Altered Notch Signaling in Dowling-Degos Disease: Additional Mutations in POGLUT1 and Further Insights into Disease Pathogenesis.

Dowling-Degos disease (DDD; MIM 179850, 615327, and 615696) is a rare autosomal-dominant hereditary pigmentation disorder. Affected individuals present with varying degrees of progressive reticulate hyperpigmentation, primarily affecting the flexures, large skin folds, trunk, face, and extremities. Genetic research has identified associations between DDD and mutations in KRT5 (Betz et al., 2006Betz R.C. Planko L. Eigelshoven S. Hanneken S. Pasternack S.M. Bussow H. et al.Loss-of-function mutations in the keratin 5 gene lead to Dowling-Degos disease.Am J Hum Genet. 2006; 78: 510-519Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar), POFUT1 (Li et al., 2013Li M. Cheng R. Liang J. Yan H. Zhang H. Yang L. et al.Mutations in POFUT1, encoding protein O-fucosyltransferase 1, cause generalized Dowling-Degos disease.Am J Hum Genet. 2013; 92: 895-903Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), POGLUT1 (Basmanav et al., 2014Basmanav F.B. Oprisoreanu A.M. Pasternack S.M. Thiele H. Fritz G. Wenzel J. et al.Mutations in POGLUT1, encoding protein O-glucosyltransferase 1, cause autosomal-dominant Dowling-Degos disease.Am J Hum Genet. 2014; 94: 135-143Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), and PSENEN (Ralser et al., 2017Ralser D.J. Basmanav F.B. Tafazzoli A. Wititsuwannakul J. Delker S. Danda S. et al.Mutations in gamma-secretase subunit-encoding PSENEN underlie Dowling-Degos disease associated with acne inversa.J Clin Invest. 2017; 127: 1485-1490Crossref PubMed Scopus (59) Google Scholar). Mutations in these genes, which are implicated in Notch signaling, underlie clinically and histologically distinct DDD subtypes (Basmanav et al., 2015Basmanav F.B. Fritz G. Lestringant G.G. Pachat D. Hoffjan S. Fischer J. et al.Pathogenicity of POFUT1 in Dowling-Degos disease: additional mutations and clinical overlap with reticulate acropigmentation of kitamura.J Invest Dermatol. 2015; 135: 615-618Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, Betz, 2017Betz R.C. A path through the reticulate pigmentation disorder jungle.Br J Dermatol. 2017; 177: 893-894Google Scholar, Ralser et al., 2017Ralser D.J. Basmanav F.B. Tafazzoli A. Wititsuwannakul J. Delker S. Danda S. et al.Mutations in gamma-secretase subunit-encoding PSENEN underlie Dowling-Degos disease associated with acne inversa.J Clin Invest. 2017; 127: 1485-1490Crossref PubMed Scopus (59) Google Scholar). Following our identification of POGLUT1 mutations in DDD (Basmanav et al., 2014Basmanav F.B. Oprisoreanu A.M. Pasternack S.M. Thiele H. Fritz G. Wenzel J. et al.Mutations in POGLUT1, encoding protein O-glucosyltransferase 1, cause autosomal-dominant Dowling-Degos disease.Am J Hum Genet. 2014; 94: 135-143Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), we studied additional DDD patients. Ethical approval was obtained from the ethics committee of the Medical Faculty of the University of Bonn. All participants provided written informed consent prior to blood sampling. The study was conducted in accordance with Declaration of Helsinki principles. All participants were examined and sub-phenotyped by a dermatologist, as suggested previously (Basmanav et al., 2015Basmanav F.B. Fritz G. Lestringant G.G. Pachat D. Hoffjan S. Fischer J. et al.Pathogenicity of POFUT1 in Dowling-Degos disease: additional mutations and clinical overlap with reticulate acropigmentation of kitamura.J Invest Dermatol. 2015; 135: 615-618Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, Betz, 2017Betz R.C. A path through the reticulate pigmentation disorder jungle.Br J Dermatol. 2017; 177: 893-894Google Scholar). The predominant presentation was reticulate hyperpigmentation confined to the non-flexural areas of the body, in particular trunk and extremities (Figure 1a–1h, Supplementary Table 2 online). All patients agreed to the publication of their photographs. Sanger sequencing revealed 12, to our knowledge previously unreported, heterozygous mutations in POGLUT1: (i) five nonsense mutations, c.20C>A (p.Ser7*), c.138C>G (p.Tyr46*), c.1051C>T (p.Gln351*), c.628C>T (p.Arg210*), and c.130G>T (p.Glu44*); (ii) three frameshift mutations, c.121_121delA (p.Arg41Glyfs*21), c.292_292delC (p.R98Gfs*21), and c.1080_1081insG (p.Asn361Glufs*5); (iii) one splice site mutation, c.320+1G>C; and (iv) three missense mutations, c.983T>G (p.Val328Gly), c.836G>A (p.Arg279Gln), and c.653G>A (p.Arg218Gln) (Figure 1i, Supplementary Figure 1 online). Interestingly, five of these mutations are found in gnomAD with an allele frequency range of 1,321e–5 to 8,153e–6 (Supplementary Table 1 online). Still, it is conceivable that these mutations