Calpain Research Articles

Caspase-8 Association with the Focal Adhesion Complex Promotes Tumor Cell Migration and Metastasis

Caspase-8 is a proapoptotic protease that suppresses neuroblastoma metastasis by inducing programmed cell death. Paradoxically, caspase-8 can also promote cell migration among nonapoptotic cells; here, we show that caspase-8 can promote metastasis when apoptosis is compromised. Migration is enhanced by caspase-8 recruitment to the cellular migration machinery following integrin ligation. Caspase-8 catalytic activity is not required for caspase-8-enhanced cell migration; rather, caspase-8 interacts with a multiprotein complex that can include focal adhesion kinase and calpain 2 (CPN2), enhancing cleavage of focal adhesion substrates and cell migration. Caspase-8 association with CPN2/calpastatin disrupts calpastatin-mediated inhibition of CPN2. In vivo, knockdown of either caspase-8 or CPN2 disrupts metastasis among apoptosis-resistant tumors. This unexpected molecular collaboration provides an explanation for the continued or elevated expression of caspase-8 observed in many tumors.

Identification and association of the single nucleotide polymorphisms in calpain3 (CAPN3) gene with carcass traits in chickens

BackgroundThe aim of this study is to screen single nucleotide polymorphisms (SNP) of chicken Calpain3 (CAPN3) gene and to analyze the potential association between CAPN3 gene polymorphisms and carcass traits in chickens. We screened CAPN3 single nucleotide polymorphisms (SNP) in 307 meat-type quality chicken from 5 commercial pure lines (S01, S02, S03, S05, and D99) and 4 native breeds from Guangdong Province (Huiyang Huxu chicken and Qingyuan Ma chicken) and Sichuan Province (Caoke chicken and Shandi Black-bone chicken), China.ResultsTwo SNPs (11818T>A and 12814T>G) were detected by single strand conformation polymorphism (SSCP) method and were verified by DNA sequencing. Association analysis showed that the 12814T>G genotypes were significantly associated with body weight (BW), carcass weight (CW), breast muscle weight (BMW), and leg muscle weight (LMW). Haplotypes constructed on the two SNPs (H1, TG; H2, TT; H3, AG; and H4, AT) were associated with BW, CW (P < 0.05), eviscerated percentage (EP), semi-eviscerated percentage (SEP), breast muscle percentage (BMP), and leg muscle percentage (LMP) (P < 0.01). Diplotype H1H2 was dominant for BW, CW, and LMP, and H2H2 was dominant for EP, SEP, and BMP.ConclusionWe speculated that the CAPN3 gene was a major gene affecting chicken muscle growth and carcass traits or it was linked with the major gene(s). Diplotypes H1H2 and H2H2 might be advantageous for carcass traits.

Investigation on variability of candidate genes for meat quality traits in Piemontese cattle

This study aimed to investigate the variability of 15 genes chosen according to their function as markers of meat quality traits in Piemontese cattle. Meat samples of Longissimus thoracis muscle and data on carcass weight (CW), shear force (SF), cooking loss (CL), and pH collected on 1,208 Piemontese young bulls progeny of 109 AI sires were available for this investigation. For each trait considered (CW, SF, CL, pH), 48 samples were chosen from each tail of normal distribution, and one or more single nucleotide polymorphisms (SNPs) were determined for the following loci: growth hormone (GH), growth hormone receptor (GHR), pro-opiomelanocortin (POMC), pituitary-specific positive transcription factor 1 (POU1F1), melanocortin-4 receptor (MC4R), corticotrophin-realising hormone (CRH), insulin-like growth factor binding protein- 3 (IGFBP3), diacylglycerol acyltransferase 1 (DGAT1), thyroglobulin (TG), carboxypeptidase E (CPE), calpain 1 (CAPN1), calpastatin (CAST), cathepsin B and D (CATB, CATD), and protein kinase adenosine monophosphate-activated 3-subunit (PRKAG3). Eight SNPs were characterized by a high, 4 by an intermediate and 2 by a low variability; 6 may be almost fixed. Based on these results, variable loci will be investigated on the entire available data set in order to ????????????study their effects on meat quality traits.

