BRAF, TERT and HLA-G Status in the Papillary Thyroid Carcinoma: A Clinicopathological Association Study.

As BRAF, TERT, HLA-G, and microRNAs have been individually associated with papillary thyroid carcinoma (PTC), we aimed to evaluate the individual and collaborative role of these markers in PTC in the same patient cohort. HLA-G and BRAF tumor expression was evaluated by immunohistochemistry. Using molecular methods, BRAFV600E and TERT promoter mutations were evaluated in thyroid fine needle aspirates. MicroRNA tumor profiling was investigated using massively parallel sequencing. We observed strong HLA-G (67.96%) while BRAF (62.43%) staining was observed in PTC specimens. BRAF overexpression was associated with poor response to therapy. The BRAFV600E (52.9%) and TERTC228T (13%) mutations were associated with extrathyroidal extension, advanced-age, and advanced-stage cancer. The TERT rs2853669 CC+TC genotypes (38%) were overrepresented in metastatic tumors. Nine modulated microRNAs targeting the BRAF, TERT, and/or HLA-G genes were observed in PTC and involved with cancer-related signaling pathways. The markers were individually associated with PTC features, emphasizing the synergistic effect of BRAFV600E and TERTC228T; however, their collaborative role on PTC outcome was not fully demonstrated. The differentially expressed miRNAs targeting the BRAF and/or HLA-G genes may explain their increased expression in the tumor milieu.

Hla-C genetic diversity and evolutionary insights in two samples from Brazil and Benin.

Human leukocyte antigen-C (HLA-C) is a classical HLA class I molecule that binds and presents peptides to cytotoxic T lymphocytes in the cell surface. HLA-C has a dual function because it also interacts with Killer-cell immunoglobulin-like receptors (KIR) receptors expressed in natural killer and T cells, modulating their activity. The structure and diversity of the HLA-C regulatory regions, as well as the relationship among variants along the HLA-C locus, are poorly addressed, and few population-based studies explored the HLA-C variability in the entire gene in different population samples. Here we present a molecular and bioinformatics method to evaluate the entire HLA-C diversity, including regulatory sequences. Then, we applied this method to survey the HLA-C diversity in two population samples with different demographic histories, one highly admixed from Brazil with major European contribution, and one from Benin with major African contribution. The HLA-C promoter and 3'UTR were very polymorphic with the presence of few, but highly divergent haplotypes. These segments also present conserved sequences that are shared among different primate species. Nucleotide diversity was higher in other segments rather than exons 2 and 3, particularly around exon 5 and the second half of the 3'UTR region. We detected evidence of balancing selection on the entire HLA-C locus and positive selection in the HLA-C leader peptide, for both populations. HLA-C motifs previously associated with KIR interaction and expression regulation are similar between both populations. Each allele group is associated with specific regulatory sequences, reflecting the high linkage disequilibrium along the entire HLA-C locus in both populations.

HLA-G Polymorphisms Are Associated with Non-segmental Vitiligo among Brazilians.

(1) Background: Vitiligo is characterized by white patches on the skin caused by loss of melanocyte activity or the absence of these cells. The available treatments minimize the symptoms by retarding the process of skin depigmentation or re-pigmenting the affected regions. New studies are required for a better comprehension of the mechanisms that trigger the disease and for the development of more efficient treatments. Studies have suggested an autoimmune feature for vitiligo, based on the occurrence of other autoimmune diseases in vitiligo patients and their relatives, and on the involvement of genes related to the immune response. (2) Methods: We evaluated, by massive parallel sequencing, polymorphisms of the HLA-G gene in vitiligo patients and control samples, to verify if variants of this gene could influence the susceptibility to vitiligo. (3) Results: We detected an association with non-segmental vitiligo regarding the haplotype Distal-010101a/G*01:01:01:01/UTR-1, adjusting for population stratification by using ancestry-informative markers (AIMs). (4) Conclusions: It remains unclear whether the HLA-G variants associated with vitiligo were detected because of the high linkage disequilibrium (LD) with HLA-A*02, or if the HLA-A variants previously reported as associated with vitiligo were detected because of the high LD with HLA-G*01:01:01:01/UTR-1, or if both genes jointly contribute to vitiligo susceptibility.

Comprehensive analysis of HFE gene in hereditary hemochromatosis and in diseases associated with acquired iron overload.

