A 32-year-old male patient from the Chinese Han population had unexplained pigmentary dilution of the hair, eyebrows, and eyelashes. He visited Beijing Tongren Hospital, Capital Medical University in June 2021 and was recruited for this study. Blood samples were obtained from the patient and his parents. This study was approved by the ethics committees of Beijing Tongren Hospital and Beijing Children's Hospital, Capital Medical University. Written informed consent was obtained in accordance with the declaration of Helsinki.

Whole exome sequencing was performed on genomic DNA from the proband. The Agilent SureSelect Human All ExonV6 Kit (Agilent Technologies, Santa Clara, CA, USA) was used to target the exonic regions of the genome. The Illumina NovaSeq 6000 platform (Illumina Inc., San Diego, CA, USA) was used for genomic DNA sequencing by Novogene Bioinformatics Technology Co., Ltd (Beijing, China) to generate 150 bp paired-end reads with a minimum coverage of 20× for ~95% of the genome (mean coverage of ~100×). The DNA sequences were analyzed by in-house quality control software to remove low-quality reads and were then aligned to the reference human genome (hs37d5) using the Burrows-Wheeler Aligner (BWA) (15), and duplicate reads were marked using Sambamba tools (16). Single nucleotide variants (SNVs) and indels were called by GATK to generate a gVCF file. The sequence variants in the proband and his parental samples were confirmed by Sanger sequencing analysis.

The human MLPH protein (NP_077006.1) sequence was aligned for analysis of the conservation of the mutated residue (p.D25H) with the sequences of the following homologous proteins: Mus musculus (NP_443748.2), Felis catus (NP_001073123.1), Ovis aries (NP_001139743.1), Oryctolagus cuniculus (NP_001284414.1), Canis lupus familiaris (NP_001096689.2), Rattus norvegicus (NP_001012135.1), and Gallus (NP_001108552.1). Conservation analysis and alignment visualization were performed by Clustal Omega (http://www.clustal.org/omega/) and Jalview software (17). The protein structure was drawn using the online tool Illustrator for Biological Sequences (IBS) (http://ibs.biocuckoo.org/). The domains of human MLPH protein referred to the structure of mouse Slac2-a/melanophilin protein (18).

The full-length cDNA of human MLPH with C terminal GFPSpark tag was synthesized by Sino Biological Inc. The human entire coding region of MLPH was subcloned into the pEGFP-C2 vector with an EGFP-tag and the pCMV-tag2B vector with a Flag-tag. The human MLPH sequence encoding the first 146 amino acids, termed Slp homology domain (SHD) (19), was subcloned into the pCMV-tag3B vector with a Myc-tag. The full-length cDNA of human RAB27A was amplified from total RNA of the human melanoma cell line MNT-1 cells (ATCC, USA) by one-step RT-PCR and the digested PCR product was cloned into the pCMV-tag2B vector with a Flag-tag. To introduce the point mutations into the MLPH or SHD, we used site-directed mutagenesis primers and high-fidelity polymerase to amplify the entire plasmid by PCR. The primers used were as follows: 5′-GTCTTGGAAGTTGTTCAACGACATTTTGACCTCCGAAGGAAAG-3′ (D25H primer; sense); 5′-CTTTCCTTCGGAGGTCAAAATGTCGTTGAACAACTTCCAAGAC-3′ (D25H primer; antisense); 5′-CGAAGGAAAGAAGAGGAATGGCTAGAGGCGTTGAAG-3′ (R35W primer; sense); and 5′-CTTCAACGCCTCTAGCCATTCCTCTTCTTTCCTTCG-3′ (R35W primer; antisense).

Briefly, MNT-1 cells (ATCC, USA) were cultured in minimal essential medium (MEM) supplemented with 20% fetal bovine serum (FBS, Invitrogen), 10% AIM-V medium (Gibco), sodium pyruvate, and non-essential amino acids at 37°C with 5% CO₂ (20). Human embryonic kidney 293 T (HEK293T) cells were cultured in DMEM (Gibco) with 10% FBS at 37°C with 5% CO₂. Subsequently, FLAG-MLPH or GFP-MLPH wild-type (WT) and MLPH mutant (p.D25H) expression plasmids were transfected into the cells using Lipofectamine 3000 (Invitrogen) in Opti-MEM (Gibco). After 6 h, the medium was changed to a fresh medium for further experiments.

