Strand Conformation Polymorphism Research Articles

Loss of p21ras, p53, and Mdm2 Protein Expression Is Indicative of Worse Prognosis in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) comprise a group of clonal disorders of the hematopoietic stem cell characterized by ineffective hematopoiesis and refractory cytopenias that can progress to acute leukemia. In order to identify molecular events implicated in the pathogenesis of MDS, we analyzed the bone marrow expression and mutational status of ras, p53 and mdm2 genes in a cohort of 25 patients with MDS. The expression of p21ras, p53, and Mdm2 proteins was studied in marrow cytopins stained by APAAP (alkaline phosphatase anti-alkaline phosphatase procedure) using monoclonal antibodies Y13-219, PAb 1801 and 2A10 to detect p21ras, p53 and mdm2 proteins, respectively. N-, K-, H-ras and p53 gene mutations were assessed by PCR-SSCP (polymerase chain reaction/single strand conformation polymorphism) and DNA sequencing to exclude the presence of gene mutations. The quantitative wild-type expression of p21ras, p53 and mdm2 proteins in patient samples (combination of number of cells and staining intensity) was compared to the values of expression of these proteins in 7 normal bone marrow samples. We found overexpression of p21ras and p53 proteins in 12/22 (55%) and 7/23 (30%) of the patients. Five of 7 patients who overexpressed wt-p53 also co-overexpressed wt-p21ras protein. We observed absence of expression of p21ras, p53 and mdm2 in 4/22, 4/23 and 9/19 cases, respectively. Overall survival of patients with absence of p21ras and p53 expression was considerably worse than that of patients with normal expression of these proteins (p=0.0006 to p21ras and p=0.004 to p53 by Log-Rank test; p=0.02 to p21ras and p=0.01 to p53 by Cox proportional hazards model after age, neutrophil and platelet count, and hemoglobin adjustment). We also observed that the patients who exhibited hypoexpression (p=0.055) and absence of mdm2 expression (p=0.07) demonstrated a tendency to have worse prognosis (Log-Rank). There was no correlation between variables p21ras, p53 and mdm2 versus hemoglobin, platelet and neutrophil counts and no association between FAB SMD subcategories and absence of p53 and mdm2 expression by Fisher test (two-tailed), but there was a strong over-representation between absence of p21ras expression and MDS with excess of blasts (p=0.0006). There was a significant correlation between p53 and mdm2 gene expression (p=0.03, Pearson). Only one patient harbored one transversion mutation in codon 12 of K-ras gene and did not correlate with p53 overexpression, but with worse prognosis. No p53 mutation was found. Overall, these findings suggest that the via of signal transduction p21ras/p53 and the auto-regulatory genes p53/mdm2 may be active and play a pivotal role in the pathogenesis of MDS. Hence, loss of bone marrow expression of p21ras, p53 and/or mdm2 proteins is an indicator of worse prognosis in MDS and may serve as early markers of leukemic transformation.

心肺停止に至った遺伝性血管性浮腫（HAE）に対してドクターカー出動による気道確保を行った1例(Performing airway securement under Doctor–car system after cardiopulmonary arrest caused by hereditary angioedema: a case report)

要旨 症例は51歳の女性。救急搬送前日より口唇や口腔内の腫脹を自覚。当日は朝から顔面・頸部の腫脹が続き，窒息様の仕草の後に倒れたため救急要請となった。救急隊到着時，家族がCPR実施中であり，CPAを確認。波形はPEA，徐脈であった。当院ドクターカー医師が到着し，喉頭展開すると咽喉頭浮腫による気道閉塞を認めた。声帯は可視できなかったが急峻なJ型に挿管チューブを形成し，経口気管挿管を試みた。声門と思われる位置で抵抗を感じたが挿管に成功した。自己心拍再開し当院搬送となった。気管支鏡検査で喉頭蓋・声門周囲の浮腫は著明であったが感染を疑う所見は乏しく，以前から誘因なく顔が腫れることがあったという既往と同様の症状を肉親も有するという家族歴より遺伝性血管性浮腫（HAE）を疑い検査を行ったところ，C4 1.3mg/dL, C1–INH活性 25%以下と共に低値であった。血管浮腫情報センター（CREATE）に遺伝子解析を依頼したところ，遺伝子の変異を認めHAEと診断された。集中治療管理を継続したが残念ながら脳蘇生が得られず死亡退院となった。HAEは未だ一般の認知度が低く迅速診断法がないこと, C1–INH製剤を常備する医療機関が限られていることなど課題も多い。しかし，早期の診断と適切な気道管理がなされれば血縁家族の罹患発見にも繋がり，心停止は回避可能な疾患である。

