Late onset of type 2 diabetes is associated with mitochondrial tRNATrp A5514G and tRNASer(AGY) C12237T mutations

Abstract Background Mitochondrial dysfunctions caused by mitochondrial DNA (mtDNA) pathogenic mutations play putative roles in type 2 diabetes mellitus (T2DM) progression. But the underlying mechanism remains poorly understood. Methods A large Chinese family with maternally inherited diabetes and deafness (MIDD) underwent clinical, genetic, and molecular assessment. PCR and sequence analysis are carried out to detect mtDNA variants in affected family members, in addition, phylogenetic conservation analysis, haplogroup classification, and pathogenicity scoring system are performed. Moreover, the GJB2, GJB3, GJB6, and TRMU genes mutations are screened by PCR‐Sanger sequencing. Results Six of 18 matrilineal subjects manifested different clinical phenotypes of diabetes. The average age at onset of diabetic patients is 52 years. Screening for the entire mitochondrial genomes suggests the co‐existence of two possibly pathogenic mutations: tRNATrp A5514G and tRNASer(AGY) C12237T, which belongs to East Asia haplogroup G2a. By molecular level, m.A5514G mutation resides at acceptor stem of tRNATrp (position 3), which is critical for steady‐state level of tRNATrp. Conversely, m.C12237T mutation occurs in the variable region of tRNASer(AGY) (position 31), which creates a novel base‐pairing (11A‐31T). Thus, the mitochondrial dysfunctions caused by tRNATrp A5514G and tRNASer(AGY) C12237T mutations, may be associated with T2DM in this pedigree. But we do not find any functional mutations in those nuclear genes. Conclusion Our findings suggest that m.A5514G and m.C12337T mutations are associated with T2DM, screening for mt‐tRNA mutations is useful for molecular diagnosis and prevention of mitochondrial diabetes.


| INTRODUC TI ON
Diabetes is a common endocrine disease in China, in particular, type 2 diabetes mellitus (T2DM) accounted for >10% of general population. 1,2 Although the pathophysiology of DM has not been fully elucidated, overwhelming evidence suggests that environmental, personal lifestyle, or nuclear genes mutations may influence T2DM pathogenesis. 3,4 Among these factors, some families are presented in maternally inherited pattern, indicating that mutations or variants in mitochondrial DNA (mtDNA) play critical roles in T2DM. 5,6 Human mitochondrial genome is a relative small molecule (16,569-bp long) which encodes 13 polypeptides, 2 rRNAs (12S rRNA and 16S rRNA), and 22 tRNAs. 7 Despite the fact that the entire mt-tRNA genes account only for approximately 10% of total mitochondrial genome, more than 2/3 mitochondrial disease-related pathogenic mutations are localized at tRNA genes. 8,9 Among these mutations, the A to G substitution at position 3243 appears to be the most common T2DM-associated pathogenic mutation. [10][11][12] Furthermore, several case-control studies indicate that tRNA Ile T4291C, 13 tRNA Glu A14692G, and T14709C mutations 14,15 are involved in the pathogenesis of T2DM.
Maternally inherited diabetes and deafness (MIDD) is a rare form of mitochondrial diabetes characterized by both DM and hearing loss. This disease can be resulted from genetic abnormalities in mtDNA, especially associated with tRNA Leu(UUR) A3243G mutation. 16 Moreover, MIDD typically affects metabolically active organs such as the endocrine pancreas and cochlea, and in some cases, also the retina, muscles, kidneys, and brain. 17 However, the pathogenesis for the MIDD needs further elucidation.
To investigate the T2DM-associated mtDNA mutations, our recently screened the mtDNA mutations in a cohort of 215 diabetic patients and 155 controls. Consequently, a four-generation family with MIDD is identified in this case-control study, to explore the contributions of mitochondrial dysfunction to DM, we perform PCR-Sanger sequencing to analyze the mutations in whole mitochondrial genome.
Moreover, more than 160 loci, around 119 genes have been identified in patients with non-syndromic hearing loss. 18 In particular, Gap junctions (GJs) are intercellular channels that allow small molecules of the cytoplasm of a cell to be directed to the adjacent cell, including ions such as K + , Na + , and Ca2 + . Connexins GJB contains 21 isoforms in humans, including GJB2 (Cx26), GJB3 (Cx31), and GJB6 (Cx30). 19 It has been suggested that several mutations such as c.235delC in GJB2, A194T in GJB3, 150-kb large deletion in GJB6 are the important causes for non-syndromic hearing loss in many populations worldwide. [20][21][22] In addition, TRMU is a nuclear gene crucial for mtDNA translation by encoding tRNA 5-methylaminomethyl-2-t hiouridylate methyltransferase, which thiolates mt-tRNA. 23 Previous study suggested that mutation in TRMU may modulate the clinical expression of deafness-associated mitochondrial 12S rRNA mutations. 24 To see whether GJB2, GJB3, GJB6, and TRMU contributed to genetic susceptibility to deafness, we analyze the mutations in these nuclear genes by direct sequencing.

