demography of the largest free-ranging cheetah population, aiming to assess whether present-day diversity may have been impacted by a historical demographic decline. Our results support the hypothesis of a historical (and most likely gradual) demographic decline over the past ~10,000 years, leading to a present-day N e ranging from 700 to 1,600 individuals. This decline was likely induced by climate-driven vegetational shifts affecting habitat suitability and possibly also interspecies interactions with prey and competitors. These results help clarify the demographic history of cheetahs in southern Africa and its impact on the current genetic diversity of this population.

Abstract Different phthalate compounds, known as emerging contaminants, such as Di-n-butyl (DBP) and di-iso-pentyl (DiPeP) phthalates, have been found in aquatic environments and are widely used as additives to increase the flexibility of plastics. This study intended to evaluate the toxicity in zebrafish embryos and larvae of DBP and DiPeP, as well as the mixture of the two compounds. Fish embryo toxicity, neurotoxicity, oxidative stress, and genotoxicity studies were carried out in Danio rerio (zebrafish) embryos and larvae. Fish were exposed to DBP and DiPeP in concentrations ranging from 0.001 to 0.125 mg/L and also a combination of compounds DBP + DiPeP (1:1 MIX). DBP caused mortality and embryo malformations from 0.062 mg/L, DiPeP at 0.125 mg/L and the mixture at 0.031 mg/L, indicating the potential toxicity of these phthalates. Isolated phthalates and in mixture induced neurotoxicity and oxidative stress, at low concentrations in zebrafish larvae. No genotoxicity was found through comet assay in larvae. DBP was more toxic than DiPeP, and the mixture was more toxic than both phthalates when tested in isolation, showing some kind of interaction.

Abstract Integrative network analysis (INA) is important for identifying gene modules or epigenetically regulated molecular pathways in diseases. This study evaluated the effect of excessive gestational weight gain (EGWG) on INA of differentially methylated regions, maternal metabolism and offspring growth. Brazilian women from “The Araraquara Cohort Study” with adequate pre-pregnancy body mass index were divided into EGWG (n=30) versus adequate gestational weight gain (AGWG, n=45) groups. The methylome analysis was performed on maternal blood using the Illumina MethylationEPIC BeadChip. Fetal-neonatal growth was assessed by ultrasound and anthropometry, respectively. Maternal lipid and glycemic profiles were investigated. Maternal triglycerides-TG (p=0.030) and total cholesterol (p=0.014); fetus occipito-frontal diameter (p=0.005); neonate head circumference-HC (p=0.016) and thoracic perimeter (p=0.020) were greater in the EGWG compared to the AGWG group. Multiple linear regression analysis showed that maternal DNA methylation was associated with maternal TG and fasting insulin, fetal abdominal circumference, and fetal and neonate HC. The DMRs studied were enriched in 142 biological processes, 21 molecular functions,and 17 cellular components with terms directed for the fatty acids metabolism. Three DMGMs were identified:COL3A1, ITGA4 and KLRK1. INA targeted chronic diseases and maternal metabolism contributing to an epigenetic understanding of the involvement of GWG in maternal metabolism and fetal-neonatal growth.