Sequence-specific Oligonucleotide Research Articles

P227 Determination of HLA -A, -B and - DRB1 alleles and HLA-A -B haplotype frequencies in Egyptians based on family study

Knowledge of allele and haplotype frequencies in human populations guides the search for an HLA- matched unrelated hematopoietic stem cell donor. A new algorithm developed by the National Marrow Donor program uses this information to predict the likelihood that a volunteer of a particular ethnicity will carry specific HLA alleles when typed at high resolution. In the present study, we determined the HLA-A,-B and -DRB1 alleles and HLA-A-B haplotype frequencies in Egyptians and compared them to various populations. Specificities of HLA-A, -B and -DRB1 loci were determined in 286 matched related donors of allogeneic hematopoeitic stem cell transplantation (AHSCT). Typing was performed using serologic and low resolution PCR- reverse sequence-specific oligonucleotide (rSSO) techniques. HLA-A-B haplotypes were defined by segregation analysis. The most frequent alleles for HLA class I HLA-A were: HLA-A2 (19.2%), A1 (15.8%), A30 (9.2%) and A24 (8.8%), for HLA-B, B41 (10.9%), B35 (10.2%), B44 (6%) and B50 (5.4%) and for DRB1∗13 (18.7%), ∗04 (15%) ,∗11(14.7%) and ∗03 (13.2%). The most frequent HLA-A-B haplotypes were: A24-B35 (3.8%), A2-B41 (3.4%), A1- B35 (3.4%) and A33-B14 (3.4%). This study report HLA allele and HLA-A-B haplotype patterns in Egyptians. These patterns were close to Jordanians and Lebanese and different from Saudi Arabians, Europeans, Americans and Asians. These data explain the difficulty of finding donors in world registries for Egyptian patients in need of a stem-cell transplant and highlight the need to for planning an Egyptian national unrelated donor registry to meet the demands of these patients.

P201 Erroneous HLA-B allele typing by sequencing-based typing method that utilizes combinations of Sanger sequencing and rSSO typing

Allele-level HLA matching reduces graft-versus-host disease and improves overall patient survival after hematopoietic stem cell transplantation (HSCT). Sanger sequencing has been the gold standard for allele-level HLA typing since 1996. The typing strategy typically employs the sequencing of exons 2 and 3 for HLA Class I and exon 2 for HLA Class II. These exons encode the antigen recognition domains and sequencing only these exons are considered to be clinically relevant for HSCT. Sanger sequencing results significant number of ambiguities in HLA typing due to partial sequencing of limited exons, inability to phase heterozygous alleles, and growing number of new HLA alleles. Additional typing methods are required to resolve phasing and ambiguity problems. Next-generation sequencing (NGS) permits sequencing of the entire HLA gene and generates unambiguous and phased-resolved HLA typing results up to four field levels. Here we describe an erroneous HLA-B allele typing by Sanger sequencing. Sanger sequencing (Genome Diagnostics B.V.) of an unrelated NMDP donor showed the following possible HLA types: B∗15:01:01,18:14 or B∗15:07:01,18:01:01 or B∗15:07:01,18:17 N or B∗15:07:03,18:01:10. The B∗15:01:01, B∗15:07:01, B∗18:01:01, and B∗18:14 are common and well-documented alleles. The high-definition reverse sequence-specific oligonucleotide (rSSO) typing (One Lambda) ruled out all possible types except B∗15:01:01,18:14. The rSSO bead for B∗15:07:01 showed reactivity below the specified cutoff. We performed NGS typing on this sample, which enabled phasing of polymorphic positions 363 and 485 in exon 3 resulted unambiguous B∗15:07:01,18:01:01 typing. In summary, the strategy of Sanger sequencing plus rSSO typing resulted in incorrect HLA-B assignment for the NMDP donor, which in turn caused the exclusion of matched donor. The incorrect HLA allele assignment can be detrimental to HSCT recipient survival. NGS provides the capability to phase polymorphisms thereby eliminating most ambiguities and provides high-resolution HLA typing without reflexive, time-consuming and expensive additional testing, and therefore NGS is the new gold standard for allele-level HLA typing.

