Allele-specific Oligonucleotide Hybridization Research Articles

Unknown Mutation Detection via Restriction Hybridization Method Instead of Using Next Generation Sequencing Method

In biology mutation is a change in the nucleotide sequences of the DNA of an organism Mainly there are three types of mutation: point mutation, deletion and insertions. Once the mutation has been defined allele specific oligonucleotide hybridization, amplification, heteroduplex formation method referred to as a diagnostic method some advance technique like CRISPR cas9 system is using for selected mutagenesis. Using restriction method system we can detect a mutation. Let’s say you have a DNA sample with fluorescent labeled from patient and you want to make sure that gene you are interested is in healthy gene. We can design different short fragment sequences to scan through DNA or find specific gene or mutation. The sequences scan the DNA if the sequences does not find targeted gene it does not bind to it its means that no fluorescence color appears under UV-light each different short fragment sequences is label with different colors. If the different short fragments sequence does not bind to the DNA or specific gene or area this means that there will be no color appear under UV light this part or gene will be separated from the DNA by using Restriction enzyme to do a Sanger sequencing gel electrophoresis. Result of the Sanger sequencing will provide the information about sequence of unknown part or gene of the DNA this method is easier and cost economic method instead of Next generation sequencing method [1-7].

Unknown Mutation Detection via Restriction Hybridization Method Instead of Using Next Generation Sequencing Method

In biology mutation is a change in the nucleotide sequences of the DNA of an organism mainly there are three types of mutation: point mutation, deletion and insertions. Once the mutation has been defined allele specific oligonucleotide hybridization, amplification, heteroduplex formation method referred to as a diagnostic method some advance technique like CRISPR cas9 system is using for selected mutagenesis. Using restriction method system we can detect a mutation. Let’s say you have a DNA sample with fluorescent labelled from patient and you want to make sure that gene you are interested is in healthy gene. We can design different short fragment sequences to scan through DNA or find specific gene or mutation. The sequences scan the DNA if the sequences does not find targeted gene it does not bind to it its means that no fluorescence color appears under UV-light each different short fragment sequences is label with different colors. If the different short fragments sequence does not bind to the DNA or specific gene or area this means that there will be no color appear under UV light this part or gene will be separated from the DNA by using Restriction enzyme to do a Sanger sequencing gel electrophoresis. Result of the Sanger sequencing will provide the information about sequence of unknown part or gene of the DNA this method is easier and cost economic method instead of Next generation sequencing method.

Retrofitting massively parallel sequencing (MPS) for HLA-DQA1 and polymarker (PM) in forensic casework.

Genotype profiling has played a major role in forensics for decades. The technology for detection and discrimination has advanced substantially, from serology to DNA sequence analysis. Currently, there may be situations where there is a need for re-analysis of forensic DNA data that was produced using methodology that is no longer available. An example of this is the allele-specific oligonucleotide hybridization assays used in the 1990s. In the study presented herein, we have developed a multiplex system combining PCR and massively parallel sequencing (MPS) technologies to identify DNA polymorphisms. Our results are consistent with those found in the widely utilized AmpliType PM + DQA1 Amplification and Typing Kit originally marketed by Perkin Elmer. During the course of our studies, it became apparent that paralogous genes for two of the loci, GYPA and HBG2 (formerly HBGG), could have confounded the interpretation of the original assays, and we describe the technical solutions we developed to overcome ambiguity in genotype assignment. This study results in a novel resource enabling the re-analysis of DNA profiling results produced decades past using current day technology.

Mutation Detection via CRISPRCas9 Micro-fluid Chip

In biology mutation is a change in the nucleotide sequences of the DNA of an organism mainly there are three types of mutation: point mutation, deletion and insertions. Once the mutation has been defined allele specific oligonucleotide hybridization, amplification, heteroduplex formation method referred to as a diagnostic method some advance technique like CRISPR cas9 system is using for selected mutagenesis. Using microfluid and CRISPR cas9 system we can detect a mutation. Let's say you have a DNA sample with fluorescent labeled from patient and you want to make sure that gene you are interested is in healthy gene. We can design a CRISPR to scan through DNA or find specific gene or mutation. The CRISPR scan the DNA if the CRISPR does not find targeted gene it does not bind to it its means that no fluorescence color appears under UV-light but its scan and find its target and this binding create a fluorescence signal its means that mutation can be occur in gene

