Identification of APC gene mutations in Italian adenomatous polyposis coli patients by PCR-SSCP analysis

The APC gene is a putative human tumor-suppressor gene responsible for adenomatous polyposis coli (APC), an inherited, autosomal dominant predisposition to colon cancer. It is also implicated in the development of sporadic colorectal tumors. The characterization of APC gene mutations in APC patients is clinically important because DNA-based tests can be applied for presymptomatic diagnosis once a specific mutation has been identified in a family. Moreover, the identification of the spectrum of APC gene mutations in patients is of great interest in the study of the biological properties of the APC gene product. We analyzed the entire coding region of the APC gene by the PCR–single-strand conformation polymorphism method in 42 unrelated Italian APC patients. Mutations were found in 12 cases. These consist of small (5–14 bp) base-pair deletions leading to frameshifts; all are localized within exon 15. Two of these deletions, a 5-bp deletion at position 3183–3187 and a 5-bp deletion at position 3926–3930, are present in 3/42 and 7/42 cases of our series, respectively, indicating the presence of mutational hot spots at these two sites.     

Inherited colorectal polyposis and cancer risk of the APC I1307K polymorphism.

Germ-line and somatic truncating mutations of the APC gene are thought to initiate colorectal tumor formation in familial adenomatous polyposis syndrome and sporadic colorectal carcinogenesis, respectively. Recently, an isoleucine-->lysine polymorphism at codon 1307 (I1307K) of the APC gene has been identified in 6%-7% of the Ashkenazi Jewish population. To assess the risk of this common APC allelic variant in colorectal carcinogenesis, we have analyzed a large cohort of unselected Ashkenazi Jewish subjects with adenomatous polyps and.or colorectal cancer, for the APC I1307K polymorphism. The APC I1307K allele was identified in 48 (10.1%) of 476 patients. Compared with the frequency in two separate population control groups, the APC I1307K allele is associated with an estimated relative risk of 1.5-1.7 for colorectal neoplasia (both P=.01). Furthermore, compared with noncarriers, APC I1307K carriers had increased numbers of adenomas and colorectal cancers per patient (P=.03), as well as a younger age at diagnosis. We conclude that the APC I1307K variant leads to increased adenoma formation and directly contributes to 3%-4% of all Ashkenazi Jewish colorectal cancer. The estimated relative risk for carriers may justify specific clinical screening for the 360,000 Americans expected to harbor this allele, and genetic testing in the setting of long-term-outcome studies may impact significantly on colorectal cancer prevention in this population.     

A new deletion polymorphism at D5S71 raises the linkage information on adenomatous polyposis coli: implications for presymptomatic diagnosis

Summary Two independent study-groups, one in Britain and the other in the United States, were the first to report linkage between APC and a TaqI restriction fragment length polymorphism (RFLP) at D5S71 (probe C11p11) on chromosome 5q. They found no recombinants in about 50 informative meioses. The same TaqI RFLP was found to be uninformative for linkage in 15 Dutch polyposis families. The recently reported four base-pair deletion polymorphism (DEL1) at D5S71 has raised the polymorphism information content of this marker from 0.17 to 0.40 in the Dutch population. Seven of 20 polyposis families screened for the DEL1 as well as the TaqI polymorphism gave a combined peak lod score of 5.68 with no recombinants in 37 informative meioses. These data, together with those so far reported in the literature, raise the peak lod score to 17.09 at a recombination fraction of 0.05, the 95% upper confidence limit being 0.09. In combination with the use of another informative marker, D5S81 (probe YN5.48) closely mapping on the other side of APC, the presymptomatic diagnosis of the disease can be made with more than 99.9% certainty. It has to be stressed, however, that the the possible existence of more than one polyposis locus cannot, as yet, be excluded.     

