Maternally inherited type 2 diabetes mellitus may be associated with mitochondrial tRNAThr mutations

Abstract

BACKGROUND

Mutations in mitochondrial DNA, especially those in mitochondrial tRNA genes, are closely related to type 2 diabetes mellitus (T2DM). However, the underlying molecular mechanisms remain largely unknown.

AIM

To investigate the potential associations between mitochondrial tRNA mutations and T2DM.

METHODS

Two Han Chinese families with T2DM underwent clinical, genetic, and biochemical analyses, and mutations in the entire mitochondrial genome were screened in the matrilineal relatives of these pedigrees. Mitochondrial function was assessed in cybrid cell lines harboring tRNA^Thr mutations.

RESULTS

The matrilineal relatives of these pedigrees expressed variable clinical phenotypes, including deafness, visual loss, and renal failure. Sequence analysis of the entire mitochondrial genomes revealed a set of genetic polymorphisms and three possible pathogenic mutations: tRNA^Thr A15951G, G15930A, and G15927A. The A15951G mutation was located adjacent to the 3’ end of tRNA^Thr, abolishing a conserved 2T-71A base-pairing. The G15930A mutation occurred at a conserved position in the variable region of tRNA^Thr, whereas the G15927A mutation disrupted the 28C-42G base pairing in the anticodon stem of tRNA^Thr. Thus, it can be speculated that these mutations may alter the tRNA structure and function. Using cybrid cell lines derived from five patients with tRNA^Thr mutations and four controls without these mutations, we found that mutant cells exhibited significantly lower adenosine triphosphate levels, membrane potential, and NAD⁺/NADH ratio, whereas reactive oxygen species were increased significantly, suggesting that the A15951G, G15930A, and G15927A mutations led to mitochondrial dysfunction, which may contribute to the progression of T2DM.

CONCLUSION

Our study revealed that tRNA^Thr is a hotspot for pathogenic mutations associated with T2DM. Screening for tRNA^Thr mutations is recommended for the prevention and diagnosis of mitochondrial diabetes.

Key Words: Type 2 diabetes mellitus; Chinese pedigree; Mitochondrial tRNA^Thr; Mutations; Mitochondrial dysfunction

Core Tip: This study identified three mitochondrial tRNA^Thr mutations—G15927A, G15930A, and A15951G—in two Han Chinese pedigrees with maternally inherited type 2 diabetes mellitus. These mutations were associated with variable clinical phenotypes, including deafness, visual impairment, and renal failure. Functional analyses using cybrid cell lines revealed that these mutations lead to mitochondrial dysfunction, as evidenced by reduced adenosine triphosphate production, decreased mitochondrial membrane potential, lower NAD⁺/NADH ratio, and increased reactive oxygen species levels. Our findings identify tRNA^Thr as a mutational hotspot in mitochondrial diabetes and support the inclusion of these mutations in genetic screening strategies for the early diagnosis and prevention of type 2 diabetes mellitus.

INTRODUCTION

Diabetes is a complex disease characterized by chronic hyperglycemia and continues to pose a major global health burden. It was estimated that 10.5% of adults worldwide had diabetes in 2021, a number projected to reach 12.2% by 2045[1]. China had the largest diabetic population globally, with over 118 million cases, accounting for roughly 22% of the global total[2]. Clinically, the two main forms are insulin-dependent type 1 diabetes and non-insulin-dependent type 2 diabetes, with type 2 diabetes mellitus (T2DM) representing a significant public health challenge that affects nearly 10% of Chinese adults[3]. Insulin resistance (IR), defined as reduced insulin efficacy in regulating glucose, protein, and lipid metabolism, plays a key role in the pathogenesis of T2DM. Accumulating evidence indicates that both genetic and environmental factors influence IR, particularly those related to energy storage and metabolism[4]. Although the precise causes of T2DM and IR remain unclear, it is widely accepted that inherited genetic components and gene–environment interactions contribute to disease development[5].

