A multi-method approach to the molecular diagnosis of overt and borderline 11p15.5 defects underlying Silver–Russell and Beckwith–Wiedemann syndromes

Background: Multiple (epi)genetic defects affecting the expression of the imprinted genes within the 11p15.5 chromosomal region underlie Silver–Russell (SRS) and Beckwith–Wiedemann (BWS) syndromes. The molecular diagnosis of these opposite growth disorders requires a multi-approach flowchart to disclose known primary and secondary (epi)genetic alterations; however, up to 20 and 30 % of clinically diagnosed BWS and SRS cases remain without molecular diagnosis. The complex structure of the 11p15 region with variable CpG methylation and low-rate mosaicism may account for missed diagnoses. Here, we demonstrate the relevance of complementary techniques for the assessment of different CpGs and the importance of testing multiple tissues to increase the SRS and BWS detection rate. Results: Molecular testing of 147 and 450 clinically diagnosed SRS and BWS cases provided diagnosis in 34 SRS and 185 BWS patients, with 9 SRS and 21 BWS cases remaining undiagnosed and herein referred to as “borderline.” A flowchart including complementary techniques and, when applicable, the analysis of buccal swabs, allowed confirmation of the molecular diagnosis in all borderline cases. Comparison of methylation levels by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) in borderline and control cases defined an interval of H19/IGF2:IG-DMR loss of methylation that was distinct between “easy to diagnose” and “borderline” cases, which were characterized by values ≤mean −3 standard deviations (SDs) compared to controls. Values ≥mean +1 SD at H19/IGF2: IG-DMR were assigned to borderline hypermethylated BWS cases and those ≤mean −2 SD at KCNQ1OT1: TSS-DMR to hypomethylated BWS cases; these were supported by quantitative pyrosequencing or Southern blot analysis. Six BWS cases suspected to carry mosaic paternal uniparental disomy of chromosome 11 were confirmed by SNP array, which detected mosaicism till 10 %. Regarding the clinical presentation, borderline SRS were representative of the syndromic phenotype, with exception of one patient, whereas BWS cases showed low frequency of the most common features except hemihyperplasia. Conclusions: A conclusive molecular diagnosis was reached in borderline methylation cases, increasing the detection rate by 6 % for SRS and 5 % for BWS cases. The introduction of complementary techniques and additional tissue analyses into routine diagnostic work-up should facilitate the identification of cases undiagnosed because of mosaicism, a distinctive feature of epigenetic disorders.