Abstract
Background and objectives:
Leigh syndrome is an important manifestation of childhood-onset primary mitochondrial disease. Panel sequencing and whole exome sequencing are cost-effective for diagnosing mitochondrial diseases; however, more than half of mitochondrial disease cases remain genetically undiagnosed. This study aimed to demonstrate that combining whole genome sequencing (WGS) and RNA sequencing (RNA-seq) analyses can identify disease-causing variants that would otherwise be missed.
Methods:
We performed WGS and RNA-seq on a patient with Leigh syndrome. Chromosomal phasing using Sanger sequencing of parental and patient blood samples was conducted to confirm compound heterozygous variants. RNA-seq data were analyzed for splicing abnormalities. Overexpression studies of wild-type NDUFA3 in patient-derived fibroblasts were performed to assess restoration of mitochondrial function.
Results:
We discovered compound heterozygous intronic variants (c.86-16_86-15del in intron2 and c.164-362G>A in intron3) of the NDUFA3 gene. RNA-seq data analysis revealed intron retention and exonization in NDUFA3. Exonization was related to a variant involving the mobile element Alu that resulted in complex abnormal splicing events. Overexpression of wild-type NDUFA3 restored mitochondrial dysfunction in patient-derived fibroblasts, confirming NDUFA3 as a Leigh syndrome causative gene.
Discussion:
This study highlights the importance of combining WGS and RNA-seq and provides new insights into detecting abnormalities in deep intronic regions, particularly those involving mobile elements, such as Alu. This approach can play a crucial role in identifying genetic variations and elucidating transcriptional control mechanisms that are not readily achieved by conventional methods, especially in the context of mobile element-induced complexities.