ADHD Atten Def Hyp Disord (2014) 6:19–23 DOI 10.1007/s12402-013-0123-9
ORIGINAL ARTICLE
Evidence of association between SNAP25 gene and attention deficit hyperactivity disorder in a Latin American sample Jubby M. Ga´lvez • Diego A. Forero • Dora J. Fonseca Heidi E. Mateus • Claudia Talero-Gutierrez • Alberto Velez-van-Meerbeke
•
Received: 5 July 2013 / Accepted: 9 December 2013 / Published online: 23 December 2013 Ó Springer-Verlag Wien 2013
Abstract Attention deficit hyperactivity disorder (ADHD) is one of the most highly heritable behavioral disorders in childhood, with heritability estimates between 60 and 90 %. Family, twin and adoption studies have indicated a strong genetic component in the susceptibility to ADHD. The synaptosomal-associated protein of molecular weight 25 kDa (SNAP25) is a plasma membrane protein known to be involved in synaptic and neural plasticity. Animal model studies have shown that SNAP25 gene is responsible for hyperkinetic behavior in the coloboma mouse. In recent studies, several authors reported an association between SNAP25 and ADHD. In this study, we used a case–control approach to analyze the possible association of two polymorphisms of SNAP25 for possible association with ADHD in a sample of 73 cases and 152 controls in a Colombian children population. Polymorphisms are located in 30 untranslated region of SNAP25, positions T1065G and T1069C. We found a significant association with the GT haplotype (rs3746554|rs1051312) of SNAP25 (p = 0.001). Evidence of association was also found for the G/G genotype of rs3746554 (p = 0.002) and C/C genotype of rs1051312 (p = 0.009). This is the first J. M. Ga´lvez C. Talero-Gutierrez A. Velez-van-Meerbeke (&) Neuroscience Department and Research Group (NeURos), School of Medicine and Health Sciences, Universidad del Rosario, Carrera 24 N63C-69, Bogota´, Colombia e-mail: [email protected] D. A. Forero School of Medicine, Universidad Antonio Narin˜o, Bogota´, Colombia D. J. Fonseca H. E. Mateus Genetic Department, School of Medicine and Health Sciences, Universidad del Rosario, Bogota´, Colombia
study in a Latin American population. Similar to other studies, we found evidence of the association of SNAP25 and ADHD. Keywords ADHD Genotype Haplotype Polymorphism rs3746554 rs1051312 SNAP25 30 UTR
Introduction Attention deficit hyperactivity disorder (ADHD) is a common psychiatric condition of childhood, characterized by persistent symptoms of inattention, hyperactivity and impulsivity. Its worldwide prevalence among children varies from 8 to 12 % (Biederman and Faraone 2005; Faraone et al. 2003) worldwide with a high social and educational morbidity. Three different subtypes are recognized according to the Diagnostic and Statistical Manual V (DSM-V) as follow: predominantly inattentive presentation, predominantly hyperactive-impulsive presentation and combined presentation, among which hyperactiveimpulsive subtype is the least prevalent (Svenaeus 2013). Environmental and genetic factors are known to be involved in the etiology of ADHD, with high heritability estimates (from 60 to 90 %) (Coolidge et al. 2000; Gizer et al. 2009). Therefore, genetic association studies have proposed candidate genes related to the etiology of ADHD, including genes involved in dopaminergic and serotoninergic pathways, as well as genes involved in neurotransmission and neuroplasticity (Faraone et al. 2005). Among several candidate genes, SNAP25 has increasingly been studied due to its role in neural plasticity. This gene encodes the synaptosomal-associated protein, 25 kDa, a presynaptic plasma membrane protein involved in synaptic
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20
vesicle exocytosis. This protein forms a core complex with synaptobrevin and syntaxin called the SNARE-complex (receptor associated with the presynaptic plasma membrane), which bridges the synaptic vesicle and plasma membranes which in turn promote the exocytosis of neurotransmitter into the synaptic cleft (Bark and Wilson 1994; Su¨dhof 1995). The first study suggesting SNAP25 as a gene involved in ADHD pathogenesis was performed in coloboma mice. These are characterized by having a heterozygous semidominant mutation that comprises a contiguous gene deletion, including SNAP25 among other genes. This deletion results in 50 % decrease in gene expression in the central nervous system. In consequence, these mice exhibited a behavioral deficit, mainly locomotor hyperactivity (Steffensen et al. 1996). Further studies demonstrated the role of SNAP25 in this phenotype, with a transgenic insertion of the gene that decreased the hyperactivity (Hess et al. 1996). Since then, multiples studies have attempted to demonstrate the relationship between SNAP25 and ADHD. Barr et al. (2000) were the first to find evidence of linkage between ADHD and SNAP25 in humans, in a sample of 127 nuclear families with 122 children with ADHD. They identified two SNPs located in the 30 untranslated region (UTR) of SNAP25. Using a transmission disequilibrium test (TDT), they found a significant biased transmission of the TC haplotype, suggesting a role of these polymorphisms in the susceptibility to ADHD. Further studies by Kustanivich et al. (2003) in a larger sample size did not replicate Barr’s results, although they showed a similar trend. Furthermore, a meta-analysis for genetics of ADHD that included 75 studies for eight common variants in five candidate genes, demonstrated a significant association between ADHD and the SNP rs3746544 in SNAP25: A pooled OR of 1.15 for the A allele, including data from seven studies (Forero et al. 2009). Other polymorphisms in SNAP25 have also being associated with ADHD, with contradictory results across different populations and studies (Renner et al. 2008; Zhang et al. 2011; Sarkar et al. 2012). The study of a short tandem repeat located in the 50 UTR, using a case–control study, performed by Zhang et al. (2011) did not detect a significant association, although the TDT did show a trend toward significance. In the same study, they investigated two other SNPs (rs362549 and rs363006) which were not associated in this sample with ADHD or its subtypes. Another study confirmed the association of additional variants (rs8636 and rs362988) with ADHD subtypes and it replicated positive results for previously reported SNPs (Sarkar et al. 2012). SNPs rs362990 and an haplotype of rs6108461, rs362990 and rs362998 have also been found to be associated with ADHD and also related to a decrease expression of SNAP25 transcript (Hawi et al. 2013).