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.

An association study of NRAMP1, VDR, MBL and their interaction with the susceptibility to tuberculosis in a Chinese population

To investigate natural-resistance-associated macrophage protein 1 (NRAMP1), mannose-binding lectin (MBL), vitamin D receptor (VDR) gene polymorphisms and their interaction with susceptibility to pulmonary tuberculosis (PTB) in a Chinese population. A case-control study was conducted in PTB (n=151), age- and sex- matched healthy controls (HCs) (n=453). Genetic polymorphisms of NRAMP1 (INT4, D543NA and 3'UTR), MBL (HL, PQ, XY and AB) and VDR (FokI and Taq) were analyzed by using PCR-restriction fragment length polymorphism (RFLP) and PCR- single- strand conformation polymorphism (SSCP) techniques. Multifactor dimensionality reduction (MDR) analysis was carried out to assess the effects of the interaction between SNPs. The distribution of NRAMP1- 3'UTR (TGTG/del), MBL- HL (H/L) and FokI (F/f) were significantly different between PTB patients and HCs (p<0.05). HPYA (OR: 1.88; 95% CI: 1.22-2.91), LPXA (OR: 3.17; 95% CI: 1.69- 5.96), LQYA (OR: 3.52; 95%CI: 1.50-8.23) and LPYB (OR: 12.37; 95%CI: 3.75- 40.85) of MBL were risk haplotypes for PTB. The TGTG- H- f (OR: 1.70; 95%CI: 1.10-2.62) and del- H-f (OR: 3.48; 95% CI: 1.45-8.37) of 3'UTR- HL- FokI were also high-risk haplotypes associated with tuberculosis. Our study suggests that genotypes of many polymorphic genes are associated with TB, it is necessary to further explore the mechanism of genotypes and gene-gene interaction in susceptibility to tuberculosis.

Genetic variations of the CTNNA1 and the CTNNB1 genes in sporadic colorectal cancer in Polish population.

Experimental as well as clinical observations have demonstrated that the E-cadherin/catenin complex is a powerful inhibitor of invasion. Abrogation of this pathway is implicated in the carcinogenesis of several malignancies, especially colorectal cancer. The aim of the study was to determine the CTNNA1 and the CTNNB1 mutations and its relationship to clinical and pathological features of sporadic colorectal cancer (CRC) in Polish patients. Paired tumor and normal tissue samples from 110 sporadic CRC patients undergoing resective surgery were prospectively studied for the alpha catenin (CTNNA1) gene and beta catenin (CTNNB1)gene mutations by PCR/single strand conformation polymorphism (SSCP). The CTNNA1 gene alteration in exon 7 were detected in 4 samples and in exon 3 of CTNNB1 gene were found in 3 samples. There was a trend at the limit of statistical significance associating younger age at diagnosis (<50) with CTNNA1 and the CTNNB1 mutations. The mutation of CTNNB1 seemed to occur more frequently in the proximal colon than distal. The CRC patients with CTNNA1 mutation had a significantly increased lymph node metastasis. On the other hand, there was no correlation between mutations and the other clinical variables (e.g. sex, grade and depth of invasion). Although we found a low frequency of mutations in the CTNNA1 and the CTNNB1 genes, but the analysis the relationship with clinical and pathological features of CRC patients may indicated an association of these mutations with the risk and progression of CRC.

