Allele-specific Research Articles

Allele-Specific QTL Fine Mapping with PLASMA

Although quantitative trait locus (QTL) associations have been identified for many molecular traits such as gene expression, it remains challenging to distinguish the causal nucleotide from nearby variants. In addition to traditional QTLs by association, allele-specific (AS) QTLs are a powerful measure of cis-regulation that are concordant with traditional QTLs but typically less susceptible to technical/environmental noise. However, existing methods for estimating causal variant probabilities (i.e., fine mapping) cannot produce valid estimates from asQTL signals due to complexities in linkage disequilibrium (LD). We introduce PLASMA (Population Allele-Specific Mapping), a fine-mapping method that integrates QTL and asQTL information to improve accuracy. In simulations, PLASMA accurately prioritizes causal variants over a wide range of genetic architectures. Applied to RNA-seq data from 524 kidney tumor samples, PLASMA achieves a greater power at 50 samples than conventional QTL-based fine mapping at 500 samples, with more than 17% of loci fine mapped to within five causal variants, compared to 2% by QTL-based fine mapping, and a 6.9-fold overall reduction in median credible set size compared to QTL-based fine mapping when applied to H3K27AC ChIP-seq from just 28 prostate tumor/normal samples. Variants in the PLASMA credible sets for RNA-seq and ChIP-seq were enriched for open chromatin and chromatin looping, respectively, at a comparable or greater degree than credible variants from existing methods while containing far fewer markers. Our results demonstrate how integrating AS activity can substantially improve the detection of causal variants from existing molecular data.

BatchPrimer3: A free web application for allele specific (SBE and allele flanking) primer design for SNPs genotyping in molecular diagnostics: A bioinformatics study

From 1990 to 2003 is the period needed to finish the human genome project. The ideas were and still introduce growing cheap, reliable and fast advance techniques for dealing with analysis of huge information of the human genome. Whereas that technique is contagious with a suitable analysis to differentiate between variants, linked to malady tendency and how drug responses. Single nucleotide polymorphism (SNP) considered the most common type of genetic variation between people. It is associated with various of diseases and sometime plays a role of prophylactic against a certain disease. Primer pairs considered the mainly implement player in polymerase chain reaction. So, they needed to design an efficient and good quality primer by many programs and applications still demanded. Many studies related to SNPs associated disease and mutations were depended on sensitive and inexpensive price method such as allele-specific (AS) polymerase chain reaction and single base extension.

Role of Transforming Growth Factor-β1 (T29C) Gene Polymorphism in Hepatic Stellate Cell Activation and Invasion and Susceptibility to Hepatitis B, C Infection in an Iraqi Patients

The interindividual varieties in the limit of changing development factor-β1 (TGF-β1) generation have been credited to hereditary polymorphisms in TGF-β1 quality. Changing development factor-beta 1 (TGFβ1) is a powerful suppressive cytokine that adds to ceaseless hepatitis B (CHB) disease. Abberations in TGFβ1 generation among people have been ascribed to TGFβ1 hereditary polymorphisms. We analyzed whether three putative polymorphisms in TGFβ1 (- 509 C/T (rs1800469), +869 C/T (rs1800470), and +11929 C/T (rs1800472)) square measure connected with CHB illness during a South-Eastern Iranian people.Methods:A total of 203 subjects with hepatitis infection (94 patients with hepatitis B virus infection and 109 patients with hepatitis C virus infection) whom admitted to Margan hospital, Center of liver diseases and gastrointestinal system were enrolled in the study. Allele specific (AS)-PCR, methods were used for assessing polymorphism of IL-10. Patients included (130 males and 73 females), with an age range (HBV: 44.6 ± 8.2), (HCV: 45.3±13.3) and (Control: 49.2 ± 9.04) years. The practical side of this study was done during the period from October 2017 to March 2018.Methods:A total of 203 subjects with hepatitis infection (94 patients with hepatitis B virus infection and 109 patients with hepatitis C virus infection) whom admitted to Margan hospital, Center of liver diseases and gastrointestinal system were enrolled in the study. Patients included (130 males and 73 females), with an age range (HBV: 44.6 ± 8.2), (HCV: 45.3±13.3) and (Con trol: 49.2 ± 9.04) years. The practical side of this study was done during the period from October 2017 to March 2018. As pathogenesis of HBV and HCV has a hereditary foundation, this primer investigation was intended to evaluate the effect of TGF-β1 (T29C) on the helplessness of Iraqi to HBV and HCV disease. Genotyping was performed utilizing single stranded polymorphism-polymerase chain response (SSP-PCR). Results: TGF-β1 T29Cgenetic polymorphism related to hepatitis B and C virus infection; revealed that the Genotype frequency of polymorphisms of (TGF) gene in Hepatitis B, C and Control, it was revealed that TC allele was higher than others 56.10% in control, 54.26% in HBV and 55.96% in HCV respectively. Results of Allele frequency showed that T allele was higher than C (69.51% in control, 57.98% HBV and 61.01% HCV).

