Denaturing High-performance Liquid Chromatography Research Articles

The impact of sensitive KIT D816V detection on recognition of Indolent Systemic Mastocytosis

Patients with Systemic Mastocytosis (SM) need a highly sensitive diagnostic test for D816V detection of the KIT receptor gene.Along with histology/cytology and flow cytometry evaluation, bone marrow (BM) from 110 consecutive adult patients referred with a suspicion of SM to Multidisciplinary Outpatient Clinic for Mastocytosis in Verona were tested both by Amplification Refractory Mutation System Reverse Transcriptase quantitative real time Polymerase Chain Reaction (ARMS-RT-qPCR) and RT-PCR+Restriction Fragment Length Polymorphism (RFLP) followed by Denaturing-High Performance Liquid Chromatography (D-HPLC) and Sanger sequencing.ARMS-RT-qPCR identified D816V mutation in 77 patients, corresponding to 100% of cases showing CD25+ mast cells (MCs) whereas RT-PCR+RFLP/D-HPLC+sequencing revealed D816V mutations in 47 patients.According to the 2008 WHO criteria 75 SM, 1 Cutaneous Mastocytosis (CM), 1 monoclonal MC activation syndrome (MMAS), and 1 SM Associated with Haematologic Non-Mast Cell Disorder (SM-AHNMD) were diagnosed. Seventeen out 75 SM patients (23%) would have not satisfied sufficient WHO criteria on the basis of the sole RT-PCR+RFLP: these patients had significantly lower serum tryptase levels and amount of CD25+ MCs.Therefore, ARMS-RT-qPCR might result particularly useful, in patients that do not fulfil major BM histological criterion, for the recognition of indolent SM with a very low MC burden.

Mycophenolic acid AUC in Thai kidney transplant recipients receiving low dose mycophenolate and its association with UGT2B7 polymorphisms.

BackgroundDespite use of a lower mycophenolate dose in Thai kidney transplant patients, acceptable graft and patient outcomes can be achieved. We therefore examined the pharmacokinetics of mycophenolic acid (MPA) by area under the curve (AUC) and investigated genetic contribution in mycophenolate metabolism in this population.MethodsKidney transplant recipients with stable graft function who were receiving mycophenolate mofetil 1,000 mg/d in combination with either cyclosporine or tacrolimus, and prednisolone were studied. The MPA concentration was measured by fluorescence polarization immunoassay (FPIA), at predose and 1, 1.5, 2, 4, 6, 8, 10, and 12 hours after dosing. Genetic polymorphisms in UGT1A8, UGT1A9, and UGT2B7 were examined by denaturing high-performance liquid chromatography (DHPLC)-based single-base extension (SBE) analysis.ResultsA total 138 patients were included in study. The mean AUC was 39.49 mg-h/L (28.39–89.58 mg-h/L), which was in the therapeutic range. The correlation between the predose MPA concentration and AUC was poor. The mean AUC in the tacrolimus group was higher than that in the cyclosporine group. Polymorphisms in UGT2B7 showed significant association with AUC.ConclusionMost of our patients with reduced mycophenolate dose had the AUC within the therapeutic range. Genetic polymorphisms in UGT2B7 may play a role in MPA metabolism in Thai kidney transplant patients.

Quantification and dynamic monitoring of EGFR T790M in plasma cell-free DNA by digital PCR for prognosis of EGFR-TKI treatment in advanced NSCLC.

