BACKGROUND Common variants in the CHRNA5-A3-B4 gene cluster have been shown to be associated with nicotine dependence and alcohol use disorders (AUDs) and related traits, including the level of response (LR) to alcohol. Recently, rare variants (MAF < 0.05) in CHRNB4 have been reported to be associated with a decreased risk of developing nicotine dependence. However, the role of rare variants in the CHRNA5-A3-B4 gene cluster to the LR to alcohol has not yet been established. METHODS To determine whether rare variants in the CHRNA5-A3-B4 gene cluster contribute to the LR to alcohol, the coding regions of these 3 genes were sequenced in 538 subjects from the San Diego Sibling Pair study. RESULTS The analyses identified 16 rare missense variants, 9 of which were predicted to be damaging using in silico analysis tools. Carriers of these variants were compared to noncarriers using a family-based design for each gene and for the gene cluster as a whole. In these analyses, a CHRNA5 carrier status was significantly associated with the phenotype related to the feeling of intoxication experienced during the alcohol challenge (p = 0.039). CONCLUSIONS These results indicate that rare genetic variation in the CHRNA5-A3-B4 gene cluster contributes modestly to the LR to alcohol in the San Diego Sibling Pair study and may protect against AUDs. However, replication studies are needed to confirm our findings.

Astrocytoma, the most common brain tumor in humans, is usually malignant and virtually incurable. Two types of malignant astrocytomas can be distinguished histopathologically: anaplastic astrocytoma and glioblastoma multiforme. Studies using DNA markers that detect restric tion fragment length polymorphisms have shown that loci on chromo somes 10 and 17p are lost frequently in tumor DNA from malignant astrocytoma patients, suggesting that tumor suppressor genes important in astrocytoma tumorigenesis may be present on 2 different chromosomes. To identify additional regions of chromosome loss, we carried out an allelotype analysis of 41 malignant astrocytoma patients using restriction fragment length polymorphism markers for each arm of every human autosome. Loss of heterozygosity was found for every autosome except chromosome 21, indicating an even greater complexity of genomic alter ations than reported previously. Many tumors showed loss of heterozy gosity for multiple chromosomes and the number of chromosomes in volved correlated with tumor histopathology. A high-resolution restriction fragment length polymorphism study of chromosome 10 loci in these patients showed that loss of broad regions of chromosome 10 was a common event, particularly in glioblastoma multiforme. An allelotype analysis has been carried out on only one other tumor, human colorectal carcinoma. Different profiles of alÃ-ele loss were observed in malignant astrocytoma and colorectal carcinoma, suggesting that the genetic events leading to these 2 human cancers may proceed along different pathways.

Previously we have conducted a genome-wide search for inflammatory bowel disease susceptibility loci in a large European cohort. Results from this study demonstrated suggestive evidence of linkage to loci at chromosomes 1q, 6p, and 10p and replicated linkages on chromosomes 12 and 16. Recently, NOD2/CARD15 on chromosome 16q12 has been found to be strongly associated with Crohn's disease. In order to determine if there are other loci in the genome that interact with the three associated functional variants in CARD15 (R702W, G908R, 1007fs), we have stratified our large inflammatory bowel disease genome scan cohort by dividing pedigrees into two groups stratified by CARD15 variant genotype. The two pedigree groups were analysed using non-parametric allele sharing methods. The group of pedigrees that contained one of the three CARD15 variants had two suggestive linkage results occurring in 6p (lod = 3.06 at D6S197, IBD phenotype) and 10p (lod=2.29 at D10S197, CD phenotype). In addition, at 16q12 where CARD15 is located, the original genome scan had a peak lod score of 2.18 at D16S415 (CD phenotype). The stratified pedigree cohort containing one of three CARD15 variants had a peak lod score of 0.90 at D16S415 (CD phenotype), accounting for approximately less than half of the genetic evidence for linkage at this locus. This result is in agreement with the existence of a substantial number of private variants at the NOD2/CARD15 locus. Interaction with NOD2/CARD15 needs to be considered in future gene identification efforts on chromosomes 6 and 10.

To identify proteins that may specifically characterize colonic polyps we have investigated the abundance of numerous proteins in epithelial cells from 15 normal colon specimens and 13 colonic polyps, using two-dimensional gel analysis to detect possible differences in expression. Silver-stained digitized images of the gels were analyzed with Melanie II 2.1 software. We consistently detected more than 700 protein spots on each gel, and found that the intensity of 59 of them was significantly altered in polyp specimens (Wilcoxon test assuming P less than or equal to 0.05). Immunostaining, microsequencing and mass spectrometry (matrix-assisted laser desorption/ionization - time of flight; MALDI-TOF) techniques were used to identify these proteins and selected others that did not show differential regulation. The expression of numatrin (nucleophosphine/B23), hsp 70, and hsp60 was increased in polyps; levels of fatty acid binding protein (L-FABP), 14-3-3 sigma, citokeratin 20, cytochrome c oxidase polypeptide Va, Rho GDP-dissociation inhibitor (Rho GDI), and beta- and gamma-actins were decreased. Although the levels of expression of a given protein often varied among Polyp specimens, it generally held true that the direction of variation (up or down) remained constant across the panel. We concluded that proteins showing constant differential regulation across all or most of the polyp specimens represent the most characteristic regulatory pathways in colon polyps, while more sporadic variations reflect characteristics of individual polyps.

