Reaction-single Strand Conformation Polymorphism Research Articles

Systematic Analyses of Clinically Relevant Single Nucleotide Polymorphisms (SNPs) of NR3C1 in Patients from the Italian Registry of Diamond Blackfan Anemia

The effects of GR on mRNA translation have mostly been studied in myocytes where its activation induces REDD1 that, by suppressing mTOR, reduces 4E-BP1(S65) and S6KA1(S235/236) inhibiting initiation of mRNA translation and protein biosynthesis (Wang JBC 2006;281:39128). It is then counterintuitive that GR agonists (glucocorticoids, GC) are effective in 60% of patients with DBA, a inherited form of pure RBC aplasia induced by mutations that impair protein biosynthesis by reducing the initial steps of mRNA translation (mRNA binding to the ribosomal subunit 40S and ribosme assembly). To address this paradox, we analyzed the phosphoproteomic profiling of erythroid cells generated in vitro either with or without the GR agonist dexamethasone by adult blood (AB) and cord blood (CB) (http://capmm.gmu.edu/data) for proteins affecting mRNA translation. By contrast with muscle cells, Dex significantly (p=0.049) increased S6KA1(S235/236) (fold change (FC)=4), eIF4G(S1108)(FC=4.16) and mTOR(S2448)(FC=1.75) in erythroid cells from CB. However, Dex significantly decreased RSK3(T356/S360)(FC=0.45) and MSK1(S360)(FC=0.52) and increased DEPTOR(FC=1.51) in AB. These results indicate that GR exerts, respectively, positive and negative regulatory functions on mRNA translation in fetal and adult erythroid cells.We previously reported that the single nucleotide polymorphism (SNP) rs6198 in NR3C1, the gene encoding GR, is present at frequency greater than normal in patients with DBA (Varricchio Blood 2011;118:473). This observation and the results described above led us to investigate whether additional clinically relevant NR3C1 SNPs are associated with disease manifestation in DBA. By polymerase chain reaction single strand conformation polymorphism analyses, we determined the frequency of 8 SNPs in 101 DBA patients, 67 from the Italian Registry and 34 from other European registries. Healthy volunteers (n=34) were analyzed as control. The SNPs investigated are: rs10482605 that decreases GRα and predicts GC resistance in major depression; rs10482616, rs7701443, rs6189/rs6190, rs860457, rs6198, that have unknown biological effects but predict GC resistance in Crohn's Disease; rs6196, that enhances expression of the dominant negative GR-β isoform predisposing to autoimmune diseases, myeloproliferative neoplasms and DBA, while reducing risks of diabetes in Cushing's Syndrome and rs33388/rs33389 that increase expression of GRγ which binds GC 10-times less efficiently than GRα and has not been associated with human diseases as yet. Cases versus control analyses suggests that the frequency of rs860457 (Table 1) and rs33388 (p=0.0689 by categorical analyses, not shown) is increased in this cohort of DBA patients. The reduced significance level of this observation may be due to the low numbers (34) of healthy controls used as comparison.Demographic, causal mutations and disease manifestation (GR responsiveness and age of anemia onset) are known for patients from the Italian Registry only. The patients were 55.6% females and 44.4% males and their average age at diagnosis was 9.14±24.56 months. The causal mutations were RPL5 (n=12), RPL11 (n=8); RPS26 (n=12), RPS19 (n=28), RPS17 (n=1) and unknown (n=6). Treatment outcome is known for 58 of the 67 patients: 14 responded, 41 did not respond while three patients underwent spontaneous remission and were excluded from the analyses. Age of disease manifestation is known for 57 patients: 27 (29.82) displayed the disease before and 40 (70.18%) after 2 months. Gender and causal mutations were neither associated with response nor with age of disease manifestation. None of the SNPs met the threshold in the response vs non-responsive groups (Table 1). However, two SNPs (rs6196, p=0.0509 and rs33386, p=0.1249) met the threshold criteria in the age of disease manifestation analyses (Table 1). Of note rs6196 predicts GC resistance in Crohn's disease and rs33386 favors expression of GRγ. No significant association was observed when SNPs that met the threshold were analyzed in a multivariable model.These results indicate that GR promotes protein biosynthesis in fetal but not in adult erythroid cells. The frequency of rs860457 which impairs GR functions is increased in DBA patients and rs6196 and rs33388 may be correlated with age to disease manifestation. These observations require further analyzes with larger cohorts of controls and/or patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

