Dermatology: Practical and Conceptual Original Article | Dermatol Pract Concept. 2024;14(3):e2024120 1 Prevalence of CDKN2A, CDK4, POT1, BAP1, MITF, ATM, and TERT Pathogenic Variants in a Single-Center Retrospective Series of Patients With Melanoma and Personal or Family History Suggestive of Genetic Predisposition Giada Ferrara1, Salvatore Paiella2, Giulio Settanni3, Melissa Frizziero4, Paolo Rosina1, Valeria Viassolo5 1 Section of Dermatology and Venereology, Department of Medicine, University of Verona, Verona, Italy 2 Pancreatic Surgery Unit, Pancreas Institute, University of Verona, Verona, Italy 3 Pathology Unit, IRCCS Sacro Cuore Don Calabria Hospital, Negrar di Valpolicella, Verona, Italy 4 Cancer Research UK Manchester Institute Cancer Biomarker Centre, Manchester, UK 5 Medical Genetics, IRCCS Sacro Cuore Don Calabria Hospital, Negrar di Valpolicella, Verona, Italy Key words: Melanoma, CDKN2A, MITF, genetic predisposition, pancreatic cancer Citation: Ferrara G, Paiella S, Settanni G, Frizziero M, Rosina P, Viassolo V. Prevalence of CDKN2A, CDK4, POT1, BAP1, MITF, ATM, and TERT Pathogenic Variants in a Single-Center Retrospective Series of Patients With Melanoma and Personal or Family History Suggestive of Genetic Predisposition. Dermatol Pract Concept. 2024;14(3):e2024120. DOI: https://doi.org/10.5826/dpc.1403a120 Accepted: February 1, 2024; Published: July 2024 Copyright: ©2024 Ferrara 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: None. Competing Interests: SP receives Consultancy Honoraria from AlphaTau. Authorship: All authors have contributed significantly to this publication. Corresponding Author: Valeria Viassolo, Medical Genetics, IRCCS Sacro Cuore Don Calabria Hospital, Via Don A. Sempreboni 5, 37024, Negrar Di Valpolicella (VR), Italy. Email: valeria.viassolo@sacrocuore.it Introduction: Approximately 20%-45% of familial melanoma (FM) cases are associated with genetic predisposition. Objectives: This single-center retrospective study aimed to assess the frequency of pathogenic variants (PV) in the main melanoma-predisposing genes in patients with cutaneous melanoma and investigate the clinical predictors of genetic predisposition. Methods: Patients included were those diagnosed with cutaneous melanoma at the Dermatology Unit of the University Hospital of Verona, Italy, from 2000 to 2022, presenting at least one of the follow- ings: multiple melanomas (≥ 3); personal/family history of pancreatic cancer (PC) (up to 2nd-degree relatives); ≥ 2 1st-degree relatives with melanoma; ≥ 1 1st-degree relatives with early-onset (<45 years) melanoma and tested for CDKN2A, CDK4, POT1, BAP1, MITF, ATM, and TERT. ABSTRACT 2 Original Article | Dermatol Pract Concept. 2024;14(3):e2024120 Introduction Melanoma is a skin cancer that originates from the neoplas- tic transformation of melanocytes. Previously considered a rare diagnosis, its incidence has grown worldwide in recent decades [1,2]. According to the patient family history, melanoma can be classified as sporadic or familial (FM). Randomly acquired genomic changes in melanocytes resulting from environmen- tal factors or aging and favored by certain phenotypic traits (eg skin color) cause sporadic melanoma [1]. Approximately 8% to 12% of patients with melanoma have at least one first-degree relative who developed this cancer [1]. Most fa- milial aggregations of melanoma are not related to a mono- genic inheritance. They can be associated with grouping sporadic cases in families with common risk factors, such as sun exposure or fair skin [1]. In up to 45% of cases, patients with FM carry a PV in a predisposing gene [3]. In this con- text, fewer somatic acquired mutations are likely required before a critical level for oncogenesis is achieved. Prevalence of moderate or high-risk alleles and pene- trance of PV in these genes vary from 15% to 45% accord- ing to geographical area and ethnicity [3-7], which is likely a reflection of a combination of environmental factors with several other inheritable genetic modifiers shared by family members. Considering the distribution of polymorphic low- risk alleles across geographical areas, no single guideline for melanoma genetic testing can be adopted worldwide. How- ever, in Southern