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CASP-9: A susceptibility locus for multiple sclerosis in Italy
Journal of Neuroimmunology 210 (2009) 100–103
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Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m
CASP-9: A susceptibility locus for multiple sclerosis in Italy V. Andreoli a,⁎, F. Trecroci a, A. La Russa a, P. Valentino b, F. Condino a, V. Latorre a, R. Nisticò b, D. Pirritano b, F. Del Giudice b, M. Canino b, R. Cittadella a, A. Quattrone a,b a b
Institute of Neurological Sciences, National Research Council, Pianolago di Mangone, Cosenza, Italy Institute of Neurology, Campus di Germaneto, University “Magna Graecia”, Catanzaro, Italy
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Article history: Received 10 December 2008 Received in revised form 24 February 2009 Accepted 5 March 2009 Keywords: Multiple sclerosis CASP-9 Polymorphism Association study Genetic susceptibility
a b s t r a c t Caspase-9 is a primary effector CASP that executes programmed cell death, which plays an important role in the development of multiple sclerosis (MS). Polymorphisms in the CASP-9 gene may influence its activity, thereby modulating the susceptibility to MS. To test this hypothesis, we evaluated a SNP in the CASP-9 gene in a set of Italian patients from Southern Italy and healthy control subjects. Our results suggest that the presence of the G/G genotype represents a higher risk factor in our MS population and a differential production of CASP-9 might be a contributory factor in determining the severity of MS. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease of undetermined aetiology, affecting mainly the white matter of the central nervous system (CNS) and is clinically characterised by progressive disability (Steinman, 1996). In recent years, several data have confirmed the presence of axonal damage in MS and have shown that the disability caused by the disease better correlates with axonal loss than it does with extensive demyelination (Trapp et al., 1998; Noseworthy et al., 2000). Although the initiating event is a matter of debate, epidemiological and genetic findings suggest that MS is an acquired autoimmune and inflammatory disease, triggered by unknown environmental factors in genetically susceptible individuals (Compston and Coles, 2002). These results demonstrate that individuals might be predisposed to MS as a result of the inheritance of many genetic factors of modest contribution that, if revealed, may present important targets for new therapies (Stewart, 1997; Oksenberg et al., 2001). What seems certain is that MS is a disease with heterogeneous pathogenic mechanisms, and several studies support the argument that MS is a primary disease of either axons, neurons or oligodendrocytes and that immune response is secondary to neurodegeneration (Trapp et al., 1998; Bö et al., 2003). Recently, neuronal apoptosis has been described in cortical MS lesions (Peterson et al., 2001) and in experimental autoimmune encephalo⁎ Corresponding author. Institute of Neurological Sciences, National Research Council, Pianolago di Mangone, 87050, Cosenza, Italy. Tel.: +39 09849801300; fax: +39 0984969306. E-mail address: [email protected] (V. Andreoli). 0165-5728/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2009.03.013
myelitis (EAE), a rat model of MS (Meyer et al., 2001). The apoptotic programme is executed by a family of essential proteases known as caspases (Nicholson and Thornberry, 1997). So far, at least two main caspase-activating cascades have been characterised: the mitochondria-mediated caspase-3 activation by caspase-9 (intrinsic pathway) and death-receptor-induced caspase-3 activation by caspase-8 (extrinsic pathway) (Zheng and Flavell, 2000). Particularly, caspase9 (CASP-9) plays a crucial role in the initiation phase of the intrinsic pathway for apoptosis. In fact, many proapoptotic stimuli engage the apoptotic machinery in the cells by causing the release of cytochromec from mitochondria, which then induces oligomerisation of a protein called Apoptotic protease activating factor-1 (Apaf-1) and recruitment of CASP-9 into a large complex known as the apoptosome. Apoptosome then activates the CASP-9 cascade downstream with effector caspases, leading to apoptosis (Srinivasula, 1998; Li et al., 1997). The mechanism is evolutionarily conserved and may play an important role in mediating neuronal death; dysregulation of this normal control mechanism then could be a contributor to various diseases characterised by excessive or inadequate cell death. These findings raise the intriguing possibility that genetic variations in the CASP-9 gene could influence susceptibility to the disease. CASP-9, mapped to the short arm of chromosome 1p36 (Hadano et al., 1999) in humans, is composed of nine exons; some polymorphisms have also been described within this gene. However, the potential role of the single nucleotide polymorphism (SNP) of the CASP-9 gene in establishing susceptibility to MS has never been clearly defined. In particular, a SNP in the coding region CASP-9 Ex5 + 32G NA causes a conservative change of a glutamine with an arginine (Q221R) (Hirano et al., 2001) and, thus, may have functional significance. In order to shed light on a