Alpha thalassemia genotypes in Kuwait

BackgroundThe frequency of the alpha thalassemia trait is approximately 40% in the Kuwaiti population, but there has been no comprehensive study of the prevalent alleles. This is a report of patients who were referred for molecular diagnosis over a 20-year period.MethodsThis is a retrospective study of the α-globin genotypes obtained in the Hemoglobin Research Laboratory of the Department of Pediatrics, Kuwait University from 1994 to 2015. Genotyping was performed by a combination of PCR, allele-specific oligonucleotide hybridization and reverse dot blot hybridization (Vienna Lab Strip Assay).ResultsFour hundred samples were characterized and analyzed from individuals aged < 1 month to 80 years, with a median of 6 years from 283 unrelated families. Most (90.8%) were Kuwaiti nationals. The commonest genotype was homozygosity for the polyadenylation-1 mutation (αPA-1α/α PA-1α) in 33.3% of the samples, followed by heterozygosity (αα/α PA-1α) for the same mutation in 32.3%. PA-1 was therefore the most frequent allele (0.59). The frequency of the α0 (−-MED) allele was 0.017. Rare alleles that were found in very low frequencies included α0 (−-FIL) in a Filipino child, Hb Constant Spring, Hb Adana, and Hb Icaria.ConclusionThere is a wide variety of alpha thalassemia alleles among Kuwaitis, but nondeletional PA-1 is by far the most common cause of the moderate to severe HbH (β4 tetramer) disease phenotype. The α0 (−MED) allele is also encountered, which has implications for premarital counseling, especially for the possibility of having babies with alpha thalassemia major (Barts hydrops fetalis).

Clinical genetic strategies for early onset neurodegenerative diseases

Methods for the genetic diagnosis of neurodegenerative disorders were reviewed, including their backgrounds and applications in the laboratory. Majority of disease-causing gene mutations were uncommon in the general population, where dominant variations could be easily identified in certain disorders. The development of molecular, next generation sequencing (NGS) and cytogenetic techniques allowed to identify multiple genetic mutations leading to diseases. Using of the accurate multivariate diagnosis of diseases would be essential for appropriate treatment of patients, genetic counseling and prevention strategies Abnormal genes play an extremely important role in the pathogenesis of neurodegenerative disorders of the nervous system. Many studies of genetic have clearly indicated that the molecular mechanisms underlying the etiology and pathogenesis of most neurodegenerative disorders until now. Mutation is necessary for life, while fidelity is necessary for DNA replication. Mistakes in DNA replication/transcription can cause cells to produce either too much or too little protein or a defective protein. Studies on mutations have revealed the normal functions of genes, messages, proteins, the causes of many diseases, and the variability of responses among individuals. Based on the presence of damaging variants, there are many genes have identified that associated with neurodegenerative disorders through to genetic analyses of patients. This declaration is interesting because also our previous analysis has shown that several types of neurodegenerative diseases associated with abnormal genes function have been well described, including Alzheimer’s disease (AD), front temporal dementia (FTD), amyotrophic lateral sclerosis (ALS), prion disease, and Parkinson’s disease (PD). Since the potential treatment strategies for these disorders may be more successful during the pre-clinical stages than in the actual clinical setup, accurate, simple, and affordable diagnostic methods are needed. A mutation can be defined as a sequence change in a test sample compared to the sequence of a reference standard. This has led to the development of methods for screening and detecting abnormal DNA, including techniques that detect previously described mutations (genotyping) and those that scan for mutations in a particular target region (mutation scanning). This review provides a broad overview of the range of currently available mutation detection techniques with special emphasis on neurodegenerative disorders in humans. We have discussed the methods used for detecting unknown mutations, such as ribonuclease, denaturing gradient-gel electrophoresis, carbodiimide, chemical cleavage, single- strand conformation polymorphism, heteroduplex, and sequencing. Furthermore, other diagnostic methods for testing mutations include allele-specific oligonucleotide hybridization; allele-specific amplification, ligation, primer extension, and the artificial introduction of restriction sites are also reviewed. In order to identify the mutation, the last of the work provides basic insights into some of the most popular in silico tools for splicing defect prediction from the viewpoint of end users.

