Review of hemophagocytic lymphohistiocytosis (HLH) in children with focus on Japanese experiences

Hemophagocytic lymphohistiocytosis (HLH) is characterized by fever and hepatosplenomegaly associated with pancytopenia, hypertriglyceridemia and hypofibrinogenemia. Increased levels of cytokines and impaired natural killer activity are biological markers of HLH. HLH can be classified into two distinct forms, including primary HLH, also referred to as familial hemophagocytic lymphohistiocytosis (FHL), and secondary HLH. Although FHL is an autosomal recessive disorder typically occurring in infancy, it is important to clarify that the disease may also occur in older patients. It is now considered that FHL is a disorder of T-cell function; moreover, clonal proliferation of T lymphocytes is observed in a few FHL patients, and cytotoxicity of these T lymphocytes for target cells is usually impaired. In 1999, perforin gene (PRF1) mutation was identified as a cause of 20-30% of FHL (FHL2) cases. In Japan, two specific mutations of PRF1 were also detected. Furthermore, in 2003, MUNC13-4 mutations were identified in some non-FHL2 patients (FHL3). Identification of other genes responsible for remaining cases is a major concern. Hematopoietic stem cell transplantation (HSCT) has been established as the only accepted curative therapy for FHL. Thus, appropriate diagnosis and prompt treatment with HSCT are necessary for FHL patients. Genetic analysis for PRF1 and MUNC13-4 and functional assay of cytotoxic T lymphocytes are recommended to be performed in each patient. In those patients displaying impaired cytotoxic function but lacking genetic defects, samples should be employed for identification of unknown genes. In the near future, an entire pathogenesis should be clarified in order to establish appropriate therapies including immunotherapy, HSCT and gene therapy.     

Late-onset cases of familial hemophagocytic lymphohistiocytosis with missense perforin gene mutations

Since the discovery of perforin gene mutations in familial hemophagocytic lymphohistiocytosis (FHL) type 2, heterogeneous features in FHL2 patients have been identified in a report of Feldmann et al. as the beginning. This study was conducted to determine the impact of characteristic gene mutations on late-onset (age > or = 7 years) hemophagocytic lymphohistiocytosis episodes. We analyzed perforin gene mutations in three late-onset cases from our registry in Japan and an additional 10 cases from the literature. Of the 13 cases with onset ages of a median of 10 (range 7-49) years, nine had homozygous and four had compound heterozygous missense mutations of the perforin gene. None had homozygous nonsense mutations. Our data suggest that nonsense perforin gene mutations yield early onset and missense mutations late onset in FHL2 cases.     

Analysis of natural killer-cell function in familial hemophagocytic lymphohistiocytosis (FHL): defective CD107a surface expression heralds Munc13-4 defect and discriminates between genetic subtypes of the disease

Natural killer (NK) cells from patients with familial hemophagocytic lymphohistiocytosis because of PRF1 (FHL2, n = 5) or MUNC13-4 (FHL3, n = 8) mutations were cultured in IL-2 prior to their use in various functional assays. Here, we report on the surface CD107a expression as a novel rapid tool for identification of patients with Munc13-4 defect. On target interaction and degranulation, FHL3 NK cells displayed low levels of surface CD107a staining, in contrast to healthy control subjects or perforin-deficient NK cells. B-EBV cell lines and dendritic cell targets reveal the FHL3 NK-cell defect, whereas highly susceptible tumor targets were partially lysed by FHL3 NK cells expressing only trace amounts of Munc13-4 protein. Perforin-deficient NK cells were completely devoid of any ability to lyse target cells. Cytokine production induced by mAb-crosslinking of triggering receptors was comparable in patients and healthy control subjects. However, when cytokine production was induced by coculture with 721.221 B-EBV cells, FHL NK cells resulted in high producers, whereas control cells were almost ineffective. This could reflect survival versus elimination of B-EBV cells (ie, the source of NK-cell stimulation) in patients versus healthy control subjects, thus mimicking the pathophysiologic scenario of FHL.     

