hyperactivity disorder in Korean population

Progress in Neuro-Psychopharmacology & Biological Psychiatry 73 (2017) 56–63

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Association of norepinephrine transporter gene polymorphisms in attention-deficit/hyperactivity disorder in Korean population So-Young Oh M.D., Ph.D. a, Yong-Ku Kim M.D., Ph.D. b,⁎ a b

Department of Psychiatry, Seoul Metropolitan Eunpyeong Hospital, Seoul, South Korea Department of Psychiatry, College of Medicine, Korea University, Seoul, South Korea

a r t i c l e

i n f o

Article history: Received 9 October 2015 Received in revised form 25 September 2016 Accepted 26 October 2016 Available online 28 October 2016 Keywords: ADHD SLC6A2 NET Polymorphism

a b s t r a c t We investigated the association of three single nucleotide polymorphisms(SNP) of the norepinephrine transporter (NET) gene SLC6A2, T-182C (rs2242446), A-3081T (rs28386840), and G-1287A (rs5569) with the prevalence of attention-deficit/hyperactivity disorder (ADHD), its clinical severity, and other disease-related characteristics in a Korean population. The genotype, allele frequency and haplotype of 103 ADHD patients and 173 controls were analyzed for these three SNPs. All participants completed the Korean version of the ADHD Rating Scale (K-ARS). The ADHD group also completed the Korean Educational Development Institute-Wechsler Intelligence Scale for Children (KEDI-WISC) and the Continuous Performance Test (CPT) in a drug-naive state. The χ2 test and logistic regression analysis revealed no significant differences in the genotype distribution or allele frequencies of each SNP between the ADHD group and the control. In the haplotype analysis, the most common T-A-G haplotype was related to an increased risk of ADHD in females (P = 0.002). There was no statistical significance between clinical features of ADHD and any specific allele of each SNP after multiple test correction except lower omission error in non-A girl carriers (GG type) of G-1287A (carrier 76.75 ± 18.74, non-carrier 55.00 ± 9.26, t = 3.026, P = 0.007, Bonferroni-corrected P = 0.042). Some values related A-3081 and G1287A showed a trend approaching the significance level when analyzed separately by gender. Even though it was not statistically meaningful after multiple test correction, G allele might have some protective effect against development of ADHD symptoms and this finding was consistent with previous studies. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Attention-deficit hyperactivity disorder (ADHD) is a childhoodonset neurodevelopmental disorder characterized by inattention, increased motor activity and impulsivity. Genetic studies consistently support the assumption that the disease is highly heritable. Twin studies, which identified the concordance rates between monozygotic and dizygotic twins, estimate that the overall heritability of ADHD is 76% (Faraone et al., 2005), which is one of the highest among psychiatric disorders (Faraone and Mick, 2010). Parents of ADHD children have a two- to eight-fold higher risk of having ADHD (Faraone, 2004), and there is a 30% prevalence of ADHD among siblings of ADHD children (Young, 2011), much higher than the prevalence of 8 to 12% in the general population (Faraone et al., 2003).

Abbreviations: ADHD, attention-deficit hyperactivity disorder; NET, norepinephrine transporter; K-ARS, Korean version of the ADHD Rating Scale; KEDI-WISC, Korean Educational Development Institute-Wechsler Intelligence Scale for Children; CPT, Continuous Performance Test. ⁎ Corresponding author at: Department of Psychiatry, Korea University Ansan Hospital, Gojan 1-dong, Danwon-gu, Ansan, Gyeonggi-Do 152-703, South Korea. E-mail address: [email protected] (Y.-K. Kim).

http://dx.doi.org/10.1016/j.pnpbp.2016.10.006 0278-5846/© 2016 Elsevier Inc. All rights reserved.

Studies on candidate genes for ADHD have mainly focused on genes involving the dopaminergic system, therefore those involving the noradrenergic system have received less attention. However, there is growing evidence that the noradrenergic system is involved in ADHD. Neuropsychological and imaging studies have shown that ADHD is associated with alterations in the prefrontal cortex (PFC) and related circuits, and noradrenergic neurotransmitter systems are thought to play important roles in this area of the brain (Arnsten, 2006). SLC6A2 gene encoding the norepinephrine transporter (NET), which is located on chromosome 16q12.2 (Brüss et al., 1993), is the most frequently investigated gene involving the noradrenergic system. NET plays a vital role in regulating catecholamine activity in the prefrontal area, which is related to the core symptoms of ADHD. Dopamine reuptake in the frontal cortex is carried out mainly by NET whereas that in the striatum is carried out by dopamine transporters; the frontal cortex has few dopamine transporters but abundant NETs, and dopamine has high affinity for the NET (Morón et al., 2002). Therefore, abnormalities in NET function may be closely related to the symptoms of ADHD, and the related genetic factors may increase the risk of ADHD. No SNP in NET has proved to be a definite risk factor for ADHD. Previous reports on the association between SLC6A2 polymorphisms and disease susceptibility are controversial. A number of studies did not

