A RAB27A 5′ untranslated region structural variant associated with late-onset hemophagocytic lymphohistiocytosis and normal pigmentation
Authors: Bianca Tesi, Jelena Rascon, Samuel C.C. Chiang, Birute Burnyte, Alexandra Löfstedt, Anders Fasth, Miriam Heizmann, Sandra Juozapaite, Rosita Kiudeliene, Egle Kvedaraite, Valdone Miseviciene, Audrone Muleviciene, Martha-Lena Müller, Magnus Nordenskjöld, Reda Matuzeviciene, Ruta Samaitiene, Carsten Speckmann, Sigita Stankeviciene, Vytautas Zekas, Matthias Voss, Stephan Ehl, Nerija Vaiciene-Magistris, Jan-Inge Henter, Marie Meeths, Yenan T. Bryceson
Journal: Journal of Allergy and Clinical Immunology (2018)

Autosomal-recessive mutations in genes required for secretory lysosome-mediated lymphocyte cytotoxicity cause primary hemophagocytic lymphohistiocytosis (HLH), an early-onset, life-threatening hyperinflammatory syndrome.1Janka G.E. Lehmberg K. Hemophagocytic syndromes—an update.Blood Rev. 2014; 28: 135-142Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar Similarly, mutations in RAB27A and LYST are associated with HLH, yet manifest hypopigmentation, because Rab27a and LYST also facilitate trafficking of pigment-containing lysosomes in melanocytes.2de Saint Basile G. Ménasché G. Fischer A. Molecular mechanisms of biogenesis and exocytosis of cytotoxic granules.Nat Rev Immunol. 2010; 10: 568-579Crossref PubMed Scopus (299) Google Scholar We detail individuals from 5 families from the Baltic area with a novel structural variant at the 5′ untranslated region (UTR) of RAB27A associated with an atypical form of Griscelli syndrome type 2 (GS2) manifesting as late-onset HLH, marked neuroinflammation, skin granulomas, lymphoma, and normal pigmentation (see Table E1 in this article's Online Repository at www.jacionline.org). In brief, patient 1 (P1) developed HLH at age 13 years after 8 years of recurrent neuroinflammation (Fig 1, A and B; see Fig E1, A, in this article's Online Repository at www.jacionline.org). Her older brother died in infancy because of severe infectious mononucleosis. Patient 2 (P2) developed fatal HLH at age 15 years. Two of his 3 sisters died at age 13 years, one of an HLH-like syndrome and the other because of brain lymphoma. Patient 3 (P3) developed HLH at age 9 years, after 3 years of recurrent neuroinflammation (Fig E1, B). Patient 4 (P4) suffered from isolated neuroinflammation, without fulfilling HLH criteria. P2, P3, and P4 also had skin granulomas (Fig 1, C). Patient 5 (P5) suffered from recurrent fever flares and EBV viremia, and developed lymphoma at age 13 years. Hypopigmentation was not evident in any of the patients (Fig E1, D). Further clinical information and methods are provided in this article's Online Repository at www.jacionline.org. Prompted by a suspicion of HLH, functional investigations of cytotoxic lymphocytes were performed on P1, P3, P4, and P5 (Table E1). Natural killer (NK)-cell cytotoxicity was defective (Fig 1, D and G). Moreover, NK- and T-cell exocytosis was impaired (Fig 1, E and F, H and I), with IL-2 stimulation inducing a partial recovery (see Fig E2, A-F, in this article's Online Repository at www.jacionline.org). Results therefore indicated primary HLH due to defective exocytosis. Despite the clinical and immunological findings, a molecular diagnosis of primary HLH was not established by exome sequencing of HLH-associated genes. A heterozygous RAB27A missense variant of unknown significance (p.Arg187Trp) was identified in P1. Moreover, previously reported heterozygous RAB27A mutations were detected in P2 (p.Arg80Thr) and P5 (p.Arg184*) (Fig 2, A).3Meeths M. Bryceson Y.T. Rudd E. Zheng C. Wood S.M. Ramme K. et al.Clinical presentation of Griscelli syndrome type 2 and spectrum of RAB27A mutations.Pediatr Blood Cancer. 