European populations, where the incidence of melanoma is historically considered low, genetic testing is generally proposed in the presence of melanoma in at least two first-degree relatives or three second-degree relatives in the same branch of the family [8]. Since the mid-1990s, many melanoma predisposition genes have been identified. CDKN2A is involved in ap- proximately 20%-40% of familial melanoma cases [9]. The CDKN2A gene consists of three exons encoding two proteins derived from alternatively framed transcripts, p16 (p16INK4A) and p14 (p14ARF), both acting as a tumor suppressor involved in cell cycle control. Genome-wide DNA sequencing studies have demon- strated that the CDKN2A locus is in a nevus-associated genomic region. Carriers of CDKN2A PV show a signifi- cantly higher number of nevi and clinically atypical nevi than non-carriers [10, 11]. Some, but not all, CDKN2A carriers exhibit a clinical phenotype consistent with atypical multiple mole melanoma syndromes (FAMMM) [5,12,13]. The detection rate of CDKN2A PV varies across dif- ferent geographical areas [4]. In 2009, CDKN2A PV were identified in about 33% of FM cases in Italy and 25% of families with only two affected first-degree relatives [13]. Recently, a large multicentric Italian study showed a lower detection rate, identifying 21 carriers of CDKN2A PV out of 373 melanoma patients having one first-degree relative with melanoma (5.63%) and ten carriers out of 101 patients with two first-degree relatives (9.9%) [14]. Increasing evidence suggests melanoma is part of larger cancer syndromes [9]. CDKN2A PV affecting p16INK4A are associated with an increased risk of developing pancre- atic cancer (PC), up to 20% within the age of 70 [15,16]. PV in other genes such as CDK4, POT1, BAP1, MITF, ATM and TERT have been associated to moderate to high- risk to develop melanoma, as well as other malignancies, and are part of multi-gene panels employed in clinic to assess the presence of an inherited melanoma predisposition syndrome [3,8,17]. The prevalence of PV in these genes in Italy is as high as 3.7% in families with at least two first-degree rela- tives with melanoma and 4% with three affected first-degree relatives [14]. Objectives This study aims to estimate the prevalence of germline PV in CDKN2A, CDK4, POT1, BAP1, MITF, ATM, and TERT and to evaluate the impact of patient’s cutaneous pheno- type and personal/family history of PC and melanomas as Results: During the study period, 35 out of 1320 patients (2.7%) underwent genetic testing. Four patients (11.4%) harbored a PV in a melanoma-predisposing gene, three in CDKN2A (8.6%), and one in MITF (2.9%). Variants currently classified as being of unknown clinical significance (VUS) were detected in CDKN2A (N = 1), MITF (N = 1), and ATM (N = 2). Family history of PC and ≥5 melanomas, personal history of ≥50 nevi, and ≥4 melanomas were significantly associated with PV in tested genes (P < 0.05). Conclusions: The prevalence of PV in predisposing genes in FM was lower than previously reported in Italian registries. Possible reasons include deleterious variants in untested intermediate/low-penetrance genes or yet-to-be-discovered high-penetrance genes and environmental risk factors. A family history of PC, a high number of nevi and melanomas predict a monogenic predisposition to melanoma. Original Article | Dermatol Pract Concept. 2024;14(3):e2024120 3 predictors of the presence of a monogenic predisposition in a single-center cohort of highly selected Italian melanoma patients. Taking into account the rising of melanoma inci- dence and the recent decrease in the prevalence of PV among FM and multiple primary melanoma cases in Italy, to select the study population we employed criteria that are more stringent than those used in the current clinical practice to propose genetic counseling and testing in Italy (Italian soci- eties such as Italian Society of Human Genetics (SIGU) [18], Italian Society of Dermatology (SIDeMaST) [19] and Italian Society of Oncology (AIOM) [20]). Methods Study Population and Ethics Statement Medical records of 1320 patients diagnosed with cutane- ous melanoma at the Dermatology Unit of the University of Verona Hospital Trust, Verona, Italy, from April 2000 