BackgroundAmong advanced non-small cell lung cancer (NSCLC) patients with an acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI), about 50% carry the T790M mutation, but this frequency in EGFR-TKI-naïve patients and dynamic change during therapy remains unclear. This study investigated the quantification and dynamic change of T790M mutation in plasma cell-free DNA (cf-DNA) of advanced NSCLC patients to assess the clinical outcomes of EGFR-TKI therapy.Materials and MethodsWe retrospectively investigated 135 patients with advanced NSCLC who obtained progression-free survival (PFS) after EGFR-TKI for >6 months for their EGFR sensitive mutations and T790M mutation in matched pre- and post-TKI plasma samples, using denaturing high-performance liquid chromatography (DHPLC), amplification refractory mutation system (ARMS), and digital-PCR (D-PCR). Real-time PCR was performed to measure c-MET amplification.ResultsDetection limit of D-PCR in assessing the T790M mutation was approximately 0.03%. D-PCR identified higher frequency of T790M than ARMS in pre-TKI (31.3% vs. 5.5%) and post-TKI (43.0% vs. 25.2%) plasma samples. Patients with pre-TKI T790M showed inferior PFS (8.9 vs. 12.1 months, p = 0.007) and overall survival (OS, 19.3 vs. 31.9 months, p = 0.001) compared with those without T790M. In patients harboring EGFR sensitive mutation, high quantities of pre-TKI T790M predicted poorer PFS (p = 0.001) on EGFR-TKI than low ones. Moreover, patients who experienced increased quantity of T790M during EGFR-TKI treatment showed superior PFS and OS compared with those with decreased changes (p = 0.044 and p = 0.015, respectively).ConclusionQualitative and quantitative T790M in plasma cf-DNA by D-PCR provided a non-invasive and sensitive assay to predict EGFR-TKI prognosis.

Relation of polymorphisms of the XPD and GSTM1 genes with susceptibility to hepatocellular carcinoma in Qinghai Tibetans

To explore the relationship of polymorphisms in the xeroderma pigmentosum group D (XPD) gene and the glutathione-S transferees M1 (GSTM1) gene with susceptibility to primary hepatic carcinoma (PHC) in Tibetans from the Qinghai region. This case-control study compared equal groups (n=102 each) of patients with PHC and healthy individuals recruited from Qinghai, Tibet.PCR and denaturing high-performance liquid chromatography (DHPLC) was used to detect each participant's genotypes for the XPD and GSTM1 genes.Non-conditional logistic regression modeling was used in multivariate analysis to evaluate the predictive value for PHC, to compare the risk of different genotypes for PHC, and to assess the risk of gene polymorphisms and environmental factors for PHC. Six factors, including smoking, carnivorous diet, alcohol consumption, hepatitis B virus (HBV) infection, immediate family members with HBV infection and immediate family members with history of PHC, were included in the logistic regression model (alpha =0.05).The XPD751C mutation genotype distribution frequencies were significantly higher in the cases than in the controls (21.6% vs. 10.8%, P=0.036). The risk of PHC increased 2.275 times (95% CI, 1.04-4.98). The frequencies of the GSTM1 genotype were remarkably higher in the cases than in the controls (60.4% vs. 39.6%, P=0.017), suggesting this as an exposure factor. Individuals with the GSTM1 genotype had 1.963 times higher risk of PHC than individuals without the GSTM1 genotype (95% CI, 1.124-3.428). With both the XPD751C mutation and the GSTM1 genotype as exposure factors, the risk incidence increased to 3.030 times (95% CI, 1.165-7.881), indicating that the combined genotypes have a synergistic effect.Application of unconditioned logistic stepwise regression analysis of the genotypes and environmental risk factors showed an interaction between the XPD751C mutation and HBV infection, alcohol consumption and immediate family members with history of PHC. In addition, an interaction between the GSTM1 genotype and HBV infection was found. Alcohol consumption, HBV infection and the presence of immediate family members with HBV infection are the main environmental risk factors of PHC in Qinghai Tibetans.Qinghai Tibetans who carry the XPD751C gene mutation and the GSTM 1 genotype are at increased risk of PHC, compared to individuals carrying only one or the other.The XPD751C mutation may increase risk of PHC when combined with the environmental factors.

Screening of Duchenne muscular dystrophy (DMD) mutations and investigating its mutational mechanism in Chinese patients.

