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Although sickle cell anemia results from homozygosity for a single mutation at position 7 of the β-globin chain, the clinical aspects of this condition are very heterogeneous. Complications include leg ulcers, which have a negative impact on patients’ quality of life and are related to the severity of the disease. Nevertheless, the complex pathogenesis of this complication has yet to be elucidated. To identify novel genes associated with leg ulcers in sickle cell anemia, we performed whole-exome sequencing of extreme phenotypes in a sample of Brazilian sickle cell anemia patients and validated our findings in another sample. Our discovery cohort consisted of 40 unrelated sickle cell anemia patients selected based on extreme phenotypes: 20 patients without leg ulcers, aged from 40 to 61 years, and 20 with chronic leg ulcers. DNA was extracted from peripheral blood leukocytes and used for whole-exome sequencing. After the bioinformatics analysis, eight variants were selected for validation by Sanger sequencing and TaqMan® genotyping in 293 sickle cell anemia patients (153 without leg ulcers) from two different locations in Brazil. After the validation, Fisher’s exact test revealed a statistically significant difference in a stop codon variant (rs12568784 G/T) in the FLG2 gene between the GT and GG genotypes ( P = 0.035). We highlight the importance of rs12568784 in leg ulcer development as this variant of the FLG2 gene results in impairment of the skin barrier, predisposing the individual to inflammation and infection. Additionally, we suggest that the remaining seven variants and the genes in which they occur could be strong candidates for leg ulcers in sickle cell anemia. Impact statement To our knowledge, the present study is the first to use whole-exome sequencing based on extreme phenotypes to identify new candidate genes associated with leg ulcers in sickle cell anemia patients. There are few studies about this complication; the pathogenesis remains complex and has yet to be fully elucidated. We identified interesting associations in genes never related with this complication to our knowledge, especially the variant in the FLG2 gene. The knowledge of variants related with leg ulcer in sickle cell anemia may lead to a better comprehension of the disease’s etiology, allowing prevention and early treatment options in risk genotypes while improving quality of life for these patients.

The use of post-alignment procedures has been suggested to prevent the identification of false-positives in massive DNA sequencing data. Insertions and deletions are most likely to be misinterpreted by variant calling algorithms. Using known genetic variants as references for post-processing pipelines can minimize mismatches. They allow reads to be correctly realigned and recalibrated, resulting in more parsimonious variant calling. In this work, we aim to investigate the impact of using different sets of common variants as references to facilitate variant calling from whole-exome sequencing data. We selected reference variants from common insertions and deletions available within the 1K Genomes project data and from databases from the Latin American Database of Genetic Variation (LatinGen). We used the Genome Analysis Toolkit to perform post-processing procedures like local realignment, quality recalibration procedures, and variant calling in whole exome samples. We identified an increased number of variants from the call set for all groups when no post-processing procedure was performed. We found that there was a higher concordance rate between variants called using 1K Genomes and LatinGen. Therefore, we believe that the increased number of rare variants identified in the analysis without realignment or quality recalibration indicated that they were likely false-positives.

For a better interpretation of variants, evidence-based databases, such as ClinVar, compile data on the presumed relationships between variants and phenotypes. In this study, we aimed to analyze the pattern of sequencing depth in variants from whole-exome sequencing data in the 1000 Genomes project phase 3, focusing on the variants present in the ClinVar database that were predicted to affect protein-coding regions. We demonstrate that the distribution of the sequencing depth varies across different sequencing centers (pair-wise comparison, p < 0.001). Most importantly, we found that the distribution pattern of sequencing depth is specific to each facility, making it possible to correctly assign 96.9% of the samples to their sequencing center. Thus, indicating the presence of a systematic bias, related to the methods used in the different facilities, which generates significant variations in breadth and depth in whole-exome sequencing data in clinically relevant regions. Our results show that methodological differences, leading to significant heterogeneity in sequencing depth, may potentially influence the accuracy of genetic diagnosis. Furthermore, our findings highlight how it is still challenging to integrate results from different sequencing centers, which may also have an impact on genomic research.

OBJECTIVE Focal cortical dysplasias (FCDs) are an important cause of drug-resistant epilepsy. In this work, we aimed to investigate whether abnormal gene regulation, mediated by microRNA, could be involved in FCD type II. METHODS We used total RNA from the brain tissue of 16 patients with FCD type II and 28 controls. MicroRNA expression was initially assessed by microarray. Quantitative polymerase chain reaction, in situ hybridization, luciferase reporter assays, and deep sequencing for genes in the mTOR pathway were performed to validate and further explore our initial study. RESULTS hsa-let-7f (p = 0.039), hsa-miR-31 (p = 0.0078), and hsa-miR34a (p = 0.021) were downregulated in FCD type II, whereas a transcription factor involved in neuronal and glial fate specification, NEUROG2 (p