to March 2022 were retrospectively reviewed. To select patients to address to genetic counseling, we employed criteria that mirror those used in countries with a high incidence of melanoma, such as Sweden [21] paying attention to the number of melanoma in case of patients with multiple or patients with a positive family history of mela- noma, to the age at diagnosis and to family history of PC. Patients with one or more of the following criteria were identified: • Multiple melanomas (≥ 3) • Personal/family history for PC (up to the second degree of kinship) • ≥ 2 melanomas among first-degree relatives • First-degree relatives with early-onset melanoma (< 45 years) Forty-six patients (3.5%) met the selection criteria, and 35 (76.1%) agreed to undergo genetic counseling and test- ing for the main melanoma predisposition genes and were included in this study. The study protocol was approved by the local Institu- tional Review Board “Comitato Etico per la Sperimentazi- one Clinica delle Province di Verona e Rovigo” on 19th of April 2023 (Prot. n. 24955, 26th of April 2023) and it was in agreement with the principles of the Declaration of Helsinki. All patients provided a signed informed consent approved by a local Institutional Review Board. For each patient, the diagnosis of melanoma was confirmed by histopathological analysis. Data Collected The following patient data were collected: environmental risk factors (eg sun exposure, history of burns), personal history of cancer (any), family history of cancer (any), in- cluding the elaboration of a three-generations family tree. Genetic counseling and testing were centralized (Medical Genetics Unit, IRCCS Sacro Cuore Don Calabria Hospital in Negrar di Valpolicella, Verona) for 29 patients (82.9%). Six patients (17.1%) were referred to other oncogenetic centers for geographical reasons. Genetic Analysis Genetic testing was performed on DNA extracted from the patient peripheral blood. Genetic testing provided in IRCCS Sacro Cuore Don Calabria Hospital in Negrar di Valpolicella, Varona analyzed CDKN2A, CDK4, POT1, BAP1, MITF, ATM, and TERT. Mutational analysis was performed by using massive parallel sequencing with Thermo Fisher Scientific S5 Prime platform, with multi- plex amplification of all exons and exon-intron junctions up to 20bp by a custom Ampliseq On-Demand Panel IAD1965574_1000 (Thermo Fisher Scientific). The reads obtained were aligned on reference genome GRCh37/hg19. Data analysis and variants annotation was performed us- ing two distinct pipelines: 1) Ion Torrent Suite v5.12, Ion- Reporter v5.12 (Thermo Fisher Scientific); 2) Ensembl Variant Effect Predictor (EMBL-EBI, Wellcome Sanger Institute). Visualization of the reads performed with IGV 2.8 (Broad Institute). Search for large deletions/insertions (Copy Number Variation, CVN) of the genes included in the panel performed by CNVs analysis with Ion Reporter v5.12 (Thermo Fisher Scientific). Genetic testing performed in other centers investi- gated a larger amplicon-based gene panel sequenced us- ing Thermo Fisher Scientific S5 Prime platform analogous to the one of our reference lab. The presence of the seven genes analyzed at the Sacro Cuore Don Calabria Hospital panel was considered mandatory to include the cases in the study cohort. Reference databases employed for clinical interpreta- tion of gene variants were: ClinVar, LOVD database (LOVD v.3.0 Build 29 - LOVD software ©2004-2023), Varsome and Franklin by Genoox. Statistical Analysis Median and interquartile range were used to present de- scriptive statistics. The association between the presence of PV in melanoma predisposition genes and various clinical characteristics was assessed using the Chi-square or Fisher exact test. The study utilized a two-sided Fisher exact test in the SPSS software to determine the statistical association. Statistical significance was set at a P value threshold of less than 0.05. Statistical analyses were performed using SPSS software ver. 25 (IBM) 4 Original Article | Dermatol Pract Concept. 