Mutational analysis of PIK3CA,JAK2,BRAF,FOXL2,IDH1,AKT1 and EZH2 oncogenes in sarcomas

Recent studies have revealed several recurrent mutations in oncogenes that could not only be underlying mechanisms of tumorigenesis, but also be potential targets for cancer therapies. Compared to carcinomas, genetic alterations of sarcomas are relatively unknown. To see whether recurrent oncogenes discovered in non-sarcomatous malignancies are present in sarcomas as well, we analyzed oncogenes with known mutations in various types of sarcomas. We performed mutational analysis of recurrent mutation sites of PIK3CA (exons 9 and 20), JAK2 (exon 14), BRAF (exon 15), FOXL2 (exon 1), IDH1 (exon 4), AKT1 (exon 3), and EZH2 (exon 16) genes in 108 sarcomas by single- strand conformation polymorphism and DNA sequencing. The sarcomas consisted of malignant fibrous histiocytomas, rhabdomyosarcomas, osteosarcomas, malignant peripheral nerve sheath tumors, leiomyosarcomas, synovial sarcomas, liposarcomas, angiosarcomas, chondrosarcomas, and Ewing sarcomas. Overall, we detected the two PIK3CA mutations and one JAK2 mutation (total: 3/108: 2.8%). Two rhabdomyosarcomas (16.7%) and one angiosarcoma (16.7%) harbored the mutations, whereas other sarcomas harbored none. The PIK3CA mutations were novel missense mutations that had not been detected in other cancers. The JAK2 mutation was an intron mutation. This study demonstrated that the somatic mutations of PIK3CA and JAK2 occurred in a small fraction of the sarcomas and that these mutations may not play a principal role in the development of sarcomas.

Association of G&gt;A transition in exon-1 of alpha crystallin gene in age-related cataracts

AimTo identify the presence of a known or novel mutation/SNP in Exon-1 (ex-1) of alpha crystallin (CRYAA) gene in different types of age-related cataract (ARC) patients.Materials and MethodsSingle strand Conformation Polymorphism (SSCP) analysis was carried for the detection of single nucleotide polymorphism (SNP) in ex-1 of alpha crystallin (CRYAA) gene which was confirmed by sequencing.ResultsThe SSCP analysis of ex-1 of CRYAA gene revealed mobility shift in patients and controls, which was due to G>A transition at 6th position in exon-1 of CRYAA gene. All the three genotypes, GG, AA and GA, were detected in patients and controls indicating that G>A substitution is polymorphic. The analysis showed significant risk for heterozygotes (GA) as compared to pooled frequencies of homozygotes (GG + AA), which was 1.81 times for all the types of cataracts in general and 2.5 times for Nuclear Cataract and twice for Cortical Cataract.ConclusionThe GA heterozygotes were at higher risk for developing NC and CC types of cataracts, where as the GG homozygotes for MT and AA homozygotes for PSC types were at risk. To our knowledge, an association of G>A transition found in ex-1 of CRYAA gene with ARC, with differential risk of genotypes for individual type of cataracts has not been reported previously.

Evolution of N-converting bacteria during the start-up of anaerobic digestion coupled biological nitrogen removal pilot-scale bioreactors treating high-strength animal waste slurry

Animal wastes have been successfully employed in anaerobic biogas production, viewed as a pragmatic approach to rationalize energy costs in animal farms. Effluents resulting from that process however are still high in nitrogen such that attempts were made to couple biological nitrogen removal (BNR) with anaerobic digestion (AD). The demand for organic substrate in such system is partitioned between the anaerobic metabolism in AD and the heterotrophic denitrification cascade following the autotrophic nitrification in BNR. Investigation of underlying N-converting taxa with respect to process conditions is therefore critical in optimizing N-removal in such treatment system. In this study, a pilot-scale intermittently aerated BNR bioreactor was started up either independently or in series with the AD bioreactor to treat high-strength swine waste slurry. The compositions of NH 3-oxidizing bacteria (AOB), NO 2 - -oxidizing bacteria (NOB) and denitrifiers ( nosZ gene) were profiled by polymerase chain reaction-capillary electrophoresis/single strand conformation polymorphism (PCR-CE/SSCP) technique and clone library analysis. Performance data suggested that these two process configurations significantly differ in the modes of biological N-removal. PCR-CE/SSCP based profiling of the underlying nitrifying bacteria also revealed the selection of distinct taxa between process configurations. Under the investigated process conditions, correlation of performance data and composition of underlying nitrifiers suggest that the stand-alone BNR bioreactor tended to favor N-removal via NO 3 - whereas the coupled bioreactors could be optimized to achieve the same via a NO 2 - shortcut.

