Associations between clinical symptoms, plasma norepinephrine and deregulated immune gene networks in subgroups of adolescent with Chronic Fatigue Syndrome

Background: Chronic Fatigue Syndrome (CFS) is one of the most important causes of disability among adolescents while limited knowledge exists on genetic determinants underlying disease pathophysiology. Methods: We analyzed deregulated immune-gene modules using Pathifier software on whole blood gene expression data (29 CFS patients, 18 controls). Deconvolution of immune cell subtypes based on gene expression profile was performed using CIBERSORT. Supervised consensus clustering on pathway deregulation score (PDS) was used to define CFS subgroups. Associations between PDS and immune, neuroendocrine/autonomic and clinical markers were examined. The impact of plasma norepinephrine level on clinical markers over time was assessed in a larger cohort (91 patients). Results: A group of 29 immune-gene sets was shown to differ patients from controls and detect subgroups within CFS. Group 1P (high PDS, low norepinephrine, low naïve CD4+ composition) had strong association with levels of serum C-reactive protein and Transforming Growth Factor-beta. Group 2P (low PDS, high norepinephrine, high naïve CD4+ composition) had strong associations with neuroendocrine/autonomic markers. The corresponding plasma norepinephrine level delineated 91 patients into two subgroups with significant differences in fatigue score. Conclusion: We identified 29 immune-gene sets linked to plasma norepinephrine level that could delineate CFS subgroups. Plasma norepinephrine stratification revealed that lower levels of norepinephrine were associated with higher fatigue. Our data suggests potential involvement of neuro-immune dysregulation and genetic stratification in CFS

Maintenance of Chronic Fatigue Syndrome (CFS) in Young CFS Patients Is Associated with the 5-HTTLPR and SNP rs25531 A > G Genotype.

Earlier studies have shown that genetic variability in the SLC6A4 gene encoding the serotonin transporter (5-HTT) may be important for the re-uptake of serotonin (5-HT) in the central nervous system. In the present study we investigated how the 5-HTT genotype i.e. the short (S) versus long (L) 5-HTTLPR allele and the SNP rs25531 A > G affect the physical and psychosocial functioning in patients with chronic fatigue syndrome (CFS). All 120 patients were recruited from The Department of Paediatrics at Oslo University Hospital, Norway, a national referral center for young CFS patients (12-18 years). Main outcomes were number of steps per day obtained by an accelerometer and disability scored by the Functional Disability Inventory (FDI). Patients with the 5-HTT SS or SLG genotype had a significantly lower number of steps per day than patients with the 5-HTT LALG, SLA or LALA genotype. Patients with the 5-HTT SS or SLG genotype also had a significantly higher FDI score than patients with the 5-HTT LALG, SLA or LALA genotype. Thus, CFS patients with the 5-HTT SS or SLG genotype had worse 30 weeks outcome than CFS patients with the 5-HTT LALG, SLA or LALA genotype. The present study suggests that the 5-HTT genotype may be a factor that contributes to maintenance of CFS

Whole blood gene expression in adolescent chronic fatigue syndrome: An exploratory cross-sectional study suggesting altered B cell differentiation and survival

Background Chronic fatigue syndrome (CFS) is a prevalent and disabling condition affecting adolescents. The pathophysiology is poorly understood, but immune alterations might be an important component. This study compared whole blood gene expression in adolescent CFS patients and healthy controls, and explored associations between gene expression and neuroendocrine markers, immune markers and clinical markers within the CFS group. Methods CFS patients (12–18 years old) were recruited nation-wide to a single referral center as part of the NorCAPITAL project. A broad case definition of CFS was applied, requiring 3 months of unexplained, disabling chronic/relapsing fatigue of new onset, whereas no accompanying symptoms were necessary. Healthy controls having comparable distribution of gender and age were recruited from local schools. Whole blood samples were subjected to RNA sequencing. Immune markers were blood leukocyte counts, plasma cytokines, serum C-reactive protein and immunoglobulins. Neuroendocrine markers encompassed plasma and urine levels of catecholamines and cortisol, as well as heart rate variability indices. Clinical markers consisted of questionnaire scores for symptoms of post-exertional malaise, inflammation, fatigue, depression and trait anxiety, as well as activity recordings. Results A total of 29 CFS patients and 18 healthy controls were included. We identified 176 genes as differentially expressed in patients compared to controls, adjusting for age and gender factors. Gene set enrichment analyses suggested impairment of B cell differentiation and survival, as well as enhancement of innate antiviral responses and inflammation in the CFS group. A pattern of co-expression could be identified, and this pattern, as well as single gene transcripts, was significantly associated with indices of autonomic nervous activity, plasma cortisol, and blood monocyte and eosinophil counts. Also, an association with symptoms of post-exertional malaise was demonstrated. Conclusion Adolescent CFS is characterized by differential gene expression pattern in whole blood suggestive of impaired B cell differentiation and survival, and enhanced innate antiviral responses and inflammation. This expression pattern is associated with neuroendocrine markers of altered HPA axis and autonomic nervous activity, and with symptoms of post-exertional malaise

Role of polymorphisms in the 5-HTT promotor region.

