Chronic Fatigue/Pain Syndromes: An Overview of Current Evidence

Several lines of evidence suggest that corticosteroid-binding globulin (CBG), long known as a cortisol-transport glycoprotein, may have broader roles in targeted-tissue hormone delivery and the neurobehavioural responses to stress. These include studies of individual kindreds with rare severe CBG gene (SERPINA6) mutations, a study of chronic fatigue patients, a community study of individuals with a relatively high prevalence of two function altering CBG gene mutations in Calabria, Italy, a study of the genetic epidemiology of chronic pain, and, finally, two separate animal CBG gene knockout models.


Introduction
Several lines of evidence suggest that corticosteroid-binding globulin (CBG), long known as a cortisol-transport glycoprotein, may have broader roles in targeted-tissue hormone delivery and the neurobehavioural responses to stress.These include studies of individual kindreds with rare severe CBG gene (SERPINA6) mutations, a study of chronic fatigue patients, a community study of individuals with a relatively high prevalence of two function altering CBG gene mutations in Calabria, Italy, a study of the genetic epidemiology of chronic pain, and, finally, two separate animal CBG gene knockout models.

Corticosteroid-binding globulin: More than just a transport glycoprotein
CBG has traditionally been considered to be a transport vehicle for the water insoluble cortisol 29 , with perhaps some role in moderating release of free cortisol in times of cortisol excess or deficiency 30 .This is in keeping with the 'free hormone hypothesis' proposed by Mendel 31 , which states that the biological activity of a hormone depends on the free rather than its protein-bound concentrations.The free steroid hormone can cross the plasma membrane of the target cell due to its small size and lipid solubility 32 .
However, there is evidence that suggests that CBG-bound cortisol could play a functional role different to unbound cortisol.While a specific CBG cell receptor has not yet been cloned, cell membrane binding sites for CBG, which share many features of a receptor, have been known for some time 33,34 .This has led some to speculate that CBG may act as a hormone and there may be a direct contribution of bound cortisol in glucocorticoid bioavailability via this yet unidentified CBG receptor 34 .Accumulation of cyclic AMP 35 occurs as a result of this CBG:cell receptor interaction.Recently, it has been shown that the NeuAc residues on the N-glycans restrict the binding of CBG to the cell receptor.Removing these NeuAc residues resulted in marked increase in cyclic AMP levels 35 .Dilution of CBG results in release of cortisol and thus suggests, at the very least, an indirect contribution of bound cortisol in glucocorticoid bioavailability 36 .
A closely related steroid binding glycoprotein in the human body is sex hormone binding globulin (SHBG), which binds testosterone and oestradiol.A role for SHBG beyond transport has been shown.Sex steroid tissue delivery involves endocytic uptake of SHBGsex steroid complexes via the LDL receptor-related protein member megalin 37 .Megalin knockout mice exhibit sexual infantilism 37 .While possible a megalin-like mechanism for endocytic uptake of CBG-cortisol complexes has not been demonstrated.