A total of 2 ml of blood was collected from each individual in sodium citrate blood collection tubes. Platelet-rich plasma was obtained by centrifugation at 150 g for 10 min at room temperature. Washed platelets were lysed in a lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.1% sodium dodecyl-sulfate (SDS), 1% Triton X-100, and 1% sodium deoxycholate) with a protease inhibitor cocktail (Sigma-Aldrich, P8340) mixture, boiled with 5× loading buffer and then separated by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE) (21). Samples were then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). The membranes were blocked with 5% milk in PBS (0.1% Tween) for 1 h. Primary antibodies, anti-MLPH (Proteintech, 10338-1-AP, 1:5,000), anti-MYO5A (Cell Signaling Technology, 34025, 1:1,000), anti-RAB27A (Santa Cruz Biotechnology, sc-81914, 1:2,000), and anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) (Cell Signaling Technology, 5174T, 1:5,000), were incubated overnight at 4°C. After washing with PBS-T, membranes were incubated with a secondary antibody (goat anti-mouse IgG or goat anti-rabbit IgG, 1:5,000; Invitrogen) for 2 h at room temperature, then developed with the ECL substrate (Thermo Scientific).

In brief, MNT-1 cells transfected with GFP-MLPH (WT) or GFP-MLPH (D25H) were grown for 24 h on coverslips. Cells were fixed with 4% paraformaldehyde for 10 min, washed with PBS, permeabilized in 0.1% Triton X-100/PBS for 10 min, and blocked in 1% bovine serum albumin (BSA)/PBS for 1 h. Coverslips were then incubated with mouse anti-TYRP1 (Covance, SIG-38150) diluted 1:500 in 1% BSA/PBS at 4°C overnight. Then, cells were washed and incubated for 2 h with a 1:500 dilution with donkey anti-mouse secondary antibody conjugated to ALEXA-594 (Invitrogen) at room temperature. After washes, cells were mounted in 4',6-diamidino-2-phenylindole (DAPI, ZSGB-BIO). Finally, confocal images were acquired using the Zeiss LSM 880 Confocal Microscope (German).

Transfected HEK293T cells with Flag-tagged pCMV-tag2B-RAB27A and Myc-tagged pCMV-tag3B-SHD (WT, D25H, or R35W) were harvested after 48 h, then were incubated with a lysis buffer (150 mM NaCl, 50 mM Tris pH 7.4, 1 mM EDTA, and 1% Triton-100) with a protease inhibitor cocktail for 30 min at 4°C. Lysates were centrifuged at 12,000× g for 10 min at 4°C and 10% of the whole-cell lysates were taken as inputs. The remaining lysates were incubated with pre-washed anti-FLAG M2-Agarose affinity gel (Sigma-Aldrich, FLAGIPT-1) at 4°C, overnight. The bead complexes were washed 5 times with a washing buffer (150 mM NaCl and 50 mM Tris pH 7.4), and the proteins were eluted in a 2× loading sample buffer and then subjected to 10% SDS-PAGE for western blotting according to the procedures in our previous study (21).

The proband was a male patient aged 32 years at the time of the examination. He had brownish and silvery-gray hair, dark gray eyebrows, and white eyelashes (Figures 1A–C). His hair shaft showed uneven melanin granules under light microscopy (Figure 1D), a typical feature observed in GS3 (22). Blood parameters, such as hemoglobin, white blood cell (WBC), and platelets were within normal ranges. The value of erythrocyte sedimentation rate (ESR) was 3 mm/h (normal range, 0~15 mm/h). Specific immunoglobulin levels (IgG, IgM, and IgA) were normal. Complement 3 (C3) was 89.3 mg/dl (normal range, 90~180 mg/dl) and C4 was normal. The ADP-induced platelet aggregation of 43.6% (normal range, 59.1~98.3%) was lower than normal. Several coagulation tests, such as prothrombin time (PT) of 11.8 s (normal range, 10.5~15 s), activated partial thromboplastin time (APTT) of 27.2 s (normal range, 22.7~31.8 s), and thrombin time (TT) of 17.5 s (normal range, 14~21 s), were within normal limits. Fibrinogen (FIB) of 1.89 g/L (normal range, 2~4 g/L) was slightly lower than normal. Brain MRI was normal. Visual acuity was 20/20 in both eyes. The fundus photograph (Supplementary Figure S1A), the anterior segment picture (Supplementary Figure S1B), and the optical coherence tomography (OCT) (Supplementary Figure S1C) were normal.

Mutational screening for more than 200 hypopigmentation-related genes using next-generation sequencing (NGS) technologies identified a novel homozygous missense variant c.73G>C (p. Asp25His) (RefSeq NM_024101.7) of the MLPH gene in the proband (Figure 2A), which was verified by Sanger sequencing (Figure 2B). Meanwhile, Sanger sequencing revealed his parents who were not consanguineous as heterozygous carriers of this mutation (Figure 2B). Using in silico tools, such as PROVEAN, PolyPhen-2, and Mutation Taster, we evaluated the pathological effects of the c.73G>C mutation on the function of MLPH, and the allele frequency of the c.73G>C variant was not available in three common databases (Table 1), predicting it as likely pathogenic (23). Conservation analysis of the protein sequence in different species showed that the Asp25 residue is highly conserved (Figure 2C). Six MLPH mutations that caused GS3, including this newly reported allele, are mainly clustered on the SHD (Figure 2D) (8, 11–14), suggesting that this region appears to be a mutational hotspot region.

