DiagnosisClinical DiagnosisThe diagnosis of familial hemophagocytic lymphohistiocytosis (FHL), a cellular immunologic disorder resulting from genetic defects in cytotoxic cell function that lead to hyperactivation, proliferation, and infiltration of macrophages and T-lymphocytes, can be established if 1 and/or 2 is present:1. Biallelic disease-causing mutations in any one of PRF1, UNC13D (also known as MUNC13-4), STX11, or STXBP2 2. At least five of the eight following criteria based on the guidelines of the Histiocyte Society [Henter et al 2007]:Prolonged fever (>7 days)Cytopenias affecting two or three of the three lineages in the peripheral blood:Hemoglobin <90 g/L (for infants age <4 weeks: Hgb <100 g/L)Platelets <100x109/LNeutrophils <1.0x109/LSplenomegalyHypertriglyceridemia and/or hypofibrinogenemiaFasting triglycerides ≥2.0 mmol/L or >3 SD of the normal value for ageFibrinogen ≤1.5 g/LHemophagocytosis. Non-malignant, mixed lymphohistiocytic accumulation in the reticuloendothelial system; the spleen, liver, lymph nodes, bone marrow, and CNS are most frequently involved.
Note: (1) Hemophagocytosis may not be apparent early in the course of the disease. (2) Hemophagocytosis is seen less often in the liver, where lymphocytic infiltration of the portal areas is typical [Kapelari et al 2005].Low or absent natural killer (NK) cell activity. This is common prior to and during active disease, as well as after remission following chemotherapy in a significant proportion of individuals with FHL. Normal or raised NK cell activity has also been observed in some affected individuals, including those with UNC13D mutations [Yamamoto et al 2004, Ishii et al 2005].
Note: (1) NK cell activity normative values are per local laboratory reference. (2) The number of circulating NK cells (CD56+/16+) is generally within normal limits. (3) In secondary (acquired or reactive) hemophagocytic lymphohistiocytosis (HLH), NK cell activity has been reported to normalize during remission [Horne et al 2005b, Ishii et al 2005].Hyperferritinemia. Serum ferritin concentration ≥500 µg/L (normal 10-290 µg/L)High plasma concentrations of soluble CD25 (soluble IL2Rα); ≥2400 U/mL
Note: (1) Normal range depends on test methodology used. (2) Results must be compared to age-matched controls.The presence of inflammatory cells in the spinal fluid in association with increased CSF protein and/or characteristic findings on brain MRI (including gross demyelination or failure of normal myelination during the first year of life, multifocal inflammation involving gray and white matter and intracranial bleeding, generalized atrophy, or brain edema are also highly suspicious for HLH [Filipovich 2011] but are not part of the formal diagnostic criteria.In addition, a positive family history of affected sibs and/or parental consanguinity in a symptomatic individual supports the diagnosis of FHL.TestingProtein analysisPerforin (the protein product of PRF1) analysis by flow cytometry:In general, absent or markedly decreased perforin protein expression is highly likely to be associated with homozygous or compound heterozygous mutations in PRF1.Of note, normal perforin protein expression may be associated with PRF1 mutations because some missense mutations alter perforin protein function without significantly changing protein expression.If perforin protein expression is normal or increased, UNC13D or STXBP2 molecular genetic testing should be considered first. Natural killer (NK) cell activity analysis:Low or absent NK cell activity is an important biomarker in persons with FHL.Low or absent NK cell activity is usually observed in persons with confirmed biallelic mutations in PRF1, UNC13D, STXBP2 or STX11 [Sieni et al 2012a].In secondary (acquired or reactive) hemophagocytic lymphohistiocytosis (HLH), NK cell activity may fluctuate over time [Horne et al 2005b, Ishii et al 2005].NK cell degranulation (CD107a) analysis:NK cell degranulation can be quantified by measurement of upregulation of surface CD107a [Bryceson et al 2007].Defective CD107a surface expression in NK cells is a frequent finding in persons with UNC13D mutations (FHL3) and STX11 mutations (FHL4), in contrast to healthy controls or persons with PRF1 mutations (FHL2) [Marcenaro et al 2006]. Individuals with mutations in STXBP2 (FHL5) also have defective CD107a expression [Zur Stadt et al 2009].Granzyme B initiates caspase-dependent and caspase-independent apoptotic killing of target cells.In most types of HLH, including PRF1 and UNC13D mutations, as well as secondary HLH, granzyme B ranges from increased to markedly increased.Increased amounts of granzyme B in persons with HLH do not necessarily indicate normal killing, but most likely indicate that the granules housing the granzymes are not able to mobilize normally.Soluble IL2R is an indicator of prolonged activation of T cells.As the IL-2 receptor (CD25) forms on the surface of T cells during activation, increasing density of the receptor causes shedding into the plasma.This soluble form of the receptor is useful as a diagnostic criterion of any of the forms of HLH and for monitoring reoccurrence of the disease.Elevated soluble IL-2 receptor levels do not differentiate between primary (genetic) and secondary HLH.It is expected for children to have different expression of CD25 and thus the soluble form of the IL2R in plasma at different ages. Therefore, when interpreting results in children, age-based reference ranges should be used.Serum ferritin concentration is a marker for generalized inflammation.It is, however, markedly elevated in the majority of persons with HLH and is a very sensitive indicator of HLH when serum concentrations are markedly increased.Increased serum ferritin concentrations make no distinction between genetic and secondary HLH.Molecular Genetic TestingGenes/loci. Five loci (FHL1, FHL2, FHL3, FHL4, and FHL5) are associated with familial hemophagocytic lymphohistiocytosis; the five disease subtypes are based on these loci (Table 1). Four genes have been identified and characterized: PRF1 (FHL2), UNC13D (FHL3), STX11 (FHL4), and STXBP2 (FHL5).Table 1. Locus Names, Genes, and Mutations Associated with Familial Hemophagocytic Lymphohistiocytosis View in own windowLocus NameGene Symbol% of FHLMutations IdentifiedFHL1N/AFour inbred Pakistani families ¹ N/AFHL2PRF1 20%-30% ^2, 3 worldwide
>50% in African American families ⁴ Multiple distinctive mutations throughout the entire coding region FHL3UNC13D(MUNC13-4)~20%-30% ^3, 5 worldwideMultiple distinctive mutations throughout the entire coding region and splicing sites
Deep intronic mutation and large inversion also reported ^{6FHL4STX11~20% of Turkish/Kurdish families 5, 7
