PCR-single Strand Conformation Polymorphism Analysis Research Articles

Sequence Variation in the Bovine Lipin-1 Gene (LPIN1) and Its Association with Milk Fat and Protein Contents in New Zealand Holstein-Friesian × Jersey (HF × J)-cross Dairy Cows.

Simple SummaryLipins are a family of proteins involved in lipid metabolism through their phosphatidate phosphatase and transcriptional co-activator activities. Of the three family members identified, lipin-1 is highly upregulated in the bovine mammary gland during lactation, and has been implicated in regulating milk production, albeit with inconsistent results in different breeds of cattle in studies. Accordingly, its role in regulating milk synthesis is not well understood. In this study, we used polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analyses to investigate variation in the bovine lipin-1 gene in New Zealand Holstein-Friesian × Jersey-cross dairy cows and found that variation in the sixth coding exon was associated with milk fat percentage and protein percentage. This suggests that lipin-1 regulates the synthesis of milk fat and milk protein.Lipin-1 is known to play a regulatory role in tissues that function in lipid metabolism. In dairy cows, the lipin-1 gene (LPIN1) is highly expressed in the mammary gland, but its function in milk production is less understood. In this study, we used PCR-single strand conformation polymorphism analysis to investigate sequence variation in three regions of bovine LPIN1 in New Zealand Holstein-Friesian × Jersey (HF × J)-cross dairy cows, including part of the 5′ non-coding region, the region containing the LPIN1β-spliced exon, and the sixth coding exon that encodes the putative transcriptional activating domain of the protein. No variation was found in the LPIN1β-spliced exon, but two sequence variants containing one single nucleotide polymorphism (SNP) were identified in the 5′ non-coding region and four sequence variants containing four non-synonymous SNPs were identified in the sixth coding exon. Among the three common variants of the sixth coding exon, variant C was found to be associated with an increase in milk fat percentage (presence 4.96 ± 0.034% vs. absence 4.81 ± 0.050%; p = 0.006) and milk protein percentage (presence 4.09 ± 0.017% vs. absence 3.99 ± 0.025%; p = 0.001), but no associations (p > 0.01) were detected for milk yield. These results suggest that variation in LPIN1 affect the synthesis of fat and proteins in milk and has potential as a gene-marker to improve milk production traits.

Variation in the Ovine Glycogen Synthase Kinase 3 Beta-Interaction Protein Gene (GSKIP) Affects Carcass and Growth Traits in Romney Sheep.

Simple SummaryThe glycogen synthase kinase 3 beta (GSK3β)-interacting protein plays a role in regulating glycogen metabolism, protein synthesis, the cell cycle, and in regulating gene expression. To date, physiological function research into the GSK3β-interacting protein has been focused on cell lines, gene ‘knockout’ models, and over-expression studies, and to our knowledge, there have been no reports on how variation in the GSK3β-interacting protein gene (GSKIP) may affect phenotypic traits. In this study, PCR-SSCP methods were used to screen for variation in exon 1 and exon 2 of GSKIP in 840 New Zealand (NZ) Romney sheep. Two variant sequences were identified in exon 1 and this variation in GSKIP was associated with variation in lamb birth weight, hot carcass weight, and fat depth at the 12th rib.The glycogen synthase kinase 3 beta (GSK3β)-interacting protein (encoded by the gene GSKIP) is a small A-kinase anchoring protein, which complexes with GSK3βand protein kinase A (PKA) and acts synergistically with cAMP/PKA signaling to inhibit GSK3β activity. The protein plays a role in regulating glycogen metabolism, protein synthesis, the cell cycle, and in regulating gene expression. In this study, PCR-single strand conformation polymorphism (PCR-SSCP) analyses were used to screen for variation in exon 1 and exon 2 of GSKIP in 840 New Zealand (NZ) Romney sheep. Two SSCP banding patterns representing two different nucleotide variants (A and B) were detected in an exon 1 region, whereas in an exon 2 region only one pattern was detected. Variants A and B of exon 1 had one non-synonymous nucleotide difference c.37A/G (p.Met13Val). The birthweight of sheep of genotype AA (5.9 ± 0.06 kg) was different (p = 0.023) to sheep of genotype AB (5.7 ± 0.06 kg) and BB (5.7 ± 0.06 kg). The hot carcass weight (HCW) of sheep of genotype AA (17.2 ± 0.22 kg) was different (p = 0.012) to sheep of genotype AB (17.6 ± 0.22 kg) and BB (18.0 ± 0.29 kg), and the fat depth at the 12th rib (V-GR) of sheep of genotype AA (7.7 ± 0.31 mm) was different (p = 0.016) to sheep of genotype AB (8.3 ± 0.30 mm) and BB (8.5 ± 0.39 mm). The results suggest that the c.37A/G substitution in ovine GSKIP may affect sheep growth and carcass traits.

