Dermatology: Practical and Conceptual Original Article | Dermatol Pract Concept. 2024;14(1):e2024050 1 Familial Melanoma Phenotype With Xeroderma Pigmentosum Group C (XP-C) Genotype - The Putative Role of MC1R Polymorphism as Modifier Franciele Antonieta Bianchi Leidenz1, Flavia Vasques Bittencourt2, Williana Garcia Braga1, Elio Magno de Sá Araújo1, Carolina Cavalieri Gomes3, Vanessa de Fatima Bernardes3, Eitan Friedman4, Luiz De Marco1 1 Departments of Surgery, School of Medicine, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil 2 Departments of Medicine, School of Medicine, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil 3 Departments of Pathology, School of Medicine, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil 4 The Preventive Personalized Medicine Center, Assuta Medical Center and the Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel Key words: Xeroderma Pigmentosum, Familial melanoma, XPC, MC1R, modifier genes Citation: Leidenz FA, Bittencourt FV, Braga WG, et al. Familial Melanoma Phenotype With Xeroderma Pigmentosum Group C (XP-C) Genotype - The Putative Role of MC1R Polymorphism as Modifier. Dermatol Pract Concept. 2024;14(1):e2024050. DOI: https://doi. org/10.5826/dpc.1401a50 Accepted: September 27, 2023; Published: January 2024 Copyright: ©2024 Leidenz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution- NonCommercial License (BY-NC-4.0), https://creativecommons.org/licenses/by-nc/4.0/, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original authors and source are credited. Funding: This work was partially funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq# 302291/2019-9), CAPES and Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG# CDS-RED-00019-16). The sponsors had no role in the design or conduct of this research. Competing interests: None. Authorship: All authors have contributed significantly to this publication. Corresponding author: L. De Marco, Department of Surgery, School of Medicine, Universidade Federal de Minas Gerais, Av Alfredo Balena 190, Belo Horizonte 30130-100, Brazil; e-mail: Ldemarco@ufmg.br. Introduction: Xeroderma pigmentosum (XP), a rare inherited condition, hallmarked by extreme sen- sitivity to sun exposure resulting in multiple skin cancers and non-malignant skin alterations is at- tributed to homozygous inactivating pathogenic variants (PVs) in DNA repair genes, predominantly the XPC gene. Objectives: Report a unique phenotypic expression of mutant XPC allele that may be compatible with a putative modifier role for MC1R polymorphism. Methods: A family of 13 siblings, seven of whom were diagnosed with at least one cutaneous melano- ma (N = 53) and non-melanoma skin cancers (N = 9) was studied. Of seven melanoma-affected cases, five consented for genetic analysis. CDKN2A revealed no PV in any case and subsequent whole-exome sequencing (WES) identified a rare homozygous missense PV (c.919C>T; p.Arg307Trp) in exon 8 of the XPC gene in all affected individuals. Notably, XPC PV carriers who co-harbored the p.I155T MC1R variant (N = 3) exhibited larger number of tumors, deeper Breslow indexes, higher rates of invasive melanomas and earlier age at diagnosis compared with non MC1R variant carriers (N = 2). Conclusions: Familial malignant melanoma phenotype may, in fact, be an unusual clinical presen- tation of XPC, and MC1R may be a genetic modifier of penetrance and phenotype of mutant XPC alleles. ABSTRACT 2 Original Article | Dermatol Pract Concept. 2024;14(1):e2024050 Introduction Xeroderma Pigmentosum (XP) is a rare autosomal recessive disorder whose estimated rates of 2.3/1,000,000 in Western countries clinically hallmarked by multitude of skin anoma- lies [1]: hyperpigmentation, premature skin aging, cutaneous (and ocular) photosensitivity, and an increased risk for de- veloping a host of skin tumors [2]. Genetically, XP is asso- ciated with homozygous pathogenic variants (PVs) in seven nucleotide excision repair pathway genes: XPA, ERCC3, XPC, ERCC2, DDB2, ERCC4, ERCC5 and one clinical vari- ant (XPV, Xeroderma Pigmentosum Variant) attributed to a mutated POLH gene [2]. The most commonly mutated gene underlying XP in Caucasians is XPC group C (MIM #278720) [3]. The clin- ical phenotype in XP due to mutant XPC