BACKGROUND: Patients with hepatitis C virus (HCV) and hepatocellular carcinoma (HCC) may or not develop iron overload (IO), which is associated with worst prognosis, because can cause serious damage to organs. HFE gene controls the iron uptake from gut, particularly in patients with hereditary hemochromatosis (HH). AIM: To identify associations between HFE coding region in patients exhibiting hereditary hemochromatosis and in diseases associated with acquired IO. METHODS: We sequenced exons 2 to 5 and boundary introns of HFE gene, evaluating all polymorphic sites in patients presenting hereditary (hemochromatosis) or acquired iron overload HCV and HCC) and in healthy controls, using Sanger sequencing. We also determined the ensemble of extended haplotype in healthy control individuals, including several major histocompatibility complex loci, using sequence specific probes. Haplotype reconstruction was performed using the Arlequin and Phase softwares, and linkage disequilibrium (LD) between histocompatibility loci and HFE gene was performed using the Haploview software. RESULTS: The HFE*003 allele was overrepresented (f = 71%) and HFE*001 allele was underrepresented (f = 14%) in HH patients compared to all groups. A strong linkage disequilibrium was observed among the H63D-G, IVS2(+4)-C and C282Y-G gene variants, particularly in HH; however, the mutation IVS2(+4)T>C was not directly associated with HH susceptibility. The HFE*001/HFE*002 genotype conferred susceptibility to HCC in HCV patients exhibiting IO (P = 0.02, OR = 14.14). Although HFE is telomeric to other histocompatibility genes, the H63D-G/IVS2(+4)-C (P ≤ 0.00001/P ≤ 0.0057) combination was in LD with HLA-B*44 allele group in healthy controls. No LD was observed between HFE alleles and other major histocompatibility loci. CONCLUSION: A differential HFE association was observed for HH and for diseases associated with acquired IO (HCV, HCC). Since HFE is very distant from other histocompatibility loci, only weak associations were observed with these alleles.

MicroRNA expression profiles discriminate childhood T- from B-acute lymphoblastic leukemia.

MicroRNAs (miRNAs) play a critical role on biological and cellular processes; the search for functional markers may be of importance for differential diagnosis, prognosis, and development of new therapeutic regimens. In this context, we evaluated the bone marrow miRNA profile of Brazilian children exhibiting T- or B-cell acute lymphoblastic leukemia (T-ALL or B-ALL), using massive parallel sequencing, using the HiSeq 2500 platform (Illumina). The differential expression analysis was conducted considering a leave-one-out approach and FDR ≤ 0.05. Machine learning algorithms were applied to search for the disease subset biomarkers. Target prediction, functional enrichment, and classification of biological categories were also performed. Sixteen miRNAs were differentially expressed between T- and B-ALL, of which 10 (miR-708-5p, miR-497-5p, miR-151a-5p, miR-151b, miR-371b-5p, miR-455-5p, miR-195-5p, miR-1266-5p, miR-574-5p, and miR-425-5p) were downregulated and six (miR-450b-5p, miR-450a-5p, miR-542-5p, miR-424-5p, miR-629-5p, and miR-29c-5p) were upregulated in childhood T-ALL. These miRNAs may be used for distinguishing childhood lymphoblastic leukemia subtypes, since it provided the clear separation of patients in these two distinct groups. Six relevant biological pathways were identified according to their role in leukemia, namely, viral carcinogenesis, cell cycle, and B-cell receptor signaling pathways for induced miRNAs and TGF-beta signaling, apoptosis, and NF-kappa B signaling for the repressed miRNAs, of which several miRNA gene targets participate in cell differentiation and hematopoiesis processes. Machine learning analysis pointed out miR-29c-5p expression as the best discriminator between childhood T- and B-ALL, which is involved in calcium signaling, critical for B-cell lymphocyte fate. Further studies are needed to assure the role of the 16 miRNAs and miR-29c-5p on acute lymphoblastic leukemia subtypes and on disease prognosis.

Biological predictors shared by dementia and bullous pemphigoid patients point out a cross-antigenicity between BP180/BP230 brain and skin isoforms.

Bullous pemphigoid (BP) following dementia diagnosis has been reported in the elderly. Skin and brain tissues express BP180 and BP230 isoforms. Dementia has been associated with rs6265 (Val66Met) polymorphism of the brain-derived neurotrophic factor (BDNF) gene and low serum BDNF. Here we investigated a possible cross-antigenicity between BP180/BP230 brain and skin isoforms. We assessed antibodies against BP180/BP230 and BDNF levels by ELISA and BDNF Val66Met SNP by PCR in three groups: 50 BP patients, 50 patients with dementia, and 50 elderly controls. Heatmap hierarchical clustering and data mining decision tree were used to analyze the patients' demographic and laboratorial data as predictors of dementia-BP association. Sixteen percent of BP patients with the lowest serological BDNF presented dementia-BP clinical association. Anti-BP180/230 positivity was unexpected observed among dementia patients (10%, 10%) and controls (14%, 1%). Indirect immunofluorescence using healthy human skin showed a BP pattern in two of 10 samples containing antibodies against BP180/BP230 obtained from dementia group but not in the control samples. Neither allelic nor genotypic BDNF Val66Met SNP was associated with dementia or with BP (associated or not with clinical manifestation of dementia). Heatmap analysis was able to differentiate the three studied groups and confirmed the ELISA results. The comprehensive data mining analysis revealed that BP patients and dementia patients shared biological predictors that justified the dementia-BP association. Autoantibodies against the BP180/BP230 brain isoforms produced by dementia patients could cross-react with the BP180/BP230 skin isoforms, which could justify cases of dementia preceding the BP disease.

