Figure 3.

Genetic and structural characterization of the MT-CYB variant. (A) Family pedigree showing the distribution of the MT-CYB variant and tissue-specific heteroplasmy levels. Squares indicate males and circles indicate females. The arrow indicates the proband. Filled symbols represent the affected individual. Heteroplasmy levels of the MT-CYB variant in blood, urine, and skeletal muscle are shown. Asterisks indicate heteroplasmy levels determined by next-generation sequencing (NGS). The maternal blood sample was analyzed by NGS, whereas the maternal urine sample was analyzed by Sanger sequencing. (B) Sanger sequencing validation of the MT-CYB variant in different tissues. The variant was detected in the proband’s blood, urine, and skeletal muscle, but was absent in the mother’s blood and urine. Red boxes indicate the variant position. (C) Evolutionary conservation analysis of the MT-CYB variant. Multiple sequence alignment of MT-CYB orthologs from different species showing the high conservation of Arg100, which is highlighted in red. (D) Structural modeling of wild-type and mutant MT-CYB proteins. Three-dimensional structural models of wild-type and p. Arg100Gln mutant MT-CYB proteins showing the local environment surrounding residue 100.