Mutational landscape of mitochondrial cytochrome b and its flanking tRNA genes associated with increased mitochondrial DNA copy number and disease risk in children with autism

Autism, the severe form of Autism Spectrum Disorder (ASD), is a genetically heterogeneous wide spectrum of impairments characterized by social dysfunction, restricted contact, and communication. Out of 80 children (aged 3 to 10 years) enrolled, 50 were diagnosed with autism (according to M-CHAT-R), while the remaining 30 were healthy children. Lactic acid and ammonia levels were measured using an autoanalyzer and children with autism had significantly higher levels than controls. The Cytb and its adjacent tRNA genes for glutamine, threonine, and proline (MT-TE, MT-TT, MT-TP) were amplified by PCR, purified, and sequenced by Sanger sequencing. A total of 57 variants (19 non-synonymous and 31 synonymous SNPs in Cytb, and seven tRNA gene variants) were identified and Cytb-specific mtDNA copy numbers were determined. There was no significant association between any Cytb variants and disease outcomes, but three of them (15067T>C, 15202C>T, 15262T>C) were linked to higher lactic acid levels and two (14753C>T, 15262T>C) to higher ammonia levels. When docked with Cytb ligands PEE, Heme1, and Heme2, 15341T>C and 15773G>A had the most detrimental impact on protein structure, with higher RMSD than the reference protein complexes. The mtDNA copy number was significantly higher in children with autism than in healthy children, although after adjusting confounding factors the difference was found to be insignificant. Out of two detrimental variants, 15341T>C, along with two additional variants, 15049C>T and 15796T>C, were found to be associated with an increased Cytb-specific mtDNA copy number. Additionally, synonymous SNPs showed codon-usage variations (RSCU). Although no association of variants in Cytb and its adjacent tRNA gene was found with autism, a cluster of variants has been found to affect protein structure and translation rate (as indicated by alteration of RSCU values), which may impair normal activity of electron transport chain (ETC) which is reflected by the significant change in lactic acid and ammonia levels. Children with autism showed increased mtDNA copy number to compensate the impact of these mutational consequences. In conclusion, the electron transport chain complexes should be investigated further in a large sample size to strengthen this finding.