Nucleotide Sequence Variation in the Insulin-Like Growth Factor 1 Gene Affects Growth and Carcass Traits in New Zealand Romney Sheep.

Insulin-like growth factor 1 (IGF1) is a mediator of the effects of growth hormone and polymorphism in the IGF1 gene (IGF1) is reported to affect fat deposition in some livestock species. In this study, nucleotide sequence variation in three regions of ovine IGF1 (part of the 5′ flanking region, the exon 3 region, and the exon 4 region) was investigated in 848 New Zealand Romney lambs using PCR-single strand conformation polymorphism (SSCP) analyses to ascertain if single nucleotide polymorphisms (SNPs) existed. Six SNPs were identified across these three regions. The effect of the sequence variation in the exon 3 and exon 4 regions on growth and carcass traits were investigated. One of the PCR-SSCP sequence variants in the exon 3 region was associated with variation in hot carcass weight, carcass fat depth at the 12th rib measured using video imaging and the percentage proportion of leg lean meat, whereas the other was associated with variation in growth rate to weaning. No associations were detected for the other gene regions analyzed. The results suggest that polymorphism in exon 3 of ovine IGF1 has potential for use as a gene-marker for some carcass and growth traits.

Compound heterozygous DUOX2 gene mutations (c.2335-1G>C/c.3264_3267delCAGC) associated with congenital hypothyroidism. Characterization of complex cryptic splice sites by minigene analysis

Iodide Organification defects (IOD) represent 10% of cases of congenital hypothyroidism (CH) being the main genes affected that of TPO (thyroid peroxidase) and DUOX2 (dual oxidasa 2). From a patient with clinical and biochemical criteria suggestive with CH associated with IOD, TPO and DUOX2 genes were analyzed by means of PCR-Single Strand Conformation Polymorphism analysis and sequencing. A novel heterozygous compound to the mutations c.2335-1G>C (paternal mutation, intron 17) and c.3264_3267delCAGC (maternal mutation, exon 24) was identified in the DUOX2 gene. Ex-vivo splicing assays and subsequent RT-PCR and sequencing analyses were performed on mRNA isolated from the HeLa cells transfected with wild-type and mutant pSPL3 expression vectors. The wild-type and c.2335-1G>C mutant alleles result in the complete inclusion or exclusion of exon 18, or in the activation of an exonic cryptic 5′ ss with the consequent deletion of 169 bp at the end of this exon. However, we observed only a band of the expected size in normal thyroid tissue by RT-PCR. Additionally, the c.2335-1G>C mutation activates an unusual cryptic donor splice site in intron 17, located at position −14 of the authentic intron 17/exon 18 junction site, with an insertion of the last 14 nucleotides of the intron 17 in mutant transcripts with complete and partial inclusion of exon 18. The theoretical consequences of splice site mutation, predicted with the bioinformatics NNSplice, Fsplice, SPL, SPLM and MaxEntScan programs were investigated and evaluated in relation with the experimental evidence. These analyses confirm that c.2335-1G>C mutant allele would result in the abolition of the authentic splice acceptor site. The results suggest the coexistence in our patient of four putative truncated proteins of 786, 805, 806 and 1105 amino acids, with conservation of peroxidase-like domain and loss of gp91phox/NOX2-like domain. In conclusion a novel heterozygous compound was identified being responsible of IOD. Cryptic splicing sites have been characterized in DUOX2 gene for the first time. The use of molecular biology techniques is a valuable tool for understanding the molecular pathophysiology of this type of thyroid defects.