S.-Y. Oh, Y.-K. Kim / Progress in Neuro-Psychopharmacology & Biological Psychiatry 73 (2017) 56–63

find any significant association (Barr et al., 2002; Cho et al., 2008; De Luca et al., 2004; McEvoy et al., 2002), but there are some reports suggesting that some SNPs are related to ADHD. The SNP rs3785157 was found to be associated with ADHD in four studies (Bobb et al., 2005; Brookes et al., 2006; Kim et al., 2008; Xu et al., 2005), but two of these studies (Brookes et al., 2006; Kim et al., 2008) found the opposite risk alleles against the earlier two studies (Bobb et al., 2005; Xu et al., 2005). The SNP rs998424 was reported to be significant in the transmission disequilibrium test in one study (Bobb et al., 2005), and another study showed a suggestive but non-significant association with ADHD (Xu et al., 2005). The latter study demonstrated a similar result for the SNP rs2242447. The SNPs rs3785143 and rs11568324 were found to be associated with ADHD in two studies (Brookes et al., 2006; Kim et al., 2008). The SNP rs28386840 (A-3081T) was also reported to be associated with ADHD in two studies by the same research group (Joung et al., 2010; Kim et al., 2006), but another study reported negative findings (Cho et al., 2008). In this study we focused on the SLC6A2 polymorphisms, T-182C (rs2242446), A-3081T (rs28386840), and G-1287A (rs5569). These three polymorphisms are relatively common and are thought to be relevant in ADHD, but previous studies have reported mixed or insufficient results. The SLC6A2 T-182C polymorphism, which is more familiarly related to major depression or the brain reward system, was investigated in two studies of ADHD (Lee et al., 2011; Rippel et al., 2006). No significant results were observed in these studies, but the subjects in one study were patients with Tourette syndrome (with/without ADHD) (Rippel et al., 2006), and the other study obtained the control data from a DNA bank, thus the childhood psychiatric history of the control group was not evaluated (Lee et al., 2011). The SLC6A2 A-3081T polymorphism is a relatively new promoter variant first reported by Kim et al. (2006). There have been two studies carried out in Korean populations (Cho et al., 2008; Joung et al., 2010), but they reported mixed results. SLC6A2 G-1287A has not been proven to be associated with ADHD in previous studies (Cho et al., 2008; Xu et al., 2005), but there have been some positive findings with regard to response to pharmacologic treatment (Yang et al., 2004) as well as in the results of computerized continuous performance testing (Song et al., 2011). Thus, we selected these three SNPs to obtain more evidence on their association with ADHD and its clinical manifestations.

2. Methods 2.1. Subjects The present study included 103 patients who visited the Department of Child and Adolescent Psychiatry at a university-affiliated hospital in the Republic of Korea. The patients were those who 1) were diagnosed with ADHD according to the DSM-IV-TR criteria (American Psychiatric Association, 1994), 2) had an IQ score above 70 based on the Korean Educational Development Institute's version of the Wechsler's Intelligence Scale for Children (KEDI-WISC), and 3) were drug-naive at the time of recruitment. We excluded children who 1) had a past history of or had recently been diagnosed with a psychotic disorder, bipolar disorder, pervasive developmental disorder, or intellectual disability, 2) were diagnosed with major depression and had evident suicidal ideation or were suspected of having a risk for selfmutilation, 3) had a neurological disorder including seizure disorder, 4) had a medical disease needing long-term treatment, 5) strongly refused to have blood drawn, and 6) had caregivers who were not able to understand the informed consent form. The control group included 173 healthy children who were all recruited from the same primary school in Korea. All of the control subjects were free of any major medical and psychiatric problems and scored lower than 19 on the Korean version of the ADHD Rating Scale

57

(K-ARS). We excluded the children who 1) had a past history of or had recently been diagnosed with a major medical, neurological, or psychiatric disorder, 2) were poorly adjusted in school or were suspected to be intellectually disabled according to the teacher's report, 3) scored 19 or more on the K-ARS, 4) strongly refused to have blood drawn, and 5) had caregivers who were not able to understand the informed consent form. Written informed consent was obtained from all participants, and the study protocol was approved by the hospital's institutional review board. 2.2. Clinical assessments Diagnostic assessments of ADHD were carried out according to DSM-IV criteria (American Psychiatric Association, 1994) with the Korean version of the Kiddie Schedule for Affective Disorders and Schizophrenia for School-Age Children, Present and Lifetime (K-SADSPL-K) by clinically experienced child–adolescent psychiatrists. The Kiddie Schedule for Affective Disorders and Schizophrenia for SchoolAge Children, Present and Lifetime (K-SADS-PL), developed by Kaufman et al. (1997), was translated into Korean and standardized by Kim et al. (2004) and its validity and reliability have been well established in the assessment of ADHD. The ADHD group and the control group completed the Koran version of the ADHD Rating Scale (K-ARS) (So et al., 2002). The ADHD Rating Scale-IV, developed by DuPaul (1991), is a behavior-rating scale consisting of nine inattention items and nine hyperactive/impulsivity items. The cutoff score is 19 when the respondent is a parent and 17 when the respondent is a teacher. The K-ARS was standardized by So et al. (2002) and is known to be highly valid and reliable. 2.3. Psychological and neurocognitive measurements All of the ADHD subjects completed the Korean Educational Development Institute-Wechsler Intelligence Scale for Children (KEDIWISC) (Park et al., 1991) and the Korean version of a computerized continuous performance test (computerized CPT) (Shin et al., 2000) in a drug-naive state. The KEDI-WISC consists of 12 subtests (information, similarities, arithmetic, vocabulary, comprehension, digit span, picture completion, picture arrangement, block design, object assembly, coding and maze) and evaluates verbal IQ, performance IQ and full-scale IQ. The computerized CPT (Greenberg and Waldman, 1993) was used to measure the inattention, impulsivity, and sustained attention deficits of the ADHD children. The Korean version of the CPT was standardized, and its validity and reliability have been well established (Shin et al., 2000). A visual stimulus was presented for 100 ms every 2 sec, with the subjects being required to respond to a square containing a triangle (target) and not respond to a square containing a circle or square (nontarget). The four major variables recorded were 1) omission errors (failure to respond to the target), which are commonly interpreted as a measure of inattention; 2) commission errors (responding inappropriately to the non-target), which are commonly interpreted as a measure of impulsivity; 3) response times for correct responses to the target, which are interpreted as a measure of information processing and motor response speed; and 4) the standard deviation of the response times for correct responses to the target (response time variability), which is interpreted as a measure of variability or consistency of attention. 2.4. DNA analysis and genotyping Genomic DNA was extracted from whole blood leukocytes using the Wizard Genomic DNA purification kit (Promega, Madison, WI, USA). The SNP was genotyped by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) methods according to the protocol originally described in previous studies (Inoue et al., 2004; Suzuki et al., 2007; Inoue et al., 2007). For T-182C, 5′-CCA TTT

