Objective: To clarify the genetic cause of autosomal recessive early-onset Parkinson’s Disease (EOPD) for which only a single heterozygous coding mutation could be identified.

Background: Mutations in PRKN and PINK1 genes are the predominant cause of autosomal recessive EOPD. Previous estimates suggested ~50% of early-onset PD patients with PINK1 or PRKN mutations have only one identifiable heterozygous variant (1). We employed comprehensive next generation sequencing and non-coding sequence interrogation to identify a second variant in patients with a strong clinical phenotype and biochemical evidence implicating PRKN.

Method: The coding regions of PRKN were sequenced by Sanger sequencing. Total protein from patient-derived fibroblasts was assessed by Western blotting to determine Parkin protein expression and function. Whole genome sequencing was complimented by analysis of PRKN non-coding regions using our novel bioinformatic tool, Introme. RT-PCR analysis of Parkin transcripts was performed on cyclohexamide-treated patient-derived fibroblasts. A custom antisense morpholino oligonucleotide was used to treat patient-derived fibroblasts for aberrant splicing correction.

Results: Sequencing of PRKN coding regions identified a 40 bp heterozygous deletion from exon 3 in four siblings presenting with EOPD, but was absent in their mother. Western blotting revealed a complete quantitative and qualitative absence of Parkin, indicating a second allelic variant ought to be present. Introme analysis of non-coding sequences revealed two candidate variants in intron 7 of PRKN that were present in both the patients and their mother. The intronic variants introduced potential canonical splice site motifs. Transcript analysis identified cryptic exon inclusion that would result in premature truncation of Parkin. An antisense oligonucleotide targeted to the causative variant was trialled for correction of the splicing defect in the hope of restoring nominal Parkin function.

Conclusion: Using whole genome sequencing and intronic sequence examination, we identified a second allelic variant to solve a case of autosomal recessive PD with only a single heterozygous variant initially identified. This approach could be applied to cases of this type for any genetic disorder and may be useful for solving a number of unresolved familial (and potentially ‘sporadic’) PD cases.

References: (1) Klein, C. et al. (2007) Deciphering the role of heterozygous mutations in genes associated with parkinsonism. Lancet Neurol 6(7):652-62

« Back to 2019 International Congress

MDS Abstracts - https://www.mdsabstracts.org/abstract/using-whole-genome-sequencing-and-intronic-sequence-analysis-to-identify-pathogenic-variants-in-parkin-related-parkinsons-disease/