Advertisement

Assessing Cutaneous Mosaicism at the Molecular Level

  • Jonathan J. Park
    Affiliations
    Department of Genetics, Yale School of Medicine, Yale University, New Haven, Connecticut, USA

    Department of Dermatology, Yale School of Medicine, Yale University, New Haven, Connecticut, USA

    Medical Scientist Training Program, Yale University, New Haven, Connecticut, USA
    Search for articles by this author
  • Keith Choate
    Correspondence
    Correspondence: Keith Choate, Department of Dermatology, Yale School of Medicine, Yale University, 333 Cedar Street, New Haven, Connecticut 06510, USA.
    Affiliations
    Department of Genetics, Yale School of Medicine, Yale University, New Haven, Connecticut, USA

    Department of Dermatology, Yale School of Medicine, Yale University, New Haven, Connecticut, USA

    Department of Pathology, Yale School of Medicine, Yale University, New Haven, Connecticut, USA
    Search for articles by this author
      Mosaicism results from postzygotic alterations during embryogenesis leading to genetically distinct populations of cells within individuals and has been historically recognized by phenotypes with visible, often patterned manifestations. Before the advent of molecular profiling assays and high-throughput sequencing, it was challenging to study mosaicism in human disease; however, the study of mosaic disorders has recently revealed unexpected and novel pathways for disease pathogenesis. In this paper, we will review the techniques for discovery of disease-causing alleles using Proteus syndrome; phakomatosis pigmentokeratotica; linear porokeratosis; and vacuoles, E1 enzyme, X-linked, autoinflammatory somatic syndrome as models. These tools represent powerful approaches for dissecting the genetic basis for human disorders.

      Abbreviations:

      indel (insertion/deletion), LP (linear porokeratosis), NGS (next-generation sequencing), PPK (phacomatosis pigmentokeratotica), PS (Proteus syndrome), SNV (single-nucleotide variant), VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic), WES (whole-exome sequencing), WGS (whole-genome sequencing)

      Summary Points

      • Mosaicism refers to genetically different populations of cells in individuals due to postzygotic alterations and can have clinical consequences depending on cell lineage, tissue distribution, variant type, variant timing, and gene function.
      • Sequence variants that would otherwise be embryonic lethal may present in isolated regions of the epidermis through somatic mosaicism, as seen in the case of the Proteus syndrome.
      • Identifying causal variants can change the paradigm for understanding diseases, as seen with the reclassification of phacomatosis pigmentokeratotica as a mosaic RASopathy.
      • Genotype-driven, phenotype-neutral strategies are modern approaches to the discovery of pathogenic variants for previously unrecognized clinical phenotype groupings, as seen with the vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic syndrome.

      Advantages

      • There are numerous technologies that can profile disorders of cutaneous mosaicism at the molecular level, including Sanger sequencing, multiplex mutation assays, whole-exome sequencing, and whole-genome sequencing.
      • Exome sequencing has emerged as a particularly powerful and cost-effective tool for unbiased discovery of pathogenic variants.
      • Identifying causal variants can serve as the starting point for understanding the biology underlying the complex phenotypes of mosaic disorders.
      • Recent developments in single-cell and CRISPR gene perturbation technologies have the potential for higher resolution analysis of affected cells and studying the pathogenesis of the mutant alleles.

      Limitations

      • Each of the molecular profiling and sequencing technologies has limitations regarding coverage, resolution, and cost.
      • Technical challenges with capturing high-quality data from DNA molecules in individual cells have limited the potential of single-cell variant or genotype-based studies.
      • Although these methods are powerful for identifying disease-associated variants, further studies are warranted to determine the mechanisms by which the alterations lead to such complex phenotypes.

