Polymorphism rs4263535 in GABRA1 intron 4 was related to deeper sedation by intravenous midazolam

Abstract

Objective

To evaluate whether polymorphisms in the gamma-aminobutyric acid A receptor α1 subunit (GABRA1) gene influence sleep induction time, bispectral index score (BIS) during sleep induction and the total dose of midazolam required to reach a Ramsay Sedation Assessment Scale (RSAS) score of 4.

Methods

Patients scheduled for elective orthopaedic surgery were enrolled. All patients received initial doses of 0.02 mg/kg intravenous midazolam. If the RSAS score did not reach 4, an additional 1-mg dose of midazolam was administered. Results were compared among groups of patients with five single-nucleotide polymorphisms (SNPs) in GABRA1: rs4263535, rs980791, rs6556562, rs998754 and rs2279020.

Results

A total of 104 patients were evaluated. Polymorphism rs4263535 was associated with the lowest BIS during sedation induction. Multinomial logistic regression analysis demonstrated that polymorphism rs4263535 was significantly associated with the total dose of midazolam required for sedation induction.

Conclusions

Polymorphism rs4263535 in GABRA1 intron 4 was associated with deeper sedation by intravenous midazolam. Patients with the A/A rs4263535 genotype required a smaller dose of midazolam.

Keywords

Benzodiazepine gamma-aminobutyric acid A receptor genetic polymorphism

Introduction

Midazolam differs from other sedative agents in its more rapid onset and shorter duration of action.¹ Clinical effects are usually observable 2–3 min after intravenous midazolam administration.¹ Comparative studies have shown that midazolam at a dose of 0.05–0.12 mg/kg produces satisfactory sedation and operating conditions as effectively as the administration of 0.06–0.2 mg/kg diazepam in patients undergoing gastroscopy, dental implantation, cataract surgery or spinal anaesthesia.^2–10 Compared with diazepam, the onset of action of midazolam is usually more rapid and the degree of amnesia is greater, but the speed of onset of the effect of midazolam without premedication varies between individuals.¹¹ Although this inter-individual variability is reduced at higher doses of the drug,⁴ these differences reduce the predictability of sedation induction using midazolam. The inter-individual variability in sedation induction time is likely influenced by several factors, including dose, rate of administration, premedication status, age, sex and American Society of Anesthesiologists (ASA) physical status score.¹¹ In addition, sensitivity to benzodiazepines such as midazolam is influenced by complex biochemical interactions that are supported by genetic studies.^12–15

Benzodiazepine-sensitive gamma-aminobutyric acid A (GABA-A) receptors are composed of an α-subunit variant (α1, α2, α3, or α5), a β-subunit (β1–3), and a γ-subunit (γ1–3) in triplet configurations including α1β2,3γ2, α2β2,3γ2, α3β2,3γ2, and α5β2,3γ2, and are regarded as the major mediators of benzodiazepine action in the brain.¹⁶ The histidine residue in GABRA1 is characteristic of all major benzodiazepine-sensitive GABA-A receptors.^17,18 Sedation, anterograde amnesia and the anticonvulsant effects produced by benzodiazepines are mediated via α1-containing GABA receptors, and anxiolysis and muscle relaxation are mediated by α2-containing GABA receptors.¹⁴ Mutations to arginine residues in the α1, α2, α3, α5, β2, β3, and γ2 subunits can produce a complete loss of potentiation of GABA-induced currents, thereby reducing benzodiazepine sensitivity.^12,19–21 Analyses of the pharmacological characteristics of recombinant GABA receptors demonstrated that mutations in the GABA receptor genetic sequences lead to benzodiazepine insensitivity.¹²

This present study hypothesized that GABA-A receptor α1 subunit (GABRA1) polymorphisms would significantly influence the pharmacological effects (e.g. onset of sedation) of midazolam. The aim of the present study was to evaluate the association between all known GABRA1 nucleotide polymorphisms (rs4263535, rs980791, rs6556562, rs998754, and rs2279020) and sensitivity to midazolam. The primary experimental endpoint was the analysis of the association between GABRA1 SNPs with the dose of midazolam required to reach a Ramsay Sedation Assessment Scale (RSAS) score of 4. Secondary experimental endpoints were the analyses of the association between each polymorphism with the time-to-reach an RSAS score of 4 (induction time) and the lowest bispectral index score (BIS) measured during sedation induction.

Patients and methods

Patients

Patients aged 20–40 years, scheduled for elective orthopaedic surgery and classified as having ASA physical status²² I or II, were enrolled sequentially in the study in the Department of Anaesthesiology and Pain Medicine, College of Medicine, Korea University, Seoul, Republic of Korea, between August 2011 and July 2012. Patients with psychiatric disorders, neurological disorders, hypothermia (<35℃), those using medications that could influence the level of sedation (such as neuromuscular blocking agents, and/or those with a history of drug abuse) were excluded. In addition, any patient who received adrenaline was also excluded because it can influence BIS.²³ The study was approved by the Korea University Hospital Institutional Review Board, Seoul, Republic of Korea (no. ED11137) and the Clinical Research Information Service, Cheongju-si, Chungcheongbuk-do, Republic of Korea (no. KCT0000848). Written informed consent was obtained from each patient for study participation and genetic analysis.

