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1The Edward Jenner Institute for Vaccine Research, Compton, Berkshire, United Kingdom. 2Immunobiology Unit, Institute for Child Health, London, United ...
American Journal of Medical Genetics 117A:255– 260 (2003)

Novel Perforin Mutation in a Patient With Hemophagocytic Lymphohistiocytosis and CD45 Abnormal Splicing James McCormick,1 Darren R. Flower,1 Stephan Strobel,2 Diana L. Wallace,1 Peter C.L. Beverley,1 and Elma Z. Tchilian1* 1

The Edward Jenner Institute for Vaccine Research, Compton, Berkshire, United Kingdom Immunobiology Unit, Institute for Child Health, London, United Kingdom

2

Hemophagocytic lymphohistiocytosis (HLH) composes a group of rare heterogenous disorders characterized by uncontrolled accumulation and infiltration of activated T lymphocytes and macrophages. Cytotoxic T and natural killer cell activity is significantly reduced or absent in these patients. Mutations in the important mediator of lymphocyte cytotoxicity perforin were identified in a number of HLH individuals. Here we report a novel missense mutation thr435met in the conserved Ca2þ binding domain of perforin in a patient with HLH. Prediction of the 3-dimensional structure of the thr435met perforin mutant using comparative molecular modeling indicates that the protein’s ability to bind Ca2þ, and therefore its cytolytic function, would be strongly compromised. In addition, this patient exhibited abnormal CD45 splicing caused by a C77G mutation in the gene encoding CD45 (PTPRC). Our findings suggest a combined role for perforin mutation and abnormal CD45 splicing as significant contributory factors in the pathogenesis of HLH. ß 2003 Wiley-Liss, Inc. KEY WORDS: perforin; HLH; CD45 splicing; PTPRC

INTRODUCTION Hemophagocytic lymphohistiocytosis (HLH) is a rare disorder characterized by disregulated activation of T

*Correspondence to: Elma Z. Tchilian, Edward Jenner Institute for Vaccine Research, Compton, Berkshire RG20 7NN, UK. E-mail: [email protected] Received 19 March 2002; Accepted 12 August 2002 DOI 10.1002/ajmg.a.10010

ß 2003 Wiley-Liss, Inc.

lymphocytes and macrophages [Arico et al., 2001]. HLH is genetically heterogenous with both familial and sporadic forms described [Janka, 1983; Dreyer et al., 1991; Dufourcq-Lagelouse et al., 1999]. The genetic defects underlying HLH have only been partially defined. Linkage of a disease gene to 9q21.3-22 (HLH type 1) was identified in four inbred Pakistani families with HLH using homozygosity mapping [Ohadi et al., 1999]. Shortly after the identification of a linkage to 10q21-22, mutations in the perforin 1 gene (PRF1) were identified [Stepp et al., 1999]. Recent reports have expanded the pattern of PRF1 mutations associated with HLH [Stepp et al., 1999; Ericson et al., 2001, Clementi et al., 2001, Kogawa et al., 2002]. Perforin is secreted by activated cytotoxic T cells and NK cells, inducing cell death by pore formation in target cells [Metkar et al., 2002]. However, perforin mutations account for a still undefined proportion of HLH cases which could be in the range of 20–40% [Ericson et al., 2001], while the majority of HLH cases are likely to be caused by defects in as yet unidentified genes with or without a environmental contribution. A patient with hemophagocytic lymphohistiocytosis with a defect in CD45 splicing has been previously described [Wagner et al., 1995]. CD45 is an abundant tyrosine phosphatase expressed on all leucocytes [Penninger et al., 2001] and recently abnormal CD45 splicing, caused by a C77G polymorphism in exon A of the gene encoding CD45 (PTPRC ) was associated with the development of multiple sclerosis and HIV-1 infection in humans [Jacobsen et al., 2000; Tchilian et al., 2001a]. Here we analyzed the perforin status in this patient and found a novel mis-sense perforin mutation thr435met in exon 3. The mutated threonine 435 is conserved between human, mouse and rat and is located in the highly conserved Ca2þ binding domain of perforin. Molecular modeling predicts that the thr435met mutation would affect the Ca2þ binding of the molecule, and therefore, impair perforin function. These findings suggest that abnormalities in CD45 splicing and perforin mutations might be associated with HLH and these associations warrant further studies.