Duchenne muscular dystrophy (DMD) is a common X-linked recessive disease of muscle degeneration and death. In order to provide accurate and reliable genetic counseling and prenatal diagnosis, we screened DMD mutations in a cohort of 119 Chinese patients using multiplex ligation-dependent probe amplification (MLPA) and denaturing high performance liquid chromatography (DHPLC) followed by Sanger sequencing. In these unrelated DMD patients, we identified 11 patients with DMD small mutations (9.2%) and 81 patients with DMD deletions/duplications (del/dup) (68.1%), of which 64 (79.0%) were deletions, 16 (19.8%) were duplications, and one (1.2%) was both deletion and duplication. Furthermore, we analyzed the frequency of DMD breakpoint in the 64 deletion cases by calculating exon-deletion events of certain exon interval that revealed a novel mutation hotspot boundary. To explore why DMD rearrangement breakpoints were predisposed to specific regions (hotspot), we precisely characterized junction sequences of breakpoints at the nucleotide level in 21 patients with exon deleted/duplicated in DMD with a high-resolution SNP microarray assay. There were no exactly recurrent breakpoints and there was also no significant difference between single-exon del/dup and multiple-exon del/dup cases. The data from the current study provided a comprehensive strategy to detect DMD mutations for clinical practice, and identified two deletion hotspots at exon 43–55 and exon 10–23 by calculating exon-deletion events of certain exon interval. Furthermore, this is the first study to characterize DMD breakpoint at the nucleotide level in a Chinese population. Our observations provide better understanding of the mechanism for DMD gene rearrangements.

Molecular Characterization of Glucose-6-Phosphate Dehydrogenase Deficiency in Abu Dhabi District, United Arab Emirates

Purpose: To investigate the frequency of glucose-6-phosphate dehydrogenase (G6PD) variants and their associated enzyme deficiencies among different age groups of individuals in Abu Dhabi, United Arab Emirates (UAE).Methods: A total of 15,995 patients (6302 UAE nationals and 9693 non-UAE nationals) who presented at Mafraq Hospital, Abu Dhabi, UAE between January 2006 and January 2009 were screened for G6PD deficiency using fluorescent spot test. Molecular analysis including polymerase chain reaction– restriction fragment length polymorphism (PCR-RFLP), denaturing high performance liquid chromatography (DHPLC) and DNA sequencing were utilized to identify common mutations in individuals with G6PD deficiency.Results: The prevalence of G6PD deficiency among UAE nationals was 7.4% and non-UAE nationals 3.8%. UAE males showed prevalence of 11.6% while for UAE females it was 3.6%. The prevalence of G6PD deficiency among non-UAE nationals was 5 and 1.7% for males and females, respectively. The Mediterranean mutation, 563C→T, was predominant in non-UAE nationals.Conclusion: G6PD Mediterranean mutation is the most prevalent mutation underlying G6PD deficiency followed by Aures mutations in both UAE nationals and non-UAE nationals. On the other hand, Africa Awas found to be more in non-UAE compared with UAE nationals.Keywords: Glucose-6-Phosphate Dehydrogenase deficiency, Mutation, Abu Dhabi, Polymerase chain reaction–restriction, Fragment length polymorphism

A novel mutation in GCH1 gene causes dopa-responsive dystonia

To identify potential mutation of the GCH1 gene in a Chinese family affected with dopa-responsive dystonia. Genomic DNA of patients was extracted from peripheral blood samples. The 6 exons of the GCH1 gene and at least 100 bp of flanking intronic sequences were amplified with PCR. Potential mutations were screened by direct sequencing. Identified mutation was verified with denaturing high performance liquid chromatography (DHPLC) in 100 healthy controls. All patients were found to be heterozygous for a novel c.597delT (p.Ala200LeufsX5) deletion in the exon 5 of the GCH1 gene. The deletion of T has resulted in formation of a shorter (203 amino acids) truncated non-functional guanosine triphosphate cyclohydrolase I. The same mutation was not found in the 100 controls. A novel GCH1 gene frameshifing mutation probably underlies the dopa-responsive dystonia in this Chinese family.

