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Contents lists available at ScienceDirect

Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet

Evaluation of miR18a-5p, miR22-3p, miR24-3p, miR106b-5p, miR107, miR125b-5p, and miR155a-5p levels in children and adolescents with attention deficit hyperactivity disorder

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Hasan Kandemir a , Mehmet Emin Erdal b , Salih Selek c,∗ , Özlem I˙ zci Ay b , I˙ brahim Fatih Karababa d , Sultan Basmacı Kandemir e , Mustafa Ertan Ay b , S¸enay Görücü Yılmaz f , Hüseyin Bayazıt d , Bahar Tas¸delen g

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Department of Child and Adolescent Psychiatry, Faculty of Medicine, Harran University, S¸anlıurfa, Turkey Department of Medical Biology, Faculty of Medicine, Mersin University, Mersin, Turkey Department of Psychiatry, Medeniyet University, Faculty of Medicine, I˙ stanbul, Turkey d Department of Psychiatry, Faculty of Medicine, Harran University, S¸anlıurfa, Turkey e Department of Psychiatry, Balıklı Göl State Hospital, S¸anlıurfa, Turkey f Department of Medical Biology and Genetics, Faculty of Medicine, Mersin University, Mersin, Turkey g Department of Biostatistics, Faculty of Medicine, Mersin University, Mersin, Turkey b c

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h i g h l i g h t s • • • •

miRNA 18a-5p, 22-3p, 24-3p, 106b-5p and 107 levels were decreased in ADHD. miRNA 155a-5p levels were increased in ADHD. miR-107 may be a candidate biomarker for ADHD. Dysregulation of circulating miRNAs may affect ADHD etiology and treatment.

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Article history: Received 21 April 2014 Received in revised form 15 July 2014 Accepted 31 July 2014 Available online xxx

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Keywords: ADHD miRNA Psychiatry Child psychiatry miR18a-5p miR22-3p miR24-3p miR106b-5p miR107 miR125b-5p miR155a-5p

Attention-deficit/hyperactivity disorder (ADHD) is one of the most prevalent childhood disorders, although disorders etiology and pathogenesis remains unknown, several theories about ADHD development have been proposed and many researchers believe that it is caused by both genetic and environmental factors. In this study we evaluated miR18a-5p, miR22-3p, miR24-3p, miR106b-5p, miR107, miR125b-5p and miR155a-5p levels in child and adolescent ADHD patients. The research sample consisted a group of 52 ADHD patients, and 52 healthy volunteer controls. There was no significant difference in age and sex between the two groups (p > 0.05). miRNA 18a-5p, 22-3p, 24-3p, 106b-5p and 107 levels were statistically significantly decreased in ADHD patients(p < 0.05). miRNA 155a-5p levels were increased in patients group (p < 0.05). The positive predictive value (PPV) and negative predictive value of miR107 was estimated for the cutoff point of 0.4480. PPV was 70% and NPV was 86.5% for the taken cut off point. There could be a close relationship between levels of circulating miRNAs and ADHD. If we could understand how the signaling pathways arranged by miRNAs, impact on CNS development, function and pathology this can improve our knowledge about ADHD etiology and treatment. © 2014 Published by Elsevier Ireland Ltd.

1. Objective

∗ Corresponding author. E-mail address: [email protected] (S. Selek).

Attention-deficit/hyperactivity disorder (ADHD) is characterized by developmentally inappropriate levels of inattention, hyperactivity, and impulsivity [1]. It is one of the most prevalent childhood disorders, occurring in 3–7% of school-aged children

http://dx.doi.org/10.1016/j.neulet.2014.07.060 0304-3940/© 2014 Published by Elsevier Ireland Ltd.

Please cite this article in press as: H. Kandemir, et al., Evaluation of miR18a-5p, miR22-3p, miR24-3p, miR106b-5p, miR107, miR125b-5p, and miR155a-5p levels in children and adolescents with attention deficit hyperactivity disorder, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.07.060

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2 Table 1 Some of the features of the studied miRNAS.

