e-Methods
Molecular study materials and methods.
After giving written informed consent, all participants provided a blood sample for genetic analyses. Genomic DNA was extracted from peripheral blood leukocytes using standard procedures and MFN2 mutation screening was performed by direct sequencing of all coding exons and exon-intron boundaries using the dideoxy terminator method on an automated ABI-3100 DNA sequencer (Applied Biosystems, Foster City, CA, USA). The sequences were compared with those of reference available at the NCBI (MNF2 genomic contig: NT_221937, cDNA: NM_014874, and protein: NP_055689; using the DNASTAR package. To exclude gene polymorphisms, sequence variants were tested in a control sample of 100 healthy and ethnically matched subjects by Denaturing High Performance Liquid Chromatography (DHPLC) (Transgenomic WAVE system).
Total RNA was isolated from peripheral blood of living family members using the PAXgene RNA Isolation Kit (Qiagen). First-strand cDNA was synthesized from DNase-treated total RNA using random primers and Super-Script II reverse transcriptase (Invitrogen). The cDNA was amplified using different primers pairs designed to specifically amplify splicing products between exon 13 and 14 of MFN2 (primer sequences and PCR conditions are available on request). PCR reactions were loaded on a vertical 9% polyacrylamide gel, each band was then excised using the QIAEX II, Gel Extraction Kit (Qiagen), purified and sequenced.
The backbone of the artificial minigene was obtained by cloning the entire genomic DNA sequence of the human beta-globin gene into the NheI and ApaI sites of pCDNA3.1 (Invitrogen). PCR conditions and primers are available on request. A genomic DNA fragment spanning from the 3’ end of intron 11 to the 5’ end of intron 14 of human MFN2 was amplified from a patient and cloned into the BsrGI site within intron 2 of beta globin. After sequencing, a clone containing the wild type allele, and a clone with the mutation, were selected for further experiments. Constructs were transfected into HeLa or U87MG cells (gift of Dr. Francesca Pistollato) using Effectene (Quiagen). After 48 hours cells were lysed using Trizol (Invitrogen). Total mRNA was extracted and retrotranscribed using the Superscript III kit (Invitrogen) and random hexameres. The artificial minigene transcripts were amplified using either primers corresponding to exon 2 and 3 of beta-globin or a forward primer on beta globin exon 2 and a reverse primer encompassing exon 13 and 14 of MFN2. After gel separation, individual bands were excised and sequenced.