**7. Interface system pathology – pain hypersensitivity (fibromyalgia syndrome) as a diseasome**

Epidemiological evidence suggests that an adverse prenatal environment permanently 'programs' physiology and increases the risk of cardiovascular, metabolic, neuroendocrine and psychiatric disorders in adulthood. Prenatal stress or exposure to excess glucocorticoids might provide the link between foetal maturation and adult pathophysiology (Turner et al., 2008). In a variety of animal models, prenatal stress, glucocorticoid exposure and inhibition (or knockout of) 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2)--the foetoplacental barrier to maternal glucocorticoids--reduce birth weight and cause increases in adult blood pressure, glucose levels, hypothalamic-pituitary-adrenal (HPA) axis activity and anxiety-related behaviours. In humans, mutations in the gene that encodes 11betahydroxysteroid dehydrogenase type 2 are associated with low birth weight. Babies with low birth weight have higher plasma cortisol levels throughout life, which indicates HPA-axis programming. In human pregnancy, severe maternal stress affects the offspring's HPA axis and is associated with neuropsychiatric disorders; moreover, maternal glucocorticoid therapy alters offspring brain function (Turner et al., 2008). Genetic variation in HPA axis genes was associated with programming might reflect permanent changes in the expression of specific transcription factors, including the glucocorticoid receptor; tissue specific effects reflect modification of one or more of the multiple alternative first exons or promoters of the glucocorticoid receptor gene. Intriguingly, some of these effects seem to be inherited by subsequent generations that are unexposed to exogenous glucocorticoids at any point in their lifespan from fertilization, which implies that these epigenetic effects persist (Turner et al., 2008). The Fibromyalgia Syndrome (FMS) can be viewed from a certain perspective as a

referred to as a 'disease module', representing a group of network components that together contribute to a cellular function and disruption which results in a particular disease

Similarly, the systematic mapping of the network based dependencies between pathophenotypes and their disease modules has culminated in the concept of the 'diseasome' which represents disease maps whose nodes are diseases and whose links represent various molecular relationships between the disease associated cellular components (Barabási et al., 2011). Understanding such links not only helps us understand how different phenotypes, often addressed by different medical subdisciplines are linked at the molecular level but can also help us to comprehend why certain groups of diseases arise together. Diseasome - based approaches can be expected to aid drug discovery, in particular when it comes to the use of approved drugs to treat molecularly linked diseases. Single target drugs can be expected to correct some dysfunctional aspects of the disease module, but they could also alter the activity of molecules that are situated in the neighbourhood of the disease module, leading to detectable side effects. Analysis of drug target networks demonstrated that many drugs are palliative, that is they do not target the actual disease associated proteins, but proteins in their network neighbourhood. Finally, using network analytic capabilities, safer and more focused multi-target combinations can be designed e.g.

**6.2 Mapping complex conditions with multiple comorbid disorders through a** 

for anti-inflammatory or anticancer drug combinations (Barabási et al., 2011).

**7. Interface system pathology – pain hypersensitivity (fibromyalgia** 

Epidemiological evidence suggests that an adverse prenatal environment permanently 'programs' physiology and increases the risk of cardiovascular, metabolic, neuroendocrine and psychiatric disorders in adulthood. Prenatal stress or exposure to excess glucocorticoids might provide the link between foetal maturation and adult pathophysiology (Turner et al., 2008). In a variety of animal models, prenatal stress, glucocorticoid exposure and inhibition (or knockout of) 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2)--the foetoplacental barrier to maternal glucocorticoids--reduce birth weight and cause increases in adult blood pressure, glucose levels, hypothalamic-pituitary-adrenal (HPA) axis activity and anxiety-related behaviours. In humans, mutations in the gene that encodes 11betahydroxysteroid dehydrogenase type 2 are associated with low birth weight. Babies with low birth weight have higher plasma cortisol levels throughout life, which indicates HPA-axis programming. In human pregnancy, severe maternal stress affects the offspring's HPA axis and is associated with neuropsychiatric disorders; moreover, maternal glucocorticoid therapy alters offspring brain function (Turner et al., 2008). Genetic variation in HPA axis genes was associated with programming might reflect permanent changes in the expression of specific transcription factors, including the glucocorticoid receptor; tissue specific effects reflect modification of one or more of the multiple alternative first exons or promoters of the glucocorticoid receptor gene. Intriguingly, some of these effects seem to be inherited by subsequent generations that are unexposed to exogenous glucocorticoids at any point in their lifespan from fertilization, which implies that these epigenetic effects persist (Turner et al., 2008). The Fibromyalgia Syndrome (FMS) can be viewed from a certain perspective as a

phenotype.