2010; 54: 563-572Crossref PubMed Scopus (78) Google Scholar To identify possible noncoding mutations, we performed whole-genome sequencing of P1 and P2. Inspection of sequencing data revealed several read pairs aberrant for insert size and orientation at the 5′UTR of RAB27A and over the first 5 exons of the adjacent PIGB, required for glycosylphosphatidylinositol anchor biosynthesis (Fig 2, A). Software-based analysis of structural variants (SVs) confirmed the presence of multiple overlapping SVs in this region, including shared events between P1 and P2 (Fig 2, A). To confirm the presence of SVs affecting copy number at the 5′UTR of RAB27A, a custom multiplex ligation-dependent probe amplification (MLPA) assay was designed. In P1, a complex SV with a duplication-normal-duplication pattern inherited from the father was confirmed, in addition to a 65-kb deletion inherited from the mother (Fig 2, A and C). P2 and P5, who carried monoallelic coding mutations, were heterozygous for the complex SV (Fig 2, C). Complex SVs are usually associated with cryptic rearrangements.4Brand H. Collins R.L. Hanscom C. Rosenfeld J.A. Pillalamarri V. Stone M.R. et al.Paired-duplication signatures mark cryptic inversions and other complex structural variation.Am J Hum Genet. 2015; 97: 170-176Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar Further analysis of split reads and discordant read pairs of the complex SV indicated that one of the duplicated regions was inverted (Fig 2, B and C). In this model, only 1 of several transcriptional start sites (TSSs) of RAB27A, encoding the transcript NM_183235.2, is disrupted by the complex SV, while a copy of PIGB remains intact (Fig 2, B). Validation of one predicted breakpoint supported this model (see Fig E3, A, in this article's Online Repository at www.jacionline.org). Together, identification of biallelic mutations in RAB27A established a diagnosis of atypical GS2 in P1, P2, and P5. The clinical resemblance and geographic proximity prompted analysis of RAB27A by MLPA in P3 and P4, patients who displayed defective lymphocyte exocytosis yet lacked a genetic diagnosis. Indeed, MLPA revealed homozygosity for the complex SV in both patients (Fig 2, C), as confirmed through sequencing of a breakpoint (Fig E3, A). Rab27a protein expression was absent in leukocytes from P3, P4, and P5 (Fig 2, D, and Fig E2, G and H). Thus, all 5 patients carried biallelic RAB27A mutations, and were at least heterozygous for the SV affecting the transcript NM_183235.2. Eight patients with GS2 and normal pigmentation have been reported to date, all with missense mutations that selectively disrupt binding of Rab27a to Munc13-4 but not to melanophilin, explaining defective lymphocyte yet normal melanocyte function.5Netter P. Chan S.K. Banerjee P.P. Monaco-Shawver L. Noroski L.M. Hanson I.C. et al.A novel Rab27a mutation binds melanophilin, but not Munc13-4, causing immunodeficiency without albinism.J Allergy Clin Immunol. 2016; 138: 599-601.e3Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 6Cetica V. Hackmann Y. Grieve S. Sieni E. Ciambotti B. Coniglio M.L. et al.Patients with Griscelli syndrome and normal pigmentation identify RAB27A mutations that selectively disrupt MUNC13-4 binding.J Allergy Clin Immunol. 2015; 135: 1310-1318.e1Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Because the complex SV disrupts only 1 of several TSSs of RAB27A, we hypothesized that lymphocytes and melanocytes might selectively use distinct RAB27A TSSs. Quantitative PCR demonstrated diminished expression of transcript NM_183235.2 in peripheral blood leukocytes from P3 (Fig 2, E), suggesting predominant usage of the TSS for this transcript. This observation is also supported by cap analysis gene expression data from the FANTOM5 project (Fig E3, C-E).7The FANTOM Consortium and the RIKEN PMI and CLST (dgt)A promoter-level mammalian expression atlas.Nature. 