The detection of mosaicism in human diseases can be challenging as the number of mosaic cells within the targeted tissue might be small. In addition, mosaic melanocytes would likely be absent inside existing SV lesions as a loss of melanocytes is the cause of vitiligo. Therefore, high-resolution methods at the single-cell level may be required to detect underrepresented cell populations.24 To detect potential mosaic cells, evaluating tissue obtained from hypochromic skin regions from individuals at the earliest stage of SV would be required (Fig. 1A). An advantage of studying the mosaicism hypothesis in SV is the possibility to use internal controls since SV is usually constrained to a unilateral segment: if the de novo mutation occurred during skin differentiation in one segment, a contralateral healthy skin could be used to establish the melanocyte’s “normal” profile (Fig. 1A). This strategy would allow controlling for confounding effects caused by interindividual variability when combining multiple SV cases.25

Figure 1.

Experimental design to test the mosaicism hypothesis in segmental vitiligo. (A) Depiction of a segmental vitiligo patient. Hypochromic areas highlighted with a full red circle would be evaluated for the presence of mosaic melanocytes. A contralateral skin sample marked with a dotted circle would be used as an internal control. (B) Skin composition for contralateral and hypochromic skin. The hypochromic skin includes residual mosaic melanocytes. (C) Subepidermal suction blister technique of normal and affected skin. The trypsinization of the blister’s roof allows the detachment of keratinocytes and melanocytes that can be together with immune infiltrated cells used in the single-cell experiments. (D) Single-cell barcoding. The pool containing cell suspension for normal and affected skin would be loaded to the single-cell channel. Individual cells would be incorporated into oil droplets and marked with unique barcoded beads, which would allow the application of different sets of single-cell approaches including scWGS, scRNA, and scATAC. (E) Cell clustering using omics data. Clustering analysis would group cells sharing similar states and identify mosaic melanocytes as well as other cell types included in the blister extract.

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Melanocytes account for ∼2.8% of the cell population in the epidermis and approximately 1200 melanocytes exist per mm2 of the skin independently of an individual ethnicity (Fig. 1B).26 To capture a representative number of viable mosaic melanocytes for the single-cell analysis, hypochromic areas of early-onset active SV would need to be determined using Wood’s lamp. Keratinocyte-melanocyte sampling of these areas could be performed with a suction blister epidermal grafting technique with subsequent trypsinization of the blister’s roof to detach the cells into a suspension (Fig. 1C).27 An advantage of collecting epidermis by using the suction blister is the widening of the sampled area, which increases the likelihood of capturing remaining viable mosaic melanocytes while being nearly a scarless technique, which facilitates patient inclusion.28 To evaluate follicular melanoblasts not captured by the blister method, a punch technique could also be used. A punch biopsy is more invasive than blistering and the overall proportion of melanoblasts captured would be small. However, methodological refinements of single cell analysis could allow in a near future the study of smaller cell populations.

To evaluate the presence of mosaic melanocytes, fluorescence-activated cell sorting selection could be used to separate the melanocyte fraction of blister extracts or melanoblasts from punch biopsies. Next, whole genome sequencing (WGS) would be performed using DNA extracted from melanocytes from hypochromic and contralateral skin from the same individuals. Variants detected in contralateral tissues would be used to exclude germline variants. Algorithms designed to detect tumor cells, such as MuTect2,29 or specific for mosaic cells, such as MosaicForecast30 and DeepMosaic,31 could be used to detect somatic mutations. The limitation of the bulk approach is the inability to separate mosaic cells from regular melanocytes and evaluate their interaction with other cells. Addressing this limitation would require single-cell (sc) sequencing technologies evaluating either the transcriptomic, epigenetic, or DNA sequence profile of individual cells (Fig. 1D).32 scWGS allows the evaluation of structural variation, CNVs, and SNVs in individual cells. A high throughput scWGS method developed to detect subclonal mutations in cancer could be used to test the hypothesis of melanocyte mosaicism.22 In SV, mosaic cells would share a state (i.e., specific genetic variants) that could be used to merge cells in clusters and define the proportion and the type of genetic variants present in mosaic cells (Fig. 1E).33 Epigenetic mosaicism in SV could be tested by using a multi-omics approach with transcriptomic (scRNA) and chromatin accessibility (scATAC) measures. Multi-omics approaches are currently being used to study a diverse set of diseases.34 Analyses of scRNA and scATAC could be used to identify mosaic cells via transcriptomic and epigenomic similarities with tools such as Seurat,35 Monocle,36 and Cicero.37 A similar scRNA approach applied to study human embryos was able to successfully detect mosaicism.20 An advantage of the single cell compared to bulk approaches is the ability to study the relationship between cells present in the blister extract or punch biopsies. In fact, scRNA study of blistering extracts has shown the recovery of melanocytes from NSV lesions.24 Applying a single-cell approach to study SV could test the mosaicism hypothesis while assessing the local immune profile as shown for NSV.24