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The complete sequence of mtDNA genes in idiopathic dilated cardiomyopathy shows novel missense and tRNA mutations
Journal of Cardiac Failure Vol. 6 No. 4 2000
The Complete Sequence of mtDNA Genes in Idiopathic Dilated Cardiomyopathy Shows Novel Missense and tRNA Mutations JOSE MARIN-GARCIA, MD,* MICHAEL J. GOLDENTHAL, PhD,* RADHA ANANTHAKRISHNAN, PhD,* MARY ELLA PIERPONT, MD, PhD† Highland Park, New Jersey; Minneapolis, Minnesota
ABSTRACT Background: Previous studies have shown that mitochondrial DNA (mtDNA) mutations are often present in patients with myocardial dysfunction. We sought to assess the prevalence and significance of heart mtDNA sequence changes in patients with idiopathic dilated cardiomyopathy (DCM). Methods and Results: DNA sequence of all the transfer ribonucleic acid (tRNA), ribosomal RNA (rRNA), and structural genes in cardiac mtDNA of 28 patients with DCM was determined and compared with a control group that had no evidence of heart disease. An increased number of point mutations were found in DCM cardiac mtDNA when compared with controls. Both novel and previously reported mutations were found in mitochondrial tRNA and structural genes. One of these mutations was heteroplasmic and resulted in changing a highly conserved nucleotide in tRNAArg. Novel, heteroplasmic mtDNA mutations (n ⫽ 4) specifying changes in moderate to highly conserved amino acid residues were found in COII, COIII, ND5, and cytb. These novel mtDNA mutations were found only in patients with severe reduction in mitochondrial enzyme activities. Conclusions: Our results indicate that a high incidence of mtDNA nucleotide sequence changes in both tRNA and structural genes are present in DCM. Five heteroplasmic mutations were detected that both changed evolutionarily conserved residues (which may impair the function of proteins or tRNAs) and were associated with specific enzymatic defects. These mutations could play an important role in the pathogenesis of cardiomyopathy. Key words: mitochondria, dilated cardiomyopathy, oxidative phosphorylation.
Idiopathic dilated cardiomyopathy (DCM) is a significant clinical entity with a relatively poor prognosis (50% to 60% survival at 2 years). Approximately 20% to 25% of cases seem to have a genetic component (1). In DCM, 2 major changes may occur at the cellular level: 1)
changes in myocardial proteins and 2) changes in cardiac energy metabolism, including defective fatty acid oxidation and/or reduced respiratory chain and oxidative phosphorylation (OXPHOS) activities. Mutations that generate either defects in myocardial structural and contractile proteins or deficiencies in myocardial bioenergetics have been postulated as primary causes for DCM (1– 4). In our laboratory, we have found a high incidence of changes in respiratory chain enzyme activities in endomyocardial biopsy samples from patients with DCM, both children and adults (5– 6). These enzymatic defects, although frequently present, are heterogeneous, affecting either single or multiple enzymes involved in the mitochondrial respiratory chain and oxidative phosphoryla-
From the *Molecular Cardiology Institute, Highland Park, New Jersey; and the †Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota. Manuscript received January 20, 2000; revised manuscript received May 5, 2000; revised manuscript accepted June 20, 2000. Reprint requests: Jose Marin-Garcia, MD, The Molecular Cardiology Institute, 75 Raritan Avenue, Highland Park, NJ 08904. Copyright © 2000 by Churchill Livingstone威 1071-9164/00/0604-0006$10.00/0 doi:10.1054/jcaf.2000.19232
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322 Journal of Cardiac Failure Vol. 6 No. 4 December 2000 tion. Enzymes with peptide subunits encoded by mitochondrial DNA (mtDNA) (eg, complex I, III, IV, and V) are more often affected than an entirely nuclear encoded enzyme such as complex II. Consequently, the mitochondrial genome has been considered a prime candidate as the potential origin for many of these deficiencies. This may be caused in part by the higher rate of mutation in the mitochondrial genome as compared with nuclear genes (presumably caused by the lack of and/or an inefficient mitochondrial DNA repair system). The mtDNA encodes 13 peptides involved in the respiratory chain, as well as 22 transfer ribonucleic acid (tRNA) and 2 ribosomal RNA (rRNA) that are involved in the regulation of mitochondrial biogenesis. Mutations in tRNA genes have been found in neurological diseases associated with cardiac involvement (eg, MELAS [mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes] and MERRF [myoclonic epilepsy and ragged-red fibers]) (7). Although the majority of mutations detected in patients with clinically isolated cardiomyopathy are in tRNA genes (8 –14), some missense mutations in structural genes have also been reported (15–18). Mutations in mtDNA that are considered pathogenic alter a residue that is evolutionarily conserved, are not present in healthy control individuals, and usually are heteroplasmic (7). In the present study, we assess the prevalence of cardiac mtDNA sequence changes in a series of patients with DCM. All 13 protein encoding genes and all 22 tRNA and rRNA genes were sequenced to determine if point mutations were present and if they correlated with specific respiratory enzyme defects.
Methods Study Patients Twenty-eight consecutive patients (15 adults and 13 children, age range from 2 weeks to 58 years) with severe DCM were clinically evaluated before undergoing orthotopic heart transplantation. All patients were on treatment with digoxin, vasodilators, and diuretics; had marked cardiomegaly; and had severe left ventricular dysfunction at cardiac catheterization (left ventricular mean ejection fraction 18.4% ⫾ 5.2% and cardiac index mean 1.95 ⫾ 0.55 L/min/m2). No evidence of familial and/or maternally inherited cardiomyopathy was found in this group. Control Group The control group consisted of 14 individuals (6 children and 8 adults, age range from 1 week to 64 years) with no history or signs of cardiac disease who died from noncardiac related causes.
Tissues Tissue samples (50 to 100 mg) were collected by biopsy at the time of transplantation in the study patients and at autopsy in the control group. They were snapfrozen and kept at ⫺70°C until used. Activity levels of respiratory complexes I to V and citrate synthase (CS) were assessed with tissue homogenates as previously described (5– 6). Respiratory complex activities were normalized with respect to CS activity. Individuals with significantly lower activity ratios (25% lower than the minimum range control value) were considered deficient for a specific complex activity. DNA Analysis DNA was extracted and purified from cardiac homogenates from each patient by using the QIAamp Tissue kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol and was amplified by polymerase chain reaction (PCR) with oligonucleotide primers of specific mtDNA sequence. Primers were designed using Right Primer software (BioDisk Inc, San Francisco, CA). The strategy for sequencing each patient’s mtDNA involved the initial amplification of roughly 700 to 1,000 base pair (bp) fragments from each region of interest. Every reaction used mtDNA specific primers with either the M13 forward or M13 reverse sequence attached (in some instances a primer set contained both M13 sequences). These amplifications were routinely performed under stringent annealing conditions (59°C or greater). After purification by the QIAquick PCR purification kit (Qiagen), each product was sequenced by using a Sequitherm EXCEL II Long-Read DNA sequencing kit (Epicentre Technologies, Madison, WI) with fluorescently labeled primers. Reactions were sequenced on a LICOR 4000 automatic sequencer (LICOR, Lincoln, NE), aligned, and analyzed by using ABI SeqEdit/Gene Prism Sequencing software (version 2.1.1; PE Applied Biosystems, Foster City, CA). All regions were sequenced in both directions. Mutations in the mtDNA sequence caused by base substitution, insertion or deletion, were determined in both patients and controls by comparison with the original Cambridge mtDNA sequence (19). We have reported only mtDNA changes in the coding sequences for proteins and tRNA genes and have avoided noncoding regions (eg, D-loop region). Patient mtDNA nucleotide sequences were also compared with control sequences previously reported in the literature, including 2 sequences from patients with myotonic dystrophy (20,21) and sequences from several normal individuals (21). In addition, all nucleotide sequences were compared with the updated MITOMAP list of mtDNA polymorphisms (22). Sequences containing heteroplasmic mutations were
mtDNA Mutations in Dilated Cardiomyopathy ●
repeatedly confirmed from independent PCR reactions and are unlikely to represent sequencing or polymerase errors. The assessment of heteroplasmy (mixed population of mutant and wild-type alleles) of any given mutation (23) was made by densitometric analysis (using NIH Image 1.33 software; National Institutes of Health, Bethesda, MD) from the sequencing profiles obtained from the LICOR automatic sequencer and Base ImageIR software (LICOR). The extent of heteroplasmy of the mutant alleles described in this study ranged from 65% to 90% relative to the wild-type allele. Evaluation of Inter-species Conservation Amino acid replacements specified by missense mutations were evaluated by comparing the protein sequences with the standard coding sequence of human (19 –21), bovine (24), rat (25), mouse (26), fin whale (27), horse (28), and pig (29) mitochondrial genomes. If a mutation altered a specific amino acid residue that was identical in all of the previously mentioned species, the evolutionary conservation was considered to be high. If the altered residue was identical in 4 or 5 of the 6 genomes, its evolutionary conservation was considered to be moderate. If a residue was identical in 3 or less of the 6 genomes, its evolutionary conservation was considered to be low. Mutations in tRNA nucleotides were compared to the tRNA genes encoded by the same mitochondrial genomes previously noted.