Beta-naphthoflavone inhibits the induction of hepatic oestrogen-dependent proteins by 17alpha-ethynylestradiol in mosquitofish (Gambusia holbrooki)

Quantification of metallothioneins (MTs) is classically associated with a cellular response to heavy metal contamination and is used in the monitoring of disturbed ecosystems. Despite the characterization of several MT genes in marine bivalves, only a few genetic studies have used MT genes as potential biomarkers of pollution. The aim of this study was to assess whether MT gene polymorphism could be used to monitor exposure of the Pacific oyster Crassostrea gigas to heavy metals and to develop specific genetic markers for population genetic studies in relation to environmental stress. The polymorphism of two exons of the C. gigas MT gene CgMT1 were studied using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) in both field populations exposed to various metals concentrations and in experimentally exposed populations. High frequencies of two SSCP types in exons 2 and 3 of the CgMT1 gene have found to be significantly associated with tolerance to metals in experimental and field oyster populations. The use of MT1 gene polymorphism in C. gigas as in the present study should therefore be of high ecological relevance. In conclusion, the analysis of the types in these two CgMT1 gene exons, which can confer a greater tolerance to heavy metals, can constitute a good biomarker of effect of the presence of heavy metals in ecosystems.     

Association of vitamin D receptor BsmI gene polymorphism with the risk of type 2 diabetes mellitus

Abstract

Relationship between vitamin D receptor (VDR) BsmI (rs1544410) gene polymorphism and the type 2 diabetes mellitus (T2DM) susceptibility is still conflicting at present. This meta-analysis was conducted to assess the association between VDR BsmI gene polymorphism and the risk of T2DM. The association studies were identified from PubMed, and Cochrane Library on 1 January 2014, and eligible investigations were included and synthesized using meta-analysis method. Eleven reports were recruited into this meta-analysis for the association of VDR BsmI gene polymorphism with T2DM susceptibility. In overall populations, B allele, BB genotype and bb genotype were not associated with T2DM risk. VDR BsmI gene polymorphism was also not associated with the T2DM risk in Asians and Caucasians. In conclusion, VDR BsmI gene polymorphism was also not associated with T2DM risk in overall populations, Asians and Caucasians. However, more studies should be conducted to confirm it.     

Detection of sequence variations in the adenomatous polyposis coli (APC) gene using denaturing high-performance liquid chromatography

We have evaluated the usefulness of denaturing high performance liquid chromatography (dHPLC) for scanning the adenomatous polyposis coli (APC) gene for point mutations, small deletions, and insertions. Our assay consists of 28 sets of primers to amplify the 15 exons of the APC gene. All PCR reactions were amplified simultaneously using the same reaction conditions in a 96-well format and then analyzed by dHPLC, using empirically determined optimum temperatures for partial fragment denaturation. Previously studied DNA specimens from 47 familial adenomatous polyposis (FAP) patients were analyzed by dHPLC and all mutations were correctly identified and confirmed by sequence analysis. This approach identified a single-base substitution in exon 6 and a 2-bp insertion in exon 15 that initially had not been detected by single-strand conformational polymorphism (SSCP) analysis. A novel mutation in exon 15 of the APC gene, 2065delG (codon 689) that had previously been undetected by the protein truncation test (PTT) was also identified by dHPLC. We present our validation studies of dHPLC technology for APC gene analysis in terms of sensitivity and specificity and compare it to current standard scanning technologies including PTT, SSCP, and conformational sensitive gel electrophoresis (CSGE).     

Two intragenic polymorphisms of the APC-gene detected by PCR and enzymatic digestion

Summary The gene causing adenomatous polyposis coli (APC) has recently been cloned. Three intragenic polymorphisms were reported to be detectable by single-strand conformation polymorphism analysis. Here, we describe an assay using polymerase-chain-reaction-based amplification and subsequent enzymatic digestion of genomic sequences to identify polymorphic sites at nucleotide positions 1458 and 5037 of the APC gene.     

Direct analysis for familial adenomatous polyposis mutations

The spectrum of disease causing mutations is immense. It just so happens that the overwhelming majority of genetic alterations in the APC gene with leads to adenomatous polyposis coli generate truncated gene products. This observation lead to the development of the in vitro synthesis protein assay (protein truncation test) which is a sensitive method to detect these truncated gene products from patient samples. This article describes the assay to detect truncated proteins for the APC gene, which can also be applied to other disease causing genetic alterations which commonly lead to truncations such in HNPCC, von Hippel-Lindau, osteogenesis imperfecta, retinoblastoma, BCRA1, beta-thalassemia, hemophilia B, Duchenene and Becker muscular dystrophy.     

