Journal of the Neurological Sciences 337 (2014) 176–179
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Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
A functional polymorphism in the promoter region of MAOA gene is associated with daytime sleepiness in healthy subjects Diego A. Ojeda a, Carmen L. Niño b, Sandra López-León c, Andrés Camargo b, Ana Adan d, Diego A. Forero a,⁎ a
Laboratory of Neuropsychiatric Genetics, Biomedical Sciences Research Group, School of Medicine, Universidad Antonio Nariño, Bogotá, Colombia School of Nursing, Universidad de Ciencias Aplicadas y Ambientales UDCA, Bogotá, Colombia Novartis Pharmaceuticals, Health Plaza, East Hanover, NJ, USA d Department of Psychiatry and Clinical Psychobiology, School of Psychology, University of Barcelona, Barcelona, Spain b c
a r t i c l e
i n f o
Article history: Received 16 September 2013 Received in revised form 26 November 2013 Accepted 3 December 2013 Available online 11 December 2013 Keywords: Neurogenetics Latin America Neuropsychiatric genetics Candidate genes Endophenotypes Daytime sleepiness Monoamine Oxidase A
a b s t r a c t Excessive daytime sleepiness (EDS) is one of the main causes of car and industrial accidents and it is associated with increased morbidity and alterations in quality of life. Prevalence of EDS in the general population around the world ranges from 6.2 to 32.4%, with a heritability of 38–40%. However, few studies have explored the role of candidate genes in EDS. Monoamine oxidase A (MAOA) gene has an important role in the regulation of neurotransmitter levels and a large number of human behaviors. We hypothesized that a functional VNTR in the promoter region of the MAOA gene might be associated with daytime sleepiness in healthy individuals. The Epworth sleepiness scale (ESS) was applied to 210 Colombian healthy subjects (university students), which were genotyped for MAOA-uVNTR. MAOA-uVNTR showed a significant association with ESS scores (p = 0.01): 3/3 genotype carriers had the lowest scores. These results were supported by differences in MAOA-uVNTR frequencies between diurnal somnolence categories (p = 0.03). Our finding provides evidence for the first time that MAOA-uVNTR has a significant association with EDS in healthy subjects. Finally, these data suggest that functional variations in MAOA gene could have a role in other phenotypes of neuropsychiatric relevance. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Excessive daytime sleepiness (EDS) is one of the main causes of car and industrial accidents around the world, being associated with decreases in performance at school or work, increased morbidity and alterations in quality of life [1,2]. The Epworth sleepiness scale was created in 1991 as a tool to quantify levels of daytime sleepiness and it has been used in a large number of clinical studies [3,4]. Prevalence of EDS in the general population, defined by the ESS, in different countries ranges from 6.2 to 32.4% [5–17] (Table 1), with a moderate heritability of approximately 38% to 40% [18–21]. This evidence highlights the importance of studying the role of genetic polymorphisms in increased diurnal somnolence [22]. However, very few studies have explored the possible association of candidate genes with differences in daytime sleepiness [23–26]. Sleep is a complex physiological process and it is fundamental to restore and maintain an adequate physical and mental health [27,28]. It is regulated by multiple environmental and molecular mechanisms, such as the circadian system [29]. In addition, many neurotransmitters are involved in the sleep process: norepinephrine, serotonin, dopamine and glutamate, among many others [28,30–32]. ⁎ Corresponding author at: Laboratory of Neuropsychiatric Genetics, School of Medicine, Universidad Antonio Nariño, Bogotá, Colombia. Tel.: +57 313 2610427. E-mail address: [email protected] (D.A. Forero). 0022-510X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jns.2013.12.005
Monoamine oxidase A (MAOA) is known to play a major role in catalyzing the oxidative deamination of norepinephrine, dopamine, and serotonin neurotransmitters [33,34], being a key enzyme for the regulation of monoamine levels. MAOA gene has a size of 90,660 bp, is located on the X chromosome at Xp.11.3–Xp11.4 and is expressed in several brain tissues [34,35]. Different polymorphisms in the MAOA gene have been identified [33–36] and a functional variable-number tandem repeat (VNTR) located in the promoter region of MAOA gene (MAOA-uVNTR) is commonly studied in neuropsychiatric genetics [35,36]. MAOA-uVNTR polymorphism consists of a 30 bp repeat sequence and functional studies have shown that this VNTR affects the transcription of MAOA, indicating that the number of repeats is related to transcriptional activity [35,36]. We hypothesized that a functional VNTR in the promoter region of the MAOA gene might be associated with differences in daytime sleepiness in healthy Colombian subjects.
