Impact of Genetic Polymorphism in the β2-Receptor Gene on Risk of Severe Hypoglycemia in Patients With Type 1 Diabetes

Impact of Genetic Polymorphism in the β2-Receptor Gene on Risk of Severe Hypoglycemia in... Abstract Context Severe hypoglycemic events are unevenly distributed in people with type 1 diabetes, making a genetic influence probable. Of the common adrenoceptor β-2 receptor gene (ADRB2) polymorphisms, the Arg16 allele is associated with receptor downregulation and reduced agonist-mediated endogenous glucose production. Objective We tested the hypothesis that the Arg16 variant is associated with severe hypoglycemia. Method A cohort of 311 patients with type 1 diabetes reported severe hypoglycemic events retrospectively in a validated questionnaire. The patients were characterized by diabetes history, state of hypoglycemia awareness, C-peptide status, HbA1c, and ADRB2 genotype. Results The ADRB2 Gly16Arg genotype distribution was in Hardy-Weinberg equilibrium. The rate of severe hypoglycemia differed among all genotypes (P = 0.01). Patients homozygous for the Arg16 genotype (AA; n = 60) had a relative rate (RR) of severe hypoglycemia of 2.2 (95% CI, 1.3 to 3.6) compared with patients homozygous for the Gly16 genotype (GG; n = 116; P = 0.002). Among patients with impaired awareness or unawareness (n = 175), those with the AA genotype (n = 33) had an RR of severe hypoglycemia of 3.2 (95% CI, 1.7 to 6.0) compared with patients with the GG genotype (n = 58; P < 0.000). Genotype was not associated with state of hypoglycemia awareness per se, as assessed by any of three classification methods. The difference was not explained by other risk factors. Conclusion Genetic polymorphism in ADRB2 is associated with risk of severe hypoglycemia in individuals with type 1 diabetes, especially in those with impaired hypoglycemia awareness. In type 1 diabetes, the metabolic and symptomatic protective responses to hypoglycemia are often impaired with time (1). An early blunting of the glucagon response is accompanied by disappearing endogenous insulin production (1, 2), rendering patients increasingly dependent upon epinephrine as their first-line protection against development of severe hypoglycemia. However, in many patients, as a result of recurrent hypoglycemia and/or long duration of diabetes, the epinephrine response to hypoglycemia also becomes blunted (i.e., hypoglycemia-associated autonomic failure) (1), leading to reduced endogenous glucose production (EGP) during hypoglycemia and impaired recognition of warning symptoms (i.e., impaired hypoglycemia awareness). Although impaired awareness is usually linked to loss of adrenergic symptoms, it is apparent that hypoglycemic warning symptoms are complex and can occur independently of the epinephrine response (3). Impaired hypoglycemia awareness, in turn, results in a grossly increased risk of severe hypoglycemia (4–7). In addition to the failing catecholamine response to hypoglycemia often seen with impaired hypoglycemia awareness, a reduced β2-adrenergic sensitivity has been reported in some (8, 9), but not all, studies (10). According to previous reports, treatment with nonspecific β-blockers with effect on the β2-receptor is associated with reduced EGP to epinephrine infusion (11) and impaired recovery from hypoglycemia (12, 13). Among patients with impaired awareness, the risk of severe hypoglycemia is varying and the fact that many patients seem chronically prone to recurrent severe hypoglycemia makes a genetic influence probable. A potential candidate gene to consider is the adrenoceptor β-2 receptor gene (ADRB2), in which several common variants are described (14). Of these, the single nucleotide polymorphism (SNP) Gly vs Arg at codon 16 (rs1042713) affects β2-receptor desensitization during β2-agonist treatment of asthma in a clinically important manner (15). Although diverse results are reported, the Arg16 variant of polymorphism in ADRB2 is associated with increased β2-receptor desensitization during agonist therapy, resulting in reduced treatment efficiency (16–18). In contrast, the ADRB2 polymorphism Gln vs Glu at codon 27 (rs1042714) is associated with increased agonist-mediated responsiveness in vasculature (16). We have shown that healthy humans who are homozygous for the ADRB2 Arg16 variant react with lesser increment in plasma glucose levels to adrenaline infusion, suggesting a reduced EGP resulting from receptor stimulation in these persons (19). This leads to the hypothesis that the ADRB2 Arg16 variant is associated with severe hypoglycemia in type 1 diabetes by either a reduced EGP during hypoglycemia or by predisposing to reduced adrenergic symptom intensity, and thus impaired hypoglycemia awareness, during hypoglycemia. We tested this hypothesis in a cohort of individuals with type 1 diabetes. Materials and Methods The study was a retrospective, observational, noninterventional cohort study. A total of 416 patients attending the outpatient clinic at Nordsjællands Hospital, Hillerød, Denmark, were invited to participate in the study. After giving written informed consent, a cohort of 311 consecutive adult patients (age >18 years) with type 1 diabetes for >1 year was recruited from November 2011 to May 2013. Type 1 diabetes was defined by insulin treatment from the time of diagnosis and unstimulated C-peptide concentration <300 pmol/L or stimulated (venous blood glucose concentration >12 mmol/L) C-peptide concentration<600 pmol/L. Pregnant women and individuals with severe concomitant diseases were excluded from participation. Information about history of diabetes, treatment, and late complications were extracted from the medical records. Hypoglycemia awareness was self-estimated using three validated methods: those of Hillerød (20) (n = 311), Gold (4) (n = 304; data were missing for 7 patients), and Clarke (5) (n = 297, data were missing for 14 patients). The Hillerød method classifies patients into three classes of hypoglycemia awareness: normal, or impaired awareness, or hypoglycemia unawareness (6, 20). In contrast, the Clarke and Gold methods divide hypoglycemia awareness into two classes (4, 5). The number of hypoglycemic events was recorded in a questionnaire based on recall within 1 year, which is well preserved and valid (20). Definition of severe hypoglycemia was “an event with low blood glucose requiring assistance of another person to actively administer carbohydrate, glucagon, or other resuscitative actions” (21), which is in accordance with the American Diabetes Association (21) and the International Hypoglycemia Study Group recommendations (22). The primary end point was number of reported events of severe hypoglycemia in the preceding year, and secondary end points were prevalence of severe hypoglycemia and self-reported hypoglycemia awareness status (via Clarke, Gold, and Hillerød methods). The study was approved by the Committee on Biomedical Ethics of the Copenhagen Capital Region (H-4–2011–043) and the Danish Data Protection Agency (2012-58-0004). Laboratory tests We measured glycated HbA1c (Tosoh G8 HPLC Analyzer; Tosoh Bioscience, San Francisco, CA) and level of C-peptide (AutoDELFIA; Wallac Oy, Turku, Finland). The patients were classified as C-peptide negative when C-peptide levels were <10 pmol/L. Purification of DNA and genotyping DNA purification and genotyping of the ADRB2 SNPs Gly16Arg (rs 042713) and Gln27Glu (rs 1042714) was carried out commercially at LGC Genomics Unit (Hoddesdon, Hertfordshire, United Kingdom) and was done blinded to patient data. DNA was extracted from frozen buffy-coat samples by the KleargeneTM extraction protocol (LGC Genomics). Genotyping was performed using the KASPTM genotyping protocol. KASPTM assays are based on competitive allele-specific PCR. The SNP-specific KASPTM Assay mix (LGC Genomics) was added to DNA samples, a thermal cycling reaction was then performed, followed by an end-point fluorescent reading. Biallelic discrimination was achieved through the competitive binding of two allele-specific forward primers, each with a unique tail sequence that corresponds with two universal fluorescence resonant energy transfer cassettes, one labeled with FAMTM dye and the other with HEXTM dye. Statistics Statistical analysis was performed using the SPSS, version 22.0 (IBM, Armonk, NY). Comparisons between variables were made by parametric and nonparametric methods as appropriate. The influence of risk factors (i.e., genotype, state of hypoglycemia awareness, duration of diabetes, and HbA1c and C-peptide status) on the number of severe hypoglycemic events was assessed by a log-linear negative binomial model, which takes into account the skewed distribution of events of severe hypoglycemia. The prevalence of severe hypoglycemia is presented as the percentage of patients having one or more events of severe hypoglycemia during the last year. The incidence of severe hypoglycemia is presented as the rate of severe hypoglycemia, given as relative rate (RR) ratio and 95% CI. To evaluate the relative impact of ADRB2 Gly16Arg genotype and known risk factors of severe hypoglycemia, a stepwise regression analysis was performed, with inclusion of significant and nearly significant (P < 0.1) univariate analysis parameters. Other values shown in the text and tables are mean and SD, or percentage, as indicated. P < 0.05 (two-sided) was regarded as significant. Results Baseline characteristics of the participants and the prevalence of the Gly16Arg genotypes are shown in Table 1. The ADRB2 Gly16Arg and the Gln27Glu genotype distribution was comparable to that of the general Danish population (23) and was in Hardy-Weinberg equilibrium (P = 0.77 and P = 0.58, respectively). No significant differences in baseline characteristics were observed between the genotypes (Table 1). A total of 19 patients