An E2 enzyme Ubc11 is required for ubiquitination of Slp1/Cdc20 and spindle checkpoint silencing in fission yeast

For ordered mitotic progression, various proteins have to be regulated by an ubiquitin ligase, the anaphase-promoting complex or cyclosome (APC/C) with appropriate timing. Recent studies have implied that the activity of APC/C also contributes to release of mitotic checkpoint complexes (MCCs) from its target Cdc20 in the process of silencing the spindle assembly checkpoint (SAC). Here we describe a temperature-sensitive mutant (ubc11-P93L) in which cell cycle progression is arrested at mitosis. The mutant grows normally at the restrictive temperature when SAC is inactivated, suggesting that the arrest is not due to abnormal spindle assembly, but rather due to prolonged activation of SAC. Supporting this notion, MCCs remain bound to APC/C even when SAC is satisfied. The ubc11⁺ gene encodes one of the two E2 enzymes required for progression through mitosis in fission yeast. Remarkably, Slp1 (a fission yeast homolog of Cdc20), which is degraded in an APC/C-dependent manner, stays stable throughout the cell cycle in the ubc11-P93L mutant lacking the functional SAC. Other APC/C substrates, in contrast, were degraded on schedule. We have also found that a loss of Ubc4, the other E2 required for progression through mitosis, does not affect the stability of Slp1. We propose that each of the two E2 enzymes is responsible for collaborating with APC/C for a specific set of substrates, and that Ubc11 is responsible for regulating Slp1 with APC/C for silencing the SAC.     

aPCR-SSCP and DNA sequencing detecting two silent SNPs at KAP8.1 gene in the cashmere goat

Keratin-associated proteins 8.1 gene (KAP8.1) is a structural gene responsible for the cashmere. KAP8.1 protein contains high glycine and tyrosine, which concerns regulation and function of the matrix structure fiber. In this study, the polymorphism of KAP8.1 gene was detected by methods of aPCR-SSCP (asymmetric polymerase chain reaction single-strand conformation polymorphism) and DNA sequencing in 791 individuals from two breeds. The results showed that there were two mutations in this gene. The mutations were described as c.63 T>G and c.66 C>G, which would result in two synonymous mutations in KAP8.1 protein. The findings go against previous research, in which there was not polymorphism at KAP8.1 gene. The reasons might be that different cashmere breeds were detected in two studies. Further analysis of results leads us to believe that the polymorphism of KAP8.1 gene might be relevant to fiber diameter.     

Association of vitamin D receptor gene polymorphism with the risk of lung cancer: a meta-analysis

Abstract

Relationship between vitamin D receptor (VDR) gene polymorphism and the risk of lung cancer from the published reports are still conflicting. This study was conducted to evaluate the relationship between VDR TaqI (rs731236), BsmI (rs1544410) and ApaI (rs7975232) gene polymorphism and the risk of lung cancer using meta-analysis method. The association studies were identified from PubMed and Cochrane Library on 1 December 2013, and eligible investigations were included and synthesized using meta-analysis method. Six reports were recruited into this meta-analysis for the association of VDR gene polymorphism with lung cancer susceptibility. In the meta-analysis for ApaI gene polymorphism, AA genotype was associated with the risk of lung cancer in Asians. In the meta-analysis for BsmI gene polymorphism, B allele, BB genotype and bb genotype were associated with lung cancer in Asians, and B allele bb genotype were associated with lung cancer risk in overall populations; furthermore, bb genotype was associated with lung cancer risk in Caucasians. In the meta-analysis for TaqI gene polymorphism, t allele and TT genotype were associated with lung cancer in overall populations and in Caucasians. In conclusion, B allele bb genotype t allele and TT genotype were associated with lung cancer risk in overall populations. AA genotype, B allele, BB genotype and bb genotype were associated with the risk of lung cancer in Asians. Furthermore, bb genotype t allele and TT genotype was associated with lung cancer risk in Caucasians. However, more studies should be conducted to confirm it.     