**'diseasome' approach** 

**syndrome) as a diseasome** 

chronic pain/inflammation problem arising subsequent to a hypersensitized pain and/or stress memory in genetically predisposed individuals probably as early as intrauterine life. First degree relatives have a significantly increased risk to develop FMS. Foetal programming is expected to result in severe pathophysiological hyperreactivity when exposed to subsequent stressful stimuli. This, and much other contemporary research implies that foetal and perinatal stress could have long lasting sequelae in adult life and that it also involves inappropriate immune upregulation. The 'at risk' FMS genotype may represent a risk for several non-classical FMS outcomes as central stress induced hypersensitisation appears to cause subsequent susceptibility to posttraumatic stress disorder-like phenomena, chronic pain syndromes, mood disorders and several other adult onset diseases. These effects often include, but are not limited to, anxiety, depression, ADHD, substance use disorders, and tobacco dependence as well as a dramatically increased risk for a variety of mental disorders (Bhadra & Petersel, 2010; González et al., 2010; Natelson 2010). There may exist cross reactions between various emotional stress and pain responses which involve both the immune and nervous systems. These share quite similar processes for pattern recognition and memory consolidation, and may represent a useful perspective from which to regard aetiological relationships between conditions consistently occurring as comorbid disorders.

#### **7.1 Chromosomal fragility in chronic fatigue/fibromyalgia syndrome (FMS)**

In 1995, during research on chromosomal fragile sites at the University of Pretoria, my cytogenetics collaborator, Ingrid Simonic, found an increased expression of common aphidicolin-inducible chromosomal fragile sites in FMS/"chronic fatigue" patients as opposed to unaffected intrafamilial controls (Fig 1) (Simonic & Gericke, Unpublished data).

Fig. 1. Higher frequencies of some aphidicolin-induced common chromosomal fragile sites in FMS/chronic fatigue individuals versus first degree controls.

Stress Shaping Brains: Higher Order DNA/Chromosome

including first degree relatives.

**8. Conclusion** 

Mechanisms Underlying Epigenetic Programming of the Brain Transcriptome 365

effect on cells) and clastogenic (causing disruption or breakages of chromosomes) effects in human lung cells. The types of aberrations seen with treatment of particulate DU are consistent with those induced by other carcinogenic metals (Wise et al., 2007). Tests of 5 Gulf War Veterans in 2007 analysed by Wayne State University Medical staff revealed the 5 Veterans studied have severe chromosome damage. The damage uncovered is 10 times the level found in the normal population. The chromosome damage is similar to that seen when exposed to alpha radiation and could be related to depleted uranium munitions exposure. In another study by Urnovitz et al., (1999), sera from Persian Gulf War veterans contained polyribonucleotides (amplicons) that ranged in size from 200 to ca. 2,000 bp. Sera from controls did not contain amplicons larger than 450 bp. DNA sequences were derived from two amplicons unique to veterans. These amplicons, which were 414 and 759 nucleotides, were unrelated to each other or to any sequence in gene bank databases. The amplicons contained short segments that were homologous to regions of chromosome 22q11.2, an antigen-responsive hot spot for genetic rearrangements. Many of these short amplicon segments occurred near, between, or in chromosome 22q11.2 Alu sequences. These results suggest that genetic alterations in the 22q11.2 region, possibly induced by exposures to environmental genotoxins during the Persian Gulf War, may have played a role in the pathogenesis of the Gulf War Syndrome. However, the data did not exclude the possibility that other chromosomes also may have been involved (Urnovitz et al., 1999). The fragile sitelike nature of all of the breakpoint sites involved in translocations with the recurrent site on 22q11.21, suggests a mechanism based on delay of DNA replication in the initiation of these chromosomal rearrangements (Gotter et al., 2007). This breakpoint is located between two AT-rich inverted repeats that form a nearly perfect palindrome (a typical common fragile site structural characteristic (Politi et al., 2010). It remains remarkable that a known interface between environmental and genomic stress, the DNA double strand break, leading to altered transcriptional activity of genes suggested here to be involved with key PWGSassociated disorders, have not been considered before as a comprehensive study approach for PGWS.. Standard cytogenetic analyses will be inadequate, and a stringently controlled study of in vitro induced breakage is proposed in war veterans and unexposed controls,

Garcia-Campayo and co-workers (2009) took the optimistic view that imaging can take advantage of developments straight from routine individual biomarkers to multiple-scale biomarker profiles. "Imaging should predict treatment response, look at stratification for specific treatment modalities, and look at the characterization of an individual patient" (Garcia-Campayo et al., 2009). Browning et al., 2011 recently reviewed the debate about the nature of somatoform disorders (such as fibromyalgia) and posed the question: "Is there evidence of altered neural function or structure that is specifically associated with somatoform disorders?" These authors described studies reporting neuroimaging findings in patients with a somatoform disorder or a functional somatic syndrome (such as fibromyalgia) and found a "relatively mature literature on symptoms of pain" (and less developed literatures on conversion and fatigue symptoms). (Browning et al., 2011). Both the hippocampus (Emad et al., 2008; Wood et al., 2009; Fayed et al., 2010) and amygdala (Lutz et al., 2008; Burgmer et al., 2009; Valdés et al., 2010) have been implicated in FMS. To summarize, the literature suggests that early life is a period of increased vulnerability, although the effects of stress may be difficult to detect for years (as seems to be the case with