123
Conversely, a study performed by Renner et al. (2008) did not show a significant preferential allele transmission to children with ADHD in a TDT of three different SNPs in SNAP25 (rs6077690, rs6039769 and rs36006). Some analyses as Pazvantoglu’s study (2013) have attempted to evaluate the role of several genes in ADHD susceptibility, indicating that not only SNAP25 is related to ADHD, but a combination of genes can act as risk factors in the pathophysiology of the disorder. In this study, we used a case–control approach to analyze a possible association of two polymorphisms (rs3746544 and rs10511312) in SNAP25 with ADHD in a sample of 73 cases and 152 controls from the Colombian children population.
Methods Study subjects Cases and controls were selected from an observational study carried out in public and private schools in Bogota´, Colombia (South America) (Ve´lez-van-Meerbeke et al. 2012). Cases were elected among children who had positive symptoms for ADHD according to the DSM-IV checklist (validated in Colombia), and if they were suspected to have ADHD according to questionnaires applied to teachers and parents. The diagnosis was confirmed applying the behavior assessment system for children (BASC) scale. Because of a low concordance in parent/ teachers scores, we separately analyzed the attention and hyperactivity scores of parents and teachers for the final sample. Therefore, children included as cases had at least one of the following: (1) children with a DSM-IV checklist applied to parents with C6 criteria of inattention and a percentile C85 in the attention score of BASC applied to parents or teachers or (2) children with a DSMIV checklist applied to parents with C6 criteria of hyperactivity and a percentile C85 in the hyperactivity score of BASC applied to parents or teachers. The Wechsler Intelligence Scale for Children-Revised was applied to all subjects, and children with a score below 70 were excluded from the study. Those children with neurological disorders such as a cognitive disability (i.e., mental retardation), cerebral palsy, severe sensory handicaps such as blindness and deafness, Tourette syndrome or psychiatric disorders were also excluded. Controls were subjects who did not have symptoms of ADHD and did not meet the criteria for ADHD. In total, 73 cases and 152 controls were recruited. This study was approved by the institutional ethical committee. Parents of patients and controls provided written informed consent.
Hyperactivity disorder in a Latin American sample
Genotyping Genomic DNA was extracted from samples of peripheral blood or oral mucosa, using standard techniques. Extracted DNA was quantified using a Nanodrop (Thermo Scientific, Wilmington, USA), and polymerase chain reaction– restriction fragment length polymorphism (PCR–RFLP) techniques were performed. The region of interest was amplified using the primers: 50 -TTCTCCTCCAA ATGCTGTCG-30 and 50 -CCACCGAGGAGAGAAAA TGAAA-30 . PCR conditions were as follow: 6.25 lL of master mix (Promega Madison, USA), 0.5 lL of each forward and reverse primers, 150 ng of DNA and 5.25 lL of clean water. PCR program was: 94 °C for 5 min, followed by 30 cycles of 94 °C for 45 s, 61 °C for 45 s and 72 °C for 45 s and a final step of 10 min at 72 °C (Agilent Thermocycler). Restriction enzyme MnlI was used to genotype the SNP rs3746544 (T1065G), and restriction enzyme DdeI was used for the SNP rs1051312 (T1069C). Conditions of both enzymatic reactions were as follow: 1 ll of the enzyme, 2 ll of the buffer, 4 ll of the PCR product and 9 ll of clean water. Product digestion was performed incubating the mix at 37 °C overnight. Digestion products were run on 1.2 % agarose gels, and genotypes were called by two independent researchers. Re-genotyping of 10 % of samples chosen randomly was performed as a quality check. Data analysis Hardy–Weinberg equilibrium was checked for the studied genetic markers. Genotype and allelic frequencies were obtained using the statistic package SNPstats (Sole´ et al. 2006). Association analysis were performed using the genetic statistic program PLINK (Purcell et al. 2007). Statistical significance was considered at p value \0.05.
