Screening the human bradykinin B2 receptor gene in patients with cardiovascular diseases: Identification of a functional mutation in the promoter and a new coding variant (T21M)

To elucidate if genetic variants in the bradykinin B₂ receptor (B₂) gene occur that could affect receptor expression and function, we screened for mutations in the promoter and in the coding region of the human B₂ gene. In our initial study we analyzed 92 consecutive, unrelated subjects (including 25 patients with hypertrophic cardiomyopathy, 18 patients with dilated cardiomyopathy (DCM), 25 patients with hypertension, 18 patients with coronary heart disease, and 6 patients with valvular heart disease) using nonradioactive polymerase chain reaction–single-strand conformation polymorphism analysis as mutation screening method. We detected eight as yet unknown polymorphic sites in the promoter region of the B₂ gene (−845 C/T, −704C/T, −649 insG, −640 T/C, −536 C/T, −412 C/G, −143 C/T and −78 C/T) with allele frequencies between 0.5 and 13%. One of them (−412 C/G) destroys a Sp1 binding site and abolishes protein binding to this Sp1 site in human umbilical vein endothelial cells and human vascular smooth muscle cells. In the protein-coding region one new coding variant (T21M) with the potential to create a truncated receptor isoform was detected. We determined the frequency of the promoter variant at position −412 (C → G) and the newly identified coding variant (T21M) in extended samples of 69 patients with HCM, 163 patients with DCM, 109 patients with hypertension, and 173 healthy anonymous blood donors. The promoter variant (−412C/G) was found in one blood donor and the T21M mutation was not found in the control population. Therefore, it appears that these mutations are rare events and the determination of clinical significance will be a demanding task in the future. Am. J. Med. Genet. 80:521–525, 1998. © 1998 Wiley-Liss, Inc.     

Establishment of lung fibroblastic cell lines from a non-human primate Tupaia belangeri and their use in a forward gene mutation assay at the hypoxanthing--guanine phosphoribosyl transferase locus

The cells obtained from a lung of a new-born male Tupaia belangeriwere maintained in mass culture for >400 days. After 55 populationdoubling levels (100 days in culture), three cell lines wereseparately established; these lines showed constant growth properties.One line, designated as T-23, was used for a mutation assay.The T-23 cells showed an absolute plating efficiency of 30–50%,and a population doubling time of 18–19 h in Dulbecco'smodified Eagle's medium containing 10% fetal bovine serum. Thecells had a modal chromosome number of 62 (pseudodiploid) withthe loss of a chromosome and the gain of an unidentified one.T-23 cells, like human cells, were much more susceptible toouabain than mouse cells but relatively less susceptible to8-azaguanine, while, unlike human cells, they were less sensitiveto 6-thioguanine (6TG). N-Methyl-N'-nitro-N-nitrosoguanidine(MNNG) was less, but 4-nitroquinoline-l-oxide (4NQO) was moretoxic to T-23 cells than to human or mouse cells. Benzo[a]pyrene-inducedtoxicity was almost comparable among the cell types. For themutation assay, we chose 6TG-resistance (100 µM) as amarker. The optimal expression time (8–13 days) and celldensity at selection to eliminate metabolic cooperation (2 x10⁴ cells/ 60-mm dish) were determined. Some of the cells selectedwith 6TG showed <0.4% of the total incorporation of [¹⁴C]hypox-anthineinto wild-type cells, suggesting the mutants under selectionwere affected at the hypoxanthine-guanine phos-phoribosyl transferaselocus. Following the optimal protocol, we performed a quantitativemutation assay with simple alky-lating agents, MNNG, N-ethyl-N'-nitro-N-nitrosoguanidine,methyl methanesulphonate, ethyl methanesulphonate and 4NQO.All induced mutants in a dose-dependent fashion. The abilityto induce mutants in T-23 cells by these mutagens was comparablewith that in a conventional V79 assay. This is the first reportof a gene mutation assay system using non-human primate cells. ¹To whom correspondence should be addressed