Profiling the Mismatch Tolerance of Argonaute 2 through Deep Sequencing of Sliced Polymorphic Viral RNAs.

Low allelic and clonal variability among endogenous RNAi targets has focused mismatch tolerance studies to RNAi-active guide strands. However, the inherent genomic instability of RNA viruses such as hepatitis C virus (HCV) gives rise to quasi-species mutants within discrete clones: this facilitates mismatch tolerance studies from a target perspective. We recently quantified the slicing imprecision of Argonaute 2 using small interfering RNA (siRNA) analogs of the DNA-directed RNAi drug TT-034 and next-generation sequencing of 5′ RNA ligase-mediated rapid amplification of cDNA ends (RACE-SEQ). Here, we present an open-source, customizable, and computationally light RACE-SEQ bioinformatic pipeline, describing adaptations that semiquantitatively report the impact of RNAi hybridization site mismatches from the target perspective. The analysis shows that Argonaute 2 has a substitution-specific, 3- to 5-log activity window between fully complementary targets and targets with mismatches across positions 10–11. It further focuses the endonucleotic Slicer imprecision around positions 13–17, demonstrating its dependence on guide strand central region complementarity, and potentiation by even a single mismatch. We further propose pharmacogenomics value in testing endogenous targets using recombinant replicon systems and RACE-SEQ to report the pharmacodynamics of sequence-specific oligonucleotide therapeutics against all possible polymorphisms in a population, in a minimally biased, patient-free manner.

Relationship between pemphigus and American tegumentary leishmaniasis: insights from serological and genetic profiles.

Antibodies against Leishmania peptides (Lbr-peps) and desmogleins (Dsgs) have been reported in pemphigus foliaceus (PF) and leishmaniasis patients, respectively. We aimed to compare serological and genetic features in a Brazilian region endemic for American tegumentary leishmaniasis (ATL) and pemphigus. Commercial anti-Dsg ELISA and in-house ELISA with Lbr-peps were used to determine the serological profile, in addition to immunoblotting (IB) and indirect immunofluorescence (IIF) assays. HLA-DRB1 and -DQA1/DQB1 alleles were characterized by PCR combined with sequence-specific oligonucleotide probes (PCR-SSOP). The serological and genetic profiles were compared using 78 PF, 62 pemphigus vulgaris (PV) and 58 ATL patients against 163 and 1592 healthy controls, respectively. Some ATL patients showed positive results for anti-Dsg1 and/or anti-Dsg3 antibodies. They also revealed 130, 160 and/or 230 kDa epidermal peptides in IB. Moreover, some ATL samples exhibited pemphigus or a bullous pemphigoid pattern in IIF. ELISA and IB assays showed Lbr-peps in pemphigus patients. HLA-DQA1*01 and -DQA1*01:02 were protective and susceptibility alleles for ATL, respectively, but the opposite for pemphigus. Anti-Dsgs in ATL may represent epiphenomena. Anti-Lbr-pep antibodies in pemphigus suggest a previous infection. A differential association of the HLA profile may contribute to the lack of co-association between pemphigus and ATL.

Development of HLA-B*57:01 Genotyping Real-Time PCR with Optimized Hydrolysis Probe Design

HLA-B*57:01 genotyping before abacavir (ABC) administration is a standard of care to avoid ABC-driven hypersensitivity reactions. Several HLA-B*57:01 tests have been developed, each with advantages and disadvantages. Some have limited accuracy, require special instrumentation, and/or are labor intensive and expensive. We developed a novel hydrolysis probe-based real-time PCR method of HLA-B*57:01 genotyping. Primer and probes were designed based on published sequence variations in exon 3 of HLA-B that distinguish HLA-B*57:01 from ABC-insensitive alleles such as HLA-B*57:03 and HLA-B*58:01. We designed PCR primers to amplify HLA-B*57:01 along with closely related alleles, such as HLA-B*57:03, directly from genomic DNA. Most ABC-insensitive alleles, including HLA-B*58:01, would not produce any products in the PCR reaction. Our hydrolysis probes enable differentiation of HLA-B*57:01 from the other amplified, but ABC-insensitive, alleles. In addition to using real-time PCR, we used restriction enzymes to generate differential digestion patterns that led to the development of an HLA-B*57:01 PCR-restriction fragment length polymorphism marker. When used to genotype a set of 75 selected clinical samples, our real-time PCR assay demonstrated 100% accuracy in distinguishing between the HLA-B*57:01-positive and -negative alleles when results were compared to those of sequence-specific oligonucleotide probe typing and reference laboratory testing. Our newly developed test will allow clinical laboratories with real-time PCR capabilities to perform HLA-B*57:01 genotyping in a timely and economical manner.

