Analyses of the PRF1 Gene in Individuals with HemophagocyticLymphohystiocytosis Reveal the Common Haplotype R54C/A91Vin Colombian Unrelated Families Associated with Late Onset Disease

Isaura P. Sánchez & Lucía C. Leal-Esteban &

Jesús A. Álvarez-Álvarez & Camilo A. Pérez-Romero &

Julio C. Orrego & Malyive L. Serna & Yadira Coll &Yolanda Caicedo & Edwin Pardo-Díaz &

Jacques Zimmer & Jack J. Bleesing & José L. Franco &

Claudia M. Trujillo-Vargas

Received: 1 December 2011 /Accepted: 22 February 2012 /Published online: 22 March 2012# Springer Science+Business Media, LLC 2012

Abstract Familial hemophagocytic lymphohistiocytosis(FHL), is a rare autosomal recessive disorder characterizedby an impairment of cytotoxic cells and uncontrolled activa-tion of macrophages. This study presents the first description

of four patients with FHL type 2 in Latin America. Patient 1fulfilled the disease diagnostic criteria since 2 months of age,whereas patients 2, 3 and 4 exhibited the typical manifesta-tions of the disease only later in their childhood. The PRF1genetic analysis in these patients revealed two previouslyreported mutations: L17fsx50 and R54C. Interestingly, sevenout of the 8 alleles evaluated here in patients carried thehaplotype R54C/A91V, suggesting that this is a highly fre-quent FHL type 2 allele in Colombia. This haplotype confersresidual cytotoxic function leading to late onset disease.Therefore, this report highlights the remarkable complexityof FHL diagnostic, emphasizing the importance of the geneticcharacterization of the disease.

Keywords NK cells . perforin . familial hemophagocyticlymphohistiocytosis type 2 .PRF1 gene

Introduction

Hemophagocytic lymphohistiocytosis (HLH; OMIM267700)is a rare but fatal disorder characterized by fever, hepatosple-nomegaly, pancytopenia, hypertriglyceridemia, hypofibrino-genemia and in some cases, central nervous system alterations[1–4]. These manifestations are the result of excessive releaseof multiple cytokines, which induces the activation of histio-cytes and other phagocytic cells. These cells ingest erythro-cytes, leukocytes, platelets, and their precursors in bonemarrow and other tissues, a process called hemophagocytosis[5, 6]. Excessive inflammatory response is the main mecha-nism responsible for extensive tissue damage ultimately

I. P. Sánchez (*) : L. C. Leal-Esteban : J. A. Álvarez-Álvarez :C. A. Pérez-Romero : J. C. Orrego :Y. Coll : J. L. Franco :C. M. Trujillo-VargasGroup of Primary Immunodeficiencies, University of Antioquia,Calle 62 #52-59 Lab.530,Medellín, Colombiae-mail: isapisan@yahoo.com

M. L. SernaPediatric Service of Hematology-Oncology,Fundación Hospitalaria San Vicente de Paúl,Medellín, Colombia

Y. CollBone Marrow Transplantation Unit, Instituto de Cancerología,Clínica Las Américas,Medellín, Colombia

Y. Caicedo : E. Pardo-DíazDepartment of Pediatrics, Hospital Universitario del Valle,Cali, Colombia

J. ZimmerLaboratory of Immunogenetics and Allergology,Centre de Recherche Public de la Santé (CRP-Santé),Luxembourg City, Luxembourg

J. J. BleesingDepartment of Pediatrics, Division of Bone MarrowTransplantation and Immune Deficiency,Cincinnati Children’s Hospital Medical Center,Cincinnati, OH, USA

J Clin Immunol (2012) 32:670–680DOI 10.1007/s10875-012-9680-5

leading to multiorgan dysfunction. In all cases, early recogni-tion of the disease and rapid implementation of specific im-mune suppressive protocols may result in variable degrees ofsymptom relief and may save lifes. However, HLH is oftenunderdiagnosed and fatal, especially in the developingcountries due to poor disease awareness by physicians anddeficient health care system.

HLH may be either primary, which is caused by anunderlying genetic defect or secondary to infections, auto-immune diseases or malignancies [7]. Viral infections arethe most common causes of all secondary forms and mayalso trigger primary HLH. Genetic forms of HLH are esti-mated to occur in 1 in 50.000 live births, have highermortality rates and worse prognosis than secondary HLH,because 70–80% of the cases manifests during the first yearof life [8, 9]. Disease-causing mutations with an autosomalrecessive mode of inheritance (designated as familial hemo-phagocytic lymphohistiocytosis, FHL) have been described.Linkage analysis initially identified a candidate genomicregion of the disease on chromosome 9q21.3–22 in fourinbred Pakistani families, which has been referred to asFHL type 1. However, the gene responsible for FHL type1 has not yet been identified [10]. Mutations in genesencoding perforin on chromosome 10q21 (PRF1; FHL type2), Munc13-4 on chromosome 17q35 (UNC13D; FHL type3), syntaxin 11 on chromosome 6q24 (STX11; FHL type 4)and Munc18-2 on chromosome 19p13 (STXBP2; FHL type5) previously have been also reported. They account forapproximately 80% of FHL cases [11–14]. Other forms ofgenetic HLH are associated with immune deficiency syn-dromes including Chediak–Higashi syndrome, Griscellisyndrome type 2, X-linked lymphoproliferative syndrome(XLP), Wiskott–Aldrich syndrome, severe combined immu-nodeficiency, lysinuric protein intolerance and Hermansky–Pudlak syndrome [15, 16]. The genes associated with FHLencode proteins that are involved either in the cytotoxicgranules fusion of NK cells (NKs) and cytotoxic T Lym-phocytes (CTLs) after the formation of the immunologicalsynapses or in the direct killing of target cells [14, 17]. Oncecytotoxic cells have recognized their targets and formed asynapse with them, cytotoxicity is induced in a multistepprocess. Firstly, recruitment and rapid polarization of themicrotubule organizing center toward the synapse areinduced, which coordinates the movement of the gran-ules towards the plasma membrane [18]. Secondly, aprocess mediated by Rab GTPases directs the tetheringand docking of the membranes [12, 18]. However,membrane’s proximity and contact is not sufficient forfusion. Instead, Munc13-4-mediated mechanisms lead tovesicle priming, which facilitates the fusion processregulated by proteins such as STX11 and STXBP2resulting in the release of perforin and serine proteasesknown as granzymes that are stored in the granules.

These molecules in turn, induce target cell destructionby apoptosis [13, 14, 19].

