Variations of the perforin gene in patients with type 1 diabetes.

OBJECTIVE: Perforin plays a key role in cell-mediated cytotoxicity. Mutations of its gene, PRF1, cause familial hemophagocytic lymphohistiocytosis but have also been associated with lymphomas and the autoimmune/lymphoproliferative syndrome. The aim of this work was to investigate the role of PRF1 variations in type 1 diabetes. RESEARCH DESIGN AND METHODS: We typed for the N252S and A91V variations in an initial population of 352 type 1 diabetic patients and 816 control subjects and a second population of 365 patients and 964 control subjects. Moreover, we sequenced the coding sequence and intron-exons boundaries in 200 patients and 300 control subjects. RESULTS: In both cohorts, allelic frequency of N252S was significantly higher in patients than in control subjects (combined cohorts: 1.5 vs. 0.4%; odds ratio 6.68 [95% CI 1.83-7.48]). Sequencing of the entire coding region detected one novel mutation in one patient, causing a P477A amino acid change not detected in 199 patients and 300 control subjects. Typing for HLA-DQA1 and DQB1 alleles showed that type 1 diabetes-predisposing DQ alpha/DQ beta heterodimers were less frequent in patients carrying N252S or P477A than in those carrying wild-type PRF1. We previously found that natural killer (NK) activity is not decreased in most N252S heterozygotes, but we detected one whose NK activity was normal at the age of 12 but strikingly low in early childhood. Here, we discovered that NK function was low in three heterozygotes in early childhood, one homozygous adult, and in the subject carrying P477A. CONCLUSIONS: These data suggest that N252S and possibly other PRF1 variations are susceptibility factors for type 1 diabetes development.

Patients of African ancestry with hemophagocytic lymphohistiocytosis share a common haplotype of PRF1 with a 50delT mutation.

Mutations of the perforin gene (PRF1) are present in a proportion of patients with hemophagocytic lymphohistiocytosis (HLH). We found that all identified infants with HLH of African descent (17 from USA, 4 from Europe) have 50delT-PRF1 (16 homozygotes, 5 compound heterozygotes), accounting for the most frequently observed PRF1 mutation. Two additional patients with HLH, self-reporting as Hispanic, carried 50delT, but no Caucasians were identified with 50delT. To test the hypothesis that this mutation represents a single haplotype, DNA from 23 patients with HLH and 30 African-American control subjects was sequenced for the PRF1 gene, including portions of the intron containing known single nucleotide polymorphisms (SNPs). The same groups were genotyped at 3 microsatellites proximal to PRF1. The SNP profiles of patients with 50delT-PRF1 were identical, and 5 novel SNPs were identified among African-American control subjects. Patients with 50delT-PRF1 were also found to have had an earlier age of disease onset than patients with other PRF1 mutations. Extent of haplotype sharing and variability of microsatellite alleles in 50delT-PRF1 chromosomes suggest that this mutation arose approximately 1000 to 4000 years ago and is restricted to patients of African descent.

Variations of the perforin gene in patients with autoimmunity/lymphoproliferation and defective Fas function.

Mutations decreasing function of the Fas death receptor cause the autoimmune lymphoproliferative syndrome (ALPS) with autoimmune manifestations, spleen/lymph node enlargement, and expansion of CD4/CD8-negative T cells. Dianzani Autoimmune Lymphoproliferative Disease (DALD) is a variant lacking this expansion. Perforin is involved in cell-mediated cytotoxicity and its biallelic mutations cause familial hemophagocytic lymphohistiocytosis (HLH). We previously described an ALPS patient carrying heterozygous mutations of the Fas and perforin genes and suggested that they concurred in ALPS. This work extends the analysis to 14 ALPS, 28 DALD, and 816 controls, and detects an N252S amino acid substitution in 2 ALPS, and an A91V amino acid substitution in 6 DALD. N252S conferred an OR = 62.7 (P = .0016) for ALPS and A91V conferred an OR = 3 (P = .016) for DALD. Copresence of A91V and variations of the osteopontin gene previously associated with DALD conferred an OR = 17 (P = .0007) for DALD. In one N252S patient, NK activity was strikingly defective in early childhood, but became normal in late childhood. A91V patients displayed lower NK activity than controls. These data suggest that perforin variations are a susceptibility factor for ALPS/DALD development in subjects with defective Fas function and may influence disease expression.

A proportion of patients with lymphoma may harbor mutations of the perforin gene.