FSHR (exon 10) gene polymorphisms and its association with fertility trait in Egyptian Ossimi sheep

For the association between of Follicle stimulating hormone receptor (FSHR) gene (partial part of exon 10) polymorphisms and litter size trait in Egyptian Ossimi sheep, polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) and DNA sequencing techniques were developed. Fifty female Ossimi sheep reared under Egyptian conditions were selected according to their litter size. DNA from blood samples of these animals was isolated to amplify 250-bp of the FSHR gene influencing litter size production trait in sheep. Based on litter size, 50 animals were selected from the highest to the lowest litter size productivity during three seasons. PCR-SSCP analysis of the FSHR gene (250-bp) showed two various genotypes AA and with frequencies 0.64 and 0.36, respectively. The frequencies of the A and B alleles were 0.82 and 0.18, respectively. PCR fragment of FSHR gene (191-bp) was sequenced only in the high and low litter size productivity animals (GenBank accession numbers from MG973191 to MG973207, sequentially). The result indicated that 6SNPs (G/71, G/72, G/77, A/110, A/111, A/191) in high fertile animals, while, 10 SNPs (T/1, C/2, T/14, A/69, A/70, A/71, A/74, G/74, A/75, A/136) have found in low fertile animals. Statistically, AA and genotypes have no significant differences (p> 0.05) on litter size trait in Ossimi sheep. FSHR (exon 10) locus was moderate polymorphic (PIC= 0.25) and it can be used for high litter size productivity in Ossimi sheep as a marker-assisted selection (MAS).

A963G single nucleotide variant of BMP15 is not common bio-marker for fecundity in goat

Bone morphogenetic protein 15 (BMP15) is crucial factor for ovulation as well as for increasing litter size. In the present investigation efforts had been carried out to assess the genetic variations in Exon 2 region of BMP15 in goat, using polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) sequencing methods and cooperated frequency distribution to discuss its possibility of related fecundity. Across the 144samples from six breeds were identified in the A963G location of BMP15 using PCR-SSCP and sequences technology. A963A genotype was the most frequent (85.4%) and G963G was the least frequent with a frequency of 4.2% and A963G is 10.4%. It revealed non significant different between high and low fecundity breed. Therefore, this single nucleotide variant is not common Bio-Marker for fecundity in Goat.

Association of MOV10L1 gene polymorphisms and male infertility in azoospermic men with complete maturation arrest.

The present research was undertaken to study probable genetic variations of MOV10L1 in 30 infertile men that had complete maturation arrest in their spermatocyte levels and 70 fertile men as the control group. We performed polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) on extracted DNAs and sequencing was used to confirm the results. Identified polymorphisms in the MOV10L1 were further subjected to a haplotype analysis. We identified eight single nucleotide polymorphisms (SNPs): one missense (rs2272837) and four nonsense polymorphisms (rs2272836, rs11704548, rs2272838, rs138271) in the exonic sequences and three polymorphisms (rs12170772, rs2272840, rs17248147) in the intronic regions. With the exception of rs2272838, there was a statistically significant association in all polymorphisms between study population (P < 0.05). The result of haplotyping analysis showed ten possible haplotypes, from which five were significantly increased in infertile patients compared with the control group. Our results suggest that MOV10L1 gene polymorphisms in the studied infertile males with complete maturation arrest are linked to infertility.

Detection of SNPs in the Cathepsin D Gene and Their Association with Yolk Traits in Chickens

CTSD (Cathepsin D) is a key enzyme in yolk formation, and it primarily affects egg yolk weight and egg weight. However, recent research has mostly focused on the genomic structure of the CTSD gene and the enzyme’s role in pathology, and less is known about the enzyme’s functions in chickens. In this paper, the correlations between CTSD polymorphisms and egg quality traits were analyzed in local Shandong chicken breeds. CTSD polymorphisms were investigated by PCR-SSCP (polymerase chain reaction single strand conformation polymorphism) and sequencing analysis. Two variants were found to be associated with egg quality traits. One variant (2614T>C), located in exon 3, was novel. Another variant (5274G>T), located in intron 4, was previously referred to as rs16469410. Overall, our results indicated that CTSD would be a useful candidate gene in selection programs for improving yolk traits.