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GGG GCA GGC GAA AGT-3′ was used as the forward primer and 5′-CGC TGA CGG GAC GCA GGG TTC CCA GCC AAG-3′ was used as the reverse primer. For A-3081T, 5′-CCT GGG GCT CTG CTG TTA GC-3′ was used as the forward primer and 5′-CCT GGA AGC AAT CGT TGG GG-3′ was used as the reverse primer. For A1287G, 5′-TTG ACT TTA TTG AAA TGC GGC-3′ was used as forward primer and 5′-TCC AGG GAG ACC CTA ATT CC-3′ was used as the reverse primer. The amplification mixture contained 0.5 μl of 100 ng/μl DNA, 2.5 μl of 10 × Taq buffer, 0.5 μl of 10 mM dNTP mixture, 1 μl primers, 19.375 μl distilled water, and 0.125 μl Taq DNA polymerase (SolGent, Korea). Samples were amplified using a thermocycler (GeneAmp PCR system 2700, Applied Biosystems, Foster City, CA, USA). An initial denaturation at 95 °C for 5 min was followed by 35 (T-182c) or 36 (A-3081T and G-1287A) cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 10 min. The reaction was terminated at 4 °C. The amplified DNA was digested with the restriction enzyme StyI (New England Biolabs) that cuts at the -182C site, BsrsI (New England Biolabs) that cuts at the -3081A site, and Sau96I (New England Biolabs) that cuts at the -1287G site. The products were electrophoresed on 3% agarose gels and stained with ethidium bromide. For T-182C, the homozygous TT genotype was identified by a single 175 bp band, CC by 145 and 30 bp bands, and TC by 175, 145, and 30 bp bands. For A-3081T, TT was identified by a single 294 bp band, AA by 198 and 96 bp bands, and AT by 294, 198, and 96 bp bands. For G-1287A, GG was identified by 113, 79, 28, and 21 bp bands, AA by 113, 100, and 28 bp bands and GA by 113, 100, 79, 28, and 21 bp bands. The 28 and 21 bp fragments were undetectable because of their small size. 2.5. Statistical analysis SPSS version 18.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for performing the χ2 test, logistic regression analysis and independent t-tests. Hardy–Weinberg equilibrium was assessed using the χ2 test for goodness of fit. Comparisons of genotype and allele frequencies between the subjects and the controls were carried out for SNP using the χ2 test. Logistic regression analysis was used to determine whether ADHD was influenced by the SLC6A2 polymorphisms after demographic factors (age and gender) were taken into account as covariates. Linkage disequilibrium and haplotype analysis were performed using SNPanalyzer (Istech, Seoul, Republic of Korea). We also examined group differences in the K-ARS, the computerized CPT, and the KEDIWISC scores in relation to the possession of a specific allele of each SNP in the ADHD group using independent t-tests. The level of statistical significance was set at P-value b 0.05, and Bonferroni-corrected P-value was used when deciding statistical significance in multiple comparison. 3. Results 3.1. Demographic data The present study included 103 children with ADHD (8.8 ± 2.4 years; range = 5–14 years) and 173 normal children (9.5 ± 1.4 years; range = 7–12 years). The mean ages of the two groups were significantly different (t = 2.729, df = 138.70, P = 0.007). The gender distribution of the two groups was also significantly different (χ2 = 27.78, P b 0.001). The male to female ratio was 3.90 (82/21) in the ADHD group and 0.90 (82/91) in the control group. 3.2. Genotype and allele frequencies of the SLC6A2 polymorphisms in ADHD patients and normal controls The distribution of the SLC6A2 T-182C, A-3081T, and G-1287A polymorphisms in the ADHD group and control group were in Hardy– Weinberg equilibrium. The χ2 test and logistic regression analysis revealed no significant differences in the genotype distribution or allele frequencies of each SNP between the two groups (Table 1). Because of