Maternal inheritance observed in certain families supports the involvement of mitochondrial DNA (mtDNA) mutations in T2DM[6]. The human mitochondrial genome encodes 13 polypeptides, 2 rRNAs, and 22 tRNAs[7]. Although mitochondrial tRNA (mt-tRNA) genes constitute only about 10% of the mitochondrial genome, they harbor more than two-thirds of the pathogenic mutations linked to mitochondrial diseases[8]. Among these, the A3243G mutation is the most common T2DM-related pathogenic mutation worldwide[9,10]. In Chinese populations, case-control studies have implicated several mt-tRNA mutations in T2DM, including tRNA^Gly T10003C[11], tRNA^Thr G15897A[12], tRNA^Trp A5514G, tRNA^Ser(AGY) C12237T[13,14], and tRNA^Cys/tRNA^Tyr A5826G[15]. These diabetes-related mtDNA mutations often occur in a heteroplasmic state and exhibit considerable clinical variability[16]. Therefore, screening for mt-tRNA mutations associated with T2DM is important for early diagnosis and prevention, especially in individuals with a family history of diabetes.

Here, we report the clinical, genetic, molecular, and biochemical characteristics of two Han Chinese pedigrees with T2DM (DM-005 and DM-010). Analysis of the mitochondrial genomes revealed three tRNA^Thr mutations, G15927A, G15930A, and A15951G, which were present exclusively in matrilineal relatives and absent in control subjects. To investigate the functional impact of these mutations, we measured adenosine triphosphate (ATP) levels, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and the NAD⁺/NADH ratio in transmitochondrial cybrid cells derived from five patients with T2DM carrying these mtDNA mutations and four controls without these mutations.

MATERIALS AND METHODS

Pedigree information

This study included two pedigrees recruited from Quzhou People’s Hospital and Hangzhou First People’s Hospital (Figure 1). A total of 280 age- and sex-matched controls (140 males and 140 females; age range: 44-66 years; mean age, 53 years) were also enrolled. The study protocol, including the informed consent forms and the consent for publication of case details, was approved by the Ethics Committee of Hangzhou First People’s Hospital (Approval No. KY-20240327-0100-01). Written informed consent and consent for publication were obtained from all enrolled participants.

Figure 1 Pedigrees of two Han Chinese families with maternally inherited type 2 diabetes mellitus. Squares indicate males, circles indicate females, filled symbols indicate affected individuals, and open symbols indicate unaffected individuals. Arrows denote the probands. DM: Diabetes mellitus.

According to the American Diabetes Association guidelines[17], the diagnostic criteria for diabetes were as follows: (1) fasting plasma glucose ≥ 7.0 mmol/L; (2) a 2-h plasma glucose level ≥ 200 mg/dL (11.1 mmol/L) during a 75-g oral glucose tolerance test; and (3) hemoglobin A1c level ≥ 6.5%.

The exclusion criteria for all participants (both patients with diabetes mellitus [DM] and healthy controls) included: (1) a history of other types of diabetes (type 1 diabetes, monogenic diabetes, or secondary diabetes due to pancreatitis or drugs); (2) pregnancy or lactation; (3) acute or chronic infections, inflammatory diseases, or malignancies; (4) severe liver or kidney dysfunction; (5) thyroid or other endocrine disorders affecting glucose metabolism; and (6) unwillingness or inability to provide written informed consent.

Clinical and laboratory analyses

Participant data—including demographics, anthropometrics, vital parameters, medical history, drug administration, medication use, and family history—were collected through personal interviews. Body mass index (BMI) was calculated as body weight in kilograms divided by height in meters squared. Obesity classification for Chinese adults followed these BMI ranges: (1) normal (18.5-24 kg/m²); (2) overweight (24-28 kg/m²); and (3) obese (≥ 28 kg/m²)[18]. Blood pressure (BP) was measured using an electronic sphygmomanometer as previously described[19]. Hypertension was defined according to the guidelines of the Joint National Committee on Detection, Evaluation and Treatment of High Blood Pressure, as a systolic BP ≥ 140 mmHg or a diastolic BP ≥ 90 mmHg[20].

