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\begin{document}

\title{Mitochondrial DNA Poly-C length heteroplasmy as a marker for risk of
critical COVID-19}



\author[1]{Eliecer Coto}%
\author[1]{Daniel Vázquez-Coto}%
\author[1]{Guillermo M. Albaiceta}%
\author[1]{Laura Amado-Rodríguez}%
\author[1]{Mar González-Fernández}%
\author[1]{Claudia García-Lago}%
\author[1]{Lucinda Velázquez-Cuervo}%
\author[1]{Elías Cuesta-Llavona}%
\author[1]{Juan Gómez}%
\affil[1]{Hospital Universitario Central de Asturias}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Mitochondria play a central role in the innate and acquired response
against viral infections. Common mtDNA variants have been associated
with severe COVID-19 and mtDNA depletion. A poly C length variation has
been associated with mtDNA instability and increased risk for several
diseases. We studied 482 patients who required treatment in the
intensive care unit and age matched population controls. The 16184-16193
poly-C and 514-523 CA-repeats were determined by fluorescent capillary
electrophoresis and Sanger sequencing of PCR fragments. We found a
significantly higher frequency of 16184-16193 mtDNA poly-C heteroplasmy
in patients aged [?]60 compared to patients aged \textgreater{}60 years.
Poly-C heteroplasmy did not differ between the age control groups.
Poly-C heteroplasmy was associated with the presence of the 16223 T
allele, that was associated with the risk of critical COVID-19 at [?]60
years. In Conclusion, heteroplasmy in the poly-C tract of the mtDNA
control region might be a marker for critical COVID-19. The 16184-16193
heteroplasmy was linked to the 16223 T allele, that was significantly
increased among patients aged [?]60 years. This finding requires
validation in other cohorts and to determine the functional link between
length variation in the mitochondrial DNA control sequence and risk of
severe SARS-CoV-2 disease.%
\end{abstract}%



\sloppy


\textbf{Mitochondrial DNA Poly-C length heteroplasmy as a marker for
risk of critical COVID-19}

Daniel V\selectlanguage{ngerman}ázquez-Coto\textsuperscript{1}, Guillermo M.
Albaiceta\textsuperscript{2,3,4,5,6}, Laura
Amado-Rodríguez\textsuperscript{2,3,4,5,6}, Mar
González-Fernández\textsuperscript{1}, Claudia
García-Lago\textsuperscript{1,3}, Lucinda
Velázquez-Cuervo\textsuperscript{1,3}, Elías
Cuesta-Llavona\textsuperscript{1,3}, Juan Gómez\textsuperscript{1,3,5},
Eliecer Coto\textsuperscript{1,3, 4}

\textsuperscript{1}Genética Molecular, Hospital Universitario Central
Asturias, Oviedo, Spain.

\textsuperscript{2}Unidad de Cuidados Intensivos Cardiológicos, Hospital
Universitario Central Asturias, Oviedo, Spain.

\textsuperscript{3}Instituto de Investigación Sanitaria del Principado
deAsturias, ISPA, Oviedo, Spain.

\textsuperscript{4}Universidad de Oviedo, Oviedo, Spain.

\textsuperscript{5}CIBER-Enfermedades Respiratorias. Instituto de Salud
Carlos III. Madrid, Spain.

\textsuperscript{6}Instituto Universitario de Oncología del Principado
de Asturias. Oviedo, Spain.

\textsuperscript{7}Neumología, Hospital Universitario Central Asturias,
Oviedo, Spain.