      Introduction

      Mosaicism refers to the presence of genetically distinct populations of cells within an organism and has broad implications for human health and disease. At the molecular level, it can result from cellular de novo alterations, including point mutations, insertion/deletions, and structural rearrangements, leading to genomic heterogeneity or epigenetic modifications that can lead to inheritable changes in gene expression (
      • Molho-Pessach V.
      • Schaffer J.V.
      Blaschko lines and other patterns of cutaneous mosaicism.
      ). Genomic alterations that confer selective advantages or disadvantages can lead to differential clonal expansion of variant cells, which in turn can have clinical consequences.
      Historically, mosaicism was recognized through phenotypes with visible, often patterned manifestations. Cutaneous lesions that follow lines of Blaschko, which represent the dorsoventral migration patterns of keratinocyte precursors, implied the presence of different clones of cells early during embryogenesis (
      • Bolognia J.L.
      • Orlow S.J.
      • Glick S.A.
      Lines of Blaschko.
      ). Other recognizable patterns include the checkerboard pattern, phylloid pattern, and patchy pattern without midline separation (
      • Happle R.
      Mosaicism in human skin. Understanding the patterns and mechanisms.
      ). In the 1980s,
      • Happle R.
      Lethal genes surviving by mosaicism: A possible explanation for sporadic birth defects involving the skin.
      advanced the idea that disorders associated with such patterns may be due to certain typically embryonic lethal mutations that can persist if acquired by cells after the zygote has formed and when intermingled with normal cells. Although animal experiments showing the rescue of lethal genotypes by chimerism with normal embryos supported this concept (
      • Bennett D.
      Rescue of a lethal T/t locus genotype by chimaerism with normal embryos.
      ), evaluating mosaicism in humans was challenging until the advent of molecular profiling technologies that could assay subpopulations of cells (
      • Biesecker L.G.
      • Spinner N.B.
      A genomic view of mosaicism and human disease.
      ) (Figure 1).
      Figure thumbnail gr1
      Figure 1Overview of molecular techniques to assess cutaneous mosaicism. (a) Example of how postzygotic, activating alterations can lead to cutaneous mosaicism phenotypes. During embryogenesis, a somatic variant may be acquired in one of the cells. As cells continue to divide, those containing both mutant and wild-type alleles propagate and contribute to the formation of tissues. Mutant cells may lead to pathologic tissue development, including cutaneous lesions that follow patterns such as lines of Blaschko. Lethal genes may survive in the epidermis through mosaicism. Other patterns of cutaneous mosaicism not shown in this figure include the checkerboard pattern, phylloid pattern, and patchy pattern without midline separation. (b) Tools to assess cutaneous mosaicism at the molecular level include those based on Sanger and next-generation sequencing technologies, each with various advantages and disadvantages. WGS is unbiased and can capture coding and noncoding variations but has a higher cost. WES enriches for coding regions that may be more likely to contain functional variants and is cost effective. Targeted molecular assays can be customized and can achieve high depth at a low cost for detection of low-frequency variants in cutaneous mosaicism phenotypes due to admixture with normal cells. All of these methods (WGS, WES, targeted assays) require the preparation of DNA isolated from affected subjects. Methods to the right focus on increasingly smaller regions of the genome but can yield more reads at a given location for less cost; methods to the left capture more of the genome for unbiased discovery but with increasing overall costs. WES, whole-exome sequencing; WGS, whole-genome sequencing.

      Whole-exome sequencing for Proteus syndrome

      Since 2005, the availability of next-generation sequencing (NGS) platforms has greatly accelerated the study of human genetics. In particular, coupling targeted capture and enrichment strategies with sequencing has allowed for cost-effective identification of coding variations in individuals by a technique called whole-exome sequencing (WES), which has been successfully applied for identifying causal alleles that underlie numerous monogenic disorders (
      • Bamshad M.J.
      • Ng S.B.
      • Bigham A.W.
      • Tabor H.K.
      • Emond M.J.
      • Nickerson D.A.
      • et al.
      Exome sequencing as a tool for Mendelian disease gene discovery.
      ).
      One of the first applications of WES for studying mosaicism in humans was in Proteus syndrome (PS), a rare disorder characterized by patchy or segmental postnatal overgrowth of multiple tissues. Manifestations included cerebriform connective tissue, epidermal nevus, disproportionate and asymmetric overgrowth with skeletal defects, dysregulated adipose tissue, vascular abnormalities, pulmonary abnormalities, and specific types of tumors (
      • Cohen Jr., M.M.
      Proteus syndrome review: molecular, clinical, and pathologic features.
      ). In 2011, a comparative genomic analysis of unaffected and affected tissue identified a somatic activating mutation c.49G>A (p.Glu17Lys) in the oncogene AKT1 in 26 of 29 patients with PS (
      • Lindhurst M.J.
      • Sapp J.C.
      • Teer J.K.
      • Johnston J.J.
      • Finn E.M.
      • Peters K.
      • et al.
      A mosaic activating mutation in AKT1 associated with the Proteus syndrome.
      ). These results supported the somatic mosaicism hypothesis advanced by Rudolf Happle decades before while implicating the activation of the phosphoinositide 3-kinase‒protein kinase B pathway in the disorder and showing the power of WES.