Results Seventy-three cases with ages between 6 and 10 years, and 152 controls were included. Cases were classified according to the DSM-IV criteria in ADHD subtypes (Table 1). Cases were classified as combined subtype (50.7 %, n = 37), inattentive subtype (35.6 %, n = 26) and hyperactive-impulsive subtype (13.7 %, n = 10). A case–control study was performed for two common variants in the 30 UTR region of SNAP25, located in positions T1065G and T1069C (rs3746554 and rs1051312) in 73 cases and 152 controls in a Colombian sample. The association analysis was carried out for single markers and for haplotypes. Both SNPs were found to be in Hardy– Weinberg Equilibrium (p = 0.33 and p = 0.84 for
21 Table 1 Demographic variables in ADHD cases and controls Variables
Cases n = 73
Controls n = 152
Age mean (SD)
8.6 (2.3)
16.9 (7.1)
Female
20.5
50.7
Male
79.5
49.3
Combined
50.7
NA
Predominately inattentive
35.6
Predominately hyperactive
13.7
Gender (%)
ADHD subtype (%)
NA not applicable
rs3746554 and rs1051312, respectively). G/G genotype of rs3746554 was found to be nominally associated with ADHD (p = 0.0025) in boys and girls, but it was only statistically significant in boys after correction for multiple testing. C/C genotype of rs1051312 (p = 0.009) was also associated in the general sample but not after gender stratification. Haplotype analysis demonstrated that GT and TC haplotypes (rs3746554|rs1051312) were significantly associated with ADHD (p = 0.001 and p = 0.050, respectively) (Table 2). The GC haplotype was not found in our sample. The same analysis was also performed for all three ADHD subtypes. Association of rs3746554 with the combined subtype with genotype (p = 0.006) and GT haplotype (p = 0.0013) was found, as well as an association of the same SNP with hyperactivity subtype (p = 0.025). We found no association of this SNP and inattention.
Discussion Association studies relating SNAP25 to ADHD susceptibility have been inconsistent. It is possible that differences in genetic background could explain discrepancies in positive results for individual SNPs between this study in a Latin American sample and previous negative works (carried out in populations of European descent). Also, discrepant results may be due to the study of different variants. A recent meta-analysis (Forero et al. 2009) evaluated two previously studied SNPs in the 30 UTR of SNAP25 in association with certain subtypes of ADHD, confirming their role in this disorder (a pooled OR of 1.15 for the A allele of rs3746544 was found, including data from seven studies). In the present study, an association of the individual SNPs rs3746554 and rs1051312 with ADHD was detected, indicating that these SNPs may confer a risk to develop ADHD. In the same way, haplotypes GT and TC were found to increase
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22 Table 2 Comparison of SNAP25 allelic and genotypic frequencies in ADHD cases and controls Polymorphism
Allele and genotype
Cases (n = 73)
Controls (n = 152)
p value*
HEW p value*
rs3746544
T
0.81
0.66
0.003a
0.331
G
0.19
0.34
G/G
0.06
0.12
rs1051312
Haplotype rs3746544|rs1051312
a
0.002a
G/T
0.28
0.44
T/T
0.67
0.44
T
0.72
0.8
C
0.28
0.2
C/C
0.1
0.02
C/T
0.36
0.35
T/T
0.54
0.63
TC GT
0.277 0.194
0.181 0.372
0.050 0.001
TT
0.527
0.445
0.229
0.050a
0.846
0.009a
NA
Bonferroni correction
* p value \0.05 considered significant HWE Hardy–Weinberg equilibrium, NA not applicable
susceptibility to this disorder. These results replicate the findings of Barr et al. (2000) and Kustanovich et al. (2003) who proposed the GT haplotype as a risk haplotype in the development of this disorder, in two different studies. Together, these results support the idea that variants in SNAP25 may play a role in the development of ADHD. In this study, the analysis of only two SNPs was performed since the inclusion of a greater number of SNPs in SNAP25 gene has not demonstrated to give additional information compared to the widely analyzed markers located in the 30 UTR region. Hawi et al. (2013) recently analyzed fifteen SNPs in this gene and found association in only three of them, forming a haplotype block. Therefore, a greater number of SNPs are not necessary in this gene in order to find association. It is important to note that these SNPs are located in the 30 UTR of the gene. Therefore, it is possible that they could be in linkage with some other variants that alter by themselves gene expression or function. However, KovacsNagy et al. (2011) suggested that the mechanism by which these polymorphisms may contribute to the disease pathology is because they may alter the function of microRNAs (miRNAs). These SNPs are located in the binding site of a miRNA (miR-641). In the mentioned study, the authors show that TT haplotype provides a perfect binding of this miRNA, whereas GT and TC haplotypes generate a single mismatch. Recently, Chang et al. (2012) performed a prioritization of genes using a computational analysis of multiple data sources. They found sixteen candidate genes as promising ADHD genes. One of these genes was STX1A which is