\textbf{Corresponding author:}

Eliecer Coto, PhD

Genética Molecular-HUCA, Oviedo, Spain

\emph{eliecer.coto@sespa.es}

\textbf{Abstract.}

Mitochondria play a central role in the innate and acquired response
against viral infections. Common mtDNA variants have been associated
with severe COVID-19 and mtDNA depletion. A poly C length variation has
been associated with mtDNA instability and increased risk for several
diseases. We studied 482 patients who required treatment in the
intensive care unit and age matched population controls. The 16184-16193
poly-C and 514-523 CA-repeats were determined by fluorescent capillary
electrophoresis and Sanger sequencing of PCR fragments. We found a
significantly higher frequency of 16184-16193 mtDNA poly-C heteroplasmy
in patients aged \selectlanguage{english}[?]60 compared to patients aged \textgreater{}60 years.
Poly-C heteroplasmy did not differ between the age control groups.
Poly-C heteroplasmy was associated with the presence of the 16223 T
allele, that was associated with the risk of critical COVID-19 at \selectlanguage{english}[?]60
years.

In Conclusion, heteroplasmy in the poly-C tract of the mtDNA control
region might be a marker for critical COVID-19. The 16184-16193
heteroplasmy was linked to the 16223 T allele, that was significantly
increased among patients aged \selectlanguage{english}[?]60 years. This finding requires
validation in other cohorts and to determine the functional link between
length variation in the mitochondrial DNA control sequence and risk of
severe SARS-CoV-2 disease.

\textbf{Key words:} COVID-19; mitochondria; haplogroups; heteroplasmy;
ageing

\textbf{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 {[}\textbf{Lee} et al.,
1998;\textbf{Wallace} 2010; \textbf{Bratic and Larsson} , 2013;
\textbf{Ziada}et 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 {[}\textbf{Wilson} and
Allard, 2004; \textbf{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 \emph{mitochondrial Eve} {[}\textbf{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
{[}\textbf{Gomez-Duran} et al., 2010; \textbf{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;
\textbf{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 {[}\textbf{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 {[}\textbf{Wu} et al., 2021;
\textbf{Dirican et al., 2022; Vazquez-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)
{[}\textbf{Santos} et al., 2008; \textbf{Li} et al.,
2010;\textbf{Klutsch} 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
(\textless{}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 {[}\textbf{Avital} et al., 2012; \textbf{Folmes} et
al., 2013; \textbf{Chae} et al., 2020{]}.

Heteroplasmy is common in poly-cytosine tracts in the mtDNA control
region {[}\textbf{Bendall} et al., 1995; \textbf{Lagerstrom-Fermer} et
al., 2001; \textbf{Shin} et al., 2006; \textbf{Zhao} et al.,
2010;\textbf{Mueller} et al., 2011; \textbf{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
{[}\textbf{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 {[} \textbf{Zhao} et al., 2010; \textbf{Mueller} et
al., 2011; \textbf{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 {[}\textbf{Stefano} et al., 2022; \textbf{Ren} et
al., 2020; \textbf{Elesela} et al., 2021; \textbf{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.

\textbf{2. Patients and Methods.}

\textbf{Study participants.} We obtained the demographic and clinical
data of 482 COVID-19 patients (age range 24-95 years) who required
admission in the intensive care unit (ICU). The study was approved by
the Ethics Committee of Principado de Asturias (Oviedo, Spain; approval
id project ISCIII-PI21/00971), and all the patients (or their next of
kin) gave informed consent to participate in the study. These patients
were hospitalised between March-2020 and December-2021, a period with
four SARS-CoV-2 pandemic waves in our community. We did not determine
the SARS-CoV-2 variant in all the patients, but the study period was
characterised by the Wuhan (pandemic waves 1-2), alpha (wave 3) and
delta (wave 4) variants.

The inclusion criteria was a severe pneumonia in need for ICU admission
with SARS-CoV-2 confirmed by PCR test, and exclusion criteria were age
\textless{}18, respiratory failure due to condition other than COVID-19,
or refusal to participate. These patients were followed till disease
remission with hospital discharge or death. Body-mass index (BMI) and
preexisting cardiovascular comorbidities (hypertension, diabetes,
hypercholesterolemia) were obtained from the clinical history at ICU
admission. Based on previous reports we compared COVID-19 patients aged
\selectlanguage{english}[?]60 and \textgreater{}60 years {[}\textbf{Nakanishi} et al., 2021{]}. At
the time of inclusion, none of the patients had a recorded diagnostic of
mitochondrial disease.

