Mitochondrial DNA Instability Is Common in HIV-Exposed Uninfected Newborns

Abstract

Worldwide, one million HIV-exposed uninfected (HEU) children are born yearly, and chronic health impairments have been reported in these children. Mitochondrial DNA (mtDNA) instability and altered mtDNA content have been evidenced in these children, but an exhaustive characterization of altered mitochondrial genomes has never been reported. We applied deep mtDNA sequencing coupled to the deletion identification algorithm eKLIPse to the blood of HEU neonates (n = 32), which was compared with healthy controls (n = 15). Dried blood spots (DBS) from African HEU children were collected seven days after birth between November 2009 and May 2012. DBS from French healthy controls were collected at birth (or <3 days of life) in 2012 and in 2019. In contrast to the absence of mtDNA instability observed at the nucleotide level, we identified significant amounts of heteroplasmic mtDNA deletions in 75% of HEU children and in none of controls. The heteroplasmy rate of the 62 mtDNA deletions identified varied from 0.01% to up to 50%, the highest rates being broadly compatible with bioenergetic defect and clinical expression. mtDNA integrity is commonly affected in HEU neonates. The nature of the deletions suggests a mechanism related to aging or tumor-associated mtDNA instability. This child population may be at risk of additional mtDNA genetic alterations considering that they will be exposed to other mitotoxic drugs including antiretroviral or anti-tuberculosis treatment.

Long-Term Persistence of Mitochondrial DNA Instability in HIV-Exposed Uninfected Children during and after Exposure to Antiretroviral Drugs and HIV

“…Out of the 24 samples collected at D7, 22 had been previously analyzed with eKLIPse version-1 algorithm [ 28 ], and analyses were repeated with the new version of the software. The percentage of children with mtDNA alterations was similar regardless the version of eKLIPse.…”

“…Lastly, we did not have the opportunity to test HUU to compare our observations to an HIV-unexposed control group. However, we previously analyzed DBS collected during the same period from healthy French newborns and found no mtDNA deletion [ 28 ]. We have previously showed that neither the use of DBS nor the duration of storage introduced artefactual deletions.…”

“…We have previously showed that neither the use of DBS nor the duration of storage introduced artefactual deletions. Of note, in this DBS validation study, half of the participants had an African mtDNA haplotype [ 28 ]. From the diagnostic laboratory archive (National Reference Center for Mitochondrial Diseases, University Angers, France), we were able to retrieve the results of 26 eKLIPse assays performed on the blood of children under ten years of age for whom mitochondrial disease was suspected.…”

“…The assay combined the deep high-throughput sequencing of mtDNA and the bioinformatic pipeline [ 11 ]. The sample flow and the quality controls were described in detail in Monnin et al, 2021 [ 28 ]. Briefly, long-range mtDNA PCR (2 overlapping fragments) was performed from 25 ng of total blood DNA.…”

“…If the quantitative evaluation of the mtDNA copy number was addressed in several contexts, qualitative investigations of mtDNA are scarce and focus on the most common deletion [ 22 , 23 , 24 , 25 , 26 ] or the mutation rate at the nucleotide level [ 22 , 23 , 27 ]. We recently applied the deep sequencing approach coupled with an eKLIPse computational pipeline to HEU at birth and reported that 75% of the HEU have deleted mtDNA molecules in blood cells, which is not found in control HUU at birth [ 28 ]. The heteroplasmy rates ranged from 0.01% to up to 50%.…”