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Journal of Neurology Research

Original Article

Volume 2, Number 1, February 2012, pages 16-24

The Possible Underworld of Chronic Fatigue Syndrome From Neurotransmitters Polymorphisms to Disease

The chronic fatigue syndrome (CFS) is characterized by intense fatigue of unknown cause, which is permanent and limits the patient's functional capacity, producing degrees of disability [1]. The pathophysiology is unknown to date; cytokines, neuropeptides, or neurotransmitters are considered to be responsible for the abnormal immune response and disrupted hypothalamo-pituitary-adrenal (HPA) axis found in patients [2, 3]. An increasing amount of neuroimaging evidence supports the hypothesis that CFS patients have a sort of neurological, structural or functional abnormalities. Moreover, neurotrophic factors, neurotransmitters and cytokines have been evaluated in order to elucidate possible mechanisms of abnormal neuropsychic findings in thus syndrome [4, 5].

The major neurotransmitters involved in descending pain-modulating systems are norepinephrine and serotonin. The serotonin transporter (SERT) is an integral membrane protein that transports serotonin from synaptic spaces into presynaptic neurons, terminates the action of serotonin and recycles it in a sodium-dependent manner. The gene encoding for SERT is called SLC6A4 and it is located on chromosome 17 (q11.1-q12). The promoter region of the SLC6A4 gene contains a polymorphism with "short" and "long" repeats in the 5-HTT-linked polymorphic region (5-HTTLPR, rs25531). The short variation leads to less transcription for SLC6A4, and it has been found that it can partly account for anxiety-related personality traits [6]. The serotonin receptors 2A is coded by the HTR2A gene located on 13q14-q21. This receptor is expressed widely throughout the central nervous system where it mediates neuronal excitation, behavioral effects, learning, anxiety. Several interesting polymorphisms have been identified for HTR2A; in particular, several studies shown links between the -1438G/A (rs6311) polymorphism and mood disorders, such as bipolar disorder [7] and major depressive disorder [8].

Deficits in neurotrophic factors have been proposed to underlie mood disorders: neurotrophins may act as critical tools in the process whereby environmental conditions guide neuronal networks to better adapt to the environment [9]. Brain derived neurotrophic factor has pleiotropic effects on neuronal development and synaptic plasticity that underlie circuit formation and cognitive function [10]. It is encoded by the BDNF gene located on chromosome 11. The Val66Met (rs6265) single nucleotide polymorphism results in a variation in the protein between Valine and Methionine at codon 66 [11].

Glycogen synthase kinase 3 (GSK3) is involved in a variety of cellular processes ranging from glycogen metabolism, insulin signaling, cell proliferation, neuronal function, oncogenesis to embryonic development. GSK3 is ubiquitously expressed with high levels in brain and is associated with a variety of neurological disease like Alzheimer’s, bipolar disorder, Huntington disease and other neurodegenerative disorders [12, 13]. In the GSK3B gene, located onchromosome 3q13.3, are described two functional SNPs: a promoter polymorphism (rs334558) and a polymorphism in intron 5 (rs6438552) [14].

Thus, we provide an association study between the SLC6A4, HTR2A and GSK3B gene polymorphisms and CFS in Italian population.

The patients were recruited through three Italian referral Centers for CFS (Oncological Referring Center-Aviano, Prof. U. Tirelli; Department of Infectious Diseases, University of Chieti, Prof. E. Pizzigallo; Department of Rheumatology, University of Pisa, Dr. L. Bazzichi) and were enrolled only patients with a certified diagnosis of CFS. A questionnaire was used to re-confirm the diagnosis and exclude any other possible causes of fatigue by symptom referral, lab tests and personal/family histories of immune and hereditary disease. All patients are Italian in order to have a homogenous genetic background between patients and controls.

A biological bank of CFS patients was established in Pavia (Laboratory of Immunogenetics, Department of Genetics and Microbiology) containing peripheral blood, mRNA, serum and red blood cells as already described elsewhere [15].

A total of 128 healthy blood donors (49 males, 94 females), matched for age, sex and ethnical origins, served as controls.

Approval for this study was obtained from the Ethics Committee of the University of Pavia. After a complete explanation of the aims and details of the study, written informed consent has been obtained from all the subjects.

Genomic DNA was extracted from EDTA-treated blood using the DNA Blood Mini Kit (Qiagen). All the SNP considered in this study were analyzed through PCR-RFLP method.

The coding and untranslated regions of each gene examined were amplified by PCR using the primers listed in Table 1. PCR amplifications were carried out in a total volume of 25 ml containing: 20 ng of genomic DNA; 0.1 ml of Taq polymerase (0.02 U/ml); 1 ml of each specific primer forward and reverse (4 ng/ml); 2.5 ml PCR Buffer 10X; 0.75 ml MgCl₂ (1.5 mM); 2.5 ml dNTPs (2 mM each); 16.15 ml of sterile H₂O. Thirty-five amplification cycles were performed, consisting of denaturation at 95 °C for 30 seconds, annealing, and elongation at 72 °C for 1 minute (Table 1). An initial denaturation step was carried out at 95°C for 5 minutes, and final elongation at 72 °C for 7 minutes. The amplified segment was run on a 2% agarose gel and read using a U.V. transilluminator.