2024;14(3):e2024120 had two melanomas, the first at 51 years, with a total nevi number >100. His father developed a PC. Genetic testing was proposed to his brother, sister, and two children; how- ever, to our knowledge, none was ultimately tested due to unwillingness. Patient #26 tested positive for c.150+5del CDKN2A variant (NM_000077.5:c.150+5del), currently classified as VUS. This patient, diagnosed with melanoma at age 26, also had a family history of melanoma since his father presented two melanomas at 32 and 49. Therefore, a genetic analysis was also performed on the patient’s father, who was a carrier of the same variant, to evaluate the segregation of this vari- ant in the family. Patient #24 had the c.272C>T (p.Pro91Leu) variant in the MITF gene (NM_001354604.2:c.272C>T) which is ab- sent in ClinVar but reported in dbSNP database with the entry rs199832302. The variant is absent in GnomAD popu- lation database and is predicted to be neutral for the protein function by in silico predictors. It is important to highlight that this variant is absent in the transcript NM_000248.4 because of its upstream position from the start codon, and this aspect may limit its clinical interest in hereditary mel- anoma. However, this patient had a potentially suggestive family history in both parental branches: his father was diagnosed with clear cell kidney cancer aged 39 years, and his mother had thyroid cancer at 50 and melanoma at 59. The father renal cancer and the mother melanoma were confirmed through medical records. On the paternal side, an uncle presented with melanoma aged 55, and a second uncle with renal cancer aged 54. We could see the father pa- tient in genetic consultation, and he accepted to undergo a genetic analysis that failed to identify the MITF c.272C>T (p.Pro91Leu), which was, in turn, of maternal origin. Two VUS were detected in ATM gene in two distinct individuals. The ATM c.8734A>G (p.Arg2912Gly) (NM_000051.4: c.8734A>G) variant found in patient #1 is currently clas- sified as a VUS based on its presence at low frequency in GnomAD database in Non-Finnish European population (0.04%) with an homozygote carrier, altered protein func- tion predicted by in silico tools and moderate aminoacid conservation score. The variant has been previously reported in ClinVar database as VUS. The ATM c.3393G>A (p.Met1131Ile) (NM_000051.4: c.3393G>A) variant found in patient #5 is a missense variant classified as VUS based on the following: lack of effect on the protein function predicted by in silico tools , presence of previous classifications on ClinVar database, and extremely low variant frequency in Non-Finnish European population in GnomAD database (0.0004%). No segregation analysis was performed in these two families. Results Genetic Testing Results Genetic testing revealed the presence of hereditary mela- noma in 4 out of 35 index cases (11.4%). In particular, three CDKN2A (8.6%) and one MITF (2.9%) PV were identi- fied in four distinct patients. Moreover, variants currently classified as VUS were detected in CDKN2A (N = 1), MITF (N = 1), and ATM (N = 2) genes in 4 distinct patients. Median age at diagnosis of first melanoma was 48.3 years (range 23-85) in the whole cohort, 51.1 (range 25-75) in patients with multiple melanomas, and 32.3 (range 25-44) in patients harboring CDKN2A PV, while the carrier of MITF PV presented his first melanoma at 51. All carriers of CDKN2A or MITF PV had a high nevus count (two had more than 100 nevi, the others between 50 and 100). Among patients with ≥ 3 melanomas, the prevalence of CDKN2A PV was 18.2%, whereas it was 4.2% in patients with one or two melanomas. All CDKN2A PV identified were missense and affected p16INK4A. Clinical phenotypes and genetic test results of patients harboring a PV or a VUS are presented in Table 1. The general characteristics of the whole cohort are shown in Table S1. Patient #7 carried the CDKN2A PV c.142C>A (p.Pro48Thr) (NM_000077.5:c.142C>A). He was diagnosed with five mel- anomas, the first at 28, with a total nevi number >100. He presented a positive family history of PC (paternal aunt) and possibly melanoma aggregation in the paternal lineage (his father developed a non-specified skin cancer in old age). Patient #8 and patient #35 harbored the well-known c.301G>T (p.Gly101Trp) CDKN2A PV (NM_000077.5: c.301G>T), which is particularly frequent in Italy and France due to a founder effect [22]. Patient #8 developed