Maternally Inherited Essential Hypertension Is Associated With the Novel

Maternally Inherited Essential Hypertension Is Associated With the Novel 4263A>G Mutation in the Mitochondrial trna Ile Gene in a Large Han Chinese Family Shiwen Wang, Ronghua Li, Andrea Fettermann, Zongbin Li, Yaping Qian, Yuqi Liu, Xinjian Wang, Anna Zhou, Jun Qin Mo, Li Yang, Pingping Jiang, Andreas Taschner, Walter Rossmanith and Min-Xin Guan Circ. Res. 2011;108;862-870 DOI: 10.1161/CIRCRESAHA.110.231811 Circulation Research is published by the American Heart Association. 7272 Greenville Avenue, Dallas, TX 72514 Copyright 2011 American Heart Association. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/cgi/content/full/108/7/862 Subscriptions: Information about subscribing to Circulation Research is online at http://circres.ahajournals.org/subscriptions/ Permissions: Permissions & Rights Desk, Lippincott Williams & Wilkins, a division of Wolters Kluwer Health, 351 West Camden Street, Baltimore, MD 21202-2436. Phone: 410-528-4050. Fax: 410-528-8550. E-mail: journalpermissions@lww.com Reprints: Information about reprints can be found online at http://www.lww.com/reprints

Maternally Inherited Essential Hypertension Is Associated With the Novel 4263A>G Mutation in the Mitochondrial trna Ile Gene in a Large Han Chinese Family Shiwen Wang,* Ronghua Li, Andrea Fettermann, Zongbin Li, Yaping Qian, Yuqi Liu, Xinjian Wang, Anna Zhou, Jun Qin Mo, Li Yang, Pingping Jiang, Andreas Taschner, Walter Rossmanith, Min-Xin Guan* Rational: Despite maternal transmission of hypertension in some pedigrees, pathophysiology of maternally inherited hypertension remains poorly understood. Objective: To establish a causative link between mitochondrial dysfunction and essential hypertension. Method and Results: A total of 106 subjects from a large Chinese family underwent clinical, genetic, molecular, and biochemical evaluations. Fifteen of 24 adult matrilineal relatives exhibited a wide range of severity in essential hypertension, whereas none of the offspring of affected fathers had hypertension. The age at onset of hypertension in the maternal kindred varied from 20 years to 69 years, with an average of 44 years. Mutational analysis of their mitochondrial genomes identified a novel homoplasmic 4263A>G mutation located at the processing site for the trna Ile 5 -end precursor. An in vitro processing analysis showed that the 4263A>G mutation reduced the efficiency of the trna Ile precursor 5 -end cleavage catalyzed by RNase P. trna Northern analysis revealed that the 4263A>G mutation caused 46% reduction in the steady-state level of trna Ile.Anin vivo protein-labeling analysis showed 32% reduction in the rate of mitochondrial translation in cells carrying the 4263A>G mutation. Impaired mitochondrial translation is apparently a primary contributor to the reductions in the rate of overall respiratory capacity, malate/glutamate-promoted respiration, succinate/glycerol- 3-phosphate-promoted respiration, or N,N,N,N -tetramethyl-p-phenylenediamine/ascorbate promoted respiration and the increasing level of reactive oxygen species in cells carrying the 4263A>G mutation. Conclusions: These data provide direct evidence that mitochondrial dysfunction caused by mitochondrial trna Ile 4263A>G mutation is involved in essential hypertension. Our findings may provide new insights into pathophysiology of maternally transmitted hypertension. (Circ Res. 2011;108:862-870.) Key Words: genetics hypertension mitochondria transcription processing Hypertension is a major public health problem, affecting approximately 1 billion worldwide. 1 The etiology of hypertension is not well understood because it is often a multifactorial condition. Hypertension can be caused by single-gene or multifactorial conditions, resulting from interactions between the environment and inherited risk factors. 2 In fact, human hypertension is a condition associated with endothelial dysfunction and oxidative stress. 3,4 Mitochondrial dysfunction has been potentially implicated in both human and experimental hypertension. 5 7 Specifically, abnormal mitochondrial respiration results in oxidative stress, uncoupling of the oxidative pathways for ATP synthesis, and subsequent failure of cellular energetic processes. 8 An inefficient metabolism caused by mitochondrial dysfunctions in skeletal and vascular smooth muscles may lead to the elevation of systolic blood pressure and therefore may be involved in the development of hypertension. 6,7,9,10 In particular, maternal transmission of hypertension has been implicated in some pedigrees, suggesting that the mutation(s) in mitochondrial (mt)dna is one of the molecular bases for this disorder. 