GENE-40439; No. of pages: 6; 4C: Gene xxx (2015) xxx–xxx
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Research paper
Association of LPHN3 rs6551665 A/G polymorphism with attention deficit and hyperactivity disorder in Korean children In Wook Hwang a,b, Myung Ho Lim b,c, Ho Jang Kwon b,d, Han Jun Jin a,b,⁎ a
Department of Nanobiomedical Science, College of Natural Science, Dankook University, Cheonan, South Korea Environmental Health Center, Dankook Medical Hospital, Cheonan, South Korea Department of Psychology, College of Public Welfare, Dankook University, Cheonan, South Korea d Department of Preventive Medicine, College of Medicine, Dankook University, Cheonan, South Korea b c
a r t i c l e
i n f o
Article history: Received 11 October 2014 Received in revised form 7 April 2015 Accepted 10 April 2015 Available online xxxx Keywords: ADHD LPHN3 rs6551665 Polymorphism Korean Children Genetic association
a b s t r a c t Attention deficit hyperactivity disorder (ADHD) is a common and highly heritable disorder of school-age children. Its heritability was estimated at 80–90% but the genetic component underpinning this disorder remains to be disclosed. Recently, a highly consistent association between latrophilin3 (LPHN3) gene and ADHD was reported. In the present study, we examined the association between the LPHN3 rs6551665 A/G polymorphism and ADHD in Korea. The samples used in the study consisted of 150 ADHD children and 322 controls. The ADHD children were diagnosed according to DSM-IV. ADHD symptoms were evaluated with Dupaul Parent ADHD Rating Scales. LPHN3 rs6551665 SNP was determined by PCR-RFLP. Hardy–Weinberg equilibrium, genotype and allele frequency differences between the case and the control, and odds ratio were examined using the chi-square and exact tests. The LPHN3 gene locus was found to have no deviation from the Hardy– Weinberg expectation. We observed a significant association between the ADHD children and control group in genotype frequency (p = 0.01) and allele frequency (p = 0.02). The ADHD children appeared to have a surplus of GG genotype (OR 2.959, 95% CI 1.416–6.184, p = 0.003) and G allele (OR 1.44, 95% CI 1.062–1.945, p = 0.02). The association was more distinctive when analysis was confined to male samples (p = 0.005), the OR of male controls and cases was 4.029 (95% CI 1.597–10.164, p = 0.002) and the OR having G allele vs. A allele was 1.46 (95% CI 1.002–2.127, p = 0.048). Thus our results imply that the LPHN3 rs6551665 GG genotype and G allele may provide a significant effect on the ADHD, although larger sample sizes and functional studies are necessary to further elucidate these findings. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Attention deficit hyperactivity disorder (ADHD) is a common childhood neuropsychiatric disorder occurring at 2–7.6% among children of school age in Korea (Kwon et al., 2014). ADHD is a heterogeneous disorder and often has effect detectable symptoms in adulthood. The disorder is characterized by behavioral problems such as attention deficit, hyperactivity, and impulsivity (American Psychiatric Association Committee on Nomenclature and Statistics, 1994). Family, adoption, and twin studies have shown that there is a strong genetic relationship of ADHD. The heritability estimates of ADHD are ranging from 80 to 90% (Faraone and Doyle, 2001; Shastry, 2004), but so far, its genetic Abbreviations: ADHD, attention deficit hyperactivity disorder; DSM-IV, Diagnostic and Statistical Manual of Mental Disorders-4th edition; SNP, single nucleotide polymorphism; PCR, polymerase chain reaction; PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism; LPHN3, latrophilin3; HWE, Hardy–Weinberg equilibrium; OR, odds ratio; CI, confidence interval. ⁎ Corresponding author at: Department of Nanobiomedical Science, Dankook University, Cheonan 330-714, South Korea. E-mail address: [email protected] (H.J. Jin).
contributions remain elusive beyond reasonable doubt from linkage and candidate gene approach (Smalley et al., 2002; Ogdie et al., 2003; Faraone, 2004) as well as genome-wide association studies (Franke et al., 2009; Banaschewski et al., 2010; Mick et al., 2010; Neale et al., 2010a,b). To elucidate the role of genetic influences in ADHD, various approaches have been used to address important issues in the occurrence of ADHD, such as twin studies, candidate gene studies, genome-wide approaches and copy number variation (CNV) studies (Schachar, 2014). Candidate gene studies have constantly showed evidence for association with DRD4, DRD5 and DAT1 of dopaminergic, SLC6A2 and 5-HTT/SLC6A4 and HTR1B serotonin receptor gene of serotonergic, NET1/SLC6A2, ADRA2A and ADRA2C of noradrenergic and CHRNA4 of nicotinergic neurotransmission and receptor function, lastly SNAP25, CHRNA4, NMDA, BDNF, NGF, NTF3, NTF4/5 and GDNF genes that are involved in neurotransmission and neuronal plasticity (Gizer et al., 2009; Schachar, 2014). However, linkage and association studies of ADHD have not reached a rigorous level for genome-wide significance since the sample sizes were far too small (Zhou et al., 2008; Neale et al., 2010a). The Psychiatric Genomics Consortium (PGC; http://pgc.unc.edu) and the ADHD Genetic Consortium
http://dx.doi.org/10.1016/j.gene.2015.04.033 0378-1119/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: Hwang, I.W., et al., Association of LPHN3 rs6551665 A/G polymorphism with attention deficit and hyperactivity disorder in Korean children, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.04.033
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I.W. Hwang et al. / Gene xxx (2015) xxx–xxx