Although it is well established that germline muta-tions in the perforin (PRF1) gene on chromosome10q21 are responsible for FHL in approximately 30 to40% of these patients [9–17], no reports have docu-mented this genetic disorder in Latin America. Ourstudy enrolled one patient with typical HLH and threewith late onset disease [2, 9]. These patients exhibited atotal absence of NKs intracellular perforin expressionand a variable impairment of cytotoxic responses, butincreased CD107a translocation in comparison with age-and/or sex-matched healthy controls. The genetic analy-sis indicated that patient 1 carried the mutation 50delT(L17fsx50) in one of her alleles. The other seven allelesevaluated here in patients carried the mutation R54Ctogether with the polymorphism A91V, suggesting thatthis is a highly frequent Colombian allele associatedwith FHL type 2. Our additional studies indicated thatA91V was only present in 3 out of 142 alleles inColombian healthy donors. Neither the L17fsx50 dele-tion nor the R54C missense mutation in the PRF1 genewas observed in the gDNA from the healthy donors.Therefore, we identify a common PRF1 haplotype inunrelated families with atypical presentation of FHLtype 2 in Colombia.

Materials and Methods

Blood Sampling and Laboratory Tests

Peripheral blood (PBs) samples were obtained frompatients, their parents and age- and sex-matched healthydonors for patients 1 and 2 and sex-matched healthydonors for patients 3 and 4. Blood tests included hemo-leucograms, serum immunoglobulins (IgG, IgM, IgA,IgE) for all the patients and specific antibody levelsagainst Respiratory Syncytial Virus (RSV), Influenza Avirus, Adenovirus, Human Immunodeficiency Virus, Her-pes Simplex Virus type I and type II, Parvovirus, DengueVirus, Hepatitis-A, -B and -C virus, Cytomegalovirus(CMV), Epstein-Barr Virus (EBV), Legionella s.p., My-coplasma s.p., Coxiella s.p., Chlamydia s.p., Toxoplasmagondii as well as VDRL in patient 1 and Leptospira s.pin patients 2 and 3. Aspartate aminotransferase (AST),alanine aminotransferase (ALT), gamma-glutamyl trans-ferase (GGT), ferritin, fibrinogen and triglycerides serumlevels were also evaluated in all the patients. This studywas approved by the Institutional Bioethical ReviewBoard of the University of Antioquia. Written informedconsent was obtained from the childrens’ parents andadults included in this study.

J Clin Immunol (2012) 32:670–680 671

Immunophenotyping and Intracellular Perforin Expressionby Flow Cytometry

The absolute number of eosinophils, neutrophils, lympho-cytes and monocytes was calculated from EDTA bloodsamples on the basis of total and differential blood cellcounts using Wright staining of blood smears and conven-tional light microscopy. Frequency and phenotype of eithernaïve (CD45RA+) or memory (CD45RO+) CD3+/CD4+and CD3+/CD8+ T cells, TCR αβ and γδ T cells, B cells(CD3-/CD19+/CD21+ lymphocytes), monocytes (CD3-/CD14+/HLA-DR+), NKs (CD3-/CD16+/CD56+), were de-termined by flow cytometry. All antibodies were purchasedfrom Becton Dickinson Biosciences (BD, San Jose, CA).Briefly, fresh peripheral blood (PB, 100 μl) was incubatedwith the corresponding monoclonal antibodies (mAbs) for20 min at room temperature (RT) in the dark. Erythrocyteswere lysed by incubation of the cell suspensions with 1XFACS lysing solution (BD) following the manufacturer’sinstructions. Finally, cells were fixed with 250 μl of 2%formaldehyde. Appropriate isotype-matched control anti-bodies were also included. Immunophenotyping of NK cellsubpopulations was defined on the basis of relative expres-sion of the markers CD16 and CD56 in peripheral bloodleucocytes (PBL). Cells were stained with fluorochrome-conjugated anti-CD3, -CD19, -CD14, -CD16, -CD56 mAbs.Lymphocytes were gated based on forward and side scatterplots. Monocytes (CD14+), T (CD3+) and B (CD19+) lym-phocytes were gated out. The remaining cells were againanalyzed for CD16 and CD56 expression: defined as theCD56dimCD16bright and the CD56brightCD16dim/- NKs.

For perforin detection, intracellular staining of NKs wasperformed using Cytofix/Cytoperm and the Perm/Wash sol-utions from BD following the manufacturer’s instructions.Briefly, after the staining of surface markers, fresh PB wasincubated with 250 μl of Cytofix/Cytoperm for 20 minutesat 4°C and then, washed twice in 1X Perm/Wash beforeantibody labeling with either Phycoerythrin (PE)- conjugat-ed anti-perforin or isotype-matched control mAb for 30minutes at 4°C. After three consecutive washes with 1XPerm/Wash, cells were resuspended in PBS (Sigma-Aldrich,St Louis, MO, USA) + 1% Fetal Bovine Serum (FBS,Lonza, Walkersville, MD, USA) prior to analysis. Flowcytometry was performed using a FACSCanto II (BD) andanalyzed using the FlowJo V8-2 software (Tree Star, Inc.Ashland, OR USA).

CD107a Translocation Assay

The CD107a translocation assay was performed as previ-ously described with minor modifications [20]. Briefly, pe-ripheral blood mononuclear cells (PBMC) were obtainedfrom heparinized blood samples by density gradient

centrifugation using Ficoll-Hypaque (Sigma-Aldrich). Via-bility of PBMC was determined by trypan blue exclusion.For cell culture, PBMC (1×106/ml) were suspended inRPMI 1640 supplemented with 2 mM of L-glutamine (Sig-ma-Aldrich), 10% of heat-inactivated fetal bovine serum,2% penicillin/streptomycin mixture (at 10.000 U/ml and10 mg/ml, respectively) (Lonza, Walkersville, MD, USA)and stimulated with/without recombinant human (rh) IL-2 orrhIL-15 (BD, San Jose, CA) for 24 hours at 37°C in 5%CO2. Subsequently, PBMC (2×105) were exposed to K562target cells (Effector:Target ratio of 1:1) in the presence ofPE-Cy5 conjugated anti-CD107a mAb (4 μl, BD). Cellswere then incubated at 37°C in 5% CO2 for 3 h addingGolgiStop™ solution (BD) after the first hour of culture. Todetect spontaneous degranulation, PBMC without targetcells were also included. Finally, cells were washed inPBS and stained with anti-CD56/CD16-FITC, anti-CD3/CD19-APC and anti-CD14-Pacific Blue specific mAbsand analyzed on a FACSCanto II cytometer. Surface expres-sion of CD107a was assessed in the CD3-/CD19-/CD14-/CD16+/CD56+ NKs subpopulation.