10 16 However, molecular pathogenesis of maternally inherited hypertension remains poorly understood. Original received September 2, 2010; revision received January 31, 2011; accepted February 1, 2011. In December 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.5 days. From the Institute of Geriatric Cardiology (S.W., Z.L., Y.L.), Chinese PLA General Hospital, Beijing, China; Divisions of Human Genetics (R.L., Y.Q., X.W., A.Z., L.Y., M.-X.G.) and Pathology (J.Q.M.), Cincinnati Children s Hospital Medical Center, OH; Institute of Genetics (P.J., M.-X.G.), College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China; Center for Anatomy and Cell Biology (A.F., A.T., W.R.), Medical University of Vienna, Austria; and Department of Pediatrics (J.Q.M., M.-X.G.), University of Cincinnati College of Medicine, OH. Both authors contributed equally to this work. Correspondence to Min-Xin Guan, PhD, Institute of Genetics, Zhejiang University, Hangzhou, Zhejiang 310058, China; E-mail gminxin88@zju.edu.cn or min-xin.guan@cchmc.org or to Shiwen Wang, MD, Institute of Geriatric Cardiology, Chinese PLA General Hospital, Beijing, China; E-mail wangshiwen4290@163.com. 2011 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.110.231811 Downloaded from circres.ahajournals.org 862 by on March 31, 2011

Wang et al Hypertension-Associated Mitochondrial DNA Mutation 863 As the part of a genetic screening program for essential hypertension in the Chinese population, 14 16 we performed clinical, genetic, molecular, and biochemical characterization of a large Han Chinese family with maternally transmitted hypertension. Fifteen of 24 adult matrilineal relatives in this 5-generation family exhibited variable severity and age at onset of hypertension. Mutational analysis of the mitochondrial genome has identified the novel 4263A G mutation in this Chinese family. This 4263A G mutation is localized at the processing site for the trna Ile 5 -end precursor, which is catalyzed by the RNase P. 17,18 The processing of precursors in mitochondrial trnas requires the precise endonucleolytic cleavage at both 5 and 3 ends. 17,18 Thus, it is anticipated that the 4263A G mutation affects the 5 -end processing of precursor in the mitochondrial trna Ile, thereby causing the mitochondrial dysfunction. Functional significance of the 4263A G mutation was evaluated by examining processing efficiency of trna Ile precursor, steady-state levels of mitochondrial trnas, including trna Ile by using lymphoblastoid cell lines derived from 3 affected matrilineal relatives carrying the 4263A G mutation and from 3 control individuals lacking the mtdna mutation. These cell lines were further assessed for the effects of the 4263A G mutation on mitochondrial protein synthesis, endogenous respiration, and substrate-dependent respiration as well as the production of reactive oxygen species (ROS). Methods An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org. Subjects A Han Chinese family (Figure 1) was ascertained at the Institute of Geriatric Cardiology of Chinese PLA General Hospital, Beijing. Informed consent, blood samples, and clinical evaluations were obtained from all participating family members, under protocols approved by the ethics committee of the Chinese PLA General Hospital and the Cincinnati Children s Hospital Medical Center Institute Review Board. Members of this family were interviewed and evaluated to identify both personal and medical histories of hypertension and other clinical abnormalities. The 342 control DNA samples were obtained from a panel of unaffected Han Chinese individuals from the same area. Members of this Chinese family underwent a physical examination, laboratory assessment of cardiovascular disease risk factors, and routine electrocardiography. A physician measured the systolic and diastolic blood pressures of subjects using a mercury column sphygmomanometer and a standard protocol. The first and the fifth Korotkoff sounds were taken as indicators of systolic and diastolic blood pressure, respectively. The average of 3 such systolic and diastolic blood pressure readings was Non-standard Abbreviations and Acronyms CCr DIG G3P IVST LVH LVMI mtdna PV ROS TMPD endogenous creatinine clearance digoxigenin succinate/glycerol-3-phosphate interventricular septal thickness left ventricular hypertrophy left ventricular mass index mitochondrial DNA premature ventricular contraction reactive oxygen species N,N,N,N -tetramethyl-p-phenylenediamine taken as the examination blood pressure. Hypertension was defined according to the recommendation of the Joint National Committee on Detection, Evaluation and Treatment of High Blood Pressure (JNC VI) 1 and the World Health Organization International Society of Hypertension 19 as a systolic blood pressure of 140 mm Hg and/or