Pure-tone audiometry was performed using an Interacoustics AD226 diagnostic audiometer in a soundproof booth, following established protocols[21]. The device was calibrated in accordance with ISO 389-1: 2017 standards, with the most recent calibration performed on June 12, 2025, and ambient noise maintained within ANSI S3.1-1999 Limits. The pure-tone average was calculated as the mean hearing threshold at 500 Hz, 1000 Hz, 2000 Hz, 4000 Hz, and 8000 Hz. Notably, the degree of hearing loss was classified as follows: (1) normal hearing, < 20 decibels (dB); (2) mild hearing impairment, 21-40 dB; (3) moderate hearing impairment, 41-70 dB; (4) severe hearing impairment, 71-95 dB; and (5) profound hearing loss, > 95 dB. Visual acuity (VA) was assessed using a Snellen chart at a distance of 5 m under 500-lux illumination, with each eye assessed separately with the participant’s corrective lenses, if applicable. VA was recorded as the smallest line read correctly and converted to decimal notation. Visual impairment was categorized by VA as follows: (1) normal, > 0.3; (2) mild, 0.3-0.1; (3) moderate, < 0.1-0.05; (4) severe, < 0.05-0.02; and (5) profound, < 0.02[22].

Blood samples were obtained from each participant after an overnight fast, between 7:00 AM and 10:00 AM. Standard laboratory methods (Hitachi, Tokyo, Japan) were used to measure serum fasting blood glucose, creatinine, lactate dehydrogenase, alanine transaminase, aspartate aminotransferase, total cholesterol, low-density lipoprotein, high-density lipoprotein, and kidney function parameters. Fasting insulin levels were assessed using a chemiluminescent immunometric assay (IMMULITE^®; Siemens Healthcare Diagnostics, Inc., Tarrytown, NY, United states). IR was calculated using the homeostasis model assessment of IR (HOMA-IR), using the formula: HOMA-IR = [fasting insulin (μU/mL) × fasting blood glucose (mmol/L)]/22.5. A HOMA-IR value ≥ 2.69 was considered indicative of IR[23].

Mutational analysis of mitochondrial genomes

To identify potential pathogenic mtDNA mutations associated with T2DM, we screened the mitochondrial genomes of matrilineal relatives from the two pedigrees [DM-005: (1) II-4; and (2) II-6; DM-010: (1) II-1; (2) II-8; and (3) III-4] using 24 primer pairs, as previously described[24]. The PCR products were purified and analyzed by direct Sanger sequencing, and the resulting sequences were compared with the revised Cambridge Reference Sequence (rCRS; GenBank accession number: NC_012920.1)[25]. The DNASTAR software package (version 5.01; Madison, WI, United States) was employed to detect mtDNA mutations and sequence variants.

Determination of the mitochondrial haplogroup

Mitochondrial haplogroups were determined based on the phylogenetic tree developed by Kong et al[26].

Phylogenetic conservation analysis

To further evaluate the pathogenic potential of the identified mtDNA mutations, a phylogenetic analysis was conducted. In brief, 17 species were selected for conservation analysis following a previously described method[27]. The conservation index for each mtDNA mutation was calculated by comparing the nucleotide at the corresponding position across the selected species. A conservation index ≥ 75% was considered to indicate functional significance[28].

Cell cultures

Lymphoblastoid cell lines were established by Epstein-Barr virus transformation as previously described[29]. The cell lines were derived from two affected individuals in pedigree DM-005 (II-4 and II-6) carrying the G15927A mutation, three affected individuals in pedigree DM-010 (II-1, II-8, and III-4) harboring both the G15930A and A15951G mutations, and four control subjects (C1, C2, C3, and C4) lacking these tRNA^Thr mutations. All cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum.