Results A total of 279 point mutations distributed relatively equally in the coding regions of cardiac mtDNA were found in our 28 patients compared with the 61 mutations found in the 14 controls. Mutations were subsequently analyzed in 3 groups: 1) point mutations found in both DCM patients and controls (at 40 loci) or in controls alone (at 5 loci), 2) point mutations solely found in DCM residing in rRNA genes or in protein-encoding structural genes (without amino acid replacement) (at 46 loci), and 3) missense and tRNA mutations found only in DCM patients (at 48 loci). The great majority of mtDNA mutations shared by both DCM and controls in the first group (Table 1) were silent mutations (at 38 loci) and have been reported as normal variants in MITOMAP (37 loci). All of the mutations in this group were homoplasmic. The 2 loci with mutations resulting in amino acid replacements (at 14766 and 13702) were at amino acid residues with low evolutionary conservation. One tRNA mutation was found in both DCM and controls at nt 15928, a previously reported polymorphic variant (22). Point mutations in the second group were mutations found only in DCM patients (total 81 mutations), the
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Table 1. Mutations in Cardiac mtDNA Genes Identified in DCM and Controls or Controls Alone
Gene ATPase 6 ATPase 6 ATPase 8 ATPase 8 COI COI COI COI COII COII COII COIII COIII COIII cytb cytb cytb cytb cytb cytb ND1 NDI ND1 ND1 ND2 ND2 ND2 ND3 ND3 ND3 ND4 ND4 ND4 ND4L ND5 ND5 ND5 ND5 ND5 ND5 ND6 16s rRNA 16s rRNA tRNA-Thr
Nucleotide Substitution
Amino Acid Residue
Number Found DCM
Control
Normal Variant*
G8856A G9123A C8428T A8563G T6413C G6023A T6866C A7325G T7624A G8251A G8020A A9377G T9540C T9716C G14905A G15043A G15301A A15607G T14783C T14766C T4117C A4143G C4242T A3480G G4985A G5417A G4655A C10400T A10397G C10181T A11350G G11914A A11812G A10550G C13173T C12633T G12372A T13161C G13368A G13702A C14167T A2706G G3010A G15928A
Ala Leu Phe Pro Asn Glu Phe Glu Thr Gly Pro Trp Leu Gly Met Gly Leu Lys Leu Ile 3 Thr Leu Arg Ser Lys Gln Gln Thr Thr Trp Phe Leu Thr Leu Met Cys Ser Leu Thr Gly Gly 3 Arg Glu na na na
2 — 1 2 2 3 3 — — 2 3 2 3 — 4 2 1 4 2 7 2 1 2 2 3 2 2 — 1 2 2 1 2 4 5 2 9 2 5 9 5 6 3 5
1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 2 1 1 2 1 1 1 2 1 1 1 1 1 1 1 1 2 2 3 1 3 1 2 5 1 2 2 1
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ novel novel ⫹ ⫹ ⫹ ⫹ novel ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ novel ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
mtDNA, mitochondrial DNA; DCM, idiopathic dilated cardiomyopathy; ⫹, previously reported; na, not applicable. * According to MITOMAP.
majority residing in structural genes with no amino acid replacement (at 34 loci) and the others in rRNA genes (at 12 loci) as shown in Table 2. A large proportion of these mutations (55%) have not been previously reported as polymorphic variants. All the mutations in this group were found to be homoplasmic. Point mutations were also found at sites in both the 12s (n ⫽ 2) and 16s (n ⫽ 10) rRNA genes (Table 2), which are less amenable to comparative analysis because these genes are less conserved. Again, all the changes were noted to be homoplasmic and likely represent polymorphic variations.
324 Journal of Cardiac Failure Vol. 6 No. 4 December 2000 Table 2. Silent Structural Gene and rRNA Mutations in DCM Gene ATPase 6 ATPase 6 COI COI COI COII COII COII COIII COIII COIII cytb cytb cytb cytb ND1 ND2 ND2 ND2 ND2 ND3 ND3 ND3 ND3 ND4 ND4L ND5 ND5 ND5 ND5 ND5 ND6 ND6 ND6 12s rRNA 12s rRNA 16s rRNA 16s rRNA 16s rRNA 16s rRNA 16s rRNA 16s rRNA 16s rRNA 16s rRNA 16s rRNA 16s rRNA
Nucleotide Substitution
AA Residue
Number Found
Normal Variant*
A9623G A9072G A7307C A7298C A7301G A7828G T7624A A7768G G9932A A9242G C9365T C15550T T15184C T15115C C15833T A3411G T5393C A5240G A5414G A4868G T10115C C10142T T10211C T10084C C11840T G10523A C13110T G12630A A13068G C13110T C13122T G14560A A14233G T14530C G709A T1189C G1719A T1738C A1811G G1888A C2332T T2352C T2416C G2758A A2768G T3197C
Ala Ser Met Val Met Leu Thr Met Trp Lys Thr Ile Ile Thr Leu Lys Ser Leu Trp Trp Ile Asn Phe Asp Leu Gly Phe Trp Ser Phe Arg Val Asp Gly na na na na na na na na na na na na
3 1 1 1 1 2 1 1 1 2 1 2 1 1 1 1 1 2 2 2 1 1 1 1 1 2 3 1 2 2 3 3 2 2 5 2 3 3 2 3 1 2 1 2 2 2
⫹ novel ⫹ novel novel ⫹ novel novel ⫹ novel ⫹ novel novel novel ⫹ ⫹ novel ⫹ novel ⫹ novel novel novel novel novel novel novel novel ⫹ novel novel ⫹ ⫹ novel ⫹ novel ⫹ ⫹ ⫹ ⫹ novel ⫹ novel ⫹ novel ⫹
rRNA, ribosomal RNA; DCM, idiopathic dilated cardiomyopathy; AA, amino acid; na, not applicable. * According to MITOMAP.
