Perforin is involved in cell-mediated cytotoxicity and mutations of its gene (PRF1) cause familial hemophagocytic lymphohistiocytosis (FLH2). PRF1 sequencing in 190 patients with multiple sclerosis and 268 controls detected two FLH2-associated variations (A91V, N252S) in both groups and six novel mutations (C999T, G1065A, G1428A, A1620G, G719A, C1069T) in patients. All together, carriers of these variations were more frequent in patients than in controls (phenotype frequency: 17 vs 9%, P=0.0166; odds ratio (OR)=2.06, 95% confidence interval (CI): 1.13–3.77). Although A91V was the most frequent variation and displayed a trend of association with multiple sclerosis (MS) in the first population of patients and controls (frequency of the 91V allele: 0.076 vs 0.043, P=0.044), we used it as a marker to confirm PRF1 involvement in MS and assessed its frequency in a second population of 966 patients and 1520 controls. Frequency of the 91V allele was significantly higher in patients than in controls also in the second population (0.075 vs 0.058%, P=0.019). In the combined cohorts of 1156 patients and 1788 controls, presence of the 91V allele in single or double dose conferred an OR=1.38 (95% CI=1.10–1.74). These data suggest that A91V and possibly other perforin variations indicate susceptibility to MS.
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system.1 Its clinical course varies; at onset, approximately 15% of patients display a primary progressive (PP) form, whereas the remainder start out with a relapsing remitting (RR) form and most of them switch to a secondary progressive (SP) form within 10–30 years.2 Both environmental and genetic factors are involved in the development/progression of MS and several studies point to a complex inheritance involving interactions between combinations of loci that may influence the immune response.3, 4
Demyelination is obviously a pathological hallmark of MS, but recent evidence has suggested that the clinically relevant cause of functional disability is injury to the axon.5 This neurodegenerative model posits that demyelination is a permissive factor that creates an environment in which the axon becomes susceptible to injury mediated either by loss of axo–glial trophic interactions or immune-mediated attack of the denuded axon. The cellular effectors responsible for injuring demyelinated axons are currently unidentified. The fact that CD8+ T cells are the most abundant lymphocytes within MS lesions6 and correlate with axon injury7 suggests that class I-restricted cytotoxic T cells (CTL) may be the culprit.
Cytolytic granules of CD8+ CTL and natural killer (NK) cells contain perforin and granzymes, and are released on the target cell upon its recognition by the cytotoxic cell. Perforin polymerizes on the target cell membrane and forms pores allowing entry of granzymes that trigger apoptosis of the target cell by cleaving caspases.8
Biallelic loss-of-function mutations of the perforin gene (PRF1) have been classically associated with about 30% of cases of familial hemophagocytic lymphohistiocytosis (FLH2), a rare life-threatening immune deficiency that occurs in infants and young adults.9, 10 FLH2 has been classically ascribed to decreased capacity of CTL and NK cells to clear viral infections; viral persistence is thought to cause the lymphoproliferative pattern. FLH2 is a recessive disease and subjects carrying heterozygous PRF1 mutations are generally healthy. However, some heterozygous variations may favor development of autoimmune diseases. This has been initially suggested for the autoimmune lymphoproliferative syndrome (ALPS), a rare pediatric autoimmune disease due to defective function of the Fas death receptor involved in both downmodulation of the immune response and cell-mediated cytotoxicity.11 ALPS is primarily due to mutations of the Fas gene or other genes involved in Fas function, but other genetic factors may concur. We have detected two FLH2-associated amino-acid substitutions of PRF1 that are associated with ALPS, that is, N252S and A91V.11 A subsequent work on patients with type 1 diabetes mellitus (T1DM) detected association with N252S, but not A91V, and a patient displayed a novel mutation causing a P477A amino-acid change decreasing NK function.12 This work was aimed to evaluate whether PRF1 also contributes to MS development in the light of recent studies showing that a region of chromosome 10q22.1, located near PRF1, may be a susceptibility locus for MS.13, 14, 15
Analysis of the whole coding region of PRF1
The entire coding region of PRF1 was sequenced in 190 MS and 268 controls to look for variations associated with FLH2 or novel variations (Figure 1). Four missense variations were detected, that is, C272T (rs35947132), A755G (rs28933375), G719A and C1069T (numerations are referred to the GenBank cDNA clone M28393, ATG=+1) causing A91V, N252S, R240H and R357W amino-acid substitutions, respectively. A91V and N252S are variations previously associated with FLH2, whereas R240H and R357W are new.
Four other novel variations, C999T, G1065A, G1428A and A1620G, were detected, but they were synonymous variations (P333P, P355P, G476G and Q540Q, respectively); analysis of their putative effect on splice sites using the Spliceview software and ESEfinder scoring matrix showed that only A1620G (Q540Q) may have an effect by creating a novel acceptor splice site (Spliceview) and a novel binding site for Ser/Arg-rich proteins (ESEfinder), a family of conserved splicing factors.