Sedation

After patients arrived at the operating room, electrocardiography, respiratory rate, noninvasive mean blood pressure, oxygen saturation, heart rate and BIS were monitored. Before initiation of sedation, 5 l/min oxygen was supplied using a facemask for 5 min. The anaesthesiologist (S.Z.Y.) asked patients to keep their eyes closed and not to roll during the study. Sedation was induced with midazolam without premedication. An initial bolus dose of 0.02 mg/kg midazolam was administered intravenously over a period of 5 s, and an anaesthesiologist (S.Z.Y.) observed each patient for a period of 3 min to evaluate the onset of sedation.²⁴ The endpoint for sedation was RSAS 4 (RSAS scores: 6, no response to stimulus; 5, sluggish response to stimulus; 4, brisk response to stimulus; 3, response to commands; 2, cooperative, properly oriented, and tranquil; 1, anxious, restless, or both).²⁵ RSAS 4 at BIS 60–80, indicating mild-to-moderate sedation, is considered an adequate sedation level for surgery. A brisk response to a light glabellar tap was consistent with the desired level of sedation. If the RSAS score did not reach 4 by 3 min after the initial bolus of midazolam, an additional 1-mg dose of midazolam was administered intravenously every 2 min until an RSAS score of 4 was achieved. The study was terminated if the oxygen saturation dropped below 95% and/or tachycardia, hypertension, or hypotension >20% greater than baseline values developed. The time-to-reach RSAS 4 and the lowest BIS during sedation induction were recorded. In addition, the initial midazolam bolus dose and the total additional amount of midazolam administered were recorded. After the study was completed, patients underwent general anaesthesia for the duration of surgery.

Genomic analysis

Forthe genetic analysis of GABRA1 polymorphisms, 2 ml of venous blood was collected prior to anaesthesia from each patient. Blood was anticoagulated with 2 mg/ml ethylenediaminetetra-acetic acid and stored at −80℃ until analysis. Genomic DNA was extracted from each blood sample using a Qiagen DNA extraction kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). GABRA1 gene fragments were amplified using specific primers (Bioneer, Daejeon, Republic ofKorea) (Table 1). Polymerase chain reaction (PCR) was carried out using 2.5 mmol/l dNTPs, 10 pmol/l of each primer, 30 ng of human genomic DNA, and 5 U Taq polymerase (iNtRON, Seongnam, Republic of Korea) in a 30 -μl reaction volume in a C1000 Touch™ Thermal Cycler (Bio-Rad, Hercules, CA, USA). The PCR cycling programme involved preliminary denaturation at 94℃ for 180 s, followed by 40 cycles of denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s, and elongation at 72℃ for 30 s, followed by a final elongation step at 72℃ for 10 min.

Table 1.

Primers used for polymerase chain reaction amplification and pyrosequencing of regions of the human gamma-aminobutyric acid A receptor α1 subunit gene.

SNP	Allele	Position	Primer	Sequence	Product size, bp
rs4263535	A > G	Intron 4	Forward	5′-GTGCAATGTCAATGAAGACAC-3′	173
			Reverse	5′-B-CATTGCTCAGCTTCTAGTTGTAA-3′
			Sequencing	5′-TTCATTCTTGTCCATAAT-3′
rs980791	A > G	Intron 9	Forward	5′-TGAAGTGAAGCAGGCATAAAAT-3′	201
			Reverse	5′-B-TGATATGGTGGTCATTAATTGG-3′
			Sequencing	5′-AAGCTTAGAAAATATCAGCA-3′
rs6556562	A > G	Intron 7	Forward	5′-ACCGTTCTCTATTAGATTCTGATT-3′	128
			Reverse	5′-B-CAGTGAAACGATCACAGTTGTAT-3′
			Sequencing	5′-ACCCGAGGGCAATGT-3′
rs998754	G > T	3′ UTR	Forward	5′-ACCTCTTTTATTTAAAGCCCTTAC-3′	257
			Reverse	5′-B-CATTTCCCTATTTTAGGCTATTAT-3′
			Sequencing	5′-CTTTTATTTAAAGCCCTTAC-3′
rs2279020	A > G	Intron 10	Forward	5′-ATGGCAAAAGTGTGGTTC-3′	124
			Reverse	5′-B-CTGCATACTCCATCCAATACC-3′
			Sequencing	5′-GAAAAGGTAAATGCTTTAAT-3′

SNP, single-nucleotide polymorphism; B, biotinylated on the 5′-end of the primer; bp, base pair.