a diastolic blood pressure of 90 mm Hg. Results Clinical Presentation The proband (III-14) developed hypertension at the age of 45 years. She presented to the Geriatric Cardiology Clinic of Chinese PLA General Hospital for further clinical evaluations at the age of 48 years. Her blood pressure was 140/ 90 mm Hg. Physical examination, laboratory assessment of cardiovascular disease risk factors, and routine electrocardiography showed no other clinical abnormalities, including diabetes, vision or hearing impairments, or renal or neurological disorders. Therefore, she exhibited a typical essential hypertension. The family originated from Shanxi in Northern China. All members of this family were interviewed and/or evaluated to identify both personal and medical histories of hypertension and other clinical abnormalities. As shown in Figure 1 and the Table, 15 of 27 matrilineal relatives had a wide range of severity in hypertension (with blood pressure greater than 140/90 mm Hg, even with treatment for hypertension), whereas only 7 of 81 nonmaternal relatives had hypertension. None of the offspring of affected fathers exhibited hypertension. As shown in the Table, the age at onset of hypertension in the maternal kindred varied from 20 years to 69 years, with an average of 44 years. Notably, the average age at onset of hypertension in this family has changed from 62 years (generation II) to 46 years (generation Figure 1. The Chinese pedigree with hypertension. Affected individuals are indicated by filled symbols. Arrowhead denotes proband.

864 Circulation Research April 1, 2011 Table. Summary of Clinical Data for Some Members in a Large Chinese Pedigree Subjects Gender Age of Test (yrs) Age of Onset (yrs) Systolic Pressure (mm Hg) Diastolic Pressure (mm Hg) IVST, mm (6 12 mm) LVMI (g/m 2 ) ECG CCr (ml/min) II-2 F 79 59 152 70 11 122.9 LVH 54.9 II-4 F 72 69 150 90 12 111.1 N 172.3 II-5 M 82 65 150 70 11 98.3 N 55.1 II-7 M 68 55 180 90 13 91.4 LVH 76.7 III-2 F 54 45 170 110 11 112.9 N 163.1 III-4 F 48 46 142 90 8 80.7 N 161.1 III-5 M 42... 128 76 9 90.4 N 89.2 III-7 M 44... 130 80 9 66.1 N 86.4 III-9 M 40 36 140 100 10 91.4 N 84.5 III-11 M 56 53 155 95 10 72.6 PV 88.3 III-14* F 48 45 140 90 10 103.1 N 122.1 III-16 F 45... 120 80 9 73.4 N 151.5 III-18* F 52 52 140 90 9 212 LVH 117.9 III-19 M 33... 120 80 12 91.2 N 178.5 IV-1 F 31 27 150 100 10 74.9 N 101.5 IV-3 F 26 23 140 95 10 90.7 N 103.3 IV-5 M 25 20 145 100 11 74.9 N 102.6 IV-6 M 24... 118 76 10 79.8 N 112.4 IV-7 M 23 23 140 90 11 80.1 LVH 119.8 IV-8 F 21... 118 85 8 77.8 N 115.9 IV-21 F 24... 95 70 7 45.1 N 118.9 IV-22 F 18... 120 80 10 74.2 N 140.8 IV-23 M 25... 120 80 9 101.8 N 148.9 V-1 M 5... 100 70............ V-2 M 6... 105 65............ V-3 F 4... 95 65............ *These patients received antihypertension treatment. This table shows pretreatment blood pressures. CCr indicates endogenous creatinine clearance; F, female; IVST, interventricular septal thickness; LVH, ECG showed left ventricular hypertrophy; LVMI, left ventricular mass index; M, male; N, electrocardiography (ECG) was normal; PV, premature ventricular contraction. III) to 23 years (generation IV) (Table). There was no evidence that any member of this family had any other cause to account for hypertension. We further examined the end organ damage on the heart and kidney among 23 matrilineal relatives of this family. As shown in the Table, 4 (II-2, II-7, III-18, and IV-7) matrilineal relatives exhibited left ventricular hypertrophy on the ECG recorded, whereas subject III-11 had premature ventricular contraction. In addition, 7 matrilineal relatives exhibited an increased interventricular septal thickness. Furthermore, the clearance of endogenous creatinine was assessed in 20 matrilineal relatives and 10 control subjects. As shown in the Table, the rates of endogenous creatinine clearance in 3 subjects (II-2, II-5, and II-7) were below the standard levels, implicating the renal dysfunction in these patients. However, none of other clinical abnormalities was observed in the maternal kindred. Identification of the 4263A>G Mutation in the Mitochondrial trna Ile Gene The maternal transmission of hypertension in this family suggested mitochondrial involvement and led us to analyze the mitochondrial genome of matrilineal relatives. For this purpose, the DNA fragments spanning the entire mtdna of the proband III-14 were PCR-amplified, and each fragment was purified and subsequently analyzed by direct sequence. As shown in Online Table I, we identified 40 variants belonging to the Eastern Asian haplogroup D4j on the maternal lineage. 