four mel- anomas, the first at age 28. He had a total nevi number >50. Family history included PC (paternal uncle). He has a sis- ter and a 32-year-old daughter, who refused to perform genetic counseling and testing. Patient #7 had four melano- mas, the first at 25 years, with a total nevi number > 50. Family history included multiple melanomas (sister) and PC (maternal grandfather). Genetic counseling was extended to the mother, sister, and daughter, and at the time of this man- uscript, the results were not available. Patient #35 developed two melanomas (at 44 and 56 years, respectively), his sister had at least four melanomas, and a paternal uncle had a mel- anoma at around 40 years. He had a total nevi number >50. The sister, the only sib of patient #35, underwent genetic testing and tested positive. Two out of his four children had genetic counseling and tested negative. Patient #33 was found to carry the MIFT PV c.952G>A (p.Glu318Lys) (NM_000248.4:c.952G>A. This patient Original Article | Dermatol Pract Concept. 2024;14(3):e2024120 5 Ta b le 1 . C lin ic al c ha ra ct er is ti cs a nd g en et ic t es t re su lt s of p at ie nt s ha rb or in g a pa th og en ic v ar ia nt o r a va ri an t cl as si fie d as v ar ia nt o f un kn ow n cl in ic al  s ig ni fic an ce Pa ti en t ID N . o f m el an o m a N o f m el an o m a in th e fa m ily a A g e at d ia g n o si s o f th e fi rs t m el an o m a FH o f PC a To ta l n ev u s co u n tb Fi tz p at ri ck sk in t yp e G en e G en et ic c h an g e (H G V S n o m en cl at u re ) Pr o te in c h an g e V ar ia n t in te rp re ta ti o n #1 3 3 64 no 3 3 A T M N M _0 00 05 1. 4: c. 87 34 A >G p. A rg 29 12 G ly V U S #5 3 4 54 no 3 3 A T M N M _0 00 05 1. 4: c. 33 93 G >A p. M et 11 31 Il e V U S #7 5 7 28 ye s 4 3 C D K N 2A N M _0 00 07 7. 5: c. 14 2C >A p. Pr o4 8T hr PV #8 4 5 25 ye s 3 3 C D K N 2A N M _0 00 07 7. 5: c. 30 1G >T p. G ly 10 1T rp PV #2 4 1 2 39 no 3 1 M IT F N M _0 01 35 46 04 .2 :c .2 72 C >T p. Pr o9 1L eu V U S #2 6 1 3 26 no 2 2 C D K N 2A N M _0 00 07 7. 5: c. 15 0+ 5d el V U S #3 3 2 2 51 ye s 4 3 M IT F N M _0 00 24 8. 4: c. 95 2G >A p. G lu 31 8L ys PV #3 5 2 7 44 no 3 3 C D K N 2A N M _0 00 07 7. 5: c. 30 1G >T p. G ly 10 1T rp PV FH = f am ily h is to ry ; P C = p an cr ea ti c ca nc er ; H G V S = H um an G en om e V ar ia ti on S oc ie ty ; V U S = va ri an t of u nk no w n cl in ic al s ig ni fi ca nc e; P V = pa th og en ic v ar ia nt a up t o 2nd d eg re e; b 1 = <1 0, 2 = 10 -5 0, 3 = 50 -1 00 , 4 = >1 00 . 6 Original Article | Dermatol Pract Concept. 2024;14(3):e2024120 lower than the 33% detection rate in the Italian population with FM reported in 2009 by the Italian Melanoma Inter- group study (25% in families with 2 melanomas and 46% in families with 3 melanomas) [13] and with a detection rate of 29.6% in the population with three or more melanomas reported in 2016 by the same group [6]. Interestingly, the authors selected patients according to Italian Society of Hu- man Genetics recommendations, using less stringent clinical criteria (≥ 2 melanomas in a single patient or within the same family lineage regardless of age at diagnosis) than those em- ployed in this study. These and other Italian studies [6,13] reported a CDKN2A mutation rate of 8.2% in patients with two melanomas, 17.6% in patients with three or more mel- anomas, 36.6% in patients with FM and two melanomas, 58.8% in patients with FM, and three or more melanomas, of 25% in families with two affected members, and of 46% in families with three affected members. In a study on multi- ple melanomas in the Veneto region, the detection rate of the CDKN2A PV for multiple melanomas was 12% [10]. Although these studies indicate a high prevalence of CDKN2A PV among FM cases in Italy, a recent Italian study showed a remarkably lower prevalence of PV (9.47%), con- sidering either CDKN2A alone or the whole gene panel [14], close to the findings of our study. The authors suggested that, in the absence of a diagnosis of at least three primary mel- anomas, personal/family history of PC, and in patients with Predictors of Genetic Predisposition We evaluated the patient phenotype (number of nevi and melanomas, age of first melanoma diagnosis) and family history of