Final Report Summary - BIOMIT (Biogenesis of the mitochondrial respiratory chain in children with severe multiorgan disorders)
The final goal of the BioMit project is to identify genes underlying the isolated or combined complex I deficiencies in children and to better understand the pathomechanisms involved in mitochondrial disorders by using the combination of the state-of-the-art methods in the field.
DNA samples from 20 Finnish patients have been analyzed by exome sequencing to assign the underlying molecular defect to genes involved in mitochondrial function. The data from the exome sequencing of the patient samples has revealed interesting candidate genes of which functional role in the pathogenesis of the disease will be further evaluated.
MTFMT. Mutations creating a premature stop codon were identified in MTFMT gene by exome sequencing from three patients with combined respiratory chain deficiency. The pathogenic mechanisms behind the MTFMT mutations were investigated and the studies showed that the patients with mutations in MTFMT lack mitochondrial immunodetectable methionyl-tRNA formyl transferase protein, which in turn leads to dysfunctional respiratory chain. In order to further confirm the pathological origin of the disease, complementation studies were carried out by creating cell lines from patient fibroblasts expressing the wild type MTFMT. The function of MTFMT seems to be highly regulated in cells since too much MTFMT leads to a very severe dominant negative effect by destroying the whole respiratory chain both in patient and in control cell lines. The deficiency of respiratory chain complexes was rescued in a patient clone with a low level of MTFMT expression confirming the origin of the disease.
Surprisingly, the mitochondrial translation was only mildly affected in patient fibroblasts with MTFMT mutations suggesting that the initiation of the mitochondrial translation may happen without the formylation of the first methionine. The strong effect of MTFMT mutations, however, on the structure of the respiratory chain could indicate that either MTFMT or the formylation itself has another role than translation initiation in mitochondria. Further studies showed that indeed MTFMT has a role in the assembly of mitochondrial respiratory chain.
Mitochondria are in an essential position in cellular metabolism. Currently, the biogenesis of mitochondrial respiratory chain is far from understood and there are, presumably, many unknown nuclear encoded factors involved in this process. Mitochondrial biogenesis is a multistep process involving the concerted expression of two different genomes. Since all the structural genes in mtDNA encode subunits of the OXPHOS enzymes, all the protein factors controlling the biogenesis at the level of replication, transcription and translation of nuclear and mitochondrial genes and the proteins responsible for import, processing and assembly into the functional subunits of cytosolic precursor proteins have to be coded by nuclear DNA. The mechanistical role of the factors identified by exome sequencing in this study from the Finnish patients with isolated or combined complex I respiratory chain deficiency will be studied in details. Currently, the exome sequencing has led to the identification of three additional very strong candidate genes whose role in disease pathogenesis is under study. In all of these genes there is either a strong correlation between patients with similar symptoms or the segregation of the mutant allele fits well for the pathogenicity within the families of these patients. As a conclusion, novel proteins have been identified as the underlying cause of the defect in Finnish pediatric patients, giving valuable information about the disease causing genes in Northern Finland. The results of this study have provided new targets for genetic analysis of patients with respiratory chain deficiency and can be utilized in the genetic counselling of the patients and their family. Further functional studies of these identified novel proteins will lead to a broader knowledge on the actors behind the mitochondrial biogenesis in a near future.