The Genetic variants of IL1RAPL2 gene associated with non-specific mental retardation in Chinese children

The present study investigated the association between the genetic variants of IL1RAPL2 gene and Non-syndromic mental retardation (NSMR) in the children of QinBa region of China. Five common SNPs (rs5962434, rs5916817, rs3764765, and rs5962298 and rs9887672) of IL1RAPL2 were chosen and examined their individual genotype frequencies using the conventional polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) method, and evaluated the association between these genetic polymorphisms and NSMR with the suitable bio-statistic software. Two SNPs (rs5962298 and rs9887672), whose alleles and genotypes distribution showed a significant differences between the control and NSMR groups (alleles: p = 0.020 and 0.017; genotypes: p = 0.025 and 0.053, respectively). Furthermore, the different gender effect was found out, when stratified the data set by the sex. Taken together, we provided substantial evidence that IL1RAPL2 conferred a NSMR susceptibility to children of Qinba region in China, and further work should been done. Key words: Non-syndromic mental retardation, molecular genetics, association analysis, genetic variants

Analyses of coding sequence point mutation and polymorphism of TGFBI gene in Chinese patients with keratoconus

To investigate the point mutations and polymorphisms of transforming growth factor beta-induced gene (TGFBI) in Chinese patients with keratoconus and discuss the relationship between the feature of gene mutations and single nucleotide polymorphisms of TGFBI gene and keratoconus. Polymerase chain reaction single strand conformation polymorphism and DNA direct sequencing were performed in 30 keratoconus cases and 30 healthy controls. All 17 exons of the TGFBI gene were analyzed for point mutations and single nucleotide polymorphisms. Totally two heterozygous nucleotide changes were identified in exon 12 of the TGFBI gene. The codon 535 is changed from GGA to TGA in 1 patient, leading to a substitution of glycine to a stop codon at the protein level (G535X). The codon 540 is changed from TTT to TTC in 2 patients and 1 control individual, resulting in a nonsense mutation (F54F), and is a single nucleotide polymorphism of the gene. Mutation and polymorphisms of the TGFBI gene were detected in Chinese patients with keratoconus in this study. The results suggest that TGFBI gene might play an important role in the pathogenesis of keratoconus.

An association study between IL1RAPL2 gene and non-specific mental retardation in Chinese children

Non-specific mental retardation (NSMR) is one of common children psychiatric diseases with a high prevalence (1–3%). Here we investigated the association between the genetic variants of IL1RAPL2 gene and NSMR in the children of QinBa region of China. We chose five common SNPs of IL1RAPL2, examined their individual genotype frequencies using the conventional polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) method, and evaluated the association between these genetic polymorphisms and NSMR with the suitable biostatistic software. The allele and genotype distributions of two SNPs (rs5962298 and rs9887672) showed significant differences between the control and NSMR groups (allele: p = 0.020 and 0.017; genotype: p = 0.025 for rs9887672 respectively). The distribution differences became more significant in girls, but disappeared in boys, suggesting a gender effect. Taken together, we provide substantial evidence that IL1RAPL2 conferred a NSMR susceptibility to children of Qinba region in China. In future, further work should be carried out to scan mutations and to investigate the specific-gender effect in this gene.

Chromosome Location and Association of Haplotypes of Insulin-like Growth Factor Binding Protein-2 with Production Performance in Swine

Insulin-like growth factor binding protein-2 is a member of the insulin-like growth factor families. Using a porcine RH panel, the gene was mapped on chromosome 15q22-23. Meanwhile, using polymerase chain reaction single strand conformation polymorphism, genotypic and allelic frequencies were analyzed in 17 pig breeds (total animals 570), together with a chi-square test of Hardy-Weinberg equilibrium. Also the association between haplotypes and production performance was analyzed in a Lantang x Landrace population family (n = 133, total 43 traits). At each locus we investigated, all the breeds showed different genotypic and allelic frequency distributions. In general, the Chinese native pig breeds carried a higher allele A frequency (over 50%) than the European pigs. For production performance, pigs with the CAG haplotype had higher fore-body and rear-body weight than those with the TGT and TAG haplotypes (P < 0.05). Also, pigs with the CAG haplotype had higher bone weight of the rear-body than those with the CAT haplotype (P < 0.05); pigs with the TGT and CAG haplotypes had higher forelimb and rearlimb weight than those with the CAT haplotype (P < 0.01 and P < 0.05, respectively); pigs with the TGG haplotype had higher leaf fat weight than those with the TGT and CAG haplotypes (P < 0.05); and pigs with the CAG haplotype had more stomach weight than those with the CAT and CGT haplotypes (P < 0.01); pigs with the TGT and CAG haplotypes had more ribs and longer body than those with the CGT-TGG, and CAT-TAG haplotypes (P < 0.05). These results suggest that IGFBP-2 is associated with production performance, but our population family was small. More studies with large samples are needed before the IGFBP-2 locus will be useful for a selection program.