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strongly correlated with SNAP25 because of their role in the synaptic function. These results demonstrated that genes related to nervous system development and gene– gene interactions form an important network that may contribute to ADHD in a combination pattern that deserves more consideration. Therefore, our study contributes to support this and other similar findings in relation to SNAP25. Although our results confirm the importance of SNAP25 in ADHD, we are aware that this gene is only one of many possibly contributing to susceptibility for this disorder. Some authors have proposed that SNAP25 plays a minor role in the general disorder but has a greater impact in some ADHD subtypes (Renner et al. 2008). Our subgroup analysis showed that SNAP25 was related mainly to hyperactivity subtype, which is in concordance with the coloboma mouse model. This result was confirmed despite a small sample size in this subgroup. Nevertheless, published studies on SNAP25 and ADHD have showed a large heterogeneity in subtype composition, for example, percentage of included inattentive cases ranged from 2 to 42 % (Forero et al. 2009). Recent studies have supported the clinical relevance of finding these gene variants for risk prediction and early treatment of affected children. A recent study demonstrated the high sensitivity and specificity of a prediction model of ADHD based on expression level of four genes, including SNAP25 (Gru¨nblatt et al. 2012). A combination of expression levels of four genes (SLC6A3, DRD5, TPH1 and SNAP25) provides a sensitivity and specificity over 80 %. Since our study is the first in a Latin America population, it gives the opportunity to further study
Hyperactivity disorder in a Latin American sample
SNAP25 and other associated genes through functional studies, in order to propose them as biomarkers than can be used in our population. Limitations of our study include a small sample size and a low sample’s power (0.645). Therefore, for further studies, it is recommended to increase the sample size. These two SNPs in SNAP25 gene are the most commonly studied, and the consolidation of large ADHD samples is a considerable challenge in developing countries.
Conclusion To our knowledge, this is the first study to demonstrate an association of SNAP25 and ADHD in a Latin American population. Our study, in conjunction with other similar reports, opens the gate to medical translational of genetic variants in SNAP25. Further studies supporting the influence of SNAP25 and other genes in this population will allow a model of risk prediction of the disorder specific for Latin American populations. Conflict of interest of interest.
The authors declare that they have no conflict
References Bark I, Wilson M (1994) Regulated vesicular fusion in neurons: snapping together the details. Proc Natl Acad Sci USA 91:4621–4624 Barr CL, Feng Y, Wigg K et al (2000) Identification of DNA variants in the SNAP-25 gene and linkage study of these polymorphisms and attention-deficit hyperactivity disorder. Mol Psychiatry 5:405–409 Biederman J, Faraone SV (2005) Attention-deficit hyperactivity disorder. Lancet 366:237–248 Chang S, Zhang W, Gao L, Wang J (2012) Prioritization of candidate genes for attention deficit hyperactivity disorder by computational analysis of multiple data sources. Protein Cell 3:526–534 Coolidge F, Thede L, Young S (2000) Heritability and the comorbidity of attention deficit hyperactivity disorder with behavioral disorders and executive function deficits: a preliminary investigation. Dev Neuropsychol 17:273–287 Faraone SV, Sergeant J, Gillberg C, Biederman J (2003) The worldwide prevalence of ADHD: is it an American condition? World Psychiatry Off J World Psychiatr Assoc WPA 2:104–113 Faraone SV, Perlis RH, Doyle AE et al (2005) Molecular genetics of attention-deficit/hyperactivity disorder. Biol Psychiatry 57:1313–1323
23 Forero DA, Arboleda GH, Vasquez R, Arboleda H (2009) Candidate genes involved in neural plasticity and the risk for attentiondeficit hyperactivity disorder: a meta-analysis of 8 common variants. J psychiatry Neurosci JPN 34:361–366 Gizer IR, Ficks C, Waldman ID (2009) Candidate gene studies of ADHD: a meta-analytic review. Hum Genet 126:51–90 Gru¨nblatt E, Geissler J, Jacob CP et al (2012) Pilot study: potential transcription markers for adult attention-deficit hyperactivity disorder in whole blood. Atten Defic Hyperact Disord 4:77–84 Hawi Z, Matthews N, Wagner J et al (2013) DNA variation in the SNAP25 gene confers risk to ADHD and is associated with reduced expression in prefrontal cortex. PLoS ONE 8:e60274 Hess EJ, Collins KA, Wilson MC (1996) Mouse model of hyperkinesis implicates SNAP-25 in behavioral regulation. J Neurosci 16:3104–3111 Kovacs-Nagy R, Sarkozy P, Hu J et al (2011) Haplotyping of putative microRNA-binding sites in the SNAP-25 gene. Electrophoresis 32:2013–2020 Kustanovich V, Merriman B, McGough J et al (2003) Biased paternal transmission of SNAP-25 risk alleles in attention-deficit hyperactivity disorder. Mol Psychiatry 8:309–315 Pazvantog˘lu O, Gu¨nes¸ S, Karabekirog˘lu K et al (2013) The relationship