<p><b>A)</b> The 5-HTT short (S) allele consists of 14 repeats at 22 bp while the long (L) allele consists of 16 repeats and a possible A to G SNP. AP2 is a transcription inhibitor found to bind the sequence if a G substitution is present [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140883#pone.0140883.ref005" target="_blank">5</a>]. <b>B)</b> Five possible allele combinations ranged by their predicted rate of transcription. Frequency of the allele combinations in the Caucasian population: SS: 20%, SL_G: 4%, L_AL_G: 5%, SL_A: 41% and L_AL_A: 26% [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140883#pone.0140883.ref006" target="_blank">6</a>]. A: adenosine, G: guanosine, AP2: activating enhancer-binding protein. <b>C)</b> 5-HTT mRNA in blood in the CFS patients.</p

The time course for outcome measures grouped as 5-HTT SS+SL_G genotype, and 5-HTT L_AL_G+SL_A+L_AL_A genotype following inclusion.

<p><b>A</b>) Number of steps (repeated measure ANOVA, F(1,116) = 7.23, p = 0.008). <b>B</b>) Functional disability inventory, FDI (repeated measure ANOVA, F(1,115) = 7.81; p = 0.006). Data are given as means ± SEM (Fisher’s LSD post hoc test using Bonferroni correction *p<0.05; **p<0.01.)</p

Characteristics of patients grouped by genotype; 5-HTT SS/SL_G versus 5-HTT L_AL_G/SL_A/L_AL_A.

<p>^aPearson Chi-square test</p><p>^bUnpaired student’s t-test.</p><p>Characteristics of patients grouped by genotype; 5-HTT SS/SL_G versus 5-HTT L_AL_G/SL_A/L_AL_A.</p

Limited geographic distribution of the novel cyclovirus CyCV-VN

A novel cyclovirus, CyCV-VN, was recently identified in cerebrospinal fluid (CSF) from patients with central nervous system (CNS) infections in central and southern Vietnam. To explore the geographic distribution of this novel virus, more than 600 CSF specimens from patients with suspected CNS infections in northern Vietnam, Cambodia, Nepal and The Netherlands were screened for the presence of CyCV-VN but all were negative. Sequence comparison and phylogenetic analysis between CyCV-VN and another novel cyclovirus recently identified in CSF from Malawian patients indicated that these represent distinct cycloviral species, albeit phylogenetically closely related. The data suggest that CyCV-VN has a limited geographic distribution within southern and central Vietnam. Further research is needed to determine the global distribution and diversity of cycloviruses and importantly their possible association with human diseas

Identification of a new cyclovirus in cerebrospinal fluid of patients with acute central nervous system infections

Acute central nervous system (CNS) infections cause substantial morbidity and mortality, but the etiology remains unknown in a large proportion of cases. We identified and characterized the full genome of a novel cyclovirus (tentatively named cyclovirus-Vietnam [CyCV-VN]) in cerebrospinal fluid (CSF) specimens of two Vietnamese patients with CNS infections of unknown etiology. CyCV-VN was subsequently detected in 4% of 642 CSF specimens from Vietnamese patients with suspected CNS infections and none of 122 CSFs from patients with noninfectious neurological disorders. Detection rates were similar in patients with CNS infections of unknown etiology and those in whom other pathogens were detected. A similar detection rate in feces from healthy children suggested food-borne or orofecal transmission routes, while high detection rates in feces from pigs and poultry (average, 58%) suggested the existence of animal reservoirs for such transmission. Further research is needed to address the epidemiology and pathogenicity of this novel, potentially zoonotic virus.IMPORTANCE Acute central nervous system (CNS) infections cause substantial morbidity and mortality, but the etiology frequently remains unknown, which hampers development of therapeutic or preventive strategies. Hence, identification of novel pathogens is essential and is facilitated by current next-generation sequencing-based methods. Using such technology, we identified and characterized the full genome of a novel cyclovirus in cerebrospinal fluid (CSF) specimens from two Vietnamese patients with CNS infections of unknown etiology, which was subsequently detected in none of 122 CSF specimens from patients with noninfectious neurological disorders but 4% of 642 CSF specimens from Vietnamese patients with suspected or confirmed CNS infections. Similar detection rates in feces from healthy children suggested food-borne or orofecal transmission routes, while frequent detection in feces from Vietnamese pigs and poultry (average, 58%) suggested the existence of animal reservoirs for such transmission. Further studies are needed to address the epidemiology and pathogenicity of this novel, potentially zoonotic virus