Characterization ofrpoBmutations associated with rifampin resistance inMycobacterium tuberculosisfrom eastern China

The aim of this study was to investigate the features of rpoB gene mutations associated with Rifampin (RIF) resistance in Mycobacterium tuberculosis (M. tuberculosis) in eastern China. The mutations of rpoB gene in 56 clinical isolates of M. tuberculosis resisted to one to four first-line drugs (rifampin, isonicotinyl hydrazide, ethambutol and streptomycin) were analysed by polymerase chain reaction single strand conformation polymorphism analysis (PCR-SSCP) and DNA sequencing. The results of PCR-SSCP showed 52 isolates were positive (existing rpoB mutation) including 47 isolates resisted to RIF. Subsequent results of DNA sequencing showed that 54 isolates had rpoB gene mutation including 49 isolates resisted to RIF. The most frequently mutated sites were at codons 526 (73.2%), 513 (10.7%) and 531 (3.5%). The rpoB codon 526 was the most frequently mutated site of RIF-resistant M. tuberculosis strains in eastern China and its frequency is significantly higher (P < 0.0001) compared with that in other areas of China and in other geographic regions worldwide. Our results reveal that geographic variation is responsible for rpoB mutations in M. tuberculosis and the resulting information will be helpful to improve a novel rapid molecular drug resistance screening approach for MDR TB.

Improvements of Polymerase Chain Reaction and Capillary Electrophoresis Single-Strand Conformation Polymorphism Methods in Microbial Ecology: Toward a High-throughput Method for Microbial Diversity Studies in Soil

The molecular signature of bacteria from soil ecosystems is an important tool for studying microbial ecology and biogeography. However, a high-throughput technology is needed for such studies. In this article, we tested the suitability of available methods ranging from soil DNA extraction to capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) for high-throughput studies. Our results showed that the extraction method does not dramatically influence CE-SSCP profiles, and that DNA extraction of a 0.25 g soil sample is sufficient to observe overall bacterial diversity in soil matrices. The V3 region of the 16S rRNA gene was amplified by PCR, and the extension time was found to be critical. We have also found that proofreading DNA polymerases generate a better signal in CE-SSCP profiles. Experiments performed with different soil matrices revealed the repeatability, efficiency, and consistency of CE-SSCP. Studies on PCR and CE-SSCP using single-species genomic DNA as a matrix showed that several ribotypes may migrate at the same position, and also that single species can produce double peaks. Thus, the extrapolation between number of peaks and number of species remains difficult. Additionally, peak detection is limited by the analysis software. We conclude that the presented method, including CE-SSCP and the analyzing step, is a simple and effective technique to obtain the molecular signature of a given soil sample.

Expression ofp16gene and Rb protein in gastric carcinoma and their clinicopathological significance