2014; 507: 462-470Crossref PubMed Scopus (556) Google Scholar In contrast, primary as well as embryonic stem cell–derived melanocytes use alternative, downstream TSSs, which were not disrupted by the complex SV (Fig E3, D and E). Quantitative PCR in primary melanocytes confirmed a smaller contribution of transcript NM_183235.2 to total RAB27A expression (Fig 2, E). Transcription of PIGB was maintained by the complex SV (Fig E3, B). Notably, an individual with a homozygous deletion encompassing all RAB27A TSSs displayed classic silvery hair.8Vincent L.M. Gilbert F. DiPace J.I. Ciccone C. Markello T.C. Jeong A. et al.Novel 47.5-kb deletion in RAB27A results in severe Griscelli syndrome type 2.Mol Genet Metab. 2010; 101: 62-65Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar Our data therefore indicate differential TSS usage between leukocytes and melanocytes, explaining the normal pigmentation observed in patients with at least 1 mutation affecting only the upstream RAB27A TSS. Nonsense mutations in RAB27A are associated with the development of HLH within the first year of life.3Meeths M. Bryceson Y.T. Rudd E. Zheng C. Wood S.M. Ramme K. et al.Clinical presentation of Griscelli syndrome type 2 and spectrum of RAB27A mutations.Pediatr Blood Cancer. 2010; 54: 563-572Crossref PubMed Scopus (78) Google Scholar Neurologic involvement affects 55% of patients at diagnosis and 67% during the course of disease.3Meeths M. Bryceson Y.T. Rudd E. Zheng C. Wood S.M. Ramme K. et al.Clinical presentation of Griscelli syndrome type 2 and spectrum of RAB27A mutations.Pediatr Blood Cancer. 2010; 54: 563-572Crossref PubMed Scopus (78) Google Scholar Three of the 5 patients reported here displayed severe and recurrent neuroinflammation resembling acute disseminated encephalomyelitis, which preceded onset of full-blown HLH by many years. Functional assays of NK- and T-cell exocytosis should therefore be considered in the diagnostic workup of patients with unexplained neuroinflammatory diseases. Late-onset HLH suggests downstream transcription in activated lymphocytes. In conclusion, we report 5 patients with atypical GS2 characterized by neuroinflammation, lymphoma, and late-onset HLH. Remarkably, 3 patients manifested skin granulomas.9Zerah M.L. DeWitt C.A. Cutaneous findings in hemophagocytic lymphohistiocytosis.Dermatology. 2015; 230: 234-243Crossref PubMed Scopus (14) Google Scholar Our results elucidate novel structural aberrations affecting the noncoding region of RAB27A, linking the lack of hypopigmentation to differential RAB27A TSS usage between lymphocytes and melanocytes. The identification of a recurrent complex SV in RAB27A suggests a founder effect in the Baltic population. Assessment of this aberration should be included in the genetic workup of patients with defective exocytosis from this area. We thank all family members for their participation. We also thank Stanley Sing Hoi Cheuk for providing melanocytes, the Department of Clinical Genetics of the Karolinska University Hospital for help with MLPA, and the Clinical Genomics unit at SciLifeLab for whole-genome sequencing. Blood samples from the patients and, when available, their parents and siblings were obtained with informed consent according to the Declaration of Helsinki. The study was approved by the Regional Ethics Review Board in Stockholm, Sweden. DNA was isolated according to standard procedure. DNA from P1 and P2 was subjected to whole-genome sequencing with a TruSeq DNA PCR-free protocol followed by sequencing on an Illumina HiSeq X machine with an average coverage of 30×. Sequencing reads were analyzed with the pipeline SpeedSeq. In brief, reads were mapped to the human genome build GRCh37 with burrows-wheeler aligner, while calling of single nucleotide variants and structural variants was performed, respectively, with FreeBayes and Lumpy.E1Chiang C. Layer R.M. Faust G.G. Lindberg M.R. Rose D.B. Garrison E.P. et al.SpeedSeq: ultra-fast personal genome analysis and interpretation.Nat Methods. 