ERAP1 and HLA-C interaction in inflammatory bowel disease in the Spanish population.

Large genome-wide analysis studies (GWAS) and meta-analyses have dramatically increased our knowledge of the genetic risk factors of inflammatory bowel disease (IBD), identifying at least 163 loci. The endoplasmic reticulum aminopeptidase-2 ( ERAP2) gene has been reported as a potential candidate gene for IBD. GWAS have also shown the potential associations between ERAP single nucleotide polymorphisms (SNP) loci and susceptibility to several autoimmune diseases, and ERAP1 and ERAP2 polymorphisms are related to HLA class I-associated diseases, including ankylosing spondylitis and Behçet's disease. Interestingly, these associations were confined to individuals carrying HLA class I-risk alleles. The aim of this study was to investigate the association of ERAP1 and ERAP2 SNPs with IBD in a Spanish population, analysing their possible interaction with specific HLA-C alleles to IBD susceptibility. A total of 367 individuals were divided into 216 IBD cases and 151 controls. SNP genotyping was performed using TaqMan® genotyping assays, whereas HLA-C typing was analysed by sequence-specific oligonucleotide probing. Herein, we report an association of the ERAP1 SNP rs30187 with the HLA-C*07 allele. The existence of shared inflammatory pathways in immunologically related diseases together with the understanding of ERAP1 function may offer clues to novel treatment strategies.

HLA-A, B, DRB1, DQA1, DQB1 alleles and haplotype frequencies in Dene and Cree cohorts in Manitoba, Canada

BackgroundFirst Nations in the Canadian province of Manitoba have disproportionately high rates of epidemic and endemic TB. Gene polymorphisms that modulate HLA Class I and II antigens are among the risk markers for TB, along with other biologic, and social determinants of health. HLA-A, B, DRB1, DQA1, DQB1 were typed in two Manitoba First Nation indigenous groups to identify and compare the frequency of gene polymorphisms that may influence susceptibility or resistance to TB. MethodsParticipants who self-identified as either Dene or Cree enrolled into the study from two First Nation communities in Manitoba, Canada. Genomic DNA was extracted from blood samples collected with informed consent from Dene (N=63) and Cree (N=42) First Nation study participants. Participants self-reported having treated active TB, treated latent TB or no TB. HLA Class I and II molecules were typed using sequence-specific oligonucleotide (SSO) probes from commercially available kits. ResultsThe rates of treated active and latent TB were marginally higher among the Dene than the Cree participants (p=0.112). Class I and II HLA loci were in Hardy-Weinberg equilibrium in both the Dene and Cree groups. In this exploratory analysis of TB and HLA allele frequencies in Dene and Cree cohorts HLA-A*03 and HLA-DQB1*05:03 were significantly associated with TB. ConclusionsThe high incidence of TB in both Dene and Cree populations in Canada requires both biomedical and socioeconomic prevention and control measures. Among the former, an understanding of HLA diversity among First Nations groups may aid the development of new effective vaccine and therapeutic modalities that depend on the interaction between small molecules and specific HLA epitopes.

Reemergence of chloroquine-sensitive pfcrt K76 Plasmodium falciparum genotype in southeastern Cameroon

