Mutation-specific Polymerase Chain Reaction Research Articles

Molecular assays for determining sulphadoxine-pyrimethamine drug resistance in India: a systematic review.

A plethora of studies analyse the molecular markers of drug resistance and hence help in guiding the evidence-based malaria treatment policies in India. For reporting mutations, a number of techniques including DNA sequencing, restriction-fragment length polymorphism and mutation-specific polymerase chain reaction have been employed across numerous studies, including variations in the methodology used. However, there is no sufficient data from India comparing these methods as well as report the prevalence of polymorphisms in SP drug resistance molecular markers independently using such methods. Therefore, all data from Indian studies available for molecular marker studies of Plasmodium falciparum drug resistance to sulphadoxine-pyrimethamine was gathered, and a systematic review was performed. This systematic review identifies the molecular methods in use in India and compares each method for detecting sulphadoxine-pyrimethamine drug resistance marker. To delay the spread of drug-resistant parasite strains, a simplified and standardized molecular method is much needed which can be obtained by analysing the performance of each method in use and answering the necessity of newer methodological approaches.

A Novel Frameshift Mutation, Deletion of HBB:c.199_202delAAAG [Codon 66/67 (-AAAG)] in β-Thalassemia Major Patients from the Western Region of Uttar Pradesh, India.

PurposeBeta thalassemia is one of the most common inherited disorders in India with heterogenous clinical phenotypes from silent carrier to clinically severe ones. Our study aimed to characterize the mutation spectrum in thalassemia patients who are coming to the hospital for follow-up from the western region of Uttar Pradesh India.Patients and MethodsFor the study, a case series of the retrospective bi-centre study was conducted. The patients from two thalassemia centers in the major hospitals (LLRMC Meerut, and JNMC, Aligarh administered by the Ministry of Health and Family Welfare (MoHFW)) in the Western Uttar Pradesh, India were considered for the study. A total of 77 blood samples were obtained from individuals (both related and unrelated) diagnosed with β-thalassemia after their consent. After DNA extraction, HBB gene amplification, mutation-specific polymerase chain reaction and gene sequencing were carried out to analyze the mutations.ResultsIn this study, seven different types of mutations were reported for the first time in Western Uttar Pradesh, India. A novel frameshift mutation, deletion of 4 nucleotides Codon 66/67 (-AAAG) in exon 2 region, is reported for the first time. IVS 1–5 (G>C) and Codon 41/42 (-CTTT) are the most frequently reported mutations. The molecular spectrum for these two cases consists of 44 and 42 alleles out of 108 alleles, respectively.ConclusionA total of 108 β-thalassemia alleles were studied from 46 homozygous and 31 compound heterozygous patients. All the individuals were from 20 districts of the Western Uttar Pradesh, India.

Minimal Residual Disease Detection in Adults with Acute Lymphoblastic Leukemia By Next Generation Sequencing: Impact on Clinical Outcomes

