Phase analysis identifies compound heterozygous deletions of the PARK2 gene in patients with early‐onset Parkinson disease

Kim SY, Seong MW, Jeon BS, Kim SY, Ko HS, Kim JY, Park SS. Phase analysis identifies compound heterozygous deletions of the PARK2 gene in patients with early‐onset Parkinson disease.

Sporadic Parkinson disease (PD) is a major type of Parkinsonism. In recent years, familial forms have been described in a subset of PD, especially earlyonset PD (EOPD) (1). At least nine monogenic causes constitute approximately 5-10% of PD: SNCA, PARK2, UCHL1, PINK1, PARK7, LRRK2, ATP13A2, GIGYF2 and HTRA2 (2). Among them, PARK2 is the most important genetic cause of EOPD with autosomal recessive inheritance (1). Molecular diagnosis of a genetic disorder with autosomal recessive inheritance requires determination of the mutational phase, in cases that are not homozygous for a deleterious mutation. It is also necessary to establish genotype-phenotype relationship.
Exon rearrangement involving one or more exons accounts for 50-60% of all PARK2 mutations (3,4) and can be easily detected by gene dosage analysis such as multiplex ligation-dependent probe amplification (MLPA) (5). However, quantitative analysis alone cannot conclusively determine the phase of exon rearrangements and the true incidence of molecularly confirmed parkin-type EOPD may be underestimated. So far, a few previous studies have analyzed the mutational phase of exon rearrangements (6,7).
In this study, we aimed to characterize the mutation spectrum of SNCA, PARK2, PINK1, and PARK7 among 114 Korean EOPD patients with a symptomatic onset age before or equal to 40 years. We performed sequencing and gene dosage analyses of these genes and analyzed the mutational phase of exon rearrangements.

Subjects
This study enrolled 114 unrelated Korean EOPD patients with onset age ≤40 years (median age, 36; range, 12-40). They were clinically diagnosed with PD based on the UK Parkinson's Disease Society Brain Bank clinical diagnostic criteria (8). No consanguineous marriages occurred within any of the participants' individual families. Normal frequency of a novel sequence variant was determined using 171 healthy subjects (median age, 63; range, 37-83). The study was approved by the institutional review board. All subjects gave their consent for participation in this study.
Genomic DNA was extracted from peripheral blood by the PureGene DNA isolation kit (Gentra Systems, Minneapolis, MN). Total RNA was isolated from the lymphoblastoid cells of each patient using the RNeasy mini kit (Qiagen, Hilden, Germany).

Sequence analyses
Target DNA was amplified by PCR and directly sequenced for detection of sequence variants in all coding exons and the flanking intronic regions of each gene (primers are available on request). Amplified products were purified by ExoSAP-IT treatment (USB, Cleveland, OH) and sequenced by the ABI PRISM 3730xl Genetic Analyzer (Applied Biosystems, Foster City, CA) using a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). Obtained sequences were analyzed using Sequencher software version 4.6 (Gene Codes Corporation, Ann Arbor, MI).

Significance assessment for novel missense variants
The significance of each novel missense variant was assessed by (i) its allelic frequency in 171 normal control subjects, (ii) interspecies amino acid conservation of the mutated amino acid, (iii) in silico prediction for the effect of novel variants, (iv) the protein domain in which the mutation is located, and (v) familial segregation.

Gene dosage analyses
Exon rearrangements were determined by MLPA, P051/P052 kit (MRC-Holland, Amsterdam, The Netherlands). DNA denaturation, probe-target sequence hybridization, ligation, and multiplex PCR were performed according to the manufacturer's protocol. Amplified products were separated using the ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems) and analyzed by GeneMarker software version 1.51 (SoftGenetics LLC, State College, PA). For the detected dosage alteration in MLPA probes, the absence of sequence variations located within probes near ligation sites was confirmed in each subject.

Reverse-transcriptase PCR (RT-PCR) and sequence analysis for phase determination
For patients carrying exon rearrangements, phase was determined by RT-PCR and subsequent sequencing. Reverse transcription was performed with 1 μg of RNA using Expand reverse transcriptase (Roche Diagnostics, Mannheim, Germany) and downstream-specific primer RNA-11R (Table S1, supporting information). Two rounds of seminested PCR were performed and the compound heterozygote was diagnosed by the presence of bands with the expected sizes of exon rearrangements in the MLPA result. Sequence analysis of the RT-PCR product reconfirmed the sequence of the deleted or duplicated exon margin.
Seven of the 17 patients harbored a contiguous multi-exon deletion and we could not determine their mutational phase based on gene dosage results alone. We analyzed the mutational phase of six patients whose RNA was available using RT-PCR and subsequent sequencing (Fig. 1). Among them, five patients were compound heterozygous for adjacent exon deletions and only one was single heterozygous for PARK2 exon rearrangement. No pathogenic point mutation or exon rearrangement was identified in the SNCA, PINK1, and PARK7 genes.