Analysis of ATP production

ATP levels in the nine cybrid cell lines were measured using the CellTiter-Glo^® Luminescent Cell Viability Assay (Promega, Madison, WI, United States; G7572), according to the manufacturer’s protocol[30].

MMP measurement

MMP was assessed using the JC-10 MMP microplate assay kit (Abcam, Cambridge, UK; ab112134), following previously described procedures[31].

ROS assessment

ROS production in mutant and control cell lines was evaluated using 2′,7′-dichlorofluorescin diacetate (Beyotime, Shanghai, China) as previously reported[32].

Determination of the NAD⁺/NADH ratio

The NAD⁺/NADH ratio was measured using a WST-8 NAD⁺/NADH Assay Kit (Beyotime), according to the manufacturer’s instructions[33].

Statistical analysis

All data are presented as the mean ± SD from at least three independent experiments. As all variables followed a normal distribution (P > 0.05), parametric tests were applied. Comparisons between mutant and control cybrid cell lines were performed using a two-tailed unpaired Student’s t-test. All analyses were performed using SPSS software version 20.0 (IBM Corp., Armonk, NY, United States). Statistical significance was set at P < 0.05.

RESULTS

Clinical presentation

Two families with maternally inherited T2DM were enrolled from Quzhou People’s Hospital and Hangzhou First People’s Hospital (Figure 1). For pedigree DM-005, a total of 12 family members across three generations were enrolled, including three matrilineal relatives (I-2, II-4, and II-6). Among them, two individuals (II-4 and II-6) were diagnosed with T2DM, while the remaining family members were unaffected. The proband (II-4), a 79-year-old woman (BMI: 25 kg/m²), visited Quzhou People’s Hospital for routine management of T2DM. As shown in Table 1, clinical evaluation revealed profound hearing loss (100 dB in the left ear and 90 dB in the right ear) and severe visual impairment (VA: 0.02 in the right eye and 0.05 in the left eye). As shown in Table 2, the patient’s hemoglobin A1c and fasting blood glucose results confirmed the diagnosis of diabetes. Her sister (II-6) also had T2DM, with disease onset at 63 years of age. She exhibited severe hearing loss (85 dB in the left ear and 75 dB in the right ear) and moderate visual impairment (VA: 0.1 for both eyes). Their mother (I-2) had died from diabetic nephropathy approximately 4 years earlier (Table 1).

Table 1 Clinical and molecular features of several affected subjects with type 2 diabetes mellitus.

Subjects	Sex	Smoking status	BMI (kg/m2)	Age at examination (years)	Age at onset (years)	BP (mmHg)	Ketoacidosis	PTA (left/right) (dB)	Degree of hearing loss	Visual acuity (left/right)	Degree of visual impairment	Functional mtDNA mutations
DM-005: II-4	Female	-	25	79	62	110/70	No	100/90	Profound	0.02/0.05	Severe	tRNAThr G15927A
DM-005: II-6	Female	-	27	74	63	110/70	Yes	85/75	Severe	0.1/0.1	Moderate	tRNAThr G15927A
DM-010: II-1	Male	+	23	72	69	113/86	No	55/20	Mild	0.3/0.2	Mild	tRNAThr G15930A and A15951G
DM-010: II-8	Female	-	21	75	64	130/89	No	66/70	Severe	0.2/0.2	Mild	tRNAThr G15930A and A15951G
DM-010: III-4	Female	+	22	50	48	107/73	Yes	20/15	Normal	0.5/0.5	Normal	tRNAThr G15930A and A15951G

BMI: Body mass index; BP: Blood pressure; dB: Decibel; DM: Diabetes mellitus; mtDNA: Mitochondrial DNA; PTA: Pure-tone average.

Table 2 Laboratory assessments of several affected patients with type 2 diabetes mellitus.

