E L S E V I E R Immunopharmacology 33 (1996) 32-35

Immunopharmacology

Polymorphisms in the gene for the human B2-bradykinin receptor. New tools in assessing a genetic risk for bradykinin-associated

diseases

Andreas Braun, Stefan Kammerer, Esther Maier, Eike B/Shme, Adelbert A. Roscher * Children's Hospital, Department of Clinical Chemistry and Biochemistry, University of Munich, Lindwurmstr. 4, D-80337 Munich,

Germany

Abstract

The B2-bradykinin receptor gene has been proposed as one of the candidate genes involved in the complex genetic underpinnings of common chronic disorders such as hypertension, ischemic heart disease or allergic asthma. Suitable genetic markers are needed to study these hypotheses. Therefore, it was our aim to identify polymorphic sites in the Bz-receptor gene. Up to now, we characterized four polymorphisms: one in the promoter region and three other ones in each of the exons. Possible biological consequences are delineated and preliminary results of allele specific different biological action are shown.

Keywords: Bradykinin; Human B2-receptor gene; Polymorphism; Genetic risk

1. Introduct ion

Bradykinin is released by limited proteolysis from kininogen precursors in the circulation, in interstitial tissue fluids or from the surface of kininogen-bind- ing cells. The large array of physiological activities is mainly mediated by specific receptors that phar- macologically classify as B2-bradykinin receptors which exhibit high affinity for kallidin (Lys- bradykinin) and bradykinin. The B2-bradykinin re- ceptor has been implicated as one of the candidate genes involved in the complex genetic underpinnings of common chronic disorders such as hypertension, ischemic heart disease or allergic asthma.

Recently, the human B2-bradykinin receptor

* Corresponding author.

cDNA was cloned (Hess et al., 1992), and by homol- ogy search this receptor was grouped to the super- family of the G-protein coupled seven transmem- brane domain receptors. For the purpose to identify polymorphic sites in the gene for the human B 2-

bradykinin receptor, we started with the analysis of the complete cDNA length and hence with the char- acterization of the genomic structure and organiza- tion of the gene. In Northern blots only a single Bz-receptor specific transcript with a length of ap- proximately 4 kb was detected. Screening of a hu- man fibroblast cDNA library resulted in one clone that overlaps 100 bp with the published cDNA (Hess et al., 1992) and exceeds the 3'-end about 2.5 kb. The clone contains a classical polyadenylation site and a poly-A tail 16 bp downstream. Screening of a human genomic DNA library with PCR derived cDNA probes resulted in the detection of several

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A. Braun et a l . / lmmunopharmacology 33 (1996) 32-35 33

clones. Exact examination of two of these clones revealed that the gene consists of three exons. The first exon is not-translated whereas the second and third one comprise the entire open reading frame and a large 3'-not-translated region. The region upstream of exon 1 was identified by luciferase reporter gene assays as the promoter/enhancer region (Kammerer et al., 1995).

2. Materials and methods

For PCR-amplification of sequences bearing poly- morphisms of the Bz-bradykinin receptor gene fol- lowing forward (F) and reverse (R) primers were used: BP-F, 5 ' -GCAGAGCTCAGCTGGAGG CG-3' and BP-R, 5'-CCTCCTCGGAGCCCAGAAG-3' for a part of the promoter region; BE1F, 5'-GCCCTi- TGAAAGATGAGCTG-3' and BE1R, 5'-AACTCC- CCACGACCACAG-3' for exon 1; BE2F2, 5'- CCATTTCTCCTCCCTGCTCGAG-3' and BE2R2, 5 ' -GGTGGGCACGGAGTCC TCTC-3' for a part of exon 2; B39F, 5 '-GAAGGTGGCCCAGTATGAGC- 3' and B35R, 5'-GAT TGGTCAGGATTTATGG-3' for a part of exon 3. For further details see Braun et al., 1995.

The luciferase reporter assays were performed as described elsewhere (Kammerer et al., 1995).

