Clinical and functional characterisation of the combined respiratory chain defect in two sisters due to autosomal recessive mutations in MTFMT

Abstract

Exome sequencing identified compound heterozygous mutations in the recently discovered mitochondrial methionyl-tRNA formyltransferase (MTFMT) gene in two sisters with mild Leigh syndrome and combined respiratory chain deficiency. The mutations lead to undetectable levels of the MTFMT protein. Blue native polyacrylamide gel electrophoresis showed decreased complexes I and IV, and additional products stained with complex V antibodies, however the overall steady state level of mt-tRNA(Met) was normal. Our data illustrate that exome sequencing is an excellent diagnostic tool, and its value in clinical medicine is enormous, however it can only be optimally exploited if combined with detailed phenotyping and functional studies.

Keywords: Leigh syndrome; Mitochondrial encephalomyopathy; Mitochondrial translation; mt-tRNA modification.

Fig. 1

A) Variant numbers following analysis pipeline of BWA (Sequence Aligner); VarScan (single base variant — SBV calling); Dindel v1.01 (Indel calling). (*) SBV — VarScan parameters min. total coverage ≥ 5-fold, min. variant coverage ≥ 3-fold, min. Quality > 10; Indel — Dindel output filter min. variant coverage ≥ 4. (a) Variants with position within targets (Illumina TruSeq 62 Mb) +/− 500 bp, seen on both (forward & reverse) strands and (SBVs only) variant allele frequency > 24%; (b) variants that match 1000 genomes (Feb 2012) and/or dbSNP135 with minor allele frequency (MAF) > 0.01; (c) rare/novel variants (MAF < 0.01) with exclusion of common variants found to be shared in an in-house panel of 91 ʻnon-respiratory complexʼ individuals; (e) MutationTaster predictions. B) Identification of pathogenic compound heterozygous mutations in MTFMT. Electropherogram of MTFMT DNA and cDNA sequence of patient 1 and a control. C) High resolution Northern blotting detected normal mt-tRNA^Met steady state levels in myoblasts of patient 1. For comparison we show also mt-tRNA^Glu and 5S rRNA. D) SDS-PAGE immunoblotting analysis in myoblasts of patient 1 showed no detectable MTFMT protein. Complex II 70 kDa subunit antibody was used as a loading control. E) Immunoblotting in myoblasts of patient 1 showed decreased steady state levels for COX II and NDUFB8. Blotting with antibodies against the complex II 70 kDa subunit and β actin showed equal loading. F) BN-PAGE in myoblasts of patient 1 showed severely decreased complexes I and IV and 3 additional bands with complex V antibodies, however complex III was normal. G) In-gel activities of complexes I and IV were decreased in myoblasts of patient 1, and complex V was slightly reduced, but showed an extra band.