V. Andreoli et al. / Journal of Neuroimmunology 210 (2009) 100–103
biologically potential role of the CASP-9 gene in susceptibility to MS, we have genotyped this SNP in a collection of MS patients and healthy control subjects from Southern Italy. This is the first study to investigate possible relationships between MS and CASP-9 gene polymorphisms in Italian patients. 2. Materials and methods 2.1. Patients and healthy individuals Our study sample consisted of 295 unrelated cases with clinically defined MS (Poser et al., 1983; McDonald et al., 2001), all Caucasians from the south of Italy (Calabria), and followed at the Institute of Neurology, University “Magna Graecia” of Catanzaro. Patients whose disease course was classified (Lublin and Reingold, 1996) as Relapsing–Remitting MS (RRMS, n = 215, 73.0%), Secondary Progressive MS (SPMS, n = 62, 21.0%) or Primary Progressive MS (PPMS, n = 18, 6.0%) were enrolled in this study. One hundred eighty-five patients were women and 110 were men. The following clinical and genetic variables were recorded for each patient: age, sex, disease duration, age at disease onset and level of disability according to the Kurtzke Expanded Disability Status Scale (EDSS) (Kurtzke, 1983). Clinical characteristics of the MS patients are summarised in Table 1. The control population included 295 unrelated healthy subjects (130 women and 165 men; mean age ± SD: 35.3 ± 8.2 years), enrolled during a previous study without history of inflammatory and/or degenerative neurological diseases. To avoid any bias attributed to the ethnic origin of the study population, only Caucasian MS patients and controls whose grandparents were all born in Calabria were included in the analysis. The differences in the sex ratio and ages between the patients and the controls were not significant (p N 0.05). Patients and controls gave their written informed consent before the examination and blood testing, and the study was approved by the local ethical committee. 2.2. CASP9 Ex5 + 32G N A genotyping Blood samples for the genomic DNA studies were obtained from peripheral blood leukocytes and DNA was extracted according to standard procedure. In principle, DNA was amplified using PCR in a total volume of 50 μl containing 15 pmol of each primer, designed according to the published sequence (Hirano et al., 2001), 200 ng genomic DNA and AmpliTaq Gold (Applied Biosystems), and using standard conditions on a PTC-100™ Programmable Thermal Controller (MJ Res. Inc. Genenco). Lastly, a 185 bp fragment containing the A → G transversion in exon 5 was amplified using the primers F:5′CGGTCCAGTCTGCATCTAGAC-3′ and R:5′-ATGCCTGCCCAGGG AACAGT3′ (annealing temperature 59 °C). Ten microlitres of the PCR product were incubated with 10 U BstUI (New England Biolabs, Beverly, MA, USA) in a total volume of 25 μl for 3 h at 60 °C. This gave products that either remained intact (allele A) or were cut into two fragments of 94
Table 1 Demographic and clinical variables of MS subjects analysed. Variable
Patients (n: 295)
Male sex: no. (%) Age (yr): mean (SD) Age at onset (yr): mean (SD) Disease duration (yr): mean (SD) Disease course: no (%) RR SP PP EDSS score: median (range)
110 (37.3) 39.8 ± 12.0 28.1 ± 9.1 13.5 ± 9.4 215 (73.0) 62 (21.0) 18 (6.0)
Data are given as means ± SD and percentage. EDSS: Expanded Disability Status Scale.
3.3 (0–8.5)
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Table 2 CASP9 Ex5 + 32G NA genotypes and alleles of cases and controls and their association with the risk of MS. Ex5 + 32G NA (Q221R)
Controls (n = 295)
Patients (n = 295)
Genotype A/A A/G G/G Allele A G
No. (%) 116 (39.3) 134 (45.4) 45 (15.3)
No. (%) 65 (22.0) 154 (52.2) 76 (25.8)
1.0 2.03 (1.34–3.07) 2.73 (1.63–4.55)
b0.001
366 (62.0) 224 (38.0)
284 (48.1) 306 (51.9)
1.0 1.68 (1.31–2.16)
b0.001
OR (95%CI)
p value
Categorical variables are expressed as frequency and percentage. The differences among groups distribution were assessed using χ2 test. Odds ratios and 95% confidence intervals were calculated according to a multivariate logistic-regression model, adjusted for age and sex.
and 91 bp (allele G). Both digested and undigested DNA fragments were visible for the respective restriction site in heterozygous samples. Amplified DNA fragments and digestion products were separated on 3% agarose gels and visualised by ethidium bromide.