Defective cytotoxic lymphocyte degranulation in syntaxin-11 deficient familial hemophagocytic lymphohistiocytosis 4 (FHL4) patients

Familial hemophagocytic lymphohistiocytosis (FHL) is typically an early onset, fatal disease characterized by a sepsislike illness with cytopenia, hepatosplenomegaly, and deficient lymphocyte cytotoxicity. Disease-causing mutations have been identified in genes encoding perforin (PRF1/FHL2), Munc13-4 (UNC13D/FHL3), and syntaxin-11 (STX11/FHL4). In contrast to mutations leading to loss of perforin and Munc13-4 function, it is unclear how syntaxin-11 loss-of-function mutations contribute to disease. We show here that freshly isolated, resting natural killer (NK) cells and CD8(+) T cells express syntaxin-11. In infants, NK cells are the predominant perforin-containing cell type. NK cells from FHL4 patients fail to degranulate when encountering susceptible target cells. Unexpectedly, IL-2 stimulation partially restores degranulation and cytotoxicity by NK cells, which could explain the less severe disease progression observed in FHL4 patients, compared with FHL2 and FHL3 patients. Since the effector T-cell compartment is still immature in infants, our data suggest that the observed defect in NK-cell degranulation may contribute to the pathophysiology of FHL, that evaluation of NK-cell degranulation in suspected FHL patients may facilitate diagnosis, and that these new insights may offer novel therapeutic possibilities.     

Temperature sensitivity of human perforin mutants unmasks subtotal loss of cytotoxicity,delayed FHL,and a predisposition to cancer

The pore-forming protein perforin is critical for defense against many human pathogens and for preventing a catastrophic collapse of immune homeostasis, manifested in infancy as Type 2 familial hemophagocytic lymphohistiocytosis (FHL). However, no evidence has yet linked defective perforin cytotoxicity with cancer susceptibility in humans. Here, we examined perforin function in every patient reported in the literature who lived to at least 10 years of age without developing FHL despite inheriting mutations in both of their perforin (PRF1) alleles. Our analysis showed that almost 50% of these patients developed at least 1 hematological malignancy in childhood or adolescence. The broad range of pathologies argued strongly against a common environmental or viral cause for the extraordinary cancer incidence. Functionally, what distinguished these patients was their inheritance of PRF1 alleles encoding temperature-sensitive missense mutations. By contrast, truly null missense mutations with no rescue at the permissive temperature were associated with the more common severe presentation with FHL in early infancy. Our study provides the first mechanistic evidence for a link between defective perforin-mediated cytotoxicity and cancer susceptibility in humans and establishes the paradigm that temperature sensitivity of perforin function is a predictor of FHL severity.     

Perforin gene analaysis in an Iranian family with familial hemophagocytic lymphohistiocytosis

Perforin gene (PRF1) mutations have been reported in 20-30% of patients with familial hemophagocytic lymphohistiocytosis (FHL), an immune disorder of infancy and early childhood. Cytotoxic T and natural killer (NK) cell activities are remarkably reduced or absent in FHL patients. We report the first cases of familial hemophagocytic lymphohistiocytosis in an Iranian family with two siblings. Exons 2 and 3 of the PRF1 gene were analyzed by polymerase chain reaction (PCR) amplification and direct sequencing. Perforin gene mutation(s) were detected in none of the cases. The result of our study indicates that not much evidence is present concerning a correlation between perforin gene defects and familial hemophagocytic lymphohistiocytosis etiology in these cases.     

