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

Abstract

BackgroundMultiple (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.ResultsMolecular 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.ConclusionsA 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0183-8) contains supplementary material, which is available to authorized users.

Diagnosis and management of Silver–Russell syndrome: first international consensus statement

“…Some patients, particularly those with upd(7)mat, have fewer typical clinical features of SRS than patients with 11p15 LOM 4,5,13,16,20,21 . In the cohort reported by Azzi and co-workers 15 , one of the nine patients scoring three of six criteria (and therefore predicted 'unlikely to have SRS') had upd(7)mat.…”

“…We, therefore, do not recommend testing for SRS in patients scoring less than three of six criteria. Of note, a small number of patients with body asymmetry have been reported to have 11p15 LOM 20,21,23 . These patients would score fewer than three of six criteria, which is insufficient to justify a clinical diagnosis of SRS in these patients.…”

“…Hypomethylation of H19/IGF2 IG-DMR is frequently incomplete and low levels of hypomethylation might elude detection. Methylation patterns might vary between different tissues and cells (leucocytes, samples from a buccal swab and skin fibroblasts) 21,33,34 and could explain cases of a negative molecular diagnosis using a blood sample.…”

Could a chimeric condition be responsible for unexpected genetic syndromes? The role of the single nucleotide polymorphism‐array analysis

“…In conclusion, many cases of whole-body chimerism are reported in literature; unfortunately most of them present insufficient molecular information allowing to clearly define the mechanism involved in the formation of individual cases. Nonetheless, several cases of proven tetragametic and parthenogenetic chimeras have been described based initially on chromosome heteromorphisms, and more recently on genotyping (Russo et al, 2016;Yamazawa et al, 2010).…”

Nomenclature and definition in asymmetric regional body overgrowth

“…Furthermore, it is usually not known whether the difference in size between the left and right body half is caused by an overdevelopment of one body half or instead underdevelopment of the other body half, or a combination of these. For example, patients with asymmetry may be proven to carry H19 hypomethylation, which is mostly associated with undergrowth [Russo et al, 2016]. We acknowledge that typically the sign that is clinically apparent (‘from the end of the bed’) is the overdevelopment, and that underdevelopment may or may not be present.…”