Perforin mutations have been demonstrated in a proportion of patients diagnosed with the familial form of hemophagocytic lymphohistiocytosis (HLH). In the present study, we evaluated whether some patients with lymphoma sharing clinical characteristics with HLH might harbor mutations of the perforin gene. We analyzed 29 patients and found that 4 patients, who developed either Hodgkin or non-Hodgkin lymphoma, had biallelic mutations of the perforin gene. One of these 4 patients, a 19-year-old female with T-cell lymphoma, had a brother carrying the same mutations who developed HLH. In 2 of the 4 patients with biallelic mutations of the perforin gene, we evaluated perforin expression by flow cytometry and natural killer (NK) activity and both were found to be absent. Moreover, we documented the presence of monoallelic mutations of the perforin gene in 4 more patients. One of these 4 latter patients also carried a mutation of the Fas gene. These data indicate that perforin deficiency, either alone or in combination with other mutations of genes involved in lymphocyte survival or functional activity, may be present in patients with lymphoma. These findings suggest that perforin also plays a key role in the mechanisms of immune surveillance that prevent tumor growth and/or development.

SAP controls the cytolytic activity of CD8+ T cells against EBV-infected cells.

The adaptor protein SAP regulates signaling through signaling lymphocytic activation molecule (SLAM)-family receptors expressed on T and natural killer (NK) cells. In patients affected by X-linked lymphoproliferative (XLP) disease, mutations in the SH2D1A gene result in defective lytic activity. However, the mechanism by which SAP controls cytotoxic activity remains unclear. T-cell-receptor (TCR) activation of CD8(+) cytotoxic T cells (CTLs) results in down-regulation of SAP, suggesting that this protein is involved in early activation events. Here, we show that SAP-deficient CTLs from patients with XLP and hemophagocytic lymphohistiocytosis (HLH) display a specific lytic defect against autologous and allogeneic Epstein-Barr virus (EBV)-positive B cells. This defect is associated with the defective polarization of 2B4, perforin, and lipid rafts at the contact area of CTLs with EBV-positive targets. Blockade of 2B4 in normal CTLs reproduces the defects in lysis and polarization observed in SAP-deficient CTLs. Expression and regulation of the SLAM-family receptors SLAM, CD84, and 2B4, as well as the lytic effectors perforin and granzyme-B are normal in SAP-deficient CTLs. In addition, TCR stimulation leads to normal proliferation and production of interleukin 2 (IL-2), IL-4, and interferon-gamma (IFN-gamma). These results demonstrate that the SAP/2B4 pathway plays a key role in CTL lytic activity against EBV-positive targets by promoting the polarization of the lytic machinery.

Inherited perforin and Fas mutations in a patient with autoimmune lymphoproliferative syndrome and lymphoma.

A 27-year-old man with the autoimmune lymphoproliferative syndrome and a large-B-cell lymphoma had heterozygous mutations in the Fas and perforin (Prf1) genes. The Fas mutation was inherited from his healthy father and was also carried by his healthy brother, whereas the Prf1 mutation was inherited from his healthy mother. The combined effect of the two mutant genes may have contributed to the development of the autoimmune lymphoproliferative syndrome and lymphoma in this patient.

Atypical features of familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHLH) is a rare, rapidly progressive disorder of early childhood characterized by uncontrolled activation of T cells and macrophages. Although perforin gene mutations have been described in a proportion of patients with FHLH, the genotype/phenotype correlation is still limited. Only a few patients with late onset clinical manifestations have been reported. The biochemical and immunologic alterations in the asymptomatic phase are not well known. We report on a family in which 2 fraternal twins both homozygous for a perforin mutation previously described as causative of the disease, markedly differed in phenotypic expression of FHLH. The twins also had a second novel heterozygous mutation. Natural killer (NK) activity was severely impaired in the patient and was normal in the asymptomatic fraternal twin. Our report highlights that FHLH may present after a long disease-free interval during which biochemical or immunologic alterations may be not evident, thus implying a role for interfering factors.

Abnormal cell surface antigen expression in individuals with variant CD45 splicing and histiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) and Langerhans cell histiocytosis (LCH) are members of a group of rare heterogenous disorders, the histiocytoses, characterized by uncontrolled accumulation of pleomorphic infiltrates of leukocytes. The etiology of these diseases is mainly unknown. CD45 is a hemopoietic cell specific tyrosine phosphatase essential for antigen receptor mediated signaling in lymphocytes and different patterns of CD45 splicing are associated with distinct functions. Recently a polymorphism (C77G) in exon 4 of CD45 causing abnormal CD45 splicing and a point mutation affecting CD45 dimerization were implicated in multiple sclerosis in humans and lymphoproliferation and autoimmunity in mice respectively. Here we show that two patients with HLH exhibited abnormal CD45 splicing caused by the C77G variant allele, while a further 21 HLH patients have normal CD45. We have also examined 62 LCH patients and found three to have the C77G mutation. Peripheral blood thymus-derived (T) CD8(+) cells from normal individuals carrying the C77G mutation show a significant decrease in the proportion of cells expressing L-selectin and increased frequency of cells with LFA-1(hi) expression. It remains to be established whether C77G is a contributing factor in these histiocytic disorders.

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.