家族性噬血细胞性淋巴组织细胞增多症一例病因和遗传学研究

目的 提高对家族性噬血性淋巴组织细胞增多症(FHL)的诊疗水平.方法 报告1例人类疱疹病毒7型(HHV7)阳性FHL2型患者的临床、病因及遗传学特征;人类疱疹病毒(HHV1～HHV8)DNA筛查采用PCR方法 ;NK细胞穿孔素(PRF1)蛋白表达采用流式细胞术检测;PRF1基因突变采用PCR技术和DNA序列分析鉴定;PRF1蛋白构象通过ExPASy和I-TASSER网站在线分析系统进行生物信息学分析;对患者亲属34例进行遗传家系分析.结果 该患者HHV7病毒DNA为350拷贝/106外周血有核细胞;PRF1阳性的NK细胞比例和PRF1表达显著降低;患者PRF1基因存在c.503G＞A/p.S168N和c.1177T＞C/p.C393R突变,其S168N突变遗传自父系,C393R突变遗传自母系.抗病毒、地塞米松、VP16及联合化疗对患者疗效短暂,经人类白细胞抗原10/10相合的非血缘异基因造血干细胞移植治疗后已健康存活9个月.结论 应加强对FHL患者免疫功能及其相关分子遗传学的研究;异基因造血干细胞移植是FHL治疗的根本措施.

Abstract：

Objective To analyze the etiological factor and genetic feature of a familial hemophagocytic lymphohistiocytosis patient with PRF1 mutation (FHL2) with human herpesvirus 7 (HHV7)infection and its family constellation. Methods Clinical characteristics, laboratory examinations of a FHL2 case with HHV7 infection were reported. HHV1-HHV8 virus DNA was screened by PCR; NK cell function was analyzed by flow cytometry; PRF1 gene mutations were analyzed by PCR and direct sequencing, structure of mutant PRF1 proteins were analyzed using ExPasy and I-TASSER server and genetics pedigree were analyzed. Results The patient's HHV7 viral was detected positive with DNA copy number of 350/106 peripheral nucleated cells. Flow cytometry analysis showed decrease both in proportion of perforin positive NK cells and perforin protein expression. Genetic testing showed PRF1 biallelic heterozygote mutations (c. 503G ＞ A/p. S168N and c. 1177T ＞ C/p. C393R) and pedigree analysis showed they were inherited. The patient was then treated with antivirus therapy, dexamethasone and VP16 therapy, but only achieved partial response. The patient was then followed by human leukocyte antigen 10/10 allele identical nonconsanguinity allogeneic hematopoietic stem cell transplantations (allo-HSCT) and soon the successful implantation of donor hematopoietic cells and persistent recovery was achieved. The patient was now surviving without recurrence for 9 months after allo-HSCT. Conclusions FHL is prone to be misdiagnosed as lymphoma. Genetic analysis of related gene mutation and herpes simplex virus detection will help in early and accurate diagnosis. Allo-HSCT is a fundamental treatment of FHL.     

Fatal immune dysregulation due to a gain of glycosylation mutation in lymphocyte perforin

Mutations in the perforin gene (PRF1) are a common cause of the fatal immune dysregulation disorder, familial hemophagocytic lymphohistiocytosis (type 2 FHL, FHL2). Here we report a female infant born with biallelic PRF1 mutations: a novel substitution, D49N, and a previously identified in-frame deletion, K285del. We assessed the effects of each mutation on the cytotoxicity of human NK cells in which the expression of endogenous perforin was ablated with miR30-based short hairpin (sh) RNAs. Both mutations were detrimental for function, thereby explaining the clinically severe presentation and rapidly fatal outcome. We demonstrate that D49N exerts its deleterious effect by generating an additional (third) N-linked glycosylation site, resulting in protein misfolding and degradation in the killer cell. Our data provide a rationale for treating some cases of type 2 familial hemophagocytic lymphohistiocytosis, based on the pharmacologic inhibition or modification of glycosylation.     

