Análise in silico do polimorfismo rs1803909 do gene ANXA2 expresso em monócitos de sangue periférico e sua relação com a osteoporose humana

Objetivou-se avaliar as possíveis alterações morfofuncionais e de estabilidade proteica decorrentes das alterações dos aminoácidos Tirosina por uma Histidina na posição 269, bem como, correlacionar com a função fisiológica da proteína e sua provável ligação com a osteoporose humana. Por meio de uma análise in silico com base nas informações disponíveis nos bancos de dados NCBI dbSNP (mudança de aminoácidos e posição) e UNIPROT (sequência na proteína). Os impactos da modificação Y269H foram analisados a partir das ferramentas SIFT, Align-GVGD, SNAP e PROVEAN (função e estrutura), e PolyPhen-2 (natureza da alteração). Além disso, utilizou-se também a ferramenta MuPRO (alterações de estabilidade na proteína). A análise in silico do polimorfismo rs1803909 demonstrou alteração funcional (ferramenta SIFT, Score= 0). Bem como, estima-se que a troca de aminoácidos pode estar relacionada a alterações danosas (PolyPhen-2, Score= 0,993) e associada a modificações na função da proteína (PROVEAN, Score= -4.015). Além disso, foram observados impactos estruturais (Align-GVGD, Score= 83,33, Classe C65) e funcionais (SNAP, Score= 57). De forma complementar, observou-se diminuição da estabilidade proteica decorrente da alteração Y269H pela ferramenta MuPRO, ∆∆G= -1.6731749. Contudo, as alterações morfofuncionais podem estar ligadas a processos danosos e a diminuição de estabilidade da proteína, dificultando assim a sua ação. Além disso, a compreensão das alterações morfofuncionais e de estabilidade do rs1803909 podem auxiliar na busca por marcadores genéticos e moleculares de diagnóstico precoce para a osteoporose em humanos.

ASSOCIAÇÃO DO POLIMORFISMO rs111426349 DO GENE TGFBR1 À SUSCEPTIBILIDADE A DIVERSOS TIPOS DE CÂNCER

O receptor 1 do fator transformador de crescimento beta (TGFBR1) é uma proteína extracelular homodimérica produzida por diversos tipos celulares, participa da formação da matriz extracelular e possui papel importante na mobilidade, proliferação e diferenciação celular. O polimorfismo rs111426349 corresponde a uma troca C> A / C> G / C> T promovendo a alteração de aminoácidos de uma Arginina por um Triptofano na posição 487.Para isso, objetivou-se avaliar as possíveis alterações morfofuncionais e de estabilidade proteica decorrentes da alteração de aminoácidos, bem como, correlacionar com a função fisiológica da proteína. Realizou-se a análise in silico com base nas informações disponíveis nos bancos de dados NCBI dbSNP (alteração de aminoácidos e posição) eUNIPROT (sequência proteica). Os efeitos da alteração daR487W foram avaliados utilizando as ferramentas SIFT e PROVEAN para avaliação funcional e PolyPhen-2 para compreensão da natureza da alteração. Além disso, as alterações de estabilidade proteica foram avaliadas com a ferramenta MuPRO.A análise in silico, demonstrou alteração funcional (SIFT, Score=0). Bem como, estima-se que troca de aminoácidos pode estar associada a alterações danosas (PolyPhen2, Score=1.000) e relacionadas a função da proteína (PROVEAN, Score=-7.192). De forma complementar, observou-se diminuição da estabilidade proteica (MuPRO, ∆∆G=-0.85). O câncer é uma doença ocasionada por uma complexa interação entre fatores genéticos e ambientais. Fatores genéticos incluem mudanças de sequência e mutações na organização do genoma da célula que podem aumentara susceptibilidade a doença. Sabe-se que avia de sinalização do TGF-βé importante na regulação de diversos processos biológicos, nos quais inclui a diferenciação, proliferação celular, migração e apoptose. As mutações na via de sinalização de TGF-β são constantemente identificadas em várias enfermidades, incluindo câncer de cólon, de mama, próstata ou de pâncreas. O TGFBR1é considerado o comunicador central da via de transdução de sinal do TGF-β,portanto as alterações morfofuncionais observadas decorrentes do rs111426349podem estar associadas a processos danosos e a diminuição da estabilidade proteica podendo comprometer a atuação correta na via de sinalização. A literatura reporta aumento de susceptibilidade a diversos tipos de câncer na presença do polimorfismo, o que ressalta a importância das análises morfofuncionais para compreensão do mecanismo fisiopatológico.Dessa forma, a análise das alterações morfofuncionais e de estabilidade podem contribuir na busca por marcadores moleculares e genéticos de diagnóstico precoce, uma vez que os polimorfismos do gene TGFBR1 estão associados à suscetibilidade ao câncer, e mais pesquisas funcionais devem ser executadas para elucidar a função do gene no desenvolvimento de diversos tipos de câncer.