Q8

miRNA

Rationale for study

Previous studies

Reference

miR18a-5p

Believed to be involved in DNA damage in ADHD

Altered in DNA damage response

miR22-3p

Believed to regulate four candidate genes: BDNF, HTR2C, MAOA, and RGS2 Believed to be related with oxidative stress which is a potential neurobiological mechanism in ADHD

Found to be altered in Panic Disorder

Libing Song, et al., miR-18a impairs DNA damage response through downregulation of ataxia telangiectasia mutated (ATM) kinase, PLoS One 6.9 (2011) e25454 [22]

miR24-3p

miR106b-5p

Believed to be related with oxidative stress which is a potential neurobiological mechanism in ADHD

Down regulated in resveratrol treatment that has antioxidant properties

miR107

Believed to be related with minimal brain change in ADHD Believed to be related with hypoxia in ADHD

Altered in traumatic brain injury and neurodegenerative diseases Altered in high altitude sickness

Believed to be related with prefrontal cortex pathophysiology ADHD

Altered in depression

miR125b-5p

miR155a-5p

48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87

Found to be altered in oxidative DNA damage and lipid peroxidation

and representing one third to one half of referrals to child mental health services [2]. ADHD is more common in boys than girls, with ratios ranging from 3:1 to 10:1 [3]. A large proportion of children with ADHD are diagnosed with another psychiatric disorder [4]. Overwhelming evidence suggests that ADHD is not a childhood condition but a lifelong disorder [5]. Although the etiology and pathogenesis of ADHD remain unknown, several theories have been proposed, and many researchers believe it is caused by both genetic and environmental factors [6]. Segregation analysis of data from neurochemical studies in families comprising genetic, twin, and adoption relationships strongly suggest a genetic etiology [7]. Substantial genetic influence on the disorder has been identified, with heritability estimates ranging from 60 to 90% [8]. Studies have identified abnormal regulation of neurotransmitter systems, particularly dopamine [9]. Preliminary molecular genetic studies have implicated several candidate genes, including the dopamine D2 and D4 (DRD4-7) receptors as well as the dopamine transporter (DAT-1) [10]. The presence of the L allele of the serotonin transporter gene 5-HTTLPR is associated with decreased serotonin levels and increased risk of ADHD [11]. MicroRNAs (miRNAs) are evolutionally conserved small noncoding RNAs that regulate approximately 30% of human protein coding gene expression at the post-transcriptional level, and they play important roles in a wide variety of biological functions [12]. miRNAs control gene expression by inhibiting translation or facilitating degradation of their target mRNAs. Computational predictions indicate that thousands of genes could be targeted by miRNAs in mammals [13]. miRNAs are important for maintaining homeostasis at the neuromuscular junction. They also play an important role in synaptic plasticity in the central nervous system, and are involved in memory and mental retardation [14]. The ability of miRNAs to affect the activity of all biological pathways may underlie some of the difficulties associated with linking psychiatric disorders to specific causative genes [15]. The involvement of multiple signaling pathways in psychiatric disease complicates both the investigation of the underlying biological causes and efforts to identify effective therapies. Focusing on the roles of miRNAs in psychiatric diseases may lead not only to an explanation of the dysregulation of multiple pathways but also to

J.D. Morrow, B. Frei, A.W. Longmire, J.M. Gaziano, S.M. Lynch, Y. Shyr, L.J. Roberts, Increase in circulating products of lipid peroxidation (F2-isoprostanes) in smokers—smoking as a cause of oxidative damage. N. Engl. J. Med. 332 (18) (1995) 1198–1203 S. Dhar, C. Hicks, A.S. Levenson, Resveratrol and prostate cancer: promising role for microRNAs. Mol. Nutr. Food Res. 55(8) (2011) 1219–1229 [30] Norman E. Buroker, et al., Circulating miRNAs from dried blood spots are associated with high altitude sickness, J. Med. Diagn. Methods (2013) [32]

novel therapies that can target entire gene networks. Perkins et al. examined the expression of 16 miRNAs in the prefrontal cortex in subjects with schizophrenia or schizoaffective disorder and found decreased expression of 15 miRNAs in patients with schizophrenia [16]. However, although miRNAs have been shown to be particularly abundant in the brain, their role in the development and activity of the nervous system remains largely unknown. In this study, we evaluated miR18a-5p, miR22-3p, miR24-3p, miR106b5p, miR107, miR125b-5p, and miR155a-5p levels in children and adolescents with ADHD. In selection of the miRNAs previous literature pointing out the potential underlying neurobiology (enzyme, carrier molecule, receptor etc.) of the disease were reviewed and potential miRNAs from the miRNA database (mirbase.org) that had both higher target scores and were available in our lab were chosen. Table 1 shows the features of the studied miRNAs.

