Integrative blood-based characterization of oxidative mitochondrial DNA damage variants implicates Mexican American’s metabolic risk for developing Alzheimer’s disease

Alzheimer’s Disease (AD) continues to be a leading cause of death in the US. As the US aging population (ages 65 +) expands, the impact will disproportionately affect vulnerable populations, e.g., Hispanic/Latino population, due to their AD-related health disparities. Age-related regression in mitochondrial activity and ethnic-specific differences in metabolic burden could potentially explain in part the racial/ethnic distinctions in etiology that exist for AD. Oxidation of guanine (G) to 8-oxo-guanine (8oxoG) is a prevalent lesion and an indicator of oxidative stress and mitochondrial dysfunction. Damaged mtDNA (8oxoG) can serve as an important marker of age-related systemic metabolic dysfunction and upon release into peripheral circulation may exacerbate pathophysiology contributing to AD development and/or progression. Analyzing blood samples from Mexican American (MA) and non-Hispanic White (NHW) participants enrolled in the Texas Alzheimer’s Research & Care Consortium, we used blood-based measurements of 8oxoG from both buffy coat PBMCs and plasma to determine associations with population, sex, type-2 diabetes, and AD risk. Our results show that 8oxoG levels in both buffy coat and plasma were significantly associated with population, sex, years of education, and reveal a potential association with AD. Furthermore, MAs are significantly burdened by mtDNA oxidative damage in both blood fractions, which may contribute to their metabolic vulnerability to developing AD.

www.nature.com/scientificreports/different mitochondrial capacity, metabolic health, and comorbidity risk such as those linked to ethnic/racial AD health disparities.
Oxidative transversion substitution mutations may be associated with increased mitochondrial dysfunction due to impaired mitochondrial capacity, metabolic health, and/or lifestyle affecting mitochondrial health, and contribute to the continuous progression of AD until a clinical endpoint, death.In this current study, our objective was to use a blood-based measurement of 8oxoG sSNVs as an indicator of impaired mitochondrial function to investigate the role of mitochondria in pathophysiology of complex disease by (1) characterizing associations to population and sex, (2) highlighting burdened genomic regions that influence mitochondrial function, and (3) determining differences in buffy coat or plasma on evaluating AD risk and/or endophenotype.