Finally, we detected the two nucleotide variations, C822T (rs885821) and T900C (rs885822), previously reported as common polymorphisms not associated with FLH2; they did not change the amino acid, nor influence the splicing sites. Their frequency was similar in the patients and the controls. Two other synonymous variations (G435A and A462G) are known to be in perfect linkage disequilibrium with N252S and were in fact only detected in the two subjects (one patient and one control) carrying this variation.11
A91V was detected 29 times in 26 patients (23 heterozygotes, 3 homozygotes) and 23 controls (heterozygotes); N252S in 1 patient and 1 control (heterozygotes); R240H in 2 patients (heterozygotes); R357W, P333P, P355P, and G476G in 1 patient each (heterozygotes); and Q540Q in 1 patient (homozygote). The R357W and P355P carriers were also heterozygous for A91V, and the two variations were found to be on different alleles by allele-specific PCR. All together, frequency of the FLH2-associated and novel variations was higher in patients than in controls (allele frequencies: 0.100 vs 0.045, P=0.0016; phenotype frequency: 17 vs 9%, P=0.0166; odds ratio (OR)=2.06, 95% confidence interval (CI): 1.13–3.77; Table 1).
The PolyPhen algorithm was used to predict the functional effect of the two novel R240H and R357W missense variations and showed that both may damage the function and structure of the protein (R240H: score=2.335; R357W: score=2.690). Therefore, we directly assessed whether R240H affects perforin function by evaluating NK activity in the 2 patients carrying the variation and 15 controls. Results showed that, at low effector/target ratios, NK activity was defective in one patient and in the low level (that is, within the first quartile) of the normal range in the other (Figure 2). This analysis was not performed in the R357W carrier because his cells were not available. Intriguingly, both patients carrying R240H displayed an early switch from the RR to the SP course (5 and 6 years from onset, respectively) and a multiple sclerosis severity score (MSSS) of 7.65 and 7.38, respectively. By contrast, this aggressive clinical evolution was not displayed by the R357W carrier.
Search for the A91V variation in a second population of patients and controls
Although A91V was the most frequent variation and displayed a trend of association with MS in the first population of patients and controls (frequency of the 91V allele: 0.076 vs 0.043, P=0.044), we used it as a marker to confirm PRF1 involvement in MS, and assessed its frequency in a second independent population of 966 patients and 1520 ethnically and geographically matched controls. The 91V allele was carried by 138 patients (131 heterozygotes and 7 homozygotes) and 168 controls (160 heterozygotes and 8 homozygotes) and its frequency was significantly higher in patients than in controls (0.075 vs 0.058%, P=0.019). In the combined cohorts of 1156 patients and 1788 controls, presence of the 91V allele in single or double dose conferred an OR=1.38 (95% CI=1.10–1.74; Table 2).
No differences were found between subjects carrying or not carrying A91V in terms of gender distribution, MS clinical form (RR, PP and SP) and MSSS (data not shown). Moreover, frequency of the MS susceptibility allele HLA-DR15 was not different in patients carrying A91V or not, as HLA-DR15 was carried by 32% of patients with the 91V allele, 29% of patients without it and 12% of the controls.
Multiple sclerosis is a complex disease that is probably the result of multiple genetic and environmental factors. Several genes have been involved in its development,4 and some of them are important in the immune response. This work shows that PRF1 may also be involved, as MS patients displayed higher frequency of PRF1 variations than the controls. This confirms data obtained by other authors showing that the chromosome region 10q22.1, where PRF1 is located, contains susceptibility genes for MS development.13, 14, 15
The most frequent variation was A91V, as frequency of the 91V allele was increased in two independent populations of MS patients than in the respective controls, and increased the risk of MS by about 1.4-fold in the combined cohorts. By contrast, A91V did not seem to influence the disease course as MSSS was not different between patients with or without A91V. Studies based on analysis of cytotoxic lymphocytes from A91V carriers or rat basophil leukemia cells transfected with variants of the perforin cDNA have shown that A91V decreases perforin function by altering its conformation, decreasing its cleavage to the active form and increasing its degradation.16, 17 Risma et al.18 classified A91V as a class 1 missense mutation with limited functional impact that allows partial maturation of the protein. Voskoboinik et al.19 have recently used a complementation assay with perforin-knockout primary CTL to show that A91V reduces both the steady-state level of perforin expression in effector cells (‘presynaptic’ dysfunction) and its intrinsic lytic capacity on target cells, and also displays some dominant-negative effect on the wild-type protein (‘postsynaptic’ dysfunction).