Subjects	FBG (mmol/L)	FINS (μU/mL)	HOMA-IR	HbA1c (%)	Serum Cr (µmol/L)	eGFR (mL/min)	LDH (U/L)	ALT (U/L)	AST (U/L)	TC (mmol/L)	LDL-C (mmol/L)	HDL-C (mmol/L)
Normal range	3.9-6.1	2.6-12.0	< 2.69	4.0-6.0	57-111	80-130	50-240	9.0-50	15-40	2.85-5.70	1.31-3.37	0.9-1.95
DM-005: II-4	8.0	31.0	11.02	9.2	122	55	255	16	49	3.58	4.55	0.66
DM-005: II-6	7.5	4.88	1.63	6.9	110	63	268	18	55	3.11	5.10	0.81
DM-010: II-1	7.0	13.90	4.32	7.5	76	95	88	29	22	4.25	3.35	1.37
DM-010: II-8	5.1	10.22	2.32	5.0	68	101	92	33	40	4.9	3.10	1.42
DM-010: III-4	4.8	3.50	0.75	5.6	92	122	105	37	43	5.0	2.9	1.28

ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; Cr: Creatinine; DM: Diabetes mellitus; eGFR: Estimated glomerular filtration rate; FBG: Fasting blood glucose; FINS: Fasting insulin; HbA1c: Hemoglobin A1c; HDL: High-density lipoprotein; HOMA-IR: Homeostasis model assessment of insulin resistance; LDH: Lactate dehydrogenase; LDL: Low-density lipoprotein; TC: Total cholesterol.

For pedigree DM-010, a total of 22 family members across four generations were enrolled, including six matrilineal relatives (I-2, II-1, II-4, II-8, III-4, and III-8). Among them, three individuals were diagnosed with T2DM (II-1, II-8, and III-4), whereas the remaining 15 family members were unaffected. The proband (II-8), a 75-year-old woman, developed T2DM at the age of 64 years and presented to Hangzhou First People’s Hospital for treatment. As shown in Tables 1 and 2, clinical examination revealed diabetes, severe hearing loss, and visual impairment. Genetic counseling revealed that matrilineal relatives II-1 and III-4 were also affected by T2DM. Additionally, I-1 and I-2 had died from cardiovascular disease several years earlier, whereas III-8 had died in a traffic accident 6 months earlier.

Screening for mtDNA mutations in the two families

Given the pattern of maternal inheritance, mtDNA mutations may be the molecular basis of T2DM. We therefore screened the mitochondrial genomes of five matrilineal relatives [DM-005: (1) II-4; and (2) II-6; DM-010: (1) II-1; (2) II-8; and (3) III-4] and 280 control subjects. As shown in Table 3, PCR amplification and direct Sanger sequencing identified multiple polymorphisms: (1) ten variants in the D-loop, three in 12S rRNA, three in 16S rRNA, three in tRNA^Thr (G15927A, G15930A and A15951G), and a common 9-bp deletion between positions 8271 and 8279; and (2) the remaining variants were located in oxidative phosphorylation (OXPHOS)-related genes. Nine missense mutations were found: (1) ND1 G4048A (Asp to Asn); (2) ND2 C5178A (Leu to Met); (3) G5460A (Ala to Thr); (4) A6 A8701G (Thr to Ala); (5) A8860G (Thr to Ala); (6) G9053A (Ser to Asp); (7) ND3 A10398G (Thr to Ala); (8) ND5 G13928C (Ser to Thr); and (9) CytB A15326G (Thr to Ala). Phylogenetic analysis across multiple species, including mouse, bovine, and Xenopus laevis[34-36], showed that, except for G15927A and A15951G, these variants were not evolutionarily conserved, suggesting that they may not actively contribute to the progression of T2DM.

Table 3 Mitochondrial DNA variants in two Han Chinese pedigrees with type 2 diabetes mellitus.