Functional GSTA1 Haplotypes Affect Clearance and Toxicity of Busulfan When Administered in 16 Doses to Pediatric Patients Undergoing Hematopoietic Stem Cell Transplantation: A Multicenter Prospective Study on Behalf of the Pediatric Disease Working Party of the European Society for Blood and Marrow Transplantation (EBMT)

Busulfan (BU) dose adjustment following therapeutic drug monitoring (TDM) contributes to better outcome of hematopoietic stem cell transplantation (HSCT). The therapeutic target window can be reached in approximately 51% to 74% of pediatric cases and 45% to 64% of infants depending on the dosing guideline used. We hypothesize that a higher proportion of pediatric patients could achieve the target BU area-under-the-curve (AUC) of 900-1500 µM.min per dose in a four times dosing schedule through genotype-guided initial BU dosing. Previous investigations by our group and others showed association of glutathione -S- transferase alpha 1 (GSTA1) with reduced BU clearance (CL), likely due to altered expression of GSTA1 enzyme, principally involved in BU metabolism.To delineate and investigate the association of each GSTA1 haplotype with the CL of BU, promoter haplotypes of GSTA1 gene were assessed in vitro with reporter gene-assay and clinically in a pediatric multi-center study (N =138) through association with BU CL, AUC, dose change and clinical outcomes. All patients received BU based myeloablative conditioning regimen. BU PK parameters from 1st dose were estimated and further doses were adjusted to have a steady state concentrations of 600-900 ng/mL and GSTA1promoter SNPs at -69, -513, -631, -1142 positions were genotyped using allele specific oligo hybridization method.Reporter-gene assays confirmed significant differences between the haplotype subgroups and supported their importance in capturing PK variability. Four GSTA1 diplotype groups (I-IV) that significantly correlated with CL (p=0.009) were distinguished. GSTA1 diplotypes (group I) underlying fast metabolizing capacity (*A2*A2&*A2*A3) had lower cumulative AUC and a higher dose increase after TDM whereas those with slow metabolizing capacity (group IV, *B1a*B1a /*B1a*B1b /*B2*B1a / *A1*B1b)had highest AUC and least dose change (p=0.02). Multivariate linear model included GSTA1 diplotype groups and non-genetic factors such as age, gender, first BU dose in mg/kg and patients origin. Dose (p<0.0005), gender (p=0.05) and GSTA1 diplotypes (p=0.01) were retained in the final model that explained 34% of overall variability. Analysis with clinical outcomes indicated higher incidences of sinusoidal obstruction syndrome in Group IV diplotype carriers (HR=7.1; 95% CI: 2.5-20.4) compared to patients with other GSTA1diplotypes. A similar association was also seen with acute graft versus host disease (aGvHD grade I-4) and combined treatment-related toxicity (TRT) (p≤0.003).In conclusion, this study has replicated and provided additional evidence for the association of GSTA1 with BU PK and clinical outcomes of HSCT. GSTA1 diplotypes can explain in some models ~20% of the variability seen in BU CL and can contribute to HSCT-related complications acting within and beyond BU metabolism. Genotyping therefore may be helpful in deciding the first BU dose, thus decreasing consequences like TRT due to inaccurate first and subsequent dosing. This may be particularly important for GSTA1 diplotype Group IV carriers. DisclosuresBittencourt:Jazz Pharmaceuticals: Consultancy, Other: Educational Grant; Seattle Genetics: Consultancy.

Genotyping of single nucleotide polymorphisms related to attention-deficit hyperactivity disorder.

Pharmacological treatment of several diseases, such as attention-deficit hyperactivity disorder (ADHD), presents marked variability in efficiency and its adverse effects. The genotyping of specific single nucleotide polymorphisms (SNPs) can support the prediction of responses to drugs and the genetic risk of presenting comorbidities associated with ADHD. This study presents two rapid and affordable microarray-based strategies to discriminate three clinically important SNPs in genes ADRA2A, SL6CA2, and OPRM1 (rs1800544, rs5569, and rs1799971, respectively). These approaches are allele-specific oligonucleotide hybridization (ASO) and a combination of allele-specific amplification (ASA) and solid-phase hybridization. Buccal swab and blood samples taken from ADHD patients and controls were analyzed by ASO, ASA, and a gold-reference method. The results indicated that ASA is superior in genotyping capability and analytical performance.