gender ratio discrepancy between ADHD patients and controls, we performed second analysis for each gender group but it did not show any statistical significance. 3.3. Linkage disequilibrium test and haplotype analysis In the linkage disequilibrium (LD) test, LD was significant between T-182C and A-3081T (D′ = 0.59, r2 = 0.179, LOD = 60.63, P b 0.05) and A-3081T and G-1287A (D′ = 0.25, r2 = 0.038, LOD = 11.12, P = 0.00001), but not between T-182C and G-1287A (D′ = 0.09, r2 = 0.007, LOD = 1.92, P = 0.059). When the boys and girls were analyzed separately, in the male population, LD was moderately significant between T-182C and A-3081T (D′ = 0.63, r2 = 0.226, LOD = 47.42, P b 0.05) and between A-3081T and G-1287A (D′ = 0.33, r2 = 0.062, LOD = 10.78, P = 0.00005) and slightly significant between T-182C and G-1287A (D′ = 0.16, r2 = 0.025, LOD = 4.37, P = 0.004). In the female population, LD was moderately significant between T-182C and A-3081T (D′ = 0.50, r2 = 0.117, LOD = 15.25, P ≤ 0.05) and not significant between A-3081T and G-1287A (D′ = 0.15, r2 = 0.013, LOD = 1.63, P = 0.083) or between T-182C and G-1287A (D′ = 0.04, r2 = 0.0004, LOD = 0.06, P = 0.755). The haplotype analysis revealed a significant association between the haplotype T-A-G and risk of ADHD (χ2 = 6.451, P = 0.011, OR = 1.573, 95% CI = 1.108–2.234) (Table 2). This association was stronger in female ADHD (χ2 = 9.394, P = 0.002, OR = 2.851, 95% CI = 1.435–5.661) but it is difficult to make a meaningful interpretation with this finding because of small sample size of the female group (21 patients, 91 controls). 3.4. K-ARS, computerized CPT, and KEDI-WISC results in SNP T-182C, A-3081T and G-1287A polymorphisms We examined the effect of a specific allele of each SNP on K-ARS, computerized CPT, and KEDI-WISC scores in the ADHD group. For each SNP, patients were classified into two groups: those possessing no copies of the allele of interest and those possessing one or two copies of the allele of interest. The results of independent t-tests indicated that there was no statistically significant effect. When the boys and girls were analyzed separately, no statistically significant differences was detected after multiple test correction except lower omission error in nonA girl carriers (GG type) of G-1287A (carrier 76.75 ± 18.74, non-carrier 55.00 ± 9.26, t = 3.026, P = 0.007, Bonferroni-corrected P = 0.042) (Tables 3, 4 & 5). 4. Discussion The question can be raised for rationale for testing SLC6A2. The pathogenesis of ADHD is not fully known yet, but decreased function in prefrontal cortex (PFC) is the most popular hypothesis to explain the core features of ADHD. Drugs prescribed for ADHD work on the catecholamine system through monoamine transporters and increase dopamine activity in brain. Methylphenidate blocks both dopamine transporter (DAT) and NET, and atomoxetine is a potent and selective presynaptic noradrenaline reuptake inhibitor, and it is 300-fold selective for NET over DAT. The PFC has few DATs and abundant NETs and dopamine affinity for NET is much higher than that of DAT (Easton et al., 2007), therefore dopamine in PFC is inactivated by NET. For this reason, NET plays an important role in ADHD therefore NET polymorphism can affect its clinical manifestation. We investigated the association of three SNPs of the norepinephrine transporter (NET) gene SLC6A2, T-182C (rs2242446), A-3081T (rs28386840), and G-1287A (rs5569) with the prevalence of attention-deficit/hyperactivity disorder (ADHD), its clinical severity, and other disease-related characteristics in a Korean population. We found no significant differences in the genotype distribution or allele frequencies of each SNP between the ADHD group and the control. In the haplotype analysis, the most common T-A-G haplotype was related to an

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Table 1 Genotype distributions and allele frequencies of SLC6A2 in the ADHD and control groups. T-182C

ADHD

Genotype

TT TC CC T C

Allele

A-3081T

AA AT TT A T

Allele

G-1287A

N

%

χ2

df

P

OR

95% CI

P

53 40 10 146 60

51.5% 38.8% 9.7% 70.9% 29.1%

86 70 17 242 104

49.7% 40.5% 9.8% 69.9% 30.1%

0.083

2

0.959

0.054

1

0.817

1.128 0.992 1 1.093 1

0.459–2.774 0.394–2.496 Reference 0.727–1.644 Reference

0.793 0.987 NA 0.668 NA

GG GA AA G A

Allele

χ2 test

Control

Logistic regression

N

%

N

%

χ2

df

P

OR

95% CI

P

36 50 17 122 84

35.0% 48.5% 16.5% 59.2% 40.8%

51 80 42 182 164

29.5% 46.2% 24.3% 52.6% 47.4%

2.520

2

0.285

2.289

1

0.130

1.538 1375 1 1.232 1

0.677–2.792 0.719–3.291 Reference 0.845–1.794 Reference

0.267 0.378 NA 0.278 NA

N

%

N

%

χ2

df

P

OR

95% CI

P

52 41 10 145 61

50.5% 39.8% 9.7% 70.4% 29.6%

78 73 22 229 117

45.1% 42.2% 12.7% 66.2% 33.8%

0.993

2

0.609

1.044

1

0.307

1.158 1.007 1 1.104 1

0.479–2.795 0.412–2.459 Reference 0.739–1.649 Reference

0.745 0.989 NA 0.628 NA

ADHD

Genotype

Logistic regression

%

ADHD

Genotype

χ2 test

Control

N

χ2 test

Control

Logistic regression

P b 0.05. The power (1-β) of the χ2 test is b0.1 for T-182C, 0.39 (genotype) and 0.48 (allele) for A-3081T, and 0.18 (genotype) and 0.25 (allele) for G-1287A. ADHD = Attention deficit-hyperactivity disorder.

increased risk of ADHD (P = 0.011). This association was stronger in females (P = 0.002) but it is difficult to make a meaningful interpretation with this finding because of small sample size of the female group (21 patients, 91 controls). The male to female ratio was 3.90 (82/21) in the ADHD group and 0.90 (82/91) in the control group. Small sample

size and demographic differences between patients and controls made a major limitation in the study. There were only 21 girls in the ADHD group, and this is a main weak point of this study. A clinic-based sample of Korean patients showed a male predominance of 6–9:1 (Cho and Shin, 1994; Kim et al., 1999). We attempted to overcome this disparity,

Table 2 Haplotype frequencies and cross-tabulation of T-182C, A-3081T and G-1287A in the ADHD and control groups. T-182C

A-3081T

T T

G-1287A

A T T

C T

G G G

A C C C

Male T

A A

G A

A C

T T T

T C T T

A A A

T T

T

G G G A A A

A T C C

Female T T T

A A

G

A A

G

T C C C

A

A T T T T

C

A A

Frequency

G G A A G A

Overall

ADHD

Control

χ2

p

OR

95% CI

0.362 0.131 0.128 0.120 0.101 0.088 0.055 0.010

0.450 0.092 0.129 0.091 0.111 0.075 0.032 0.019

0.311 0.126 0.156 0.096 0.136 0.096 0.068 0.010

6.451 3.234 0.0 1.053 0.067 0.828 0.718 0.02

0.011 0.072 0.996 0.305 0.795 0.363 0.397 0.886

1.573 0.594 0.999 0.725 1.069 0.745 0.695 0.839

1.108–2.234 0.336–1.053 0.571–1.745 0.392–1.343 0.648–1.763 0.394–1.407 0.299–1.618 0.076–9.311