Gene expression analysis of terminal differentiation of human melanoma cells highlights global reductions in cell cycle-associated genes

Defects in differentiation are frequently observed in cancer cells. By appropriate treatment specific tumor cell types can be induced to terminally differentiate. Metastatic HO-1 human melanoma cells treated with IFN-β plus mezerein (MEZ) undergo irreversible growth arrest and terminal differentiation followed by apoptosis. In order to define the molecular changes associated with this process, changes in gene expression were analyzed by cDNA microarray hybridization and by semi-quantitative and quantitative RT-PCRs of representative 44 genes. The expression of 210 genes was changed more than two-fold at either 8 or 24 h post-treatment (166 up and 44 down). Major biological processes associated with the up-regulated genes were response to endogenous/exogenous stimuli (38%), cell proliferation (13%), cell death (16%) and development (30%). Approximately 34% of the down-regulated genes were associated with cell cycle, 9% in DNA replication and 11% in chromosome organization, respectively. Suppression of cell cycle associated genes appeared to directly correlate with growth arrest observed in the terminal differentiation process. Expression of Calpain 3 (CAPN3) variant 6 was suppressed by the combined treatment and maintained high in various melanoma cell lines. However, over-expression of the CAPN3 did not significantly affect growth kinetics and cell viability, suggesting that up-regulation of CAPN3 alone may not be a causative, but an associated change with melanoma development. This analysis provides further insights into the spectrum of up-regulated and the first detailed investigation of down-regulated gene changes associated with and potentially causative of induction of loss of proliferative capacity and terminal differentiation in human melanoma cells.

Gene Expression Profiling in Limb-Girdle Muscular Dystrophy 2A

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in calpain 3 (CAPN3). Calpain 3 plays different roles in muscular cells, but little is known about its functions or in vivo substrates. The aim of this study was to identify the genes showing an altered expression in LGMD2A patients and the possible pathways they are implicated in. Ten muscle samples from LGMD2A patients with in which molecular diagnosis was ascertained were investigated using array technology to analyze gene expression profiling as compared to ten normal muscle samples. Upregulated genes were mostly those related to extracellular matrix (different collagens), cell adhesion (fibronectin), muscle development (myosins and melusin) and signal transduction. It is therefore suggested that different proteins located or participating in the costameric region are implicated in processes regulated by calpain 3 during skeletal muscle development. Genes participating in the ubiquitin proteasome degradation pathway were found to be deregulated in LGMD2A patients, suggesting that regulation of this pathway may be under the control of calpain 3 activity. As frizzled-related protein (FRZB) is upregulated in LGMD2A muscle samples, it could be hypothesized that β-catenin regulation is also altered at the Wnt signaling pathway, leading to an incorrect myogenesis. Conversely, expression of most transcription factor genes was downregulated (MYC, FOS and EGR1). Finally, the upregulation of IL-32 and immunoglobulin genes may induce the eosinophil chemoattraction explaining the inflammatory findings observed in presymptomatic stages. The obtained results try to shed some light on identification of novel therapeutic targets for limb-girdle muscular dystrophies.

Changes of physiological pain threshold in gonadectomized rats

Objective:To investigate the modulatory effect of gonadal hormone on peripheral pain.Methods:Orchiectomized and ovariectomized rats models were established.Radio heater and Von-Frey hair were used to determine the peripheral mechani- cal and thermal pain threshold in gonadectomized rats and their corresponding sham-controls.Results:The body weight was in- creased and the uterus weight was decreased after ovariectomy in female rats(P0.01).There was no significant change in the body weight of male rats after orchiectomization.The 100% hind paw withdrawal threshold to Von-Frey hair stimulation was decreased significantly after ovariectomization in female rats,with no significant change in the thermal pain threshold.There was no significant changes in the 100% hind paw withdrawal threshold after Von-Frey hair stimulation or thermal pain threshold af- ter orchidectomy in male rats.Conclusion.These results suggest that changes in androgen level of male rats have no influence on the peripheral basal pain threshold,and female gonadal hormone may inhibit the peripheral pain signaling and has no effect on thermal pain threshold.