Molecular gate keepers succumb to gene aberrations in colorectal cancer in Kashmiri population, revealing a high incidence area

Background/Aim:Colorectal cancer (CRC) is one of the leading malignancies worldwide and has been reported to show geographical variation in its incidence, even within areas of ethnic homogeneity. The aim of this study was to identify p53 and K-ras gene mutations in CRC patients in a Kashmiri population, and to assess whether these mutations are linked with clinicopathological parameters.Materials and Methods:Paired tumor and normal tissue samples from a consecutive series of 53 patients undergoing resective surgery for CRC were prospectively studied for p53 and K-ras gene mutations by PCR/single strand conformation polymorphism (SSCP).Results:Less than half (45%, 19/42) of the patients presented mutations in the p53 gene. Twenty eight mutations were found in the p53 gene, which comprised of 23 substitutions (17 transitions + 6 transversions), and five insertions. The 23 substitutions constituted 18 missense mutations, two nonsense mutations, and three silent mutations. Of the 28 mutations (7.14%) observed in this study, 2 were not previously reported for CRC samples and were identified as novel p53 mutations. A few patients (22.64%, 12/53) presented with mutations in K-ras, constituting 13 missense mutations, out of which 11 were G→A transitions, one was a G→C transversion, and one a G→T transversion. More than half (61.5%) of the mutations occurred in codon 12 whereas a few (38.5%) occurred in codon 13. One tumor contained missense mutations in both codons. Comparison of the mutation profiles of our patients with those of other ethnic populations and regions reflected both differences and similarities, indicating co-exposure to a unique set of risk factors.Conclusion:Mutations of the p53 and K-ras genes are some of the most common genetic changes in the development of human CRC. The high frequency of p53 gene mutations implicates p53 as a predominant factor for CRC in the high-risk ethnic Kashmiri population.

Association of DAZL haplotypes with spermatogenic failure in infertile men

To identify novel DAZL single nucleotide polymorphisms (SNPs) and to compare allele/genotype frequencies, linkage disequilibrium (LD) characteristics, and DAZL haplotypes between fertile and infertile men. Prospective case study. University genetics laboratory and reproductive clinics. Two hundred thirty-one infertile men and 191 men with proven fertility. Single strand conformation polymorphism and sequence analysis for DAZL gene polymorphism screening were done for all subjects enrolled. Novel SNPs, allele/genotype frequencies, LD characteristics, and DAZL haplotypes between fertile and infertile men. Five SNPs were identified: 260A>G, 386A>G, 520+34c>a, 584+28c>t and 796+36g>a. SNP 386A>G was significantly associated with spermatogenic failure and was mainly heterozygous in infertile patients. The major haplotypes in infertile men were AACTA (45.8%), followed by AACCG, AAATA, AAACG, and GGACG for 260A>G/386A>G/520+34c>a/584+28c>t/796+36g>a. The major haplotypes for the control subjects were AACCG (41.7 %), AAATA, and AACTA. Of all haplotypes, five showed significant differences in frequency between infertile men and control subjects. Haplotypes AACTA, AAACG, and GGACG were overtransmitted in patients with spermatogenic failure, whereas haplotypes AACCG and AAATA were undertransmitted in these patients. Our study suggests the association of autosomal DAZL haplotypes with human spermatogenic failure.