Occurrence of low frequency PIK3CA and AKT2 mutations in gastric cancer

The PI3K/AKT signal transduction pathway has distinct functional roles in tumor progression. PIK3CA was reported to harbor the hot-spot in many types of tumor. Akt, the downstream of PI3K, its family members especially AKT2 activation in human cancer has been extensively studied, but its activation by mutation was less reported. The occurrence of PIK3CA and AKT2 mutations in a variety of cancers indicates their important involvement in carcinogenesis. Therefore, we investigated their mutation frequencies in gastric cancer (GC) in China. In our study, we selected hot-spot related exons 9, 18 and 20 of PIK3CA and kinase domain exons 6–14 of AKT2 genes were screened in 10 GC cell lines, 100 advanced primary GC and matched normal tissues. Denaturing high performance liquid chromatography (DHPLC) and DNA sequencing were used to analyze the mutations in the two genes. Two point mutations in the PIK3CA gene were identified in 4 of 10 GC cell lines and in 4 of 100 GC primary tumors. Two polymorphisms in AKT2 were detected in 19 of 100 GC primary tumors. One point mutation in AKT2 was detected in 1 of 10 GC cell lines and 3 of 100 GC primary tumors but no hot spot variation was detected. Our results indicate that PIK3CA and AKT2 mutations occurred at low frequency in GC, and suggest that the PIK3CA/AKT2 pathway might engage other events during gastric carcinogenesis.

Genetype distribution of mycoplasma pneumoniae by denaturing high-performance liquid chromatography

Objective To investigate the genetype distribution of mycoplasma pneumoniae (MP) by denaturing high-performance liquid chromatography (DHPLC). Methods A total of 300 cases nasopharyngeal aspirate were collected from our hospital. The MP genes of standard strains and clinical specimens isolates were amplified by PCR followed by DHPLC and genetype determination. Results A total of 110 cases were positive after 24 hours fermentation from 300 cases with pharyngeal swab. By the specific primers of MP-129, MP-FH standard strain and specimens, 2 280 bp and 2 580 bp gene fragments were made out respectively. One hundred and ten strains of clinical isolates were detected by DHPLC. One hundred and seven strains of P1-Ⅰ were 1b subtype, 3 were type P1-Ⅱ which were all 2a subtype. Conclusion The genetype of MP infection in children from our hospital is P1-Ⅰ, 1b subtype by using DHPLC technology. Key words: Denaturing high-performance liquid chromatography; Mycoplasma pneumoniae; Genetype

Discordant diagnoses obtained by different approaches in antithrombin mutation analysis

ObjectivesIn hereditary antithrombin (AT) deficiency it is important to determine the underlying mutation since the future risk of thromboembolism varies considerably between mutations. DNA investigations are in general thought of as flawless and irrevocable, but the diagnostic approach can be critical. We therefore investigated mutation results in the AT gene, SERPINC1, with two different approaches. Design and methodsSixteen patients referred to the Centre for Thrombosis and Haemostasis, Odense University Hospital, with biochemical indications of AT deficiency, but with a negative denaturing high-performance liquid chromatography (DHPLC) mutation screening (routine approach until recently) were included. As an alternative mutation analysis, direct sequencing of all exons and exon–intron boundaries without pre-selection by DHPLC was performed. ResultsOut of sixteen patients with a negative DHPLC mutation screening, discordant results were found in ten patients (62.5%) when using direct sequencing: Eight had the Basel mutation (c.218C>T), while two had the Cambridge II mutation (c.1246G>T). For seven of the ten patients this meant an altered clinical risk-assessment for future thromboses. ConclusionsAwareness must be drawn to the possibility of differences in DNA diagnostics in general and advances when using newer techniques in particular. One should consider re-analysis of results obtained by earlier sequencing strategies, as clinically important information can be overlooked.

