Syndrome
Rooij, A.M. de
Citation
Rooij, A. M. de. (2010, April 27). Genetic and epidemiological aspect of Complex Regional Pain Syndrome. Retrieved from https://hdl.handle.net/1887/15335
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
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Summary, conclusions and future plans
Knowledge about genes involved in CRPS may provide important information on biological pathways involved in the pathogenesis of the syndrome. Such information could potentially open up new avenues for diagnostics and therapeutic options.
However, prior to starting extensive gene finding studies, it is import to establish to what extent genetic factors contribute to CRPS. While the pathogenesis of CRPS is incompletely understood, the role of environmental factors is well established. In approximately 90% of cases the onset of the syndrome is preceded by tissue injury, such as a fracture or a surgical procedure.1 In a recent study an association was found between the use of Angiotensin-Converting Enzyme (ACE) inhibitors and the risk of developing CRPS,2 providing the first molecular insight in the pathophysiology of CRPS.
Little information is available on the role of genetic factors in CRPS. Hence, it is unclear what determines the inter-individual differences in susceptibility of subjects to develop CRPS after exposure to tissue injury. As with probably all diseases, it is very likely that susceptibility to develop CRPS should have some genetic basis. In fact, it becomes increasingly evident that this indeed is the case. First, CRPS patients with a more severe phenotype have a substantially younger age at onset compared with patients in whom the disease remits or stabilizes.3,4 Second, CRPS may cluster in families. There are a few reports of familial occurrence of CRPS with two or three affected members.5,6,7,8,9,10 Finally, there are now several positive genetic associations between CRPS and alleles of the HLA gene complex on chromosome 6.11,12,13,14
Collectively, available information points towards a multifactorial origin of CRPS, i.e., a disease in which both genetic and environmental factors play a role. To date, little research has been done aimed at identifying genetic factors in CRPS.
Therefore, the first aim of this thesis was to study the contribution of genetic factors in CRPS. CRPS may develop without a noxious event and it has been suggested that such patients have a larger genetic predisposition (i.e., a higher genetic load) to develop the syndrome than patients that require a trauma for disease-onset. Although the currently most frequently used diagnostic criteria sets do not require the presence of a trauma, the existence of spontaneous-onset CRPS is highly debated and often these patients are excluded from studies. To identify if CRPS patients with a spontaneous onset differ from patients with a trauma-induced onset, phenotypic characteristics of both groups were compared in Chapter 2. Data of 537 CRPS patients which were followed up at Departments of Anesthesiology of four Dutch hospitals revealed that 39 out of 537 (7%) patients developed CRPS without a known
eliciting event. There where no significant differences between the two groups with respect to gender, or onset in upper or lower limb or left or right side of the body.
Compared with CRPS patients with a trauma-induced onset, spontaneous-onset cases were on average 9 years younger at disease-onset, possibly indicating an increased susceptibility to develop the condition. The median disease duration in spontaneous- onset cases was 1.4 years longer, which may indicate reluctance to make this diagnosis in the absence of a clear initiating event or reflect the poorer prognosis in these patients. No significant differences were observed between the clinical characteristics of CRPS patients in whom the syndrome developed with or without a preceding trauma. In conclusion, CRPS may develop with and without a precipitating noxious event. The younger age at onset of the syndrome in patients in whom the syndrome initiated spontaneously, may suggest that genetic factors play a larger role in these patients than in those in whom the initiation of syndrome requires a trauma.
The familial occurrence of CRPS was assessed in Chapter 3. Families were recruited through the Dutch Association of CRPS patients and through referral by clinicians.
The number of affected members per family, the phenotypic expression, and the mode of inheritance were assessed. Demographic and clinical characteristics of familial CRPS (fCRPS) patients were compared with those of sporadic CRPS (sCRPS) patients from a Dutch population-based study, and with a group of sCRPS patients that was proportionally matched to the type of referral center of the fCRPS probandi to control for referral bias. Thirty-one CRPS families with two or more affected relatives were identified, including two families with five, four with four, eight with three, and seventeen with two affected relatives. In comparison with sCRPS patients, fCRPS patients had a younger age at onset and more often had multiple affected extremities and dystonia. We conclude that CRPS may occur in a familial form, but did not observe a clear inheritance pattern, so, based on these data, no definite conclusions can be made about a possible mode of inheritance. Additionally, patients with fCRPS developed the disease at a younger age and had a more severe phenotype than sporadic cases, suggesting a genetic predisposition to develop CRPS.