PC and melanoma among 1st- and 2nd-degree rel- atives as clinical predictors of the presence of a genetic pre- disposition to melanoma. As shown in Table 2, three out of the four patients harboring CDKN2A or MITF PV had a positive family history of PC (75%), whereas, among 31 pa- tients without PV in tested genes, five had a 1st- or 2nd-degree relative with PC (16.1%) (P = 0.0302). In addition, having ≥ 50 nevi or at least four melanomas and observing ≥ 5 mel- anomas among patient and 1st- or 2nd-degree relatives were statistically significantly associated with PV in tested genes (P < 0.05), whereas a personal history of fewer than four melanomas and a young age (< 45 years) at first melanoma diagnosis did not predict a monogenic predisposition. Conclusions Given the stringent inclusion criteria employed, a higher per- centage of patients carrying a PV in a melanoma- predisposing gene was expected in the presented series. However, the de- tection rate was only 11.4%. The prevalence of CDKN2A PV was 8.6% in this study and was 18.2% considering patients with ≥ 3 melanomas and 4.2% in patients with 1 or 2 melanomas. This is remarkably Table 2. Detection rate according to patient cutaneous phenotype and family history among first- and second-degree relatives Patient phenotype and FH PV (any gene), N (%) No PV, N (%) Total, N (%) Two-sided Fisher exact test Negative family history of PC 1 (3.07%) 26 (96.30%) 27 (100%) P = 0.0302 Positive family history of PC 3 (37.50%) 5 (62.50%) 8 (100%) Personal history of ≥ 50 nevi 4 (23.53%) 13 (76.47%) 17 (100%) P = 0.0455 Personal history of < 50 nevi 0 (0%) 18 (100%) 18 (100%) < 45 years at first melanoma 3 (20.00%) 12 (80.00%) 15 (100%) P = 0.2924 ≥ 45 years at first melanoma 1 (5.00%) 19 (95.00%) 20 (100%) Personal history of ≥ 3 melanomas 2 (18.18%) 9 (81.82%) 11 (100%) P = 0.5748 Personal history of < 3 melanomas 2 (8.33%) 22 (91.67%) 24 (100%) Personal history of ≥ 4 melanomas 2 (100%) 0 (0%) 2 (100%) P = 0.0101 Personal history of < 4 melanomas 2 (6.06%) 31 (93.94%) 33 (100%) Personal history/family history of ≥ 4 melanomas 3 (27.27%) 8 (72.73%) 11 (100%) P = 0.0819 Personal history/family history of < 4 melanomas 1 (4.17%) 23 (95.83%) 24 (100%) Personal history/family history ≥ 5 melanomas 3 (50.00%) 3 (50.00%) 6 (100%) P = 0.0114 Personal history/family history < 5 melanomas 1 (3.45%) 28 (96.55%) 29 (100%) FH = family history; PC = pancreatic cancer; PV = pathogenic variant Original Article | Dermatol Pract Concept. 2024;14(3):e2024120 7 in GnomAD database, with one homozygote reported, the presence of this variant in control cases and its co- segregation with other PV strictly related to the observed phenotype in some studies suggests a more careful classification in VUS. No segregation studies have been performed for this vari- ant in our series, but it is something to take in serious ac- count for the future in order to investigate the potential re-classification. In line with the findings of previous studies [14], we found a statistically significant association between the pres- ence of PV in the panel of melanoma-predisposing genes tested and a family history of PC in up to second-degree rel- atives (P = 0.0302). Early diagnosis of PC seems possible thanks to ongoing research on surveillance in high-risk in- dividuals with a hereditary predisposition. Surveillance with annual magnetic-resonance cholangiopancreatography or endoscopic ultrasound is recommended for individuals with known CDKN2A PV [15,16] starting from 40 or 30 in Italy [29,30]. The three carriers of CDKN2A PV in this study were referred to Centers dedicated to PC surveillance. This study has limitations. First, it is a small study, with only 35 out of 46 eligible patients accepting to undergo ge- netic counseling and testing. Moreover, while the diagno- ses of melanoma in the patients enrolled in this study were pathologically confirmed, it was not so for the diagnoses of PC or melanoma in family members and might not be ac- curate. Furthermore, the prevalence of CDKN2A and ATM PV is conditioned by the clinical classification of the CDKN2A c.150+5del variant and the ATM  c.8734A>G (p.Arg2912Gly) variant that is, at present, unknown. Fur- ther functional studies, segregation, and