Analysis of activity in fMRI data using affinity propagation clustering

Clustering analysis is a promising data-driven method for the analysis of functional magnetic resonance imaging (fMRI) data. The huge computation load, however, makes it difficult for the practical use. We use affinity propagation clustering (APC), a new clustering algorithm especially for large data sets to detect brain functional activation from fMRI. It considers all data points as possible exemplars through the minimisation of an energy function and message-passing architecture, and obtains the optimal set of exemplars and their corresponding clusters. Four simulation studies and three in vivo fMRI studies reveal that brain functional activation can be effectively detected and that different response patterns can be distinguished using this method. Our results demonstrate that APC is superior to the k-centres clustering, as revealed by their performance measures in the weighted Jaccard coefficient and average squared error. These results suggest that the proposed APC will be useful in detecting brain functional activation from fMRI data.     

A PvuII polymorphism of the low density lipoprotein receptor gene is not associated with plasma concentrations of low density lipoproteins including LP(a)

Lipoprotein(a) [Lp(a)] is a low density lipoprotein (LDL), in which apolipoprotein B-100 (apo B-100) is attached to apolipoprotein(a) [apo(a)], a glycoprotein of variable size. Lp(a) may be as atherogenic as LDL. In normal populations, Lp(a) concentrations in plasma are largely determined by the apo(a) gene locus on chromosome 6, but regulation of synthesis and catabolism of Lp(a) is poorly understood. In some studies, a PvuII restriction fragment length polymorphism (RFLP) in the LDL receptor gene seems to affect concentrations of LDL in plasma, and other studies have indicated that Lp(a) catabolism could be mediated by the LDL receptor. We therefore expected that the PvuII polymorphism in the LDL receptor gene might be associated with Lp(a) levels in 170 Caucasian men aged 40 years, selected to have a high representation of low molecular weight apo(a) phenotypes. However, plasma concentrations of cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides and Lp(a) were all unrelated to the LDL receptor gene PvuII polymorphism both in the group as a whole and when it was subgrouped by apo(a) phenotype. Therefore our data do not support the concept that this particular LDL receptor gene polymorphism is associated with LDL receptor function, and our data therefore neither support nor rule out a role for the LDL receptor in Lp(a) catabolism.     

Identification of a human specificAlu insertion in the factor XIIIB gene

The factor XIIIB gene was examined to determine the nature of a previously described 300 bp restriction fragment length polymorphism (RFLP) seen in the human population. Polymerase chain reaction analysis of different regions within the factor XIIIB gene was carried out to define a high resolution map of the region encompassing the polymorphism, followed by DNA sequence analysis. AnAlu insertion was found to be the source of this variation. ThisAlu repeat is a member of the human specific-1 (HS-1) subfamily, although one of the five diagnostic nucleotides is a cattarhine specific (CS) subfamily mutation, suggesting that it may represent an intermediate form in the evolution between these two subfamilies. Subsequently, we developed a PCR-based assay to detect the polymorphism, rendering it a more useful marker for genetic linkage studies and genome mapping. This insertion is also a valuable polymorphism for human population studies, as demonstrated by the large variations in allele frequencies seen in three population groups.     

Association of vitamin D receptor BsmI (rs1544410) gene polymorphism with the chronic kidney disease susceptibility

Abstract

Association of vitamin D receptor (VDR) BsmI (rs1544410) gene polymorphism with the chronic kidney disease (CKD) susceptibility from the published reports are still conflicting. This meta-analysis was performed to evaluate the relationship between VDR BsmI (rs1544410) gene polymorphism and the risk of CKD. The association studies were identified from PubMed, Cochrane Library and China Biological Medicine Database on 1 March 2014, and eligible investigations were included and synthesized using meta-analysis method. Nine reports were recruited into this meta-analysis for the association of VDR BsmI gene polymorphism with CKD susceptibility. In this meta-analysis for overall populations, the BsmI B allele BB genotype and bb genotype were not associated with the risk of CKD (B allele: OR?=?1.12, 95% CI: 0.88–1.44, p?=?0.36; BB genotype: OR?=?1.15, 95% CI: 0.81–1.62, p?=?0.43; bb genotype: OR?=?0.86, 95% CI: 0.61–1.20, p?=?0.36). Furthermore, VDR BsmI gene polymorphism was not associated with CKD susceptibility in Asians and in Caucasians. In conclusion, the BsmI gene polymorphism was not associated with CKD susceptibility in overall populations, in Asians and in Caucasians. However, more studies should be conducted to confirm it.     