have raised a question about the small sample size in the genetic analyses of ADHD. To overcome the issue on the sample size, the PGC analyzed GWAS data from over 19 countries for 5 major psychiatric disorders including ADHD (ADHD, ASD, major depression, bipolar disorder and schizophrenia), by examining GWAS data in 33,332 cases and 27,888 controls of European ancestry. In this analysis four SNPs remained significant at the genome-wide significance level (p b 5 × 10−8): regions on chromosomes 3p21 and 10q24, and SNPs related with CACNA1c and CACNB2, the two L-type voltage-gated calcium channel subunits (Cross-Disorder Group of the Psychiatric Genomics Consortium et al., 2013). The drawback of the GWAS studies is that the study uses high levels for the statistical significance since they analyze a large number of statistical tests, this rigorous criteria for significance could miss the signal of association from genes that have moderate individual contribution to ADHD (Schachar, 2014). The candidate gene approaches, therefore, have its own virtue on the analyses of genetic contribution for ADHD. In a recent study, Arcos-Burgos et al. (2010) reported an association between polymorphisms within latrophilin 3 (LPHN3) and adult ADHD. It has been also reported that a common variant of the LPHN3 increases the risk of developing ADHD by 1.2 fold (Acosta et al., 2011). LPHN3 is a member of the LPHN subfamily that is related with G-protein coupled receptors (GPCRs) and the receptor has been reported to be important for the regulation in the exocytosis of neurotransmitters particularly for norepinephrine (Davletov et al., 1998; Rahman et al., 1999; Silva et al., 2009; Choudhry et al., 2012). In the functional studies, the LPHN3 is expressed in key brain regions where it closely related to attention and activity. Its variants likely affect metabolism in neural circuits involved in ADHD and are also involved in responses to stimulant medication (Arcos-Burgos et al., 2010; Ribases et al., 2010; Acosta et al., 2011). To the best of our knowledge, the genetic relationship between LPHN3 gene and ADHD in Korean population has not been examined. The present study investigated an association between LPHN3 rs6551665 A/G polymorphism and genetic contribution of developing ADHD in a sample of population based Korean ADHD children relative to the normal control children. 2. Materials and methods 2.1. Subject We analyzed a total of 472 samples from the Children's Health and Environmental Research (CHEER) cohort study and precious study (Kwon et al., 2014). Of these samples, 120 ADHD children and 322 control individuals were subset of CHEER cohort study. In addition, 30 ADHD children were a subset of the samples analyzed in Kwon et al. (2014). The CHEER study was carried out on elementary school children of 10 cities in Korea from 2005 to 2010 biennially. An interview was randomly performed to children with Korean version of the Dupaul Attention Deficit Hyperactivity Disorder Rating Scales (K-ARS) (Kim et al., 2002) including 18 items based on the DSM-IV diagnostic criteria for ADHD. In this study, the K-ARS score for the ADHD samples was 19 or higher, and the score for the control group was less than 19. For these 18 items, questions 1–9 are for inattention and questions 10–18 are for hyperactive/impulsive. According to these criteria, we classified the subtype of ADHD samples as follows; combined (ADHD/C), predominantly inattentive (ADHD/I) and predominantly hyperactive/impulsive (ADHD/HI) (Morgan et al., 1996; Dupaul et al., 1997; Gaub and Carlson, 1997a; Barkley and Murphy, 1998; Lahey et al., 1998). Each clinical interview was conducted by researchers with at least 1 parent. Informed consent was obtained from all the participants of this study. The sex and age for ADHD children and the control group were matched to avoid possible biases of the study. A clinical evaluation and the DSMIV diagnosis (American Psychiatric Association Committee on Nomenclature and Statistics, 1994) were performed by a child psychiatrist. The number of ADHD children is 150 [97 boys (64.7%) and 53 girls
(35.3%)], and the mean age was 8.05 ± 1.04. The number of samples in the control group was 322 [191 boys (59.3%) and 131 girls (40.7%)], and the mean age was 8.22 ± 1.48. There was no significant difference in the sex and age between the ADHD children and control group (Table 1). The study protocol was approved by the Ethics Committee of the Dankook University Hospital. 2.2. DNA extraction and genotyping DNA was extracted from leukocytes using the G-DEX™ llb Genomic DNA Extraction kit (Intron Biotechnology, Korea) or the GeneAll Exgene Clinic SV mini kit (GeneALL, Korea). A primer set was designed for the determination of the rs6551665 A/G polymorphism and the SNP was located within the intron region in the LPHN3 gene (Fig. 1). A BLAST search for the homologous sequence data was performed by using the BLASTN program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). A pair of PCR primer was designed by Primer3Plus based on blasted sequence (Untergasser et al., 2007). Genomic DNA was amplified using the new designed primers as follows: forward, 5′-ATCAGCATGCAGTAGCCCTC3′ and reverse 5′-GAGCAAGAGTTCTCAAACATGGT-3′. Each PCR reaction was performed in a total volume of 20 μl containing 10 ng of genomic DNA, 10 pM each primer, 0.2 mM dNTPs, 2.0 mM MgCl2, 10 × PCR buffer and 1.0 U NV DNA polymerase (NAVI BioTech, Korea). The cycling conditions used an initial denaturation step at 95 °C for 5 min, and then 35 cycles at 94 °C for 1 min, 58 °C for 1 min, 72 °C for 1 min, and a final extension at 72 °C for 10 min. The PCR product was digested with 1.0 U Hph I restriction enzyme (New England Biolabs, USA) for 3 h at 37 °C and electrophoresed in 3% agarose gel (Lonza, USA). The polymorphic Hph I site was detected by restriction fragment length polymorphism in producing fragments of 203 and 99 bp (A allele) or 139, 99 and 64 bp (G allele) (Fig. 2). 2.3. Data analyses Statistical analyses were performed using SPSS 21 Statistics (IBM Korea, Korea) to determine frequencies, cross tabulation analyses, and descriptive statistics. Chi-squared tests were used to assess Hardy– Weinberg equilibrium (HWE). In addition, a test of proportion and odds ratio (OR) with 95% confidence intervals (CI), and Fisher's exact test in a 2 × 2 table were calculated using the statistical analysis on the internet (SISA, http://www.quantitativeskills.com/sisa/). A p value of b0.05 was considered statistically significant. It has been reported that a sample size of 210 subjects is required to obtain a 95% or higher power in the chi-square calculation between the controls and ADHD case samples in the Korean population (Kwon et al., 2014). Our study was conducted in 472 subjects, therefore we expected that this study has enough power to assess an association of the LPHN3 rs6551665 polymorphism and ADHD in the Korean population. Table 1 Characteristics of ADHD children (n = 150) and control (n = 322) group. Characteristics Ageb Gender
Male Female
ADHD (n = 150)a
Control (n = 322)
F or χ2
p-value
8.05 ± 1.04 97 (64.7%) 53 (35.3%)
8.22 ± 1.48 191 (59.3%) 131 (40.7%)
46.61 1.23
0.13 0.27c
ADHD (n = 120)d Subtype
Combined Inattentive Hyperactive/impulsive
78 (65%) 32 (27%) 10 (8%)
a CHEER study with 120 ADHD samples and 30 ADHD samples analyzed in Kwon et al. (2014). b These data represent mean ± SD, by independent t-test. c The Chi-square p-value. d CHEER study with 120 ADHD samples.