BackgroundChloroquine had been used extensively during the last five decades in Cameroon. Its decreasing clinical effectiveness, supported by high proportions of clinical isolates carrying the mutant pfcrt haplotype (CVIET), led the health authorities to resort to amodiaquine monotherapy in 2002 and artemisinin-based combination therapy (ACT) in 2004 (artesunate–amodiaquine, with artemether–lumefantrine as an alternative since 2006) as the first-line treatment of uncomplicated malaria. The aim of the present study was to investigate whether the withdrawal of chloroquine was associated with a reduction in pfcrt mutant parasite population and reemergence of chloroquine-sensitive parasites in southeastern Cameroon between 2003 and 2012.MethodsThe frequency of pfcrt haplotypes at positions 72–76 in Plasmodium falciparum isolates collected from individuals in 2003 and 2012 in southeastern Cameroon was determined by sequence specific oligonucleotide probes-enzyme linked immunosorbent assay (SSOP-ELISA).ResultsThe proportions of parasites carrying the mutant haplotype CVIET and the wild-type CVMNK were 53.0 and 28.0% in 2003, respectively. The proportion of the mutant haplotype in samples collected 9 years later decreased to 25.3% whereas the proportion of parasites carrying the wild-type CVMNK haplotype was 53.7%.ConclusionsEven though the proportion of chloroquine-sensitive parasites seems to be increasing in southeastern Cameroon, a reintroduction of chloroquine cannot be recommended at present in Cameroon. The current national anti-malarial drug policy should be implemented and reinforced to combat drug-resistant malaria.

Is HLA the cause of the high incidence of type 1 diabetes in the Canary Islands? Results from the Type 1 Diabetes Genetics Consortium (T1DGC)

IntroductionIncidence of childhood-onset type 1 diabetes mellitus in the Canary Islands is the highest reported so far in Spain, and among the highest worldwide. The HLA region accounts for approximately half the genetic risk of type 1 diabetes. Our aim was to assess distribution of high-risk and protective HLA haplotypes in the Canarian families included in the T1DGC, as compared to the rest of Spain. MethodsThe T1DGC study, an international project to study the genetics and pathogenesis of type 1 diabetes, enrolled more than 3000 families with type 1 diabetes worldwide. Spain provided 149 of these families, of whom 42 were from Tenerife and Gran Canaria. HLA was genotyped centrally using a PCR-based, sequence-specific oligonucleotide probe system. Haplotypes were reconstructed using the deterministic algorithm alleHap in the R programming environment. Based on prior T1DGC results in Caucasian population, haplotypes DRB1*0405-DQA1*0301-DQB1*0302, DRB1*0401-DQA1*0301-DQB1*0302, DRB1*0301-DQA1*0501-DQB1*0201, DRB1*0402-DQA1*0301-DQB1*0302 and DRB1*0404-DQA1*0301-DQB1*0302 were considered high-risk. DRB1*0701-DQA1*0201-DQB1*0303, DRB1*1401-DQA1*0101-DQB1*0503, DRB1*1501-DQA1*0102-DQB1*0602, DRB1*1101-DQA1*0501-DQB1*0301, DRB1*1104-DQA1*0501-DQB1*0301, DRB1*1303-DQA1*0501-DQB1*0301, DRB1*1301-DQA1*0103-DQB1*0603 and DRB1*0403-DQA1*0301-DQB1*0302 were considered protective. The distribution of protective, high-risk, and other haplotypes in the (first two) affected siblings and unaffected parents from Canarian and non-Canarian Spanish families was compared (Chi-square test). ResultsNo significant differences were found between the regions in distribution of the HLA haplotypes in the affected siblings or in the non-affected parents. ConclusionsThe high incidence of childhood-onset type 1 diabetes in the Canarian population does not appear to be explained by a greater prevalence of high-risk class II HLA haplotypes in families with the disease. However, sample size limits the differences that can be detected in this study.

DNA damage response inhibition at dysfunctional telomeres by modulation of telomeric DNA damage response RNAs

The DNA damage response (DDR) is a set of cellular events that follows the generation of DNA damage. Recently, site-specific small non-coding RNAs, also termed DNA damage response RNAs (DDRNAs), have been shown to play a role in DDR signalling and DNA repair. Dysfunctional telomeres activate DDR in ageing, cancer and an increasing number of identified pathological conditions. Here we show that, in mammals, telomere dysfunction induces the transcription of telomeric DDRNAs (tDDRNAs) and their longer precursors from both DNA strands. DDR activation and maintenance at telomeres depend on the biogenesis and functions of tDDRNAs. Their functional inhibition by sequence-specific antisense oligonucleotides allows the unprecedented telomere-specific DDR inactivation in cultured cells and in vivo in mouse tissues. In summary, these results demonstrate that tDDRNAs are induced at dysfunctional telomeres and are necessary for DDR activation and they validate the viability of locus-specific DDR inhibition by targeting DDRNAs.