      Targeted molecular assays for phakomatosis pigmentokeratotica

      Targeted molecular techniques such as direct Sanger sequencing or sensitive multiplex mutation assays can also be used to study mosaicism, as demonstrated for phacomatosis pigmentokeratotica (PPK) (
      • Groesser L.
      • Herschberger E.
      • Sagrera A.
      • Shwayder T.
      • Flux K.
      • Ehmann L.
      • et al.
      Phacomatosis pigmentokeratotica is caused by a postzygotic HRAS mutation in a multipotent progenitor cell.
      ). Phakomatoses are a group of congenital neurocutaneous disorders characterized by nevi and hamartomas, and PPK was identified in 1996 as a distinct type of epidermal nevus syndrome characterized by co-occurrence of nevus sebaceous and papular nevus spilus (
      • Happle R.
      • Hoffmann R.
      • Restano L.
      • Caputo R.
      • Tadini G.
      Phacomatosis pigmentokeratotica: a melanocytic-epidermal twin nevus syndrome.
      ).
      Previous studies had shown that multiplex detection of hotspot mutations could screen for many sequence variants simultaneously in a rapid and cost-effective manner (
      • Kompier L.C.
      • Lurkin I.
      • Aa MNM van der
      • Rhijn BWG van
      • Kwast TH van der
      • Zwarthoff E.C.
      FGFR3, HRAS, KRAS, NRAS and PIK3CA mutations in bladder cancer and their potential as biomarkers for surveillance and therapy.
      ), and this approach identified postzygotic mosaic HRAS and KRAS mutations as the cause of nevus sebaceous and Schimmelpenning syndrome, respectively (
      • Groesser L.
      • Herschberger E.
      • Ruetten A.
      • Ruivenkamp C.
      • Lopriore E.
      • Zutt M.
      • et al.
      Postzygotic HRAS and KRAS mutations cause nevus sebaceous and Schimmelpenning syndrome.
      ). Using SNaPshot assays and Sanger sequencing and focusing on RAS, FGFR3, PIK3CA, and BRAF in six patients with PPK, a heterozygous HRAS c.37G>C (p.Gly13Arg) mutation was found in four patients, and a heterozygous HRAS c.182A>G (p.Gln61Arg) mutation was found in two (
      • Groesser L.
      • Herschberger E.
      • Sagrera A.
      • Shwayder T.
      • Flux K.
      • Ehmann L.
      • et al.
      Phacomatosis pigmentokeratotica is caused by a postzygotic HRAS mutation in a multipotent progenitor cell.
      ). This upended the prevailing hypothesis of nonallelic twin spotting whereby two different homozygous recessive mutations give rise to the nevus sebaceous and papular nevus spilus and showed that a single heterozygous dominant-activating mutation in HRAS can cause PPK, categorizing the syndrome as a mosaic RASopathy.

      Second-hit loss of heterozygosity in linear porokeratosis

      Although cutaneous mosaicism can arise by dominant, heterozygous, somatic variants in otherwise wild-type individuals, another mechanism is by somatic mutations in a subpopulation of cells in individuals carrying a germline heterozygous mutation (
      • Happle R.
      The categories of cutaneous mosaicism: A proposed classification.
      ). This phenomenon was illustrated in linear porokeratosis (LP), a disorder of keratinization where porokeratotic plaques are distributed along the lines of Blaschko (
      • Atzmony L.
      • Khan H.M.
      • Lim Y.H.
      • Paller A.S.
      • Levinsohn J.L.
      • Holland K.E.
      • et al.
      Second-hit, postzygotic PMVK and MVD mutations in linear porokeratosis.
      ). To test the hypothesis that postzygotic somatic alterations underlie LP, WES on unaffected and affected tissues of three cases with LP was performed, identifying the combinations of germline mutations and second-hit postzygotic somatic mutations (
      • Atzmony L.
      • Khan H.M.
      • Lim Y.H.
      • Paller A.S.
      • Levinsohn J.L.
      • Holland K.E.
      • et al.
      Second-hit, postzygotic PMVK and MVD mutations in linear porokeratosis.
      ). All were found to have germline, heterozygous mutations in PMVK or MVD, with somatic second mutation or allelic loss. One case had a copy-neutral loss of heterozygosity, showing the importance of analyzing copy-number changes. PMVK and MVD both encode key enzymes in the mevalonate pathway, suggesting a pathogenesis-based avenue for therapeutic intervention as is currently being explored with topical cholesterol/lovastatin (
      • Atzmony L.
      • Lim Y.H.
      • Hamilton C.
      • Leventhal J.S.
      • Wagner A.
      • Paller A.S.
      • et al.
      Topical cholesterol/lovastatin for the treatment of porokeratosis: a pathogenesis-directed therapy.
      ).