3. Results and discussion

For the identification of polymorphic sites in the human Bz-bradykinin receptor gene we examined a part of the promoter region and all exons with flanking intron sequences by PCR followed by single strand conformation polymorphism electrophoresis or by agarose electrophoresis. Four polymorphisms were identified.

In the promoter region a cytosine to thymidine transition occurs 58 bp upstream of the putative transcription start site (named BP-58). The two dif- ferent alleles could clearly be demonstrated by single strand conformation polymorphism (data not shown). In exon 1 we detected a tandem repeat polymor- phism that is located 12 hp downstream of the putative transcription start site and consists of three

different alleles. One allele, named BE1-2G, bears two repeat units of nine base pairs (GGTGGGGAC) whereas the other two alleles, named BE1-3G and BE1-3T, consist of three repeat units. In the allele BE1-3T a G to T transversion occurs in the interme- diate one of the repeat units (GGTGGTGAC). All three alleles were also differentiated by single strand conformation polymorphism and the patterns ob- tained are highly reproducible (data not shown). In exon 2 a C to T transition was found in the coding region which results in an arginine to cysteine substi- tution at the 14th amino acid position (R14C). To demonstrate this polymorphism we established a PCR method utilizing mutated primers (see Materials and methods: mutated nucleotides are underlined) fol- lowed by Taq I-restriction digest. In order to control the completeness of the restriction digest the mutated forward primer was designed to create an invariant Taq I site. The mutated reverse primer creates a Taq I-restriction site only if the cytosine is present but not if the thymidine is present in the template. Therefore, the Taq I digest of the PCR product results in a 145 bp fragment in the presence of the C allele and in a 165 bp fragment for the T allele. In exon 3 we detected a more complex tandem repeat polymorphism that is located in the 3'-not-translated region of the mRNA. One repeat unit is character- ized by the consensus sequence TGGA(A)GG- GCTAGAACC which is 15 or 16 bp in length, respectively. In expansion to recently published data (Braun et al., 1995), the further examination of different alleles by sequencing revealed that the most common allele in the German population consists of 43 (instead of 48) repeat units and the second com- mon allele of 33 (instead of 35) units, respectively. We were also able to identify two rare variant alleles whose lengths have not been estimated by sequenc- ing. All four polymorphic sites were evaluated in a population study utilizing a collective of unrelated individuals of South German origin. The allelic dis- tributions are given in Table 1. The allelic frequen- cies indicated that all of these loci are indeed clearly polymorphic. The statistical analysis revealed that the various genotypes are in equilibrium according to the Hardy-Weinberg principles. This means that the proportions of the various genotypes are likely to remain constant from one generation to another un- der the assumption that in a given population there is

34 A. Braun et al . / Immunopharmacology 33 (1996) 32-35

Table 1 Allelic frequencies of the four polymorphic sites in the human B2-bradykinin receptor gene in a sample of unrelated individuals of southern Germany

Alleles Frequency Statistical analysis

Promoter - Polymorphism (BP-58, n = 100) BP-58-C 0.595 X2-value 0.44 BP-58-T 0.405 exact p-value * 0.80 Exon 1 - Polymorphism (BE1, n = 179) BE1-2G 0.5307 X2-value 1.293 BE1-3T 0.2458 exact p-value * 0.730694 BE1-3G 0.2235 Exon 2 - Polymorphism (BE2, n = 179) BE2-C 0.8911 X 2 -value 2.675 BE2-T 0.1089 exact p-value * 0.12 Exon 3 - Polymorphism (BE3, n = 179) BE3-R43 0.8409 X2-value 2.009 BE3-R33 0.1397 exact p-value * 0.9189 BE3-RV1 0.0197 BE3-RV2 0.0059

* According to the method of Miiller and Clerget-Darpoux (1991).

random mating, no gene flow from racial admixture and a constant rate of mutation.