The adenomatous polyposis coli (APC) gene is considered as the true gatekeeper of colonic epithelial proliferation: It is mutated in the majority of colorectal tumors, and mutations occur at early stages of tumor development in mouse and man. These mutant proteins lack most of the seven 20-amino-acid repeats and all SAMP motifs that have been associated with down-regulation of intracellular beta-catenin levels. In addition, they lack the carboxy-terminal domains that bind to DLG, EB1, and microtubulin. APC also appears to be essential in development because homozygosity for mouse Ape mutations invariably results in early embryonic lethality. Here, we describe the generation of a mouse model carrying a targeted mutation at codon 1638 of the mouse Ape gene, Apc1638T, resulting in a truncated Ape protein encompassing three of the seven 20 amino acid repeats and one SAMP motif, but missing all of the carboxy-terminal domains thought to be associated with tumorigenesis. Surprisingly, homozygosity for the Apc1638T mutation is compatible with postnatal life. However, homozygous mutant animals are characterized by growth retardation, a reduced postnatal viability on the B6 genetic background, the absence of preputial glands, and the formation of nipple-associated cysts. Most importantly, Apc(1638T/1638T) animals that survive to adulthood are tumor free. Although the full complement of Apc1638T is sufficient for proper beta-catenin signaling, dosage reductions of the truncated protein result in increasingly severe defects in beta-catenin regulation. The SAMP motif retained in Apc1638T also appears to be important for this function as shown by analysis of the Apc1572T protein in which its targeted deletion results in a further reduction in the ability of properly controlling beta-catenin/Tcf signaling. These results indicate that the association with DLG, EB1, and microtubulin is less critical for the maintenance of homeostasis by APC than has been suggested previously, and that proper beta-catenin regulation by APC appears to be required for normal embryonic development and tumor suppression.

. Inherited mutations of the APC gene predispose carriers to multiple adenomatous polyps of the colon and rectum and to colorectal cancer. Mutations located at the extreme 5' end of the APC gene, however, are associated with a less severe disease known as attenuated adenomatous polyposis coli (AAPC). Many individuals with AAPC develop relatively few colorectal polyps but are still at high risk for colorectal cancer. We report here the identification of a 5' APC germline mutation in five separately ascertained AAPC families from Newfoundland, Canada. This disease-causing mutation is a single base-pair change (G to A) in the splice-acceptor region of APC intron 3 that creates a mutant RNA without exon 4 of APC . The observation of the same APC mutation in five families from the same geographic area demonstrates a founder effect. Furthermore, the identification of this germline mutation strengthens the correlation between the 5' location of an APC disease-causing mutation and the attenuated polyposis phenotype.

Most inherited mutant alleles of the adenomatosis polyposis coli gene ( APC ) cause the appearance of large numbers of colon polyps 1 , 2 , 3 , 4 , the familial polyposis syndrome. (These mutant alleles are designated APC P alleles.) A subset of APC mutations, the attenuated or APC AP alleles, predispose to only a few colon polyps 5 . This leads to the hypothesis that if mutation of the inherited normal allele is rate limiting in polyp development, the increased number of polyps associated with the APC P allele indicates that the frequency of mutations that can lead to polyp formation is higher among APC P carriers than among APC AP carriers. We have previously suggested that the APC protein might modulate the frequency of mutations, such as loss of heterozygosity 6 (LOH), necessary for colon polyp formation 5 . We thus reasoned that tumours from patients who carry an APC AP allele might show a reduced frequency of LOH compared with tumours from patients who carry an APC P allele. Loss of AAPC mutant alleles is designated as LOH AP . Screening of tumours from APC AP carriers revealed a reduction of LOH compared with that of an unselected group of polyposis patients 7 . In fact, no loss of the inherited APC N allele was observed, although sequencing showed that the inherited APC N allele had frequently undergone point mutations and small deletions in the tumours. A low frequency loss of the inherited APC AP allele was seen. These findings support the suggestion that the APC AP allele has residual gene activity and that this activity modulates the spectrum and frequency of mutations that lead to adenoma formation.