Influence Of GST Gene Polymorphisms On Busulfan Pharmacokinetics and Outcome Of Hematopoietic Stem Cell Transplantation In Thalassemia Pediatric Patients

IntroductionThe only curative therapy for thalassemia remains the replacement of the defective erythropoiesis by allogeneic hematopoietic stem cell transplantation (HSCT). Although largely successful, HSCT can have serious adverse effects such as; veno-occlusive disease (VOD), graft failure and graft versus host disease (GVHD) associated mainly with the conditioning regimen. Most commonly used conditioning regimens in children comprise the alkylating agent busulfan (BU). Interindividual differences in Bu pharmacokinetics (PK) have been observed and may be an important cause of resistance to treatment and toxicity. It has been hypothesized that some of this variability in treatment might be predicted by genetic variants of enzymes involved in the metabolism of BU. Identifying pharmacogenetic determinants of BU may allow for prospective identification of patients with suboptimal drug responses allowing for complementation of traditional treatment protocols by genotype-based BU dose adjustment. Since BU is mainly metabolized by glutathione S-transferase (GST), we studied the relationship between functional variants within GSTA1, GSTM1 and first dose pharmacokinetics (PK) and outcomes of intravenous (IV) busulfan (Bu). MethodsThe study was performed on 44 thalassemia (thalassemia major = 35 and thalassemia intermediate = 9) pediatric patients (median age = 8, Female = 47.7%) of Middle Eastern origin, who underwent allogeneic HSCT with IV BU as part of a myeloablative-conditioning regimen. According to the standard, 5 different IV Bu doses were given based on weight: 1.0mg/kg for <9 kg, 1.2 mg/kg for 9-16 kg, 1.1 mg/kg for 16-23 kg, 0.95 mg/kg for 23-34 kg, and 0.8 mg/kg for >34 kg. Bu was administered as a 2-hour IV infusion every 6 hours for a total of 16 doses from day -9 to day -6. Bu levels following the first Bu dose were measured using an established analytical method and the PK parameters were estimated using non-compartmental analysis. Bu dose levels were adjusted at dose 4 to achieve Bu Css of 600-900 ng/mL. Genomic DNA collected prior to myeloablation was used for genotyping GSTM1 del by gel electrophoresis and GSTA1promoter variants (-69C>T, -513T>C, -631T>G and -1142) by allele-specific oligonucleotide hybridization. Statistical analysis was performed by using Linear regression to test gene-dosage effect and to assess the effect on genotype on (maximum concentration (Cmax), area under the curve (AUC), Css and Clearance (CL) in a univariate model. The cumulative incidence of aGVHD in relation to GST genotypes was estimated by one-survival curves using Kaplan-Meier analysis and log-rank test. Univariate Cox regression analysis was used to estimate hazard ratio (HR) with 95% confidence interval (CI). ResultsNeutrophil, platelet recoveries, grade 1-4 acute GVHD, grade 2-4 acute GVHD and VOD incidences were 93.2%, 88.6%, 34.1%, 11.4% and 2% respectively. Overall survival rate was 90.9%, events free survival was 84%, while 13.6% of patients had primary and secondary graft failure. Gene-dosage effect between GSTA1 T-69 and G-1142 alleles (defining haplotypes *B and *B1, respectively) and higher Bu exposure (higher Bu levels, p≤ 0.001 and lower clearance, p=0.001) was seen. An association was also seen with GSTM1, where patients with GSTM1 null genotype had a higher occurrence of GVHD grade 1-4 (p=0.01), see Figure 1. [Display omitted] Discussion/ConclusionA strong association between GSTA1*B and BU PK was observed in accordance with the functionality of this variant and our previous analysis in a different patient cohort. We were unable to test whether this variant increases BU related-VOD due to the low VOD cases. GSTA1 and GSTM1 variants could potentially open the possibility to tailor first Bu dose according to patient genetics. Disclosures:No relevant conflicts of interest to declare.

Megaloblastic Anemia and Mitochondriopathy Caused by a Homozygous Mutation in Sideroflexin-4.