20 Thirty-nine variants were the known polymorphisms, 13 whereas a novel adenine-to-guanine substitution at position 4263 (4263A G) (Figure 2A) changed the stop codon TAA of the ND1 mrna to an equivalent TAG stop codon, 21 and, at the same time, caused an A-to-G transition at the 5 end of the trna Ile gene (Figure 2B). 22 In fact, the 4263A G mutation lies in the processing site for the trna Ile 5 end precursor, which is catalyzed by the RNase P 23 and is important for trna identity. 24 The processing of precursors in mitochondrial trnas requires the precise endonucleolytic cleavage at both 5 and 3 ends. 17,18 Thus, the 4263A G mutation may affect the reaction efficiency of the RNase P involved in trna Ile 5 end metabolism. To determine whether the 4263A G mutation is present in homoplasmy in all matrilineal relatives, the fragments spanning the trna Ile gene were PCR-amplified and subsequently digested with StyI because the 4263A G mutation created

Wang et al Hypertension-Associated Mitochondrial DNA Mutation 865 Figure 2. Identification and qualification of the 4263A>G mutation in the trna Ile.A,Partial sequence chromatograms of trna Ile gene from an affected individual (III-14) and a married-incontrol (III-13). Arrow indicates the base change at position 4263. B, Scheme of location of 4263A G in the precursors of trna Ile gene. Cloverleaf structure of human mitochondrial trna Ile was derived from Florentz et al (2003). 22 C, Quantification of the mtdna 4263A G mutation in 6 members of the Chinese family. PCR products were digested with StyI and analyzed by electrophoresis in a 7% polyacrylamide gel stained with ethidium bromide. Patients and control individuals are indicated. the site for this restriction enzyme. As shown in Figure 2C, there was no detectable wild type DNA in all available matrilineal relatives, indicating that the 4263A G mutation was present in homoplasmy in these matrilineal relatives. In addition, this mutation was absent in 342 Han Chinese controls and 100 affected matrilineal relatives from other 50 Chinese families with maternally transmitted essential hypertension. To examine if the 4263A G mutation affects the trna Ile coding sequence, the 5 and 3 ends of the mitochondrial trna Ile from a Chinese control subject A32 and the proband III-14 cell lines were sequenced after cdna synthesis, PCR amplification, and cloning as described elsewhere. 25 Indeed, both sequences of trna Ile carried either adenine or guanine at position at 4263, indicating that the 4263A G mutation did not affect the trna Ile coding sequence. Alteration in the 5 -End Processing of Mitochondrial trna Ile Precursor We used an in vitro processing system to determine whether the primary defect arising from the 4263A G mutation is the perturbed processing of the 5 end of the trna by RNase P. The wild-type and mutant trna Ile precursors corresponding to mtdna at positions 4235 to 4350 (Figure 3A) were prepared by in vitro transcription. Mitochondrial RNase P was reconstituted from purified recombinant proteins MRPP1, MRPP2, and MRPP3. 23 The in vitro processing kinetics of the wild-type and mutant substrates were determined as detailed elsewhere. 23 No qualitative processing alteration of the mutant trna Ile precursor was observed (data not shown). However, the processing efficiency of the mutant trna Ile transcript decreased to 70% of that of the wild type (Figure 3B). These data indicate that the 4263A G mutation has a quantitative effect on the 5 end processing of trna Ile precursor, but does not lead to any aberrant processing product. Marked Decrease in the Level of Mitochondrial trna Ile To investigate whether the impaired processing of the trna Ile precursor caused by the 4263A G mutation affects this steady-state level of trna, we subjected total mitochondrial RNA from lymphoblastoid cell lines to Northern blots and hybridized them with a digoxigenin (DIG)-labeled oligodeoxynucleotide probes specific for trna Ile and other trnas. The other probes were specific for trna Leu(UUR), trna Leu(CUN), trna Lys, and trna Met as representatives of the whole Heavy (H)-strand transcription unit and trna Ser(UCN) and trna Gln derived from the Light (L)-strand transcription unit. 17,18 As shown in Figure 4A, the amount of trna Ile in mutant cells was markedly decreased, as compared to those in control cells. For comparison, the average level of each trna in control and mutant cell lines was normalized to the average levels in the same cell line for reference 5S RNA encoded by nuclear genome. 26 As shown in Figure 4B, 46% reduction in the steady-state level of trna Ile was observed in the mutant III-14 cell line carrying the 4263A G mutation, as compared with that of a wild type A32 cell line belonging to the same mtdna haplogroup. In contrast, the average steady state levels of trna Gln, trna Leu(UUR), trna Leu(CUN), trna Ser(UCN), trna Met and trna Lys in the mutant cell line ranged from 75% to 90% of those in the wild-type cell line. Mitochondrial Protein Synthesis Defect To examine whether the failure in trna metabolism caused by the 4263A G mutation impairs mitochondrial translation, cells from cell lines derived from the proband and 2 affected matrilineal relatives carrying the 4263A G mutation and 3