Development of duplex-crossed allele-specific PCR targeting of TPMT*3B and *3C using crossed allele-specific blockers to eliminate non-specific amplification

Prospective testing for variants in the thiopurine S-methyltransferase (TPMT) is considered a key process in the development of thiopurine therapy. This testing is done to avoid toxicity and side effects in the management of diverse immunological and malignant conditions. Real-time fluorescent PCR techniques using duplex-crossed allele-specific primers in a single tube (DCAS-PCR) were developed in this study to genotype the common loss-of-function TPMT*3B c.460G > A (rs1800460) and TPMT*3C c.719A > G (rs1142345) usually occurring in individuals of Chinese ethnicity. In this method, several integrated strategies were used to completely eliminate the non-specific amplification that is commonly presented in traditional allele-specific (AS) PCR. These strategies include using AS-primers (ASP) that both are artificially mismatched in the penultimate positions and phosphorothioate modifications in the 5′-termini positions. In the assay, an AS-blocker was used, locus-specific TaqMan (LST) probes were used and we used at least two fragments were simultaneously amplified in a single tube which satisfy the thermodynamic characteristics of DNA polymerase to eliminate non-specific amplification. In a group of 200 unselected subjects, the results showed that 8 samples were heterozygous of TPMT*3C, and all samples possessed wild-type TPMT*3B. There was no non-specific amplification, and the genotypes were 100% consistent with Sanger sequencing.

Association of EGFR 1 Gene Alteration and their Association with Lung Adenocarcinoma Patients

Background:The epidermal growth factor receptor 1 (EGFR1) plays a significant role in cell proliferation and development. Its regulation in humans is very critical and incompletely understood in Non small cell lung cancer (NSCLC).Methods:100 newly diagnosed NSCLC (lung adenocarcinoma) patients and 100 healthy controls were included and allele specific (AS) polymerase chain reaction (PCR) was used to genotype and expression was analyzed by quantitative real time PCR. Overall survival of patients was analyzed by Kaplan-Meier method and for prognostic significance ROC curve was plotted.Results:A statistically significant difference (p<0.0001) in CC, AA and CA genotypes distribution among patients and healthy controls was observed. Compared to the CC genotype as reference, OR was 30.40 (95%CI 1.75- 524.9, p=0.0002) and 3.97 (95%CI 1.49-10.52, p=0.003) for the homozygous AA and heterozygous CA genotypes respectively. Kaplan-Meier survival analysis was also performed to analyze the relationship of EGFR1 (-191C/A) genotypes with progression free median survival of NSCLC patients and the difference was found to be significantly (p=0.0002) associated with different genotypes. In the ROC curve with respect to TNM stage at optimal cut-off value of 9.88 fold increase in EGFR1 mRNA expression, sensitivity and specificity were 92.9%, 83.3% respectively (AUC=0.95, p<0.0001). ROC curve w.r.t. distant metastases at optimal cut-off value of 13.5 fold change EGFR1 mRNA expression, sensitivity and specificity were 68.2%, 71.4% respectively (AUC=0.81, p<0.0001). In ROC curve w.r.t to presence/ absence of pleural effusion at optimal cut-off value of 14.8 fold change EGFR1 mRNA expression sensitivity and specificity were 66.7%, 68.2% respectively (AUC=0.71, p=0.009).Conclusions:Study concluded EGFR1 promoter polymorphism could be a risk factor associated with disease and may be used as prognostic marker for patients’ survival and predictor for disease worseness.