Germ-line mutations in the APC gene cause adenomatous polyposis coli (APC), a syndrome in which patients develop hundreds to thousands of precancerous adenomatous colorectal polyps, We described previously an attenuated form of APC (AAPC) resulting from very 5' mutations in APC in which affected patients exhibit fewer colorectal polyps and a later age of onset of colorectal cancer. However, because striking variations in colorectal polyp numbers occur among patients carrying identical AAPC mutations, alleles of another gene may modify the expression of the APC disease phenotype, We tested the hypothesis that loss of function of human cyclooxygenase 2 (COX-2), known to modify the APC phenotype in the Apc(Delta 716) mouse, results in a decreased tumor burden in AAPC patients that develop very few colorectal polyps. Genomic DNA sequence analysis of human COX-2 revealed a silent mutation in exon 3 that was evenly distributed between two classes of patients with AAPC, those with small or Large numbers of colorectal polyps. We also found no difference in Levels of COX-2 mRNA in transformed blood lymphocytes among AAPC patients of either class or patients with classical APC, and no alterations that correlated with a lesser or greater number of colorectal polyps were detectable within approximately the first 1 kb of the promoter sequence. Therefore, mutation of the human COX-2 gene does not appear to be responsible for a low tumor burden among AAPC subjects.

Mutation of the adenomatous polyposis coli (APC) gene is an early step in the initiation of colon cancer. Because the distribution pattern of a protein within the cell can provide important clues as to function, we have used a combination of immunofluorescence microscopy and biochemical fractionation to determine the location of APC protein in epithelial cells. Immunofluorescence microscopy placed full-length APC protein in both the nucleus and the cytoplasm. The nuclear APC protein was concentrated in discrete subnuclear regions, including nucleoli, whereas the cytoplasmic APC protein concentrated at the leading edge of migrating cells. Colocalization of APC protein with rRNA confirmed a nucleolar localization. These immunocytochemical findings have been supported by cell fractionation, which demonstrated that full-length APC protein was located in both the membrane/cytoskeletal and the nuclear fractions.

Human colon carcinoma cell line HCT116 harbors an oncogenic Ki-ras gene. Introduction of an exogenous full-length NF1 gene or its GTPase-activating protein (GAP)-related domain suppressed the tumor-forming ability of this cell line in nude mice. A GAP-related domain peptide carrying a K1423E mutation, which shows greatly diminished GAP activity but a normal binding affinity for p2lras-GTP, was also tested. This construct was able to suppress tumor formation by the HCT116 cell line, thus ruling out the possibility that the observed tumor suppression is due to the GAP activity of NF1. Reduced Raf-1 kinase activity in cells which expressed these NF1 constructs suggested that neurofibromin may interfere with the interaction between Ras and Raf. Introduction of a mutationally activated Raf-1 kinase domain reversed tumor suppression by neurofibromin, implicating Raf-1 as the primary downstream transducer of the oncogenic Ras signal. An increase in apoptotic cell death, which could be delayed by activated Raf-1 kinase, was also seen in cells carrying the exogenous NF1 gene.

DOC-2 is a human gene originally identified as a 767-bp cDNA fragment isolated from normal ovarian epithelial cells by differential display against ovarian carcinoma cells. We have now determined the complete cDNA sequence of the 3.2-kb DOC-2 transcript and localized the gene to chromosome 5. A 12.5-kb genomic fragment at the 5′-end of DOC-2 has also been sequenced, revealing the intron–exon structure of the first eight exons (788 bases) of the DOC-2 gene. Translation of the DOC-2 cDNA predicts a hydrophobic protein of 770 amino acid residues with a molecular weight of 82.5 kDa. Comparison of the DNA and amino acid sequences of DOC-2 to publicly accessible sequence databases revealed 83% identity to p96, a murine protein of similar size, thought to be a mitogen-responsive phosphoprotein. In addition, about 45% identity was observed between the first 140 N-terminal residues of DOC-2 and theCaenorhabditas elegansM110.5 andDrosophila melanogasterDab genes.

While germline mutations in the adenomatous polyposis coli (APC) gene cause the hereditary colon cancer syndrome (familial adenomatous polyposis (FAP)), the role of common germline APC variants in sporadic adenomatous polyposis remains unclear. We studied the association of eight APC single nucleotide polymorphisms (SNPs), possibly associated with functional consequences, and previously identified gene–environment (dietary fat intake and hormone replacement therapy (HRT) use) interactions, in relation to advanced colorectal adenoma in 758 cases and 767 sex- and race-matched controls, randomly selected from the screening arm of the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. Cases had at least one verified advanced adenoma of the distal colon; controls, a negative sigmoidoscopy. We did not observe an association between genotypes for any of the eight APC SNPs and advanced distal adenoma risk (Pglobal gene-based = 0.92). Frequencies of identified common haplotypes did not differ between cases and controls (Pglobal haplotype test = 0.97). However, the risk for advanced distal adenoma was threefold higher for one rare haplotype (cases: 2.7%; controls: 1.6%) (odds ratio (OR) = 3.27; 95% confidence interval (CI) = 1.08–9.88). The genetic association between D1822V and advanced distal adenoma was confined to persons consuming a high-fat diet (Pinteraction = 0.03). Similar interactions were not observed with HRT use. In our large, nested case-control study of advanced distal adenoma and clinically verified adenoma-free controls, we observed no association between specific APC SNPs and advanced adenoma. Fat intake modified the APC D1822V-adenoma association, but further studies are warranted.

