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Genetic variants in mitochondrial tRNA genes are associated with essential hypertension in a Chinese Han population
Clinica Chimica Acta 410 (2009) 64–69
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Genetic variants in mitochondrial tRNA genes are associated with essential hypertension in a Chinese Han population Hai-Yan Zhu a,b, Shi-Wen Wang a,⁎, Li Liu c, Rui Chen a, Lin Wang a, Xing-Lai Gong d, Min-Lu Zhang e a
Institute of Geriatric Cardiology, General Hospital of Chinese PLA, Beijing 100853, China Emergency Department, General Hospital of Chinese PLA, Beijing 100853, China Department of National Center for Cardiovascular Disease, Cardiovascular Institute and Fuwai Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100037, China d Department of Mathematical Sciences, University of Cincinnati, Cincinnati, OH 45221-0025, USA e Department of Computer Science, University of Cincinnati, Cincinnati, OH 45221-0030, USA b c
a r t i c l e
i n f o
Article history: Received 20 July 2009 Received in revised form 9 September 2009 Accepted 10 September 2009 Available online 22 September 2009 Keywords: Essential hypertension Mitochondrial tRNA genes Pathogenesis
a b s t r a c t Background: Of multiple factors contributing to essential hypertension, mitochondrial variants exhibited the trends for serving as molecular and genetic markers for the disease in last five years. However, previous studies focused on African-American or Caucasian pedigrees, knowledge of mitochondrial tRNA genes and population-based Chinese hypertensives were limited. Methods: We performed sequence analysis in tRNA genes, hot spots for cardiovascular diseases, in 270 Chinese Han essential hypertensives and 270 controls. Lymphoblastoid cell lines were immortalized by transformation with the Epstein–Barr virus. Rates of oxygen consumption in intact cells were determined with a YSI 5300 oxygraph (Yellow Springs Instruments) on samples, harboring variants in tRNA genes. Results: There were 26 variants in tRNA genes that were found in hypertensives and these variants were not in controls. Functional analysis found that these variants may lead to deficiencies in tRNA 3′ end metabolism and/or impairment of critical subunits of the respiratory chain. Most importantly, the oxygen consumption rate in cells harboring variants T4454C (P = 0.0010) and A4263G (P = 0.0001) decreased as compared to the average level of control cell lines. Conclusions: Variants located in mitochondrial tRNA genes may have biologic plausibility to implicate in the pathogenesis of Chinese essential hypertension. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Essential hypertension has been a common concern for years in China and in the world due to its high morbidity and severe outcomes of complications including heart failure, cardiac arrhythmia and left ventricular hypertrophy, etc [1,2]. While the nuclear genome has been studied extensively with respect to hypertension, few studies have examined the impact of mitochondrial genomic variants in essential hypertension [3–5]. Mitochondria are important in many diseases as they supply greater than 90% of the energy to the human body through oxidative phosphorylation, produce reactive oxidative species harmful to cells and induce apoptosis [6]. In particular, mitochondrial variants exhibited the trends for serving as molecular and genetic markers for essential hypertension in last five years [7–9]. In mitochondrial genome, more than 50% of the characterized pathology-related mtDNA mutations concentrate within the tRNA ⁎ Corresponding author. Institute of Geriatric Cardiology, General Hospital of Chinese PLA, 28 Fuxing Road, Beijing 100853, China. Tel.: +86 10 66936761; fax: +86 10 88270497. E-mail address: [email protected] (S.-W. Wang). 0009-8981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2009.09.023
genes which were of less than 10% of the whole genome [10]. These tRNA genes have the essential role in the synthesis of proteins involved in energy metabolism. So far, 139 pathogenic and 243 polymorphic mitochondrial tRNA variants, have been described and they have become the foreground of numerous case reports [10]. Herein, mitochondrial tRNA genes were of particular significance in the pathogenesis of hypertension. It is worth mentioning that five tRNA genes (tRNALeu(UUR), tRNALys, tRNAMet, tRNAGln and tRNAIle) were hot spots for cardiovascular diseases, as described previously [10,11]. However, previous studies focused on nucleic genes and family-based mitochondrial screening in Caucasian and AfricanAmerican pedigrees, knowledge of mitochondrial tRNA genes and population-based Chinese hypertensives was limited [6,7,12–14]. Thus, our objective was to determine the relationship between mitochondrial tRNA genes and Chinese essential hypertension. We performed a systematic and extensive screening of mitochondrial genes in 3 regions, including tRNALeu(UUR), tRNALys, tRNAMet, tRNAGln and tRNAIle in a Chinese Han cohort. We focused on this Chinese Han population because of the high morbidity of essential hypertension in Chinese adults (nearly 11.8%) [2] and the limited amount of research on this racial group. We used a population instead of family-based
H.-Y. Zhu et al. / Clinica Chimica Acta 410 (2009) 64–69
strategy for two reasons: 1) Large numbers of hypertensives without family history are not detected in China, and they might be overlooked for lacking of medical knowledge and regular checks. 2) The morbidity of essential hypertension is not totally family-based; 50–60% of hypertensives are population-based distributed [2].
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typically harvested for RNA isolation 3 days after a split while the cultures were in logarithmic growth phase. Cell lines derived from 26 probands harboring variants in tRNA genes, respectively, and three genetically unrelated control individuals (1,2,3), grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS).