Introduction: Measuring minimal residual disease (MRD) is an essential component of directing treatment and predicting outcomes in patients with acute lymphoblastic leukemia (ALL). Current methods used to detect MRD in patients with ALL include multiparameter flow cytometry (Flow), allele-specific and mutation-specific quantitative polymerase chain reaction, and more recently next-generation sequencing (NGS)-based methods. While flow cytometry remains standard of care for ALL MRD evaluation in most institutions, quantification of MRD with higher sensitivity assays could potentially improve early detection of clinical relapse resulting in better survival outcomes. The clonoSEQ NGS-based platform (Adaptive Biotechnologies, Seattle WA) tracks tumor specific V(D)J rearrangements in lymphoid malignancies and can detect MRD at less than 10-6 (0.0001%) level. Here we describe a real-world practice-based comparison of the impact of MRD testing by clonoSEQ and Flow on clinical outcomes in ALL. Methods: We retrospectively evaluated 45 adult patients with confirmed ALL admitted to the Norris Cancer Center (University of Southern California) between November 2017 and June 2020. In all patients, the presence/absence [(+)/(-)] of MRD was evaluated on bone marrow aspirates by both the clonoSEQ assay (clonoSEQ MRD) and by a flow cytometry-based COG ALL MRD assay (Flow). Descriptive statistics were used to characterize the cohort. The Fisher's exact test was used to describe groups with categorical outcomes. A p-value of < 0.05 was considered statistically significant. Results: Forty-five patients included in our study were aged 20 to 67 (median age 38), with 67% (30/45) males, 82% diagnosed with B-ALL (13% Ph-positive, 36% Ph-like, 51% Ph- negative),13% with T-ALL, and 5% with mixed-phenotype leukemia. Three patients were excluded from the analysis due to insufficient sampling. Among 38% (16/42) of patients with original Flow(-)/clonoSEQ(+) MRD, 31% (5/16) eventually became Flow(+) and later clinically relapsed, and 19% (3/16) received blinatumomab as a bridge to allogeneic hematopoietic cell transplantation (allo-HCT) and 2 remain in clinical remission (CR), 1 died from other medical complications. 11/14 Ph-like patients were analyzed post-induction. Post-induction clonoSEQ MRD status was used to direct these Ph-like B-ALL patients to allo-HCT (if positive, 8/11 patients) or to continue chemotherapy (if negative, 3/11 patients). Among 8/11 patients who underwent allo-HCT, 3/8 relapsed, while 5/8 remain in CR. All 3/11 patients who continued chemotherapy without allo-HCT, based on the clonoSEQ(-) MRD, remain in CR. Comparative post-induction clonoSEQ/Flow MRD data was available for 12 patients. Among these, 25% (3/12) were Flow(+)/clonoSEQ(+), 25% (3/12) were Flow(-)/clonoSEQ(+), and 50% (6/12) were Flow(-)/clonoSEQ(-), with the 12-month disease-free survival (DFS) rates of 33%, 100%, and 50%, respectively. Conclusions: Adult ALL is commonly associated with high relapse rates, often requiring therapy intensification with other agents or allo-HCT. We observed a trend towards a lower relapse rate and better outcomes with early treatment with blinatumomab and/or allo-HCT. Patients with post-induction clonoSEQ(-) MRD, who continued chemotherapy without allo-HCT or immunotherapy maintained molecular CR for > 1 year after diagnosis. Our data supports the clinical benefit of early molecular MRD evaluation with clonoSEQ to guide subsequent additional treatment (to reduce/eliminate MRD) or defer allo-HCT in MRD(-) patients. It is also important to note that a substantial subset of our unique adult population had high risk disease, whereas previous studies have focused on pediatric populations. Due to the small number of patients included, this analysis was mainly descriptive, and a larger patient cohort would be needed to better evaluate the clinical impact of MRD testing in adult ALL. Disclosures Yaghmour: Jazz:Consultancy, Speakers Bureau;Astellas:Consultancy, Speakers Bureau;Takeda:Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Alexion:Consultancy, Speakers Bureau;Agios:Consultancy, Membership on an entity's Board of Directors or advisory committees;Novartis:Consultancy, Membership on an entity's Board of Directors or advisory committees.Varnavski:Adaptive Biotechnologies:Current Employment.

Melanoma-Derived BRAF(V600E) Mutation in Peritumoral Stromal Cells: Implications for in Vivo Cell Fusion.