Analysis and interpretation of mixture DNA using AS-PCR of mtDNA.

Semi-nested PCR with allele-specific (AS) primers and sequencing of mitochondrial DNA (mtDNA) were performed to analyze and interpret DNA mixtures, especially when biological materials were degraded or contained a limited amount of DNA. SNP-STR markers were available to identify the minor DNA component using AS-PCR; moreover, SNPs in mtDNA could be used when the degraded or limited amounts of DNA mixtures were not successful with SNP-STR markers. Five pairs of allele-specific primers were designed based on three SNPs (G15043A, T16362C, and T16519C). The sequence of mtDNA control region of minor components was obtained using AS-PCR and sequencing. Sequences of the amplification fragments were aligned and compared with the sequences of known suspects or databases. When this assay was used with the T16362C and T16519C SNPs, we found it to be highly sensitive for detecting small amounts of DNA (∼30 pg) and analyzing DNA mixtures of two contributors, even at an approximately 1‰ ratio of minor and major components. An exception was tests based on the SNP G15043A, which required approximately 300 pg of a 1% DNA mixture. In simulated three contributor DNA mixtures (at rate of 1:1:1), control region fragments from each contributor were detected and interpreted. AS-PCR combined with semi-nested PCR was successfully used to identify the mtDNA control region of each contributor, providing biological evidence for excluding suspects in forensic cases, especially when biological materials were degraded or had a limited amount of DNA.

Rapid detection of drug-resistant Mycobacterium tuberculosis directly from clinical specimens using allele-specific polymerase chain reaction assay.

Background & objectives:Rapid detection of drug resistance in Mycobacterium tuberculosis (MTB) is essential for the efficient control of tuberculosis. Hence, in this study a nested-allele-specific (NAS) PCR, nested multiple allele-specific PCR (NMAS-PCR) and multiple allele-specific (MAS) PCR assays were evaluated that enabled detection of the most common mutations responsible for isoniazid (INH) and rifampicin (RIF) resistance in MTB isolates directly from clinical specimens.Methods:Six pairs of primers, mutated and wild type, were used for the six targets such as codon 516, 526 and 531 of rpoB, codon 315 of katG and C15-T substitution in the promoter region of mabA-inhA using allele-specific (AS) PCR assays (NAS-PCR, NMAS-PCR and MAS-PCR). The performance of AS PCR method was compared with phenotypic drug susceptibility testing (DST).Results:The usefulness of AS PCR assays was evaluated with 391 clinical specimens (251 Acid fast bacilli smear positive and MTB culture positive; 93 smear negative and MTB culture positive; 47 smear positive and MTB culture negative) and 344 MTB culture positive isolates. With culture-based phenotypic DST as a reference standard, the sensitivity and specificity of the NAS-PCR, NMAS-PCR and MAS-PCR assay for drug resistance-related genetic mutation detection were 98.6 and 97.8 per cent for INH, 97.5 and 97.9 per cent for RIF and 98.9 and 100 per cent for multidrug resistance (MDR).Interpretation & conclusions:The performance of AS PCR assays showed that those could be less expensive and technically executable methods for rapid detection of MDR-TB directly from clinical specimens.