clinical data of other family clusters could help define the impact of these gene variants. Upon reviewing the obtained results, according to recent data from the Italian Melanoma Intergroup, we believe that clinical criteria to propose genetic counseling and testing to melanoma patients in Italy should be re-evaluated, adopting criteria in use in high-incidence melanoma countries. More- over, despite its limitations, this series supports the genetic assessment for oncological hereditary predisposition in mel- anoma patients with a positive family history of PC up to the 2nd degree of kinship. References 1. Truderung OA, Sagi JC, Semsei AF, Szalai C. Melanoma suscep- tibility: an update on genetic and epigenetic findings. Int J Mol Epidemiol Genet. 2021;12(5):71-89. PMID: 34853632 PMCID: PMC8611230. 2. Garbe C, Keim U, Gandini S, et al. Epidemiology of cutaneous mel- anoma and keratinocyte cancer in white populations 1943-2036. Eur J Cancer. 2021;152:18-25. DOI: 10.1016/j.ejca.2021 .04.029. Epub 2021 May 29. PMID: 34062483. melanoma diagnosed after 60, the benefit of genetic testing should be carefully evaluated due to the low detection rate. Our data support this position and emphasize that the in- clusion criteria for hereditary melanoma genetic testing in Italy may need to be reconsidered. We suggest that the as- sessment for genetic counseling referral should be proposed with caution in cases involving sporadic multiple melanomas with only two occurrences and those with a late age of onset, especially for patients with I-II skin type and environmental risk factors. We speculate that the main burden of familial/ multiple melanomas in our region may be linked to PV in untested moderate to low penetrance genes or in yet-to-be- discovered cancer predisposition genes, combined with envi- ronmental risk factors. In line with the literature, all CDKN2A deleterious vari- ants identified in our study were missense variants affecting p16INK4A [23] The percentage of patients carrying MITF PV in this study was 2.9%, comparable to data from the literature [3,24]. MITF is a transcription factor involved in the homeo- stasis of melanocytes and other cell types. MITF c.952G>A (p.Glu318Lys) is a well-known PV conferring a moderate risk of developing melanoma. Moreover, multiple primary melanomas, increased nevus count, early-onset melanoma, and family history of melanoma have been reported in car- riers of MITF c.952G>A (p.Glu318Lys) [24-26]. Although some authors have suggested a possible association between MITF c.952G>A (p.Glu318Lys) and PC and renal cancer, currently, there is not sufficient evidence to support clinical and radiological surveillance for cancers other than mela- noma [14,25,27,28]. Focusing on VUS with an interesting potential of re- classification in “likely pathogenic”, the c.150+5del vari- ant falls in intron 1 of the CDKN2A gene and meets criteria for suspicion as it involves an intronic deletion, not at the splice donor/acceptor consensus site, but at a nearby site. This variant has been reported only once in global genetics databases. It has suspicious characteristics confirmed by the ACMG pathogenicity classification tool, which predicts this variant to be likely pathogenic. Together with the patient’s personal and family history, these elements suggest a delete- rious effect of the CDKN2A c.150+5del variant. However, in the absence of evidence of the impact of this variant on mRNA splicing, its classification remains pending. We also reported in a distinct patient the ATM c.8734A>G (p.Arg2912Gly) variant, currently classified as a VUS. It is interesting to note that the Varsome ACMG classification tool classifies it as a “Likely Pathogenic” variant, based on disrupted protein function predicted by in silico tools, presence of functional and segregation studies that supports this prediction, involvement of a nucleotide, which falls in an hotspot sequence rich of PVs. However, its prevalence 8 Original Article | Dermatol Pract Concept. 2024;14(3):e2024120 Pathogenic Variants in ACD, ATM, BAP1, and POT1. Cancers 2020, 12(4). PMID: 32325837 PMCID: PMC7226507. 18. Bianchi-Scarrà G, Grammatico M, Genuardi B, et al. 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