      Genotype-driven approaches for vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic syndrome

      The widespread availability of DNA sequencing has led to genotype-driven approaches to delineating human disease, whereby sequenced individuals are studied independently of phenotype to identify potential subsets with previously unrecognized clinical characteristic groupings and common underlying genetic variants (
      Deciphering Developmental Disorders Study
      Large-scale discovery of novel genetic causes of developmental disorders.
      ). This approach may help to characterize rare disorders that are not well-defined as discrete clinical entities.
      In 2020, a genotype-driven approach was employed to study inflammatory syndromes (
      • Beck D.B.
      • Ferrada M.A.
      • Sikora K.A.
      • Ombrello A.K.
      • Collins J.C.
      • Pei W.
      • et al.
      Somatic mutations in UBA1 and severe adult-onset autoinflammatory disease.
      ). Analysis of 2,560 exomes of patients referred for undiagnosed inflammatory symptoms or otherwise affected by atypical, unclassified disorders identified three men with novel, predicted deleterious, apparently heterozygous variants in methionine-41 (p.Met41) of the X-linked gene UBA1, the major E1 enzyme that initiates ubiquitylation. Additional phenotypically similar cases were identified, and ultimately 25 cases were confirmed to have somatic UBA1 mutations by Sanger sequencing. All had a treatment-refractory inflammatory syndrome that developed in adulthood characterized by progressive hematologic abnormalities, recurrent fevers, pulmonary involvement, neutrophilic dermatoses, and cutaneous vasculitis. The authors named the disorder vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome.
      Further characterization included transcriptome analyses of peripheral blood, cytokine profiling, immunoblotting experiments, immunohistochemistry, electron microscopy, and the establishment of CRISPR-Cas9‒edited zebrafish models. The authors also found that participants with UBA1 mosaic mutations had predominantly wild-type lymphocytes but predominantly mutant myeloid cells, which drives the inflammation in patients with VEXAS syndrome. They note that the p.Met41 mutation is likely only compatible with life owing to somatic mosaicism in lineage-restricted cell types. Altogether, the VEXAS syndrome story represents an example of a genotype-driven approach made possible by the modernization of sequencing and molecular profiling technologies.

      Novel biology revealed by study of cutaneous diseases

      Molecular investigation of cutaneous disorders can be used as a basis for discovering new biology. For example, WES in nevus comedonicus, a disorder characterized by comedones and inflammatory cysts in linear configurations, identified somatic variants in NEK9, a gene not previously implicated in follicular biology (Figure 2). Affected tissue demonstrating loss of markers of follicular differentiation with a gain of a marker of interfollicular differentiation suggests that NEK9 may play a role in follicular cell‒fate decisions (
      • Levinsohn J.L.
      • Sugarman J.L.
      • Yale Center for Mendelian Genomics
      • McNiff J.M.
      • Antaya R.J.
      • Choate K.A.
      Somatic mutations in NEK9 cause nevus comedonicus.
      ). Similarly, WES in hepatic hemangiomas and cutaneous venous malformations revealed a recurrent GJA4 mutation causing changes in cell morphology and noncanonical activation of SGK1, a regulator of cell proliferation and apoptosis, thereby identifying a new pathway for vascular anomalies (
      • Ugwu N.
      • Atzmony L.
      • Ellis K.T.
      • Panse G.
      • Jain D.
      • Ko C.J.
      • et al.
      Cutaneous and hepatic vascular lesions due to a recurrent somatic GJA4 mutation reveal a pathway for vascular malformation.
      ). Finally, WES identified PLCD1 mutations in trichilemmal (pilar) cysts, which are benign, keratin-filled cysts from the outer root sheath of hair follicles typically seen on the scalp. These findings implicated a monoallelic second hit genetic mechanism causing dysregulated calcium signaling and subsequent cyst formation, highlighting the importance of PLCD1 in hair follicle homeostasis (
      • Hörer S.
      • Marrakchi S.
      • Radner F.P.W.
      • Zolles G.
      • Heinz L.
      • Eichmann T.O.
      • et al.
      A monoallelic two-hit mechanism in PLCD1 explains the genetic pathogenesis of hereditary trichilemmal cyst formation.
      ). In all of these studies, using WES to discover disease-causing variants and then performing follow-up experiments in appropriate models, new insights were gained into the molecular pathobiology of the skin.
      Figure thumbnail gr2
      Figure 2Identifying somatic NEK9 mutations in nevus comedonicus. (a) Clinical and histologic features of nevus comedonicus. Three individuals with nevus comedonicus presented with linear patches of comedones on distinct body sites. Histopathologic examination of excision tissue revealed follicles with dilated ostia, acanthosis and papillomatosis of the outer root sheath, and cystic structures filled with keratin adjacent to normal follicles. (b) WES was performed on DNA isolated from the tissue and blood of individuals with nevus comedonicus, and tissue-specific somatic SNVs and indels were identified and ranked. Mutations were found in NEK9 for all the three individuals and confirmed with Sanger sequencing. Reprinted with permission from
      • Levinsohn J.L.
      • Sugarman J.L.
      • Yale Center for Mendelian Genomics
      • McNiff J.M.
      • Antaya R.J.
      • Choate K.A.
      Somatic mutations in NEK9 cause nevus comedonicus.
      . indel, insertion/deletion; NLS, nuclear localization sequence; ref, reference; SNV, single-nucleotide variant; WES, whole-exome sequencing.