Melanoma often recurs in patients after the removal of the primary tumor, suggesting the presence of recurrent tumor-initiating cells that are undetectable using standard diagnostic methods. As cell fusion has been implicated to facilitate the alteration of a cell’s phenotype, we hypothesized that cells in the peritumoral stroma having a stromal phenotype that initiate recurrent tumors might originate from the fusion of tumor and stromal cells. Here, we show that in patients with BRAFV600E melanoma, melanoma antigen recognized by T-cells (MART1)-negative peritumoral stromal cells express BRAFV600E protein. To confirm the presence of the oncogene at the genetic level, peritumoral stromal cells were microdissected and screened for the presence of BRAFV600E with a mutation-specific polymerase chain reaction. Interestingly, cells carrying the BRAFV600E mutation were not only found among cells surrounding the primary tumor but were also present in the stroma of melanoma metastases as well as in a histologically tumor-free re-excision sample from a patient who subsequently developed a local recurrence. We did not detect any BRAFV600E mutation or protein in the peritumoral stroma of BRAFWT melanoma. Therefore, our results suggest that peritumoral stromal cells contain melanoma-derived oncogenic information, potentially as a result of cell fusion. These hybrid cells display the phenotype of stromal cells and are therefore undetectable using routine histological assessments. Our results highlight the importance of genetic analyses and the application of mutation-specific antibodies in the identification of potentially recurrent-tumor-initiating cells, which may help better predict patient survival and disease outcome.

Detecting EGFR alterations in clinical specimens—pitfalls and necessities

We investigated the epidermal growth factor receptor (EGFR) status in early stage lung cancer in Southern Sweden, a population for which there are no previous reports on the EGFR mutation frequency. Three hundred fifty small cell lung cancers, adenocarcinomas (AC), squamous cell carcinomas (SqCC), and large cell carcinomas were analyzed using a combination of techniques for the analysis of protein expression, gene copy numbers, and mutations. Immunohistochemical (IHC) staining with antibodies for the EGFR mutations L858R and del E746-A750 revealed intratumoral heterogeneity and several discrepant cases when compared to mutation-specific polymerase chain reaction (PCR)-based analysis. The frequencies of these two mutations, when considering IHC staining with mutation-specific antibodies in a cohort of 298 cases and subsequent confirmation by PCR, were 10 % in AC and <2 % in SqCC. Furthermore, screening by sequencing of EGFR in a cohort of 52 lung AC and squamous carcinomas demonstrated a more diverse mutation spectrum, not covered by the mutation-specific antibodies. High expression of total EGFR protein was correlated to high gene copy numbers but did not reflect the mutational status of the tumors. We believe that the mutation spectra in a Southern Swedish population is too diverse to be covered by the mutation-specific antibodies, and we also raise some other issues regarding the use of the mutation-specific antibodies, for example concerning heterogeneous expression of the mutated protein, optimal antibody dilution, and discrepancies between staining results and PCR.

Association of Common Variants of Vascular Endothelial Growth Factor and Interleukin-18 Genes with Allograft Survival in Renal Transplant Recipients of North India

Increased vascular endothelial growth factor (VEGF) production promotes enhanced endothelial permeability, enhanced leukocyte migration into the allograft, thereby leading to a clinically recognized rejection episode. Interleukin-18 (IL-18), a potent proinflammatory cytokine, may also be involved in mechanisms of kidney allograft rejection. The present study was, therefore, undertaken to investigate the association of functional polymorphisms in VEGF (2578C>A, 1154A>G) and IL-18 (607C>A, 137G>C) genes with risk of allograft rejection in renal transplant recipients of North India. Two hundred renal transplant recipients, 150 matched recipients-donors, and 200 unrelated healthy individuals were genotyped by amplification refractory mutation specific polymerase chain reaction and by polymerase chain reaction-restriction fragment length polymorphism. Variant allele VEGF 1154A>G (p = 0.56; odds ratio [OR] = 1.32) and variant allele (p = 0.004, OR = 1.54) and variant genotype (p = 0.007, OR = 3.26) of IL-18 607C>A, GC of IL-18 137G>C (p = 0.043, OR = 0.63) were significantly different in healthy individuals as compared with the patients with renal transplant. When 114 nonrejectors were compared with 36 rejectors (150 recipients) for association with allograft rejection, significant association was observed in heterozygous genotype of VEGF 2578C>A (p = 0.033), VEGF 1154A>G (p = 0.024). In IL-18 137G>C, CC genotype, C allele showed protective association with allograft rejection. Kaplan-Meier analysis indicated a higher mean time for first rejection episode in CA genotype carriers (31 months) as compared with AA (29 months) for VEGF 2578C>A (log p = 0.035). In VEGF, the haplotypes A-A and A-G (2578-1154) were associated with reduced risk and in IL-18 607A-137G, they were associated with high risk for allograft rejection. This observation suggests these polymorphisms are an ideal marker for prediction of pretransplant allograft outcome.