Genetic Analysis

Genomic DNA (gDNA) was isolated from blood samplesusing the DNA Isolation Kit provided by Puregene™(Gentra Systems, Minneapolis, MN) following the man-ufacturer’s instructions. The entire coding region andexon/intron boundaries of the PRF1 gene (10q21-q22)were analyzed by PCR and direct bi-directional sequenc-ing. Primer design and synthesis as well as PCR productamplification and sequencing from patients 1 and 2 wereperformed by the Molecular Genetics Laboratory of Cin-cinnati Children’s Hospital Medical Center (Cincinnati,OH). For patients 3 and 4, the entire coding region andexon/intron boundaries of the exon 2 of the PRF1 genewere amplified from the gDNA using the followingprimers: forward, 5′-CCCTTCCATGTGCCCTGATAATC-3′ and reverse, 5′-AAGCAGCCTCCAAGTTTGATTG-3 as previously reported [7]. DNA from 71healthy blood donors was used as control. Sequencingwas performed by Macrogen Inc (Seoul, Korea). Homol-ogy search against the human genome database at NCBIwas performed using nucleotide Blast (Blast-n version2.2.24) using the reference M31951 (for PRF1 gDNA)and M28393 (mRNA). Alignments were performed usingthe program ClustalW version 2.0 (http://www.ebi.ac.uk/clustalw/index.html). Targeted analysis of the specificmutations in the gDNA in the patients’ parents was alsoperformed. Mutation nomenclature is based on the rec-ommendation by the American College of Medical Ge-netics that nucleotide +1 is designated the A of the ATG-translation initiation codon.

672 J Clin Immunol (2012) 32:670–680

Results

Clinical Features of the Patients Included in This Study

Patient 1 was a 2 month-old female, first child from unre-lated healthy parents from Medellin (Antioquia, Colombia)with no family history of FHL. At the age of 9 weeks, thepatient was hospitalized with a several day history of par-oxysmal coughing, fever and occasional wheezing. No ane-mia or neutropenia but severe thrombocytopenia (29.000platelets/μl) was observed. She also exhibited RSV-specific immunoglobulin G (IgG) antibodies. Antibiotictreatment with ceftriaxone was then initiated for 6 daysand after that, she went into remission. Two weeks later,she was again admitted at the hospital with fever, bicytope-nia (57.000 platelets/μl and 500 neutrophils/μl), jaundiceand generalized mucocutaneous pallor and hepatosplenome-galy. Serum IgM, IgA, IgE levels were normal but increasedIgG levels were observed. Infectious disease screening onlyshowed the presence of Parvovirus B19-specific immuno-globulin M (IgM). The biochemical laboratory values whichinclude liver enzymes were elevated with an AST of 846 UI/L, ALT of 1.948 UI/L, GGT of 787 UI/L. Hyperferritinemia(19.741 ng/ml), hypofibrinogenemia (45 mg/dl), and slight-ly increased blood triglycerides levels (249 mg/dl) were alsoobserved. The cerebrospinal fluid (CSF) examinationshowed elevated protein concentration (64 mg/dl) andslightly increased leukocyte counts (10/μl) without clinicalevidence of neurological dysfunction. Two weeks after, shepresented with generalized tonic-clonic seizures and pro-gressive increase in CSF leucocytes and therefore, intrathe-cal methotrexate treatment was initiated. The histology of

bone marrow aspirate revealed lymphocytes engulfed byhistiocytes in multiple microscopic fields (Fig. 1). ChestX-rays were normal and the abdominal ultrasound studyshowed hepatosplenomegaly. Since these findings wereconsistent with FHL according to the Histiocyte Society’sdiagnostic criteria [2, 9], the patient was treated according tothe HLH 2004 protocol [2, 9] and we started all studies ofNKs function as discussed below. When genetic diagnosticwas confirmed, the patient underwent hematopoietic stemcell transplantation (HSCT) from unrelated umbilical cordblood but she died a few days later due to sepsis andsystemic organic dysfunction. At the time of death, therewas no evidence of engraftment.

Patient 2 was a 5 year-old male at the time of confirma-tive diagnosis, first son from unrelated healthy parents fromRionegro (Antioquia-Colombia) with no family history ofFHL. He has a healthy maternal sibling. Before the overtmanifestation of the disease, the patient was hospitalized sixtimes from 1 to 3-years old due to episodes of fever, hep-atosplenomegaly, micropapular exanthem, respiratory dis-tress, anasarca, pancytopenia, anemia, cervical adenopathyand high levels of ferritin, triglycerides and alkaline phos-phatase (ALP). At 2.5 years of age, initial bone marrowaspirate and cervical lymph nodes biopsies showed no ab-normalities. However, at 3.2 years of age, mild and occa-sional erythrophagocytosis was shown in a subsequent bonemarrow aspirate. Liver, spleen and retroperitoneal lymphnode biopsies also showed lymphocyte infiltrate and prolif-eration. A second cervical lymph node biopsy at this age,led to a tentative diagnosis of Hodgkin’s disease but henever received specific treatment. Additionally, pneumoniawith persistent pancytopenia was diagnosed, which was

Fig. 1 Hemophagocytosis inone of our FHL patients. Wrightstaining of bone marrow cellsfrom patient 1. White arrowsindicate histiocytes which havephagocytosed lymphocytes.(magnification power: 1000X)

J Clin Immunol (2012) 32:670–680 673

treated with piperacillin/tazobactam for 13 days. After theseepisodes, the patient went into a long interval of remission.Two years later, the patient was admitted to the hospitalagain with fever, hepatosplenomegaly, jaundice, dyspneaand pancytopenia (700 neutrophils/μl, 400 lymphocytes/μl, 41.000 platelets/μl). During hospitalization, the patientshowed anasarca, respiratory distress, hepatic dysfunctionwith high blood levels of total and conjugated bilirubin,AST of 589 UI/L, ALT of 472 UI/L, GGT of 657 UI/L andlactate dehydrogenase of 1.097 UI/L. Hyperferritinemia(696 ng/ml), hypofibrinogenemia (97 mg/dl), and slightlyincreased blood triglycerides levels (308 mg/dl) also wereobserved. Serum IgM, IgA, IgE levels were normal butincreased IgG levels were observed. Serology against dif-ferent infections was persistently negative, but he mani-fested a positive viral load for EBV (5.397 DNA copies/ml). All together, these findings were consistent with FHLaccording to the Histiocyte Society’s diagnostic criteria [2,9]. The patient started chemotherapy treatment followingthe HLH-2004 protocol [2, 9]. As he remained asymptom-atic and entered remission under HLH treatment, he wasrecently subjected to HSCT from his mother’s blood.