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Table 1. Primer Pairs and Restriction Enzyme Used for Genetic Analysis

The amplified segments were digested using specific restriction enzyme (Table 1) repentantly for 4 hours at 37 °C and the digest run on a 3% agarose gel and read using a U.V. transilluminator.

A χ² test for goodness of fit was used to verify whether the observed allele frequencies agreed with those expected under Hardy-Weinberg equilibrium. Allelic and genotypic distribution were estimated by χ² test and differences considered statistically significant when the P-value was <0.05. Crude and adjusted odds ratios (ORs) are reported with their 95% confidence intervals (CIs) using univariate analyses.

We observed a bias towards female sex (3:1 ratio) in agreement with international literature [16-19]. The mean age at CFS onset ± SD was 32.2 ± 12.9 and 33.9 ± 12.5 years for male and female patients (P = 0.62), respectively (Table 2). In any polymorphisms of SLC6A4, HTR2A, BDNF and GSK3B genes analyzed, the allelic and genotypic frequencies is not significant different in patients and controls (Table 3). Genotypes were in Hardy-Weinberg equilibrium both in the patients and controls. For the same polymorphisms in the CFS patients population, divided according to gender, no statistically significant data were found (Table 4).

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Table 2. Clinical Characteristics and Presence of XMRV of 70 Italian CFS Patients

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Table 3. Allelic and Genotypic Distribution Between CFS Patients and Controls

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Table 4. Allelic and Genotypic Distribution Between Male and Female CFS Patients

Arthralgia was the main symptom among CFS patients (89%). Noteworthy, statistical significant differences in frequencies for arthralgia were found between female (95%) and male (77%) patients (OR = 5.62, 95% CI 1.13 - 28.02). Clinical variables were not significantly different between gender. Myalgia had the lowest frequency among symptoms (8%). Concerning comorbidities, 3% of patients had type 2 diabetes, 4% had hypothyroidism, one patient had Rheumatoid Arthritis (1%), and in another patient (1%) the diagnosis of psoriasis was confirmed. Data shown in Table 2.

The 43% of the CFS patients presents depressive symptoms (Table 2). In the subgroup of CFS patients with depressive symptoms the presence of female sex is significant higher (88%) than in the not depressed patients (35%). On the contrary, the presence of male gender is significant higher in patient without depressive symptoms (65% vs 12%; P = 0.0002). The distribution of the serotonin transporter and receptor 2A, BDNF and GSK3B polymorphisms in depressed and non-depressed CFS patients is shown in Table 5. The genotypic and allelic frequencies of the HTR2A -1438G/A polymorphism showed a difference (P = 0.05) between the genotype AA, while no difference was observed between the frequencies of the heterozygous AG and homozygous GG genotypes. In particular, the frequency of the AA genotype was higher in the depressed patients (48%) compared to the patients without depressive symptoms (21%). The crude odds ratio for the presence of CFS associated with depression in subjects bearing the homozygous AA genotype was 3.56 (95% CI, 1.13 - 11.17).

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Table 5. Allelic and Genotypic Distribution Between CFS Patients With and Without Depressive Symptoms

CFS is a complex, chronic disorder of unknown aetiology, characterized by the presence of intense and disabling fatigue, which interferes with daily activities, and is usually associated to systemic, physical and neuropsychological manifestations [1]. The aetiology and the pathogenic mechanisms of CFS are not fully understood; several hypotheses have been postulated [20]. One of these is the neuroendocrinological theory and an increasing amount of neuroimaging evidence supports the hypothesis that CFS patients have structural or functional abnormalities within the brain [4].

Genetic studies [21, 22] have revealed that a polymorphism in the regulatory region of SLC6A4 gene is associated with CFS. SLC6A4 govern the serotonin neuronal system and regulates the duration and strength of the interactions between serotonin and its receptors through reuptake of serotonin from the extracellular space. The polymorphisms in the 5’ upstream region (5-HTTLPR) is composed of either 14 (S) or 16 (L) and, although infrequent, 18 and 20 repetitive elements (XL) [23]. In the Japanese population a significant increase of longer (L and XL) allelic variants was found in the CFS patients compared to the controls. The L allele is believed to retain higher transcriptional activity, which causes decreased concentration of serotonin in the extracellular space in CFS patients [23]. In Italian population we did not confirm this association; the frequency of L allele in CFS patients and controls is similar and also the distribution of the genotype in the two group is not different. It is believed that extensive molecular and anatomic diversity among serotonin receptors make the serotonergic system able to regulate pain, inflammation, memory, sleep, appetite, thermoregulation, and various neuroendocrine functions as well as depression, anxiety, and fatigue [24]. We studied a polymorphism in the promoter region, position -1438, of serotonin receptor 2A. The -1438 G/A polymorphism has been implicated in neuropsychiatric disorders such as schizophrenia [25] and seasonal affective disorder [26]. In our CFS population we did not found any statistical significant difference in frequencies between patients and controls. The differences between our results and those cited above may be due to ethnic genetic differences between the populations analyzed. On the basis of these results, we cannot exclude the involvement of the serotonergic system in the pathogenesis of this disease: both considered genes are presents other polymorphism that should be associated with this disease.