Genes	Position	Replacement	Conservation (H/B/M/X)1	rCRS	DM-005	DM-010	Previously reported2
D-loop	73	A to G		A	G	G	Yes
	153	A to G		A	G		Yes
	199	A to G		A		G	Yes
	249	Del A		A		Del A	Yes
	263	A to G		A	G		Yes
	310	T to CTC/TC		T	CTC	TC	Yes
	524	Del C		C		Del C	Yes
	16183	T to C		T	C		Yes
	16189	T to C		T		C	Yes
	16311	T to C		T	C		Yes
12S rRNA	709	G to A	G/G/A/-	G		A	Yes
	750	A to G	A/A/A/-	A	G	G	Yes
	1438	A to G	A/A/A/G	A	G	G	Yes
16S rRNA	2706	A to G	A/G/A/A	A	G	G	Yes
	3010	G to A	G/G/A/A	G	A		Yes
	3107	Del N		N	Del N	Del N	Yes
ND1	3552	T to A		T		A	Yes
	3970	C to T		C	T	T	Yes
	4048	G to A (Asp to Asn)		G		A	Yes
	4071	C to T		C	T		Yes
ND2	4715	A to G		A	G		Yes
	4769	A to G		A	G	G	Yes
	4883	C to T		C	T	T	Yes
	5178	C to A (Leu to Met)	L/T/T/T	C	A		Yes
	5460	G to A (Ala to Thr)	A/V/I/I	G	A		Yes
CO1	6053	C to T		C	T		Yes
	6392	T to C		T		C	Yes
	7028	C to T		C	T	T	Yes
	7196	C to A		C		A	Yes
CO2	8020	G to A		G	A		Yes
NC7	8271-8279	9-bp Del			9-bp Del		Yes
A6	8701	A to G (Thr to Ala)	T/S/L/Q	A	G	G	Yes
	8860	A to G (Thr to Ala)	T/A/A/T	A	G	G	Yes
	9053	G to A (Ser to Asp)	S/G/G/T	G	A		Yes
CO3	9540	T to C		T	C	C	Yes
ND3	10310	G to A		G	A		Yes
	10398	A to G (Thr to Ala)	T/T/T/A	A		G	Yes
	10400	C to T		C		T	Yes
ND4	10873	T to C		T		C	Yes
	11719	G to A		G	A	A	Yes
ND5	12705	C to T		C	T	T	Yes
	13928	G to C (Ser to Thr)		G	C		Yes
ND6	14668	C to T		C	T	T	Yes
CytB	14783	T to C		T	C		Yes
	15043	G to A		G	A	A	Yes
	15301	G to A		G	A	A	Yes
	15326	A to G (Thr to Ala)	T/M/I/I	A	G	G	Yes
tRNAThr	15927	G to A	G/G/G/G	G	A		Yes
	15930	G to A	G/G/A/A	G		A	Yes
	15951	A to G	A/A/A/A	A		G	Yes

¹Conservation assessment of mutations in tRNA, rRNA and OXPHOS-encoding genes among human (H), bovine (B), mouse (M), and Xenopus laevis (X).

²Please see the Mitomap (HYPERLINK) and mtDB (HYPERLINK) databases.

DM: Diabetes mellitus; OXPHOS: Oxidative phosphorylation; rCRS: Revised Cambridge Reference Sequence.

As shown in Figures 2 and 3, the homoplasmic G15927A mutation at position 42 in the anticodon stem of tRNA^Thr disrupted the conserved 28C-42G base pairing. The A15951G mutation, located adjacent to the 3′ end (position 71) of tRNA^Thr, abolished the conserved 2T-71A Watson-Crick base pairing. These structural alterations likely impaired tRNA structure, function, and metabolism[37].

Figure 2  Identification of the mitochondrial tRNA^Thr G15927A, G15930A, and A15951G mutations by direct Sanger sequencing.

Figure 3  Predicted cloverleaf structures of wild-type and mutant tRNA^Thr. The G15927A, G15930A, and A15951G mutations are indicated by arrows.

The mtDNA haplogroup analysis

According to Phylotree and the East Asian phylogenetic classification systems[26], the mtDNA haplogroups of pedigrees DM-005 and DM-010 were B5b and D4b, respectively.