Detection of IDH1 and IDH2 Mutation in Formalin-fixed Paraffin-embedded Gliomas Using Allele-specific COLD-PCR and Probe Melting Curve Analysis

Many tumors, especially gliomas, contain an isocitrate dehydrogenase (IDH) mutation that can be used for clinical diagnosis and prognosis. Our study aimed to develop a new reliable detection assay for IDH1 and IDH2 mutations for clinical diagnosis based on the allele-specific (AS) coamplification with lower denaturing-polymerase chain reaction (COLD-PCR) and probe melting curve analysis (PMCA). The method includes 3 elements allowing for the sensitive detection of low-abundance mutations: (1) PCR amplification of the target fragments with AS primers; (2) COLD-PCR; and (3) PMCA for differentiating the different mutations after amplification. We conducted a blinded study with 45 paraffin-embedded gliomas specimens and 13 fresh specimens screened for IDH mutations using Sanger sequencing. Concordance between the results of our AS-COLD-PCR/PMCA assay and Sanger sequencing was 100%. Our assay appeared to be superior to direct sequencing with a much higher sensitivity of 0.4% mutations. In summary, our assay is a cost-effective, convenient, and sensitive method for detecting IDH mutations and could be applied in the clinical setting to assess small brain biopsies.

Development and validation of an allele-specific PCR assay for genotyping a promoter and exonic single nucleotide polymorphisms of MGMT gene.

DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) specifically remove the methyl/alkyl group from the O6-position of guanine and restore the guanine to its normal form without causing DNA strand breaks. Relationship between MGMT activity and resistance to alkylating therapeutic agents is well established. Non-availability of simple, cost-effective and efficient methods of genotyping may hinder investigations on genotype-phenotype associations. No simple genotyping procedures such as allele-discrimination Taqman Assays were available for two genetic variations in MGMT gene that had previously demonstrated to be affecting its function and expression. These two variants were included to genotype in a clinical study (Clinicaltrail.gov ID: NCT01257854). Hence, the present study is aimed at developing, validating a rapid and simple allele-specific PCR method that genotypes exonic variant rs2308321 (c.520A>G) and a promoter variant rs113813075 (c.-459C>A) with standard PCR instruments. Web-based allele-specific (AS) primer design application called web-based allele-specific primer was used to design primers. Genomic DNA of lymphoblastoid cell line obtained from the Coriell repository with known genotypes were used to standardize the genotyping procedure. The PCR products were analyzed by 3% Agarose gel electrophoresis and by DNA Screen Tape assay with the Agilent 4200 TapeStation. The allele-specific PCR assay described here is a suitable strategy for efficient and reliable genotyping for difficult variants. This method offers cost-effective strategy for genotyping in clinical cohort studies provided positive controls established by Sanger sequencing are available for the variant.

Molecular Identification of Four Members of the Anopheles dirus Complex Using the Mitochondrial Cytochrome C Oxidase Subunit I Gene.

Precise mosquito species identification is an essential step for proper management and control of malaria vectors. Misidentification of members in the Anopheles dirus complex, some which are primary malaria vectors in Thailand and mainland Southeast Asia, remains problematic because of indistinguishable or overlapping morphological characters between sibling species. Moreover, there is a need for alternative methods, since the existing molecular techniques in the literature are not entirely satisfactory in differentiating all members in the An. dirus complex. The nucleotide polymorphisms in the mitochondrial cytochrome c oxidase subunit I (COI) sequences were developed to identify the 4 species within the An. dirus complex using an allele-specific (AS) multiplex polymerase chain reaction (PCR). The identified primers amplified and clearly differentiated the 4 members of the complex found in Thailand, Anopheles dirus, An. cracens, An. scanloni, and An. baimaii with PCR products 428/104, 236, 625, and 428 bp, respectively. These results demonstrate that an AS-PCR based on the COI region can accurately identify 4 members of An. dirus complex and would be useful as an alternative PCR-based method for accurate species identification.

P099 Virtual crossmatching successfully extended donor pool for highly sensitized patients with allele specific antibodies