Point mutations in the third group were found at 48 loci (Table 3), resulting in either amino acid changes in structural genes (missense mutations) or nucleotide changes in mitochondrial transfer genes (tRNA) in DCM patients; they were not present in controls. The observed mtDNA defects included 28 missense and 20 tRNA mutations. In general, the mutations found in the study patients and not in the control group were widely distributed throughout the mtDNA map (Figure 1). None of the mutations previously noted in association with hy-
pertrophic cardiomyopathy (HCM) (10 –14,17) were present in our patients. Sequence changes were found in all 13 of the mtDNA genes encoding proteins. Of the 28 missense mutations that were found only in the study patients, 16 have been previously reported, and the majority of these were designated polymorphic variations (22). This designation may not be accurate in each patient because a number of those polymorphisms have been detected in patients with cardiomyopathy (15,16,23). Of the 16 missense mutations previously reported, 7 reside in sequences encoding nonconserved amino acid residues, whereas 9 alter moderately to highly conserved residues. The latter group includes mutations at nt 11718 (ND4), 10575 (ND4L), 9775 (COIII), 15218 (cytb), 5931 (CO1), 7325 (CO1), 5301 (ND2), and 4824 (ND2). Of the 12 novel missense mutations found in this study, 5 alter nonconserved amino acid residues, whereas 7 alter moderately to highly conserved residues, including mutations at 11999 (ND4), 14069 (ND5), 9216 (COIII), 7923 (COII), 10738 (ND4L), 10237 (ND3), and 15508 (cytb). Four of these novel structural gene mutations (ie, 7923, 9216, 14069, and 15508) are heteroplasmic and likely pathogenic. Two other structural gene heteroplasmic changes noted in this cohort were found in 10398 (ND3), a previously reported mutation resulting in a change in a weakly conserved amino acid, and 10554 (ND4L), which alters a nonconserved residue. In our patients, 20 mutations (5 occurring more than once) were present in 15 of the 22 mitochondrial tRNA genes (ie, Phe, Val, Ile, Trp, Ala, Asn, Cys, Asp, Lys, Gly, Arg, His, Ser, Leu, and Thr). Eight of these have been previously reported. Several mutations including nt 12246 (Ser), 12308 (Leu), 15924 (Thr), and 15951 (Thr) have been found in association with neuromuscular mitochondrial diseases (22). Novel mutations were found at nt 602 (Phe), 1604 (Val), 4312 (Ile), 5558 (Trp), 5655 (Ala), 5721 (Asn), 5819 (Cys), 7578 (Asp), 8331 (Lys), 10424 (Arg), 12181 (His), and 12246 (Leu). Of these tRNA gene defects, 3 mutations were heteroplasmic including nt 5655 (Ala), 10424 (Arg), and 12181 (His). Only one of these mutations resulted in changing a highly conserved nucleotide (10424 [Arg]). Its location in the stem of the tRNA D-loop region may destabilize the cloverleaf secondary structure of this molecule and affect its function. It is important to note that the number of missense/ tRNA point mutations found in each individual was variable (ranging from 1 to 9). The distribution of mutations with regard to the patient’s age (Table 4) shows that the number of mutations harbored by each patient did not vary with age. The maximum number of mutations found in a single individual (not including numerous neutral/silent mutations or mutations found in our
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Table 3. Missense and tRNA Mutations in mtDNA in DCM Site 602 1604 3338 4025 4312 4824 5301 5558 5655 5721 5819 5931 7325 7389 7521 7578 7853 7923 8331 8414 8701 9216 9775 10001 10084 10237 10398 10424 10463 10554 10575 10738 11718 11999 12181 12246 12302 12308 14069 14178 14798 15218 15314 15452 15508 15907 15924 15951
Gene
NT Change
AA Change
No. Ind
Novel/Rept
Het/Hom
Conserved
tRNA-Phe tRNA-Val ND1 ND1 tRNA-Ile ND2 ND2 tRNA-Trp tRNA-Ala tRNA-Asn tRNA-Cys COI COI COI tRNA-Asp tRNA-Asp COII COII tRNA-Lys ATP8 ATP6 COIII COIII tRNA-Gly ND3 ND3 ND3 tRNA-Arg tRNA-Arg ND4L ND4L ND4L ND4 ND4 tRNA-His tRNA-Ser tRNA-Leu tRNA-Leu ND5 ND6 cytb cytb cytb cytb cytb tRNA-Thr tRNA-Thr tRNA-Thr
C3G G3T T3C C3T C3T A3G A3G T3C T3C A3C T3C A3G A3C T3C G3A A3T G3A A3G T3A C3T A3G A3G A3G T3C T3C T3C A3G T3C T3C T3G A3G T3G G3A A3G C3G C3G A3G A3G C3T T3C T3C A3G G3A C3A C3G A3G A3G A3G
NA NA Val 3 Ala Thr 3 Met NA Thr 3 Ala Ile 3 Val NA NA NA NA Thr 3 Ala Glu 3 Asp Tyr 3 His NA NA Val 3 Ile Tyr 3 Cys NA Leu 3 Phe Thr 3 Ala Gln 3 Glu Glu 3 Gly NA Ile 3 Thr Ile 3 Thr Thr 3 Ala NA NA Ser 3 Ala Met 3 Val Val 3 Gly Gly 3 Glu Thr 3 Ala NA NA NA NA Ser 3 Leu Ile 3 Val Phe 3 Leu Thr 3 Ala Ala 3 Thr Leu 3 Ile Asp 3 Glu NA NA NA
1 1 1 2 1 2 1 1 1 1 2 1 1 1 2 1 2 1 1 2 2 1 1 1 1 1 4 1 3 1 1 2 1 2 2 1 2 5 2 1 3 1 1 4 3 2 2 1
Novel Novel Rept Rept Novel Rept Rept Novel Novel Novel Novel Rept Rept Novel Rept Novel Rept Novel Novel Rept Rept Novel Rept Rept Novel Novel Rept Novel Rept Novel Rept Novel Rept Novel Novel Rept Novel Rept Novel Novel Rept Rept Novel Rept Novel Rept Rept Rept
Hom Hom Hom Hom Hom Hom Hom Hom Het Hom Hom Hom Hom Hom Hom Hom Hom Het Hom Hom Hom Het Hom Hom Hom Hom Het Het Hom Het Hom Hom Hom Hom Het Hom Hom Hom Het Hom Hom Hom Hom Hom Het Hom Hom Hom
High Con ND Non Con Non Con Non Con Mod Con Mod Con ND Non Con Mod Con Non Con Mod Con Mod Con Non Con Non Con High Con Non Con High Con Mod Con Non Con Non Con High Con Con High Con Non Con High Con Low Con High Con High Con Non Con Low Con Mod Con High Con High Con Low Con Non Con Non Con High Con Mod Con Non Con High Con Mod Con Non Con Non Con Mod Con Non Con Mod Con High Con
NT, nucleotide; AA, amino acid; NA, not applicable; Con, conserved; Mod, moderate; Rept, reported in literature; Ind, individual; Hom, homoplasmic; Het, heteroplasmic; tRNA, transfer RNA; mtDNA, mitochondrial DNA; DCM, idiopathic dilated cardiomyopathy.
controls) was 9. Although every patient had at least 1 point mutation, none of the mutations found only in our patients were common to all patients. Comparative analysis of cardiac mtDNA sequences was performed with normal control individuals. None of the potentially pathogenic, heteroplasmic mutations in conserved mtDNA sequences (eg, nt 10424, 7923, 9216, 14069, and 15508) described in DCM patients with enzymatic defects were detected in this group of controls or in those patients with HCM (n ⫽ 6) or ischemic cardiomyopathy (n ⫽ 9) who were similarly analyzed in our laboratory.
Respiratory Enzyme Defects Specific OXPHOS enzymatic activities and the extent to which they are reduced (relative to age-matched control levels) in each patient are shown in Table 4. Enzymatic activities were within normal limits for all the controls. Reduced levels of at least 1 OXPHOS enzyme activity compared with age-matched controls were found in 22 of the 28 patients. Each patient with heteroplasmic mtDNA mutations altering conserved residues (also highlighted in Table 4) displayed reduced activity
326 Journal of Cardiac Failure Vol. 6 No. 4 December 2000
Fig. 1. A linear representation of the circular 16,569 base pair human mtDNA molecule showing the location of the 22 tRNAs identified by their cognate amino acid using the single letter code (F, V, L, I, Q, M, W, A, N, C, Y, S, D, K, G, R, H, S, L, E, T, P), the 2 ribosomal RNA genes (12s and 16s), and all 13 protein-encoding genes (ND1–ND6, COI–COIII, cytb, ATPase6, and ATPase8). The key further identifies the functional roles (ie, the respiratory complexes and associated enzymatic function) played by the proteins encoded by each of the 13 structural genes. Also shown are the location of the heteroplasmic mutant alleles detected in DCM, including point mutations in tRNAArg, COII, COIII, ND4L, ND5, and cytb genes. The positions of the origin of replication for the heavy strand (OH) contained within the noncoding D-loop (D-L) region and of the light strand replication origin (OL) are also shown. The numbering is according to Anderson et al (19).
levels for at least 1 cardiac mitochondrial respiratory complex. Mitochondrial Morphology Of the 28 patients in this study, 6 harbored abnormal mitochondria structure (Table 4), including bizarrely shaped giant mitochondria with unusual cristae formation and electron-dense deposits.
Discussion Abnormalities in the structure and function of mitochondria have been described in a considerable number
of patients with cardiomyopathy (2). Although only 6 patients (21%) of the study patients showed evidence of structural mitochondrial defects, 22 (78%) displayed marked mitochondrial dysfunction at the level of respiratory enzyme activity. Although the molecular basis of these mitochondrial defects has not yet been fully established, mitochondrial DNA mutations (both point mutations and deletions) are increasingly being identified and associated with cardiac disease. To draw valid conclusions concerning their role in the pathophysiology of cardiomyopathy, one must exclude the possibility of polymorphic variations (30) by comparison to previously established mtDNA sequences and to sequences of nor-
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Table 4. Patient Age, Enzyme and Morphological Data, and Specific mtDNA Point Mutations Patient No.
