Perforin gene mutations in adult-onset hemophagocytic lymphohistiocytosis

Perforin gene (PRF1) mutations cause the primary form of hemophagocytic lymphohistiocytosis (HLH). We report a genetic defect of PRF1 in a 62-year-old Japanese man with recurrent episodes of HLH. Sequencing of PRF1 from both peripheral blood mononuclear cells and nail clippings showed compound heterozygous mutation, including deletion of two base pairs at codons 1090 and 1091 (1090-1091delCT) and guanine-to-adenine conversion at nucleotide position 916 (916GAEA). Although primary HLH has been detected in infants and children, genetic mutation of PRF1 or other genes should be considered a differential diagnosis of HLH even in the elderly.     

Perforin expression in cytotoxic lymphocytes from patients with hemophagocytic lymphohistiocytosis and their family members.

Mutations in the perforin gene have been described in some patients with hemophagocytic lymphohistiocytosis (HLH), but the role of perforin defects in the pathogenesis of HLH remains unclear. Four-color flow cytometric analysis was used to establish normal patterns of perforin expression for control subjects of all ages, and patterns of perforin staining in cytotoxic lymphocytes (natural killer [NK] cells, CD8(+) T cells, CD56(+) T cells) from patients with HLH and their family members were studied. Eleven unrelated HLH patients and 19 family members were analyzed prospectively. Four of the 7 patients with primary HLH showed lack of intracellular perforin in all cytotoxic cell types. All 4 patients showed mutations in the perforin gene. Their parents, obligate carriers of perforin mutations, had abnormal perforin-staining patterns. Analysis of cytotoxic cells from the other 3 patients with primary HLH and remaining family members had normal percentages of perforin-positive cytotoxic cells. On the other hand, the 4 patients with Epstein-Barr virus-associated HLH typically had depressed numbers of NK cells but markedly increased proportions of CD8(+) T cells with perforin expression. Four-color flow cytometry provides diagnostic information that, in conjunction with evidence of reduced NK function, may speed the identification of life-threatening HLH in some families and direct further genetic studies of the syndrome.     

Hypercytokinemia in familial hemophagocytic lymphohistiocytosis

Familial hemophagocytic lymphohistiocytosis (FHL) is a frequently missed and almost uniformly fatal childhood disorder. It is characterized by fever, hepatosplenomegaly, cytopenia, coagulopathy, and hypertriglyceridemia. The pathogenesis of FHL is not known but the above clinical and laboratory findings are compatible with reported in vitro and in vivo effects of several inflammatory cytokines. We measured circulating interferon-gamma (IFN-gamma), tumor necrosis factor/cachectin (TNF), and interleukin-6 (IL-6) in nine children with FHL. During active disease, elevated IFN-gamma was detected in seven of seven children, TNF in six of six, and IL-6 in two of six children studied. Thus, important inflammatory cytokines are augmented in active FHL and may contribute to the pathogenesis of the disease. Soluble CD8 was also increased in seven of seven children, which suggests a pathophysiologic importance of cytotoxic T lymphocytes. Because FHL appears to be associated with a systemic hypercytokinemia, our results also indicate that studies of FHL may contribute to the understanding of cytokine effects in vivo. Moreover, FHL is a hereditary disorder, suggesting that the hypercytokinemia is caused by a genetic defect in cytokine regulation.     

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.     

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.     

An inframe perforin gene deletion in familial hemophagocytic lymphohistiocytosis is associated with perforin expression