Extended HLA-G genetic diversity and ancestry composition in a Brazilian admixed population sample: Implications for HLA-G transcriptional control and for case-control association studies.

Human leukocyte antigen-G (HLA-G) is a nonclassical Major Histocompatibility Complex (MHC) molecule with immunomodulatory function and restricted tissue expression. The genetic diversity of HLA-G has been extensively studied in several populations, however, the segment located upstream -1406 has not yet been evaluated. We characterized the nucleotide variation and haplotype structure of an extended distal region (-2635), all exons and the 3'UTR segment of HLA-G by next-generation sequencing (NGS) in a sample of 335 Brazilian individuals. We detected 29 variants at the HLA-G distal promoter region, arranged into 19 haplotypes, among which we identified sites that may influence transcription factor targeting. Although the variation pattern in the distal region resembled the one observed in the conventional promoter segment, molecular signature for balancing selection was observed in the promoter segment from -1406 to -1 (Tajima's D = 2.315, P = 0.017), but not in this distal segment (D = 1.049, P = 0.118). Furthermore, the ancestry composition of this Brazilian population sample was determined by the analysis of SNPforID 34-plex ancestry informative marker (AIM) SNP panel. The distribution of HLA-G haplotypes was ancestry-dependent, corroborating previous findings and emphasizing the importance of considering the ancestry information in association studies.

Polymorphic sites at the 3' untranslated region of the HLA-G gene are associated with differential hla-g soluble levels in the Brazilian and French population.

HLA-G molecule has well-recognized tolerogenic properties, and the encoding gene shows lower frequency of polymorphism at the coding region but higher variability at regulatory 5' and 3' untranslated (3'UTR) regions. At least three 3'UTR polymorphic sites have been associated with HLA-G mRNA regulation, including the 14 base pair (14bp) Insertion/Deletion, +3142C-G and +3187A-G. We studied the association of polymorphic sites at 3'UTR (sequencing analysis, encompassing the 14bp Ins-Del/+3003T-C/+3010C-G/+3027C-A/+3035C-T/+3142C-G/+3187A-G/+3196C-G polymorphic sites) with plasma soluble HLA-G levels (sHLA-G, detected by ELISA) in 187 French and 153 Brazilian healthy individuals. Allele and genotype frequencies were closely similar in both populations; however, Brazilians showed a higher HLA-G 3'UTR haplotype diversity. Considering sHLA-G levels in both populations altogether, individuals presenting 14bp Del/Del showed higher levels compared to 14bpIns/Ins genotype (P <0.05); those presenting +3010C/G showed higher levels compared to the +3010C-C genotype (P< 0.05); those presenting +3027C-C showed higher levels than the +3027A-A genotype (P< 0.05); and those bearing +3035C-C showed higher levels compared to the +3035C-T (P < 0.01) and +3035T-T (P < 0.05) genotypes. The analyses of 3'UTR haplotypes showed that UTR-1 (DelTGCCCGC) was associated with higher expression of sHLA-G, whereas UTR-5 (InsTCCTGAC) and UTR-7 (InsTCATGAC) with lower expression and other UTRs (UTR-2/3/4/6) exhibited intermediate levels. Since the differential expression of HLA-G may be beneficial or harmful depending on the underlying condition, the identification of individuals genetically programmed to differentially express HLA-G may help on defining novel strategies to control the immune response against the underlying disorder.

Insights on the HLA-G evolutionary history provided by a nearby Alu insertion.

The AluyHG element belongs to the AluYb8 subfamily. It is a polymorphic insertion, located approximately 20 kb from the HLA-G 3'-untranslated region (3'-UTR), which has been used for evolution studies because it exhibits identity for descendants and it is still polymorphic in the human genome. To understand the evolutionary mechanisms acting on HLA-G, we evaluated the presence or absence of the AluyHG element, associating this variable site with others observed at HLA-G coding, 3'-UTR, or both regions in four distinct populations (Brazilian, French, Congolese, and Senegalese). The results were compared with the 1000Genomes Consortium data. The worldwide AluyHG frequencies showed an increment, starting lower in Africa and increasing following distance and time of human dispersion out of Africa. The same haplotype pattern was observed in all populations, indicating that most of the HLA-G haplotypes already detected were originated earlier in Africa, before Homo sapiens dispersion. The AluyHG insertion was associated with the G*01:01:01:01/UTR-1 haplotype, with rare recombinants. Despite its high frequency in worldwide populations, the G*01:01:01:01/UTR-1 haplotype should be very recent. The low frequency of recombinants indicates that the rate of recombination at the HLA-G gene is very low.