between the presence of ADHD and certain candidate gene polymorphisms in a Turkish sample. Gene 528:320–327 Purcell S, Neale B, Todd-Brown K et al (2007) PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 81:559–575 Renner TJ, Walitza S, Dempfle A et al (2008) Allelic variants of SNAP25 in a family-based sample of ADHD. J Neural Transm 115:317–321 Sarkar K, Bhaduri N, Ghosh P et al (2012) Role of SNAP25 explored in eastern Indian attention deficit hyperactivity disorder probands. Neurochem Res 37:349–357 Sole´ X, Guino´ E, Valls J et al (2006) SNPStats: a web tool for the analysis of association studies. Bioinformatics 22:1928–1929 Steffensen SC, Wilson MC, Henriksen SJ (1996) Coloboma contiguous gene deletion encompassing Snap alters hippocampal plasticity. Synapse 22:281–289 Su¨dhof TC (1995) Synaptic core complex of synaptobrevin, syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site. J Biol Chem 270:2213–2217 Svenaeus F (2013) Diagnosing mental disorders and saving the normal: American Psychiatric Association, 2013. Diagnostic and statistical manual of mental disorders, 5th edn. American Psychiatric Publishing, Washington, DC, Med Health Care Philos p 991 Ve´lez-van-Meerbeke A, Zamora IP, Guzma´n G et al (2012) Evaluating executive function in schoolchildren with symptoms of attention deficit hyperactivity disorder. Neurologia 28:348–355 Zhang H, Zhu S, Zhu Y et al (2011) An association study between SNAP-25 gene and attention-deficit hyperactivity disorder. Eur J Paediatr Neurol 15:48–52
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ORIGINAL ARTICLE
Evidence of association between SNAP25 gene and attention deficit hyperactivity disorder in a Latin American sample Jubby M. Ga´lvez • Diego A. Forero • Dora J. Fonseca Heidi E. Mateus • Claudia Talero-Gutierrez • Alberto Velez-van-Meerbeke
•
Received: 5 July 2013 / Accepted: 9 December 2013 / Published online: 23 December 2013 Ó Springer-Verlag Wien 2013
Abstract Attention deficit hyperactivity disorder (ADHD) is one of the most highly heritable behavioral disorders in childhood, with heritability estimates between 60 and 90 %. Family, twin and adoption studies have indicated a strong genetic component in the susceptibility to ADHD. The synaptosomal-associated protein of molecular weight 25 kDa (SNAP25) is a plasma membrane protein known to be involved in synaptic and neural plasticity. Animal model studies have shown that SNAP25 gene is responsible for hyperkinetic behavior in the coloboma mouse. In recent studies, several authors reported an association between SNAP25 and ADHD. In this study, we used a case–control approach to analyze the possible association of two polymorphisms of SNAP25 for possible association with ADHD in a sample of 73 cases and 152 controls in a Colombian children population. Polymorphisms are located in 30 untranslated region of SNAP25, positions T1065G and T1069C. We found a significant association with the GT haplotype (rs3746554|rs1051312) of SNAP25 (p = 0.001). Evidence of association was also found for the G/G genotype of rs3746554 (p = 0.002) and C/C genotype of rs1051312 (p = 0.009). This is the first J. M. Ga´lvez C. Talero-Gutierrez A. Velez-van-Meerbeke (&) Neuroscience Department and Research Group (NeURos), School of Medicine and Health Sciences, Universidad del Rosario, Carrera 24 N63C-69, Bogota´, Colombia e-mail: [email protected] D. A. Forero School of Medicine, Universidad Antonio Narin˜o, Bogota´, Colombia D. J. Fonseca H. E. Mateus Genetic Department, School of Medicine and Health Sciences, Universidad del Rosario, Bogota´, Colombia
study in a Latin American population. Similar to other studies, we found evidence of the association of SNAP25 and ADHD. Keywords ADHD Genotype Haplotype Polymorphism rs3746554 rs1051312 SNAP25 30 UTR
Introduction Attention deficit hyperactivity disorder (ADHD) is a common psychiatric condition of childhood, characterized by persistent symptoms of inattention, hyperactivity and impulsivity. Its worldwide prevalence among children varies from 8 to 12 % (Biederman and Faraone 2005; Faraone et al. 2003) worldwide with a high social and educational morbidity. Three different subtypes are recognized according to the Diagnostic and Statistical Manual V (DSM-V) as follow: predominantly inattentive presentation, predominantly hyperactive-impulsive presentation and combined presentation, among which hyperactiveimpulsive subtype is the least prevalent (Svenaeus 2013). Environmental and genetic factors are known to be involved in the etiology of ADHD, with high heritability estimates (from 60 to 90 %) (Coolidge et al. 2000; Gizer et al. 2009). Therefore, genetic association studies have proposed candidate genes related to the etiology of ADHD, including genes involved in dopaminergic and serotoninergic pathways, as well as genes involved in neurotransmission and neuroplasticity (Faraone et al. 2005). Among several candidate genes, SNAP25 has increasingly been studied due to its role in neural plasticity. This gene encodes the synaptosomal-associated protein, 25 kDa, a presynaptic plasma membrane protein involved in synaptic
