Biomedical subjects

R R Freimuth

Publications and source records attributed to R R Freimuth.

6 recordsLinked to original sources

PubMed2005

Interethnic variability of ERCC2 polymorphisms.

Excision Repair Cross-Complementing Rodent Repair Group 2 (ERCC2) plays an important role in DNA repair by eliminating bulky DNA adducts produced by platinum agents during the nucleotide excision repair pathway. Several studies have associated polymorphisms in ERCC2 with response to platinum therapy, lung cancer risk, and DNA repair capacity. This study examined ERCC2 polymorphisms and haplotype structure across 18.9 kb in 95 European, 95 African, and 95 Asian individuals. Single-nucleotide polymorphisms (SNPs) (ERCC2 -9164 A>T, -1989 A>G, -516 G>A, 468 C>A [Arg156Arg], 1737 C>T [Val579Val], 2133 C>T [Asp711Asp], and 2251 T>G [Lys751Gln]) were mined and mapped using Golden Path, PolyMAPr, and Promolign. Genotyping was performed using PCR and pyrosequencing. Allele frequencies ranged from 0 to 0.47 (Europeans), 0.05 to 0.72 (Africans), and 0 to 0.47 (Asians). The synonymous cSNP at codon 579 could not be confirmed in our populations. There were significant differences in haplotype structure and frequency between populations. This information on ERCC2 genomic structure will allow the construction of definitive studies to clarify the clinical role of this important gene.

Adult↗

PubMed2004

Human cytosolic sulfotransferase database mining: identification of seven novel genes and pseudogenes.

A total of 10 SULT genes are presently known to be expressed in human tissues. We performed a comprehensive genome-wide search for novel SULT genes using two different but complementary approaches, and developed a novel graphical display to aid in the annotation of the hits. Seven novel human SULT genes were identified, five of which were predicted to be pseudogenes, including two processed pseudogenes and three pseudogenes that contained introns. Those five pseudogenes represent the first unambiguous SULT pseudogenes described in any species. Expression-profiling studies were conducted for one novel gene, SULT6B1, and a series of alternatively spliced transcripts were identified in the human testis. SULT6B1 was also present in chimpanzee and gorilla, differing at only seven encoded amino-acid residues among the three species. The results of these database mining studies will aid in studies of the regulation of these SULT genes, provide insights into the evolution of this gene family in humans, and serve as a starting point for comparative genomic studies of SULT genes.

Amino Acid Sequence↗

PubMed2002

Human sulfotransferase SULT2A1 pharmacogenetics: genotype-to-phenotype studies.

SULT2A1 catalyzes the sulfate conjugation of dehydroepiandrosterone (DHEA) as well as other steroids. As a step toward pharmacogenetic studies, we have 'resequenced' SULT2A1 using 60 DNA samples from African-American and 60 samples from Caucasian-American subjects. All exons, splice junctions and approximately 370 bp located 5' of the site of transcription initiation were sequenced. We observed 15 single nucleotide polymorphisms (SNPs), including three non-synonymous coding SNPs (cSNPs) that were present only in DNA from African-American subjects. Linkage analysis revealed that two of the nonsynonymous cSNPs were tightly linked. Expression constructs were created for all nonsynonymous cSNPs observed, including a 'double variant' construct that included the two linked cSNPs, and those constructs were expressed in COS-1 cells. SULT2A1 activity was significantly decreased for three of the four variant allozymes. Western blot analysis demonstrated that decreased levels of immunoreactive protein appeared to be the major mechanism responsible for decreases in activity, although apparent Km values also varied among the recombinant allozymes. In addition, the most common of the nonsynonymous cSNPs disrupted the portion of SULT2A1 involved with dimerization, and this variant allozyme behaved as a monomer rather than a dimer during gel filtration chromatography. These observations indicate that common genetic polymorphisms for SULT2A1 can result in reductions in levels of both activity and enzyme protein. They also raise the possibility of ethnic-specific pharmacogenetic variation in SULT2A1-catalyzed sulfation of both endogenous and exogenous substrates for this phase II drug-metabolizing enzyme.

Base Sequence↗

PubMed2001

Human sulfotransferase SULT1C1 pharmacogenetics: gene resequencing and functional genomic studies.

