Harvey W. Mohrenweiser

Challenges and complexities in estimating both the functional impact and the disease risk associated with the extensive genetic variation in human DNA repair genes

Individual risk and the population incidence of disease result from the interaction of genetic susceptibility and exposure. DNA repair is an example of a cellular process where genetic variation in families with extreme predisposition is documented to be associated with high disease likelihood, including syndromes of premature aging and cancer. Although the identification and characterization of new genes or variants in cancer families continues to be important, the focus of this paper is the current status of efforts to define the impact of polymorphic amino acid substitutions in DNA repair genes on individual and population cancer risk. There is increasing evidence that mild reductions in DNA repair capacity, assumed to be the consequence of common genetic variation, affect cancer predisposition. The extensive variation being found in the coding regions of DNA repair genes and the large number of genes in each of the major repair pathways results in complex genotypes with potential to impact cancer risk in the general population. The implications of this complexity for molecular epidemiology studies, as well as concepts that may make these challenges more manageable, are discussed. The concepts include both experimental and computational approaches that could be employed to develop predictors of disease susceptibility based on DNA repair genotype, focusing initially on studies to assess functional impact on individual proteins and pathways and then on molecular epidemiology studies to assess exposure-dependent health risk. In closing, we raise some of the non-technical challenges to the utilization of the full richness of the genetic variation to reduce disease occurrence and ultimately improve health care.

Polymorphisms of XRCC1 and XRCC3 genes and susceptibility to breast cancer

Mammalian cells are constantly exposed to a wide variety of genotoxic agents from both endogenous and exogenous sources. Genetic variability in DNA repair may contribute to human cancer risk. We used a case-control study design (162 cases and 302 controls) to test the association between three amino acid substitution variants of DNA repair genes (XRCC1 Arg194Trp, XRCC1 Arg399Gln, and XRCC3 Thr241Met) and breast cancer susceptibility. We found a weak association between the XRCC1 194Trp allele and breast cancer risk (adjusted odds ratio (OR)=1.98; 95% confidence interval (CI)=0.85-4.63). We also found a potential gene-gene interaction between the XRCC1 194Trp allele and XRCC3 241Met allele and breast cancer risk (adjusted OR=8.74; 95% CI=1.13-67.53). Although larger studies are needed to validate the study results, our data suggest that amino acid substitution variants of XRCC1 and XRCC3 genes may contribute to breast cancer susceptibility.

Organization and evolution of the cytochrome P450 CYP2A-2B-2F subfamily gene cluster on human chromosome 19

Cytochrome P450 genes from the CYP2A, CYP2B, and CYP2F subfamilies form a tight cluster which we have localized on the detailed physical map of human chromosome 19. The corresponding three gene subfamilies are also clustered in the mouse genome, on the region of chromosome 7 known to be syntenic to human chromosome 19. One hundred eight cosmid clones from the human P450 region were assembled into a single contig of 350 kb, restriction mapped, and probed with cDNAs from the three gene subfamilies. A total of 11 genes were identified in humans, including five from the 2A subfamily, three from the 2B subfamily, and three from the 2F subfamily; at least six of the 11 are pseudogenes. The organization of the genes, with members of the three subfamilies intermixed, indicates that the evolution of this gene cluster has been complex. The modern gene arrangement in humans is probably the result of a series of tandem duplications, plus at least one inverted duplication. The identification of all genes and pseudogenes in this cluster also makes it possible to determine the origins of some previously known variant P450 transcripts.

Mapping of the chromosome 19 q-arm glioma tumor suppressor gene using fluorescence in situ hybridization and novel microsatellite markers

Allelic loss of chromosome arm 19q is a frequent event in human diffuse glioma, suggesting the presence of a tumor suppressor gene. Previous loss of heterozygosity (LOH) analyses have mapped this gene to a 1.4-megabase interval, between the genetic markers D19S412 and STD. Further narrowing of this interval has been limited by the resolution of mapped polymorphic markers. In the present study, we have used genomic clones mapped to 19q as fluorescence in situ hybridization (FISH) probes to map the breakpoints of 13 gliomas with 19q13.3 deletion boundaries. In addition, we have developed three new polymorphic microsatellite markers (D19S1180, D19S1181, and D19S1182) that map between D19S412 and STD and have used these new markers to identify two gliomas with small deletions between the D19S412 and STD markers. Collectively, these data suggest that the region of common deletion may be as narrow as 150 kb and should facilitate future efforts to identify the glioma 19q tumor suppressor gene.

Genomic Organization, Chromosomal Localization, Tissue Distribution, and Biophysical Characterization of a Novel MammalianShaker-related Voltage-gated Potassium Channel, Kv1.7

We report the isolation of a novel mouse voltage-gated Shaker-related K+ channel gene,Kv1.7 (Kcna7/KCNA7). Unlike other known Kv1 family genes that have intronless coding regions, the protein-coding region of Kv1.7 is interrupted by a 1.9-kilobase pair intron. The Kv1.7 gene and the related Kv3.3(Kcnc3/KCNC3) gene map to mouse chromosome 7 and human chromosome 19q13.3, a region that has been suggested to contain a diabetic susceptibility locus. The mouse Kv1.7 channel is voltage-dependent and rapidly inactivating, exhibits cumulative inactivation, and has a single channel conductance of 21 pS. It is potently blocked by noxiustoxin and stichodactylatoxin, and is insensitive to tetraethylammonium, kaliotoxin, and charybdotoxin. Northern blot analysis reveals ∼3-kilobase pair Kv1.7transcripts in mouse heart and skeletal muscle. In situhybridization demonstrates the presence of Kv1.7 in mouse pancreatic islet cells. Kv1.7 was also isolated from mouse brain and hamster insulinoma cells by polymerase chain reaction.