Patient 3 was a 12 year-old male at the time of confirma-tive diagnosis, son from unrelated healthy parents fromCerrito (Valle-Colombia). He had a brother that died previ-ously with an unspecific diagnosis of histiocytosis. Beforethe overt manifestation of the disease, the patient presentedtwo mild episodes of pneumonia at 3 and 5 years of age witha favorable treatment response. Six and a half years later, hewas admitted to the hospital with a 2 month history of feverassociated with asthenia, adynamia and weight loss. Uponexamination, the patient was reported to have multiple cer-vical adenopathies, generalized skin pallor, hepatospleno-megaly and pancytopenia (450 neutrophils/μl, 31.200platelets/μl and 3.430.000 erythrocytes/μl). This led to atentative diagnosis of a lymphoproliferative syndrome. Cul-tures from urine, blood and bone marrow aspirates werenegative for bacteria and fungi and did not report hemopha-gocytosis. Infectious disease screening only showed thepresence of CMV-specific IgG antibodies. Serum IgA, IgGlevels were upon normal ranges but he had increased serumIgE and diminished IgM levels according to age. The bio-chemical laboratory values indicated elevated total and con-jugated bilirubin. He presented elevated AST (228 UI/L),ALT (260 UI/L) and alkaline phosphatase (ALP, 471 U/L).Hyperferritinemia (1.437 ng/ml), increased blood triglycer-ides levels (472 mg/dl) and normal fibrinogen levels(322 mg/dl) were also observed. Abdominal ultrasound scanshowed hepatosplenomegaly and increased retroperitoneallymph nodes. Chest X-rays showed pneumonic infiltratesbut whole-body gammagraphy was normal. Additionally, aliver biopsy showed no abnormalities. Flow cytometry stud-ies of bone marrow aspirate demonstrated neither

lymphoproliferative disorder nor acute leukemia. Antibiotictreatment with cefepimem, meropenem, amikacin, gentami-cine and amphotericin B was then initiated during the firstmonth of hospitalization and suspended during the follow-ing 20 days due to poor treatment response. Then, treatmentwith meropenem, vancomycin, amphotericin B and fluco-nazole was initiated at the second month of hospitalizationbecause of exacerbation of pneumonia symptoms. Due to adeteriorated clinical state, the patient underwent treatmentfor tuberculosis and fungi again without a favorable re-sponse. After assessment by the hematology-oncology ser-vice, clinicians concluded that the patient fulfilled four ofthe diagnostic criteria for HLH [2, 9]. All the NKs function-al studies were performed and the patient underwent treat-ment according to the HLH 2004 protocol [2, 9]. After15 days of treatment, the patient presented a transitorycentral nervous system alteration, presumably as a cyclo-sporine side effect. However, at present he remains asymp-tomatic and into remission under HLH treatment waiting fora matched stem cell donor.

Patient 4 was a 6-year old male, second son from unre-lated healthy parents from Sonson (Antioquia-Colombia)with no family history of FHL. The mother only had spon-taneous abortion at the twelfth week of pregnancy. Thepatient has a healthy 4 year-old brother. At 2-weeks ofage, the patient presented with a varicella infection, withoutcomplications. Then, he was admitted to hospital at 4 yearsof age, with a 2 month history of ataxia, nystagmus, bilateralsensorineural hearing loss and mastoiditis. The patient pre-sented with splenomegaly and an accessory spleen, and inaddition, leukopenia (3.100 leucocytes/μl), neutropenia(800 neutrophils/μl), bilateral panuveitis. Toxoplasma-specific IgG antibodies were demonstrated at time of pre-sentation. Tuberculosis, brucellosis, fungal infection, Coganand Vogt-Koyanagi-Harada syndrome and neoplastic dis-ease were ruled out. A one-month treatment with Sulfadox-ine and Pyrimethamine was initiated and corticoids wereadministered for 2 months. After this treatment, the patientwent into a long interval of remission. Two years later, thepatient was again hospitalized with asthenia, hyporexia,generalized skin pallor and night cough episodes associatedwith occasional fever and hearing loss. Abdominal ultra-sound scan confirmed the hepatosplenomegaly and the ac-cessory spleen and chest X-rays showed multiple pulmonarynodules. PCR in bronchoalveolar lavage fluid was positivefor Mycobacterium tuberculosis and two bone marrow bi-opsies reported no abnormalities. Specific treatment fortuberculosis was initiated. Rapid respiratory deteriorationdue to the pneumonia was observed. Moreover, he presentedwith an oral infection by Candida albicans. Treatment withmeropenem, vancomycin, trimethoprim/sulfamethoxazoleand fluconazole was initiated. Serum IgA, IgE levels werenormal but diminished IgM and IgG levels were observed.

674 J Clin Immunol (2012) 32:670–680

Persistent pancytopenia, hyperferritinemia (581 ng/ml), in-creased blood triglycerides (397 mg/dl) and slightly dimin-ished fibrinogen levels (126 mg/dl) were reported. At thistime, the histology of the bone marrow aspirate showedincreased histiocytes and erythrophagocytosis. Treatmentwith trimethoprim/sulfamethoxazole and meropenem wassuspended and the patient was treated according to theHLH-2004 protocol [2, 9]. Currently, the patient remainsasymptomatic and in remission, under HLH treatment waitingfor a matched stem cell donor

Low Numbers and Abnormalities of Lymphocyte Subsetsin Patients

PB phenotyping of patient 1 before the initiation of theHLH-2004 protocol, showed conspicuously decreasednumbers of CD3+CD4+ and CD3+CD8+ T cells. Likewise,abnormalities of T cells subsets were observed with reduc-tion of naïve (CD4+CD45RA+/CD8+CD45RA+) and in-crease of efector/memory T cells (CD4+CD45RO+/CD8+CD45RO+) in comparison to the values of age-matched healthy donors reported in the literature (Data notshown) [21, 22]. TCRαβ+ and TCRγδ+ T cell percentageswere within normal values according to age [23, 24]. Inaddition, numbers of B and NKs subpopulations (CD56bright

and CD56dim NKs) in PB were within normal range andincreased percentages of monocytes were observed (Datanot shown) [25, 26]. After the initiation of the HLH-2004protocol, the patient exhibited only a mild decrease in theabsolute PB NK cell numbers (Data not shown). In turn,patient 2 showed a marked leukopenia not only before butalso after the initiation of the HLH-2004 protocol. However,the percentages and absolute numbers of total lymphocytesand NKs subpopulations were always among normal rangesin comparison to the values of age-matched healthy donorsreported in the literature (Data not shown) [21, 25]. Noanalyses of naïve vs memory and TCRαβ+ vs TCRγδ+ Tcells subsets could be performed in patient 2. Analyses ofleukocytes in PB of patient 3 before initiation of the HLH-2004 protocol showed a marked leukopenia but increasedtotal lymphocytes. NKs subpopulations percentages andnumbers were comparable to age-matched healthy donors,reported in the literature [21, 25]. Patient 4 also showeddiminished numbers of leukocytes and total lymphocytes inPB. Increased percentages but normal numbers of NKs wereobserved before initiation of the HLH-2004 protocol (Datanot shown).

