Edited by: Antonio Condino-Neto, University of São Paulo, Brazil
Reviewed by: Alain Fischer, Institut National de la Santé et de la Recherche Médicale (INSERM), France; Hirokazu Kanegane, Tokyo Medical and Dental University, Japan
*Correspondence: Mitsunori Fukuda,
This article was submitted to Primary Immunodeficiencies, a section of the journal Frontiers in Immunology
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Griscelli syndrome type 2 (GS-2) is an inborn error of immunity characterized by partial albinism and episodes of hemophagocytic lymphohistiocytosis (HLH). It is caused by
Griscelli syndrome type 2 (GS-2; MIM#607624) is an inborn error of immunity (IEI) characterized by partial albinism and the occurrence of acute phases of hemophagocytic lymphohistiocytosis (HLH) (
RAB27A is highly expressed in melanocytes and a variety of secretory cells, including lymphocytes (
In cytotoxic T lymphocytes (CTLs), RAB27A controls secretion of cytolytic granules by binding the priming factor MUNC13-4 (
Genetic and clinical characteristics of patients with GS-2 sine albinism.
Family | Patient | Age (year) | Sex | Mutation | Protein change | Inheritance | RAB27A interaction with MUNC13-4 | RAB27A interaction with MLPH | NK cytotoxicity | NK degranulation | Clinical | Treatment | Outcome | Ref |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
I | 1 | 2.7 | M | c.428T>C | Val143Ala | Homo | Disrupted | Nl | Defective | Defective | CNS HLH | HLH-2004 |
Died | Current study |
II | 2 | 0.5 | M | c.422-424delGAG | Arg141—Val142delinsIle | Homo | Disrupted | Nl | ND | ND | HLH | – | Died | J Allergy Clin Immunol. 2015 May;135(5):1310-8 |
3 | 0.5 m | M | c.422-424delGAG | Arg141—Val142delinsIle | Homo | Disrupted | Nl | Defective | ND | HLH | HSCT | Died | J Allergy Clin Immunol. 2015 May;135(5):1310-8 | |
4 | 10.7 | F | c.422-424delGAG | Arg141—Val142delinsIle | Homo | Disrupted | Nl | Defective | Defective | HLH | HSCT | Alive | J Allergy Clin Immunol. 2015 May;135(5):1310-8 | |
III | 5 | 7.3 | M | c.422-424delGAG |
Arg141—Val142delinsIle |
Compound hetero | Disrupted |
Nl |
Defective | Defective | HLH | HSCT | Alive | J Allergy Clin Immunol. 2015 May;135(5):1310-8 |
IV | 6 | 4 | F | c.227C>T |
Ala76Val |
Compound Hetero | Disrupted |
Nl |
Defective | Defective | HLH | HSCT | Alive | J Allergy Clin Immunol. 2015 May;135(5):1310-8 |
V | 7 | 5 | M | c.422-424delGAG |
Arg141—Val142delinsIle |
Compound Hetero | Disrupted |
Nl |
Defective | Defective | HLH | HSCT | Alive | J Allergy Clin Immunol. 2015 May;135(5):1310-8 |
VI | 8 | 9 | F | c.244C>T | Arg82Cys | Homo | Disrupted | Nl | Defective | Defective | CNS HLH |
DEX |
Died | J Allergy Clin Immunol. 2016 Aug;138(2):599-601 |
9 | 8 | M | c.244C>T | Arg82Cys | Homo | Disrupted | Nl | Defective | Defective | No | No | Alive | J Allergy Clin Immunol. 2016 Aug;138(2):599-601 | |
10 | 5 | M | c.244C>T | Arg82Cys | Homo | Disrupted | Nl | Defective | Defective | No | No | Alive | J Allergy Clin Immunol. 2016 Aug;138(2):599-601 | |
VII | 11 | 14 | F | Large 5’ UTR dup/inv/del |
SV |
Compound Hetero | ND | ND | Defective | Defective | Neuroinflammation |
HLH-2004 | Died | J Allergy Clin Immunol. 2018 Jul;142(1):317-321 |
VIII | 12 | 14.5 | M | Large 5’ UTR dup/inv |
SV |
Compound Hetero | ND | ND | ND | ND | Neuroinflammation |
HLH-2004, ATG | Died | J Allergy Clin Immunol. 2018 Jul;142(1):317-321 |
IX | 13 | 9 | M | Large 5’ UTR dup/inv | SV | Homo | ND | ND | ND | Defective | Neuroinflammation |
HLH-2004 |
Alive | J Allergy Clin Immunol. 2018 Jul;142(1):317-321 |
X | 14 | 8 | F | Large 5’ UTR dup/inv | SV | Homo | ND | ND | Defective | Defective | Neuroinflammation |
Steroid |
Alive | J Allergy Clin Immunol. 2018 Jul;142(1):317-321 |
XI | 15 | 13 | M | Large 5’ UTR dup/inv |
Arg184* | Compound Hetero | ND | ND | Defective | Defective | Hodgkin lymphoma | GPOH-HD 2002 | Alive | J Allergy Clin Immunol. 2018 Jul;142(1):317-321 |
XII | 16 | 14 | M | c.400-401delAA |
Lys134Glufs*2 |
Compound Hetero | ND | ND | Defective | Defective | Large B cell Lymphoma |
HLH-94 |
Died | Pediatr Blood Cancer. |
ATG, anti-thymocyte globulin; DEX, dexamethasone; CNS, central nervous system; GPOH-HD, German Society of Pediatric Oncology and Hematology-Hodgkin’s Disease; HLH, hemophagocytic lymphohistiocytosis; HSCT, hematopoietic stem cell transplantation; LIP, lymphocytic interstitial pneumonitis; MFM, mycophenolate mofetile; ND, not determined; Nl, normal; SV, structural variant.
