Catalog of 668 SNPs detected among 31 genes encoding potential drug targets on the cell surface.

We have been publishing a series of detailed maps of single-nucleotide polymorphisms (SNPs) detected within the genomic loci of 145 genes encoding drug-metabolizing enzymes and transporters. As an addition to the maps reported earlier, we provide here high-density SNP maps of 31 genes encoding various receptors and adhesion molecules of medical importance. By examining a total of approximately 382 kb of genomic DNA encompassing these 31 genes, we identified 668 SNPs among 48 healthy Japanese individuals: 86 in 5' flanking regions, 27 in 5' untranslated regions, 45 in coding regions, 399 in introns, 47 in 3' untranslated regions, and 64 in 3' flanking regions. We also discovered 113 variations of other types. Of the 668 SNPs, 371 (55.5%) appeared to be novel, on the basis of comparisons with the dbSNP database of the National Center for Biotechnology Information (US) or with previous publications. The maps constructed in this study will serve as an additional resource for studies of complex genetic diseases and drug-response phenotypes to be mapped by linkage-disequilibrium analyses.

Molecular cloning and characterization of chymopasin, a novel serine protease from rat pancreas.

INTRODUCTION: Pancreas secretes many enzymes for food digestion into the pancreatic juice. We cloned a novel serine protease, chymopasin, from rat pancreas. AIMS: To know the localization of this enzyme in the pancreas and to analyze the enzymatic characteristics. METHODOLOGY: We cloned chymopasin cDNA using 3' and 5' RACEs. Northern blot and in situ hybridization were used to study the expression of this enzyme. Recombinant chymopasin protein produced by was analyzed by Western blot using specific antibody, and its enzymatic characteristics were examined using commercially available synthetic substrates, fibrin and gelatin. RESULTS: The open reading frame of rat chymopasin consisted of 792 bp encoding 264 amino acid residues. The deduced amino acid sequence contained the essential catalytic triad characteristic of the serine protease family. There was no putative N-glycosylation site. The amino acid sequence of rat chymopasin showed 54.5% identity to rat chymotrypsin B. Northern blot analysis showed that the transcript was strongly expressed in the pancreas. In situ hybridization with digoxigenin-labeled cRNA probe showed that the positive signals were observed in the acinar cells, but not in the islet or duct cells. Chymopasin protein was detected in the pancreas homogenate and bile-pancreatic juice. Further, cerulein stimulated the secretion of rat chymopasin into bile-pancreatic juice. CONCLUSION: These results suggested that rat chymopasin might be a digestive enzyme secreted from the acinar cells. From the enzyme assay using synthetic substrates, the purified recombinant chymopasin expressed in showed chymotrypsin-like activity. In addition, rat recombinant chymopasin showed fibrinolytic and gelatinolytic activities. These results suggested a role in the pathogenesis of pancreatic damage.

Catalog of 86 single-nucleotide polymorphisms (SNPs) in three uridine diphosphate glycosyltransferase genes: UGT2A1, UGT2B15, and UGT8.

We report here three high-density maps of variations found among 48 Japanese individuals in three uridine diphosphate glycosyltransferase (UGT) genes, UGT2A1, UGT2B15, and UGT8. A total of 86 single-nucleotide polymorphisms (SNPs) were identified through systematic screening of genomic regions containing these genes: 8 in 5' flanking regions, 7 in coding regions, 67 in introns, 3 in 3' untranslated regions, and 1 in a 3' flanking region. We also discovered 14 variations of other types. Of the 86 SNPs, 63 (73%) were considered to be novel on the basis of comparison of our data with the Database of SNPs (dbSNP) of the National Center for Biotechnology Information. Among the seven SNPs identified in exonic sequences, five were non-synonymous changes that would result in amino-acid substitutions. The collection of SNPs derived from this study will serve as an additional resource for studies of complex genetic diseases and responsiveness to drug therapy.

Catalog of 605 single-nucleotide polymorphisms (SNPs) among 13 genes encoding human ATP-binding cassette transporters: ABCA4, ABCA7, ABCA8, ABCD1, ABCD3, ABCD4, ABCE1, ABCF1, ABCG1, ABCG2, ABCG4, ABCG5, and ABCG8.