2. Methods 2.1. Participants Two hundred and ten unrelated young subjects (university students) living in Bogotá, Colombia were included. Participants were recruited in two private Universities, through posters and informative talks. This
D.A. Ojeda et al. / Journal of the Neurological Sciences 337 (2014) 176–179 Table 1 Prevalence of excessive daytime sleepiness (EDS), using the Epworth sleepiness scale, in general populations around the worlda. Author, year
Country
Sample size
Age range
% EDS
Pahwa, 2012 Wu, 2012 Mume, 2011 Joo, 2009 Bouscoulet, 2008 Bouscoulet, 2008 Bouscoulet, 2008 Bouscoulet, 2008 Pallesen, 2007 Sanford, 2006 Gander, 2005 Moo-Estrella, 2005 Soldatos, 2005 Soldatos, 2005 Soldatos, 2005 Soldatos, 2005 Soldatos, 2005 Soldatos, 2005 Soldatos, 2005 Soldatos, 2005 Soldatos, 2005 Soldatos, 2005 Walsleben, 2004 Shin, 2003 Souza, 2002 Average EDS
Canada China Nigeria Korea Chile Mexico Uruguay Venezuela Norway USA New Zealand Mexico Austria Belgium Brazil China Germany Japan Portugal Slovakia South Africa Spain USA Korea Brazil
283 3214 634 4405 1173 1062 941 1357 2301 703 5441 638 490 6832 1999 10,079 2016 10,424 784 502 202 1999 470 3871 408
18–97 18–80 17–31 49 ± 8 56.8 ± 0.5 55.9 ± 0.5 60.2 ± 0.5 55.1 ± 0.3 18–90 20–98 30–60 20 ± 3 44.3 ± 18.8 40.0 ± 16.3 37.4 ± 15.0 37.1 ± 14.9 49.8 ± 18.2 36.1 ± 10.7 44.5 ± 22.8 38.4 ± 16.0 37.7 ± 15.6 47.7 ± 18.4 40–91 Mean age: 16.8 N18 years 17.2%
20.8 22.2 11.2 12.2 22.7 17.7 9.5 14.7 17.7 32.4 21.6 31.6 9.2 17.5 14.3 6.2 7.2 12.4 18.3 13.7 24.5 12.7 23.9 15.9 18.9
a Included papers used a cutpoint of ESS scores N 10 to define the presence of excessive daytime sleepiness.
research study was advertised as a genetic study of circadian phenotypes in healthy subjects. Subjects completed the questionnaires, after receiving detailed information from researchers trained in assessment of circadian phenotypes. There were 147 women (70%), with an age range between 18 and 30 years (mean = 20.8; SD = 2.7). The population living in Bogotá, the capital city of Colombia, is the result of a historical admixture of Southern Europeans with Amerindians [37]. STRUCTURE program (Version 2.3.4) was used for an analysis of ancestry informative markers and did not show evidence of significant population stratification in the samples used in this work [23,38]. We excluded subjects with selfreport of personal history of neuropsychiatric diseases. Institutional Ethics Committees of each participant institution approved this study and all subjects provided written informed consent [39]. 2.2. Measurement instruments Participants completed a self-administered questionnaire, which included socio-demographic variables such as age, sex and personal and familial history of neuropsychiatric disorders. We applied the Epworth sleepiness scale (ESS) [3], it is a self-completed questionnaire, broadly used and validated, to quantify the general level of daytime sleepiness. ESS is an 8-item questionnaire with a Likert-type response and it evaluates the level of general sleepiness during eight daily situations [3]. It ranges from 0 to 24, with higher scores related to more sleepiness [4]. A Spanish version of the ESS has been previously validated and used in studies from Colombia and other Latin American countries [8,10,23,40]. The internal consistency of ESS (Cronbach's alpha) in this study was 0.712.