2. Method The research sample consisted a group of 52 patients from Harran University Faculty of Medicine Research Hospital, Child and Adolescent Psychiatry Clinic who were referred. The clinic for the first time and diagnosed with ADHD, and 52 healthy volunteer controls. All patients were diagnosed as ADHD by a child psychiatrist according to DSM-IV-TR diagnostic criteria and they were treatment naive. In this study, the ADHD module of K-SADS-PL was used to make the diagnosis of ADHD [17]. Patients with a history of cardiovascular disorders, epilepsy, diabetes mellitus, psychotic disorders, pervasive developmental disorders or severe head injury were excluded. The healthy controls were recruited from healthy child outpatient unit. After complete description of the study to the subjects, a written informed consent was obtained from the parents as well as the assent of children and adolescents. All of the study procedures were in accordance with the Declaration of Helsinki. Ethics committee of the Harran University Medical School approved the trial. Also a semi-structured form was used to detect several socio demographic and clinical variables of the patients. The final patient and control groups displayed similar distribution in age and gender. Study was funded by Harran University Board of Scientific Research Projects (Funding Number: 12010). This study

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Table 2 Primer/probe sequences of the miR analyzed by quantitative RT–PCR. microRNA name

Gene ID*

NCBI reference sequence number**

Primer/probe sequence

hsa-miR-26b-5p

407017

NR 029500.1

hsa-miR-18a-5p

406953

NR 029488.1

hsa-miR-22-3p

407004

NR 029494.1

hsa-miR-24-3p

407012

NR 029496.1

hsa-miR-106b-5p

406900

NR 029831.1

hsa-miR-107

406901

NR 029524.1

hsa-miR-125b-5p

406911

NR 029671.1

hsa-miR-155-5p

406947

NR 030784.1

hsa-miR-26b-5p-RT 5 -GTCGTATGCAGTGCAGGGTCCGAGGTATTCGCACTGCATACGACACCTAT-3 hsa-miR-26b-5p-F 5 -GCCGCTTCAAGTAATTCAGG-3 hsa-miR-26b-5p-PR 5 -FAM-TG(pdC)ATA(pdC)GA(pdC)A(pdC)CTATCC-ZNA4-BHQ-1-3 hsa-miR-18a-5p-RT 5 -GTCGTATGCAGTGCAGGGTCCGAGGTATTCGCACTGCATACGACCTATCT-3 hsa-miR-18a-5p-F 5 -GCCGCTAAGGTGCATCTAGTG-3 hsa-miR-18a-5p-PR 5 -FAM-TG(pdC)ATA(pdC)GA(pdC)CTAT(pdC)TGC-ZNA4-BHQ-1-3 hsa-miR-22-3p-RT 5 -GTCGTATGCAGTGCAGGGTCCGAGGTATTCGCACTGCATACGACACAGTT-3 hsa-miR-22-3p-F 5 -GCCGCAAGCTGCCAGTT-3 hsa-miR-22-3p-PR 5 -FAM-TG(pdC)ATA(pdC)GA(pdC)A(pdC)AGTT(pdC)T-ZNA4-BHQ-1-3 hsa-miR-24-3p-RT 5 -GTCGTATGCAGTGCAGGGTCCGAGGTATTCGCACTGCATACGACCTGTTC-3 hsa-miR-24-3p-F 5 -GCCGCTGGCTCAGTTCAG-3 hsa-miR-24-3p-PR 5 -FAM-TG(pdC)ATA(pdC)GA(pdC)CTGTTCCT-ZNA4-BHQ-1-3 hsa-miR-106b-5p-RT 5 -GTCGTATGCAGTGCAGGGTCCGAGGTATTCGCACTGCATACGACATCTGC-3 hsa-miR-106b-5p-F 5 -GCCGCTAAAGTGCTGACAGT-3 hsa-miR-106b-5p-PR 5 -FAM-TG(pdC)ATA(pdC)GA(pdC)ATCTGCAC-ZNA4-BHQ1-3 hsa-miR-107-RT 5 -GTCGTATGCAGTGCAGGGTCCGAGGTATTCGCACTGCATACGACTGATAG-3 hsa-miR-107-F 5 -GCCGCAGCAGCATTGTACAGGG-3 hsa-miR-107-PR 5 -FAM-TG(pdC)ATA(pdC)GA(pdC)TGATAG(pdC)C-ZNA4-BHQ-1-3 hsa-miR-125b-5p-RT 5 -GTCGTATGCAGTGCAGGGTCCGAGGTATTCGCACTGCATACGACTCACAA-3 hsa-miR-125b-5p-F 5 -GCCGCTCCCTGAGACCCTAAC-3 hsa-miR-125b-5p-PR 5 -FAM-TG(pdC)ATA(pdC)GA(pdC)T(pdC)A(pdC)AAGT-ZNA4-BHQ1-3 hsa-miR-155-5p-RT 5 -GTCGTATGCAGTGCAGGGTCCGAGGTATTCGCACTGCATACGACACCCCT-3 hsa-miR-155-5p-F 5 -GCCGCTTAATGCTAATCGTGAT-3 hsa-miR-155-5p-PR 5 -FAM-TG(pdC)ATA(pdC)GA(pdC) A(pdC)C(pdC)(pdC)TAT-BHQ-1-3 miR-Universal-R 5 -GTGCAGGGTCCGAGGTAT-3

