Abstract
<title>Abstract</title> <p> Silver-Russell syndrome (SRS) is an imprinting disorder primarily associated with growth retardation. A molecular diagnosis is achieved in about 60% of patients. The most common defect is loss of methylation (LoM) at the <italic>H19/IGF2</italic> imprinting locus on chromosome (chr) 11p15.5, followed by maternal uniparental disomy of chromosome 7 (upd(7)mat). Less frequent causes include single-nucleotide variants and copy-number variants in several other chromosome regions. About 40% of cases remain undiagnosed. We investigated 61 Italian patients fulfilling the Netchine-Harbinson clinical scoring system (NH-CSS) criteria. Multi-locus DNA methylation analysis identified molecular alterations in 29 patients, including 24 with LoM at the <italic>H19</italic> / <italic>IGF2</italic> imprinted locus, 3 with upd(7)mat, 1 case with methylation abnormalities affecting chr14, and 1 case with an extremely rare maternal duplication involving the telomeric domain of chr11p15.5. Whole-exome sequencing (WES) of 32 molecularly negative patients identified pathogenic/likely pathogenic variants in genes associated with growth retardation in 12 cases. Two of these genes were absent from conventional gene panels. Genome-wide methylation analysis was performed in 48 SRS cases. We observed differentially methylated probes in promoter regions of genes implicated in growth, craniofacial, and skeletal development, supporting epigenetic deregulation extending beyond canonical imprinted loci. Our findings highlight the molecular heterogeneity of SRS and support integrated diagnostic approaches. Alterations outside chr11 and chr7 represent the second most frequent molecular defect in our cohort. Genome-wide methylation profiling may also help identify genes involved in somatic growth across SRS molecular subgroups. Overall, these results support redefining SRS as a molecular rather than clinical diagnosis. </p>