Human leukocyte antigen allele linkage disequilibrium and haplotype structure in volunteer bone marrow donors of Paraná State.

BackgroundBone marrow transplantation has been used in the treatment of various diseases, especially hematologic diseases. The success of this treatment, among other factors, requires human leukocyte antigens (HLA) compatibility between patient and donor. Knowing the human leukocyte antigens allele group and haplotype frequencies as well as the linkage disequilibrium between alleles of different human leukocyte antigens loci can shorten the search time for a compatible bone marrow donor.ObjectiveTo assemble and analyze data on human leukocyte antigens frequencies available in the Laboratory of Immunogenetics and Histocompatibility (LIGH) database of the Universidade Federal do Paraná adding an estimation of the Hardy–Weinberg equilibrium and linkage disequilibrium.MethodsThe sample was composed of seven populations grouped by self-declared ancestry or inferred from the surname as follows: Laboratory of Immunogenetics and Histocompatibility database (all groups), descendants of Italians, Poles, and Asians, Afro-Brazilians, Mulattos (mixed ancestry) and Amerindians. Human leukocyte antigens genotyping was carried out using the polymerase chain reaction-sequence specific primers (PCR-SSP) and -sequence specific oligonucleotide (PCR-SSO) technologies.ResultsThere were high frequencies of the HLA-A*02, HLA-B*35 and HLA-DRB1*13 allelic groups in all groups. The same was observed for the HLA-A*01-B*08-DRB1*03 haplotype except for Asian descendants. It was observed that the human leukocyte antigens Laboratory of Immunogenetics and Histocompatibility database and the Asian group are not in Hardy–Weinberg equilibrium. The Italian, Polish, Asian, Mulatto and Amerindian descendants showed haplotypes in complete linkage disequilibrium. Our results were compared with data on the human leukocyte antigens in the Paraná population available from the Brazilian Voluntary Bone Marrow Donor Registry (REDOME) and data published on the population of Curitiba and the northern region of Paraná.ConclusionsHaplotypes frequent in the Asian group were not the most frequently observed in the Laboratory of Immunogenetics and Histocompatibility database and the National Bone Marrow Donor Registry for the state of Paraná. Linkage disequilibrium information may prove useful in the search for bone marrow donors for patients awaiting a suitable donor.

Is HLA the cause of the high incidence of type 1 diabetes in the Canary Islands? Results from the Type 1 Diabetes Genetics Consortium (T1DGC)

IntroductionIncidence of childhood-onset type 1 diabetes mellitus in the Canary Islands is the highest reported so far in Spain, and among the highest worldwide. The HLA region accounts for approximately half the genetic risk of type 1 diabetes. Our aim was to assess distribution of high-risk and protective HLA haplotypes in the Canarian families included in the T1DGC, as compared to the rest of Spain. MethodsThe T1DGC study, an international project to study the genetics and pathogenesis of type 1 diabetes, enrolled more than 3000 families with type 1 diabetes worldwide. Spain provided 149 of these families, of whom 42 were from Tenerife and Gran Canaria. HLA was genotyped centrally using a PCR-based, sequence-specific oligonucleotide probe system. Haplotypes were reconstructed using the deterministic algorithm alleHap in the R programming environment. Based on prior T1DGC results in Caucasian population, haplotypes DRB1*0405-DQA1*0301-DQB1*0302, DRB1*0401-DQA1*0301-DQB1*0302, DRB1*0301-DQA1*0501-DQB1*0201, DRB1*0402-DQA1*0301-DQB1*0302 and DRB1*0404-DQA1*0301-DQB1*0302 were considered high-risk. DRB1*0701-DQA1*0201-DQB1*0303, DRB1*1401-DQA1*0101-DQB1*0503, DRB1*1501-DQA1*0102-DQB1*0602, DRB1*1101-DQA1*0501-DQB1*0301, DRB1*1104-DQA1*0501-DQB1*0301, DRB1*1303-DQA1*0501-DQB1*0301, DRB1*1301-DQA1*0103-DQB1*0603 and DRB1*0403-DQA1*0301-DQB1*0302 were considered protective. The distribution of protective, high-risk, and other haplotypes in the (first two) affected siblings and unaffected parents from Canarian and non-Canarian Spanish families was compared (Chi-square test). ResultsNo significant differences were found between the regions in distribution of the HLA haplotypes in the affected siblings or in the non-affected parents. ConclusionsThe high incidence of childhood-onset type 1 diabetes in the Canarian population does not appear to be explained by a greater prevalence of high-risk class II HLA haplotypes in families with the disease. However, sample size limits the differences that can be detected in this study.