Biallelic mutations identified in other ethnic groups, albeit at a low frequency 8Three recurrent mutations identified in Turkish/Kurdish families
Additional mutations identified in other populationsFHL5STXBP216% in Central Europeans, Turks, and Saudis 9~20% in North American patients with FHL 10Multiple distinctive mutations throughout the entire coding region and splicing sites 1. Linkage analysis using homozygosity mapping in four inbred families of Pakistani descent with hemophagocytic lymphohistiocytosis (HLH) identified a locus (FHL1) on chromosome 9q21.3 22 [Ohadi et al 1999]. No gene in which mutation is causative has been identified at this locus.2. Voskoboinik et al [2004] 3. Ishii et al [2005] 4. Molleran Lee et al [2004], Lee et al [2006] 5. Zur Stadt et al [2005] 6. Meeths et al [2011]7. Zur Stadt et al [2006]8. Marsh et al [2010b]; Weitzman, personal communication (2010)9. Zur Stadt et al [2009]10. Johnson et al [2010]Evidence for locus heterogeneity. Approximately 30% of individuals diagnosed with FHL do not have identified mutations in any of the four genes listed (see Table 1). Ménasché et al [2005] provide evidence that additional genetic loci may be responsible for FHL.Clinical testingTable 2. Summary of Molecular Genetic Testing Used in Familial Hemophagocytic LymphohistiocytosisView in own windowLocus Name / Gene Symbol% of FHL Attributed to Mutations in This GeneTest MethodMutations DetectedTest AvailabilityFHL2 / PRF120%-30% 1>50% 2Sequence analysis / mutation scanning 3Sequence variants 4, 5Clinical FHL3 / UNC13D20%-30% 1Sequence analysis / mutation scanningSequence variants 4, 5, 6Clinical UnknownTargeted mutation analysis 7253-kb inversion 7FHL4 / STX11~20% in Turks/Kurds 81% in North Americans 9 and 5% in Central Europeans 8Sequence analysis / mutation scanning 3Sequence variants 4Clinical UnknownDeletion / duplication analysis 10Exonic and whole-gene deletion 11FHL5 / STXBP2~20% in North Americans 12Sequence analysisSequence variants 4, 5Clinical 1. Mutations in both PRF1 and UNC13D have been identified with almost equal frequency (~20%-30%) in individuals of western European and Japanese descent [Göransdotter Ericson et al 2001, Molleran Lee et al 2004, Ishii et al 2005, Lee et al 2006].2. For individuals of African American descent [Molleran Lee et al 2004]3. Sequence analysis and mutation scanning of the entire gene can have similar mutation detection frequencies; however, mutation detection rates for mutation scanning may vary considerably between laboratories depending on the specific protocol used.4. Examples of mutations detected by sequence analysis may include small intragenic deletions/insertions and missense, nonsense, and splice site mutations; typically, exonic or whole-gene deletions/duplications are not detected.5. Single mutations have been identified in PRF1 [Johnson, unpublished data (2012)], UNC13D [Zur Stadt et al 2005; Qian, personal communication (2012)], and STXBP2 [Johnson, unpublished (2012)] in a minority of individuals with HLH. These data suggest that a second mutation in (a) the other allele not detectable by direct sequencing, or in (b) a different HLH-related gene may be responsible for a minority of cases. Based on family studies, there is no evidence of a dominant model of disease in FHL2, FHL3, FHL4, or FHL5. 6. A deep intronic mutation has been reported; this must be included in design of sequence analysis approach (see Molecular Genetics) [Meeths et al [2011].7. Targeted PCR specific for the breakpoints of the 253-kb inversion (see Molecular Genetics) [Meeths et al [2011]8. Rudd et al [2006], Zur Stadt et al [2006] 9. Marsh et al [2010a]10. Testing that identifies deletions/duplications not readily detectable by sequence analysis of the coding and flanking intronic regions of genomic DNA; included in the variety of methods that may be used are: quantitative PCR, long-range PCR, multiplex ligation-dependent probe amplification (MLPA), and chromosomal microarray (CMA) that includes this gene/chromosome segment.11. A whole-gene deletion in STX11 has been reported [Zur Stadt et al 2005]. 12. Johnson et al [2010]Interpretation of test resultsFor issues to consider in interpretation of sequence analysis results, click here.Some individuals with FHL have only one identified mutation; it is possible that the second allele is mutated in the promoter region, deeper in the introns in other non-coding regions of the gene, or is a large complex rearrangement and is thus undetectable by methods in current use. Alternately, a second mutation could be present in a different HLH-related gene for which clinical testing is not currently possible.Approximately 30% of North American individuals with FHL do not have mutations in PRF1, UNC13D, STX11 or STXBP2 [Johnson, unpublished data (2012)]; therefore, normal test results do not exclude the diagnosis of FHL.Testing StrategyTo confirm/establish the diagnosis in a proband. The recommended genetic workup of a person with HLH is complex and depends on the individual’s race/ethnicity, immunologic test results, and clinical presentation, among other factors (see Genotype/Phenotype Correlations). A testing algorithm (pdf) is available to assist clinicians in the prioritization of testing [Jordan et al 2011]. No unique clinical findings distinguish between FHL2, FHL3, FHL4, and FHL5. However, immunologic testing may be useful in directing the genetic workup of a person with FLH (see Genotype/Phenotype Correlations and testing algorithm).PRF1 sequence analysis/mutation scanning is generally performed first, as more than 80% of African Americans and 20% of North Americans of northern European background with FHL have at least one PRF1 mutation [Johnson, unpublished data (2012)]. Sequence analysis typically does not detect gross deletions (exonic, multiexonic, or whole-gene deletions), insertions, or some complex gene rearrangements. However, no gross deletions, insertions, or complex gene rearrangements have been identified in PRF1 to date.In persons of northern European background, UNC13D molecular genetic testing is generally performed next. UNC13D mutations are rare in African Americans. Sequencing of the entire coding region and exon/intron boundaries of UND13D including targeted analyses of the deep intronic splicing mutation and the large inversion should be performed. Approximately 1%-2% of North Americans of northern European background have the deep intronic splicing mutation or the large inversion [Qian, personal communication (2012)].STXBP2 mutations account for about 20% of mutations in North American persons with FHL and have been identified in all ethnic/racial groups tested [Johnson, unpublished data (2012)]. Therefore, sequence