Aim Even with the new Kidney Allocation System, patients with 98–100% cPRA possessing additional allelic antibodies (i.e., the antibody is directed against a polymorphism(s) present in some members of an antigenic group but not others) remain disadvantaged to receive donor organs. Listing the parent antigens as unacceptable (UA) limits an already shallow donor pool. This study presents our experience in expanding the donor pool for this group of patients using a virtual crossmatch (vXM) approach. Methods 38 recipients transplanted with 98–100% cPRA were included. When patients had allele specific antibodies, parent antigens were not considered UA. Instead, when donors possessing parent antigens were offered, a vXM was performed. Donor organs whose allelic specificities were not the targets of the allelic antibodies were transplanted. Patients were followed post-transplant for de novo DSA (dDSA), C4d deposition, acute cellular rejection (ACR), antibody mediated rejection (AMR), and at 1 year creatinine levels ( Results 11 out of 38 (29%) recipients with allele specific antibodies were transplanted with parent antigen positive/allele specificity negative donor kidneys. All vXMs and their corresponding physical crossmatches were negative (Table 1A). The median MFI value for the top strong allelic antibodies was 4013 (range 2314–15603). Transplant outcomes among these patients were compared to the control group of recipients who had no allelic antibodies. Outcomes were identical between the two groups (Table 1B). Conclusions For highly sensitized patients with allele specific antibodies, parent antigens to which patients have allele specific antibodies do not need to be considered as UA. Accurate vXMs can predict donor/recipient compatibility based on allelic DSA. This approach maximizes the donor offers in a timely manner to a particularly disadvantaged group of highly sensitized patients without negative impact to transplant outcomes. Download : Download high-res image (468KB) Download : Download full-size image

OR19 MIRNA mediated post transcriptional regulation of HLA transcripts

Aim MicroRNAs (miRNAs) regulate the post-transcriptional expression of targeted mRNA transcripts, however little is known about the influence of HLA polymorphisms on altered miRNA target specificity & allele specific post-transcriptional regulation. We utilize a custom developed bioinformatics platform, utilizing both miRNA transcript expression data and computational miRNA target site prediction to study the influence of HLA polymorphisms on altered miRNA target specificity and differential post-transcriptional regulation of HLA alleles within B and T lymphocytes. Methods Computational predicted miRNA target sites for all known miRNA transcripts (miRBase release 21) within the 3 ′ UTR of every known full-length HLA allele for HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, HLA-DPB1 and HLA-DRB1 (IMGT version 3.27) were determined using miRanda with a minimum heteroduplex energy of −20 Kcal/mol. Tissue specific miRNA expression profiles were subsequently used to filter miRNA-target interactions in order to determine the tissue specific differential miRNA target specificity HLA alleles. Results Our data demonstrate that endogenously expressed miRNA within both B and T lymphocytes display differential target specificity across HLA alleles, driven by polymorphisms present within the 3 ′ UTR of HLA alleles. Interestingly, we find differential targeting of endogenously expressed miRNA within polymorphic regions of HLA-DRB1 and HLA-G that have been associated with altered HLA expression and implicated in GVHD, preeclampsia, oncogenesis and allograft rejection. We also observe that several endogenously expressed miRNAs target the 3 ′ UTR of numerous HLA genes, acting as a potential master, universal regulator of HLA expression with B and T lymphocytes. Conclusions The developed bioinformatics platform identifies target sites of all known miRNA on the 3 ′ UTR of every annotated full-length HLA allele. This atlas is further refined using tissue specific miRNA expression patterns to generate tissue specific target maps, revealing altered miRNA target specificity both across tissue types and across HLA alleles, driven by polymorphisms across HLA alleles. This powerful platform may be used to test novel hypothesis related to altered HLA expression as it relates to a variety of disease states.

Molecular authentication of two medicinal plants Ligularia fischeri and Ligularia stenocephala using allele-specific PCR (AS-PCR) strategy

Ligularia fischeri (Gom-chi) and Ligularia stenocephala (Gon-dal-bi) are popular edible herbs in Korea. L. fischeri is used to treat jaundice, hepatitis, rheumatoid arthritis, and scarlet fever, while L. stenocephala is used to treat anxiety, weakness, and menstrual disorders. The herbal medicinal activities of these two herbs differ, but they are very difficult to distinguish based on their morphologies, especially in their dried forms. In an effort to distinguish these two plant species, we sequenced three barcoding genes in plastids, matK, rbcL, and trnH-psbA. From the analysis of sequence variations, we detected five single nucleotide polymorphisms (SNPs) between two the species. Allele specific (AS)-primers in the SNPs were employed in discrimination of the two species. Of the five AS-primer sets, one primer pair in matK gene showed reproducibly distinguishable PCR amplification between plants of L. fischeri and L. stenocephala. The method is reproducible and efficient, and is the first reported molecular method to discriminate between L. fischeri and L. stenocephala.