For the pyrosequencing, biotinylated PCR template (20 μl) was immobilized by incubation (with shaking at 1400 r.p.m. for 10 min at room temperature) with a mixture of 5 μl streptavidin beads (GE Healthcare Biosciences, Uppsala, Sweden) in 40 μl PyroMark Binding Buffer (Qiagen Korea, Seoul, Republic of Korea). For primer annealing, 40 μl PyroMark Annealing Buffer (Qiagen Korea) containing 0.4 μM sequencing primer was incorporated into each well. The bead–PCR product was transferred onto a probe with a Qiagen Vacuum Prep Workstation (Qiagen Korea), according to the manufacturer’s instructions. The captured beads on probes were incubated in 70% ethanol for 5 s and the solution was flushed through the filters for 5 s. The beads were then treated with a denaturing solution (0.2 M NaOH) that was flushed through the filters for 5 s. A wash buffer (10 mM Tris-acetate, pH 7.6) was used to rinse the beads for 5 s. All liquid was completely drained from the probes, after which the beads were transferred into Pyromark™ Wash Buffer (Qiagen Korea) containing the sequencing primer, which was heated at 85℃ for 2 min then cooled to room temperature (15–25℃ for ≥5 min. Pyrosequencing was performed on a Qiagen PyroMark Q96 ID instrument (Qiagen Korea) according to the manufacturer’s instructions.

Statistical analyses

Correlation coefficients between continuous variables such as body mass index (BMI), total midazolam dose, time to reach RSAS 4, and lowest BIS during sedation induction were obtained using Spearman’s rank correlation analysis. Deviation of the frequency of polymorphisms from the Hardy–Weinberg equilibrium was assessed using the χ²-test.

Median differences in total midazolam dose, time to reach RSAS 4, and lowest BIS during sedation induction between the groups with various GABRA1 polymorphisms were evaluated using the Kruskal–Wallis test. The Mann–Whitney U-test was performed to evaluate differences between the A/A, G/A, and G/G genotypes, and P-values were corrected with the Bonferroni correction according to the number of comparisons.

Multinomial logistic regression was applied to whole-genome single-nucleotide polymorphism (SNP) data to find SNPs associated with the responses of interest. Factors were identified that related to midazolam sensitivity and GABRA1 polymorphisms at rs4263535 using multinomial logistic regression analysis with total midazolam dose, lowest BIS during sedation induction, time-to-reach RSAS 4, age, sex, BMI and ASA physical status as covariates. However, the associations of age, sex, BMI, and ASA physical status with the effects of midazolam in patients with various GABRA1 genotypes were not statistically significant. Therefore, only midazolam dose, lowest BIS during sedation induction, and time to reach RSAS 4 were considered as risk factors using the backward variable selection method.

The required sample size for this study was determined based on a pilot study on the effect of the rs4263535 genotype because no data were available on the effect of midazolam in patients with varying GABRA1 genotypes. A power analysis was performed using G*Power software (version 3.1; Dusseldorf University, Dusseldorf, Germany) to determine whether the population means of the total dose of midazolam for the three assessed genotypes were equivalent.²⁶ The minimum sample size was determined to be 104 based on an α-value of 0.05, a power of 0.80, and an effect size of 0.313 (calculated from the pilot data from 28 patients). Assuming a 10% loss to follow-up observations, 114 patients were calculated to be required.

Statistical significance was defined as P < 0.05 and the statistical analyses were performed with SPSS® software, version 20.0 (SPSS Inc., Chicago, IL, USA) for IBM computers (IBM, Armonk, NY, USA). Calculations of allele frequency and heterozygosity using the Hardy–Weinberg equilibrium test were performed using R software version 2.15.2 (The R Foundation for Statistical Computing, Vienna, Austria).

Results

A total of 104 patients (42 male and 62 female) were enrolled in this study. A flow diagram of the progression through the study is shown in Figure 1. Patients who refused to participate or those undergoing psychiatric management were excluded (n = 10). Characteristics of included patients are presented in Table 2. Correlations between BMI, total dose of midazolam, lowest BIS during sedation induction, and time to reach RSAS 4 are shown in Table 3. Total dose of midazolam was positively correlated with time to reach RSAS 4 (r = 0.781, P < 0.001) and BMI (r = 0.204, P = 0.041), but not with BIS during sedation induction (r = 0.165).

Figure 1.

Flow diagram of the progression of patients undergoing routine elective orthopaedic surgical procedures through the study.

Table 2.

Characteristics of patients (n = 104) undergoing routine elective orthopaedic surgical procedures who were enrolled in the present study.