Functional consequences of perforin gene mutations in 22 patients with familial haemophagocytic lymphohistiocytosis

Familial haemophagocytic lymphohistiocytosis (FHL), an inherited form of haemophagocytic lymphohistiocytosis (HLH) syndrome, is characterized by the overwhelming activation of T lymphocytes and macrophages invariably leading to death in the absence of treatment. FHL is a heterogeneous autosomal recessive disorder, with one known causative gene which codes for perforin, a cytotoxic effector protein. In this study, we have characterized the genotype and phenotype of 14 unrelated families with perforin deficiency. Four new missense mutations of the perforin gene were identified. In every case, perforin gene mutations led to undetectable intracellular perforin expression within cytotoxic cells, while some residual T-cell cytotoxic activity could be associated with certain missense mutations. Clinical and biological analyses did not differentiate between patients with nonsense or missense mutations, although age at diagnosis, which tended to be similar within members of the same family, was delayed in patients from two families belonging to the second group. In one case, consequences of perforin deficiency, diagnosed at birth, could be assessed prior to onset of clinical manifestations. No evidence for T-cell activation could be shown, suggesting that an exogenous event is required to trigger the disease manifestation. Control assessment of perforin expression and cytotoxic assays by lymphocytes from young children led to the conclusion that perforin content of natural killer cells could be a reliable diagnostic test at any age. Altogether, these data enabled a better characterization of perforin deficiency and its consequences, and defined reliable diagnostic tools.     

Perforin and lymphohistiocytic proliferative disorders

Perforin is critical for cytotoxicity mediated by granules present in natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Perforin-deficient mice have impaired cytotoxicity by NK cells and CTLs, resulting in failure to control infections with certain viruses or bacteria. Infection of perforin-deficient mice with lymphocytic choriomeningitis virus results in haemophagocytic lymphohistiocytosis and elevated levels of pro-inflammatory cytokines. Mutations throughout the perforin gene have been identified in patients with familial haemophagocytic lymphohistiocytosis (FHL) type 2. These patients present with fever, hepatosplenomegaly, pancytopenia, have marked elevations of T-helper type 1 and type 2 cytokines, and have impaired NK cell and CTL cytotoxicity. A number of infectious pathogens have been implicated as triggering the onset of disease. Identification of mutations in perforin as the cause of FHL should allow prenatal diagnosis of the disorder. While stem cell transplantation is curative, gene therapy might be effective in the future.     

Haemophagocytic lymphohistiocytosis: proposal of a diagnostic algorithm based on perforin expression

Haemophagocytic lymphohistiocytosis (HLH) is a rare, fatal disorder of early infancy. Mutations of the PRF1 gene have been identified in a subset of patients. However, the distinction between the different genetically determined and environmental subtypes of the disease remains a major issue to be solved. This may result in delayed or inappropriate application of bone marrow transplantation (BMT). We propose an algorithm that uses a combination of three rapid laboratory tests, i.e. perforin expression by peripheral lymphocytes, assessment of the behaviour of the 2B4 lymphocyte receptor and natural killer (NK) cell activity, to identify the different subgroups of HLH. In 19 patients diagnosed according to current criteria, we tested perforin expression, 2B4 receptor function and NK cell activity. PRF1 mutations were found in all seven patients showing absent perforin expression. In one male with abnormal behaviour of the 2B4 receptor, SH2D1A mutation confirmed the diagnosis of X-linked lymphoproliferative disease. Four patients with normal NK cell activity had evidence of associated infections. Of the seven with impaired NK cell activity, two had a probable genetically determined subtype of HLH and five appeared as sporadic, infection-associated cases. Improving the diagnostic approach may restrict the use of BMT, the only recognized curative treatment, to HLH patients with a documented poor prognosis while patients with milder disorders may be treated less intensively. Our flow chart could also lead to better selection of patients for specific gene analysis.     