Distinct susceptibility of induction of methylation of p16ink4a and p19arf CpG islands by X-radiation and chemical carcinogen in mice

Inactivation of the tumor suppressor genes p16ink4a and p19arf/p14arf by hypermethylation of promoter CpG islands occurs frequently in various tumors. The aim of this study is to investigate the difference of susceptibility of methylation induced by carcinogens between p16ink4a and p19arf. The methylation status of both genes was analyzed by denaturing high performance liquid chromatography (DHPLC) and bisulfite-sequencing, respectively. The expression level of P16 protein was analyzed by immunohistochemistry. Results showed that p16ink4a methylation was detected in the glandular stomach, small intestine and other organs of mice following X-radiation and subsequent bone marrow transplantation (BMT), but not in mock control mice. We found that the intestinal tract was the most sensitive organ for X-ray induced p16ink4a methylation. Loss of P16 protein expression was observed in the intestinal tissues of X-irradiated mice, but not in the mock control mice. Interestingly, p19arf methylation was not observed in the gastrointestinal tissues of the negative control mice following X-radiation/BMT. However, administration of N-nitrosomethylurea and/or Helicobacter felis infection promoted methylation of p19arf CpG islands in the gastrointestinal tracts, but did not promote p16ink4a methylation. In addition, p16ink4a methylation was detected not only in the X-irradiated GFP-negative tissue cells, but also in the GFP-positive bone marrow-derived cells that were transplanted into the BMT mice after X-radiation. In conclusion, the methylation susceptibility of p16ink4a and p19arf to carcinogen treatments was remarkably different: X-radiation indirectly induces systemic p16ink4a methylation, especially in the intestine; whereas N-nitrosomethylurea and/or H. felis infection induce p19arf methylation in their target organs.

Analysis and carrier screening for copy numbers of SMN and NAIP genes in children with spinal muscular atrophy

To assess the association of copy number variations of SMN1, SMN2, NAIP, GTF2H2 and H4F5 genes with clinical classification of spinal muscular atrophy in children, and determine the copy number of the SMN gene among pregnant women. A carrier screening was also performed in Sichuan province. The copy number variations of the above genes among 53 confirmed SMA patients were determined with MLPA technique. The copy number variations were analyzed by the Fisher's exact test. Deletion of exon 7 in the SMN1 gene was screened with denaturing high performance liquid chromatography (DHPLC) for 427 pregnant women. Among the 53 cases of type I, II, and III SMA patients, the rate of homozygous deletion of both exons 7 and 8 of the SMN1 gene were 100%, 94.44% and 87.50%, respectively, whereas those of homozygous deletion of exon 7 of SMN1 gene were 0, 5.56%, and 12.50%, respectively. The patients with 1, 2, 3, and 4 copies of exon 7 of the SMN2 gene were 11.32%, 67.92%, 13.21% and 7.55%, respectively. The patients with 0, 1, and 2 copies of exon 5 of NAIP gene were 11.32%, 62.26%, and 26.42%, respectively. No deletion was detected in GTF2H2 or H4F5 genes. The heterozygous loss rate of exon 7 in SMN gene in the pregnant women population of Sichuan region was approximately 2.11%. Copy number variations of SMN2 and NAIP genes in patients are related to SMA clinical types (P < 0.05). In contrast, there was no relationship between SMA clinical types and deletion of exons 7 and 8 in the SMN1 gene (P > 0.05). Analysis of copy number change in SMN1 gene can assist SMA carrier screening. However, when the general population without SMA family history is screened for disease-causing genes, it should be noted that the type "2+0" carriers may affect the screening result, and the result should be interpreted with caution.

Distinct polymicrobial populations in a chronic foot ulcer with implications for diagnostics and anti-infective therapy