2.3. Data analysis The Hardy–Weinberg equilibrium was tested using Pearson's χ2 goodness of fit test for CASP-9 Ex5 + 32G NA polymorphism. Using univariate analyses for this bi-allelic marker individual p-values were calculated using standard 3 × 2 and 2 × 2 χ2 contingency tables comparing genotype and allele counts in MS cases against controls. Categorical variables were expressed as counts and percentages, continuous variables were shown by mean and standard deviation and discrete variables by median and range. Relative risks for MS, estimated as the odds ratios [ORs] and 95% confidence intervals (95% CI), were calculated by multivariate logistic-regression analysis, adjusting for sex and age. To evaluate possible differences in clinical features among each +32G NA polymorphism variant in the patients group, one-way analysis of variance (ANOVA test) or Kruskal–Wallis test was used. Differences in sex distributions among groups were evaluated with the χ2 test. In all tests, a p-value less than 0.05 denoted the presence of a statistically significant difference. Statistical analyses were performed with Statistical Package for Social Sciences software (SPSS version, 12.0, Chicago, IL, USA) for Windows.
3. Results 3.1. CASP9 Ex5 + 32G N A genotypes and susceptibility to MS In our study, control and patient sample genotype frequencies were distributed according to Hardy–Weinberg equilibrium (controls, p = 0.54; MS, p = 0.48). Table 2 depicts the allele and genotype frequency distribution and association of the studied polymorphism in MS individuals. We found that the frequency of the G/G homozygote in MS patients (25.8%) was significantly higher than in the controls (15.3%), and the subjects who carried this genotype had a 2.73-fold increased risk for developing MS (p b 0.001, OR = 2.73, 95% CI = 1.63–4.55). In the public databases the A allele is reported to be the largest allele of this SNP (rs1052576) in the European population. Here, CASP-9 A allele was observed to be significantly high in our controls (62.0%), providing a reduced risk; the G allele was also overrepresented in MS patients (51.9%) in comparison with the controls (38.0%, p b 0.001, OR = 1.68, 95% CI = 1.31–2.16). Our results show that the CASP-9-G allele was associated with increased predisposition to MS. These data emphasize that the striking associations observed for this genetic variation in CASP-9 genes may play an important role in the aetiology of MS.
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3.2. Gender, genotype and MS risk The entire sample consisted of 590 subjects (315 women and 275 men). Interestingly, in men the + 32 G/G genotype (CASP9 Ex5 + 32G NA polymorphism) was associated with a very significantly increased risk of MS (p b 0.001, OR = 4.77, 95% CI = 2.15–10.52). In addition, among women the same G/G genotype was associated with a less pronounced but still significantly increased risk of MS (p = 0.012, OR = 2.67, 95% CI = 1.35–5.26). 3.3. Clinical characteristics and genotypes Stratifying MS patients according to the course of the disease, a trend towards an increase in the allelic frequency of this SNP in RRMS patients compared with controls (50.5 versus 37.1%; p b 0.001) was observed. Indeed, these results were derived from a significantly increased number of individuals, with the G/G genotype in this MS population, compared with healthy subjects (26.0 versus 14.0%; p b 0.001; OR = 2.78, 95% CI = 1.66–4.64). As the disease course in MS can be defined with greater certainly after 10 years from onset, the association was also analysed in patients with a disease duration of more than 10 or 20 years. Interestingly, the analysis confirmed the association between the GG genotype and milder disability in MS patients, with a disease duration of more than 10 years (p b 0.001; OR = 2.46, 95% CI = 1.38–4.38). Moreover, OR analyses in patients with a disease duration of more than 20 years showed the association to become stepwise increasingly marked over the years (p b 0.001, OR = 3.31, 95% CI = 1.39–7.84) (Table 3). This suggests that the genetic influence of CASP-9 on MS risk and progression may become more prominent with increasing duration of the disease. Finally, no association between genotypes or alleles of the CASP9 Ex5 + 32G NA variant and age at disease onset or EDSS score (Kurtzke, 1983) was detected (data not showed). 