Age
Specific OXPHOS Enzyme Defects*
1 2 3 4 5 6 7 8 9 10
0.5 y 1.3 y 1.4 y 1.4 y 0.5 y 1y 6 wk 3 wk 15 y 14 y
III (35) I (90), III (30), IV (50) I (20), III (25) none IV (30) none none I (90), III (90), V (75) I (100), IV (50) V (95)
11 12
15 y 14 y
none I (70), III (75)
13 14 15 16 17 18
14 22 21 39 33 50
y y y y y y
I (100), IV (25) III (25), IV (75), V (25) I (35), III (25), IV (40) none I (70) III (25), V (85)
19 20 21 22 23
48 46 49 48 43
y y y y y
III (30) III (100), IV (35), V (100) none IV (35) IV (35)
24 25 26 27 28
46 42 58 55 56
y y y y y
IV (70) III (40), IV (30) V (60) V (100) III (100), IV (80)
mtDNA Missense, rRNA, and tRNA Gene Mutations† 15508 (cytb), 602 (PHE), 709 (12s), 15452 (cytb) 10463 (ARG), 709 (12s), 15452 (cytb), 5301 (ND2), 1738 (16s), 3197 (16s) 10463 (ARG), 10554 (ND4L), 709 (12s), 5558 (TRP), 1888 (16s) 1604 (VAL) 10084 (ND3), 5721 (ASN), 12308 (LEU) 7521 (ASP), 14178 (ND6), 7389 (COI), 1738 (16s), 2885 (16s), 2768 (16s) 15907 (THR), 12308 (LEU) 15314 (cytb), 10424 (ARG) 15218 (cytb), 12308 (LEU), 4824 (ND2), 2758 (16s) 15924 (THR), 14798 (cytb), 12308 (LEU), 1811 (16s), 8331 (LYS), 8701 (ATP6), 1189 (12s) 5819 (CYS), 5931 (COI), 9775 (COIII), 2758 (16s) 709 (12s), 1719 (16s rRNA), 1738 (16s), 8414 (ATP8), 2768 (16s), 5655 (ALA), 2885 (16s), 10398 (ND3), 14069 (ND5) 7853 (COII), 11999 (ND4), 3338 (ND1) 12246 (SER), 12302 (LEU), 9216 (COIII), 1888 (16s) 7853 (COII), 11999 (ND4), 2352 (16s), 14069 (ND5) 10398 (ND3), 14798 (cytb), 12308 (LEU), 12181 (HIS), 1811 (16s) 10001 (GLY), 8701 (ATP6), 4824 (ND2) 10398 (ND3), 15951 (THR), 15907 (THR), 4025 (ND1), 2332 (16s), 2416 (16s), 7521 (ASP) 10463 (ARG), 709 (12s), 10738 (ND4L) 10398 (ND3), 15508 (cytb), 14798 (cytb), 15452 (cytb) 10237 (ND3), 12181 (HIS) 11718 (ND4), 4025 (ND1), 1888 (16s) 10398 (ND3), 10575 (ND4L), 15452 (cytb), 14928 (cytb), 7325 (COI), 2352 (16s) 7923 (COII) 1189 (12s), 10738 (ND4L), 12302 (LEU) 1719 (16s rRNA), 15924 (THR) 7578 (ASP), 12308 (LEU), 3197 (16s) 15508 (cytb), 5819 (CYS), 4312 (ILE)
Mitochondrial Morphology‡ normal abnormal normal normal normal normal normal abnormal normal abnormal normal normal abnormal normal normal normal normal abnormal normal normal normal normal normal normal normal normal normal abnormal
mtDNA, mitochondrial DNA; rRNA, ribosomal RNA; tRNA, transfer RNA. * Number in bracket in OXPHOS column represents the % reduction of control activity. † Bold characters in mtDNA point mutation column represent potential pathogenic mutations. ‡ Abnormalities in mitochondrial shape, size and cristae were noted.
mal individuals. However, the definition of what is truly a mtDNA mutation is not so simple. A number of sequence alterations in mtDNA (compared to the standard Cambridge sequence [19]) may occur (21). Some of these represent simple mistakes in the original mtDNA sequence. For example, alterations in sequence at nt 14199, 15236, 14272, 14368, 3106, 8860 listed as normal variants in MITOMAP were found in all of our patients and controls. Also, the definition of what constitutes a normal or polymorphic variant itself is not truly rigorous (ie, a number of putative controls showing mtDNA sequence variation may develop cardiac disorder at a later time). Our approach involved the complete sequencing of all of the coding genes of cardiac mtDNA in each of 28 individuals with DCM. Our rationale for this extensive effort stemmed from our previous experience with DCM, which showed a high incidence of specific enzyme activity defects but of which molecular basis was undefined (5,6). Only by using such a comprehensive analysis
might the effect of single and multiple mutations be elucidated. Comparison With Previous Studies Recent studies documenting the presence of mtDNA mutations have focused on mutant tRNA alleles and their flanking sequences, analyzing only selected regions of the mtDNA genome in addition to restriction length polymorphism (23,31). In their study, Arbustini et al (23) limited their analysis to patients harboring mitochondrial ultrastructural abnormalities, which raises the warning that DCM patients with nonspecific mitochondrial structural abnormalities may carry mtDNA mutations. In spite of these differences, our study also showed a wide distribution of mutations throughout the entire mitochondrial genome. Interpretation of Study Findings We found heteroplasmic mutations in highly conserved regions in cytb, COII, COIII, and in tRNAArg and
328 Journal of Cardiac Failure Vol. 6 No. 4 December 2000 ND5. The prevailing view that heteroplasmic mutations are more likely to occur in pathogenic mutations than in normal polymorphisms is therefore consistent with these mutations being considered pathogenic. In addition, a number of previously unreported and reported mtDNA mutations in highly conserved sequences that seemed to be homoplasmic (ie, containing either all mutant or all wild-type alleles) were identified. Although it is likely that the majority of these homoplasmic mutations represent polymorphisms, some of these nucleotide changes may also be pathogenic because deleterious homoplasmic mtDNA mutations can be present in affected tissues (30,31). It is also noteworthy that the point mutations detected in previous studies were not found in our study and vice versa. This is consistent with the heterogeneity of enzymatic defects observed in DCM patients and supports the uniqueness of most of the reported mtDNA mutations involved in mitochondrial disease. Moreover, it is clear that the task of routine screening for pathogenic mtDNA mutations is particularly difficult because relatively few mutations are shared. A high incidence of OXPHOS enzymatic activity defects was found in the study patients. These defects were found in any of the 4 respiratory complex I, III, IV, and V activities (with at least 1 mtDNA-encoded subunit) and not in complex II activity, whose peptide subunits are entirely nuclear-encoded. Moreover, the enzymatic findings are consistent with the assignment of pathogenecity for mutant alleles of COII, COIII, and cytb because mutations in these genes were accompanied by marked reductions in the respiratory activities of complex IV (correlated with the presence of the COII and COIII alleles) and a reduced level of complex III (with the presence of cytb). In addition, patients harboring the heteroplasmic tRNA mutation (eg, Arg10424) display multiple defects in respiratory activities. The molecular basis of the mitochondrial dysfunction and presumably of the cardiac pathology (ie, the presence of heteroplasmic mutations and their correlation with specific enzymatic reductions) was therefore identified in a total of 9 of the 28 DCM patients. The basis for the reduced respiratory enzymatic activity levels observed in 13 patients, however, remains unidentified and potentially could be because of any of a variety of nuclear DNA defects. In addition, some of the other mtDNA mutations identified in this study as likely polymorphisms could have a contributory role in pathogenesis either acting as deleterious homoplasmic mutations or acting in combination. Our data also suggest that in the study group, no difference was found in the incidence or type of mtDNA point mutations as a function of age. Although the number of patients of different ages in this study is too limited for a statistical analysis of age, our findings of 3 children with pathogenic mtDNA mutations (2 less than
a year old) suggest that at least a subset of the detected mtDNA mutations are primary to mitochondrial dysfunction and not likely a consequence of long-standing disease. This contrasts with the age-dependent accumulation of large-scale mtDNA deletions (32) in somatic cells, proposed as a potential scenario (33) in the development of DCM. Studies directed toward further understanding both the significance and the mechanism for generating the higher incidence of mtDNA point mutations in DCM are clearly warranted and will be necessary in addressing the critical questions of cause and effect. Such studies should also include the determination of the extent of somatic mutations in cardiac mtDNA of each individual.
Conclusion Our data suggest that mtDNA point mutations seem to be more prevalent in DCM. This finding may be related to the severity of myocardial disease in the cases presented (ie, those requiring cardiac transplant). It also could be influenced by the genetic constitution of the patient and control populations for which we have limited data. The role of an accumulated load of mtDNA mutations in the genesis and progression of myocardial disease and mitochondrial pathology clearly warrants further examination. In addition, several of the mutations identified in the study patients seem to be pathogenic because of the following: 1) they change highly conserved residues that may modulate the function of proteins or tRNAs, 2) they are heteroplasmic, 3) they are associated with specific enzymatic defects, and 4) they are neither present in our controls nor in the literature. In vivo methods of assessing mitochondrial enzyme function with specifically altered mitochondrial genes (to test the potential pathogenicity of each mutation) seem warranted. Also, secondary structure analysis of proteins and tRNA with and without point mutations might be informative in predicting the effects on protein function.