Impaired Perforin Expression of NKs in the Patients

Taking into account that the four patients included herefulfilled the FHL diagnostic criteria, according to the His-tiocyte Society, and considering that perforin deficiency is

the most frequent genetic defect in FHL [9, 17], NKs frompatients PBs were analyzed for perforin expression by flowcytometry. Figure 2 shows a severe decrease in perforinexpression, observed in NKs from PBs in patients 1 and 2.Similar results were found in patients 3 and 4 (Data notshown). Of note, although disease onset and severity weredifferent between the patients included in this study, nodifferences in perforin expression were observed amongthem. We also attempted to evaluate the cytotoxic activityof PBMC against K562 target cells upon stimulation withcytokines in the four patients by a flow-based method.These results indicated that cytotoxicity was severely im-paired in patient 1 and greatly reduced in patients 2 to 4 incomparison to this response in age and sex-matched healthycontrols (Data not shown).

NKs from Patients Exhibit Increased Translocationof CD107a to the Cell Surface

To further characterize the cytotoxicity defects in thepatients included in this study, the CD107a translocationassay was performed. CD107a (LAMP-1) is a transmem-brane protein of cytotoxic granules, which is exposed at thecell surface after degranulation. Defects in the translocationof this molecule to the cell surface strongly suggest defectsin the tethering, docking and fusion of the cytolytic gran-ules, which directs the diagnosis of either FHL type 3, type4 or type 5 defects. Our results show no differences in theexpression of CD107a in unstimulated NKs from patients 1and 2 and age- and sex-matched healthy donors (Fig. 3).Nevertheless, when PBMC were cocultured with cytokinesand exposed to K562 target cells, augmented translocationof CD107a in NKs were observed in both patients comparedwith CD107a expression in NKs from healthy donors(Fig. 3). Similar results were observed in NKs from patients3 and 4 (Data not shown).

Genetic Diagnosis of FHL Type 2 due to PRF1 GeneDefects in the Patients

After the perforin-deficiency was identified in the patientsand impairment in cellular cytotoxicity was observed, wedetermined whether the patients and their parents exhibitedmutations in the PRF1 gene. Genetic analysis of genomicDNA revealed that patient 1 was a compound heterozygotefor two previously reported mutations in exon 2 of the PRF1gene: one allele carried a deletion of nucleotide 50 (thymi-dine) of the coding sequence (CDS) resulting in a frameshiftat amino acid 17 (leucine) of the perforin protein and apremature stop codon 99 base pairs after the deletion(L17fsx50). This defect leads to a severely truncated proteinwhen translated from the first allele. The second allelecarried a C > T substitution at nucleotide 160 of the CDS

J Clin Immunol (2012) 32:670–680 675

leading to the R54C amino acid change of the perforinprotein (Fig. 4a). Sequence analysis of the patients’ parentsgDNA revealed that the 50delT mutation had maternalorigin. Moreover, the presence of the R54C mutation wasfound in one allele of the father’s PFR1 gene (Fig. 4b). Onthe other hand, patients 2, 3 and 4 were found to be homo-zygous for the mutation 160 C > T (R54C) (Fig. 4a and b).The carrier status of the patients’ parents for the mutationR54C was also confirmed with the exception of patient 3’sfather who was not available for testing (Fig. 4b).

Presence of the A91V Variant in the PRF1 Gene fromPatients, Their Parents and Healthy Donors

As the common variant sequence C > T at the nucleotide272 of the PRF1 CDS leading to a change from a small(Alanine) into a large (Valine) hydrophobic amino acid(A91V) in the perforin protein has been previously reportedas a genetic susceptibility factor for FHL, we evaluated thefrequency of this variant in gDNA from our patients andtheir parents. Our analysis revealed that patient 1 was het-erozygous for this polymorphism, inherited from his father(Fig. 4a and b). Patients 2, 3 and 4 were homozygous forA91V in the PRF1 gene (Fig. 4a and b). The heterozygousstate of the parents from these three patients was also

confirmed with the exception of the father from patient 3.We further evaluated the frequency of A91V in the exon 2 ofthe PRF1 gene from 71 healthy Colombian individuals.Interestingly, we found that 3 out of 71 individuals wereheterozygous for this polymorphism. Neither the L17fsx50deletion nor the R54C missense mutation in the PRF1 genewas observed in the gDNA from the healthy donors (Datanot shown).

Discussion

The present study reports the first four cases of FHL type 2in Colombia. Patient 1 was a typical FHL case whereaspatients 2, 3 and 4 presented with only mild manifestationsof disease during the first years of age and the confirmativediagnosis of FHL was performed later in life after severalyears of remission. Importantly from a diagnostic perspec-tive, the patients showed total absence of NKs intracellularperforin expression and impairment of granule-mediatedcytotoxic responses. Genetic analysis revealed that patient1 was a compound heterozygote for the mutations 50delT(L17fsx50) and 160 C > T (R54C) of the perforin gene.Moreover, the common sequence variant 272 C > T (A91V)was also present in one of her alleles. On the other hand,

Fig. 2 Perforin expression ofNKs from the FHL patients.Peripheral blood leukocytes(PBL) from the patients includedin this study and age/sex-matchedhealthy donors were stained withfluorochrome-conjugated anti-CD3, -CD19, -CD14, -CD16, -CD56 mAbs. Thereafter, cellswere fixed, permeabilized,stained with anti-perforin PEmAb and analyzed by flowcytometry. Lymphocytes weregated based on forward and sidescatter plots. Monocytes (CD14+), T (CD3+) and B (CD19+)lymphocytes were gated out. Theremaining cells were againanalyzed for CD16 and CD56expression. NKs were definedphenotypically as CD3-CD19-CD14-/CD16 + CD56+ cells.Filled histograms representperforin expression in NKs frompatients 1 and 2 and age- andsex-matched healthy donors,open histograms represent theisotype control mAb. Similarresults were found in patients 3and 4 (Data not shown). MFI:Mean Fluorescence Intensityvalues

676 J Clin Immunol (2012) 32:670–680

patients 2, 3 and 4 were homozygous not only for themutation R54C but also for the sequence variant A91V ofthe perforin gene. The carrier status of the patients’ parentswas confirmed with the exception of the father from patient3. Therefore, this study describes that the R54C/A91V hap-lotype is highly frequent in the PRF1 gene of familiesaffected with atypical FHL in Colombia.