123
J. M. Ga´lvez et al.
20
vesicle exocytosis. This protein forms a core complex with synaptobrevin and syntaxin called the SNARE-complex (receptor associated with the presynaptic plasma membrane), which bridges the synaptic vesicle and plasma membranes which in turn promote the exocytosis of neurotransmitter into the synaptic cleft (Bark and Wilson 1994; Su¨dhof 1995). The first study suggesting SNAP25 as a gene involved in ADHD pathogenesis was performed in coloboma mice. These are characterized by having a heterozygous semidominant mutation that comprises a contiguous gene deletion, including SNAP25 among other genes. This deletion results in 50 % decrease in gene expression in the central nervous system. In consequence, these mice exhibited a behavioral deficit, mainly locomotor hyperactivity (Steffensen et al. 1996). Further studies demonstrated the role of SNAP25 in this phenotype, with a transgenic insertion of the gene that decreased the hyperactivity (Hess et al. 1996). Since then, multiples studies have attempted to demonstrate the relationship between SNAP25 and ADHD. Barr et al. (2000) were the first to find evidence of linkage between ADHD and SNAP25 in humans, in a sample of 127 nuclear families with 122 children with ADHD. They identified two SNPs located in the 30 untranslated region (UTR) of SNAP25. Using a transmission disequilibrium test (TDT), they found a significant biased transmission of the TC haplotype, suggesting a role of these polymorphisms in the susceptibility to ADHD. Further studies by Kustanivich et al. (2003) in a larger sample size did not replicate Barr’s results, although they showed a similar trend. Furthermore, a meta-analysis for genetics of ADHD that included 75 studies for eight common variants in five candidate genes, demonstrated a significant association between ADHD and the SNP rs3746544 in SNAP25: A pooled OR of 1.15 for the A allele, including data from seven studies (Forero et al. 2009). Other polymorphisms in SNAP25 have also being associated with ADHD, with contradictory results across different populations and studies (Renner et al. 2008; Zhang et al. 2011; Sarkar et al. 2012). The study of a short tandem repeat located in the 50 UTR, using a case–control study, performed by Zhang et al. (2011) did not detect a significant association, although the TDT did show a trend toward significance. In the same study, they investigated two other SNPs (rs362549 and rs363006) which were not associated in this sample with ADHD or its subtypes. Another study confirmed the association of additional variants (rs8636 and rs362988) with ADHD subtypes and it replicated positive results for previously reported SNPs (Sarkar et al. 2012). SNPs rs362990 and an haplotype of rs6108461, rs362990 and rs362998 have also been found to be associated with ADHD and also related to a decrease expression of SNAP25 transcript (Hawi et al. 2013).
123
Conversely, a study performed by Renner et al. (2008) did not show a significant preferential allele transmission to children with ADHD in a TDT of three different SNPs in SNAP25 (rs6077690, rs6039769 and rs36006). Some analyses as Pazvantoglu’s study (2013) have attempted to evaluate the role of several genes in ADHD susceptibility, indicating that not only SNAP25 is related to ADHD, but a combination of genes can act as risk factors in the pathophysiology of the disorder. In this study, we used a case–control approach to analyze a possible association of two polymorphisms (rs3746544 and rs10511312) in SNAP25 with ADHD in a sample of 73 cases and 152 controls from the Colombian children population.