Familial hemophagocytic lymphohistiocytosis is an autosomal recessive disease of early childhood manifested by hypercytokinemia and organ infiltration of macrophages and activated lymphocytes, and it is characterized by a fulminant clinical course. The molecular mechanism underlying this disease appears to be a deregulation of apoptosis of activated T cells and macrophages. Approximately 20-40% of patients with familial hemophagocytic lymphohistiocytosis reported worldwide had a perforin gene mutation. We report herein a novel perforin variant in the homozygous state in an Omani boy who was diagnosed 44 days after birth. Sequence analysis of the perforin gene coding region revealed a 12-base pair deletion (codon 284-287) resulting in the deletion of four amino acids in the membrane attack complex domain of the protein. This deletion maintains the reading frame of the perforin mRNA. Both parents were heterozygotes for this molecular defect. Flow-cytometric analysis revealed intracellular perforin expression at the lower end of the normal range in the cytotoxic T cells (CD3+/CD8+) and (CD3+/CD56+) and in around 50% of the natural killer cells (CD3-/CD56+). This is an additional example of a perforin variant which is associated with a significant level of cellular perforin expression and thus confirms that drastic reduction in its expression is not a constant feature in familial hemophagocytic lymphohistiocytosis type 2.     

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.     

A novel mutation of the transcobalamin II gene in an infant presenting with hemophagocytic lymphohistiocytosis

Transcobalamin II (TC II) deficiency is a rare disorder of cobalamin (CBL, vitamin B12) metabolism that occurs due to mutations in transcobalamin gene (TCN2). Hemophagocytic lymphohistiocytosis (HLH) in contrast is a syndrome characterized by uncontrolled immune response with hyperinflammation. A 2-month-old male baby was admitted with complaints of fever, cough, diarrhea, and respiratory distress. The parents were first cousins. The baby exhibited five of the eight diagnostic criteria for HLH-2004 and was diagnosed as HLH. A second bone marrow aspiration demonstrated megaloblastic changes in the erythroid series. The patient’s vitamin B12 level was normal; however, hyperhomocysteinemia was present. A genetic deficiency of TC II was suspected. The patient and his parents were tested for TCN2 mutation. He had a homozygote mutation that was not included in Human ‘Gene Mutation Database Cardiff’. The patient was treated with intramuscular vitamin B12, which was followed by improvement in both clinical and laboratory findings. He was 12 months old at the time of this report, with normal physical and neuromotor development. In this case presenting with the clinical and laboratory findings of HLH, TC II deficiency was diagnosed. A new mutation was found that was not reported before. Potential causative mechanisms of HLH induced by defects of cobalamin synthesis merit further investigation.     

Unusual immunophenotype of CD8+ T cells in familial hemophagocytic lymphohistiocytosis

Familial hemophagocytic lymphohistiocytosis (FHL) is an inherited, fatal disorder of infancy. We report here a 17-day-old female infant who presented with high fever, hepatosplenomegaly, hypertriglyceridemia, hypofibrinogenemia, thrombocytopenia, and liver failure. Leukocytosis was detected with circulating "atypical" lymphoid cells. Flow cytometric studies revealed expanded subpopulations of CD8+ T cells with unusual immunophenotypic features, including a subset that lacked CD5 expression. A liver biopsy showed hemophagocytic lymphohistiocytosis with exuberant infiltrates of CD8+ T cells that lacked perforin. Mutational studies revealed a 666C-->A (H222Q) missense mutation in the perforin gene. T-cell receptor studies on flow-sorted T-cell subpopulations revealed no evidence of monoclonality. Analysis of T-cell receptor excision circle levels indicated long proliferative history in the aberrant CD8+ T-cell subsets. This case provides an instructive example of uncontrolled reactive proliferation of CD8+ T cells in FHL, resulting in atypical morphology and unusual immunophenotypic features that might suggest malignancy in other clinical settings.     

Reduced-intensity conditioning in unrelated donor cord blood transplantation for familial hemophagocytic lymphohistiocytosis