866 Circulation Research April 1, 2011 Figure 3. In vitro assay for the processing of mitochondrial trna Ile precursor. A, Human mitochondrial trna Ile precursors. Twenty-eight nucleotides (nt) of 5 -end leader and 19 nt of 3 -end trailer are shown, including the 4263A G substitution. B, In vitro processing assays. Processing assays with mitochondrial RNase P were carried out in parallel for wild-type and mutant substrates. Samples were withdrawn and stopped after 4, 8, 12, 16, or 30 minutes, respectively. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis and detected by phosphor storage autoradiography. The Image-Quant program was used for relative quantification of reaction products. The relative processing efficiency was calculated from the initial phase of the reaction. Graph shows the results of a representative experiment. controls were labeled for 30 minutes with [ 35 S]methionine [ 35 S]cysteine in methionine-free regular DMEM medium in the presence of 100 g/ml of emetine which was used to inhibit cytosolic protein synthesis. 27 Figure 5A shows typical electrophoretic patterns of the mitochondrial translation products of the mutant and control cell lines. Patterns of the mtdna-encoded polypeptides of the cells carrying the 4263A G mutation were qualitatively identical to those of the control cells, in terms of electrophoretic mobility of the various polypeptides. However, cell lines carrying the 4263A G mutation trended to a decrease in the total rate of labeling of the mitochondrial translation products relative to those of the control cell line. Figure 5B shows a quantification of the results of a large number of labeling experiments and electrophoretic runs, which were carried out using Image- Quant program analysis of appropriate exposures of the fluorograms and normalization to data obtained for the 143B.TK sample. In fact, the overall rates of labeling of the mitochondrial translation products in the cell lines derived from 3 affected individuals (III-11, III-18, III-14) carrying the 4263A G mutation were decreased 25%, 30% and 40%, with an average of 32% (P 0.026) relative to the mean value measured in the control cell lines. Respiration Deficiency The endogenous respiration rates of cell lines derived from 3 affected individual (III-11, III-14, III-18) carrying the 4263A G mutation and 3 controls were measured by determining the O 2 consumption rate in intact cells, as described previously. 28 As shown in Figure 6A, the rate of total O 2 consumption in the lymphoblastoid cell lines derived from 3 affected individuals ranged between 74.9% and 80.6%, with an average reduction of 77.8% (P 0.003) relative to the mean value measured in the control cell lines. To investigate which of the enzyme complexes of the respiratory chain was affected in the mutant cell lines, O 2 consumption measurements were carried out on digitoninpermeabilized cells, using different substrates and inhibitors. 29 As shown in Figure 6B, in the cell lines derived from 3 affected individuals, the rate of malate/glutamate-driven respiration, which depends on the activities of NADH:ubiquinone oxidoreductase (complex I), ubiquinol cytochrome c reductase (complex III), and cytochrome c oxidase (complex IV), but usually reflects the rate-limiting activity of complex I, 29 was very significantly decreased, relative to the average rate in the control cell lines, by 77% to 80% ( 78% on the average; P 0.009). Similarly, the rate of succinate/glycerol- Figure 4. Northern blot analysis of mitochondrial trna. A, Equal amounts (2 g) of total mtrna samples from the various cell lines were electrophoresed through a denaturing polyacrylamide gel, were electroblotted, and were hybridized with the DIG-labeled oligonucleotide probes specific for the trna Ile, trna Gln, trna Leu(UUR), trna Leu(CUN), trna Ser(UCN), trna Met, trna Lys, and 5S RNA, respectively. B, Quantification of the trna levels. Average relative trna Ile, trna Gln, trna Leu(UUR), trna Leu(CUN), trna Ser(UCN), trna Met, and trna Lys content per cell was normalized to the average content per cell of 5S RNA in the control cell line (A32) and in the mutant cell line (III-14), respectively. The values for the latter are expressed as percentages of the average values for the control cell line. The calculations were based on 3 independent determinations in each cell line. Error bars indicate 2 SEM.