alleles dependent on sun exposure and the mutational background, hence on the geographical origin [4]. The incidence of skin cancers is 1,000 times higher in XP patients compared with average risk population and a major contributor to the substantially decreased life expectancy in XP cases, around 30 years [5]. These patients have an estimated 10,000-fold increased risk of non-melanoma skin cancer, a 2,000-fold increased risk of melanoma under the age of 20 [1] and the first skin lesions may appear as early as eight years of age [5]. Familial melanoma syndrome (FMS) is a term used to describe the presence of two or more cases of cutaneous mel- anoma in first- or second-degree relatives [6]. An alternative classification [7] refers to these cases as Melanoma dominant syndrome when melanoma is the first or the predominant clinical manifestation in a family. Objectives Here we report a family with cutaneous melanomas with a seemingly autosomal dominant inheritance pattern with- out clear clinical characteristics of XP, where a homozygous XPC inactivating PV co-segregated with the phenotype, and a mutant MC1R allele seemingly affected the clinical mela- noma phenotype. Methods The proband (Figure 1), individual II.11, was referred in 2000 at age 28, after a diagnosis of melanoma and a history of death of his older brother from metastatic melanoma of the lower lip at age 38. Since then, a total of 24 melanomas have been confirmed in the proband. These melanomas were mostly located in the head (N = 19) and neck (N = 2) regions, including the scalp (Figure 2), and upper trunk (N = 3). The majority (19/24) were in situ, three were thin melanomas (Breslow 0.45 mm, 0.47 mm and 0.9 mm), one was 1.4 mm, and one was 2.0 mm. Physical examination revealed brown hair and brown eyes, Fitzpatrick phototype III, sun damage on exposed areas with some pigmented changes, such as solar lentigines. Throughout 20 years of follow-up, he also presented two basal cell carcinomas (BCC) and one baso- squamous carcinoma of the head and neck. History of un- protected excessive sun exposure was reported. Dermoscopy of pigmented lesions did not show any specific patterns. All consenting siblings (12) were examined, and five of these sib- lings were diagnosed with melanoma, (a total of 7/13 mela- noma affected siblings). Of melanoma patients, the majority had multiple atypical nevi (three had more than 20, one had fewer than 10 and one had none), all have a relevant his- tory of unprotected sun exposure throughout life. They did not get sunburned easily and did not have a recollection of many blistering despite having a long history of excessive un- protected sun exposure. Most of them have freckles or solar melanosis, confirming the sun-damaged skin. Clinical char- acteristics of affected individuals are shown in Table 1. All melanoma cases reported a history of significant sun expo- sure, Fitzpatrick type III, and solar lentigines in sun exposed areas. At the time of reporting, five of the six living affected siblings have had multiple primary melanomas, varying from three to 24 tumors per person. Age range at first melanoma Figure 1. Pedigree of proband (II.11) family. Individuals affected by cutaneous melanoma are represented in black-filled shapes, while white shapes represent individuals without history of melanoma. Original Article | Dermatol Pract Concept. 2024;14(1):e2024050 3 diagnosis was 22-45 years of age. Neither visceral metastasis nor lymph node disease have been reported in any of the six affected siblings, and no adjuvant therapies were given. Interestingly, no relevant ophthalmologic condition has been diagnosed in the affected family members. The siblings were born to consanguineous (second-degree cousins) phenotyp- ically healthy parents; the father died at age 58 without any history of skin cancer. The mother is still alive (age 86), in good health and never had any skin cancer. Occupational ex- posure to sun was suggested by the fact that they were mostly farm laborers. Figure 2 demonstrates some of their clinical features. This study was approved by the Institutional Re- view Board of Universidade Federal de Minas Gerais (CAAE 0472.0.203.240-11). Prior to enrolment, written informed consent was obtained from