0.395 0.126 0.124 0.116 0.104 0.072 0.053 0.009

0.422 0.152 0.117 0.110 0.095 0.054 0.028 0.022

0.369 0.101 0.132 0.122 0.111 0.090 0.075 0.000

0.449 1.123 0.03 0.025 0.04 1.618 1.051 1.003

0.503 0.290 0.861 0.875 0.841 0.203 0.305 0.317

1.161 1.459 0.941 0.952 0.922 0.577 0.585 0.0

0.75–1.8 0.724–2.941 0.475–1.865 0.513–1.765 0.419–2.028 0.245–1.358 0.207–1.648 0.0–0.0

0.317 0.143 0.140 0.132 0.114 0.078 0.058 0.017

0.563 0.073 0.000 0.025 0.150 0.134 0.054 0.000

0.263 0.154 0.173 0.148 0.106 0.072 0.064 0.018

9.394 0.842 5.347 5.692 0.0 1.824 0.002 0.466

0.002* 0.359 0.021 0.017 0.999 0.177 0.961 0.495

2.851 0.559 0.0 0.124 1.0 2.0 0.961 0.0

1.435–5.661 0.159–1.965 0.0–0.0 0.016–0.933 0.383–2.609 0.72–5.557 0.2–4.621 0.0–0.0

Additive model, *P b 0.006 (Bonferroni correction). The power (1-β) of the χ2 test is 0.99, 0.31, 0.19, 0.06, 0.18, 0.18, 0.54 and 0.25 in orders; 0.29, 0.58, 0.09, 0.08, 0.10, 0.36, 0.63 and 1.00 in orders for males; 0.99, 0.31, 0.84, 0.61, 0.15, 0.34, 0.06 and 0.13 in orders for females. ADHD = Attention deficit-hyperactivity disorder.

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Table 3 Comparison of clinical variables between specific allele carriers and non-carriers in the ADHD group. Clinical variables

T-182C

A-3081T

t K-ARS

Inattention

Hyperactivity-Impulsivity

Total

CPT

Omission error

Commission error

Response time

Response variability

KEDI-WISC

Verbal IQ

Performance IQ

Total IQ

T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C

P 14.03 ± 5.96 13.40 ± 5.66 13.75 ± 5.85 14.17 ± 6.00 12.66 ± 5.40 11.40 ± 5.68 12.83 ± 5.57 12.25 ± 5.29 26.87 ± 9.51 24.80 ± 11.01 26.92 ± 10.06 26.42 ± 9.31 71.06 ± 22.26 71.70 ± 24.99 74.02 ± 26.57 68.50 ± 17.71 75.74 ± 22.76 66.90 ± 25.29 75.55 ± 22.96 74.21 ± 23.32 51.13 ± 16.90 58.90 ± 13.35 53.15 ± 21.07 50.81 ± 11.49 79.71 ± 24.93 78.90 ± 21.72 80.30 ± 27.58 79.02 ± 21.64 103.02 ± 14.40 101.80 ± 10.44 104.04 ± 12.06 101.85 ± 15.64 101.21 ± 14.20 98.80 ± 13.47 101.46 ± 14.88 100.52 ± 13.42 102.50 ± 13.26 100.30 ± 10.20 103.15 ± 12.22 101.48 ± 13.68

G-1287A

t

0.320

0.749

−0.357

0.722

0.695

0.489

0.537

0.592

0.642

0.522

0.255

0.799

−0.086

0.932

1.203

0.232

1.152

0.252

0.288

0.774

−1.402

0.164

0.676

0.501

0.098

0.922

0.258

0.797

0.260

0.795

0.781

0.437

0.512

0.610

0.332

0.741

0.507

0.613

0.640

0.524

P

A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T

14.07 ± 5.93 13.47 ± 5.92 13.37 ± 5.35 15.18 ± 6.82 12.41 ± 5.50 13.12 ± 5.04 12.15 ± 5.35 13.30 ± 5.53 26.67 ± 9.60 26.59 ± 10.05 25.76 ± 9.43 28.48 ± 9.91 70.26 ± 22.03 75.29 ± 24.34 72.26 ± 23.98 68.85 ± 19.04 74.02 ± 23.62 78.82 ± 20.22 75.80 ± 22.40 72.94 ± 24.52 51.76 ± 16.26 52.71 ± 19.15 52.36 ± 18.97 51.03 ± 10.97 79.83 ± 24.83 78.65 ± 23.70 79.29 ± 25.78 80.30 ± 22.17 102.63 ± 14.31 104.24 ± 12.79 103.01 ± 13.21 102.67 ± 15.73 101.35 ± 14.19 99.12 ± 13.79 101.55 ± 14.33 99.79 ± 13.69 102.35 ± 13.10 101.94 ± 12.65 102.57 ± 12.36 101.70 ± 14.29