Methods Study subjects Cases and controls were selected from an observational study carried out in public and private schools in Bogota´, Colombia (South America) (Ve´lez-van-Meerbeke et al. 2012). Cases were elected among children who had positive symptoms for ADHD according to the DSM-IV checklist (validated in Colombia), and if they were suspected to have ADHD according to questionnaires applied to teachers and parents. The diagnosis was confirmed applying the behavior assessment system for children (BASC) scale. Because of a low concordance in parent/ teachers scores, we separately analyzed the attention and hyperactivity scores of parents and teachers for the final sample. Therefore, children included as cases had at least one of the following: (1) children with a DSM-IV checklist applied to parents with C6 criteria of inattention and a percentile C85 in the attention score of BASC applied to parents or teachers or (2) children with a DSMIV checklist applied to parents with C6 criteria of hyperactivity and a percentile C85 in the hyperactivity score of BASC applied to parents or teachers. The Wechsler Intelligence Scale for Children-Revised was applied to all subjects, and children with a score below 70 were excluded from the study. Those children with neurological disorders such as a cognitive disability (i.e., mental retardation), cerebral palsy, severe sensory handicaps such as blindness and deafness, Tourette syndrome or psychiatric disorders were also excluded. Controls were subjects who did not have symptoms of ADHD and did not meet the criteria for ADHD. In total, 73 cases and 152 controls were recruited. This study was approved by the institutional ethical committee. Parents of patients and controls provided written informed consent.
Hyperactivity disorder in a Latin American sample
Genotyping Genomic DNA was extracted from samples of peripheral blood or oral mucosa, using standard techniques. Extracted DNA was quantified using a Nanodrop (Thermo Scientific, Wilmington, USA), and polymerase chain reaction– restriction fragment length polymorphism (PCR–RFLP) techniques were performed. The region of interest was amplified using the primers: 50 -TTCTCCTCCAA ATGCTGTCG-30 and 50 -CCACCGAGGAGAGAAAA TGAAA-30 . PCR conditions were as follow: 6.25 lL of master mix (Promega Madison, USA), 0.5 lL of each forward and reverse primers, 150 ng of DNA and 5.25 lL of clean water. PCR program was: 94 °C for 5 min, followed by 30 cycles of 94 °C for 45 s, 61 °C for 45 s and 72 °C for 45 s and a final step of 10 min at 72 °C (Agilent Thermocycler). Restriction enzyme MnlI was used to genotype the SNP rs3746544 (T1065G), and restriction enzyme DdeI was used for the SNP rs1051312 (T1069C). Conditions of both enzymatic reactions were as follow: 1 ll of the enzyme, 2 ll of the buffer, 4 ll of the PCR product and 9 ll of clean water. Product digestion was performed incubating the mix at 37 °C overnight. Digestion products were run on 1.2 % agarose gels, and genotypes were called by two independent researchers. Re-genotyping of 10 % of samples chosen randomly was performed as a quality check. Data analysis Hardy–Weinberg equilibrium was checked for the studied genetic markers. Genotype and allelic frequencies were obtained using the statistic package SNPstats (Sole´ et al. 2006). Association analysis were performed using the genetic statistic program PLINK (Purcell et al. 2007). Statistical significance was considered at p value \0.05.
Results Seventy-three cases with ages between 6 and 10 years, and 152 controls were included. Cases were classified according to the DSM-IV criteria in ADHD subtypes (Table 1). Cases were classified as combined subtype (50.7 %, n = 37), inattentive subtype (35.6 %, n = 26) and hyperactive-impulsive subtype (13.7 %, n = 10). A case–control study was performed for two common variants in the 30 UTR region of SNAP25, located in positions T1065G and T1069C (rs3746554 and rs1051312) in 73 cases and 152 controls in a Colombian sample. The association analysis was carried out for single markers and for haplotypes. Both SNPs were found to be in Hardy– Weinberg Equilibrium (p = 0.33 and p = 0.84 for
21 Table 1 Demographic variables in ADHD cases and controls Variables
Cases n = 73
Controls n = 152
Age mean (SD)
8.6 (2.3)
16.9 (7.1)
Female
20.5
50.7
Male
79.5
49.3
Combined
50.7
NA
Predominately inattentive
35.6
Predominately hyperactive
13.7
Gender (%)
ADHD subtype (%)
NA not applicable
rs3746554 and rs1051312, respectively). G/G genotype of rs3746554 was found to be nominally associated with ADHD (p = 0.0025) in boys and girls, but it was only statistically significant in boys after correction for multiple testing. C/C genotype of rs1051312 (p = 0.009) was also associated in the general sample but not after gender stratification. Haplotype analysis demonstrated that GT and TC haplotypes (rs3746554|rs1051312) were significantly associated with ADHD (p = 0.001 and p = 0.050, respectively) (Table 2). The GC haplotype was not found in our sample. The same analysis was also performed for all three ADHD subtypes. Association of rs3746554 with the combined subtype with genotype (p = 0.006) and GT haplotype (p = 0.0013) was found, as well as an association of the same SNP with hyperactivity subtype (p = 0.025). We found no association of this SNP and inattention.