4. Discussion In this report, we provide evidence that a variation of the CASP-9 gene is directly associated with susceptibility to MS in our Italian patients. The major finding of the present study is that the producer genotype AA is preventative against the patient developing the severe form of MS, while the high-producer GG is associated with different degrees of severity, significantly so in all patients with a disease duration of more than 20 years. It should be noted that CASP-9 Ex5 + 32G NA polymorphism encodes for a glutamine to arginine amino acid change at codon 221 of the protein, located at the border of the helix region of CASP-9. In particular, the Q221R variant might lead to conformational changes in the molecule, resulting in an alteration of its function. However, the contribution of this polymorphisms remains unknown at this stage and the possible functional role of this variant should be investigated. Altogether, it is possible that the mutated allele modifies the affinity of this protein to Apaf-1, influencing the amount of neuronal damage in the lesions and thus the progression of disability in MS. Hence, direct functional effects of the polymorphism studied here are biologically plausible because CASP-9, as an initiator CASP, plays an important role in the apoptosome-driven apoptosis pathway, which is essential for eliminating mutated or transformed neural progenitor cells from the brain Table 3 Relative risks for MS, estimated as the odds ratios [ORs] and 95% confidence intervals [CI], as analysed by logistic regression in all patients and in patients with disease duration (dd) more than 10 and 20 years. Genotype G/G
%
ORs
95% CI
p value
Total group Patients with dd ≥ 10 Patients with dd ≥ 20
25.8 25.0 30.9
2.73 2.46 3.31
1.63–4.55 1.38–4.38 1.39–7.84
b0.001 b0.001 b0.001
(Yin et al., 2006; Hakem et al., 1998). In theory, at the protein level the GG genotype of CASP-9 Ex5 + 32G NA may produce higher levels of the CASP-9 protein and therefore up-regulate apoptotic processes in MS as it increases the apoptosome formation under normal physiological conditions. In addition, evidence of neuronal apoptosis in MS is currently emerging. During the past few years several death receptor/ ligand systems mediating apoptosis have been discovered. Tumour necrosis factor (TNF), CD95 (APO-1/Fas) ligand and other ligands, such as TNF-related apoptosis-inducing ligand (TRAIL), interact with their respective receptors to induce apoptotic cell death and were found to be responsible for MS risk (Amirzargar et al., 2007; Kantarci et al., 2004; Kikuchi et al., 2005). New structural evidence suggests that CSF from MS patients induces apoptotic cell death of cultured neurons (Alcazar et al., 1998, 2000), which may also have a correlation with clinical disability (Cid et al., 2002). More specifically, apoptotic neurons have recently been identified in both cortical MS lesions (Meyer et al., 2001) and EAE (Nicholson and Thornberry, 1997). Evidence from CASP-9 knock-out mouse models and CASP-9 null cell lines show that CASP-9 is essential to the regulation of cell homeostasis through cleavage of many key players involved in apoptosis. Moreover, these mouse models of mutant CASP-9 mice have been associated with perinatal and postnatal death (Kuida et al., 1998; Hakem et al., 1998). Interestingly, more recent studies have demonstrated that aberrant apoptosome function, potentially arising from a lack of CASP-9 bound to oligomerised Apaf-1 (Liu et al., 2002; Wolf et al., 2001), contributes not only to carcinogenesis (Shivapurkar et al., 2003) but it has also been implicated in the inappropriate apoptosis characteristic of various neurodegenerative disorders (Sang et al., 2005). If apoptosome activity is necessary for the cell death characteristic of neurodegeneration, then inactivation of the apoptosome might provide a therapeutic avenue for treating these disorders as well as MS. The latter is consistent with the finding that one recently identified Apaf-1-interacting inhibitor, AIP (a splice variant of caspase-9 that is expressed endogenously in the brain), was found to be capable of protecting neurons from cell death induced by cerebral ischemia (Cao et al., 2004). So far, the best therapeutic options for neuroprotection in MS are the immunomodulatory agent glatiramer acetate and interferon beta drugs (Dhib-Jalbut, 2002). Although beneficial, these two options fail to prevent the progression of disease in some patients; hence, there is the need to explore new strategies that not only limit inflammation and demyelination in the CNS but also prevent axonal loss (Costello et al., 2007). Currently, minocycline (MIN), a tetracycline antibiotic under evaluation for use in neurodegenerative diseases, has shown remarkable benefit in animal models with several neurological disorders, including MS (Yong, 2004). Moreover, Maier et al. (2007) have very recently shown that treatment with MIN also exerts neuroprotective effects independent of its anti-inflammatory properties when initiated at the onset of clinical signs and could therefore attenuate the disease course. This minocycline-induced neuroprotection is related to a direct antagonism of multiple mechanisms leading to neuronal apoptosis, including the inhibition of CASP-9 activity (Yong et al., 2004). This is particularly provocative if we consider the possibility that halting inflammation in white matter lesions may not prevent chronic irreversible neurological disability in MS patients. In fact, MS may be a classic neurodegenerative disease with a silent stage of neuronal loss preceding symptoms or detection of lesions by MRI. Early neurodegeneration is silent largely due to the brain's remarkable ability to compensate for neuronal loss, through various mechanisms. Thus, the primacy of neurodegeneration in MS is plausible and MS may become the disease of choice for development of neuroprotective therapies because the inflammatory lesions identify the patient before significant neuronal loss or axonal transection occurs (Dhib-Jalbut et al., 2006). Finally, mechanistic insight of the CASP-9 activation may also provide a new means to better understand the neuronal apoptosis in MS, in which normal apoptotic machinery, including the caspase-activation