Single-nucleotide polymorphisms (SNPs) at some gene loci are useful as markers of individual risk for adverse drug reactions or susceptibility to complex diseases. We have been focusing on identifying SNPs in and around genes encoding drug-metabolizing enzymes and transporters, and have constructed several high-density SNP maps of such regions. Here we report SNPs at additional loci, specifically 13 genes belonging to the superfamily of ATP-binding cassette transporters ( ABCA4, ABCA7, ABCA8, ABCD1, ABCD3, ABCD4, ABCE1, ABCF1, ABCG1, ABCG2, ABCG4, ABCG5, and ABCG8). Sequencing a total of 416 kb of genomic DNA from 48 Japanese volunteers identified 605 SNPs among these 13 loci: 14 in 5' flanking regions, 5 in 5' untranslated regions, 37 within coding elements, 529 in introns, 8 in 3' untranslated regions, and 12 in 3' flanking regions. By comparing our data with SNPs deposited in the dbSNP database of the National Center for Biotechnology Information (US) and with published reports, we determined that 491 (81%) of the SNPs reported here were novel. We also detected 107 genetic variations of other types among the loci examined (insertion-deletions or mono- di-, or trinucleotide polymorphisms). The high-density SNP maps we constructed on the basis of these data should provide useful information for investigating associations between genetic variations and common diseases or responsiveness to drug therapy.

Relation between morphologic changes in the main pancreatic duct and exocrine pancreatic function after a secretin test.

INTRODUCTION: Because pancreatic exocrine function testing methods are problematic, both imaging and functional tests are important in the diagnosis of chronic pancreatitis. AIM: To evaluate the usefulness of ultrasonographic monitoring of the main pancreatic duct after a secretin test. METHODOLOGY: A total of 70 subjects (30 control subjects, 26 patients with probable chronic pancreatitis, and 14 patients with definite chronic pancreatitis) were selected. The main pancreatic duct diameters were measured serially after an injection of secretin (100 IU/body). The relation between the magnitude of the duct dilation and exocrine pancreatic function on the secretin test was evaluated. RESULTS: The main pancreatic duct dilated immediately after a bolus injection of secretin, showed a peak after 2-5 minutes, and recovered gradually. The response curve of the definite group had a flatter pattern than that of the other groups. For the maximal to basal duct diameter ratio, statistically significant differences were found between the control and definite groups and between the control and probable groups. In addition, the ratio correlated significantly with the maximal bicarbonate concentration and secretory volume on the secretin test. CONCLUSIONS: The results of the current study indicate that exocrine pancreatic function and the morphologic changes of the main pancreatic duct are significantly related. Dynamic ultrasonographic findings may reflect pancreatic function; consequently, this test may be a useful tool in the diagnosis of chronic pancreatitis.

Thirteen single-nucleotide polymorphisms (SNPs) in the alcohol dehydrogenase 4 (ADH4) gene locus.

The human alcohol dehydrogenase 4 (ADH4) gene encodes the class II ADH4 pi subunit, which contributes to the metabolization of a wide variety of substrates, including ethanol, retinol, other aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. Here we report the results of systematic screening for single-nucleotide polymorphisms (SNPs) in the ADH4 gene by means of direct sequencing combined with a polymerase chain reaction method. A total of 16 genetic variations including 13 SNPs were found; 4 in the 5' flanking region, 4 in the 5' untranslated region, and 8 within introns. No variation was found in coding, 3' untranslated, or 3' flanking regions. Eight of the 13 SNPs were not reported in the NCBI dbSNP database or any previous publications. Our SNP map presented here should provide tools to evaluate the role of ADH4 in complex genetic diseases and a variety of pharmacogenetic effects.

Catalog of 77 single-nucleotide polymorphisms (SNPs) in the carbohydrate sulfotransferase 1 (CHST1) and carbohydrate sulfotransferase 3 (CHST3) genes.

Individual phenotypes with respect to drug response or toxicity often result from genetic variations that alter drug metabolism. We have been focusing on genomic loci that encode various enzymes and transporters involved in the metabolism of drugs, and have described more than 1200 single-nucleotide polymorphisms (SNPs) and other variations. Regarding the carbohydrate sulfotransferase (CHST) gene family, we have already constructed high-density SNP maps of three genomic segments that included CHST2, CHST4, and CHST5, providing a total of 28 SNPs for those loci. In the present study, we screened DNA from 48 healthy Japanese volunteers for SNPs at the CHST1 and CHST3 gene loci, by means of direct sequencing combined with a polymerase chain reaction method for amplifying genomic DNA, and characterized 77 SNPs and four insertion-deletion polymorphisms. The collection of human variations presented here adds to the archive of tools now available for investigating complex genetic diseases, population migration patterns, and a variety of pharmacogenetic possibilities.