Mutation of Pla2g2a, a secretory phospholipase A(2) gene, dramatically increases the number of intestinal polyps that develop in the multiple intestinal neoplasia (Min) mouse, a murine model for adenomatous polyposis coli in humans. We tested the hypothesis that mutation of the human homologue(s) of this gene might be responsible for the more severe phenotype (hundreds of polyps) seen in a subset of individuals with attenuated adenomatous polyposis coli (AAPC). DNA sequence analysis demonstrated that alterations of PLA2G2A, as well as related genes PLA2G2C and PLA2G5, were evenly distributed between three classes of AAPC subjects: those with small, intermediate, and large numbers of adenomatous colonic polyps. Among 67 additional unrelated AAPC subjects, a stop mutation in PLA2G2C did not correlate with an increased burden of adenomatous polyps. Therefore, mutation of the human homologue(s) of murine Pla2g2a does not appear to be responsible for phenotypic variation among subjects with AAPC.

Reverse transcription-PCR combined with either (a) restriction enzyme digestion and repeat PCR or (b) ligase chain reaction has identified two new alternatively spliced transcripts of the adenomatous polyposis coli (APC) gene. In one of these transcripts exons 1-4 and the first 16 bases of exon 5 are deleted; in the other exons 2-4 and the first 16 bases of 5 are deleted. Both transcripts use an intraexonic splice acceptor in exon 5. These transcripts delete exons mutated in attenuated APC (3 and 4) and could account for the reduction in severity of this variant.

Genetic linkage analyses with genotypic data obtained from four CEPH reference families initially assigned 24 new PCR-based markers to chromosome 17 and located the markers at specific intervals of an existing genetic map of chromosome 17p. Each marker was additionally genotyped with an ordered set of obligate, phase-known recombinant chromosomes. The breakpoint-mapping panels for each family consisted of two parents, one sib with a nonrecombinant chromosome, and one or more sibs with obligate recombinant chromosomes. The relative order of markers was determined by sorting segregation patterns of new markers and ordered anchor markers and by minimizing double-recombination events. Consistency of segregation patterns with multiple flanking loci constituted support for order. A genetic map of chromosome 17p was completed with 39 markers in 23 clusters, with an average space of 3 cM between clusters. The collection of informative genotypes was highly efficient, requiring fivefold fewer genotypes than would be collected with all the CEPH families. Given the availability of large numbers of highly informative PCR-based markers, meiotic breakpoint mapping should facilitate construction of a human genomic map with 1-cM resolution.

Six novel polymorphic short sequence repeats were identified and localized on the linkage map of human chromosome 21 by genotyping the CEPH reference pedigrees. One of these markers, the tetrameric (AAAG)n repeat D21S1245, was found to be hypermutable. In the DNAs from lymphoblastoid cell lines of members of the 40 CEPH families a total of 18 new alleles were detected. These new alleles, sometimes appearing in mosaic forms, arose equally in paternal and maternal DNAs, and could be equally larger or smaller than the alleles from which they were derived. The larger alleles of D21S1245 are more prone to be converted to new alleles. None of the new alleles with mosaicism were present in the corresponding genomic blood DNA, and therefore originated during or after the establishment of the lymphoblastoid cell lines; half of the new alleles without mosaicism were also found in genomic blood DNA of the appropriate CEPH individuals. The range of germline mutation rate observed in the 716 meioses examined was 0.56-1.4 x 10(-2); the range of somatic mutations observed in the 405 cell lines examined was 1.96-3.46 x 10(-2). This is one of the most hypermutable microsatellite repeat polymorphism in the human genome detected to date. D21S1245, is highly polymorphic (heterozygosity of 0.96) and maps between D21S231 and D21S198.

The dominant retinitis pigmentosa locus RP9 has previously been localized to 7p13-p15, in the interval D7S526-D7S484. We now report refinement of the locus to the interval D7S795-D7S484 and a YAC contig of approximately 4.8 Mb spanning this region and extending both distally and proximally from it. The contig was constructed by STS content mapping and physically orders 29 STSs in 28 YAC clones. The order of polymorphic markers in the contig is consistent with a genetic map that has been assembled using haplotype data from the CEPH pedigrees. This contig will provide a primary resource for the construction of a transcriptional map of this region and for the identification of the defective gene causing this form of adRP.