More than 70 germline mutations in the PRF1 gene havebeen associated with FHL type 2 including microdeletions,non-sense and missense mutations. They span mainly theCDS, with a high proportion located at the transmembraneprotein domain within the membrane attack complex

perforin domain (MACPRF) [7, 27–38]. Interestingly, theL17fsx50 and R54C mutations reported in our patientsoccur in the extreme N terminal of the protein within thelytic peptide region. This region is proposed to weaken themembrane through electrostatic interactions facilitating in-sertion of the active perforin that finally forms barrel-shapedpores on the target cell [39]. The L17fsx50 mutation, whichresults in a severely truncated protein, has been reportedmainly in African American patients. However, some caseshave been described in Hispanic patients [7, 35, 40]. Intra-genic polymorphism analysis of PRF1 gene has shown thatL17fsx50 mutation in African American patients is

Fig. 3 CD107a translocation assay in NKs from the FHL patients.PBMC from the patients included in this study and age/sex-matchedhealthy donors were incubated with or without rhIL-2 or rhIL-15 for 24hours and then, exposed to K562 cells in the presence of the CD107amAb and monensin for 3 hours. Then, PBMC were stained withfluorochrome-conjugated anti-CD3, -CD19, -CD14, -CD16, -CD56mAbs. Lymphocytes were gated based on forward and side scatterplots. Monocytes (CD14+), T (CD3+) and B (CD19+) lymphocytes

were gated out. The remaining cells were again analyzed for CD16 andCD56 expression. Flow cytometry pseudocolor plots show the percent-age of CD3-CD19-CD14-/CD16 + CD56+ NKs that express surfaceCD107a (in the plots upper right quadrant) in unstimulated cells, cellsexposed only to K562 target cells, cells stimulated with either rhIL-2 orrhIL-15 and exposed to K562 cell line from patients 1 and 2 and age-and sex-matched healthy donors. Similar results were found in patients3 and 4 (Data not shown)

J Clin Immunol (2012) 32:670–680 677

associated with homozygosity of the PRF1 H300H poly-morphism, which has been the most frequent polymorphismobserved in the perforin gene. Moreover, undetectable orreduced perforin expression and absent or minimal NKscytotoxic function (<1%) have been observed in all homo-zygous L17fsx50 patients and in high frequency of hetero-zygous patients carrying an additional missense mutation inthe second allele [7, 35, 40]. In accordance with thesereports, the clinical phenotype from patient 1 reported herewas severe and associated with undetectable perforin expres-sion and complete impairment of NKs cytotoxic response.Interestingly, even though patient 1 was Hispanic and carriedthe R54C missense mutation in the second allele out ofL17fsx50 (compound heterozygote), the H300H polymor-phism was not found in exon 3 of the gene.

On the other hand, the R54C mutation has been describedin compound heterozygous patients, diagnosed with FHLtype 2 at age older than 1 year, linked to partial perforindeficiency in NKs and complete absence of this protein inCD8+ and CD56+ T cells [34]. It should be mentioned thatwhenever the A91V variant was present, the disease onsetwas always beyond infancy (between the ages of 3 and22 years) [30, 34, 37]. In the present study, patients 2, 3and 4 who were homozygous for R54C/A91V, had also amilder phenotype and residual NK cell cytotoxic activityeven though they exhibited almost no baseline PB expres-sion of perforin. Notably, patients 3 and 4 survived to CMVand Varicella infections, respectively. It is also important tonote that most patients manifest symptoms of FHL withinthe first months of life with few cases of late onset disease[31]. Late onset FHL has a more diverse clinical phenotype,easily misdiagnosed as disorders, including common vari-able immunodeficiency, non-Hodgkin lymphoma or as anisolated neurologic disorder [41–43]. These atypical FHL

presentations have been reported in adolescents and even inadults as old as 62 years of age [5, 27, 28, 31, 32, 42, 44,45]. They also may be associated with milder and oftenrecurrent HLH episodes and prolonged survival in the ab-sence of HSCT, which is unusual in patients with the typicaldisease phenotype [42]. Atypical FHL is usually linked withmissense or splice-site mutations in the affected genes [28,37, 42, 46]. According to the parameters used to defineatypical cases of FHL, patients 2, 3, and 4 from the presentstudy may be included in this group because, and althoughthey presented with milder episodes of HLH during the firstyears of life, their clinical phenotype was diverse and notconclusive. Additionally, they remained asymptomatic forseveral years, until developing overt clinical symptoms ofHLH. However, it is important to mention that negativefamily history FHL and limited access to NK cell functiontests may also have delayed the FHL diagnosis.

Interestingly, we found the allele R54C/A91V in 4 unre-lated families from Colombia and previous studies havereported the same genotype related with delayed onset ofFHL type 2 in patients with Hispanic ethnicity. In a cohortof 50 North American families, a 3 year-old patient withHispanic ethnicity was reported, carrying R54C/A91Vinherited from his father [7]. This patient demonstratedmarkedly decreased perforin protein expression and mini-mal, but detectable, NKs cytotoxic activity at high Effector:Target ratios [7]. More importantly, a Japanese study hadpreviously reported a Colombian offspring with atypicalFHL who carried the R54C and the nonsense mutation285delK and also the A91V variant. The R54C and A91Voriginated from his asymptomatic Colombian mother and285delK inherited from his Japanese father [37]. Furthergenotyping analyses are necessary to determine if thisdisease-linked haplotype is shared among several Colombian

Fig. 4 Genetic analysis of the perforin-deficient patients. a. Schematicrepresentation of PRF1 gene which spans the three exons, two ofwhich are part of the CDS (Exons 2 and 3). Part of the CDS andpeptide wild type sequences indicating the positions are shown inblack. Changes in the maternal allele of patient 1 are shown in blue:

50 del T (L17fsX50). Changes in the paternal allele are shown in red:160 C > T (R54C) and 272 C > T (A91V). Patients 2, 3 and 4 werehomozygous for the mutations 160 C > T (R54C) and 272 C > T(A91V). b. Pedigree of the family members from the four patients. Theasterisks denote individuals available for testing

678 J Clin Immunol (2012) 32:670–680

families, as previously reported for other haplotypes [7, 35,47].

In addition to the mutations found in our patients, we alsofound the A91V variant within the perforin gene in all thepatients included in this study; in patient 1 in a heterozygousstate and in patient 2, 3 and 4 in a homozygous state. A91Vis by far the most widely debated PRF1 variant because it iscommonly found in healthy white Caucasians in a hetero-zygous state [9, 27, 48] and A91V heterozygous carriersexhibit normal baseline NKs cytotoxic function despite de-creased perforin expression [7]. Only two cases of homozy-gous asymptomatic individuals for A91V have beenreported in the literature [30, 32]. In FHL patients, theA91V variant together with nonsense perforin mutationsalways led to null perforin expression and activity. Howev-er, residual NKs cytotoxic activity, although markedly re-duced, can often be detected when A91V is found inassociation with perforin missense mutations [32]. Anotherstudy also identified the A91V transition in the heterozy-gous state in 7/202 individuals (3%) [7]. Interestingly, ourreport describes for the first time the association ofL17fsx50, R54C and the variant A91V in one patient, elu-cidating the impact of high penetrance of L17fsx50 muta-tion in PRF1 gene to affect the protein function. Moreover,our study also identified 3 heterozygous Colombian indi-viduals for the A91V variant out of 71 healthy donorsanalyzed, which suggests a frequency of 4.2% of this poly-morphism in our population.

