Griscelli Syndrome Type 2 Sine Albinism: Unraveling Differential RAB27A Effector Engagement

Abstract

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 RAB27A mutations that encode RAB27A, a member of the Rab GTPase family. RAB27A is expressed in many tissues and regulates vesicular transport and organelle dynamics. Occasionally, GS-2 patients with RAB27A mutation display normal pigmentation. The study of such variants provides the opportunity to map distinct binding sites for tissue-specific effectors on RAB27A. Here we present a new case of GS-2 without albinism (GS-2 sine albinism) caused by a novel missense mutation (Val143Ala) in the RAB27A and characterize its functional cellular consequences. Using pertinent animal cell lines, the Val143Ala mutation impairs both the RAB27A-SLP2-A interaction and RAB27A-MUNC13-4 interaction, but it does not affect the RAB27A-melanophilin (MLPH)/SLAC2-A interaction that is crucial for skin and hair pigmentation. We conclude that disruption of the RAB27A-MUNC13-4 interaction in cytotoxic lymphocytes leads to the HLH predisposition of the GS-2 patient with the Val143Ala mutation. Finally, we include a review of GS-2 sine albinism cases reported in the literature, summarizing their genetic and clinical characteristics.

Keywords: Griscelli syndrome type 2 sine albinism; MLPH/SLAC2-A; MUNC13-4; RAB27A; hemophagocytic lymphohistiocytosis; inborn error of immunity; whole-exome sequencing.

Figure 1

Clinical and genetic findings of the patient (A). Normal complexion with left-sided Bell’s palsy (B). Fairly normal hair pigmentation of the patient (Val143Ala) determined by light microscopy compared to (C) abnormal clumps of melanin characteristic of GS-2 (Lys22Arg) (D, E). Pedigree of the family showing the familial segregation of RAB27A mutation and chromatograms pertinent to each family member.

Figure 2

Impaired degranulation and killing of patient cytotoxic T cells (A). Ex vivo degranulation of cultured cytotoxic T cells from the patient, heterozygous mother, and a healthy control after incubation with medium (dotted line) or with 1 µg/ml anti-CD3 + L1210 cells was measured by surface expression of CD107a using flow cytometry (B). Collected mean intensity fluorescence (MFI) of 3 independent experiments showing the difference (delta MFI) between unstimulated and stimulated cells (C). Lytic granule mobilization to an artificial immunological synapse was normal of patient and mother CTLs. Microscope slides were coated with 10 µg/ml anti-CD3, CTLs were settled for 10 min, PFA fixed, permeabilized with saponin, and stained for actin (white), pericentrin (yellow), and lytic granules (red). Shown are single slices from the artificial synapse interface formed on the microscope slide and a 90°C rotation of all sliced showing the location of the lytic granules close to the pericentrin. Bottom of the microscope slide in the 90°C side view (plate-bound CD3 localization), is indicated by a red line (D). Percentage lysis of P815-NucLight Red targets over time measured by IncuCyte killing assay using two healthy controls (CTR), the mother and patient CTLs overtime measured for 4 h (E). Protein analysis by western blotting showed normal protein expression of RAB27A in the mother and the patient’s CTLs. To show specificity of the RAB27A antibody a human RAB27A knockout (RAB27-/-) CD8⁺ cell line was run in parallel.

Figure 3

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.

Figure 4

RAB27A (Val143Ala) partially restored peripheral melanosome distribution in RAB27A-deficient melanocytes (A). Melan-ash cells stably expressing either EGFP alone (top panels), EGFP-RAB27A (WT, wild-type) (middle panels), or EGFP-RAB27A (Val143Ala) (indicated as V143A; bottom panels) by retrovirus infection (EGFP in green and DAPI in blue). The insets show magnified views of the boxed areas (melanosomes are pseudo-colored in magenta) and indicate the association of EGFP-RAB27A (Val143Ala) with melanosomes in melan-ash cells. Note that expression of wild-type RAB27A in melan-ash cells completely rescued the perinuclear aggregation phenotype (i.e., peripheral melanosome distribution), whereas expression of RAB27A (Val143Ala) did partially. Scale bars, 20 µm (B). Quantification of the cells with peripheral melanosome distribution shown in (A). The results are expressed as the percentages of infected cells exhibiting peripheral melanosome distribution (means ± S.E. of three determinations; n > 25 in each determination). ***p < 0.001 (Tukey’s test) (C). The protein expression level of EGFP-RAB27A (WT) and EGFP-RAB27A(Val143Ala) in blasticidin-selected melan-ash cells. Cell lysates were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting with the antibodies indicated. The positions of the molecular mass markers (in kilodaltons) are shown on the left.

Figure 5

RAB27A effector binding activities of RAB27A(Val143Ala). Interaction between T7-MUNC13-4 and FLAG-RAB27A (WT or Val143Ala; indicated as V143A) (A), T7-SLP2-A and FLAG-RAB27A (WT or Val143A) (B), or T7-SLAC2-A and FLAG-RAB27A (WT or Val143Ala) (C), in COS-7 cell lysates was analyzed by co-immunoprecipitation assays using anti-FLAG tag antibody-conjugated agarose beads (A, B) or anti-T7 tag antibody-conjugated agarose beads (C). Co-immunoprecipitated T7-MUNC13-4 and T7-SLP2-A (or FLAG-RAB27A) and immunoprecipitated FLAG-RAB27A (or T7-MLPH/SLAC2-A) were detected by immunoblotting with HRP-conjugated anti-T7 tag antibody and anti-FLAG tag antibody (or anti-FLAG tag antibody and anti-T7 tag antibody), respectively. The positions of the molecular mass markers (in kilodaltons) are shown on the left. Note that the Val143Ala mutation of RAB27A dramatically reduced the binding activity toward MUNC13-4 and SLP2-A.

Figure 6

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 (https://www.cgl.ucsf.edu/chimera/). Val143 and other amino acids that were mutated in patients with GS-2 sine albinism were highlighted in green and red, respectively.