For each tumor and germline sample, five highly polymorphic microsatellite loci in and around PRKN (D6S1599, D6S305, and D6S1581) and FBXO4 (D5S418 and D5S2082) were amplified using fluorophore-tagged primers, whose sequences are available through the UCSC Genome Browser (41). Each reaction contained 25 ng template DNA, 1 U polymerase, 0.4 μM each of forward and reverse primers, 250 μM dNTPs, and 2.5 mM of MgCl2 in a 15 μL reaction. PCR was conducted as follows: denaturation for 12 min at 95°C; 10 cycles of denaturation for 15 s at 95°C, annealing for 15 s at 55°C, and extension for 30 s at 72°C; 20 cycles of denaturation for 15 s at 89°C, annealing for 15 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 2% (w/v) agarose, 0.6 μg/mL ethidium bromide gel at 120 volts for approximately 30 min. PCR product that yielded clear bands was submitted to fragment analysis (GENEWIZ, Inc., South Plainfield, NJ, USA). Allele peaks for each microsatellite locus were called with GeneMarker software (SoftGenetics, LLC, State College, PA, USA) (42). The allelic ratio (AR) of the fluorescent signals of an individual’s discrete alleles were calculated as follows:

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The ratio of fluorescent signal from discreet alleles A and B was compared between tumor (T) and matched germline (G) samples. Consistent with prior studies (43, 44, 45, 46, 47), cases with allelic ratios above 2 or below 0.5 – indicative of a two-fold or greater change in allele signal ratio in the tumor sample compared to the germline sample – were scored as having undergone LOH.

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