Alternative start codon selection shapes mitochondrial function and rare human diseases.

Rare genetic diseases collectively affect millions of individuals. A common target of many rare diseases is the mitochondria, intracellular organelles that originated through endosymbiosis. Eukaryotic cells require related proteins to function both within the mitochondria and in the host cell. By analyzing N-terminal protein isoforms generated through alternative start codon selection, we identify hundreds of differentially localized isoform pairs, including dual-localized isoforms that are essential for both mitochondrial and host cell function. Subsets of dual mitochondria-localized isoforms emerged during early eukaryotic evolution, coinciding with mitochondrial endosymbiosis. Importantly, we identify dozens of rare disease alleles that affect these alternative protein variants with unique molecular and clinical consequences. Alternative start codon selection can bypass pathogenic nonsense and frameshift mutations, thereby selectively eliminating specific isoforms, which we term isoform-selective alleles (ISAs). Together, our findings illuminate the evolutionary and pathological relevance of alternative translation, offering insights into the molecular basis of rare human diseases.