analysis for STXBP2 is appropriate in any person with suspected FHL for whom molecular genetic testing of PRF1 and UNC13D is normal. Of note, no gross deletions, insertions, or complex rearrangements have been identified in STXBP2 to date.Mutations in STX11 account for about 1% of the disease-causing mutations in North American individuals and about 5% of central European individuals with HLH. Sequence analysis or mutation scanning of STX11 should follow PRF1, UNC13D and STXBP2 mutation analyses that do not identify at least one disease-causing mutation. If sequence analysis is normal, deletion/duplication analysis of STX11 can be considered, as gross deletions have been reported in STX11. Mutations in STX11 have been identified in most racial/ethnic groups [Marsh et al 2010b]. In symptomatic individuals not found to have mutations in PRF1, UNC13D, STX11, or STXBP2, sequencing of RAB27A for Griscelli syndrome type 2 (see Differential Diagnosis) should be considered. Note: Approximately 2%-3% of North American individuals with FHL have mutations in RAB27A. These individuals may not show evidence of partial albinism or other pigmentary abnormalities typically reported with Griscelli syndrome type 2 [Johnson, unpublished data (2012)].In males with symptoms of HLH not found to have mutations in PRF1, UNC13D, STX11, STXBP2, or RAB27A, molecular genetic testing of XIAP (also known as BIRC4) and SH2D1A should be considered (see Differential Diagnosis). Carrier testing for at-risk relatives requires prior identification of two disease-causing mutations in the family.Note: Carriers are heterozygotes for this autosomal recessive disorder and are not at risk of developing the disorder. Predictive testing for at-risk asymptomatic sibs of a proband requires prior identification of two disease-causing mutations in the family.Prenatal diagnosis and preimplantation genetic diagnosis (PGD) for at-risk pregnancies require prior identification of two disease-causing mutations in the family.Genetically Related (Allelic) DisordersPRF1. There are reports of other phenotypes associated with mutations in PRF1; the pathologic consequences are not well defined.Clementi et al [2005] reported PRF1 sequence variants in a proportion of persons with lymphoma:Two of four individuals with lymphoma had two PRF1 sequence variants, one of which is c.272C>T (p.Ala91Val), found in about 3% of controls [Molleran Lee et al 2004] and for which understanding of the pathogenic effect is still controversial (see Molecular Genetics).Solomou et al [2007] reported an association between PRF1 mutations and acquired aplastic anemia. Three of five reported individuals had the c.272C>T variant described above; a fourth had the c.11G>A (p.Arg4His) variant, a common benign variant in African Americans [Molleran Lee et al 2004]; the fifth had a novel sequence variant. All affected individuals were reported to have absent or markedly reduced perforin expression and absent perforin staining on microscopy; four of the five had hemophagocytosis in bone marrow. Chia et al [2009] examined temperature-sensitive PRF1 missense mutations in a subset of individuals with biallelic PRF1 mutations who had not developed hemophagocytic lymphohistiocytosis (HLH) by age ten years. 50% developed at least one hematologic malignancy by adulthood.Orilieri et al [2008] reported an association between the p.Asn252Ser (NM_001083116.1:c.755A>G) variant in PRF1 and type 1 diabetes mellitus.Cannella et al [2007] described 44 persons with childhood anaplastic large-cell lymphoma, 27.3% of whom had sequence variants in PRF1 compared to 10.2% in the general population (p<.01).UNC13D. Zhang et al [2008] reported an association between UNC13D mutations and a specific haplotype with sJIA with macrophage activation syndrome (MAS). However, Donn et al [2008] failed to confirm an association between systemic onset sJIA/MAS and sequence variants or specific haplotypes in PRF1, GZMB, UNC13D, or RAB27A.STX11. No other phenotypes are known to be associated with mutations in STX11.STXBP2. A secondary phenotype consisting of colitis, bleeding disorders, and hypogammaglobulinemia has been described in approximately one third of individuals with mutations in STXBP2 [Meeths et al 2010b]. This phenotype may precede the development of symptoms of HLH; however, data are insufficient to determine if all individuals with mutations in STXBP2 who have this secondary phenotype will develop symptoms of HLH.}

Natural HistorySymptoms of familial hemophagocytic lymphohistiocytosis (FHL) are usually evident within the first months or years of life [Aricò et al 1996] and may even develop in utero [Malloy et al 2004]. However, symptomatic presentation throughout childhood and even into adulthood has been observed in some cases [Aricò et al 1996, Allen et al 2001, Clementi et al 2002, Zhang et al 2011, Sieni et al 2012b].FHL usually presents in childhood as an acute illness with prolonged fever (>7 days), cytopenias, and hepatosplenomegaly. Rash and lymphadenopathy are less frequently observed. Liver dysfunction (elevated serum aminotransferase and bilirubin), neurologic dysfunction (irritability or lethargy, hypotonia or hypertonia, seizures, ataxia, and cranial nerve involvement presenting as nerve palsy and blindness), and bone marrow hemophagocytosis complete the typical presentation of the disorder.Oftentimes, FHL is associated with documented infection, especially herpes viral infections [Imashuku et al 2005] and leishmaniasis [Filipovich 2011].In adults, FHL may present with acute onset of full-blown HLH, or as a more insidious illness with recurrent bouts of nonspecific symptoms of HLH which may resolve spontaneously or with steroid treatment alone. HLH in adults is often erroneously attributed to an infectious etiology without a full genetic workup being undertaken.Common signs and symptomsFever is frequently indolent and protracted but may decline spontaneously. In a few children, it may develop late in the course of the disease.Splenomegaly and hepatomegaly are usually pronounced and progressive.Skin rash is uncharacteristic, transient, and often associated with high fever.Lymph node enlargement develops in fewer than 50% of individuals but may occasionally be significant.Neurologic abnormalities may be present at initial clinical presentation or in many instances may develop later in the course of the disease. The presentation may include irritability, bulging fontanel in infants, neck stiffness, hypotonia, hypertonia, and convulsions. Cranial nerve VI and/or VII palsy, ataxia, hemiplegia, quadriplegia, blindness, and coma may occur, as well as nonspecific signs of increased intracranial pressure.