References 1. Kelly DP, Strauss AW: Inherited cardiomyopathies. N Engl J Med 1994;330:913–9 2. Marin-Garcia J, Goldenthal MJ: Mitochondrial DNA defects in cardiomyopathy. Cardiovasc Pathol 1998;7:205–11 3. Olson TM, Michels VV, Thibodeau SN, Tai YS, Keating MT: Actin mutations in dilated cardiomyopathy, a heritable form of heart failure. Science 1998;280:750 –2 4. Mestroni L, Rocco C, Gregori D, Sinagra G, Di Lenarda A, Miocic S, Vatta M, Pinamonti B, Muntoni F, Caforio AL, McKenna WJ, Falaschi A, Giacca M, Camerini F: Familial dilated cardiomyopathy: Evidence for genetic and phenotypic heterogeneity. J Am Coll Cardiol 1999;34:181–90 5. Marin-Garcia J, Goldenthal MJ, Pierpont ME, Anan-
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The Complete Sequence of mtDNA Genes in Idiopathic Dilated Cardiomyopathy Shows Novel Missense and tRNA Mutations JOSE MARIN-GARCIA, MD,* MICHAEL J. GOLDENTHAL, PhD,* RADHA ANANTHAKRISHNAN, PhD,* MARY ELLA PIERPONT, MD, PhD† Highland Park, New Jersey; Minneapolis, Minnesota
ABSTRACT Background: Previous studies have shown that mitochondrial DNA (mtDNA) mutations are often present in patients with myocardial dysfunction. We sought to assess the prevalence and significance of heart mtDNA sequence changes in patients with idiopathic dilated cardiomyopathy (DCM). Methods and Results: DNA sequence of all the transfer ribonucleic acid (tRNA), ribosomal RNA (rRNA), and structural genes in cardiac mtDNA of 28 patients with DCM was determined and compared with a control group that had no evidence of heart disease. An increased number of point mutations were found in DCM cardiac mtDNA when compared with controls. Both novel and previously reported mutations were found in mitochondrial tRNA and structural genes. One of these mutations was heteroplasmic and resulted in changing a highly conserved nucleotide in tRNAArg. Novel, heteroplasmic mtDNA mutations (n ⫽ 4) specifying changes in moderate to highly conserved amino acid residues were found in COII, COIII, ND5, and cytb. These novel mtDNA mutations were found only in patients with severe reduction in mitochondrial enzyme activities. Conclusions: Our results indicate that a high incidence of mtDNA nucleotide sequence changes in both tRNA and structural genes are present in DCM. Five heteroplasmic mutations were detected that both changed evolutionarily conserved residues (which may impair the function of proteins or tRNAs) and were associated with specific enzymatic defects. These mutations could play an important role in the pathogenesis of cardiomyopathy. Key words: mitochondria, dilated cardiomyopathy, oxidative phosphorylation.
Idiopathic dilated cardiomyopathy (DCM) is a significant clinical entity with a relatively poor prognosis (50% to 60% survival at 2 years). Approximately 20% to 25% of cases seem to have a genetic component (1). In DCM, 2 major changes may occur at the cellular level: 1)
changes in myocardial proteins and 2) changes in cardiac energy metabolism, including defective fatty acid oxidation and/or reduced respiratory chain and oxidative phosphorylation (OXPHOS) activities. Mutations that generate either defects in myocardial structural and contractile proteins or deficiencies in myocardial bioenergetics have been postulated as primary causes for DCM (1– 4). In our laboratory, we have found a high incidence of changes in respiratory chain enzyme activities in endomyocardial biopsy samples from patients with DCM, both children and adults (5– 6). These enzymatic defects, although frequently present, are heterogeneous, affecting either single or multiple enzymes involved in the mitochondrial respiratory chain and oxidative phosphoryla-
From the *Molecular Cardiology Institute, Highland Park, New Jersey; and the †Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota. Manuscript received January 20, 2000; revised manuscript received May 5, 2000; revised manuscript accepted June 20, 2000. Reprint requests: Jose Marin-Garcia, MD, The Molecular Cardiology Institute, 75 Raritan Avenue, Highland Park, NJ 08904. Copyright © 2000 by Churchill Livingstone威 1071-9164/00/0604-0006$10.00/0 doi:10.1054/jcaf.2000.19232
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322 Journal of Cardiac Failure Vol. 6 No. 4 December 2000 tion. Enzymes with peptide subunits encoded by mitochondrial DNA (mtDNA) (eg, complex I, III, IV, and V) are more often affected than an entirely nuclear encoded enzyme such as complex II. Consequently, the mitochondrial genome has been considered a prime candidate as the potential origin for many of these deficiencies. This may be caused in part by the higher rate of mutation in the mitochondrial genome as compared with nuclear genes (presumably caused by the lack of and/or an inefficient mitochondrial DNA repair system). The mtDNA encodes 13 peptides involved in the respiratory chain, as well as 22 transfer ribonucleic acid (tRNA) and 2 ribosomal RNA (rRNA) that are involved in the regulation of mitochondrial biogenesis. Mutations in tRNA genes have been found in neurological diseases associated with cardiac involvement (eg, MELAS [mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes] and MERRF [myoclonic epilepsy and ragged-red fibers]) (7). Although the majority of mutations detected in patients with clinically isolated cardiomyopathy are in tRNA genes (8 –14), some missense mutations in structural genes have also been reported (15–18). Mutations in mtDNA that are considered pathogenic alter a residue that is evolutionarily conserved, are not present in healthy control individuals, and usually are heteroplasmic (7). In the present study, we assess the prevalence of cardiac mtDNA sequence changes in a series of patients with DCM. All 13 protein encoding genes and all 22 tRNA and rRNA genes were sequenced to determine if point mutations were present and if they correlated with specific respiratory enzyme defects.
Methods Study Patients Twenty-eight consecutive patients (15 adults and 13 children, age range from 2 weeks to 58 years) with severe DCM were clinically evaluated before undergoing orthotopic heart transplantation. All patients were on treatment with digoxin, vasodilators, and diuretics; had marked cardiomegaly; and had severe left ventricular dysfunction at cardiac catheterization (left ventricular mean ejection fraction 18.4% ⫾ 5.2% and cardiac index mean 1.95 ⫾ 0.55 L/min/m2). No evidence of familial and/or maternally inherited cardiomyopathy was found in this group. Control Group The control group consisted of 14 individuals (6 children and 8 adults, age range from 1 week to 64 years) with no history or signs of cardiac disease who died from noncardiac related causes.
Tissues Tissue samples (50 to 100 mg) were collected by biopsy at the time of transplantation in the study patients and at autopsy in the control group. They were snapfrozen and kept at ⫺70°C until used. Activity levels of respiratory complexes I to V and citrate synthase (CS) were assessed with tissue homogenates as previously described (5– 6). Respiratory complex activities were normalized with respect to CS activity. Individuals with significantly lower activity ratios (25% lower than the minimum range control value) were considered deficient for a specific complex activity. DNA Analysis DNA was extracted and purified from cardiac homogenates from each patient by using the QIAamp Tissue kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol and was amplified by polymerase chain reaction (PCR) with oligonucleotide primers of specific mtDNA sequence. Primers were designed using Right Primer software (BioDisk Inc, San Francisco, CA). The strategy for sequencing each patient’s mtDNA involved the initial amplification of roughly 700 to 1,000 base pair (bp) fragments from each region of interest. Every reaction used mtDNA specific primers with either the M13 forward or M13 reverse sequence attached (in some instances a primer set contained both M13 sequences). These amplifications were routinely performed under stringent annealing conditions (59°C or greater). After purification by the QIAquick PCR purification kit (Qiagen), each product was sequenced by using a Sequitherm EXCEL II Long-Read DNA sequencing kit (Epicentre Technologies, Madison, WI) with fluorescently labeled primers. Reactions were sequenced on a LICOR 4000 automatic sequencer (LICOR, Lincoln, NE), aligned, and analyzed by using ABI SeqEdit/Gene Prism Sequencing software (version 2.1.1; PE Applied Biosystems, Foster City, CA). All regions were sequenced in both directions. Mutations in the mtDNA sequence caused by base substitution, insertion or deletion, were determined in both patients and controls by comparison with the original Cambridge mtDNA sequence (19). We have reported only mtDNA changes in the coding sequences for proteins and tRNA genes and have avoided noncoding regions (eg, D-loop region). Patient mtDNA nucleotide sequences were also compared with control sequences previously reported in the literature, including 2 sequences from patients with myotonic dystrophy (20,21) and sequences from several normal individuals (21). In addition, all nucleotide sequences were compared with the updated MITOMAP list of mtDNA polymorphisms (22). Sequences containing heteroplasmic mutations were
mtDNA Mutations in Dilated Cardiomyopathy ●
repeatedly confirmed from independent PCR reactions and are unlikely to represent sequencing or polymerase errors. The assessment of heteroplasmy (mixed population of mutant and wild-type alleles) of any given mutation (23) was made by densitometric analysis (using NIH Image 1.33 software; National Institutes of Health, Bethesda, MD) from the sequencing profiles obtained from the LICOR automatic sequencer and Base ImageIR software (LICOR). The extent of heteroplasmy of the mutant alleles described in this study ranged from 65% to 90% relative to the wild-type allele. Evaluation of Inter-species Conservation Amino acid replacements specified by missense mutations were evaluated by comparing the protein sequences with the standard coding sequence of human (19 –21), bovine (24), rat (25), mouse (26), fin whale (27), horse (28), and pig (29) mitochondrial genomes. If a mutation altered a specific amino acid residue that was identical in all of the previously mentioned species, the evolutionary conservation was considered to be high. If the altered residue was identical in 4 or 5 of the 6 genomes, its evolutionary conservation was considered to be moderate. If a residue was identical in 3 or less of the 6 genomes, its evolutionary conservation was considered to be low. Mutations in tRNA nucleotides were compared to the tRNA genes encoded by the same mitochondrial genomes previously noted.