Genotyping analysis of the Pax9 Gene in patients with maxillary canine impaction

Background: Paired-box gene 9 (PAX9) mutation is potentially associated with impaction in some patient populations. Here, we analyzed the relationship between PAX9 polymorphism and the occurrence of maxillary canine impaction. Methods: Patients with and without maxillary canine impaction were selected based on specific inclusion criteria, and samples of genomic DNA were obtained from a buccal mucosa swab. DNA was amplified by polymerase chain reaction and sequenced for further bioinformatics analysis to identify single nucleotide polymorphism (SNP) genotypes. Genotype and allele counting was performed in both case and control groups prior to conducting statistical analysis. Results: Four SNPs were identified in patients with maxillary canine impaction, with relative confidence determined based on chromatogram-peak assessment. All SNPs were located in exon 3 of PAX9 and in the region sequenced by the primer pair −197Fex3 and +28Rex3. Three of the SNPs (rs375436662, rs12881240, and rs4904210) were reported previously and are annotated in NCBI (dbSNP version 150), whereas another SNP mapped to chromosome 14 has not been reported. Patients with a CC genotype at SNP 3 [odds ratio (OR): 2.61 vs. TT; 1.28 vs. CT] and a CC genotype at SNP 4 [OR: 0.71 vs. GG; 0.79 vs. CG] were more likely to have maxillary canine impaction. Conclusions: These results demonstrated that the presence of SNPs 3 and 4 is associated with increased likelihood of suffering from maxillary canine impaction.

Extracting allelic read counts from 250,000 human sequencing runs in Sequence Read Archive

The Sequence Read Archive (SRA) contains over one million publicly available sequencing runs from various studies using a variety of sequencing library strategies. These data inherently contain information about underlying genomic sequence variants which we exploit to extract allelic read counts on an unprecedented scale. We reprocessed over 250,000 human sequencing runs (>1000 TB data worth of raw sequence data) into a single unified dataset of allelic read counts for nearly 300,000 variants of biomedical relevance curated by NCBI dbSNP, where germline variants were detected in a median of 912 sequencing runs, and somatic variants were detected in a median of 4,876 sequencing runs, suggesting that this dataset facilitates identification of sequencing runs that harbor variants of interest. Allelic read counts obtained using a targeted alignment were very similar to read counts obtained from whole genome alignment. Analyzing allelic read count data for matched DNA and RNA samples from tumors, we find that RNA-seq can also recover variants identified by WXS, suggesting that reprocessed allelic read counts can support variant detection across different library strategies in SRA. This study provides a rich database of known human variants across SRA samples that can support future meta-analyses of human sequence variation.

P.134 Infantile Onset Multisystem Neurologic, Endocrine and Pancreatic Disease: case series and review

Background: We report three brothers born to consanguineous parents of Syrian descent with a novel homozygous c.324G>A (p.W108*) mutation in PTRH2 that encodes mitochondrial peptidyl-tRNA hydrolase 2. Mutations in PTRH2 have recently been identified in the autosomal recessive condition, Infantile Onset Multisystem Neurologic, Endocrine and Pancreatic Disease (IMNEPD). To our knowledge, this is the first case of IMNEPD described in a Canadian centre. Methods: Clinical phenotyping enabled a targeted approach in which all exons of PTRH2 were sequenced. We identified a novel mutation and compared our patients with those recently described. Results: We identified a homozygous nonsense mutation in PTRH2, c.324G>A (p.W108*). This G to A mutation results in a premature stop at codon 108 that produces a truncated protein, removing most of the amino acids at the enzymatic active site. This mutation is not listed in the human Gene Mutation Database Cardiff, NCBI dbSNP, 1000 Genomes, Exome Variant Server or ClinVar and is a rare variant listed in gnomAD. Conclusions: In IMNEPD, nonsense mutations in PTRH2 appear to cause severe disease with postnatal microcephaly, neurodevelopmental regression, and ataxia with additional features of seizures, peripheral neuropathy, and pancreatic dysfunction, whereas missense mutations may produce a milder phenotype. The spectrum exhibited by our patients suggests variable expressivity with PTRH2 mutations.