177
and their products were separated at 140 V in a 2% agarose gel stained with SYBR®-safe (Invitrogen, Carlsbad, CA, USA). MAOA-uVNTR genotyping was carried out using two primers (F: 5-ACA GCC TGA CCG TGG AGA AG-3 and R: 5-GAA CGG ACG CTC CAT TCG GA-3) [36]. PCR program consisted of a 5-min denaturation step at 94 °C (1 cycle), 94 °C for 20s, 64 °C for 20s, 72 °C for 40s (35 cycles) and 72 °C for 7 min (1 cycle). Polymerase chain reactions contained 2 μl of genomic DNA (~50 ng), 1.5 mM MgCl2, 10× reaction Buffer, 1.0 μM of each primer, 1 mM of dNTPs, 1 M de Betaine, and 0.8 U of Taq polymerase (Bioline, London, United Kingdom) in a total volume of 20 μl. For MAOA-uVNTR, five alelles have been reported: 2 (294 bp), 3 (324 bp), 4 (354 bp), 5 (384 bp) and 3.5 (340 bp) [36]. Fragment sizes were determined by comparison to molecular weight markers (HyperLadder V, Bioline, London, United Kingdom). All genotypes were independently checked by two different researchers in order to confirm and validate the results. Additionally, to verify consistency in the genetic results, a random subsample (10% of subjects) was reanalyzed. 2.4. Statistical data analysis Genotype frequencies were assessed for Hardy–Weinberg equilibrium, only in females, with a χ2 test. ESS scores were tested for normal distribution using the Shapiro–Wilk test. The 75th percentile of the ESS score distribution was used as a cut-off point to determine Normal (scores ≤ 14) or High (scores ≥ 15) sleepiness categories for diurnal somnolence in our sample. We used an approach based on percentiles for the present sample, related to the possibility that it could be more useful than the implementation of a fixed cut-off [16,23]. A statistical analysis of ESS scores between genotypes was carried out with PLINK software (version 1.07), using a linear regression model. For this analysis, ESS scores and genotypes were the dependent and independent variables, respectively. For comparisons of genotype frequencies between diurnal somnolence categories a logistic regression was carried out in PLINK 1.07 software. In these analyses, male hemizygous subjects were combined with female homozygous subjects [42,43]. A nominal value of p b 0.05 was considered as statistically significant. 3. Results ESS scores ranged from 0 to 21, with a mean value of 11.32 (SD: 4.36). Shapiro–Wilk test showed that the ESS scores in our sample had a normal distribution (non-significant p value). In our sample, two MAOA-uVNTR alleles were found (3 and 4 repeats) and this polymorphism was in Hardy–Weinberg equilibrium (p = 0.84, in females). Frequency of the 3-repeat allele was 0.36 in females and 0.34 in males. Allele frequencies for the MAOA-uVNTR observed in this study were similar to those reported in another sample of Latin American subjects (χ2 = 0.03, p = 0.86) [44]. A linear regression model showed a statistically significant association between MAOA-uVNTR and ESS scores (p = 0.01). We observed that MAOA 3/3 genotype carriers (mean 9.62) had the lowest scores for the ESS scale (Table 2). In addition, we found a significant association between diurnal somnolence categories (normal and high) and genotype frequencies for the MAOA-uVNTR (p = 0.03) using a logistic regression (Table 2). Frequency of the MAOA 3/3 genotype was higher in the normal diurnal somnolence category (22.7 vs. 8.9%).
