Characterizing C2orf69 as mitochondrial protein involved in OXPHOS glycogen storage and cell cycle

Inborn errors of metabolism (IEM) is a generic term for a group of more than 1,000 diseases. Starting with 4 IEMs in 1902, today, new inherited metabolic diseases become more and more easier to identify by omics techniques. Although the human genome is encoded since 2003, there are still many uncharacterized genes left. One way to elucidate the function of these genes is the investigation of families with rare inherited diseases, as performed in this study. Here, for the first time, homozygous loss-of-function mutations in the uncharacterized gene C2orf69 were identified in patients with a complex pediatric multisystem disorder exhibiting symptoms of a mitochondrial disease and glycogen storage disease type IV. The affected gene product, C2orf69, localizes to mitochondria via a N-terminal signal peptide and Y2H screen revealed mitochondrial proteins as interaction partners. Impairment of respiratory chain complex activity was observed in patient derived tissues and a CRISPR-Cas9 knockout (KO) cell model. The KO cells further revealed markedly elevated reactive oxygen species levels and reduced proliferation. RNA-seq also exposed alterations in mitochondrial pathways, proliferation and cell cycle. KO cells show reduced proliferation and exhibit slowed G2/M phase progression, in line with the high expression of C2orf69 in the M phase of the cell cycle. Immunological pathways were also altered, resembling the patients phenotype of recurrent auto-inflammation. Simultaneously, C2orf69 appears to regulate glycogen storage, as indicated by reduced glycogen branching enzyme (GBE1) activity and deposits of PAS-positive material in tissues from C2orf69 patients. However, the exact mechanism that leads to impairment of these two energy providing systems, mitochondrial OXPHOS and glycogen storage, remains subject of further investigations