all patients. Sanger sequencing for the CDKN2A gene, the most com- mon high penetrance melanoma susceptibility gene, was ini- tially performed. Subsequently, peripheral blood DNA from individual II.11 was submitted to Whole-Exome Sequencing (WES). The VCF file was analyzed using Mendel, MD soft- ware (http://mendel.medicina.ufmg.br) [8] as well as by In- genuity® Variant AnalysisTM software (www.ingenuity.com/ variants). Captured sequences were aligned with the human reference genome GRCh37 and a sequence of filters applied. A flow chart is shown in Figure 3. Given the autosomal re- cessive inheritance pattern in the studied family we focused Figure 2. Clinical images of affected individuals. (A) Note skin photoaging and solar lentigines on sun exposed areas of the proband with a large scar from a previous removed melanoma and a new melanoma on the left side of the scar. (B) Melanoma on the scalp in the proband, in a relatively sun protected area. (C) Two melanomas on the forehead of individual II.8. (D) One melanoma in the left cervical region of individual II.13 on exposed area showing solar lentigines, surrounded by pigmentary changes. Table 1 Familial melanoma and their characteristics Affected individuals Age of first melanoma Number of melanomas Tumor(s) localization(s) Breslow thickness Sun exposure Total nevus body count Other skin cancers II.4 45 3 Head (3) All in situ Excessive unprotected <20 None II.7 33 11 Head (3), neck (1), limbs (2) and upper trunk (5) All in situ Excessive unprotected 20-100 Two BCC II.8 35 7 Head (3), neck (2), and chest (2) 0.24mm and 6 in situ Excessive unprotected 20-100 Three BCC II.10 48 1 Head (scalp) in situ Excessive unprotected 20-100 None II.11 28 24 Head (19), neck (2), and upper trunk (3) 0.45mm; 0.47mm; 0.9mm; 1.4mm; 2.0mm and 19 in situ Excessive unprotected >100 Two BCC, one basosquamous carcinoma II.13 22 7 Head (3), neck (3) and upper trunk (1) 0.23mm; 0.36 mm; 2.3 mm and 4 in situ Excessive unprotected >100 One BCC 4 Original Article | Dermatol Pract Concept. 2024;14(1):e2024050 homozygous variants only, it was narrowed down to eight genes, of whom XPC was the strongest candidate. Sequencing of XPC demonstrated that the clinically un- affected mother carried the heterozygous XPC variant p.Ar- g307Trp (c.919C>T) in exon 8. All five affected individuals studied were homozygous (T/T) for this sequence variant. Another known pathogenic variant (c.464T>C; p.Ile155Thr) was detected in the MC1R gene (NM_002386.4) by WES. Three (II.7, II. 11 and II.13) of the five affected melanoma cases who carried the XPC homozygous variant harbored this heterozygous PV. These three co-carriers (XPC and MC1R) carriers displayed significantly worse disease phenotype compared with the two non-co-carriers: higher number of melanomas per person (14 versus 5), earlier age of melanoma diagnosis (average 27.6 years versus 40 years) and thicker melanomas (three invasive in co-carriers ver- sus noninvasive melanomas in non-co-carriers, with mean on homozygous variants. All seemingly candidate variants were confirmed by Sanger sequencing and co- segregation was subsequently carried out in all consenting cases (N = 15) using Sanger sequencing. To further assess the putative clinical significance of the detected XPC sequence variant and the possibility of residual protein function, we performed immunohistochemical anal- ysis using XPC antibody (D-10: sc-74410, Santa Cruz Bio- technology) both on melanoma tissue from the proband and on normal mucosal tissue from the same individual mucosa (serving as control). Results CDKN2A gene genotyping yielded no PVs. WES showed 46,452 variants and, after a series of filtering, exclusion of variants in our control exomes bank and selection of Figure 3. Flowchart of variant analyses. Original Article | Dermatol Pract Concept. 