Sulfotransferase (SULT) enzymes catalyze an important phase II reaction in the biotransformation of many drugs and other xenobiotics. We previously cloned the human SULT1C1 cDNA and gene as steps toward pharmacogenetic studies. We have now 'resequenced' the exons, portions of introns flanking exons and approximately 315 bp of the 5' flanking region of SULT1C1 in 89 DNA samples from Caucasian subjects to identify common genetic polymorphisms. Nineteen separate polymorphisms were observed, including four nonsynonymous coding region single nucleotide polymorphisms (cSNPs) and five insertions/deletions. These data were also used to determine and/or infer common SULT1C1 haplotypes. Three of the four nonsynonymous cSNPs had allele frequencies greater than 1%, including one with a frequency of 6.7%. Expression constructs were created for all of the nonsynonymous cSNPs observed, and those constructs were used to transfect COS-1 cells. Three of the four SULT1C1 variant allozymes had significantly reduced enzyme activity when compared with the wild-type enzyme. Among the variant allozymes, apparent Km values for 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the sulfate donor for the reaction, varied 7-fold, and quantitative Western blot analysis showed variable levels of immunoreactive protein when compared to the wild-type enzyme. Therefore, mechanisms responsible for decreased activity involved both alterations in levels of enzyme protein and alterations in substrate kinetics. In summary, application of a 'genotype to phenotype' strategy has resulted in the identification of a series of functionally significant common genetic polymorphisms for SULT1C1. It will now be possible to evaluate the possible contribution of these polymorphisms to variation in the sulfate conjugation of drugs, other xenobiotics and/or disease pathophysiology.

Animals↗

PubMed2000

Human sulfotransferases SULT1C1 and SULT1C2: cDNA characterization, gene cloning, and chromosomal localization.

Sulfate conjugation catalyzed by sulfotransferase (SULT) enzymes is an important pathway in the biotransformation of many drugs, other xenobiotics, neurotransmitters, and hormones. We previously described a human cDNA, SULT1C1, that encoded a protein similar in sequence to that of rat ST1C1. Subsequently, a related human cDNA, SULT1C2, was reported. In the present study, we set out to characterize further the human SULT1C1 cDNA and then to clone, structurally characterize, and map its gene. As an initial step, we performed 5'- and 3'-RACE with SULT1C1 cDNA. Those experiments demonstrated that a small number of SULT1C1 transcripts contained an "insert," which we later showed resulted from alternative splicing that involved an Alu sequence in intron 3 of SULT1C1. We then cloned and structurally characterized the SULT1C1 gene from a human genomic BAC library. Because the sequence of SULT1C2 was closely related to that of SULT1C1 and because the genes for other human SULT paralogues occur in clusters, we screened the BAC clones that had been positive for SULT1C1 to search for SULT1C2 and discovered a clone that contained both genes. That BAC was used to sequence and structurally characterize SULT1C2. SULT1C1 and SULT1C2 were approximately 21 and 10 kb in length, respectively. Both genes contained seven exons that encoded protein, and both had structures that were similar to those of other genes that encode members of the SULT1 family. Finally, human SULT1C1 and SULT1C2 mapped to 2q11.2 by fluorescence in situ hybridization. The cloning and structural characterization of SULT1C1 and SULT1C2 will now make it possible to perform molecular genetic and pharmacogenomic studies of these sulfate-conjugating enzymes in humans.

Animals↗

PubMed2000

Human 3'-phosphoadenosine 5'-phosphosulfate synthetase 1 (PAPSS1) and PAPSS2: gene cloning, characterization and chromosomal localization.

Sulfae conjugation is an important pathway in the metabolism of a large number of exogenous and endogenous compounds. These reactions are catalyzed by sulfotransferase (SULT) enzymes that utilize 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfate donor. PAPS is synthesized from ATP and inorganic sulfate by PAPS synthetase (PAPSS). Two separate PAPSS cDNAs, PAPSS1 and PAPSS2, have been identified in human tissues. We have cloned and characterized the genes for human PAPSS1 and PAPSS2 to make it possible to study the pharmacogenomics of these enzymes. Both genes consisted of 12 exons with virtually identical exon-intron splice junction locations. All splice junctions conformed to the "GT-AG" rule. The total length of PAPSS1 was approximately 108 kb, while that of PAPSS2 was greater than 37 kb. The 5'-flanking region of PAPSS1 did not include a TATA box sequence near the site of transcription initiation, but PAPSS2 had a TATA motif located 21 bp upstream from the site of transcription initiation. Northern blot analysis showed that the major PAPSS1 and PAPSS2 transcripts were approximately 2.7 and 4.2 kb in length, respectively. PAPSS1 mapped to human chromosome band 4q24 while PAPSS2 mapped to 10q22-23 by fluorescence in situ hybridization analysis. Cloning and structural characterization of PAPSS1 and PAPSS2 will make it possible to perform molecular genetic and pharmacogenomic studies of these important enzymes in humans.

Blotting, Northern↗