2. Methods 2.5. Oxygen consumption measurements 2.1. Patients We evaluated 270 individuals with hypertension; all participants were 1) Beijing residents who were out-patients or in-patients at the Institute of Geriatric Cardiology, Chinese People's Liberation Army (PLA) General Hospital from January 2006 to January 2007; 2) N18 y; 3) diagnosed as primary hypertension. Exclusion criteria: Participants were diagnosed as 1) secondary hypertension (primary aldosteronism, renal arterial sterosis, aortic coarctation, etc.); 2) congenital heart diseases; 3) organic valve diseases. All participants were Han Chinese evaluated and underwent a thorough examination including medical record review, clinical evaluation, biochemical assay as well as genetic analysis. The protocol was approved by the Ethics Committee of Chinese PLA General Hospital and Institute Review Board of Cincinnati Children's Hospital Medical Center; all participants gave informed consent. Controls were healthy Beijing residents who accepted annual checkup in Chinese People's Liberation Army (PLA) General Hospital from January 2006 to January 2007. None of the control subjects had a family history of hypertension, and all had a SBP of b130 mm Hg and a DBP of b85 mm Hg. 2.2. Blood pressure assessment Blood pressure was measured in triplicate by a single physician who was expert in the evaluation of hypertension, with an appropriate arm cuff and a mercury sphygmomanometer with the subject in sitting position after 5 min rest. The arithmetic mean of the last 2 measurements was calculated. Korotkoff phase I was taken for systolic BP and V for diastolic BP. Hypertension was defined as systolic BP (SBP) ≥140 mm Hg or diastolic BP (DBP) ≥90 mm Hg on 3 consecutive day according to the Seventh Report of the Joint National Committee [1].
Rates of oxygen consumption in intact cells were determined with YSI 5300 oxygraph (Yellow Springs Instruments) on samples, harboring variants in tRNA genes. Nearly 1 × 107 cells of each sample were suspended in 1.5 ml of special Dulbecco modified Eagel medium (DMEM) lacking glucose, supplemented with 10% dialyzed fetal bovine serum (FBS) [17]. Each cell was measured 6 times. 2.6. Computer analysis Continuous variables were expressed as mean ± SD. Discrete variables in groups were expressed as frequency. Relations between continuous variables were assessed by the unpaired, 2-tailed Student's t-test, and discrete variables were analyzed by Pearson's χ2 test, using the software Stata 7.0 (StataCorp, College Station, TX), P b 0.05 was considered statistically different. 3. Results 3.1. Clinical evaluation Descriptive information for the 270 age and gender balanced cases–controls was provided in Table 1. The average age (66.6 ± 11.4 vs 65.1 ± 17.5 y, P = NS), gender (33.7% vs. 29.6%, P = 0.NS) and body mass index (24.54 ± 2.90 vs. 24.16 ± 2.83, P = NS) were similar to the controls. Biochemical values including fast blood sugar (5.31 ± 0.98 vs. 5.42 ± 1.35, P = NS), total cholesterol (4.70 ± 0.99 vs. 4.90 ± 1.11, P = 0.05), triglyceride (1.59 ± 1.01 vs. 1.50 ± 0.56, P = NS), high density lipoprotein (1.31 ± 0.29 vs. 1.32 ± 0.30, P = NS) and low density lipoprotein (2.54 ± 0.78 vs. 2.61 ± 0.82, P = NS) didn't exhibit any difference between the 2 groups. 3.2. Mitochondrial DNA analysis
2.3. Mitochondrial DNA analysis DNA was isolated from whole blood using Promega Wizard® Genomic DNA Purification Kit (Madison, WI), 3 fragments: 3150–3600 (including tRNALeu(UUR)), 3796–4654 (including tRNAIle, tRNAMet and tRNAGln), 7937–8797 (including tRNALys) were purified. The entire mitochondrial genome of DNA harboring variants A4263G and T4454C were PCR amplified in 24 overlapping fragments, by using sets of the light (L) strand and the heavy (H) strand oligonucleotide primers and subsequently analyzed by direct sequencing in an ABI 3700 automated DNA sequencer, using the BigDye Terminator Cycle sequencing reaction kit and analyzed with SeqWeb program GAP (GCG) according to the updated consensus Cambridge sequence [15]. The primers for 3 fragments are as follows: 3243F: 5′-AGGACAAGAGAAATAAGGCCT-3′, 3243R: 5′-TAAATAGGAGGCCTAGGTTG-3′, 6F: 5′TGCTCCTTTAACCTCTCCA-3′, 6R: 5′-AAGGATTATGGATGCGGTTG-3′, 12F: 5′-ACGAGTACACCGACTACGGC-3′, 12R: 5′-TGGGTGGTTGGTGTAAATGA3′. 2.4. Cell cultures Lymphoblastoid cell lines were immortalized by transformation with the Epstein–Barr virus [16]. Briefly, cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, and 1% penicillin/ streptomycin. Cultures were split 1:2 every 3–4 days and cells were
A list of pathogenic variants was taken from MitoMap (http:// www.mitomap.org/) and a list of polymorphisms from mtDB [18,19]. In the 270 hypertensives, 26 changes in tRNA genes (tRNALeu(UUR), tRNALys, tRNAMet, tRNAGln and tRNAIle) were found and these variants were not in controls (Fig. 1). Except for C4456T, other variants were homoplasmic changes including 5 novel base pair substitutions (A4263G, T4277C, T4353C, C4410A, and T8311C). The frequency of the variants varied from 1/270 to 10/270. As seen in Table 2, further
Table 1 Clinical manifestations of the hypertensives and the controls.
Women, n (%) Age, y Body mass index, kg/m2 Systolic blood pressure, mm Hg Diastolic blood pressure, mm Hg Fasting blood glucose, mmol/l Total cholesterol, mmol/l Triglyceride, mmol/l High density lipoprotein, mmol/l Low density lipoprotein, mmol/l a
Represents statistically significant.