Association of wool traits with variation in the ovine KAP1-2 gene in Merino cross lambs

The impact of variation in KRTAP1-2 on wool traits was investigated in 383 Merino cross sheep, sired by six different rams. DNA samples from these sheep were genotyped at KRTAP1-2 using PCR-single strand conformational polymorphism (PCR-SSCP) analysis. Seven previously reported gene variants (A–C, E–H) and two newly identified variants (J and K) were detected, with a frequency of 25.5%, 10.3%, 1.4%, 0.1%, 39.4%, 12.8%, 9.1%, 0.4% and 0.3%, respectively. The effects of variants E, J and K on wool traits were not investigated as they were found at a very low frequency. Of the other six variants; A, B and C were found to be associated with a number of wool traits, but the strongest association was with three correlated traits clean fleece weight (CFW), greasy fleece weight (GFW) and yield. The presence of A was associated with an increase in GFW, CFW and yield. The presence of B was also associated with an increase in GFW, CFW and yield. The presence of C was associated with a decrease in GFW and CFW, but increased yield. Genotypes AF and AG had a higher GFW than FG and FH; AF and AG had a higher CFW than FG; and sheep of AF, AG and BF had a higher yield than those of FF and FG. These results suggest that consideration of ovine KRTAP1-2 may be useful when developing breeding programs based on wool weight.

ATM gene mutations in sporadic breast cancer patients from Brazil.

PurposeThe Ataxia-telangiectasia mutated (ATM) gene encodes a multifunctional kinase, which is linked to important cellular functions. Women heterozygous for ATM mutations have an estimated relative risk of developing breast cancer of 3.8. However, the pattern of ATM mutations and their role in breast cancer etiology has been controversial and remains unclear. In the present study, we investigated the frequency and spectrum of ATM mutations in a series of sporadic breast cancers and controls from the Brazilian population.MethodsUsing PCR-Single Strand Conformation Polymorphism (SSCP) analysis and direct DNA sequencing, we screened a panel of 100 consecutive, unselected sporadic breast tumors and 100 matched controls for all 62 coding exons and flanking introns of the ATM gene.ResultsSeveral polymorphisms were detected in 12 of the 62 coding exons of the ATM gene. These polymorphisms were observed in both breast cancer patients and the control population. In addition, evidence of potential ATM mutations was observed in 7 of the 100 breast cancer cases analyzed. These potential mutations included six missense variants found in exon 13 (p.L546V), exon 14 (p.P604S), exon 20 (p.T935R), exon 42 (p.G2023R), exon 49 (p.L2307F), and exon 50 (p.L2332P) and one nonsense mutation in exon 39 (p.R1882X), which was predicted to generate a truncated protein.ConclusionsOur results corroborate the hypothesis that sporadic breast tumors may occur in carriers of low penetrance ATM mutant alleles and these mutations confer different levels of breast cancer risk.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-0787-z) contains supplementary material, which is available to authorized users.

Infrequent ERBB2 mutations in Chinese patients with non-small cell lung cancer.

ERBB2 mutations have been reported to occur in a subset of patients with lung adenocarcinomas or lung squamous cell carcinomas for some ethnicities, but it is unclear for Chinese patients with lung squamous cell carcinomas up to now. We retrospectively evaluated the status of ERBB2 mutations in a large cross-sectional cohort of 212 Chinese patients with non-small cell lung cancer (NSCLC) diagnosed in several hospitals from southern China during a time period of 1.5 years by polymerase chain reaction (PCR)-based direct sequencing and PCR-single strand conformation polymorphism (PCR-SSCP) analysis. ERBB2 mutation was found in 1 of 49 lung adenocarcinomas (2.0%) and none in lung squamous cell carcinomas and lung adenosquamous carcinomas. It implies the occurrence of ERBB2 mutations is infrequent in Chinese patients with NSCLC, especially in lung squamous cell carcinomas.