To analyze the correlation between the protein expression of p16 and Rb genes in gastric carcinoma (GC), to investigate the role of p16 gene in invasion and lymph node metastasis of GC, and to examine the deletion and mutation in exon 2 of p16 gene in GC. The protein expression of p16 and Rb genes was examined by streptavidin-peroxidase conjugated method (S-P) in normal gastric mucosa, dysplastic gastric mucosa and GC. The deletion and mutation of p16 gene were examined by polymerase chain reaction (PCR) and polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) respectively in normal gastric mucosa and GC. The positive rates of P16 and Rb protein expression respectively were 96% (77/80) and 99% (79/80) in normal gastric mucosa, 92% (45/50) and 80% (40/50) in dysplastic gastric mucosa, 48% (58/122) and 60% (73/122) in GC. The positive rates of P16 and Rb protein expression in GC were significantly lower than that in normal gastric mucosa and dysplastic gastric mucosa (P<0.05). The positive rate of P16 protein expression in mucoid carcinoma (10%, 1/10) was significantly lower than that in poorly differentiated carcinoma (51%, 21/41), undifferentiated carcinoma (58%, 15/26) and signet ring cell carcinoma (62%, 10/16) (P<0.05). The positive rates of P16 protein in 30 cases of paired primary and lymph node metastatic GC were 47% (14/30) and 17% (5/30) respectively, being significantly lower in the later than in the former (P<0.05). There was no mutation in exon 2 of p16 gene in the 25 freshly resected primary GCs. But five cases in the 25 freshly resected primary GCs displayed deletion in exon 2 of p16 gene. The positive rate of both P16 and Rb proteins was 16% (14/90), and the negative rate of both P16 and Rb proteins was 8% (7/90) in 90 GCs. The rate of positive P16 protein with negative Rb protein was 33% (30/90). The rate of negative P16 protein with positive Rb protein was 43% (39/90). There was reverse correlation between P16 and Rb expression in 90 GCs (P<0.05). The loss protein expression of p16 and Rb genes is related to GC. The loss expression of P16 protein is related to the histopathologic subtypes and lymph node metastasis of GC. Expression of P16 and Rb proteins in GC is reversely correlated. The deletion but not mutation in exon 2 of p16 gene may be involved in GC.

Relationship between polymorphism of class II transactivator gene promoters and chronic hepatitis B

To investigate the relationship between the polymorphism of class II transactivator (CIITA) gene promoters and chronic hepatitis B (CHB). Genomic DNA was prepared from peripheral blood leukocytes. Promoters I, III and IV of gene were analyzed respectively with polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) in 65 patients with CHB, 26 patients with acute hepatitis B (AHB) and 85 normal controls. No abnormal migration was found in PCR-SSCP analysis of the three promoters in the three groups. Also, no sequential difference was observed at the three promoters among the CHB patients, AHB patients and normal controls. No polymorphism in promoters I, III and IV of CIITA gene exists in CHB patients, ABH patients and normal controls, suggesting that the promoter of CIITA gene might be a conserved domain.

두경부 종양에서 DHPLC를 이용한 p53체세포 돌연변이 검출 연구

두경부 편평 세포암종(HNSCC: head and neck squamous cell carcinoma) 의 발생과 관련하여 p53 종양 억제 유전자 (tumor suppressor gene) 의 돌연변이는 높은 비율로 나타나는 것으로 보고 되고 있다. 단국대학교 병원에서 두경부 종양으로 진단 받고 수술 받은 환자의 조직 50개를 대상으로 p53 종양 억제 유전자의 exon 5-8 까지의 영역에서 DNA를 추출하여 PCR-SSCP(polymerase chain reaction single strand conformational polymorphism) 방법과 DHPLC(denaturing high performance liquid chromatography) 방법으로 p53체세포 돌연변이(somatic mutation)를 비교 분석하였다. 그 결과 SSCP 분석 방법은 16개(32%), DHPLC 분석 방법은 17개(34%) 를 검출하였고 그 중 SSCP와 DHPLC 분석 방법 모두 exon 8번에서 결실(deletion) 형태의 돌연변이를 확인하였으며 최종적으로 자동 염기 서열 분석기(automatic DNA sequencer) 를 통하여 모든 돌연변이를 확인하였다. DHPLC 분석방법이 SSCP 방법보다 분석 시간이나 노력이 덜 소모되며 보다 더 정확한 돌연변이 검출 방법임을 확인하였다. Mutation of p53 tumor suppressor gene in HNSCC (head and neck squamous cell carcinoma) has been proposed high rate. We extracted genomic DNA from 50 head and neck cancer. The DNA was amplified by PCR at exon 5-8 in p53 tumor suppressor gene. We have compared single strand conformation polymorphism (SSCP) and denaturing high performance liquid chromatography (DHPLC) method for analysis of p53 somatic mutation. As a result, 16 deleted mutations (32%) were detected by SSCP analysis and 17 deleted mutations (34%) were detected by DHPLC analysis at exon 8. All of 17 mutations were proved by sequencing. We conclude that DHPLC is a fast and simple screening method rather than SSCP analysis.