BDNF, a member of the growth factor family of neurotrophins, contributes to the activity-dependent synaptic development and survival of serotonergic neurons [27, 28]. The Val66Met polymorphisms analyzed in this study seems to be not associated with the disease. However BDNF it is known to have crucial roles during brain development as well as in adults brain by regulating synaptic transmission and plasticity and it regulates neuronal survival in the central nervous system via AKT pathway. Activated AKT regulates a number of cell survival-related proteins by phosphorylation, such as glycogen synthase kinase 3 beta (GSK3B) [29]. GSK3B plays a key role in the phosphorylation and regulation of metabolic enzymes and many transcription factors, and it is involved in a variety of cellular processes ranging from glycogen metabolism, insulin signaling, cell proliferation, neuronal function, oncogenesis to embryonic development. The known biochemical pathways in which GSK3B is involved, suggests a role as a susceptibility locus for multiple diseases [30]. In Italian CFS patients we do not find a significant associations with the polymorphisms (rs334558 and rs6438552) analyzed and the disease. These results do not exclude the possible role of these neurological markers in the pathophysiology of CFS.

It is possible to envisage an epigenetic interaction between genes or between these genes and environmental stressors that lead to the development of the disease. There is considerable evidence that there are complex interactions between genes, environment, that increase risk or protect against the neurobiological alterations. For example in humans the 5-HTTLPR gene and environment interaction has an effect on the vulnerability of individuals for the onset of depression in the face of stressful life events in adulthood [31]. In addiction the HPA axis has bidirectional relationships with the serotonergic and noradrenergic systems further complicating the biological picture.

Several factors have been related to the pathology of CFS, like anti-virus and autoantibodies [32], but the results from different studies are controversial and conflicting. It is therefore important to stratify the study groups in terms of symptoms, age, duration of disease, andtreatments for other disorders, and to investigate gene expression.

It is firmly established that women experience of depression at roughly twice the rate of men. Contemporary research has indicated that sex hormones comprise crucial orchestrators of the differences in susceptibility associated to sex in depression, as well as in certain infectious and autoimmune diseases. Interestingly, it has been suggested that altered functioning of the immune system may be implicated in the medical morbidity of this affective disorder [33]. In our study population the 44% of the CFS patients presents depressive symptoms and the presence of female in this subgroup is significant higher (P = 0.0002). The distribution ofSLC6A4, BDNF and GSK3B polymorphisms are not different in CFS patients divided according to presence of depressive symptoms. The only difference concerns the distribution of genotypic frequencies of -1438G/A polymorphism in HTR2A gene. In particular, homozygous genotype AA was higher in the depressed patients compared to the patients without depressive symptoms and the crude odds ratio for the presence of CFS associated with depression in subjects bearing the homozygous AA genotype was 3.56 (95% CI, 1.13 - 11.17). However, our sample size was appropriately powered based upon the observed frequency of the homozygous AA genotype in our control population. The -1438A/G SNP lies upstream of two alternative promoters of HTR2A and has the potential to positively modulate the HTR2A promoter in the presence of A allele in vitro [34]. Smith et al., 2008 reported that HTR2A -1438 A allele was more common in CFS subjects (48.7%) when compared to controls (27.4%) in American population. In addition, secondary analyses sought to evaluate quantitative measures of functional impairment, fatigue, and symptoms with HTR2A polymorphisms, revealed that the -1438 A allele was associated with a reduction in general health, vitality, and social function. However, examination of -1438G/A allelic variants revealed that the A allele creates a consensus binding site for the transcription factor Th1/E47. Whereas promoter activity was significantly greater with the A allele relative to the G allele it is possible to speculate that CFS patients with depressive symptoms with AA genotype have greater promoter activity of HTR2A gene. References of increased promoter activity [35], mRNA and protein levels [36] and receptor binding [37] with this promoter polymorphism and the association of the A allele with CFS sustain a hyperactive serotonergic system in the disease.

In conclusion, yet CFS remains an aetiopathogenic enigma; the neuroendocrine system remains an intriguing field of research in CFS. Globally, there is evidence for a reduced cortisol output and HPA axis hypofunction in a proportion of patients with CFS. In addition, recent insights suggest a neuroendocrine-immune interface [38-40]. Cytokines, neuropeptides, or neurotransmitters are considered to be responsible for the abnormal immune response and disrupted hypothalamo-pituitary-adrenal (HPA) axis found in the patients [2, 3]. Furthermore, additional neuroendocrine-immune studies in humans are also require to compare CFS with mood disorders since evidence is occurring that all these condition may be characterized by inflammation.

Conflict of Interest

None.

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