Results
Descriptive statistics for the cohort analyzed for cellular mitochondrial 8oxoG variants are displayed in Table 1.As anticipated, MMSE, CDR sum, and years of education in both populations had significantly different means based on cognitive status.Age significantly differed in MAs between cognitive groups and years of education was lower compared to NHWs.
Directly genotyped and imputed APOE frequencies for each population is shown in Supplementary Table 1.Genotype frequencies for APOE and OGG1 by cognitive status in each population are shown in Supplementary Table 2. Hardy-Weinberg proportions for APOE and OGG1 in both populations separately and together indicates Table 1.Descriptive statistics of NHW and MA participants categorized by population and cognitive phenotype in the Texas Alzheimer's Research and Care Consortium for buffy coat mitochondrial DNA oxidative mutational load.NC normal control, MCI mild cognitive impairment, AD Alzheimer's Disease.a The mean difference is significant at 0.05.b NC vs.MCI 0.016, NC vs. AD < 0.001, MCI vs. AD < 0.001.c NC vs.MCI <0.001, NC vs. AD < 0.001, MCI vs. AD < 0.001.d NC vs.MCI 0.003, NC vs. AD 0.042, MCI vs. AD 0.542.e NC vs.MCI 0.028, NC vs. AD < 0.001, MCI vs. AD 0.017.f NC vs.MCI <0.001, NC vs. AD < 0.001, MCI vs. AD < 0.001.g NC vs.MCI <0.001, NC vs. AD < 0.001, MCI vs. AD < 0.001.h NC vs.MCI 0.001, NC vs. AD 0.273, MCI vs. AD 0.540.Evaluation of 8oxoG variant count in the buffy coat of MA and NHW TARCC participants.In the MA population 8oxoG variant count was significantly reduced for subjects reporting depression compared to those without depression; mean = 6.548 and 7.704, respectively (Supplemental Fig. 1).Tobacco abuse demonstrated an approach for significance in association with 8oxoG demonstrating a higher variant load compared to non-smokers in MAs; mean = 7.935 and 7.048, respectively (Supplemental Fig. 2).These trends were not observed in the NHW cohort.Multiple linear regression model predictions in the whole cohort were performed to assess the associations with 8oxoG and to determine if there are predictive interactions.Sex regarding females (P = 0.0007), years of education (P = 0.0055), BMI (P = 0.0288), and tobacco abuse (P = 0.0086) were significantly associated with 8oxoG variant count, and the population-sex interaction demonstrated a significant interaction effect (P = 0.0038) (Supplementary Table 5).Further analysis of 8oxoG variant count in both population and sex via two-way ANOVA indicates population is significantly associated (P < 0.0001), while sex was marginally significant (P = 0.0922).
In the subsequent multiple linear regression model, a diabetes × cognition interaction was evaluated and showed a significant association with population (P < 0.0001), sex (P = 0.0429), years of education (P = 0.0109), BMI (P = 0.0254), and tobacco abuse (P = 0.0155); although the interaction effect was not significant (Supplementary Table 6).Cognitive status with respect to AD displayed a suggestive association compared to controls (P = 0.0556).
Previously derived 8oxoG variant load for each subject corresponding to 8oxoG "hotspots" 24 were further analyzed via multiple linear regression prediction models assessing a population × sex and years of education interaction effect, and a diabetes × cognition interaction effect was less informative than analyzing total 8oxoG variant count (Supplementary Tables 7 and 8).Population stratification lost significant statistical associations (Supplementary Tables 9 and 10) that were observed in Supplementary Table 7 and 8.
Multiple linear regression modelling in MAs indicated that total 8oxoG was significantly associated with cognitive status, sex, years of education, and tobacco abuse (Table 2).BMI did not show significant association in MAs as compared to the regression models investigating interactive effects (Supplementary Tables 5-6).Modelling within NHWs did not demonstrate any associations (Table 3).
Additional prediction modelling used cognitive status as a binary variable to combine the effects of AD and MCI compared to NCs and showed similar results to the models with greater resolution on cognitive status (Supplementary Tables 11-18).
Assessment of ccf-mtDNA 8oxoG variant count in MA and NHW TARCC participants.The subset of participants included for the ccf-mtDNA 8oxoG variants were selected from subjects included in the buffy coat analysis to compare the blood fractions collected from the same visit (Table 4).Age, sex, and years of education were considered confounding variables for CI and were utilized with the aim to pairwise match AD with NCs to help reduce the risk of confounders influencing false associations to AD due to the smaller sample size.
Genotype frequencies obtained and imputed for APOE in each population and genotype frequencies for APOE and OGG1 distributed by cognitive status in each population are shown (Supplementary Table 19 and 20).Testing for Hardy-Weinberg Equilibrium for APOE and OGG1 were insignificant for the total cohort and within each population, indicating that the genotype frequencies are in equilibrium (Supplementary Tables 21 and 22).www.nature.com/scientificreports/Although our attempt to match samples based on age was unsuccessful; a Pearson's correlation was performed to exclude age as a potential cofounder and showed age does not need to be considered a covariate in our dataset as it was not correlated with total ccf-mtDNA 8oxoG variant count (Supplementary Fig. 3).