G.P.14.01 Immunohistochemical analysis of calpain 3: Advantages and limitations in diagnosing LGMD2A

The limb-girdle muscular dystrophies (LGMD) are difficult to differentiate on clinical grounds alone. Accurate diagnoses require comprehensive clinical assessment and laboratory testing to guide mutation analysis. Immunoblot analysis is currently the gold standard to identify patients where there is a clinical suspicion of LGMD2A (calpainopathy). Monoclonal antibodies directed against the muscle-specific calpain 3 (CAPN3) produce characteristic patterns of bands on immunoblot: full length CAPN3 (94kDa), plus additional degradation fragments at approximately 60 or 30kDa.

Novel role of calpain-3 in the triad-associated protein complex regulating calcium release in skeletal muscle

Calpain-3 (CAPN3) is a non-lysosomal cysteine protease that is necessary for normal muscle function, as mutations in CAPN3 result in an autosomal recessive form of limb girdle muscular dystrophy type 2A. To elucidate the biological roles of CAPN3 in skeletal muscle, we performed a search for potential substrates and interacting partners. By yeast-two-hybrid analysis we identified the glycolytic enzyme aldolase A (AldoA) as a binding partner of CAPN3. In co-expression studies CAPN3 degraded AldoA; however, no accumulation of AldoA was observed in total extracts from CAPN3-deficient muscles suggesting that AldoA is not an in vivo substrate of CAPN3. Instead, we found CAPN3 to be necessary for recruitment of AldoA to one specific location, namely the triads, which are structural components of muscle responsible for calcium transport and excitation-contraction coupling. Both aldolase and CAPN3 are present in the triad-enriched fraction and are able to interact with ryanodine receptors (RyR) that form major calcium release channels. Levels of triad-associated AldoA and RyR were decreased in CAPN3-deficient muscles compared with wild-type. Consistent with these observations we found calcium release to be significantly reduced in fibers from CAPN3-deficient muscles. Together, these data suggest that CAPN3 is necessary for the structural integrity of the triad-associated protein complex and that impairment of calcium transport is a phenotypic feature of CAPN3-deficient muscle.

Study on association of single nucleotide polymorphism of CAPN1 gene with muscle fibre and carcass traits in quality chicken populations

This study was aimed at investigating the effect of the calpain 1 (CAPN1) gene on carcass and meat quality traits in eight meat-type chicken populations, including five pure lines (developed from Chinese local breeds) and three cross-breeds. Primer pairs for the Coding Sequence (CDS) region in CAPN1 were designed from the chicken genomic sequence database. Polymorphisms were detected by polymerase chain reaction (PCR)-Single Strand Conformation Polymorphism (SSCP) and DNA sequencing. Three single nucleotide polymorphisms (SNP; C2546T, G3535A and C7198A) were detected among individuals in each population. The associations of their haplotypes (H1 = CGA, H2 = CGC, H3 = CAA, H4 = CAC, H5 = TGA and H7 = TAA) with chicken breast muscle fibre and carcass traits were analysed. Results showed that the haplotypes were associated with live weight (LW), carcass weight (CW), breast muscle weight (BMW) and leg muscle weight (LMW) (p < 0.05), and were also related to eviscerated percentage (%EP) and breast muscle fibre density (p < 0.01). H1H3 haplotype was dominant for LW, CW and BMW; H1H5 haplotype was dominant for EP; H3H4 haplotype was dominant for LMW and H1H1 haplotype was dominant for BFD. It was concluded that the CAPN1 gene may be a major gene affecting meat quality traits of chicken or it is linked with the major gene. H1H3, H1H5 and H3H4 were the most advantageous haplotypes for carcass traits whereas H1H1 was the positive haplotype for breast muscle fibre trait.

Variation at the Calpain 3 gene is associated with meat tenderness in zebu and composite breeds of cattle

BackgroundQuantitative Trait Loci (QTL) affecting meat tenderness have been reported on Bovine chromosome 10. Here we examine variation at the Calpain 3 (CAPN3) gene in cattle, a gene located within the confidence interval of the QTL, and which is a positional candidate gene based on the biochemical activity of the protein.ResultsWe identified single nucleotide polymorphisms (SNP) in the genomic sequence of the CAPN3 gene and tested three of these in a sample of 2189 cattle. Of the three SNP genotyped, the CAPN3:c.1538+225G>T had the largest significant additive effect, with an allele substitution effect in the Brahman of α = -0.144 kg, SE = 0.060, P = 0.016, and the polymorphism explained 1.7% of the residual phenotypic variance in that sample of the breed. Significant haplotype substitution effects were found for all three breeds, the Brahman, the Belmont Red, and the Santa Gertrudis. For the common haplotype, the haplotype substitution effect in the Brahman was α = 0.169 kg, SE = 0.056, P = 0.003. The effect of this gene was compared to Calpastatin in the same sample. The SNP show negligible frequencies in taurine breeds and low to moderate minor allele frequencies in zebu or composite animals.ConclusionThese associations confirm the location of a QTL for meat tenderness in this region of bovine chromosome 10. SNP in or near this gene may be responsible for part of the overall difference between taurine and zebu breeds in meat tenderness, and the greater variability in meat tenderness found in zebu and composite breeds. The evidence provided so far suggests that none of these tested SNP are causative mutations.

