Appendix e-1

Detailed Materials and Methods

Whole-exome sequencing

Index patients of 26 families underwent whole-exome sequencing as described 4. Sequencing was performed on a Genome Analyzer HiSeq 2000 system (Illumina, San Diego, CA) after in-solution enrichment of exonic and adjacent intronic sequences (SureSelect Human all Exon 50 Mb kit (Agilent, Santa Clara, USA) and indexing of samples for multiplex-sequencing (Multiplexing Sample Preparation Oligonucleotide Kit, Illumina). Read alignment was performed with BWA (version 0.5.8) to the human genome assembly hg19. Single nucleotide variants and small insertions and deletions were called with SAMtools (version 0.1.7). We filtered variants to exclude HapMap-SNPs present in dbSNP132 with an average heterozygosity greater than 0.02 and those present in more than two of >2000 in-house exomes from individuals with unrelated diseases. Variants annotated in known dHMN and CMT2 genes were identified in the resulting data sets and confirmed by Sanger sequencing.

RNA analysis

RNA isolation was performed as described 6. Briefly, human blood (7.5 ml) was diluted to 45 ml with RBC buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA), incubated for 10 min at room temperature (RT) and then centrifuged at 600x g for 10 min. The cell pellet was washed once in RBC buffer and once in PBS. The cells were resuspended in 1.2 ml Trizol solution and RNA was further purified according to the protocol provided by the supplier (Life Technologies, Darmstadt, Germany). Reverse transcription was done using the Qiagen Quantitect Reverse Transcription Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. PCR primers for amplification of a part of the HSJ1 transcript were localized in the fourth exon (forward) and the fifth exon (reverse). Primer sequences and PCR conditions are available upon request. Bands were excised from agarose gels for Sanger sequencing in order to confirm specific amplification.

Fibroblast cell culture

Fibroblast cell cultures were performed as described 7. After giving informed consent, a 1-mm skin punch biopsy was taken from subject IV.1 of family 1. Biopsy material was transferred to DMEM medium containing 20% fetal calf serum, penicillin/streptomycin and glutamine (D20 medium). The tissue was transferred to a 60 mm plastic Pasteur dish and submersed in D20 medium at standard conditions. After a lawn of fibroblasts had grown, biopsy tissues were removed and the cultures were grown and passaged 3-4 times before proteins were isolated for western blotting.

Western blotting

Western blotting was conducted as described 8. Briefly, fibroblast cultures were lysed with lysis buffer containing 25 mM Tris, 1 mM NaVO3, 1% SDS, and 2 mM EDTA. Protein concentrations were determined by a BCA assay (Thermo Scientific, Rockford, USA). Samples of 20 μg of protein were loaded onto 10% SDS-polyacrylamide gels. After electrophoresis, proteins were transferred to PVDF membranes (Millipore, Darmstadt, Germany). Membranes were blocked with 5% milk powder and incubated with the primary antibodies against HSJ1 (rabbit polyclonal, 1:500, Proteintech Group, Chicago, USA) and β-actin (rabbit polyclonal, 1:15.000, Santa Cruz Biotechnology, Santa Cruz, USA) and appropriate HRP-conjugated secondary antibodies (1:20,000, Life Technologies). Bound antibodies were visualized using chemiluminescence (Thermo Scientific).

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