AbstractAbstract 79Megaloblastic anemias are characterized by impaired DNA metabolism, often due to deficiencies in vitamin B12 or folate. Genes underlying hereditary forms of megaloblastic anemia not caused by vitamin B12 or folate deficiencies, however, remain largely unknown. Here we characterize a genetic deficiency in a patient with infantile-onset megaloblastic anemia, developmental delays, and a mitochondrial disorder of unknown etiology. Analysis of peripheral blood smears from the patient revealed hypersegmented neutrophils and erythroid macrocytes, classic features of megaloblastic anemias. The patient's vitamin B12 and folate levels are normal, eliminating their deficiency as potential causes of the disease.Whole-exome sequencing of the proband cDNA identified a homozygous, single nucleotide deletion (c.231delC) in Sideroflexin-4 (SFXN4), a predicted mitochondrial multi-spanning transmembrane protein. We experimentally verified the mitochondrial localization of SFXN4 using a combination of western analyses on mitochondrial lysates and confocal fluorescence immunohistochemistry. Using trypsin-sensitivity assays on isolated mitoplasts, we further determined the submitochondrial localization of SFXN4 to the inner mitochondrial membrane.Bioinformatic analyses predict that the mutation introduces a frame shift and a premature stop codon (p.Pro78Leufs*25), resulting in a severely truncated polypeptide. To determine whether the mutant mRNA were expressed in vivo, we used qRT-PCR to assess the steady state level of SFXN4 mRNA in cultured fibroblasts from the proband. qRT-PCR revealed a 92% reduction in SFXN4 expression, consistent with nonsense-mediated decay of the mutant transcript. Genotyping of the index patient and 3 generations of her nuclear family using both Sanger sequencing and allele-specific oligonucleotide hybridization showed that the mutant allele is inherited in an autosomal recessive manner (Fig. A), the result of a presumed founder effect. [Display omitted] We used complementary zebrafish and human fibroblast systems to model the megaloblastic anemia and mitochondrial disease in the patient, respectively. Using splice-blocking antisense morpholino oligomers (MO) targeting sfxn4, we induced a loss-of-function phenotype in zebrafish embryos (hereafter, referred to as “morphants”). qRT-PCR confirmed the efficient knockdown of sfxn4, as morphants have <10% sfxn4 mRNA. Knockdown of sfxn4 in transgenic, Tg(globin LCR:eGFP) zebrafish showed a gross reduction in GFP+ erythrocytes and hemoglobinized cells stained by o-dianisidine (Fig. B, top), while quantification of the red cell population by flow cytometry showed a 60% reduction in the red cell mass. To characterize the anemia, we performed cytospins of flow-sorted erythroid cells from sfxn4 morphants and analyzed their morphology. Wright staining revealed that sfxn4 morphants have red cells with large nuclei containing non-condensed chromatin (Fig. B, bottom), consistent with the features of megaloblastic anemia observed in the index patient. Enumeration of the nuclear: cytoplasmic area ratios showed that red cells from sfxn4 morphants have a nearly 3-fold increase in the ratio of nuclear to cytoplasmic size.We also investigated the mitochondrial disorder using patient fibroblasts, which showed a severe reduction in complex I (37%) and complex I+III (7%) activity. The over-expression of wild-type human SFXN4 in proband fibroblasts completely rescued the respiratory defect of complex I+III, while transfection of the mutant c.231delC SFXN4 construct failed to increase complex I+III activity. In a complementary strategy, the over-expression of wild-type SFXN4 cRNA from either zebrafish or human partially rescues the anemia in morphant embryos, validating their functional orthologous relationship.In summary, a recessive loss-of-function mutation in SFXN4, a previously uncharacterized gene, causes the megaloblastic anemia and mitochondrial disorder described in the index patient. Genetic complementation studies in patient fibroblasts and sfxn4-silenced zebrafish morphants validate the pathogenicity of the mutation. Our findings: (1) demonstrate the requirement of SFXN4 for mitochondrial homeostasis and erythropoiesis, and (2) establish SFXN4 as a new candidate gene for mitochondriopathies and megaloblastic anemias. Disclosures:No relevant conflicts of interest to declare.

Study of KRAS new predictive marker in a clinical laboratory

The presence of somatic mutations in the KRAS gene has been identified as a reliable strong negative predictor for the response to targeting the epidermal growth factor receptor (EGFR), in patients with metastatic colorectal cancer and the use of anti-EGFR monoclonal antibodies such as Cetuximab and Panitumumab is now restricted to patients with no detectable KRAS mutations. Between 30 and 40% of colorectal cancers contain a mutated KRAS oncogene. The aim of this study was to evaluate concordance between three methods to analyze KRAS mutational status in regard to clinical testing. We analyzed KRAS mutations in codons 12 and 13 of exon 2 in one hundred formalin-fixed paraffin-embedded (FFPE) colorectal cancer samples by three different methods: Direct Sequencing and two commercial kits on allele-specific oligonucleotide hybridization (KRAS StripAssay, Vienna Lab.) and Amplification Refractory Mutation System/Scorpions (ARMS/S; TheraScreen KRAS Mutation kit DxS) based on q-PCR. We have found similar frequencies of KRAS mutations by TheraScreen and Strip-Assay (44 and 48%), with a κ value of 0.90, indicating almost perfect agreement between methods. The frequency by direct sequencing was much lower (26%) and the κ values were 0.67 (compared to TheraScreen) and 0.57 (compared to Strip-Assay) indicating low sensitivity. On analyzing KRAS mutation in FFPE tumor samples, direct sequencing sensitivity is too low to be used in a clinical setting. Choosing between ARMS/S; TheraScreen KRAS Mutation kit DxS and KRAS StripAssay, Vienna Lab, will depend on laboratory facilities and expertise.