Associations between RAS Gene Polymorphisms, Environmental Factors and Hypertension in Mongolian People

To explore the association between RAS system genes (AGT, ACE and AT(1)R) polymorphisms, environmental factors and hypertension in Mongolian people. On the basis of cross-sectional study, a case-control study with 299 hypertensives and 281 nomotensives was conducted, and the conditions of environmental factors were acquired by questionnaire. Serum lipid and insulin were detected by using biochemical experiments. Six single nucleotide polymorphisms of RAS system were genotyped by polymerase chain reaction/restriction fragment length polymorphism and polymerase chain reaction/single strand conformation polymorphism. Overweight or obesity, high serum TG and insulin resistance were risk factors of hypertension by single factor analysis. All the RAS genotype distributions were compatible with Hardy-Weinberg expectations. There were no significant differences to be found between cases and controls for genotype frequencies or allele frequencies of the six polymorphisms of RAS system, except in men group, OR value of men carried ACE ID+DD genotype vs. men carried II genotype was 2.20 (95%CI 1.21-4.02), and OR of people who carried both ACE ID (or DD) and AGT M235T MT (or MM) vs. people with both ACE ID (or DD) and AGT M235T TT was 1.59 (95%CI 1.06-2.38). Overweight or obesity, dyslipidemia and insulin resistance were risk factors of hypertension in Mongolian people. ACE gene ID+DD genotype was the risk factor of hypertension in men group. People who carried both ACE ID (or DD) and AGT M235T MT (or MM) had more risk to have hypertension.

Molecular markers for systematic identification and population genetics of the invasive Ponto‐Caspian freshwater gammarid Dikerogammarus villosus (Crustacea, Amphipoda)

AbstractThe Ponto‐Caspian amphipod, Dikerogammarus villosus, is an invasive species of many European rivers. First, we show that size difference of nrDNA ITS1 allows discriminating D. villosus from Dikerogammarus bispinosus, a closely related but morphologically hardly distinguishable species. Second, we present two types of polymorphic markers for D. villosus, three microsatellites and two single nucleotide polymorphisms (SNPs) of mtDNA COI gene, which were scored by polymerase chain reaction‐single strand conformational polymorphism (PCR‐SSCP). These markers will be very useful in studying population genetics of D. villosus.

TP53 and p16INK4A, but not H‐KI‐Ras, are involved in tumorigenesis and progression of pleomorphic adenomas

The putative role of TP53 and p16(INK4A) tumor suppressor genes and Ras oncogenes in the development and progression of salivary gland neoplasias was studied in 28 cases of pleomorphic adenomas (PA), 4 cases of cystic adenocarcinomas, and 1 case of carcinoma ex-PA. Genetic and epigenetic alterations in the above genes were analyzed by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP) and sequencing and by Methylation Specific-PCR (MS-PCR). Mutations in TP53 were found in 14% (4/28) of PAs and in 60% (3/5) of carcinomas. Mutations in H-Ras and K-Ras were identified in 4% (1/28) and 7% (2/28) of PAs, respectively. Only 20% (1/5) of carcinomas screened displayed mutations in K-Ras. p16(INK4A) promoter hypermethylation was found in 14% (4/28) of PAs and 100% (5/5) carcinomas. All genetic and epigenetic alterations were detected exclusively in the epithelial and transitional tumor components, and were absent in the mesenchymal parts. Our analysis suggests that TP53 mutations and p16(INK4A) promoter methylation, but not alterations in the H-Ras and K-Ras genes, might be involved in the malignant progression of PA into carcinoma.

Discrepancies in p21ras, p53, and Mdm2 Protein Expression Predict Worse Prognosis in Acute Myeloid Leukemia.

