1. Introduction
Mitochondria contain their own genome, a circular double-strand DNA molecule (mtDNA) of 16,569-bp that encodes for proteins of the mitochondrial respiratory chain and mitochondrial tRNAs and rRNAs. Because mtDNA is inherited from the mother, germ-line mutations in the mtDNA are associated with rare maternal inherited diseases. The mtDNA is prone to acquire nucleotide changes that accumulate with age or under exposure to environmental toxics [Lee et al., 1998;Wallace 2010; Bratic and Larsson , 2013; Ziadaet al., 2019].
In addition to rare pathogenic variants the mtDNA contains many common variants that originated in individuals from particular populations and spread with worldwide migrations. Specific combinations of these variants classify the mitochondrial haplogroups, with frequencies that are characteristic of each human population [Wilson and Allard, 2004; Torroni et al., 2006; Brotherton et al., 2013] . For instance, haplogroup H is defined by 7028C (among other nucleotide changes) and is the most common among Europeans while is absent among individuals of African or East Asian ascent. These mtDNA haplogroups are transmitted from mother to offspring and their worlwide distribution permitted to trace the migration of humans outside Africa, raising the concept of a mitochondrial Eve [Pakendorf andStoneking , 2005].
Mitochondrial DNA variants/haplogroups have been associated with differences in physiological processes such as energy production, ageing, regulation of apoptosis or pathogen immune-mediated responses [Gómez-Durán et al., 2010; Chen et al., 2012;Kenney et al., 2014; Krzywanski et al., 2016; Friedrich et al., 2022]. As a consequence, these haplogroups have been associated with adaptation to exercise or susceptibility to develop several traits such as diabetes, cardiovascular disease, or infectious diseases [Castro et al., 2007; Yonova-Doing et al., 2022]. In reference to infections these variants might be associated with the risk of sepsis or the severity of HIV and herpex disease, among others [Hendrickson et al., 2008; Yang et al., 2008; Hart et al., 2013; Levinson et al, 2016 ]. Haplogroups might also play a role in the risk for severe COVID-19, the disease caused by SARS-CoV-2 [Wu et al., 2021; Dirican et al., 2022; Vázquez-Coto et al., 2022; Kumari et al. 2023].
Each cell has a variable number of mitochondria and each mitochondria contains several copies of the mtDNA. For a particular nucleotide position, the mitochondria from each individual may exhibit the same variant (homoplasmy) or different alleles (heteroplasmy) [Santos et al., 2008; Li et al., 2010;Klütsch et al., 2011]. Heteroplasmy is commonly inherited from the mother and for disease-related variants the degree of heteroplasmy in the different tissues determines the extent of the symptoms. Rare highly penetrant mutations cause monogenic disorders that often affect the nervous system, muscles, heart, and endocrine organs, and many healthy individuals carry low levels of heteroplasmy (<1% of mtDNA with the mutation) either inherited from the mother or acquired. An increased burden of heteroplasmy contributes to increased risk for diseases such as MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes), diabetes mellitus, and others [Avital et al., 2012; Folmes et al., 2013; Chae et al., 2020].
Heteroplasmy is common in poly-cytosine tracts in the mtDNA control region [Bendall et al., 1995; Lagerström-Fermér et al., 2001; Shin et al., 2006; Zhao et al., 2010;Mueller et al., 2011; Shen et al., 2015]. One of these is located between nucleotides 16184-16193, that contains the origin for replication of the mtDNA heavy (H) chain. These poly-C tracts are prone to length instability that would increase the risk of mtDNA loss and impairment of mitochondrial regulated processes [Chiaratti et al., 2022]. Among others, the T16189C mtDNA polymorphism increases the risk for poly-C instability and has been associated with diabetes, cancer, and coronary artery disease (CAD), among other diseases [ Zhao et al., 2010; Mueller et al., 2011; Shen et al., 2015]. Length heteroplasmy might be at low level in resting while increases in cells subjected to extensive division, such as the immune cells under chronic inflammation. Increased heteroplasmy might thus represent a marker of a deleterious immune-response that would increase the risk of developing severe infectious diseases [Stefano et al., 2022; Ren et al., 2020; Elesela et al., 2021; Li et al., 2021].
Due to the overactivation of the immune system among individuals infected by SARS-CoV-2 we hypothesised that blood leukocytes from patients with severe COVID-19 might exhibit a different profile of poly-C heteroplasmy. To address this issue we characterised the mtDNA region containing the 16184-16193 poly-C tract in patients with critical COVID-19 and age-matched population controls.