PRF1 Mutations Research Articles

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

AbstractNatural 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.

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.

A91V perforin variation in healthy subjects and FHLH patients

Familial haemophagocytic lymphohistiocytosis (FHLH) is a heterogeneous autosomal recessive disorder characterized by hyperactivation of monocytes/macrophages. Perforin (PRF1) gene alterations have been documented in 40% of patients with FHLH. Although several mutations have been identified, a clear correlation between the individual molecular alteration and the phenotypic expression of the disease is still unclear. In particular, the role that the A91V substitution plays in the pathogenesis of the disease is still controversial. In the effort to make a conclusive remark to this issue, we here report on the frequency of the A91V mutation in a group of unrelated healthy families obtained from the "Centre d'Etude du Polymorphisme Humain" (CEPH), which are considered representative of the worldwide population. This frequency was compared to that observed in FHLH patients recruited through the Italian National Registry. The frequency in CEPH healthy subjects is 3.7%, thus indicating that the alteration represents a polymorphism. However, the frequency of this alteration in FHLH patients associated with PRF1 mutation is much higher than that observed in controls (26.2%, P = 0.0002), suggesting that the alteration is an important genetic susceptibility factor.

Novel Munc13-4 Mutations in Patients with Hemophagocytic Lymphohistiocytosis.

Familial hemophagocytic Lymphohistiocytosis (FLH) is a rare disorder characterized by fever, hepatosplenomegaly, cytopenia, hypertriglyceridemia, hypofibrinogenemia and reduced natural killer (NK) cell activity. Several genetic defects have been recognized as associated with FHL. After identification of perforin gene (PRF1) mutation as the cause of about one third of cases of FHL (Stepp,1999), in 2003 the same group reported that another gene involved in cellular cytotoxicity, Munc13-4, encoding for a protein involved in granule release in cytotoxic lymphocyte, is associated with a subset of patients with FHL. We have analyzed for Munc13-4 mutations 16 families of patients diagnosed with FHL according to current criteria, in whom PRF1 mutations had been excluded; 14 were Italian, 1 from USA, one from UK. The 32 exons and their proximal flanking regions of Munc13-4 gene were amplified from genomic DNA and amplification products were directly sequenced by cycle sequencing approach (BigDye Terminator Applied Biosystems). Sequences of primers utilized for the amplification reactions were designed with dedicated software (Primer Express) according to the gene sequence retrived from Genebank. A total of 13 mutations were identified in 12 families; 3 had homozygous mutations, 5 had combined heterozygous mutations, and 4 had only one detectable mutated allele. Mutation 753+IG in exon 9 (splice donor site) had been already reported (Feldman 2003, zurStadt 2005); The following novel mutations were found: G175A in exon 3 (A59T), delC532 in exon 6 (178 fs), A610G in exon 7 (M204V), G1241T in exon 14 (R414L), A1847G in exon 20 (E616G), del GGAG 2346 (782 fs) in exon 24, A2599G (K867E) in exon 27, C2650T in exon 28 (Q884stop), C2782T in exon 29 (R928C), C2896T in exon 30 (R966W), 3082delC in exon 31 (1028fs), insG3226 in exon 32 (1076fs). Mutation A2599 was observed in 6 families; mutation A1847G in 3 families; C2782T and G1241T in 2 families. We have also identified the polymorphisms C279T, and A3198G. Mutations were scattered over different exons and almost entirely different from those reported so far. A2599G mutation was frequently observed and its role deserves further investigation. Screening of Munc13-4 mutations is a powerful tool to confirm the diagnosis, to refine the therapeutic choice including indication to HSCT, selection of familiar donor, and identification of carriers and prenatal diagnosis in most of the families with FHL not associated with PRF1 mutations. Due to lack of immunologic or flow-cytometric tools for rapid screening of Munc13-4 defective patients, the high number of coding exons and lack of mutation clustering, screening of Munc13-4 mutations, although expensive and time consuming, remains necessary and allowed us to identify the genetic defect in the majority of our patients with FHL not associated with PRF1 mutations.