The second aim of this thesis was to obtain an estimate of the size of the contribution of genetic factors involved in the etiology of CRPS. A measure for this contribution is the sibling recurrence risk ratio. This is the ratio of the risk of disease for a person given that a sibling is affected, compared with the risk to develop the disease in the general population.15,16 Values higher than 1 are indicative of familial aggregation. The sibling recurrence risk ratio (λsibling) for CRPS was calculated in Chapter 4. We surveyed 405 CRPS patients to collect information on the occurrence of CRPS in their siblings, and
compared this risk with the population risk to develop the syndrome. Information on disease status was collected from 1242 siblings, of which 24 were possibly affected according to their siblings. The diagnosis was confirmed in 16 patients, rejected in two, and could not be verified in the remaining six. Age-specific risk ratios were calculated for younger (<50 years) and older (≥50 years) age groups. The λsibling
including all siblings reported affected was significantly increased to 1.8 (95% CI: 1.1 to 2.7). Using only confirmed affected siblings, the λsibling was 1.3 (95% CI: 0.8 to 2.1), but this increase did not reach significance. The strongest evidence for familial aggregation was seen in the younger age group with a λsibling for possibly affected and confirmed cases of 5.6 (95% CI: 3.0 to 9.8) and 3.4 (95% CI: 1.5 to 6.8), respectively.
We concluded that this study yielded no indications for an overall increased risk of developing CRPS for siblings of CRPS patients. However, when the population is stratified in categories that are based on age at onset of the syndrome, patients with an age at onset younger than 50 years had a substantially higher risk to have an affected sibling, which may indicate that genetic factors play a more pronounced role in patients in whom the syndrome develops at a younger age. These findings also suggest that the chance for success in identifying genetic factors in future studies may be enhanced by selecting young onset cases.
The third aim of this thesis was to identify possible susceptibility and causative genes for CRPS. For this purpose, we performed three genetic studies.
In the first study, we aimed to evaluate if susceptibility genes in the human leukocyte antigen (HLA) complex play a role in CRPS patients with fixed dystonia. The HLA complex comprises a gene family that has important immunologic functions. Prior studies have found associations between CRPS and the HLA complex, but these studies generally lacked the power to draw meaningful conclusions. In Chapter 5 we performed the most extensive study to date investigating the role of HLA alleles (i.e., HLA-A, HLA-B, HLA-DRB1, and HLA-DQB1) in 150 CRPS patients, who also had fixed dystonia. Significant associations were found with CRPS and dystonia and two HLA alleles (i.e., HLA-B62 and HLA-DQ8). The associations remained significant after correction for multiple testing. The involvement of HLA-B62 and HLA-DQ8 in CRPS with dystonia may indicate that these HLA loci are implicated in the susceptibility to develop the syndrome or in its phenotypical expression. Because of the phenotype under study, it is not possible to indicate if the identified associations are related to susceptibility to develop CRPS or dystonia, or perhaps to processes underlying chronification of the syndrome. Future studies should explore the role of
HLA factors in conferring susceptibility to develop CRPS or expression of particular subtypes of the syndrome.
In the second study we evaluated the presence of causative mutations in known primary dystonia genes in CRPS patients with dystonia. A role for genetic factors in the etiology of primary dystonia has long been recognized. To date at least 17 subtypes of dystonia can be distinguished on a genetic basis. For ten of these subtypes a causative gene has been identified. Approximately 20% of all CRPS patients develop dystonia.
