The coding regions of PRKN and FBXO4 and intron-exon boundaries were amplified by PCR using self-designed primers (Supplementary Table 1, see section on supplementary materials given at the end of this article), AmpliTaq Gold DNA Polymerase with Buffer II and MgCl2 (Applied Biosystems). Each reaction contained 25 ng template DNA, 1 U polymerase, 0.5 μM each of forward and reverse primers, 200 μM dNTPs, and 1.5 mM of MgCl2 in a 20 μL reaction. PCR was conducted as follows: denaturation for 10 min at 95°C; 35 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 55°C, and extension for 30 s at 72°C; and a final extension step for 10 min at 72°C. Gel electrophoresis of PCR product was run on a 1.5% (w/v) agarose, 0.6 μg/mL ethidium bromide gel at 120 volts for approximately 30 min. PCR product that yielded clear bands was enzymatically purified with ExoSAP-IT (Applied Biosystems) and Sanger sequenced in forward and reverse directions (GENEWIZ, Inc., South Plainfield, NJ, USA). Sequence data were aligned to NCBI reference sequences (PRKN, NM_004562.3; FBXO4, NM_033484.2) and analyzed with Sequencher software (Gene Codes Corporation, Ann Arbor, MI, USA). The entire coding sequence and intron-exon boundaries were examined for mutations. Variant databases dbSNP (33), COSMIC (34), and ClinVar (35) were queried for any identified variants. Variants were assessed by predictive modeling tools SIFT (36) and Poly-Phen (37), and meta prediction tools REVEL (38) and MetaLR/dbNSFP (39), all available through Ensembl (40).

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