Our previous work showed that frequency of the 91V allele was also increased in an incomplete variant of ALPS, whereas N252S was associated with the typical form of ALPS and T1DM.11, 12 The functional significance of N252S is debated, but we showed that it may be associated with decreased NK activity in the early childhood.11, 12 Although frequency of N252S was apparently not different in MS and controls, it is possible that PRF1 variations favor development of several autoimmune diseases, with differences reflecting their effects on perforin function. A91V has also been associated with other immune diseases, such as lymphomas and acute childhood lymphocytic leukemia, and atypical (late-onset) FLH2.20, 21
Besides A91V and N252S, we detected two new missense PRF1 mutations in MS patients that cause R240H and R357W amino-acid substitutions. R240H occurs nearby N252S within the membrane-attack complex, a region critically involved in the pore-forming activity of perforin.22 However, analysis of NK activity in the two R240H carriers showed that it was near the low limit of the normal range. Although both carriers were heterozygous, we suggest that R240H causes a mild decrease of perforin function without exerting a dominant-negative effect on the wild-type form. R357W is located in the same domain, but we could not evaluate its functional effect because fresh cells from the carrier were not available. However, both R357W and R240H were predicted to damage perforin function and structure by in silico analysis with the PoliPhen program.
Four other novel mutations were detected in MS patients, but they were synonymous (P333P, P355P, G476G, Q540Q). Q540Q may have an effect on RNA splicing, as it seems to create a new acceptor splice site. The others did not influence canonical splicing sites, but they might theoretically influence perforin expression by disturbing exonic splicing enhancers, mRNA processing and transport, efficiency of codon usage by tRNA stability of mRNA secondary structure, protein folding or interaction with microRNA.23, 24, 25, 26, 27, 28 An alternative possibility is that they do not have a direct effect, but they are linkage disequilibrium with other unknown PRF1 mutations in the 5′ UTR. However, we could not assess perforin expression because fresh cells from the carriers were not available.
Besides A91V, the other mutations are too rare to draw conclusions about their individual association with MS, but they raise the possibility that the overall effect of PRF1 variations on MS development may be substantially higher than that detected by A91V alone. In line with this possibility, these variations conferred a global OR=2.06 for MS development in the first population of patients and controls whose entire PRF1-coding region was sequenced. It is intriguing that two patients were compound heterozygous for A91V, and R357W or P355P, respectively, and another patient was homozygous for Q540Q, which raises the possibility that the biallelic variations may have contributed to their MS. The MS association with several rare PRF1 variations is in line with reports on systemic lupus erythematosus and inflammatory bowel disease indicating that private/rare variations as well as common polymorphisms of other genes may be important in common complex diseases.29, 30
Perforin-mediated cytotoxicity has been classically associated with clearance of virus-infected cells. Therefore, it is possible that defects of perforin activity favor MS development by delaying virus clearance, which may favor development of crossreactions between viral and self-antigens by molecular mimicry. In this context, it is noteworthy that EBV infections are crucial in FLH2 pathogenesis, and have also been suggested to be important as triggering factors in MS.31, 32
On the other hand, an increasing bulk of data suggests that perforin and cell-mediated cytotoxicity may also be involved in downmodulation of the immune response. This regulatory activity may involve several mechanisms including perforin-mediated killing of effector lymphocytes and antigen-presenting cells. Defective immune response switching off may favor both lymphocyte accumulation and autoimmunity.8, 33, 34, 35, 36 It is noteworthy that involvement of inherited defects of the immune response switching off in MS development may not be limited to PRF1, but may also involve defective apoptosis of activated lymphocytes induced through the Fas or the activation-induced cells death (AICD) mechanisms. This possibility is suggested by our previous work showing that substantial proportions of MS patients carry inherited defects of Fas function similar to those displayed by ALPS patients.37 Moreover, several reports detected high serum levels of osteopontin, a cytokine capable to inhibit AICD, in MS patients and we found that this is partly associated with variants of the osteopontin gene.38, 39, 40, 41
In conclusion, this work suggests that PRF1 variations may be a predisposing factor for MS by affecting either the antiviral response or the immune response switching off. Defects of both of these functions may favor development of autoimmunity by prolonging the immune response and increasing the risk of crossreactions between viral and self-antigens. Similar defects may be caused also by alterations of other genes and may be a general predisposing factor for autoimmunity.
Materials and methods
We analyzed two independent cohorts of Italian patients (391 men, 765 women; M/F: 1/1.96) with MS, diagnosed according to McDonald et al.'s criteria42 and randomly selected ethnically matched healthy controls. The first population was composed of 190 patients and 268 controls, the second by 966 patients and 1520 controls.
