Geoff Joslyn

Identification and characterization of the familial adenomatous polyposis coli gene

DNA from 61 unrelated patients with adenomatous polyposis coli (APC) was examined for mutations in three genes (DP1, SRP19, and DP2.5) located within a 100 kb region deleted in two of the patients. The intron-exon boundary sequences were defined for each of these genes, and single-strand conformation polymorphism analysis of exons from DP2.5 identified four mutations specific to APC patients. Each of two aberrant alleles contained a base substitution changing an amino acid to a stop codon in the predicted peptide; the other mutations were small deletions leading to frameshifts. Analysis of DNA from parents of one of these patients showed that his 2 bp deletion is a new mutation; furthermore, the mutation was transmitted to two of his children. These data have established that DP2.5 is the APC gene.

Identification of deletion mutations and three new genes at the familial polyposis locus

Small (100-260 kb), nested deletions were characterized in DNA from two unrelated patients with familial adenomatous polyposis coli (APC). Three candidate genes located within the deleted region were ascertained and a previous candidate gene, MCC, was shown to be located outside the deleted region. One of the new genes contained sequence identical to SRP19, the gene coding for the 19 kd component of the ribosomal signal recognition particle. The second, provisionally designated DP1 (deleted in polyposis 1), was found to be transcribed in the same orientation as MCC. Two other cDNAs, DP2 and DP3, were found to overlap, forming a single gene, DP2.5, that is transcribed in the same orientation as SRP19.

Genomewide search for type 2 diabetes susceptibility genes in four American populations.

Type 2 diabetes is a serious, genetically influenced disease for which no fully effective treatments are available. Identification of biochemical or regulatory pathways involved in the disease syndrome could lead to innovative therapeutic interventions. One way to identify such pathways is the genetic analysis of families with multiple affected members where disease predisposing genes are likely to be segregating. We undertook a genomewide screen (389-395 microsatellite markers) in samples of 835 white, 591 Mexican American, 229 black, and 128 Japanese American individuals collected as part of the American Diabetes Associations GENNID study. Multipoint nonparametric linkage analyses were performed with diabetes, and diabetes or impaired glucose homeostasis (IH). Linkage to diabetes or IH was detected near markers D5S1404 (map position 77 cM, LOD = 2.80), D12S853 (map position 82 cM, LOD = 2.81) and GATA172D05 (X-chromosome map position 130 cM, LOD = 2.99) in whites, near marker D3S2432 (map position 51 cM, LOD = 3.91) in Mexican Americans, and near marker D10S1412 (map position 14 cM, LOD = 2.39) in African Americans mainly collected in phase 1 of the study. Further analyses showed evidence for interactions between the chromosome 5 locus and region on chromosome 12 containing the MODY 3 gene (map position 132 cM) and between the X-chromosome locus and region near D12S853 (map position 82 cM) in whites. Although these results were not replicated in samples collected in phase 2 of the GENNID study, the region on chromosome 12 was replicated in samples from whites described by Bektas et al. (1999).

p53 Mutations in Barrett's adenocarcinoma and high-grade dysplasia

BACKGROUND/AIMS Allelic losses of chromosome 17p and overexpression of p53 protein have been reported in Barretts adenocarcinomas. This study aimed to determine the stage in which p53 mutations arise in neoplastic progression in Barretts esophagus and their relationship to the clonal evolution of cancer. METHODS Fourteen patients with high-grade dysplasia, adenocarcinoma, or both arising in Barretts esophagus were evaluated. Flow cytometric cell sorting was used to obtain purified populations of neoplastic cells for analysis of p53 mutations. DNA was extracted, and exons 5 through 9 of the p53 gene were amplified by polymerase chain reaction. Amplified DNA was sequenced and analyzed by automated sequencing. RESULTS Nine of the 14 patients had p53 mutations. Six of the 9 patients had regions of high-grade dysplasia that could be evaluated; all 6 had p53 mutations in high-grade dysplasia. In 3 patients, the same p53 mutations were found in both high-grade dysplasia and adenocarcinoma. All 14 patients had aneuploidy. In 4 patients, diploid cell populations could also be evaluated for p53 mutations; 3 of the 4 patients had p53 mutations in diploid cell populations. In 2 patients, the same p53 mutation was found in multiple aneuploid cell populations within a cancer. CONCLUSIONS p53 mutations occur frequently in Barretts adenocarcinomas. They develop in diploid cell populations. The same p53 mutations are then found in aneuploid cell populations in high-grade dysplasia, in cancer, and in multiple aneuploid cell populations in cancer.