On examination of CSF, elevated protein, increased number of mononuclear cells, and occasionally hemophagocytosis are characteristic findings.
MRI may show gross demyelination or failure of normal myelination during the first year of life. MRI may also show multifocal inflammation involving gray and white matter. Bleeding, generalized atrophy, and brain edema may be observed. Hyperdense areas on CT may be falsely interpreted as calcifications.Life expectancy. Median survival in typical FHL, without treatment, is less than two months. Progressive hemophagocytic lymphohistiocytosis (HLH) and invasive infection account for the majority of deaths in untreated children [Sung et al 2002]. Although children can survive without hematopoietic cell transplantation (HCT) [Henter et al 2002], the five-year survival of children treated with chemotherapy alone was only 10.1% in one large series. Allogeneic hematopoietic stem cell transplantation is potentially curative (see Management). The course of the disease and life expectancy are not well studied in adults with FHL.

Genotype-Phenotype CorrelationsBoth Feldmann and Molleran Lee documented poor correlation between phenotype and genotype in FHL2 with different classes of PRF1 mutations [Feldmann et al 2002, Molleran Lee et al 2004]. In general, age of onset tends to be younger in those with nonsense versus missense PRF1 mutations [Clementi et al 2002, Feldmann et al 2002, Zur Stadt et al 2006, Trizzino et al 2008]. In particular, homozygosity for the PRF1 p.Leu17Argfs*34 mutation, commonly seen in affected individuals of African descent, is associated with an earlier onset (median age 3 months; range 1.5-10 months) compared to other PRF1 missense mutations [Lee et al 2006]. However, affected individuals within the same family may develop evidence of disease at different ages [Allen et al 2001].In a study of 35 Japanese individuals with FHL, Ishii et al [2005] concluded that persons with PRF1 mutations had earlier disease onset than those with UNC13D mutations or FHL for whom no genetic basis was established. Deficient NK cell activity persisted after chemotherapy in all persons with FHL2, whereas some individuals with FHL3 or the non-FHL2/FHL3 subtype showed partial recovery of NK cell activity during remission.Alloantigen-specific CTL-mediated cytotoxicity was deficient in individuals with FHL2 with PRF1 nonsense mutations and very low in individuals with FHL3, but was only moderately reduced in individuals with FHL2 with PRF1 missense mutations [Ishii et al 2005].Ueda et al [2006] found that the incidence of deficient NK cell activity was higher in those with PRF1 mutations than in those with UNC13D or uncharacterized disease-causing mutations. In addition, nonsense mutations in PRF1 were associated with significantly higher levels of ferritin and soluble Il2Ra than observed in other subtypes of FHL.Horne et al [2008] concluded that persons with mutations in PRF1 had earlier onset of symptoms than those with STX11 mutations, and that those with STX11 mutations had a somewhat milder disease course than those with PRF1 mutations, UNC13D mutations, or uncharacterized mutations. Rudd et al [2006] reported long remissions without specific treatments in some individuals with STX11 mutations. They also reported a higher than expected incidence of psychomotor retardation and myelodysplastic syndrome or acute myelogenous leukemia in their small group with STX11 mutations. Marsh et al [2010b] noted that missense mutations in STX11 were associated with later onset and preserved NK cell function compared to nonsense mutation in STX11, which resulted in abrogation of NK cell function. In general the age of onset of disease tends to be later in individuals with STXBP2 mutations [Meeths et al 2010b] as compared to individuals with PRF1 or UNC13D mutations, but is variable, even within the same family. Later age at onset in persons with the common c.1247-1G>C mutation in STXBP2 (both in the homozygous and compound heterozygous states) in comparison to individuals with various missense mutations has been reported [Zur Stadt et al 2009, Pagel et al 2012]. In contrast, the authors have observed onset of HLH in infancy in over half of individuals with the c.1247-1G>C mutation, as well as considerable discordance in phenotype and age at onset between siblings with the same genotype [Johnson, unpublished data (2012)]. Gastrointestinal disorders, bleeding diathesis, and hypogammaglobulinemia, with onset often prior to symptoms of HLH, has been reported in a subset of individuals with STBXP2 mutations [Meeths et al 2010b].Adult onset of disease in individuals with FHL-causing mutation(s) has been well described [Allen et al 2001, Clementi et al 2002, Ueda et al 2006]. Missense and splice site variants in PRF1, UNC13D and STXBP2 were recently reported in 14% of adults with HLH [Zhang et al 2011]. Similarly, Sieni et al [2012b] identified 11 adults with FHL secondary to mutations in PRF1, UNC13D, STXBP2, and SH2D1A. In both studies, the p.Ala91Var variant in PRF1 was the most commonly identified mutation. While p.Ala91Var is not by itself pathogenic, it does have an effect on cytotoxic function [Voskobionik et al 2005] and may function as a genetic modifier of disease in some affected individuals.