Genetic studies of the leptin receptor gene in morbidly obese French Caucasian families

Family studies have shown that in some populations up to 75% of the variation of body mass index can be explained by genetic factors. However, in humans, no major obesity gene has been identified to date. In contrast, there are a number of genetically well defined animal models for obesity. In two of those models (ob/ob and db/db), defects in the same pathway are responsible for obesity. Recently, some evidence has been found for the OB gene also being involved in human obesity. In this study we investigated the potential role of the OB receptor (OBR) in the etiology of massive obesity in humans using familial linkage analyses and case-control association studies. The typing of two microsatellite markers (D1S198 and D1S209), flanking the OBR gene, in 256 sib pairs showed no evidence for linkage with obesity. In order to be able to detect small gene effects, association studies with a 3′-UTR insertion/deletion polymorphism were carried out. The results of these analyses remained non-significant (χ² = 3.442, P = 0.18). However, subjects heterozygous for the insertion/deletion polymorphism showed a slight trend towards lower insulin values 30 min after an oral glucose load compared to homozygous individuals (P = 0.02). In summary, our results do not support a major role of the human OBR gene in the development of morbid obesity in our population. Received: 4 December 1996 / Accepted: 25 June 1997     

Linkage disequilibrium predicts physical distance in the adenomatous polyposis coli region.

To test the reliability of linkage-disequilibrium analysis for gene mapping, we compared physical distance and linkage disequilibrium among seven polymorphisms in the adenomatous polyposis coli (APC) region on chromosome 5. Three of them lie within the APC gene, and two lie within the nearby MCC (mutated in colon cancer) gene. One polymorphism lies between the two genes, and one is likely to be 5' of MCC. Five of these polymorphisms are newly reported. All polymorphisms were typed in the CEPH kindreds, yielding 179-205 unrelated two-locus haplotypes. Linkage disequilibrium between each pair of polymorphisms is highly correlated with physical distance in this 550-kb region (correlation coefficient -.80, P < .006). This result is replicated in both the Utah and non-Utah CEPH kindreds. There is a tendency for greater disequilibrium among pairs of polymorphisms located within the same gene than among other pairs of polymorphisms. Trigenic, quadrigenic, three-locus, and four-locus disequilibrium measures were also estimated, but these measures revealed much less disequilibrium than did the two-locus disequilibrium measures. A review of 19 published disequilibrium studies, including this one, shows that linkage disequilibrium nearly always correlates significantly with physical distance in genomic regions > 50-60 kb but that it does not do so in smaller genomic regions. We show that this agrees with theoretical predictions. This finding helps to resolve controversies regarding the use of disequilibrium for inferring gene order. Disequilibrium mapping is unlikely to predict gene order correctly in regions < 50-60 kb in size but can often be applied successfully in regions of 50-500 kb or so in size. It is convenient that this is the range in which other mapping techniques, including chromosome walking and linkage mapping, become difficult.     