Wang et al Hypertension-Associated Mitochondrial DNA Mutation 867 Figure 5. Mitochondrial translation assay. A, Electrophoretic patterns of the mitochondrial translation products of lymphoblastoid cell lines and of 143B.TK cells labeled for 30 minutes with [ 35 S]methionine in the presence of 100 g/ml emetine. Samples containing equal amounts of protein (30 g) were run in SDS/polyacrylamide gradient gels. COI, COII, and COIII indicate subunits I, II, and III of cytochrome c oxidase; ND1, ND2, ND3, ND4, ND4L, ND5, and ND6, subunits 1, 2, 3, 4, 4L, 5, and 6 of the respiratory chain reduced nicotinamideadenine dinucleotide dehydrogenase; A6 and A8, subunits 6 and 8 of the H -ATPase; and CYTb, apocytochrome b. B, Quantification of the rates of the mitochondrial translation labeling. The rates of mitochondrial protein labeling, as detailed elsewhere, 18,27 were expressed as percentages of the value for 143B.TK in each gel, with error bars representing 2 SEMs. A total of 3 independent labeling experiments and 3 electrophoretic analyses of each labeled preparation were performed on cell lines. 3-phosphate (G3P)-driven respiration, which depends on the activities of complexes III and IV, but usually reflects the rate-limiting activity of complex III, was significantly affected in the mutant cell lines, relative to the average rate in the control cell lines, by 76% to 81% ( 78% on the average; P 0.0063). Furthermore, the rate of N,N,N,N -tetramethylp-phenylenediamine (TMPD)/ascorbate-driven respiration, which reflects the activity of complex IV, exhibited a 78% to 82% reduction in complex IV activity ( 80% on the average) in the mutant cell lines relative to the average rate in the control cell lines. ROS Production Increases The levels of the ROS generation in the vital cells derived from 3 affected matrilineal relatives carrying the 4263A G mutation and 3 control individuals lacking the mutation were measured with flow cytometry under normal and H 2 O 2 stimulation. 30,31 Geometric mean intensity was recorded to measure the rate of ROS of each sample. The ratio of geometric mean intensity between unstimulated and stimulated with H 2 O 2 in each cell line was calculated to delineate the reaction on increasing level of ROS under oxidative stress. As shown in Figure 7, the levels of ROS generation in the lymphoblastoid cell lines derived from 3 affected individuals carrying 4263A G mutation ranged from 113% and 121%, with an average 117% (P 0.0007) of the mean value measured in the control cell lines. Discussion In the present study, we performed clinical, genetic, and molecular characterizations of a five-generation large Chinese family with essential hypertension. Hypertension as the sole clinical phenotype only presented in the maternal lineage of this pedigree. In particular, the 65% penetrance of hypertension among adults in the maternal lineage was higher than those in other families with maternally transmitted hypertension. 11,15,16 This suggests that a mtdna mutation is the molecular basis for this disorder. Molecular analysis of mitochondrial genome identified a homoplasmic 4263A G mutation in the trna Ile gene. The following evidence suggests that the 4263A G mutation is a pathogenic mtdna mutation that causes a genetic predisposition to essential hypertension. This mutation is only present in the maternal lineage of this pedigree but not in other members of this family and 342 Chinese controls. The 4263A G mutation is localized at the processing site for the trna Ile 5 -end precursor, 17,18 perturbing the processing of the trna Ile 5 -end precursor. Finally, lymphoblastoid cell lines derived from 3 affected matrilineal relatives of the Chinese family carrying the 4263A G mutation, compared with 3 wild-type cell lines, exhibited marked reduction in the steady state level of affected trna Ile, impairment of mitochondrial translation and deficient respiration. All 22 human mitochondrial trnas including trna Ile are transcribed from the precursors of the H- or L-strand polycistronic transcripts. 17,32 The processing of precursors in mitochondrial trnas requires the precise endonucleolytic cleavage at both 5 and 3 ends. Extra nucleotides at their 5 termini are removed by RNase P, 17,23 whereas the excision of trnas from primary polycistronic mitochondrial transcripts at their 3 end is catalyzed by the 3 endonuclease. 