BackgroundPolymicrobial infections caused by combinations of different bacteria are being detected with an increasing frequency. The evidence of such complex infections is being revealed through the use of novel molecular and culture-independent methods. Considerable progress has been made in the last decade regarding the diagnostic application of such molecular techniques. In particular, 16S rDNA-based sequencing and even metagenomic analyses have been successfully used to study the microbial diversity in ecosystems and human microbiota. Here, we utilized denaturing high-performance liquid chromatography (DHPLC) as a diagnostic tool for identifying different bacterial species in complex clinical samples of a patient with a chronic foot ulcer.Case presentationA 45-year-old female suffered from a chronic 5x5cm large plantar ulcer located in the posterior calcaneal area with subcutaneous tissue infection and osteomyelitis. The chronic ulcer developed over a period of 8 years. Culture and DHPLC revealed a distinct and location-dependent polymicrobial infection of the ulcer. The analysis of a superficial biopsy revealed a mixture of Staphylococcus aureus, Proteus vulgaris, and Fusobacterium nucleatum, whereas the tissue-deep biopsy harbored a mixture of four different bacterial species, namely Gemella morbillorum, Porphyromonas asaccharolytica, Bacteroides fragilis, and Arcanobacterium haemolyticum.ConclusionsThis clinical case highlights the difficulties in assessing polymicrobial infections where a mixture of fastidious, rapid and slow growing bacteria as well as anaerobes exists as structured communities within the tissue architecture of chronic wound infections. The diagnosis of this multilayered polymicrobial infection led to a microbe-adapted antibiotic therapy, targeting the polymicrobial nature of this infection in addition to a standard local wound treatment. However, a complete wound closure could not be achieved due to the long-lasting extensive destruction of tissue.

Interference of Co-Amplified Nuclear Mitochondrial DNA Sequences on the Determination of Human mtDNA Heteroplasmy by Using the SURVEYOR Nuclease and the WAVE HS System

High-sensitivity and high-throughput mutation detection techniques are useful for screening the homoplasmy or heteroplasmy status of mitochondrial DNA (mtDNA), but might be susceptible to interference from nuclear mitochondrial DNA sequences (NUMTs) co-amplified during polymerase chain reaction (PCR). In this study, we first evaluated the platform of SURVEYOR Nuclease digestion of heteroduplexed DNA followed by the detection of cleaved DNA by using the WAVE HS System (SN/WAVE-HS) for detecting human mtDNA variants and found that its performance was slightly better than that of denaturing high-performance liquid chromatography (DHPLC). The potential interference from co-amplified NUMTs on screening mtDNA heteroplasmy when using these 2 highly sensitive techniques was further examined by using 2 published primer sets containing a total of 65 primer pairs, which were originally designed to be used with one of the 2 techniques. We confirmed that 24 primer pairs could amplify NUMTs by conducting bioinformatic analysis and PCR with the DNA from 143B-ρ0 cells. Using mtDNA extracted from the mitochondria of human 143B cells and a cybrid line with the nuclear background of 143B-ρ0 cells, we demonstrated that NUMTs could affect the patterns of chromatograms for cell DNA during SN-WAVE/HS analysis of mtDNA, leading to incorrect judgment of mtDNA homoplasmy or heteroplasmy status. However, we observed such interference only in 2 of 24 primer pairs selected, and did not observe such effects during DHPLC analysis. These results indicate that NUMTs can affect the screening of low-level mtDNA variants, but it might not be predicted by bioinformatic analysis or the amplification of DNA from 143B-ρ0 cells. Therefore, using purified mtDNA from cultured cells with proven purity to evaluate the effects of NUMTs from a primer pair on mtDNA detection by using PCR-based high-sensitivity methods prior to the use of a primer pair in real studies would be a more practical strategy.

Use of denaturing high-performance liquid chromatography (DHPLC) to characterize the bacterial and fungal airway microbiota of cystic fibrosis patients