Construction of cybrid cell lines

Lymphoblastoid cell lines derived from two affected individuals in pedigree DM-005 (II-4 and II-6) carrying the G15927A mutation, three affected individuals in pedigree DM-010 (II-1, II-8, and III-4) carrying the mutations G15930A and A15951G, and four controls without these mutations were fused with mtDNA-less human ρ 206 cells (derived from the 143B.TK⁻ cell line)[38]. Cybrid clones were selected in Dulbecco's modified Eagle’s medium containing 5-bromo-2′-deoxyuridine (BrdU) without uridine. PCR and Sanger sequencing confirmed the presence of the G15927A, G15930A, and A15951G mutations in the corresponding cybrid cells.

Decreased ATP production in mutant cells

OXPHOS capacity was assessed by measuring ATP production using the CellTiter-Glo^® assay. As shown in Figure 4A, the average ATP production in the mutant cell lines was 62.5% of that in the control cell lines (P = 0.0001).

Figure 4 Assessment of mitochondrial functions in mutant and control cybrid cell lines. A: Adenosine triphosphate production; B: Mitochondrial membrane potential; C: Reactive oxygen species levels; D: NAD⁺/NADH ratio. ATP: Adenosine triphosphate; DM: Diabetes mellitus; MMP: Mitochondrial membrane potential; ROS: Reactive oxygen species.

Reduced MMP in mutant cell lines

MMP, generated by proton pumping through respiratory chain complexes I, III and IV, is critical for energy storage during OXPHOS[39]. As shown in Figure 4B, the mean MMP in the mutant cell lines was 60.33% of that in the control cell lines (P < 0.0001).

Increased ROS levels in mutant cells

The ROS levels in patients with tRNA^Thr mutations and controls were determined using fluorometry. As shown in Figure 4C, ROS levels were approximately 40.6% higher in the mutant cell lines than in the control cell lines (P < 0.0001).

Decreased NAD⁺/NADH ratio in mutant cells

The NAD⁺/NADH ratio regulates the intracellular redox state, particularly in the mitochondria and nucleus[40]. To assess whether the tRNA^Thr mutations affected cellular redox balance, we measured this ratio in mutant and control cell lines. As shown in Figure 4D, the mutant cells exhibited a significantly lower NAD⁺/NADH ratio than the control cell lines (P < 0.0001).

DISCUSSION

This study provides the clinical, genetic, and biochemical characterization of two families with maternally inherited T2DM carrying tRNA^Thr mutations. Notably, T2DM affected only matrilineal relatives, supporting the hypothesis that mtDNA mutation-induced mitochondrial dysfunction is the molecular basis of this disorder. The age at disease onset ranged from 48 to 69 (mean 61) years. In pedigree DM-005, the two affected individuals had deafness, diabetes, and visual impairment. In pedigree DM-010, two patients (II-1 and II-8) had diabetes and myopia, whereas one (III-4) had diabetes alone.

Whole mitochondrial genome sequencing of matrilineal relatives from pedigrees DM-005 and DM-010 identified three tRNA^Thr mutations (G15927A, G15930A, and A15951G), which were detected only in affected matrilineal relatives and were absent in controls (Table 4). At the molecular level, the homoplasmic G15927A mutation affected a highly conserved nucleotide (G42) critical for tRNA stability and identity[41]. Disruption of the 28C-42G base pairing likely altered the secondary structure and function of tRNA^Thr, similar to the effect of the tRNA^Ile A4300G mutation[42]. Furthermore, the G15927A mutation reduced the steady-state level of tRNA^Thr and caused an approximately 53% reduction in mitochondrial protein translation[43,44], supporting its role as a primary mutation associated with T2DM.

Table 4 Molecular characteristics of type 2 diabetes mellitus-associated G15927A, G15930A, and A15951G mutations.

