β₂-Adrenergic Receptor Gene Variation and Hypertension in Subjects With Type 2 Diabetes

Methods

For the case-control association study, we enrolled 291 unrelated hypertensive patients without type 2 diabetes and with age at onset of ≤60 years and 265 unrelated healthy control subjects without medication, personal history of hypertension, first-degree family history of hypertension, or type 2 diabetes from southern Sweden.²⁰ The subjects identified in the Skaraborg Hypertension and Diabetes Project consisted of 2 cohorts. One cohort included 1149 patients with hypertension and/or diabetes and the other cohort included an age-stratified, random sample (1400 invited, 1109 participating) from the population 40 years of age and older. Hypertensive patients and normotensive spouses from 250 families were recruited from healthcare centers in the Scania region. In addition, 124 unrelated hypertensive patients with type 2 diabetes were ascertained from the Skaraborg Hypertension and Diabetes project.²¹ Three subjects were excluded because of genotyping failure and misclassification. Hypertension was defined as 3 consecutive blood pressure measurements ≥160 mm Hg (systolic blood pressure) and/or ≥90 mm Hg (diastolic blood pressure) or ongoing antihypertensive treatment. The control subjects had systolic and diastolic blood pressures ≤150 mm Hg and ≤80 mm Hg, respectively. The diagnosis of type 2 diabetes was based on criteria from the World Health Organization.²² The participants were categorized as follows:¹ all hypertensives are patients with hypertension, regardless of whether they had type 2 diabetes or not (n=415);² hypertensives without type 2 diabetes (n=291);³ hypertensives with type 2 diabetes (n=124); and⁴ healthy controls (n=265) (Table 1⇓).

For the genotype-discordant sib pair study, 494 normotensive siblings were ascertained from 118 families with type 2 diabetes in Botnia, Finland,²³ and they were genotyped for the B2AR gene Arg16Gly and Gln27Glu polymorphisms to identify genotype discordant pairs. In total, we identified 72 sib pairs in which 1 of the siblings was homozygous for the Gly16 allele and the other sibling carried either 1 or 2 Arg16 alleles. Similarly, we identified 40 sib pairs in which 1 of the siblings was homozygous for the Glu27 allele whereas the other sibling had 1 or 2 Gln27 alleles. The 72 sib pairs who were discordant for Arg16Gly polymorphism originated from 46 sibships that comprised 118 individuals, and the 40 sib pairs who were discordant for the Gln27Glu polymorphism originated from 26 sibships and included 64 individuals (Table 1⇑). The ethics committees of the Gothenburg and Lund University approved the study. Informed consent was obtained from all the participants.

Blood pressure was measured with a sphygmomanometer after 5 minutes of rest in the supine position in the Swedish subjects and twice within 10 minutes after 15 minutes of rest in the sitting position in the Finnish subjects, the values given are the averages of the 2 measurements. Body weight and height were measured and body mass index (BMI) was calculated. BMI ≥27 kg/m² was considered as overweight. Total genomic DNA was extracted from the whole blood by standard methods.²⁴

The Arg16Gly and Gln27Glu polymorphisms were genotyped by polymerase chain reaction and restriction fragment-length polymorphism methods. The Arg16Gly polymorphism was amplified in a genomic 203-bp fragment using primers β₂ to 16-MM-F (5′-CGCCTTCTTGCTGGCACGCAAT) and β₂ to 16/27-R (5′-CCAGTGAAGTGATGAAGTAGTT), of which the former contains a nucleotide mismatch (underlined) to create a BsrDl recognition site in case of the Gly16 allele. The Gln27Glu polymorphism was amplified in a genomic 169-bp fragment with primers β₂ to 27-MM-F (5′-CCGGACCACGACGTCACCCAG) and β₂ to 16/27-R, of which the former contains a nucleotide mismatch (underlined) to create a BstNl recognition site in case of the Glu27 allele. The primers were designed according to the published B2AR cDNA sequence.¹ PCR reactions were performed with 50 ng of genomic DNA in a total volume of 20 μL containing 10 pmol of each primer, 4 nmol dNTPs, 0.5 U Taq polymerase (Pharmacia) in 1x (NH₄)₂SO₄-buffer (16 mmol/L [NH₄]₂SO₄; 67 mmol/L Tris, pH 8.8; 0.01% Tween), with 3% formamide, and 1.5 mmol/L MgCl₂. PCR conditions were as follows: initial denaturation at 94°C for 5 minutes, followed by 30 cycles of denaturation (94°C for 30 seconds), annealing (60°C for 30 seconds), and extension (72°C for 30 seconds), with the final extension at 72°C for 10 minutes. The PCR products were digested for 2 hours at 60°C using the buffers recommended by the manufacturer. The product containing the Arg16Gly polymorphism was digested with 1 U of BsrDl (New England Biolabs), which produced 2 fragments of 22 and 181 bps in case of the Gly16 allele, leaving the Arg16 allele uncut. The product containing the Gln27Glu polymorphism was digested with 1 U of BstNl (New England Biolabs), which resulted in 2 fragments of 95 and 75 bps in case of Gln27 allele and 3 fragments of 20, 58, and 95 bps in case of the Glu27 allele. The fragments were separated on 4.5% multipurpose agarose gel (Appligene) with ethidium bromide and visualized under ultraviolet light.

