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. 2022 Mar 12;11(6):974.
doi: 10.3390/cells11060974.

Biallelic Variants in ENDOG Associated with Mitochondrial Myopathy and Multiple mtDNA Deletions

Alessia Nasca  1 , Andrea Legati  1 , Megi Meneri  2   3 , Melisa Emel Ermert  1   3 , Chiara Frascarelli  1 , Nadia Zanetti  1 , Manuela Garbellini  2 , Giacomo Pietro Comi  3   4 , Alessia Catania  1 , Costanza Lamperti  1 , Dario Ronchi  2   3 , Daniele Ghezzi  1   3
Affiliations

Biallelic Variants in ENDOG Associated with Mitochondrial Myopathy and Multiple mtDNA Deletions

Alessia Nasca et al. Cells. .

Abstract

Endonuclease G (ENDOG) is a nuclear-encoded mitochondrial-localized nuclease. Although its precise biological function remains unclear, its proximity to mitochondrial DNA (mtDNA) makes it an excellent candidate to participate in mtDNA replication, metabolism and maintenance. Indeed, several roles for ENDOG have been hypothesized, including maturation of RNA primers during mtDNA replication, splicing of polycistronic transcripts and mtDNA repair. To date, ENDOG has been deemed as a determinant of cardiac hypertrophy, but no pathogenic variants or genetically defined patients linked to this gene have been described. Here, we report biallelic ENDOG variants identified by NGS in a patient with progressive external ophthalmoplegia, mitochondrial myopathy and multiple mtDNA deletions in muscle. The absence of the ENDOG protein in the patient's muscle and fibroblasts indicates that the identified variants are pathogenic. The presence of multiple mtDNA deletions supports the role of ENDOG in mtDNA maintenance; moreover, the patient's clinical presentation is very similar to mitochondrial diseases caused by mutations in other genes involved in mtDNA homeostasis. Although the patient's fibroblasts did not present multiple mtDNA deletions or delay in the replication process, interestingly, we detected an accumulation of low-level heteroplasmy mtDNA point mutations compared with age-matched controls. This may indicate a possible role of ENDOG in mtDNA replication or repair. Our report provides evidence of the association of ENDOG variants with mitochondrial myopathy.

Keywords: ENDOG; endonuclease G; mitochondrial DNA; mitochondrial myopathy; multiple mtDNA deletions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
mtDNA studies in the patient (Pt) and controls (Cts). (A) Southern blot analysis of mtDNA from muscle samples. Arrows indicate multiple mtDNA deleted species. (B) Long-range PCR analysis of mtDNA (wild-type amplicon: 10.5 kbp). Arrows indicate multiple mtDNA deleted species; “Ct+” indicates samples obtained from patients with biallelic POLG1 and C1QBP mutations, used as positive controls. (C) Quantitative PCR analysis of muscle mtDNA content normalized to nuclear DNA (mtDNA/nDNA) and 7S mtDNA levels normalized to total mtDNA in patient (Pt, red) and control (Ct, black) samples. Error bars indicate standard deviation.
Figure 2
Figure 2
Genetic and protein studies on ENDOG. (A) Pedigree of the patient’s family, with segregation analysis of the ENDOG variants. (B) Snapshots from IGV software (Version 2.3.72) of the ENDOG variants identified in the proband. The changes c.734G > T and c.737C > T were always present on the same reads, indicating they are on the same allele. (C) Western blot analysis of ENDOG in skeletal muscle lysates from control individuals (Ct) and the patient described in the manuscript (Pt). Two different exposure times were used for the ENDOG antibody (shorter exposure in the upper panel, longer exposure in the middle panel). GAPDH (lower panel) was used as a loading control, and for normalization of the ENDOG amount. Error bars indicate standard deviation. (D) Western blot analysis of ENDOG in fibroblasts from a control individual (Ct) and the patient (Pt). Two different exposure times were used for the ENDOG antibody (shorter exposure in the upper panel, longer exposure in the middle panel). GAPDH (lower panel) was used as a loading control, and for normalization of the ENDOG amount. Error bars indicate standard deviation.
Figure 3
Figure 3
Steady-state levels of OXPHOS complex subunits. (A) Western blot analysis of OXPHOS subunits in skeletal muscle lysates from control individuals (Cts), a patient harboring biallelic C1QBP mutations (Ct+) and the patient described in the manuscript (Pt). OXPHOS subunit-specific antibodies were used against NDUFB8 or NDUFA9 (CI); SDHA or SDHB (CII); UQCRC2 (CIII); COXII or COXIV (CIV); and ATP5A (CV). Cytosolic b-actin (ACTB) was used as a loading control. (B) Graph reporting protein level normalized to ACTB (in percentage, 100% being the mean value of controls), for the Western blot depicted in panel A.
Figure 4
Figure 4
Functional studies on the patient’s fibroblasts. (A) Depletion/repopulation experiment in fibroblasts from the patient (Pt), a control individual (Ct) and a patient harboring biallelic RNASEH1 mutations (Ct+). Depletion was achieved with the addition of EtBr to the culture medium for 4 days (EtBr), and recovery was followed up for 14 days (R + 14) after the removal of the drug. The percentage of mtDNA/nDNA was determined by qPCR; for each subject, an initial value was set up to 100%, before starting treatment (0). (B) Number of heteroplasmic variants detected by NGS in whole mtDNA from the patient (Pt) and control individuals (Ct). Three different ranges of heteroplasmy were considered: above 5%, between 2 and 5%, between 1 and 2%.
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