Functional validation of a novel ATM variant in a patient with multiple melanoma: implications for personalized prevention and treatment

Background: Multiple melanoma (MM) is a rare condition characterized by the development of multiple independent primary cutaneous melanomas and is associated with both genetic predisposition and environmental factors. Although several high- and intermediate-penetrance susceptibility genes have been identified, the genetic basis of MM remains incompletely understood. The ATM gene has recently emerged as a candidate low-to-intermediate penetrance melanoma susceptibility gene. Methods: To investigate the genetic determinants of MM, we screened a cohort of 32 patients presenting with at least three independent primary melanomas using a customized next-generation sequencing panel including established and candidate melanoma susceptibility genes, as well as ATM. Identified variants were evaluated for their potential functional relevance. In patient MM17, carrying two ATM variants, the effect of a previously unreported variant was further investigated by RNA analysis of peripheral blood mononuclear cells (PBMCs). Structural analyses were also performed to assess the potential impact of the identified ATM variants on protein structure. Results: Germline variants were identified in 6 of 32 patients (20%). Among these, only the POT1 variant was classified as pathogenic according to ACMG criteria, while four of the six variant-positive patients (67%) carried germline variants in ATM. Patient MM17 carried two ATM variants, including one not previously described in melanoma patients. Functional analysis demonstrated that this variant creates an additional splice site in exon 58, resulting in a transcript with a 19-bp deletion. RT-PCR analysis of RNA isolated from the patient's PBMCs confirmed the aberrant transcript. The deletion introduces a premature stop codon, leading to a truncated, nonfunctional ATM protein. Structural modelling of the identified ATM missense variants suggested that the amino acid substitutions themselves do not significantly affect overall protein folding or substrate binding. Conclusions: Our findings support a potential role for germline ATM alterations in susceptibility to multiple melanoma and highlight the importance of functional characterization of variants whose pathogenic significance cannot be established from sequence analysis alone. The identification of an ATM variant causing aberrant splicing and loss of protein function in a patient with MM may have implications for personalized surveillance and prevention strategies. In particular, approaches targeting the consequences of ATM loss of function could potentially be explored for chemoprevention in patients carrying the same variant.