2015; 12: 966-968Crossref PubMed Scopus (281) Google Scholar The genomic region spanning RAB27A and PIGB was visualized with the Integrative Genomics Viewer.E2Thorvaldsdóttir H. Robinson J.T. Mesirov J.P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration.Brief Bioinform. 2013; 14: 178-192Crossref PubMed Scopus (4917) Google Scholar Integrative Genomics Viewer was also used to visualize discordant and split reads at the breakpoints. Split reads were mapped to the reference genome using the BLAT function of the University of California, Santa Cruz (UCSC) genome browser. To confirm the presence of the SV, primers were designed to PCR amplify the breakpoints of the mutant allele based on the information from discordant and split reads. PCR products were sequenced on an ABI3730 Genetic Analyzer. The breakpoint C-invE (Fig 2, B) was captured with the following primers: the forward 5′-CAACAGTTATGCGGCTCTCA-3′ and the reverse 5′-TGCGTGCAGACTGGATAAAG-3′. To validate the presence of copy number alterations affecting RAB27A and PIGB, we used a custom-designed MLPA assay. MLPA probes were designed to cover the regions involved by the structural variants together with control probes to be used for normalization of the data. Probe sequences are listed in Table E2. The MLPA procedure was carried out according to the manufacturer's instructions (MRC-Holland, Amsterdam, The Netherlands). PCR products were separated on an ABI3500 XL Genetic Analyzer and analyzed with Peak Scanner 2.0 software. PBMCs from the families of P3 and P4, and healthy unrelated controls, were isolated within 24 hours of venipuncture and analyzed by flow cytometry for NK-cell and cytotoxic T-cell exocytosis at resting state and after 36 hours of 500U IL-2 stimulation (R&D Systems, Minneapolis, Minn) as previously described.E3Chiang S.C.C. Theorell J. Entesarian M. Meeths M. Mastafa M. Al-Herz W. et al.Comparison of primary human cytotoxic T-cell and natural killer cell responses reveal similar molecular requirements for lytic granule exocytosis but differences in cytokine production.Blood. 2013; 121: 1345-1356Crossref PubMed Scopus (100) Google Scholar NK-cell–mediated cytotoxicity was assessed via standard 51Cr 4-hour assay, using labeled K562 cells as target, as previously described.E4Meeths M. Chiang S.C.C. Wood S.M. Entesarian M. Schlums H. Bang B. et al.Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) caused by deep intronic mutation and inversion in UNC13D.Blood. 2011; 118: 5783-5793Crossref PubMed Scopus (85) Google Scholar FlowJo (v9.9, Treestar, Ashland, Ore) was used for flow cytometry and Prism (v7, Graphpad, La Jolla, Calif) for statistical analyses and plotting. Quantitative PCR was used to quantify RAB27A and PIGB transcript in PBMC-derived RNA from P3 and his parents, compared with unrelated healthy controls. Similarly, quantitative PCR was used to quantify transcripts in RNA isolated from melanocytes of healthy donors (n = 2). For RAB27A, one primer pair was designed to capture all isoforms, and another to capture only the isoform NM_183235.2, disrupted by the SV. For PIGB, primer pairs were designed to target the mRNA before (upstream) and after (downstream) the breakpoint. Publicly available FANTOM5 data were downloaded from http://fantom.gsc.riken.jp/5/data/ (on June 26, 2017). The data set consists of cap analysis gene expression peaks for human samples in the form of relative log expression normalized data.E5The FANTOM Consortium and the RIKEN PMI and Clst (dgt)A promoter-level mammalian expression atlas.Nature. 