First patient with bloom syndrome caused by a deep intronic variant leading to pseudoexon activation

Bloom Syndrome is a rare genetic autosomal recessive disorder characterized by genome instability, cancer susceptibility, immune abnormalities, severe growth delay, various skin lesions and dysmorphic facial features. It is caused by germline mutations in the BLM/RECQL3 gene, which encodes the RecQL3 helicase. This gene plays a role in the homologous recombination (HR) repair pathway. Mutations in this gene are associated with sensitivity to DNA damaging agents like MMC (mitomycin C). We report a Belgian boy presenting with severe growth delay, microcephaly, facial dysmorphism and several immune defects. Increased sister chromatid exchanges (SCEs) were observed, indicating genomic instability, a pathognomonic sign for Bloom Syndrome. Molecular analysis had revealed heterozygosity for a nonsense variant (c.3379C>T, p.(Gln1127*)), never described before, inherited from his father. Sequencing at the gDNA level had not revealed a second variant. Here, we present the identification of a deep-intronic variant by cDNA analysis. From EDTA blood samples a phytohemagglutinin stimulated short‐term lymphocyte culture was established. We established two cultures, allowing to treat one with puromycin prior to RNA extraction. Total RNA was extracted using the QIAamp® RNeasy Mini Kit. cDNA was synthesized using Superscript® III Reverse Transcriptase. Primers were developed to span the complete coding region of the BLM gene, followed by RT-PCR and Sanger sequencing. cDNA analysis revealed an aberrant transcript encompassing a part of intron 15 of the BLM gene. Sequencing of the relevant part of genomic DNA revealed a deep intronic substitution (c.3032-258A>G, p.(?)) predicted to create a cryptic donor splice site. The patient inherited this variant from his mother. This deep intronic substitution gives rise to two aberrant transcripts: r.[3019_3020ins3020-414_3020-259](in frame) and r.[3019_3020ins3020-424_3020-259](out-of-frame). We are currently evaluating the residual protein levels and if sensitivity to MMC can be established. In summary, we report a Belgian boy compound heterozygous for a novel nonsense variant in exon 18 and a deep intronic variant in intron 15. This mechanism has never been described before in the BLM gene and further functional validations are ongoing. This case highlights the role of non-coding variations associated with genetic syndromes and the added-value of RNA-based approaches in patients with a clear phenotype lacking a molecular diagnosis.