Molecular characterization of mitochondrial transferRNAGln and transferRNAMet A4401G mutations in a Chinese family with hypertension

  • Authors:
    • Shuai‑Shuai Yu
    • Ji‑Mei Du
    • Zhi‑De Tang
    • Zhi‑Feng He
  • View Affiliations

  • Published online on: February 17, 2017     https://doi.org/10.3892/mmr.2017.6216
  • Pages: 1832-1836
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Abstract

Mutations in mitochondrial (mt)transfer (t)RNA (mt‑tRNA) have been reported to serve important roles in hypertension. To determine the underlying molecular mechanisms of mt‑tRNA mutations in hypertension, the present study screened for mt‑tRNA mutations in a Chinese family with a high incidence of essential hypertension. Sequence analysis of the mt‑tRNA genes in this family revealed the presence of an A4401G mutation in the glycine‑and methionine‑tRNA genes, and a G5821A mutation in the cysteine‑tRNA (tRNACys) gene. The G5821A mutation was located at a position conserved in various species, and disrupted G6‑C67 base‑pairing. It was hypothesized that the G5821A mutation may decrease the baseline expression levels of tRNACys, and consequently result in failure of tRNA metabolism. The A4401G mutation was reported to cause the mitochondrial dysfunction responsible for hypertension. Thus, the combination of G5821A and A4401G mutations may contribute to the high incidence of hypertension in this family. Mt‑tRNA mutations may serve as potential biomarkers for hypertension.

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 (24). 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).

Table I.

Primers for polymerase chain reaction amplification of the mt-tRNA genes.

Table I.

Primers for polymerase chain reaction amplification of the mt-tRNA genes.

Target genePrimer namePrimer sequence (5′-3′)Product size (bp)
tRNAPheMT-1F CTCCTCAAAGCAATACACTG802
MT-1R TGCTAAATCCACCTTCGACC
tRNAValMT-2F CGATCAACCTCACCACCTCT802
MT-2R TGGACAACCAGCTATCACCA
tRNALeu(UUR)MT-4F AAATCTTACCCCGCCTGTTT887
MT-4R AGGAATGCCATTGCGATTAG
tRNAIleMT-6F TGGCTCCTTTAACCTCTCCA898
tRNAGln
tRNAMetMT-6R AAGGATTATGGATGCGGTTG814
tRNAAlaMT-8F CTAACCGGCTTTTTGCCC
tRNAAsn
tRNACysMT-8R ACCTAGAAGGTTGCCTGGCT987
tRNASer(UCN)MT-11F ACGCCAAAATCCATTTCACT
tRNAAspMT-11R CGGGAATTGCATCTGTTTTT
tRNALysMT-12F ACGAGTACACCGACTACGGC900
MT-12R TGGGTGGTTGGTGTAAATGA
tRNAGlyMT-15F TCTCCATCTATTGATGAGGGTCT891
tRNAArgMT-15R AATTAGGCTGTGGGTGGTTG
tRNAHisMT-18F TATCACTCTCCTACTTACAG866
tRNASer(AGY)
tRNALeu(CUN)MT-18R AGAAGGTTATAATTCCTACG938
tRNAGluMT-21F GCATAATTAAACTTTACTTC
MT-21R AGAATATTGAGGCGCCATTG
tRNAThrMT-22F TGAAACTTCGGCTCACTCCT1,162
tRNAProMT-22R GAGTGGTTAATAGGGTGATAG

[i] F, forward; R, reverse; mt, mitochondrial; t, transfer; Leu, leucine; UUR, codons UUR; Phe, phenylalanine; Val, valine; Ile, isoleucine; Gln, glutamine; Met, methionine; Ala, alanine; Asn, asparagine; Cys, cysteine; Ser, serine; UCN, codons UCN; AGY, codons AGY; Asp, aspartic acid; Lys, lysine; His, histidine; Thr, threonine; Pro, proline; CUN, codons CUN.

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.

Table II.

Clinical features of the Han Chinese family with essential hypertension.

Table II.

Clinical features of the Han Chinese family with essential hypertension.

SubjectGenderAge at onsetAge when assessedSystolic pressure (mm/Hg)Diastolic pressure (mm/Hg)
I-2Female708314595
II-6Female505914085
II-3Male515715095
III-4Female333514090
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).

Table III.

Pathogenicity scoring system for the A4401 and G5821A mutations.

Table III.

Pathogenicity scoring system for the A4401 and G5821A mutations.

Scoring criteriaA4401G mutationScore/20G5821AmutationScore/20
More than one independent reportYes  2Yes2
Evolutionary conservation of the base pairNo changes  2No changes2
Variant heteroplasmyNo  0No0
Segregation of the mutation with diseaseYes  2Yes2
Histochemical evidence of mitochondrial diseaseStrong evidence  2No evidence0
Biochemical defect in complex I, III or IVYes  2No0
Evidence of mutation segregation with biochemical defect from single-fiber studiesNo  0No0
Mutant mt-tRNA steady-state level or evidence of pathogenicity in trans-mitochondrial cybrid studiesStrong evidence  5Weak evidence2
Maximum scoreDefinitely pathogenic15Possibly pathogenic8

[i] Scoring system classification: ≥11 points, definitely pathogenic; 7–10 points, possibly pathogenic; ≤6 points, neutral polymorphisms.

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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Spandidos Publications style
Yu SS, Du JM, Tang ZD and He ZF: Molecular characterization of mitochondrial transferRNAGln and transferRNAMet A4401G mutations in a Chinese family with hypertension. Mol Med Rep 15: 1832-1836, 2017
APA
Yu, S., Du, J., Tang, Z., & He, Z. (2017). Molecular characterization of mitochondrial transferRNAGln and transferRNAMet A4401G mutations in a Chinese family with hypertension. Molecular Medicine Reports, 15, 1832-1836. https://doi.org/10.3892/mmr.2017.6216
MLA
Yu, S., Du, J., Tang, Z., He, Z."Molecular characterization of mitochondrial transferRNAGln and transferRNAMet A4401G mutations in a Chinese family with hypertension". Molecular Medicine Reports 15.4 (2017): 1832-1836.
Chicago
Yu, S., Du, J., Tang, Z., He, Z."Molecular characterization of mitochondrial transferRNAGln and transferRNAMet A4401G mutations in a Chinese family with hypertension". Molecular Medicine Reports 15, no. 4 (2017): 1832-1836. https://doi.org/10.3892/mmr.2017.6216