Increased prevalence of mutant null alleles that cause hereditary fructose intolerance in the American population

Mutations in the aldolase B gene (ALDOB) impairing enzyme activity toward fructose-1-phosphate cleavage cause hereditary fructose intolerance (HFI). Diagnosis of the disease is possible by identifying known mutant ALDOB alleles in suspected patients; however, the frequencies of mutant alleles can differ by population. Here, 153 American HFI patients with 268 independent alleles were analyzed to identify the prevalence of seven known HFI-causing alleles (A149P, A174D, N334K, Delta4E4, R59Op, A337V, and L256P) in this population. Allele-specific oligonucleotide hybridization analysis was performed on polymerase chain reaction (PCR)-amplified genomic DNA from these patients. In the American population, the missense mutations A149P and A174D are the two most common alleles, with frequencies of 44% and 9%, respectively. In addition, the nonsense mutations Delta4E4 and R59Op are the next most common alleles, with each having a frequency of 4%. Together, the frequencies of all seven alleles make up 65% of HFI-causing alleles in this population. Worldwide, these same alleles make up 82% of HFI-causing mutations. This difference indicates that screening for common HFI alleles is more difficult in the American population. Nevertheless, a genetic screen for diagnosing HFI in America can be improved by including all seven alleles studied here. Lastly, identification of HFI patients presenting with classic symptoms and who have homozygous null genotypes indicates that aldolase B is not required for proper development or metabolic maintenance.

Isoelectrofocusing and PCR Amplification-Reverse Hybridization Assay in Evaluation of Alpha-1-Antitrypsin Deficiency

Isoelectrofocusing and PCR Amplification-Reverse Hybridization Assay in Evaluation of Alpha-1-Antitrypsin DeficiencyAlpha-1-antitrypsin deficiency is a potentially lethal genetic disorder, which has pulmonary and liver manifestations. The standardized biochemical and molecular diagnostic protocol for detection of clinically relevant alleles is needed. The paper summarizes current concepts about AATD, describes the potentials of isoelectric focusing and PCR amplification-reverse allele specific oligonucleotide hybridization assay in the detection of affected individuals and shortly presents our experiences in the evaluation of AATD. We conclude that the systematic clinical laboratory approach to AATD might be based on the combination of mentioned methods, coordinated by alpha-1-antritrypsin quantification. Additionally, its complete medical implementation is achieved through teamwork between clinical chemists, molecular biologists and clinicians.

Semi-quantitative discrimination of HBV mutants using allele-specific oligonucleotide hybridization with Handy Bio-Strand

The analysis of hepatitis B virus (HBV) mutations is important for understanding HBV progression and for deciding on appropriate clinical treatments. However, it is difficult to determine the quantitative abundance of various mutants in heterogeneous mixtures by conventional methods such as direct sequencing or the TaqMan assay. In this study, we investigated the possibility of using both allele-specific oligonucleotide hybridization (ASOH) and allele-specific oligonucleotide competitive hybridization (ASOCH) with the Handy Bio-Strand system for the quantitative identification of three well-defined HBV variants: the basal core promoter (BCP) mutations (nt1762 and nt1764), the pre-core (PC) mutation (nt1896), and variance at nt1858. Using standardized mixtures of wild-type and mutant DNA, optimal hybridization conditions for ASOH and ASOCH were determined. Next, the performance of these methods was evaluated using actual serum DNAs from HBV patients. Excellent reproducibility was obtained both in the analysis of internal positive controls and in the semi-quantitative categorization of heterogeneous viral mixtures into five abundance groups (0%, 25%, 50%, 75%, and 100% mutant virus). Combined with real-time PCR to determine the HBV viral load, this hybridization method offers a new tool with applications both in HBV clinical research and treatment.