are pathogenic due to (i) the relatively late age of onset; and (ii) the comparatively mild clinical phenotype. Therefore, DDD might be more common than reported to date. The nonsense and frameshift mutations are likely to result in either nonsense-mediated mRNA decay or the formation of a truncated protein, as exemplarily demonstrated in Supplementary Figure 3a and 3b online. To determine the structural consequences of the splice site mutation c.320+1G>C, exon trapping was used (Ralser et al., 2017Ralser D.J. Basmanav F.B. Tafazzoli A. Wititsuwannakul J. Delker S. Danda S. et al.Mutations in gamma-secretase subunit-encoding PSENEN underlie Dowling-Degos disease associated with acne inversa.J Clin Invest. 2017; 127: 1485-1490Crossref PubMed Scopus (59) Google Scholar). This revealed complete skipping of exon 3, resulting in disruption of the functionally important POGLUT1 N-terminal domain (Supplementary Figure 2a, 2b online). To assess the consequences of two of the here-described missense mutations and the previously reported mutation p.Arg279Trp (Basmanav et al., 2014Basmanav F.B. Oprisoreanu A.M. Pasternack S.M. Thiele H. Fritz G. Wenzel J. et al.Mutations in POGLUT1, encoding protein O-glucosyltransferase 1, cause autosomal-dominant Dowling-Degos disease.Am J Hum Genet. 2014; 94: 135-143Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), wild-type POGLUT1 and the mutants p.Arg279Gln, p.Val328Gly, and p.Arg279Trp were each fused to a C-terminal MycHis epitope and overexpressed in HEK293T cells. The enzymatic activities resulting from the wild-type and mutant POGLUT1 constructs were evaluated using an enzymatic assay, as described elsewhere (Takeuchi et al., 2012Takeuchi H. Kantharia J. Sethi M.K. Bakker H. Haltiwanger R.S. Site-specific O-glucosylation of the epidermal growth factor-like (EGF) repeats of notch: efficiency of glycosylation is affected by proper folding and amino acid sequence of individual EGF repeats.J Biol Chem. 2012; 287: 33934-33944Crossref PubMed Scopus (61) Google Scholar). Western blot showed that both p.Arg279Gln and p.Arg279Trp resulted in a protein product, the secretion level of which is similar to that of wild-type POGLUT1, indicating that it is folded. However, this mutant protein was enzymatically dysfunctional (Figure 2a, Supplementary Figures 4–6 online). In contrast, the construct encoding p.Val328Gly was detected in cell lysates but not in media, suggesting that the mutant protein is unstable and eventually degraded (Supplementary Figures 4–6). To demonstrate that loss of function of the mutant p.Val328Gly protein did not result from the introduction of an additional mutation during PCR-mediated site-directed mutagenesis, a revertant was constructed. Secretion levels of the revertant and the wild-type were comparable (Supplementary Figures 4 and 6). These findings were supported by protein modeling based on the structures of human POGLUT1 co-crystallized with substrates (PDB-code: 5L0T) (Li et al., 2017Li Z. Fischer M. Satkunarajah M. Zhou D. Withers S.G. Rini J.M. Structural basis of Notch O-glucosylation and O-xylosylation by mammalian protein-O-glucosyltransferase 1 (POGLUT1).Nat Commun. 2017; 8: 185Crossref PubMed Scopus (28) Google Scholar) (Figure 2b–2f). Arg279 is involved in the high-affinity binding of the donor substrate (Figure 2b). Substitution of glutamine for both, Arg218 and Arg279, respectively, likely decreases substrate affinity, thereby impairing POGLUT1 catalytic function (Figure 2c, 2d). Val328 is part of a hydrophobic cluster of POGLUT1 (Figure 2e), which stabilizes the C-terminal end of a helix formed by residues 320–331 (Li et al., 2017Li Z. Fischer M. Satkunarajah M. Zhou D. Withers S.G. Rini J.M. Structural basis of Notch O-glucosylation and O-xylosylation by mammalian protein-O-glucosyltransferase 1 (POGLUT1).Nat Commun. 2017; 8: 185Crossref PubMed Scopus (28) Google Scholar). The substitution of glycine for valine likely causes loss of the helix and destabilization of the entire protein (Figure 2f). Conceivably, this unstable POGLUT1 variant is prone to rapid degradation, as observed in our immunoblotting experiments (Supplementary Figures 4–6). POGLUT1 is involved in the post-translational modification of Notch proteins (Acar et al., 2008Acar M. Jafar-Nejad H. Takeuchi H. Rajan A. Ibrani D. Rana N.A. et al.Rumi is a CAP10 domain glycosyltransferase that modifies Notch and is required for Notch signaling.Cell. 2008; 132: 247-258Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, Basmanav et al., 2014Basmanav F.B. Oprisoreanu A.M. Pasternack S.M. Thiele H. Fritz G. Wenzel J. et al.Mutations in POGLUT1, encoding protein O-glucosyltransferase 1, cause autosomal-dominant Dowling-Degos disease.Am J Hum Genet. 