Genetic Drivers of Epigenetic and Transcriptional Variation in Human Immune Cells

SummaryCharacterizing the multifaceted contribution of genetic and epigenetic factors to disease phenotypes is a major challenge in human genetics and medicine. We carried out high-resolution genetic, epigenetic, and transcriptomic profiling in three major human immune cell types (CD14+ monocytes, CD16+ neutrophils, and naive CD4+ T cells) from up to 197 individuals. We assess, quantitatively, the relative contribution of cis-genetic and epigenetic factors to transcription and evaluate their impact as potential sources of confounding in epigenome-wide association studies. Further, we characterize highly coordinated genetic effects on gene expression, methylation, and histone variation through quantitative trait locus (QTL) mapping and allele-specific (AS) analyses. Finally, we demonstrate colocalization of molecular trait QTLs at 345 unique immune disease loci. This expansive, high-resolution atlas of multi-omics changes yields insights into cell-type-specific correlation between diverse genomic inputs, more generalizable correlations between these inputs, and defines molecular events that may underpin complex disease risk.

Abstract 2097: Translating a mutant p53 reactivating drug (ZMC1) in murine pancreatic cancer models

Abstract Pancreatic cancer therapy suffers from a lack of effective chemotherapy. TP53 is second only to KRAS as the most commonly mutated gene in pancreatic cancer with point mutations occurring in 75% of patients. We identified ZMC1 as an allele specific mutant p53 reactivator and lead compound for mutant p53 targeted drug development. ZMC1 restores wildtype structure and function by functioning as a zinc metallochaperone to restore zinc binding to mutant p53 proteins with impaired zinc binding. Our aim was to translate this novel mechanism in vivo using murine pancreatic cancer models. We investigated the pharmacokinetics (PK) and pharmacodynamics (PD) of ZMC1 and performed efficacy studies for allele specificity in nude mice with subcutaneous tumors from murine pancreatic cancer cell lines derived from a genetically engineered mouse model (KPC) expressing mutant KrasG12D and different alleles of TP53 (WT, null, p53R172H, p53R270H). Tumor growth inhibition became apparent only in KPCp53-R172H xenografts but not in KPCp53-R270H. We then performed efficacy studies in the autochthonous KPC model and found that, ZMC1 extended the median survival from 15 to 26 days in the KPCp53-R172H mice (p = 0.05) but not in the KPCp53-R270H mice. We sought to improve the efficacy of ZMC1 by synthesizing it complexed with zinc (Zn-1) in a 2:1 molar ratio. Zn-1 significanlty increased the median survival of KPCp53-R172H mice from 15 to 35 days (p = 0.0042). The apoptosis rate in the tumors (by Immunohistochemistry staining with Cleaved Caspase 3) was also increased by treatment of ZMC1 and Zn-1. These studies indicate that ZMC1 can function in vivo as a mutant p53 targeted anti-cancer drug at doses that are well tolerated. Furthermore, ZMC1 can be optimized by synthesizing it complexed with zinc. Citation Format: Xin Yu, Ashley T. Tsang, Tracy Withers, John Gelleran, David Augeri, S. David Kimball, Darren R. Carpizo. Translating a mutant p53 reactivating drug (ZMC1) in murine pancreatic cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2097.