Tumor progress is a multistep process in which clones of cells become abnormal by accumulating growth alterations until they are transformed and/or immortalized. In order to verify a potential role for p53, mdm2, and Ras genes in acute leukemogenesis, we investigated the expression and mutational status of these genes in a cohort of 35 adult patients with acute myeloid leukaemia (AML) at diagnosis with a minimum follow up of 4,5 years. The expression of p21ras, p53, and Mdm2 proteins was studied in bone marrow cytospins stained by APAAP (alkaline phosphatase anti-alkaline phosphatase procedure) using monoclonal antibodies Y13-219, PAb 1801 and 2A10, respectively. N-, K-, H-ras and p53 gene mutations were assessed by PCR-SSCP (polymerase chain reaction/single strand conformation polymorphism) and DNA sequencing to exclude the presence of mutations. We found 2 N-Ras gene and 2 p53 gene mutations in 4 patients. Stratification of AML patients by different cytoplasmic expression patterns (p21ras/p53, p53/mdm2, or p21ras/mdm2, and p21ras/mdm2/p53) revealed that patients with phenotypes p53+/mdm2+ (n=2) had significant best prognosis, patients p53—/mdm2— (n=16) an intermediate prognosis and patients with discrepant phenotypes p53—/mdm2+ or p53+/mdm2— (n=6) a worse prognosis (p=0.003 by Log Rank and p=0.008 by Wilcoxon). Patients with phenotypes p21ras+/mdm2+ (n=2) showed significant best prognosis, patients p53—/mdm2— (n=16) an intermediate prognosis and patients with discrepant phenotypes p53—/mdm2+ or p53+/mdm2— (n=8) a worse prognosis (p=0.07 by Log Rank and p=0.02 by Wilcoxon). Patients with phenotypes p21ras+/p53+/mdm2+(n=2) had a tendency to exhibit best prognosis, patients p21ras—/p53—/mdm2— (n=13) a tendency to intermediate prognosis and patients with discrepant phenotypes p21ras/p53/mdm2 (n=8) had a tendency to worse prognosis (p=0.06 by Log Rank and p=0.06 by Wilcoxon). However, stratification of AML patients by different expression values 0(—), 1(+) or 2(++) for each isolated variable p21ras, p53 and mdm2 using the same survival analysis demonstrated that there were no statistically significant difference among these groups. We found that discrepancies in cytoplasmic expression of p53/mdm2, p21ras/mdm2 and p21ras/p53/mdm2 confer worse long term prognosis to these categories of AML patients. These findings suggest that the signalization pathways p53/mdm2, ras/mdm2 and ras/p53/mdm2 may be active in AML and that an intact p53/Mdm2 axis is important in the prognosis of these patients.

High frequency of somatic missense mutation of BRCA2 in female breast cancer from Taiwan

Somatic mutation of BRCA2 has been thought to be rare in breast cancers, though common allelic deletions in the BRCA2 locus (13q12-q13) imply an important role of somatic mutation in these tumors. Reasons of the reported rare incidence could be related to very few studies focusing on the mutational analysis of BRCA2 in sporadic tumors. The mutational status of the BRCA2 gene in exon11, the largest exon harboring the RAD51 interacting BRC domains which are critical for BRCA2 function, was screened by polymerase chain reaction/single strand conformation polymorphism (PCR-SSCP) analysis followed by direct sequencing. Tumor and paired normal tissue sample from 175 patients unselected for family history or age were taken after mastectomy for breast cancer and evaluated. There were 20 mutations of BRCA2 gene in exon 11 in 15 cases (15/175, 8.6%). Most mutations we identified were point mutation (19/20, 95%), except for one nucleotide insertion. Furthermore, among these mutations, missense mutations comprised 80% (16/20) of the BRCA2 mutations. All mutations we found were novel mutation after searched in the BIC database. There were three recurrent mutations at codon 1904; and two recurrent mutations at 1907, 1936, 1937 and 1968, respectively. The mutations were associated with ductal carcinoma in situ ( P=0.038) and borderline with low histological grade ( P=0.072). Besides, there were three cases possessing multiple mutations in the region we studied and one of them demonstrated aggressive lymph node metastasis. These findings implicated that somatic mutations of BRCA2 genes may play a significant role in the pathogenesis of breast carcinoma in Taiwan. In addition, those events were associated with some early clinicopathological features.