Differential DiagnosisSecondary (acquired or reactive) hemophagocytic lymphohistiocytosis (HLH) is difficult to distinguish from familial (primary) HLH by clinical or histologic findings alone. Given the rapid advances in genetic diagnosis of FHL, molecular genetic testing is recommended even in HLH suspected to be acquired.The diagnosis of secondary HLH is usually made in association with infection by viruses, bacteria, fungi, or parasites or in association with lymphoma, autoimmune disease, or metabolic disease [Imashuku et al 2000, Imashuku et al 2005]. Acquired HLH may have decreased, normal, or increased NK cell activity.Secondary HLH appears to be self-limited because some affected individuals are able to fully recover having received supportive medical treatment (i.e., IV immunoglobulin) alone. However, long-term remission without the use of cytotoxic and immune-suppressive therapies is highly unlikely in adults with HLH and in individuals with CNS involvement [Imashuku et al 1999].Secondary HLH is usually associated with:Infection, particularly involving the herpes virus group, usually in older children and adolescents [Fisman 2000]. An example is EBV (Epstein-Barr virus)-associated HLH, which is more common in Asians than in whites or Africans [Ma et al 2001]. Also in one case, compound heterozygosity for two missense mutations in PRF1 was associated with chronic active EBV infection with hemophagocytic lymphohistiocytosis [Katano et al 2004].‘Macrophage activation syndrome’ (MAS), the most serious and life-threatening complication of systemic-onset juvenile idiopathic arthritis (sJIA). MAS was first described by Hadchouel et al [1985] as a hemorrhagic syndrome associated with mental status changes, hepatosplenomegaly, increased serum concentrations of liver enzymes, and a sharp fall in blood counts. The term MAS was coined by Stéphan et al [1993] in reference to the bone marrow histologic findings of numerous, well-differentiated macrophages (or histiocytes) actively phagocytosing hematopoietic elements. Such cells may infiltrate many organs in MAS. MAS responds to therapies similar to those typically effective in HLH. The amount of perforin expressed in NK cells from individuals with severe sJRA is less than in normal controls [Normand et al 2000], suggesting a mechanism of immune susceptibility to hemophagocytic lymphohistiocytosis (HLH) similar to FHL2. More recent studies also show that natural killer cell dysfunction is a distinguishing feature of sJIA and MAS [Villanueva et al 2005].Autoimmune diseases, such as rheumatologic disorders, treated with immune suppressive agents [Janka et al 1998]. Secondary HLH is most commonly associated with sJIA, but has also been observed in systemic lupus erythematosus [Muiesan et al 1998].Inborn errors of metabolism including biotinidase deficiency [Kardas et al 2012], lysinuric protein intolerance [Duval et al 1999], galactosemia [Marcoux et al 2005], multiple sulfatase deficiency, Gaucher disease, Pearson syndrome, galactosialidosis, methylmalonic acidemia, and propionic academia [Gokce et al 2012] have all been reported in association with secondary hemophagocytic lymphohistiocytosis in some individuals. It is not known how these metabolic disorders lead to HLH. Inherited immune disorders can be associated with highly lethal hemophagocytic syndromes, sometimes triggered by exposure to EBV or other viruses. These include the following:X-linked lymphoproliferative disease (XLP)XLP1, caused by SAP deficiency, results from hemizygous mutations in SH2D1A. The three main phenotypes of this type of XLP are an inappropriate immune response to Epstein-Barr virus (EBV) infection resulting in unusually severe and often fatal infectious mononucleosis, dysgammaglobulinemia, and/or lymphoproliferative disorders typically of B cell origin. Clinical manifestations of XLP1 vary, even among affected family members. The most common presentation is a near-fatal or fatal EBV infection associated with an unregulated and exaggerated immune response with widespread proliferation of cytotoxic T cells, EBV-infected B cells, and macrophages. Mortality is greater than 90%. In about one third of males with XLP1, hypogammaglobulinemia of one or more immunoglobulin subclasses is diagnosed prior to EBV infection or in rare survivors of EBV infection. The prognosis for males with this phenotype is more favorable if they are managed with regular IV IgG. Lymphomas or other lymphoproliferative disease occur in about one third of males with XLP1, some of whom have hypogammaglobulinemia or have survived an initial EBV infection. The lymphomas seen in XLP1 are typically high-grade B cell lymphomas, non-Hodgkin type, often extranodal, and particularly involving the intestine. Allogeneic HSCT is the only curative therapy for XLP1 and should be undertaken in confirmed cases of XLP1 as early in life as feasible. Average life expectancy without curative bone marrow transplantation has been estimated as less than ten years. Inheritance is X-linked recessive. XLP2, caused by XIAP deficiency and resulting from mutations in XIAP (aka BIRC4), is located (as the name implies) on the X chromosome [Rigaud et al 2006]. The most common manifestation of XIAP deficiency is the development of HLH, which is often recurrent. Because of this, it has recently been suggested that XIAP deficiency may be best classified as a cause of X-linked HLH [Marsh et al 2010a]. Hypogammaglobulinemia and colitis have also been described in individuals with XIAP deficiency. In contrast to XLP1, no cases of lymphoma have been observed in individuals with XIAP deficiency [Pachlopnik Schmid et al 2011]. Patient management includes treatment of HLH when it occurs, immunoglobulin replacement if needed, and consideration of allogeneic bone marrow transplant.Chediak-Higashi syndrome is a complex syndrome characterized by partial albinism, abnormal platelet function, and severe immunodeficiency. This disorder is caused by mutations in CHS1, which encodes a protein involved in intracellular vesicle formation, resulting in a failure to fuse lysosomes properly with phagosomes. Chediak-Higashi syndrome can be differentiated from FHL based on the presence of huge secretory lysosomes in the neutrophils and lymphocytes and giant melanosomes on skin biopsy. Inheritance is autosomal recessive.Griscelli syndrome type 2 (GS2) is a disorder of cytotoxic T lymphocytes caused by mutations in a small GTPase, RAB27A, which controls the movement of vesicles within cells [Ménasché et al 2000]. GS2 is usually associated with neurologic abnormalities in addition to partial albinism with fair skin and silvery-grey hair and has an extremely high incidence of HLH. A recent Swedish study identified RAB27A mutations in 5% of individuals with primary HLH and without a known diagnosis of GS2 [Meeths et al 2010a]. GS2 and FHL3 may share a common pathway: Neeft et al [2005] showed that the protein encoded by UNC13D is a direct partner of the protein RAB27A, and that the RAB27A/UNC13D complex is essential in the regulation of secretory granule fusion with the plasma membrane in hematopoietic cells. Inheritance is autosomal recessive.Note to clinicians: For a patient-specific ‘simultaneous consult’ related to this disorder, go to , an interactive diagnostic decision support software tool that provides differential diagnoses based on patient findings (registration or institutional access required).