Hypertensives
Controls
(n = 270)
(n = 270)
91 (33.7) 66.56 ± 11.39 24.54 ± 2.90 135.39 ± 16.45 79.09 ± 11.74 5.31 ± 0.98 4.70 ± 0.99 1.59 ± 1.01 1.31 ± 0.29 2.54 ± 0.78
80 (29.6) 65.07 ± 17.46 24.16 ± 2.83 120.56 ± 12.03 74.45 ± 6.47 5.42 ± 1.35 4.90 ± 1.11 1.50 ± 0.56 1.32 ± 0.30 2.61 ± 0.82
P-value
NS NS NS 0.0001a 0.0001a NS 0.05 NS NS NS
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Fig. 1. Mitochondrial tRNA variants in 270 Chinese Han hypertensives. Of 26 variants in tRNA genes (tRNALeu(UUR), tRNALys, tRNAMet, tRNAGln and tRNAIle), 21 previously reported polymorphisms or mutations are indicated by arrow, 5 novel variants are shown by arrow plus pentagon.
H.-Y. Zhu et al. / Clinica Chimica Acta 410 (2009) 64–69 Table 2 Sequence analysis of mitochondrial tRNA genes in 270 hypertensives. Position tRNAs 3254 3277 3278 3290 4263 4277 4295 4314 4316 4317 4343 4345 4353 4363 4387 4388 4392 4395 4410 4435 4454 4456 8311 8337 8347 8348 a
tRNALeu(UUR) tRNALeu(UUR) tRNALeu(UUR) tRNALeu(UUR) I I I I I I Q Q Q Q Q Q Q Q M M M M K K K K
Replacement Conservation Previously Hypertensives (n = 270) (H/B/M/X a) reported C to T G to A T to C T to C A to G T to C A to G T to C A to TA A to G A to G C to T T to C T to C C to A A to G C to T A to G C to A A to G T to C C to C/T T to C T to C A to G A to G
C//T/T/T G/T/C/A T/C/C/T T/A/A/A A/A/A/A T/C/T/– A/A/A/A T/C/C/C A/A/A/A A/A/G/A A/A/A/A C/C/T/T T/C/C/C T/T/T/A C/T/A/T A/A/A/A C/C/C/C A/A/A/T C/C/C/C A/A/A/A T/T/A/A C/T/T/C T/T/G/T T/T/A/T A/A/A/C A/T/A/A
Yes Yes Yes Yes No No Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes No Yes Yes Yes No Yes Yes Yes
2 3 1 10 1 1 2 3 1 1 3 1 1 3 1 1 1 1 1 1 4 1 1 1 1 3
H/B/M/X means Human/Bovine/Mouse/Xenopus.
evaluation by phylogenetic analysis of these variants and sequences from other organisms including mouse, bovine and xenopus laevis showed evolutionary conservation in positions 4263, 4295, 4316, 4343, 4388, 4392, 4410 and 4435. Over 24 overlapping fragments spanning through the entire mitochondrial DNA, mtDNAs harboring variants A4263G and T4454C were PCR amplified, sequenced and compared with the Cambridge consensus sequence. A series of nucleotide changes were detected (Table 3). In mtDNA with variant A4263G, there were 10 polymorphisms in the D-loop region, 2 variants in the 12S rRNA gene, 2 variants in the 16S rRNA gene, and 6 variants in protein-encoding genes. In mtDNA with variant T4454C, there were 5 polymorphisms in the D-loop region, 2 variants in the 12S rRNA gene, 2 variants in the 16S rRNA gene, 10 variants in protein-encoding genes. Except A4263G, C3435T, C3497T, A7076G, C8609T were novel, other variants were previously reported. The overall odds ratio for mitochondria tRNA variants in hypertensives compared to controls is 5.23 (95% CI 2.27, 12.05). 3.3. Measurement of oxygen consumption rate To measure the endogenous respiration rates of mutant cell lines, oxygen consumption rates in intact mutant cells derived from lymphoblastoid cell lines were investigated using the classic method described previously [20]. The oxygen consumption rate in cells harboring homogenetic variants T4454C (P = 0.0010) and A4263G (P = 0.0001) decreased as compared to the average level of control cell lines (Fig. 2, Table 4). The other cells were at the same level compared to the controls (P N 0.05). 4. Discussion In our study, environmental factors including age, gender, BMI, fasting blood sugar and blood lipids, contributing a lot to high blood pressure [21,22] did not exhibit difference between cases and controls, as seen in Table 1. It was presumed that hereditary factors may associate with the pathogenesis of hypertension in the Chinese Han population. Actually, 26 mitochondrial tRNA base pair changes
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were detected in the hypertensives and these variants were not found in 270 controls. Of them, 21 were previously reported polymorphisms and pathologic-related mutations. They may lead to deficiencies in tRNA 3′ end metabolism (3′ end cleavage, CCA addition and aminoacylation) and/or impairment of critical subunits of the respiratory chain, which help understanding of molecular mechanisms of mitochondrial tRNA genes in essential hypertension as follows: C3254T and A8348G mildly reduced the efficiency of aminoacyl-tRNA synthetase (aaRS) [23]; A4295G reduced efficiency of tRNA 3′-processing endoribonuclease (tRNAseZ) and aaRS [24]; A4317G reorganized T-stem which markedly reduced the efficiency of tRNA 3′-processing endoribonuclease tRNAseZ, aaRS, and tRNA nucleotidyl transferase (CCAse) [25]. The other possible effects of tRNA mutations on tRNA modifications may be the reduction of efficiency of the binding properties of aminoacyl-tRNAs to mitochondrial elongation factor Tu (Ef–Tu) [26]. All of the above variants influenced the process of aminoacylation which affected tRNA metabolism and impaired the synthesis of protein ultimately. Most importantly, eight of these variants were highly conserved from bacteria to human beings (A4263G, A4295G, A4316TA, A4343G, A4388G, C4392T, C4410A and A4435G) thus minor changes in charge or structure may result in impairment of translation and affect the synthesis of protein. In addition, A4295G, A4435G and T4363C all localized at the 3′ end adjacent to the anticodon, which contributed to