The aim of this study was to evaluate the use of denaturing high-performance liquid chromatography (DHPLC) to characterize cystic fibrosis (CF) airway microbiota including both bacteria and fungi. DHPLC conditions were first optimized using a mixture of V6, V7 and V8 region 16S rRNA gene PCR amplicons from 18 bacterial species commonly found in CF patients. Then, the microbial diversity of 4 sputum samples from 4 CF patients was analyzed using cultural methods, cloning/sequencing (for bacteria only) and DHPLC peak fraction collection/sequencing. DHPLC analysis allowed identifying more bacterial and fungal species than the classical culture methods, including well-recognized pathogens such as Pseudomonas aeruginosa. Even if a lower number of bacterial Operational Taxonomic Units (OTUs) was identified by DHPLC, it allowed to find OTUs unidentified by cloning/sequencing. The combination of both techniques permitted to correlate the majority of DHPLC peaks to defined OTUs. Finally, although Aspergillus fumigatus detection using DHPLC can still be improved, this technique clearly allowed to identify a higher number of fungal species versus classical culture-based methods. To conclude, DHPLC provided meaningful additional data concerning pathogenic bacteria and fungi as well as fastidious microorganisms present within the CF respiratory tract. DHPLC can be considered as a complementary technique to culture-dependent analyses in routine microbiological laboratories.

α-Internexin: A Novel Biomarker for Pancreatic Neuroendocrine Tumor Aggressiveness

We aimed to test whether α-internexin could be a molecular biomarker of tumor aggressiveness and prognosis in pancreatic neuroendocrine tumors (PNETs). Using immunohistochemical staining and Western blot, we detected the expression of α-internexin in 350 tumors from 343 patients, of whom 257 were followed up. Methylation of α-internexin promoter was examined by bisulfite sequencing to identify the crucial region that determines gene expression. Methylation of gene promoter in tumors was quantitatively measured by denaturing high performance liquid chromatography (DHPLC). We correlated α-internexin expression with clinicopathological characteristics. α-Internexin was expressed in 53% of 350 PNETs. The reduced expression of α-internexin was significantly associated with advanced stage (P < .0001), metastases (P < .0001), and recurrence (P = .003). α-Internexin expression was found in 57.1% of 212 surviving patients and in 17.1% of 35 deceased patients (P < .0001). Reduced expression of α-internexin was associated with shorter overall survival in PNET patients (log rank P < .0001) as well as in patients with noninsulinoma and nonfunctional (NF)-PNETs (log rank P = 0.0073 and P = 0.010, respectively). The crucial region of α-internexin promoter (-149 to +96 nucleotides [nt]) was identified, and the hypomethylation of this area in PNETs was significantly associated with gene expression (P = .015). α-Internexin can be a useful prognostic biomarker for PNETs.

Efficient detection of factor IX mutations by denaturing high-performance liquid chromatography in Taiwanese hemophilia B patients, and the identification of two novel mutations

Hemophilia B (HB) is an X-linked recessive disorder characterized by mutations in the clotting factor IX (FIX) gene that result in FIX deficiency. Previous studies have shown a wide variation of FIX gene mutations in HB. Although the quality of life in HB has greatly improved mainly because of prophylactic replacement therapy with FIX concentrates, there exists a significant burden on affected families and the medical care system. Accurate detection of FIX gene mutations is critical for genetic counseling and disease prevention in HB. In this study, we used denaturing high-performance liquid chromatography (DHPLC), which has proved to be a highly informative and practical means of detecting mutations, for the molecular diagnosis of our patients with HB. Ten Taiwanese families affected by HB were enrolled. We used the DHPLC technique followed by direct sequencing of suspected segments to detect FIX gene mutations. In all, 11 FIX gene mutations (8 point mutations, 2 small deletions/insertions, and 1 large deletion), including two novel mutations (exon6 c.687–695, del 9 mer and c.460–461, ins T) were found. According to the HB pedigrees, 25% and 75% of our patients were defined as familial and sporadic HB cases, respectively. We show that DHPLC is a highly sensitive and cost-effective method for FIX gene analysis and can be used as a convenient system for disease prevention.