      Study design for cutaneous mosaicism disorders

      Strategies for tissue sampling, data processing and analysis, and potential validation experiments should be considered carefully when undertaking human genomic sequencing studies of mosaic disease (Figure 3). To begin, unless using a genotype-driven approach, investigators must typically first identify unaffected and affected tissue. For limited cutaneous disease, blood or saliva DNA is often used as the source of unaffected genomic data, but data from the cutaneous-skeletal hypophosphatemia syndrome (
      • Lim Y.H.
      • Ovejero D.
      • Sugarman J.S.
      • DeKlotz C.M.C.
      • Maruri A.
      • Eichenfield L.F.
      • et al.
      Multilineage somatic activating mutations in HRAS and NRAS cause mosaic cutaneous and skeletal lesions, elevated FGF23 and hypophosphatemia.
      ) reveal that multisystem mosaicism can be sporadic and discontinuous. These findings raise the possibility that blood or saliva could rarely also harbor the same genomic changes as affected skin. For this reason, investigators should consider additional sources of unaffected DNA, such as from adjacent, clinically unaffected tissue. On the basis of clinical presentation and histology, investigators can identify the affected cell type. However, within affected tissue, abundance of mutant cells must be considered because admixture with germline DNA can limit detection capacity. For proliferative/hamartomatous disorders, mutant cells predominate, and direct preparation of DNA from tissue is typically adequate. For disorders in which mutant cells are likely to represent <20% of tissue, other purification approaches or much deeper sequencing must be considered. To purify mutant cells, selective cell culture, FACS of affected cells, and laser-capture microscopy can be used. Which tissues to sample, the relative abundance of mutant cells, whether additional controls should be obtained, and whether purification must be performed are all ideally considered before study initiation.
      Figure thumbnail gr3
      Figure 3Flowchart of sample preparation. Flowchart detailing the overall workflow for the different molecular profiling technologies for cutaneous mosaicism. First, samples are typically isolated from affected and unaffected tissues, although investigators may not have paired control DNA in the initial steps of a genotype-driven approach. Examples of sampling methods include skin biopsies, peripheral blood draws, and saliva swabs. Further purification methods may include selective cell culture, FACS, and laser capture microscopy. DNAs are then extracted and prepared for NGS or Sanger sequencing. Note that samples may have low mutant allele frequencies due to admixture. Preparation for NGS may include DNA fragmentation, adaptor ligation, and target capture and enrichment strategies. Specific techniques may enrich for exomes, chromatin accessibility, methylation status, and single cells. Preparation for targeted molecular assays and Sanger sequencing may include multiplex primer design and PCRs such as with SNaPshot multiplex systems. After sequencing is performed, in silico analysis is performed to identify causal variants. Once variants are identified, disease pathogenesis may be further evaluated with downstream functional experiments. NGS, next-generation sequencing.
      Once samples are obtained, appropriate sequencing parameters, bioinformatic approaches, and statistical analyses should be used to ensure that the research objectives are met. Examples of genomic and bioinformatic tools include the Burrows‒Wheeler Aligner for alignment of reads to the human genome, HaplotypeCaller for identifying germline single-nucleotide variants (SNVs) and insertions/deletions (indels), Mutect2 for identifying somatic SNVs and indels, and ANNOVAR for variant functional annotation (
      • Van der Auwera G.A.
      • Carneiro M.O.
      • Hartl C.
      • Poplin R.
      • del Angel G.
      • Levy-Moonshine A.
      • et al.
      From FastQ data to high confidence variant calls: the genome analysis toolkit best practices pipeline.
      ). Statistical tools such as a Fisher exact test can also be used to assess the enrichment of mutant reads in comparison with that of wild-type reads. In general, identified genes that contain somatic variants, which are significantly enriched in affected tissues and predicted to be damaging and in which other somatic variants cause a consistent cutaneous phenotype, constitute genetic proof of pathogenesis.