the stabilization of structure thus lead to the decrease of tRNA metabolism. Otherwise, a novel variant A4263G at the initial part of tRNAIle may influence the transcription of tRNA herein affect the steady-level of protein synthesis. It also possible that such mutations could affect the tRNAs correct decoding of the mRNA in the ribosome (by increase in frameshifting for example) and, particularly in the case of T4363C, could also interfere with aminoacylation, since glutaminyl-tRNA synthetases are known to interrogate the anticodon loop of tRNAGln during catalysis [27]. And some other novel tRNA variants (T4277C, T4353C, C4410A, T8311C) in tRNALeu(UUR), tRNAIle, tRNAMet, tRNAGln and tRNALys need to be further investigated in the future. Interestingly, functional analysis found significant reduction of oxygen consumption rate in two mutant cell lines carrying variants T4454C and A4263G. Oxygen consumption is a classical means of assessing energy expenditure, one component of energy balance [28,29]. Cells and organisms are able to trigger an adaptive response to hypoxic conditions that is aimed to help them to cope with these threatening conditions [30]. Failing to keep the balance of oxygen consumption and production may cause cardiovascular diseases and is of particular significance in the pathogenesis of essential hypertension. A variant in mitochondrial tRNAIle has been identified in a single family which segregated with hypertension and appeared causal [14]. Interestingly, we deduced that except the A4263G and T4454C variants, there were other amino acid changes that may contribute to hypertension by entire mitochondrial genome sequencing of the cells, described in Table 3. This finding would suggest that there may be a threshold effect with some of these mtDNA variants. Such that often variability at a single locus will not be sufficient to increase hypertension risk. This is consistent with previous work that essential hypertension is controlled by multiple genetic loci, each with a relatively weak effect in the population at large [31]. In conclusion, we reported a systematic screen for mitochondrial tRNA variants and their effect on Chinese Han essential hypertension. In the Chinese Han hypertensives, variants located in mitochondrial tRNA genes have biologic plausibility to implicate in the pathogenesis of the disease. Acknowledgements Authors thank the participants of the investigation and sophisticated clinical-data collecting of Chunlin Zeng. The work was supported by grants from Chinese National Natural Science Funds (Capital Developmental Grant—Key Project No: 2003-2019).
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Table 3 Full sequence analysis of mtDNAs harboring variants T4454C or A4263G. Gene
Position
Replacement
D-loop
73 263 310 489 514 515 16223 16260 16362 16519 750 1438 2706 3010 3435 3497 4454 4263 4491 4769 4883 5178 7028 7076 8271 8271-79 8609 8701 8860 9540 10398 10400 10873 11696 11719 12705 13759 13928 14305 14668 14783 15043 15301 15326
A to G A to G T to CTC T to C Del C Del A C to T C to T T to C T to C A to G A to G A to G G to A C to T C to T T to C A to G G to A A to G C to T C to A C to T A to G ACCCCCTCTA to A 9-bp del C to T A to G A to G T to C A to G C to T T to C G to A G to A C to T G to A G to C G to A C to T T to C G to A G to A A to G
12SrRNA 16SrRNA ND1 tRNAMet tRNAIle ND2
CO1 NC7 A6
CO3 ND3 ND4
ND5
ND6 CYTB
a
Amino acid changement
Conservation (H/B/M/X)a
A/A/A/G A/G/A/A G/G/A/A Ala to Val
Val to Ile
Leu to Met
A/A/L/S T/A/A/A A/A/A/A V/I/I/V
L/T/T/T
Pro to Leu Thr to Ala Thr to Ala
P/S/S/F T/S/L/Q T/A/A/T
Thr to Ala
T/T/T/A
Val to Ile
V/T/T/M
Ala to Thr Ser to Thr
A/T/T/I S/T/S/T
Thr to Ala
Previously reported Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes No Yes Yes Yes Yes Yes No Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
T/M/I/I
MtDNA harboring T4454C
MtDNA harboring A4263G G G CTC C Del C Del A T T C C G G G A
G CTC C
T C G G G A T T C
G A G T A T
G T A T G
A 9-bp del T G G C G T C A A T
G C G T C A A A C
A T C
T C A
A G
G
Conservation of amino acid for polypeptides or nucleotide for rRNAs in human (H), bovine (B), mouse (M), and Xenopus laevis (X).
Table 4 Oxygen consumption rate in lymphoblastoid cell lines carrying mutation and the control cell lines, relative to mean of controls (%). Mutant cell linesa
Control cell lines 1
2
3
4b
5c
103.61 ± 7.51
97.08 ± 16.8
93.1 ± 15.01
81.44 ± 5.15
60.31 ± 9.28
Numbers 1, 2, 3 represent control cell lines; 4 represents the cell line harboring mutation T4454C; 5 indicates the cell line harboring A4263G. a P-value 0.0001, significant difference between the mutant cell lines and the controls. b P-value 0.0010, significant difference between the mutant cell line harboring the variant T4454C and the controls. c P-value 0.0001, significant difference between the mutant cell line harboring the variant A4263G and the controls.
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Fig. 2. Average rates of endogenous oxygen consumption per cell measured in different lymphoblastoid cell lines are shown, with error bars representing standard errors of the mean (SEM). The six determinations were made for each cell line. Numbers 1,2,3 represent control cell lines; 4 represents the cell line harboring variant T4454C; 5 indicates the cell line harboring A4263G.
