key: cord-0758494-tzzoa4at authors: Dirican, Ebubekir; Savrun, Şeyda Tuba; Aydın, İsmail Erkan; Gülbay, Gonca; Karaman, Ülkü title: Analysis of mitochondrial DNA cytochrome‐b (CYB) and ATPase‐6 gene mutations in COVID‐19 patients date: 2022-03-22 journal: J Med Virol DOI: 10.1002/jmv.27704 sha: 7bd93d395dcdbb951a6a690c6b9e465933952e26 doc_id: 758494 cord_uid: tzzoa4at Coronavirus disease of 2019 (COVID‐19) is a pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Mutations of mitochondrial DNA (mtDNA) are becoming increasingly common in various diseases. This study aims to investigate mutations in the cytochrome‐b (CYB) and adenosine triphosphatase‐6 (ATPase‐6) genes of mtDNA in COVID‐19 patients. The association between mtDNA mutations and clinical outcomes is investigated here. In the present study, mutations of the mtDNA genes CYB and ATPase‐6 were investigated in COVID‐19 (+) (n = 65) and COVID‐19 (−) patients (n = 65). First, we isolated DNA from the blood samples. After the PCR analyses, the mutations were defined using Sanger DNA sequencing. The age, creatinine, ferritin, and CRP levels of the COVID 19 (+) patients were higher than those of the COVID‐19 (−) patients (p = 0.0036, p = 0.0383, p = 0.0305, p < 0.0001, respectively). We also found 16 different mutations in the CYB gene and 14 different mutations in the ATPase‐6 gene. The incidences of CYB gene mutations A15326G, T15454C, and C15452A were higher in COVID‐19 (+) patients than COVID‐19 (−) patients; p < 0.0001: OR (95% CI): 4.966 (2.215−10.89), p = 0.0226, and p = 0.0226, respectively. In contrast, the incidences of A8860G and G9055A ATPase‐6 gene mutations were higher in COVID‐19 (+) patients than COVID‐19 (−) patients; p < 0.0001: OR (95%CI): 5.333 (2.359−12.16) and p = 0.0121 respectively. Yet, no significant relationship was found between mtDNA mutations and patients' age and biochemical parameters (p > 0.05). The results showed that the frequency of mtDNA mutations in COVID‐19 patients is quite high and it is important to investigate the association of these mutations with other genetic mechanisms in larger patient populations. The coronavirus disease of 2019 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a serious public health threat globally, endangering millions of people in a growing number of countries. 1 COVID-19 began in the Chinese city of Wuhan and has since extended to almost all countries of the world. 2 Many recent studies have described the epidemiological and clinical characteristics of symptomatic patients infected with SARS-CoV-2 remains largely unknown. 2 SARS-CoV-2 causes numerous cellular and systemic events that significantly impact the intracellular and extracellular mitochondrial activities and can lead to disease progression and severity. 3 Mitochondria play an essential role in the host's response to viral infection and immunity, which is the key to antiviral signaling and exacerbating inflammatory processes. Mitochondria have been identified as potential targets in SARS-CoV-2 infection. 4 The relationship between mitochondrial DNA (mtDNA) damage and COVID-19 infection is based on oxidative damage. mtDNA is vulnerable and exposed to oxidative stress as a result of metabolic function. In infection, increased reactive oxygen species (ROS) production can affect cell organelles including mitochondria. 5 Mitochondria are crucial for cellular energy production. 6 mtDNA is a closed circular molecule that encodes 13 polypeptides which form oxidative phosphorylation complexes in humans. 7 mtDNA mutations and deletions are associated with oxidative stress, mitochondrial malfunction, and cell death. 7 mtDNA mutations can be occasional, genetic, or Mendelian in nature. Moreover, they can include mtDNA rearrangements such as deletions, inversions, or duplications, as well as point mutations. 8 mtDNA damage plays a key role in human aging, cancer, and neurological disorders. Point mutations of single bases or deletions of the 16.5-kb mitochondrial genome are the leading footprints of mtDNA damage. 9 Because the human mitochondrial genome is so tiny compared with the nuclear genome, mitochondrial genetics poses unique clinical and research questions. 