All the participants were of European ancestry from the region of
Asturias (Northern Spain, total population 1 million). The controls
(N=363) were individuals from the general population recruited with the
main purpose of defining the DNA variant frequencies. These controls
were not serologicaly studied (presence of anti-SARS-CoV-2 antibodies)
to exclude previous asymptomatic infection. In order to exclude the
posibility of age-bias in the genotype frequencies we compared patients
and controls within the same age-range.

\textbf{Genetic analysis.} Patients and controls were studied for mtDNA
single nucleotide polymorphisms (SNPs) C7028T and C16223T by Sanger
sequencing of PCR fragments. The 7028 polymorphism differentiates the
major European haplogroup H (7028C) from no-H (7028T), while 16223
differentiates the ancestral African macro-haplogroup N (16223T) from
the out-of-Africa macro-haplogroup R (16223C) (\emph{www.mitomap.org)} .
This method allowed to determine the presence of heteroplasmy at the
7028 and 16223 nucleotides.

To define the degree of length heteroplasmy at the 16184-16193 poly C
tract we performed a fluorescent capillary electrophoresis of PCR
fragments generated with primers FAM-5'CTGCCAGCCACCATGAATATTGTACGG and
5'GTGGCTTTG GAGTTGCAGTTGATGTGTGA (annealing at 65oC). The forward primer
was labelled with FAM and fragments were thus visualised as fluorescent
peaks after capillary electrophoresis (\textbf{Figure 1A} ). Homoplasmic
fragments corresponded to single peaks while the presence of additional
peaks was considered as heteroplasmy. We also characterised the degree
of heteroplasmy at the CA-repeat between nucleotides 514-523 by
fluorescent capillary electrophoresis of PCR fragments with primers
fam-5' CACTTTTAACAGTCACCCCCCAACTAAC and 5'TTCGGGGTATGGGGTTA GCAGCG
(annealing at 65oC) (\textbf{Figure 1B} ). To discrimitate true from PCR
stutter peaks (characteristic of amplicons containing nucleotide
repeats) we condidered heteroplasmy when the additional peaks had a
heigh \textgreater{}10\% relative to the main fluorescent peak
{[}\textbf{Walsh et al., 2016{]}} . The accuracy of this method was
validated by sequencing PCR fragments with different degrees of
heteroplasmy (\textbf{suppl figure} ).

\textbf{Statistical analysis.} All the values were collected in an
excell file. The statistical analysis was performed with the R-software
(\emph{www.r-project.org}). Logistic regression (linear generalized
model, LGM) was used to compare mean values and frequencies between the
groups.

\textbf{3. Results.}

In \textbf{table 1} we show the main characteristics of the critical
COVID-19 patients aged \selectlanguage{english}[?]60 years or older. We found a higher frequency
of male in the two age groups. As expected the frequency of
hypertensives, diabetics, and hyperlipaemics was higher among the
patients \textgreater{}60 years. There were a total of 103 deaths
(\textbf{table 1} ). The frequency of mtDNA 7028C that characterises
haplogroup H was significantly lower in the \selectlanguage{english}[?]60 years patients (38\% vs
48\%). The frequency of 16223T was significantly higher in the \selectlanguage{english}[?]60 years
group (18\% vs 10\%). The 7028C and 16223T did not differ between the
two age control groups (\textbf{table 2} ). Thus, we concluded that the
difference between pagtients aged \selectlanguage{english}[?]60 and \textgreater{}60 years would
not be a consequence of a survival-effect in the general population, and
these mtDNA markers could thus represent risk factors for age-dependent
critical COVID-19.