Reduced frequency of two activating KIR genes in patients with sepsis

Natural killer (NK) cell activity is regulated by activating and inhibitory signals transduced by killer cell immunoglobulin-like receptors (KIR). Diversity in KIR gene repertoire among individuals may affect disease outcome. Sepsis development and severity may be influenced by genetic factors affecting the immune response. Here, we examined sixteen KIR genes and their human leucocyte antigen (HLA) class I ligands in critical patients, aiming to identify patterns that could be associated with sepsis. Male and female patients (ages ranging between 14 and 94years-old) were included. DNA samples from 211 patients with sepsis and 60 controls (critical care patients with no sepsis) collected between 2004 and 2010 were included and genotyped for KIR genes using the polymerase chain reaction method with sequence-specific oligonucleotide (PCR-SSO), and for HLA genes using the polymerase chain reaction method with sequence-specific primers (PCR-SSP). The frequencies of activating KIR2DS1 and KIR3DS1 in sepsis patients when compared to controls were 41.23% versus 55.00% and 36.49% versus 51.67% (p=0.077 and 0.037 respectively before Bonferroni correction). These results indicate that activating KIR genes 2DS1 and 3DS1 may more prevalent in critical patients without sepsis than in patients with sepsis, suggesting a potential protective role of activating KIR genes in sepsis.

HLA Genotyping in Patients with End-Stage Renal Disease Waiting For Cadaveric Renal Transplantation in Federation of Bosnia and Herzegovina.

AIM:The research was conducted by genotyping two Human Leukocyte Antigen (HLA) gene classes. The main objective of this research was to investigate distribution and frequency of the allelic groups, genotypes and haplotypes in the gene loci of HLA class I (HLA-A*, -B*, -C*) and HLA class II (HLA-DRB1*, -DQB1*) in patients included in the program of cadaveric renal transplantation.MATERIAL AND METHODS:Our study covered 186 blood samples of patients who are registered on the list for cadaveric renal transplantation in Federation of Bosnia and Herzegovina and included 59 control, healthy unrelated individuals. For the HLA typing, we have used three different methods: micro lymphocyte cytotoxicity test (MLCT), Polymerase Chain Reaction (PCR) – Sequence Specific Primers (SSP) and PCR – Sequence-Specific Oligonucleotides (SSO) or Luminex technology. All patients and cadaveric donors were tested using the three methods because the system is polymorphic.RESULTS:Analysis of the results of genotyping HLA class I gene loci identified dominant HLA-A*02, HLA-B*35, HLA-C*07 allelic groups. Analysis of the HLA class II gene loci genotyping showed that HLA-DRB1*11 and HLA-DQB1*03 loci had the highest incidence in HLA class II.CONCLUSION:Based on our results and previous research, there were no observed differences between allelic frequencies and genotypes of healthy people and people with ESRD. Differences between allelic groups occurred, but they were not statistically significant, except HLA-C*01 (p = 0.020).

Managing the sequence-specificity of antisense oligonucleotides in drug discovery.

All drugs perturb the expression of many genes in the cells that are exposed to them. These gene expression changes can be divided into effects resulting from engaging the intended target and effects resulting from engaging unintended targets. For antisense oligonucleotides, developments in bioinformatics algorithms, and the quality of sequence databases, allow oligonucleotide sequences to be analyzed computationally, in terms of the predictability of their interactions with intended and unintended RNA targets. Applying these tools enables selection of sequence-specific oligonucleotides where no- or only few unintended RNA targets are expected. To evaluate oligonucleotide sequence-specificity experimentally, we recommend a transcriptomics protocol where two or more oligonucleotides targeting the same RNA molecule, but with entirely different sequences, are evaluated together. This helps to clarify which changes in cellular RNA levels result from downstream processes of engaging the intended target, and which are likely to be related to engaging unintended targets. As required for all classes of drugs, the toxic potential of oligonucleotides must be evaluated in cell- and animal models before clinical testing. Since potential adverse effects related to unintended targeting are sequence-dependent and therefore species-specific, in vitro toxicology assays in human cells are especially relevant in oligonucleotide drug discovery.