t

0.381

0.704

−1.449

0.151

−0.490

0.625

−1.003

0.318

0.034

0.973

−1.335

0.185

−0.842

0.402

0.712

0.478

−0.780

0.437

−0.581

0.563

−0.213

0.832

0.442

0.659

0.180

0.858

−0.193

0.847

−0.429

0.669

0.116

0.908

0.594

0.554

0.587

0.558

0.118

0.907

0.314

0.754

G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A

P 13.64 ± 5.83 16.90 ± 6.03 14.24 ± 5.06 13.70 ± 6.68 12.26 ± 5.45 15.00 ± 4.50 12.72 ± 5.43 12.34 ± 5.44 26.08 ± 9.62 31.90 ± 8.45 26.96 ± 9.21 26.36 ± 10.11 70.18 ± 22.90 79.50 ± 15.93 74.59 ± 20.54 67.72 ± 23.82 73.72 ± 23.63 84.90 ± 14.03 77.24 ± 24.78 72.50 ± 21.19 51.78 ± 16.55 53.20 ± 18.73 51.96 ± 19.63 51.88 ± 13.40 78.44 ± 24.75 90.20 ± 20.48 81.35 ± 24.01 77.94 ± 25.14 103.63 ± 14.02 96.30 ± 12.79 100.74 ± 14.72 105.06 ± 13.06 101.67 ± 13.65 94.70 ± 17.00 100.24 ± 15.49 101.70 ± 12.63 103.10 ± 12.80 94.90 ± 12.65 100.62 ± 13.71 103.94 ± 12.07

−1.670

0.098

0.456

0.650

−1.533

0.129

0.350

0.727

−1.836

0.069

0.310

0.757

−1.251

0.214

1.536

0.128

−1.463

0.147

1.025

0.308

−0.255

0.799

0.023

0.981

−1.446

0.151

0.689

0.492

1.582

0.117

−1.552

0.124

1.494

0.138

−0.517

0.607

1.924

0.057

−1.285

0.202

Independent t-test. Significance level of P b 0.05. ADHD = Attention deficit-hyperactivity disorder; K-ARS = Korean ADHD Rating Scale; CPT = Continuous Performance Test; KEDI-WISC = Korean Educational Development Institute-Wechsler Intelligence Scale for Children.

and our sample had 3.9:1 male predominance, close to the male to female ratio found in a community sample. Nevertheless, the small sample size is a crucial weak point. Because of this gender ratio discrepancy between ADHD patients and controls, we performed second analysis for each gender group but it did not show any statistical significance except lower omission error in non-A girl carriers (GG type) of G-1287A (carrier 76.75 ± 18.74, non-carrier 55.00 ± 9.26, t = 3.026, P = 0.007, Bonferroni-corrected P = 0.042). The present study has several other limitations: First, we used the cut-off score 19 when using K-ARS for screening ADHD cases for the control group and some false-negative cases might be included in the control group. Second, we were unable to detect individuals with a past history of ADHD and other clinical properties that may be affected by SLC6A2 such as comorbidities or character traits among the controls, thereby increasing the heterogeneity of the group. Third, KEDI-WISC and CPT were performed only in patient group, therefore it made a limitation for assessing differences in neurocognitive functions according to the genotype. Due to these limitation, especially small sample size, it is difficult to make a significant conclusion in this study. However, there were some findings that should be investigated in a future study. Even though we cannot find any statistically meaningful result after multiple test corrections, some values showed a trend approaching the significance level

when analyzed separately by gender. In assessing the association between genotype or allele frequency and ADHD, we found this trend in A-3081T in female group. Compared with the T allele, the A allele had an odds ratio of 2.223 (95% CI: 1.081–4.570, P = 0.030). Two reports on A-3081T in a Korean ADHD population have been published; Cho et al. (2008) reported no association, and Joung et al. (2010) reported an association between A-3081T with ADHD in boys, but they indicated the opposite allele to ours as a risk factor. This discrepancy should be clarified by future studies. One possible explanation for the discrepancy is that hereditability could differ between genders, and these differences between genders could have an opposing effect in a study including both boys and girls. We also found that female non-T carriers (AA type) of A-3081T with ADHD showed lower omission error scores on the computerized CPT than T carriers (P = 0.028), which is in agreement with a previous study (Cho et al., 2008). This finding means that non-T carriers have fewer attention problems despite the fact that their genetic risk may be higher. There are several possible explanations for this. 1) In girls with ADHD, the AA genotype of A-3081T may have an influence on hyperactivity-impulsivity rather than inattention. 2) In girls with ADHD, the attention problem may be caused by factors other than A-3081T. 3) The female sample in the present study may suffer from selection bias in that girls with more prominent and easily observed symptoms (i.e., hyperactivity-impulsivity vs.

S.-Y. Oh, Y.-K. Kim / Progress in Neuro-Psychopharmacology & Biological Psychiatry 73 (2017) 56–63

61

Table 4 Comparison of clinical variables between specific allele carriers and non-carriers in the ADHD group, in male. Clinical variables

T-182C

A-3081 T

t K-ARS

Inattention

Hyperactivity-Impulsivity

Total

CPT

Omission error

Commission error

Response time

Response variability

KEDI-WISC

Verbal IQ

Performance IQ

Total IQ

T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C

P 13.93 ± 6.14 13.25 ± 6.21 13.51 ± 5.84 14.24 ± 6.44 13.07 ± 5.46 12.38 ± 5.71 12.93 ± 5.43 13.08 ± 5.54 27.23 ± 9.64 25.63 ± 11.83 26.83 ± 9.88 27.32 ± 9.85 71.75 ± 22.91 73.25 ± 28.06 75.02 ± 27.67 68.53 ± 17.08 78.70 ± 22.75 70.75 ± 27.10 77.32 ± 23.32 78.53 ± 23.29 52.03 ± 17.05 57.63 ± 14.59 53.46 ± 20.11 51.66 ± 12.56 82.39 ± 24.71 76.75 ± 23.83 80.17 ± 28.71 83.61 ± 19.26 103.41 ± 14.43 103.50 ± 10.65 104.73 ± 12.58 102.00 ± 15.50 101.07 ± 13.97 96.50 ± 14.09 102.02 ± 14.05 99.08 ± 13.88 102.72 ± 13.44 100.00 ± 11.16 103.85 ± 12.12 100.92 ± 14.26

