# DNA Methylation Episignature as a Novel Diagnostic Tool for Diamond‐Blackfan Anemia Syndrome

## TL;DR

This study introduces DNA methylation profiling as a new diagnostic tool for Diamond-Blackfan Anemia Syndrome, offering a reliable biomarker for all patients and subtypes.

## Contribution

The study identifies a distinct DNA methylation episignature for Diamond-Blackfan Anemia Syndrome and its subtypes, enabling accurate diagnosis and differentiation from similar disorders.

## Key findings

- A DNA methylation episignature was identified that differentiates DBAS from Fanconi anemia and other disorders.
- A DNAm classifier was developed for the DBA6 and DBA7 subtypes, aiding in accurate diagnosis.
- Methylation profiling successfully identified DBAS in molecularly uncharacterized cases and validated through genome sequencing.

## Abstract

Diamond‐Blackfan Anemia Syndrome (DBAS) is a rare inherited bone marrow failure syndrome (IBMFS) characterized by impaired erythropoiesis and significant genetic heterogeneity. Diagnosis can be challenging due to clinical variability and the lack of sensitive and specific biomarkers. We investigated the evidence for a DNA methylation (DNAm) episignature in a cohort of 80 DBAS patients with causative variants in various ribosomal protein genes: DBA1 (RPS19, n = 30), DBA4 (RPS17, n = 6), DBA5 (RPL35A, n = 8), DBA6 (RPL5, n = 15), DBA7 (RPL11, n = 13), DBA10 (RPS26, n = 8). We identified a distinct and highly accurate episignature biomarker for DBAS, clearly differentiating it from both Fanconi anemia and a broad spectrum of other episignature‐positive disorders. Furthermore, we developed a specific DNAm classifier for the clinically similar DBA6 and DBA7 subtypes. Applying the DBAS episignature analysis to six molecularly uncharacterized cases, three exhibited the DBAS pattern. Subsequent genome sequencing identified causative genetic variants in two (RPL5: c.325‐380A>G:p.?; RPL26: c.‐6 + 3_‐6 + 25del:p.?), validating the test robustness. Methylation profiles from two revertant cases (RPS19:P47L and RPS17 full gene deletion) exhibited the DBAS episignature, suggesting it to be a stable epigenetic mark associated with the underlying genetic mutation, likely established early in development. In conclusion, we propose DNAm profiling as a robust diagnostic tool for DBAS, providing a biomarker applicable to all patients with clinical suspicion of the disease and critically aiding in the resolution of variants of uncertain significance and molecularly uncharacterized cases.

Diamond‐Blackfan Anemia Syndrome (DBAS) is a congenital bone marrow failure disorder characterized by defective erythropoiesis and a spectrum of congenital anomalies, including craniofacial malformations, limb abnormalities, and cardiac and renal defects. DBAS is primarily caused by mutations in genes encoding proteins of the small and large ribosomal subunits. These genetic defects are associated with a distinct DNA methylation profile that differentiates individuals with DBAS from healthy controls. Notably, a specific episignature has been identified in patients belonging to the DBA6 and DBA7 molecular subgroups. DNA methylation profiling has proven useful for distinguishing patients with suspected DBAS but without known molecular alterations from those affected by other conditions that phenotypically resemble DBAS.