2.3. DNA extraction and genetic analysis
4. Discussion
Peripheral blood samples were collected from participants into ethylene diamine tetra acetic acid (EDTA) tubes (Vacutainer, Becton Dickinson, Rutherford, NJ, USA) and genomic DNA was extracted using a salting out method [23,41]. PCR reactions were carried out in a Labnet MultiGene thermal cycler (Labnet International Inc., Edison, NJ, USA)
Excessive daytime sleepiness is quite common around the world (Table 1), being associated with increases in accidents, morbidity and impairments in quality of life [1]. Although it has been shown that genetic factors have an important role in EDS [18,20], very few studies have explored the role of polymorphisms in candidate genes, such as
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D.A. Ojeda et al. / Journal of the Neurological Sciences 337 (2014) 176–179
Table 2 Association of Epworth sleepiness scale (ESS) scores and diurnal somnolence categories (Normal or High) with MAOAu-VNTR. MAOA groups
N (%)
Mean ESS scores (SD)
MAOA groups
Normal sleepiness n (%)
High sleepiness n (%)
3/3 3/4 4/4 Total p value: 0.01a
39 (18.6) 71 (33.8) 100 (47.6) 210 (100)
9.62 (3.99) 11.83 (4.11) 11.63 (4.55) 11.32 (4.36)
3/3 3/4 4/4 Total p value: 0.03b
35 (22.7) 49 (31.8) 70 (45.5) 154 (100)
5 (8.9) 21 (37.5) 30 (53.6) 56 (100)
a b
p value for comparison of scores between genotype groups, using a linear regression model. p value for comparison of MAOA frequencies between normal and high somnolence groups, using a logistic regression.
PER3 gene, on diurnal somnolence [23–26]. In our sample, we found a high mean ESS score (11.32), which could be consistent with previous evidence of a higher prevalence of diurnal sleepiness in Latin American countries (Table 1) [5,8,10]. In the present study, we found that a common functional VNTR in the promoter region of MAOA gene is significantly associated with individual differences in daytime sleepiness in a sample of healthy Colombian subjects. In our study, MAOA 3/3 genotype carriers had the lowest scores for the ESS scale. In addition, these results were supported by a significant association between diurnal somnolence categories and frequencies for the MAOA-uVNTR. In the context of genetic studies of diurnal somnolence in healthy subjects, the use of percentiles could be more useful than the analysis based on a predefined cutoff point (fixed cut-off points could be more useful in epidemiological studies, for example to compare prevalence rates between different populations). Our results could be interpreted in the context of previous knowledge for MAOA biology [35]. MAOA gene encodes a key enzyme for the regulation of several neurotransmitters, such as dopamine, noradrenaline, and serotonin [28,30–32], which are fundamental for the regulation of sleep and other behavioral phenotypes. In line with this, mice that are deficient for MAOA show sleep alterations [45]. It has been demonstrated that the 4-repeat allele of the human MAOA-uVNTR is transcriptionally and enzymatically more active (2 to 10 times) than the 3-repeat allele [35,36]. MAOA-uVNTR has been extensively studied as a possible candidate polymorphism for a large number of neuropsychiatric disorders [33–35] However, our findings provide evidence for the first time that a functional MAOA-uVNTR has a significant association with daytime sleepiness in a sample of healthy subjects. These data suggest that functional variations in MAOA gene could also affect sleep and behavioral circadian phenotypes and disorders [23]. These findings should be replicated in other ethnic groups, using larger samples, in order to confirm and understand the role of MAOA-uVNTR in sleep disturbances. Conflict of interest S. L-L is employee of Novartis Pharmaceuticals. All authors declare that there is no conflict of interest. Acknowledgments This study was supported by grants from Colciencias (Contract#7652011), Universidad Antonio Nariño (VCTI-UAN) and Universidad de Ciencias Aplicadas y Ambientales (UDCA). The authors thank all those who assisted with recruitment of subjects. References [1] Pagel JF. Excessive daytime sleepiness. Am Fam Physician 2009;79:391–6. [2] Guilleminault C, Brooks SN. Excessive daytime sleepiness: a challenge for the practising neurologist. Brain 2001;124:1482–91. [3] Johns MW. A new method for measuring daytime sleepiness: the Epworth sleepiness scale. Sleep 1991;14:540–5. [4] Miletin MS, Hanly PJ. Measurement properties of the Epworth sleepiness scale. Sleep Med 2003;4:195–9.
