A case-based evaluation of SRD5A1, SRD5A2, AR, and ADRA1A as candidate genes for severity of BPH.

In men with a clinical diagnosis of benign prostatic hyperplasia (BPH), polytomous logistic regression analysis was conducted to evaluate associations between two silent polymorphisms in SRD5A1 (codon positions 30 and 116), two polymorphisms in SRD5A2 (Val89Leu substitution and C to T transition in intron 1), a trinucleotide (CAG)n repeat in androgen receptor (AR), and an Arg492Cys substitution in ADRA1A and clinical parameters that characterize severity of BPH. Candidate gene selection was based on two mechanistic pathways targeted by pharmacotherapy for BPH: (1) androgen metabolic loci contributing to prostate growth (static obstruction); and (2) factors affecting smooth muscle tone (dynamic obstruction). Polymorphisms in SRD5A2 were not associated with severity of BPH; however, SRD5A1 polymorphisms were associated with severity of BPH. The process(es) in which these silent single-nucleotide polymorphisms (SNPs) influence BPH phenotypes is unknown and additional studies will be needed to assess whether these SNPs have direct functional consequences. The characterization of additional molecular factors that contribute to static and dynamic obstruction may help predict response to pharmacotherapy and serve to identify novel drug targets for the clinical management of BPH.

Recombinant fibrinogen studies reveal that thrombin specificity dictates order of fibrinopeptide release.

During cleavage of fibrinogen by thrombin, fibrinopeptide A (FpA) release precedes fibrinopeptide B (FpB) release. To examine the basis for this ordered release, we synthesized A'beta fibrinogen, replacing FpB with a fibrinopeptide A-like peptide, FpA' (G14V). Analyses of fibrinopeptide release from A'beta fibrinogen showed that FpA release and FpA' release were similar; the release of either peptide followed simple first-order kinetics. Specificity constants for FpA and FpA' were similar, demonstrating that these peptides are equally competitive substrates for thrombin. In the presence of Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, the rate of FpB release from normal fibrinogen was reduced 3-fold, consistent with previous data; in contrast, the rate of FpA' release from A'beta fibrinogen was unaffected. Thus, with A'beta fibrinogen, fibrinopeptide release from the beta chain is similar to fibrinopeptide release from the alpha chain. We conclude that the ordered release of fibrinopeptides is dictated by the specificity of thrombin for its substrates. We analyzed polymerization, following changes in turbidity, and found that polymerization of A'beta fibrinogen was similar to that of normal fibrinogen. We analyzed clot structure by scanning electron microscopy and found that clots from A'beta fibrinogen were similar to clots from normal fibrinogen. We conclude that premature release of the fibrinopeptide from the N terminus of the beta chain does not affect polymerization of fibrinogen.

Rapid identification of RT-PCR clones containing translation-terminating mutations.

The technique of in vitro transcription/translation (IVTT) has become an important method of detecting mutations that result in a prematurely terminated protein. Subsequent characterization of the mutations by cloning and sequencing the RT-PCR products, however, is often difficult and time consuming. This is due in large part to the altered metabolism to which transcripts containing translation terminating mutations are subject. Recent data has shown that mRNAs with nonsense or frame shift mutations are often selectively degraded, so that mutation bearing transcripts are significantly less abundant that wild-type transcripts and, after cloning, mutant clones are correspondingly scarce. We have developed a reliable method of identifying the cDNA clones containing translation terminating mutations by a 'second round' of IVTT. Clones are subjected to PCR and IVTT using similar conditions as in the initial IVTT reaction and are identified unequivocally as either wild-type or mutant prior to sequencing. Wasteful 'blind' sequencing is thus avoided as well as possible misidentification of taq polymerase errors as the mutation of interest.

Identification of DNA mismatch repair gene mutations in hereditary nonpolyposis colon cancer patients.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common autosomal dominant disease caused by germline mutations in DNA mismatch repair genes. The mutational spectrum in these genes appears to be diverse, in both the distribution and the nature of the mutations. However, most described mutations generate a premature stop codon and ultimately result in the synthesis of a truncated protein. We have employed an in vitro transcription/translation assay to identify germline mutations in DNA mismatch repair genes from patients suspected of belonging to HNPCC kindreds. Our results suggest that this approach will be highly effective in identifying mutations in these patients and may lead to a reliable diagnostic test for the pre-symptomatic identification of HNPCC.