Chronic fatigue/pain syndromes, the concept of 'allostasis' and the role of hypothalamo-pituitary-adrenal axis
Chronic fatigue/pain syndromes are common.Epidemiological studies have estimated the point prevalence of chronic widespread pain (CWP) in the community to be up to 11% 38 , and about 9% of the total population will experience significant chronic fatigue at any one time 39 .It should be noted, however, that up to two-thirds of these patients complaining of chronic fatigue will not meet the criteria for chronic fatigue syndrome (CFS) 40,41 .A 71 considerable overlap exists, however, in terms of both clinical and biochemical characteristics, and perhaps the pathogenesis of chronic fatigue and chronic pain 42 .
CFS is a clinical diagnosis, the hallmark of which is disabling fatigue for over six months with prolonged (>24hrs) post-exertional exacerbation along with other symptoms which include impaired short-term memory and concentration, sore throat, tender lymph nodes, myalgia, arthralgia, headaches and unrefreshing sleep 43 .Since the term 'chronic fatigue syndrome' was proposed in 1988 to replace the prior 'chronic Epstein Barr virus syndrome' (based on the realization that not all chronic fatigue cases were post-infective in nature) 44 , newer aetiological models based on neuroendocrine 45 , psychiatric 46 , evolutionary 47 , immunological 48 and non-infective inflammatory 49 mechanisms have been described.A well-accepted explanation for the development of CFS or even the relative contribution of the different possible mechanisms, however, remains elusive.CFS, a debilitating disease sharing many features with fibromyalgia 50 , CWP and similar idiopathic chronic fatigue syndromes, significantly impairs a patient's quality of life 51 , 52 , social 53 and emotional well being 54 , 55 , besides putting considerable economic strain on the community 56 .It is, therefore, imperative that a better understanding of the causation of CFS and related disorders is achieved to enable development of effective therapeutic options, which are currently lacking 57 .
A hereditary component to CFS has also been suggested [58][59][60] .Recently, an analysis of the Utah population database (UPDB) looking at the genetic relationships of CFS patients was published 61 .811 patients diagnosed with CFS by the US CDC or the Fukuda criteria 43 underwent genealogical analysis.A significant excess in CFS relative risk among first (2.70,95% CI: 1.56-4.66),second (2.34, 95% CI: 1.31-4.19),and third degree relatives (1.93, 95% CI: 1.21-3.07)was observed.The human stress system includes the two effector arms, the hypothalamic-pituitary-adrenal axis (HPA axis) and the sympathetic nervous system, with their chief hormonal products, cortisol and noradrenaline/adrenaline, respectively.The stress system responds in a highly coordinated and stress-specific manner to stressors, which may be defined as threats to homeostasis or the stable internal milieu of the organism.Stressors such as infection, inflammation, trauma, and psychic disturbance such as fear or anxiety act via inflammatory cytokines or internal CNS influences to produce a range of physiological responses designed to protect the body from stress, such as elevated blood pressure, redirection of blood flow, mobilization of metabolic substrates and CNS arousal.The stress system has basal tone and it has been proposed that altered chronic stress system activity, which may be produced by excessive stress at key developmental stages such as intrauterine, childhood and adolescence, may be detrimental to health.For example, excessive stress system activity may lead to metabolic deterioration such as hypertension, diabetes, central adiposity, osteoporosis and mental illness, which together comprise a high proportion of modern chronic illnesses.Chronically altered stress system activity may be described as a state of allostasis representing stability but with risk of long term tissue damage 62 .
On the other hand, reduced stress system activity, another form of allostasis, may be expected to produce a state of hypo-arousal and lack of anti-nociceptive activity centrally, leading to the many chronic pain and fatigue based disorders (CFS, fibromyalgia, irritable bowel syndrome, migraine and many others).A number of studies have shown relative hypocortisolism in pain/fatigue disorders [63][64][65] .In patients with CFS, studies have demonstrated low levels of cortisol in plasma 63,64 (in morning 63 as well as in the evening 66 ), urine 63,[67][68][69][70] and saliva [70][71][72][73] .Corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) challenge tests, which test adequacy of the HPA axis also show similar results in CFS patients 63 although not consistently 45 .Hypocortisolism has also been shown in patients with fibromyalgia [74][75][76] and chronic pain syndromes 77,78 .Chronic fatigue syndrome has a strikingly high female preponderance (up to 75%) and it has been shown that the glucocorticoid sensitivity of pro-inflammatory cytokine production after psychological stress is different among the sexes 79 .

CBG gene mutation: Kindred studies
Four major function altering mutations of the CBG gene have been described in humans.These include CBG Leuven, CBG Lyon, CBG null and a CBG non-cortisol binding variant.Old and new genetic nomenclature for these mutations is shown in Table 1.CBG Leuven (c.344T>A, p.Leu115His) reduces CBG:cortisol binding three-fold 15,80 .CBG Lyon has been described in three kindreds and reduces cortisol binding affinity 4-fold (c.1165G>A, p.Asp389Asn) 15,81 .CBG null (c.32G>A, p.Trp11X) prevents CBG synthesis and homozygotes are completely CBG deficient 82 .Both CBG Lyon and null are associated with fatigue and chronic pain and were described together in single kindred where the phenotype was similar 82 .The description of a kindred with a non-cortisol binding variant of CBG included an index case with fatigue 83 .