A label-free electrochemical DNA biosensor for breast cancer marker BRCA1 based on self-assembled antifouling peptide monolayer

Electrochemical assays potentially offer a highly cheap, automated, portable and sensitive method for a great deal of disease biomarker detections, and minimization of nonspecific adsorption is a critical issue for their application in natural complex media. We present herein, the development and application of a highly sensitive and selective electrochemical DNA hybridization biosensor for breast cancer marker BRCA1, based on a zwitterionic peptide self-assembled monolayer (SAM) support serving as the low fouling substrate, the 19-mer BRCA1 related sequence-specific oligonucleotides, and the sensitively label-free sensing technique of electrochemical impedance spectroscopy (EIS). The capability of such peptide SAM in resisting nonspecific protein adsorption was surveyed using EIS, and the biosensor fabrication process and sensing performance were also investigated with X-Ray photoelectron spectroscopy (XPS) and EIS. Significantly, this developed biosensing strategy achieved a linear detection range from 1.0fM to 10.0pM with a limit of detection of 0.3fM, associated with satisfactory sensing properties of facile fabrication, desired sensitivity, high selectivity, and favourable reproducibility and antifouling ability, manifesting its promising potential in practical application.

Detection of KRAS Mutations in Plasma DNA Using a fully Automated Rapid Detection System in Colorectal Cancer Patients

KRAS mutations have been recognized as predictive markers of primary resistance to anti-EGFR-antibodies in colorectal cancer patients. In addition, newly detected KRAS mutations have been reported to be related with acquired resistance to chemotherapy containing anti-EGFR antibody. Considering this evidence, monitoring of KRAS mutations is indispensable for making treatment decisions, and the method should be non-invasive allowing repeated examinations. Recently, we established a novel automated sensitive detection system for KRAS mutations, named mutation-biased PCR quenching probe system (MBP-QP). The goal of our study was to investigate the potential for monitoring KRAS mutations during treatment with anti-EGFR antibodies. The detection limit of MBP-QP using a control plasmid containing KRAS mutations was 1–9 copies, and 0.05–0.3% mutant plasmid was detectable in a mixture of wild type and mutants. One-hundred twenty colorectal cancer patients were genotyped for KRAS mutations with MBP-QP as well as polymerase chain reaction reverse sequence-specific oligonucleotide (PCR-rSSO), which has already been applied to cancer tissue samples in the clinical setting. Concordance rates between plasma DNA and cancer tissues were 68% with MBP-QP and 66% with PCR-rSSO, indicating that these systems are equivalent in terms of detecting KRAS mutations with plasma DNA. KRAS mutations in plasma DNA were frequently observed in systemic metastatic cancer patients, and in three patients KRAS mutations appeared after chemotherapy containing anti-EGFR antibody. A prospective study is needed for clarifying whether KRAS mutations detected in plasma DNA are predictive markers of treatment efficacy with anti-EGFR antibody.

Investigation of human leukocyte antigen in osteoarticular brucellosis

Background/aim: To determine the relationship between human leukocyte antigen (HLA) antigens and osteoarticular brucellosis by evaluating HLA Class I and II antigens in control subjects and patients developing osteoarticular brucellosis in Turkey.Materials and methods: The study included 28 patients with osteoarticular involvement diagnosed with brucellosis and 100 controls. The HLA Class I and II antigens were studied in isolated DNA samples using sequence-specific oligonucleotide procedure.Results: The mean age of the 28 patients and 100 controls was 42.3 ± 20.2 years and 50.1 ± 16.3 years, respectively. When the frequency of HLA Class I was examined, HLA-A*02 and HLA-B*27 antigens were detected in 50% and 28.57% of the patients, respectively. However, there was no significant difference when compared to the control group. Among the HLA Class I antigens, HLA-Cw*10 was determined in 35.71% of the patients and 17% of the controls; the difference was significant (P = 0.039).Conclusion: In the development of brucellosis, the frequency of HLA-Cw*10 among HLA Class I antigens was observed to be increased, and HLA-Cw*10 was considered likely to cause predisposition to brucellosis. Further studies to be performed on a higher number of patients and controls could demonstrate that genetic factors play a role in the diagnosis of osteoarticular brucellosis.