ManagementEvaluations Following Initial DiagnosisTo establish the extent of disease in an individual diagnosed with hemophagocytic lymphohistiocytosis (HLH), as well as to determine the appropriate therapy, the following evaluations are recommended:Physical examination to evaluate for rashes, lymphadenopathy, hepatosplenomegaly, and neurologic dysfunctionEvaluation of blood and bone marrow compartments (CBC and BM biopsy)Determination of the extent of liver involvement by measuring serum concentration of transaminases, bilirubin, triglycerides, sodium, and lactate dehydrogenaseIdentification of potential infectious co-factors, especially viral infection or reactivation, which would require specific treatmentEstablishing the presence or extent of CNS involvement by evaluating the CSF and performing neuroimaging and neuropsychological assessmentTesting of NK cell activity, intracellular perforin and granzyme B expression, and CD107a mobilization if these tests are availableEvaluation of inflammatory factors such as serum concentrations of ferritin, sIL2Rα, and other cytokinesEvaluation and monitoring of PT, PTT, and fibrinogenMedical genetics consultationMolecular genetic testing, if not performed previously, which may help to determine if an affected individual is a candidate for bone marrow transplantation:Genetic testing for PRF1, UNC13D, STX11, and STXBP2 mutations Consideration of testing for RAB27A mutations in any individual with HLH, even with no obvious evidence of depigmentationConsideration of testing for XIAP (formerly BIRC4) and SH2D1A mutations in any male with HLHCollection of materials for future characterization of underlying genetic defects, most significantly in individuals with terminal diseaseTreatment of ManifestationsFor a detailed explanation of treatment for HLH, see Jordan et al [2011] (full text). HLH-1994. To improve survival, in 1994 the Histiocyte Society initiated a prospective international collaborative therapeutic study. It combined chemotherapy and immunotherapy (etoposide, corticosteroids, cyclosporine A, and intrathecal methotrexate for individuals with CNS diseases), followed by HSCT in persistent, recurring, and/or familial disease. Although HLH-94 was primarily designed for the treatment of FHL, it was also open to all individuals with non-familial HLH.HLH-2004. The HLH-2004 protocol was opened on January 1, 2004, and was designed for individuals with FHL and non-familial HLH. This protocol was based on the HLH-94 protocol with minor therapeutic modifications such as initiation of cyclosporine from onset of induction therapy.The two protocols developed by the Histiocyte Society are available from the Histiocyte Society Web site.Given the poor prognosis of individuals with HLH without prompt and effective treatment, it is recommended that treatment be initiated when clinical suspicion is high, even if all recommended studies are incomplete. In general, treatment involves the following:Chemotherapy and immunotherapy to achieve a clinically stable resolution prior to hematopoietic cell transplantation (HCT)Antibiotics or antiviral agents to treat or prevent infections that may have triggered the exaggerated inflammatory responseAllogeneic HCT, the only curative therapy, which should be undertaken in children with confirmed FHL as early in life as feasible: Presymptomatically if status is confirmed by family history of clinical HLH, or After achievement of clinical remission in simplex cases (i.e., a single occurrence in a family) Use of HCT has improved survival significantly [Henter et al 2002]. Initially, three-year-survival in children who received HLH-94 therapy was approximately 64% [Horne et al 2005a]. More recently, reduced intensity regimens prior to HCT have diminished the early transplant mortality and increased three-year-survival rates to 92% [Marsh et al 2010c]. Long-term follow up reveals that after HCT most children treated early in the disease course return to normal or near-normal quality of life. Brain atrophy secondary to steroid therapy often identified on neuroimaging studies during the acute phase of HLH gradually resolves after HCT [Shuper et al 1998]. However, children who experienced serious CNS involvement may have irreversible neurologic problems and learning disabilities despite adequate disease control following HCT.Prevention of Primary ManifestationsAllogeneic HCT is the only curative therapy and should be undertaken in children with confirmed familial HLH as early in life as possible.Prevention of Secondary ComplicationsPrompt treatment of HLH with infection prophylaxis is indicated in immunocompromised individuals.SurveillanceThe following are recommended annually after HCT:Neuropsychological evaluationRegular follow-up by transplant specialists to monitor for late complications relating to growth and hormone functionAgents/Circumstances to AvoidThe following should be avoided:Live vaccinationsExposure to infectionsEvaluation of Relatives at RiskMolecular genetic testing of at-risk sibs for the family-specific mutations is appropriate to identify those who are affected before symptoms occur for the purpose of early medical management and consideration of presymptomatic bone marrow transplantation.See Genetic Counseling for issues related to testing of at-risk relatives for genetic counseling purposes.Pregnancy Management To date, pregnancy in a woman with primary FHL has not been described. Secondary hemophagocytic lymphohistiocytosis is a rare complication of pregnancy and is beyond the scope of this review. Primary hemophagocytic lymphohistiocytosis in the fetus is a (reportedly) rare but recognized entity which is typically characterized by nonimmune fetal hydrops [Malloy et al 2004, Nitta et al 2007, Woods et al 2009], preterm delivery [Woods et al 2009], and/or fetal demise. In utero chemotherapy followed by post-natal HCT has been described [Shah et al 2009]. Therapies Under InvestigationSearch ClinicalTrials.gov for access to information on clinical studies for a wide range of diseases and conditions. Note: There may not be clinical trials for this disorder.OtherThe treatment of FHL is also the most effective treatment for secondary HLH and the hemophagocytic syndrome associated with other inherited immunodeficiencies.