Molecular analysis distinguishes metastatic disease from second cancers in patients with retinoblastoma

The pediatric ocular tumor retinoblastoma readily metastasizes, but these lesions can masquerade as histologically similar pediatric small round blue cell tumors. Since 98% of retinoblastomas have RB1 mutations and a characteristic genomic copy number "signature", genetic analysis is an appealing adjunct to histopathology to distinguish retinoblastoma metastasis from second primary cancer in retinoblastoma patients. Here, we describe such an approach in two retinoblastoma cases. In patient one, allele-specific (AS)-PCR for a somatic nonsense mutation confirmed that a temple mass was metastatic retinoblastoma. In a second patient, a rib mass shared somatic copy number gains and losses with the primary tumor. For definitive diagnosis, however, an RB1 mutation was needed, but heterozygous promoter→exon 11 deletion was the only RB1 mutation detected in the primary tumor. We used a novel application of inverse PCR to identify the deletion breakpoint. Subsequently, AS-PCR designed for the breakpoint confirmed that the rib mass was metastatic retinoblastoma. These cases demonstrate that personalized molecular testing can confirm retinoblastoma metastases and rule out a second primary cancer, thereby helping to direct the clinical management.

Detection of HER2 polymorphism and expression using circulating DNA and RNA as a tool in lung adenocarcinoma patients: a case control study.

Circulating DNA and RNA is an important prognostic tool for noninvasive malignant disease detection and in disease prognosis. Study aimed to evaluate the possible prognostic role of HER2 (-3444C/T) promoter polymorphism and its mRNA expression in Lung adenocarcinoma patients using circulating DNA and RNA. One hundred newly diagnosed lung adenocarcinoma patients and 100 age and sex matched healthy controls were included and allele specific (AS) polymerase chain reaction (PCR) was used for genotyping and expression was analyzed by quantitative real time PCR. Overall survival of patients was analyzed by Kaplan-Meier method. We observed a statistically significant difference in the frequency of HER2 CC, CT, and CT genotype among lung adenocarcinoma cases vs. healthy controls (P=0.001). Compared to the CC genotype, OR 2.51 (1.4-4.51), 5.97 (1.17-30.41) and RR 1.56 (1.17-2.07), 2.83 (0.82-9.73) for heterozygous CT and homozygous TT genotypes suggesting possible dominant effect on risk of lung adenocarcinoma. Cases with CC genotype showed 9.29 fold increased mRNA expression while cases with heterozygous CT and homozygous TT genotype showed 16.26, 16.72 fold increased mRNA expression (P<0.0001). We observed 13.92 fold increased HER2mRNA expression Lung adenocarcinoma patients. Patients in different TNM stages showed significant difference in HER2 mRNA expression which was found to be significantly associated (P<0.0001). Patients with distant metastases and without distant metastases had 17.44 and 11.16 fold increased HER2 mRNA expression was also found to be significantly associated (P<0.0001). It was also observed that patients with pleural effusion and without pleural effusion showed significant difference in HER2 mRNA expression (P=0.03). We also analysed patients with CC, TT, CT (P=0.02) and CT + TT (P=0.008) genotype showed 15.8, 7.9, 9.5 and 7.9 months of overall median survival time and found to be significantly associated, respectively. Patients with >13 and ≤13 fold increased HER mRNA expression also showed 7.9 and 11.5 months of overall median survival time was also found to be significantly associated (P=0.01). Our work provides evidence that circulating DNA and RNA may be a potential prognostic tool in Lung adenocarcinoma patients. Promoter polymorphism of HER2 (-3444C/T) gene had significant impact on higher HER2 mRNA expression could be a predictive factor for patients' worse overall survival and metastatic behaviour.

The influence of CYP2C8*3 on carbamazepine serum concentration in epileptic pediatric patients.

The aim of the present study was to investigate the distribution of CYP2C8 variants *3 and *5, as well as their effect on carbamazepine pharmacokinetic properties, in 40 epileptic pediatric patients on carbamazepine treatment. Genotyping was conducted using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and allele-specific (AS)-PCR methods, and steady-state carbamazepine plasma concentrations were determined by high performance liquid chromatography (HPLC). The CYP2C8 *3 and *5 polymorphisms were found at frequencies of 17.5 and 0.0%, respectively. After dose adjustment, there was a difference in daily dose in CYP2C8*3 carriers compared to non carriers [mean ± standard deviation (SD): 14.19 ± 5.39 vs. 15.46 ± 4.35 mg/kg; p = 0.5]. Dose-normalized serum concentration of carbamazepine was higher in CYP2C8*3 (mean ± SD: 0.54 ± 0.18 vs. 0.43 ± 0.11 mg/mL, p = 0.04), and the observed correlation between weight-adjusted carbamazepine dose and carbamazepine concentration after dose adjustment was significant only in CYP2C8*3 non carriers (r = 0.52, p = 0.002). However, the population pharmacokinetic analysis failed to demonstrate any significant effect of CYP2C8 *3 polymorphism on carbamazepine clearance [CL L/h = 0.215 + 0.0696*SEX+ 0.000183*DD]. The results indicated that the CYP2C8*3 polymorphism might not be of clinical importance for epilepsy treatment in pediatric populations.

Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function.

Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare autosomal dominant disease caused by heterozygous mutations in the p63 gene and characterized by limb defects, orofacial clefting, ectodermal dysplasia, and ocular defects. Patients develop progressive total bilateral limbal stem cell deficiency, which eventually results in corneal blindness. Medical and surgical treatments are ineffective and of limited benefit. Oral mucosa epithelial stem cells (OMESCs) represent an alternative source of stem cells capable of regenerating the corneal epithelium and, combined with gene therapy, could provide an attractive therapeutic avenue. OMESCs from EEC patients carrying the most severe p63 mutations (p.R279H and p.R304Q) were characterized and the genetic defect of p.R279H silenced using allele-specific (AS) small interfering RNAs (siRNAs). Systematic screening of locked nucleic acid (LNA)-siRNAs against R279H-p63 allele in (i) stable WT-ΔNp63α-RFP and R279H-ΔNp63α-EGFP cell lines, (ii) transient doubly transfected cell lines, and (iii) p.R279H OMESCs, identified a number of potent siRNA inhibitors for the mutant allele, which had no effect on wild-type p63. In addition, siRNA treatment led to longer acquired life span of mutated stem cells compared to controls, less accelerated stem cell differentiation in vitro, reduced proliferation properties, and effective ability in correcting the epithelial hypoplasia, thus giving rise to full thickness stratified and differentiated epithelia. This study demonstrates the phenotypic correction of mutant stem cells (OMESCs) in EEC syndrome by means of siRNA mediated AS silencing with restoration of function. The application of siRNA, alone or in combination with cell-based therapies, offers a therapeutic strategy for corneal blindness in EEC syndrome. Stem Cells 2016;34:1588-1600.

MG-127 Residual disease monitoring in a retinoblastoma patient by pcr of a novel deletion breakpoint

Background Allele-specific (AS)-PCR for known point mutations is a high-sensitivity method to monitor for minimal residual disease (MRD) in metastatic retinoblastoma. However, this approach is difficult to apply to large RB1 gene deletions. Objective We describe a novel, high sensitivity molecular assay for MRD monitoring of a large RB1 deletion. Method The proband’s retinoblastoma RB1 deletion was identified using quantitative multiplex (QM)-PCR. Array comparative genomic hybridization (aCGH) was used to map the boundaries of the tumour RB1 deletion. Inverse PCR was then applied to capture the unique deletion breakpoint, and AS primers were optimised for high sensitivity surveillance for MRD. Result Full RB1 gene testing revealed no mutation in blood of this patient. Using QM-PCR, we found one RB1 mutation in the retinoblastoma tumour (del P- >11). aCGH confirmed an ~238 kb hemizygous deletion extending centromeric from RB1. The precise deletion breakpoint was determined using inverse PCR to amplify from the known two-copy flanking sequence to identify a PCR product in tumour DNA that was absent from the patient’s blood DNA. AS primers based on the breakpoint detected a tumour-specific PCR product with a sensitivity of 1 in 1000 cells. The original bone marrow (BM) prior to therapy was strongly positive, but all subsequent BM, CSF, and cells harvested for stem cell transplant have been negative for the tumour-specific marker. Conclusion aCGH followed by inverse PCR provides a cost- and time-effective way to map QM-PCR detected RB1 breakpoints for high sensitivity molecular surveillance for MRD.