Expansion of donor-derived hematopoietic stem cells with PIGA mutation associated with late graft failure after allogeneic stem cell transplantation

AbstractA small population of CD55−CD59− blood cells was detected in a patient who developed donor-type late graft failure after allogeneic stem cell transplantation (SCT) for treatment of aplastic anemia (AA). Chimerism and PIGA gene analyses showed the paroxysmal nocturnal hemoglobinuria (PNH)–type granulocytes to be of a donor-derived stem cell with a thymine insertion in PIGA exon 2. A sensitive mutation-specific polymerase chain reaction (PCR)–based analysis detected the mutation exclusively in DNA derived from the donor bone marrow (BM) cells. The patient responded to immunosuppressive therapy and achieved transfusion independence. The small population of PNH-type cells was undetectable in any of the 50 SCT recipients showing stable engraftment. The de novo development of donor cell–derived AA with a small population of PNH-type cells in this patient supports the concept that glycosyl phosphatidylinositol–anchored protein–deficient stem cells have a survival advantage in the setting of immune-mediated BM injury.

NOTCH1 mutation can be an early, prenatal genetic event in T-ALL

NOTCH1 mutations are common in T-lineage acute lymphoblastic leukemia (T-ALL). Twin studies and retrospective screening of neonatal blood spots provide evidence that fusion genes and other chromosomal abnormalities associated with pediatric leukemias can originate prenatally. Whether this is also the case for NOTCH1 mutations is unknown. Eleven cases of T-ALL were screened for NOTCH1 mutations and 4 (36%) had mutations in either the heterodimerization (HD) or proline glutamic acid/serine/threonine (PEST) domains. Of these 4, 3 could be amplified by mutation-specific polymerase chain reaction primers. In one of these 3, with the highest sensitivity, NOTCH1 mutation was detected in neonatal blood spots. In this patient, the blood spot was negative for SIL-TAL1 fusion, present concomitant with NOTCH1 mutation, in the diagnostic sample. We conclude that NOTCH1 can be an early or initiating event in T-ALL arising prenatally, to be complemented by a postnatal SIL-TAL1 fusion.

Analysis of sulphadoxine/pyrimethamine resistance-conferring mutations of Plasmodium falciparum from Mozambique reveals the absence of the dihydrofolate reductase 164L mutant

BackgroundPlasmodium falciparum is the predominant human malaria species in Mozambique and a lead cause of mortality among children and pregnant women nationwide. Sulphadoxine/pyrimethamine (S/P) is used as first line antimalarial treatment as a partner drug in combination with artesunate.MethodsA total of 92 P. falciparum-infected blood samples, from children with uncomplicated malaria attending the Centro de Saude de Bagamoyo in the Province of Maputo-Mozambique, were screened for S/P resistance-conferring mutations in the pfdhfr and pfdhps genes using a nested mutation-specific polymerase chain reaction and restriction digestion (PCR-RFLP). The panel of genetic polymorphisms analysed included the pfdhfr 164L mutation, previously reported to be absent or rare in Africa.ResultsThe frequency of the S/P resistance-associated pfdhfr triple mutants (51I/59R/108N) and of pfdhfr/pfdhps quintuple mutants (51I/59R/108N + 437G/540E) was 93% and 47%, respectively. However, no pfdhfr 164L mutants were detected.ConclusionThe observation that a considerably high percentage of P. falciparum parasites contained S/P resistance-associated mutations raises concerns about the validity of this drug as first-choice treatment in Mozambique. On the other hand, no pfdhfr 164L mutant was disclosed, corroborating the view that that this allele is still rare in Africa.