17,33 Thus, it was anticipated that the A-to-G transition at position 4263 in the H-strand led to defective trna Ile 5 -end processing in the H-strand transcripts. The observation that the 4263A G mutation caused 30% reduction in the efficiency of the 5 -end processing of trna Ile precursor strongly indicated that the primary defect arising from the 4263A G mutation was the perturbed processing of the trna Ile 5 -end precursor. There is increasing evidence showing that the 5 - and 3 -end processing defects arising from pathogenic mitochondrial trna mutations could contribute to clinical abnormalities. The deafness-associated 7445T C mutation in the precursor of the trna Ser(UCN) gene and the cardiomyopathiesassociated 4269A G and 4295A G mutations in the trna Ile gene altered 3 -end processing efficiency of corresponding trnas. 34,35 Similarly, the mitochondrial encepha-

868 Circulation Research April 1, 2011 Figure 6. Respiration assays. A, Average rates of endogenous O 2 consumption per cell measured in different cell lines are shown, with error bars representing 2 SEM. A total of 4 determinations were made on each of lymphoblastoid cell lines. B, Polarographic analysis of O 2 consumption in digitonin-permeabilized cells of the various cell lines using different substrates and inhibitors. The activities of the various components of the respiratory chain were investigated by measuring on 1 10 7 digitonin-permeabilized cells the respiration dependent on malate/glutamate, on succinate/g3p and on TMPD/ascorbate. A total of 4 determinations were made on each of the lymphoblastoid cell lines. Graph details and symbols are explained in the legend to Figure 4. mal/glu indicates malate/glutamatedependent respiration; succ/g-3-p, succinate/g3p-dependent respiration; and asc/tmpd, TMPD/ascorbate-dependent respiration. lomyopathy, lactic acidosis, stroke-like symptoms (MELAS)- associated 3243A G and 3271T C mutations and mitochondrial myopathy-associated 3302A G mutation in the trna Leu(UUR) led to the trna 5 -end processing defects. 36 38 In the present investigation, 46% reduction in the steadystate level of trna Ile was observed in the mutant cell line Figure 7. The ROS production assays. The rates of production in ROS from 3 affected matrilineal relatives and 3 control individuals were analyzed by BD-LSR II flow cytometer system with or without H 2 O 2 stimulation. The relative ratio of intensity (stimulated vs unstimulated with H 2 O 2 ) was calculated. The average of 3 determinations for each cell line is shown, with error bars representing 2 SEM. carrying the 4263A G mutation, relative to that of a wild type cell line belong to the same mtdna haplogroup. The reduced level of trna Ile in cells carrying the 4263A G mutation most likely resulted from a defect in 5 -end processing of trna Ile precursor, similar to the 4401A G mutation in the junction between trna Met and trna Gln genes. 15 Alternatively, the mutant trna Ile may be metabolically less stable and more subject to degradation, thereby lowering the steady state level of trna Ile. It is interesting to note that the steady state levels of trna Gln, trna Leu(UUR), trna Leu(CUN), trna Ser(UCN), trna Met, and trna Lys in the mutant cell line reduced from 10% to 25%, as compared with those in the wild-type cell line. It is likely that the reduced level of trna Ile may mediate mitochondrial trna metabolism, thereby lowering the levels of those mitochondrial trnas, as in the case of the trna Leu(UUR) A3243G mutation. 38 Furthermore, the mutation in TRMU, encoding a 5-methylaminomethyl-2-thiouridylate-methyltransferase responsible for the biosynthesis of 5-taurinomethyl-2- thiouridine ( m5s2u) of mitochondrial trna Lys, trna Glu, and trna Gln in the wobble position, not only lowered the steady-state levels of trna Lys, trna Glu, and trna Gln but also those of other trnas, such as trna Leu(UUR), trna Ser(UCN), trna Met, and trna His. 39 A shortage of the trna Ile leads to the reduced rate of mitochondrial protein synthesis. These defects appear to be responsible for the reduced activities of the mitochondrial respiration chain. Subsequently, these defects result in the reduction of ATP production and an increase of reactive oxygen species production. These mitochondrial dysfunctions may contribute to the development of hypertension. 7,10,40 44 The homoplasmic form, mild mitochondrial dysfunction, late onset, and incomplete penetrance of hypertension observed in this Chinese family carrying the 4263A G mutation suggest that the mutation is an inherited risk factor necessary for the