were treated with β-blockers. Excluding these from the analysis did not significantly change the results; therefore, they were not excluded from the cohort. Table 1. Baseline Characteristics and Hypoglycemia Awareness Classification According to ADRB2 Polymorphisms Characteristic GG GA AA Total P Patients, no. (%) 116 (37) 135 (43) 60 (19) 311 (100) — Male patients, % 65 59 52 60 0.20 Age, mean ± SD, y 53 ± 13 52 ± 15 53 ± 14 52 ± 14 0.74 Duration of diabetes, mean ± SD, y 23 ± 12 23 ± 15 24 ± 15 23 ± 14 0.93 HbA1c, mean ± SD, mmol/mol 61 ± 11 63 ± 13 62 ± 10 62 ± 11 0.50 C-peptide negative status, %a 77 78 73 77 0.80 Hypoglycemia awareness, %  Hillerød classification, aware/impaired/unaware 50/34/16 38/49/13 45/43/12 43/42/14 0.22  Gold classification,b aware/impaired 73/27 79/21 76/24 76/24 0.57  Clarke classification,c aware/unclassified/impaired 75/9/16 73/9/18 71/11/18 74/9/17 0.98 Characteristic GG GA AA Total P Patients, no. (%) 116 (37) 135 (43) 60 (19) 311 (100) — Male patients, % 65 59 52 60 0.20 Age, mean ± SD, y 53 ± 13 52 ± 15 53 ± 14 52 ± 14 0.74 Duration of diabetes, mean ± SD, y 23 ± 12 23 ± 15 24 ± 15 23 ± 14 0.93 HbA1c, mean ± SD, mmol/mol 61 ± 11 63 ± 13 62 ± 10 62 ± 11 0.50 C-peptide negative status, %a 77 78 73 77 0.80 Hypoglycemia awareness, %  Hillerød classification, aware/impaired/unaware 50/34/16 38/49/13 45/43/12 43/42/14 0.22  Gold classification,b aware/impaired 73/27 79/21 76/24 76/24 0.57  Clarke classification,c aware/unclassified/impaired 75/9/16 73/9/18 71/11/18 74/9/17 0.98 a Patients were classified as C-peptide negative when C-peptide levels were <10 pmol/L. b Gold classification (n = 304, data missing for seven patients; distribution of patients according to genotype group: GG, n = 114; GA, n = 131; AA, n =59). c Clarke classification (n = 297, data missing for 14 patients; distribution of patients according to genotype group: GG, n = 113; GA, n = 128; AA, n = 56). View Large Table 1. Baseline Characteristics and Hypoglycemia Awareness Classification According to ADRB2 Polymorphisms Characteristic GG GA AA Total P Patients, no. (%) 116 (37) 135 (43) 60 (19) 311 (100) — Male patients, % 65 59 52 60 0.20 Age, mean ± SD, y 53 ± 13 52 ± 15 53 ± 14 52 ± 14 0.74 Duration of diabetes, mean ± SD, y 23 ± 12 23 ± 15 24 ± 15 23 ± 14 0.93 HbA1c, mean ± SD, mmol/mol 61 ± 11 63 ± 13 62 ± 10 62 ± 11 0.50 C-peptide negative status, %a 77 78 73 77 0.80 Hypoglycemia awareness, %  Hillerød classification, aware/impaired/unaware 50/34/16 38/49/13 45/43/12 43/42/14 0.22  Gold classification,b aware/impaired 73/27 79/21 76/24 76/24 0.57  Clarke classification,c aware/unclassified/impaired 75/9/16 73/9/18 71/11/18 74/9/17 0.98 Characteristic GG GA AA Total P Patients, no. (%) 116 (37) 135 (43) 60 (19) 311 (100) — Male patients, % 65 59 52 60 0.20 Age, mean ± SD, y 53 ± 13 52 ± 15 53 ± 14 52 ± 14 0.74 Duration of diabetes, mean ± SD, y 23 ± 12 23 ± 15 24 ± 15 23 ± 14 0.93 HbA1c, mean ± SD, mmol/mol 61 ± 11 63 ± 13 62 ± 10 62 ± 11 0.50 C-peptide negative status, %a 77 78 73 77 0.80 Hypoglycemia awareness, %  Hillerød classification, aware/impaired/unaware 50/34/16 38/49/13 45/43/12 43/42/14 0.22  Gold classification,b aware/impaired 73/27 79/21 76/24 76/24 0.57  Clarke classification,c aware/unclassified/impaired 75/9/16 73/9/18 71/11/18 74/9/17 0.98 a Patients were classified as C-peptide negative when C-peptide levels were <10 pmol/L. b Gold classification (n = 304, data missing for seven patients; distribution of patients according to genotype group: GG, n = 114; GA, n = 131; AA, n =59). c Clarke classification (n = 297, data missing for 14 patients; distribution of patients according to genotype group: GG, n = 113; GA, n = 128; AA, n = 56). View Large ADRB2 Gly16Arg genotype and severe hypoglycemia The overall prevalence of severe hypoglycemia was 22%, and the rate of severe hypoglycemia was 0.54 events per patient per year. The prevalence of one or more events of severe hypoglycemia experienced during the last year according to genotype was 35% in the people homozygous for the Arg16 allele (AA), which was almost twofold higher than in patients homozygous for the Gly16 allele (GG; 18%; P = 0.013; Fig. 1). The RR of severe hypoglycemia during the last year was increased in AA patients (2.2; 95% CI, 1.3 to 3.6) compared with GG patients (P < 0.002; Fig. 2). Figure 1. View largeDownload slide Prevalence of severe hypoglycemia according to the Gly16Arg ADRB2 polymorphisms and hypoglycemia awareness status. Difference in severe hypoglycemia (i.e., one or more events) experienced during the last year, according to genotype, GG, GA, and AA, in the total population (N = 311; P = 0.027)*, in patients with normal hypoglycemia awareness (n = 136; P = 0.93), and in patients with impaired hypoglycemia awareness status or hypoglycemia unawareness according to the Hillerød classification (n = 175; P = 0.009)**. Figure 1. View largeDownload slide Prevalence of severe hypoglycemia according to the Gly16Arg ADRB2 polymorphisms and hypoglycemia awareness status. Difference in severe hypoglycemia (i.e., one or more events) experienced during the last year, according to genotype, GG, GA, and AA, in the total population (N = 311; P = 0.027)*, in patients with normal hypoglycemia awareness (n = 136; P = 0.93), and in patients with impaired hypoglycemia awareness status or hypoglycemia unawareness according to the Hillerød classification (n = 175; P = 0.009)**. Figure 2. View largeDownload slide RR of severe hypoglycemia according to the ADRB2 Gly16Arg polymorphism and hypoglycemia awareness status (using the Hillerød method). The difference in RR (black bars indicate 95% CI) of reported events of severe hypoglycemia during the last year, according to awareness status and genotype: GG (light gray dots), GA (dark gray dots), and AA (black dots). A significant difference between genotypes was found in the total population (N = 311; P = 0.01)* and in patients with either hypoglycemia unawareness or impaired awareness (n = 175; P < 0.002)**. No significant difference was found between genotype groups in patients with normal hypoglycemia awareness (n = 136; P = 0.087). Figure 2. View largeDownload slide RR of severe hypoglycemia according to the ADRB2 Gly16Arg polymorphism and hypoglycemia awareness status (using the Hillerød method). The difference in RR (black bars indicate 95% CI) of reported events of severe hypoglycemia during the last year, according to awareness status and genotype: GG (light gray dots), GA (dark gray dots), and AA (black dots). A significant difference between genotypes was found in the total population (N = 311; P = 0.01)* and in patients with either hypoglycemia unawareness or impaired awareness (n = 175; P < 0.002)**. No significant difference was found between genotype groups in patients with normal hypoglycemia awareness (n = 136; P = 0.087). ADRB2 Gly16Arg genotype and hypoglycemia awareness Overall, according to the Hillerød classification, 14% of patients were classified as having hypoglycemia unawareness. Using the Gold classification, the prevalence was 24%; it was 17% when using the Clarke classification (Table 1). We found no significant difference in the distribution of awareness classes between the genotype groups with any of the three classification methods (Table 1). ADRB2 Gly16Arg genotype, severe hypoglycemia, and hypoglycemia awareness Prevalence of severe hypoglycemia according to Gly16Arg ADRB2 polymorphisms and hypoglycemia awareness status according to the Hillerød method is shown in Fig. 1. There was no difference in the prevalence of severe hypoglycemia between genotype groups among patients with normal hypoglycemia awareness (P = 0.93). Among patients with impaired awareness or unawareness according to the Hillerød classification, the prevalence of severe hypoglycemia was 55% in AA patients compared with 28% in GG patients (P = 0.009; Fig. 1). Prevalence of severe hypoglycemia according to Gly16Arg ADRB2 polymorphisms and hypoglycemia awareness status according to the Gold and Clarke classifications showed the same trend as seen using the Hillerød classification. The RR of severe hypoglycemia according to Gly16Arg ADRB2 polymorphism and hypoglycemia awareness status according to the Hillerød method is shown in Fig. 2. There was no difference in the RR of severe hypoglycemia between genotype groups among patients with normal awareness (P = 0.087). Among patients having either impaired awareness or unawareness of hypoglycemia, AA patients had an increased rate of severe hypoglycemia (3.2; 95% CI, 1.7 to 6.0; P < 0.000) compared with GG patients (Fig. 2). ADRB2 Gly16Arg genotype and other risk factors, and multivariate analyses The impact of known risk factors on the incidence of severe hypoglycemia was tested by univariate analysis (Table 2). Increasing levels of HbA1c, C-peptide negative status, and hypoglycemia unawareness status according to all three methods were significantly associated with increased incidence of severe hypoglycemia, whereas increased duration of diabetes showed a trend toward increasing the incidence of severe hypoglycemia, but it did not reach statistically significance (Table 2). Table 2. Univariate Analysis of Known Risk Factors of Severe Hypoglycemia Univariate Analysis Variable RR (95% CI) P AA genotype 2.2 (1.3–3.6) 0.002 GA genotype 1.4 (0.89–2.2) 0.15 Duration of diabetes, y 1.01 (1.0–1.02) 0.09 HbA1c, mmol/mol 1.02 (1.00–1.03) <0.03 C-peptide negativea 3.1 (1.7–5.6) <0.000 Hillerød classification  Hypoglycemia unaware 7.3 (4.2–12.7) <0.000  Impaired awareness 2.6 (1.6–4.2) <0.000 Gold classificationb  Hypoglycemia unaware 3.0 (2.0–4.5) <0.000 Clarke classificationc  Hypoglycemia unaware 5.4 (3.5–8.3) <0.000 Univariate Analysis Variable RR (95% CI) P AA genotype 2.2 (1.3–3.6) 0.002 GA genotype 1.4 (0.89–2.2) 0.15 Duration