A genomic 172-bp fragment containing the 5′LC-Arg19Cys polymorphism was PCR amplified with a forward primer 5′-GAATGAGGCTTCCAGGCGTCC and a reverse primer 5′- GTCGTGGTCCGGCGCATGGC. PCR reactions were performed with 50 ng of genomic DNA in a total volume of 20 μL containing 10 pmol of each primer, 2 nmol dNTPs, 0.5 U Taq polymerase (Amersham Pharmacia) in a PCR buffer recommended by the manufacturer (Pharmacia) with 1.5% formamid. PCR conditions were as follows: initial denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation (94°C for 30 seconds), annealing (65°C for 30 seconds), and extension (72°C for 30 seconds), with the final extension at 72°C for 10 minutes. The PCR products were digested in a total volume of 30 μL for 3 hours at 37°C with 1 U of MspA1I restriction endonuclease (Promega) in a buffer recommended by the manufacturer. The MspA1I cuts the 5′LC-Arg19 allele to fragments of 144 and 54 bps, whereas the 5′LC-Cys19 allele remains intact. The fragments were separated on 4.5% multipurpose agarose gel (Appligene) with ethidium bromide and visualized under ultraviolet light.

Results

In the Swedish population, the genotype frequencies for the Arg16Gly polymorphism in the B2AR gene were 13.4% for Arg16Arg, 45.6% for Arg16Gly, and 41.0% for the Gly16Gly genotype, and the genotype frequencies for the Gln27Glu polymorphism were 30.1% for Gln27Gln, 48.5% for Gln27Glu, and 21.3% for the Glu27Glu genotype. This is in accordance with the genotype frequencies reported in other European populations.⁴ The observed genotype frequencies in the different study groups were in Hardy-Weinberg equilibrium. The codon 16 and 27 polymorphisms were in linkage disequilibrium in the Swedish population: 100% of the Arg16-carrying chromosomes had Gln at position 27, whereas 28% of the Gly16-carrying chromosomes had Gln and 72% had Glu in position 27. The 5′LC-Arg19Cys polymorphism was in tight linkage disequilibrium with the Gln27Glu polymorphism; the Cys19 allele was associated with the Gln27 allele in all but 1 (n=1112) chromosome.

There was a significant increase in the OR (OR 2.14; 95% CI, 1.05 to 4.33, P=0.035) for hypertension in the Arg16Arg genotype carriers with type 2 diabetes (Table 2⇓). Similarly, there was an increase in the OR for hypertension in the Gln27Gln genotype carriers (OR 1.55; 95% CI, 1.00 to 2.41, P=0.050). Neither the Arg16 nor the Gln27 allele conferred an increased risk for hypertension in subjects without type 2 diabetes. The carriers of the different genotypes showed no statistically significant difference between the serum creatinine values and the occurrence of proteinuria. The Arg16Arg genotype was associated with hypertension, especially in the nonobese (BMI<27 kg/m²) subgroup of patients with hypertension and type 2 diabetes (OR 3.47; 95% CI, 1.06 to 11.33, P=0.040).

Among the sib pairs discordant for the Arg16Gly polymorphism, those siblings carrying 1 or 2 Arg16 alleles had higher systolic blood pressure compared with individuals homozygous for the Gly16 allele (P=0.049, Table 3⇓). Among the nondiabetic pairs (n=54), siblings with 1 or 2 Arg16 alleles had a higher BMI than siblings homozygous for Gly16 (P=0.026). This was due to the male siblings (11 pairs), 25.9 kg/m² versus 24.4 kg/m² (P=0.018). Figure 1⇓ shows the difference in blood pressure and BMI of the individuals in the sib pair combinations used in the permutation test. There was a significant difference in systolic blood pressure between the siblings and in BMI in nondiabetic siblings homozygous for the Gly16 allele and carriers of 1 or more Arg16 alleles.