2014; 94: 135-143Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, Takeuchi et al., 2011Takeuchi H. Fernandez-Valdivia R.C. Caswell D.S. Nita-Lazar A. Rana N.A. Garner T.P. et al.Rumi functions as both a protein O-glucosyltransferase and a protein O-xylosyltransferase.Proc Natl Acad Sci USA. 2011; 108: 16600-16605Crossref PubMed Scopus (65) Google Scholar). To gain further insights into the pathogenesis of DDD, POGLUT1 was downregulated in the melanocyte-derived cell lineage MZ7-MEL using small interfering RNA–mediated knockdown. Adequate POGLUT1 knockdown was confirmed by quantitative PCR (Figure 2g). An mRNA expression analysis of selected Notch pathway genes revealed altered Notch signaling (Figure 2g). This is analogous to findings from recent POFUT1 knockdown experiments in zebrafish larvae and HaCaT cells (Li et al., 2013Li M. Cheng R. Liang J. Yan H. Zhang H. Yang L. et al.Mutations in POFUT1, encoding protein O-fucosyltransferase 1, cause generalized Dowling-Degos disease.Am J Hum Genet. 2013; 92: 895-903Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), and PSENEN mutation carriers (Pavlovsky et al., 2018Pavlovsky M. Sarig O. Eskin-Schwartz M. Malchin N. Bochner R. Mohamad J. et al.A phenotype combining hidradenitis suppurativa with Dowling-Degos disease caused by a founder mutation in PSENEN.Br J Dermatol. 2018; 178: 502-508Google Scholar). These findings support the hypothesis that dysfunctional Notch signaling is a common pathomechanism in DDD (Frank et al., 2018Frank J. Ralser D.J. Betz R.C. Intra- and interfamilial phenotype variability associated with mutations in gamma-secretase subunit-encoding PSENEN.J Invest Dermatol. 2018; 138: 1215-1218Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). Interestingly, mRNA expression of MITF, which encodes microphthalmia-associated transcription factor, was elevated in POGLUT1-deficient cells (Figure 2g). MITF is important for melanocyte development by regulating the expression of TYR, which encodes the enzyme tyrosinase. Tyrosinase is directly involved in melanin production by catalyzing the conversion of tyrosine to melanin (Hsiao and Fisher, 2014Hsiao J.J. Fisher D.E. The roles of microphthalmia-associated transcription factor and pigmentation in melanoma.Arch Biochem Biophys. 2014; 563: 28-34Crossref PubMed Scopus (88) Google Scholar, Kawakami and Fisher, 2017Kawakami A. Fisher D.E. The master role of microphthalmia-associated transcription factor in melanocyte and melanoma biology.Lab Invest. 2017; 97: 649-656Crossref PubMed Scopus (133) Google Scholar, Yasumoto et al., 1995Yasumoto K. Yokoyama K. Shibata K. Tomita Y. Shibahara S. Microphthalmia-associated transcription factor as a regulator for melanocyte-specific transcription of the human tyrosinase gene.Mol Cell Biol. 1995; 15: 1833Google Scholar). The mRNA expression of TYR was also elevated in POGLUT1-deficient MZ7-MEL cells (Figure 2g). These data indicate a connection between altered Notch signaling and elevated levels of MITF and TYR, resulting in an increased production and misdirected deposition of melanin, as observed in patients with DDD. Detailed studies on these processes are needed. In conclusion, our analyses demonstrate a gene-phenotype correlation in DDD patients with POGLUT1 mutations, enabling clinical sub-phenotyping, as suggested previously (Basmanav et al., 2015Basmanav F.B. Fritz G. Lestringant G.G. Pachat D. Hoffjan S. Fischer J. et al.Pathogenicity of POFUT1 in Dowling-Degos disease: additional mutations and clinical overlap with reticulate acropigmentation of kitamura.J Invest Dermatol. 2015; 135: 615-618Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, Betz, 2017Betz R.C. A path through the reticulate pigmentation disorder jungle.Br J Dermatol. 2017; 177: 893-894Google Scholar). The data presented here reveal 12, to our knowledge previously unreported, POGLUT1 mutations in DDD, and evidence for the causal involvement of missense mutations. The association between altered Notch signaling and elevated mRNA levels of MITF and TYR, which are directly involved in melanogenesis, may be implicated in DDD hyperpigmentation. Jorge Frank: http://orcid.org/0000-0003-1439-8577 Regina C. Betz: http://orcid.org/0000-0001-5024-3623 The authors state no conflict of interest. We thank all patients for their participation. The study was supported by local funding (BONFOR to RCB and DJR) and National Institutes of Health funding (GM061126 to RSH). RCB is a member of the DFG-funded Excellence Cluster ImmunoSensation, and is a past recipient of a Heisenberg-Professorship from the DFG (BE 2346/4-2). We dedicate this article to the memory of Martin Leverkus, who died while this work was in progress. Download .pdf (.44 MB) Help with pdf files Supplementary Data