Molecular characterization of mitochondrial transferRNAGln and transferRNAMet A4401G mutations in a Chinese family with hypertension
- Authors:
- Published online on: February 17, 2017 https://doi.org/10.3892/mmr.2017.6216
- Pages: 1832-1836
Abstract
Introduction
Hypertension is a primary public health problem, affecting ~1 billion individuals worldwide (1). To date, the etiology of hypertension remains to be fully elucidated due to its multi-factorial nature. Hypertension may be due to one or numerous hereditary, environmental and individual factors. A maternal inheritance pattern of hypertension has previously been identified in certain families, suggesting that a mutation in mitochondrial (mt) DNA is a cause of this disease (2–4). Previously, various mtDNA point mutations have been associated with hypertension, including the A4435G mutation in the methionine-transfer RNA (tRNAMet) gene (5), A4295G and A4263G mutations in the isoleucine-tRNA (tRNAIle) gene (6,7), anA1555G mutation in the 12S ribosomal RNA gene (8), a T3308C mutation in the nicotinamide adenine dinucleotide dehydrogenase (ND), subunit 1 gene and a C3303T mutation in the leucine-tRNA, codons UUA/G gene (9,10). Leucine-tRNA, codons CUN A12330G combined with ND, subunit 5 T12338C mutations have previously been reported to associate with hypertension in a Han Chinese family (11).
The present study investigated the involvement of the mitochondrial genome in the pathogenesis of hypertension in the Chinese population. During a systematic and extended mutational screening of mtDNA in a large cohort of subjects admitted to The First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China) with hypertension, a single Chinese family with maternally inherited hypertension was identified. The mutational screening of the mt-tRNA gene led to the identification of a homoplasmic A4401G mutation in the conjunction of glycine-tRNA(tRNAGln) and tRNAMet, and a G5821A mutation in cysteine-tRNA (tRNA-Cys) was additionally identified.
Materials and methods
Subjects
As part of genetic screening program for hypertension, a Han Chinese family (Fig. 1) was recruited from The First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China). Informed consent, blood samples and clinical evaluations were obtained from all participating family members, under protocols approved by the ethics committee of Wenzhou Medical University. A total of 300 control DNA samples were obtained from a panel of unaffected Han Chinese individuals from the same area. Members of this family were interviewed and evaluated to determine personal and medical histories, including hypertension and other clinical abnormalities. In addition, a physical examination, laboratory assessment of cardiovascular disease risk factors and routine electrocardiography were performed. A physician measured the systolic and diastolic blood pressures of subjects using a mercury column sphygmomanometer, following a standard protocol. The first and the fifth Korotkoff sounds were measured as indicators of systolic and diastolic blood pressure, respectively. Hypertension was defined according to the recommendation of the Joint National Committee on Detection, Evaluation and Treatment of High Blood Pressure and the World Health Organization International Society of Hypertension as a systolic blood pressure of ≥140 mm Hg and/or a diastolic blood pressure of ≥90 mm Hg (12,13).
Genotype analysis of mt-tRNA mutations
Genomic DNA was isolated from the whole blood of subjects using a Gentra Puregene Blood kit (Qiagen, Inc., Valencia, CA, USA). DNA fragments spanning the mt-tRNA genes were amplified by polymerase chain reaction (PCR) using oligodeoxynucleotides, as previously described (14) (Table I). Each fragment was purified and subsequently analyzed by direct sequencing using the ABI PRISM®3700 Genetic Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and BigDye Terminator version 3.1 Cycle Sequencing kit (Thermo Fisher Scientific, Inc.). The resultant sequence data were compared with the updated Cambridge Reference Sequence (GenBank accession no. NC_012920).
Assigning pathogenicity to the mt-tRNA mutation
The pathogenicity classifications of the A4401G and G5821A mutations were assigned using an updated version of a previously validated scoring system (15). This pathogenicity scoring system uses weighted criteria covering a variety of molecular and genetic data, from which an overall pathogenicity score was obtained. The scoring system for variants was as follows: ≥11 points, ‘definitely pathogenic’; 7–10, ‘possibly pathogenic’ and <6, ‘neutral polymorphism’.