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Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
A functional polymorphism in the promoter region of MAOA gene is associated with daytime sleepiness in healthy subjects Diego A. Ojeda a, Carmen L. Niño b, Sandra López-León c, Andrés Camargo b, Ana Adan d, Diego A. Forero a,⁎ a
Laboratory of Neuropsychiatric Genetics, Biomedical Sciences Research Group, School of Medicine, Universidad Antonio Nariño, Bogotá, Colombia School of Nursing, Universidad de Ciencias Aplicadas y Ambientales UDCA, Bogotá, Colombia Novartis Pharmaceuticals, Health Plaza, East Hanover, NJ, USA d Department of Psychiatry and Clinical Psychobiology, School of Psychology, University of Barcelona, Barcelona, Spain b c
a r t i c l e
i n f o
Article history: Received 16 September 2013 Received in revised form 26 November 2013 Accepted 3 December 2013 Available online 11 December 2013 Keywords: Neurogenetics Latin America Neuropsychiatric genetics Candidate genes Endophenotypes Daytime sleepiness Monoamine Oxidase A
a b s t r a c t Excessive daytime sleepiness (EDS) is one of the main causes of car and industrial accidents and it is associated with increased morbidity and alterations in quality of life. Prevalence of EDS in the general population around the world ranges from 6.2 to 32.4%, with a heritability of 38–40%. However, few studies have explored the role of candidate genes in EDS. Monoamine oxidase A (MAOA) gene has an important role in the regulation of neurotransmitter levels and a large number of human behaviors. We hypothesized that a functional VNTR in the promoter region of the MAOA gene might be associated with daytime sleepiness in healthy individuals. The Epworth sleepiness scale (ESS) was applied to 210 Colombian healthy subjects (university students), which were genotyped for MAOA-uVNTR. MAOA-uVNTR showed a significant association with ESS scores (p = 0.01): 3/3 genotype carriers had the lowest scores. These results were supported by differences in MAOA-uVNTR frequencies between diurnal somnolence categories (p = 0.03). Our finding provides evidence for the first time that MAOA-uVNTR has a significant association with EDS in healthy subjects. Finally, these data suggest that functional variations in MAOA gene could have a role in other phenotypes of neuropsychiatric relevance. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Excessive daytime sleepiness (EDS) is one of the main causes of car and industrial accidents around the world, being associated with decreases in performance at school or work, increased morbidity and alterations in quality of life [1,2]. The Epworth sleepiness scale was created in 1991 as a tool to quantify levels of daytime sleepiness and it has been used in a large number of clinical studies [3,4]. Prevalence of EDS in the general population, defined by the ESS, in different countries ranges from 6.2 to 32.4% [5–17] (Table 1), with a moderate heritability of approximately 38% to 40% [18–21]. This evidence highlights the importance of studying the role of genetic polymorphisms in increased diurnal somnolence [22]. However, very few studies have explored the possible association of candidate genes with differences in daytime sleepiness [23–26]. Sleep is a complex physiological process and it is fundamental to restore and maintain an adequate physical and mental health [27,28]. It is regulated by multiple environmental and molecular mechanisms, such as the circadian system [29]. In addition, many neurotransmitters are involved in the sleep process: norepinephrine, serotonin, dopamine and glutamate, among many others [28,30–32]. ⁎ Corresponding author at: Laboratory of Neuropsychiatric Genetics, School of Medicine, Universidad Antonio Nariño, Bogotá, Colombia. Tel.: +57 313 2610427. E-mail address: [email protected] (D.A. Forero). 0022-510X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jns.2013.12.005
Monoamine oxidase A (MAOA) is known to play a major role in catalyzing the oxidative deamination of norepinephrine, dopamine, and serotonin neurotransmitters [33,34], being a key enzyme for the regulation of monoamine levels. MAOA gene has a size of 90,660 bp, is located on the X chromosome at Xp.11.3–Xp11.4 and is expressed in several brain tissues [34,35]. Different polymorphisms in the MAOA gene have been identified [33–36] and a functional variable-number tandem repeat (VNTR) located in the promoter region of MAOA gene (MAOA-uVNTR) is commonly studied in neuropsychiatric genetics [35,36]. MAOA-uVNTR polymorphism consists of a 30 bp repeat sequence and functional studies have shown that this VNTR affects the transcription of MAOA, indicating that the number of repeats is related to transcriptional activity [35,36]. We hypothesized that a functional VNTR in the promoter region of the MAOA gene might be associated with differences in daytime sleepiness in healthy Colombian subjects.
