Banca de DEFESA: HOZANA BEATRIZ DANTAS BARBALHO
Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.STUDENT : HOZANA BEATRIZ DANTAS BARBALHO
DATE: 28/07/2026
TIME: 09:00
LOCAL: Videoconferência
TITLE:
Screening for epigenetic biomarkers for Silver-Russell Syndrome: an integrative analysis of public methylomes and an ex vivo pilot test
KEY WORDS:
Silver-Russell syndrome; Imprinting; Hypomethylation; Methylome; Epigenetic biomarker; Molecular diagnosis; MS-MLPA; qPCR.
PAGES: 68
BIG AREA: Ciências Biológicas
AREA: Bioquímica
SUMMARY:
Silver-Russell Syndrome (SRS) is a rare condition characterized by pre- and postnatal growth restriction associated with alterations in genomic imprinting. Approximately 50% of cases are caused by hypomethylation at imprinting center 1 (IC1), located at the 11p15.5 locus; IC1 regulates the expression of the H19 and IGF2 genes. Currently, molecular diagnosis is primarily performed using the methylation-sensitive Multiplex Ligation-dependent Probe Amplification (MS-MLPA) technique; however, despite its widespread use, this method relies on HhaI enzyme binding sites, thereby assessing only seven specific cytosines within IC1. Given this, the present study aimed to identify differentially methylated cytosines (DMCs) in the 11p15.5 region through an integrative analysis of public methylomes, using these findings to guide the design of a pilot test for detecting hypomethylation in individuals with suspected SRS. A differential methylation analysis was conducted using the GSE104451 dataset via simple linear regression models (limma package, R) applying thresholds of effect size ≥0.1 and p ≤ 4.3 × 10−5, followed by validation in two independent public cohorts (from studies in England and Japan) and the use of MethBank for negative control samples. Sixty differentially methylated probes were identified within IC1; of these, 36 exhibited hypomethylation in the validation cohorts, with IGF2 cytosines showing the most significant hypomethylation. Based on these findings, primer pairs targeting 27 DMCs were designed for H19, CTCF, and IGF2, adhering to the criteria established by Wojdacz et al. (2008). A pilot qPCR with melting curve assay was subsequently conducted as a proof of concept. Genomic DNA from participants with suspected RSS (n=4) and controls (n=5) was treated with sodium bisulfite and amplified via SYBR Green qPCR, followed by melting curve analysis. The IGF2 target performed best, showing a statistically significant difference between the groups (Wilcoxon rank-sum test: W=0, p=0.03038), in contrast to H19, which failed to differentiate the groups (W=1, p=0.0606). These experimental findings align with in silico observations, where IGF2 was shown to harbor the cytosines most relevant to the loss-of-methylation scenario. Concurrently, a science communication project regarding the syndrome was developed on Instagram, demonstrating potential to facilitate access to information about RSS for diverse audiences (laypeople and professionals). The results indicate that integrating public methylation data is an effective strategy for identifying novel diagnostic biomarkers for RSS. Validating differentially methylated cytosines (DMCs) across ethnically diverse cohorts supports the universality of their application; meanwhile, the pilot assay, despite limitations regarding sample size and the use of SYBR Green, shows promise for enabling more accessible diagnostics.
COMMITTEE MEMBERS:
Presidente - 1046922 - LEONARDO CAPISTRANO FERREIRA
Interna - 350647 - SELMA MARIA BEZERRA JERONIMO
Externo à Instituição - ALEXANDER AUGUSTO DE LIMA JORGE - USP