Applera bigdye version 3

To confirm the MUC1 VNTR mutations in other family members, Sanger sequencing was performed. For Sanger sequencing, the PCR products of the VNTR region were amplified with intronic primers (5’-ACAGGATGTCACTCTGGC-3’) and sequenced using Applera BigDye version 3.1 (Applied Biosystems, CA) with 5× high-GC buffer (Tsingke Biotech) and an ABI 3730XL Avant Genetic Analyzer automated sequencer (Thermo Fisher Scientific). Two researchers analysed the sequences independently. For individuals with MUC1 VNTR mutations and normal renal function (as assessed by estimated glomerular filtration rate (eGFR) measurement), we re-collected blood samples and performed Sanger sequencing again at a different company.

Duplication of the fragment that contains PMP22 was performed with multiplex ligation-dependent probe amplification (SALSA MLPA kit P033, MRC-Holland). The coding and splicing of the site-flanking regions of PMP22, MFN2, GJB1, and MPZ were PCR amplified with intronic primers and directly sequenced using Applera BigDye version 3.1 (Applied Biosystems, Foster City, CA, USA) and the automated sequencer ABI 3730XL (Applied Biosystems). The amplicon sequences were aligned by SeqMan Pro to the published human gene sequences in the NCBI database² with 7.1.0 (DNASTAR Inc., Madison, WI, USA). All nucleotide differences were compared to dbSNP³ and to the human genetic mutation databases IPNMDB⁴ and HGVS⁵. The in silico pathogenicity prediction tools Provean⁶, PolyPhen⁷ and Mutation Taster⁸ were used to further validate the mutations.