pdC: substitution of C-5 propynyl-dC (pdC) for dC is an effective strategy to enhance base pairing. Using these base substitutions, duplex stability and melting temperatures are raised by C-5 propynyl-C 2.8◦ per substitution. Zip nucleic acids (ZNA): ZNA probes provide broad flexibility in assay design and represent an effective alternative to minor groove binder (MGB)- and locked nucleic acid (LNA)-containing oligonucleotides (C. Paris, et al. Zip nucleic acids are potent hydrolysis probes for quantitative PCR. Nucl. Acids Res. (2010). doi: 10.1093/nar/gkp1218). http://nar.oxfordjournals.org/cgi/content/abstract/gkp1218. * http://www.ncbi.nlm.nih.gov/gene. ** http://www.ncbi.nlm.nih.gov/RefSeq/. 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159

was a cross-sectional study and blood sampling was made once from both controls and patients. Total RNA was extracted from Peripheral Whole Blood using Tri-Reagent (Sigma). Reverse transcriptase reactions contained 5 ␮l of extracted total RNA, 50 nM stem–loop RT primer, 1× RT buffer, 0.25 mM each of dNTPs, 50 unit of modified M-MuLV Reverse Transcriptase (Thermo Scientific, Vilnius, Lithuania), 25 unit of RiboLock RNase inhibitor (Thermo Scientific, Vilnius, Lithuania) and nuclease-free water to a total reaction volume of 15 ␮l. The reaction was performed on an automated Thermal Cycler (Techne Flexigene, Cambridge, UK). RT-PCR conditions for 30 min at 16 ◦ C, 30 min at 42 ◦ C, 5 min at 85 ◦ C and then held at 4◦ . QuantitativeComparative CT (CT ) Real-time PCR was performed in an ABI Prism 7500 Real-Time PCR System (Applied Biosystems) using the SDS 2.0.6 software. The specific primers and fluorogenic ZNATM probes for the microRNAs were designed using Primer Express 3.0 software (Applied Biosystems) and are listed in Table 2. The hsamiR-26b-5p is used as control according to the Applied Biosystems application note cms 044972 (Applied Biosystems: http://www3. appliedbiosystems.com/cms/groups/mcb marketing/documents/ generaldocuments/cms 044972.pdf). The mixed RNAs generated from the control group was used as a Reference RNA sample. Primers and probes were purchased from Metabion International AG, D-82152 Martinsried/Deutschland. The 25 ␮l PCR included 3 ␮l RT-PCR product, 12.5 ␮l of 2× TaqMan Universal PCR Master Mix (Applied Biosystems), 900 nmol of each primer (Primer F and Universal Primer R) and 200 nmol TaqMan® probe. The reactions were incubated in a 96-well plate of preincubation at 50 ◦ C for 2 min and at 95 ◦ C for 10 min, followed by 40 cycles at 95 ◦ C for 15 s and at 60 ◦ C for 90 s. Amplifications and analysis were performed in an ABI Prism 7500 Real-Time PCR System (Applied Biosystems), using the SDS 2.0.6 software for allelic discrimination (Applied Biosystems). All reactions were run in triplicate. Relative expression of miRNA was calculated using 2−ct method. Higher ct values indicate lower expression rates.