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Genetic variants in mitochondrial tRNA genes are associated with essential hypertension in a Chinese Han population Hai-Yan Zhu a,b, Shi-Wen Wang a,⁎, Li Liu c, Rui Chen a, Lin Wang a, Xing-Lai Gong d, Min-Lu Zhang e a
Institute of Geriatric Cardiology, General Hospital of Chinese PLA, Beijing 100853, China Emergency Department, General Hospital of Chinese PLA, Beijing 100853, China Department of National Center for Cardiovascular Disease, Cardiovascular Institute and Fuwai Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100037, China d Department of Mathematical Sciences, University of Cincinnati, Cincinnati, OH 45221-0025, USA e Department of Computer Science, University of Cincinnati, Cincinnati, OH 45221-0030, USA b c
a r t i c l e
i n f o
Article history: Received 20 July 2009 Received in revised form 9 September 2009 Accepted 10 September 2009 Available online 22 September 2009 Keywords: Essential hypertension Mitochondrial tRNA genes Pathogenesis
a b s t r a c t Background: Of multiple factors contributing to essential hypertension, mitochondrial variants exhibited the trends for serving as molecular and genetic markers for the disease in last five years. However, previous studies focused on African-American or Caucasian pedigrees, knowledge of mitochondrial tRNA genes and population-based Chinese hypertensives were limited. Methods: We performed sequence analysis in tRNA genes, hot spots for cardiovascular diseases, in 270 Chinese Han essential hypertensives and 270 controls. Lymphoblastoid cell lines were immortalized by transformation with the Epstein–Barr virus. Rates of oxygen consumption in intact cells were determined with a YSI 5300 oxygraph (Yellow Springs Instruments) on samples, harboring variants in tRNA genes. Results: There were 26 variants in tRNA genes that were found in hypertensives and these variants were not in controls. Functional analysis found that these variants may lead to deficiencies in tRNA 3′ end metabolism and/or impairment of critical subunits of the respiratory chain. Most importantly, the oxygen consumption rate in cells harboring variants T4454C (P = 0.0010) and A4263G (P = 0.0001) decreased as compared to the average level of control cell lines. Conclusions: Variants located in mitochondrial tRNA genes may have biologic plausibility to implicate in the pathogenesis of Chinese essential hypertension. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Essential hypertension has been a common concern for years in China and in the world due to its high morbidity and severe outcomes of complications including heart failure, cardiac arrhythmia and left ventricular hypertrophy, etc [1,2]. While the nuclear genome has been studied extensively with respect to hypertension, few studies have examined the impact of mitochondrial genomic variants in essential hypertension [3–5]. Mitochondria are important in many diseases as they supply greater than 90% of the energy to the human body through oxidative phosphorylation, produce reactive oxidative species harmful to cells and induce apoptosis [6]. In particular, mitochondrial variants exhibited the trends for serving as molecular and genetic markers for essential hypertension in last five years [7–9]. In mitochondrial genome, more than 50% of the characterized pathology-related mtDNA mutations concentrate within the tRNA ⁎ Corresponding author. Institute of Geriatric Cardiology, General Hospital of Chinese PLA, 28 Fuxing Road, Beijing 100853, China. Tel.: +86 10 66936761; fax: +86 10 88270497. E-mail address: [email protected] (S.-W. Wang). 0009-8981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2009.09.023
genes which were of less than 10% of the whole genome [10]. These tRNA genes have the essential role in the synthesis of proteins involved in energy metabolism. So far, 139 pathogenic and 243 polymorphic mitochondrial tRNA variants, have been described and they have become the foreground of numerous case reports [10]. Herein, mitochondrial tRNA genes were of particular significance in the pathogenesis of hypertension. It is worth mentioning that five tRNA genes (tRNALeu(UUR), tRNALys, tRNAMet, tRNAGln and tRNAIle) were hot spots for cardiovascular diseases, as described previously [10,11]. However, previous studies focused on nucleic genes and family-based mitochondrial screening in Caucasian and AfricanAmerican pedigrees, knowledge of mitochondrial tRNA genes and population-based Chinese hypertensives was limited [6,7,12–14]. Thus, our objective was to determine the relationship between mitochondrial tRNA genes and Chinese essential hypertension. We performed a systematic and extensive screening of mitochondrial genes in 3 regions, including tRNALeu(UUR), tRNALys, tRNAMet, tRNAGln and tRNAIle in a Chinese Han cohort. We focused on this Chinese Han population because of the high morbidity of essential hypertension in Chinese adults (nearly 11.8%) [2] and the limited amount of research on this racial group. We used a population instead of family-based
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strategy for two reasons: 1) Large numbers of hypertensives without family history are not detected in China, and they might be overlooked for lacking of medical knowledge and regular checks. 2) The morbidity of essential hypertension is not totally family-based; 50–60% of hypertensives are population-based distributed [2].
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typically harvested for RNA isolation 3 days after a split while the cultures were in logarithmic growth phase. Cell lines derived from 26 probands harboring variants in tRNA genes, respectively, and three genetically unrelated control individuals (1,2,3), grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS).