Results
Clinical features
The proband (II-6) was a 59-year-old woman from Wenzhou, Zhejiang. She began to suffer from hypertension at age 50. Her blood pressure was 145/95 mmHg and she attended The First Affiliated Hospital of Wenzhou Medical University for treatment. A physical examination revealed the absence of other clinical abnormalities, including diabetes mellitus, loss of vision or deafness. As presented in Fig. 1, the family of the proband exhibited a typical pattern of maternally-inherited hypertension, with a penetrance of 80%. The clinical features of this Han Chinese family are listed in Table II.
mt-tRNA analysis
The maternal inheritance pattern of hypertension suggested the involvement of mitochondria; therefore, the mitochondrial genome of matrilineal relatives was investigated. As mt-tRNA genes have previously been demonstrated to be hotspots for pathogenic mutations associated with hypertension (16), the present study focused on mt-tRNA variants. As presented in Table I, 11 primers that spanned the entire mt-tRNA genome were designed. Following this, putative mt-tRNA mutations were screened; the comparison of the resultant sequences with the reversed Cambridge consensus sequences identified two candidate tRNA mutations: The A4401G mutation in the conjunction of tRNAGln and tRNAMet and a mutation G5821A in the tRNACys gene (Figs. 2 and 3). These tRNA mutations were further assessed by phylogenetic analysis of sequences from other organisms, including mouse (17), bovine (18) and Xenopus Laevis (19). The present study identified that these mutations are highly evolutionarily conserved and may therefore possess significant functions.
Pathogenicity scoring of the A4401G and G5821A mutations
According to the pathogenicity scoring system (15), the present study classified the A4401G mutation as ‘definitely pathogenic’ with a total score of 15 points, whereas the G5821A mutation was identified to be ‘possibly pathogenic’ with a total score of 8 points (Table III).
Discussion
The present study conducted clinical, genetic and molecular analysis of a three-generation Han Chinese family with a high incidence of essential hypertension. Hypertension, with no accompanying conditions, was identified in matrilineal relatives of this family, suggesting that mtDNA mutations were the underlying molecular basis. The age-of-onset of hypertension varied from 33 to 70 years, with an average of 51 years, whereas matrilineal relatives exhibited early-onset hypertension, suggesting that mitochondrial sequence variants may be the risk factor for this disease.
Screening for mt-tRNA mutations identified homoplasmic mutations in the A4401G and G5821A genes. The absence of A4401G and G5821A mutations in the 300 healthy controls indicated that these mutations may be involved in the pathogenesis of hypertension. Notably, the A to G transition at position 4401 was located at the 5′ end of the tRNAMet and tRNAGln genes (20,21). A at the 4401 position is highly conserved among various primates. In addition, this mutation was present only in matrilineal relatives of this family but not in healthy controls, indicating that it may contribute to the pathogenesis of essential hypertension. The A4401G mutation was first described in a patient with left ventricular hypertrophy (22). Functional analysis demonstrated that cytoplasmic hybrid cells containing the A4401G mutation have a 30% reduction in tRNAMet and tRNAGln expression levels. The reduced levels of tRNAMet and tRNAGln in cells with the A4401G mutation potentially results from a defect in the processing of tRNAMet and tRNAGln precursors at the 5′ end. Therefore, A4401G may be regarded as a pathogenic mutation associated with cardiovascular diseases.
Sequence analysis of the mt-tRNA genes identified the presence of a homoplasmic G5821A mutation in the tRNACys gene. The G5821A mutation was located in extremely conserved nucleotides in the acceptor arm of tRNACys, and disrupted the highly conserved base-pairing (G6-C67). A failure in tRNACys function may aggravate the mitochondrial dysfunction caused by the A4401G mutation. A decrease in tRNAMet, tRNAGln and tRNACys may result in reduced mitochondrial protein synthesis, decreased activities of the mitochondrial respiration chain, and consequently a reduction of adenosine triphosphate production and an increase of reactive oxygen species generation. In conclusion, the results of the present study suggested that the tRNACys G5821A gene mutation may regulate the phenotypic expression of hypertension-associated A4401G mutations in tRNAMet and tRNAGln. mt-tRNA gene mutations may therefore be useful biomarkers for the molecular detection or prevention of maternally inherited hypertension. Further studies involving the use of additional samples are required to verify this conclusion.
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