CBG null
We have described a 39 member Italian-Australian kindred with a novel null (complete lossof-function) CBG mutation, an exon 2 mutation causing premature termination codon corresponding to residue-12 (c121GA) 82 (Fig. 1).The 48 year-old female proband was found to have low total plasma cortisol levels but normal 24-hour urinary free cortisol.She had an elevated plasma cortisol fraction and undetectable CBG levels.CBG gene sequencing of the family revealed two null homozygotes, 19 null heterozygotes, three Lyon heterozygotes and two compound (Null/Lyon) heterozygotes.CBG levels were also undetectable in the other two CBG null homozygotes.There was a 50% CBG reduction in the null heterozygotes and an even greater reduction in the compound heterozygotes.Five

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Corticosteroid-Binding Globulin Gene Mutations and Chronic Fatigue/Pain Syndromes: An Overview of Current Evidence 73 members of the family, including the female proband, met the United States' Centre for Disease Control (CDC) criteria 43 for chronic fatigue syndrome.In addition, 12 out of the 14 members with heterozygote mutation and two out of three with homozygous mutation were found to have idiopathic chronic fatigue.Pain syndromes were observed in six subjects with null mutation -four were null heterozygotes while two were homozygotes.One of these pain affected null subjects also fulfilled the criteria for CFS.Prior to finding this family with CBG null deficiency it had been thought that complete CBG deficiency was incompatible with life 9,84 .

CBG Lyon
The CBG variant produced due to a missense mutation, an aspartic acid to aspargine substitution at residue 367 (Asp 367  Asn) was first described in a 43 year old woman of northwest African origin in Lyon, France, and is known as the CBG Lyon mutation 81 .Her main symptoms included chronic asthenia, depressive mood and hypotension.She was found to have very low total plasma cortisol but normal ACTH and urine free cortisol levels.A low free cortisol concentration suggested an abnormality in CBG binding and was later confirmed.Her four children were all found to heterozygous for this mutation.
Since then, CBG Lyon has been described in two other kindreds 82,85 .The mutation has been reported in a 40 year-old white Brazilian woman presenting with chronic fatigue and hypotension.The family members screened, including her parents and her children, were found to be heterozygous for the mutation but did not complain of chronic fatigue 85 .We have also reported CBG Lyon mutation in the family members of a proband with CBG null mutation 82 .Three family members were CBG Lyon heterozygotes, while two had coinheritance of CBG Lyon and CBG null (compound heterozygotes).The family members with co-inheritance had clearly low CBG levels.All available family members with CBG Lyon mutation, both heterozygotes and compound heterozygotes reported significant fatigue 82 .

CBG non-binding Gly 237 Val
This CBG gene polymorphism, involving a c.776g>t transversion in exon 3 of the SERPINA6 gene resulting in a p.Gly237Val substitution, was described recently in a 26 year-old Pakistani-British woman presenting with fatigue and hypotension.This CBG variant lacks any steroid binding activity.Two siblings were found homozygous for this mutation and two more family members (including the proband's mother) were found to be heterozygous.The homozygous members had reduced CBG levels (about 50% for the proband) by RIA measurements but undetectable CBG when measured with cortisolbinding capacity assays.However, aside from the proband, none of the family members, including two siblings found homozygous for the mutation, reported symptoms of chronic pain or fatigue.The only biochemical finding that differentiated the proband from the other homozygous members was the increased cortisol pulsatility 83 .