Genetics of fat tissue accumulation in pigs: a comparative approach

Fatness traits are important in pig production since they influence meat quality and fattening efficiency. On the other hand, excessive fat accumulation in humans has become a serious health problem due to worldwide spread of obesity. Since the pig is also considered as an animal model for numerous human diseases, including obesity and metabolic syndrome, comparative genomic studies may bring new insights into genetics of fatness/obesity. Input of genetic factors into phenotypic variability of these traits is rather high and the heritability coefficient (h ²) of these traits oscillates around 0.5. Genome scanning revealed the presence of more than 500 QTLs for fatness in the pig genome. In addition to QTL studies, many candidate gene polymorphisms have been analyzed in terms of their associations with pig fatness, including genes encoding leptin (LEP) and its receptor (LEPR), insulin-like growth factor 2 (IGF-2), fatty acid-binding proteins (FABP3 andFABP4), melanocortin receptor type 4 (MC4R), and theFTO (fat mass and obesity-associated) gene. Among them, a confirmed effect on pig fatness was found for a well-known polymorphism of theIGF-2 gene. In humans the strongest association with predisposition to obesity was shown for polymorphism of theFTO gene, while in pigs such an association seems to be doubtful. The development of functional genomics has revealed a large number of genes whose expression is associated with fat accumulation and lipid metabolism, so far not studied extensively in terms of the association of their polymorphism with pig fatness. Recently, epigenomic mechanisms, mainly RNA interference, have been considered as a potential source of information on genetic input into the fat accumulation process. The rather limited progress in studies focused on the identification of gene polymorphism related with fatness traits shows that their genetic background is highly complex.     

Relationship between PPARγ Pro12Ala gene polymorphism and type 2 diabetic nephropathy risk in Asian population: results from a meta-analysis

Abstract

The relationship between peroxisome proliferator-activated receptor gamma (PPARγ) Pro12Ala gene polymorphism and type 2 diabetic nephropathy (T2DN) risk in Asians is still unclear. This study was performed to evaluate if there was an association between the PPARγ Pro12Ala gene polymorphism and T2DN risk in Asians using meta-analysis. The relevant reports were searched and identified from PubMed, Cochrane Library and CBM-disc (China Biological Medicine Database) on 1 October 2013, and eligible studies were included and synthesized. Ten reports were recruited into this meta-analysis for the association of the PPARγ Pro12Ala gene polymorphism with T2DN risk. The Pro12Ala gene polymorphism in the Asian population was shown to be not associated with T2DN risk (Ala/Ala: OR?=?0.67, 95% CI: 0.22–2.00, p?=?0.47; Pro/Pro: OR?=?1.77, 95% CI: 0.82–1.65, p?=?0.39; Ala allele: OR?=?0.74, 95% CI: 0.47–1.16, p?=?0.19). In the sensitivity analysis according to Hardy-Weinberg equilibrium (HWE), the control source from hospital, the control source from population, the genotyping methods using PCR-RFLP, the genotyping methods using Taqman, sample size of case (≥100), the association of the PPARγ Pro12Ala gene polymorphism with T2DN risk was also not found. Interestingly, in the sensitivity analysis according to sample size of case (<100), Ala allele was associated with T2DN risk, but not the Pro/Pro genotype. However, the sample size for sensitivity analysis according to sample size of case (<100) was relatively small and therefore, the results should be interpreted with care. In conclusion, the PPARγ Pro12Ala gene polymorphism was not associated with T2DN risk in Asians. However, Ala allele was associated with T2DN risk when the sample size of case was less than 100. Nonetheless, additional studies are required to firmly establish a correlation between the PPARγ Pro12Ala gene polymorphism and T2DN risk in Asians.     

Prion protein gene polymorphism in healthy and BSE-affected Slovak cattle

Variation of thePrP gene was examined in healthy and BSE-affected Slovak cattle. According to previous studies, the 23-bp indel polymorphism is supposed to be associated with higher susceptibility to BSE. We investigated 301 samples from healthy cattle of various Slovak breeds and 24 samples obtained from tissues of BSE-affected cattle in Slovakia. We examined thePrP gene for the 23-bp indel polymorphism in the putative promoter region, 12-bp indel polymorphism in the first intron of thePrP gene, variations in number of octapeptide repeat units, and presence of the silent AAC>AAT transition in codon 192 within the protein-coding region of thePrP gene. Altogether we found 23 different genotypes in the group of healthy cattle and only 6 genotypes in the group of BSE-affected cattle. Comparison of homozygotes for the 23-bp insertion and heterozygotes showed significant differences (P < 0.05) in genotype distribution between the examined groups. Thereby the homozygous insertion genotype at the 23-bp indel polymorphism site in the promoter region of the prion protein gene seems to have a protective effect against BSE.