PO14 Comprehensive genomic testing uncovers Multiple Molecular Diagnoses in patients with RASopathies

Background RASopathies are among the most common monogenic rare diseases and predispose to hypertrophic cardiomyopathy, congenital heart defects, and other cardiovascular anomalies, resulting in syndromic complex cardiac phenotypes. The advent of high-throughput genomic technologies has significantly expanded the diagnostic yield, enabling identification of an increasing number of genetically determined conditions and revealing that approximately 4% of patients carry more than one molecularly confirmed genetic disorder (multiple molecular diagnosis, MMD). MMDs can present in several ways: as distinct MMD, where the manifestations of each disorder are clinically separable; as blended MMD, where overlapping features make the contribution of each disorder partially indistinguishable; and as indistinguishable MMD, where both conditions belong to the same nosological group and all clinical features can be attributed to either condition with equal likelihood. Purpose This study aimed to determine the prevalence of RASopathies in a phenotypically selected cohort and, within this subgroup, to evaluate the frequency and spectrum of MMD. Methods We analyzed 886 patients referred for genetic testing in 2023–2024. All underwent whole-exome sequencing (WES) with single-nucleotide variant (SNV) and copy-number variant (CNV) calling; in selected cases, trio or parental analysis was performed. Results A molecular diagnosis was established in 228 patients (yield 25.7%). RASopathies were identified in 23 (2.6%), most commonly Noonan syndrome type 1 due to recurrent PTPN11 variants. Other causative genes included BRAF, SOS1, PPP1CB, RIT1, MAP2K1, and MAP2K2 (with novel segregating variants in RIT1 and MAP2K2). MMD was found in 11 patients (4.8% of positives), including 3 with RASopathies: (i) PTPN11-related Noonan syndrome with SPAST and APOB variants, (ii) PTPN11-related Noonan syndrome with an STS deletion (both distinct MMD), and (iii) SOS1-related Noonan syndrome with FBN1-related Marfan syndrome (blended phenotype). Conclusions Comprehensive genomic testing, including CNV detection, improves diagnostic yield, uncovers MMD, and refines the interpretation of complex phenotypes, leading to better clinical management and genetic counseling. Our data confirm that RASopathies are often reported in combination with other genetic disorders. In one remarkable case, the coexistence of SOS1-related Noonan syndrome type 4 and FBN1-related Marfan syndrome resulted in a blended phenotype in which features of both conditions were so tightly interwoven that they effectively constituted a novel syndromic presentation with a complex cardiac phenotype. This case underscores the importance of genome-wide testing and detailed phenotype-genotype correlation, which allow the re-interpretation of previously fragmented or misleading clinical diagnoses (e.g., skeletal dysplasia or kyphoscoliotic Ehlers-Danlos syndrome) into a unified molecularly defined entity.