The BAX gene maps to the glioma candidate region at 19q13.3, but is not altered in human gliomas

The bax protein regulates apoptosis in a cellular pathway that involves both bcl-2 and p53, two molecules associated with human glioma tumorigenesis. We therefore evaluated the possibility that BAX functions as a glioma tumor suppressor gene. Somatic cell hybrid panels, fluorescence in situ hybridization and cosmid mapping localized the BAX gene to 19q13.3, approximately 300 kb centromeric to HRC. Thus BAX maps to the region of chromosome 19 most frequently deleted in gliomas. Routine and pulsed-field gel electrophoresis/Southern blotting studies, however, failed to reveal large-scale deletions or rearrangements of the BAX gene in gliomas. In addition, single strand conformation polymorphism analysis of all six BAX exons and flanking intronic sequences did not disclose mutations in 20 gliomas with allelic loss of the other copy of 19q. A C/T polymorphism was detected in intron 3 and was common in the general population. Therefore, although BAX maps to the glioma candidate region on the long arm of chromosome 19, BAX is probably not the 19q glioma tumor suppressor gene.

Chromosome 19q Deletions in Human Gliomas Overlap Telomeric to D19S219 and May Target a 425 kb Region Centromeric to D19S112

Chromosome 19q harbors a tumor suppressor gene that is involved in astrocytoma, oligodendroglioma and mixed glioma tumorigenesis. We had previously mapped this gene to an approximately 5 megabase region of chromosome 19q13.2- 13.3 between APOC2 and HRC. To narrow the location of this tumor suppressor further, we studied 138 gliomas for loss of allelic heterozygosity at six microsatellite polymorphisms between APOC2 and HRC, including a newly described polymorphism in the ERCC2 gene. Allelic loss occurred in 48 gliomas (35%), including 25 of 41 oligodendroglial tumors (61%). Four cases had proximal breakpoints within the APOC2-HRC region, two telomeric to ERCC2 and two telomeric to D19S219. In addition, one of the latter tumors had an interstitial deletion between D19S219 and D19S112, a distance of only 425 kilobases surrounding the DM (myotonic dystrophy) gene. These findings suggest that the glioma tumor suppressor on chromosome 19q maps to 19q13.3, telomeric to D19S219 and perhaps centromeric to D19S112. The data exclude a number of candidate genes from 19q13.2-13.3, including a putative phosphatasc gene and the DNA repair/metabolism genes ERCC1, ERCC2 and probably LIG1.

Order and genomic distances among members of the carcinoembryonic antigen (CEA) gene family determined by fluorescence in situ hybridization

Fluorescence in situ hybridization was used to establish the order of, and to estimate genomic distances among, members of the carcinoembryonic antigen (CEA) and pregnancy-specific glycoprotein (PSG) subgroups on chromosome 19. Fluorescence in situ hybridization to metaphase chromosomes localized the PSG subgroup telomeric to the CEA subgroup. Cosmid clones containing sequences for individual genes in the CEA and PSG subgroups were also hybridized to human sperm pronuclear and somatic interphase nuclear chromatin targets. The mapping results lead to the gene order cen-CGM7-CEA-NCA-CGM1-BGP-CGM9-CGM8-PSG-te l. The genomic distances between selected pairs of gene family members were estimated from the physical distances between hybridization sites measured in pronuclei. The CEA-PSG gene family region is estimated to span 1.1 to 1.2 Mb.

Polymorphisms in the genes encoding members of the tristetraprolin family of human tandem CCCH zinc finger proteins.

The three known mammalian CCCH tandem zinc finger proteins of the tristetraprolin (TTP) class have recently been demonstrated to be mRNA-binding proteins. The prototype, TTP, functions in normal physiology to promote the instability of the tumor necrosis factor alpha (TNFalpha) and granulocyte-macrophage colony-stimulating factor mRNAs. Conversely, these mRNAs are stabilized in TTP-deficient mice, leading to an inflammatory phenotype characterized by overproduction of these cytokines. To explore sequence variations in TTP and its two related proteins, we sequenced genomic DNA encoding the TTP protein (ZFP36) and those of its two known mammalian relatives, ZFP36L1 and ZFP36L2, from 72 to 92 anonymous human subjects from various geographical and ethnic backgrounds. We also sequenced ZFP36 in genomic DNA from 92 subjects exhibiting evidence of excessive TNFalpha action. The resequencing strategy identified 13 polymorphisms in the protein-coding regions of these three genes, of which six would result in amino acid changes; other putative polymorphisms were identified by EST searches. One mutation in ZFP36L1 was a dinucleotide substitution that would prevent splicing of the single intron. This mutation was identified in only one allele of the original 144 sequenced from an adult female Aka Pygmy from the Central African Republic; a second individual with the same variant allele was found by genotyping 58 additional Aka DNA samples. Analysis of mRNA from one of these subjects lymphoblasts confirmed that ZFP36L1 mRNA levels were approximately 50% of those in a comparable sample without the mutation. The functional significance of this and the other polymorphisms identified remains to be determined by both biochemical and population linkage studies.

Assignment of the gene encoding DNA ligase I to human chromosome 19q13.2–13.3

The gene encoding DNA ligase I has been mapped on human chromosome 19 by analysis of rodent-human somatic cell hybrids informative for this chromosome and by two-color fluorescence in situ hybridization. The DNA ligase I gene (LIG1) is localized to 19q13.2-13.3 and is distal to ERCC1, the most telomeric of three DNA repair genes on this chromosome.