HPCDb: an integrated database of pancreatic cancer

We have established a database of Human Pancreatic Cancer (HPCDb) through effectively mining, extracting, analyzing, and integrating PC-related genes, single-nucleotide polymorphisms (SNPs), and microRNAs (miRNAs), now available online at http://www.pancancer.org/. Data were extracted from established databases, ≥5 published literature (PubMed), and microarray chips (screening of differentially expressed genes using limma package in R, |log2 fold change (FC)| > 1). Further, protein–protein interactions (PPIs) were investigated through the Human Protein Reference Database. miRNA–target relationships were also identified using the online software TargetScan. Currently, HPCDb contains 3284 genes, 120 miRNAs, 589 SNPs, 10,139 PPIs, and 3904 miRNA–target pairs. The detailed information on PC-related genes (e.g., gene identifier (ID), symbol, synonyms, full name, chip sets, expression alteration, PubMed ID, and PPIs), miRNAs (e.g., accession number, chromosome location, related disease, PubMed ID, and miRNA–target interactions), and SNPs (e.g., SNP ID, allele, gene, PubMed ID, chromosome location, and disease) is presented through user-friendly query interfaces or convenient links to NCBI GEO, NCBI PubMed, NCBI Gene, NCBI dbSNP, and miRBase. Overall, HPCDb provides biologists with relevant information on human PC-related molecules at multiple levels, helping to generate new hypotheses or identify candidate markers.

Structural and functional impact of SNPs in P-selectin gene: A comprehensive in silico analysis

P-selectin is an adhesion molecule which plays an important role in the development of inflammation. It is encoded by the SELP gene located on chromosome 1q21-q24. Various single nucleotide polymorphisms (SNPs) ofSELPhave been reported to be associated with various inflammatory disease conditions. The genetics behind these diseases could be better understood by knowing the structural and functional impact of various genetic determinants ofSELP. So far, this is the first comprehensive and systematicin silicoanalysis of SNPs inSELP. A total of 2780 SNPs ofSELPwere retrieved from NCBI dbSNP. Only conserved and validated SNPs with minor allele frequency (MAF) ≥ 0.05 were subjected to further analysis. Based on these criteria, we selected 4 non-synonymous SNPs (nsSNPs) and 119 non-coding SNPs (ncSNPs). The nsSNPs were analyzed for deleterious effects using SIFT, Polyphen-2, nsSNPAnalyzer, SNP & Go, SNPs3, Mutperd and I-mutant web tools. The template prediction for variant structure modeling was performed using MUSTER and SWISS-MODEL. The functional impact of ncSNPs was analyzed by SNPinfo and RegulomeDB. Thein silicoanalysis predicted 3 nsSNPs and 21 ncSNPs as potential candidates for future case-control association studies and functional analysis ofSELP.

A Role for TET2 Mutations in Paroxysmal Nocturnal Hemoglobinuria (PNH)