Severe and progressive encephalitis as a presenting manifestation of a novel missense perforin mutation and impaired cytolytic activity

Mutations in the perforin gene cause familial hemophagocytic lymphohistiocytosis (FHL). The first symptoms of FHL are usually intractable fever, hepatosplenomegaly, and pancytopenia. Most FHL patients subsequently develop central nervous system (CNS) manifestations due to infiltration of tissues by activated lymphocytes and macrophages. We report 2 FHL patients with an atypical phenotype characterized by isolated severe neurologic symptoms mimicking chronic encephalitis and leading to an early death. Functional and molecular analyses revealed the same novel missense mutation in the perforin gene in both patients; this mutation affected the calcium-binding domain of the protein. This missense mutation did not affect perforin maturation or expression in cytotoxic cells but impaired in vitro cytotoxic activity. Diagnosis was delayed in both patients because of the initial neurologic expression and the normal expression of perforin in circulating lymphocytes. This emphasizes the importance of early diagnosis of this atypical form of FHL, as CNS involvement causes severe, irreversible encephalopathy. This observation also raises the question of the role of some mutations in the neurologic expression of FHL.     

Novel spectrum of perforin gene mutations in familial hemophagocytic lymphohistiocytosis in ethnic Omani patients

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive immune disorder, characterized by fever, hepatosplenomegaly, pancytopenia, hypertriglyceridemia, hypofibrinogenemia, markedly elevated levels of inflammatory cytokines, and impaired cytotoxic activity of lymphocytes. FHL is often fatal in early infancy. Histologic features include organ infiltration by activated macrophages and lymphocytes. Four genetic loci (FHL1, 2, 3, and 4) have been identified, of which FHL2 involves mutations in the perforin gene and is present in 20-50% of patients with FHL. We herein report the first comprehensive molecular analysis of 16 unrelated cases of FHL in ethnic Omanis. Using direct DNA sequencing analysis in 11 families, seven different mutations were identified in the coding region of the perforin gene, of which five were novel. Perforin gene defects do not seem to be involved in one-third of the cases of FHL in ethnic Omanis.     

Characterization of PRF1, STX11 and UNC13D genotype-phenotype correlations in familial hemophagocytic lymphohistiocytosis

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare autosomal recessive lethal condition characterized by fever, cytopenia, hepatosplenomegaly and hemophagocytosis. The hallmark of FHL is defect apoptosis triggering and lymphocyte cellular cytotoxicity. Thus far three disease-causing genes (PRF1, UNC13D, STX11) have been identified. We performed a genotype-phenotype study in a large, multi-ethnic cohort of 76 FHL patients originating from 65 unrelated families. Biallelic mutations in PRF1, UNC13D and STX11 were demonstrated in 13/74 (18%), 6/61 (10%) and 14/70 (20%) patients, respectively. In 27/60 (45%) patients analyzed for all three genes, no molecular diagnosis was established. STX11 mutations were most common in Turkish families (7/28, 25%), whereas in Middle East families, PRF1 mutations were most frequent (6/13, 46%). No biallelic mutation was identified in most families of Nordic origin (13/14, 93%). Patients carrying PRF1 mutations had higher risk of early onset (age <6 months) compared to patients carrying STX11 mutations [adjusted odds ratio 8.23 (95% confidence interval [CI] = 1.20-56.40), P = 0.032]. Moreover, patients without identified mutations had increased risk of pathological cerebrospinal fluid (CSF) at diagnosis compared to patients with STX11 mutations [adjusted odds ratio 26.37 (CI = 1.90-366.82), P = 0.015]. These results indicate that the disease-causing mutations in FHL have different phenotypes with regard to ethnic origin, age at onset, and pathological CSF at diagnosis.     

A functional analysis of the putative polymorphisms A91V and N252S and 22 missense perforin mutations associated with familial hemophagocytic lymphohistiocytosis

Up to 60% of cases of the autosomal recessive immunodeficiency hemophagocytic lymphohistiocytosis (HLH) are associated with mutations in the perforin (PRF1) gene. In this study, we expressed wild-type and mutated perforin in rat basophil leukemia cells to study the effect on lytic function of the substitutions A91V and N252S (commonly considered to be neutral polymorphisms) and 22 perforin missense substitutions first identified in HLH patients. Surprisingly, we found that A91V perforin was expressed at reduced levels compared with wild-type perforin, resulting in partial loss of lytic capacity. In contrast, expression and function of N252S-substituted perforin were normal. Most HLH-associated mutations resulted in protein degradation (probably due to misfolding) and complete loss of perforin activity, the exception being R232H, which retained approximately 30% wild-type activity. Several other mutated proteins (H222Q, C73R, F157V, and D313V) had no detectable lytic activity but were expressed at normal levels, suggesting that their functional defect might map downstream at the level of the target cell membrane. One further perforin substitution identified in an HLH patient (V183G) was normally expressed and displayed normal lysis. This report represents the first systematic functional analysis of HLH-associated missense mutations and the 2 most common perforin polymorphisms.     