\textbf{Control region length heteroplasmy.} Fluorescent capillary
electrophoresis of the amplified fragments showed multiple peaks
(heteroplasmy) at the 16184-16193 poly-C tract significantly more
frequent among the \selectlanguage{english}[?]60 years patients compared to age matched controls
(19\% vs 9\%; p=0.002). Heteroplasmy was also more common among the \selectlanguage{english}[?]60
years compared to the older patients (19\% vs 10\%; p=0.02), without
differences between patients and controls \textgreater{}60 years
(\textbf{table 2} ). We found a maximum frequency of length heteroplasmy
among patients aged \textless{}50 years, suggesting that this
mitochondrial genomic event was significantly associated with critical
COVID-19 at younger age (\textbf{Figure 2; suppl. Table 1} ).

The 16184-16193 poly-C tract that increases length heteroplasmy has been
associated with several multifactorial disorders, including metabolic
and cardiovascular. Because critical COVID-19 was significantly
associated with hypertension, diabetes, dyslipidaemia, and male sex, we
determined whether length heteroplasmy was higher among patients with
these conditions. We did not find statistical differences between the
groups (\textbf{suppl. table 2} ). We also compared death and survivors,
without significant difference. Moreover, after correcting by multiple
variables advanced age was the only significantly associated with the
risk of death (p\textless{}0.001).

We also determined the frequencies of the control region CAn-repeat. We
did not find significant differences between patients and controls in
the two age-groups. Heteroplasmy at this region was found in 3-6\% of
the study cohorts, without significant difference between the groups.

\textbf{Poly-C heteroplasmy was linked to 16223 T.} We found a higher
frequency of poly-C heteroplasmy among patients 16223 T compared to
16223 C, in the two age groups (\textbf{suppl. table 3} ). The 16223 T
is the ancestral African allele and is present in less than 10\% of
current Europeans with the rare IXW haplogroups (\textbf{suppl. table 4}
). This pointed to the possibility that the higher frequency of 16189 T
and 162184-16193 poly-C heteroplasmy were a consequence of the linkage
between 16223 T and 16189 C, a variant that increases the risk of poly-C
instability (\textbf{suppl. table 5} ).

\textbf{4. Discussion.}

The main finding of our study was a significantly increased frequency of
length heteroplasmy at the poly-C tract in the mtDNA region that
contains the replication start site of the H strand. This sequence,
between nucleotides 16184-16193, contains a poly-C tract interrupted by
C\textgreater{}T changes, and the presence of C could results in
replication instability with multiple poly-C sequences.

The poly-C heteroplasmy might be inherited from the mother with cellular
heterogeneity between different tissues in the same individual. In
addition, the degree of heteroplasmy might increase with age or under
exposure to environmental toxics, drugs, bacterial and viral infections,
among others. An increased length heteroplasmy at this poly-C region has
been reported among cancer, diabetes and coronary artery disease
patients {[}\textbf{Zhao} et al., 2010; \textbf{Mueller} et al.,
2011;\textbf{Shen} et al., 2015{]}. This associations might be explained
by a mechanism that links the poly-C tract with an impaired replication
of the mtDNA that could result in a reduction of the number of mtDNA
copies and the loss of mitochondrial functions.~In a study with blood
samples from 837 healthy adults \textbf{Liou} et al. found that the
number of mtDNA copies was significantly reduced among individuals with
uninterrupted poly-C {[}\textbf{Liou} et al, 2010{]}. Thus, variants
that increased the amount of length heteroplasmy might cause alteration
of mtDNA copy number in human blood cells compared to T-interrupted
tracts {[}\textbf{Amo} et al., 2017{]}. Interestingly, length
heteroplasmy at the control region was also associated with
significantly lower copy number of mtDNA in leukocytes from breast
cancer patients {[}\textbf{Zhao et al., 2010} {]}.