Please cite this article as: Hwang, I.W., et al., Association of LPHN3 rs6551665 A/G polymorphism with attention deficit and hyperactivity disorder in Korean children, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.04.033
I.W. Hwang et al. / Gene xxx (2015) xxx–xxx
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Fig. 1. Genomic organization of the Chr 4:61,843,840-61,895,000 region surrounding the rs6551665. The map is based on genome build GRCh38.p2 http://www.ensembl.org/. (A) LPHN3 gene is located in 4q13.1 region. (B) The sequence variant rs6551665, located in the intron region of LPHN3 gene (Chr 4: 61,873,823), is not associated with a binding site for the transcription factors. The red square represents a transcription factor binding site. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
3. Results 3.1. Demographic characteristics of the subjects A total of 472 children were analyzed for the study. The children in both the ADHD group and the control group had never taken any psycho-stimulant in advance. There was no difference in age (F = 46.61, p = 0.13) and sex (F = 1.23, p = 0.27) between the control group and ADHD case individuals (Table 1). 3.2. Comparison of frequency of the genotypes and alleles with genetic polymorphism of LPHN3 rs6551665 A/G between the control and ADHD groups LPHN3 rs6551665 genotyping data from the control and ADHD groups are summarized in Table 2. Genotype distribution of all
control (A/A 55.9%, A/G 39.4% and G/G 4.7%) and ADHD groups (A/A 48.7%, A/G 39.3% and G/G 12%) were in agreement with the Hardy–Weinberg equilibrium (HWE) (Table 2). Significant genotype and allele frequency differences were observed between the controls and the cases (p = 0.01 and 0.02, respectively). We also found a significant association (p = 0.02), when the samples with hyperactivity were compared with control groups (10 cases vs 322 controls) (Supplementary Table S1). Table 3 shows the results for the association of LPHN3 rs6551665 polymorphism. The OR of ADHD children having the GG genotype compared with controls was 2.959 (95% CI 1.416–6.184, p = 0.003). The result also remains significant when the analysis was performed by allele frequencies. The OR having the G allele versus A allele was 1.44 (95% CI 1.062–1.945, p = 0.02) (Table 3). This association was more distinctive when the analysis was confined to male samples (p = 0.005) (Table 2). The OR of ADHD male samples and male controls was 4.029 (95% CI 1.597–10.164, p = 0.002) and the OR having G allele vs. A allele was 1.46 (95% CI 1.002–2.127, p = 0.048) (Supplementary Table S2). The associations between all subtypes of ADHD and control group were analyzed (Supplementary Table S1). 4. Discussion
Fig. 2. Detection of genotype and allelic variations at the rs6551665 in LPHN3 gene. PCR products were electrophoresed after digesting with Hph I. Lane M: 1 kb + DNA ladder; lanes 1, 2, 4, 5, 6, 8, 10, 11, and 13: AG alleles; lanes 3, 9, 12, and 14: AA alleles; lane 7: GG alleles.
It has been reported that multiple environmental risk factors significantly related to the development of ADHD. In addition, there is now enormous evidence for substantial genetic influences on the etiology and pathogenesis of ADHD (Pineda et al., 2007; Gizer et al., 2009; Waldman and Gizer, 2006; Arcos-Burgos and Muenke, 2010). Recently, common variants of the LPHN3 gene located on chromosome 4 were recognized as new candidate gene to analyze the genetic susceptibility for ADHD in several ethnic groups (Arcos-Burgos et al., 2010; Acosta et al., 2011; Fallgatter et al., 2013). LPHN3, unlikely from the latrophilin1 and latorphilin2 that serve as receptors for alpha-latrotoxin, a component of the black widow spider (Latrodectus mactans), is the most exclusively expressed in the brain (Sugita et al., 1998; Ichtchenko et al., 1998). LPHN3 gene showed significantly increased expression of
Please cite this article as: Hwang, I.W., et al., Association of LPHN3 rs6551665 A/G polymorphism with attention deficit and hyperactivity disorder in Korean children, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.04.033
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Table 2 Genotype and allele frequencies of LPHN3 rs6551665 in the ADHD children and control group. Group
Total ADHD Control Male ADHD Control Female ADHD Control
n
p⁎⁎
Genotype (%) A/A
A/G
G/G
150 322
73(48.7%) 180(55.9%)
59(39.3%) 127(39.4%)
18(12.0%) 15(4.7%)
97 191
47(48.4%) 101(52.9%)
35(36.1%) 82(42.9%)
15(15.5%) 8(4.2%)
53 131
29(54.7%) 79(60.3%)
21(39.6%) 45(34.4%)
3(5.7%) 7(5.3%)
Allele frequency (%)
p⁎⁎⁎
HWE⁎⁎⁎
Allele A
Allele G
0.01⁎
205(68.3%) 487(75.6%)
95(31.7%) 157(24.4%)
0.02⁎
0.26 0.21
0.005⁎
129(66.5%) 284(74.3%)
65(33.5%) 98(25.7%)
0.048⁎
0.06 0.08
0.54
79(74.5%) 203(77.5%)
27(25.5%) 59(22.5%)
0.54
0.75 0.86
HWE Hardy–Weinberg equilibrium. ⁎ p b 0.05. ⁎⁎ The Fisher's exact test p-value. ⁎⁎⁎ The Chi-square p-value.
mRNA in the region of human amygdala, caudate nucleus, cerebellum, and cerebral cortex, in contrast the lower and no expression was detected in corpus callosum, hippocampus, whole brain extract, occipital pole, frontal lobe, temporal lobe putamen, thalamus, medulla and spinal cord. It has been reported that the regions where LPHN3 gene are highly expressed are in key regions of brain for attention and activity (ArcosBurgos et al., 2010). Lange et al. (2012) validated the function of LPHN3 gene in the brain by examining LPHN3 orthologous lphn3.1 gene during zebrafish development. Loss of lphn3.1 function in zebrafish showed reduction and misplacement of dopamine positive neurons in the ventral diencephalon and a hyperactive/impulsive motor phenotype. Moreover the normal phenotype was restored by the methylphenidate and atomoxetine that are ADHD treatment drugs (Lange et al., 2012). Similar observation has been reported from a transgenic mice model experiment (Wallis et al., 2012; Kotepui et al., 2012). In the experiment, LPHN3 KO mice had profoundly disrupted multiple monoamine-signaling levels, which causes ADHD like symptom. Therefore, these reports give us a clue for the function of LPHN3 gene in brain and its etiology of developing ADHD. Another function of LPHN3 gene has been also reported that increased mRNA expression of LPHN3 gene in breast-cancer tissues is significantly correlated with axillary lymph node positivity, along with increased mRNA expression of MMP13 (Kotepui et al., 2012). This report is particularly intriguing since it has reported that the expression of LPHN3 is confined within brain (Sugita et al., 1998; Ichtchenko et al., 1998). Kotepui et al. (2012) reported that mRNA expression of LPHN3 and MMP13 is closely related with tumor aggressiveness since commonly the metastasis of breast carcinoma is spread through the axillary lymph nodes and may be modulated by the expression of LPHN3 and MMP13. Thus the role of LPHN3 in axillary-node metastasis in breast cancer, as well as in ADHD, is a subject for further analysis. This study showed the significant correlation between the LPHN3 rs6551665 A/G polymorphism and ADHD phenotype (p = 0.01).
Table 3 The odds ratio of LPHN3 rs6551665 genotypes and alleles between ADHD children and control group. Total
Genotype AAb AG GG Allele Ab/G
ADHD children versus control OR
(95% CI)
p-valuea
1.000 1.146 2.959
(0.759–1.729) (1.416–6.184)
– 0.52 0.003⁎
1.440
(1.062–1.945)
0.02⁎
OR odds ratio, CI confidence interval. ⁎ p b 0.05. a The Chi-square p-value. b Reference.