Patients were consecutive patients enrolled from the Multiple Sclerosis Centers of the ‘Amedeo Avogadro’ University of Eastern Piedmont (Novara), the University of Milan, IRCCS Maggiore Policlinico Hospital (Milan), the Don Gnocchi Institute (Milan), the Santa Croce Hospital (Cuneo), the University of Rome ‘La Sapienza’, S Andrea Hospital (Rome) and the University of Bari (Bari). Their clinical and demographic features were similar to those of other series.43, 44 Controls were consecutive Italian donors obtained from the transfusion services of the respective hospitals. Patients and controls were unrelated, Caucasian and Italian, matched for age and gender, and analyzed as follows:45
RR: Occurrence of exacerbations, each lasting at least 24 h and separated by at least 1 month of inactivity, with full recovery or sequelae (n=852).
PP: Steady worsening of symptoms and signs from onset for at least 6 months, whether superimposed with relapses or not, with occasional plateau and temporary minor improvements (n=92).
SP: Initial RR course followed by steady worsening of symptoms and signs for at least 6 months, whether superimposed with relapses or not, with minor remissions and plateaux (n=212).
Progression of disability was assessed with the MSSS.46 In RR patients, MSSS score was assessed in remission phase.
All patients gave their informed consent according to the Declaration of Helsinki.47 The research was approved by the Novara ethical committee.
Amplification of PRF1 and mutation detection
Genomic DNA was isolated from peripheral blood mononuclear cells (PBMCs) using standard methods and exons 2 and 3 of the PRF1-coding region were amplified in standard PCR conditions. PCR products were purified with the EXO/SAP kit (GE Healthcare, Piscataway, NJ, USA). In the first population, the entire coding region was sequenced with the ABI PRISMR BigDye Terminator kit (Applied Biosystems, Foster City, CA, USA) on an automatic sequencer (Applied Biosystems 3100 Genetic Analyser) according to the manufacturer's instructions. Figure 1 shows primers used for amplification, sequencing and typing. Briefly, exon 2 was amplified with primers A+B (755 bp) and sequenced with the same oligonucleotides. Exon 3 was amplified with C+D (1289 bp) and sequenced with these and with two additional internal primers (E and F). In the second population, the +272 C/T (A91V) variation was typed by sequencing (233 patients and 548 controls) or by the TaqMan 5′-allelic discrimination assay (733 patients and 972 controls; Applied Biosystems). Allelic-specific primers and probes used for discrimination have been previously described.20 Genotyping of each sample was automatically attributed by the SDS 1.3 software for allelic discrimination. Similar results were obtained in patients typed by the two methods. All variations were confirmed twice by sequencing independent DNA samples. The genotypic distribution of the variation did not deviate significantly from the Hardy–Weinberg equilibrium in any group.
The wild-type (91A) and mutant (91V) alleles were separately amplified using specific PCR amplification of genomic DNA (forward primer: G or H; reverse primer: D). PCR products were typed for P355P and R357W by sequencing with the ABI PRISMR BigDye Terminator kit on the 3100 Genetic Analyser using the internal primer E.
Patients and controls were specifically typed for DRB1*1501 allele as previously described.48
Natural killer activity of PBMC was assessed by a standard 4 h 51Cr-release assay with K562 cells as the target. Results are expressed as specific lysis % calculated as follows: (sample 51Cr release−spontaneous release)/(maximal release−spontaneous release) × 100.
Phenotype frequencies were calculated as the number of individuals carrying an allele (either homozygotes or heterozygotes) divided by the total number of individuals.
Allelic and phenotype frequencies were compared with the χ2-test with the Yates correction. All P-values are two-tailed and the significance cutoff was P<0.05. Putative effect of the variation on splicing sites was evaluated using the SpliceView program on the WebGene website (http://www.itb.cnr.it/sun/webgene/) and the ESEfinder scoring matrix (http://www.rulai.cshl.edu/tools/ESE). Putative functional significance of the missense variations was evaluated with the PolyPhen program (http://genetics.bwh.harvard.edu/pph).
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This work was partially supported by FISM grant 2005/R/10 (Genoa), Telethon grant E1170 (Rome), AIRC (Milan), PRIN Project (MIUR, Rome), Compagnia di San Paolo (Turin), Fondazione Cassa di Risparmio di Cuneo (Cuneo), Regione Piemonte (Ricerca Sanitaria Finalizzata Project and Ricerca Sanitaria Applicata-CIPE Project), Associazione ‘Amici di Jean’ (Turin), Fondazione Lagrange (Turin) and Centro Dino Ferrari (Milan).
The authors report no conflict of interest.
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Cappellano, G., Orilieri, E., Comi, C. et al. Variations of the perforin gene in patients with multiple sclerosis. Genes Immun 9, 438–444 (2008). https://doi.org/10.1038/gene.2008.35
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