Abnormalities of epidermal growth factor receptor in lung squamous‐cell carcinomas, adenosquamous carcinomas, and large‐cell carcinomas

Tyrosine kinase domain (TKD) gene mutations of the epidermal growth factor receptor gene (EGFR) have proven to be clinically significant in nonsmall-cell lung cancer (NSCLC), particularly in adenocarcinoma. However, TKD mutations together with deletion mutations in the extracellular domain of EGFR (EGFRvIII) have not been fully investigated in NSCLC except for adenocarcinoma. The present study sought to gain further insight into the significance of EGFR mutations in NSCLC by focusing on nonadenocarcinoma NSCLC. EGFR TKD mutations were investigated using direct sequencing and mutation-specific polymerase chain reaction (PCR), and EGFRvIII mutations were examined using reverse transcriptase-PCR in samples from 42 NSCLC patients and 6 NSCLC cell lines excluding adenocarcinoma. EGFR TKD mutations were detected in 1 of 7 (14%) squamous-cell carcinomas with an adenocarcinoma component and 2 of 4 (50%) adenosquamous carcinomas. In contrast, EGFR TKD mutations were not identified in 24 pure squamous-cell carcinomas without any adenocarcinoma component, 7 large-cell carcinomas, or 6 cell lines. EGFRvIII was detected solely in 1 of 7 large-cell carcinomas (14%), but not in 31 squamous-cell carcinomas, 4 adenosquamous carcinomas, or 6 cell lines. These results suggest that EGFR TKD mutations are found in NSCLCs with an adenocarcinoma element. Patients with such lesions are thus considered candidates for molecular therapies targeting EGFR.

A Simple and Sensitive Method for Detecting Major Mutations Within the Tyrosine Kinase Domain of the Epidermal Growth Factor Receptor Gene in Non???small-cell Lung Carcinoma

The detection of mutations in the epidermal growth factor receptor (EGFR) gene impacts therapeutic decision-making for non-small-cell lung carcinoma (NSCLC). Although direct sequencing has been most frequently used to detect EGFR mutations, this method displays several disadvantages. We set up simple mutation-specific polymerase chain reaction (PCR) for common delE746-A750 and L858R mutations of EGFR using primers specific to these mutations. Both mutation-specific PCR and direct sequencing methods were used to investigate 62 samples of NSCLC, and the results were compared. To evaluate the sensitivity of mutation-specific PCR, DNA mixtures containing various proportions of mutant alleles were analyzed. Mutation-specific PCR revealed delE746-A750 in 8 samples and L858R in 14 samples. All samples with either delE746-A750 or L858R mutation revealed by direct sequencing also displayed positive results for mutation-specific PCR. Conversely, mutations in 3 samples revealed by L858R-specific PCR were barely detectable by direct sequencing. In DNA mixture analysis, DNA mixtures containing 2.5% of delE746-A750 allele or 0.25% of L858R allele yielded positive results with mutation-specific PCR. Our mutation-specific PCR showed satisfactory sensitivity and reliability for detecting major EGFR mutations in clinical NSCLC samples. Given the practical availability, this method could be widely applicable to the treatment of lung cancers.

Viral latency and reservoirs: relevance for mother-to-child transmission and resistance.