2014; 507: 462-470Crossref PubMed Scopus (1244) Google Scholar Data for biological replicates (n = 3) were averaged. Further analysis and plotting was done in R version 3.3.1. Cryopreserved PBMCs of patients (P3 and P4) and family members as well as local and transport controls were used to analyze Rab27a protein expression levels. To this end, freshly thawed cells were counted, sedimented, and resuspended in ice-cold lysis buffer (150 mM sodium chloride, 50 mM Tris, 2 mM EDTA, pH 7.6) supplemented with 1% (v/v) Triton X-100, 1% (v/v) Igepal CA-630 (Sigma-Aldrich, St Louis, Mo), and protease inhibitors (ThermoFisher, Waltham, Mass). Postnuclear lysates were separated by SDS-PAGE under reducing conditions (NuPage, ThermoFisher) and transferred to nitrocellulose membranes using the iBlot transfer system (ThermoFisher). Rabbit pAbs against Rab27a and Calnexin were purchased from ProteinTech Group and Enzo Life Science, respectively. For western blot performed on PBMCs from patient 5, the following antibodies were used: anti-RAB27A rabbit polyclonal antibodies #5415 and #5416, diluted 1:5000 (kind gift from Gillian Griffiths) and anti-beta-Actin mouse mAb, diluted 1:10,000 (Clone AC-15, Sigma Aldrich #A1978). Signals were visualized using ECL chemistry (ThermoFisher) and acquired using the Li-Cor Odyssey Fc system or the Fusion Imaging System (Vilber, Collégien, France). P1, a girl of Lithuanian origins, presented at the age of 6 years with acute multiple vomiting episodes, headache, weakness, ataxia, and confusion. Cerebrospinal fluid analysis showed minimal pleocytosis and normal protein level. Brain magnetic resonance imaging (MRI) revealed multiple T2 hyperintensive lesions in cerebellum, periventricular regions, corpus callosum, and brain stem, prompting a diagnosis of acute disseminated encephalomyelitis (Fig E1, A). She subsequently experienced 8 relapses within the next 2 years managed with intravenous and oral steroids. During remissions she suffered from mild ataxia and tremor. At the age of 8 years, she was diagnosed with remitting-relapsing multiple sclerosis and started to receive preventive treatment with monthly intravenous immunoglobulin infusions for 18 months and achieved long-lasting remission. During follow-up, she underwent MRI investigations that demonstrated similar patterns, and in addition development of cerebellar atrophy. At age 13 years, following herpes simplex labialis infection, the patient presented with fever, jaundice, hepato- and splenomegaly, pancytopenia, hyperferritinemia, hypofibrinogenemia, and hypoalbuminemia, followed by ascites, pleural effusion, and encephalopathy. Liver enzymes were markedly elevated. Bone marrow biopsy showed no hemophagocytosis. Analysis of cytotoxic lymphocyte function showed defective cytotoxicity and exocytosis (Table E1). Neuroimaging showed nonspecific multifocal hyperintense white matter lesions in both hemispheres in FLAIR MRI (Fig 1, B). Nineteen days later the patient fulfilled the HLH diagnostic criteria (Table E1). Treatment according to HLH-2004 protocol was initiated but the patient's condition deteriorated and she died 48 days later because of massive central nervous system (CNS) lesions provoked by CNS infection (EBV reactivation and Aspergillus fumigatus). Her older brother died at the age of 1 year because of infectious mononucleosis. The patient displayed no signs of oculocutaneous albinism. P2, a boy of Swedish origin, was diagnosed with benign granuloma of the skin at the age of 2 years. At the age of 2.5 years he suffered from rapidly progressive sensorineuronal hearing loss. At age 3 years, he developed fever, breathing difficulties with interstitial lung infiltrates, hepatosplenomegaly, increased liver enzymes, deteriorating motoric abilities, and low immunoglobulins. A primary EBV infection was detected. Recovery was obtained with antibiotic and antimycotic treatment, and immunoglobulin replacement. He suffered from recurrent episodes regarded as fungal pneumonias, although a fungal infection was never verified. At the age of 14.5 years, he deteriorated and was admitted to the hospital because of fever and coughing. An X-ray revealed interstitial lung disease. He also