Fig. 2
Analysis of methylation status of imprinted SNRPN locus and heterozygous RC-L1 insertions. a Schematic showing location of binding sites of primers in the SNRPN locus used to PCR amplify its CpG island as an internal control for the successful enrichment of methylated and unmethylated gDNA fractions. Blue box –exon. b Example gel image of PCR product (263 bp) amplified from SNRPN CpG island in methylated and unmethylated fractions of DNA from all three tissues. (Cer cerebellum, MCX motor cortex, M methylated fraction of DNA, U unmethylated fraction of DNA). c Box-Whisker plot demonstrating methylation status of the CpG island of the imprinted SNRPN locus in cerebellum, motor cortex and blood of 9 individuals. Methylation status was determined by comparing the band intensity of the PCR product in the methylated and unmethylated fractions of gDNA. The average methylation of the CpG island of the SNRPN locus in cerebellum, motor cortex and blood comprises 55%, 48% and 55%, respectively. Cerebellum, n = 6 (controls = 2, FALS = 2, SALS = 2); MCX, n = 9 (controls = 2, FALS = 2, SALS = 5); blood, n = 3 (SALS = 3). d Schematic showing the position of binding sites of primers used to amplify the genomic 5′ junction of the selected full length RC-L1 loci when present (filled site) and the empty site when the respective RC-L1 is absent. The L1 5′UTR-specifc primer binds position 223–206 of the L1.3 reference sequence TSD, target site duplication; UTR, untranslated region; ORF1 and ORF2, open reading frame 1 and 2; An, polyA stretch; e PCR analysis of 5′ junctions of RC-L1 loci L1-chr6-q24.1 and L1-chrX-p22.2 and the empty sites of heterozygous carriers in the methylated and unmethylated gDNA fractions from blood and motor cortex of same individual. f The 5′ junction of the RC-L1s is enriched in the methylated fraction of DNA, while the empty sites (absent allele) lack any meaningful methylation in heterozygous carriers. Average methylation status of 5′ junctions RC-L1 loci and empty sites comprises 79% and 2%, respectively (14 heterozygous RC-L1 loci). Two-tailed t-test *** p < 0.001 X = mean