Discussion Association studies relating SNAP25 to ADHD susceptibility have been inconsistent. It is possible that differences in genetic background could explain discrepancies in positive results for individual SNPs between this study in a Latin American sample and previous negative works (carried out in populations of European descent). Also, discrepant results may be due to the study of different variants. A recent meta-analysis (Forero et al. 2009) evaluated two previously studied SNPs in the 30 UTR of SNAP25 in association with certain subtypes of ADHD, confirming their role in this disorder (a pooled OR of 1.15 for the A allele of rs3746544 was found, including data from seven studies). In the present study, an association of the individual SNPs rs3746554 and rs1051312 with ADHD was detected, indicating that these SNPs may confer a risk to develop ADHD. In the same way, haplotypes GT and TC were found to increase
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22 Table 2 Comparison of SNAP25 allelic and genotypic frequencies in ADHD cases and controls Polymorphism
Allele and genotype
Cases (n = 73)
Controls (n = 152)
p value*
HEW p value*
rs3746544
T
0.81
0.66
0.003a
0.331
G
0.19
0.34
G/G
0.06
0.12
rs1051312
Haplotype rs3746544|rs1051312
a
0.002a
G/T
0.28
0.44
T/T
0.67
0.44
T
0.72
0.8
C
0.28
0.2
C/C
0.1
0.02
C/T
0.36
0.35
T/T
0.54
0.63
TC GT
0.277 0.194
0.181 0.372
0.050 0.001
TT
0.527
0.445
0.229
0.050a
0.846
0.009a
NA
Bonferroni correction
* p value \0.05 considered significant HWE Hardy–Weinberg equilibrium, NA not applicable
susceptibility to this disorder. These results replicate the findings of Barr et al. (2000) and Kustanovich et al. (2003) who proposed the GT haplotype as a risk haplotype in the development of this disorder, in two different studies. Together, these results support the idea that variants in SNAP25 may play a role in the development of ADHD. In this study, the analysis of only two SNPs was performed since the inclusion of a greater number of SNPs in SNAP25 gene has not demonstrated to give additional information compared to the widely analyzed markers located in the 30 UTR region. Hawi et al. (2013) recently analyzed fifteen SNPs in this gene and found association in only three of them, forming a haplotype block. Therefore, a greater number of SNPs are not necessary in this gene in order to find association. It is important to note that these SNPs are located in the 30 UTR of the gene. Therefore, it is possible that they could be in linkage with some other variants that alter by themselves gene expression or function. However, KovacsNagy et al. (2011) suggested that the mechanism by which these polymorphisms may contribute to the disease pathology is because they may alter the function of microRNAs (miRNAs). These SNPs are located in the binding site of a miRNA (miR-641). In the mentioned study, the authors show that TT haplotype provides a perfect binding of this miRNA, whereas GT and TC haplotypes generate a single mismatch. Recently, Chang et al. (2012) performed a prioritization of genes using a computational analysis of multiple data sources. They found sixteen candidate genes as promising ADHD genes. One of these genes was STX1A which is
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strongly correlated with SNAP25 because of their role in the synaptic function. These results demonstrated that genes related to nervous system development and gene– gene interactions form an important network that may contribute to ADHD in a combination pattern that deserves more consideration. Therefore, our study contributes to support this and other similar findings in relation to SNAP25. Although our results confirm the importance of SNAP25 in ADHD, we are aware that this gene is only one of many possibly contributing to susceptibility for this disorder. Some authors have proposed that SNAP25 plays a minor role in the general disorder but has a greater impact in some ADHD subtypes (Renner et al. 2008). Our subgroup analysis showed that SNAP25 was related mainly to hyperactivity subtype, which is in concordance with the coloboma mouse model. This result was confirmed despite a small sample size in this subgroup. Nevertheless, published studies on SNAP25 and ADHD have showed a large heterogeneity in subtype composition, for example, percentage of included inattentive cases ranged from 2 to 42 % (Forero et al. 2009). Recent studies have supported the clinical relevance of finding these gene variants for risk prediction and early treatment of affected children. A recent study demonstrated the high sensitivity and specificity of a prediction model of ADHD based on expression level of four genes, including SNAP25 (Gru¨nblatt et al. 2012). A combination of expression levels of four genes (SLC6A3, DRD5, TPH1 and SNAP25) provides a sensitivity and specificity over 80 %. Since our study is the first in a Latin America population, it gives the opportunity to further study
Hyperactivity disorder in a Latin American sample
SNAP25 and other associated genes through functional studies, in order to propose them as biomarkers than can be used in our population. Limitations of our study include a small sample size and a low sample’s power (0.645). Therefore, for further studies, it is recommended to increase the sample size. These two SNPs in SNAP25 gene are the most commonly studied, and the consolidation of large ADHD samples is a considerable challenge in developing countries.
Conclusion To our knowledge, this is the first study to demonstrate an association of SNAP25 and ADHD in a Latin American population. Our study, in conjunction with other similar reports, opens the gate to medical translational of genetic variants in SNAP25. Further studies supporting the influence of SNAP25 and other genes in this population will allow a model of risk prediction of the disorder specific for Latin American populations. Conflict of interest of interest.
The authors declare that they have no conflict
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