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MATERIALS AND METHODS Materials Fresh blood was obtained from the previously described family W. [Wagner et al., 1995] from the Immunobiology Unit, Institute for Child Health, London, UK. PBMC were isolated by centrifugation on a FicollPaque (Amersham Pharmacia Biotech, Buckinghamshire, UK) density gradient and genomic DNA was extracted by standard procedures [Sambrook et al., 1989]. Mutation Analysis of the Perforin Gene The perforin gene was amplified by PCR from genomic DNA as previously described [Stepp et al., 1999; Ericson et al., 2001]. Briefly, exon 2 was amplified using PF1 forward (50 -CCCTTCCATGTGCCCTGATAATC-30 ) and PF2 reverse (50 -AGGAGCCTCCAAGTTTGAG-30 ) primers. Exon 3 was amplified using PF3 forward (50 -ACTGCTCCCGGCCAGGATCATTG-30 ) and PF8 reverse (50 -GGCTCCCACTGTGAGA-30 ) primers. Each reaction mixture contained 0.5 ml of both forward and reverse primers (20 pmol), 0.5 ml dNTP (10mM), 5 ml 10  PCR buffer (PE Applied Biosystems, Cheshire, UK), 4 ml MgCl2 (25 mM), and 2.5 U of AmpliTaq Gold (PE Applied Biosystems). The PCR parameters used were denaturation at 968C for 15 min followed by 35 cycles (968C for 30 sec, 588C for 20 sec and 728C for 30 sec) and a final extension at 728C for 2 min. The purified PCR products were subjected to direct automated sequencing using an ABI Prism 377 (PE Applied Biosystems). The sequences obtained were compared to the published human sequence (PRF1 Genbank accession number M28393). Sequence analysis of the perforin gene in family W. showed the presence of a previously described silent heterozygous C to T polymorphism at position 900 (his300his) [Ericson et al., 2001] and a novel heterozygous C to T transition at position 1304 (C1304T) resulting in a change of threonine 435 to methionine (thr435met). The presence of the C1304T mutation was further confirmed by restriction digestion since this transition changes the sequence from CCACGG to CCATGG which can be detected by digestion with NcoI (Gibco Life Technologies, Paisley, UK). Primers PF6 forward (50 -GTCACCACCCAGGACTGCT-30 ) and PF8 reverse (see above), flanking the C1304T mutation were used to generate a 526 bp product and the mutant allele was detected by the presence of two fragments of 425 bp and 101 bp after digestion with NcoI. Molecular Modeling Sequence analysis shows that the C-terminal region of perforin contains a Ca2þ binding C2 domain [Schultz et al., 2000]. The structures of a number of C2 domains have now been experimentally determined [Rizo and Sudhof, 1998] and are comprised of a compact b-sandwich fold composed of two orthogonally aligned fourstranded b-sheets. The structures of C2 domains fall into one of two classes—the S and P variants—that are

related by a circular permutation of their folding topology. The three-dimensional structures of four representatives of each of the two variant classes were extracted from the protein databank [Berman et al., 2000]: 3RPB, 1RSY, 1DSY, and 1DQV for variant S and 1E8X, 1RLW, 1D5R, and 1DJX for variant P. The MALIGN3D function of the MODELLER suite [Sali and Blundell, 1993] was used to align the four representative three-dimensional structures for each of the two variants. The sequence of the perforin C2 domain was then manually aligned to the resultant multiple alignments using CINEMA [Parry-Smith et al., 1998]. Because of significantly closer sequence similarity in the region of the Ca2þ binding site between perforin and the S variant multiple alignment, the S type C2 domain was used as a template for modeling the three dimensional structure of the C2 domain of perforin using MODELLER. For the sake of simplicity, it is this model which is described below, although similar results were obtained with the P variant model. The model was briefly optimized in a solvent bath using the molecular mechanics program AMBER [Cornell et al., 1995]. The resulting structure was then analyzed by visual inspection using the molecular graphics program SYBYL. RESULTS Analysis of Perforin Status in Patient W. We obtained material from a patient W. with HLH, previously described as exhibiting CD45 abnormal splicing as characterized by the lack of the single CD45RO þ T cell population [Wagner et al., 1995]. No information was available on the perforin status of this patient. Patient W. was the third child of healthy unrelated British Caucasian parents [Wagner et al., 1995]. He presented at age 3 months with fever, diarrhea, pallor, increasing irritability and marked cervical lymphoadenopathy and hepatosplenomegaly. Laboratory investigations revealed pancytopenia, coagulopathy, and hypertryglyceridemia. The diagnosis of HLH was made from the bone marrow aspirate, which showed hemophagocytosis. There was a good response to initial treatment with dexamethasone and etoposide and he underwent allogeneic bone marrow transplantation from his HLA identical brother. The perforin gene (PRF1) is located on chromosome 10 (10q21-22) and is organized into three exons, the first of which is not translated [Lichtenheld and Podack, 1989]. We analyzed exons 2 and 3 of the perforin gene in patient W. and found two mutations in exon 3. The first is a previously reported silent heterozygous C to T polymorphism at position 900 (his300his) [Ericson et al., 2001]. The second is a novel mis-sense heterozygous point mutation at nucleotide position 1304 (C1304T) resulting in a change of threonine 435 to methionine (thr435met). The presence of C1304T was analyzed in all family members by sequencing. Furthermore, this mutation introduces a new restriction site for NcoI which was used to confirm the genotype of the family members (Fig. 1). Restriction analysis revealed that the patient, his mother and two siblings are heterozygous for the mutant C1304T (thr435met) allele (Lanes 2 and

