Genomic instability in the PARK2 locus is associated with Parkinson's disease

Abstract

Parkinson's disease (PD) is a common neurodegenerative disorder affecting mostly elderly people, although there is a group of patients developing so-called early-onset PD (EOPD). Mutations in the PARK2 gene are a common cause of autosomal recessive EOPD. PARK2 belongs to the family of extremely large human genes which are often localised in genomic common fragile sites (CFSs) and exhibit gross instability. PARK2 is located in the centre of FRA6E, the third most mutation-susceptible CFS of the human genome. The gene encompasses a region of 1.3 Mbp and, among its mutations, large rearrangements of single or multiple exons account for around 50%. We performed an analysis of the PARK2 gene in a group of 344 PD patients with EOPD and classical form of the disease. Copy number changes were first identified using multiplex ligation probe amplification (MLPA), with their ranges characterised by array comparative genomic hybridisation (aCGH). Exact breakpoints were mapped using direct sequencing. Rearrangements were found in eight subjects, including five deletions and three duplications. Rearrangements were mostly non-recurrent and no repetitive sequences or extended homologies were identified in the regions flanking breakpoint junctions. However, in most cases, 1-3 bp microhomologies were present, strongly suggesting that microhomology-mediated mechanisms, specifically non-homologous end joining (NHEJ) and fork stalling and template switching (FoSTeS)/microhomology-mediated break-induced replication (MMBIR), are predominantly involved in the rearrangement processes in this genomic region.

Keywords: Common fragile sites; FRA6E; Genomic rearrangements; PARK2; Parkinson’s disease.

Fig. 1

Representation of the ranges of all PARK2 rearrangements found in the study. Exons of the PARK2 gene are represented by numbered boxes. Identified deletions are depicted in black and duplications in grey. Rearrangements encompassed from one to four exons in the region between exons 2 and 7. Mutations Ex4_7del and Ex2dup were identified in two subjects

Fig. 2

Four early-onset Parkinson’s disease (EOPD) pedigrees showing the PARK2 mutations’ inheritance in families. Solid symbols individuals with EOPD; open symbols unaffected individuals without identified mutations, mutations carriers on one or, like in case 6 III-2, on both alleles in the presymptomatic stage at the moment of analysis; index patients are denoted by arrows

Fig. 3

DNA sequences spanning eight identified rearrangements aligned with corresponding normal intronic regions. Homology regions across junctions are bold and underlined. The grey boxes indicate inserted sequences. The arrows indicate palindromic sequences able to form DNA hairpins and the asterisks indicate TTTAAA sequence known to be able to induce a curvature in the DNA molecule. 1–3-bp microhomologies were found in 6/8 cases. In the case of subject ID3, both heterozygous deletions (Ex4-7del and Ex3del) are presented. Breakpoints of the Ex4_7del mutation are identical in cases ID2 and ID3. In the case of subject ID6, the duplication is represented by two separate alignments (at the 5′ and 3′ sites) due to an inverted orientation. In cases ID3, ID6 and ID8, the insertions are of unknown origin

Fig. 4

Predicted fork stalling and template switching (FoSTeS)/microhomology-mediated break-induced replication (MMBIR) mechanism of the Ex2-5dup mutation in subject ID6. Upon replicating, the first exon of the PARK2 gene replication fork stalled and one strand invaded either the sister molecule or the homologue chromosome in inverted orientation (1), resulting in inverted duplication. Subsequently, the original forks were restored, but primed upstream of the point where it first stalled (2), resulting in triplication of the grey-highlighted region