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Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m
CASP-9: A susceptibility locus for multiple sclerosis in Italy V. Andreoli a,⁎, F. Trecroci a, A. La Russa a, P. Valentino b, F. Condino a, V. Latorre a, R. Nisticò b, D. Pirritano b, F. Del Giudice b, M. Canino b, R. Cittadella a, A. Quattrone a,b a b
Institute of Neurological Sciences, National Research Council, Pianolago di Mangone, Cosenza, Italy Institute of Neurology, Campus di Germaneto, University “Magna Graecia”, Catanzaro, Italy
a r t i c l e
i n f o
Article history: Received 10 December 2008 Received in revised form 24 February 2009 Accepted 5 March 2009 Keywords: Multiple sclerosis CASP-9 Polymorphism Association study Genetic susceptibility
a b s t r a c t Caspase-9 is a primary effector CASP that executes programmed cell death, which plays an important role in the development of multiple sclerosis (MS). Polymorphisms in the CASP-9 gene may influence its activity, thereby modulating the susceptibility to MS. To test this hypothesis, we evaluated a SNP in the CASP-9 gene in a set of Italian patients from Southern Italy and healthy control subjects. Our results suggest that the presence of the G/G genotype represents a higher risk factor in our MS population and a differential production of CASP-9 might be a contributory factor in determining the severity of MS. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease of undetermined aetiology, affecting mainly the white matter of the central nervous system (CNS) and is clinically characterised by progressive disability (Steinman, 1996). In recent years, several data have confirmed the presence of axonal damage in MS and have shown that the disability caused by the disease better correlates with axonal loss than it does with extensive demyelination (Trapp et al., 1998; Noseworthy et al., 2000). Although the initiating event is a matter of debate, epidemiological and genetic findings suggest that MS is an acquired autoimmune and inflammatory disease, triggered by unknown environmental factors in genetically susceptible individuals (Compston and Coles, 2002). These results demonstrate that individuals might be predisposed to MS as a result of the inheritance of many genetic factors of modest contribution that, if revealed, may present important targets for new therapies (Stewart, 1997; Oksenberg et al., 2001). What seems certain is that MS is a disease with heterogeneous pathogenic mechanisms, and several studies support the argument that MS is a primary disease of either axons, neurons or oligodendrocytes and that immune response is secondary to neurodegeneration (Trapp et al., 1998; Bö et al., 2003). Recently, neuronal apoptosis has been described in cortical MS lesions (Peterson et al., 2001) and in experimental autoimmune encephalo⁎ Corresponding author. Institute of Neurological Sciences, National Research Council, Pianolago di Mangone, 87050, Cosenza, Italy. Tel.: +39 09849801300; fax: +39 0984969306. E-mail address: [email protected] (V. Andreoli). 0165-5728/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2009.03.013
myelitis (EAE), a rat model of MS (Meyer et al., 2001). The apoptotic programme is executed by a family of essential proteases known as caspases (Nicholson and Thornberry, 1997). So far, at least two main caspase-activating cascades have been characterised: the mitochondria-mediated caspase-3 activation by caspase-9 (intrinsic pathway) and death-receptor-induced caspase-3 activation by caspase-8 (extrinsic pathway) (Zheng and Flavell, 2000). Particularly, caspase9 (CASP-9) plays a crucial role in the initiation phase of the intrinsic pathway for apoptosis. In fact, many proapoptotic stimuli engage the apoptotic machinery in the cells by causing the release of cytochromec from mitochondria, which then induces oligomerisation of a protein called Apoptotic protease activating factor-1 (Apaf-1) and recruitment of CASP-9 into a large complex known as the apoptosome. Apoptosome then activates the CASP-9 cascade downstream with effector caspases, leading to apoptosis (Srinivasula, 1998; Li et al., 1997). The mechanism is evolutionarily conserved and may play an important role in mediating neuronal death; dysregulation of this normal control mechanism then could be a contributor to various diseases characterised by excessive or inadequate cell death. These findings raise the intriguing possibility that genetic variations in the CASP-9 gene could influence susceptibility to the disease. CASP-9, mapped to the short arm of chromosome 1p36 (Hadano et al., 1999) in humans, is composed of nine exons; some polymorphisms have also been described within this gene. However, the potential role of the single nucleotide polymorphism (SNP) of the CASP-9 gene in establishing susceptibility to MS has never been clearly defined. In particular, a SNP in the coding region CASP-9 Ex5 + 32G NA causes a conservative change of a glutamine with an arginine (Q221R) (Hirano et al., 2001) and, thus, may have functional significance. In order to shed light on a