2. Methods 2.5. Oxygen consumption measurements 2.1. Patients We evaluated 270 individuals with hypertension; all participants were 1) Beijing residents who were out-patients or in-patients at the Institute of Geriatric Cardiology, Chinese People's Liberation Army (PLA) General Hospital from January 2006 to January 2007; 2) N18 y; 3) diagnosed as primary hypertension. Exclusion criteria: Participants were diagnosed as 1) secondary hypertension (primary aldosteronism, renal arterial sterosis, aortic coarctation, etc.); 2) congenital heart diseases; 3) organic valve diseases. All participants were Han Chinese evaluated and underwent a thorough examination including medical record review, clinical evaluation, biochemical assay as well as genetic analysis. The protocol was approved by the Ethics Committee of Chinese PLA General Hospital and Institute Review Board of Cincinnati Children's Hospital Medical Center; all participants gave informed consent. Controls were healthy Beijing residents who accepted annual checkup in Chinese People's Liberation Army (PLA) General Hospital from January 2006 to January 2007. None of the control subjects had a family history of hypertension, and all had a SBP of b130 mm Hg and a DBP of b85 mm Hg. 2.2. Blood pressure assessment Blood pressure was measured in triplicate by a single physician who was expert in the evaluation of hypertension, with an appropriate arm cuff and a mercury sphygmomanometer with the subject in sitting position after 5 min rest. The arithmetic mean of the last 2 measurements was calculated. Korotkoff phase I was taken for systolic BP and V for diastolic BP. Hypertension was defined as systolic BP (SBP) ≥140 mm Hg or diastolic BP (DBP) ≥90 mm Hg on 3 consecutive day according to the Seventh Report of the Joint National Committee [1].
Rates of oxygen consumption in intact cells were determined with YSI 5300 oxygraph (Yellow Springs Instruments) on samples, harboring variants in tRNA genes. Nearly 1 × 107 cells of each sample were suspended in 1.5 ml of special Dulbecco modified Eagel medium (DMEM) lacking glucose, supplemented with 10% dialyzed fetal bovine serum (FBS) [17]. Each cell was measured 6 times. 2.6. Computer analysis Continuous variables were expressed as mean ± SD. Discrete variables in groups were expressed as frequency. Relations between continuous variables were assessed by the unpaired, 2-tailed Student's t-test, and discrete variables were analyzed by Pearson's χ2 test, using the software Stata 7.0 (StataCorp, College Station, TX), P b 0.05 was considered statistically different. 3. Results 3.1. Clinical evaluation Descriptive information for the 270 age and gender balanced cases–controls was provided in Table 1. The average age (66.6 ± 11.4 vs 65.1 ± 17.5 y, P = NS), gender (33.7% vs. 29.6%, P = 0.NS) and body mass index (24.54 ± 2.90 vs. 24.16 ± 2.83, P = NS) were similar to the controls. Biochemical values including fast blood sugar (5.31 ± 0.98 vs. 5.42 ± 1.35, P = NS), total cholesterol (4.70 ± 0.99 vs. 4.90 ± 1.11, P = 0.05), triglyceride (1.59 ± 1.01 vs. 1.50 ± 0.56, P = NS), high density lipoprotein (1.31 ± 0.29 vs. 1.32 ± 0.30, P = NS) and low density lipoprotein (2.54 ± 0.78 vs. 2.61 ± 0.82, P = NS) didn't exhibit any difference between the 2 groups. 3.2. Mitochondrial DNA analysis
2.3. Mitochondrial DNA analysis DNA was isolated from whole blood using Promega Wizard® Genomic DNA Purification Kit (Madison, WI), 3 fragments: 3150–3600 (including tRNALeu(UUR)), 3796–4654 (including tRNAIle, tRNAMet and tRNAGln), 7937–8797 (including tRNALys) were purified. The entire mitochondrial genome of DNA harboring variants A4263G and T4454C were PCR amplified in 24 overlapping fragments, by using sets of the light (L) strand and the heavy (H) strand oligonucleotide primers and subsequently analyzed by direct sequencing in an ABI 3700 automated DNA sequencer, using the BigDye Terminator Cycle sequencing reaction kit and analyzed with SeqWeb program GAP (GCG) according to the updated consensus Cambridge sequence [15]. The primers for 3 fragments are as follows: 3243F: 5′-AGGACAAGAGAAATAAGGCCT-3′, 3243R: 5′-TAAATAGGAGGCCTAGGTTG-3′, 6F: 5′TGCTCCTTTAACCTCTCCA-3′, 6R: 5′-AAGGATTATGGATGCGGTTG-3′, 12F: 5′-ACGAGTACACCGACTACGGC-3′, 12R: 5′-TGGGTGGTTGGTGTAAATGA3′. 2.4. Cell cultures Lymphoblastoid cell lines were immortalized by transformation with the Epstein–Barr virus [16]. Briefly, cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, and 1% penicillin/ streptomycin. Cultures were split 1:2 every 3–4 days and cells were
A list of pathogenic variants was taken from MitoMap (http:// www.mitomap.org/) and a list of polymorphisms from mtDB [18,19]. In the 270 hypertensives, 26 changes in tRNA genes (tRNALeu(UUR), tRNALys, tRNAMet, tRNAGln and tRNAIle) were found and these variants were not in controls (Fig. 1). Except for C4456T, other variants were homoplasmic changes including 5 novel base pair substitutions (A4263G, T4277C, T4353C, C4410A, and T8311C). The frequency of the variants varied from 1/270 to 10/270. As seen in Table 2, further
Table 1 Clinical manifestations of the hypertensives and the controls.
Women, n (%) Age, y Body mass index, kg/m2 Systolic blood pressure, mm Hg Diastolic blood pressure, mm Hg Fasting blood glucose, mmol/l Total cholesterol, mmol/l Triglyceride, mmol/l High density lipoprotein, mmol/l Low density lipoprotein, mmol/l a
Represents statistically significant.