In Chapter 6 we hypothesized that genes responsible for monogenic primary dystonia could play a role in the susceptibility to develop dystonia in CRPS patients. To increase the chance of finding gene mutations, we investigated only those DYT genes with a role in biological pathways involved in disease mechanisms that are considered to play a role in CRPS, or disease mechanisms that potentially may express one or more features encountered in CRPS. Hence, DYT genes that are known to play a role in oxidative stress 17, aberrant inflammation,18,19,20,21,22 and/or aberrant neuroplasticity23,24,25,26,27 were investigated. Also DYT genes that potentially may play a role in the development of pain and susceptibility to external triggers in initiating the phenotype were included in this study. We therefore chose to sequence all coding exons and adjacent intronic sequences of the following genes: DYT1, DYT5a, DYT5b, DYT6, DYT11 DYT12, and DYT16 in 44 young-onset CRPS patients with fixed dystonia aiming to identify high-penetrant causal gene mutations. No such mutations were identified, indicating that these genes do not seem to play a major role in CRPS. However, the possibility remains that promoter mutations and larger deletions may have been missed, as our analysis method is not suited to identify such mutations. Also it is possible that one or more polymorphisms in these genes are associated with the susceptibility to develop dystonia, but this was not tested.
In the third study the SCN9A gene was scanned for high-penetrant mutations.
Mutations in the SCN9A gene that encodes the α1 subunit of voltage-gated NaV1.7 Na+ channels have been linked to primary erythromelalgia and paroxysmal extreme pain disorder. In these two syndromes pain sensitivity is altered and vasomotor changes are prominent. Similar clinical features are also observed in CRPS, which makes this gene an attractive candidate for genetic studies in CRPS. In Chapter 7 we investigated the involvement of the SCN9A gene in familial CRPS. NaV1.7 channels are predominantly expressed within nociceptors and are able to amplify sub-threshold stimuli to a level that they can pass the threshold, thereby setting the gain on nociceptors.28,29,30,31,32 We performed a mutation analysis of the gene using genomic DNA of the index cases from four CRPS families. All 26 coding exons and adjacent sequences of the SCN9A gene were analyzed by direct sequencing analysis. No causal
gene mutations were identified in the SCN9A gene in any of the four probands. We did not find any evidence that the SCN9A gene plays a major role in CRPS. It cannot be excluded, however, that the SCN9A gene plays a role in sporadic CRPS, or perhaps even a minor role in familial CRPS. For the latter, many more familial cases need to be investigated and/ or additional genetic methods need to be used to test for gene mutations that remain undetected with direct sequencing.
Taken together, no causative gene mutations in CRPS were found. The implications of the association between CRPS with dystonia and HLA-B62 and HLA-DQ8 are still unclear, but may implicate that immune mechanisms are involved in the etiology of CRPS. Further studies are needed to evaluate the role of HLA-B62 and HLA-DQ8 in different subtypes of CRPS.
Problems in gene identification in CRPS
There are several problems that make gene identification especially difficult in CRPS.
First, there is no golden standard in the diagnosis of CRPS; the diagnosis of CRPS is made on the basis of clinical diagnostic criteria. Different sets of criteria are applied, all with different sensitivity and specificity. The most widely used criteria, also used in the studies described in this thesis, are those proposed by the International Association for the Study of Pain (IASP).33 The IASP criteria have high sensitivity, but low specificity,34 and it is therefore possible that false-positive patients are included in studies. To decrease the number of false-positives and increase clinical homogeneity, it may be preferred to use diagnostic criteria with the highest specificity, for instance the criteria developed by Harden and Bruehl.35,36 However, even patients with long disease duration, who initially fulfilled criteria with the largest specificity, may no longer fulfill these criteria at the time of evaluation, and thus will be excluded. Many young-onset patients will eventually fall in this chronic group. Interestingly, our studies indicate that particularly in young-onset cases genetic factors may contribute to the etiology of CRPS. Excluding these cases because they no longer qualify for CRPS criteria may effectively reduce the chances of finding genetic factors in CRPS. Nevertheless, to increase the chance of finding genetic factors in CRPS, it will be important to restrict patient selection to very homogeneous subgroups and use more stringent diagnostic criteria.
Second, clinical manifestations of CRPS are very heterogeneous. It is not implausible that different disease mechanisms underlie the different patient subgroups, and, accordingly, that different genetic factors are responsible for those disease mechanisms. Consequently, identification of genetic factors may become increasing difficult or even impossible in studies that allow inclusion of clinically heterogeneous patient groups. The recruitment of homogenous subgroups will contribute to a better coherency between clinical manifestations, pathophysiology and genetic mechanisms.