This review summarizes recent data regarding the development of HIV resistance among women provided with single-dose nevirapine, alone or in combination with other antiretroviral agents to prevent mother-to-child transmission. Traditional sequencing methods and methods to detect low levels of mutations are reviewed and contrasted with methods to detect archived resistance within resting CD4 T cells. Nevirapine resistance can be detected in 18-69% of plasma-based sequencing assays. Highly sensitive, mutation-specific polymerase chain reaction methods have found nevirapine-resistant virus in up to 89% of samples immediately after nevirapine administration and at lower levels for up to 12 months after exposure. Studies to assess the presence of nevirapine-resistant virus archived in cells have yielded inconclusive results. Rigorous analysis to uncover the archiving of non-nucleoside reverse transcriptase inhibitor resistance mutations in the latent cellular reservoir after single-dose nevirapine is lacking. Prevention of the transmission of HIV from mother to child is an important public health problem for resource-limited countries. Simple and effective methods to reduce transmission such as single-dose nevirapine given to the mother during labor and to the infant are practical interventions, but a majority of women will subsequently develop HIV resistance mutations. There are limited, but compelling data to suggest that single-dose nevirapine can lead to persistent, and possibly archived, non-nucleoside reverse transcriptase inhibitor resistance, thus placing women at risk of virological failure when rechallenged with non-nucleoside reverse transcriptase inhibitor-based antiretroviral therapy.

Chimiosensibilité de Plasmodium falciparum importé des Comores à Marseille en 2002–2003

The aim of this work was to study the chemosensitivity of Plasmodium falciparum strains isolated from patients presenting with malaria after having returned from Comoros Islands in 2002-2003, and hospitalized at the North University Hospital, in Marseilles, France. In vitro drug susceptibility (for strains maintained in culture) and mutation-specific polymerase chain reaction (PCR) assays (for all strains) were performed. Out of 23 strains kept in culture, 50% were shown to be resistant in vitro to chloroquine, 50% were resistant to pyrimethamine, 40% to cycloguanil, 25% to atovaquone, and 7% to mefloquine. However all these strains were susceptible to quinine, halofantrine, and artemether. Moreover, 48 strains were tested by molecular methods. As a result, 69% were shown to have the Asp108 mutation in the dihydrofolate reductase gene (Pfdhfr), the basic mutation associated with antifolate resistance, and 54% had additional mutations Ile51 plus Arg59, associated with a high level of resistance. Furthermore, 90% of the 20 strains tested in 2003 were shown to have the point mutation Pfcrt76 in the P. falciparum chloroquine resistance transporter (Pfcrt) gene recently proposed as a molecular marker of chloroquine-resistance. Obtaining plasmodium strains from Comoros to be tested in Marseilles, where all laboratory facilities are available, is a unique opportunity to establish a surveillance of falciparum drug resistance in the Comoros islands.

Molecular Analysis to Detect Pancreatic Ductal Adenocarcinoma in High-Risk Groups

Screening of high-risk groups for pancreatic cancer has not been adopted because of concerns regarding specificity and sensitivity. Suitability of a combination of 3 novel molecular screening techniques was investigated. Pancreatic juice was extracted from 146 patients with pancreatic ductal adenocarcinoma, chronic pancreatitis, or biliary tract stones. p53 mutations were analyzed by using a modified yeast functional assay, K-ras status was analyzed using mutation-specific real-time PCR and the proportion of p16(INK4a) promoter methylation was estimated using comparative methylation-specific real-time PCR. p53 mutations were detected in 20 of 48 (42%) cancer cases, none of 49 controls, and 2 of 49 (4%) patients with pancreatitis. K-ras mutations were detected in 31 of 57 (54%) cancer patients, 13 of 61 (21%) controls, and 23 of 67 (34%) patients with pancreatitis. Twenty-six of 42 (62%) cancer patients had promoter methylation levels > 12%, compared with 3 of 24 (13%) controls, and 2 of 26 (8%) with pancreatitis. Mutations in p53 or high-level p16(INK4a) promoter methylation occurred in 29 of 36 (80%) patients with cancer, 3 of 24 (13%) controls, and 3 of 22 (13%) with pancreatitis. Three patients (8%) of 36 with cancer; 14 of 24 (58%) controls, and 13 of 22 (59%) patients with pancreatitis had no marker. The gallstone disease patients had a high rate of positive K-ras mutations, possibly reflecting the fact that they were not disease free. Combination molecular analysis increased the discrimination between patients with malignant and benign disease. This level of discrimination would allow patients in high-risk groups to be stratified from negligible risk to over 50% probability of an early cancer.