Mitochondrial DNA mutations in the parotid gland of cigarette smokers and non-smokers

It has previously been demonstrated that mitochondrial DNA (mtDNA) mutations accumulate in the lung and increase in frequency with age. It has also been shown that the level of mtDNA mutations including deletions and base substitutions are elevated in lung tissue of smokers relative to non-smokers. We have previously shown that the ‘common’ 4977 bp mtDNA deletion is present in the parotid (salivary) gland of smokers and non-smokers and that there is a significant increase in the level of this deletion in Warthins tumour, an oncocytoma of the parotid gland. In this study we used semi-quantitative PCR to confirm the presence of 4977 bp mtDNA deletion in the parotid gland of non-smokers and smokers. Importantly, we show that the deletion accumulates with age regardless of smoking status and that there was no significant difference in the level of the 4977 bp deletion in parotid tissue of smokers and non-smokers. Using strand conformational polymorphism (SSCP) and direct sequencing we also found 5/23 smokers had parotid tissue specific base substitutions: either an A/T to G/C transition at A4767 or a G/C to A/T transition at G4853. These results are evidence of age related increase in the 4977 bp deletion and a higher level of mutations, probably due to oxidative damage, in the parotid gland of smokers.

Effect of ALDH2 and CYP2E1 Gene Polymorphisms on Drinking Behavior and Alcoholic Liver Disease in Japanese Male Workers

Aims: We examined the relationships of ALDH2 and CYP2E1 genotypes on drinking behavior and the incidence of alcoholic liver disease in Japanese male workers.Methods: Two hundred and eighty‐seven Japanese men were selected from one metal company to adjust for similar economic and social backgrounds. Drinking behavior was assessed from a self‐assessment questionnaire. Genotypes of ALDH2 and CYP2E1 were analyzed with the polymerase chain reaction‐single strand conformation polymorphism and with the polymerase chain reaction‐restriction fragment length polymorphism, respectively.Results: The frequency of the ALDH2 genotype was 55% for typical homozygotes, 42% for heterozygotes, and 4% for atypical homozygotes. The frequency of the CYP2E1 genotype was 62% for c1 homozygotes, 35% for heterozygotes, and 3% for c2 homozygotes. The ALDH2 genotype closely influenced drinking habits, but not the CYP2E1 genotype. Among habitual drinkers, ALDH2 typical homozygotes consumed significantly larger amounts of ethanol than ALDH2 heterozygotes, whereas CYP2E1 genotypes did not influence daily alcohol consumption. Sixteen men (5.6%) were diagnosed with alcoholic liver disease. In terms of ALDH2 genotypes, 12 cases (7.6%) were typical homozygotes and 4 (3.4%) were heterozygotes, whereas the incidence of alcoholic liver disease was not different between c1/c1 homozygotes and c1/c2 heterozygotes. When the interactive contribution of the ALDH2 and CYP2E1 genotypes on drinking behavior and the incidence of alcoholic liver disease were examined, there were no significant differences in the CYP2E1 genotype among the subjects with the same ALDH2 genotype.Conclusion: The ALDH2 genotype is strongly associated with individual alcohol drinking behavior and the development of alcoholic liver disease in Japanese male workers, but the CYP2E1 genotype is not.

Can p53 alterations be used to predict tumour response to pre-operative chemo-radiotherapy in locally advanced rectal cancer?

Purpose: To examine whether p53 tumour suppressor gene alterations can be used to predict tumour response to pre-operative chemo-radiation in locally advanced rectal cancer in terms of reduction in tumour size and local failure. Methods: p53 alterations were studied in pre-treatment biopsy specimens of rectal carcinomas from 48 patients by immunohistochemistry (IHC) and polymerase chain reaction/single strand conformation polymorphism (PCR-SSCP) gene mutation analysis. Pre-operative pelvic radiotherapy was delivered with four fields, 45 Gy to the ICRU point in 25 fractions over 5 weeks. A radio-sensitising dose of 5-fluorouracil (500 mg/m 2) was delivered concurrently for 6 days of the 5-week schedule (days 1, 2, 3 and days 22, 23 and 24). Total meso-rectal excision was planned 4 to 6 weeks from completion of pre-operative treatment. Response to therapy was assessed by macroscopic measurement of the surgical specimen by a pathologist who was unaware of the pre-treatment tumour size or of the p53 status. Results: IHC evidence of p53 protein accumulation was found in 40% of tumours, p53 gene mutation in 35% and p53 alteration (either or both changes) in 46%. The average reduction in tumour size was 53% in the group with ‘wild-type’ p53 ( IHC−/SSCP−) and 63% in the group with altered p53 (either IHC+ or SSCP+; P=0.18). No significant differences in tumour size reduction or local failure were observed in the groups with p53 overexpression or p53 mutation compared with normal. Conclusions: p53 alteration detected by IHC or SSCP analysis is not a clinically useful predictor of local response to pre-operative adjuvant therapy in advanced rectal carcinoma.