In line with this, it is not surprising that in the sibling recurrence risk study the overall risk of developing CRPS for siblings of CRPS patients was low.
Third, our data indicate that on a population level, the role of genetic factors in CRPS is negligible. However, in line with other studies of this thesis, the heritability of CRPS was substantially higher in young-onset cases. Therefore, future genetic studies should focus on specific subgroups, thereby increasing the chance of finding genetic factors.
Fourth, CRPS has a relatively low incidence, rendering the recruitment of sufficiently large numbers of patients of specific subgroups of the syndrome difficult. To obtain these large numbers, it will be essential to have an international consortium that uses essentially the same criteria for diagnosing patients.
Finally, to date there are no known candidate genes that play a role in CRPS.
Possibilities of identifying genetic factors in CRPS, for instance by linkage studies, are limited as linkage studies are very dependent on the certainty with which individuals can be classified as affected. As mentioned earlier, it is difficult to make an accurate diagnosis in CRPS. Although the percentage of spontaneous-onset CRPS is higher in familial CRPS, most cases still require exposure to a trauma to develop the condition.
As a result, the inheritance pattern in CRPS families may not be useful for a linkage approach, simply because genetically predisposed subjects are clinically unaffected because they have not been exposed to a trauma of sufficient severity to trigger the syndrome (i.e., reduced penetrance). Although the problem of reduced penetrance is observed with many diseases, it may be particularly problematic in CRPS.
Genome wide association studies (GWAS) nowadays are a popular alternative genetic approach to identify gene variants for multifactorial diseases to understand the pathways involved in disease.37 Major breakthroughs in genetic methodology have made it possible, and cost-effective, to test hundreds of thousands of polymorphisms in cases and controls with samples sizes of one to several thousand. Over the last couple of years, an impressive number of causal gene variants has been identified, for many diseases, including neurological diseases.38 Interestingly, there are also success stories of diseases in which most cases, like in CRPS, are sporadic, such as Amyotrophic Lateral Sclerosis.39 Unfortunately, the very low incidence of CRPS combined with the uncertainties in the clinical diagnosis, make GWAS not very appealing for CRPS.
Future plans
As indicated before, it is crucial to obtain very homogeneous subgroups to increase the chances of success in future genetic studies. To date, the most frequently used method to define subgroups is to focus on a prominent clinical characteristic, such as the presence of dystonia or skin temperature characteristics. Another potentially beneficial approach is to use data-driven techniques to identify subtypes of the syndrome. By using cluster analysis techniques, data can be analyzed in an unbiased way, that is, without an a priori hypothesis. This methodology explores whether individuals can be classified into specific groups in such a way that patients within groups are rather similar, while the difference between groups is large. Since patients in the same subgroup are clinically more similar, they are more likely to share disease mechanisms. Another potentially promising method to distinguish subgroups of CRPS patients (i.e., endophenotypes) is the use of biomarkers obtained from body fluids such as urine, cerebral spinal fluid, or, very relevant for CRPS, artificially drawn blister fluid. Biomarkers may not only help in creating subgroups, but may also provide direct indications of the involved biological pathways and thus help to select potential candidate genes for genetic testing. Finally, there is a promising approach to select candidate genes on the basis of gene expression profiling results. With this methodology, variations in gene expression of RNA obtained from white blood cells can be compared between CRPS patients and controls; for instance after first stimulating the white blood cells with a compound that induces a strong response from the immune system. The results of such a study may provide information on potentially involved biological pathways and thus genes involved in these pathways.
In conclusion, CRPS is a complex multifactorial disease and there is increasing evidence for a role of genetic factors in its etiology. The search for causative genes has not been successful yet, although a potential genetic factor seems to reside in the HLA locus. To increase the chances of successful gene identification studies in the future, well-defined subgroups of CRPS must be selected. The use of new methodologies, such as cluster analysis or gene expression profiling, can be very helpful to achieve this goal. As genome wide association studies are beginning to yield gene variants for complex diseases, it seems only a matter of time before this novel methodology will be used on (very) large numbers of CRPS patients. It is expected that the identification of genes in CRPS will provide more insight in the etiology of the syndrome and eventually may contribute to the development of new treatments.
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