Specific detection of Flt3 point mutations by highly sensitive real-time polymerase chain reaction in acute myeloid leukemia

Among activating class III receptor tyrosine kinase (Flt3) mutations, internal tandem duplications of Flt3 (Flt3-ITD) are detected in about 25% of patients with acute myeloid leukemia (AML). In contrast, mutations within the tyrosine kinase domain of Flt3 (Flt3-TKD mutations) are less frequent (approximately 7%), and there are only limited data on the frequency of recently demonstrated activating Flt3 point mutation at codon 592 (Flt3-V592A mutation). We evaluated a new approach for rapid screening of Flt3-TKD and Flt3-V592A mutations using the fluorescence resonance energy transfer (FRET) principle in a group of 122 patients. Based on individual Flt3-TKD mutations, we designed patient-specific primers to perform a highly sensitive polymerase chain reaction (PCR) assay for rapid detection of minimal residual disease (MRD). We also used a model system with MonoMac-6 cells carrying the Flt3-V592A mutation to establish a mutation-specific real-time PCR approach also for this molecular aberration. We identified 9 cases (8%) of Flt3-TKD mutations (5 cases of mutation D835Y, 3 cases of mutation D835H, and 1 case of mutation Del836), and no cases of Flt3-V592A mutation. Screening for Flt3-TKD mutations with fluorescent probes is equivalent to conventional screening using standard PCR followed by EcoRV restriction. We present a real-time PCR protocol that can be used for MRD analyses based on individual Flt3-TKD mutations. Examples of MRD analyses are presented for all 3 subtypes of Flt3-TKD mutation identified in this study. In summary, we demonstrate new methodological approaches for rapid screening of Flt3 point mutations and for detection of MRD based on patient-specific Flt3-TKD mutations.

Chronic renal failure rats are highly sensitive to aristolochic acids, which are nephrotoxic and carcinogenic agents

Aristolochic acid (AA), a component of some Chinese herbal medicines, may cause Chinese Herbs Nephropathy (CHN) and multi-systemic tumors by the formation of AA–DNA adducts. In this study, we established an animal model to further characterize the mechanisms of AA-induced diseases. Our results indicated that AA significantly inhibited rat growth in terms of weight gain. By measuring the serum creatinine levels, AA resulted in considerable damage to the rat renal system, not only for those in which chronic renal failure (CRF) was induced but also for normal healthy rats. Mutation-specific polymerase chain reaction (PCR) and XbaI restriction fragment length polymorphism (RFLP) revealed the CAA→CTA transversion mutation at codon 61 of the H- ras proto-oncogene from the stomach tissues of CRF rats fed with AA, but not from other tissues of rats in the same experimental group. In addition, no such mutations were found in the tissues of CRF rats without AA treatment or healthy rats fed with AA. Our results strongly demonstrated that AA was in fact nephrotoxic and carcinogenic, especially to those CRF rats.

A Multi-Exonic BRCA1 Deletion Identified in Multiple Families through Single Nucleotide Polymorphism Haplotype Pair Analysis and Gene Amplification with Widely Dispersed Primer Sets

The identification of intragenic rearrangements is important for a comprehensive understanding of mutations that occur in some clinically important genes. Single nucleotide polymorphism haplotypes obtained from clinical sequence data have been used to identify patients at high risk for rearrangement mutations. Application of this method identified a novel 26-kb deletion of BRCA1 exons 14 through 20 in patients from multiple families with hereditary breast and ovarian cancer. Clinical sequence data from 5911 anonymous patients were screened for genotypes that were inconsistent with known pairs of canonical haplotypes in BRCA1 that could be explained by hemizygous deletions involving exon 16. Long-range polymerase chain reaction demonstrated that two of six samples identified by this search contained a deletion in the expected region encompassing exons 14 through 20. The breakpoint was fully characterized by DNA sequencing and demonstrated that the deletion resulted from Alu-mediated recombination. This mutation was also identified twice in a set of 982 anonymous specimens that had negative clinical test results, but uninformative haplotypes. Three additional occurrences of this mutation were found by testing 10 other patients with the indicative genotype. An assay for this mutation was added to a comprehensive clinical breast/ovarian cancer test and eight more instances were found in 20,649 probands. This multiexon deletion has therefore been detected in 15 different North American families with hereditary breast/ovarian cancer. In conclusion, this primarily computational approach is highly effective and identifies specimens using existing data that are enriched for deletion mutations.