Alterations in thermal niches have been widely associated with the Anthropocene erosion of reptiles' diversity. They entail potential physiological constraints for organisms' performance, which can lead to activity restrictions and impact fitness and demography. Reptiles are ectotherms which rely on seasonal periodicity to maximize the performance of biological functions. Despite it, the ecological implications of shifts in local temperatures are barely explored at the seasonal scale. This study aims to assess how changes in air temperature and substrate temperature affect the activity, body temperature (Tb) and thermoregulation patterns of the sand lizard, Liolaemus arambarensis (an endangered, microendemic species from southern Brazil), throughout a four-year period. Field surveys were conducted monthly on a restricted population in a sand-dune habitat. The annual fluctuations of the seasonal temperatures led to significant changes in the activity and Tb of L. arambarensis and shaped thermoregulation trends, suggesting biological plasticity as a key factor in the face of such variability. Lizards tended to maintain seasonal Tb in mild and harsh seasons through increased warming/cooling efforts. Anomalous winter conditions seemed especially critical for individual performance due to their apparent high impact favouring/constraining activity. Activity and thermoregulation were inhibited in frigid winters, probably due to a vulnerable physiology to intense cold spells determined by higher preferred body temperatures than Tb. Our results warn of a complex sensitivity in lizards to anomalous seasonal temperatures, which are potentially enhanced by climate change. The current work highlights the importance of multiannual biomonitoring to disentangle long-term responses in the thermal biology of reptiles and, thereby, to integrate conservation needs in the scope of global change.

Background Despite advances in treatments for endometriosis, some symptoms persist owing to the chronic inflammation observed in this disease. Objective To identify resources, methods, and/or complementary treatments to alleviate the pain symptoms of endometriosis, and to identify adverse effects of treatments. Search strategy Lilacs, Scielo, PEDro, Scopus, Pubmed, CENTRAL Cochrane, Science Direct, and Google Scholar were searched for studies published in Portuguese, English, and Spanish to July 31, 2017, using the terms "physical therapy" OR "complementary treatment" AND "endometriosis". Selection criteria Randomized controlled trials relating to complementary pelvic pain treatment and adverse effects. Data collection and analysis Eight studies were identified; two studies were included in the meta-analysis. Main results The complementary interventions studied were acupuncture, exercise, electrotherapy, and yoga. All were inconclusive in affirming benefit, but demonstrated a positive trend in the treatment of symptoms of endometriosis. Meta-analysis of acupuncture showed a significant benefit in pain reduction as compared with placebo (P=0.007). Conclusions Numerous complementary treatments have been used to alleviate the symptoms of endometriosis, but only acupuncture has demonstrated a significant improvement in outcomes. Nevertheless, other approaches demonstrated positive trends toward improving symptoms; this should encourage investigators to design controlled studies to support their applicability.

Background: Despite resulting from homozygosity of a single mutation at position 6 of the beta-globin locus, the clinical aspects of sickle cell anemia (SCA) are very heterogeneous. Leg ulcer is one of the many resulting complications, exerting a quite negative impact in the quality of life of patients and is related to the severity of the disease. The pathogenesis of such complication is complex and still not well explained. Objective: Seeking for novel genes associated with leg ulcer in SCA, we proposed exome sequencing of extreme phenotypes in a sample of the Brazilian population. Methods: Our sample consisted of 40 unrelated patients with SCA from the Hematology and Hemotherapy Center of the University of Campinas (Unicamp), Campinas, SP, Brazil. The cohort was composed by 20 patients who didn't have leg ulcer, with ages varying from 40 to 61 years (it is unusual the appearance of this disorder at such age) and 20 patients with chronic leg ulcer. DNA was collected from peripheral blood leukocytes and submitted to exome sequencing. Capture and enrichment were performed with the Nextera Rapid Capture Exome kit (Illumina) and samples were loaded in the Illumina HiSeq2500. The bioinformatics process was based on the GATK Pipeline and manual filters in vcftools and excel. Briefly, we prioritized the variants according to some conditions, like score quality, depth, Hardy-Weinberg equilibrium and minor allele frequency greater than 0.1. Subsequently, through PLINK, we selected the variants with p value less than 0.05 from Fisher association test. The annotation was made through wAnnovar. We excluded the synonymous variants, remaining with 244 variants at this point. Finally, the deleterious variants were selected, in addition to those not classified by SIFT, PolyPhen2 and FATHMM. Twenty-one variants remained, all of them in genes beyond the usual set of a priori biological candidate genes for this phenotype. We picked up 6 variants with the lowest p values: rs4857302 (CRYBG3), rs3782489 (KRT77), rs11800462 (TNFRSF25), rs13428956 (FOXD4L1), rs201853154 (UBTFL1) and rs11454536 (VWDE). We suggest that these variants and their genes could potentially be related to the development of leg ulcer in SCA. However, they should all be validated in other populations for confirmation. Disclosures No relevant conflicts of interest to declare.