Hypertensives
Controls
(n = 270)
(n = 270)
91 (33.7) 66.56 ± 11.39 24.54 ± 2.90 135.39 ± 16.45 79.09 ± 11.74 5.31 ± 0.98 4.70 ± 0.99 1.59 ± 1.01 1.31 ± 0.29 2.54 ± 0.78
80 (29.6) 65.07 ± 17.46 24.16 ± 2.83 120.56 ± 12.03 74.45 ± 6.47 5.42 ± 1.35 4.90 ± 1.11 1.50 ± 0.56 1.32 ± 0.30 2.61 ± 0.82
P-value
NS NS NS 0.0001a 0.0001a NS 0.05 NS NS NS
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Fig. 1. Mitochondrial tRNA variants in 270 Chinese Han hypertensives. Of 26 variants in tRNA genes (tRNALeu(UUR), tRNALys, tRNAMet, tRNAGln and tRNAIle), 21 previously reported polymorphisms or mutations are indicated by arrow, 5 novel variants are shown by arrow plus pentagon.
H.-Y. Zhu et al. / Clinica Chimica Acta 410 (2009) 64–69 Table 2 Sequence analysis of mitochondrial tRNA genes in 270 hypertensives. Position tRNAs 3254 3277 3278 3290 4263 4277 4295 4314 4316 4317 4343 4345 4353 4363 4387 4388 4392 4395 4410 4435 4454 4456 8311 8337 8347 8348 a
tRNALeu(UUR) tRNALeu(UUR) tRNALeu(UUR) tRNALeu(UUR) I I I I I I Q Q Q Q Q Q Q Q M M M M K K K K
Replacement Conservation Previously Hypertensives (n = 270) (H/B/M/X a) reported C to T G to A T to C T to C A to G T to C A to G T to C A to TA A to G A to G C to T T to C T to C C to A A to G C to T A to G C to A A to G T to C C to C/T T to C T to C A to G A to G
C//T/T/T G/T/C/A T/C/C/T T/A/A/A A/A/A/A T/C/T/– A/A/A/A T/C/C/C A/A/A/A A/A/G/A A/A/A/A C/C/T/T T/C/C/C T/T/T/A C/T/A/T A/A/A/A C/C/C/C A/A/A/T C/C/C/C A/A/A/A T/T/A/A C/T/T/C T/T/G/T T/T/A/T A/A/A/C A/T/A/A
Yes Yes Yes Yes No No Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes No Yes Yes Yes No Yes Yes Yes
2 3 1 10 1 1 2 3 1 1 3 1 1 3 1 1 1 1 1 1 4 1 1 1 1 3
H/B/M/X means Human/Bovine/Mouse/Xenopus.
evaluation by phylogenetic analysis of these variants and sequences from other organisms including mouse, bovine and xenopus laevis showed evolutionary conservation in positions 4263, 4295, 4316, 4343, 4388, 4392, 4410 and 4435. Over 24 overlapping fragments spanning through the entire mitochondrial DNA, mtDNAs harboring variants A4263G and T4454C were PCR amplified, sequenced and compared with the Cambridge consensus sequence. A series of nucleotide changes were detected (Table 3). In mtDNA with variant A4263G, there were 10 polymorphisms in the D-loop region, 2 variants in the 12S rRNA gene, 2 variants in the 16S rRNA gene, and 6 variants in protein-encoding genes. In mtDNA with variant T4454C, there were 5 polymorphisms in the D-loop region, 2 variants in the 12S rRNA gene, 2 variants in the 16S rRNA gene, 10 variants in protein-encoding genes. Except A4263G, C3435T, C3497T, A7076G, C8609T were novel, other variants were previously reported. The overall odds ratio for mitochondria tRNA variants in hypertensives compared to controls is 5.23 (95% CI 2.27, 12.05). 3.3. Measurement of oxygen consumption rate To measure the endogenous respiration rates of mutant cell lines, oxygen consumption rates in intact mutant cells derived from lymphoblastoid cell lines were investigated using the classic method described previously [20]. The oxygen consumption rate in cells harboring homogenetic variants T4454C (P = 0.0010) and A4263G (P = 0.0001) decreased as compared to the average level of control cell lines (Fig. 2, Table 4). The other cells were at the same level compared to the controls (P N 0.05). 4. Discussion In our study, environmental factors including age, gender, BMI, fasting blood sugar and blood lipids, contributing a lot to high blood pressure [21,22] did not exhibit difference between cases and controls, as seen in Table 1. It was presumed that hereditary factors may associate with the pathogenesis of hypertension in the Chinese Han population. Actually, 26 mitochondrial tRNA base pair changes
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were detected in the hypertensives and these variants were not found in 270 controls. Of them, 21 were previously reported polymorphisms and pathologic-related mutations. They may lead to deficiencies in tRNA 3′ end metabolism (3′ end cleavage, CCA addition and aminoacylation) and/or impairment of critical subunits of the respiratory chain, which help understanding of molecular mechanisms of mitochondrial tRNA genes in essential hypertension as follows: C3254T and A8348G mildly reduced the efficiency of aminoacyl-tRNA synthetase (aaRS) [23]; A4295G reduced efficiency of tRNA 3′-processing endoribonuclease (tRNAseZ) and aaRS [24]; A4317G reorganized T-stem which markedly reduced the efficiency of tRNA 3′-processing endoribonuclease tRNAseZ, aaRS, and tRNA nucleotidyl transferase (CCAse) [25]. The other possible effects of tRNA mutations on tRNA modifications may be the reduction of efficiency of the binding properties of aminoacyl-tRNAs to mitochondrial elongation factor Tu (Ef–Tu) [26]. All of the above variants influenced the process of aminoacylation which affected tRNA metabolism and impaired the synthesis of protein ultimately. Most importantly, eight of these variants were highly conserved from bacteria to human beings (A4263G, A4295G, A4316TA, A4343G, A4388G, C4392T, C4410A and A4435G) thus minor changes in charge or structure may result in impairment of translation and affect the synthesis of protein. In addition, A4295G, A4435G and T4363C all localized at the 3′ end adjacent to the anticodon, which contributed to the stabilization of