MAs, especially females, have a greater burden in total ccf-8oxoG variant count.
In the whole cohort ccf-8oxoG variant count was significantly elevated in the MA population compared to NHWs; mean = 0.8500 and 0.7160, respectively (Fig. 1).Ccf-8oxoG variant count did not significantly differ based on cognitive status or sex (Supplementary Figs. 4 and 5).A significant population × sex interaction was not observed (Fig. 2); however, ccf-8oxoG variant count was significantly elevated in MA females compared to NHW females (mean = 0.8702 and 0.7771, respectively).Despite cognitive phenotype, MAs had an elevated 8oxoG sSNV burden compared to NHWs (Fig. 3), yet there was not a significant difference for 8oxoG variant count in each population when assessing for sex and and cognitive phenotype (Fig. 4).
Results of multiple linear regression modelling of ccf-mtDNA 8oxoG variant count in the whole cohort with respect to population-sex and education interactive effects while considering APOE status as a dosage effect (i.e., no ε4 allele, one ε4 allele, two ε4 alleles) indicated a significant positive association for APOE and the population × sex interaction (Supplementary Table 23).When assessing for a diabetes × cognition interaction, a significant association between ccf-8oxoG variant count and both population and APOE was determined (Supplementary Table 24).
Additional multiple linear regression models were performed to characterize associations with 8oxoG "hotspots" in the whole cohort (Supplementary Fig. 6 and Supplementary Tables 25-26).The regression modelling for both interaction effects, population × sex and education, and diabetes × cognition in the whole cohort did not observe the same associations for 8oxoG "hotspots", indicating these variants may not be informative in this context.

Population-specific associations to ccf-8oxoG variant count.
Population stratification for the multiple linear regression models did not demonstrate any statistical significance for ccf-8oxoG count in MAs (Table 5); however, in the NHW population AD and diabetes was marginally significant (Table 6).Further, the model showed significant statistical association with sex and age, as well.
Multiple linear regression models for 8oxoG "hotspots" in each population were performed and no associations were observed in the MA population; however, in the NHW population a significant negative association to age was discerned (Supplementary Tables 27-28).Although the significant association to age in the NHW population was previously observed in the non-"hotspot" stratified regression model, generally the "hotspot" stratified analyses were less informative.