Characteristic	Overall n = 104	Sex		Statistical significance^a
Characteristic	Overall n = 104	Male n = 42	Female n = 62	Statistical significance^a
Age, years	30.9 ± 5.8	30.1 ± 6.3	31.4 ± 6.6	NS
ASA, I/II	97 (93.3)/7 (6.7)	38 (90.5)/4 (9.5)	59 (95.2)/3 (4.8)	NS
Body weight, kg	64.4 ± 14.7	74.5 ± 12.9	58.1 ± 11.9	P < 0.001
Height, cm	165.3 ± 8.0	172.5 ± 5.9	160.7 ± 5.4	P < 0.001
BMI, kg/m²	23.7 ± 4.8	25.1 ± 4.4	22.9 ± 4.8	P = 0.028
Total midazolam dose, mg	4.0 (2.0–11.0)	4.5 (2.0–10.0)	4.0 (2.0–11.0)	P = 0.042
Lowest BIS during induction	70 (50–82)	70 (62–82)	70 (50–74)	NS
Time-to-reach RSAS 4, s	259 (61–950)	236.5 (61–820)	270 (67–950)	NS

Values presented as mean ± SD, median (range) or n (%).

Between-group differences in continuous variables were analysed using Student’s t-test or Mann–Whitney U-test. Differences in categorical variables were analysed using χ²-test or Fisher’s exact test as appropriate.

ASA, American Society of Anesthesiologists physical status; BMI, body mass index; BIS, bispectral index; RSAS, Ramsay Sedation Assessment Scale; NS, no statistically significant difference (P ≥ 0.05).

Table 3.

Results of the Spearman’s rank correlation coefficient analysis to test the association between two ranked patient characteristics.

Parameter	Total dose of midazolam	Lowest BIS during induction time	Time-to-reach RSAS 4
BMI	0.204^a	−0.170	−0.149
Total dose of midazolam		0.165	0.781^a
Lowest BIS during induction			0.181

P < 0.05 when data were analysed using Spearman’s rank correlation coefficient.

BIS, bispectral index; RSAS, Ramsay Sedation Assessment Scale; BMI, body mass index.

Genotype and allele frequencies of the GABRA1 polymorphisms are shown in Table 4. All of the allele frequencies of the genetic polymorphisms assessed in the study population fitted the Hardy–Weinberg equilibrium.

Table 4.

Genotypes with allele frequencies and heterozygosity of the human gamma-aminobutyric acid A receptor α1 subunit gene polymorphisms.

SNP	Genotype	Frequency	Allele	Frequency	Heterozygosity
rs4263535	G/G	25 (24)	A	105 (50)	0.502
	G/A	53 (51)	G	103 (50)
	A/A	26 (25)
rs980791	A/A	14 (13)	G	120 (58)	0.490
	A/G	60 (58)	A	88 (42)
	G/G	30 (29)
rs6556562	G/G	43 (41)	G	136 (65)	0.455
	G/A	50 (48)	A	72 (35)
	A/A	11 (11)
rs998754	G/G	19 (18)	T	115 (55)	0.497
	G/T	55 (53)	G	93 (45)
	T/T	30 (29)
rs2279020	G/G	30 (29)	G	115 (55)	0.497
	G/A	55 (53)	A	93 (45)	0.502
	A/A	19 (18)

Values were presented as n (%).

SNP, single-nucleotide polymorphism.

All P-values for the Hardy–Weinberg Equilibrium were not significant (P ≥ 0.05).

Table 5 presents the medians (with ranges) of the total dose of midazolam, lowest BIS recorded during sedation induction and time to reach RSAS 4 in groups of patients with A/A, G/A, and G/G rs4263535 genotypes. The Kruskal–Wallis test showed that the rs4263535 polymorphism was associated with the lowest BIS during sedation induction (P = 0.025), and statistically significant median differences were observed in the lowest BIS measured during induction between the patients with the A/A and G/G rs4263535 genotypes (P = 0.042; Mann–Whitney U-test). Polymorphisms rs980791, rs6556562, rs998754, and rs2279020 were not significantly associated with BIS during induction, total administered dose of midazolam, or time to reach RSAS.

Table 5.

Evaluation of the equivalence of the population medians of total midazolam dose, lowest bispectral index (BIS) during sedation induction, and time to reach Ramsay Sedation Assessment Scale (RSAS) 4 across the three evaluated single-nucleotide polymorphism (SNP) genotypes of the human gamma-aminobutyric acid A receptor α1 subunit gene using the Kruskal–Wallis test.