Multiplex genotyping and allele frequency estimation in pooled DNAs using non-gel capillary electrophoresis

We present a novel approach of single-nucleotide polymorphism (SNP) analysis in which allele-specific oligonucleotide hybridization is followed by non-gel capillary electrophoresis (ASOH–NGCE) in conjunction with laser-induced fluorescence (LIF). This allows rapid multiplex allelotyping and allele frequency estimation. This method, based on site separation of the hybridization duplexes, retains the simplicity and specificity of ASOH and the homogeneous feature of NGCE with poly( N,N-dimethylacrylamide) (PDMA) as a sieving medium. ASOH–NGCE can be applied to multiplex SNP loci genotyping with excellent separation of hybridization mixtures. Average relative standard deviations (RSDs) were low for within-day (1.10%) and between-day (2.41%) reproducibility. Moreover, the allele frequencies in pooled DNAs were accurately determined from peak areas and equilibrium dissociation constants. Our method was highly sensitive in detecting alleles with frequency as low as 1% and in distinguishing allele frequencies differing by 1% between pools. The average value of differences between real and estimated frequencies (accuracy) was only 0.004.

Typing of 48 autosomal SNPs and amelogenin with GenPlex SNP genotyping system in forensic genetics

GenPlex (Applied Biosystems) is a new SNP genotyping system based on an initial PCR amplification followed by an oligo ligation assay (OLA). The OLA consists of the hybridization of allele and locus specific oligonucleotides (ASOs and LSOs) to PCR products and posterior ligation of ASOs and LSOs. The ligation products are immobilized to microtitre plates and reporter oligonucleotides (ZipChute ® probes) are hybridized to the ligation products. ZipChute ® probes are subsequently eluted and detected using capillary electrophoresis. Applied Biosystems developed the GenPlex SNP genotyping system with amelogenin and 48 of the 52 SNPs used in the 52 SNP-plex assay developed by the SNP forID consortium. The system requires equipment that is usually found in forensic genetic laboratories. The use of a robot for performance of the pipetting steps is highly recommendable. A total of 286 individuals from Denmark, Somalia and Greenland were investigated with GenPlex using a Biomek ® 3000 (Beckman Coulter) robot. The results were compared to results obtained with an ISO 17025 accredited SNP typing assay based on single base extension (SBE). With the GenPlex SNP genotyping system, full SNP profiles were obtained in 97.6% of the investigations. Perfect concordance was obtained in duplicate investigations and the SNP genotypes obtained with the GenPlex system were concordant with those of the accredited SBE based SNP typing system except for one result in rs901398 in one of 286 individuals most likely due to a mutation 6 bp downstream of the SNP. Reproducible SNP genotypes were obtained from as little as 250 pg of DNA.

Glypican-3 is overexpressed in lung squamous cell carcinoma, but not in adenocarcinoma

Glypican-3 is a membrane-bound proteoglycan whose expression has been linked to malignancies through the existence of both mutations and aberrant protein expression. Reports on glypican-3 expression in lung cancer were limited, with some evidence for loss of expression, which suggested a tumor-suppressor role. We sought to evaluate glypican-3 expression in lung cancer at the protein and mRNA levels and correlate it with clinical, histological and genomic characteristics such as RAS mutation status. We used immunohistochemistry on tissue microarray to study glypican-3 expression in 97 patients, evaluated glypican-3 mRNA levels by quantitative polymerase chain reaction in 143 patients and identified RAS mutations by allele-specific oligonucleotide hybridization. We correlated the results with clinical and histological data. Glypican-3 immunostaining was negative in all normal lung tissues, but positive in 23% of lung carcinoma samples. High protein and mRNA expression was associated with squamous histology (positive stain in 55% of squamous cell carcinoma vs 8% of adenocarcinoma, P<0.0001 for both immunostaining and mRNA). RAS mutations were highly associated with adenocarcinoma and low glypican-3 mRNA expression (P<0.0001 for both). Among smokers, glypican-3 mRNA expression was reduced in adenocarcinoma patients (P=0.013), and was elevated in those with squamous cell carcinoma (P=0.03, interaction P=0.0009). These opposing associations also correlated with the smoking burden. Patients with tumors staining positively for glypican-3 smoked significantly more than patients with tumors staining negatively (P=0.013). No association was found between glypican-3 expression and patient outcome. In conclusion, glypican-3 was overexpressed in cancerous compared with normal lung tissue. Adenocarcinoma and squamous cell carcinoma had differential expression of glypican-3, with predilection to squamous cell carcinoma patients who smoked. Glypican-3 expression in squamous cell carcinoma as an oncofetal protein renders it a potential candidate marker for early detection of lung squamous cell carcinoma.

