Asthma is a chronic inflammatory disorder of the lungs, specifically affecting the bronchi and bronchioles. It is characterized by intermittent airflow obstruction, airway hyper-responsiveness, and recurrent episodes of breathlessness, wheezing, chest tightness, and coughing. If left untreated, it can lead to irreversible airway remodelling and intractable airflow limitation (1, 2). Asthma represents a major global health concern, affecting an estimated 262 million individuals worldwide, and its prevalence continues to increase (3).

In Kuwait, asthma prevalence is notably high, with recent estimates between 11%–15% among adults, 18% in children, and an overall adjusted rate of approximately 9.5%—the highest in the Gulf region (4, 5). Asthma-related hospital admissions and mortality also remain significant. Although hospitalization rates declined by 49.5% and mortality by 77.6% from 2000 to 2014 (6), recent WHO data (7) reported an age-adjusted mortality rate of 1.12 per 100,000, reflecting a persistent disease burden despite improved management. Kuwaiti nationals experience significantly higher risks than non-Kuwaitis for both hospitalizations and mortality, as indicated by adjusted rate ratios exceeding 1.8 and 1.2, respectively. This disparity arises from environmental factors (e.g., dust storms, historical oil-related pollution, humidity, indoor allergens like incense), behavioural risks (e.g., smoking prevalence of 20%–30%, household second-hand smoke), socioeconomic issues (e.g., stigma, delayed care, emergency service overuse), and clinical factors (e.g., high comorbidity rates such as allergic rhinitis in 70%–80%, obesity around 40%, and poor adherence to medications) (6, 8). Non-Kuwaitis, often expatriates, may be underdiagnosed or repatriated in severe cases, artificially reducing reported rates (6). Psychological stressors, including war-related trauma, further increase risks among Kuwaitis (9). Compared to other populations, Kuwait's asthma prevalence (9.5%–18%) is generally lower than Caucasian cohorts (10%–20%) but higher than East African populations (∼5%–6%). Differences are likely due to varying environmental exposures and genetic factors (5, 10). Kuwait's unique context—with high consanguinity rates (∼50%) increasing the expression of recessive genetic variants in key immune pathways and significant environmental stressors—exacerbates asthma susceptibility, severity, and allergic phenotypes, highlighting the need for targeted research and interventions (8, 11).

Asthma is a multifactorial disorder where genetic and environmental factors interact to influence disease susceptibility and severity (12). Numerous genome-wide association studies (GWAS) have identified key autosomal genetic loci of asthma, including genes involved in immune response and inflammation pathways, such as TSLP, TNFSF4, ADORA1, CHIT1, GATA3, and MUC5AC (1, 2, 13, 14). These genes primarily regulate immune responses, epithelial barrier function, and inflammatory processes associated with asthma pathogenesis. However, despite these insights, a parental history of allergy—particularly from the maternal side—remains a notably strong predisposing factor (15–18). Children of asthmatic mothers are at higher risk of developing asthma compared to those with non-asthmatic mothers (19, 20), suggesting that maternal inheritance, including mitochondrial DNA (mtDNA), could contribute to asthma susceptibility.

Mitochondria are specialized organelles responsible for cellular energy production and are inherited exclusively from the mother. They contain their own genome, encoding key proteins and RNA molecules essential for oxidative phosphorylation (OXPHOS), which drives ATP production and supports cellular metabolic balance. In addition to generating energy, mitochondria play crucial roles in regulating calcium homeostasis, apoptosis, and the production of reactive oxygen species (ROS). Excessive ROS production can lead to oxidative stress and activate inflammatory pathways, contributing to chronic inflammation and tissue damage (21). Mitochondrial dysfunction, resulting from mutations or variants in mtDNA, can impair OXPHOS efficiency, leading to increased ROS levels and promoting inflammatory responses—central mechanisms implicated in asthma pathogenesis (22).

Previous studies have investigated the potential role of mitochondrial genetic background in asthma. Raby et al. (23) identified an association between mitochondrial haplogroup U and elevated total serum IgE levels in children with asthma, primarily in Caucasian populations. Similarly, Zifa et al. (24) reported specific mtDNA mutations more frequent in asthmatics than in controls in a Greek cohort. Studies conducted in Chinese (25) and German (26) populations also identified mtDNA variants associated with asthma risk or severity. More recently, studies in admixed and African ancestry populations, such as those by Vergara et al. (27) and Xu et al. (28), have reported associations between maternal African mitochondrial ancestry or mtDNA copy number and asthma-related traits. Collectively, these findings support the role of mitochondrial genetic variation in asthma susceptibility across multiple ancestries.

However, the specific contributions of mtDNA in Middle Eastern populations—particularly those with substantial African and Asian haplogroup components—remain largely unexplored. This study addresses this gap by investigating mitochondrial variation in a Kuwaiti cohort, providing novel insights into population-specific asthma risk mechanisms.