2024;14(1):e2024050 5 a family with atypical XP presentation age in the proband. Late onset of tumors and late diagnosis of XP are uncommon but there are a few previously reported cases with distinct phenotypes [10,11]. To our knowledge, there are two reports of XPC-related XP phenotype being diagnosed in late adult- hood: an 83-year-old French woman with multiple melano- mas whose unusual long-term survival was attributed to a lower UV-radiation exposure and regular clinical follow-up and a 42-year-old Caucasian man with multiple melanomas and a missense PV in the XPC gene that still retained some XPC protein function and, seemingly contributing to late XP diagnosis [4,11]. XP typically leads to a shortened life span, with cancer related death usually occurring at 30-40 years of age [12]. In the case described herein all but one patient are alive aged 41-62 years, at the time of reporting (Figure 1). The less se- vere phenotype and the less pronounced effect on early age mortality in this family suggests that the XPC mutated pro- tein is still capable of exerting some residual activity and re- pair DNA damage, as previously suggested [4]. Being a recessive condition, it is expected that around 25% of the offspring would be affected. In this family, seven of the 13 siblings are affected (53.8%), with an extraordi- nary higher number of melanomas (N = 43) compared with non-melanoma skin cancers (n= 9), an unusual tumor distri- bution to the one usually seen in XP patients – carcinomas are five times more common than melanomas [1]. Additional clinically unique features in this family that have led to XP not even being considered as a possible diagnosis prior to genetic analysis, were the fact that melanomas were not only Breslow thickness of 0.17 mm in carriers, versus 0.017 mm in non-carriers). Our data shows that the median number of non-melanoma skin cancer (NMSC) per person among MC1R wild-type XP-C individuals was 1.5 (three BCC in two subjects). In contrast, among carriers of the I155T vari- ant, the median number of NMSC was 2.0, with six tumors occurring in three individuals. The XPC c.919C>T;p.Arg307Trp variant is predicted to be pathogenic by several different bioinformatics tools: dis- ease causing by MutationTaster (http://www.mutationtaster .org/), probably damaging by PolyPhen-2 with a score of 1.0 (http://genetics.bwh.harvard.edu/pph2/) and deleteri- ous by the Protein Variation Effect Analyzer (PROVEAN, http://provean.jcvi.org/index.php), with a score of –6.872. The pathogenicity of the c.919C>T;p.Arg307Trp sequence variant is also inferred by its rarity (prevalence in GnomAD (https://gnomad.broadinstitute.org/) 0.00002422 with none reported in Brazilian databanks (https://abraom.ib.usp.br/). Variant classification according to 2015 ACMG guidelines is PM2 (moderate evidence of pathogenicity) and with addi- tional supporting evidence of pathogenicity (PP1). Immunohistochemistry analysis demonstrated no XPC staining in the melanoma tissue compared with normal non cancer surrounding dermal tissue, as is predicted by func- tional studies [9] (Figure 4). Conclusions XP is usually diagnosed in childhood heralded by the pres- ence of typical skin lesions [5]. The current study describes Figure 4. Immunohistochemistry assay. (A) Melanoma tissue from proband stained with H&E. (B) Melanoma tissue from proband very lightly stained by the XPC antibody, suggesting there is no significant expression of this protein in this tissue. The dark color is mostly due to melanin. (C) XPC antibody in a control tissue (mucosa) showing high positivity, therefore, significant quantity of XPC protein in this normal tissue. (D) the same melanoma tissue from proband highly colored by a control anti-tubulin antibody, showing that this tissue is indeed viable. 6 Original Article | Dermatol Pract Concept. 2024;14(1):e2024050 3. Ali MZ, Blatterer J, Khan MA, et al. Identification of a novel protein truncating mutation p.Asp98* in XPC associated with xeroderma pigmentosum in a consanguineous Pakistani fam- ily. Mol Genet Genomic Med. 2020;8(2):e1060. DOI: 10.1002 /mgg3.1060. PMID: 31923348. PMCID: PMC7005610. 4. Meneses M, Chavez-Bourgeois M, Badenas C, et al. Atypical Clinical Presentation of Xeroderma Pigmentosum in a Patient Harboring a Novel Missense Mutation in the XPC Gene: The Importance of Clinical Suspicion. Dermatology. 2015;231(3): 217-221. DOI: 10.1159/000433527. PMID: 26278556. 5. Ribeiro MG, Zunta GL, Santos JS, Moraes AM, Lima CSP, Ortega MM. Clinical features related to xeroderma pigmento- sum in a Brazilian patient diagnosed at advanced age. Appl Clin Genet. 2018;11:89-92. DOI: 10.2147/TACG.S155083. PMID: 30127633. PMCID: PMC6089094. 