Calpain 3 is a modulator of the dysferlin protein complex in skeletal muscle

Muscular dystrophies comprise a genetically heterogeneous group of degenerative muscle disorders characterized by progressive muscle wasting and weakness. Two forms of limb-girdle muscular dystrophy, 2A and 2B, are caused by mutations in calpain 3 (CAPN3) and dysferlin (DYSF), respectively. While CAPN3 may be involved in sarcomere remodeling, DYSF is proposed to play a role in membrane repair. The coexistence of CAPN3 and AHNAK, a protein involved in subsarcolemmal cytoarchitecture and membrane repair, in the dysferlin protein complex and the presence of proteolytic cleavage fragments of AHNAK in skeletal muscle led us to investigate whether AHNAK can act as substrate for CAPN3. We here demonstrate that AHNAK is cleaved by CAPN3 and show that AHNAK is lost in cells expressing active CAPN3. Conversely, AHNAK accumulates when calpain 3 is defective in skeletal muscle of calpainopathy patients. Moreover, we demonstrate that AHNAK fragments cleaved by CAPN3 have lost their affinity for dysferlin. Thus, our findings suggest interconnectivity between both diseases by revealing a novel physiological role for CAPN3 in regulating the dysferlin protein complex.

NF‐NF‐κBB‐dependent expression of the antiapoptotic factor c‐FLIP is regulated by calpain 3, the protein involved in limb‐girdle muscular dystrophy type 2A

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in the cysteine protease calpain 3 (CAPN3) that leads to selective muscle wasting. We previously showed that CAPN3 deficiency is associated with a profound perturbation of the NF-kappaB/IkappaB alpha survival pathway. In this study, the consequences of altered NF-kappaB/IkappaB alpha pathway were investigated using biological materials from LGMD2A patients. We first show that the antiapoptotic factor cellular-FLICE inhibitory protein (c-FLIP), which is dependent on the NF-kappaB pathway in normal muscle cells, is down-regulated in LGMD2A biopsies. In muscle cells isolated from LGMD2A patients, NF-kappaB is readily activated on cytokine induction as shown by an increase in its DNA binding activity. However, we observed discrepant transcriptional responses depending on the NF-kappaB target genes. IkappaB alpha is expressed following NF-kappaB activation independent of the CAPN3 status, whereas expression of c-FLIP is obtained only when CAPN3 is present. These data lead us to postulate that CAPN3 intervenes in the regulation of the expression of NF-kappaB-dependent survival genes to prevent apoptosis in skeletal muscle. Deregulations in the NF-kappaB pathway could be part of the mechanism responsible for the muscle wasting resulting from CAPN3 deficiency.

Transcriptional explorations of CAPN3 identify novel splicing mutations, a large‐sized genomic deletion and evidence for messenger RNA decay

Mutations in the gene encoding calpain-3 (CAPN3) cause autosomal recessive limb-girdle muscular dystrophy type 2A (LGMD2A) and idiopathic eosinophilic myositis. Accurate diagnosis and genetic counselling are based on the identification of disease-causing mutations on both alleles of CAPN3 in the patients. In the present study, we used transcriptional analysis as a complementary approach for patients suspected of being affected with LGMD2A, in whom initial denaturing high-performance liquid chromatography genomic mutation screening evidenced no or only one CAPN3 mutation obviously considered as disease causing. This allowed to identify and characterize cDNA deletions. Further genomic analysis allowed to determine the origin of these deletions, either as splicing defects caused by intronic mutations or as an internal multi-exonic deletion. In particular, we report two novel CAPN3 mutations (c.1745 + 4_1745 + 7delAGTG in IVS13 and c.2185-16A>G in IVS20) and a recurrent large-sized genomic deletion including exons 2-8 for which genomic breakpoints have been characterized. In addition, our results indicate nonsense-mediated messenger RNA decay as a mechanism for under-expression of CAPN3 associated to some specific variations.