A Novel Perforin Mutation in HLH

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Fig. 1. Identification of perforin C1304T transition in family W. A: Family tree indicating the perforin phenotype in each member of family W. The patient with HLH (5) is indicated by asterisks. B: Primers flanking the mutation were used to amplify a 526 bp fragment. The C to T transition introduces an NcoI restriction site which cleaves the mutant PCR product into two fragments of 425 and 101 bp. The patient (lane 5), his mother and two siblings (lanes 2, 4, and 6) are heterozygous for the C1304T mutant perforin allele, while the father and older brother (lanes 1 and 3) have only the 526 bp undigested wild type allele. The perforin mutation co-segregated in the family with the C77G mutation in the gene encoding CD45.

smaller Ca binding domain (C2 domain) is a protein module widely used in nature. In perforin, calcium binding by the C2 domain triggers a profound conformational change in the overall structure of the C9homologous region of the molecule. This probably exposes an otherwise hidden hydrophobic region of the originally hydrophilic protein, leading to aggregation and the formation of cytolytic pores. The novel thr435met mutation identified in family W. is part of the highly conserved C2 domain of perforin. Threonine 435 is conserved between human, mouse and rat (Fig. 2A). Molecular modeling suggests that the thr435met mutation is located within the Ca2þ binding site of the C2 domain (Fig. 2B,C). The increased hydrophobicity and altered polarizability of the methionine residue is likely to affect severely the carefully controlled dielectric environment of this site, which is crucial to the tight binding of calcium ions. Moreover, the increased steric bulk of methionine is also likely to cause significant disruption to the arrangement of amino acid side-chains in this region. The carefully arranged geometry of an array of negatively charged acidic amino acid side chains is an absolute requirement to properly co-ordinate the positively charged calcium ions within the binding site. Both effects of the thr435met mutation will impinge directly on the ability of the protein to bind Ca2þ ions and, through the conformational change that this triggers within the protein, will severely compromise its cytolytic activity. DISCUSSION

4–6, Fig. 1) while the father and the oldest brother were homozygous for the normal perforin allele (Lanes 1 and 3, Fig. 1). The other perforin polymorphism C900T (his300his) showed an identical pattern of inheritance to the C1304T (thr435met) mutation in the family W. (data not shown). Thr435met Disrupts the Ca2þ Binding Activity of Perforin The perforin gene (PRF1) encodes a 534 amino acid polypeptide that is constitutively expressed by natural killer cells [Yu et al., 1999], while cytotoxic T-cells express the protein only when stimulated by effectortarget cell interactions. This interaction induces the cytotoxic effector cell to release cytoplasmic granules into the intercellular space. In the presence of Ca2þ perforin inserts into the cell membrane and then polymerises to form poly-perforin pores, each consisting of 12-18 monomeric units. Pore formation leads to destruction of cells by osmotic lysis and by allowing the entry of apoptosis inducing granzymes [Tschopp and Nabholz, 1990; Lowin et al., 1995; Darmon et al., 1995]. Perforin is composed of two functional domains. The first is a region of about 330 amino acids which is highly homologous to regions of the C6, C7, C8 and C9 components of the complement cascade. This represents the membrane channel-forming region also seen in the membrane attack complex of complement. The second