Genetic analysis of the human Insulin-like 3 gene: absence of mutations in a Greek paediatric cohort with testicular maldescent.

This study investigated the hypothesis that genetic alterations of the human insulin-like 3 (INSL3) gene are associated with testicular maldescent (TMD). Genomic DNA was extracted and amplified from peripheral blood samples of 170 unrelated children with all possible phenotypical expressions of TMD and 50 volunteers with normal external genitalia from the general paediatric population (controls). PCR-single strand conformation polymorphism analysis was used to screen INSL3 gene for genetic variants. For rapid screening of a detected nonsilent genetic alteration, restriction assay using endonuclease Eag I was further employed. Products were analysed on 2% agarose gel and restriction patterns were visualised by ethidium bromide. Differences in genotype and allelic distributions of nonsilent genetic alterations were evaluated between (i) patients-controls, (ii) familial-sporadic, (iii) bilateral-unilateral and (iv) intra-abdominal-inguinal cases of TMD. No mutations were detected. Three common INSL3 gene polymorphisms (27G>A, 126G>A, 178G>A) unrelated to any particular phenotype of TMD were detected both in patients and controls. These results indicate that INSL3 gene mutations are not a common cause of TMD in the human.

Analysis of the Fungal Flora in Environmental Dust Samples by PCR–SSCP Method

Conventional microbiological techniques yield only limited information on the composition of fungal communities in dust. The aim of this study was to establish and optimize PCR-single strand conformation polymorphism (PCR-SSCP) analysis for investigation of fungal diversity in rural dust samples. Three different DNA extraction protocols were tested on 38 fungal cultures. A total of six known universal fungal primer pairs were tested targeting the 18S rRNA gene, the 28S rRNA gene and the ITS region, respectively. Objective evaluation was performed with respect to the following parameters: efficiency to amplify all 38 strains; separation of seven species from different phylogenetic groups on the SSCP gel; additional bands in PCR-SSCP analysis; possibility to classify the amplified gene fragments to species level. Primer ITS1/ITS4 and PowerSoil™ DNA isolation showed the best performance in most cases and were chosen for further analysis. The detection limit of the developed system was 200 CFU/g dust. Moreover, the reproducibility of the system could be demonstrated, leading to average profile similarities of 94.94% [SD = 2.51] within gels, 93.03% [SD = 4.69] between different days and 87.66% [SD = 6.62] between different gels when testing shed and mattress dust samples. Sequencing allowed identification on species level, in detail: Alternaria alternata, Cladosporium sphaerospermum, Cladosporium cladosporioides as well as the yeasts Candida cabralensis and Candida catenulata. This demonstrates the adaptability of the method. In this study, a standardized system for fungal community analysis was developed that provides reproducible results applicable for epidemiological purposes.

Specific-mutational patterns of p53 gene in bladder transitional cell carcinoma among a group of Iraqi patients exposed to war environmental hazards

BackgroundTo unfold specific-mutational patterns in TP53 gene due to exposures to war environmental hazards and to detect the association of TP53 gene alteration with the depth of bladder cancer.MethodsTwenty-nine bladder carcinomas were analyzed for TP53 alterations. PCR-single strand conformational polymorphism analysis, DNA sequencing and immunohistochemical analysis using monoclonal mouse anti-human p53 antibody (Clone DO-7) were employed.ResultsTP53 gene mutations occurred in 37.9% of the cases while TP53 overexpression occurred in 58.6%. Both of them were associated with deep invasive-tumors. Single mutations were seen in 63.6%, whereas only 27.3% have shown double mutations. Four mutations were frameshifted (30.8%); two of them showed insertion A after codon 244. There was no significant association between TP53 mutations and protein overexpression (P>0.05), while a significant association was observed between TP53 alterations and tumors progression (P ≤ 0.01).ConclusionThe infrequent TP53mutations, especially insertion A and 196 hotspot codon, may represent the specific-mutational patterns in bladder carcinoma among the Iraqi patients who were exposed to war environmental hazards. TP53 alteration associated with bladder cancer progression should be analyzed by both mutational and protein expression analysis.