Gene	Nucleotide changes	CI (%)	Number of nucleotides in tRNA	Location in tRNA	Watson-Crick base pairing	Disease association
tRNAThr	G15927A	100	42	Anticodon stem	Disrupt 28C-42G base pairing	NSHL; LHON; CHD; DCM
tRNAThr	G15930A	50	45	Variable region	-	NSHL
tRNAThr	A15951G	100	71	Acceptor arm	Disrupts 2T-71A base pairing	LHON

CHD: Coronary heart disease; CI: Conservation index; DCM: Dilated cardiomyopathy; LHON: Leber hereditary optic neuropathy; NSHL: Nonsyndromic hearing loss.

The A15951G mutation has been associated with Leber hereditary optic neuropathy[45]. It is located near the 3′ end of tRNA^Thr[46], and nucleotide position 71 is important for recognition by the cognate aminoacyl-tRNA synthetase[46]. Previous studies have shown that the A15951G mutation reduces tRNA^Thr levels by approximately 35%, likely owing to improper aminoacylation or pre-tRNA processing defects, as reported for the A3243G mutation in tRNA^Leu(UUR)[47]. Functional studies confirmed that the A15951G mutation reduces the steady-state level of tRNA^Thr[45], leading to impaired tRNA metabolism and mitochondrial dysfunction. The G15930A mutation occurs in a variable region of tRNA^Thr, inducing thermodynamic changes, and has been implicated in nonsyndromic hearing loss[48].

Transmitochondrial cybrid technology is commonly used to evaluate the effects of mtDNA genetic polymorphisms on OXPHOS while controlling for differences in the nuclear genetic background. In this study, we established two mutant cell lines carrying the G15927A mutation, three mutant cybrid cell lines carrying both the A15951G and G15930A mutations, and four control cybrid cells without these mutations. Compared with the control cell lines, the mutant cell lines exhibited worse mitochondrial dysfunction, characterized by reduced ATP, MMP, and NAD⁺/NADH ratio, along with significantly elevated ROS levels.

MMP is essential for maintaining cellular health and viability, and its loss promotes apoptosis and reduces ATP production[49]. Abnormal MMP may also increase ROS production. ROS, which are by-products of oxidative stress, include peroxides, superoxides, hydroxyl radicals, and singlet oxygen[50]. High ROS levels can damage proteins, lipids, RNA, and DNA, leading to irreversible functional changes that can affect mitochondrial function, apoptosis, and cellular signaling[51]. Additionally, the NAD⁺/NADH ratio regulates the intracellular redox state, antioxidant capacity, and cell signaling[52]. The marked decrease in this ratio observed in the mutant cells indicates greater oxidative stress and more severe mitochondrial dysfunction compared with controls.

Several limitations of this study should be acknowledged. First, the sample size was relatively small, comprising only two Han Chinese pedigrees and five affected individuals carrying tRNA^Thr mutations. Therefore, the findings may not be generalizable to all patients with t2DM harboring tRNA^Thr mutations. Second, although we used cybrid cell lines to eliminate the effects of the nuclear genetic background, we did not perform rescue experiments (e.g., transducing wild-type tRNA^Thr into mutant cybrids) to directly confirm that the observed mitochondrial dysfunction was solely attributable to the G15927A, G15930A, and A15951G mutations. Third, the two pedigrees carried multiple other mtDNA variants (in ND1, ND2, CytB), and although phylogenetic analysis suggested that these variants were not evolutionarily conserved and were likely non-pathogenic, their potential synergistic or modifying effects cannot be completely excluded. Fourth, this study lacked in vivo validation using animal models, which would help establish a causal relationship between these tRNA^Thr mutations and T2DM. Finally, the cross-sectional design and absence of long-term follow-up data limited our ability to assess the dynamic progression of T2DM and its complications in these pedigrees. Future studies with larger cohorts, functional rescue experiments, and animal models were warranted to further validate our findings.

CONCLUSION

In conclusion, our study suggests that tRNA^Thr mutations are associated with T2DM. The G15927A, A15951G, and G15930A mutations may serve as risk factors for T2DM. These findings provide novel insights into the molecular mechanisms, early detection, and prevention of mitochondrial diabetes.