The number of mitochondria and mtDNA copies vary between cells and
tissues and might be critical for a proper physiological response to
situations that require a high energy demand and other
mitochondrial-mediated responses {[}\textbf{Stefano} et al.,
2022;\textbf{He} et al., 2010; \textbf{Stefano} et al., 2017{]}. A high
degree of heteroplasmy might impairs the maintenance of ATP levels in
response to strong physiological demand, such as the required by the
immunological cells after infections {[}\textbf{Koshiba}
2013;\textbf{Moore and Ting} , 2008; \textbf{Schilf} et al.,
2021;\textbf{Shenoy} 2020{]}. The effect of heteroplasmy in mtDNA copy
number and mitochondrial function has been validated in mouse strains
with different mtDNA variants {[}\textbf{Hu et al., 2019} {]}. These
engineered mice showed a non-random segregation of mtDNA copy numbers
that suggested a pressure to reduce the degree of heteroplasmy when this
was detrimental for covering the cellular demands. Situations that
increase the degree of heteroplasmy, such as aging, exposure to
environmental compounds, infections, etc, might result in an impaired
capacity to respond through mitochondrial pathways, increasing the risk
for disease.

Our study was based on whole blood cells and would thus be
representative of the degree of heteroplasmy among leukocytes in a
disease (COVID-19) characterised by an exacerbated inflammatory response
with over-activation of immune-cells to respond to the infection. These
cells are thus under strong metabolic demand that would require an
increase in the number of mitochondria and mtDNA copies. It is tempting
to speculate that a similar dynamic of mtDNA length heteroplasmy occurs
in other cells and tissues such as lung, brain, heart. This possibility
might be investigated in the context of persistent (long-COVID)
symptoms.

The role of mitochondria in the pathophysiology of acute and chronic
inflammation has been extensively studied. Immune-competent cells such
as monocytes, macrophages, antigen-presenting cells, are under
programmed changes in mitochondrial bioenergetics in response to innate
and adaptive immunological processes {[}\textbf{Angajala} et al.,
2018;\textbf{Zuo} et al., 2019{]}. The degree of mtDNA length
heteroplasmy might contribute to define the extent of these
immune-mediated responses, making some individuals more susceptible to
an impaired capacity to fight infection or to regulate the extent of the
proinflammatory stimuli {[}\textbf{Lechuga-Vieco} et al.,
2020;\textbf{Pollara} et al., 2018; \textbf{Zhu} et al., 2018{]}.
Several studies have shown the capacity of viruses, including
SARS-CoV-2, to hijack oxidative phosphorylation, ATP production, and
other mitochondrial functions {[}\textbf{Stefano} et al.,
2022;\textbf{Stefano} et al., 2020{]}. Key mitochondrial mediated
processes might be also affected by viral proteins that reduced the
antiviral innate immune responses, thus promoting the extent of
infection and disease severity {[}\textbf{Chen} et al, 2007;
\textbf{Yoshizumi} et al., 2014; \textbf{Choi} et al., 2018{]}. Some
authors have suggested that SARS-CoV-2 infection and replication was
improved by the takeover of mitochondrial processes, and viral proteins
would suppress mitochondrial functions reducing the innate and adaptive
immune responses {[}\textbf{Singh} et al., 2020{]}. In this context,
heteroplasmy at the poly-C tract might reduce the individual's capacity
to exhibit a proper mitochondria mediated immune response increasing the
risk for severe COVID-19.

A functional link between 16189C and increased risk for disease has been
suggested by some authors. Park et al. identified 16189C as a risk
factor for diabetes and used chromatin immunoprecipitation in cybrid
cells to identify the mitochondrial single-stranded DNA-binding protein
(mtSSB) as a candidate protein bound to the 16189 region
{[}\textbf{Park} et al., 2008{]}. MtSSB has a lower binding affinity for
the 16189C variant and because this nucleotide lies in the control
region of mtDNA replication and transcription the variant might affect
mtDNA replication and recovery after mitochondrial damage
{[}\textbf{Takamatsu} et al., 2002; \textbf{Park} et al., 2008{]}. In
this context, several studies have reported increased circulating mtDNA
as a marker of COVID-19 severity and mortality {[}\textbf{Scozzi} et
al., 2021; \textbf{Valdes-Aguayo} et al., 2022; \textbf{Streng} et al.,
2022{]}. Studies to determine whether mtDNA variants and leght
heteroplasmy were associated with reduced number of mtDNA copies and
increased circulating mtDNA would be of special interest.