Abstract 1262 PNH is characterized by circulating blood cells that are deficient in the surface expression of proteins that require glycosylphosphatidylinositol (GPI). The loss of the GPI-linked complement inhibitors CD55 and CD59 on the red cell results in complement mediated hemolysis, and this defect on the surface of platelets is likely responsible for the marked hypercoagulable state seen in PNH. The loss of GPI-linked proteins is due to the clonal expansion of a stem cell with an acquired somatic mutation in the PIG-A gene, which is responsible for an early step in the production of GPI. In PNH, as in aplastic anemia, there is a decreased stem cell and erythroid progenitor pool, there are oligoclonal T cell expansions, there is an HLA allele association, and cytopenias can occur, which respond to immunosuppression. Normal individuals harbor occult populations of cells with the PNH phenotype and genotype, and there is evidence that PIG-A mutations are growth neutral in animals. These findings are all supportive of the immune escape model, which posits that the abnormal environment associated with marrow injury represents the “second hit” which selects in favor of the PNH clone. However, there is a long-standing interest in finding second genetic hits. Indeed, others have reported rearrangements of HMGA2 in PNH; we have reported a series where 24% of patients had a cytogenetic abnormality and another series where 3 out of 29 patients with PNH had the JAK2V617F mutation and an MPN/PNH overlap syndrome. Here we have investigated a series of 17 patients with PNH to see whether we would find mutations in TET2, which is commonly mutated in myeloid disorders. We separated granulocytes from patients with at least 40% GPI-negative granulocytes, extracted DNA, which was subjected to whole genome amplification, followed by bi-directional sequencing using a dye terminator approach. In 11 out of 17 patients, we identified the 5284A>G, I1762V variant, with an allele frequency of 41% (95% confidence interval 25% to 58%) compared with 22% in the NCBI dbSNP database. In 4 of the patients we identified the 5162T>G, L1721W variant, with an allele frequency of 12%, which was not significantly different from the NCBI dbSNP database (9%). In 3 patients, we identified the 1088C>T, P363L variant, with an allele frequency of 9%, which also was not significantly different from the database (3%). However, one remarkable patient was heterozygous for all three of these SNPs– and was also heterozygous for a previously undescribed nonsense mutation, 2697T>A, Y899X. This mutation was seen in 3 separate sequencing reactions, and the alleles were present in approximately a 1:1 ratio. This mutation occurs at the 3' end of exon 3, a region where chain terminating mutations have been previously reported. Of note, it has been recently reported that heterozygous disruption of Tet2 in a mouse model results in an increase in the stem cell compartment as determined by a competitive repopulation assay, suggesting that this patient's TET2 mutation may have partly contributed to the advantage that the PNH clone demonstrated. Because 100% of her granulocytes were GPI–negative, it was not possible to be certain which abnormality came first. This patient's diagnosis of severe hemolytic PNH had been first confirmed 13 years ago, and she has a prior history of intra-abdominal thromboses and a DVT. She is now doing well on both coumadin and eculizumab. Unlike patients with a concurrent JAK2 mutation, she does not have features of an MPN, but her blood counts are higher than most of the other patients in this series, with a WBC of 7.7 compared with a median of 3.7 for the other patients, a platelet count of 321 compared with a median of 131, and a reticulocyte count of 247,000 compared with a median of 132,000. In conclusion, inactivating TET2 mutations, like activating JAK2 mutations, may contribute to the expansion of PNH clones in a subset of patients. Disclosures: No relevant conflicts of interest to declare.

Impact of Factor × Mutations on the Activity of Edoxaban

Abstract 3321 Background: Edoxaban is a direct inhibitor of factor Xa (FXa), and its efficacy as an oral anti-coagulant agent is less likely to be affected by food intake or drug-drug interaction. This profile of edoxaban suggests a good compliance in clinical use.However it is not clear whether genetic variations of FX influence the efficacy of edoxaban. Objectives: To investigate the possible inter-patient variability in the efficacy of edoxaban stemming from SNPs in the FX gene, we characterized the enzyme activity of FXa derived from wild type FX (FX-WT) and from FX with two known mutations, A152T (FX-A152T) and G192R (FX-G192R). The impact of FX mutations were also tested on the pharmacological activity of edoxaban. Methods: Among known FX SNPs in the NCBI dbSNP database, two non-synonymous SNPs are located inside mature FX, rs3211772 (allele frequency: 0.006) and rs3211783 (allele frequency: 0.022), corresponding to A152T and G192R. The former located inside the light chain and the latter located inside the activation peptide of FX. We selected these two SNPs and examined whether they might influence on the efficacy of edoxaban. We prepared recombinant FX proteins of FX-WT, FX-A152T and FX-G192R. We measured the enzyme activities of these FXa and the anti-FXa and anticoagulant effects of edoxaban on these FXa. Recombinant FX proteins were activated with Russell's viper venom factor × activator and FXa activity was measured using a chromogenic substrate S-2222. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were measured using FX-deficient plasma supplemented with recombinant FX proteins with a coagulometer CA-50. Results: Km values of FX-WT, FX-A152T and FX-G192R FXa were 0.55, 0.53 and 0.54 nM, respectively, Vmax of FX-WT, FX-A152T and FX-G192R FXa were 21.0, 21.8 and 21.4 mOD/min, respectively. PTs of plasma containing these mutations were 25.2 (FX-WT), 24.2 (FX-A152T) and 24.1 (FX-G192R) seconds. aPTTs of plasma containing the mutated FXs were 76.7 (FX-WT), 77.3 (FX-A152T) and 72.6 (FX-G192R) seconds. These data indicated that these mutations do not affect the basal FXa catalytic activity and coagulation activity. The Ki values of edoxaban for the mutated Fxas, the concentrations of edoxaban required to double the PT (PTCT2) and aPTT (aPTTCT2) in plasma containing the mutated FXs did not affected by two FX mutarions (Table 1). These data demonstrated that those mutations have no impact on the anticoagulant activity of edoxaban. Conclusions: Two FX mutations, A152T and G192R, do not affect the basal FXa catalytic activity and coagulation activity. Edoxaban acts equally on FX-WT, FX-A152T and FX-G192R. It is suggested that edoxaban has little inter-patient variability stemming from SNPs in FX gene. Disclosures: Noguchi: Daiichi Sankyo Co., Ltd.: Employment. Takahashi:Daiichi Sankyo Co., Ltd.: Employment. Ishihara: Daiichi Sankyo Co., Ltd.: Employment. Morishima:Daiichi Sankyo Co., Ltd.: Employment. Shibano:Daiichi Sankyo Co., Ltd.: Employment.