Evaluation of a species-specific PCR assay for the Anopheles funestus group from eleven African countries and Madagascar

A newly published cocktail polymerase chain reaction (PCR) assay can identify five members of the Anopheles funestus group: An. funestus, An. vaneedeni, An. parensis, An. leesoni and An. rivulorum. The assay was evaluated on specimens from 11 African countries: Angola, Cote d’Ivoire, Ethiopia, Kenya, Malawi, Mozambique, Namibia, South Africa, Tanzania, Uganda and Zambia; and the island of Madagascar. The polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) and the internal transcribed spacer PCR (ITS2-PCR) assays were used as a priori identification methods on 900 specimens. Of these, 96.4% were correctly identified using the new cocktail PCR method. The remaining 3.5% (32) from Malawi failed to amplify. The failure to identify these samples was not significantly different from the samples from the other countries (Chi-square; P>0.05). The results suggest that the new species-specific PCR assay is an efficient and effective means of identifying 5 members of the An. funestus group in a single reaction. It is also less time-consuming than the molecular methods previously used on the group.

P53 mutation in breast cancer. Correlation with cell kinetics and cell of origin.

Several studies have investigated the expression of the cytokeratins (CKs), vimentin, the epithelial growth factor receptor (EGFR), the oestrogen receptor (ER), and the progesterone receptor (PgR), in breast cancer, but no study has directly compared p53 mutations with these phenotypic and differentiation markers in the same case. The present study was designed to provide some of this information. The expression of the p53 and bcl-2 proteins was evaluated by immunohistochemistry in relation to phenotypic characteristics and cellular kinetic parameters (mitotic index and apoptotic index) in 37 cases of ductal carcinoma in situ (DCIS) and 27 cases of infiltrating ductal carcinoma (IDC) of the breast. In addition, p53 gene mutation was examined by polymerase chain reaction single strand conformation polymorphism analysis (SSCP). Thirteen cases (eight DCIS and five IDC) showed expression of CK8, CK14, CK18, vimentin, and EGFR, consistent with a stem cell phenotype, whereas 44 cases (27 DCIS and 17 IDC) showed expression of CK8 and CK1, weak or negative expression of CK18, but were negative for vimentin and EGFR, consistent with a luminal cell phenotype. DCIS and IDC cases with a stem cell phenotype were ER/PgR negative and intermediately or poorly differentiated. In contrast, the cases with luminal cell phenotype were ER/PgR positive and well or intermediately differentiated. In addition, intermediately or poorly differentiated cases with a stem cell phenotype showed higher proliferative activity (per cent of MIB-l positive cells) than did intermediately or well differentiated cases with a luminal cell phenotype. Both DCIS and IDC cases with a stem cell phenotype were p53 positive and bcl-2 negative by immunohistochemistry. In IDC, p53 expression was associated with a reduction of both mitotic index and apoptotic index compared with DCIS. Most of the tumours showing a more differentiated phenotype (luminal) were p53 negative and bcl-2 positive. In these cases, cell kinetic parameters increased from DCIS to IDC. These data suggest the existence of subsets of DCIS and IDC that, because of their phenotypic characteristics, could be derived from subpopulations of normal breast cells having different control mechanisms of cell proliferation and neoplastic progression. These results are compatible with the hypothesis that the phenotype of the cell of origin constrains both tumour phenotype and the choice of genetic events; however, the occurrence of p53 mutants by chance during neoplastic transformation cannot be excluded.

A second locus and new alleles in the major histocompatibility complex class II (ELA-DQB) region in the horse

More than two nucleotide sequences of the second exon of the ELA-DQB region retrieved from a single animal and two different sequences isolated from horses homozygous in the major histocompatibility complex (MHC) region by descent indicated the existence of at least two ELA-DQB loci at the genomic level. New alleles detected by polymerase chain reaction single strand conformation polymorphism (SSCP) and defined by nucleotide sequencing of the second exon of the DQB gene(s) were described. Based on the level of nucleotide sharing, at least two groups of alleles were shown to exist. The newly defined alleles belonged preferentially to one of the groups. However, their specific locus assignment was not possible from the data collected. At least one of these alleles was shown to be transcribed. No frame-shift mutations were identified among the new alleles, although one pseudoallele containing a stop codon was identified at the genomic DNA level.

Topoisomerase IIalpha gene expression is regulated by the p53 tumor suppressor gene in nonsmall cell lung carcinoma patients.