Genetic subtypes of familial hemophagocytic lymphohistiocytosis: correlations with clinical features and cytotoxic T lymphocyte/natural killer cell functions

AbstractMutations of the perforin (PRF1) and MUNC13-4 genes distinguish 2 forms of familial hemophagocytic lymphohistiocytosis (FHL2 and FHL3, respectively), but the clinical and biologic correlates of these genotypes remain in question. We studied the presenting features and cytotoxic T lymphocyte/natural killer (CTL/NK) cell functions of 35 patients for their relationship to distinct FHL subtypes. FHL2 (n = 11) had an earlier onset than either FHL3 (n = 8) or the non-FHL2/FHL3 subtype lacking a PRF1 or MUNC13-4 mutation (n = 16). Deficient NK cell activity persisted after chemotherapy in all cases of FHL2, whereas some patients with FHL3 or the non-FHL2/FHL3 subtype showed partial recovery of this activity during remission. Alloantigen-specific CTL-mediated cytotoxicity was deficient in FHL2 patients with PRF1 nonsense mutations, was very low in FHL3 patients, but was only moderately reduced in FHL2 patients with PRF1 missense mutations. These findings correlated well with Western blot analyses showing an absence of perforin in FHL2 cases with PRF1 nonsense mutations and of MUNC13-4 in FHL3 cases, whereas in FHL2 cases with PRF1 missense mutations, mature perforin was present in low amounts. These results suggest an association between the type of genetic mutation in FHL cases and the magnitude of CTL cytolytic activity and age at onset.

Mutation spectrum in children with primary hemophagocytic lymphohistiocytosis: molecular and functional analyses ofPRF1, UNC13D, STX11, andRAB27A

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal-recessive disease that affects young children. It presents as a severe hyperinflammatory syndrome with activated macrophages and T lymphocytes. Mutations in the perforin 1 gene (PRF1) were found in FHL-2 in 15-50% of all cases. Defective granule exocytosis caused by mutations in the hMunc13-4 gene (UNC13D) has been described in FHL-3. FHL-4 patients have mutations in STX11, a t-SNARE involved in intracellular trafficking. We analyzed a large group of 63 unrelated patients with FHL of different geographic origins (Turkey:32; Germany:23; others:8) for mutations in STX11, PRF1, and UNC13D. We identified mutations in 38 samples (20 in PRF1, 12 in UNC13D, and six in STX11). Of 32 patients from Turkey, 14 had mutations in PRF1, six had mutations in UNC13D, and six had mutations in STX11. The mutation Trp374X in PRF1 was found in 12 patients from Turkey and was associated with a very early onset of the disease below the age of 3 months in all cases. In contrast, three of 23 and four of 23 patients from Germany, and three of eight and two of eight from other origins showed mutations in PRF1 and UNC13D, respectively, but none in STX11. Thus, FHL-2, FHL-3, and FHL-4 account for 80% of the HLH cases of Turkish origin, and for 30% of German patients. Furthermore, we identified mutations in RAB27A in three patients with FHL-related Griscelli syndrome type 2. In functional studies using a mammalian two-hybrid system we found that missense mutations Ala87Pro in Rab27a and Leu403Pro in hMunc13-4 each prevented the formation of a stable hMunc13-4/Rab27a complex in vitro. Our findings demonstrate extensive genetic and allelic heterogeneity in FHL and delineate an approach for functionally characterizing missense mutations in RAB27A and UNC13D.

UNC13D and PRF1 Mutations in Childhood Patients with Hemophagocytic Lymphohistiocytosis.