P53 expression, p53 and Ha- ras mutation and telomerase activation during nitrosamine-mediated hamster pouch carcinogenesis

Squamous cell carcinomas (SCC) induced in hamster buccal pouch (HBP) by 22 weeks of topical N-methyl-N-benzylnitrosamine (MBN) treatment (twice-weekly, 10 mg MBN/ml propylene glycol) were evaluated for: (i) altered expression of p53 using immunohistochemistry (IHC); (ii) mutations in Ha-ras and p53 using PCR/single strand conformation polymorphism (SSCP); (iii) telomerase activity using the telomerase repeat amplification protocol (TRAP). Precancerous lesions were also evaluated using p53 IHC. Hamsters were killed for lesion analysis at either 3 days (group A, eight hamsters, 89 carcinomas) or 7 weeks (group B, six hamsters, 105 carcinomas) following the final MBN application. Between 3 days and 7 weeks post-treatment the proportion of tumors exhibiting p53 IHC activity (at least 10% of nuclei stained using D07 antibodies for detection of both mutant and wild-type p53) fell from 91 to 50%. However, during this same post-treatment period the frequency of tumors analyzed exhibiting confirmed sequence alterations in the conserved exons (E5-E8) of p53 remained constant (5/15 = 33% in group A versus 14/45 = 31% in group B). Heightened expression of wild-type p53 resulting from DNA damage in the immediate post-treatment period is likely to have contributed to the high proportion of group A tumors exhibiting p53 IHC activity. Nearly 80% of the identified p53 mutations were G-->A and C-->T transitions. The identified p53 point mutations occurred at or near (within three codons) of the corresponding hot-spot codons (175, 245, 248 and 273) of human oral SCC. The proportion of group A and group B tumors analyzed exhibiting Ha-ras mutations was 1/15 (7%) and 7/45 (16%), respectively. Only four of the observed eight Ha-ras mutations occurred in codons known to result in activation of this gene. Telomerase activation was demonstrated in 11 of 13 group A tumors (85%) and in 23 of 24 (96%) group B tumors analyzed. The alterations in p53, Ha-ras and telomerase activity observed in this HBP-MBN model are similar in many respects to those observed in the analogous human lesions of the head and neck. This model may be particularly useful for development of cancer chemoprevention regimens and multimodality cancer therapies.

Mutation of the SRC gene in endometrial carcinoma.

Recently, an activating mutation of the SRC Gene has been implicated in about one‐tenth of advanced colon cancers. The SRC 531 mutation results in truncation of SRC directly C‐terminal to the regulatory Tyr 530 and appears to activate the Tyr 530. To investigate whether mutation of SRC plays an important role in the development and progression of gynecological tumors, we performed mutational analysis of the entire coding region of SRC in 70 ovarian carcinomas, 68 endometrial carcinomas and 3 endometrial stromal sarcomas by means of polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) followed by nucleotide sequencing and restriction fragment length polymorphism (RFLP) analysis. We found one truncated mutation at codon 531 (Gln to Stop) in an endometrial carcinoma. However, we found no mutation of this gene in ovarian carcinoma or endometrial stromal sarcoma. Our results suggest that mutation of SRC may be implicated in a small proportion of endometrial carcinomas.