Founder effects in two predominant intronic mutations of UNC13D, c.118-308C>T and c.754-1G>C underlie the unusual predominance of type 3 familial hemophagocytic lymphohistiocytosis (FHL3) in Korea

Familial hemophagocytic lymphohistiocytosis (familial HLH or FHL) is a potentially fatal autosomal recessive disorder. Our previous study demonstrated that UNC13D mutations (FHL3) account for ～90 % of FHL in Korea with recurrent splicing mutation c.754-1G>C (IVS9-1G>C). Notably, half of the FHL3 patients had a monoallelic mutation of UNC13D. Deep intronic mutations in UNC13D were recently reported in patients of European descent. In this study, we performed targeted mutation analyses for deep intronic mutations and investigated on the founder effect in FHL3 in Korean patients. The study patients were 72 children with HLH including those with FHL3 previously reported to have a monoallelic UNC13D mutation. All patients were recruited from the Korean Registry of Hemophagocytic Lymphohistiocytosis. In addition to conventional sequencing of FHL2-4, targeted tests for c.118-308C>T and large intronic rearrangement mutations of UNC13D were performed. Haplotype analysis was performed for founder effects using polymorphic markers in the FHL3 locus. FHL mutations were detected in 20 patients (28 %). Seventeen patients had UNC13D mutations (FHL3, 85 %) and three had PRF1 mutations (FHL2, 15 %). UNC13D:c.118-308C>T was detected in ten patients, accounting for 38 % of all mutant alleles of UNC13D, followed by c.754-1G>C (26 %). Haplotype analyses revealed significantly shared haplotypes in both c.118-308C>T and c.754-1G>C, indicating the presence of founder effects. The deep intronic mutation UNC13D:c.118-308C>T accounts for the majority of previously missing mutations and is the most frequent mutation in FHL3 in Korea. Founder effects of two recurrent intronic mutations of UNC13D explain the unusual predominance of FHL3 in Korea.     

Distinct mutations in STXBP2 are associated with variable clinical presentations in patients with familial hemophagocytic lymphohistiocytosis type 5 (FHL5)

Familial hemophagocytic lymphohistiocytosis (FHL) is a genetically determined hyperinflammatory syndrome caused by uncontrolled immune response mediated by T-lymphocytes, natural killer (NK) cells, and macrophages. STXBP2 mutations have recently been associated with FHL5. To better characterize the genetic and clinical spectrum of FHL5, we analyzed a cohort of 185 patients with suspected FHL for mutations in STXBP2. We detected biallelic mutations in 37 patients from 28 families of various ethnic origins. Missense mutations and mutations affecting 1 of the exon 15 splice sites were the predominant changes detectable in this cohort. Patients with exon 15 splice-site mutations (n = 13) developed clinical manifestations significantly later than patients with other mutations (median age, 4.1 year vs 2 months) and showed less severe impairment of degranulation and cytotoxic function of NK cells and CTLs. Patients with FHL5 showed several atypical features, including sensorineural hearing deficit, abnormal bleeding, and, most frequently, severe diarrhea that was only present in early-onset disease. In conclusion, we report the largest cohort of patients with FHL5 so far, describe an extended disease spectrum, and demonstrate for the first time a clear genotype-phenotype correlation.     