Discussion
Ethnic/racial differences in developing cognitive impairment are known to exist, yet reports investigating biological, behavioral, and lifestyle factors that lead to differential mechanisms of neurodegeneration in populations more heavily burdened by cognitive decline are limited.Here, we investigated the predictability of 8oxoG variant count from two blood fractions in assessing risk for cognitive decline in two populations.We hypothesized that   www.nature.com/scientificreports/indirectly evaluating mitochondrial dysfunction through cellular mitochondrial 8oxoG sSNVs may serve as a better biomarker for MAs due to their observed metabolic endophenotype and burden, while ccf-mtDNA 8oxoG variants may provide improved utility as a biomarker for NHWs due to their inflammatory endophenotype.Altogether, our results confirm MAs, especially females, show greater mtDNA oxidative damage compared to NHWs.Tobacco abuse trended for significance with increasing cellular 8oxoG mutational load in MAs; however, interestingly, non-depressed individuals showed elevated cellular 8oxoG variant burden.Stratified regression analysis by population in buffy coat PBMCs demonstrated an association with AD, sex, education, and tobacco abuse, while this was not observed in NHWs.Similarly, ccf-8oxoG variant load was significantly higher in MAs, and MA females had elevated levels compared to males.Ccf-8oxoG stratified regression analysis for NHWs showed a suggestive association with normal cognition in younger aged males without diabetes.This may indicate that oxidative variants from ccf-mtDNA are reduced in older NHW females with AD and diabetes comorbidity; however, the potential reason for this is unclear.There is growing evidence indicating the protective effects of estrogen against OS related damage 47,48 .Estrogen deficiency post menopause is associated with increased levels of OS, higher blood levels of free fatty acids, and reduced antioxidant defense 47,48 , so it is expected that aging females would demonstrate elevated levels of oxidative damage.Nonetheless, due to our observation it begs the question if there are sex-related differences for the age-related decline in mitophagy.
Depression is a known risk factor for developing MCI and AD, and a study demonstrated a depressive endophenotype of MCI and AD in MAs 49 .Furthermore, numerous studies report MAs experience more depressive symptoms compared to other Hispanic/Latino subpopulations 50,51 , as well as NHWs [52][53][54][55][56][57] .Accumulating evidence  www.nature.com/scientificreports/indicates that individuals with depression have higher levels of 8oxoG and oxidative damage, as OS encompasses a critical role in depression pathophysiology through the activity of ROS 58,59 .Substantial evidence indicates a higher prevalence of depression and depressive symptoms among MAs, yet our data denoted that non-depressed MAs exhibited elevated levels of 8oxoG variants in buffy coat PBMCs.However, depression appeared to be underrepresented because of limited reports of depression in MAs with MCI and AD.Subsequent regression models in the whole cohort and MA population show a negative trend between 8oxoG variant count in buffy PBMCs and non-depressed MAs.Surveying for the presence or absence of depression may have poor resolution when investigating cognitive associations compared to assessing for a collection of depressive symptoms.Previous studies report distinct clustering of depressive symptoms is imperative when studying the connection between cognition and depression 60,61 .Our results for 8oxoG and depression among MAs warrants further investigation by implementing depressive symptoms and/or other indicators of depression.
Our results revealed increased levels of 8oxoG variants in buffy PMBCs of MAs with a history of tobacco abuse.Smoking tobacco and exposure to tobacco smoke has been shown to cause elevated levels of 8oxoG compared to non-smokers [62][63][64][65] because of the various carcinogens contained within 66,67 .As previously mentioned, carcinogens readily form DNA adducts and can lead to OS through the production of ROS.Additionally, cigarette smoke has been recognized to cause chronic inflammation leading to increasing OS which further results in accumulating oxidative damage 66,68 .A recent study reported that smoking tobacco increased risk for cognitive decline in aging MAs 69 .Following linear regression models including MAs all demonstrated a significant link with tobacco abuse.These results seem to indicate tobacco abuse as a strong modifiable risk factor for increased mitochondrial oxidative damage in MAs and demonstrates the importance of addressing such behaviors to prevent increased risk for CI in this population.
Our higher resolution linear regression models in the whole cohort from buffy coat PBMCs established that population (MA), tobacco abuse, BMI, education, and population × sex were statistically associated with 8oxoG somatic variants.It is well-established that social determinants of health can play a significant role in risk for disease, and the association between socioeconomic status and health outcome becomes more prominent with age 70 .When considering education as an associated factor in this context it is logical to question the influence of occupation and/or other socioeconomic factors (e.g., environmental exposure to pollutants) have on risk for cognitive impairment.There are numerous reports indicating that educational attainment is associated with healthy aging [70][71][72][73][74] , which may be attributed to educational opportunities influencing healthy behaviors, improved access to quality healthcare, better neighborhood quality, and employment outlook including careers with limited exposure to hazardous environments and/or strenuous labor, which can potentially translate to higher pay/income and life fulfillment 74 .Previous research has shown evidence that cognitive requirements from one's predominant occupation is associated with age-related rate of cognitive decline (e.g., individuals that had lifetime careers that necessitated greater amounts of cognitive requirements had better cognitive function later in life) [75][76][77][78] .Within the framework that higher educational attainment leads to a better occupation and income which is likely to influence housing and surrounding environmental conditions, it would be expected that the inclusion of occupation and pollution would demonstrate significance as well in our linear regression models.Thus, occupation and pollution are likely important mediators in reducing the risk for cognitive impairment in the MA population similar to education, emphasizing racial inequities experienced by Mexican Americans as a result of structural racism.Structural racism contributes to environmental differences including but not limited to neighborhood and education quality, access to quality healthcare, pollutant and violence exposure, workforce opportunities and safety, and generational wealth 1,[79][80][81] .Future studies investigating the relationship between cognitive decline and occupation, as well as pollution, in MAs may be crucial in demonstrating the necessity of programs that focus on mitigating the effects of structural racism.www.nature.com/scientificreports/AD was suggestively associated with cellular 8oxoG sSNVs when assessing an interaction between diabetes and cognitive status.These results were not observed in the NHW population, and perhaps of interest is the fact that many of the coefficients were in the opposite direction (though not significant).Increasing evidence connects AD and T2D, showing a greater risk for cognitive decline due to T2D, and robust correlations indicate high blood sugar is associated with the presence of Aβ plaques 82 .Brain dysfunction is frequently observed in earlier stages of T2D, and hemoglobin A1C (established biomarker for T2D) has been related to decline in functional memory and hippocampal size 82 .Links between T2D and AD implicate mitochondrial dysfunction as a participating factor in the development and/or progression of neurodegeneration and may be of exceptional importance for ethnic/racial differences in disease severity and manifestation.This evidence further suggests that mitochondrial health could be a contributor to the unexplained disparities in CI among MAs, especially since MAs are at great risk for metabolic disorders.
It is important to note that this study has its limitations including (1) measuring 8oxoG lesions indirectly, (2) small sampling for the plasma dataset, (3) lacking biochemical, metabolic, and inflammatory phenotypes, (4) missing most nDNA variants, (5) solely sampling blood tissue, (6) evaluating in one cohort, and (7) APOE in the NHW model could have reduced power due to a larger number of missingness compared to MAs.Additionally, another limitation that could be considered is the absence of cell composition data to adjust for differences in mitochondrial content that may introduce variability in variant allele frequency among both cell and ethnic/ racial populations.To better understand the biological and mechanistic roles of mitochondrial dysfunction and oxidative DNA damage in this context, future studies should incorporate larger sampling, include more biological markers indicative of metabolic health and systemic inflammation, determine if utilizing MMSE, CDR sum, and/or other neuropsychological tests for cognitive function strengthens our power to further support/ validate our results, and characterize the nuclear genetic background associated with the mtDNA for each subject.Future studies should also investigate the mechanistic role of mitochondrial processes, such as mitophagy and mitochondrial quality control and sensing, play in contributing to the observed differences in cellular and ccf-mtDNA 8oxoG sSNVs between MAs and NHWs in relation to cognitive decline.Additionally, future studies will aim to validate the applicability of peripheral cellular and cell-free pathophysiological phenotypes as biomarkers for assessing brain pathology, disease risk, and/or disease stage.These studies will also investigate expression of DNA repair machinery, the role of sex hormones, and validate oxidative mtDNA load using alternative methods.
Uniquely, our data specifically point to novel, population-based effects in 8oxoG damage in cellular and cellfree mtDNA.Overall, our results indicate both cellular and ccf-8oxoG variants mtDNA are significantly elevated in MAs compared to NHWs and indicate sex-differences (Fig. 5).Notably, our results from evaluating oxidized cellular mtDNA compared to ccf-mtDNA presents the possibility that these biomarkers may have significant predictive capability in MAs, especially females, compared to NHWs due to the observed statistical significance of assessing various independent variables.This evidence implies mitochondrial dysfunction in cellular mtDNA may be distinctly related to disease pathology in MAs with cognitive decline, whereas in ccf-mtDNA displayed poor associations in MAs compared to NHWs.This cumulative evidence supports the notion that (1) blood-based signatures of mitochondrial dysfunction differ between ethnic/racial populations, (2) cellular and ccf-mtDNA possess different functionality in potentially developing pathophysiological conditions, and (3) ethnic/racial differences exist in the manifestation of neurodegeneration through the assessment of mitochondrial oxidative DNA damage from different blood fractions.