We previously demonstrated the effectiveness of next-generation sequencing (NGS) whole-exome data for capturing the mitochondrial genome. Our comparative analyses using duplicate samples sequenced via whole-genome and Sanger sequencing revealed high concordance in identifying mitochondrial variants and haplogroups, validating the utility of exome data for mitochondrial genetic analysis (29, 30).

Building on this background, we investigated mitochondrial DNA variation and haplogroup distribution in a Kuwaiti cohort to assess potential associations with asthma susceptibility.

Kuwait's unique genetic and environmental context provides a window into how mtDNA haplogroups and variants can influence asthma. The 1991 oil fires and ongoing pollution have imposed chronic oxidative stress on the population (44–46). These environmental stressors likely interact with genetic factors –particularly mitochondrial variants– to modulate OXPHOS efficiency and inflammatory responses (21, 22).

In this context, we investigated the association between mtDNA haplogroups and variants with asthma risk by analysing indirect whole-exome sequences from 287 Kuwaiti individuals, with an average mtDNA coverage of 27× Principal component analysis revealed that individuals sharing the same maternal haplogroup clustered together, supporting the accuracy of our haplogroup profiling and indicating minimal confounding factors.

The maternal haplogroup association analysis revealed mitochondrial haplogroup M as a significant risk factor for asthma in the Kuwaiti population. Individuals in our study carrying maternal haplogroup M had a threefold increased risk of developing asthma (OR = 3.371; 95% CI = 1.091–10.416; P = 0.035) after adjusting for covariates. To our knowledge, this is the first study to report such an association. Haplogroup M is predominantly found in Asian and African populations compared to European populations (47) and may confer asthma risk due to specific mitochondrial variants that contribute to mitochondrial dysfunction, oxidative stress, and inflammation—pathways strongly implicated in asthma pathogenesis (22).

Kuwait's population has a distinctive genetic makeup shaped by admixture from settlers of Saudi Arabian, Iranian, and other Arabian Peninsula origins, with a significant African contribution observed among its Bedouin subgroups (48). Thus, the identification of haplogroup M as an asthma risk factor in this study likely reflects unique genetic backgrounds interacting with specific regional environmental exposures, such as elevated pollution levels and allergen prevalence.

Our finding contrasts notably with previous studies focusing primarily on European-ancestry cohorts, where haplogroup U rather than M has been implicated. For instance, Raby et al. (23) observed that haplogroup U carriers in North America exhibited significantly higher serum IgE levels and greater atopic sensitization compared to non-carriers. Similarly, Zifa et al. (24) found a significant association between haplogroup U and asthma severity in a Greek cohort, where 27.6% of asthma patients belonged to haplogroup U compared to only 4% of controls. These earlier studies suggest a mitochondrial maternal lineage effect underlying asthma susceptibility and severity. In our Kuwaiti cohort, haplogroup U showed no significant association with asthma, nor did it follow frequency trends previously observed in Western populations; its frequency was marginally higher (by approximately 1%) in controls compared to asthmatics. This discrepancy highlights the complexity of mitochondrial genetic factors in asthma and emphasizes the importance of investigating diverse populations. While genetic background and environmental context may contribute to population-specific associations, the lack of replication of previously reported haplogroup U associations may also reflect limited statistical power in our modest cohort. Therefore, the novel association between haplogroup M and asthma identified here likely arises from mitochondrial polymorphisms within haplogroup M sub-clades that may confer neutrality or selective advantage in their original geographic context but predispose carriers to asthma in Kuwait's specific environmental conditions.

Beyond haplogroup comparisons, we identified 10 exonic mitochondrial variants associated with asthma risk through two complementary analyses: case-control association testing (Table 3) and exclusivity analysis (Table 4), within genes encoding subunits of Complex I (MT-ND1, MT-ND3, MT-ND5), Complex III (Cytochrome b gene, MT-CYB), Complex IV (Cytochrome c oxidase genes, MT-CO1, MT-CO2), and the D-loop region upstream of the mitochondrial tRNA-Pro (MT-TP) gene. Complex I mediates electron transfer from NADH to ubiquinone, Complex III facilitates electron transfer from ubiquinol to cytochrome c, and Complex IV catalyses electron transfer to molecular oxygen. Genetic variants within these complexes can disrupt mitochondrial electron transport, resulting in mitochondrial dysfunction, elevated ROS production, oxidative stress, and inflammatory responses—key mechanisms underlying asthma pathogenesis (49–53). Notably, both missense and synonymous variants were identified in these critical genes. Although our study did not directly assess functional impact, prior research has shown that similar types of variants in these same mitochondrial genes can disrupt mRNA stability, translation, or electron transport, potentially leading to excess ROS and inflammation (49, 54, 55). These mechanisms may underlie the biological relevance of the observed associations in asthma. Previous research by Fukuda et al. (56) identified altered expression of mitochondrial respiratory chain genes, specifically subunits of Complex I (NADH dehydrogenase) and Complex IV (Cytochrome c oxidase II and III), in allergic airway conditions, collectively reinforcing the relevance of mitochondrial genetic variants in asthma susceptibility.