2. Methods 2.1. Participants Two hundred and ten unrelated young subjects (university students) living in Bogotá, Colombia were included. Participants were recruited in two private Universities, through posters and informative talks. This
D.A. Ojeda et al. / Journal of the Neurological Sciences 337 (2014) 176–179 Table 1 Prevalence of excessive daytime sleepiness (EDS), using the Epworth sleepiness scale, in general populations around the worlda. Author, year
Country
Sample size
Age range
% EDS
Pahwa, 2012 Wu, 2012 Mume, 2011 Joo, 2009 Bouscoulet, 2008 Bouscoulet, 2008 Bouscoulet, 2008 Bouscoulet, 2008 Pallesen, 2007 Sanford, 2006 Gander, 2005 Moo-Estrella, 2005 Soldatos, 2005 Soldatos, 2005 Soldatos, 2005 Soldatos, 2005 Soldatos, 2005 Soldatos, 2005 Soldatos, 2005 Soldatos, 2005 Soldatos, 2005 Soldatos, 2005 Walsleben, 2004 Shin, 2003 Souza, 2002 Average EDS
Canada China Nigeria Korea Chile Mexico Uruguay Venezuela Norway USA New Zealand Mexico Austria Belgium Brazil China Germany Japan Portugal Slovakia South Africa Spain USA Korea Brazil
283 3214 634 4405 1173 1062 941 1357 2301 703 5441 638 490 6832 1999 10,079 2016 10,424 784 502 202 1999 470 3871 408
18–97 18–80 17–31 49 ± 8 56.8 ± 0.5 55.9 ± 0.5 60.2 ± 0.5 55.1 ± 0.3 18–90 20–98 30–60 20 ± 3 44.3 ± 18.8 40.0 ± 16.3 37.4 ± 15.0 37.1 ± 14.9 49.8 ± 18.2 36.1 ± 10.7 44.5 ± 22.8 38.4 ± 16.0 37.7 ± 15.6 47.7 ± 18.4 40–91 Mean age: 16.8 N18 years 17.2%
20.8 22.2 11.2 12.2 22.7 17.7 9.5 14.7 17.7 32.4 21.6 31.6 9.2 17.5 14.3 6.2 7.2 12.4 18.3 13.7 24.5 12.7 23.9 15.9 18.9
a Included papers used a cutpoint of ESS scores N 10 to define the presence of excessive daytime sleepiness.
research study was advertised as a genetic study of circadian phenotypes in healthy subjects. Subjects completed the questionnaires, after receiving detailed information from researchers trained in assessment of circadian phenotypes. There were 147 women (70%), with an age range between 18 and 30 years (mean = 20.8; SD = 2.7). The population living in Bogotá, the capital city of Colombia, is the result of a historical admixture of Southern Europeans with Amerindians [37]. STRUCTURE program (Version 2.3.4) was used for an analysis of ancestry informative markers and did not show evidence of significant population stratification in the samples used in this work [23,38]. We excluded subjects with selfreport of personal history of neuropsychiatric diseases. Institutional Ethics Committees of each participant institution approved this study and all subjects provided written informed consent [39]. 2.2. Measurement instruments Participants completed a self-administered questionnaire, which included socio-demographic variables such as age, sex and personal and familial history of neuropsychiatric disorders. We applied the Epworth sleepiness scale (ESS) [3], it is a self-completed questionnaire, broadly used and validated, to quantify the general level of daytime sleepiness. ESS is an 8-item questionnaire with a Likert-type response and it evaluates the level of general sleepiness during eight daily situations [3]. It ranges from 0 to 24, with higher scores related to more sleepiness [4]. A Spanish version of the ESS has been previously validated and used in studies from Colombia and other Latin American countries [8,10,23,40]. The internal consistency of ESS (Cronbach's alpha) in this study was 0.712.