HLH is a rapidly fatal disease if left untreated. It is characterized by the infiltration of activated lymphocytes and histiocytes (macrophages) into the spleen, liver, lymph nodes, bone marrow and the central nervous system. Mutations in perforin were recently identified in a number of HLH individuals [Stepp et al., 1999; Ericson et al., 2001, Clementi et al., 2001; Kogawa et al., 2002]. In this study, we have analyzed the perforin status in a patient with HLH [Wagner et al., 1995] and identified a novel perforin mis-sense mutation thr435met in exon 3. Threonine 435 is conserved between human, mouse and rat. Other mis-sense heterozygous perforin mutations have been previously reported, although not all of them involve conserved amino acids [Stepp et al., 1999; Ericson et al., 2001]. Patients harboring these mutations have been shown to display substantially reduced levels of expression of perforin protein as well as reduced cytolytic activity [Stepp et al., 1999; Kogawa et al., 2002]. This is the first description of thr435met mutation and further studies of the expression and function of this mis-sense mutation will be required to determine how it may affect perforin function. Because of the lack of further clinical material, we were prevented from undertaking these studies. The mutation is, however, located in the highly conserved Ca2þ binding domain of the perforin molecule and molecular modeling suggests that thr435met would affect the ability of perforin to undergo conformational change and membrane insertion in the presence of Ca2þ. Such a drastic mutation would impair perforin function,

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Fig. 2. A: Protein alignment of the Ca2þ binding C2 domain of human, mouse and rat perforin. The C2 domain is highly conserved between species. This conservation includes the position of the mutated Thr435 residue in the HLH patient W. (indicated by arrow). B: Three-dimensional model of perforin C2 domain, built using the structures from PDB entries 3RPB, 1RSY, 1DSY, and 1DQV as a template using MODELLER, and demonstrating the location of the thr435met mutation (shown in green). The structure is shown using a Ribbon representation and displayed using ALTER and

POVRAY [Flower, 1997]. The direction of the b-pleated sheets is indicated by the direction of the arrows. C: A more detailed atomic presentation of the calcium-binding site of non-mutated perforin. The positively charged calcium ions are presented as purple spheres and the mutated residue in yellow. The surrounding environment consists of acidic amino acids. The mutation inserts a large sulfur-containing hydrophobic residue (methionine), deforming both the geometry and dielectrics of the local atomic environment and thus severely compromising the binding of Ca2þ.

without necessarily greatly affecting the expression or overall folding of the protein. Finally, it is very difficult to predict the role of the thr435met mutation in disease pathogenesis since the patient and three other healthy members of the family are heterozygotes. As the patient here shows a typical clinical picture of HLH, we can not

exclude the possibility that he carries either further unidentified mutations in the non-coding regions of PRF1 or mutations in as yet unidentified genes that may contribute to disease pathogenesis, but it remains much more likely that the disease is primarily a result from the perforin mutation we observed.

A Novel Perforin Mutation in HLH

In this respect, it is interesting that the patient exhibits abnormal CD45 splicing [Wagner et al., 1995]. We confirmed that the C77G polymorphism in exon A of the gene encoding CD45 is the cause for the observed variant splicing in patient W., which is in accordance with our previous studies in which C77G was identified as the common cause of abnormal CD45 splicing [Tchilian et al., 2001b]. Interestingly, the perforin thr435met and the CD45 exon A (C77G) mutations cosegregate together in family W., so that the patient together with the mother and two healthy sibs exhibit both perforin thr435met and CD45 C77G mutations. We postulate that the C77G mutation is a factor provoking the disease along with the perforin mutation. This is plausible since studies with transgenic mice suggest that expression of a high molecular weight CD45 isoform alone causes immunodeficiency and these mice cannot generate any cytotoxic T cell responses or neutralising antibodies after viral infection [Kozieradzki et al., 1997]. Sporadic HLH cases are often provoked by viral infection later in childhood [Dreyer et al., 1991] and one might speculate that individuals with abnormal CD45 splicing may have a higher risk of viral infection. This is further supported by our recent findings that abnormal CD45 splicing and C77G polymorphism is associated with HIV-1 infection [Tchilian et al., 2001a] suggesting that individuals with abnormal CD45 splicing may have increased susceptibility to viral infection or impaired anti-viral responses particularly in infancy. Our data has shown that PRF1 mutations can occur in the Ca2þ binding domain encoded by exon 3 and, in the absence of the necessary but unavailable material required for further study, molecular modeling provides a powerful molecular rationale for its impaired function. Although mutations in the genes encoding perforin and CD45 are associated with HLH, the primary cause may be due to mutations in one or more as yet unidentified genes or, as seems more likely, the disease is the result of a number of gene mutations, including those identified here, interacting to produce the disease phenotype. In any case, the identification of these genetic defects may have important diagnostic, prognostic, and therapeutic implications in the screening and future treatment of HLH. ACKNOWLEDGMENT We thank the patient family, clinicians, and nurses for help with patient samples. REFERENCES

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