of diabetes, y 1.01 (1.0–1.02) 0.09 HbA1c, mmol/mol 1.02 (1.00–1.03) <0.03 C-peptide negativea 3.1 (1.7–5.6) <0.000 Hillerød classification  Hypoglycemia unaware 7.3 (4.2–12.7) <0.000  Impaired awareness 2.6 (1.6–4.2) <0.000 Gold classificationb  Hypoglycemia unaware 3.0 (2.0–4.5) <0.000 Clarke classificationc  Hypoglycemia unaware 5.4 (3.5–8.3) <0.000 The impact of known risk factors on the incidence of severe hypoglycemia was tested by univariate analysis (N = 311). RR values represent the following: Gly16Arg genotype, RR of AA and GA patients compared with GG patients; HbA1c level, RR per one increment in mmol/mol in HbA1c. For C-peptide status RR, C-peptide–negative subjects (C-peptide levels <10 pmol/L) are compared with C-peptide–positive subjects. Hypoglycemia awareness status (Hillerød method), RR of patients with hypoglycemia unawareness or impaired awareness is compared with those having normal hypoglycemia awareness. Hypoglycemia awareness status according to Gold and Clarke methods: RR of patients with hypoglycemia unawareness is compared with subjects having normal hypoglycemia awareness. a C-peptide status (n = 308, data missing for three patients). b Gold classification (n = 304, data missing for seven patients). c Clarke classification (n = 297, data missing for 14 patients). There was a group of 27 patients who were unclassified according to the Clarke classification (not shown). View Large Table 2. Univariate Analysis of Known Risk Factors of Severe Hypoglycemia Univariate Analysis Variable RR (95% CI) P AA genotype 2.2 (1.3–3.6) 0.002 GA genotype 1.4 (0.89–2.2) 0.15 Duration of diabetes, y 1.01 (1.0–1.02) 0.09 HbA1c, mmol/mol 1.02 (1.00–1.03) <0.03 C-peptide negativea 3.1 (1.7–5.6) <0.000 Hillerød classification  Hypoglycemia unaware 7.3 (4.2–12.7) <0.000  Impaired awareness 2.6 (1.6–4.2) <0.000 Gold classificationb  Hypoglycemia unaware 3.0 (2.0–4.5) <0.000 Clarke classificationc  Hypoglycemia unaware 5.4 (3.5–8.3) <0.000 Univariate Analysis Variable RR (95% CI) P AA genotype 2.2 (1.3–3.6) 0.002 GA genotype 1.4 (0.89–2.2) 0.15 Duration of diabetes, y 1.01 (1.0–1.02) 0.09 HbA1c, mmol/mol 1.02 (1.00–1.03) <0.03 C-peptide negativea 3.1 (1.7–5.6) <0.000 Hillerød classification  Hypoglycemia unaware 7.3 (4.2–12.7) <0.000  Impaired awareness 2.6 (1.6–4.2) <0.000 Gold classificationb  Hypoglycemia unaware 3.0 (2.0–4.5) <0.000 Clarke classificationc  Hypoglycemia unaware 5.4 (3.5–8.3) <0.000 The impact of known risk factors on the incidence of severe hypoglycemia was tested by univariate analysis (N = 311). RR values represent the following: Gly16Arg genotype, RR of AA and GA patients compared with GG patients; HbA1c level, RR per one increment in mmol/mol in HbA1c. For C-peptide status RR, C-peptide–negative subjects (C-peptide levels <10 pmol/L) are compared with C-peptide–positive subjects. Hypoglycemia awareness status (Hillerød method), RR of patients with hypoglycemia unawareness or impaired awareness is compared with those having normal hypoglycemia awareness. Hypoglycemia awareness status according to Gold and Clarke methods: RR of patients with hypoglycemia unawareness is compared with subjects having normal hypoglycemia awareness. a C-peptide status (n = 308, data missing for three patients). b Gold classification (n = 304, data missing for seven patients). c Clarke classification (n = 297, data missing for 14 patients). There was a group of 27 patients who were unclassified according to the Clarke classification (not shown). View Large To evaluate the relative impact of ADRB2 Gly16Arg genotype and known risk factors of severe hypoglycemia, a stepwise regression analysis was performed, with inclusion of ADRB2 Gly16Arg genotype, HbA1c level, C-peptide status, diabetes duration, and hypoglycemia awareness status (Table 3). In this analysis, the ADRB2 Gly16Arg genotype remained associated with events of severe hypoglycemia, with the AA genotype having an increased RR of 2.0 (95% CI, 1.2 to 3.5; P = 0.01; Table 3). Data analysis also revealed an increased RR for patients having impaired hypoglycemia awareness (RR, 2.2; 95% CI, 1.3 to 3.6; P < 0.004) and hypoglycemia unawareness (RR 6.8; 95% CI, 3.7 to 12.3; P < 0.000; Table 3). Table 3. Multivariate Analysis and Relative Impact of ADRB2 Gly16Arg Genotype and Known Risk Factors of Severe Hypoglycemia Multivariate Analysis Variable RR (95% CI) P Hillerød method (n = 308)a  AA genotype 2.0 (1.2–3.5) 0.01  GA genotype 1.1 (0.7–1.9) 0.60  Duration of diabetes, y 1.0 (0.98–1.01) 0.38  HbA1c, mmol/mol 1.02 (1.00–1.04) 0.046  C-peptide negative status 2.6 (1.3–5.2) 0.006   Hypoglycemia awareness status    Impaired hypoglycemia awareness 2.2 (1.3–3.6) 0.004    Hypoglycemia unaware 6.8 (3.7–12.3) <0.000 Gold method (n = 301)b  AA genotype 2.4 (1.4–4.2) 0.001  GA genotype 1.6 (0.97–2.6) 0.066  Duration of diabetes, y 1.0 (0.98–1.01) 0.69  HbA1c - mmol/mol 1.02 (1.00–1.04) 0.046  C-peptide negative status 3.0 (1.5–5.7) 0.001   Hypoglycemia awareness status    Hypoglycemia unaware 3.0 (2.0–4.6) <0.000 Clarke method (n = 294)c  AA genotype 2.1 (1.2–3.7) 0.01  GA genotype 1.1 (0.7–1.9) 0.62  Duration of diabetes, y 1.0 (0.98–1.02) 0.89  HbA1c - mmol/mol 1.01 (1.00–1.03) 0.15  C-peptide negative status 2.4 (1.3–4.7) 0.009   Hypoglycemia awareness status    Hypoglycemia unaware 4.8 (3.0–7.7) <0.000 Multivariate Analysis Variable RR (95% CI) P Hillerød method (n = 308)a  AA genotype 2.0 (1.2–3.5) 0.01  GA genotype 1.1 (0.7–1.9) 0.60  Duration of diabetes, y 1.0 (0.98–1.01) 0.38  HbA1c, mmol/mol 1.02 (1.00–1.04) 0.046  C-peptide negative status 2.6 (1.3–5.2) 0.006   Hypoglycemia awareness status    Impaired hypoglycemia awareness 2.2 (1.3–3.6) 0.004    Hypoglycemia unaware 6.8 (3.7–12.3) <0.000 Gold method (n = 301)b  AA genotype 2.4 (1.4–4.2) 0.001  GA genotype 1.6 (0.97–2.6) 0.066  Duration of diabetes, y 1.0 (0.98–1.01) 0.69  HbA1c - mmol/mol 1.02 (1.00–1.04) 0.046  C-peptide negative status 3.0 (1.5–5.7) 0.001   Hypoglycemia awareness status    Hypoglycemia unaware 3.0 (2.0–4.6) <0.000 Clarke method (n = 294)c  AA genotype 2.1 (1.2–3.7) 0.01  GA genotype 1.1 (0.7–1.9) 0.62  Duration of diabetes, y 1.0 (0.98–1.02) 0.89  HbA1c - mmol/mol 1.01 (1.00–1.03) 0.15  C-peptide negative status 2.4 (1.3–4.7) 0.009   Hypoglycemia awareness status    Hypoglycemia unaware 4.8 (3.0–7.7) <0.000 Three stepwise regression analyses of ADRB2 Gly16Arg genotype, hypoglycemia awareness status [Hillerød classification (model 1), Gold classification (model 2), Clarke classification (model 3)], duration of diabetes, HbA1c level, and C-peptide status were performed. RR values represent the following: for Gly16Arg genotype, RR of AA and GA patients compared with GG patients; for HbA1c level, RR per one increment in mmol/mol in HbA1c; C-peptide status: C-peptide–negative subjects (C-peptide levels below 10 pmol/l) are compared to C-peptide–positive subjects. Hypoglycemia awareness status (Hillerød method): RR of patients with hypoglycemia unawareness or impaired awareness is compared with patients having normal hypoglycemia awareness. Hypoglycemia awareness status according to Gold and Clarke methods: RR of patients with hypoglycemia unawareness is compared with patients having normal hypoglycemia awareness. a n=308: data missing on C-peptide status for three patients. b n = 301: data missing on Gold hypoglycemia awareness classification for 7 patients and on C-peptide status for three patients. c n = 294: data missing on Clarke classification for 14 patients and on C-peptide status for 3 patients. A group of 27 subjects were unclassified according to the Clarke hypoglycemia awareness classification (not shown). View Large Table 3. Multivariate Analysis and Relative Impact of ADRB2 Gly16Arg Genotype and Known Risk Factors of Severe Hypoglycemia Multivariate Analysis Variable RR (95% CI) P Hillerød method (n = 308)a  AA genotype 2.0 (1.2–3.5) 0.01  GA genotype 1.1 (0.7–1.9) 0.60  Duration of diabetes, y 1.0 (0.98–1.01) 0.38  HbA1c, mmol/mol 1.02 (1.00–1.04) 0.046  C-peptide negative status 2.6 (1.3–5.2) 0.006   Hypoglycemia awareness status    Impaired hypoglycemia awareness 2.2 (1.3–3.6) 0.004    Hypoglycemia unaware 6.8 (3.7–12.3) <0.000 Gold method (n = 301)b  AA genotype 2.4 (1.4–4.2) 0.001  GA genotype 1.6 (0.97–2.6) 0.066  Duration of diabetes, y 1.0 (0.98–1.01) 0.69  HbA1c - mmol/mol 1.02 (1.00–1.04) 0.046  C-peptide negative status 3.0 (1.5–5.7) 0.001   Hypoglycemia awareness status    Hypoglycemia unaware 3.0 (2.0–4.6) <0.000 Clarke method (n = 294)c  AA genotype 2.1 (1.2–3.7) 0.01  GA genotype 1.1 (0.7–1.9) 0.62  Duration of diabetes, y 1.0 (0.98–1.02) 0.89  HbA1c - mmol/mol 1.01 (1.00–1.03) 0.15  C-peptide negative status 2.4 (1.3–4.7) 0.009   Hypoglycemia awareness status    Hypoglycemia unaware 4.8 (3.0–7.7) <0.000 Multivariate Analysis Variable RR (95% CI) P Hillerød method (n = 308)a  AA genotype 2.0 (1.2–3.5) 0.01  GA genotype 1.1 (0.7–1.9) 0.60  Duration of diabetes, y 1.0 (0.98–1.01) 0.38  HbA1c, mmol/mol 1.02 (1.00–1.04) 0.046  C-peptide negative status 2.6 (1.3–5.2) 0.006   Hypoglycemia awareness status    Impaired hypoglycemia awareness 2.2 (1.3–3.6) 0.004    Hypoglycemia unaware 6.8 (3.7–12.3) <0.000 Gold method (n = 301)b  AA genotype 2.4 (1.4–4.2) 0.001  GA genotype 1.6 (0.97–2.6) 0.066  Duration of diabetes, y 1.0 (0.98–1.01) 0.69  HbA1c - mmol/mol 1.02 (1.00–1.04) 0.046  C-peptide negative status 3.0 (1.5–5.7) 0.001   Hypoglycemia awareness status    Hypoglycemia unaware 3.0 (2.0–4.6) <0.000 Clarke method (n = 294)c  AA genotype 2.1 (1.2–3.7) 0.01  GA genotype 1.1 (0.7–1.9) 0.62  Duration of diabetes, y 1.0 (0.98–1.02) 0.89  HbA1c - mmol/mol 1.01 (1.00–1.03) 0.15  C-peptide negative status 2.4 (1.3–4.7) 0.009   Hypoglycemia awareness status    Hypoglycemia unaware 4.8 (3.0–7.7) <0.000 Three stepwise regression analyses of ADRB2 Gly16Arg