Splice-site variant in the RPS7 5'-UTR leads to a decrease in the mRNA level and development of Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by erythroid aplasia. Pathogenic variants in ribosomal protein (RP) genes, GATA1, TSR2, and EPO, are considered to be the etiology of DBA. Variants in 5'-untranslated regions (UTRs) of these genes are poorly studied and can complicate the variant interpretation. We investigated the functional consequences NM_001011.4:c.-19 + 1G > T variant in the donor splice-site of the RPS7 5'-UTR. This variant was found in a family where two sons with DBA were carriers. Father, who also had this variant, developed myelodysplastic syndrome, which caused his death. Search for candidate causal variants and copy number variations in DBA-associated genes left RPS7 variant as the best candidate. Trio whole exome sequencing analysis revealed no pathogenic variants in other genes. Functional analysis using luciferase expression system revealed that this variant leads to disruption of splicing. Also, a decrease in the levels of mRNA and protein expression was detected. In conclusion, the established consequences of 5'-UTR splice-site variant c.-19 + 1G > T in the RPS7 gene provide evidence that it is likely pathogenic.

Upstream ORF frameshift variants in the PAX6 5'UTR cause congenital aniridia.

Congenital aniridia (AN) is a severe autosomal dominant panocular disorder associated with pathogenic variants in the PAX6 gene. Previously, we performed a molecular genetic study of a large cohort of Russian patients with AN and revealed four noncoding nucleotide variants in the PAX6 5'UTR. 14 additional PAX6-5'UTR variants were also reported in the literature, but the mechanism of their pathogenicity remained unclear. In the present study, we experimentally analyze five patient-derived PAX6 5'UTR-variants: four variants that we identified in Russian patients (c.-128-2delA, c.-125dupG, c.-122dupG, c.-118_-117del) and one previously reported (c.-52+5G>C). We show that the variants lead to a decrease in the protein translation efficiency, while mRNA expression level is not significantly reduced. Two of these variants also affect splicing. Furthermore, we predict and experimentally validate the presence of an evolutionarily conserved small uORF in the PAX6 5'UTR. All studied variants lead to the frameshift of the uORF, resulting in its extension. This extended out-of-frame uORF overlaps with the downstream CDS and thereby reduces its translation efficiency. We conclude that the uORF frameshift may be the main mechanism of pathogenicity for at least 15 out of 18 known PAX6 5'UTR variants. Moreover, we predict additional uORFs in the PAX6 5'UTR.