G.P.4.15 CAPN3 mutations in patients with idiopathic eosinophilic myositis: A predystrophic stage of LGMD2A?

Objective and Background: Mutations in the gene encoding calpain-3 (CAPN3) cause autosomal-recessive limb girdle muscular dystrophy type 2A (LGMD2A). In six children, we recently identified CAPN3 mutations as a genetic cause of idiopathic eosinophilic myositis (EM), a rare pathological entity characterised by eosinophilic infiltration of skeletal muscles. Here, we further evaluated the hypothesis of EM being a possible histopathological pre-dystrophic developmental stage of LGMD2A. Methods: We screened CAPN3 for mutations using DHPLC and direct sequencing, in five unrelated patients (four children and one young adult), recruited on the basis of EM diagnosed after histological examination of muscle biopsy samples, without any identified aetiological factor. In addition, information on the presence of eosinophils was retrospectively assessed in 17 genetically confirmed LGMD2A patients and inflammatory changes by analysing haematoxylin-eosin stained cryosections from muscle samples (mean age of biopsy 28 years). Results: Two disease-causing CAPN3 mutations were identified in the five patients affected with idiopathic EM. In one additional patient, only one disease-causing CAPN3 mutation was identified. Additionally, we found a sparse presence of eosinophils in muscle biopsies from 5/17 patients with inflammatory (n = 3 mild focal, n = 2 moderate focal) and myopathic changes (n = 2 mild, n = 2 moderate, n = 1 severe). Conclusion: Our findings confirm mutations in CAPN3 as a genetic cause of eosinophilic myositis. The presence of eosinophils has been discussed before as a component of muscular dystrophy (Cai et al. 2000). The presence of marked eosinophilic infiltrates in EM patients, together with the findings of our retrospective analysis of biopsies from LGMD2A patients might suggest the following possibilities (1) EM associated with CAPN3 mutations could be a distinct phenotype, (2) eosinophilic infiltration might represent an early or transient developmental stage in the inflammation associated with primary calpainopathy.

Epistasis Between Calpain 1 and Its Inhibitor Calpastatin Within Breeds of Cattle

The calpain gene family and its inhibitors have diverse effects, many related to protein turnover, which appear to affect a range of phenotypes such as diabetes, exercise-induced muscle injury, and pathological events associated with degenerative neural diseases in humans, fertility, longevity, and postmortem effects on meat tenderness in livestock species. The calpains are inhibited by calpastatin, which binds directly to calpain. Here we report the direct measurement of epistatic interactions of causative mutations for quantitative trait loci (QTL) at calpain 1 (CAPN1), located on chromosome 29, with causative mutations for QTL variation at calpastatin (CAST), located on chromosome 7, in cattle. First we identified potential causative mutations at CAST and then genotyped these along with putative causative mutations at CAPN1 in >1500 cattle of seven breeds. The maximum allele substitution effect on the phenotype of the CAPN1:c.947G>C single nucleotide polymorphism (SNP) was 0.14 sigma(p) (P = 0.0003) and of the CAST:c.155C>T SNP was also 0.14 sigma(p) (P = 0.0011) when measured across breeds. We found significant epistasis between SNPs at CAPN1 and CAST in both taurine and zebu derived breeds. There were more additive x dominance components of epistasis than additive x additive and dominance x dominance components combined. A minority of breed comparisons did not show epistasis, suggesting that genetic variation at other genes may influence the degree of epistasis found in this system.