The involved sites included areas harbouring NF-KB genes, which are proneuroinflammatory factors (Fig 2). Several studies indicate that the nuclear factor-kappa B (NF-kappaB) -activation cascade plays a crucial role not only in immune esponses, inflammation, and apoptosis but also in the development and processing of pathological pain (Niederberger & Geisslinger, 2008). This could represent one aspect of a FMS diseasome.

Fig. 2. Examples of induced chromosomal fragile sites in regions harbouring NF-KB genes.

#### **7.2 A diseasome overlap with Persian Gulf War syndrome?**

Chronic fatigue syndrome (CFS) and Persian Gulf War Ilness (PGI)/Persian Gulf War Syndrome (PGWS) and Fibromyalgia syndrome (FMS) are overlapping symptom complexes without objective markers or known pathophysiology according to the literature. When the cerebrospinal fluid proteome was examined to find proteins that were differentially expressed in this CFS-spectrum of illnesses compared to control subjects an identical set of central nervous system, innate immune and amyloidogenic proteins in cerebrospinal fluids were identified in two independent cohorts of subjects with overlapping CFS, PGWS and fibromyalgia. Although syndrome names and definitions were different, the proteome and presumed pathological mechanism(s) appear to be shared (Baraniuk et al., 2005). PGWS may be a syndrome especially occurring in FMS susceptible genotypes when exposed to specific genotoxicants or the stress of warfare. Of the 130 veterans who were evaluated clinically, 103 had unexplained fatigue, and 44 veterans met the 1994 U.S. Centers for Disease Control criteria for CFS (Mc Cauley et al., 2002). The number of Gulf War veterans who have developed the so-called Gulf War syndrome has risen to about one-third of the 800,000 U.S. forces deployed, and unknown proportions of those involved in the subsequent wars. Uncounted civilians and personnel of other nations that fought in Iraq and other wars since 1991 have also been afflicted (Bertell, 2006). Particulate depleted uranium (DU), widely suspected as one of the prime causes of PGWS associated pathologies, compounds were demonstrated to induce time and concentration-dependent cytotoxic (producing a toxic effect on cells) and clastogenic (causing disruption or breakages of chromosomes) effects in human lung cells. The types of aberrations seen with treatment of particulate DU are consistent with those induced by other carcinogenic metals (Wise et al., 2007). Tests of 5 Gulf War Veterans in 2007 analysed by Wayne State University Medical staff revealed the 5 Veterans studied have severe chromosome damage. The damage uncovered is 10 times the level found in the normal population. The chromosome damage is similar to that seen when exposed to alpha radiation and could be related to depleted uranium munitions exposure. In another study by Urnovitz et al., (1999), sera from Persian Gulf War veterans contained polyribonucleotides (amplicons) that ranged in size from 200 to ca. 2,000 bp. Sera from controls did not contain amplicons larger than 450 bp. DNA sequences were derived from two amplicons unique to veterans. These amplicons, which were 414 and 759 nucleotides, were unrelated to each other or to any sequence in gene bank databases. The amplicons contained short segments that were homologous to regions of chromosome 22q11.2, an antigen-responsive hot spot for genetic rearrangements. Many of these short amplicon segments occurred near, between, or in chromosome 22q11.2 Alu sequences. These results suggest that genetic alterations in the 22q11.2 region, possibly induced by exposures to environmental genotoxins during the Persian Gulf War, may have played a role in the pathogenesis of the Gulf War Syndrome. However, the data did not exclude the possibility that other chromosomes also may have been involved (Urnovitz et al., 1999). The fragile sitelike nature of all of the breakpoint sites involved in translocations with the recurrent site on 22q11.21, suggests a mechanism based on delay of DNA replication in the initiation of these chromosomal rearrangements (Gotter et al., 2007). This breakpoint is located between two AT-rich inverted repeats that form a nearly perfect palindrome (a typical common fragile site structural characteristic (Politi et al., 2010). It remains remarkable that a known interface between environmental and genomic stress, the DNA double strand break, leading to altered transcriptional activity of genes suggested here to be involved with key PWGSassociated disorders, have not been considered before as a comprehensive study approach for PGWS.. Standard cytogenetic analyses will be inadequate, and a stringently controlled study of in vitro induced breakage is proposed in war veterans and unexposed controls, including first degree relatives.