Polymorphisms in Multidrug Resistance-Associated Protein Genes Are Associated with Worse Outcome in Childhood Acute Lymphoblastic Leukemia.

Methotrexate (MTX) is a key compound of chemotherapeutic regimens used in the treatment of childhood acute lymphoblastic leukemia (ALL). Inter-individual differences in response to this drug may cause treatment failures and adverse drug reactions. In particular, transporters of the adenosine triphosphate-binding cassette (ABC) family such as the ABCC (multidrug resistance-related protein, MRP) are involved in the efflux of this drug. The elimination of MTX may arise, among other factors, to be a major determinant of chemoresistance in leukemic blast cells. It has been demonstrated that ALL patients with high MRP expression had an unfavourable prognosis with relapsed patients showing a higher expression of MRP genes. The polymorphisms in MRP genes, particularly those in promoter region may explain differences in expression and thereby the inter-individual differences in clinical outcome for ALL children. We analyzed polymorphisms in five MRP genes (MRP1-MRP5). Two polymorphisms in MRP1, 7 in MRP2, 4 in MRP3, 8 in MRP4 and 3 in MRP5 gene, located in regulatory and coding gene regions were selected from NCBI dbSNP database. The polymorphisms were analyzed in 50 healthy individuals to estimate allele frequency, linkage disequilibrium, and haplotype phase. Variants sufficient to infer most common haplotypes were further analyzed in 243 children with de novo ALL, treated at Ste-Justine Hospital with multi-agent chemotherapy according to the Dana-Farber Cancer Institute protocols 87-01, 91-01, 95-01 and 2000-01. These polymorphisms/haplotypes were investigated for association with disease outcome. The polymorphisms of two genes, MRP1 and MRP3, appeared associated with event free survival (EFS). Individuals with the AA -genotype of A -1665G promoter polymorphism in MRP1 gene had worse EFS (69% vs. 85%, p=0.007) compared to patients with the GG or AG genotypes (Hazard ratio, HR=2.2 (IC 95%: 1.2–3.9). Carriers of A allele of G-1696A and A-189T polymorphisms in the MRP3 gene had a worse EFS (68% vs. 81%, p=0.016) compared to those with the GG or TT genotype respectively (HR=2,1 IC 95%: 1.1–3.8). The A alleles of both polymorphisms belong to the same haplotype. A-189 is uniquely tagging this haplotype, therefore the association between this haplotype and ALL outcome was also found. A-1696 is shared among this and additional haplotype, the latter not associated with EFS, rendering A-1696 allele less relevant for the prediction of ALL outcome. All associations remained significant in multivariate analysis after inclusion of the known prognostic factors. Similar results were obtained if disease free and overall survival were analyzed instead of EFS. As MRPs are also expressed in the blood-brain barrier, a possible correlation between these polymorphisms and type of relapse (isolated bone marrow or central nervous system) was analysed. No significant difference between the type of relapse and MRP1 or MRP3 described polymorphisms was seen. The variants MRP1 A-1665, and MRP3 A-189 localised in the regulatory region of these genes, have an impact on the outcome in ALL children and could predict differences in MTX response. Further analysis between these polymorphisms and MTX levels as well as combined effect with other at risk genotypes of the folate cycle should be analysed to determine the effect of MRP variants on the outcome of children with ALL.