structure thus lead to the decrease of tRNA metabolism. Otherwise, a novel variant A4263G at the initial part of tRNAIle may influence the transcription of tRNA herein affect the steady-level of protein synthesis. It also possible that such mutations could affect the tRNAs correct decoding of the mRNA in the ribosome (by increase in frameshifting for example) and, particularly in the case of T4363C, could also interfere with aminoacylation, since glutaminyl-tRNA synthetases are known to interrogate the anticodon loop of tRNAGln during catalysis [27]. And some other novel tRNA variants (T4277C, T4353C, C4410A, T8311C) in tRNALeu(UUR), tRNAIle, tRNAMet, tRNAGln and tRNALys need to be further investigated in the future. Interestingly, functional analysis found significant reduction of oxygen consumption rate in two mutant cell lines carrying variants T4454C and A4263G. Oxygen consumption is a classical means of assessing energy expenditure, one component of energy balance [28,29]. Cells and organisms are able to trigger an adaptive response to hypoxic conditions that is aimed to help them to cope with these threatening conditions [30]. Failing to keep the balance of oxygen consumption and production may cause cardiovascular diseases and is of particular significance in the pathogenesis of essential hypertension. A variant in mitochondrial tRNAIle has been identified in a single family which segregated with hypertension and appeared causal [14]. Interestingly, we deduced that except the A4263G and T4454C variants, there were other amino acid changes that may contribute to hypertension by entire mitochondrial genome sequencing of the cells, described in Table 3. This finding would suggest that there may be a threshold effect with some of these mtDNA variants. Such that often variability at a single locus will not be sufficient to increase hypertension risk. This is consistent with previous work that essential hypertension is controlled by multiple genetic loci, each with a relatively weak effect in the population at large [31]. In conclusion, we reported a systematic screen for mitochondrial tRNA variants and their effect on Chinese Han essential hypertension. In the Chinese Han hypertensives, variants located in mitochondrial tRNA genes have biologic plausibility to implicate in the pathogenesis of the disease. Acknowledgements Authors thank the participants of the investigation and sophisticated clinical-data collecting of Chunlin Zeng. The work was supported by grants from Chinese National Natural Science Funds (Capital Developmental Grant—Key Project No: 2003-2019).
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Table 3 Full sequence analysis of mtDNAs harboring variants T4454C or A4263G. Gene
Position
Replacement
D-loop
73 263 310 489 514 515 16223 16260 16362 16519 750 1438 2706 3010 3435 3497 4454 4263 4491 4769 4883 5178 7028 7076 8271 8271-79 8609 8701 8860 9540 10398 10400 10873 11696 11719 12705 13759 13928 14305 14668 14783 15043 15301 15326
A to G A to G T to CTC T to C Del C Del A C to T C to T T to C T to C A to G A to G A to G G to A C to T C to T T to C A to G G to A A to G C to T C to A C to T A to G ACCCCCTCTA to A 9-bp del C to T A to G A to G T to C A to G C to T T to C G to A G to A C to T G to A G to C G to A C to T T to C G to A G to A A to G
12SrRNA 16SrRNA ND1 tRNAMet tRNAIle ND2
CO1 NC7 A6
CO3 ND3 ND4
ND5
ND6 CYTB
a
Amino acid changement
Conservation (H/B/M/X)a
A/A/A/G A/G/A/A G/G/A/A Ala to Val
Val to Ile
Leu to Met
A/A/L/S T/A/A/A A/A/A/A V/I/I/V
L/T/T/T
Pro to Leu Thr to Ala Thr to Ala
P/S/S/F T/S/L/Q T/A/A/T
Thr to Ala
T/T/T/A
Val to Ile
V/T/T/M
Ala to Thr Ser to Thr
A/T/T/I S/T/S/T
Thr to Ala
Previously reported Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes No Yes Yes Yes Yes Yes No Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
T/M/I/I
MtDNA harboring T4454C
MtDNA harboring A4263G G G CTC C Del C Del A T T C C G G G A
G CTC C
T C G G G A T T C
G A G T A T
G T A T G
A 9-bp del T G G C G T C A A T
G C G T C A A A C
A T C
T C A
A G
G
Conservation of amino acid for polypeptides or nucleotide for rRNAs in human (H), bovine (B), mouse (M), and Xenopus laevis (X).
Table 4 Oxygen consumption rate in lymphoblastoid cell lines carrying mutation and the control cell lines, relative to mean of controls (%). Mutant cell linesa
Control cell lines 1
2
3
4b
5c
103.61 ± 7.51
97.08 ± 16.8
93.1 ± 15.01
81.44 ± 5.15
60.31 ± 9.28
Numbers 1, 2, 3 represent control cell lines; 4 represents the cell line harboring mutation T4454C; 5 indicates the cell line harboring A4263G. a P-value 0.0001, significant difference between the mutant cell lines and the controls. b P-value 0.0010, significant difference between the mutant cell line harboring the variant T4454C and the controls. c P-value 0.0001, significant difference between the mutant cell line harboring the variant A4263G and the controls.
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Fig. 2. Average rates of endogenous oxygen consumption per cell measured in different lymphoblastoid cell lines are shown, with error bars representing standard errors of the mean (SEM). The six determinations were made for each cell line. Numbers 1,2,3 represent control cell lines; 4 represents the cell line harboring variant T4454C; 5 indicates the cell line harboring A4263G.
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