SNP	Genotype	Total dose of midazolam, mg	Lowest BIS during induction time	Time-to-reach RSAS 4, s
rs4263535	A/A	4.0 (2.0–10.0)	70.0 (64.0–82.0)^a	250.0 (90.0–626.0)
	G/A	4.5 (2.0–11.0)	70.0 (55.0–79.0)^a	292.0 (61.0–950.0)
	G/G	4.0 (2.8–8.5)	70.0 (50.0–70.0)^a	245.0 (85.0–820.0)
rs980791	A/A	3.8 (2.0–8.5)	70.0 (55.0–70.0)	257.5 (67.0–820.0)
	A/G	4.5 (2.0–11.0)	70.0 (50.0–82.0)	254.0 (71.0–950.0)
	G/G	4.0 (2.0–9.0)	70.0 (65.0–71.0)	296.0 (61.0–807.0)
rs6556562	A/A	4.0 (2.5–5.5)	70.0 (59.0–70.0)	200.0 (67.0–440.0)
	G/A	4.0 (2.0–10.0)	70.0 (55.0–82.0)	257.5 (90.0–950.0)
	G/G	4.5 (2.0–11.0)	70.0 (50.0–79.0)	287.5 (61.0–807.0)
rs998754	G/G	4.0 (2.0–8.5)	70.0 (55.0–70.0)	222.0 (67.0–820.0)
	G/T	4.5 (2.0–11.0)	70.0 (50.0–82.0)	256.0 (71.0–950.0)
	T/T	4.0 (2.0–9.0)	70.0 (65.0–71.0)	301.0 (61.0–807.0)
rs2279020	A/A	4.0 (2.0–8.5)	70.0 (55.0–70.0)	222.0 (67.0–820.0)
	G/A	4.5 (2.0–11.0)	70.0 (50.0–82.0)	256.0 (71.0–950.0)
	G/G	4.5 (2.0–9.0)	70.0 (65.0–71.0)	301.0 (61.0–807.0)

Values shown as median (range).

The rs4263535 polymorphism was associated with the lowest BIS during sedation induction (P = 0.025; Kruskal–Wallis test); and multiple comparisons for the rs4263535 polymorphism showed significant median differences in the lowest BIS measured during induction between patients with A/A and G/G rs4263535 genotypes (P = 0.042; Mann–Whitney U-test). P-values for multiple comparisons were corrected with the Bonferroni correction to account for the number of comparisons.

As shown in Table 6, multinomial logistic regression analysis with backward variable selection comparing the A/A and G/G groups revealed that the rs4263535 polymorphism was significantly associated with the total administered dose of midazolam for the A/A group (P = 0.040). Moreover, the odds ratio for A/A relative to G/G would be expected to decrease by a factor of 0.671 if the other variables in the model are held constant and a patient was to increase the total dose of midazolam by 1 IU. In other words, if a patient was to increase his or her total dose of midazolam, the probability of the patient having the G/G rs4263535 GABRA1 genotype would be greater than the probability of that patient having the A/A rs4263535 GABRA1 genotype. Using multinomial logistic regression analysis with backward variable selection, patients with the G/G rs4263535 GABRA1 genotype were found to be more resistant to the effects of midazolam compared with thos with the A/A phenotype.

Table 6.

Multinomial logistic regression analysis of the association between the human gamma-aminobutyric acid A receptor α1 subunit gene polymorphism rs4263535 genotypes with other factors.

		Univariate logistic regression				Multivariate logistic regression using backward variable selection				Reference category
		Statistical significance		95% CI		Statistical significance^a		95% CI
Genotype	Covariate	Statistical significance	OR	Lower	Upper	Statistical significance^a	OR	Lower	Upper
A/A	Total midazolam	NS	0.837	0.610	1.148	P = 0.040	0.671	0.459	0.981	G/G
	Lowest BIS during sedation induction	P = 0.038	1.246	1.012	1.533	NS	1.271	0.996	1.621
	Time-to-reach RSAS 4	NS	0.999	0.996	1.002
G/A	Total midazolam	NS	1.028	0.801	1.318	NS	0.940	0.707	1.249	G/G
	Lowest BIS during sedation induction	NS	1.128	0.976	1.303	NS	1.132	0.973	1.318
	Time-to-reach RSAS 4	NS	1.001	0.999	1.004
G/G	Total midazolam	NS	1.228	0.928	1.626	P = 0.049	1.400	1.001	1.959	A/A
	Lowest BIS during sedation induction	NS	0.905	0.753	1.089	NS	0.891	0.705	1.126
	Time-to-reach RSAS 4	NS	1.002	0.999	1.005

CI, confidence Interval; OR, odds ratio; BIS, bispectral index; RSAS, Ramsay Sedation Assessment Scale; NS, no statistically significant difference (P ≥ 0.05).

Multinomial logistic regression analysis with backward variable selection comparing the A/A and G/G groups revealed that the rs4263535 polymorphism was significantly associated with the total administered dose of midazolam for the A/A group (P = 0.040) and the G/G group (P = 0.049).