Methods
Sample acquisition and description.Cohort.The Texas Alzheimer's Research and Care Consortium (TARCC) is a population-based collaborative longitudinal research initiative that has expanded between several Texas medical research institutions 83 .TARCC explores factors that may attribute to the development and progression of cognitive impairment due to AD in the MA population compared to their NHW counterparts.
Participants.The study received institutional review board approval under the University of North Texas Health Science Center IRB #1330309-1 and all experiments were performed in accordance with relevant guidelines and regulations.Informed written consent was obtained from participants and/or their legally authorized proxies to take part in the study and allow publication of findings before data collection.Volunteer aging participants enrolled in TARCC annually complete a medical evaluation, clinical interview, neuropsychological testing, and blood draw.Eligible participants obtained categorical clinical diagnoses of ' Alzheimer's disease' , 'Mild Cognitive Impairment' , and 'Normal Control' based on the criteria provided by the National Institute for Neurological Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association 84 .Additional information regarding the inclusion and exclusionary criteria of TARCC has been discussed elsewhere 49 .This study included NHW and MA subjects (N = 559; Table 1) diagnosed with Alzheimer's Disease (n = 104), Mild Cognitive Impairment (n = 127), or normal cognition (n = 328).Obtained buffy coat samples from NHWs (n = 261) and MAs (n = 299) and a subset of plasma samples from 62 NHWs and 57 MAs (N = 119; Table 4) collected at the same visit were selected to match the distribution of subjects with respect to age, sex, and type-2 diabetes among both populations.The plasma subset did not include individuals diagnosed with MCI.These samples were analyzed to characterize cellular and circulating cell-free mtDNA (ccf-mtDNA) oxidative damage from blood.Whole mtDNA amplification.The whole mitochondrial genome and large mtDNA fragments for each sample were amplified using the REPLI-g ® Human Mitochondrial DNA kit (Qiagen, Venlo, Netherlands) following the manufacturer's protocol.This kit uses the high fidelity proofreading phi29 DNA polymerase capable of both rolling circle and multiple displacement amplification in combination of random hexamers 85 .Mitochondrial genome amplification was performed in order to increase mtDNA levels relative to nuclear DNA to enhance mtDNA coverage for whole genome sequencing.Amplified product was quantified via Qubit dsDNA BR assay on the Qubit 4 fluorometer (Invitrogen TM , Thermo Fisher Scientific, Waltham, MA) for each sample and a small test size of approximately 12 samples were evaluated to determine the distribution of amplicon sizes using the 4200 TapeStation System (Agilent Technologies, Santa Clara, CA) following the manufacturer's protocol.The Genomic DNA ScreenTape and corresponding reagents were used to determine the presence of mtDNA fragments from 200 bp to the whole genome.