displayed splenomegaly, pericardial fluid, and elevated liver enzymes. One and a half week later he was again hospitalized with fever and low white blood cells. Spleen biopsy showed infiltration of histiocytic cells and Aspergillus fumigatus. A bone marrow examination did not show signs of malignancy and no or very little hemophagocytosis. He received antibiotics, antimycotics, and corticosteroids. At this point, fulfilling diagnostic criteria (Table E1), a diagnosis of HLH was established, and a secondary form of HLH was suspected. His condition deteriorated, with respiratory difficulties and increased inflammation. He received cyclosporin, antithymocyte globulin, and corticosteroids and later also etoposide. However, he continued to deteriorate and later died in the intensive care unit because of cardiac arrest. Genetic sequencing of PRF1 was performed at the time without findings. One of his 3 sisters died at age 13 years because of an intracerebral tumor, later diagnosed as T-cell lymphoma (immunoblastic lymphoma). Another sister died at age 13 years in a clinical picture of interstitial pneumonia, aplastic anemia, aspergillus, liver disease, and progressive paresis. She also suffered from hearing loss. The third sister is healthy. The patient displayed no signs of oculocutaneous albinism. P3 is a second child of healthy nonconsanguineous parents of Lithuanian origin. At the age of 5 years, the boy was admitted because of acute ataxia, strabismus, diplopia, sleepiness, and papilledema. Three weeks earlier the patient had an episode of acute respiratory infection. There were multiple focal demyelinating lesions on brain MRI (Fig E1, B). He was diagnosed as having acute demyelinating encephalomyelitis, and intravenous methylprednisolone pulse markedly improved symptoms. Eight months later he was admitted to the intensive care unit because of acute respiratory failure, fever, and reappearance of previously observed neurological symptoms. He also had a maculopapular rash. Skin biopsy showed granulomatous dermatitis, CD3/CD4- and CD3/CD8-positive cells, and CD68-positive histiocytes (Fig 1, C). Lung computed tomography showed focal confluent infiltration and enlarged mediastinal lymph nodes (1-1.3 cm; Fig E1, C). A lung biopsy showed focal lymphocyte and macrophage infiltration (predominance of CD3 T lymphocytes, 20% CD20-positive cells, CD68 macrophages). Slightly decreased immunoglobulin G subtypes were found, but routine test results for cellular immunity were normal. The patient became steroid-dependent for the subsequent 3 years, after which, at the age of 9 years, following tonsillitis, he developed persistent fever with elevated liver enzymes, hepatomegaly, neutropenia, and anemia. Analysis of cytotoxic lymphocyte function showed defective cytotoxicity and exocytosis (Fig 1, D-F). A diagnosis of HLH was established (Table E1) and treatment according to HLH-2004 protocol was initiated. At the age of 10 years, P3 received a hematopoietic stem cell transplantation (HSCT) from his HLA-identical brother. A reduced intensity conditioning based on fludarabine (180 mg/m2) and melphalan (140 mg/m2) was used. For graft-versus-host disease (GvHD) prophylaxis, cyclosporine A and mycophenolate mofetil were used. The patient developed an acute GvHD grade II (skin +++, GI +), which later developed into a chronic form with skin involvement that has resolved. The patient displayed no signs of oculocutaneous albinism (Fig E1, D). P4 is the second child of healthy nonconsanguineous parents from Lithuania. P4 developed skin granulomas at the age of 16 months. At the age of 1 year 9 months, she developed ataxia, impaired walking, and tremor after varicella infection. The symptoms improved following a course of prednisone, but subsequent similar attacks followed. In 2 exacerbation episodes EBV reactivation was detected. MRI showed numerous demyelinating