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biologically potential role of the CASP-9 gene in susceptibility to MS, we have genotyped this SNP in a collection of MS patients and healthy control subjects from Southern Italy. This is the first study to investigate possible relationships between MS and CASP-9 gene polymorphisms in Italian patients. 2. Materials and methods 2.1. Patients and healthy individuals Our study sample consisted of 295 unrelated cases with clinically defined MS (Poser et al., 1983; McDonald et al., 2001), all Caucasians from the south of Italy (Calabria), and followed at the Institute of Neurology, University “Magna Graecia” of Catanzaro. Patients whose disease course was classified (Lublin and Reingold, 1996) as Relapsing–Remitting MS (RRMS, n = 215, 73.0%), Secondary Progressive MS (SPMS, n = 62, 21.0%) or Primary Progressive MS (PPMS, n = 18, 6.0%) were enrolled in this study. One hundred eighty-five patients were women and 110 were men. The following clinical and genetic variables were recorded for each patient: age, sex, disease duration, age at disease onset and level of disability according to the Kurtzke Expanded Disability Status Scale (EDSS) (Kurtzke, 1983). Clinical characteristics of the MS patients are summarised in Table 1. The control population included 295 unrelated healthy subjects (130 women and 165 men; mean age ± SD: 35.3 ± 8.2 years), enrolled during a previous study without history of inflammatory and/or degenerative neurological diseases. To avoid any bias attributed to the ethnic origin of the study population, only Caucasian MS patients and controls whose grandparents were all born in Calabria were included in the analysis. The differences in the sex ratio and ages between the patients and the controls were not significant (p N 0.05). Patients and controls gave their written informed consent before the examination and blood testing, and the study was approved by the local ethical committee. 2.2. CASP9 Ex5 + 32G N A genotyping Blood samples for the genomic DNA studies were obtained from peripheral blood leukocytes and DNA was extracted according to standard procedure. In principle, DNA was amplified using PCR in a total volume of 50 μl containing 15 pmol of each primer, designed according to the published sequence (Hirano et al., 2001), 200 ng genomic DNA and AmpliTaq Gold (Applied Biosystems), and using standard conditions on a PTC-100™ Programmable Thermal Controller (MJ Res. Inc. Genenco). Lastly, a 185 bp fragment containing the A → G transversion in exon 5 was amplified using the primers F:5′CGGTCCAGTCTGCATCTAGAC-3′ and R:5′-ATGCCTGCCCAGGG AACAGT3′ (annealing temperature 59 °C). Ten microlitres of the PCR product were incubated with 10 U BstUI (New England Biolabs, Beverly, MA, USA) in a total volume of 25 μl for 3 h at 60 °C. This gave products that either remained intact (allele A) or were cut into two fragments of 94
Table 1 Demographic and clinical variables of MS subjects analysed. Variable
Patients (n: 295)
Male sex: no. (%) Age (yr): mean (SD) Age at onset (yr): mean (SD) Disease duration (yr): mean (SD) Disease course: no (%) RR SP PP EDSS score: median (range)
110 (37.3) 39.8 ± 12.0 28.1 ± 9.1 13.5 ± 9.4 215 (73.0) 62 (21.0) 18 (6.0)
Data are given as means ± SD and percentage. EDSS: Expanded Disability Status Scale.
3.3 (0–8.5)
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Table 2 CASP9 Ex5 + 32G NA genotypes and alleles of cases and controls and their association with the risk of MS. Ex5 + 32G NA (Q221R)
Controls (n = 295)
Patients (n = 295)
Genotype A/A A/G G/G Allele A G
No. (%) 116 (39.3) 134 (45.4) 45 (15.3)
No. (%) 65 (22.0) 154 (52.2) 76 (25.8)
1.0 2.03 (1.34–3.07) 2.73 (1.63–4.55)
b0.001
366 (62.0) 224 (38.0)
284 (48.1) 306 (51.9)
1.0 1.68 (1.31–2.16)
b0.001
OR (95%CI)
p value
Categorical variables are expressed as frequency and percentage. The differences among groups distribution were assessed using χ2 test. Odds ratios and 95% confidence intervals were calculated according to a multivariate logistic-regression model, adjusted for age and sex.
and 91 bp (allele G). Both digested and undigested DNA fragments were visible for the respective restriction site in heterozygous samples. Amplified DNA fragments and digestion products were separated on 3% agarose gels and visualised by ethidium bromide.