      Advantages and limitations of molecular techniques

      The molecular techniques described in this paper have various advantages and disadvantages. WES enables unbiased discovery of the genetic etiologies and has advantages over whole-genome sequencing (WGS) with lower cost and decreased analytic burden. However, protein-coding regions represent only ∼2% of the genome, and WGS can identify causal variants that may lie in noncoding regions. Targeted mutation assays for specific gene panels are rapid and cost effective compared with NGS-based approaches but are biased and limited to specific mutations and genes of interest. One strength of panel-based approaches containing likely candidate genes is that very high-depth sequencing can be performed at a low cost. This is a key advantage because mosaics often have to be sequenced deeply to detect low mutant allele frequencies in admixed samples. Genotype-driven approaches for variant characterization are also powerful and do not require a defined clinical phenotype but may require potentially prohibitively large populations of sequenced individuals for appropriate statistical power. The specific technique to use for any given study will depend on the phenotype observed, capacity to identify and isolate the affected cell type, and available resources. Given the relative accessibility of skin samples, these techniques are well-suited for studying cutaneous mosaicism and other diseases in dermatology.

      Single-cell genomics, genomic perturbations, and future directions

      Given that mosaicism involves cellular and genetic heterogeneity, assessing individual cell genomes is potentially a powerful approach to interrogating the clonal dynamics of variant and normal cells. Indeed, single-cell sequencing studies have shown widespread mosaicism in apparently normal tissues (
      • McConnell M.J.
      • Lindberg M.R.
      • Brennand K.J.
      • Piper J.C.
      • Voet T.
      • Cowing-Zitron C.
      • et al.
      Mosaic copy number variation in human neurons. Science.
      ). However, single-cell DNA sequencing has not seen widespread adaptation owing to technical challenges with obtaining high-quality genotype data from single molecules of DNA from individual cells (
      • Gawad C.
      • Koh W.
      • Quake S.R.
      Single-cell genome sequencing: current state of the science.
      ). Further development of higher resolution molecular profiling assays can provide deeper insights into cellular states and behaviors.
      CRISPR-based technologies have enabled facile manipulation of genomic sequences for numerous applications (
      • Guitart J.R.
      • Johnson J.L.
      • Chien W.W.
      Research techniques made simple: the application of CRISPR-Cas9 and genome editing in investigative dermatology.
      ), including induction of somatic mosaicism and allele complexity with cutaneous phenotypes in mouse models (
      • Yen S.T.
      • Zhang M.
      • Deng J.M.
      • Usman S.J.
      • Smith C.N.
      • Parker-Thornburg J.
      • et al.
      Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes.
      ). CRISPR can be used to efficiently generate cell lines and in vivo models of identified novel variants for further study. The rapid generation of in vivo zebrafish models to assess UBA1 variants in VEXAS syndrome showed the power of genomic perturbation technologies.
      Finally, WGS has seen limited application compared with WES in large part owing to cost. The focus on coding variation is justified by their enrichment for functional or deleterious consequences (
      • Bamshad M.J.
      • Ng S.B.
      • Bigham A.W.
      • Tabor H.K.
      • Emond M.J.
      • Nickerson D.A.
      • et al.
      Exome sequencing as a tool for Mendelian disease gene discovery.
      ). However, sequence variants in noncoding regions are increasingly being recognized as having important phenotypic consequences; large-scale sequencing and analytic efforts are underway to characterize novel, noncoding signatures of mutational processes for cancer (
      • Campbell P.J.
      • Getz G.
      • Korbel J.O.
      • Stuart J.M.
      • Jennings J.L.
      • Stein L.D.
      • et al.
      Pan-cancer analysis of whole genomes.
      ). As sequencing costs continue to decrease, studying how noncoding variations can drive cutaneous mosaicism phenotypes may yield further insights into disease pathogenesis.