A limitation of our study was the absence of over-time comparison of the
degree of poly-C heteroplasmy within the same individual. Heteroplasmy
is commonly inherited and could thus be present in all the individual's
cells, but it is well known that de degree of heteroplasmy might
increase with age or in response to environmental exposures. It is
possible that SARS-CoV-2 proteins that target the mitochondria enhance
the miss-replication of mtDNA promoting impairment of innate and
acquired immunity and increasing the risk for critical COVID-19. To
verify this hypothesis it is necessary to determine the degree of
heteroplasmy before and after infection.

Finally, the risk of developing severe COVID-19 has been associated with
some mitochondrial haplogroups {[}\textbf{Wu} et al.,
2021;\textbf{Vazquez-Coto} et al., 2022{]}. We previously reported a
decreased frequency of the common European haplogroup H (7028 C) that
could be protective for critical disease at younger age. In addition,
16223 T might be associated with an increased risk of severe disease.
Interestingly, 16189 C is more common among individuals with haplogroups
with 16223 T (such as the European X) than among haplogroup H
(characterised by 16223 C) (\textbf{supplementary table} )
{[}\textbf{Laricchia} et al., 2022{]}. Thus, the association of
haplogroups with several diseases might be explained by its linkage to
16189 C and other variants that could increase the risk for poly-C
length heteroplasmy.

In conclusion, we report an increased frequency of poly-C length
heteroplasmy at the mtDNA control region that contains the replication
start site of the H-strand among patients with critical COVID-19. Length
heteroplasmy at this region has been associated with a higher risk of
cancer, diabetes, coronary artery disease, and viral diseases, among
others. The poly-C heteroplasmy could increase mtDNA instability and
reduction of the mtDNA copies per cell, that might contribute to
manifest an exacerbated inflammatory response and impaired activation of
the innate and acquired immunological response to viral infection.
Further research to confirm the association are necessary, as well as
functional studies to uncover the linkage between mtDNA length
heteroplasmy and viral disease.

\textbf{Acknowledgements.} This work was supported by a grant from the
Spanish Plan Nacional de I+D+I Ministerio de Economia y Competitividad
and the European FEDER, grants ISCIII-PI21/00971 (E.C.), RICOR2040-
RD21/0005/0011 (E.C.), and PI22/00705 (J.G.).

\textbf{Competing interests.} None of the authors have competing
interests related to this work.

\textbf{AUTHOR CONTRIBUTIONS} . \textbf{Lead researchers} : EC, GMA,
JG;\textbf{study design} : EC, GMA, JG; \textbf{patient assessment and
data acquisition} : GMA, LAR, MGC, ECLL, JG; \textbf{database:} DVC, EC,
GMA, JG; \textbf{genotyping:} DVC, CGL, MGF, LVC, EC; \textbf{data
filtering and analysis:} EC, DVC; \textbf{statistical analysis:} EC,
DVC;\textbf{analysis of results:} EC, DVC; \textbf{writing the
manuscript:}EC; \textbf{revision of manuscript:} all the authors.

\textbf{ETHICS AND CONSENT.} This study was approved by the clinical
research ethics committee of Hospital Universitario Central Asturias
(HUCA) (project approval id ISCIII-PI21/00971). All the participants or
they next of kin gave written or verbal consent. Data were handled in
observance of Spanish legislation on data protection. The study complies
with the principles of the Declaration of Helsinki (``Recommendations
guiding doctors in biomedical research involving human subjects'').

\textbf{DATA AVAILABILITY STATEMENT} . The data that support the
findings of this study are available from the corresponding author upon
reasonable request. An Excel file with the raw data would be available
for meta-analysis research.

\textbf{Table 1.} Main values in the critical COVID-19 patients aged \selectlanguage{english}[?]60
years and older.