Therefore, these polymorphisms can be utilized as

Table 2

Hypotheses on the biological consequences of the polymorphic alleles of the B2-bradykinin receptor gene

Polymorphism Effect on:

promoter region ~ transcription rate exon 1 ~ transcription rate exon 2 R14C = receptor affinity

= efficiency of signal transduction receptor stability

exon 3 = mRNA processing/transport mRNA stability

new experimental tools in association or linkage studies evaluating the Bz-receptor gene as a suscepti- bility marker or a candidate disease gene.

Although speculative at the present stage, a vari- ety of hypotheses may be discussed with respect to the biological consequences of these polymorphisms (Table 2).

The polymorphism of the promoter region may influence the transcription rate of the gene. A similar effect may also result from the exon 1 polymor- phism, since this site is located 12 bp downstream of

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VP(C) vP('r) BE1-2G(C) BE1-3G(C) BE1-3T(C) BE1-3T(T) pXP-

Fig. 1. Luciferase reporter gene assay for the human B2-bradykinin receptor promoter and exon 1 region in the human embryonic kidney cell line 293. The relative light units (rlu) represent a scale for the luciferase expression under the control of different alleles of the regulatory region: two promoter alleles of approximately 1.2 kb in length upstream of the repeat polymorphism (VP(C) and VP(T)) and four fragments of approximately 1.3 kb in length. In these constructs the promoter region was combined with various alleles of the exon 1 (BE1-2G(C), BE1-3G(C), BE1-3T(C), and BE1-3T(T)). The vector containing no insert (pXP - ) represents the background of the assay. The positive control construct pRSV, possessing a strong RSV promoter, generates a light emission of about 13 million relative light units (data not shown). The mean + 1 S.D. of three different experiments is shown.

A. Braun et al . / bnmunopharmacology 33 (1996) 32-35 35

the putative transcription start site. The polymor- phism of exon 2 (coding region) results in two different allelic polypeptides that could alter either

the affinity of the receptor for bradykinin or the

efficiency of the signal transduction pathway. It may also influence the biological half time of the recep- tor. The T-untranslated region of genes has been shown to participate in the regulation of mRNA

stability by a variety of mechanisms (Ross, 1995). Therefore the bradykinin receptor polymorphism in exon 3 could theoretically affect the processing and the transport of the mRNA from the nucleus to the r ibosomes or the stability of the mRNA.

As a first attempt to prove these hypotheses, we initiated in vitro transfection experiments in human

embryonic kidney cells. We performed luciferase reporter gene assays to examine the effect of the different alleles of the promoter and exon 1 on the

transcription rate (Fig. 1). The two different alleles of the promoter region (VP(C) and VP(T)) did not show significant differences in the extent of lu- ciferase expression. However, by combining the pro- moter region with various alleles of the exon 1 (BE1-2G(C), BEI-3G(C) , BE1-3T(C), and BE1- 3T(T)) in all of the combinations a reduction of the

transcriptional activity was observed. The decrease was most pronounced (to about 50%) by using the promoter in combination with the BE1-3T(C) allele.

These preliminary experiments represent of course only one example of the variety of biological alter- ations that may possibly occur as consequences of

these polymorphisms. Further in vitro transfection experiments or association and linkage studies of defined 'disease populations ' are needed to substan-

tiate these hypotheses.

References

Braun A, Kammerer S, BiShme E, Mtiller B, Roscher A.A. Identification of polymorphic sites of the human bradykinin B 2 receptor gene. Biochem Biophys Res Comm 1995; 211: 234-240.

Hess JF, Borkowski JA, Young GS, Strader CD, Ransom RW. Cloning and pharmacological characterization of a human bradykinin (BK-2) receptor. Biochem Biophys Res Comm 1992; 184: 260-268.

Kammerer S, Braun A, Arnold N, Roscher AA The human bradykinin B 2 receptor gene: full length cDNA, genomic organization and identification of the regulatory region. Biochem Biophys Res Comm 1995; 211: 226-233.

Mtiller B, Clerget-Darpoux F. A test based on the exact probabil- ity distribution of the chi-square statistic-incorporation into the MASC method. Ann Hum Genet 1991; 55: 69-75.

Ross J. mRNA stability in mammalian cells (review). Microbiol Rev 1995; 59: 423-450.