Our findings confirm that the testing of perforin expres-sion and NKs cell function are required not only for HLHpatients in early infancy but also in older children and youngadults who accomplish the HLH diagnostic criteria. Thiswould facilitate an earlier diagnosis of FHL and help toguide treatment decisions including HSCT at the propertime. It is the first time that four cases of FHL type 2 arereported in Latin America. A remarkable finding from ourstudy is the presence of the same allele (R54C/A91V) in 4unrelated families, suggesting that this is a frequent FHLtype 2 allele in Colombia. Our findings encourage us tocontinue with the active search of FHL in Colombia and toestablish appropriate diagnostic algorithms to achieve themolecular diagnosis of this disease and to improve thequality of life of patients that can be potentiallymisdiagnosed.

Acknowledgements We are grateful to the patients’ families andhealthy control volunteers involved in this study for their participationand cooperation. We thank the Pediatric Physician Luisa FernandaRojas for her generous contribution in this work. This study wassupported by the Colombian Institute for the Development of Scienceand Technology, COLCIENCIAS (Grant #111540820536), Foundationfor the promotion of Research and Technology – Banco de la Repúb-lica (Grant #2361) and the Estrategia de Sostenibilidad 2009–2011,Vicerrectoria de Investigaciones-Universidad de Antioquia, Medellin-Colombia.

Disclosure of Conflict of Interest None of the authors has anypotential financial conflict of interest related to this manuscript.

References

1. Farquhar JW, Claireaux AE. Familial haemophagocytic reticulosis.Arch Dis Child. 1952;27(136):519–25.

2. Henter JI, Horne A, Aricó M, Egeler RM, Filipovich AH, ImashukuS, et al. HLH-2004: Diagnostic and therapeutic guidelines for hemo-phagocytic lymphohistiocytosis. Pediatr Blood Cancer. 2007;48(2):124–31.

3. Arico M, Janka G, Fischer A, Henter JI, Blanche S, Elinder G, etal. Hemophagocytic lymphohistiocytosis. Report of 122 childrenfrom the International Registry. FHL Study Group of the Histio-cyte Society. Leukemia. 1996;10(2):197–203.

4. Ishii E, Ohga S, Imashuku S, Yasukawa M, Tsuda H, Miura I, et al.Nationwide survey of hemophagocytic lymphohistiocytosis in Ja-pan. Int J Hematol. 2007;86(1):58–65.

5. Henter JI, Elinder G, Ost A. Diagnostic guidelines for hemopha-gocytic lymphohistiocytosis. The FHL Study Group of the Histio-cyte Society. Semin Oncol. 1991;18:29–33.

6. Henter JI, Aricó M, Egeler RM, Elinder G, Favara BE, FilipovichAH, et al. HLH-94: a treatment protocol for hemophagocyticlymphohistiocytosis. HLH study Group of the Histiocyte Society.Med Pediatr Oncol. 1997;28(5):342–7.

7. Molleran Lee S, Villanueva J, Sumegi J, Zhang K, Kogawa K,Davis J, et al. Characterisation of diverse PRF1 mutations leadingto decreased natural killer cell activity in North American familieswith haemophagocytic lymphohistiocytosis. J Med Genet. 2004;41(2):137–44.

8. Henter J-I, Elinder G, Söder Q, Öst A. Incidence and clinicalfeatures of familial hemophagocytic lymphohistiocytosis in Swe-den. Acta Pediatr Scand. 1991;80(4):428–35.

9. Gholam C, Grigoriadou S, Gilmour KC, Gaspar HB. Familialhaemophagocytic lymphohistiocytosis: advances in the geneticbasis, diagnosis and management. Clin Exp Immunol. 2011;163(3):271–83.

10. Ohadi M, Lalloz MR, Sham P, Zhao J, Dearlove AM, Shiach C, etal. Localization of a gene for familial hemophagocytic lymphohis-tiocytosis at chromosome 9q21.3-22 by homozygosity mapping.Am J Hum Genet. 1999;64(1):165–71.

11. Stepp SE, Dufourcq-Lagelouse R, Le Deist F, Bhawan S, CertainS, Mathew PA, et al. Perforin gene defects in familial hemopha-gocytic lymphohistiocytosis. Science. 1999;286(5446):1957–9.

12. Feldmann J, Callebaut I, Raposo G, Certain S, Bacq D, Dumont C,et al. Munc13–4 is essential for cytolytic granules fusion and ismutated in a form of familial hemophagocytic lymphohistiocytosis(FHL3). Cell. 2003;115(4):461–73.

13. Zur Stadt U, Schmidt S, Kasper B, Beutel K, Diler AS, Henter JI,et al. Linkage of familial hemophagocytic lymphohistiocytosis(FHL) type-4 to chromosome 6q24 and identification of mutationsin syntaxin 11. Hum Mol Genet. 2005;14(16):827–34.

14. Côte M, Ménager MM, Burgess A, Mahlaoui N, Picard C, SchaffnerC, et al. Munc18-2 deficiency causes familial hemophagocytic lym-phohistiocytosis type 5 and impairs cytotoxic granule exocytosis inpatient NK cells. J Clin Invest. 2009;119(12):3765–73.

15. Bryceson YT, Rudd E, Zheng C, Edner J, Ma D, Wood SM, et al.Defective cytotoxic lymphocyte degranulation in syntaxin-11 de-ficient familial hemophagocytic lymphohistiocytosis 4 (FHL4)patients. Blood. 2007;110(6):1906–15.

16. Ishii E, Ohga S, Imashuku S, Kimura N, Ueda I, Morimoto A, et al.Review of hemophagocytic lymphohistiocytosis (HLH) in childrenwith focus on Japanese experiences. Crit Rev Oncol Hematol.2005;53(3):209–23.

J Clin Immunol (2012) 32:670–680 679

17. Marcenaro S, Gallo F, Martini S, Santoro A, Griffiths GM, AricóM, et al. Analysis of natural killer–cell function in familial hemo-phagocytic lymphohistiocytosis (FHL): defective CD107a surfaceexpression heralds Munc13-4 defect and discriminates betweengenetic subtypes of the disease. Blood. 2006;108(7):2316–23.

18. Cullen SP, Martin SJ. Mechanisms of granule-dependent killing.Cell Death and Differ. 2008;15(2):251–62.

19. Klenchin VA, Martin TFJ. Priming in exocytosis: Attaining fusion-competence after vesicle docking. Biochimie. 2000;82(5):399–407.

20. Betts MR, Brenchley JM, Price DA, De Rosa SC, Douek DC,Roederer M, et al. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranula-tion. J Immunol Methods. 2003;281(1–2):65–78.

21. Comans-Bitter WM, de Groot R, van den Beemd R, Neijens HJ,Hop WC, Groeneveld K, et al. Immunophenotyping of bloodlymphocytes in childhood. Reference values for lymphocyte sub-populations. J Pediatr. 1997;130(3):388–93.

22. Neubert R, Delgado I, Abraham K, Schuster C, Helge H. Evalua-tion of the age-dependent development of lymphocyte surfacereceptors in children. Life Sci. 1998;62(12):1099–110.