genotype, hypoglycemia awareness status [Hillerød classification (model 1), Gold classification (model 2), Clarke classification (model 3)], duration of diabetes, HbA1c level, and C-peptide status were performed. RR values represent the following: for Gly16Arg genotype, RR of AA and GA patients compared with GG patients; for HbA1c level, RR per one increment in mmol/mol in HbA1c; C-peptide status: C-peptide–negative subjects (C-peptide levels below 10 pmol/l) are compared to C-peptide–positive subjects. Hypoglycemia awareness status (Hillerød method): RR of patients with hypoglycemia unawareness or impaired awareness is compared with patients having normal hypoglycemia awareness. Hypoglycemia awareness status according to Gold and Clarke methods: RR of patients with hypoglycemia unawareness is compared with patients having normal hypoglycemia awareness. a n=308: data missing on C-peptide status for three patients. b n = 301: data missing on Gold hypoglycemia awareness classification for 7 patients and on C-peptide status for three patients. c n = 294: data missing on Clarke classification for 14 patients and on C-peptide status for 3 patients. A group of 27 subjects were unclassified according to the Clarke hypoglycemia awareness classification (not shown). View Large ADRB2 Gln27Glu genotype, severe hypoglycemia, and hypoglycemia awareness, There was no significant association between the ADRB2 Gln27Glu genotype (rs1042714) and the prevalence of one or more events of hypoglycemia (P = 0.21), the incidence of hypoglycemia (P = 0.52) or hypoglycemia awareness status according to the Hillerød classification (P = 0.62), the Gold classification (P = 0.88), or the Clarke classification (P = 0.95). Discussion In this study, we tested the hypothesis that the Arg16Gly polymorphism in ADRB2 is associated with severe hypoglycemia in patients with type 1 diabetes. The ADRB2 may be a key mediator of EGP and generation of hypoglycemic warning symptoms (11–13, 19). The prevalence and the incidence rate of severe hypoglycemia were increased in individuals with type 1 diabetes who were homozygous for the ADRB2 Arg16 allele, particularly in those with impaired hypoglycemia awareness and, thereby, predisposed to severe hypoglycemia due to impaired warning symptoms. However, no direct association was observed between genotype and hypoglycemia awareness status per se, suggesting that the effect of the Arg16 allele is due to a reduced EGP during hypoglycemia rather than a reduction of warning symptom intensity. In accordance, the Arg16 allele has been associated with decreased endogenous glucose mobilization during stimulation with epinephrine in healthy humans at normoglycemia (19). Sufficient EGP is crucial during daily-life asymptomatic hypoglycemia, and is particularly important among patients with impaired awareness (24), to go between meals without developing severe hypoglycemia. The finding of an increased risk of severe hypoglycemia in AA patients can hypothetically be attributed to reduced EGP during hypoglycemia due to an increased agonist-mediated receptor downregulation induced by recurrent hypoglycemic epinephrine release. This is in accordance with in vivo findings (25) in which AA healthy subjects demonstrated increased agonist-mediated receptor downregulation in vasculature (16) and AA patients with asthma using long-acting β2-adrenergic receptor agonists demonstrate reduced treatment efficacy (15). This conflicts with the findings of a Dutch study (26) that showed recurrent hypoglycemia increases β-2 adrenergic cardiovascular sensitivity in healthy AA subjects. Glucose responses, however, were not assessed in that study. Our findings also appear to conflict with two other studies from the same Dutch group in, respectively, 85 and 486 patients with type 1 diabetes (27, 28), which reported an association between the GG individuals and unawareness assessed by a modified Clarke classification. Neither study found an association between ADRB2 Gly16Arg genotype and prevalence of severe hypoglycemia (27, 28). In accordance with the Nijmegen group findings (27, 28), our study shows a slight trend toward increased hypoglycemia unawareness in the GG group, using the Hillerød and Gold classifications (but not the traditional Clarke classification). All the hypoglycemia unawareness status trends were far from statistically significant, which is also in accordance with the Nijmegen group finding of a very weak association between GG frequencies and hypoglycemia awareness status (odds ratio, 1.49; 95% CI, 1.01 to 2.20; P = 0.046), which, in addition, was not corrected for multiple testing (28). Unfortunately, a direct comparison between our study and the Dutch studies is hampered by the fact that in the latter, rates of severe hypoglycemia were not reported and heterozygous and homozygous Arg16 patients were pooled; a potential recessive effect of the Arg16 allele as observed in our study cannot be assessed. Hypoglycemia unawareness is the most important known risk factor of severe hypoglycemia (6); therefore, it is paradoxical that the association between GG genotype and hypoglycemia unawareness observed in the Dutch studies did not translate into an increased prevalence of severe hypoglycemia (27, 28). The findings in our study are in line with the hypothesis that in AA individuals with type 1 diabetes, the EGP resulting from epinephrine release during hypoglycemia is reduced, leading to an increased risk of severe hypoglycemia, particularly in those with impaired hypoglycemia awareness. Mechanistic studies aimed at testing this hypothesis are warranted. Strengths and limitations Among the strengths of this study is the use of a validated questionnaire and three classification methods for estimating hypoglycemia awareness status. There was accordance among the analyses of awareness status (i.e., Hillerød, Gold, and Clarke methods), which decreases the risk for a false-negative finding. The study cohort is in Hardy-Weinberg equilibrium, making selection bias less probable. The thorough determination of the hypoglycemic phenotype by registration of prevalence and of incidence rate of severe hypoglycemia, recording of other risk factors, the finding of expected effect of impaired awareness, and the robustness of the main genotype findings in the multivariate analysis altogether increase the strength of the genotype effect analysis. Such data have not been presented in earlier studies of this genotype, to our knowledge. The size of the cohort, on the other hand, is a limitation and does not permit an analysis of the impact of potential haplotype effects. Conclusion A clinically significant increased risk of severe hypoglycemia was found among people with type 1 diabetes who were homozygous for the Arg16 allele of ADRB2. This was not explained by differences in hypoglycemia awareness between genotype groups. The difference between genotypes was amplified in patients with impaired awareness but absent in people with normal awareness. This supports the theory that the association is mediated by a reduced epinephrine-induced EGP in AA patients during daily-life, asymptomatic hypoglycemia, which is most common in patients with impaired awareness. Our findings need to be confirmed in independent cohorts and require further mechanistic studies to explore the influence of the Gly16Arg polymorphism of ADRB2 on responses during hypoglycemia and the association with agonist-mediated receptor downregulation after recurrent hypoglycemia. Abbreviations: Abbreviations: AA homozygous for the Arg16 allele ADRB2 adrenoceptor β-2 receptor gene EGP endogenous glucose production GA heterozygous for the Gly16 and Arg16 allele GG homozygous for the Gly16 allele RR relative rate SNP single nucleotide polymorphism Acknowledgments Financial Support: The study was funded by a grant from the Research Foundation of Nordsjællands University Hospital Hillerød, the Beckett Foundation, and the Foundation of Captain Lieutenant Harald Jensen and Wife. B.T. received core funding from Nordsjællands University Hospital Hillerød. Author Contributions: K.Z.R., N.V.O., B.T., and U.P.-B. initiated the study. L.F., B.T., and U.P.-B. designed the study. L.F. collected the data. K.Z.R., P.L.K., and U.P.-B. analyzed the data. K.Z.R. drafted the first version of the manuscript. All authors contributed to preparation of the paper. Disclosure Summary: L.F. is employed by Novo Nordisk. B.T. served on advisory boards for Eli Lilly. U.P.-B. served on advisory boards for AstraZeneca/Bristol-Myers Squibb and Novo Nordisk, and received lecture fees from AstraZeneca/Bristol-Myers Squibb, Sanofi Aventis, and Novo Nordisk. The remaining authors have nothing to disclose. References 1. Cryer PE . Hypoglycemia in Diabetes: Pathophysiology, Prevalence, and Prevention . Alexandria, VA : American Diabetes Association ; 2009 . 2. Cryer PE . The barrier of hypoglycemia in diabetes . Diabetes . 2008 ; 57 ( 12 ): 3169 – 3176 . Google Scholar CrossRef Search ADS PubMed 3. Dagogo-Jack S , Rattarasarn C , Cryer PE . Reversal of hypoglycemia unawareness, but not defective glucose counterregulation, in IDDM . Diabetes . 1994 ; 43 ( 12 ): 1426 – 1434 . 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Gjesing AP , Sparsø T , Borch-Johnsen K , Jørgensen T , Pedersen O , Hansen T , Olsen NV . No consistent effect of ADRB2 haplotypes on obesity, hypertension and quantitative traits of body fatness and blood pressure among 6,514 adult Danes . PLoS One . 2009 ; 4 ( 9 ): e7206 . Google Scholar CrossRef Search ADS PubMed 24. Schopman JE , Geddes J , Frier BM . Frequency of symptomatic and asymptomatic hypoglycaemia in type 1 diabetes: effect of impaired awareness of hypoglycaemia . Diabet Med . 2011 ; 28 ( 3 ): 352 – 355 . Google Scholar PubMed 25. Leineweber K , Brodde OE . Beta2-adrenoceptor polymorphisms: relation between in vitro and in vivo phenotypes . Life Sci . 2004 ; 74 ( 23 ): 2803 – 2814 . Google Scholar CrossRef Search ADS PubMed 26. Schouwenberg BJ , Smits P , Tack CJ , de Galan BE . The effect of antecedent hypoglycaemia on β2-adrenergic sensitivity in healthy participants with the Arg16Gly polymorphism of the β2-adrenergic receptor . Diabetologia . 2011 ; 54 ( 5 ): 1212 – 1218 . Google Scholar CrossRef Search ADS PubMed 27. Schouwenberg BJ , Veldman BA , Spiering W , Coenen MJ , Franke B , Tack CJ , de Galan BE , Smits P . The Arg16Gly variant of the beta2-adrenergic receptor predisposes to hypoglycemia unawareness in type 1 diabetes mellitus . Pharmacogenet Genomics . 2008 ; 18 ( 4 ): 369 – 372 . Google Scholar CrossRef Search ADS PubMed 28. Schouwenberg BJ , Coenen MJ , Paterson AD , Tack CJ , Smits P , Kramers C , de Galan BE . Genetic determinants of impaired awareness of hypoglycemia in type 1 diabetes . Pharmacogenet Genomics . 2017 ; 27 ( 9 ): 323 – 328 . Google Scholar CrossRef Search ADS PubMed Copyright © 2018 Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Clinical Endocrinology and Metabolism Oxford University Press