Measurement
mtDNA sequencing.The Nextera XT TM DNA Library Preparation kit (Illumina, San Diego, CA) was used to prepare the sample library for sequencing following the manufacturer's protocol.All samples were sequenced on the NextSeq 550 Sequencer (Illumina) platform with high output kit v2.5 generating paired-end reads of 150 bp for 300 cycles.The buffy coat samples had an average read depth of 1855X, while the plasma samples had an average read depth of 3970X.www.nature.com/scientificreports/mtDNA sequence mapping/alignment and variant calling.The methods section of Reid, et al., outlines sequence mapping/alignment and mtDNA variant identification for the processing of raw mtDNA gzipped FASTQ pairs generated for each sample 24 .

Identification of biological variants indicative of oxidative damage.
Considering possible bias from the potential presence of basecalls derived from technical oxidative damage as explained in Reid, et al., basecalls were evaluated and manually reviewed as detailed in Reid, et al. 24 .Variants indicative of 8oxoG damage for each subject from the buffy coat portion were summed and normalized by accounting for read depth (variant count per 1000 read depth) to evaluate group differences based on the following variables: population, cognition, sex, type-2 diabetes, comorbidity (cognitive impairment and diabetes), both APOE and OGG1 genotype, and lifestyle factors.Variants indicative of 8oxoG damage from plasma was summed and normalized by accounting for read depth and were log10 transformed to evaluate group differences with the same variables described above.8oxoG variant "hotspots" were identified as previously indicated in Reid et al. 24 .
APOE and OGG1 genotyping imputation.Genome-wide SNP profiles were generated using the Illumina Infinium Multi-Ethnic Global Array which types 1.7million SNPs.Standard filtering based on SNP missingness, individual missingness, and minor allele frequency (5%) was conducted according to Anderson et al., 2010 86 .Genetic imputation of APOE (rs7412 and rs429358 for individuals missing APOE genotypes) and OGG1 (rs1052133) was performed using Impute2 based on the 1000 Genomes Project Phase 3 data; probabilistic genotypes for were called at a threshold of 0.8.
Statistical analyses.Statistical analyses were performed using Microsoft Excel, IBM SPSS software (v.27.0), R software (v.4.2.0), and GraphPad Prism software (v.9.4.0).Welch's t-test (two-tailed) and two-way ANOVA were performed on 8oxoG mutational load to compare between both population groups.Multiple linear regression analysis was performed to evaluate the relationship between cognition, sex, age, education, diabetes and depression status, and tobacco abuse with 8oxoG variant count both within the whole study cohort and in stratified analyses of MAs and NHWs.