Discussion

This study is the first to evaluate the influence of genetic variation on the induction time for midazolam sedation. Current results show that the polymorphism rs4263535 in intron 4 of the human GABRA1 gene is associated with deeper sedation by intravenous midazolam, compared with other polymorphisms in this gene. Furthermore, this current study showed that patients with the A/A rs4263535 GABRA1 genotype required smaller doses of midazolam to achieve sedation, and showed a lower BIS value during sedation induction, compared with patients with the G/G rs4263535 GABRA1 genotype.

Benzodiazepines such as midazolam have hypnotic, sedative, anxiolytic, amnesic, anticonvulsant and centrally produced muscle relaxant properties.² The mechanism of action of benzodiazepines is reasonably well understood and does not involve release, synthesis, or altered metabolism of GABA.^1–3 Instead, benzodiazepines potentiate the inhibitory action of GABA by increasing the flow of chloride ions through ion channels, which reduces the ability of the affected cell to generate an action potential.⁷ The hypnotic effect of midazolam is known to be associated with GABA accumulation and benzodiazepine receptor occupation.²⁷ Midazolam interferes with GABA reuptake, which results in GABA accumulation.⁴ In addition, the occupation of GABA receptors with benzodiazepines such as midazolam increases membrane hyperpolarization and neuronal inhibition.^12,28 Midazolam also causes phasic inhibition by prolonging inhibitory post-synaptic currents, and its action at extra-synaptic GABA-A receptors produces tonic inhibition, which is a continuous increased inhibitory current.^5,29 These midazolam-induced changes lead to neuronal hyperpolarization that reduces neuronal excitability.²⁹ In addition, midazolam reduces thalamic and cerebral cortical blood flow, as well as metabolic activity, in those regions.¹² These well-established effects of midazolam in the brain produce altered neuronal activity that changes the level of consciousness of patients administered this drug.²⁹

Research has suggested that mutations and polymorphisms in GABRA1 are associated with alcohol-related behavioural alterations including excitement, sleep, withdrawal symptoms and dependence.³⁰ A polymorphism in GABRA1 intron 9 (rs980791) has been associated with alcohol dependence (P = 0.01) and susceptibility to blackout (P < 0.01).³¹ A polymorphism in GABRA1 intron 4 (rs4263535) has been associated with the age of first drunkenness (P < 0.01), and a polymorphism in GABRA1 intron 10 (rs2279020) has been associated with blackout (P = 0.02) and alcohol dependence (P = 0.03).³¹ Essential tremors,³² juvenile myoclonic epilepsy,³³ autism³⁴ and schizophrenia³⁵ have also been associated with GABRA1 mutations. In the same manner, GABRA1 polymorphism rs4263535 seems to be associated with determining the pharmacodynamics and pharmacokinetics of benzodiazepine in this study.

As shown in Table 5, the A/A rs4263535 GABRA1 genotype was associated with the lowest BIS measured during sedation induction in this study. However, the total dose of midazolam required to reach RSAS 4 did not vary significantly among the rs4263535 genotypes, indicating that the level of sedation, as estimated by BIS, in all patients after an identical dose of midazolam may not be an equivalent effect. Patients reached the same RSAS after midazolam administration, but the level of consciousness as measured by the BIS was likely to be higher in those with the A/A rs4263535 GABRA1 genotype, suggesting that such patients were more resistant to the effects of midazolam.

The level of consciousness of a patient is generally assessed by subjective clinical scales such as the observer assessment of alertness/sedation score or the RSAS score,^36,37 which broadly categorize sedation levels as mild, moderate, or deep. More recently developed BIS methodology has provided an additional means of assessing the level of consciousness of a patient: the BIS is regarded as a valuable addition to the clinician’s repertoire of methods for monitoring the level of consciousness in patients undergoing midazolam sedation.^7–10,15 BIS monitoring provides an objective real-time measurement of the depth of anaesthesia via a weighted sum of electroencephalographic subparameters that are transformed into numeric values ranging from 0 (no electrical activity in the brain) to 100 (fully conscious). As the state of consciousness evolves from awake to sedated, or exhibits burst suppression, the BIS decreases. As an anaesthesia guideline, a BIS of 80–90 is considered to be indicative of conscious sedation, a BIS of 60–80 is indicative of moderate or deep sedation that produces anxiolysis and/or analgesia,³⁸ and a BIS of 40–60 is required for maintenance of general anaesthesia.³⁹ BIS values observed during the present study were consistent with increased brain activity indicative of higher levels of consciousness in patients with the A/A genotype for polymorphism rs4263535, compared with patients with the G/G genotype for this polymorphism.