      Conclusions

      Technological advances in high-throughput sequencing and molecular assays have greatly accelerated the discovery of novel allelic variants that drive cutaneous mosaic disorders. Comparative exome sequencing, sensitive multiplex mutation assays, and genotype-driven approaches have all seen successful applications. The development of more advanced molecular profiling modalities such as single-cell approaches and genetic perturbation systems such as CRISPR enables higher resolution investigation into the pathogenesis and function of these genetic variants, and further functional experiments can yield insights into the biology of cutaneous mosaicism and potentially identify novel therapeutic approaches.

      Graphical illustrations

      Certain graphical illustrations were made with BioRender (biorender.com).

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      JJP is supported by the National Institutes of Health Medical Scientist Training Program training grant ( T32GM007205 ). KC has received funding from the National Institutes of Health R01AR071491 . JJP is an MD-PhD student, and KC is faculty.

      Author Contributions

      Supervision: KC; Writing - Original Draft Preparation: JJP; Writing - Review and Editing: JJP, KC

      Supplementary Material

      References

        • Atzmony L.
        • Khan H.M.
        • Lim Y.H.
        • Paller A.S.
        • Levinsohn J.L.
        • Holland K.E.
        • et al.
        Second-hit, postzygotic PMVK and MVD mutations in linear porokeratosis.
        JAMA Dermatol. 2019; 155: 548-555
        • Atzmony L.
        • Lim Y.H.
        • Hamilton C.
        • Leventhal J.S.
        • Wagner A.
        • Paller A.S.
        • et al.
        Topical cholesterol/lovastatin for the treatment of porokeratosis: a pathogenesis-directed therapy.
        J Am Acad Dermatol. 2020; 82: 123-131
        • Bamshad M.J.
        • Ng S.B.
        • Bigham A.W.
        • Tabor H.K.
        • Emond M.J.
        • Nickerson D.A.
        • et al.
        Exome sequencing as a tool for Mendelian disease gene discovery.
        Nat Rev Genet. 2011; 12: 745-755
        • Beck D.B.
        • Ferrada M.A.
        • Sikora K.A.
        • Ombrello A.K.
        • Collins J.C.
        • Pei W.
        • et al.
        Somatic mutations in UBA1 and severe adult-onset autoinflammatory disease.
        N Engl J Med. 2020; 383: 2628-2638
        • Bennett D.
        Rescue of a lethal T/t locus genotype by chimaerism with normal embryos.
        Nature. 1978; 272 (539–539)
        • Biesecker L.G.
        • Spinner N.B.
        A genomic view of mosaicism and human disease.
        Nat Rev Genet. 2013; 14: 307-320
        • Bolognia J.L.
        • Orlow S.J.
        • Glick S.A.
        Lines of Blaschko.
        J Am Acad Dermatol. 1994; 31 (quiz 190): 157-190
        • Campbell P.J.
        • Getz G.
        • Korbel J.O.
        • Stuart J.M.
        • Jennings J.L.
        • Stein L.D.
        • et al.
        Pan-cancer analysis of whole genomes.
        Nature. 2020; 578: 82-93
        • Cohen Jr., M.M.
        Proteus syndrome review: molecular, clinical, and pathologic features.
        Clin Genet. 2014; 85: 111-119
        • Deciphering Developmental Disorders Study
        Large-scale discovery of novel genetic causes of developmental disorders.
        Nature. 2015; 519: 223-228
        • Gawad C.
        • Koh W.
        • Quake S.R.
        Single-cell genome sequencing: current state of the science.
        Nat Rev Genet. 2016; 17: 175-188
        • Groesser L.
        • Herschberger E.
        • Ruetten A.
        • Ruivenkamp C.
        • Lopriore E.
        • Zutt M.
        • et al.
        Postzygotic HRAS and KRAS mutations cause nevus sebaceous and Schimmelpenning syndrome.
        Nat Genet. 2012; 44: 783-787
        • Groesser L.
        • Herschberger E.
        • Sagrera A.
        • Shwayder T.
        • Flux K.
        • Ehmann L.