Choudhry et al. (2012) reported a highly significant interaction between four LPHN3 tag SNPs including rs6551665, rs1947274, rs6858066 and rs2345039 and maternal stress during pregnancy. Of these SNPs, rs6551665 was replicated in the present study, although the design of the studies was distinct. It has been reported that with an estimated prevalence of 4% to 12% in school-age children, ADHD seems like more common in boys than girls (American Academy of Pediatrics, 2000; Quinn and Wigal, 2004). The ratio of boy to girl ranged from 9:1 to 6:1 in clinical samples and in community-based population study samples are 3:1 (Gaub and Carlson, 1997b; Quinn and Wigal, 2004). Given the known gender-specific trait of ADHD, boy and girl samples were analyzed separately. The analysis of girl samples provided no evidence for an association between LPHN3 polymorphism and ADHD (p = 0.54). But a high frequency of GG genotype in the ADHD boys (15/97, 15.5%) compare to control group (8/191, 4.2%) was observed (p = 0.005). Despite previous reports about the gender differences on the occurrence of ADHD, the reasons for this are unclear. Further analyses with a large number of samples are necessary to have conclusive understanding for this gender specific relationship of ADHD. We also performed analyses according to their subtypes of ADHD, the samples were divided into inattentive, hyperactive/impulsive and combined type. Significant association was detected between the hyperactive/impulsive subtypes and control group (p = 0.02). However, the sample size of hyperactive/impulsive group was too small to get relevant decision, even though they were reached at the significant level (p b 0.05). The correlation of hyperactive/impulsive subtypes in ADHD deserves further analysis. Especially, the candidate gene approaches have a substantial risk of generating false positive results since most of gene associations were analyzed individually without considering the total number of plausible associations and the possibility of bias toward publishing positive results but not negative. Therefore, the positive associations detected from candidate studies were often not replicated in genome-wide association studies (Bosker et al., 2011). These results implicate that single association studies should be thoroughly replicated (Sullivan, 2007; Schachar, 2014). The present study has inherent deficiency that the number of the studied sample was relatively small. The samples of this study were 150 ADHD children and 322 individuals for the control group. The next limitation is that the only one SNP was investigated in this study, even though there are various SNPs on the genes related with the various ADHD phenotypes. Although it is clear that a lot of genetic factors cause the increased ADHD vulnerability, it was not considered the interaction with other risk factors. In contrast to the methodological limitation described previously, the study has advantages. First, the case and the control groups were matched for sex and age so that there were no significant differences. ADHD is much more prevalent in males and in the period of adolescence, therefore sex and age characteristics can give a great influence. Second, this study used population-based samples. In the previous
Please cite this article as: Hwang, I.W., et al., Association of LPHN3 rs6551665 A/G polymorphism with attention deficit and hyperactivity disorder in Korean children, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.04.033
I.W. Hwang et al. / Gene xxx (2015) xxx–xxx
studies, subjects were usually ADHD children who visited hospitals for their clinical symptoms thus, it is hard to consider that the samples were representing the general population. In the present study, the subjects of the risk individuals were selected by the questionnaire survey from the whole population within a region and sampling of case and controls were completely random. For these reason, the samples of the study were possibly more adequate to the analysis of a general population than those samples of the study tested with patients who visited the local hospitals. Third, the Koreans are reported relatively homogeneous (Jin et al., 2009, 2013), so the study could compare homogeneous groups unlike the studies that performed analyses in other countries with subjects from various ethnic origins and nations. Fourth, all the sample individuals analyzed here underwent clinical evaluation and DSM-IV diagnosis by child psychiatrists, under strict criteria for inclusion and exclusion samples. The patient group, therefore, was composed of pure ADHD-diagnosed children. In conclusion, our results imply that the LPHN3 rs6551665 A/G polymorphism may provide a significant effect on the occurrence of ADHD, although larger sample sizes and functional studies are necessary to further elucidate these findings. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2015.04.033. Conflicts of interests No competing financial interest exists. Acknowledgments This work was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI13C0747). References Acosta, M.T., Velez, J.I., Bustamante, M.L., Balog, J.Z., Arcos-Burgos, M., Muenke, M., 2011. A two-locus genetic interaction between LPHN3 and 11q predicts ADHD severity and long-term outcome. Transl. Psychiatry 1, e17. American Academy of Pediatrics, 2000. Clinical practice guideline: diagnosis and evaluation of the child with attention-deficit/hyperactivity disorder. Pediatrics 105 (5), 1158–1170. American Psychiatric Association Committee on Nomenclature and Statistics, 1994. Diagnostic and Statistical Manual of Mental Disorders (DSM-IV). 4th ed. American Psychiatric Association Press, Washington, DC. Arcos-Burgos, M., Muenke, M., 2010. Toward a better understanding of ADHD: LPHN3 gene variants and the susceptibility to develop ADHD. Atten. Defic. Hyperact. Disord. 2 (3), 139–147. Arcos-Burgos, M., Jain, M., Acosta, M.T., Shively, S., Stanescu, H., Wallis, D., Domene, S., Velez, J.I., Karkera, J.D., Balog, J., et al., 2010. A common variant of the latrophilin 3 gene, LPHN3, confers susceptibility to ADHD and predicts effectiveness of stimulant medication. Mol. Psychiatry 15 (11), 1053–1066. Banaschewski, T., Becker, K., Scherag, S., Franke, B., Coghill, D., 2010. Molecular genetics of attention-deficit/hyperactivity disorder: an overview. Eur. Child Adolesc. Psychiatry 19 (3), 237–257. Barkley, R.A., Murphy, K., 1998. Attention-Deficit Hyperactivity Disorder: A Clinical Workbook. 2nd ed. Guilford Press, New York. Bosker, F.J., Hartman, C.A., Nolte, I.M., Prins, B.P., Terpstra, P., Posthuma, D., van Veen, T., Willemsen, G., DeRijk, R.H., de Geus, E.J., Hoogendijk, W.J., Sullivan, P.F., Penninx, B.W., Boomsma, D.I., Snieder, H., Nolen, W.A., 2011. Poor replication of candidate genes for major depressive disorder using genome-wide association data. Mol. Psychiatry 16 (5), 516–532. Choudhry, Z.1., Sengupta, S.M., Grizenko, N., Fortier, M.E., Thakur, G.A., Bellingham, J., Joober, R., 2012. LPHN3 and attention-deficit/hyperactivity disorder: interaction with maternal stress during pregnancy. J. Child Psychol. Psychiatry 53 (8), 892–902. Cross-Disorder Group of the Psychiatric Genomics Consortium, et al., 2013. Genetic relationship between five psychiatric disorders estimated from genome-wide SNPs. Nat. Genet. 45 (9), 984–994. Davletov, B.A., Meunier, F.A., Ashton, A.C., Matsushita, H., Hirst, W.D., Lelianova, V.G., Wilkin, G.P., Dolly, J.O., Ushkaryov, Y.A., 1998. Vesicle exocytosis stimulated by alpha-latrotoxin is mediated by latrophilin and requires both external and stored Ca2+. EMBO J. 17 (14), 3909–3920. DuPaul, G.J., Anastopoulos, A.D., McGoey, K.E., Power, T.J., Reid, R., Ikeda, M.J., 1997. Teacher ratings of attention deficit hyperactivity disorder symptoms: factor structure and normative data. Psychological. Assessment 9 (4), 436–444.