10 Mitochondrial ATPase 6 (mt-ATP6) is a component of ATP synthase, a large enzyme that catalyzes the last stage of oxidative phosphorylation and is encoded by the mitochondrial genome. 11,12 mtDNA encodes three subunits of these complexes (I, III, and IV). The mitochondrial cytochrome-b (mt-CytB) gene encodes the mt-CytB protein, which is the only component of the respiratory complex III encoded by the mitochondrial genome that plays a key role in the electron transport system. 13 Thus, the present study was performed to evaluate mitochondrial cytochrome-b (CYB) and ATPase-6 gene mutations in COVID-19-positive and -negative patients. We also investigated the association between these gene mutations and the clinical biochemical demographic features in COVID-19 patients. The Eco→Tech DNA isolation kit (Cat no: EcoBGD-50x) was used to isolate genomic DNA from blood. For DNA isolation, 200 µl of blood was used for each sample and the isolation was successfully performed by following the protocol recommended by the manufacturer of the corresponding kit. First, 200 µl of EcoSpin Lysis Buffer was added to each 200 µl whole-blood sample and mixed well. Then, 20 µl RNase A was added to the mixture from Step 1 and mixed well, which was incubated for 3 min at room temperature. Next, 20 µl Proteinase K was added to the mixture and mixed well, which was incubated for 10 min at 55°C. Then, 400 µl EcoSpin Binding Buffer was added and mixed well. After the washing and elution steps, the isolation was successfully completed. The NanoDrop instrument Take3 Plate (BioTek) was used to measure the concentration of the DNA samples. The absorption ratios at 260 and 280 nm were used to evaluate the DNA purity. A ratio of approximately 1.8 is universally considered "pure" for DNA. All of the DNA samples were stored at −20°C before PCR. The SensoQuest Labcycler device (thermalcycler) was used for the PCR stage. For each PCR reaction, 25 µl of EcoTaq 2× PCR Master Mix, 2 µl of forward primer (10 µM), 2 µl of reverse primer (10 µM), 10 pg −500 µg template DNA, and ddH 2 O were used. Preoptimized primers were preferred 14 (Table 1) . PCR conditions were set as follows: 5 min at 95°C, 2 min at 94°C, 1 min at 61°C, 2 min at 72°C, 10 min at 72°C, and pause at 4°C for CYB and ATPase-6 genes. After PCR analysis, PCR products were run with 3 µl of ethidium bromide on a 2% gel. A 50 bp marker was used. Bands of 675 and 1064 bp amplified for CYB and ATPase-6 genes, respectively, were visualized in UV light. The ABI3500 (Applied Biosciences) instrument was used for DNA sequencing. Before performing sequence analysis, the PCR products were cleared with exoSAP. After Sanger sequencing, analysis was performed using MITOMAP and Chromas Lite 2.1 (Technelysium) software. We used seven bioinformatics tools, Polymorphism Phenotyping v2 The program GraphPad Prism 7.04 was used for all of the statistical analyses. The normal distribution of the data was demonstrated by the Shapiro−Wilk normality test. We used mean ± SD to describe the normally distributed variables. The association between COVID-19 (+) and COVID-19 (−) patients and clinical parameters was examined using the Mann−Whitney U test. A χ 2 test was conducted to calculate the association between the mutation types. The association between mutations and clinical parameters was also examined using the Mann−Whitney U test. Values for p < 0.05 were accepted as statistically significant. Table 2 . In all COVID-19 (+) and COVID-19 (−) patients included in the study, the mtDNA CYB and ATPase-6 genes were amplified by PCR. The 1064 bp (CYB) and 675 bp (ATPase-6) PCR products were analyzed in a 2% agarose gel ( Figure 1A , B). The human mitochondrial genome sequence used to identify mutations was "Cambridge Reference Series" (http://www.mitomap. org) and analyzed using Chromas Lite software. The sequence analysis of the detected mutations is shown in Table 3 . Sixteen (Table 3) . A15326G and A8860G mutations were the most frequent mutation types in both the COVID-19 patient and control groups. We used seven different in silico variants prediction