A novel frameshift variant in PURA syndrome: role of NMD pathway in disease mechanism

Abstract

Background

PURA syndrome is a rare genetic disorder characterized by obvious hypotonia, feeding difficulties, apnea, and drowsiness, which is caused by variants in the Purine Rich Element Binding Protein A (PURA) on chromosome 5q31.2-q31.3, and is inherited in an autosomal dominant manner. This condition presents challenges for early diagnosis and effective treatment. This study investigates its pathogenic mechanisms and potential therapeutic strategies.

Methods

Peripheral blood samples were collected from a Chinese newborn with severe hypotonia and his parents. Whole-exome sequencing (WES) and Sanger validation identified a novel PURA variant, which had not been previously reported. To explore the potential involvement of nonsense-mediated mRNA decay (NMD) in PURA syndrome, wild-type (WT) and variant (Var) PURA expression vectors were constructed and transiently transfected into 293 T cells via liposomal transfection. Real-time quantitative polymerase chain reaction (PCR) and Western blotting were performed to analyze the PURA expression. The levels of PURA mRNA and protein were assessed following treatment with the NMD inhibitor cycloheximide (CHX) and the small interfering RNA targeting Up-frameshift protein 1 (siRNA-UPF1), a key NMD factor.

Results

The WES identified a novel heterozygous frameshift variant in exon 1 of PURA (NM_005859.5): c.632_651dup; p.(Leu218Trpfs*14), which introduces a premature termination codon (PTC). However, no such variant was detected in his parents. Functional validation assays revealed that the variant construct expressed significantly lower levels of PURA mRNA and protein compared to the WT. CHX treatment and siRNA-UPF1 transfection significantly increased the mRNA and protein expression levels of PURA in the variant construct.

Conclusion

The novel PURA variant, c.632_651dup; p.(Leu218Trpfs*14), is a pathogenic heterozygous frameshift. The NMD pathway is involved in the degradation of its aberrant transcript. Our research expands the genotypic spectrum of pathogenic PURA variants, and offers a new perspective for potential therapeutic intervention.