Molecular GeneticsInformation in the Molecular Genetics and OMIM tables may differ from that elsewhere in the GeneReview: tables may contain more recent information. —ED.Table A. Hemophagocytic Lymphohistiocytosis, Familial: Genes and DatabasesView in own windowLocus NameGene SymbolChromosomal LocusProtein NameLocus SpecificHGMDFHL1UnknownUnknownUnknown FHL2PRF110q22​.1Perforin-1Resource of Asian Primary Immunodeficiency Diseases (RAPID)
CCHMC - Human Genetics Mutation DatabasePRF1FHL3UNC13D17q25​.1Protein unc-13 homolog DResource of Asian Primary Immunodeficiency Diseases (RAPID)
CCHMC - Human Genetics Mutation DatabaseUNC13DFHL4STX116q24​.2Syntaxin-11Resource of Asian Primary Immunodeficiency Diseases (RAPID)
CCHMC - Human Genetics Mutation DatabaseSTX11FHL5STXBP219p13​.2Syntaxin-binding protein 2CCHMC - Human Genetics Mutation DatabaseSTXBP2Data are compiled from the following standard references: gene symbol from HGNC; chromosomal locus, locus name, critical region, complementation group from OMIM; protein name from UniProt. For a description of databases (Locus Specific, HGMD) to which links are provided, click here.Table B. OMIM Entries for Hemophagocytic Lymphohistiocytosis, Familial (View All in OMIM) View in own window 170280PERFORIN 1; PRF1 267700HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS, FAMILIAL, 1; FHL1 601717SYNTAXIN-BINDING PROTEIN 2; STXBP2 603552HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS, FAMILIAL, 4; FHL4 603553HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS, FAMILIAL, 2; FHL2 605014SYNTAXIN 11; STX11 608897UNC13, C. ELEGANS, HOMOLOG OF, D; UNC13D 608898HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS, FAMILIAL, 3; FHL3 613101HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS, FAMILIAL, 5; FHL5PRF1Normal allelic variants. PRF1 has three exons; exons 2 and 3 encode a 555-amino acid polypeptide. Genomic DNA of PRF1 spans about 5 kb. To date, several normal allelic variants have been observed (see Table 3). These have been evaluated by biochemical and functional testing in vitro and are highly unlikely to have pathologic effects on perforin expression.Pathologic allelic variants. To date, more than 115 pathologic variants have been identified. Mutations have been found throughout the entire coding region. Mutations in PRF1 include nonsense and missense mutations, small deletions, and small insertions [Voskoboinik et al 2005]. To date, no gross deletions, splicing site mutations, or complex rearrangements have been reported.The variant c.272C>T has been reported as a disease-causing mutation [Clementi et al 2001]. This sequence variant is identified in about 3% of the North American general population [Molleran Lee et al 2004] and in a much higher proportion of patients with FHL [Busiello et al 2006, Zhang et al 2007]. Earlier studies have shown that the sequence change c.272C>T results in reduced levels of perforin expression [Voskoboinik et al 2005] and that p.Ala91Val perforin has reduced cytotoxicity in CTL and NK cells [Trambas et al 2005]. In addition, Voskoboinik showed that the c.272C>T (p.Ala91Val) substitution in perforin not only causes reduced steady state levels of expression in effector cells, but also results in a reduced intrinsic capacity for lysis and even had some dominant-negative effect on the wild type perforin [Voskoboinik et al 2007]. Even though c.272C>T by itself is not disease causing, these cumulative data suggest that the sequence variant may be an important genetic susceptibility factor and may play a role in the disease process. Table 3. Selected PRF1 Allelic VariantsView in own windowClass of Variant AlleleDNA Nucleotide ChangeProtein Amino Acid Change
(Alias ¹) ² Reference SequencesNormalc.11G>Ap.Arg4HisNM_001083116​.1
NP_001076585​.1c.368G>Ap.Arg123Hisc.726C>Tp.(=)
(Cys242Cys)c.822C>Tp.(=)
(Ala274Ala)c.900C>Tp.(=)
(His300His)Pathologicc.272C>T ^{3p.Ala91Val 3c.50delTp.Leu17Argfs*34See Quick Reference for an explanation of nomenclature. GeneReviews follows the standard naming conventions of the Human Genome Variation Society (www​.hgvs.org).1. Variant designation that does not conform to current nomenclature2. p.(=) designates that protein has not been analyzed, but no change is expected3. Not disease-causing, but proposed as a genetic susceptibility factor or disease modifier (see PRF1 Pathologic allelic variants)Normal gene product. PRF1 encodes the cytolytic effector perforin, which is expressed by cytolytic T lymphocytes and NK cells [Stepp et al 1999]. Perforin expression is higher in resting than in activated cells. Perforin induces apoptotic cell death in response to granzymes (serine esterases that represent most of the granule content of T cytotoxic cells) independent from the FAS-mediated apoptotic machinery.Abnormal gene product. PRF1 mutations affect cellular cytotoxicity, resulting in impaired antiviral defense and dysregulation of apoptotic mechanisms involved in the regulation of immune responses to inappropriate proliferation of cells, such as T cells and macrophages. Although the mechanisms of regulating perforin expression are incompletely understood, it is known that methylation of a promoter-specific region does affect the level of perforin expression.UNC13D (MUNC13-4)Normal allelic variants. UNC13D consists of 32 exons ranging from 36 to 235 bp in size. A dozen normal allelic variants, which are highly unlikely to have pathologic effects on UNC13D protein expression, have been observed.Pathologic allelic variants. To date, more than 100 pathologic variants have been identified throughout the entire coding region and the exon/intron boundaries of UNC13D. Mutations that have been identified include missense, nonsense, and splice site mutations and small deletions and/or insertions. One deep intronic mutation (c.118-308C>T) and one large gene inversion have been detected [Meeths et al 2011]. A large 253-kb inversion straddles the UNC13D 3’ end and adjacent sequences; the breakpoints have been mapped and it is detectable by targeted mutation analysis (Table 2). The deep intronic mutation lies in a region typically not sequenced; detection will require specific primers.Normal gene product. The protein encoded by UNC13D has 1090 amino acids and is a member of the Munc13 protein family, which is an essential effector of