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and their products were separated at 140 V in a 2% agarose gel stained with SYBR®-safe (Invitrogen, Carlsbad, CA, USA). MAOA-uVNTR genotyping was carried out using two primers (F: 5-ACA GCC TGA CCG TGG AGA AG-3 and R: 5-GAA CGG ACG CTC CAT TCG GA-3) [36]. PCR program consisted of a 5-min denaturation step at 94 °C (1 cycle), 94 °C for 20s, 64 °C for 20s, 72 °C for 40s (35 cycles) and 72 °C for 7 min (1 cycle). Polymerase chain reactions contained 2 μl of genomic DNA (~50 ng), 1.5 mM MgCl2, 10× reaction Buffer, 1.0 μM of each primer, 1 mM of dNTPs, 1 M de Betaine, and 0.8 U of Taq polymerase (Bioline, London, United Kingdom) in a total volume of 20 μl. For MAOA-uVNTR, five alelles have been reported: 2 (294 bp), 3 (324 bp), 4 (354 bp), 5 (384 bp) and 3.5 (340 bp) [36]. Fragment sizes were determined by comparison to molecular weight markers (HyperLadder V, Bioline, London, United Kingdom). All genotypes were independently checked by two different researchers in order to confirm and validate the results. Additionally, to verify consistency in the genetic results, a random subsample (10% of subjects) was reanalyzed. 2.4. Statistical data analysis Genotype frequencies were assessed for Hardy–Weinberg equilibrium, only in females, with a χ2 test. ESS scores were tested for normal distribution using the Shapiro–Wilk test. The 75th percentile of the ESS score distribution was used as a cut-off point to determine Normal (scores ≤ 14) or High (scores ≥ 15) sleepiness categories for diurnal somnolence in our sample. We used an approach based on percentiles for the present sample, related to the possibility that it could be more useful than the implementation of a fixed cut-off [16,23]. A statistical analysis of ESS scores between genotypes was carried out with PLINK software (version 1.07), using a linear regression model. For this analysis, ESS scores and genotypes were the dependent and independent variables, respectively. For comparisons of genotype frequencies between diurnal somnolence categories a logistic regression was carried out in PLINK 1.07 software. In these analyses, male hemizygous subjects were combined with female homozygous subjects [42,43]. A nominal value of p b 0.05 was considered as statistically significant. 3. Results ESS scores ranged from 0 to 21, with a mean value of 11.32 (SD: 4.36). Shapiro–Wilk test showed that the ESS scores in our sample had a normal distribution (non-significant p value). In our sample, two MAOA-uVNTR alleles were found (3 and 4 repeats) and this polymorphism was in Hardy–Weinberg equilibrium (p = 0.84, in females). Frequency of the 3-repeat allele was 0.36 in females and 0.34 in males. Allele frequencies for the MAOA-uVNTR observed in this study were similar to those reported in another sample of Latin American subjects (χ2 = 0.03, p = 0.86) [44]. A linear regression model showed a statistically significant association between MAOA-uVNTR and ESS scores (p = 0.01). We observed that MAOA 3/3 genotype carriers (mean 9.62) had the lowest scores for the ESS scale (Table 2). In addition, we found a significant association between diurnal somnolence categories (normal and high) and genotype frequencies for the MAOA-uVNTR (p = 0.03) using a logistic regression (Table 2). Frequency of the MAOA 3/3 genotype was higher in the normal diurnal somnolence category (22.7 vs. 8.9%).