Aberrant methylation and decreased expression of the RIZ1 gene are frequent in adult acute lymphoblastic leukemia of T-cell phenotype

Retinoblastoma protein-interacting zinc finger, RIZ1, is a tumor suppressor gene that is inactivated in various solid tumors. However, the role of the RIZ1 gene has not been well examined in adult acute lymphoblastic leukemia (ALL). We analyzed the expression and promoter methylation status of the RIZ1 gene in patients with newly diagnosed ALL by quantitative real-time reverse transcription polymerase chain reaction (PCR) and methylation-specific PCR, respectively. RIZ1 expression in 67 cases of ALL (mean 1.043) was decreased compared with that in normal bone marrow (mean 1.471) (p = 0.030). Methylation was detected in 11 of 71 patients (15.5%) but not in healthy controls. Methylation was associated with decreased RIZ1 expression in many ALL cases examined, but this was not statistically significant. In T-ALL, RIZ1 methylation was more frequent (63.6%) than in B-ALL (6.7%) (p < 0.0001) and the decrease of RIZ1 expression was more significant than in B-ALL (p = 0.045). 5-Aza-2′-deoxycytidine treatment of MOLT-4 cells with RIZ1 methylation induced demethylation of RIZ1 and restoration of expression. Forced RIZ1 expression in T-ALL cell lines suppressed cell growth accompanied by G2/M arrest and apoptosis. No mutations were found by PCR-single strand conformation polymorphism analysis in hotspots of the gene. These results suggest that RIZ1 is inactivated in adult ALL, and this inactivation is associated with methylation in T-ALL.

High diversity of the fungal community structure in naturally-occurring Ophiocordyceps sinensis.

Background Ophiocordyceps sinensis (syn. Cordyceps sinensis), which is a parasite of caterpillars and is endemic to alpine regions on the Tibetan Plateau, is one of the most valuable medicinal fungi in the world. “Natural O. sinensis specimens” harbor various other fungi. Several of these other fungi that have been isolated from natural O. sinensis specimens have similar chemical components and/or pharmaceutical effects as O. sinensis. Nevertheless, the mycobiota of natural O. sinensis specimens has not been investigated in detail.Methodology/Principal FindingsBased on the technique of PCR-single-strand conformation polymorphism (PCR-SSCP), the mycobiota of three different sections (stromata, sclerotia, and mycelial cortices) from natural O. sinensis specimens were investigated using both culture-dependent and -independent methods. For the culture-dependent method, 572 fungal strains were isolated, and 92 putative operational taxonomic units (OTUs) were identified from 226 sequenced strains with the threshold of 97%. For the culture-independent method, 490 fungal clones were identified from about 3000 clones of ITS fragments from the whole-community DNA; based on PCR-SSCP analyses, 266 of these clones were selected to be sequenced, and 118 putative OTUs were detected. The overwhelming majority of isolates/clones and OTUs were detected from mycelial cortices; only a few were detected from stromata and sclerotia. The most common OTUs detected with both methods belonged to Ascomycota; however, only 13 OTUs were detected simultaneously by both methods. Potential novel lineages were detected by each of the two methods.Conclusions/SignificanceA great number of fungal species present in the mycobiota of naturally-occurring O. sinensis specimens were detected, and many of them may represent undescribed lineages. That only a few of the same OTUs were detected by both methods indicated that different methods should be used. This study increased our understanding about the fungal community structure of this valuable medicinal herb.