Results A total of 279 point mutations distributed relatively equally in the coding regions of cardiac mtDNA were found in our 28 patients compared with the 61 mutations found in the 14 controls. Mutations were subsequently analyzed in 3 groups: 1) point mutations found in both DCM patients and controls (at 40 loci) or in controls alone (at 5 loci), 2) point mutations solely found in DCM residing in rRNA genes or in protein-encoding structural genes (without amino acid replacement) (at 46 loci), and 3) missense and tRNA mutations found only in DCM patients (at 48 loci). The great majority of mtDNA mutations shared by both DCM and controls in the first group (Table 1) were silent mutations (at 38 loci) and have been reported as normal variants in MITOMAP (37 loci). All of the mutations in this group were homoplasmic. The 2 loci with mutations resulting in amino acid replacements (at 14766 and 13702) were at amino acid residues with low evolutionary conservation. One tRNA mutation was found in both DCM and controls at nt 15928, a previously reported polymorphic variant (22). Point mutations in the second group were mutations found only in DCM patients (total 81 mutations), the
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Table 1. Mutations in Cardiac mtDNA Genes Identified in DCM and Controls or Controls Alone
Gene ATPase 6 ATPase 6 ATPase 8 ATPase 8 COI COI COI COI COII COII COII COIII COIII COIII cytb cytb cytb cytb cytb cytb ND1 NDI ND1 ND1 ND2 ND2 ND2 ND3 ND3 ND3 ND4 ND4 ND4 ND4L ND5 ND5 ND5 ND5 ND5 ND5 ND6 16s rRNA 16s rRNA tRNA-Thr
Nucleotide Substitution
Amino Acid Residue
Number Found DCM
Control
Normal Variant*
G8856A G9123A C8428T A8563G T6413C G6023A T6866C A7325G T7624A G8251A G8020A A9377G T9540C T9716C G14905A G15043A G15301A A15607G T14783C T14766C T4117C A4143G C4242T A3480G G4985A G5417A G4655A C10400T A10397G C10181T A11350G G11914A A11812G A10550G C13173T C12633T G12372A T13161C G13368A G13702A C14167T A2706G G3010A G15928A
Ala Leu Phe Pro Asn Glu Phe Glu Thr Gly Pro Trp Leu Gly Met Gly Leu Lys Leu Ile 3 Thr Leu Arg Ser Lys Gln Gln Thr Thr Trp Phe Leu Thr Leu Met Cys Ser Leu Thr Gly Gly 3 Arg Glu na na na
2 — 1 2 2 3 3 — — 2 3 2 3 — 4 2 1 4 2 7 2 1 2 2 3 2 2 — 1 2 2 1 2 4 5 2 9 2 5 9 5 6 3 5
1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 2 1 1 2 1 1 1 2 1 1 1 1 1 1 1 1 2 2 3 1 3 1 2 5 1 2 2 1
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ novel novel ⫹ ⫹ ⫹ ⫹ novel ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ novel ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
mtDNA, mitochondrial DNA; DCM, idiopathic dilated cardiomyopathy; ⫹, previously reported; na, not applicable. * According to MITOMAP.
majority residing in structural genes with no amino acid replacement (at 34 loci) and the others in rRNA genes (at 12 loci) as shown in Table 2. A large proportion of these mutations (55%) have not been previously reported as polymorphic variants. All the mutations in this group were found to be homoplasmic. Point mutations were also found at sites in both the 12s (n ⫽ 2) and 16s (n ⫽ 10) rRNA genes (Table 2), which are less amenable to comparative analysis because these genes are less conserved. Again, all the changes were noted to be homoplasmic and likely represent polymorphic variations.
324 Journal of Cardiac Failure Vol. 6 No. 4 December 2000 Table 2. Silent Structural Gene and rRNA Mutations in DCM Gene ATPase 6 ATPase 6 COI COI COI COII COII COII COIII COIII COIII cytb cytb cytb cytb ND1 ND2 ND2 ND2 ND2 ND3 ND3 ND3 ND3 ND4 ND4L ND5 ND5 ND5 ND5 ND5 ND6 ND6 ND6 12s rRNA 12s rRNA 16s rRNA 16s rRNA 16s rRNA 16s rRNA 16s rRNA 16s rRNA 16s rRNA 16s rRNA 16s rRNA 16s rRNA
Nucleotide Substitution
AA Residue
Number Found
Normal Variant*
A9623G A9072G A7307C A7298C A7301G A7828G T7624A A7768G G9932A A9242G C9365T C15550T T15184C T15115C C15833T A3411G T5393C A5240G A5414G A4868G T10115C C10142T T10211C T10084C C11840T G10523A C13110T G12630A A13068G C13110T C13122T G14560A A14233G T14530C G709A T1189C G1719A T1738C A1811G G1888A C2332T T2352C T2416C G2758A A2768G T3197C
Ala Ser Met Val Met Leu Thr Met Trp Lys Thr Ile Ile Thr Leu Lys Ser Leu Trp Trp Ile Asn Phe Asp Leu Gly Phe Trp Ser Phe Arg Val Asp Gly na na na na na na na na na na na na
3 1 1 1 1 2 1 1 1 2 1 2 1 1 1 1 1 2 2 2 1 1 1 1 1 2 3 1 2 2 3 3 2 2 5 2 3 3 2 3 1 2 1 2 2 2
⫹ novel ⫹ novel novel ⫹ novel novel ⫹ novel ⫹ novel novel novel ⫹ ⫹ novel ⫹ novel ⫹ novel novel novel novel novel novel novel novel ⫹ novel novel ⫹ ⫹ novel ⫹ novel ⫹ ⫹ ⫹ ⫹ novel ⫹ novel ⫹ novel ⫹
rRNA, ribosomal RNA; DCM, idiopathic dilated cardiomyopathy; AA, amino acid; na, not applicable. * According to MITOMAP.