Familial hemophagocytic lymphohistiocytosis (FHL) is a disorder of immune homeostasis characterized by fever, cytopenias, hepatosplenomegaly, and coagulopathy. We studied the outcomes of 13 FHL patients who underwent the first unrelated cord blood transplantation (UCBT) after non-myeloablative conditionings. The major regimen consisted of fludarabine (FLU; n = 12)+melphalan (MEL; n = 11)± low-dose total body irradiation (TBI 2-4 Gy; n = 6). The median age at presentation and period to UCBT were 6 and 5 months, respectively. Central nervous system (CNS) disease developed in one infant at diagnosis, and in two others until UCBT. HLH activity was controlled in all but one at the time of UCBT. Ten patients had early engraftment on median day 21 with no grade >2 treatment-related toxicity and two controllable grade >2 acute GVHD. Two patients with early rejection successfully underwent subsequent UCBT after myeloablative conditioning. Two others had late graft failure following mixed donor chimerism. Two deaths occurred from HLH; early liver failure and late CNS disease. Of 11 FLU+MEL-conditioned patients, the frequency of disease-free complete engraftment was higher for MEL (≥120 mg/m(2) )+TBI, or high-dose MEL (180 mg/m(2) ) than for others (83% vs. 25%, p = 0.036). The FLU+MEL-based non-myeloablative regimen was acceptable for FHL infants undergoing UCBT, although further studies will be needed for confirmation.     

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.     

Treatment of familial hemophagocytic lymphohistiocytosis with antithymocyte globulins, steroids, and cyclosporin A

Familial hemophagocytic lymphohistiocytosis (FHL) is a potentially fatal disease characterized by diffuse infiltration by histiocytes and T lymphocytes. Treatment with myelotoxic drugs, such as etoposide, brings about remission in most patients, but problems of toxicity remain, and the development of disease resistance can cause secondary relapses. We have used an alternative approach, based on the suggested primary role of T-cell activation in FHL, comprising combined treatment with steroids (2 to 5 mg/kg/d methylprednisolone intravenously, followed by progressive tapering) and rabbit antithymocyte globulins (10 mg/kg/d for 5 days), followed by maintenance therapy with cyclosporine A (CSA). In a pilot study of six patients (four with a family history of FHL), all showed systemic remission within 7 days, which was complete in five cases; despite treatment with intrathecal methotrexate, one patient died of severe brain involvement. Two patients received T-cell--depleted HLA--non-identical bone marrow transplants, which was successful in one case. The other three patients, who have been on CSA maintenance therapy for periods of 6 to 24 months, are in complete remission. We have observed no side-effects (there has been no persisting T-cell immunodeficiency). These results suggest that nonmyelotoxic treatments for FHL may be safe, effective, and worthy of further investigation; they also support the key role of T lymphocytes in the disease.     

Germline heterozygous variants in genes associated with familial hemophagocytic lymphohistiocytosis as a cause of increased bleeding

Familial hemophagocytic lymphohistiocytosis (FHL) is caused by biallelic variants in genes regulating granule secretion in cytotoxic lymphocytes. In FHL3–5, the affected genes UNC13D, STX11 and STXBP2 have further been shown to regulate the secretion of platelet granules, giving rise to compromised platelet function. Therefore, we aimed to investigate platelet degranulation in patients heterozygous for variants in UNC13D, STX11 and STXBP2. During the work-up of patients referred to the Coagulation Unit, Skåne University Hospital, Malmö, Sweden and the Department of Hematology, Rigshospitalet, Copenhagen, Denmark due to bleeding tendencies, 12 patients harboring heterozygous variants in UNC13D, STX11 or STXBP2 were identified using targeted whole exome sequencing. Transmission electron microscopy (TEM) was used to assess the secretion of platelet dense granules following thrombin stimulation. Platelet degranulation, activation and aggregation were further assessed by flow cytometry (FC) and light transmission aggregometry (LTA) with lumi-aggregometry. In total, eight out of twelve (67%) patients showed impaired degranulation by at least one of the assays (TEM, FC and LTA). In the 12 patients, eight different heterozygous variants were identified. One variant was strongly associated with impaired degranulation, while four of the variants were associated with impaired granule secretion to a slightly lesser extent. One additional variant was found in six out of the twelve patients, and was associated with varying degrees of degranulation impairment. Accordingly, six out of the eight (75%) identified variants were associated with impaired platelet degranulation. Our results suggest that heterozygous variants in UNC13D, STX11 and STXBP2 are sufficient to cause platelet secretion defects resulting in increased bleeding.