Heterozygous TP63 mutation in a Chinese patient with ectrodactyly-ectodermal dysplasia clefting syndrome without clefting

To determine if alteration in TP63 is responsible for a Chinese patient with ectrodactyly-ectodermal dysplasia clefting (EEC) syndrome, but without cleft palate/lip. Screening of TP63 gene was performed in the patient with EEC syndrome and his family members using PCR-single strand conformational polymorphism (SSCP) analysis, then performed by direct sequencing of the coding region. A C > T substitution at nucleotide position 838 in exon 7 was detected in the patient, and the change predicted a heterozygous missense mutation, Arg280Cys. His parents showed the wild type. The results indicate that the de novo mutation Arg280Cys of the TP63 gene observed in the patient maybe contribute to his EEC syndrome.

PCR-Single-Strand Conformational Polymorphism Method for Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis : Systematic Review and Meta-Analysis

The reference standard methods for drug susceptibility testing of Mycobacterium tuberculosis, such as culture on Lowenstein-Jensen or Middlebrook 7H10/11 medium, are very slow to give results; and due to the emergence of multidrug-resistant M. tuberculosis and extensively drug-resistant M. tuberculosis, there is an urgent demand for new, rapid, and accurate drug susceptibility testing methods. PCR-single-strand conformational polymorphism (PCR-SSCP) analysis has been proposed as a rapid method for the detection of resistance to rifampin, but its accuracy has not been systematically evaluated. We performed a systematic review and meta-analysis to evaluate the accuracy of PCR-SSCP analysis for the detection of rifampin-resistant tuberculosis. We searched the Medline, Embase, Web of Science, BIOSIS, and LILACS databases and contacted authors if additional information was required. Ten studies met our inclusion criteria for rifampin resistance detection. We applied the summary receiver operating characteristic (SROC) curve to perform the meta-analysis and to summarize diagnostic accuracy. The sensitivity of PCR-SSCP analysis for the rapid detection of rifampin-resistant tuberculosis was 0.79 (95% confidence interval [CI], 0.75 to 0.82), the specificity was 0.96 (95% CI, 0.94 to 0.98), the positive likelihood ratio was 16.10 (95% CI, 5.87 to 44.13), the negative likelihood ratio was 0.20 (95% CI, 0.10 to 0.40), and the diagnostic odds ratio was 100.93 (95% CI, 31.95 to 318.83). PCR-SSCP analysis is a sensitive and specific test for the rapid detection of rifampin-resistant M. tuberculosis. Additional studies in countries with a high prevalence of multidrug-resistant M. tuberculosis and also cost-effectiveness analysis are required in order to obtain a complete picture on the utility of this method for rapid drug resistance detection in M. tuberculosis.

Abstract B55: The impact of inactivation of p53 and PTEN tumor suppressors on promotion and progression of human non-small cell lung carcinoma

Abstract Lung cancer is the major cause of cancer-related death worldwide and the five-year survival rate is not improving significantly, remaining at about 15% for all stages. Inactivation of p53 and PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressors are molecular events highly related to the pathogenesis of lung cancer, particularly to non-small cell lung carcinoma (NSCLC). However, we still do not have clear image of the sequence of events in NSCLC promotion and progression, that is, whether inactivation of either of these tumor suppressors is early or late event and what the impact of these events is on NSCLC pathogenesis and patients survival. Therefore, we analyzed mutational inactivation of p53 and inactivation of PTEN by loss of heterozygosity (LOH) in 30 NSCLC patients and correlated obtained data with histopathological parameters that are indicators of initiation and progression of the disease: histological subtype, tumor grade, tumor stage, lymph node invasion and necrosis. PCR-single strand conformation polymorphism (PCR-SSCP) analyses revealed and sequencing procedures confirmed that 33.3% of patients had mutationaly inactivated p53 while fragment analysis of PTEN showed that 43.3% had inactivated PTEN by LOH. In total population of analyzed patients, 13.3% of them carried inactivated p53 only, 23.3% had inactivated PTEN only, while 20.0% of patients had inactivated both tumor suppressors. These data suggest that inactivation of PTEN, either alone or in combination with the inactivation of p53, prevalently occurs in NSCLC. Correlation analyses imply that inactivation of either p53 or PTEN is an early event in pathogenesis of NSCLC, while inactivation of both tumor-suppressors contributes to further tumor progression. In addition, correlation analysis of inactivated PTEN and p53 with the degree of genomic instability of each patient (obtained in previous study) disclosed that mutated p53 is associated with high genomic instability while inactivated PTEN was more frequent in patients with low genomic instability confirming important role of p53 in maintaining the stability of the genome. Finally, Kaplan &amp; Meyer survival analyses revealed that patients with inactivated p53 have much worse prognoses. In order to conclude the study of p53 and PTEN inactivation, analyses of hypermetylation of PTEN promoter and LOH of p53 gene are underway. Citation Information: Cancer Res 2009;69(23 Suppl):B55.

