Identification of two novel non-coding variants in Diamond-Blackfan Anemia Syndrome patients by whole genome sequencing

• DBAS is characterized by pure red cell aplasia with 30% of cases undiagnosed likely due to non-coding variants.

• Molecular diagnoses of DBAS require careful analysis beyond the coding regions, typically not fully covered or analyzed by clinical testing.

Diamond-Blackfan Anemia Syndrome (DBAS) is a rare congenital disorder with variable penetrance and expressivity, characterized by pure red cell aplasia that typically manifests as early-onset chronic macrocytic or normocytic anemia and is often associated with other congenital anomalies. DBAS is etiologically heterogeneous, with over 20 known DBAS-associated genes encoding small and large ribosomal protein subunits, and an inheritance pattern largely as autosomal dominant or sporadic. We report two DBAS cases with prior negative genetic testing, which included targeted gene panels, karyotype analysis, chromosome breakage analysis, and whole exome sequencing (WES). Although clinical whole genome sequencing (WGS) was initially negative, in-depth reanalysis identified two novel non-coding variants in the RPS gene family: a maternally inherited splicing variant at the end of the first non-coding exon in RPS7 (NM_001011.4, c.-19G>C) in Family 1, and a deep intronic de novo variant in RPS19 (NM_001022.4, c.172+350C>T) in Family 2. In Family 1, several maternal relatives were identified as sharing the same variant through cascade testing; clinically, they exhibited variable degrees of anemia and elevated erythrocyte adenosine deaminase (eADA) activity, a marker for DBAS. RNA sequencing analysis demonstrated deleterious functional consequences for both non-coding variants. In Case 1, hematopoietic stem cell transplant (HSCT) with an unaffected matched sibling donor, who did not carry the variant, successfully cured the congenital anemia. This study identified novel non-coding variants and underscores the clinical utility of WGS in accelerating diagnosis and improving care for rare genetic disorders, particularly when timely treatment decisions are critically important.