Introduction: Familial Hemophagocytic Lymphohistiocytosis (FHLH) is an autosomal recessive disease affecting young children. It manifests itself as a severe hyperinflammatory syndrome with activated macrophages and T-lymphocytes. Mutations in Perforin (PRF1) are responsible for FHL-2 in about 15 to 50 % of the patients. Defective granule exocytosis due to mutations in UNC13D have recently been described as the genetic defect underlying FHL-3. Both types are phenotypically not distinguishable from one another.Patients and Methods: Genetic analysis was performed in either the 2 or the 32 exons of PRF1 and UNC-13D, respectively, in 61 patients with hemophagocytic lymphohistiocytosis (HLH) of different geographic origin (28 from Germany, 24 from Turkey and 9 from other countries).Results: We identified mutations in 28 samples investigated (18 in PRF1, 10 in UNC-13D). Besides several known mutations novel deletions, missense and nonsense mutations were detected in both genes scattering the whole coding regions. The Trp374stop mutation (11/13 patients with a PRF1 mutation from Turkey) was the only single mutation repeatedly observed in several patients with a common geographic origin. In HLH patients from Germany, 7/28 patients showed homozygous or compound heterozygous mutations in either PRF1 (3) or UNC13D (4). In 24 HLH patients from Turkey 13 patients with a mutation in PRF1 and 4 patients with a mutation in UNC13D were identified. In addition, four out of 9 patients with a different geographic origin showed mutations in one of this genes. In 10 patients only one heterozygous mutation in UNC13D was detected so far. In these cases, control samples or regulatory regions of the gene have to be analysed to ascertain the relevance of the underlying mutation.DefectTurkeyGermanOtherAllUNC13D44210PRF1133218Unknown721533Discussion: Overall, UNC-13D and PRF1 mutations were detectable in 45% of the cases. Our results indicate that FHL-2 and FHL-3 account for more than 60% of the HLH cases of Turkish origin with only a small number involving UNC-13D. In contrast, gene defects were identified in only 25% of the patients with a German ancestry. This work was supported by the Foerdergemeinschaft Kinderkrebszentrum Hamburg e. V.

Classification of Familial Hemophagocytic Lymphohistiocytosis Based on Genetic and Immunologic Analyses.

Background: Familial hemophagocytic lymphohistiocytosis (FHL) is a disorder with an autosomal recessive mode of inheritance in infancy. Recently, mutations of the perforin (PRF1) and MUNC13–4 genes have been identified in FHL patients. Since natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) involve mechanisms to kill virus-infected and malignant cells via perforin/granzyme-mediated granule exocytosis pathway, cytotoxic functions of NK cells and CTLs are impaired in FHL. In the present study, we analyzed the correlation between clinical features, genetic abnormalites and cytotoxic functions of CTLs in patients with different FHL subtypes.FHL families: In Japan HLH Study Group, total 57 Japanese patients fulfilled the diagnostic criteria of FHL and their family members were registered and the clinical data were collected from each family. Since the majority of FHL patients in Japan were registered in our study, it is thought that our data reflect the epidemiology of FHL in Japanese.Results: Among 57 patients registered in this study, PRF1 mutations were detected in 11 patients (FHL2), whereas 8 showed mutations of MUNC13–4 gene (FHL3). When the clinical findings were compared, the FHL2 patients with PRF1nonsense mutation showed early onset, while the onset of those with both PRF1 missense mutation and MUNC13–4 mutation was late. Alloantigen-specific CD8+ CTL lines were established from the FHL patients and their healthy parents. The degrees of cytotoxicity mediated by CTL lines generated from the patients with various FHL subtypes were compared. CTLs generated from the FHL2 patients with PRF1 nonsense mutation exerted no antigen-specific cytotoxicity. The cytotoxicity of CTLs from the patients with MUNC13–4 mutation was low but still remained, whereas cytotoxicity of CTLs from those with PRF1 missence mutation appeared to be moderate. By Western blot analysis, the missense mutant PRF1 gene also produced full length protein, but the active mature form was markedly reduced, indicating that the proteolytic cleavage of perforin is inhibited in these patients.Conclusion: The present study demonstrates that the subtypes of FHL can be classified by the patterns of genetic mutation and dysfunction of CTLs responsible for the pathogenesis of FHL. Although it has been established that allogeneic hematopoietic stem cell transplantation (HSCT) is a sole curative therapy for FHL, early identification of genetic defects and cytotoxic activity of CTLs may confirm whether HSCT should be really needed in each patient.

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.

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.