Wang et al Hypertension-Associated Mitochondrial DNA Mutation 869 development of hypertension but may by itself be insufficient to produce a clinical phenotype. Indeed, the incomplete penetrance of other clinical abnormalities arises from homoplasmic mtdna mutations such as hypertensionassociated mtdna 4435A G and 4401A G mutations, 15,16 deafness-associated 12S rrna 1555A G mutation, 45 and Leber s hereditary optic neuropathy-associated ND4 11778G A mutation. 46 These homoplasmic mtdna mutations only exhibited mild mitochondrial dysfunction. 15,16,45,47 The other modifier factors such as nuclear modifier genes, environmental and epigenetic factors, and personal lifestyles 43,48 may also contribute to the development of hypertension in these subjects carrying the 4263A G mutation. In particular, the tissue specificity of this mutation is likely attributed to tissue-specific RNA processing or the involvement of nuclear modifier genes. In summary, the present study provides the clinical, genetic, molecular, and biochemical evidence that the novel mitochondrial trna Ile 4263A G mutation is associated with essential hypertension in a Chinese family. However, the tissue specificity of this mutation is likely attributable to the tissue-specific RNA processing or the contribution of nuclear modifier genes. The 4263A G mutation should be added to the list of inherited risk factors for future molecular diagnosis. Thus, our findings may provide the new insights into the understanding of pathophysiology and valuable information for management and treatment of maternally inherited hypertension. Future research should further explore the emerging link among hypertension, mitochondrial dysfunction, and their cause/effect relationship. Sources of Funding This work was supported by NIH grants R01DC05230 and R01DC07696 from the National Institute on Deafness and Other Communication Disorders (to M.X.G.), the Key Project of Natural Science Foundation of China 81030002 and 2007CB07403 from the National Key Basic Research and Development Project Fund (to S.W.), and Austrian Science Fund (FWF) grant P20213 (to W.R.). None. Disclosures References 1. Guidelines Subcommittee. 1999 World Health Organization-International Society of Hypertension Guidelines for the Management of Hypertension. Guidelines Subcommittee. J Hypertens. 1999;17:151 183. 2. Lifton RP, Gharavi AG, Geller DS. Molecular mechanisms of human hypertension. Cell. 2001;104:545 556. 3. Romero JC, Reckelhoff JF. State-of-the-Art lecture. Role of angiotensin and oxidative stress in essential hypertension. Hypertension. 1999;34: 943 949. 4. Redon J, Oliva MR, Tormos C, Giner V, Chaves J, Iradi A, Saez GT. 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Novelty and Significance What Is Known? Maternal transmission of hypertension has been observed in a subset of familial systemic arterial hypertension. Matrilineal pattern of transmission is attributable to the inheritance of mitochondria from oocyte during fertilization. Mitochondrial dysfunction may be involved in the development of systemic hypertension. What New Information Does This Article Contribute? We describe a large Chinese family with a matrilineal pattern of inheritance of hypertension and show that 65% of the adults exhibited systemic hypertension as the sole clinical phenotype. We identified a new trna Ile 4263A G mutation that perturbed the processing of the trna Ile 5 -end precursor and was associated with a lower level of trna Ile. Reduced trna Ile level was associated with impaired protein translation in the mitochondria and reduced activity of the mitochondrial respiration chain. These defects were associated with reduced ATP production and an increase in the production of reactive oxygen species. Although maternal transmission of systemic hypertension has been observed in some pedigrees, the pathophysiology of maternally inherited hypertension remains poorly understood. Here, we provide direct evidence that mitochondrial dysfunction caused by the mitochondrial trna Ile 4263A G mutation is responsible for systemic hypertension in a single large Chinese family. These data may provide new insights into the understanding of pathophysiology and valuable information for management and treatment of maternally inherited systemic arterial hypertension.