DNA Methylation Analysis of 4qA D4Z4 Repeats

DNA methylation was analyzed by BSS assay. BS conversion was performed on 1 μg of genomic DNA using the EpiTect Bisulfite Kit (Qiagen) as per the manufacturer’s instructions, and 200 ng of converted genomic DNA was used per PCR. For the 4qA BSS analysis, converted DNA was amplified by nested PCR using oligonucleotide primers and thermocycling conditions that amplify 4qA but not 4qB; the initial PCR was performed with oligonucleotide primers BSS1438F (5’-GTTTTGTTGGAGGAGTTTTAGGA) and BSS3742R (5’-AACATTCAACCAAAATTTCACRAAA) and then followed by nested PCR with oligonucleotide primers BSS1438F and BSS3626R (5’-AACAAAAATATACTTTTAACCRCCAAAAA) using 10% of the first PCR product as template. Polymorphic nucleotide changes that preferentially amplify the 4A subtelomeric region are underlined. The BSS3742R sequence does not exist in 4B or 10B and utilizes a polymorphic change at bp 7946 in FJ439133 to eliminate 10A166, and BSS3626R utilizes polymorphic changes at bp 7827 in FJ439133 to eliminate 10A, 4B, and 10B [15 (link)]. All PCRs were performed using GoTaq Hot Start Polymerase (Promega) as follows: 94°C for 2 min, 25 cycles of 94°C for 15 sec, 58°C for 20 sec, and 72°C for 50 sec, followed by a final extension at 72°C for 10 min. The 593-bp PCR product spans the end of full-length DUX4 exon 1 to the beginning of DUX4 exon 3, therefore allowing specific analysis of the methylation status of the most distal 4qA D4Z4 repeat, which contains 57 CpGs (Additional file 1: Figure S1A). For the 4qA-L BSS analysis, converted DNA was similarly amplified by nested PCR. The initial PCR was performed with oligonucleotide primers BSS4qALF (5’-TTATTTATGAAGGGGTGGAGTTTGTT) and BSS3742R, and then followed by nested PCR with oligonucleotide primers 4qALF and BSS3626R using 10% of the first PCR product as template. All PCRs were performed using GoTaq Hot Start Polymerase (Promega) as follows: 94°C for 2 min, 25 cycles of 94°C for 15 sec, 58°C for 20 sec, and 72°C for 30 sec followed by a final extension at 72°C for 10 min. The 354-bp PCR product spans the 3’ end of the extended 4qA-L D4Z4 repeat to the beginning of DUX4 exon 3, therefore allowing specific analysis of the methylation status of the most distal 4qA D4Z4 repeat sequence, which contains 30 CpGs (Additional file 1: Figure S1D). When no PCR product was obtained with either the 4qA- or 4qA-L-specific BS PCRs, DNA methylation status of same distal D4Z4 region was analyzed using primer BSS3702R (5’-AAAACCAACRAACTCCCTTACAC) instead of BSS3626R. BSS3702R amplifies distal D4Z4 from both 10A and 4A. For the DUX4 5’ region, BS-converted DNA was amplified by nested PCR as described above. The initial PCR was performed with oligonucleotide primers BSS167F (5’-TTTTGGGTTGGGTGGAGATTTT) and BSS1036R (5’-AACACCRTACCRAACTTACACCCTT), and then followed by nested PCR with oligonucleotide primers BSS475F (5’-TTAGGAGGGAGGGAGGGAGGTAG) and BSS1036R. A polymorphic nucleotide change at bp 6748 in FJ439133 (underlined) was used to preferentially amplify the 4A subtelomeric region. This 578-bp PCR product contains 61 CpGs to preferentially analyze the methylation status of the DUX4 5’ region of chromosome 4-type D4Z4 repeats (Additional file 1: Figure S1E).
All BS PCR products were cloned into the pGEM-T Easy Vector system I (Promega) for sequencing analysis. At least 10 clones were sequenced for each subject and their methylation status was analyzed using web-based analysis software BISMA (http://biochem.jacobs-university.de/BDPC/BISMA/) [36 (link)] with the default parameters. Default parameters have a lower threshold of 90% identity to the reference sequence, a lower threshold of BS conversion rate of 95%, and remove identical sequences derived from the same genomic template based on conversion artifacts. To remove PCR amplification bias, 1 CpG in BSS3626R primer and 2 CpGs in BSS1036R primer were removed from the analysis; therefore, a total of 56 CpGs, 30 CpGs, and 59 CpGs were analyzed for the 4qA, 4qA-L, and DUX4 5’ region, respectively. The “R” designation in primer sequences represents a purine (A or G).