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Please cite this article as: Hwang, I.W., et al., Association of LPHN3 rs6551665 A/G polymorphism with attention deficit and hyperactivity disorder in Korean children, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.04.033
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Research paper
Association of LPHN3 rs6551665 A/G polymorphism with attention deficit and hyperactivity disorder in Korean children In Wook Hwang a,b, Myung Ho Lim b,c, Ho Jang Kwon b,d, Han Jun Jin a,b,⁎ a
Department of Nanobiomedical Science, College of Natural Science, Dankook University, Cheonan, South Korea Environmental Health Center, Dankook Medical Hospital, Cheonan, South Korea Department of Psychology, College of Public Welfare, Dankook University, Cheonan, South Korea d Department of Preventive Medicine, College of Medicine, Dankook University, Cheonan, South Korea b c
a r t i c l e
i n f o
Article history: Received 11 October 2014 Received in revised form 7 April 2015 Accepted 10 April 2015 Available online xxxx Keywords: ADHD LPHN3 rs6551665 Polymorphism Korean Children Genetic association
a b s t r a c t Attention deficit hyperactivity disorder (ADHD) is a common and highly heritable disorder of school-age children. Its heritability was estimated at 80–90% but the genetic component underpinning this disorder remains to be disclosed. Recently, a highly consistent association between latrophilin3 (LPHN3) gene and ADHD was reported. In the present study, we examined the association between the LPHN3 rs6551665 A/G polymorphism and ADHD in Korea. The samples used in the study consisted of 150 ADHD children and 322 controls. The ADHD children were diagnosed according to DSM-IV. ADHD symptoms were evaluated with Dupaul Parent ADHD Rating Scales. LPHN3 rs6551665 SNP was determined by PCR-RFLP. Hardy–Weinberg equilibrium, genotype and allele frequency differences between the case and the control, and odds ratio were examined using the chi-square and exact tests. The LPHN3 gene locus was found to have no deviation from the Hardy– Weinberg expectation. We observed a significant association between the ADHD children and control group in genotype frequency (p = 0.01) and allele frequency (p = 0.02). The ADHD children appeared to have a surplus of GG genotype (OR 2.959, 95% CI 1.416–6.184, p = 0.003) and G allele (OR 1.44, 95% CI 1.062–1.945, p = 0.02). The association was more distinctive when analysis was confined to male samples (p = 0.005), the OR of male controls and cases was 4.029 (95% CI 1.597–10.164, p = 0.002) and the OR having G allele vs. A allele was 1.46 (95% CI 1.002–2.127, p = 0.048). Thus our results imply that the LPHN3 rs6551665 GG genotype and G allele may provide a significant effect on the ADHD, although larger sample sizes and functional studies are necessary to further elucidate these findings. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Attention deficit hyperactivity disorder (ADHD) is a common childhood neuropsychiatric disorder occurring at 2–7.6% among children of school age in Korea (Kwon et al., 2014). ADHD is a heterogeneous disorder and often has effect detectable symptoms in adulthood. The disorder is characterized by behavioral problems such as attention deficit, hyperactivity, and impulsivity (American Psychiatric Association Committee on Nomenclature and Statistics, 1994). Family, adoption, and twin studies have shown that there is a strong genetic relationship of ADHD. The heritability estimates of ADHD are ranging from 80 to 90% (Faraone and Doyle, 2001; Shastry, 2004), but so far, its genetic Abbreviations: ADHD, attention deficit hyperactivity disorder; DSM-IV, Diagnostic and Statistical Manual of Mental Disorders-4th edition; SNP, single nucleotide polymorphism; PCR, polymerase chain reaction; PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism; LPHN3, latrophilin3; HWE, Hardy–Weinberg equilibrium; OR, odds ratio; CI, confidence interval. ⁎ Corresponding author at: Department of Nanobiomedical Science, Dankook University, Cheonan 330-714, South Korea. E-mail address: [email protected] (H.J. Jin).
contributions remain elusive beyond reasonable doubt from linkage and candidate gene approach (Smalley et al., 2002; Ogdie et al., 2003; Faraone, 2004) as well as genome-wide association studies (Franke et al., 2009; Banaschewski et al., 2010; Mick et al., 2010; Neale et al., 2010a,b). To elucidate the role of genetic influences in ADHD, various approaches have been used to address important issues in the occurrence of ADHD, such as twin studies, candidate gene studies, genome-wide approaches and copy number variation (CNV) studies (Schachar, 2014). Candidate gene studies have constantly showed evidence for association with DRD4, DRD5 and DAT1 of dopaminergic, SLC6A2 and 5-HTT/SLC6A4 and HTR1B serotonin receptor gene of serotonergic, NET1/SLC6A2, ADRA2A and ADRA2C of noradrenergic and CHRNA4 of nicotinergic neurotransmission and receptor function, lastly SNAP25, CHRNA4, NMDA, BDNF, NGF, NTF3, NTF4/5 and GDNF genes that are involved in neurotransmission and neuronal plasticity (Gizer et al., 2009; Schachar, 2014). However, linkage and association studies of ADHD have not reached a rigorous level for genome-wide significance since the sample sizes were far too small (Zhou et al., 2008; Neale et al., 2010a). The Psychiatric Genomics Consortium (PGC; http://pgc.unc.edu) and the ADHD Genetic Consortium
http://dx.doi.org/10.1016/j.gene.2015.04.033 0378-1119/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: Hwang, I.W., et al., Association of LPHN3 rs6551665 A/G polymorphism with attention deficit and hyperactivity disorder in Korean children, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.04.033