2.3. DNA extraction and genetic analysis
4. Discussion
Peripheral blood samples were collected from participants into ethylene diamine tetra acetic acid (EDTA) tubes (Vacutainer, Becton Dickinson, Rutherford, NJ, USA) and genomic DNA was extracted using a salting out method [23,41]. PCR reactions were carried out in a Labnet MultiGene thermal cycler (Labnet International Inc., Edison, NJ, USA)
Excessive daytime sleepiness is quite common around the world (Table 1), being associated with increases in accidents, morbidity and impairments in quality of life [1]. Although it has been shown that genetic factors have an important role in EDS [18,20], very few studies have explored the role of polymorphisms in candidate genes, such as
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Table 2 Association of Epworth sleepiness scale (ESS) scores and diurnal somnolence categories (Normal or High) with MAOAu-VNTR. MAOA groups
N (%)
Mean ESS scores (SD)
MAOA groups
Normal sleepiness n (%)
High sleepiness n (%)
3/3 3/4 4/4 Total p value: 0.01a
39 (18.6) 71 (33.8) 100 (47.6) 210 (100)
9.62 (3.99) 11.83 (4.11) 11.63 (4.55) 11.32 (4.36)
3/3 3/4 4/4 Total p value: 0.03b
35 (22.7) 49 (31.8) 70 (45.5) 154 (100)
5 (8.9) 21 (37.5) 30 (53.6) 56 (100)
a b
p value for comparison of scores between genotype groups, using a linear regression model. p value for comparison of MAOA frequencies between normal and high somnolence groups, using a logistic regression.
PER3 gene, on diurnal somnolence [23–26]. In our sample, we found a high mean ESS score (11.32), which could be consistent with previous evidence of a higher prevalence of diurnal sleepiness in Latin American countries (Table 1) [5,8,10]. In the present study, we found that a common functional VNTR in the promoter region of MAOA gene is significantly associated with individual differences in daytime sleepiness in a sample of healthy Colombian subjects. In our study, MAOA 3/3 genotype carriers had the lowest scores for the ESS scale. In addition, these results were supported by a significant association between diurnal somnolence categories and frequencies for the MAOA-uVNTR. In the context of genetic studies of diurnal somnolence in healthy subjects, the use of percentiles could be more useful than the analysis based on a predefined cutoff point (fixed cut-off points could be more useful in epidemiological studies, for example to compare prevalence rates between different populations). Our results could be interpreted in the context of previous knowledge for MAOA biology [35]. MAOA gene encodes a key enzyme for the regulation of several neurotransmitters, such as dopamine, noradrenaline, and serotonin [28,30–32], which are fundamental for the regulation of sleep and other behavioral phenotypes. In line with this, mice that are deficient for MAOA show sleep alterations [45]. It has been demonstrated that the 4-repeat allele of the human MAOA-uVNTR is transcriptionally and enzymatically more active (2 to 10 times) than the 3-repeat allele [35,36]. MAOA-uVNTR has been extensively studied as a possible candidate polymorphism for a large number of neuropsychiatric disorders [33–35] However, our findings provide evidence for the first time that a functional MAOA-uVNTR has a significant association with daytime sleepiness in a sample of healthy subjects. These data suggest that functional variations in MAOA gene could also affect sleep and behavioral circadian phenotypes and disorders [23]. These findings should be replicated in other ethnic groups, using larger samples, in order to confirm and understand the role of MAOA-uVNTR in sleep disturbances. Conflict of interest S. L-L is employee of Novartis Pharmaceuticals. All authors declare that there is no conflict of interest. Acknowledgments This study was supported by grants from Colciencias (Contract#7652011), Universidad Antonio Nariño (VCTI-UAN) and Universidad de Ciencias Aplicadas y Ambientales (UDCA). The authors thank all those who assisted with recruitment of subjects. References [1] Pagel JF. Excessive daytime sleepiness. Am Fam Physician 2009;79:391–6. [2] Guilleminault C, Brooks SN. Excessive daytime sleepiness: a challenge for the practising neurologist. Brain 2001;124:1482–91. [3] Johns MW. A new method for measuring daytime sleepiness: the Epworth sleepiness scale. Sleep 1991;14:540–5. [4] Miletin MS, Hanly PJ. Measurement properties of the Epworth sleepiness scale. Sleep Med 2003;4:195–9.
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