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Figure 1.

Clinical characteristics of the sib pairs discordant for the Arg16Gly polymorphism of the β₂-adrenergic receptor gene. *P=0.049; **P=0.026.

There were no significant differences in blood pressure levels or BMI between siblings discordant for the Gln27Glu polymorphism.

Discussion

The key finding of the present study was an increased risk for hypertension in type 2 diabetic carriers of the Arg16 and Gln27 alleles of the B2AR gene. This increased risk was not due to differences in nephropathy between the carriers. We also found that normotensive siblings from families with type 2 diabetes carrying 1 or 2 Arg16 alleles had a higher systolic blood pressure and a higher BMI compared with siblings homozygous for Gly16 allele. Because the B2AR regulates blood pressure as well as glucose and fat metabolism, we tested the B2AR polymorphisms for association with hypertension as well as diabetes and obesity. Some caution in interpretation of the data is warranted because we performed more than 1 test. The observed association between hypertension and the Arg16 allele is in accordance with a previous report in Scandinavians.⁹ In addition, a recent study in German twins showed that the Arg16 allele was associated with higher systolic blood pressure levels.¹⁰ On the other hand, Gratze et al found that young normotensive white male subjects homozygous for the Gly16 allele had higher blood pressure and decreased peripheral vasodilatation in response to salbutamol infusion.¹³ In addition, an association between the Gly16 allele and hypertension has been reported in African Caribbeans.⁸ Salt-sensitive individuals show a greater downregulation of the B2AR in response to high-salt intake.²⁷ Black Africans are known to be more salt sensitive than whites,²⁸ which has been proposed to explain the association between hypertension and the Gly16 allele in African Caribbeans.⁸

There are several other polymorphisms in the vicinity of the Arg16Gly polymorphism, such as the 5′LC-Arg19Cys, Gln27Glu, and Thr164Ile. Unfortunately, previous studies have not taken linkage disequilibrium between these polymorphisms into account. In vitro the 5′LC-Cys19 allele has been associated with an increase in B2AR gene expression, the Glu27 allele has been associated with a resistance to downregulation, the Ile164 allele has been associated with an impaired G_s-protein coupling,¹² and the Arg16 allele has been associated with decreased receptor density on the surface of cultured human fibroblasts.²⁹ Liggett and coworkers reported that the Gly16 allele is associated with increased agonist-induced downregulation of the B2AR in Chinese hamster fibroblasts and that the effect of the Gly16 allele dominates when combined with the Glu27 allele.¹² Interestingly, the Glu27 allele has otherwise been shown to be resistant to agonist-induced downregulation.³⁰

In this study we found that the haplotype LC-Cys19-Arg16-Gln27 was associated with increased risk for hypertension in the case-control association study and with elevated systolic blood pressure and higher BMI in genotype-discordant sib pairs. The allele frequencies of the polymorphisms in the B2AR gene differ between different populations.³¹ The findings that the Arg16 allele is associated with hypertension and higher blood pressure levels in Scandinavians, whereas the Gly16 allele is associated with hypertension in other populations,^{8 13} may suggest that it is not the Gly16Arg polymorphism per se but rather some polymorphism in linkage disequilibrium with the Gly16Arg polymorphism that is responsible for the association.

Because of the well-established link between hypertension and obesity, we also analyzed BMI in relation to codon 16 and 27 genotypes. We observed that the Arg16 allele was associated with a higher BMI in the sib-pair study. This is not surprising because of the strong linkage disequilibrium between the Gly16Arg and Gln27Glu polymorphisms. Several studies have shown that the Glu27 allele is associated with obesity.^{6 15 19} However, other studies have found the Gln27 allele to be associated with obesity in males.^{7 32} Our data are in accordance with a predominant role of the B2AR gene in promoting obesity in males. The difference in BMI in sib pairs was restricted to male subjects with the Arg16 allele.

In conclusion, the Arg16 and Gln27 alleles of the B2AR gene confer an increased risk for hypertension in nonobese patients with type 2 diabetes. This association was replicated in a sib-pair analysis showing that siblings with 1 or 2 Arg16 alleles had higher systolic blood pressure levels and BMI than siblings homozygous for the Gly16 allele.