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have raised a question about the small sample size in the genetic analyses of ADHD. To overcome the issue on the sample size, the PGC analyzed GWAS data from over 19 countries for 5 major psychiatric disorders including ADHD (ADHD, ASD, major depression, bipolar disorder and schizophrenia), by examining GWAS data in 33,332 cases and 27,888 controls of European ancestry. In this analysis four SNPs remained significant at the genome-wide significance level (p b 5 × 10−8): regions on chromosomes 3p21 and 10q24, and SNPs related with CACNA1c and CACNB2, the two L-type voltage-gated calcium channel subunits (Cross-Disorder Group of the Psychiatric Genomics Consortium et al., 2013). The drawback of the GWAS studies is that the study uses high levels for the statistical significance since they analyze a large number of statistical tests, this rigorous criteria for significance could miss the signal of association from genes that have moderate individual contribution to ADHD (Schachar, 2014). The candidate gene approaches, therefore, have its own virtue on the analyses of genetic contribution for ADHD. In a recent study, Arcos-Burgos et al. (2010) reported an association between polymorphisms within latrophilin 3 (LPHN3) and adult ADHD. It has been also reported that a common variant of the LPHN3 increases the risk of developing ADHD by 1.2 fold (Acosta et al., 2011). LPHN3 is a member of the LPHN subfamily that is related with G-protein coupled receptors (GPCRs) and the receptor has been reported to be important for the regulation in the exocytosis of neurotransmitters particularly for norepinephrine (Davletov et al., 1998; Rahman et al., 1999; Silva et al., 2009; Choudhry et al., 2012). In the functional studies, the LPHN3 is expressed in key brain regions where it closely related to attention and activity. Its variants likely affect metabolism in neural circuits involved in ADHD and are also involved in responses to stimulant medication (Arcos-Burgos et al., 2010; Ribases et al., 2010; Acosta et al., 2011). To the best of our knowledge, the genetic relationship between LPHN3 gene and ADHD in Korean population has not been examined. The present study investigated an association between LPHN3 rs6551665 A/G polymorphism and genetic contribution of developing ADHD in a sample of population based Korean ADHD children relative to the normal control children. 2. Materials and methods 2.1. Subject We analyzed a total of 472 samples from the Children's Health and Environmental Research (CHEER) cohort study and precious study (Kwon et al., 2014). Of these samples, 120 ADHD children and 322 control individuals were subset of CHEER cohort study. In addition, 30 ADHD children were a subset of the samples analyzed in Kwon et al. (2014). The CHEER study was carried out on elementary school children of 10 cities in Korea from 2005 to 2010 biennially. An interview was randomly performed to children with Korean version of the Dupaul Attention Deficit Hyperactivity Disorder Rating Scales (K-ARS) (Kim et al., 2002) including 18 items based on the DSM-IV diagnostic criteria for ADHD. In this study, the K-ARS score for the ADHD samples was 19 or higher, and the score for the control group was less than 19. For these 18 items, questions 1–9 are for inattention and questions 10–18 are for hyperactive/impulsive. According to these criteria, we classified the subtype of ADHD samples as follows; combined (ADHD/C), predominantly inattentive (ADHD/I) and predominantly hyperactive/impulsive (ADHD/HI) (Morgan et al., 1996; Dupaul et al., 1997; Gaub and Carlson, 1997a; Barkley and Murphy, 1998; Lahey et al., 1998). Each clinical interview was conducted by researchers with at least 1 parent. Informed consent was obtained from all the participants of this study. The sex and age for ADHD children and the control group were matched to avoid possible biases of the study. A clinical evaluation and the DSMIV diagnosis (American Psychiatric Association Committee on Nomenclature and Statistics, 1994) were performed by a child psychiatrist. The number of ADHD children is 150 [97 boys (64.7%) and 53 girls
(35.3%)], and the mean age was 8.05 ± 1.04. The number of samples in the control group was 322 [191 boys (59.3%) and 131 girls (40.7%)], and the mean age was 8.22 ± 1.48. There was no significant difference in the sex and age between the ADHD children and control group (Table 1). The study protocol was approved by the Ethics Committee of the Dankook University Hospital. 2.2. DNA extraction and genotyping DNA was extracted from leukocytes using the G-DEX™ llb Genomic DNA Extraction kit (Intron Biotechnology, Korea) or the GeneAll Exgene Clinic SV mini kit (GeneALL, Korea). A primer set was designed for the determination of the rs6551665 A/G polymorphism and the SNP was located within the intron region in the LPHN3 gene (Fig. 1). A BLAST search for the homologous sequence data was performed by using the BLASTN program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). A pair of PCR primer was designed by Primer3Plus based on blasted sequence (Untergasser et al., 2007). Genomic DNA was amplified using the new designed primers as follows: forward, 5′-ATCAGCATGCAGTAGCCCTC3′ and reverse 5′-GAGCAAGAGTTCTCAAACATGGT-3′. Each PCR reaction was performed in a total volume of 20 μl containing 10 ng of genomic DNA, 10 pM each primer, 0.2 mM dNTPs, 2.0 mM MgCl2, 10 × PCR buffer and 1.0 U NV DNA polymerase (NAVI BioTech, Korea). The cycling conditions used an initial denaturation step at 95 °C for 5 min, and then 35 cycles at 94 °C for 1 min, 58 °C for 1 min, 72 °C for 1 min, and a final extension at 72 °C for 10 min. The PCR product was digested with 1.0 U Hph I restriction enzyme (New England Biolabs, USA) for 3 h at 37 °C and electrophoresed in 3% agarose gel (Lonza, USA). The polymorphic Hph I site was detected by restriction fragment length polymorphism in producing fragments of 203 and 99 bp (A allele) or 139, 99 and 64 bp (G allele) (Fig. 2). 2.3. Data analyses Statistical analyses were performed using SPSS 21 Statistics (IBM Korea, Korea) to determine frequencies, cross tabulation analyses, and descriptive statistics. Chi-squared tests were used to assess Hardy– Weinberg equilibrium (HWE). In addition, a test of proportion and odds ratio (OR) with 95% confidence intervals (CI), and Fisher's exact test in a 2 × 2 table were calculated using the statistical analysis on the internet (SISA, http://www.quantitativeskills.com/sisa/). A p value of b0.05 was considered statistically significant. It has been reported that a sample size of 210 subjects is required to obtain a 95% or higher power in the chi-square calculation between the controls and ADHD case samples in the Korean population (Kwon et al., 2014). Our study was conducted in 472 subjects, therefore we expected that this study has enough power to assess an association of the LPHN3 rs6551665 polymorphism and ADHD in the Korean population. Table 1 Characteristics of ADHD children (n = 150) and control (n = 322) group. Characteristics Ageb Gender
Male Female
ADHD (n = 150)a
Control (n = 322)
F or χ2
p-value
8.05 ± 1.04 97 (64.7%) 53 (35.3%)
8.22 ± 1.48 191 (59.3%) 131 (40.7%)
46.61 1.23
0.13 0.27c
ADHD (n = 120)d Subtype
Combined Inattentive Hyperactive/impulsive
78 (65%) 32 (27%) 10 (8%)
a CHEER study with 120 ADHD samples and 30 ADHD samples analyzed in Kwon et al. (2014). b These data represent mean ± SD, by independent t-test. c The Chi-square p-value. d CHEER study with 120 ADHD samples.