Topoisomerase IIalpha (Topo IIalpha) is an essential nuclear enzyme for chromosome segregation during mitosis. Previous experimental studies using cell lines reported that Topo IIalpha expression was negatively regulated by wild-type p53 through the gene's promoter region. Surgically resected tumor specimens from 98 nonsmall cell lung carcinoma (NSCLC) patients who were not treated with preoperative chemotherapy were studied. Quantitative reverse-transcription polymerase chain reaction analysis was done to evaluate Topo IIalpha gene expression. Polymerase chain reaction single strand conformation polymorphism following sequencing was performed to investigate mutations of p53. Topo IIalpha gene expression in squamous cell carcinomas was significantly higher than in adenocarcinomas (P = 0.0007). Topo IIalpha gene expression in moderately differentiated tumors and poorly differentiated tumors was significantly higher than in well differentiated tumors (P = 0.0032 and P = 0.0005, respectively). Thirty nine tumors (40%) had mutations of p53. Topo IIalpha gene expression in tumors with mutant p53 was significantly higher than in those with wild-type p53 (P = 0.0224). The current study suggests that Topo IIalpha gene expression is regulated by p53 gene status in NSCLC patients and that the overexpression of Topo IIalpha induced by mutant p53 might cause more aggressive carcinogenesis.

Detection of exon polymorphisms in the human lactoferrin gene.

We previously demonstrated that lactoferrin gene polymorphisms occur in cancer cells of patients with leukemia and breast cancer. In this study, we established a non-radioactive polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, one of the most sensitive and simplest methods to detect polymorphisms and mutations of the human lactoferrin gene. We optimized the PCR conditions for nine different DNA templates and 16 pairs of exon primers for SSCP analysis. The DNA templates used in the analyses were prepared from a cosmid clone (CT6-1) that contains the human lactoferrin gene, human placental tissue, leukocytes from 10 normal volunteers, leukemic cells of two patients, and previously established three breast and two leukemic cell lines. With the appropriate exon-primer sets, PCR products from exon I to exon 16 of the lactoferrin gene were generated from the DNA templates and analyzed by SSCP. Compared with the homogenous cloned DNA, lactoferrin gene polymorphisms were detected within exons 2, 5, 7, 9, 13, 14, and 15 of the normal placental and leukocyte DNA. In addition, abnormal migration patterns of the lactoferrin gene in cancer cells were detected in exons 4, 5, 13, 14, and 15. The PCR-SSCP band migration patterns can be attributed either to gene polymorphism in normal cells or to DNA mutations in cancer cells and the employed method cannot distinguish between them. Nonetheless, the present analysis suggests that genetic polymorphisms of the lactoferrin gene exist in selected exons and additional mutations of the lactoferrin gene do occur in the cancer cells.

An insight into the genetic pathway of adenocarcinoma of the small intestine.

Although the adenoma to carcinoma pathway in colorectal cancer is well described, the mechanisms of carcinogenesis in the small intestine remain unclear. The aim of this study was to investigate candidate genes in the genetic pathway of adenocarcinoma of the small intestine. A total of 21 non-familial, non-ampullary adenocarcinomas of the small intestine were analysed. DNA was extracted from formalin fixed paraffin wax embedded tissue using standard techniques. The replication error (RER) status was determined by amplification of BAT26. The mutation cluster region (MCR) of the adenomatous polyposis coli (APC) gene was screened using polymerase chain reaction single strand conformational polymorphism and direct sequencing. Immunohistochemistry was performed on formalin fixed paraffin wax embedded tissue using monoclonal antibodies for hMLH1, hMSH2, beta-catenin, E-cadherin, and p53. Fourteen male and seven female patients with a median age of 64 years (range 21-85) presented with adenocarcinoma of the duodenum (10), jejunum (7), and ileum (4). One cancer (5%) was found to be RER+, and all tumours stained positive for hMLH1 and hMSH2. No mutations were detected in the MCR of the APC gene. beta-Catenin showed increased nuclear expression with loss of membranous staining in 10 cancers (48%). Absent or decreased membrane expression of E-cadherin was found in eight cancers (38%). Strong staining of p53 was found in the nucleus of five cancers (24%). We did not detect mutations in the MCR of the APC gene, and this suggests that adenocarcinoma of the small intestine may follow a different genetic pathway to colorectal cancer. Abnormal expression of E-cadherin and beta-catenin was common and reflects an early alternative to APC in this pathway in which mutations may be found in adenocarcinoma of the small intestine.