Analysis of microsatellite instability in medulloblastoma

Medulloblastoma is the most common malignant brain tumor in children. The presence of microsatellite instability (MSI) in brain tumors, particularly medulloblastomas, has not been properly addressed. The aim of the present study was to evaluate the role of MSI in medulloblastoma carcinogenesis. MSI status was determined in 36 patients using a pentaplex PCR of quasimonomorphic markers (NR27, NR21, NR24, BAT25, and BAT26). Methylation status of mismatch repair (MMR) genes was achieved by methylation-specific multiplex ligation-dependent probe amplification (MLPA). In addition, MutS homolog 6 (MSH6) expression was determined by immunohistochemistry. Mutations of 10 MSI target genes (TCF4, XRCC2, MBD4, MRE11, ATR, MSH3, TGFBR2, RAD50, MSH6, and BAX) were studied by pentaplex PCR followed by analysis with GeneScan 3.7 software. Mutation analysis of hotspot regions of beta-catenin (CTNNB1) and BRAF (v-raf murine sarcoma viral oncogene homolog B1) oncogenes was performed by PCR single-strand conformation polymorphism analysis followed by direct sequencing. Among the 36 tumors, we found four (11%) cases with instability, one with high MSI and three with low MSI. Methylation analysis of MMR genes in cases presenting shifts on the MSI markers revealed mild hypermethylation of MSH6 in 75% of cases, yet MSH6 was expressed in all the tumors. The MSI target genes MBD4 (methyl-CpG binding domain protein 4) and MRE11 (meiotic recombination 11 homolog A) were mutated in two different tumors. No CTNNB1 or BRAF mutations were found. This study is the most comprehensive analysis of MSI in medulloblastomas to date. We observed the presence of MSI together with mutations of MSI target genes in a small fraction of cases, suggesting a new genetic pathway for a role in medulloblastoma development.

The relationship of P47phox C923T (Ala308Val) polymorphism to cerebral infarction and its effect on plasma lipid levels

To investigate the relationship between C923T (Ala308Val) polymorphism in the exon10 of P47(phox) gene and cerebral infarction and to evaluate the effect of C923T polymorphism on plasma lipid levels. Peripheral blood samples were collected from 110 patients with cerebral infarction, 100 sex and age-matched healthy controls, and 102 members of 10 cerebral infarction pedigrees 19 of which were cerebral infarction patients. The polymorphism in P47(phox) gene was determined by PCR-single strand conformation polymorphism analysis and DNA sequencing. Plasma lipid levels were measured by routine methods. Three gene types, CC, CT, and TT were found in the C923T (Ala308Val) polymorphism site. No statistically significant differences were found in the frequencies of genotypes and alleles of C923T polymorphism between the controls and cerebral infarction patients (all P > 0.05). The serum levels of triglyceride of cerebral infarction patients and controls with the CT genotype were markedly higher than those of the cerebral infarction patients and controls with the CC genotype (both P < 0.05). There is no association between the C923T polymorphism and cerebral infarction. C923Tpolymorphism is associated with plasma lipid metabolism.

