Screening for nuclear genetic defects in the ATP synthase-associated genes TMEM70, ATP12 and ATP5E in patients with 3-methylglutaconic aciduria

Abstract

Supporting Information The following Supporting information is available for this article: Fig. S1. Complex V analysis. Blue Native polyacrylamide gel electrophoresis (BN-PAGE) analysis of mitochondrial respiratory complexes in the muscle of patient 3. Mitochondrial particles were separated on a 5–15% BN-PAGE. Western blot (left panel) was performed with antibodies against complex II: 30-kDa subunit (CII), complex III: core protein 2 subunit (CIII), complex IV: COXI subunit (CIV), complex V: alpha subunit (CV). In gel activity, staining (right panel) of complex V of the patient's muscle. Fig. S2. Common TMEM70 homozygous missense mutation c.317–2A>G. Sequence analysis of genomic DNA of patient 1 and a control individual. The site of the mutation is indicated by an arrow. Fig. S3. Polymerase chain reaction (PCR) analysis and identification of a genomic DNA deletion. (a) PCR with different sets of oligonucleotides narrowed the region involved in the genomic deletion. (b) PCR with long-distance primers amplified specific products smaller than the expected size as compared to controls, indicating the presence of an intragenic deletion. Asterisks indicate the specific bands in agarose gels. Oligonucleotides used in each PCR are labeled in bold. Fig. S4. The c.580G>A substitution is a new TMEM70 polymorphic variant. (a) Heterozygous c.580G>A substitution in patient 3 and her parents. Mutation is indicated by an arrow. (b) Reverse transcriptase-polymerase chain reaction (PCR) and sequence analysis in fibroblasts from the maternal lineage showed no abnormalities in the TMEM70 transcript. (c) Restriction fragment length polymorphism (RFLP) analysis showed the presence of the c.580G>A substitution in 2 out of 100 alleles from healthy controls. C1–C5, control individuals; P3, patient 3. Arrowheads indicate the restriction sites for HpyCH4III; asterisks indicate the samples heterozygous for the c.580G>A substitution. Table S1. Oligonucleotides used in this study Additional Supporting information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.