Horseradish peroxidase (HRP)-conjugated anti-FLAG tag mouse monoclonal (M2) antibody, and anti-FLAG tag antibody-conjugated agarose beads were obtained from Sigma-Aldrich (St. Louis, MO, USA). HRP-conjugated anti-T7 tag mouse monoclonal antibody and anti-T7 tag antibody-conjugated agarose beads were from Novagen™, Merck KGaA (Darmstadt, Germany). Anti-GFP rabbit polyclonal antibody and anti-β-actin mouse monoclonal antibody (C043) were also obtained from MBL (Nagoya, Japan) and Applied Biological Materials (Richmond, BC, Canada), respectively.
Blood and hair samples were obtained with informed consent according to the Institutional Review Boards’ guidelines of the Children’s Medical Center. Genomic DNA was obtained from whole blood by the conventional salting-out method. Whole exome sequencing was performed on a patient sample, as previously described (
Cytotoxic T cell culture (CTL) were prepared by stimulating peripheral blood mononuclear cells (PBMCs) with PHA (1.25 μg/ml) and ∼100 U/ml human IL-2 (produced from a transfected cell line) in the presence of irradiated (30 Gy for 5 min) allogeneic PBMCs as feeder cells. RPMI (Gibco, ThermoFisher, UK) with 5% human serum (Sigma-Aldrich, USA) and ∼100 U/ml human IL-2 was used as culture medium. Every 14 to 18 days, T cells were re-stimulated as above.
T cell cultures were washed in ice-cold sterile PBS and lysed at 2 × 107/ml in lysis buffer [50 mM Tris–HCl (pH7.4), 150 mM NaCl, 1% Triton X-100, 1% NP-40, 2 mM EDTA, and 1x Halt™ protease inhibitors (Thermo Fisher)] for 30 min on ice before cell debris was pelleted by centrifugation at 13,000 for 25 min at 4°C. 22.5 µl cell lysate was mixed with 7.5µl 4x NuPAGE™ LDS reducing sample buffer [Tris base (141 mM), Tris HCl (106 mM), LDS (2%), EDTA (0.51 mM), and 50mM DTT] and heated at 95°C for 5 min before separation on a 4–12% NuPAGE™ Bis-Tris gel in MES running buffer [MES (50 mM), Tris base (50 mM), sodium dodecyl sulfate (SDS) (0.1%), EDTA (1 mM), pH 7.3 (all Thermo Fisher)]. Precision Plus Protein Kaleidoscope Prestained Protein Standards (Bio-Rad, Hercules, CA, USA) were also run. Proteins were transferred to nitrocellulose membranes [Trans-Blot® Turbo™ Mini Nitrocellulose Transfer Packs (Bio-Rad) using the mixed molecular weight program of a semi-dry Trans-Blot Turbo Transfer System (Bio-Rad)]. Membranes were blocked in TBS, 5% non-fat dried milk, and 0.05% Tween-20 (Sigma-Aldrich). Membranes were incubated with primary antibodies (rabbit anti-RAB27A (see ref.12) at 1:1,000 in blocking buffer at 4°C overnight or 1:1,000 rabbit anti-calnexin (C4731, Sigma-Aldrich) for 1 h at room temperature. Membranes were washed 4× for 5 min in TBS, 0.05% Tween, and incubated with 1:10,000 goat anti-rabbit (H+L) HRP labeled secondary antibodies (Thermo Fisher) in blocking buffer for 45 min at room temperature. Membranes were washed as before, developed using ECL Prime Western Blotting solution (Amersham), and imaged using a Bio-Rad ChemiDoc.