| Pedigree information and clinical assessments
From January 2019 to January 2021, we enrolled 215 subjects with diabetes and 155 controls from Hangzhou First People's Hospital, as shown in Figure 1, a large Chinese pedigree with T2DM was ascertained during this mutational screening program. We first invited the members of this family to participate for this study, the blood samples, family history, and detailed personal information were collected. This study was approved by the Ethics Committee of Hangzhou First People's Hospital (Approval Number: 2020-004-01), and each participant provided their written informed consent.
Moreover, 155 healthy subjects including 70 males and 85 females, aged from 38 to 50 years were recruited from the Healthy Examination Center of our hospital as controls. These controls were healthy subjects without any diseases; whereas the subjects had a family history of mitochondrial diseases will be excluded. Body mass index (BMI) was calculated by as the body weight (kg) divided by the square of the height (m 2 ). Obesity was defined using the BMI for Chinese adults: normal: 18.5-24 (kg/m 2 ), overweight: 24-28 (kg/m 2 ), and obese ≥28 (kg/m 2 ). Moreover, we monitored the blood pressure (BP) by using an electronic sphygmomanometer, according to the protocol as previously described. 26 The systolic BP ≥140 mmHg or the diastolic BP ≥90 mmHg was regarded as hypertension. 27 For biochemical assessment, serum FPG was determined by the regular laboratory methods (Beckman Coulter AU5800). In addition, the OGTT was carried out by measurement of plasma glucose concentrations at 0 and 2-h after 75-g glucose administration, while plasma insulin (0 h) and C-peptide (0 h) were measured by chemiluminescent immunometric assay (IMMULITE ® , Siemens). 28 Moreover, the audiological examination was assessed to evaluate the hearing function, which was calculated on the basis of results of pure-tone audiometry (PTA). The degrees of hearing loss were divided into 5 groups: (1) PTA<26 Decibel (dB): normal hearing; (2) 26 dB<PTA<40 dB: mild;

| Screening for mtDNA mutations or variants
To detect the mtDNA variants, genomic DNA was extracted from blood of each participant by using Paxgene Blood DNA Isolation kits (QIAGEN). Briefly, 24 primers were used to amplify whole mitochondrial genomes from affected subjects (II-4, II-6, III-5, III-10, III-18, and III-22), according to the protocol as described previously. 31 Furthermore, the ABI 3700 DNA instrument was employed to analyze the sequences by comparing with the revised Cambridge sequences (rCRS, GenBank accession number: NC_012920.1). 32 The DNA STAR software package version 5.01 (Madison) was used to detect mtDNA mutations or variants.

| Evolutionary conservation assessment
To analysis the potential pathogenicity of a candidate mtDNA mutation, phylogenetic conservation assessment was carried out. We chose 12 vertebrate species and then compared with human mtDNA variant at a certain position to see the degree of conservation index (CI). 33 The CI ≥75% was regarded to be functional potential. 34 Furthermore, mitochondrial haplogroup was classified according to the study by Kong et al. 35

| The pathogenicity scoring system
To identify the potential pathogenic mt-tRNA mutations, we used the following criteria: (1) presented in <1% of the healthy controls; (2) CI ≥75%, as proposed by Ruiz-Pesini and Wallace 34 ; (3) potential to cause structural and functional alterations; and (4) a score of ≥7 points under an established pathogenicity scoring system. 38 If the total scores of a mt-tRNA mutation were less than 6, it belonged to "neutral polymorphism," if the scores were 7-10, it was "possible pathogenic," whereas the scores were more than 11, it was classified as "definitely pathogenic."