Perforin gene mutations in patients with acquired aplastic anemia

Perforin is a cytolytic protein expressed mainly in activated cytotoxic lymphocytes and natural killer cells. Inherited perforin mutations account for 20% to 40% of familial hemophagocytic lymphohistiocytosis, a fatal disease of early childhood characterized by the absence of functional perforin. Aplastic anemia, the paradigm of immune-mediated bone marrow failure syndromes, is characterized by hematopoietic stem cell destruction by activated T cells and Th1 cytokines. We examined whether mutations in the perforin gene occurred in acquired aplastic anemia. Three nonsynonymous PRF1 mutations among 5 unrelated patients were observed. Four of 5 patients with the mutations showed some hemophagocytosis in the bone marrow at diagnosis. Perforin protein levels in these patients were very low or absent, and perforin granules were completely absent. Natural killer (NK) cell cytotoxicity from these patients was significantly decreased. Our data suggest that PRF1 genetic alterations help explain the aberrant proliferation and activation of cytotoxic T cells and may represent genetic risk factors for bone marrow failure.     

Characteristic perforin gene mutations of haemophagocytic lymphohistiocytosis patients in Japan

Perforin gene (PRF1) mutations appear to occur in about 30% of patients with haemophagocytic lymphohistiocytosis (HLH). We tested perforin expression and gene mutations in 14 HLH patients and six patients with Epstein-Barr virus-associated HLH (EBV-HLH) in Japan. Five of the 14 HLH patients had perforin abnormalities. The presence of PRF1 genetic abnormality correlated well with the lack of perforin expression as determined by flow cytometry. Sequencing showed that four patients had a compound heterozygous mutation while the fifth patient had a homozygous mutation. Three of the mutations we detected were novel. In contrast, none of the six EBV-HLH patients showed perforin abnormalities. Our data, combined with the PRF1 mutations in three previously reported Japanese patients, suggest that the 1090-1091delCT and 207delC mutations of the perforin gene are frequently present in Japanese HLH patients (62.5% and 37.5% respectively). Examination of the geographical origins of the ancestors in the perforin-mutant HLH patients revealed that they mostly came from the Western part of Japan, suggesting that the present-day cases may largely derive from a common ancestor.     

Reduced perforin expression in systemic juvenile idiopathic arthritis is restored by autologous stem-cell transplantation

OBJECTIVES: Familial haemophagocytic lymphohistiocytosis (FHL) is a disorder characterized by deficient cytotoxic T-cell function and activated macrophages, owing to a defect in the perforin gene and absent perforin expression. Because symptoms of patients with systemic juvenile idiopathic arthritis (sJIA) are sometimes clinically very similar to those with FHL, we studied whether perforin expression in sJIA patients would be reduced also. METHODS: We determined the perforin expression levels on two subsets of CD8(+) cells (CD8(+)CD28(-)CD45RA(-) and CD8(+)CD28(-)CD45RA(+)) and natural killer (NK) cells from patients with sJIA under conventional treatment as well as before and after autologous stem-cell transplantation (ASCT). RESULTS: CD45RA(-) cytotoxic effector cells of sJIA patients (n=13) express significantly lower levels of perforin than polyarticular juvenile idiopathic arthritis (pJIA, n=9) patients [sJIA mean fluorescence intensity (MFI) 34.6; pJIA MFI 98.0] or control donors (MFI 124.6, n=5). A similar pattern was seen in the CD45RA(+) subset. Also NK cells from sJIA patients expressed significantly less intracellular perforin (sJIA MFI 398.4; controls MFI 972.4). In four patients with sJIA who were treated with ASCT, a clear increase in perforin expression was found at 12 months after ASCT in both cytotoxic effector cell subsets (CD45RA(-) subset before ASCT MFI 13.2; 12 months after ASCT MFI 172.3). CONCLUSION: We conclude that perforin expression can be severely reduced in sJIA. This finding may implicate defective cytotoxicity and haemophagocytosis and could thus explain why sJIA may be complicated by macrophage activation syndrome. ASCT leads to a reconstitution of the (T cell) immune system with a normal expression of perforin.