Please cite this article as: Hwang, I.W., et al., Association of LPHN3 rs6551665 A/G polymorphism with attention deficit and hyperactivity disorder in Korean children, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.04.033
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Fig. 1. Genomic organization of the Chr 4:61,843,840-61,895,000 region surrounding the rs6551665. The map is based on genome build GRCh38.p2 http://www.ensembl.org/. (A) LPHN3 gene is located in 4q13.1 region. (B) The sequence variant rs6551665, located in the intron region of LPHN3 gene (Chr 4: 61,873,823), is not associated with a binding site for the transcription factors. The red square represents a transcription factor binding site. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
3. Results 3.1. Demographic characteristics of the subjects A total of 472 children were analyzed for the study. The children in both the ADHD group and the control group had never taken any psycho-stimulant in advance. There was no difference in age (F = 46.61, p = 0.13) and sex (F = 1.23, p = 0.27) between the control group and ADHD case individuals (Table 1). 3.2. Comparison of frequency of the genotypes and alleles with genetic polymorphism of LPHN3 rs6551665 A/G between the control and ADHD groups LPHN3 rs6551665 genotyping data from the control and ADHD groups are summarized in Table 2. Genotype distribution of all
control (A/A 55.9%, A/G 39.4% and G/G 4.7%) and ADHD groups (A/A 48.7%, A/G 39.3% and G/G 12%) were in agreement with the Hardy–Weinberg equilibrium (HWE) (Table 2). Significant genotype and allele frequency differences were observed between the controls and the cases (p = 0.01 and 0.02, respectively). We also found a significant association (p = 0.02), when the samples with hyperactivity were compared with control groups (10 cases vs 322 controls) (Supplementary Table S1). Table 3 shows the results for the association of LPHN3 rs6551665 polymorphism. The OR of ADHD children having the GG genotype compared with controls was 2.959 (95% CI 1.416–6.184, p = 0.003). The result also remains significant when the analysis was performed by allele frequencies. The OR having the G allele versus A allele was 1.44 (95% CI 1.062–1.945, p = 0.02) (Table 3). This association was more distinctive when the analysis was confined to male samples (p = 0.005) (Table 2). The OR of ADHD male samples and male controls was 4.029 (95% CI 1.597–10.164, p = 0.002) and the OR having G allele vs. A allele was 1.46 (95% CI 1.002–2.127, p = 0.048) (Supplementary Table S2). The associations between all subtypes of ADHD and control group were analyzed (Supplementary Table S1). 4. Discussion
Fig. 2. Detection of genotype and allelic variations at the rs6551665 in LPHN3 gene. PCR products were electrophoresed after digesting with Hph I. Lane M: 1 kb + DNA ladder; lanes 1, 2, 4, 5, 6, 8, 10, 11, and 13: AG alleles; lanes 3, 9, 12, and 14: AA alleles; lane 7: GG alleles.
It has been reported that multiple environmental risk factors significantly related to the development of ADHD. In addition, there is now enormous evidence for substantial genetic influences on the etiology and pathogenesis of ADHD (Pineda et al., 2007; Gizer et al., 2009; Waldman and Gizer, 2006; Arcos-Burgos and Muenke, 2010). Recently, common variants of the LPHN3 gene located on chromosome 4 were recognized as new candidate gene to analyze the genetic susceptibility for ADHD in several ethnic groups (Arcos-Burgos et al., 2010; Acosta et al., 2011; Fallgatter et al., 2013). LPHN3, unlikely from the latrophilin1 and latorphilin2 that serve as receptors for alpha-latrotoxin, a component of the black widow spider (Latrodectus mactans), is the most exclusively expressed in the brain (Sugita et al., 1998; Ichtchenko et al., 1998). LPHN3 gene showed significantly increased expression of
Please cite this article as: Hwang, I.W., et al., Association of LPHN3 rs6551665 A/G polymorphism with attention deficit and hyperactivity disorder in Korean children, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.04.033
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Table 2 Genotype and allele frequencies of LPHN3 rs6551665 in the ADHD children and control group. Group
Total ADHD Control Male ADHD Control Female ADHD Control
n
p⁎⁎
Genotype (%) A/A
A/G
G/G
150 322
73(48.7%) 180(55.9%)
59(39.3%) 127(39.4%)
18(12.0%) 15(4.7%)
97 191
47(48.4%) 101(52.9%)
35(36.1%) 82(42.9%)
15(15.5%) 8(4.2%)
53 131
29(54.7%) 79(60.3%)
21(39.6%) 45(34.4%)
3(5.7%) 7(5.3%)
Allele frequency (%)
p⁎⁎⁎
HWE⁎⁎⁎
Allele A
Allele G
0.01⁎
205(68.3%) 487(75.6%)
95(31.7%) 157(24.4%)
0.02⁎
0.26 0.21
0.005⁎
129(66.5%) 284(74.3%)
65(33.5%) 98(25.7%)
0.048⁎
0.06 0.08
0.54
79(74.5%) 203(77.5%)
27(25.5%) 59(22.5%)
0.54
0.75 0.86
HWE Hardy–Weinberg equilibrium. ⁎ p b 0.05. ⁎⁎ The Fisher's exact test p-value. ⁎⁎⁎ The Chi-square p-value.
mRNA in the region of human amygdala, caudate nucleus, cerebellum, and cerebral cortex, in contrast the lower and no expression was detected in corpus callosum, hippocampus, whole brain extract, occipital pole, frontal lobe, temporal lobe putamen, thalamus, medulla and spinal cord. It has been reported that the regions where LPHN3 gene are highly expressed are in key regions of brain for attention and activity (ArcosBurgos et al., 2010). Lange et al. (2012) validated the function of LPHN3 gene in the brain by examining LPHN3 orthologous lphn3.1 gene during zebrafish development. Loss of lphn3.1 function in zebrafish showed reduction and misplacement of dopamine positive neurons in the ventral diencephalon and a hyperactive/impulsive motor phenotype. Moreover the normal phenotype was restored by the methylphenidate and atomoxetine that are ADHD treatment drugs (Lange et al., 2012). Similar observation has been reported from a transgenic mice model experiment (Wallis et al., 2012; Kotepui et al., 2012). In the experiment, LPHN3 KO mice had profoundly disrupted multiple monoamine-signaling levels, which causes ADHD like symptom. Therefore, these reports give us a clue for the function of LPHN3 gene in brain and its etiology of developing ADHD. Another function of LPHN3 gene has been also reported that increased mRNA expression of LPHN3 gene in breast-cancer tissues is significantly correlated with axillary lymph node positivity, along with increased mRNA expression of MMP13 (Kotepui et al., 2012). This report is particularly intriguing since it has reported that the expression of LPHN3 is confined within brain (Sugita et al., 1998; Ichtchenko et al., 1998). Kotepui et al. (2012) reported that mRNA expression of LPHN3 and MMP13 is closely related with tumor aggressiveness since commonly the metastasis of breast carcinoma is spread through the axillary lymph nodes and may be modulated by the expression of LPHN3 and MMP13. Thus the role of LPHN3 in axillary-node metastasis in breast cancer, as well as in ADHD, is a subject for further analysis. This study showed the significant correlation between the LPHN3 rs6551665 A/G polymorphism and ADHD phenotype (p = 0.01).
Table 3 The odds ratio of LPHN3 rs6551665 genotypes and alleles between ADHD children and control group. Total
Genotype AAb AG GG Allele Ab/G
ADHD children versus control OR
(95% CI)
p-valuea
1.000 1.146 2.959
(0.759–1.729) (1.416–6.184)
– 0.52 0.003⁎
1.440
(1.062–1.945)
0.02⁎
OR odds ratio, CI confidence interval. ⁎ p b 0.05. a The Chi-square p-value. b Reference.