| Analysis of GJB2, GJB3, GJB6, and TRMU genes mutations
To see the contributions of nuclear genes to deafness expression, However, these subjects did not have other clinical disorders, including coronary heart disease, cancer, or infectious diseases (Table 1).

| Screening for mtDNA mutations
Owing to the maternally inheritance, we screened the mtDNA mu-  bovine, 45 and Xenopus laevis. 46 We found that except for tRNA Trp A5514G, tRNA Ser(AGY) C12237T mutations (Figures 2-4), other mtDNA variants may not be pathogenic since they either occurred in control group or had very low degrees of CIs.
As shown in Figure 3 and Table 3, the A-to-G substitution at po-

| m.A5514G and m.C12237T mutations affected tRNAs secondary structure
To see the effects of m.A5514G and m.C12237T mutations on tRNAs structure, we used RNA Fold program to analyze the secondary structure of tRNA Trp and tRNA Ser(AGY) with and without these mutations. 36 As shown in Figures 5 and 6, we noticed that m.A5514G  3.4 | m.A5514G and m.C12237T mutations were "possibly pathogenic" associated with MIDD As shown in Table 4, based on the classic pathogenicity scoring system, 38 the total scores of m.A5514G and m.C12237T mutations were both 8 points, which belonged to "possibly pathogenic" mutations for MIDD.

| Mutational analysis of nuclear genes
To see whether nuclear genes (GJB2, GJB3, GJB6, and TRMU) mutations played active roles in clinical expression of hearing impairment, we initiated a mutational analysis of the exons of GJB2, GJB3, GJB6, and TRMU in matrilineal relatives of this pedigree. However, we did not find any functional variants in these genes.

| DISCUSS ION
Mutations in mtDNA were the important causes for MIDD, currently, the clinical features of MIDD were often variable due to the heteroplasmy and subsequent segregation of the mutated mtDNA. 47 Diagnosis and prediction of MIDD prognosis were difficult for providers based on phenotypic features alone because of the large variation of heteroplasmic mtDNA inheritance. 48 At the cellular level, the β-cell required large amounts of ATP to produce insulin.
The impaired mitochondrial functions caused by mtDNA mutations decreased ATP production and increased ROS level leading to ab- Moreover, m.C12237T mutation occurred at position 31 in the variable region of tRNA Ser(AGY) and was expected to form a novel Watson-Crick base-pairing (11A-31T). Interestingly, m.A12308G mutation which was located at the position 31 in tRNA Leu(CUN) , had been found to be associated with cardiomyopathy, 54 metabolic syndrome, 55 and increasing the risk of stroke. 56 Therefore, we believed that the m.C12237T mutation, which was similar to m.A12308G mutation, most probably led to the failure in tRNAs metabolism via affecting its secondary structure, and subsequently impair the mitochondrial functions. 57 Based on these observations, we proposed that the possible molecular mechanisms underlying m.A5514G and m.C12237T mutations in the phenotypic expression of MIDD may be as follows: first, the mutations disrupted the secondary structures of tRNAs and subsequently resulted the failure in tRNAs metabolism, such as reducing tRNA steady-state level, aminoacylation ability, affecting 3' end processing, or its chemical modifications. 58 These biochemical processes will lead to the impairment of mitochondrial protein translation and influence the respiratory chain functions. As a result, these mutations led to mitochondrial dysfunctions which caused the pancreatic β-cell apoptosis or necrosis, 59,60 and involved in the pathogenesis of MIDD in this pedigree. At the same time, the absent of functional variants in GJB2, GJB3, GJB6, and TRMU genes suggested that these genes may not play putative roles in the phenotypic manifestation of MIDD, therefore, the combination of tRNA Trp A5514G and tRNA Ser(AGY) C12237T mutations may be responsible for MIDD in this pedigree. The main limitation of this study was the relatively small sample sizes, further studies including more patients with DM were needed to verify the conclusions.

ACK N OWLED G M ENT
This study was supported by the grants from Zhejiang Natural Science Foundation (No. LY18H160062) and Health Commission of Zhejiang Province (No.2021RC022).

CO N FLI C T O F I NTE R E S T
None.

E TH I C S A PPROVA L
The Ethics Committee of Hangzhou First People's Hospital approved this study (No. 2020-004-01).

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed during this study are included in