t

0.297

0.768

−0.524

0.602

0.340

0.735

−0.123

0.902

0.435

0.664

−0.219

0.827

−0.172

0.864

1.244

0.217

0.920

0.360

−0.230

0.818

−0.891

0.376

0.474

0.637

0.615

0.541

−0.619

0.538

−0.017

0.986

0.863

0.391

0.877

0.383

0.936

0.352

0.550

0.584

0.987

0.327

G-1287A P

A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T

14.10 ± 6.24 12.94 ± 5.67 13.09 ± 5.32 15.63 ± 7.44 12.94 ± 5.56 13.25 ± 5.17 12.40 ± 5.25 14.38 ± 5.76 27.29 ± 9.76 26.19 ± 10.24 25.78 ± 9.25 30.00 ± 10.59 71.40 ± 23.12 73.88 ± 24.52 71.47 ± 24.82 72.88 ± 19.72 77.60 ± 23.86 79.06 ± 20.86 78.00 ± 23.08 77.67 ± 23.85 53.13 ± 16.77 50.50 ± 17.40 53.02 ± 18.64 51.63 ± 11.96 83.06 ± 24.84 76.94 ± 23.37 80.73 ± 26.79 84.33 ± 18.62 103.38 ± 14.41 103.56 ± 12.89 104.00 ± 13.07 102.08 ± 16.27 101.25 ± 14.10 98.06 ± 13.52 101.76 ± 14.14 97.96 ± 13.45 102.79 ± 13.43 101.06 ± 12.51 103.27 ± 12.51 100.54 ± 14.73

t

0.675

0.502

−1.716

0.090

−0.204

0.839

−1.493

0.139

0.398

0.692

−1.783

0.078

−0.378

0.706

−0.245

0.807

−0.224

0.824

0.058

0.954

0.555

0.580

0.337

0.737

0.891

0.376

−0.598

0.551

−0.046

0.964

0.556

0.580

0.815

0.418

1.116

0.268

0.467

0.642

0.845

0.401

G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A

P 13.48 ± 6.06 17.25 ± 5.83 13.76 ± 4.92 13.95 ± 7.05 12.62 ± 5.49 16.38 ± 3.82 12.97 ± 5.43 13.02 ± 5.53 26.32 ± 9.80 33.63 ± 7.52 26.73 ± 9.03 27.36 ± 10.54 71.06 ± 23.94 79.38 ± 15.29 73.89 ± 21.29 70.14 ± 25.02 77.08 ± 23.83 85.13 ± 15.15 79.78 ± 25.78 76.24 ± 20.76 52.82 ± 17.05 50.63 ± 15.54 53.24 ± 19.73 52.02 ± 13.99 80.94 ± 24.78 89.63 ± 22.03 83.35 ± 23.53 80.48 ± 25.58 104.52 ± 13.87 93.63 ± 12.29 101.81 ± 15.27 104.83 ± 12.88 101.42 ± 13.48 93.38 ± 16.99 100.05 ± 15.47 101.10 ± 12.645 103.54 ± 12.98 92.75 ± 11.61 101.22 ± 14.633 103.52 ± 11.84

−1.674

0.098

−0.141

0.888

−1.878

0.064

−0.041

0.967

−2.037

0.045

−0.282

0.779

−0.958

0.341

0.712

0.479

−0.930

0.355

0.677

0.501

0.347

0.729

0.320

0.750

−0.948

0.346

0.517

0.606

2.128

0.037

−0.954

0.343

1.560

0.123

−0.329

0.743

2.249

0.027

−0.774

0.441

Independent t-test. Significance level of P b 0.05. ADHD = Attention deficit-hyperactivity disorder; K-ARS = Korean ADHD Rating Scale; CPT = Continuous Performance Test; KEDI-WISC = Korean Educational Development Institute-Wechsler Intelligence Scale for Children.

inattention) have a relatively higher likelihood of being seen in a psychiatric setting, meaning that the girls in this sample may have hyperactivity-impulsivity as their primary symptoms rather than inattention. In addition, findings for G-1287A appear that the G allele might have some protective effect against the development of ADHD symptoms. Male non-G carriers (AA type) of G-1287A with ADHD had a higher KARS-T score (carrier 26.32 ± 9.80, non-carrier 33.63 ± 7.52, t = −2.037, P = 0.045) and lower verbal IQ and total IQ scores (verbal IQ: carrier 104.52 ± 13.87, non-carrier 93.63 ± 12.29, t = 2.128, P = 0.037; total IQ: carrier 103.54 ± 12.98, non-carrier 92.75 ± 11.61, t = 2.249, P = 0.027) than G carriers. In addition, female non-A carriers (GG type) of G-1287A showed lower omission error (carrier 76.75 ± 18.74, non-carrier 55.00 ± 9.26, t = 3.026, P = 0.007) and commission error (carrier 69.42 ± 20.41, non-carrier 52.88 ± 9.76, t = 2.122, P = 0.048) than A carriers. This finding is consistent with the CPT data reported by Song et al. (2011) that indicated those with the GG genotype of G-1287A showed lower commission error scores compared with A carriers. A few studies have suggested a gender-specific association in ADHD. Biederman et al. reported that the genetic association of SLC6A2 with ADHD may be stronger in females (Biederman et al., 2008), and Cho et al. found that the BDNF rs11030101 polymorphism was significantly

associated with ADHD in Korean girls (Cho et al., 2010). Gender differences in the prevalence, course, and clinical features of ADHD are well-known. These differences may be the outcome of hormonal influences on gene expression and regulation or may be due to other non-genetic factors related to gender. The mechanism by which sex hormones act in gene expression and regulation in ADHD is unclear, and this should be a focus of future studies. In conclusion, we found no statistically significant association between each SNP in SLC6A2 and ADHD but the most common T-A-G haplotype was related to an increased risk of ADHD in female. In female, GG genotype of G-1287A showed lower omission error than A carriers. Some values related A-3081 and G-1287A showed a trend approaching the significance level when analyzed separately by gender. Even though it was not statistically meaningful after multiple test correction, G allele might have some protective effect against development of ADHD symptoms and this finding was consistent with previous studies.