foci in the cerebellum, and a massive confluent conglomerate in the splenium involving both hemispheres and the spinal cord. At the age of 7 years 8 months, the biopsy of CNS lesions was performed showing nonmalignant reactive lymphoid infiltrates (CD3+, CD4+, CD8+, GranB+, CD30−), nonspecific granulomatous microfoci. No signs characteristic of HLH were detected (Table E1). Analysis of cytotoxic lymphocyte function showed defective cytotoxicity and exocytosis (Fig 1, G-I). At the age of 8 years, P4 received an HSCT from a matched unrelated donor. A reduced-intensity conditioning based on fludarabine (150 mg/m2) and treosulphan (42 g/m2) was used. For GvHD prophylaxis, alemtuzumab (0.6 mg/kg), cyclosporine A, and mycophenolate mofetil were used. At the time of report, the patient is 2 months after HSCT. The patient displayed no signs of oculocutaneous albinism. P5 is a 14-year-old boy from Russia. He had uncomplicated infectious mononucleosis at age 2 years without signs of HLH and good clinical recovery. At the age of 10 years, he developed recurrent fever flares lasting up to 3 weeks without obvious trigger. The overall condition was not significantly impaired. There were no significant cytopenias and the patient never met diagnostic criteria for HLH. At the age of 11.3 years, the patient rapidly developed splenomegaly with pancytopenia. Splenectomy corrected the cytopenia and diminished the frequency and intensity of fever flares. Enlargement of abdominal lymph nodes was noted and a biopsy showed only follicular hyperplasia. At the age of 12.8 years, he had sudden onset of cervical lymphoadenopathy and was diagnosed with Hodgkin lymphoma, stage IVA. He responded well to treatment according to the GPOH-HD 2002 protocol and is currently in full remission. The combination of recurrent fever flares, splenomegaly, and lymphoma prompted further immunological investigations. NK-cell and cytotoxic T lymphocytes cytotoxicity and exocytosis were defective (Table E1). The patient displayed no signs of oculocutaneous albinism.Fig E2A, NK-cell cytotoxicity assay in IL-2–stimulated PBMCs from P3 and relatives. B, NK-cell degranulation assay in IL-2–stimulated PBMCs from P3 and relatives. C, CD8+CD57+ T-cell degranulation assay in IL-2–stimulated PBMCs from P3 and relatives. D, NK-cell cytotoxicity assay in IL-2–stimulated PBMCs from P4 and relatives. E, NK-cell degranulation assay in IL-2–stimulated PBMCs from P4 and relatives. F, CD8+CD57+ T-cell degranulation assay in IL-2–stimulated PBMCs from P4 and relatives. G, Western blot analysis of Rab27a in P4 and relatives. H, Western blot analysis of Rab27a in P5.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3A, Sanger trace for breakpoint C-invE in all patients. B, Quantitative PCR of PIGB to quantify RNA from PBMCs isolated in P3 and relatives. C, UCSC Genome browser screenshot showing localization of the RAB27A TSS according to FANTOM5 CAGE data from human cells. p1 is the TSS for the isoform NM_183235.2. D, Tags per million at the different RAB27A TSS from the FANTOM5 CAGE data. E, Tags per million at the different RAB27A TSS during differentiation of human embryonic stem cells into pigmented melanocytes (d, day). CAGE, Cap analysis gene expression.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Clinical and laboratory findings at diagnosis of HLHFamily12345PatientP1P2P3P4P5Ethnical originLithuaniaSwedenLithuaniaLithuaniaRussiaFamilial diseaseYesYesNoNoNoParental consanguinityNoNoNoNoNoSexFemaleMaleMaleFemaleMaleRAB27A allele 1Deletion, c.559C>T (p.Arg187Trp)Dup-InvDup-InvDup-InvDup-InvRAB27A allele 2Dup-Invc.239G>C (p.Arg80Thr)Dup-InvDup-Invc.550C>T (p.Arg184*)Age at diagnosis of HLH (y)1414.59No HLHNo HLHFeverYesYesYesNoYes (intermittent)SplenomegalyYesYesNoNoYes, chronic (splenectomized)HepatomegalyYesYesYesNoTransient, self-limitingHemoglobin (g/L)838790Within normalNDNeutrophils (109/L)0.450.800.14Within normalNDPlatelets (109/L)3554194Within normalNDTriglycerides (mmol/L)2.495.82.71Within normalNDFibrinogen (g/L)0.60.40.61.51NDHemophagocytosisNoNoYesNDNDFerritin (μg/L)24514900012000Within normalNDSoluble CD25 (pg/mL)20512.4ND164371760NDNK-cell activityNDNDDefectiveDefectiveDefectiveNK-cell degranulationDefective, 3.1% ΔCD107aNDDefective, 6% ΔCD107aDefective, 3.3% ΔCD107aDefective, 0.6% ΔCD107aNeurological manifestationsYes, before HLH onsetYesYes, before HLH onsetYesNoPathological CSFYesNDYesYesNDTreatment active diseaseHLH-2004Cortico, CSA, ATGHLH-2004Cortico, MMFRemission at 2 moNoNoYesNAAge at HSCTNot done107Outcome and follow-upDeceased 48 d after HLH onsetAlive 19 mo after HSCTAlive at 7 yAlive at 16 yOther manifestationsNoSkin granuloma, lung infiltratesSkin granuloma, lung infiltratesSkin granuloma, isolated CNS involvementHodgkin lymphoma at 13 y, recurrent fever episodes from 10 yATG, Antithymocyte globulin; Cortico, corticosteroids; CSA, cyclosporine A; CSF, cerebrospinal fluid; Dup-Inv, Duplication-Inversion; MMF, mycophenolate mofetil; NA, not applicable/available; ND, no data. Open table in a new tab Table E2MLPA probesGeneSize (bp)Used for Figure 2, CGABRA4_fam-pilot84Control probeRAB27A_probe587Probe C in Figure 2, CRAB27A_probe690RAB27A_probe793RAB27A_probe896Probe D in Figure 2, CRAB27A_probe499Probe B in Figure 2, CRAB27A_probe3102Stern PCLN13q105Control probeRAB27A_probe10108Probe E in Figure 2, CRAB27A_probe9111RAB27A_probe1114Probe A in Figure 2, CPIGB_probe11117Probe F in Figure 2, CRB1ex23129Control probeMRPL41_e1_132132Control probeSRY135Control probeGABRA4_fam-pilot_AgggttccctaagggttggaCAGCCTGTTGTCATAACCATCGGABRA4_fam-pilot_B/5Phos/AGCAAACTGTCCAGGATGCGtctagattggatcttgctggcacStern PCLN13q_AgggttccctaagggttggaGACACAAGGGTGTAAAATGCACGStern PCLN13q_B/5Phos/TTTCAGGGTGTGTTTGCATATGATTTAATCAATCAGTATGtctagattggatcttgctggcacRB1ex23_AgggttccctaagggttggaGTCACCAATACCTCACATTCCTCGAAGCCCTTACAAGTTTCCTRB1ex23_B/5Phos/AGTTCACCCTTACGGATTCCTGGAGGGAACATCTATATTTCACCtctagattggatcttgctggcacMRPL41_e1_132_AgggttccctaagggttggaGACCCTGACAACCTGGAAAAGTACGGCTTCGAGCCCACACAGGAGMRPL41_e1_132_B/5Phos/GGAAAGCTCTTCCAGCTCTACCCCAGGAACTTCCTGCGCTAGCTGtctagattggatcttgctggcacSRY_AgggttccctaagggttggaCAGTGCAAAGGAAGGAAGAGCTTCTCCGGAGAGCGGGAATATTCTSRY_B/5Phos/CTTGCACAGCTGGACTGTAATCATCGCTGTTGAATACGCTTAACATAGtctagattggatcttgctggcacRAB27A_p5_AgggttccctaagggttggaGGACTTCAGGAAGCTGCAATGTTTRAB27A_p5_B/5Phos/GCTTTTGTGAATTTCCTTCCCtctagattggatcttgctggcacRAB27A_p6_AgggttccctaagggttggaCACAGAACCTCAGAGAAGCCTTGARAB27A_p6_B/5Phos/GGGGCAACTGGTCAACCAATTAGGtctagattggatcttgctggcacRAB27A_p7_AgggttccctaagggttggaCAGACCTCGTGGTTCCCACTCAGARAB27A_p7_B/5Phos/GGCAGCCAAGTTCATCCCTCTCAGTGGtctagattggatcttgctggcacRAB27A_p8_AgggttccctaagggttggaGCCTTCTAAGCCGTCTCGCTGACTTGTRAB27A_p8_B/5Phos/GTCTACCTCCACCGCAAATTCCAGCTGtctagattggatcttgctggcacRAB27A_p4_AgggttccctaagggttggaGATCCAACTGCTCCCTTCAAGAAGTTRAB27A_p4_B/5Phos/GGTAATTAGGGTGAGGTGGAATGATGTACTCtctagattggatcttgctggcacRAB27A_p3_AgggttccctaagggttggaCAACTGGCCAGCTGTCACTCAAATGCTAATTRAB27A_p3_B/5Phos/GTGTCTATCATCTGCTTTCTCTAATAGCCtctagattggatcttgctggcacRAB27A_p10_AgggttccctaagggttggaGTGGCAAGATGGGTGGTAAGTCCTAAATACTTTARAB27A_p10_B/5Phos/GAAGCTGTATGCCAGTTATTTCGTTCCTATGGtctagattggatcttgctggcacRAB27A_p9_AgggttccctaagggttggaGCTTTAAGAATGGTGTGGAGGGACCAGAGGTCACTARAB27A_p9_B/5Phos/CTGTGCCTTACAAGGAGCCAACCAGAGCAGCAGtctagattggatcttgctggcacRAB27A_p1_AgggttccctaagggttggaGAGGCATGACCATTTGATCGCACCACTCCTTCAGGAATRAB27A_p1_B/5Phos/CCAGGACTTGTCCACACACCGTTCCATTCGCTTCtctagattggatcttgctggcacPIGB_p_11_AgggttccctaagggttggaGAGGACCATTTATGTTTCCGGAACAGAATACCAATGCTAPIGB_p_11_B/5Phos/CAGAATGTTGAGTCCCCCTACTGACCTACTTCCCTCtctagattggatcttgctggcac Open table in a new tab ATG, Antithymocyte globulin; Cortico, corticosteroids; CSA, cyclosporine A; CSF, cerebrospinal fluid; Dup-Inv, Duplication-Inversion; MMF, mycophenolate mofetil; NA, not applicable/available; ND, no data.