Polymorphism at the ovine β3-adrenergic receptor locus (ADRB3) and its association with lamb mortality1

The beta3-adrenergic receptors (ADRB3) are guanine nucleotide-binding protein (G-protein)-coupled receptors predominantly found on the surface of adipocytes. They are major mediators of the lipolytic and thermogenic effects of high catecholamine concentrations, in particular norepinephrine. Recently, variation in the ovine beta3-adrenergic receptor gene (ADRB3) has been associated with lamb survival in Merino sheep. In this study, PCR-single strand conformational polymorphism analysis of part of the ADRB3 intron was used to genotype 13,420 lambs (Borderdale, n = 351; Corriedale, n = 4,260; Coopworth, n = 1,225; Dorset Down, n = 663; Dorset Down x Coopworth, n = 264; Hampshire, n = 230; Merino, n = 4,488; Merino x Coopworth, n = 329; Merino x Polwarth, n = 226; Polwarth, n = 928; Poll Dorset, n = 241; and Suffolk, n = 215) born on 22 farms throughout the South Island of New Zealand. Univariate and multivariate odds ratios calculated for each of the 8 alleles revealed weak to moderate associations of the A and E alleles with cold survival and alleles C and F with cold-related mortality, and a strong association of the D allele with cold-related mortality and total mortality. This variation at the ADRB3 locus can possibly be exploited as a gene marker to increase selection accuracy when breeding for more cold-tolerant lambs.

P53 immunostaining is correlated with reduced survival and is not correlated with gene mutations in resected pulmonary large cell carcinomas

Malignancy of pulmonary large cell carcinomas (LCC) increases from classic LCC through LCC with neuroendocrine morphology (LCCNM) to large cell neuroendocrine carcinomas (LCNEC). However, the histological classification has sometimes proved to be difficult. Because the malignancy of LCC is highly dependent on proteins with functions in the cell cycle, DNA repair, and apoptosis, p53 has been targeted as a potentially useful biological marker. p53 mutations in lung cancers have been shown to result in expression and protein expression also occurs in the absence of mutations. To validate the importance of both p53 protein expression (by immunostaining) and p53 gene mutations in lung LCC (by PCR-single strand conformational polymorphism analysis of exons 5, 6, 7, and 8) and to study their relationships with clinical factors and sub-classification we investigated the correlation of p53 abnormalities in 15 patients with LCC (5 classic LCC, 5 LCNEC, and 5 LCCNM) who had undergone resection with curative intent. Of these patients, 5/15 expressed p53 and none had mutant p53 sequences. There was a negative survival correlation with positive p53 immunostaining (P = 0.05). After adjustment for stage, age, gender, chemotherapy, radiotherapy, and histological subtypes by multivariate analysis, p53 expression had an independent impact on survival. The present study indicates that p53 assessment may provide an objective marker for the prognosis of LCC irrespective of morphological variants and suggests that p53 expression is important for outcome prediction in patients with the early stages of LCC. The results reported here should be considered to be initial results because tumors from only 15 patients were studied: 5 each from LCC, LCNEC and LCCNM. This was due to the rarity of these specific diseases.

Evaluation of PCR–Single-Strand Conformational Polymorphism Analysis for Identification of Gram-Negative Histamine-Producing Bacteria Isolated from Fish

In this study, the previously established PCR–single-strand conformation polymorphism (SSCP) system for detecting and identifying gram-negative histamine-producing bacteria was evaluated. This system can detect and identify histamine-producing bacteria directly from seafood by the use of sequence polymorphisms of the histidine decarboxylase gene (hdc). First, we isolated 81 histamine-producing strains of bacteria from fish samples and analyzed the hdc gene by the PCR-SSCP system. The 22 newly obtained SSCP banding patterns were added to our database, and the utility of our modified database was tested in a second experiment consisting of 18 strains of histamine-producing bacteria isolated from 25 fish samples. Approximately 80% of the histamine-producing strains corresponded to those in the new database. Use of the database for PCR-SSCP analysis, including the band patterns newly added in this study, for the hdc gene makes it possible to more accurately identify histamine producers.