2.3. Data analysis The Hardy–Weinberg equilibrium was tested using Pearson's χ2 goodness of fit test for CASP-9 Ex5 + 32G NA polymorphism. Using univariate analyses for this bi-allelic marker individual p-values were calculated using standard 3 × 2 and 2 × 2 χ2 contingency tables comparing genotype and allele counts in MS cases against controls. Categorical variables were expressed as counts and percentages, continuous variables were shown by mean and standard deviation and discrete variables by median and range. Relative risks for MS, estimated as the odds ratios [ORs] and 95% confidence intervals (95% CI), were calculated by multivariate logistic-regression analysis, adjusting for sex and age. To evaluate possible differences in clinical features among each +32G NA polymorphism variant in the patients group, one-way analysis of variance (ANOVA test) or Kruskal–Wallis test was used. Differences in sex distributions among groups were evaluated with the χ2 test. In all tests, a p-value less than 0.05 denoted the presence of a statistically significant difference. Statistical analyses were performed with Statistical Package for Social Sciences software (SPSS version, 12.0, Chicago, IL, USA) for Windows.
3. Results 3.1. CASP9 Ex5 + 32G N A genotypes and susceptibility to MS In our study, control and patient sample genotype frequencies were distributed according to Hardy–Weinberg equilibrium (controls, p = 0.54; MS, p = 0.48). Table 2 depicts the allele and genotype frequency distribution and association of the studied polymorphism in MS individuals. We found that the frequency of the G/G homozygote in MS patients (25.8%) was significantly higher than in the controls (15.3%), and the subjects who carried this genotype had a 2.73-fold increased risk for developing MS (p b 0.001, OR = 2.73, 95% CI = 1.63–4.55). In the public databases the A allele is reported to be the largest allele of this SNP (rs1052576) in the European population. Here, CASP-9 A allele was observed to be significantly high in our controls (62.0%), providing a reduced risk; the G allele was also overrepresented in MS patients (51.9%) in comparison with the controls (38.0%, p b 0.001, OR = 1.68, 95% CI = 1.31–2.16). Our results show that the CASP-9-G allele was associated with increased predisposition to MS. These data emphasize that the striking associations observed for this genetic variation in CASP-9 genes may play an important role in the aetiology of MS.
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3.2. Gender, genotype and MS risk The entire sample consisted of 590 subjects (315 women and 275 men). Interestingly, in men the + 32 G/G genotype (CASP9 Ex5 + 32G NA polymorphism) was associated with a very significantly increased risk of MS (p b 0.001, OR = 4.77, 95% CI = 2.15–10.52). In addition, among women the same G/G genotype was associated with a less pronounced but still significantly increased risk of MS (p = 0.012, OR = 2.67, 95% CI = 1.35–5.26). 3.3. Clinical characteristics and genotypes Stratifying MS patients according to the course of the disease, a trend towards an increase in the allelic frequency of this SNP in RRMS patients compared with controls (50.5 versus 37.1%; p b 0.001) was observed. Indeed, these results were derived from a significantly increased number of individuals, with the G/G genotype in this MS population, compared with healthy subjects (26.0 versus 14.0%; p b 0.001; OR = 2.78, 95% CI = 1.66–4.64). As the disease course in MS can be defined with greater certainly after 10 years from onset, the association was also analysed in patients with a disease duration of more than 10 or 20 years. Interestingly, the analysis confirmed the association between the GG genotype and milder disability in MS patients, with a disease duration of more than 10 years (p b 0.001; OR = 2.46, 95% CI = 1.38–4.38). Moreover, OR analyses in patients with a disease duration of more than 20 years showed the association to become stepwise increasingly marked over the years (p b 0.001, OR = 3.31, 95% CI = 1.39–7.84) (Table 3). This suggests that the genetic influence of CASP-9 on MS risk and progression may become more prominent with increasing duration of the disease. Finally, no association between genotypes or alleles of the CASP9 Ex5 + 32G NA variant and age at disease onset or EDSS score (Kurtzke, 1983) was detected (data not showed). 4. Discussion In this report, we provide evidence that a variation of the CASP-9 gene is directly associated with susceptibility to MS in our Italian patients. The major finding of the present study is that the producer genotype AA is preventative against the patient developing the severe form of MS, while the high-producer GG is associated with different degrees of severity, significantly so in all patients with a disease duration of more than 20 years. It should be noted that CASP-9 Ex5 + 32G NA polymorphism encodes for a glutamine to arginine amino acid change at codon 221 of the protein, located at the border of the helix region of CASP-9. In particular, the Q221R variant might lead to conformational changes in the molecule, resulting in an alteration of its function. However, the contribution of this polymorphisms remains unknown at this stage and the possible functional role of this variant should be investigated. Altogether, it is possible that the mutated allele modifies the affinity of this protein to Apaf-1, influencing the amount of neuronal damage in the lesions and thus the progression of disability in MS. Hence, direct functional effects of the polymorphism studied here are biologically plausible because CASP-9, as an initiator CASP, plays an important role in the apoptosome-driven apoptosis pathway, which is essential for eliminating mutated or transformed neural progenitor cells from the brain Table 3 Relative risks for MS, estimated as the odds ratios [ORs] and 95% confidence intervals [CI], as analysed by logistic regression in all patients and in patients with disease duration (dd) more than 10 and 20 years. Genotype G/G