Chromosome 15q25.1 genetic markers associated with level of response to alcohol in humans

As with other genetically complex common psychiatric and medical conditions, multiple genetic and environmental components contribute to alcohol use disorders (AUDs), which can confound attempts to identify genetic components. Intermediate phenotypes are often more closely correlated with underlying biology and have often proven invaluable in genetic studies. Level of response (LR) to alcohol is an intermediate phenotype for AUDs, and individuals with a low LR are at increased risk. A high rate of concurrent alcohol and nicotine use and dependence suggests that these conditions may share biochemical and genetic mechanisms. Genetic association studies indicate that a genetic locus, which includes the CHRNA5-CHRNA3-CHRNB4 gene cluster, plays a role in nicotine consumption and dependence. Genetic association with alcohol dependence was also recently shown. We show here that two of the markers from the nicotine studies also show an association (multiple testing corrected P < 0.025) with several LR phenotypes in a sample of 367 siblings. Additional markers in the region were analyzed and shown to be located in a 250-kb expanse of high linkage disequilibrium containing three additional genes. These findings indicate that LR intermediate phenotypes have utility in genetic approaches to AUDs and will prove valuable in the identification of other genetic loci conferring susceptibility to AUDs.

Human Variation in Alcohol Response Is Influenced by Variation in Neuronal Signaling Genes

BACKGROUND Alcohol use disorders (AUD) exhibit the properties shared by common conditions and diseases classified as genetically complex. The etiology of AUDs is heterogeneous involving mostly unknown interactions of environmental and heritable factors. A persons level of response (LR) to alcohol is inversely correlated with a family history and the development of AUDs. As an AUD endophenotype, alcohol LR is hypothesized to be less genetically complex and closer to the primary etiology of AUDs. METHODS A genome wide association study (GWAS) was performed on subjects characterized for alcohol LR phenotypes. Gene Set Enrichment Analysis (GSEA) of the GWAS data was performed to determine whether, as a group, genes that participate in a common biological function (a gene set) demonstrate greater genetic association than would be randomly expected. RESULTS The GSEA analysis implicated variation in neuronal signaling genes, especially glutamate signaling, as being involved in alcohol LR variability in the human population. CONCLUSIONS These data, coupled with cell and animal model data implicating neuronal signaling in alcohol response, support the conclusion that neuronal signaling is mechanistically involved in alcohols cellular and behavioral effects. Further, these data suggest that genetic variation in these signaling pathways contribute to human variation in alcohol response. Finally, this concordance of the cell, animal, and human findings supports neuronal signaling, particularly glutamate signaling, as a prime target for translational studies to understand and eventually modulate alcohols effects.

Functional characterization of human variants of the mu-opioid receptor gene

Opioids and their receptors have an important role in analgesia and alcohol and substance use disorders (ASUD). We have identified several naturally occurring amino acid changing variants of the human mu-opioid receptor (MOR), and assessed the functional consequences of these previously undescribed variants in stably expressing cell lines. Several of these variants had altered trafficking and signaling properties. We found that an L85I variant showed significant internalization in response to morphine, in contrast to the WT MOR, which did not internalize in response to morphine. Also, when L85I and WT receptor were coexpressed, WT MOR internalized with the L85I MOR, suggesting that, in the heterozygous condition, the L85I phenotype would be dominant. This finding is potentially important, because receptor internalization has been associated with development of tolerance to opiate analgesics. In contrast, an R181C variant abolished both signaling and internalization in response to saturating doses of the hydrolysis-resistant enkephalin [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAMGO). Coexpression of the R181C and WT receptor led to independent trafficking of the 2 receptors. S42T and C192F variants showed a rightward shift in potency of both morphine and DAMGO, whereas the S147C variant displayed a subtle leftward shift in morphine potency. These data suggest that these and other such variants may have clinical relevance to opioid responsiveness to both endogenous ligands and exogenous drugs, and could influence a broad range of phenotypes, including ASUD, pain responses, and the development of tolerance to morphine.

Examination of Rare Missense Variants in the CHRNA5‐A3‐B4 Gene Cluster to Level of Response to Alcohol in the San Diego Sibling Pair Study

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.

Glypican Gene GPC5 Participates in the Behavioral Response to Ethanol: Evidence from Humans, Mice, and Fruit Flies

Alcohol use disorders are influenced by many interacting genetic and environmental factors. Highlighting this complexity is the observation that large genome-wide association experiments have implicated many genes with weak statistical support. Experimental model systems, cell culture and animal, have identified many genes and pathways involved in ethanol response, but their applicability to the development of alcohol use disorders in humans is undetermined. To overcome the limitations of any single experimental system, the analytical strategy used here was to identify genes that exert common phenotypic effects across multiple experimental systems. Specifically, we (1) performed a mouse linkage analysis to identify quantitative trait loci that influence ethanol-induced ataxia; (2) performed a human genetic association analysis of the mouse-identified loci against ethanol-induced body sway, a phenotype that is not only comparable to the mouse ethanol-ataxia phenotype but is also a genetically influenced endophenotype of alcohol use disorders; (3) performed behavioral genetic experiments in Drosophila showing that fly homologs of GPC5, the member of the glypican gene family implicated by both the human and mouse genetic analyses, influence the fly’s response to ethanol; and (4) discovered data from the literature demonstrating that the genetically implicated gene’s expression is not only temporally and spatially consistent with involvement in ethanol-induced behaviors but is also modulated by ethanol. The convergence of these data provides strong support to the hypothesis that GPC5 is involved in cellular and organismal ethanol response and the etiology of alcohol use disorders in humans.