        • et al.
        Phacomatosis pigmentokeratotica is caused by a postzygotic HRAS mutation in a multipotent progenitor cell.
        J Invest Dermatol. 2013; 133: 1998-2003
        • Guitart J.R.
        • Johnson J.L.
        • Chien W.W.
        Research techniques made simple: the application of CRISPR-Cas9 and genome editing in investigative dermatology.
        J Invest Dermatol. 2016; 136: e87-e93
        • Happle R.
        Lethal genes surviving by mosaicism: A possible explanation for sporadic birth defects involving the skin.
        J Am Acad Dermatol. 1987; 16: 899-906
        • Happle R.
        Mosaicism in human skin. Understanding the patterns and mechanisms.
        Arch Dermatol. 1993; 129: 1460-1470
        • Happle R.
        The categories of cutaneous mosaicism: A proposed classification.
        Am J Med Genet A. 2016; 170A: 452-459
        • Happle R.
        • Hoffmann R.
        • Restano L.
        • Caputo R.
        • Tadini G.
        Phacomatosis pigmentokeratotica: a melanocytic-epidermal twin nevus syndrome.
        Am J Med Genet. 1996; 65: 363-365
        • Hörer S.
        • Marrakchi S.
        • Radner F.P.W.
        • Zolles G.
        • Heinz L.
        • Eichmann T.O.
        • et al.
        A monoallelic two-hit mechanism in PLCD1 explains the genetic pathogenesis of hereditary trichilemmal cyst formation.
        J Invest Dermatol. 2019; 139: 2154-2163.e5
        • Kompier L.C.
        • Lurkin I.
        • Aa MNM van der
        • Rhijn BWG van
        • Kwast TH van der
        • Zwarthoff E.C.
        FGFR3, HRAS, KRAS, NRAS and PIK3CA mutations in bladder cancer and their potential as biomarkers for surveillance and therapy.
        PLoS One. 2010; 5e13821
        • Levinsohn J.L.
        • Sugarman J.L.
        • Yale Center for Mendelian Genomics
        • McNiff J.M.
        • Antaya R.J.
        • Choate K.A.
        Somatic mutations in NEK9 cause nevus comedonicus.
        Am J Hum Genet. 2016; 98: 1030-1037
        • Lim Y.H.
        • Ovejero D.
        • Sugarman J.S.
        • DeKlotz C.M.C.
        • Maruri A.
        • Eichenfield L.F.
        • et al.
        Multilineage somatic activating mutations in HRAS and NRAS cause mosaic cutaneous and skeletal lesions, elevated FGF23 and hypophosphatemia.
        Hum Mol Genet. 2014; 23: 397-407
        • Lindhurst M.J.
        • Sapp J.C.
        • Teer J.K.
        • Johnston J.J.
        • Finn E.M.
        • Peters K.
        • et al.
        A mosaic activating mutation in AKT1 associated with the Proteus syndrome.
        N Engl J Med. 2011; 365: 611-619
        • McConnell M.J.
        • Lindberg M.R.
        • Brennand K.J.
        • Piper J.C.
        • Voet T.
        • Cowing-Zitron C.
        • et al.
        Mosaic copy number variation in human neurons. Science.
        Science. 2013; 342: 632-637
        • Molho-Pessach V.
        • Schaffer J.V.
        Blaschko lines and other patterns of cutaneous mosaicism.
        Clin Dermatol. 2011; 29: 205-225
        • Ugwu N.
        • Atzmony L.
        • Ellis K.T.
        • Panse G.
        • Jain D.
        • Ko C.J.
        • et al.
        Cutaneous and hepatic vascular lesions due to a recurrent somatic GJA4 mutation reveal a pathway for vascular malformation.
        HGG Adv. 2021; 2 ([published correction appears in HGG Adv 2021;3:100061])100028
        • Van der Auwera G.A.
        • Carneiro M.O.
        • Hartl C.
        • Poplin R.
        • del Angel G.
        • Levy-Moonshine A.
        • et al.
        From FastQ data to high confidence variant calls: the genome analysis toolkit best practices pipeline.
        Curr Protoc Bioinformaatics. 2013; 43 (11.10.1-11.10.33)
        • Yen S.T.
        • Zhang M.
        • Deng J.M.
        • Usman S.J.
        • Smith C.N.
        • Parker-Thornburg J.
        • et al.
        Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes.
        Dev Biol. 2014; 393: 3-9