6. Sá BCS, Moredo LF, Gomes EE, Araújo ESS, Duprat JP. Hereditary melanoma: a five-year study of Brazilian patients in a cancer referral center - phenotypic characteristics of probands and pathological features of primary tumors. An Bras Dermatol. 2018;93(3):337-340. DOI: 10.1590/abd1806-4841.20186201. PMID: 29924249. PMCID: PMC6001076. 7. Abdo JF, Sharma A, Sharma R. Role of Heredity in Mela- noma Susceptibility: A Primer for the Practicing Surgeon. Surg Clin North Am. 2020;100(1):13-28. DOI: 10.1016/j.suc .2019.09.006. PMID: 31753108. 8. G C C L Cardenas R, D Linhares N, L Ferreira R, Pena SDJ. Mendel,MD: A user-friendly open-source web tool for analyz- ing WES and WGS in the diagnosis of patients with Mendelian disorders. PLoS Comput Biol. 2017;13(6):e1005520. DOI: 10.1371/journal.pcbi.1005520. PMID: 28594829. PMCID: PMC5464533. 9. Budden T, Davey RJ, Vilain RE, et al. Repair of UVB-induced DNA damage is reduced in melanoma due to low XPC and global genome repair. Oncotarget. 2016;7(38):60940-60953. DOI: 10.18632/oncotarget.10902. PMID: 27487145. PMCID: PMC5308628. 10. Fassihi H, Sethi M, Fawcett H, et al. Deep phenotyping of 89 xeroderma pigmentosum patients reveals unexpected hetero- geneity dependent on the precise molecular defect. Proc Natl Acad Sci U S A. 2016 1;113(9):E1236- E1245. DOI: 10.1073 /pnas.1519444113. PMID: 26884178. PMCID: PMC4780618. 11. Macke EL, Morales-Rosado JA, Gupta A, et al. A novel missense variant and multiexon deletion causing a delayed presentation of xeroderma pigmentosum, group C. Cold Spring Harb Mol Case Stud. 2020;6(4):a005165. DOI: 10.1101/mcs.a005165. PMID: 32843428. PMCID: PMC7476405. 12. Jacobelli S, Soufir N, Lacapere JJ, et al. Xeroderma pigmentosum group C in a French Caucasian patient with multiple melanoma and unusual long-term survival. Br J Dermatol. 2008;159(4): 968-973. DOI: 10.1111/j.1365-2133.2008.08791.x. PMID: 18717677. diagnosed in chronically sun exposed areas, such as scalp of hairy individuals, lack of typical XP-associated skin changes such as actinic keratosis, atrophy, telangiectasias and marked skin photoaging. In the present study, having both the XPC homozygous variant and a variant MC1R allele (p.I155T) may have dele- teriously affected the phenotype, with those harboring both variants exhibiting seemingly a more aggressive clinical and pathological phenotype. Few studies reported the effect of co-harbouring MC1R alleles in genetically proven XP cases. A previous study of 17 Nepalese XP patients suggested that the p.R163Q MC1R variant in XPC patients was associated with younger age at first carcinoma and more numerous cu- taneous carcinomas, although this latter difference was not statistically significant [1]. Additionally, it has been specu- lated that the coexistence of XPC mutation with another MC1R variant (p.V60L) was associated with multiple pri- mary melanoma phenotype in a single French patient who had 10 melanomas and six BCC before being diagnosed with XP [12]. Although the putative modifier effect on MC1R variant on the phenotype of the homozygous deleterious variant in the XPC gene is intriguing, it remains currently speculative at best and awaits further functional studies and/ or additional families displaying similar effect. In conclusion, a familial melanoma phenotype with a presumed autosomal recessive inheritance pattern was shown to be atypical in a genetically proven XP. The role, if any, of MC1R alleles in modifying the clinical tumor pheno- type in XPC mutation carriers is suggested. Validation and extension of these preliminary observations are needed. Ethics statement: This study was approved by the Insti- tutional Review Board of Universidade Federal de Minas Gerais (CAAE 0472.0.203.240-11). Prior to enrolment, written informed consent was obtained from all patients. References 1. Espi P, Parajuli S, Benfodda M, et al. Clinical and genetic charac- teristics of xeroderma pigmentosum in Nepal. J Eur Acad Der- matol Venereol. 2018;32(5):832-839. DOI: 10.1111/jdv.14717. PMID: 29178624. 2. Ijaz A, Basit S, Gul A, et al. XPC gene mutations in families with xeroderma pigmentosum from Pakistan; prevalent founder effect. Congenit Anom (Kyoto). 2019;59(1):18-21. DOI: 10.1111/cga .12281. PMID: 29569758.