Impact of Genetic Polymorphism in the β2-Receptor Gene on Risk of Severe Hypoglycemia in Patients With Type 1 Diabetes

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Oxford University Press
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Copyright © 2018 Endocrine Society
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0021-972X
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1945-7197
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10.1210/jc.2017-02739
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Abstract

Abstract Context Severe hypoglycemic events are unevenly distributed in people with type 1 diabetes, making a genetic influence probable. Of the common adrenoceptor β-2 receptor gene (ADRB2) polymorphisms, the Arg16 allele is associated with receptor downregulation and reduced agonist-mediated endogenous glucose production. Objective We tested the hypothesis that the Arg16 variant is associated with severe hypoglycemia. Method A cohort of 311 patients with type 1 diabetes reported severe hypoglycemic events retrospectively in a validated questionnaire. The patients were characterized by diabetes history, state of hypoglycemia awareness, C-peptide status, HbA1c, and ADRB2 genotype. Results The ADRB2 Gly16Arg genotype distribution was in Hardy-Weinberg equilibrium. The rate of severe hypoglycemia differed among all genotypes (P = 0.01). Patients homozygous for the Arg16 genotype (AA; n = 60) had a relative rate (RR) of severe hypoglycemia of 2.2 (95% CI, 1.3 to 3.6) compared with patients homozygous for the Gly16 genotype (GG; n = 116; P = 0.002). Among patients with impaired awareness or unawareness (n = 175), those with the AA genotype (n = 33) had an RR of severe hypoglycemia of 3.2 (95% CI, 1.7 to 6.0) compared with patients with the GG genotype (n = 58; P < 0.000). Genotype was not associated with state of hypoglycemia awareness per se, as assessed by any of three classification methods. The difference was not explained by other risk factors. Conclusion Genetic polymorphism in ADRB2 is associated with risk of severe hypoglycemia in individuals with type 1 diabetes, especially in those with impaired hypoglycemia awareness. In type 1 diabetes, the metabolic and symptomatic protective responses to hypoglycemia are often impaired with time (1). An early blunting of the glucagon response is accompanied by disappearing endogenous insulin production (1, 2), rendering patients increasingly dependent upon epinephrine as their first-line protection against development of severe hypoglycemia. However, in many patients, as a result of recurrent hypoglycemia and/or long duration of diabetes, the epinephrine response to hypoglycemia also becomes blunted (i.e., hypoglycemia-associated autonomic failure) (1), leading to reduced endogenous glucose production (EGP) during hypoglycemia and impaired recognition of warning symptoms (i.e., impaired hypoglycemia awareness). Although impaired awareness is usually linked to loss of adrenergic symptoms, it is apparent that hypoglycemic warning symptoms are complex and can occur independently of the epinephrine response (3). Impaired hypoglycemia awareness, in turn, results in a grossly increased risk of severe hypoglycemia (4–7). In addition to the failing catecholamine response to hypoglycemia often seen with impaired hypoglycemia awareness, a reduced β2-adrenergic sensitivity has been reported in some (8, 9), but not all, studies (10). According to previous reports, treatment with nonspecific β-blockers with effect on the β2-receptor is associated with reduced EGP to epinephrine infusion (11) and impaired recovery from hypoglycemia (12, 13). Among patients with impaired awareness, the risk of severe hypoglycemia is varying and the fact that many patients seem chronically prone to recurrent severe hypoglycemia makes a genetic influence probable. A potential candidate gene to consider is the adrenoceptor β-2 receptor gene (ADRB2), in which several common variants are described (14). Of these, the single nucleotide polymorphism (SNP) Gly vs Arg at codon 16 (rs1042713) affects β2-receptor desensitization during β2-agonist treatment of asthma in a clinically important manner (15). Although diverse results are reported, the Arg16 variant of polymorphism in ADRB2 is associated with increased β2-receptor desensitization during agonist therapy, resulting in reduced treatment efficiency (16–18). In contrast, the ADRB2 polymorphism Gln vs Glu at codon 27 (rs1042714) is associated with increased agonist-mediated responsiveness in vasculature (16). We have shown that healthy humans who are homozygous for the ADRB2 Arg16 variant react with lesser increment in plasma glucose levels to adrenaline infusion, suggesting a reduced EGP resulting from receptor stimulation in these persons (19). This leads to the hypothesis that the ADRB2 Arg16 variant is associated with severe hypoglycemia in type 1 diabetes by either a reduced EGP during hypoglycemia or by predisposing to reduced adrenergic symptom intensity, and thus impaired hypoglycemia awareness, during hypoglycemia. We tested this hypothesis in a cohort of individuals with type 1 diabetes. Materials and Methods The study was a retrospective, observational, noninterventional cohort study. A total of 416 patients attending the outpatient clinic at Nordsjællands Hospital, Hillerød, Denmark, were invited to participate in the study. After giving written informed consent, a cohort of 311 consecutive adult patients (age >18 years) with type 1 diabetes for >1 year was recruited from November 2011 to May 2013. Type 1 diabetes was defined by insulin treatment from the time of diagnosis and unstimulated C-peptide concentration <300 pmol/L or stimulated (venous blood glucose concentration >12 mmol/L) C-peptide concentration<600 pmol/L. Pregnant women and individuals with severe concomitant diseases were excluded from participation. Information about history of diabetes, treatment, and late complications were extracted from the medical records. Hypoglycemia awareness was self-estimated using three validated methods: those of Hillerød (20) (n = 311), Gold (4) (n = 304; data were missing for 7 patients), and Clarke (5) (n = 297, data were missing for 14 patients). The Hillerød method classifies patients into three classes of hypoglycemia awareness: normal, or impaired awareness, or hypoglycemia unawareness (6, 20). In contrast, the Clarke and Gold methods divide hypoglycemia awareness into two classes (4, 5). The number of hypoglycemic events was recorded in a questionnaire based on recall within 1 year, which is well preserved and valid (20). Definition of severe hypoglycemia was “an event with low blood glucose requiring assistance of another person to actively administer carbohydrate, glucagon, or other resuscitative actions” (21), which is in accordance with the American Diabetes Association (21) and the International Hypoglycemia Study Group recommendations (22). The primary end point was number of reported events of severe hypoglycemia in the preceding year, and secondary end points were prevalence of severe hypoglycemia and self-reported hypoglycemia awareness status (via Clarke, Gold, and Hillerød methods). The study was approved by the Committee on Biomedical Ethics of the Copenhagen Capital Region (H-4–2011–043) and the Danish Data Protection Agency (2012-58-0004). Laboratory tests We measured glycated HbA1c (Tosoh G8 HPLC Analyzer; Tosoh Bioscience, San Francisco, CA) and level of C-peptide (AutoDELFIA; Wallac Oy, Turku, Finland). The patients were classified as C-peptide negative when C-peptide levels were <10 pmol/L. Purification of DNA and genotyping DNA purification and genotyping of the ADRB2 SNPs Gly16Arg (rs 042713) and Gln27Glu (rs 1042714) was carried out commercially at LGC Genomics Unit (Hoddesdon, Hertfordshire, United Kingdom) and was done blinded to patient data. DNA was extracted from frozen buffy-coat samples by the KleargeneTM extraction protocol (LGC Genomics). Genotyping was performed using the KASPTM genotyping protocol. KASPTM assays are based on competitive allele-specific PCR. The SNP-specific KASPTM Assay mix (LGC Genomics) was added to DNA samples, a thermal cycling reaction was then performed, followed by an end-point fluorescent reading. Biallelic discrimination was achieved through the competitive binding of two allele-specific forward primers, each with a unique tail sequence that corresponds with two universal fluorescence resonant energy transfer cassettes, one labeled with FAMTM dye and the other with HEXTM dye. Statistics Statistical analysis was performed using the SPSS, version 22.0 (IBM, Armonk, NY). Comparisons