Degranulation of cultured T cells was analyzed at day10 after re-stimulation, and T cells were starved overnight without IL-2 (
This was as described in (
Multiwell microscopy slides were cleaned with 70% ethanol for 15 min at room temperature and were coated with 0.01% poly-L-lysine (Millipore Sigma, UK) for 15 min, washed with PBS, and coated with 10 μg/ml hamster humanized anti-CD3 antibody (ChAgly, a gift from Herman Waldmann). Cultured T cells were washed and added in FCS-free IMDM and were allowed to adhere for 12 min. Cells were fixed for 15 min in 4% paraformaldehyde (PFA) (15710-S, Electron Microscopy Systems, USA), permeabilized in 0.1% Triton X-100, and blocked in 2% BSA in PBS (40 min). Samples were labeled for 1 h with primary antibodies [pericentrin (Abcam, UK)] and LAMP1 (H4A3, hybridoma supernatant) and phalloidin followed by fluorophore-conjugated secondary antibodies (donkey anti-mouse 488 and goat anti-rabbit 647, Invitrogen, UK) (1 h at RT) together with phalloidin 568 (Invitrogen, UK). Nuclei were stained for 5 min at RT with Hoechst 33342 (H3570, Thermo Fisher, UK), and samples were mounted in ProLong Diamond Antifade Reagent (P36961, Thermo Fisher, UK). Images were taken with an IX81 Olympus microscope equipped with an Andor Revolution system fitted with a CSU-X1 spinning-disk unit (Yokogawa, UK).
Mutant human RAB27A expression plasmids carrying a Val-to-Ala mutation at amino acid position 143 were produced by inverse PCR techniques essentially as described previously (
The black mouse-derived immortal melanocyte cell line melan-a and
Two immortal mouse melanocyte cell lines were cultured on coverslips and fixed with 4% PFA for 10 min. The coverslips were incubated for 1 h with DAPI. The samples were mounted using ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, Waltham, MA). Infected cells were identified by EGFP fluorescence, and their fluorescence images, together with the corresponding bright-field images, were captured at random with an FV1000D confocal fluorescence microscope and Fluoview software (Olympus, Tokyo, Japan). Melanosome distribution was assessed by examination of images of infected melan-a/ash cells (more than 25 cells/dish, three independent dishes for each transfection). Cells in which more than 50% of the melanosomes were present around the nucleus were judged to be aggregated as described previously (
COS-7 cells were co-transfected for 24 h with pEF-FLAG-RAB27A (wild-type or a Val143Ala mutant) and pEF-T7-MUNC13-4, pEF-T7-SLP2-A, or pEF-T7-MLPH/SLAC2-A by using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific). The transfected cells were lysed with a lysis buffer [50 mM HEPES-KOH, pH 7.2, 150 mM NaCl, 1 mM MgCl2, and 1% Triton X-100 supplemented with complete EDTA-free protease inhibitor mixture (Roche, Basel, Switzerland)]. The cell lysates were incubated for 1 h at 4°C with anti-FLAG or anti-T7 tag antibody-conjugated agarose beads. The beads were washed three times with a washing buffer (50 mM HEPES-KOH, pH7.2, 150 mM NaCl, 1 mM MgCl2, and 0.1% Triton X-100), and proteins bound to the beads were analyzed by 10% SDS-polyacrylamide gel electrophoresis (PAGE) followed by immunoblotting with HRP-conjugated anti-FLAG and anti-T7 tag antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence.
Statistical tests were performed using Tukey’s test, and
The patient was a boy of Iranian origin and was born to consanguineous parents. Informed consent was obtained from the parents. He had no albinism, and his hair shafts showed fairly normal pigmentation (
Clinical and genetic findings of the patient
Whole exome sequencing revealed a homozygous missense c.428T>C (Val143Ala) variant in exon 6 of
In line with other homozygous mutations in the
Impaired degranulation and killing of patient cytotoxic T cells
To evaluate the effect of the Val143Ala mutation on the melanosomal localization of RAB27A, an EGFP (enhanced green fluorescence protein)-tagged RAB27A (Val143Ala) mutant was stably expressed in black mouse-derived melanocytes [melan-a cells (
Subcellular localization of RAB27A(Val143Ala) mutant and its effect on melanosome distribution in wild-type melanocytes. Melan-a cells stably expressing either EGFP alone (top panels), EGFP-RAB27A (WT, wild-type) (middle panels), or EGFP-RAB27 (Val143Ala) (indicated as V143A; bottom panels) (EGFP in green and DAPI in blue). The insets show magnified views of the boxed areas (melanosomes are pseudo-colored in magenta). Note that RAB27A (Val143Ala) was mostly present in the cytosol and only weakly co-localized with melanosomes, whereas wild-type RAB27A was present on melanosomes. Scale bars, 20 µm.