Acknowledgement This study received a research scholarship from Prof. Lee Byoung Yoon fund. The article is based on the doctoral dissertation of Dr. S-Y Oh.

62

S.-Y. Oh, Y.-K. Kim / Progress in Neuro-Psychopharmacology & Biological Psychiatry 73 (2017) 56–63

Table 5 Comparison of clinical variables between specific allele carriers and non-carriers in the ADHD group, in female. Clinical variables

T-182C

A-3081 T

t K-ARS

Inattention

Hyperactivity-Impulsivity

Total

CPT

Omission error

Commission error

Response time

Response variability

KEDI-WISC

Verbal IQ

Performance IQ

Total IQ

T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C T Non-T C Non-C

P 14.42 ± 5.35 14.00 ± 4.24 15.14 ± 6.15 14.00 ± 4.80 11.11 ± 4.99 7.50 ± 4.95 12.29 ± 6.78 10.00 ± 3.88 25.53 ± 9.15 21.50 ± 9.19 27.43 ± 11.87 24.00 ± 7.44 68.33 ± 19.86 65.50 ± 3.54 67.17 ± 17.51 68.43 ± 19.99 64.06 ± 19.18 51.50 ± 4.95 63.50 ± 17.38 62.50 ± 19.71 47.61 ± 16.27 64.00 ± 7.07 51.00 ± 29.03 48.50 ± 7.82 69.11 ± 23.53 87.50 ± 9.19 81.17 ± 20.11 66.57 ± 23.52 101.58 ± 14.56 95.00 ± 8.48 100.00 ± 7.90 101.43 ± 16.60 101.74 ± 15.41 108.00 ± 5.66 98.14 ± 20.08 104.43 ± 11.67 101.68 ± 12.89 101.50 ± 7.78 99.00 ± 12.91 103.00 ± 12.30

t

0.107

0.916

0.469

0.645

0.973

0.343

0.991

0.334

0.592

0.561

0.816

0.425

0.197

0.846

−0.134

0.895

0.902

0.379

0.107

0.916

−1.383

0.184

0.208

0.843

−1.074

0.297

1.322

0.203

0.619

0.543

−0.267

0.792

−0.560

0.582

−0.914

0.372

0.020

0.985

-0.690

0.498

G-1287A P

A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T A Non-A T Non-T

14.00 ± 4.984 22.00 14.67 ± 5.52 14.00 ± 4.95 10.75 ± 5.11 11.00 11.00 ± 5.89 10.44 ± 3.75 24.75 ± 9.05 33.00 25.67 ± 10.66 24.44 ± 6.73 66.47 ± 17.93 98.00 76.18 ± 19.73 58.11 ± 12.37 62.16 ± 18.87 75.00 64.82 ± 15.10 60.33 ± 22.87 47.21 ± 13.86 88.00 49.09 ± 21.23 49.44 ± 8.14 69.11 ± 22.13 106.00 72.09 ± 19.38 69.56 ± 28.11 105.25 ± 14.06 115.00 98.50 ± 13.47 104.22 ± 15.00 101.65 ± 14.83 116.00 100.58 ± 15.79 104.67 ± 13.90 100.95 ± 12.22 116.00 99.33 ± 11.57 104.78 ± 13.35

t

−1.566

0.134

0.286

0.778

−0.048

0.962

0.247

0.808

−0.890

0.385

0.301

0.767

−1.713

0.104

2.385

0.028

−0.663

0.515

0.527

0.605

−2.870

0.010

−0.047

0.963

−1.625

0.122

0.238

0.814

−1.024

0.319

−0.918

0.370

−0.944

0.357

−0.616

0.545

−1.202

0.244

−1.000

0.330

G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A G Non-G A Non-A

P 14.26 ± 4.98 15.50 ± 9.19 15.62 ± 5.39 12.38 ± 4.34 10.89 ± 5.21 9.50 ± 2.12 12.00 ± 5.55 8.75 ± 3.24 25.16 ± 9.11 25.00 ± 11.31 27.62 ± 10.05 21.13 ± 5.33 66.72 ± 18.42 80.00 ± 25.46 76.75 ± 18.74 55.00 ± 9.26 60.44 ± 17.83 84.00 ± 12.73 69.42 ± 20.41 52.88 ± 9.76 47.67 ± 14.11 63.50 ± 34.65 48.00 ± 19.61 51.13 ± 10.41 68.56 ± 22.64 92.50 ± 19.09 75.17 ± 25.47 64.63 ± 18.67 100.32 ± 14.45 107.00 ± 11.31 97.69 ± 13.09 106.25 ± 14.87 102.58 ± 14.63 100.00 ± 22.63 100.77 ± 16.15 104.88 ± 12.89 101.47 ± 12.32 103.50 ± 17.68 98.92 ± 11.01 106.13 ± 13.83

−0.315

0.756

1.433

0.168

0.369

0.717

1.497

0.151

0.023

0.982

1.675

0.110

−0.944

0.358

3.026

0.007

−1.797

0.089

2.122

0.048

−1.331

0.200

−0.411

0.686

−1.431

0.170

1.001

0.330

−0.629

0.537

−1.383

0.183

0.229

0.821

−0.608

0.550

−0.215

0.832

−1.322

0.202

Independent t-test. Significance level of P b 0.05. ADHD = Attention deficit-hyperactivity disorder; K-ARS = Korean ADHD Rating Scale; CPT = Continuous Performance Test; KEDI-WISC = Korean Educational Development Institute-Wechsler Intelligence Scale for Children.

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