the cytolytic secretory pathway. UNC13D differs from the other Munc13 proteins, as it is not expressed in the brain but rather is highly expressed in hematopoietic tissues including T- and B-lymphocytes and monocytes and in non-hematopoietic tissues, such as the lung and the placenta. UNC13D is involved in vesicle-plasma membrane fusion during exocytosis of perforin- and granzyme-containing granules by cytotoxic T cells and NK cells [Feldmann et al 2002, Ménager et al 2007], which follows granule docking and precedes plasma granule membrane fusion [Ménasché et al 2005].UNC13D is a direct partner of RAB27A. The two proteins are highly expressed in cytotoxic T-cells (CTLs) and mast cells, where they colocalize on secretory lysosomes. Together, they assist priming the vesicles for exocytosis [Ménager & de Saint Basile 2007]. The region comprising the Munc13 homology domains is essential for the localization of UNC13D to secretory lysosomes [Neeft et al 2005].Abnormal gene product. Mutations in UNC13D prevent interaction with RAB27A and abolish cytolytic secretion [Neeft et al 2005]. Individuals with UNC13D mutations have impaired lymphocyte cytotoxic function with normal or increased perforin expression. Interestingly, the c.118-308C>T mutation impairs transcription in lymphocytes, but not some other cell types [Meeths et al 2011].Table 4. Selected UNC13D Pathologic Allelic Variants View in own windowDNA Nucleotide ChangeProtein Amino Acid ChangeReference Sequencesc.118-308C>TNANM_199242​.2
NP_954712​.1(253-kb inversion)NANA = not applicableSTX11Normal allelic variants. STX11 consists of two exons. Exon 2 encodes 287 amino acid residues. Genomic DNA of STX11 spans about 37 kb. To date, three normal allelic variants have been identified [Zhang, unpublished (2010)]; they are highly unlikely to have pathologic effects on STX11 protein expression. See Table 5.Pathologic allelic variants. A total of nine distinct mutations have been reported; they include three families in highly inbred Turkish/Kurdish FHL4 kindreds. STX11 mutations have been found in individuals with FHL from other ethnic backgrounds, albeit at a low frequency [Marsh et al 2010b]. See Table 5. Nonsense and missense STX11 mutations have been reported in most affected individuals to date. A whole-gene deletion in STX11 has been reported [Zur Stadt et al 2005].Table 5. Selected STX11 Allelic VariantsView in own windowClass of Variant
AlleleDNA Nucleotide ChangeProtein Amino Acid ChangeReference SequencesNormalc.146G>Ap.Arg49GlnNM_003764​.3
NP_003755​.2c.546G>Ap.Glu182Glnc.651G>Tp.Leu217LeuPathologicc.[369_370delAG; 374_379delCGC]
(5-bp deletion)p.Val124Glyfs*60c.802C>Tp.Gln268*g.25560_44750del
(19.2-kb deletion)--AL135917See Quick Reference for an explanation of nomenclature. GeneReviews follows the standard naming conventions of the Human Genome Variation Society (www​.hgvs.org).Normal gene product. The gene product of STX11 is known for its function in vesicle transport. It is a member of soluble N-ethylmaleimide sensitive factor attachment protein receptors present on target membranes (t-SNAREs) [Zur Stadt et al 2005]. STX11 is expressed in peripheral mononuclear cells, as well as in unstimulated NK cells and CD8+ T cells [Bryceson et al 2007].Abnormal gene product. The absence of the STX11 protein has been observed in monocytes and lymphocytes in persons with FHL4. The abrogation of degranulation and cytotoxicity by resting PBLs observed in this disorder are believed to be caused by the defects in STX11 [Arneson et al 2007].STXBP2Normal allelic variants. STXBP2 is located on chromosome 19p13.3-p13.2 and its genomic DNA spans about 11 kb. STXBP2 comprises 19 exons. To date, more than two dozen normal allelic variants have been identified [Zhang, unpublished (2012)]; they are highly unlikely to have pathologic effects on STXBP2 protein expression. For example, the normal allelic variant p.Ala433Val did not affect binding between syntaxin-11 and UNC13B [Zur Stadt et al 2009].Pathologic allelic variants. To date, 27 pathologic mutations have been identified throughout the entire coding region and the exon/intron boundaries of STXBP2. Identified mutations include missense, nonsense, and splice site mutations as well as small deletions and/or insertions. Gross deletions, insertions, and other complex mutations have not been observed to date [Zhang, unpublished (2012)].Table 6. Selected STXBP2 Allelic Variants View in own windowClass of Variant AlleleDNA Nucleotide ChangeProtein Amino Acid ChangeReference SequencesNormalc.1298C>Tp.Ala433ValNM_006949​.2
NP_008880​.2Pathologicc.1247-1G>CNASee Quick Reference for an explanation of nomenclature. GeneReviews follows the standard naming conventions of the Human Genome Variation Society (www​.hgvs.org). NA = not applicableNormal gene product. STXBP2 encodes syntaxin-11, a polypeptide of 593 amino acids and a member of the STXBP/Munc-18/Sec1 family. Syntaxin-11, formerly known as UNC18B, is expressed predominantly as a 2.4-kb message in placenta, lung, liver, kidney, and pancreas, as well as in peripheral blood lymphocytes [Ziegler et al 1996]. It interacts with soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxins, and regulates intracellular vesicle trafficking [Côte et al 2009]. Like other Sec/Munc18 family members, STXBP2 has three domains and forms an arch-shaped structure. The central cavity, formed by domains 1 and 3a, provide the binding surface for syntaxin-11. In addition, STXBP2 and STX11 share binding sites and are colocalized in CD8+ T and NK cells. Stimulation with IL2 can further enhance colocalization in CD8+ and NK cells [Zur Stadt et al 2009]. Abnormal gene product. Zur Stadt et al [2009] showed that missense mutations in STXBP2 can lead to a complete loss of the ability to bind to syntaxin-11. Cultured NK cells from patients with mutations in STXBP2 exhibit impaired cytotoxic granule exocytosis with decreased or absent CD107 expression. Ex vivo cytotoxicity in NK and cytotoxic T-cell were both decreased [Meeths et al 2010b]. However, degranulation and cytotoxicity could be at least partially corrected by in vitro stimulation with IL2 [Zur Stadt et al 2009]. Defects in STXBP2 can reduce or completely abolish syntaxin-11 expression. In contrast, STXBP2 expression does not require syntaxin-11 [Côte et al 2009].}