23. Nicolas L, Monneret G, Debard AL, Blesius A, Gutowski MC,Salles G, et al. Human gammadelta T cells express a higher TCR/CD3 complex density than alphabeta T cells. Clin Immunol.2001;98(3):358–63.

24. Kamath AB, Wang L, Das H, Li L, Reinhold VN, Bukowski JF.Antigens in tea-beverage prime human Vgamma 2Vdelta 2 T cellsin vitro and in vivo for memory and nonmemory antibacterialcytokine responses. Proc Natl Acad Sci U S A. 2003;100(10):6009–14.

25. Poli A, Michel T, Thérésine M, Andrès E, Hentges F, Zimmer J.CD56bright natural killer (NK) cells: an important NK cell subset.Immunology. 2009;126(4):458–65.

26. Berrio M, Correa CM, Jimenez ME. El hemograma: Análisis einterpretación con las tres generaciones. Escuela de Bacteriología yLaboratorio Clínico. Ed. Universidad de Antioquia. Colombia,2003; 41

27. Trambas C, Gallo F, Pende D, Marcenaro S, Moretta L, De FuscoC, et al. A single amino acid change, A91V, leads to conforma-tional changes that can impair processing to the active form ofperforin. Blood. 2005;106(3):932–7.

28. Busiello R, Adriani M, Locatelli F, Galgani M, Fimiani G, ClementiR, et al. Atypical features of familial hemophagocytic lymphohistio-cytosis. Blood. 2004;103(12):4610–2.

29. Menasche G, Feldmann J, Fischer A, de Saint Basile G. Primaryhemophagocytic syndromes point to a direct link between lympho-cyte cytotoxicity and homeostasis. Immunol Rev. 2005;203:165–79.

30. Risma KA, Frayer RW, Filipovich AH, Sumegi J. Aberrant matu-ration of mutant perforin underlies the clinical diversity of hemo-phagocytic lymphohistiocytosis. J Clin Invest. 2006;116(1):182–92.

31. Clementi R, Emmi L, Maccario R, Liotta F, Moretta L, DanesinoC, et al. Adult onset and atypical presentation of hemophagocyticlymphohistiocytosis in siblings carrying PRF1 mutations. Blood.2002;100(6):2266–7.

32. Feldmann J, Le Deist F, Ouachee-Chardin M, Certain S, AlexanderS, Quartier P, et al. Functional consequences of perforin genemutations in 22 patients with familial haemophagocytic lympho-histiocytosis. Br J Haematol. 2002;117(4):965–72.

33. Goransdotter EK, Fadeel B, Nilsson-Ardnor S, Söderhäll C,Samuelsson A, Janka G, et al. Spectrum of perforin gene mutationsin familial hemophagocytic lymphohistiocytosis. Am J Hum Genet.2001;68(3):590–7.

34. Kogawa K, Lee SM, Villanueva J, Marmer D, Sumegi J, FilipovichAH. Perforin expression in cytotoxic lymphocytes from patientswith hemophagocytic lymphohistiocytosis and their familymembers.Blood. 2002;99(1):61–6.

35. Lee SM, Sumegi J, Villanueva J, Tabata Y, Zhang K, ChakrabortyR, et al. Patients of African ancestry with hemophagocytic lym-phohistiocytosis share a common haplotype of PRF1 with a 50delTmutation. J Pediatr. 2006;149(1):134–7.

36. Suga N, Takada H, Nomura A, Ohga S, Ishii E, Ihara K, et al.Perforin defects of primary haemophagocytic lymphohistiocytosisin Japan. Br J Haematol. 2002;116(2):346–9.

37. Ueda I, Kurokawa Y, Koike K, Ito S, Sakata A, Matsumora T, et al.Late-onset cases of familial hemophagocytic lymphohistiocytosiswith missense perforin gene mutations. Am J Hematol. 2007;82(6):427–32.

38. Zur Stadt U, Beutel K, Kolberg S, Schneppenheim R, Kabisch H,Janka G, et al. Mutation spectrum in children with primary hemo-phagocytic lymphohistiocytosis: molecular and functional analysesof PRF1, UNC13D, STX11, and RAB27A. Hum Mutat. 2006;27(1):62–8.

39. Natarajan K, Cowan JA. Solution structure of a synthetic lyticpeptide: the perforin amino terminus. Chem Biol. 1998;5(3):147–54.

40. Trizzino A, Zur Stadt U, Ueda I, Risma K, Janka G, Ishii E, et al.Genotype–phenotype study of familial haemophagocytic lympho-histiocytosis due to perforin mutations. J Med Genet. 2008;45(1):15–21.

41. Feldmann J, Ménasché G, Callebaut I, Minard-Colin V, Bader-Meunier B, Clainche LL, et al. Severe and progressive encephalitisas a presenting manifestation of a novel missense perforin muta-tion and impaired cytolytic activity. Blood. 2005;105(7):2658–63.

42. Rohr J, Beutel K, Maul-Pavicic A, Vraetz T, Thiel J, Warnatz K, etal. Atypical familial hemophagocytic lymphohistiocytosis due tomutations in UNC13D and STXBP2 overlaps with primary immu-nodeficiency diseases. Haematologica. 2010;95(12):2080–7.

43. Mancebo E, Allende LM, Guzman M, Paz-Aratl E, Gil J, Urrea-Moreno R, et al. Familial hemophagocytic lymphohistiocytosis inan adult patient homozygous for A91V in the perforin gene, withtuberculosis infection. Haematologica. 2006;91(9):1257–60.

44. Allen M, De Fusco C, Legrand F, Clementi R, Conter V, DanesinoC, et al. Familial hemophagocytic lymphohistiocytosis: how latecan the onset be? Haematologica. 2001;86(5):499–503.

45. Nagafuji K, Nonami A, Kumano T, Kikushige Y, Yoshimoto G,Takenaka K, et al. Perforin gene mutations in adult-onset hemopha-gocytic lymphohistiocytosis. Haematologica. 2007;92(7):978–81.

46. Ueda I, Ishii E, Morimoto A, Ohga S, Sako M, Imashuku S.Correlation between phenotypic heterogeneity and gene mutation-al characteristics in familial hemophagocytic lymphohistiocytosis(FHL). Pediatr Blood Cancer. 2006;46(4):482–8.

47. Danielian S, Basile N, Rocco C, Prieto E, Rossi J, Barsotti D, et al.Novel Syntaxin 11 Gene (STX11) Mutation in Three ArgentineanPatients with Hemophagocytic Lymphohistiocytosis. J Clin Immu-nol. 2010;30(2):330–7.

48. Zur Stadt U, Beutel K, Weber B, Kabisch H, Schneppenheim R,Janka G. A91V is a polymorphism in the perforin gene not caus-ative of an FHLH phenotype. Blood. 2004;104:1909–10.

680 J Clin Immunol (2012) 32:670–680