tools, PolyPhen-2, PANTHER, SIFT, PROVEAN, Mutation Assessor, SNAP, and CADD, to predict the functional effects of the variants of CYB and ATPase-6 genes. As a result of the in silico analysis, we found that 13 missense types (nonsynonymous substitution) were found to have various effects on diseases (Table 4 ). G15431A and T15674C were predicted to be deleterious variants in the CYB gene by in silico programs. Moreover, G9055A, A8836G, and A8860G were predicted to be deleterious variants in the ATPase-6 gene. The age, hematological, and biochemical test results of the samples with and without (wild-type) CYB and ATP mutations are shown in Mutations in the human mitochondrial genome are linked to several diseases, most of which are inherited from the mother and all of which are related to abnormalities in oxidative energy metabolism. 15 These diseases are currently incurable and virtually untreatable and have a wide range of penetrance, symptoms, and prognosis. 16 mtDNA mutations have been detected in many body fluids, including urine and saliva 17 and serum. 18 Mutations in OXPHOS mtDNA genes do not always result in alterations in the encoded protein. 19 Viruses affect mitochondrial function, metabolism, and innate immune signaling. 20 Metabolism, calcium regulation, airway contractility in the lung, gene and protein balance, oxidative stress, and apoptosis are all affected by mitochondrial dysfunction. Mitochondrial dysfunction affects homeostatic cellular processes such as aging and senescence. 21 Mitochondria are proving to be significant in COVID-19 pathogenesis due to their function in innate antiviral immunity and inflammation. 22 To the best of our knowledge, no previous research has investigated the association between CYB and ATPase-6 gene 29 Although the heterogeneity of the patients does not reflect the results confirmed by the literature, we can say that the laboratory findings were related to the severity of disease in our patients. F I G U R E 1 ATPase-6 and CYB gel electrophoresis image More mutations occur in mtDNA than in nuclear DNA, and a correlation has been found between ROS increase and the agerelated increase in mutant mtDNA. 30 Considering that SARS-CoV-2 indirectly produces the production of ROS, the cells of elderly people might be exposed to more ROS than those of healthy younger people when infected with this virus. 5 (+) patients had A8860G missense mutation in the ATPase-6 gene. In another study, the frequencies of transversions in the ATP6 and CytB genes were found to be 96% and 97%, respectively, while the frequency of transversions in the ATP6 gene was found to be 4% and 3% in the CytB gene. 13 Pirola et al. 36 Moreover, G9055A, A8836G, and A8860G were predicted to be deleterious variants in the ATPase-6 gene by in silico programs. Our study indicates that these variants may change the secondary structure of the CYB and ATPase-6 proteins and subsequently can reduce their enzyme activity. The alteration of the enzyme activity can affect ROS and disease severity. In addition, these data suggest that more studies will need to be conducted to reveal the effects of these A8860G and A15326G mtDNA mutations in COVID-19 disease. The current study has some limitations that should be mentioned. We examined CYB and ATPase-6 mutations and clinical parameters of patients with COVID-19. First, we were unable to analyze other mtDNA genes (ND1 and D310). Second, we did not have the opportunity to work with many patients. Therefore, additional research and study with larger patient populations are required to substantiate our findings and demonstrate their relevance. To the best of our knowledge, there is no publication in the relevant literature that focuses on the association between CYB and ATPase-6 mutations and COVID-19 patients. As this is a pilot study, more data on mtDNA variants need to be collected to highlight the association between known mitochondrial variants and COVID-19 patients. The high prevalence of mtDNA mutations in COVID-19 patients suggests that they play a key role in the disease and alter patients' energy metabolism. 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