Novel deep intronic mutation in PLA2G6 causing early-onset Parkinson's disease with brain iron accumulation through pseudo-exon activation.

PLA2G6 is the causative gene for a group of autosomal recessive neurodegenerative disorders known as PLA2G6-associated neurodegeneration (PLAN). We present a case with early-onset parkinsonism, ataxia, cognitive decline, cerebellar atrophy, and brain iron accumulation. Sequencing of PLA2G6 coding regions identified only a heterozygous nonsense variant, but mRNA analysis revealed the presence of an aberrant transcript isoform due to a novel deep intronic variant (c.2035-274G > A) leading to activation of an intronic pseudo-exon. These results expand the genotypic spectrum of PLAN, showing the paramount importance of detecting possible pathogenic variants in deep intronic regions in undiagnosed patients.

In silico identification of pseudo-exon activation events in personal genome and transcriptome data.

Causative mutations for human genetic disorders have mainly been identified in exonic regions that code for amino acid sequences. Recently, however, it has been reported that mutations in deep intronic regions can also cause certain human genetic disorders by creating novel splice sites, leading to pseudo-exon activation. To investigate how frequently pseudo-exon activation events occur in normal individuals, we conducted in silico identification of such events using personal genome data and corresponding high-quality transcriptome data. With rather stringent conditions, on average, 2.6 pseudo-exon activation events per individual were identified. More pseudo-exon activation events were found in 5' donor splice sites than in 3' acceptor splice sites. Although pseudo-exon activation events have sporadically been reported as causative mutations in genetic disorders, it is revealed in this study that such events can be observed in normal individuals at a certain frequency. We estimate that human genomes typically contain on average at least 10 pseudo-exon activation events. The actual number should be higher than this, because we used stringent criteria to identify pseudo-exon activation events. This suggests that it is worth considering the possibility of pseudo-exon activation when searching for causative mutations of genetic disorders if candidate mutations are not identified in coding regions or RNA splice sites.

Missing heritability in Bloom syndrome: First report of a deep intronic variant leading to pseudo-exon activation in the BLM gene.

Pathogenic biallelic variants in the BLM/RECQL3 gene cause a rare autosomal recessive disorder called Bloom syndrome (BS). This syndrome is characterized by severe growth delay, immunodeficiency, dermatological manifestations and a predisposition to a wide variety of cancers, often multiple and very early in life. Literature shows that the main mode of BLM inactivation is protein translation termination. We expanded the molecular spectrum of BS by reporting the first deep intronic variant causing intron exonisation. We describe a patient with a clinical phenotype of BS and a strong increase in sister chromatid exchanges (SCE), who was found to be compound heterozygous for a novel nonsense variant c.3379C>T, p.(Gln1127Ter) in exon 18 and a deep intronic variant c.3020-258A>G in intron 15 of the BLM gene. The deep intronic variant creates a high-quality de novo donor splice site, which leads to retention of two intron segments. Both pseudo-exons introduce a premature stop codon into the reading frame and abolish BLM protein expression, confirmed by Western Blot analysis. These findings illustrate the role of non-coding variation in Mendelian disorders and herewith highlight an unmet need in routine testing of Mendelian disorders, being the added value of RNA-based approaches to provide a complete molecular diagnosis.