Figure 1 .
Figure 1.ccf-mtDNA 8oxoG variant count is significantly elevated in the Mexican American population.(a) Log transformed ccf-mtDNA 8oxoG variant count grouped by population using an unpaired, two-tailed t-test (n = 122, t-statistic = 4.666, df = 120, P ≤ 0.0001).Error bars represent standard error of the mean.(b) Violin plot demonstrating distribution of 8oxoG variant count in MAs and NHWs (n = 122) with effect size and confidence interval plotted on right y-axis.Dashed lines indicate the mean and dotted lines represent the 1st and 3 rd quartile.The triangle represents the difference of the means.

Figure 2 .
Figure 2. ccf-mtDNA 8oxoG variant count is significantly higher in Mexican American females compared to non-Hispanic White females.Log transformed plasma 8oxoG variant count by population and sex was analyzed using a two-way ANOVA (n = 122, p = 0.1275, F-statistic = 2.356, df = 118).Significant p-value for MA-Female 8oxoG variant count compared to NHW-Female (p = 2.515e-005).Error bars represent standard error of the mean.

Figure 3 .
Figure3.ccf-mtDNA 8oxoG variant count is significantly higher in Mexican Americans compared to non-Hispanic Whites despite cognitive phenotype.Log transformed plasma 8oxoG variant count by population and cognitive status was analyzed using a two-way ANOVA with interaction terms for population-cognition (n = 122, P = 0.4434, F-statistic = 0.5916, df = 118).There is no significant population × cognition interaction.Significant source of variation was caused by population (P ≤ 0.0001).Significant p-value for MA-NC 8oxoG variant count compared to NHW-NC (P = 0.0301) and MA-AD compared to NHW-AD (P = 0.0014).Error bars represent standard error of the mean.

Figure 4 .
Figure 4. ccf-mtDNA 8oxoG variant count by cognitive phenotype and sex in each population.(a) Grouped bar graph of log transformed plasma 8oxoG variant count by cognition and sex in MAs testing for cognition × sex interaction via two-way ANOVA (n = 57, P = 0.3633, F-statistic = 0.8408, df = 53).(b) Grouped bar graph of log transformed plasma 8oxoG variant count by cognition and sex in NHWs testing for cognition × sex interaction via two-way ANOVA (n = 65, P = 0.6476, F-statistic = 0.2110, df = 61).Error bars represent standard error of the mean.

Figure 5 .
Figure5.Hypothetical schematic representing the potential mechanism(s) observed from cellular and ccf-mtDNA 8oxoG variants in MA vs NHW participants of TARCC.In the buffy coat PBMC blood fraction (left) observations in cellular 8oxoG variant count were found to be significant in MAs and the effects were not observed in NHWs.In buffy coat PBMCs, cellular 8oxoG variant count was significantly elevated in MAs, especially females, and was associated with CI and tobacco abuse.Modifiable risk factors, biological processes, and genetics such as lifestyle, environment, social determinants of health, immune cell function, and mito-nuclear interactions are theorized to contribute to elevated oxidative damage to mtDNA in MAs from TARCC.In the blood plasma fraction (right) observations of ccf-8oxoG variant count were significantly reduced in NHW females and were associated with AD and diabetes.These results suggest possible alterations in mitochondrial quality control and/or lack of sensing in NHW females.Overall results from cellular and ccf-mtDNA 8oxoG variant loads suggest mitophagy may play a role in ethnic/racial differences in AD etiology.This figure was created with BioRender.com.

Table 2 .
Multiple linear regression prediction model in the Mexican American population considering total cellular 8oxoG variant count.Italics and bolding indicate a P-value of significance, while italics alone indicate a p-value approaching significance.

Table 3 .
Multiple linear regression prediction model in non-Hispanic Whites considering total cellular 8oxoG variant count.

Table 5 .
Multiple linear regression results for ccf-8oxoG variant count within Mexican Americans.Italics and bolding indicate a P-value of significance, while italics alone indicate a P-value approaching significance.

Table 6 .
Multiple linear regression results for ccf-8oxoG variant count within non-Hispanic Whites.Italics and bolding indicate a P-value of significance, while italics alone indicate a P-value approaching significance.