In vitro transcription/translation assay for the screening of hMLH1 and hMSH2 mutations in familial colon cancer.

BACKGROUND & AIMS: Hereditary nonpolyposis colorectal cancer (HNPCC) has been linked recently to a defect in repairing mismatched nucleotides in DNA. The aim of this study was to screen for germline mutations that result in prematurely truncated proteins in two of the mismatch repair genes identified at this time, hMLH1 and hMSH2, in a consecutive series of patients belonging to familial aggregations of colorectal cancer. METHODS: Nineteen individuals with colorectal cancer from 19 families were consecutively referred because of a strong positive family history of colorectal cancer. Premature truncation mutations in hMLH1 and hMSH2 were sought from lymphocyte RNA by using an in vitro transcription/translation (IVTT) assay. RESULTS: Protein truncating mutations in the hMLH1 or hMSH2 genes were found in 50% of families with HNPCC (6 of 12) but were not observed in any of the remaining familial aggregations that did not fulfill the standard criteria for HNPCC. In some of the IVTT-positive samples, the mutations were characterized by genomic sequencing. CONCLUSIONS: IVTT may be a practical method to accomplish primary screening of germline mutations in DNA mismatch pair genes in HNPCC; however, a broader approach is necessary to obtain a more complete picture of the mutational spectrum in HNPCC and other familial aggregations of colorectal cancer.

Distribution of 13 truncating mutations in the neurofibromatosis 1 gene.

Neurofibromatosis 1 (NF1) is a common genetic disorder characterized by abnormalities of tissues derived from the neural crest. To define germ-line mutations in the NF1 gene, we studied 20 patients with familial or sporadic cases of NF1 diagnosed clinically and one patient with only café-au-lait spots and no other diagnostic criteria. A protein truncation assay identified abnormal polypeptides synthesized in vitro from five RT-PCR products that represented the entire NF1 coding region. Truncated polypeptides were observed in 14 individuals. The mutations responsible for the generation of abnormal polypeptides were characterized by DNA sequencing. Thirteen previously unpublished mutations were characterized in the 14 individuals. The mutation 2027insC was observed in two unrelated individuals; the other 12 mutations were unique. The sequence changes included seven nonsense and four frameshift mutations that created premature translation termination signals, and two large in-frame deletions that led to the synthesis of truncated polypeptides. One of the mutations was found in the child with a single clinical diagnostic criterion, providing her with a presumptive diagnosis of NF1. Our results confirm that truncating mutations are frequent in both familial and sporadic NF1 cases. The identification of mutations in 14 of 21 individuals studied (67%) suggests that the use of protein truncation assays will rapidly accelerate the rate of identification of NF1 mutations. Because we scanned the entire NF1 coding region in each individual, the distribution of NF1 truncating mutations was discerned for the first time. The mutations were relatively evenly distributed throughout the coding region with no evidence for clustering.

Purification and characterization of recombinant human fibrinogen.

Our laboratory has been using protein engineering to study the relationship of primary structure to fibrinogen function. In order to examine genetically altered domains in the context of the intact, functional fibrinogen molecule, we have expressed recombinant human fibrinogen in Chinese Hamster Ovary (CHO) cells. The cDNA for each fibrinogen chain was individually cloned into the same expression vector. Each vector was cotransfected with the selection vector pRSVneo into CHO cells. In addition, the plasmids encoding A alpha and gamma were cotransfected with pRSVneo. Cells resistant to G418, a neomycin analogue, were isolated and clonal lines developed. Analysis of these lines demonstrated that CHO cells express and secrete free gamma chain, and an A alpha-gamma complex. To obtain recombinant fibrinogen, the A alpha-gamma G418-resistant clones were transfected with the B beta expression plasmid and a second selection vector, pMSVhis. Colonies resistant to neomycin and histidinol were selected and clonal lines obtained. These clones secreted biologically active recombinant human fibrinogen, which was purified from serum-free culture media by protamine-Sepharose chromatography. Analysis of the purified protein on SDS-polyacrylamide gels demonstrated a pattern indistinguishable from plasma fibrinogen. Removal of Asn-linked carbohydrate with glycosidase F revealed the presence of carbohydrate on the B beta and gamma chains, as is seen for plasma fibrinogen.