To evaluate the impact of the D25H mutation on MLPH, the MLPH expression level in the platelets of the patient was determined by Western blotting. HEK293T cells were transfected with Flag-MLPH and our results showed both Flag and MLPH antibodies recognized the same specific band, which confirmed the specificity of the MLPH antibody (Figure 3A). As shown in Figure 3B, the protein levels of MLPH, MYO5A, and RAB27A were not altered in the patients with GS3 compared with an unaffected control. Next, we examined the localization pattern of the MLPH (D25H) protein in MNT-1 cells. We observed that both the WT and mutant MLPH proteins were associated primarily with punctate structures located throughout the cell body and peripheral dendrites (Figures 4a,e, 5a,d), similar to the MLPH (WT) distribution described for melan-a melanocytes (5). Therefore, the D25H mutation did not interfere with the expression and localization of the MLPH protein.

The RAB27A protein binds to the surface of the melanosome and participates in actin-dependent melanosome movement via direct interaction with its effector MLPH and indirect interaction with MYO5A (24, 25). To further define whether the D25H substitution compromises the RAB27A-MLPH interaction, an SHD (D25H)-MLPH construct was used to express the D25H mutant. Flag-RAB27A and Myc-tagged SHD (WT), SHD (D25H), or SHD (R35W) were co-transfected into HEK293T cells, respectively. Cell lysates were immunoprecipitated with anti-Flag beads, followed by immunoblotting with anti-Flag and anti-Myc antibodies, respectively (Figure 3C). The results showed that the SHD (D25H)-MLPH variant reduced the binding to RAB27A protein, compared with the control SHD (WT)-MLPH (Figure 3C). As a positive control, the interaction between SHD (R35W)-MLPH and RAB27A was also decreased, as previously reported (8). These data suggest that the conserved D25 residue of MLPH plays role in the interaction with RAB27A.

It was previously described that mutations in MLPH cause the clustered perinuclear distribution observed in leaden mice (26). The pathologic defect in a GS3 patient with the MLPH (R35W) substitution induced aggregation of melanosomes in the perinuclear area of the patient's melanocytes (27). To test whether MLPH (D25H) is disease-causing, light microscopy and immunofluorescence confocal microscopy were used to examine the distribution of melanosomes. Green fluorescence showed the plasmid was successfully transfected into the cells (Figures 4a,e, 5a,d). Both melanocytes transfected with green fluorescence protein (GFP)-MLPH (WT) (Figure 4c, red arrows) and un-transfected cells from the same field of view (Figure 4c, red arrowhead) had a normal distribution of melanosomes at the periphery of the cells. In contrast, melanocytes overexpressed GFP-MLPH (D25H) showed melanosome accumulation in the perinuclei (Figure 4g, red arrow) and a lack of melanosomes in the peripheral dendrites (Figure 4h, red arrow), while those adjacent un-transfected cells exhibited a normal melanosome distribution (Figure 4g, red arrowheads). Quantification of melanosome distribution showed that more melanosomes were clustered in the perinuclear region but less in the periphery (Figure 4i).

Next, we used laser scanning confocal microscopy to study the localization of the melanosome-specific protein TYRP1 (Figures 5b,e), which is involved in the biosynthesis of melanin and the maintenance of melanosome structures (28, 29). In melanocytes transfected with or without GFP-MLPH (WT) (Figure 5b), TYRP1 staining showed a punctate pattern in the cell periphery and dendrites (Figure 5b, white arrowhead and arrow), with a slight aggregation around the nuclei. MLPH colocalized with TYRP1 in the cell's periphery dendritic tip (Figure 5c). By comparison, melanocytes overexpressed GFP-MLPH (D25H) showed a perinuclear accumulation (Figure 5e, white arrowhead) and the dendrites were devoid of TYRP1 staining (Figure 5e, white rectangle). While un-transfected cells displayed a normal localization of TYRP1 (Figure 5e, arrow). However, there was almost no colocalization between the MLPH (D25H) and TYRP1 in the cell periphery (Figure 5f). Taken together, these results demonstrated that the D25H mutation resulted in perinuclear aggregation of melanosomes, suggesting the impeded movement of melanosomes toward the cell periphery, which underlies the pathological effects of the patient with GS3.