This present study had some limitations. Various factors affect benzodiazepine pharmacodynamics, and the effect or function of a drug is related to serum levels of the drug at its site of action, which can be estimated by pharmacokinetic methodology. Using plasma concentration data and pharmacokinetics simulations, it has been estimated that a benzodiazepine receptor occupancy of <20% may be sufficient to produce an anxiolytic effect, while sedation is observed with 30–50% receptor occupancy, and unconsciousness requires ≥60% occupancy.⁴⁰ Therefore, lipophilicity, receptor affinity, density, occupancy and receptor reserves all affect the pharmacodynamics of benzodiazepines. In addition, metabolic stability, heterogeneity of other receptors, distribution of receptors in the brain and receptor pathology can also influence the effects of medications. In addition, any other genetic variations that affect the pharmacokinetics of midazolam need to be considered. The pharmacokinetics of midazolam might affect the amount of midazolam that reaches the receptor, therefore it might also have an effect on the intensity of sedation. Generally, high-frequency electroencephalography (EEG) activity decreases as consciousness level decreases in drug-free patients. However, sedative benzodiazepines increase beta activity.^41,42 In this present study, it was not possible to analyse the beta ratio, signal quality index and an electromyography level in patients because BIS data were collected manually. In future, EEG analysis will be used to more precisely evaluate the effect of GABRA1 polymorphisms on the pharmacodynamics of midazolam. In addition, this present study may have been underpowered because the minimum sample size was determined to be 114, but only 104 patients were analysed.

In conclusion, results of the present study show that the rs4263535 polymorphism in intron 4 of the human GABRA1 gene was associated with deeper sedation by intravenous midazolam, and patients with the A/A rs4263535 genotype required smaller doses of midazolam. Thus, patients with the A/A genotype might require a smaller dose of midazolam to achieve RSAS 4 during the sedation induction period, compared with other patients. This finding should contribute to the ability of clinicians to induce optimal anaesthesia with midazolam by allowing them to customize dosing regimens based on patient genotypes.

Footnotes

Acknowledgements

This report contains a portion of the doctoral dissertation of Shin Yuong Lee.

Declaration of conflicting interest

The authors declare that there are no conflicts of interest.

Funding

This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI13C2181). No funding other than personal was used in conducting the audit as well as writing the manuscript.

References

Khanderia

Pandit

. Use of midazolam hydrochloride in anesthesia. Clin Pharm 1987; 6: 533–547.

Reves

Fragen

Vinik

. Midazolam: pharmacology and uses. Anesthesiology 1985; 62: 310–324.

Parker

Mahan

Giugliano

. Efficacy and safety of intravenous midazolam and ketamine as sedation for therapeutic and diagnostic procedures in children. Pediatrics 1997; 99: 427–431.

Berggren

Eriksson

. Midazolam for induction of anaesthesia in outpatients: a comparison with thiopentone. Acta Anaesthesiol Scand 1981; 25: 492–496.

Burns

Shelly

Park

. The use of sedative agents in critically ill patients. Drugs 1992; 43: 507–515.

Kollef

Levy

Ahrens

. The use of continuous i.v. sedation is associated with prolongation of mechanical ventilation. Chest 1998; 114: 541–548.

Lera dos Santos

Maluf-Filho

Chaves

. Deep sedation during gastrointestinal endoscopy: propofol-fentanyl and midazolam-fentanyl regimens. World J Gastroenterol 2013; 19: 3439–3446.

Fan

Islam

. Comparison of dexmedetomidine and midazolam for conscious sedation in dental surgery monitored by bispectral index. Br J Oral Maxillofac Surg 2013; 51: 428–433.

Khurana

Agarwal

Verma

. Comparison of midazolam and propofol for BIS-guided sedation during regional anaesthesia. Indian J Anaesth 2009; 53: 662–666.

10.

Apan

Doganci

Ergan

. Bispectral index-guided intraoperative sedation with dexmedetomidine and midazolam infusion in outpatient cataract surgery. Minerva Anestesiol 2009; 75: 239–244.

11.

Dundee

Halliday

Harper

. Midazolam. A review of its pharmacological properties and therapeutic use. Drugs 1984; 28: 519–543.

12.

Benson

Low

Keist

. Pharmacology of recombinant gamma-aminobutyric acidA receptors rendered diazepam-insensitive by point-mutated alpha-subunits. FEBS Lett 1998; 431: 400–404.

13.

Mohler

Richards

. The benzodiazepine receptor: a pharmacological control element of brain function. Eur J Anaesthesiol Suppl 1988; 2: 15–24.

14.

Mohler

Fritschy

Rudolph

. A new benzodiazepine pharmacology. J Pharmacol Exp Ther 2002; 300: 2–8.

15.

Amrein

Hetzel

. Pharmacology of Dormicum (midazolam) and Anexate (flumazenil). Acta Anaesthesiol Scand Suppl 1990; 92: 6–15.

16.

Fritschy

Mohler

. GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits. J Comp Neurol 1995; 359: 154–194.

17.

Kim

Lim

. Effects of genetic polymorphisms of OPRM1, ABCB1, CYP3A4/5 on postoperative fentanyl consumption in Korean gynecologic patients. Int J Clin Pharmacol Ther 2013; 51: 383–392.