Characterization of purified recombinant fibrinogen: partial phosphorylation of fibrinopeptide A.

Human fibrinogen has been expressed in Chinese hamster ovary (CHO) cells using a novel two-step procedure which permits efficient synthesis of engineered variant fibrinogens. CHO cells secreting recombinant fibrinogen were grown in roller bottles and maintained in serum-free media for several months. Recombinant protein was purified from media containing 2-4 micrograms/mL fibrinogen using protamine-Sepharose chromatography. Recombinant fibrinogen was identical to plasma fibrinogen when examined on Coomassie-stained SDS gels run under reducing conditions, and on SDS gels when run under nonreducing conditions after partial or complete plasmin degradation, indicating normal chain assembly, disulfide bond formation, and overall protein conformation. Thrombin digestion of purified fibrinogen led to clot formation with release of normal fibrinopeptides, as identified by HPLC. Fibrinopeptide A released from recombinant fibrinogen was partially phosphorylated (22%), similar to the degree of phosphorylation found for human plasma fibrinogen (20-25%), indicating that partial phosphorylation in inherent in fibrinogen synthesis.

A synthetic analog of fibrinogen alpha 27-50 is an inhibitor of thrombin.

Binding of the synthetic peptide AAKDSDWPFAS-DEDWNYKAPSGAR, a fibrinogen alpha 27-50 analog, to thrombin was studied by inhibition assays and affinity chromatography. Peptide alpha 27-50 corresponds to a segment of human fibrinogen downstream from the thrombin cleavage site, with cysteine residues at positions 28, 36, 45 and 49 replaced by alanine. The peptide inhibited clotting of fibrinogen with an inhibition constant of 190-400 microM. Cleavage of fibrinopeptides A and B was inhibited by the peptide and the peptide was competitive with fibrinogen for thrombin. Inhibition of the small substrate tosyl-Gly-Pro-Arg-p-nitroaniline was not observed indicating that the peptide did not block the active site of the enzyme. Peptide alpha 27-50 that was covalently linked to Sepharose bound active site-inhibited thrombin at low ionic strength and was eluted at higher salt concentration. The peptide was not cleaved on overnight exposure to thrombin as determined by reverse phase HPLC. In summary, the peptide bound to, but was not a substrate for thrombin. These results suggest that this region of fibrinogen contributes to binding of thrombin.

Analysis of fibrinogen alpha residues 27-50: interactions with thrombin.

In order to further understand the unusual specificity of thrombin activity, we have examined the interactions of thrombin with fibrinogen alpha-chain residues 27-50 by two complementary approaches. Using genetically engineered constructions, we have synthesized a fusion protein containing fibrinogen A alpha-residues 1-50 and seven variants of this protein with substitutions in alpha-residues 33-41. These fusion proteins were purified and analysed as thrombin inhibitors and substrates. Substitution of Phe35----Leu of Asp40----Gly substantially increased Ki, but all the fusion proteins were equally good substrates when assayed at concentrations equivalent to Ki. These results indicate that Phe35 and Asp40 are important for thrombin binding, but not catalysis. We have also synthesized a peptide analogous to fibrinogen alpha 27-50. This peptide was a competitive inhibitor of thrombin-catalysed cleavage of fibrinogen, but was not an inhibitor of hydrolysis of small chromogenic substrates. These data further indicate that residues alpha 27-50 are important in the thrombin-fibrinogen interaction.

Inhibition of thrombin by synthetic hirudin peptides.

To investigate the role of different regions of hirudin in the interaction with the proteinase thrombin, segments of hirudin containing 15-51 residues were synthesized. The C-terminal segment 40-65 inhibited the fibrinogen clotting activity of thrombin but not amidolysis of tosyl-Gly-Pro-Arg-p-nitroanilide. Central peptide 15-42 was insoluble at pH 7, but peptide 15-65 inhibited fibrinogen clotting and amidolysis to an equal extent. The N-terminal loop peptide 1-15 had no inhibitory activity and did not affect the potency of peptide 15-65. These data suggest that the central region inhibits catalysis.