Genetic variability in beta-lactoglobulin, calpastain and calpain loci in Kurdi sheep

DNA-based molecular methods have made possible genotyping of animals of any age and sex for milk and meat genes, thus providing a potentially more efficient and flexible selection tool. Among specific genes that may affect economically important traits in sheep, the Beta-lactoglobulin (BLG) locus has been extensively studied [Barillet et al., 2005]. The genotype BB of BLG seems to be associated with higher milk yield; on the other hand genotypes AA and AB seem to be superior in protein and casein content and crude yield [Garzon et al., 1992]. Another genes intensively investigated in farm animal are that of calpastatin (CAST) and calpain (CAPN). Calpastatin and calpain (CAPN) deserves special attention because of their major role in meat production. The calpain-calpastatin system (CCS) comprises a family of calcium dependent neutral proteases. The CAPNs have been shown to play the major role in post mortem tenderization in beef, lamb, and pork by degrading specific muscle structural proteins. The aim of the present study was to identify genotypes of BLG, CAST and CAPN genes in Kurdi sheep breed by PCR.

Do the calpains degrade actin, α‐actinin, or myosin?

Do the calpains degrade actin, α‐actinin, or myosin?

Genetic Variability and Population Structure in Beta-lactoglobulin, Calpastain and Calpain Loci in Iranian Kurdi Sheep

The genotypes for Beta-Lactoglobulin (BLG) and calpastatin (CAST) were determined by Polymerase Chain Reaction (PCR) and restriction enzyme digestion and genotyped for calpain (CAPN) by PCR-SSCP method in an Iranian breed sheep, Kurdi. Blood samples were collected from 100 pure bred Kurdi sheep from Kurdi breeding station located in Shirvan, Mashhad. The extraction of genomic DNA was based on Guanidin Thiocyanate-Silica gel method. After PCR reaction, amplicons were digested with restriction enzymes MspI and RsaI for beta-lactoglobulin and calpastatin genes, respectively. The beta-lactoglobulin locus had 3 genotypes with frequencies of 0.24, 0.54 and 0.22 for AA, AB and BB, respectively; calpastatin genotypes had 2 genotypes with frequencies of 0.76 and 0.24 for MM and MN genotypes, respectively. Calpain genotypes were analyzed with SSCP method, which had 2 genotypes with frequencies of 0.92 and 0.08 for AA and AB, respectively. Heterozygosity value for beta-lactoglobulin locus was 49% and for calpastatin and calpain loci was very low (24 and 8%, respectively). chi2 test confirmed the Hardy-Weinberg equilibrium for three loci in this population. These data provide evidence that Iranian's Kurdi sheep breed have a variability, which opens interesting prospects for future selection programs, especially marker-assistant selection between different genotypes of different locus and milk and cheese characteristics, gain and meat traits and also for preservation strategies.

AAV-mediated delivery of a mutated myostatin propeptide ameliorates calpain 3 but not α-sarcoglycan deficiency

Myostatin is a negative regulator of muscle mass whose inhibition has been proposed as a therapeutic strategy for muscle-wasting conditions. Indeed, blocking myostatin action through different strategies has proved beneficial for the pathophysiology of the dystrophin-deficient mdx mouse. In this report, we tested the inhibition of myostatin by AAV-mediated expression of a mutated propeptide in animal models of two limb-girdle muscular dystrophies: LGMD2A caused by mutations in the calpain 3 (CAPN3) gene and LGMD2D caused by mutations in the alpha-sarcoglycan gene (SGCA). In the highly regenerative Sgca-null mice, survival of the alpha-sarcoglycan-deficient muscle fibers did not improve after transfer of the myostatin propeptide. In calpain 3-deficient mice, a boost in muscle mass and an increase in absolute force were obtained, suggesting that myostatin inhibition could constitute a therapeutic strategy in this predominantly atrophic disorder.

A large deletion and novel point mutations in the calpain 3 gene (CAPN3) in Bulgarian LGMD2A patients

Limb-girdle muscular dystrophy type 2A (LGMD2A) is caused by mutations in the calpain 3 (CAPN3) gene. The clinical diagnoses of these cases in Bulgaria are very complicated, no protein analysis on muscular biopsy is available in our country, and genetic tests are the only possibility to clarify the diagnoses in clinically ambiguous cases. We screened 48 unrelated Bulgarian cases with preliminary diagnoses of different types of muscular dystrophy for mutations in the CAPN3 gene. Altogether, 20 families (42%) were found to carry mutations in the CAPN3 gene. Several misdiagnosed cases were clarified. Three novel and six recurrent mutations were identified. In total, 40% of the patients are homozygous for c.550delA, and 70% carry it at least on one allele. The affected group of women in our sample shows later onset, milder clinical manifestation, slower progression, and later invalidization.