Analysis of mitochondrial DNA sequences in childhood encephalomyopathies reveals new disease-associated variants

Abstract

Background: Mitochondrial encephalomyopathies are a heterogeneous group of clinical disorders generally caused due to mutations in either mitochondrial DNA (mtDNA) or nuclear genes encoding oxidative phosphorylation (OXPHOS). We analyzed the mtDNA sequences from a group of 23 pediatric patients with clinical and morphological features of mitochondrial encephalopathies and tried to establish a relationship of identified variants with the disease.

Methodology/principle findings: Complete mitochondrial genomes were amplified by PCR and sequenced by automated DNA sequencing. Sequencing data was analyzed by SeqScape software and also confirmed by BLASTn program. Nucleotide sequences were compared with the revised Cambridge reference sequence (CRS) and sequences present in mitochondrial databases. The data obtained shows that a number of known and novel mtDNA variants were associated with the disease. Most of the non-synonymous variants were heteroplasmic (A4136G, A9194G and T11916A) suggesting their possibility of being pathogenic in nature. Some of the missense variants although homoplasmic were showing changes in highly conserved amino acids (T3394C, T3866C, and G9804A) and were previously identified with diseased conditions. Similarly, two other variants found in tRNA genes (G5783A and C8309T) could alter the secondary structure of Cys-tRNA and Lys-tRNA. Most of the variants occurred in single cases; however, a few occurred in more than one case (e.g. G5783A and A10149T).

Conclusions and significance: The mtDNA variants identified in this study could be the possible cause of mitochondrial encephalomyopathies with childhood onset in the patient group. Our study further strengthens the pathogenic score of known variants previously reported as provisionally pathogenic in mitochondrial diseases. The novel variants found in the present study can be potential candidates for further investigations to establish the relationship between their incidence and role in expressing the disease phenotype. This study will be useful in genetic diagnosis and counseling of mitochondrial diseases in India as well as worldwide.

Figure 1. Ethidium Bromide stained polyacrylamide gels of PCR amplified products encompassing heteroplasmic variants G4812C, A4136G, A9194G, A10149T and T11916A analyzed by PCR-RFLP in patients and their respective mothers.

Mutation G4812C causes a loss of Dde I site and hence shows a single band after digestion whereas wild type fragment shows 2 bands (bp). Variant A4136G creates an Nla III site and after digestion results in two bands of 43 bp and 107 bp. The heteroplasmic mutation has three bands (43 bp, 107 bp and 150 bp) as shown. Similarly other variants e.g. A9194G (creates Hha I site), A10149T (creates Dde I site) and T11916A (creates Rsa I site) and all result in 3 bands after digestion and separation on 12% PAGE. The homoplasmic mutations should yield only 2 bands lacking the wildtype band after separation on PAGE.

Figure 2. Kyte-Doolittle Hydropathy Plot of wild type and mutated NADH Dehydrogenase Subunits (ND1).

Mutation T3866C (Isoleucine-Valine) showing a significant decrease in hydrophobicity at amino acid position 187 in the ND1 protein. SPLIT 4.0 plots predicting Tran membrane regions and alterations in them due to mutation T3866C. Black line is indicative of wild type whereas red line indicates the mutated polypeptide.

Figure 3. Changes in the secondary structure of tRNA lysine caused by variant C8309T identified in Patient-13.

The free energy of the cloverleaf structure is significantly increased in the mutated tRNA lysine.

Figure 4. Evolutionary conservation of novel and known amino acid changes in the studied patients.

The mtDNA sequences for ND1, ND2, ND3, ND4, COX 3, ATPase 6 and ATPase 8 protein subunits of different mammalian species were converted to protein sequence by Gene Runner software. The amino acid sequences were aligned by Clustal×programme. Accession numbers for the mtDNA sequences used for alignment are: Human (NC_001807), Gorilla (NC_001645), Chimpanzee (NC_001643), Orangutan (NC_001646), Bovine (NC_006853), Dog (NC_002008), Pig (NC_000845), Rat (NC_001665), and Mouse (NC_005089).