| Aims: The present study aimed to examine mitochondrial function in peripheral blood mononuclear cells(PBMCs)isolated from patients with m.3243A>G mutation and to analyze the associations between mitochondrial function,heteroplasmy levels and the severity of clinical phenotypes evaluated by precise measurements.We also explored the alterations in topographical information and mechanical properties of PBMCs isolated from patients with m.3243A>G mutation by atomic force microscopy(AFM).Methods: A total of 17 patients with m.3243A>G mutation were recruited from the department of endocrinology and metabolism of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital between December 2015 and December 2017.17 sex-and age-matched controls were also enrolled.Heteroplasmy levels of the m.3243A>G mutation in peripheral blood leukocytes,saliva and urine sediment were determined by pyrosequencing in 17 patients with m.3243A>G mutation.Detailed clinical evaluation included endocrinological,audiological,neurological and cardiac examinations.Mitochondrial function was examined in PBMCs isolated from patients with m.3243A>G mutation.Furthermore,biomechanical properties of PBMCs isolated from patients with m.3243A>G mutation were determined by AFM at nanometer range.Results: We found that the most frequent clinical manifestations of m.3243A>G mutation included diabetes(15/17,88.2%),neuroimaging abnormality(13/16,81.3%),sensorineural hearing loss(12/15,80.0%),osteopenia or osteoporosis(8/15,53.3%),left ventricular diastolic dysfunction(4/15,26.7%)underweight(4/17,23.5%),and retinal pigment epithelium changes(3/15,20.0%).Heteroplasmy levels in urine sediment were higher than those in leukocytes and saliva.There were significant ultrastructural abnormalities of the mitochondria in mutant PBMCs.We also observed decreased levels of ND4 L m RNA,ATP production,mitochondrial membrane potential(ΔΨm)and increased ROS production in mutant PBMCs compared to normal PBMCs(P < 0.05).Linear correlation analysis demonstrated the heteroplasmy levels in leukocytes were positively correlated to the levels of GA and Hb A1 c,but negatively correlated with the level of BMD T-score on total hip and the age at onset of hearing loss(P < 0.05).Our results demonstrated that the ΔΨm in mutant PBMCs was negatively correlated with the levels of BLA and heteroplasmy levels in leukocytes,but positively correlated with the level of BMD T-score on neck of femur and the age at onset of hearing loss(P < 0.05).Additionally,levels of ND4 L m RNA in mutant PBMCs was negatively correlated with the levels of BLA,but positively correlated with the level of BMD T-score on neck of femur and the age at onset of diabetes(P < 0.05).The morphological analysis revealed that the cell height was lower and the cell membrane was much rougher in PBMCs of diabetic patients than those in PBMCs of healthy controls(P < 0.05).Compared to control PBMCs,there were significant increases in adhesion force(384%)and Young’s modulus(205%)in PBMCs isolated from diabetic patients,indicating that mutant PBMCs were much stiffer than control PBMCs.Conclusions: The m.3243A>G mutation in mitochondrial DNA could result in various clinical manifestations and abnormalities of mitochondrial morphology and function.The ΔΨm in PBMCs and heteroplasmy levels in leukocytes could reflect the severity of the clinical phenotypes in the m.3243A>G mutation.In addition,there were significant alterations of topographical and mechanical properties in mutant PBMCs. |