In addition to exonic variants, our study identified variants in the mitochondrial control region (MT:16248C > T, MT:16319G > A) and upstream of the tRNA-Pro gene (MT:16359T > C) associated with asthma susceptibility. The control region variants (MT:16248C > T, MT:16319G > A), located in hypervariable region 1 (HVR1), are traditionally linked to haplogroup-defining polymorphisms but may also influence mtDNA replication or transcription efficiency. Alterations in control region activity could modulate mitochondrial copy number or nucleoid organization, indirectly exacerbating oxidative stress in airway cells—a mechanism implicated in asthma pathogenesis (28). While the upstream MT:16359T > C variant does not directly alter the tRNA-Pro gene, previous studies have demonstrated that mitochondrial tRNA mutations are significantly more frequent in asthmatic patients than in controls (24–26). Although our study did not detect the MT:3243A > G mutation in tRNA-Leu(UUR)—a variant classically linked to MELAS syndrome but reported in other asthma cohorts (57)—the observed enrichment of tRNA-Pro variants in our cohort supports the broader role of mitochondrial tRNA destabilization in airway disease. Such tRNA defects impair respiratory chain biogenesis, elevate ROS production, and amplify inflammatory responses in airway epithelia (52). Interestingly, one MT-TP variant (MT:16519C > T) initially exhibited a protective effect against asthma in our cohort (OR = 0.46; P = 0.045); however, this association did not remain significant after adjusting for covariates. This suggests that the protective effect may be influenced by confounding factors, warranting further investigation.

While SIFT and PolyPhen-2 predicted low pathogenicity for the variants identified in our study, these tools were developed for nuclear genes and may not reliably assess mitochondrial variants. Wang et al. (58) reported only ∼57% concordance between predictions and known mtDNA variant classifications, highlighting the need for cautious interpretation of such results. Moreover, ClinVar does not annotate any mtDNA variants for asthma, including those identified here, across all clinical classifications. However, some are linked to other conditions sharing mitochondrial dysfunction, inflammation, or oxidative stress with asthma pathogenesis (59). For example, familial breast cancer and gastric cancer involve chronic inflammation and ROS overproduction (60, 61); major depressive disorder exhibits comorbidity with asthma via cytokine dysregulation (62); and Leber hereditary optic neuropathy features mitochondrial impairment with elevated ROS (63), akin to airway oxidative stress in asthma.

This study has certain constraints that warrant acknowledgment. The primary limitation is the modest cohort size (n = 287, including 48 asthmatic individuals), which limits the statistical power to detect associations with rare mitochondrial haplogroups or low-frequency variants. Post hoc power analysis indicated moderate power for key findings (56%–68% for the haplogroup M association and ∼66% for representative mtDNA variants), highlighting the need for replication in larger, independent cohorts. The retrospective nature of the study and its nesting within a broader population-based genetic cohort resulted in a fixed case-control ratio. To address potential bias from this imbalance, we performed a sensitivity analysis using Firth logistic regression, which showed that the primary associations remained statistically significant. Additionally, asthma status was self-reported, precluding stratification by disease severity or immunological subtypes (e.g., eosinophilic asthma) and limiting adjustment for clinical covariates such as IgE levels. While WES is not primarily designed for mtDNA capture, previous validations—including our own—support its utility in detecting homoplasmic variants and assigning haplogroups. Our analysis showed consistent coverage across the mitochondrial genome (mean depth ∼27×) without regional dropout; meeting depth thresholds commonly used for reliable variant calling. However, this depth is suboptimal for detecting low-frequency heteroplasmic variants, which require higher coverage and targeted sequencing to distinguish from technical artifacts (33). Heteroplasmy is also tissue-specific; thus, blood-derived mtDNA may not fully represent disease-relevant mitochondrial variation in airway tissues (64). Multiple testing correction was not applied, as conventional methods (e.g., Bonferroni) can be overly conservative for mitochondrial data due to its linked, non-recombining structure, potentially masking cumulative effects in a multifactorial disease like asthma. Future studies should incorporate larger cohorts, deeper tissue-targeted sequencing, and orthogonal validation methods to more accurately characterize mtDNA heterogeneity in asthma.

Despite its limitations, this study provides novel insights into the role of mitochondrial genetics in asthma susceptibility within the Kuwaiti population. We identified mitochondrial haplogroup M as a significant risk factor, along with several mtDNA variants in genes directly involved in mitochondrial dysfunction and ROS production—both of which are implicated in asthma pathogenesis. These findings emphasize the interplay between maternal lineage, mitochondrial biology, and asthma risk, offering a foundation for future research. Further functional studies—including in vitro or in vivo experiments—in larger Middle Eastern cohorts are necessary to validate these associations and to explore how mitochondrial genetic profiles interact with environmental stressors to influence asthma outcomes.