Choudhry et al. (2012) reported a highly significant interaction between four LPHN3 tag SNPs including rs6551665, rs1947274, rs6858066 and rs2345039 and maternal stress during pregnancy. Of these SNPs, rs6551665 was replicated in the present study, although the design of the studies was distinct. It has been reported that with an estimated prevalence of 4% to 12% in school-age children, ADHD seems like more common in boys than girls (American Academy of Pediatrics, 2000; Quinn and Wigal, 2004). The ratio of boy to girl ranged from 9:1 to 6:1 in clinical samples and in community-based population study samples are 3:1 (Gaub and Carlson, 1997b; Quinn and Wigal, 2004). Given the known gender-specific trait of ADHD, boy and girl samples were analyzed separately. The analysis of girl samples provided no evidence for an association between LPHN3 polymorphism and ADHD (p = 0.54). But a high frequency of GG genotype in the ADHD boys (15/97, 15.5%) compare to control group (8/191, 4.2%) was observed (p = 0.005). Despite previous reports about the gender differences on the occurrence of ADHD, the reasons for this are unclear. Further analyses with a large number of samples are necessary to have conclusive understanding for this gender specific relationship of ADHD. We also performed analyses according to their subtypes of ADHD, the samples were divided into inattentive, hyperactive/impulsive and combined type. Significant association was detected between the hyperactive/impulsive subtypes and control group (p = 0.02). However, the sample size of hyperactive/impulsive group was too small to get relevant decision, even though they were reached at the significant level (p b 0.05). The correlation of hyperactive/impulsive subtypes in ADHD deserves further analysis. Especially, the candidate gene approaches have a substantial risk of generating false positive results since most of gene associations were analyzed individually without considering the total number of plausible associations and the possibility of bias toward publishing positive results but not negative. Therefore, the positive associations detected from candidate studies were often not replicated in genome-wide association studies (Bosker et al., 2011). These results implicate that single association studies should be thoroughly replicated (Sullivan, 2007; Schachar, 2014). The present study has inherent deficiency that the number of the studied sample was relatively small. The samples of this study were 150 ADHD children and 322 individuals for the control group. The next limitation is that the only one SNP was investigated in this study, even though there are various SNPs on the genes related with the various ADHD phenotypes. Although it is clear that a lot of genetic factors cause the increased ADHD vulnerability, it was not considered the interaction with other risk factors. In contrast to the methodological limitation described previously, the study has advantages. First, the case and the control groups were matched for sex and age so that there were no significant differences. ADHD is much more prevalent in males and in the period of adolescence, therefore sex and age characteristics can give a great influence. Second, this study used population-based samples. In the previous
Please cite this article as: Hwang, I.W., et al., Association of LPHN3 rs6551665 A/G polymorphism with attention deficit and hyperactivity disorder in Korean children, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.04.033
I.W. Hwang et al. / Gene xxx (2015) xxx–xxx
studies, subjects were usually ADHD children who visited hospitals for their clinical symptoms thus, it is hard to consider that the samples were representing the general population. In the present study, the subjects of the risk individuals were selected by the questionnaire survey from the whole population within a region and sampling of case and controls were completely random. For these reason, the samples of the study were possibly more adequate to the analysis of a general population than those samples of the study tested with patients who visited the local hospitals. Third, the Koreans are reported relatively homogeneous (Jin et al., 2009, 2013), so the study could compare homogeneous groups unlike the studies that performed analyses in other countries with subjects from various ethnic origins and nations. Fourth, all the sample individuals analyzed here underwent clinical evaluation and DSM-IV diagnosis by child psychiatrists, under strict criteria for inclusion and exclusion samples. The patient group, therefore, was composed of pure ADHD-diagnosed children. In conclusion, our results imply that the LPHN3 rs6551665 A/G polymorphism may provide a significant effect on the occurrence of ADHD, although larger sample sizes and functional studies are necessary to further elucidate these findings. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2015.04.033. Conflicts of interests No competing financial interest exists. Acknowledgments This work was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI13C0747). References Acosta, M.T., Velez, J.I., Bustamante, M.L., Balog, J.Z., Arcos-Burgos, M., Muenke, M., 2011. A two-locus genetic interaction between LPHN3 and 11q predicts ADHD severity and long-term outcome. Transl. Psychiatry 1, e17. American Academy of Pediatrics, 2000. Clinical practice guideline: diagnosis and evaluation of the child with attention-deficit/hyperactivity disorder. Pediatrics 105 (5), 1158–1170. American Psychiatric Association Committee on Nomenclature and Statistics, 1994. Diagnostic and Statistical Manual of Mental Disorders (DSM-IV). 4th ed. American Psychiatric Association Press, Washington, DC. Arcos-Burgos, M., Muenke, M., 2010. Toward a better understanding of ADHD: LPHN3 gene variants and the susceptibility to develop ADHD. Atten. Defic. Hyperact. Disord. 2 (3), 139–147. Arcos-Burgos, M., Jain, M., Acosta, M.T., Shively, S., Stanescu, H., Wallis, D., Domene, S., Velez, J.I., Karkera, J.D., Balog, J., et al., 2010. A common variant of the latrophilin 3 gene, LPHN3, confers susceptibility to ADHD and predicts effectiveness of stimulant medication. Mol. Psychiatry 15 (11), 1053–1066. Banaschewski, T., Becker, K., Scherag, S., Franke, B., Coghill, D., 2010. Molecular genetics of attention-deficit/hyperactivity disorder: an overview. Eur. Child Adolesc. Psychiatry 19 (3), 237–257. Barkley, R.A., Murphy, K., 1998. Attention-Deficit Hyperactivity Disorder: A Clinical Workbook. 2nd ed. Guilford Press, New York. Bosker, F.J., Hartman, C.A., Nolte, I.M., Prins, B.P., Terpstra, P., Posthuma, D., van Veen, T., Willemsen, G., DeRijk, R.H., de Geus, E.J., Hoogendijk, W.J., Sullivan, P.F., Penninx, B.W., Boomsma, D.I., Snieder, H., Nolen, W.A., 2011. Poor replication of candidate genes for major depressive disorder using genome-wide association data. Mol. Psychiatry 16 (5), 516–532. Choudhry, Z.1., Sengupta, S.M., Grizenko, N., Fortier, M.E., Thakur, G.A., Bellingham, J., Joober, R., 2012. LPHN3 and attention-deficit/hyperactivity disorder: interaction with maternal stress during pregnancy. J. Child Psychol. Psychiatry 53 (8), 892–902. Cross-Disorder Group of the Psychiatric Genomics Consortium, et al., 2013. Genetic relationship between five psychiatric disorders estimated from genome-wide SNPs. Nat. Genet. 45 (9), 984–994. Davletov, B.A., Meunier, F.A., Ashton, A.C., Matsushita, H., Hirst, W.D., Lelianova, V.G., Wilkin, G.P., Dolly, J.O., Ushkaryov, Y.A., 1998. Vesicle exocytosis stimulated by alpha-latrotoxin is mediated by latrophilin and requires both external and stored Ca2+. EMBO J. 17 (14), 3909–3920. DuPaul, G.J., Anastopoulos, A.D., McGoey, K.E., Power, T.J., Reid, R., Ikeda, M.J., 1997. Teacher ratings of attention deficit hyperactivity disorder symptoms: factor structure and normative data. Psychological. Assessment 9 (4), 436–444.
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