between variables were made by parametric and nonparametric methods as appropriate. The influence of risk factors (i.e., genotype, state of hypoglycemia awareness, duration of diabetes, and HbA1c and C-peptide status) on the number of severe hypoglycemic events was assessed by a log-linear negative binomial model, which takes into account the skewed distribution of events of severe hypoglycemia. The prevalence of severe hypoglycemia is presented as the percentage of patients having one or more events of severe hypoglycemia during the last year. The incidence of severe hypoglycemia is presented as the rate of severe hypoglycemia, given as relative rate (RR) ratio and 95% CI. To evaluate the relative impact of ADRB2 Gly16Arg genotype and known risk factors of severe hypoglycemia, a stepwise regression analysis was performed, with inclusion of significant and nearly significant (P < 0.1) univariate analysis parameters. Other values shown in the text and tables are mean and SD, or percentage, as indicated. P < 0.05 (two-sided) was regarded as significant. Results Baseline characteristics of the participants and the prevalence of the Gly16Arg genotypes are shown in Table 1. The ADRB2 Gly16Arg and the Gln27Glu genotype distribution was comparable to that of the general Danish population (23) and was in Hardy-Weinberg equilibrium (P = 0.77 and P = 0.58, respectively). No significant differences in baseline characteristics were observed between the genotypes (Table 1). A total of 19 patients were treated with β-blockers. Excluding these from the analysis did not significantly change the results; therefore, they were not excluded from the cohort. Table 1. Baseline Characteristics and Hypoglycemia Awareness Classification According to ADRB2 Polymorphisms Characteristic GG GA AA Total P Patients, no. (%) 116 (37) 135 (43) 60 (19) 311 (100) — Male patients, % 65 59 52 60 0.20 Age, mean ± SD, y 53 ± 13 52 ± 15 53 ± 14 52 ± 14 0.74 Duration of diabetes, mean ± SD, y 23 ± 12 23 ± 15 24 ± 15 23 ± 14 0.93 HbA1c, mean ± SD, mmol/mol 61 ± 11 63 ± 13 62 ± 10 62 ± 11 0.50 C-peptide negative status, %a 77 78 73 77 0.80 Hypoglycemia awareness, %  Hillerød classification, aware/impaired/unaware 50/34/16 38/49/13 45/43/12 43/42/14 0.22  Gold classification,b aware/impaired 73/27 79/21 76/24 76/24 0.57  Clarke classification,c aware/unclassified/impaired 75/9/16 73/9/18 71/11/18 74/9/17 0.98 Characteristic GG GA AA Total P Patients, no. (%) 116 (37) 135 (43) 60 (19) 311 (100) — Male patients, % 65 59 52 60 0.20 Age, mean ± SD, y 53 ± 13 52 ± 15 53 ± 14 52 ± 14 0.74 Duration of diabetes, mean ± SD, y 23 ± 12 23 ± 15 24 ± 15 23 ± 14 0.93 HbA1c, mean ± SD, mmol/mol 61 ± 11 63 ± 13 62 ± 10 62 ± 11 0.50 C-peptide negative status, %a 77 78 73 77 0.80 Hypoglycemia awareness, %  Hillerød classification, aware/impaired/unaware 50/34/16 38/49/13 45/43/12 43/42/14 0.22  Gold classification,b aware/impaired 73/27 79/21 76/24 76/24 0.57  Clarke classification,c aware/unclassified/impaired 75/9/16 73/9/18 71/11/18 74/9/17 0.98 a Patients were classified as C-peptide negative when C-peptide levels were <10 pmol/L. b Gold classification (n = 304, data missing for seven patients; distribution of patients according to genotype group: GG, n = 114; GA, n = 131; AA, n =59). c Clarke classification (n = 297, data missing for 14 patients; distribution of patients according to genotype group: GG, n = 113; GA, n = 128; AA, n = 56). View Large Table 1. Baseline Characteristics and Hypoglycemia Awareness Classification According to ADRB2 Polymorphisms Characteristic GG GA AA Total P Patients, no. (%) 116 (37) 135 (43) 60 (19) 311 (100) — Male patients, % 65 59 52 60 0.20 Age, mean ± SD, y 53 ± 13 52 ± 15 53 ± 14 52 ± 14 0.74 Duration of diabetes, mean ± SD, y 23 ± 12 23 ± 15 24 ± 15 23 ± 14 0.93 HbA1c, mean ± SD, mmol/mol 61 ± 11 63 ± 13 62 ± 10 62 ± 11 0.50 C-peptide negative status, %a 77 78 73 77 0.80 Hypoglycemia awareness, %  Hillerød classification, aware/impaired/unaware 50/34/16 38/49/13 45/43/12 43/42/14 0.22  Gold classification,b aware/impaired 73/27 79/21 76/24 76/24 0.57  Clarke classification,c aware/unclassified/impaired 75/9/16 73/9/18 71/11/18 74/9/17 0.98 Characteristic GG GA AA Total P Patients, no. (%) 116 (37) 135 (43) 60 (19) 311 (100) — Male patients, % 65 59 52 60 0.20 Age, mean ± SD, y 53 ± 13 52 ± 15 53 ± 14 52 ± 14 0.74 Duration of diabetes, mean ± SD, y 23 ± 12 23 ± 15 24 ± 15 23 ± 14 0.93 HbA1c, mean ± SD, mmol/mol 61 ± 11 63 ± 13 62 ± 10 62 ± 11 0.50 C-peptide negative status, %a 77 78 73 77 0.80 Hypoglycemia awareness, %  Hillerød classification, aware/impaired/unaware 50/34/16 38/49/13 45/43/12 43/42/14 0.22  Gold classification,b aware/impaired 73/27 79/21 76/24 76/24 0.57  Clarke classification,c aware/unclassified/impaired 75/9/16 73/9/18 71/11/18 74/9/17 0.98 a Patients were classified as C-peptide negative when C-peptide levels were <10 pmol/L. b Gold classification (n = 304, data missing for seven patients; distribution of patients according to genotype group: GG, n = 114; GA, n = 131; AA, n =59). c Clarke classification (n = 297, data missing for 14 patients; distribution of patients according to genotype group: GG, n = 113; GA, n = 128; AA, n = 56). View Large ADRB2 Gly16Arg genotype and severe hypoglycemia The overall prevalence of severe hypoglycemia was 22%, and the rate of severe hypoglycemia was 0.54 events per patient per year. The prevalence of one or more events of severe hypoglycemia experienced during the last year according to genotype was 35% in the people homozygous for the Arg16 allele (AA), which was almost twofold higher than in patients homozygous for the Gly16 allele (GG; 18%; P = 0.013; Fig. 1). The RR of severe hypoglycemia during the last year was increased in AA patients (2.2; 95% CI, 1.3 to 3.6) compared with GG patients (P < 0.002; Fig. 2). Figure 1. View largeDownload slide Prevalence of severe hypoglycemia according to the Gly16Arg ADRB2 polymorphisms and hypoglycemia awareness status. Difference in severe hypoglycemia (i.e., one or more events) experienced during the last year, according to genotype, GG, GA, and AA, in the total population (N = 311; P = 0.027)*, in patients with normal hypoglycemia awareness (n = 136; P = 0.93), and in patients with impaired hypoglycemia awareness status or hypoglycemia unawareness according to the Hillerød classification (n = 175; P = 0.009)**. Figure 1. View largeDownload slide Prevalence of severe hypoglycemia according to the Gly16Arg ADRB2 polymorphisms and hypoglycemia awareness status. Difference in severe hypoglycemia (i.e., one or more events) experienced during the last year, according to genotype, GG, GA, and AA, in the total population (N = 311; P = 0.027)*, in patients with normal hypoglycemia awareness (n = 136; P = 0.93), and in patients with impaired hypoglycemia awareness status or hypoglycemia unawareness according to the Hillerød classification (n = 175; P = 0.009)**. Figure 2. View largeDownload slide RR of severe hypoglycemia according to the ADRB2 Gly16Arg polymorphism and hypoglycemia awareness status (using the Hillerød method). The difference in RR (black bars indicate 95% CI) of reported events of severe hypoglycemia during the last year, according to awareness status and genotype: GG (light gray dots), GA (dark gray dots), and AA (black dots). A significant difference between genotypes was found in the total population (N = 311; P = 0.01)* and in patients with either hypoglycemia unawareness or impaired awareness (n = 175; P < 0.002)**. No significant difference was found between genotype groups in patients with normal hypoglycemia awareness (n = 136; P = 0.087). Figure 2. View largeDownload slide RR of severe hypoglycemia according to the ADRB2 Gly16Arg polymorphism and hypoglycemia awareness status (using the Hillerød method). The difference in RR (black bars indicate 95% CI) of reported events of severe hypoglycemia during the last year, according to awareness status and genotype: GG (light gray dots), GA (dark gray dots), and AA (black dots). A significant difference between genotypes was found in the total population (N = 311; P = 0.01)* and in patients with either hypoglycemia unawareness or impaired awareness (n = 175; P < 0.002)**. No significant difference was found between genotype groups in patients with normal hypoglycemia awareness (n = 136; P = 0.087). ADRB2 Gly16Arg genotype and hypoglycemia awareness Overall, according to the Hillerød classification, 14% of patients were classified as having hypoglycemia unawareness. Using the Gold classification, the prevalence was 24%; it was 17% when using the Clarke classification (Table 1). We found no significant difference in the distribution of awareness classes between the genotype groups with any of the three classification methods (Table 1). ADRB2 Gly16Arg genotype, severe hypoglycemia, and hypoglycemia awareness Prevalence of severe hypoglycemia according to Gly16Arg ADRB2 polymorphisms and hypoglycemia awareness status according to the Hillerød method is shown in Fig. 1. There was no difference in the prevalence of severe hypoglycemia between genotype groups among patients with normal hypoglycemia awareness (P = 0.93). Among patients with impaired awareness or unawareness according to the Hillerød classification, the prevalence of severe hypoglycemia was 55% in AA patients compared with 28% in GG patients (P = 0.009; Fig. 1). Prevalence of severe hypoglycemia according to Gly16Arg ADRB2 polymorphisms and hypoglycemia awareness status according to the Gold and Clarke classifications showed the same trend as seen using the Hillerød classification. The RR of severe hypoglycemia according to Gly16Arg ADRB2 polymorphism and hypoglycemia awareness status according to the Hillerød method is shown in Fig. 2. There was no difference in the RR of severe hypoglycemia