Point mutations in the third group were found at 48 loci (Table 3), resulting in either amino acid changes in structural genes (missense mutations) or nucleotide changes in mitochondrial transfer genes (tRNA) in DCM patients; they were not present in controls. The observed mtDNA defects included 28 missense and 20 tRNA mutations. In general, the mutations found in the study patients and not in the control group were widely distributed throughout the mtDNA map (Figure 1). None of the mutations previously noted in association with hy-
pertrophic cardiomyopathy (HCM) (10 –14,17) were present in our patients. Sequence changes were found in all 13 of the mtDNA genes encoding proteins. Of the 28 missense mutations that were found only in the study patients, 16 have been previously reported, and the majority of these were designated polymorphic variations (22). This designation may not be accurate in each patient because a number of those polymorphisms have been detected in patients with cardiomyopathy (15,16,23). Of the 16 missense mutations previously reported, 7 reside in sequences encoding nonconserved amino acid residues, whereas 9 alter moderately to highly conserved residues. The latter group includes mutations at nt 11718 (ND4), 10575 (ND4L), 9775 (COIII), 15218 (cytb), 5931 (CO1), 7325 (CO1), 5301 (ND2), and 4824 (ND2). Of the 12 novel missense mutations found in this study, 5 alter nonconserved amino acid residues, whereas 7 alter moderately to highly conserved residues, including mutations at 11999 (ND4), 14069 (ND5), 9216 (COIII), 7923 (COII), 10738 (ND4L), 10237 (ND3), and 15508 (cytb). Four of these novel structural gene mutations (ie, 7923, 9216, 14069, and 15508) are heteroplasmic and likely pathogenic. Two other structural gene heteroplasmic changes noted in this cohort were found in 10398 (ND3), a previously reported mutation resulting in a change in a weakly conserved amino acid, and 10554 (ND4L), which alters a nonconserved residue. In our patients, 20 mutations (5 occurring more than once) were present in 15 of the 22 mitochondrial tRNA genes (ie, Phe, Val, Ile, Trp, Ala, Asn, Cys, Asp, Lys, Gly, Arg, His, Ser, Leu, and Thr). Eight of these have been previously reported. Several mutations including nt 12246 (Ser), 12308 (Leu), 15924 (Thr), and 15951 (Thr) have been found in association with neuromuscular mitochondrial diseases (22). Novel mutations were found at nt 602 (Phe), 1604 (Val), 4312 (Ile), 5558 (Trp), 5655 (Ala), 5721 (Asn), 5819 (Cys), 7578 (Asp), 8331 (Lys), 10424 (Arg), 12181 (His), and 12246 (Leu). Of these tRNA gene defects, 3 mutations were heteroplasmic including nt 5655 (Ala), 10424 (Arg), and 12181 (His). Only one of these mutations resulted in changing a highly conserved nucleotide (10424 [Arg]). Its location in the stem of the tRNA D-loop region may destabilize the cloverleaf secondary structure of this molecule and affect its function. It is important to note that the number of missense/ tRNA point mutations found in each individual was variable (ranging from 1 to 9). The distribution of mutations with regard to the patient’s age (Table 4) shows that the number of mutations harbored by each patient did not vary with age. The maximum number of mutations found in a single individual (not including numerous neutral/silent mutations or mutations found in our
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Table 3. Missense and tRNA Mutations in mtDNA in DCM Site 602 1604 3338 4025 4312 4824 5301 5558 5655 5721 5819 5931 7325 7389 7521 7578 7853 7923 8331 8414 8701 9216 9775 10001 10084 10237 10398 10424 10463 10554 10575 10738 11718 11999 12181 12246 12302 12308 14069 14178 14798 15218 15314 15452 15508 15907 15924 15951
Gene
NT Change
AA Change
No. Ind
Novel/Rept
Het/Hom
Conserved
tRNA-Phe tRNA-Val ND1 ND1 tRNA-Ile ND2 ND2 tRNA-Trp tRNA-Ala tRNA-Asn tRNA-Cys COI COI COI tRNA-Asp tRNA-Asp COII COII tRNA-Lys ATP8 ATP6 COIII COIII tRNA-Gly ND3 ND3 ND3 tRNA-Arg tRNA-Arg ND4L ND4L ND4L ND4 ND4 tRNA-His tRNA-Ser tRNA-Leu tRNA-Leu ND5 ND6 cytb cytb cytb cytb cytb tRNA-Thr tRNA-Thr tRNA-Thr
C3G G3T T3C C3T C3T A3G A3G T3C T3C A3C T3C A3G A3C T3C G3A A3T G3A A3G T3A C3T A3G A3G A3G T3C T3C T3C A3G T3C T3C T3G A3G T3G G3A A3G C3G C3G A3G A3G C3T T3C T3C A3G G3A C3A C3G A3G A3G A3G
NA NA Val 3 Ala Thr 3 Met NA Thr 3 Ala Ile 3 Val NA NA NA NA Thr 3 Ala Glu 3 Asp Tyr 3 His NA NA Val 3 Ile Tyr 3 Cys NA Leu 3 Phe Thr 3 Ala Gln 3 Glu Glu 3 Gly NA Ile 3 Thr Ile 3 Thr Thr 3 Ala NA NA Ser 3 Ala Met 3 Val Val 3 Gly Gly 3 Glu Thr 3 Ala NA NA NA NA Ser 3 Leu Ile 3 Val Phe 3 Leu Thr 3 Ala Ala 3 Thr Leu 3 Ile Asp 3 Glu NA NA NA
1 1 1 2 1 2 1 1 1 1 2 1 1 1 2 1 2 1 1 2 2 1 1 1 1 1 4 1 3 1 1 2 1 2 2 1 2 5 2 1 3 1 1 4 3 2 2 1
Novel Novel Rept Rept Novel Rept Rept Novel Novel Novel Novel Rept Rept Novel Rept Novel Rept Novel Novel Rept Rept Novel Rept Rept Novel Novel Rept Novel Rept Novel Rept Novel Rept Novel Novel Rept Novel Rept Novel Novel Rept Rept Novel Rept Novel Rept Rept Rept
Hom Hom Hom Hom Hom Hom Hom Hom Het Hom Hom Hom Hom Hom Hom Hom Hom Het Hom Hom Hom Het Hom Hom Hom Hom Het Het Hom Het Hom Hom Hom Hom Het Hom Hom Hom Het Hom Hom Hom Hom Hom Het Hom Hom Hom
High Con ND Non Con Non Con Non Con Mod Con Mod Con ND Non Con Mod Con Non Con Mod Con Mod Con Non Con Non Con High Con Non Con High Con Mod Con Non Con Non Con High Con Con High Con Non Con High Con Low Con High Con High Con Non Con Low Con Mod Con High Con High Con Low Con Non Con Non Con High Con Mod Con Non Con High Con Mod Con Non Con Non Con Mod Con Non Con Mod Con High Con
NT, nucleotide; AA, amino acid; NA, not applicable; Con, conserved; Mod, moderate; Rept, reported in literature; Ind, individual; Hom, homoplasmic; Het, heteroplasmic; tRNA, transfer RNA; mtDNA, mitochondrial DNA; DCM, idiopathic dilated cardiomyopathy.
controls) was 9. Although every patient had at least 1 point mutation, none of the mutations found only in our patients were common to all patients. Comparative analysis of cardiac mtDNA sequences was performed with normal control individuals. None of the potentially pathogenic, heteroplasmic mutations in conserved mtDNA sequences (eg, nt 10424, 7923, 9216, 14069, and 15508) described in DCM patients with enzymatic defects were detected in this group of controls or in those patients with HCM (n ⫽ 6) or ischemic cardiomyopathy (n ⫽ 9) who were similarly analyzed in our laboratory.
Respiratory Enzyme Defects Specific OXPHOS enzymatic activities and the extent to which they are reduced (relative to age-matched control levels) in each patient are shown in Table 4. Enzymatic activities were within normal limits for all the controls. Reduced levels of at least 1 OXPHOS enzyme activity compared with age-matched controls were found in 22 of the 28 patients. Each patient with heteroplasmic mtDNA mutations altering conserved residues (also highlighted in Table 4) displayed reduced activity
326 Journal of Cardiac Failure Vol. 6 No. 4 December 2000
Fig. 1. A linear representation of the circular 16,569 base pair human mtDNA molecule showing the location of the 22 tRNAs identified by their cognate amino acid using the single letter code (F, V, L, I, Q, M, W, A, N, C, Y, S, D, K, G, R, H, S, L, E, T, P), the 2 ribosomal RNA genes (12s and 16s), and all 13 protein-encoding genes (ND1–ND6, COI–COIII, cytb, ATPase6, and ATPase8). The key further identifies the functional roles (ie, the respiratory complexes and associated enzymatic function) played by the proteins encoded by each of the 13 structural genes. Also shown are the location of the heteroplasmic mutant alleles detected in DCM, including point mutations in tRNAArg, COII, COIII, ND4L, ND5, and cytb genes. The positions of the origin of replication for the heavy strand (OH) contained within the noncoding D-loop (D-L) region and of the light strand replication origin (OL) are also shown. The numbering is according to Anderson et al (19).
levels for at least 1 cardiac mitochondrial respiratory complex. Mitochondrial Morphology Of the 28 patients in this study, 6 harbored abnormal mitochondria structure (Table 4), including bizarrely shaped giant mitochondria with unusual cristae formation and electron-dense deposits.
Discussion Abnormalities in the structure and function of mitochondria have been described in a considerable number
of patients with cardiomyopathy (2). Although only 6 patients (21%) of the study patients showed evidence of structural mitochondrial defects, 22 (78%) displayed marked mitochondrial dysfunction at the level of respiratory enzyme activity. Although the molecular basis of these mitochondrial defects has not yet been fully established, mitochondrial DNA mutations (both point mutations and deletions) are increasingly being identified and associated with cardiac disease. To draw valid conclusions concerning their role in the pathophysiology of cardiomyopathy, one must exclude the possibility of polymorphic variations (30) by comparison to previously established mtDNA sequences and to sequences of nor-
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Table 4. Patient Age, Enzyme and Morphological Data, and Specific mtDNA Point Mutations Patient No.