%
ORs
95% CI
p value
Total group Patients with dd ≥ 10 Patients with dd ≥ 20
25.8 25.0 30.9
2.73 2.46 3.31
1.63–4.55 1.38–4.38 1.39–7.84
b0.001 b0.001 b0.001
(Yin et al., 2006; Hakem et al., 1998). In theory, at the protein level the GG genotype of CASP-9 Ex5 + 32G NA may produce higher levels of the CASP-9 protein and therefore up-regulate apoptotic processes in MS as it increases the apoptosome formation under normal physiological conditions. In addition, evidence of neuronal apoptosis in MS is currently emerging. During the past few years several death receptor/ ligand systems mediating apoptosis have been discovered. Tumour necrosis factor (TNF), CD95 (APO-1/Fas) ligand and other ligands, such as TNF-related apoptosis-inducing ligand (TRAIL), interact with their respective receptors to induce apoptotic cell death and were found to be responsible for MS risk (Amirzargar et al., 2007; Kantarci et al., 2004; Kikuchi et al., 2005). New structural evidence suggests that CSF from MS patients induces apoptotic cell death of cultured neurons (Alcazar et al., 1998, 2000), which may also have a correlation with clinical disability (Cid et al., 2002). More specifically, apoptotic neurons have recently been identified in both cortical MS lesions (Meyer et al., 2001) and EAE (Nicholson and Thornberry, 1997). Evidence from CASP-9 knock-out mouse models and CASP-9 null cell lines show that CASP-9 is essential to the regulation of cell homeostasis through cleavage of many key players involved in apoptosis. Moreover, these mouse models of mutant CASP-9 mice have been associated with perinatal and postnatal death (Kuida et al., 1998; Hakem et al., 1998). Interestingly, more recent studies have demonstrated that aberrant apoptosome function, potentially arising from a lack of CASP-9 bound to oligomerised Apaf-1 (Liu et al., 2002; Wolf et al., 2001), contributes not only to carcinogenesis (Shivapurkar et al., 2003) but it has also been implicated in the inappropriate apoptosis characteristic of various neurodegenerative disorders (Sang et al., 2005). If apoptosome activity is necessary for the cell death characteristic of neurodegeneration, then inactivation of the apoptosome might provide a therapeutic avenue for treating these disorders as well as MS. The latter is consistent with the finding that one recently identified Apaf-1-interacting inhibitor, AIP (a splice variant of caspase-9 that is expressed endogenously in the brain), was found to be capable of protecting neurons from cell death induced by cerebral ischemia (Cao et al., 2004). So far, the best therapeutic options for neuroprotection in MS are the immunomodulatory agent glatiramer acetate and interferon beta drugs (Dhib-Jalbut, 2002). Although beneficial, these two options fail to prevent the progression of disease in some patients; hence, there is the need to explore new strategies that not only limit inflammation and demyelination in the CNS but also prevent axonal loss (Costello et al., 2007). Currently, minocycline (MIN), a tetracycline antibiotic under evaluation for use in neurodegenerative diseases, has shown remarkable benefit in animal models with several neurological disorders, including MS (Yong, 2004). Moreover, Maier et al. (2007) have very recently shown that treatment with MIN also exerts neuroprotective effects independent of its anti-inflammatory properties when initiated at the onset of clinical signs and could therefore attenuate the disease course. This minocycline-induced neuroprotection is related to a direct antagonism of multiple mechanisms leading to neuronal apoptosis, including the inhibition of CASP-9 activity (Yong et al., 2004). This is particularly provocative if we consider the possibility that halting inflammation in white matter lesions may not prevent chronic irreversible neurological disability in MS patients. In fact, MS may be a classic neurodegenerative disease with a silent stage of neuronal loss preceding symptoms or detection of lesions by MRI. Early neurodegeneration is silent largely due to the brain's remarkable ability to compensate for neuronal loss, through various mechanisms. Thus, the primacy of neurodegeneration in MS is plausible and MS may become the disease of choice for development of neuroprotective therapies because the inflammatory lesions identify the patient before significant neuronal loss or axonal transection occurs (Dhib-Jalbut et al., 2006). Finally, mechanistic insight of the CASP-9 activation may also provide a new means to better understand the neuronal apoptosis in MS, in which normal apoptotic machinery, including the caspase-activation
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