.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\textbf{[?]60 years} \textbf{N=164}\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\textbf{\textgreater{}60 years} \textbf{N=318}\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
p-value\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\textbf{Male} \textbf{Female}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
118 (72\%) 46 (28\%)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
235 (73\%) 83 (27\%)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
0.85\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{Age Median years} \textbf{(range)}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
53 (18-60)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
71 (61-92)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\textbf{BMI median} \textbf{(range)}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
29(19-54)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
31(19-55)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
0.01\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{BMI \textgreater{}30}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
69 (42\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
151 (47\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.01\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{Diabetes}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
21 (13\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
76 (24\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.005\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{Hypercholesterolemia}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
54 (33\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
162 (51\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
0.0004\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{Hypertension}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
66 (40\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
203 (64\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{Death}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
17 (10\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
86 (28\%)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textless{}0.0001\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2.} Frequency of the mtDNA variants in the critical
COVID-19 patients and population controls. All them were genotyped for
the 7028 variant (C=haplogroup H) and the 16223 variant (T=haplogroup N;
C=haplogroup R).\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{[?]60 years}\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{[?]60 years}\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{\textgreater{}60 years}\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{\textgreater{}60 years}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Covid-19} \textbf{N=164}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Controls} \textbf{N=182}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Covid-19} \textbf{N=318}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Controls} \textbf{N=181}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{7028 C (H)}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
62 (38\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
86 (47\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
153 (48\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
82 (45\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{7028 T (no-H)}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
102 (62\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
96 (53\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
165 (52\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
99 (55\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{p-value (T)} \textbf{OR (95\%CI)}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.11 OR=1.43 (0.92-2.22)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.11 OR=1.43 (0.92-2.22)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.19 OR=0.75 (0.49-1-15)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.19 OR=0.75 (0.49-1-15)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{16223 T (N)}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
27 (16\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
9 (5\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
31 (10\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
9 (5\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{16223 C (R)}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
133 (81\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
170 (93\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
283 (89\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
169 (93\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{16223 T/C} \textbf{Heteroplasmy}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
4 (3\%)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
3 (2\%)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
4 (1\%)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
3 (2\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{p-value (T)} \textbf{OR (95\%CI)}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.002 OR=3.36 (1.49-7.54)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.002 OR=3.36 (1.49-7.54)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.39 OR=1.47 (0.60-3.59)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.39 OR=1.47 (0.60-3.59)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{514-523 CAn} \textbf{CA\textsubscript{4}}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
21 (13\%)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
20 (11\%)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
32 (10\%)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
21 (12\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{CA\textsubscript{5}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
128 (79\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
140 (77\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
258 (81\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
145 (80\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{CA\textsubscript{6}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
6 (3\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
9 (5\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
12 (4\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
7 (4\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{CA\textsubscript{7}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2 (1\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
4(1\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
4 (2\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{CA\textsubscript{heteroplasmy}}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
9 (5\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
11 (6\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
12 (4\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
4 (3\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{16184-16193} \textbf{Heteroplasmy}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
32 (19\%)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
16 (9\%)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
31 (10\%)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
24 (13\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Homoplasmy}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
132 (71\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
176 (91\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
287 (90\%)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
157 (87\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.002 OR=2.67 (1.40-5.06)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.002 OR=2.67 (1.40-5.06)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.67 OR=0.89 (0.51-1.53)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
p=0.67 OR=0.89 (0.51-1.53)\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

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\textbf{Figure 1.} Capillary electrophoresis of the 16184-16193 poly-C
tract (A) and the 514 CAn repeat (B). Heteroplasmy (A2, A3, A4, B3): in
addition to the main alleles all the fragments showed an additional peak
known as DNA polymerase slippage product or stutter band. This peak
would have a height \textless{}10\% relative to the main peak
(\textbf{85} ). To confirm the accuracy of the capillary electrophoresis
we confirmed the homoplasmy/heteroplasmy of PCR fragments by Sanger
sequencing (see supplementary figures).

\textbf{Figure 2.} Frequencies of the poly-C lenght heteroplasmy and
16223 T patients in four age groups. Frequency of poly-C heteroplasmy
and 16223 T were higher in the youngest (\textless{}50 years) group.\selectlanguage{english}
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