Molecular analysis of HLA-DQ A alleles in coeliac disease lack of a unique disease-associated sequence.

Susceptibility to coeliac disease is strongly associated with some HLA class II antigens, encoded by the HLA-D region. Since the HLA-DQ locus seems to be primarily involved, we have analysed by polymerase chain reaction amplification and allele-specific oligonucleotide hybridization the most polymorphic region of the HLA-DQ A1 gene. No difference was observed between the 20 coeliac patients and 20 HLA-D-matched healthy controls who took part in the study. Furthermore, in patients and controls, the restriction fragment length polymorphism analysis of the HLA-DQ A gene using the restriction enzyme BglII did not disclose any specific disease-associated fragment. Our results are not consistent with a unique DQ A coeliac disease-associated sequence, but rather with the hypothesis that some polymorphic residues or allelic hypervariable regions, although found also in the normal population, can predispose to coeliac disease due to their higher frequency in this condition.

The molecular heterogeneity of β-thalassemia in Greece

β-thalassemia is the most predominant genetic defect in Greece. In this study, we investigated the heterogeneity and the frequency of β-thalassemia mutations among 3796 heterozygotes detected in the course of DNA based diagnoses. The diagnostic strategy included Denaturing Gradient Gel Electrophoresis (DGGE), Allele Specific Oligonucleotide Hybridization (ASO), GAP PCR, Restriction Enzyme (RE) analysis and direct sequencing and led to 100% identification of the underlying molecular lesion. Six out of 33 different β-globin defects identified accounted for more than 91.4% of the total β-thalassemia chromosomes in Greece. The β-globin gene mutations cd29 C→T, IVS-I-2 T→C, IVS-I-5 G→T, cd37 G→A and poly A Kurdish AATAAA→AATAAG are for the first time reported in Greece, whereas cd7 GAG→TAG is a new β 0-thalassemia mutation detected in an adult man from Albania residing in Greece. Three DNA single nucleotide polymorphisms (IVS-I-85 T→C, IVS-I-91 C→T and IVS-I-108 T→C) were also revealed; among these, IVS-I-85 T→C and IVS-I-91 C→T are new and described for the first time worldwide.

Downregulation of PAR‐4, a pro‐apoptotic gene, in pancreatic tumors harboring K‐ras mutation

Oncogenic ras is known to inhibit cell death and growth inhibitory genes and activate prosurvival genes. Proapoptotic gene PAR-4, has been found to be downregulated by oncogenic ras. Since pancreatic tumors harbor a high incidence of K-ras point mutations, we hypothesized that oncogenic K-ras might influence the function and expression of PAR-4. PAR-4 expression levels were analyzed in 4 established pancreatic tumor cell lines, 10 normal pancreatic tissues, 44 frozen tumor tissues and 25 paraffin-embedded pancreatic adenocarcinoma samples by Real Time RT-PCR, Western blot analysis and immunohistochemistry. K-ras mutational status was analyzed by allele-specific oligonucleotide-hybridization. Expression levels of PAR-4 were correlated with the K-ras mutational status and clinical characteristics. Further, modulation of endogenous PAR-4 was tested by transiently expressing oncogenic ras in a wild-type K-ras pancreatic cancer cell line, BxPC-3. Three cell lines with K-ras mutations showed low levels of PAR-4 when compared to a normal pancreatic tissue. Of 44 frozen tumors, 16 showed appreciable upregulation of Par mRNA and 27 showed significant downregulation of PAR-4 mRNA when compared to normal pancreatic tissue and 1 had levels equivalent to normal pancreatic tissue. Of 25 paraffin-embedded tumors, 9 showed downregulation of PAR-4 protein and this downregulation of PAR-4 correlated significantly with K-ras mutational status (p < 0.00002). In addition, the presence of PAR-4 mRNA or protein expression in pancreatic tumors correlated with prolonged survival. Transient overexpression of oncogenic ras in wild-type K-ras BxPC-3 cells significantly downregulated the endogenous PAR-4 protein levels and conferred accelerated growth. Thus, downregulation or loss of PAR-4 expression by oncogenic ras may provide a selective survival advantage for pancreatic tumors, through inhibition of proapoptotic pathway mediated by PAR-4.