Partial melanosomal localization of EGFP-RAB27A (Val143Ala) in melan-a cells further prompted us to investigate whether this mutant supports melanosome transport in the absence of an endogenous mouse RAB27A. To this end, we stably expressed the Val143Ala variant in RAB27A-deficient immortalized melanocytes [melan-ash cells (
RAB27A (Val143Ala) partially restored peripheral melanosome distribution in RAB27A-deficient melanocytes
Because RAB27A is known to regulate actin-based melanosome transport in melanocytes and lytic granule exocytosis in cytotoxic T lymphocytes through interaction with cell-type-specific or tissue-specific effectors (
RAB27A effector binding activities of RAB27A(Val143Ala). Interaction between T7-MUNC13-4 and FLAG-RAB27A (WT or Val143Ala; indicated as V143A)
In the present study, we biochemically and functionally analyzed a novel homozygous
The effect of the Val143Ala mutation in melanocytes was clearly different from that of two previously characterized GS-2 mutations (Lys22Arg and Ile44Thr) with partial albinism in our previous studies. The Lys22Arg mutation caused cytosolic localization of EGFP-RAB27A (Lys22Arg) because of its defect in GTP binding activity, whereas the Ile44Thr mutation had no effect on the melanosomal localization of RAB27A (Ile44Thr) (
We noted the fact that Val143 of human RAB27A is located at a bend region between the β5 strand and the α4 helix of the common RAB GTPase structure and that it is not located in the consensus phosphate/magnesium-binding motifs or guanine base-binding motifs. However, Val in this position is invariant among the RAB27 subfamily proteins from different species (
To date, a total of 16 patients from twelve families have been reported to present with GS-2 sine albinism (
Structure of human RAB27A and localization of mutations in patients with GS-2 sine albinism. A 3D model of human RAB27A complexed with GTP was depicted by UCSF CHIMERA (
In conclusion, the results of our analyses of the Val143Ala mutation of RAB27A can explain the phenotype of the GS-2 patient without albinism. The Val143Ala mutation impairs both the RAB27A–SLP2-A interaction and RAB27A–MUNC13-4 interaction, but it has no effect on the RAB27A–MLPH/SLAC2-A interaction that is crucial for skin and hair pigmentation (
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below:
The studies involving human participants were reviewed and approved by The IRB of Children’s Medical Center affiliated to TUMS approved this study (IR.TUMS.CHMC.REC.1399.080). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the minor(s)’ legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.
YO performed the research on melanocytes and binding assays in COS-7 cells, analyzed data, and wrote the manuscript. SA performed killing, artificial synapse, and degranulation assays and wrote the manuscript. VZ, PA, and AR followed the patient clinically. KS and MG performed killing and degranulation assays. CA performed patient western blot. MS performed whole-exome sequencing and Sanger sequencing. GG supervised the CTL derivation, killing, and degranulation assays. SE supervised degranulation and artificial synapse assay. MF supervised the melanocyte research and wrote the manuscript. NP followed the patient, proposed the study, and wrote the manuscript. All authors contributed to the article and approved the submitted version.
This work was supported in part by Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (grant number 19H03220 to MF), and by Japan Science and Technology Agency (JST) CREST (grant number JPMJCR17H4 to MF). Also, funding supporting SA, KS, and GG from the Wellcome Trust 10390 and 100140. SE and MG were funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—SFB1160/2—Project A01. NP is an associate professor of pediatrics at the Tehran University of Medical Sciences (TUMS).
SA worked as a scientific advisor for Sobi. The IRB of Children’s Medical Center affiliated to TUMS approved this study (IR.TUMS.CHMC.REC.1399.080).
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The reviewer HK declared a past co-authorship with one of the authors SE to the handling editor.
The reviewer AF declared a past co-authorship with one of the authors SE to the handling editor.
We thank Jonathan Kaufman for helping to configure the RAB27A 3D structure, Nasrin Alipour for bioinformatics assistance, and Herman Waldmann and Toshio Kitamura for kindly donating materials.