Use of PCR-DHPLC with fluorescence detection for the characterization of the bacterial diversity during cassava (Manihot esculenta crantz) fermentation

Denaturing high-performance liquid chromatography (DHPLC) has been described as a suitable method to study DNA polymorphisms. Here, cassava (Manihot esculenta Crantz) fermentation liquor was examined using DHPLC analysis to characterize the bacterial diversity during the fermentation process. GC-clamped amplicons corresponding to a variable region of the bacterial community 16S rDNA were synthesized using polymerase chain reaction (PCR) and then resolved on a base-composition basis using preparative DHPLC. Eluate fractions were collected at random and used as a source of whole community DNA that could be used to determine the bacterial diversity. As a first approach, GC-clamps were removed from the eluted DNA fragments using PCR to avoid the possible bias these clamps could cause during the construction of clone libraries. As a second approach, a clone library of each eluate sample was constructed, preserving the GC-clamps of the DNA fragments. The first approach generated 132 bacterial rDNA sequences with an average size of 200 bp, 45% of which had similarity to unculturable or non-classified bacteria. The second approach produced 194 sequences identified as Proteobacteria (48%), uncultured or non-classified environmental bacteria (40%) and Firmicutes (12%). We detected a remarkably greater bacterial diversity using the first approach than the second approach. The DHPLC-PCR method allowed for the fast and non-laborious detection of a vast bacterial diversity that was associated with cassava fermentation, and we conclude that it is a promising alternative for the characterization of the overall microbial diversity in complex samples.

Drug resistance and BCR‐ABL kinase domain mutations in Philadelphia chromosome–positive acute lymphoblastic leukemia from the imatinib to the second‐generation tyrosine kinase inhibitor era: The main changes are in the type of mutations, but not in the frequency of mutation involvement

Patients with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) frequently relapse on imatinib with acquisition of BCR-ABL kinase domain (KD) mutations. To analyze the changes that second-generation tyrosine kinase inhibitors (TKIs) have brought in mutation frequency and type, a database review was undertaken of the results of all the BCR-ABL KD mutation analyses performed in the authors' laboratory from January 2004 to January 2013. Interrogation of the database retrieved 450 mutation analyses in 272 patients with Ph+ ALL. Prescreening of samples was performed with denaturing high-performance liquid chromatography (D-HPLC), followed by direct sequencing of D-HPLC-positive cases. BCR-ABL KD mutations were detected in 70% of imatinib-resistant patients, with T315I, E255K, and Y253H mutations accounting for 75% of cases. Seventy-eight percent of the patients reported to be resistant to second-generation TKIs after imatinib failure were positive for mutations, and 58% of them had multiple mutations. Analysis of patients relapsing on dasatinib revealed a newly acquired T315I mutation in almost two-thirds of the cases. Direct sequencing detected no mutations at diagnosis, even in patients who relapsed after a few months. Second-generation TKIs ensure a more rapid debulking of the leukemic clone and have much fewer insensitive mutations, but long-term disease control remains a problem, and the T315I mutation is revealed to be an even more frequent enemy. BCR-ABL KD mutation screening of patients with Ph+ ALL who are receiving imatinib or second-generation TKIs would be a precious ally for timely treatment optimization. In contrast, the clinical usefulness of conventional direct sequencing at diagnosis seems to be very low. American Cancer Society.

Carrier screening for spinal muscular atrophy in 4719 pregnant women in Shanghai region

Spinal muscular atrophy (SMA) is a common and fatal autosomal recessive disorder. Approximately 94% of SMA patients are caused by homozygous deletion of SMN1 gene. SMA carrier screening is recommended considering the high carrier frequency (1 in 35-50) as well as severity of the disease. A prospective population-based cohort study was carried out on 4719 pregnant women from Shanghai region. Copy numbers of SMN1 and SMN2 genes were effectively determined with denaturing high performance liquid chromatography (DHPLC) technique. The method has detected 94% of SMA cases with deletion or conversion of the SMN1 genes. Ninety SMA carriers with only one copy of the SMN1 gene were identified among the 4719 pregnant woman. The carrier rate was 1.9%. Respectively, 1.2% and 0.6% of the carriers were caused by SMN1 gene deletion and SMN1 gene conversion. Through this study, we have determined the frequency of SMA mutation carriers in a population of pregnant women. The result may provide a basis for genetic counseling in order to reduce the rate of SMA affected births.