The data were processed and analyzed using the statistical package SPSS-11.5 for Windows. Normality of 2−DDCT values was checked by Shapiro Wilk test. In case of non-normal distribution, 2−DDCT values were expressed as median, first quartile (25th percentile), third quartile (75th percentile), and the comparison between groups was performed using the Mann–Whitney test. Box-plot graph was used to represent data distribution of miR18a5p, miR22-3p, miR24-3p, miR106b-5p, miR107, miR125b-5p and miR155a-5p variables according to the groups. Significant differences (two-tailed p) less than 0.05 were regarded as significant. 3. Results This study included 52 ADHD subjects with a mean age of 10.09 ± 2.36 years (range, 7–17 years) and 52 control subjects with a mean age of 10.92 ± 2.96 years (range, 7–17 years). No statistically significant differences were detected in age and sex between the two groups (p > 0.05; Table 3). The levels of MiR18a-5p, miR22-3p, miR24-3p, miR106b-5p, and miR107 were significantly decreased in the ADHD subjects compared with controls (p < 0.05). Additionally, decreased levels of miR-125b levels were observed, but this trend was not statistically significant (p > 0.05). MiR155a-5p levels were increased in the patient group (p < 0.05; Table 4). The receiver operator characteristic (ROC) graph was drawn for miR107. The positive predictive value (PPV) and negative predictive value of miR107 was estimated for the cutoff point of 0.4480. PPV was 70% and NPV was 86.5% for the taken cut off point. 4. Discussion To our knowledge, this is the first study to evaluate miRNA levels in ADHD subjects; therefore, we compared our results with those in other psychiatric disorders. We found decreased levels of miR18a-5p, miR22-3p, miR24-3p, miR106b-5p, and miR107 in ADHD subjects compared with controls. In a study examining

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4 Table 3 Sociodemographic and clinical characteristic of patients.

ADHD (n = 52) Sex: male/female (n) Age: mean ± SD (year) Age range(year) ADHD subtypes (N) Attention deficit Hyperactivity/impulsivity Combined

42/10 10.09 ± 2.36 7–17

Control (n = 52)

Comparison p = 0.174 (x2 = 1.846) p = 0.119 (t = −1.574)

36/16 10.92 ± 2.96 7–17

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Table 4 Comparison of miRNA levels* . Controls

hsa-miR-18a-5p hsa-miR22-3p hsa-miR-24-3p hsa-miR-106b-5p hsa-miR-107 hsa-125b-5p hsa-miR-155-5p *

191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231

Patients

p Value

Median

25–75%

Median

25–75%

2.1366 0.7884 1.1988 1.7052 1.8769 1.9763 0.3331

1.3001–3.6987 0.2737–1.9878 0.5765–2.1922 1.1714–2.7165 0.7394–3.6685 0.9575–4.6276 0.1693–0.7744

0.3264 0.1939 0.4239 0.7376 0.2162 1.7163 0.9312

0.1266–1.1465 0.0850–0.8438 0.1951–1.9272 0.3246–1.9879 0.0569–0.5804 0.7222–6.4082 0.2169–2.5472