The incidence of HLA-DQ2/DQ8 in Turkish children with celiac disease and a comparison of the geographical distribution of HLA-DQ.

IntroductionCeliac disease (CD) is an auto-immune enteropathy that occurs in genetically pre-disposed people as a result of the consumption of gluten-containing foods.AimTo identify the incidence of HLA-DQ2 and HLA-DQ8 observed in children with CD.Material and methodsIn this study, we focused on children ranging in age from 2 to 18 years and diagnosed with celiac disease. In our patients diagnosed with CD, in addition to tissue transglutaminase antibodies (anti-tTG), we also evaluated HLA-DQ2 B1 and HLA-DQ8 B1 alleles using the method of polymerase chain reaction (PCR)/sequence-specific oligonucleotide probes (Luminex®). The detection of 0201/0202 for HLA-DQ2 allele and 0302 for HLA-DQ8 allele was accepted as a positive result.ResultsThe mean age of our patients with celiac disease was 7.42 ±3.18 years, and the female/male ratio was 1.5/1. Seventy-six percent of our patients were HLA-DQ2 and/or HLA-DQ8 positive, 67% were HLA-DQ2 positive, and 25% were HLA-DQ8 positive. Nevertheless, 24% of them were HLA-DQ2 and HLA-DQ8 negative. The incidence of HLA-DQ2 in the control group was 18.8% with a significant difference compared to the HLA-DQ2 incidence in the patient group (67%) (p < 0.05). Similarly the HLA-DQ8 incidence in the control group (5.7%) was significantly lower than the incidence in the patient group (25%) (p < 0.05).ConclusionsThe incidence of the patients diagnosed with CD, who are HLA-DQ2 and HLA-DQ8 negative, varies among different populations.

Association of human leukocyte antigen DRB1 with anti-cyclic citrullinated peptide autoantibodies in Saudi patients with rheumatoid arthritis.

BACKGROUNDThe genetic association between human leukocyte antigen (HLA)-DRB1 alleles and the risk of development of autoantibodies has been investigated, but there are few studies from the Gulf region.OBJECTIVESTo investigate the association between the HLA-DRB1 shared epitope and the risk for development of autoantibodies in rheumatoid arthritis (RA) patients in a Saudi population.DESIGNAnalytical cross-sectional study.SETTINGTertiary care hospital in Riyadh, Saudi Arabia.PATIENTS AND METHODSWe enrolled consecutive Saudi RA patients attending the rheumatology clinic between January and April 2015. Previously published data on HLA typing on unmatched healthy controls were used for comparison. HLA typing was performed using sequence-specific oligonucleotide probes (SSOP). Rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP) antibodies, and antinuclear antibodies (ANA) were also measured. Logistic regression analysis was used to study the autoantibodies as possible explanatory variables for the presence of the HLA-DRB1 shared epitope.MAIN OUTCOME MEASURE(S)The association between the presence of the shared epitope and the risk of developing anti-CCP antibodies, ANA, and RF.RESULTSIn 76 patients with RA, carrying the shared epitope was associated with a significantly higher risk of having RA [OR=2.65, 95% CI (1.42–4.94), P=.0009]. However, only HLA-DRB1*04:05 was significantly associated with RA [OR=3.73, 95% CI (1.61–8.96), Pc=.016]. In the logistic regression analysis, only anti-CCP was significantly associated with the shared epitope [OR=14.51, 95% CI (1.53–137.49), P=.02].CONCLUSIONSOur analysis indicates that the presence of the HLA-DRB1 shared epitope is strongly associated with the development of anti-CCP antibodies in Saudi patients with RA.LIMITATIONSA larger sample size is needed to confirm our finding.