Genetic epidemiology studies
Given the evidence from the kindred studies, we hypothesized that CBG polymorphisms could act as a genetic risk factor for patients with CFS.Two hundred and forty eight patients with CFS and an equal number of control subjects had full CBG gene sequencing.An exon 3 polymorphism (c.825G-->T, Ala-Ser224) was more commonly observed in CFS patients than expected by chance at the trend level (P<0.07),suggesting that homozygosity for the serine allele of the CBG gene may predispose to CFS 86 .
We also conducted a population-based study in Calabria, Italy, the region our Italian-Australian null/Lyon kindred originated from, to look at the prevalence of CBG mutations in the local community.The results showed a high prevalence of CBG null and Lyon mutations (3.6%).Chronic widespread pain, but not chronic fatigue, was found to be common in subjects with CBG mutation 87 .
Genetic influences have been postulated to account for 50% of the variance as well as the reduced pain thresholds seen in chronic pain syndromes 88,89 .The prospective populationbased cohort study EPIFUND (Epidemiology of functional disorders), examined if genetic variation within the HPA axis genes was associated with susceptibility to musculoskeletal pain.The CBG gene (SERPINA6) and six other HPA axis genes CRH, CRH receptor 1 (CRHR1), CRH binding protein (CRHBP), the ACTH precursor pro-opiomelanocortin (POMC) and its receptor (MC2R), the glucocorticoid receptor (NR3C1) were examined.Seventy-five single nucleotide polymorphisms (SNPs) were detected in 164 CWP patients and 172 pain-free controls.Amongst the seven HPA axis genes, the most notable genetic variation was in the SERPINA6 gene.Two SNPs in SERPINA6 (rs 941601 and rs 8022616), located within a single haplotype block, were significantly associated with CWP.Moreover, in patients reporting pain, four SNPs of SERPINA6 were associated with the maximum number of pain sites 88 .This finding assumes significance given that there was no association with SNPs in CRH, CRHR1, CRHBP, POMC or NR3C1 and CWP was observed and only a single SNP in MC2R, rs11661134, was associated with increased odds of having CWP.

CBG gene knockout mice models
The effect of a gene deletion can also be studied in the laboratory setting by producing 'knockout gene' mouse models, achieved by a homologous recombination between DNA sequences in the existing chromosome and the newly introduced DNA into pluripotent embryo-derived stem cells 90 .In the study reported by Richard et al, the CBG gene knockout mouse was created by 'floxing' -contraction for flanking the lox p sites -exon 2 of the SERPINA6 gene 91 .The learned helplessness paradigm 92 , an animal model of depression, was used to evaluate behavioural changes following intense stress.HPA axis dysregulation has previously been linked to helpless behavior 93,94 .The CBG deficient mice (Cbg -/-) showed increased immobility in the forced-swimming test and markedly enhanced learned helplessness after prolonged uncontrollable stress (footshock) as well as markedly reduced total circulating corticosterone in both rested and stressed states.Responses to milder stressors was not altered.In another CBG knockout mice study 95 , Cbg -/-mice had a reduction in CBG levels and a correspondingly ten-fold increase in levels of free cortisol.Despite this, there was no evidence of enhanced glucocorticoid activity, suggesting the role of CBG in mediating corticosteroid functions.More importantly, Cbg -/-mice exhibited a possible fatigue syndrome, characterised by reduced activity levels compared with the control group.The elevated cortisol and reduced activity levels were not seen in the study of Richard et al 91 .Taken together, however, these findings suggest an important hitherto unanticipated role for CBG in the neurobehavioural aspects of stress system function.

Conclusion
There is an unequivocal role for CBG as a transport molecule for cortisol, and altered levels of CBG are generally met with unaltered levels of free cortisol, confirming it is free cortisol which is actively regulated in blood.
However, recent studies have linked rare CBG gene mutations, which alter CBG levels or binding affinity, to pain/fatigue syndromes.This association is not universal suggesting that other genetic or environmental factors influence the phenotype.Genetic epidemiology studies point to the CBG gene and its variants as having a role in the risk of developing a chronic pain phenotype.Animal studies have also shown that CBG genetic deletions can produce altered neurobehavioural responses to stress.This mounting evidence suggests a role for CBG in tissue delivery or other elements of stress system function, although the precise mechanisms await elucidation.

Table 1 .
Old and new nomenclature for known mutations and polymorphisms in CBG