Incidence of HBV variants with a mutation at nt551 among hepatitis B patients in Nanjing and its neighbourhood

To investigate the epidemiology of hepatitis B virus (HBV) strains with a mutation at nt551 in surface gene among hepatitis B patients in Nanjing and its neighbourhood. By using mutation-specific polymerase chain reaction (msPCR) established by our laboratory for amplifying HBV DNAs with a mutation at nt551, 117 serum samples taken from hepatitis B patients were detected. The results showed that 112 samples were positive for nt551A, 4 samples were positive for nt551G. One sample was positive for nt551T. No nt551C of HBV DNA was found. The incidence of HBsAg mutants with G, C, T, A at nt551 among 117 samples was 3.42%, 0%, 0.85%, 95.73%, respectively. In Nanjing and its neighbourhood, hepatitis B patients are mainly infected with wild genotype HBV. The incidence of mutants with a mutation at nt551 in HBV genome is significantly lower than that in wild genotype HBV DNA (P<0.01). The necessity of adding components of HBsAg mutants to HBV vaccine needs further investigation.

A Novel GγAγ(δβ)0-Thalassemia with a 27 kb Deletion

A new GγAγ(δβ)0-thalassemia (thal) was found in six unrelated Japanese individuals, and characterized by a method employing only polymerase chain reaction (PCR) and direct sequencing. This GγAγ(δβ)0-thal mutation has removed a fragment of about 27 kb of DNA, that starts approximately 2.8 kb downstream of the Aγ-globin gene and ends in the L1 repeat sequence, 7.0 kb downstream of the β-globin gene. The 5′ breakpoint is similar to that of the previously reported Japanese GγAγ(δβ)0-thal (called here Jpn type 1 for convenience). However, the 3′ endpoint is quite different. This new Japanese δβ-thal, designated as Japanese type 2 (Jpn type 2), shows a deletion rather similar to Turkish type 3 δβ-thal but with 5′ and 3′ breakpoints located inside the deletion of Turkish type 3. A mutation-specific gap PCR was designed to diagnose patients with the Jpn type 2 GγAγ(δβ)0-thal. The identified carriers exhibited a thalassemia minor.

Lack of Association between Leber’s Hereditary Optic Neuropathy Primary Point Mutations and Multiple Sclerosis in Iran

The hypothesis that mitochondrial genes may implicate susceptibility to multiple sclerosis (MS) is supported by an increasing number of case reports on Leber’s hereditary optic neuropathy (LHON)-associated mitochondrial DNA (mtDNA) point mutations in patients with MS. A number of mtDNA mutations with primary pathogenic significance for LHON, a maternally inherited disease causing severe bilateral visual loss predominantly in young men, have been detected in patients with an MS-like phenotype. To evaluate the link between MS and LHON primary point mutations, we investigated 31 non-related Iranian clinically definite MS patients (23 females and 8 males) with optic nerve involvement, as well as 25 patients (16 females and 9 males) without involvement of the optic nerve as controls. Three patients had severe bilateral visual loss without any recovery. We searched for the presence of LHON mitochondrial mutations at nucleotide positions (np) 11,778, 3,460, and 14,484 by mutation-specific polymerase chain reaction and restriction fragment length polymorphism. Our results suggest that there is no association between Iranian patients with MS and mtDNA point mutations at np 11,778, 3,460, and 14,484.