between genotype groups among patients with normal awareness (P = 0.087). Among patients having either impaired awareness or unawareness of hypoglycemia, AA patients had an increased rate of severe hypoglycemia (3.2; 95% CI, 1.7 to 6.0; P < 0.000) compared with GG patients (Fig. 2). ADRB2 Gly16Arg genotype and other risk factors, and multivariate analyses The impact of known risk factors on the incidence of severe hypoglycemia was tested by univariate analysis (Table 2). Increasing levels of HbA1c, C-peptide negative status, and hypoglycemia unawareness status according to all three methods were significantly associated with increased incidence of severe hypoglycemia, whereas increased duration of diabetes showed a trend toward increasing the incidence of severe hypoglycemia, but it did not reach statistically significance (Table 2). Table 2. Univariate Analysis of Known Risk Factors of Severe Hypoglycemia Univariate Analysis Variable RR (95% CI) P AA genotype 2.2 (1.3–3.6) 0.002 GA genotype 1.4 (0.89–2.2) 0.15 Duration of diabetes, y 1.01 (1.0–1.02) 0.09 HbA1c, mmol/mol 1.02 (1.00–1.03) <0.03 C-peptide negativea 3.1 (1.7–5.6) <0.000 Hillerød classification  Hypoglycemia unaware 7.3 (4.2–12.7) <0.000  Impaired awareness 2.6 (1.6–4.2) <0.000 Gold classificationb  Hypoglycemia unaware 3.0 (2.0–4.5) <0.000 Clarke classificationc  Hypoglycemia unaware 5.4 (3.5–8.3) <0.000 Univariate Analysis Variable RR (95% CI) P AA genotype 2.2 (1.3–3.6) 0.002 GA genotype 1.4 (0.89–2.2) 0.15 Duration of diabetes, y 1.01 (1.0–1.02) 0.09 HbA1c, mmol/mol 1.02 (1.00–1.03) <0.03 C-peptide negativea 3.1 (1.7–5.6) <0.000 Hillerød classification  Hypoglycemia unaware 7.3 (4.2–12.7) <0.000  Impaired awareness 2.6 (1.6–4.2) <0.000 Gold classificationb  Hypoglycemia unaware 3.0 (2.0–4.5) <0.000 Clarke classificationc  Hypoglycemia unaware 5.4 (3.5–8.3) <0.000 The impact of known risk factors on the incidence of severe hypoglycemia was tested by univariate analysis (N = 311). RR values represent the following: Gly16Arg genotype, RR of AA and GA patients compared with GG patients; HbA1c level, RR per one increment in mmol/mol in HbA1c. For C-peptide status RR, C-peptide–negative subjects (C-peptide levels <10 pmol/L) are compared with C-peptide–positive subjects. Hypoglycemia awareness status (Hillerød method), RR of patients with hypoglycemia unawareness or impaired awareness is compared with those having normal hypoglycemia awareness. Hypoglycemia awareness status according to Gold and Clarke methods: RR of patients with hypoglycemia unawareness is compared with subjects having normal hypoglycemia awareness. a C-peptide status (n = 308, data missing for three patients). b Gold classification (n = 304, data missing for seven patients). c Clarke classification (n = 297, data missing for 14 patients). There was a group of 27 patients who were unclassified according to the Clarke classification (not shown). View Large Table 2. Univariate Analysis of Known Risk Factors of Severe Hypoglycemia Univariate Analysis Variable RR (95% CI) P AA genotype 2.2 (1.3–3.6) 0.002 GA genotype 1.4 (0.89–2.2) 0.15 Duration of diabetes, y 1.01 (1.0–1.02) 0.09 HbA1c, mmol/mol 1.02 (1.00–1.03) <0.03 C-peptide negativea 3.1 (1.7–5.6) <0.000 Hillerød classification  Hypoglycemia unaware 7.3 (4.2–12.7) <0.000  Impaired awareness 2.6 (1.6–4.2) <0.000 Gold classificationb  Hypoglycemia unaware 3.0 (2.0–4.5) <0.000 Clarke classificationc  Hypoglycemia unaware 5.4 (3.5–8.3) <0.000 Univariate Analysis Variable RR (95% CI) P AA genotype 2.2 (1.3–3.6) 0.002 GA genotype 1.4 (0.89–2.2) 0.15 Duration of diabetes, y 1.01 (1.0–1.02) 0.09 HbA1c, mmol/mol 1.02 (1.00–1.03) <0.03 C-peptide negativea 3.1 (1.7–5.6) <0.000 Hillerød classification  Hypoglycemia unaware 7.3 (4.2–12.7) <0.000  Impaired awareness 2.6 (1.6–4.2) <0.000 Gold classificationb  Hypoglycemia unaware 3.0 (2.0–4.5) <0.000 Clarke classificationc  Hypoglycemia unaware 5.4 (3.5–8.3) <0.000 The impact of known risk factors on the incidence of severe hypoglycemia was tested by univariate analysis (N = 311). RR values represent the following: Gly16Arg genotype, RR of AA and GA patients compared with GG patients; HbA1c level, RR per one increment in mmol/mol in HbA1c. For C-peptide status RR, C-peptide–negative subjects (C-peptide levels <10 pmol/L) are compared with C-peptide–positive subjects. Hypoglycemia awareness status (Hillerød method), RR of patients with hypoglycemia unawareness or impaired awareness is compared with those having normal hypoglycemia awareness. Hypoglycemia awareness status according to Gold and Clarke methods: RR of patients with hypoglycemia unawareness is compared with subjects having normal hypoglycemia awareness. a C-peptide status (n = 308, data missing for three patients). b Gold classification (n = 304, data missing for seven patients). c Clarke classification (n = 297, data missing for 14 patients). There was a group of 27 patients who were unclassified according to the Clarke classification (not shown). View Large To evaluate the relative impact of ADRB2 Gly16Arg genotype and known risk factors of severe hypoglycemia, a stepwise regression analysis was performed, with inclusion of ADRB2 Gly16Arg genotype, HbA1c level, C-peptide status, diabetes duration, and hypoglycemia awareness status (Table 3). In this analysis, the ADRB2 Gly16Arg genotype remained associated with events of severe hypoglycemia, with the AA genotype having an increased RR of 2.0 (95% CI, 1.2 to 3.5; P = 0.01; Table 3). Data analysis also revealed an increased RR for patients having impaired hypoglycemia awareness (RR, 2.2; 95% CI, 1.3 to 3.6; P < 0.004) and hypoglycemia unawareness (RR 6.8; 95% CI, 3.7 to 12.3; P < 0.000; Table 3). Table 3. Multivariate Analysis and Relative Impact of ADRB2 Gly16Arg Genotype and Known Risk Factors of Severe Hypoglycemia Multivariate Analysis Variable RR (95% CI) P Hillerød method (n = 308)a  AA genotype 2.0 (1.2–3.5) 0.01  GA genotype 1.1 (0.7–1.9) 0.60  Duration of diabetes, y 1.0 (0.98–1.01) 0.38  HbA1c, mmol/mol 1.02 (1.00–1.04) 0.046  C-peptide negative status 2.6 (1.3–5.2) 0.006   Hypoglycemia awareness status    Impaired hypoglycemia awareness 2.2 (1.3–3.6) 0.004    Hypoglycemia unaware 6.8 (3.7–12.3) <0.000 Gold method (n = 301)b  AA genotype 2.4 (1.4–4.2) 0.001  GA genotype 1.6 (0.97–2.6) 0.066  Duration of diabetes, y 1.0 (0.98–1.01) 0.69  HbA1c - mmol/mol 1.02 (1.00–1.04) 0.046  C-peptide negative status 3.0 (1.5–5.7) 0.001   Hypoglycemia awareness status    Hypoglycemia unaware 3.0 (2.0–4.6) <0.000 Clarke method (n = 294)c  AA genotype 2.1 (1.2–3.7) 0.01  GA genotype 1.1 (0.7–1.9) 0.62  Duration of diabetes, y 1.0 (0.98–1.02) 0.89  HbA1c - mmol/mol 1.01 (1.00–1.03) 0.15  C-peptide negative status 2.4 (1.3–4.7) 0.009   Hypoglycemia awareness status    Hypoglycemia unaware 4.8 (3.0–7.7) <0.000 Multivariate Analysis Variable RR (95% CI) P Hillerød method (n = 308)a  AA genotype 2.0 (1.2–3.5) 0.01  GA genotype 1.1 (0.7–1.9) 0.60  Duration of diabetes, y 1.0 (0.98–1.01) 0.38  HbA1c, mmol/mol 1.02 (1.00–1.04) 0.046  C-peptide negative status 2.6 (1.3–5.2) 0.006   Hypoglycemia awareness status    Impaired hypoglycemia awareness 2.2 (1.3–3.6) 0.004    Hypoglycemia unaware 6.8 (3.7–12.3) <0.000 Gold method (n = 301)b  AA genotype 2.4 (1.4–4.2) 0.001  GA genotype 1.6 (0.97–2.6) 0.066  Duration of diabetes, y 1.0 (0.98–1.01) 0.69  HbA1c - mmol/mol 1.02 (1.00–1.04) 0.046  C-peptide negative status 3.0 (1.5–5.7) 0.001   Hypoglycemia awareness status    Hypoglycemia unaware 3.0 (2.0–4.6) <0.000 Clarke method (n = 294)c  AA genotype 2.1 (1.2–3.7) 0.01  GA genotype 1.1 (0.7–1.9) 0.62  Duration of diabetes, y 1.0 (0.98–1.02) 0.89  HbA1c - mmol/mol 1.01 (1.00–1.03) 0.15  C-peptide negative status 2.4 (1.3–4.7) 0.009   Hypoglycemia awareness status    Hypoglycemia unaware 4.8 (3.0–7.7) <0.000 Three stepwise regression analyses of ADRB2 Gly16Arg genotype, hypoglycemia awareness status [Hillerød classification (model 1), Gold classification (model 2), Clarke classification (model 3)], duration of diabetes, HbA1c level, and C-peptide status were performed. RR values represent the following: for Gly16Arg genotype, RR of AA and GA patients compared with GG patients; for HbA1c level, RR per one increment in mmol/mol in HbA1c; C-peptide status: C-peptide–negative subjects (C-peptide levels below 10 pmol/l) are compared to C-peptide–positive subjects. Hypoglycemia awareness status (Hillerød method): RR of patients with hypoglycemia unawareness or impaired awareness is compared with patients having normal hypoglycemia awareness. Hypoglycemia awareness status according to Gold and Clarke methods: RR of patients with hypoglycemia unawareness is compared with patients having normal hypoglycemia awareness. a n=308: data missing on C-peptide status for three patients. b n = 301: data missing on Gold hypoglycemia awareness classification for 7 patients and on C-peptide status for three patients. c n = 294: data missing on Clarke classification for 14 patients and on C-peptide status for 3 patients. A group of 27 subjects were unclassified according to the Clarke hypoglycemia awareness classification (not shown). View Large Table 3. Multivariate Analysis and Relative Impact of ADRB2 Gly16Arg Genotype and Known Risk Factors of Severe Hypoglycemia Multivariate Analysis Variable RR (95% CI) P Hillerød method (n = 308)a  AA genotype 2.0 (1.2–3.5) 0.01  GA genotype 1.1 (0.7–1.9) 0.60  Duration of diabetes, y 1.0 (0.98–1.01) 0.38  HbA1c, mmol/mol 1.02 (1.00–1.04) 0.046  C-peptide negative status 2.6 (1.3–5.2) 0.006   Hypoglycemia awareness status    Impaired hypoglycemia awareness 2.2 (1.3–3.6) 0.004    Hypoglycemia unaware 6.8 (3.7–12.3) <0.000 Gold method (n = 301)b  AA genotype 2.4 (1.4–4.2) 0.001  GA genotype 1.6 (0.97–2.6) 0.066  Duration of diabetes, y 1.0 (0.98–1.01) 0.69  HbA1c - mmol/mol 