Age
Specific OXPHOS Enzyme Defects*
1 2 3 4 5 6 7 8 9 10
0.5 y 1.3 y 1.4 y 1.4 y 0.5 y 1y 6 wk 3 wk 15 y 14 y
III (35) I (90), III (30), IV (50) I (20), III (25) none IV (30) none none I (90), III (90), V (75) I (100), IV (50) V (95)
11 12
15 y 14 y
none I (70), III (75)
13 14 15 16 17 18
14 22 21 39 33 50
y y y y y y
I (100), IV (25) III (25), IV (75), V (25) I (35), III (25), IV (40) none I (70) III (25), V (85)
19 20 21 22 23
48 46 49 48 43
y y y y y
III (30) III (100), IV (35), V (100) none IV (35) IV (35)
24 25 26 27 28
46 42 58 55 56
y y y y y
IV (70) III (40), IV (30) V (60) V (100) III (100), IV (80)
mtDNA Missense, rRNA, and tRNA Gene Mutations† 15508 (cytb), 602 (PHE), 709 (12s), 15452 (cytb) 10463 (ARG), 709 (12s), 15452 (cytb), 5301 (ND2), 1738 (16s), 3197 (16s) 10463 (ARG), 10554 (ND4L), 709 (12s), 5558 (TRP), 1888 (16s) 1604 (VAL) 10084 (ND3), 5721 (ASN), 12308 (LEU) 7521 (ASP), 14178 (ND6), 7389 (COI), 1738 (16s), 2885 (16s), 2768 (16s) 15907 (THR), 12308 (LEU) 15314 (cytb), 10424 (ARG) 15218 (cytb), 12308 (LEU), 4824 (ND2), 2758 (16s) 15924 (THR), 14798 (cytb), 12308 (LEU), 1811 (16s), 8331 (LYS), 8701 (ATP6), 1189 (12s) 5819 (CYS), 5931 (COI), 9775 (COIII), 2758 (16s) 709 (12s), 1719 (16s rRNA), 1738 (16s), 8414 (ATP8), 2768 (16s), 5655 (ALA), 2885 (16s), 10398 (ND3), 14069 (ND5) 7853 (COII), 11999 (ND4), 3338 (ND1) 12246 (SER), 12302 (LEU), 9216 (COIII), 1888 (16s) 7853 (COII), 11999 (ND4), 2352 (16s), 14069 (ND5) 10398 (ND3), 14798 (cytb), 12308 (LEU), 12181 (HIS), 1811 (16s) 10001 (GLY), 8701 (ATP6), 4824 (ND2) 10398 (ND3), 15951 (THR), 15907 (THR), 4025 (ND1), 2332 (16s), 2416 (16s), 7521 (ASP) 10463 (ARG), 709 (12s), 10738 (ND4L) 10398 (ND3), 15508 (cytb), 14798 (cytb), 15452 (cytb) 10237 (ND3), 12181 (HIS) 11718 (ND4), 4025 (ND1), 1888 (16s) 10398 (ND3), 10575 (ND4L), 15452 (cytb), 14928 (cytb), 7325 (COI), 2352 (16s) 7923 (COII) 1189 (12s), 10738 (ND4L), 12302 (LEU) 1719 (16s rRNA), 15924 (THR) 7578 (ASP), 12308 (LEU), 3197 (16s) 15508 (cytb), 5819 (CYS), 4312 (ILE)
Mitochondrial Morphology‡ normal abnormal normal normal normal normal normal abnormal normal abnormal normal normal abnormal normal normal normal normal abnormal normal normal normal normal normal normal normal normal normal abnormal
mtDNA, mitochondrial DNA; rRNA, ribosomal RNA; tRNA, transfer RNA. * Number in bracket in OXPHOS column represents the % reduction of control activity. † Bold characters in mtDNA point mutation column represent potential pathogenic mutations. ‡ Abnormalities in mitochondrial shape, size and cristae were noted.
mal individuals. However, the definition of what is truly a mtDNA mutation is not so simple. A number of sequence alterations in mtDNA (compared to the standard Cambridge sequence [19]) may occur (21). Some of these represent simple mistakes in the original mtDNA sequence. For example, alterations in sequence at nt 14199, 15236, 14272, 14368, 3106, 8860 listed as normal variants in MITOMAP were found in all of our patients and controls. Also, the definition of what constitutes a normal or polymorphic variant itself is not truly rigorous (ie, a number of putative controls showing mtDNA sequence variation may develop cardiac disorder at a later time). Our approach involved the complete sequencing of all of the coding genes of cardiac mtDNA in each of 28 individuals with DCM. Our rationale for this extensive effort stemmed from our previous experience with DCM, which showed a high incidence of specific enzyme activity defects but of which molecular basis was undefined (5,6). Only by using such a comprehensive analysis
might the effect of single and multiple mutations be elucidated. Comparison With Previous Studies Recent studies documenting the presence of mtDNA mutations have focused on mutant tRNA alleles and their flanking sequences, analyzing only selected regions of the mtDNA genome in addition to restriction length polymorphism (23,31). In their study, Arbustini et al (23) limited their analysis to patients harboring mitochondrial ultrastructural abnormalities, which raises the warning that DCM patients with nonspecific mitochondrial structural abnormalities may carry mtDNA mutations. In spite of these differences, our study also showed a wide distribution of mutations throughout the entire mitochondrial genome. Interpretation of Study Findings We found heteroplasmic mutations in highly conserved regions in cytb, COII, COIII, and in tRNAArg and
328 Journal of Cardiac Failure Vol. 6 No. 4 December 2000 ND5. The prevailing view that heteroplasmic mutations are more likely to occur in pathogenic mutations than in normal polymorphisms is therefore consistent with these mutations being considered pathogenic. In addition, a number of previously unreported and reported mtDNA mutations in highly conserved sequences that seemed to be homoplasmic (ie, containing either all mutant or all wild-type alleles) were identified. Although it is likely that the majority of these homoplasmic mutations represent polymorphisms, some of these nucleotide changes may also be pathogenic because deleterious homoplasmic mtDNA mutations can be present in affected tissues (30,31). It is also noteworthy that the point mutations detected in previous studies were not found in our study and vice versa. This is consistent with the heterogeneity of enzymatic defects observed in DCM patients and supports the uniqueness of most of the reported mtDNA mutations involved in mitochondrial disease. Moreover, it is clear that the task of routine screening for pathogenic mtDNA mutations is particularly difficult because relatively few mutations are shared. A high incidence of OXPHOS enzymatic activity defects was found in the study patients. These defects were found in any of the 4 respiratory complex I, III, IV, and V activities (with at least 1 mtDNA-encoded subunit) and not in complex II activity, whose peptide subunits are entirely nuclear-encoded. Moreover, the enzymatic findings are consistent with the assignment of pathogenecity for mutant alleles of COII, COIII, and cytb because mutations in these genes were accompanied by marked reductions in the respiratory activities of complex IV (correlated with the presence of the COII and COIII alleles) and a reduced level of complex III (with the presence of cytb). In addition, patients harboring the heteroplasmic tRNA mutation (eg, Arg10424) display multiple defects in respiratory activities. The molecular basis of the mitochondrial dysfunction and presumably of the cardiac pathology (ie, the presence of heteroplasmic mutations and their correlation with specific enzymatic reductions) was therefore identified in a total of 9 of the 28 DCM patients. The basis for the reduced respiratory enzymatic activity levels observed in 13 patients, however, remains unidentified and potentially could be because of any of a variety of nuclear DNA defects. In addition, some of the other mtDNA mutations identified in this study as likely polymorphisms could have a contributory role in pathogenesis either acting as deleterious homoplasmic mutations or acting in combination. Our data also suggest that in the study group, no difference was found in the incidence or type of mtDNA point mutations as a function of age. Although the number of patients of different ages in this study is too limited for a statistical analysis of age, our findings of 3 children with pathogenic mtDNA mutations (2 less than
a year old) suggest that at least a subset of the detected mtDNA mutations are primary to mitochondrial dysfunction and not likely a consequence of long-standing disease. This contrasts with the age-dependent accumulation of large-scale mtDNA deletions (32) in somatic cells, proposed as a potential scenario (33) in the development of DCM. Studies directed toward further understanding both the significance and the mechanism for generating the higher incidence of mtDNA point mutations in DCM are clearly warranted and will be necessary in addressing the critical questions of cause and effect. Such studies should also include the determination of the extent of somatic mutations in cardiac mtDNA of each individual.
Conclusion Our data suggest that mtDNA point mutations seem to be more prevalent in DCM. This finding may be related to the severity of myocardial disease in the cases presented (ie, those requiring cardiac transplant). It also could be influenced by the genetic constitution of the patient and control populations for which we have limited data. The role of an accumulated load of mtDNA mutations in the genesis and progression of myocardial disease and mitochondrial pathology clearly warrants further examination. In addition, several of the mutations identified in the study patients seem to be pathogenic because of the following: 1) they change highly conserved residues that may modulate the function of proteins or tRNAs, 2) they are heteroplasmic, 3) they are associated with specific enzymatic defects, and 4) they are neither present in our controls nor in the literature. In vivo methods of assessing mitochondrial enzyme function with specifically altered mitochondrial genes (to test the potential pathogenicity of each mutation) seem warranted. Also, secondary structure analysis of proteins and tRNA with and without point mutations might be informative in predicting the effects on protein function.
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