Unclassified variants (UVs) and polymorphism
Two novel missense variants were identified in this study, including PINK1 : c.835C>T (p.Arg279Cys) and SNCA: c.158C>T (p.Ala53Val) ( Table 3 and Fig. S1, supporting information). The PINK1 variant (c.835C>T) was located in the protein kinase domain and the amino acid was highly conserved across species. However, it was classified as a sequence variant of unknown significance because it was also identified in normal controls (EOPD vs normal controls, 0.43 vs 0.58%) and was in a single heterozygous state. The SNCA variant (c.158C>T) was identified in healthy family members as well as normal controls, and the amino acid was poorly conserved. Therefore, it was classified as a rare polymorphism in the causative gene for autosomal dominant EOPD.
The PARK2 : c.814C>A (p.Leu272Ile) has been previously reported and was also classified as a UV in this study because the amino acid at this codon was highly conserved and the sequence variant was not identified in our normal controls.

Discussion
This study suggests that a significant portion of EOPD patients with apparent contiguous multiexon deletions may actually be compound heterozygous for two different adjacent exon deletions. By phase determination, more than 80% (5 of 6) of patients with contiguous multi-exon deletions and 30% (5 of 18) of all PARK2 mutation carriers were diagnosed as compound heterozygotes, respectively. Therefore, quite a number of contiguous multi-exon deletions or duplications that were reported in previous studies and were unknown for phase are likely to be compound heterozygous ones. This study also indicates that the RT-PCR alone can determine the mutational phase of many patients with contiguous multi-exon deletions, even when familial segregation analysis is not available for them.
Undetermined mutational phase could lead to a false conclusion on the contribution of single heterozygous mutations to the development or clinical severity of EOPD (6,11,12). In our study, considering contiguous exon rearrangements as single heterozygotes, onset age was statistically different between single heterozygotes and patients without mutation. However, this difference between the two patient groups was no longer observed after determination of the mutational phase.
The mutation spectrum revealed in this study was different from those in other studies in Korean population (13,14). The proportion of molecularly confirmed cases with two pathogenic mutant alleles was very high (4.2-5.5 vs 12.3%). One study reported that 5.5% (3/55) of patients had two PARK2 mutations (13). The other reported that 4.2% (3/72) of patients had two mutations and 12.5% (9/72) of patients had one or two mutations (14). In this study, 15.8% (18/114) were mutation-carrying patients and 12.3% (14/114) were molecularly confirmed cases with two PARK2 mutant alleles. Our higher mutation identification rate is probably based on the followings: we analyzed larger number of EOPD patients and they were more strictly selected according to an earlier age of onset (≤40 years) than those in previous studies.
The PARK2 gene was the most predominant genetic cause and exon rearrangement was far more common than point mutation in this population. We identified 20 alleles with single exon rearrangement and nine alleles with multi-exon rearrangement. Exon rearrangements were clustered between exons 2 and 6, and single deletions of exon 3 or 4 and contiguous exon 3-4 deletions were most common in this population (Table 1). These locations of frequent exon rearrangements in Koreans were similar to those in previous reports of other ethnicities (15,16). Allele frequency of exon rearrangements was about ten times higher than that of point mutations in all mutation carriers (0.91 vs 0.09). This high contribution of exon rearrangements has to be considered in the molecular diagnosis of EOPD in this population.
In conclusion, this study shows that phase determination is prerequisite to molecular diagnosis for autosomal recessive EOPD, especially in subjects with PARK2 exon rearrangements.

Supporting Information
The following Supporting information is available for this article: Fig. S1. Three unclassified variants identified in this study: PINK1 :c.835C>T (p.Arg279Cys), SNCA:c.158C>T (p.Ala53Val), and PARK2 :c.814C>A (p.Leu272Ile). (a) Electropherograms of these sequence variants (arrow): the upper panel shows the sequence of the normal control and the lower panel shows the sequence of the patient. (b) Amino acid conservation across species: arginine at codon 279 in the PINK1 and leucine at codon 272 in the PARK2 are highly conserved across most species, but alanine at codon 53 in SNCA is poorly conserved. Table S1. Primers and PCR conditions for semi-nested RT-PCR of the PARK2 gene. Additional Supporting information may be found in the online version of this article. Please note: Wiley-Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.