1.02 (1.00–1.04) 0.046  C-peptide negative status 3.0 (1.5–5.7) 0.001   Hypoglycemia awareness status    Hypoglycemia unaware 3.0 (2.0–4.6) <0.000 Clarke method (n = 294)c  AA genotype 2.1 (1.2–3.7) 0.01  GA genotype 1.1 (0.7–1.9) 0.62  Duration of diabetes, y 1.0 (0.98–1.02) 0.89  HbA1c - mmol/mol 1.01 (1.00–1.03) 0.15  C-peptide negative status 2.4 (1.3–4.7) 0.009   Hypoglycemia awareness status    Hypoglycemia unaware 4.8 (3.0–7.7) <0.000 Multivariate Analysis Variable RR (95% CI) P Hillerød method (n = 308)a  AA genotype 2.0 (1.2–3.5) 0.01  GA genotype 1.1 (0.7–1.9) 0.60  Duration of diabetes, y 1.0 (0.98–1.01) 0.38  HbA1c, mmol/mol 1.02 (1.00–1.04) 0.046  C-peptide negative status 2.6 (1.3–5.2) 0.006   Hypoglycemia awareness status    Impaired hypoglycemia awareness 2.2 (1.3–3.6) 0.004    Hypoglycemia unaware 6.8 (3.7–12.3) <0.000 Gold method (n = 301)b  AA genotype 2.4 (1.4–4.2) 0.001  GA genotype 1.6 (0.97–2.6) 0.066  Duration of diabetes, y 1.0 (0.98–1.01) 0.69  HbA1c - mmol/mol 1.02 (1.00–1.04) 0.046  C-peptide negative status 3.0 (1.5–5.7) 0.001   Hypoglycemia awareness status    Hypoglycemia unaware 3.0 (2.0–4.6) <0.000 Clarke method (n = 294)c  AA genotype 2.1 (1.2–3.7) 0.01  GA genotype 1.1 (0.7–1.9) 0.62  Duration of diabetes, y 1.0 (0.98–1.02) 0.89  HbA1c - mmol/mol 1.01 (1.00–1.03) 0.15  C-peptide negative status 2.4 (1.3–4.7) 0.009   Hypoglycemia awareness status    Hypoglycemia unaware 4.8 (3.0–7.7) <0.000 Three stepwise regression analyses of ADRB2 Gly16Arg genotype, hypoglycemia awareness status [Hillerød classification (model 1), Gold classification (model 2), Clarke classification (model 3)], duration of diabetes, HbA1c level, and C-peptide status were performed. RR values represent the following: for Gly16Arg genotype, RR of AA and GA patients compared with GG patients; for HbA1c level, RR per one increment in mmol/mol in HbA1c; C-peptide status: C-peptide–negative subjects (C-peptide levels below 10 pmol/l) are compared to C-peptide–positive subjects. Hypoglycemia awareness status (Hillerød method): RR of patients with hypoglycemia unawareness or impaired awareness is compared with patients having normal hypoglycemia awareness. Hypoglycemia awareness status according to Gold and Clarke methods: RR of patients with hypoglycemia unawareness is compared with patients having normal hypoglycemia awareness. a n=308: data missing on C-peptide status for three patients. b n = 301: data missing on Gold hypoglycemia awareness classification for 7 patients and on C-peptide status for three patients. c n = 294: data missing on Clarke classification for 14 patients and on C-peptide status for 3 patients. A group of 27 subjects were unclassified according to the Clarke hypoglycemia awareness classification (not shown). View Large ADRB2 Gln27Glu genotype, severe hypoglycemia, and hypoglycemia awareness, There was no significant association between the ADRB2 Gln27Glu genotype (rs1042714) and the prevalence of one or more events of hypoglycemia (P = 0.21), the incidence of hypoglycemia (P = 0.52) or hypoglycemia awareness status according to the Hillerød classification (P = 0.62), the Gold classification (P = 0.88), or the Clarke classification (P = 0.95). Discussion In this study, we tested the hypothesis that the Arg16Gly polymorphism in ADRB2 is associated with severe hypoglycemia in patients with type 1 diabetes. The ADRB2 may be a key mediator of EGP and generation of hypoglycemic warning symptoms (11–13, 19). The prevalence and the incidence rate of severe hypoglycemia were increased in individuals with type 1 diabetes who were homozygous for the ADRB2 Arg16 allele, particularly in those with impaired hypoglycemia awareness and, thereby, predisposed to severe hypoglycemia due to impaired warning symptoms. However, no direct association was observed between genotype and hypoglycemia awareness status per se, suggesting that the effect of the Arg16 allele is due to a reduced EGP during hypoglycemia rather than a reduction of warning symptom intensity. In accordance, the Arg16 allele has been associated with decreased endogenous glucose mobilization during stimulation with epinephrine in healthy humans at normoglycemia (19). Sufficient EGP is crucial during daily-life asymptomatic hypoglycemia, and is particularly important among patients with impaired awareness (24), to go between meals without developing severe hypoglycemia. The finding of an increased risk of severe hypoglycemia in AA patients can hypothetically be attributed to reduced EGP during hypoglycemia due to an increased agonist-mediated receptor downregulation induced by recurrent hypoglycemic epinephrine release. This is in accordance with in vivo findings (25) in which AA healthy subjects demonstrated increased agonist-mediated receptor downregulation in vasculature (16) and AA patients with asthma using long-acting β2-adrenergic receptor agonists demonstrate reduced treatment efficacy (15). This conflicts with the findings of a Dutch study (26) that showed recurrent hypoglycemia increases β-2 adrenergic cardiovascular sensitivity in healthy AA subjects. Glucose responses, however, were not assessed in that study. Our findings also appear to conflict with two other studies from the same Dutch group in, respectively, 85 and 486 patients with type 1 diabetes (27, 28), which reported an association between the GG individuals and unawareness assessed by a modified Clarke classification. Neither study found an association between ADRB2 Gly16Arg genotype and prevalence of severe hypoglycemia (27, 28). In accordance with the Nijmegen group findings (27, 28), our study shows a slight trend toward increased hypoglycemia unawareness in the GG group, using the Hillerød and Gold classifications (but not the traditional Clarke classification). All the hypoglycemia unawareness status trends were far from statistically significant, which is also in accordance with the Nijmegen group finding of a very weak association between GG frequencies and hypoglycemia awareness status (odds ratio, 1.49; 95% CI, 1.01 to 2.20; P = 0.046), which, in addition, was not corrected for multiple testing (28). Unfortunately, a direct comparison between our study and the Dutch studies is hampered by the fact that in the latter, rates of severe hypoglycemia were not reported and heterozygous and homozygous Arg16 patients were pooled; a potential recessive effect of the Arg16 allele as observed in our study cannot be assessed. Hypoglycemia unawareness is the most important known risk factor of severe hypoglycemia (6); therefore, it is paradoxical that the association between GG genotype and hypoglycemia unawareness observed in the Dutch studies did not translate into an increased prevalence of severe hypoglycemia (27, 28). The findings in our study are in line with the hypothesis that in AA individuals with type 1 diabetes, the EGP resulting from epinephrine release during hypoglycemia is reduced, leading to an increased risk of severe hypoglycemia, particularly in those with impaired hypoglycemia awareness. Mechanistic studies aimed at testing this hypothesis are warranted. Strengths and limitations Among the strengths of this study is the use of a validated questionnaire and three classification methods for estimating hypoglycemia awareness status. There was accordance among the analyses of awareness status (i.e., Hillerød, Gold, and Clarke methods), which decreases the risk for a false-negative finding. The study cohort is in Hardy-Weinberg equilibrium, making selection bias less probable. The thorough determination of the hypoglycemic phenotype by registration of prevalence and of incidence rate of severe hypoglycemia, recording of other risk factors, the finding of expected effect of impaired awareness, and the robustness of the main genotype findings in the multivariate analysis altogether increase the strength of the genotype effect analysis. Such data have not been presented in earlier studies of this genotype, to our knowledge. The size of the cohort, on the other hand, is a limitation and does not permit an analysis of the impact of potential haplotype effects. Conclusion A clinically significant increased risk of severe hypoglycemia was found among people with type 1 diabetes who were homozygous for the Arg16 allele of ADRB2. This was not explained by differences in hypoglycemia awareness between genotype groups. The difference between genotypes was amplified in patients with impaired awareness but absent in people with normal awareness. This supports the theory that the association is mediated by a reduced epinephrine-induced EGP in AA patients during daily-life, asymptomatic hypoglycemia, which is most common in patients with impaired awareness. Our findings need to be confirmed in independent cohorts and require further mechanistic studies to explore the influence of the Gly16Arg polymorphism of ADRB2 on responses during hypoglycemia and the association with agonist-mediated receptor downregulation after recurrent hypoglycemia. Abbreviations: Abbreviations: AA homozygous for the Arg16 allele ADRB2 adrenoceptor β-2 receptor gene EGP endogenous glucose production GA heterozygous for the Gly16 and Arg16 allele GG homozygous for the Gly16 allele RR relative rate SNP single nucleotide polymorphism Acknowledgments Financial Support: The study was funded by a grant from the Research Foundation of Nordsjællands University Hospital Hillerød, the Beckett Foundation, and the Foundation of Captain Lieutenant Harald Jensen and Wife. B.T. received core funding from Nordsjællands University Hospital Hillerød. Author Contributions: K.Z.R., N.V.O., B.T., and U.P.-B. initiated the study. L.F., B.T., and U.P.-B. designed the study. 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Published: Aug 1, 2018

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