18.

Wieland

Lüddens

Seeburg

. A single histidine in GABAA receptors is essential for benzodiazepine agonist binding. J Biol Chem 1992; 267: 1426–1429.

19.

Knoflach

Benke

Wang

. Pharmacological modulation of the diazepam-insensitive recombinant gamma-aminobutyric acidA receptors alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2. Mol Pharmacol 1996; 50: 1253–1261.

20.

Hadingham

Garrett

Wafford

. Cloning of cDNAs encoding the human gamma-aminobutyric acid type A receptor alpha 6 subunit and characterization of the pharmacology of alpha 6-containing receptors. Mol Pharmacol 1996; 49: 253–259.

21.

Wafford

Thompson

Thomas

. Functional characterization of human gamma-aminobutyric acidA receptors containing the alpha 4 subunit. Mol Pharmacol 1996; 50: 670–678.

22.

American Society of Anesthesiologists Physical Status Classification System. http://www.asahq.org/resources/clinical-information/asa-physical-status-classification-system (2014, accessed 1 June 2015).

23.

Andrzejowski

Sleigh

Johnson

. The effect of intravenous epinephrine on the bispectral index and sedation. Anaesthesia 2000; 55: 761–763.

24.

Pratila

Fischer

Alagesan

. Propofol versus midazolam for monitored sedation: a comparison of intraoperative and recovery parameters. J Clin Anesth 1993; 5: 268–274.

25.

Ramsay

Savege

Simpson

. Controlled sedation with alphaxalone-alphadolone. Br Med J 1974; 2: 656–659.

26.

Faul

Erdfelder

Lang

. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods 2007; 39: 175–191.

27.

Olkkola

Ahonen

. Midazolam and other benzodiazepines. Handb Exp Pharmacol 2008; 182: 335–360.

28.

Rudolph

Crestani

Benke

. Benzodiazepine actions mediated by specific gamma-aminobutyric acid(A) receptor subtypes. Nature 1999; 401: 796–800.

29.

Bai D, Zhu G, Pennefather P, et al. Distinct functional and pharmacological properties of tonic and quantal inhibitory postsynaptic currents mediated by gamma-aminobutyric acid(A) receptors in hippocampal neurons.

30.

Hellevuo

Kiianmaa

Korpi

. Effect of GABAergic drugs on motor impairment from ethanol, barbital and lorazepam in rat lines selected for differential sensitivity to ethanol. Pharmacol Biochem Behav 1989; 34: 399–404.

31.

Dick

Plunkett

Wetherill

. Association between GABRA1 and drinking behaviors in the collaborative study on the genetics of alcoholism sample. Alcohol Clin Exp Res 2006; 30: 1101–1110.

32.

Deng

Xie

. Genetic analysis of the GABRA1 gene in patients with essential tremor. Neurosci Lett 2006; 401: 16–19.

33.

Cossette

Liu

Brisebois

. Mutation of GABRA1 in an autosomal dominant form of juvenile myoclonic epilepsy. Nat Genet 2002; 31: 184–189.

34.

Whitehead

Menold

. Identification of significant association and gene-gene interaction of GABA receptor subunit genes in autism. Am J Hum Genet 2005; 77: 377–388.

35.

Petryshen

Middleton

Tahl

. Genetic investigation of chromosome 5q GABAA receptor subunit genes in schizophrenia. Mol Psychiatry 2005; 10: 1074–1088.

36.

Chernik

Gillings

Laine

. Validity and reliability of the observer’s assessment of alertness/sedation scale: study with intravenous midazolam. J Clin Psychopharmacol 1990; 10: 244–251.

37.

Sessler

Riker

Ramsay

. Evaluating and monitoring sedation, arousal, and agitation in the ICU. Semin Respir Crit Care Med 2013; 34: 169–178.

38.

Liu

Singh

White

. Electroencephalographic bispectral index correlates with intraoperative recall and depth of propofol-induced sedation. Anesth Analg 1997; 84: 185–189.

39.

Glass

Bloom

Kearse

. Bispectral analysis measures sedation and memory effects of propofol, midazolam, isoflurane, and alfentanil in healthy volunteers. Anesthesiology 1997; 86: 836–847.

40.

Amrein

Hetzel

Hartmann

. Clinical pharmacology of flumazenil. Eur J Anaesthesiol Suppl 1988; 2: 65–80.

41.

Breimer

Hennis

Burm

. Quantification of the EEG effect of midazolam by aperiodic analysis in volunteers. Pharmacokinetic/pharmacodynamic modelling. Clin Pharmacokinet 1990; 18: 245–253.

42.

Loughnan

Sebel

Thomas

. Evoked potentials following diazepam or fentanyl. Anaesthesia 1987; 42: 195–198.