Cardiac transplantation in an incomplete Kearns-Sayre syndrome with mitochondrial DNA deletion

Vol. 3, No. 5/6. pp. 561 566. 1993 Copyrightt" 1994ElsevierScienceLtd Printed in Great Britain. All rightsreserved 096(~8966/93$6.00+ .00

Neuromusc. Disord.,

Pergamon

CARDIAC SAYRE

TRANSPLANTATION SYNDROME

WITH

IN AN

INCOMPLETE

MITOCHONDRIAL

DNA

KEARNSDELETION

C. TRANCHANT,* B. MOUSSON,t M . MOHR,3~ R. DUMOULIN,t M. WELSCH,§ C. WEESS,* G . STEPIEN

and J. M. WARTER* *Service des Maladies du Syst6me Nerveux et du Muscle, C H U , Strasbourg; #Centre d'Etude des Maladies M&aboliques, HOpital Debrousse, Lyon; ++Institut d'Anatomie Pathologique, Facult6 de M6decine, Strasbourg; §Service d'Hypertension et des Maladies Vasculaires, C H U , Strasbourg; IIUMR 106, CNRS, U C B Lyon I, France

Abstract--A 38-yr-old man with external ophthalmoplegia, cardiac conduction abnormalities, hearing loss, and ragged-red fibres in skeletal muscle biopsy, developed severe signs of cardiac failure within a few months. Echocardiography and angiography demonstrated a dilated cardiomyopathy. Ubiquinone 140 mg day-' did not stop the worsening of the cardiac status and cardiac transplantation was performed. Molecular analysis showed a heteroplasmic 4.5 kb mitochondrial DNA deletion in endomyocardial tissue. Eighteen months later, cardiac evolution is good and neurological status is stable. Key words: Kearns-Sayre syndrome, cardiac transplantation, mitochondrial DNA deletion.

modified Gomori trichrome, periodic acidSchiff (PAS), oil red O, ATPase, N A D H and succinic dehydrogenase stains, as well as electron microscopy.

INTRODUCTION

Syndromes characterized by the presence of ragged-red fibres in muscle biopsy stained with the modified Gomori trichrome constitute a clinically heterogeneous group of diseases. Cardiac conduction abnormalities can occur in these diseases, for example, in Kearns-Sayre syndrome (KSS). KSS is defined by the triad: onset before age 20, progressive external ophthalmoplegia (PEO), and pigmentary degeneration of the retina, plus one of the following: heart block, cerebellar syndrome or high cerebrospinal fluid protein content (over 100 mg dl- ~). Abnormalities of myocardiac ultrastructure and clinical signs of cardiomyopathy are rare. We report a patient with incomplete KSS who developed dilated cardiomyopathy with cardiac failure which required heart transplantation. Molecular analysis demonstrated a heteroplasmic mitochondrial D N A (mtDNA) deletion in myocardium.

Enzymological studies Enzymological studies of mitochondrial respiratory complexes were performed in explanted myocardium.

Southern blot analysis Total D N A (5 /tg) derived from the frozen heart muscle was digested with 20 U of Pvu II ('single cutter' enzyme restriction), subjected to electrophoresis on a 0.8% agarose gel, transferred to Hybond + (Amersham) and probed with labelled m t D N A (generous gift of A. R6tig, I N S E R M U12, H6pital des Enfants Malades, Paris). To identify the site of the deletion, the D N A was digested with appropriate restriction enzymes: Ava II, BamHl, EcoRI, Hind III, Pst I.

METHODS

Polymerase chain reaction (PCR) method

Histological analysis

Previously extracted DNA (0.5 #g) was submitted to PCR amplification with three combinations of primers: nucleotides (nt) (L)8150-8166 + (H)16159-16142, nt (L)53175333 + (H)13832-13812, nt (L)8150-8166 + (H) 15613-15594. The reaction was carried out

A standard battery of histological and histochemical stains were performed on deltoid muscle biopsy and on explanted myocardial muscle: haematoxylin and eosin stain on paraffin-embedded sections and on cryostat sections, 561

562

C. TRANCHANTet

with 200 pM of each primer and 2.5 U of Taq polymerase (Perkin Elmer Cetus) (30 cycles; denaturation 30 s at 94°C; annealing 30 s at 55°C; primer extension 90 s at 72°C). The amplified DNA fragment was digested by restriction enzymes: Ava II, BamHl, EcoRI, Hind III, Xba I, Hpa I. CASE REPORT

A 17-yr-old man without familial history presented in 1969, after a pharyngitis, with haematuria. Renal biopsy was normal and did not show any sign of glomerulonephritis. In 1985, during a routine medical examination, an elevated blood pressure was found. An electrocardiogram showed a left anterior branch block. Basal echocardiography was normal and with the ejection fraction at 0.63. Routine biochemistry and renal function tests were normal. Beta-blockers and angiotensin converting enzyme inhibitors improved blood pressure. In March 1990, at age 38, he developed unsteadiness and ear buzzing. Neurological examination showed a complete external ophthalmoplegia which, in fact, had been present for several years, and a sensorineural hypoacousia. Strength,

al.

sensibility, deep tendon reflexes were normal and plantar responses were flexor. Fundal examination was nol:mal. Routine haematology and biochemistry including muscular enzymes and lactate level were normal. The protein level in the cerebrospinal fluid was 0.83 g 1 t. Electromyogram and nerve conductions were normal. Cardiac function was unchanged. A deltoid muscle biopsy was performed. Light microscopy demonstrated fibre size variation and internal nuclei, numerous ragged-red fibres and excessive oxidative activity. Electron microscopy showed abnormal mitochondria (Fig. 1). Neurological evolution was stable, but in the following months cardiac failure signs appeared: effort dyspnoea, ventricular extrasystolia. In November 1990, echocardiography demonstrated global hypokinetic cardiomyopathy. Angiography with right and left catheterism confirmed a dilated cardiomyopathy with an ejection fraction at 0.30. Pulmonary pressures and coronarography were still normal. In parallel with tonicardiac treatment, ubiquinone (Ubiten) 140 mg day -~ was given. Clinical evolution was stable until March 1991. At this time, after a rhinopharyngitis, he developed several episodes of cardiac failure. Echocardiography demonstrated a low transval-

Fig. 1. Electronmicrographof the deltoideusmuscle.Subsarcolemmalaggregateof abnormal mitochondria with paracrystallineinclusions ( x 10,600).

Cardiac Transplantation in KSS

Fig. 2. Left ventricle myocardium from the explanted heart. Two fibres are "ragged-red" with modified Gomori trichrome stain (x 250).

Fig. 3. Electron micrograph of the explanted myocardium. Normhl mitochondria moderately increased in number ( x 16,200).

563

564

C. TRANCHANT e/ a[,

F Pm

O. 15887 I 7S DNA

12s rRNA

V

PL

16s rRNA

14149

f 16142

/

15613 L (UUR) lqD5 NDI I M

-L (L-'~ 12337 -S (AGY) 12137

Q 11680

ND2

10256 lqD4

W

10766 "R D3

S COClq) COl .~Onl D

COH

KI

10404

999O

ATPue 6

ATPase 8

Fig. 4. Mitochondrial DNA function map indicating the position of the KSS deletion in explanted endomyocardial tissue. OH and OL, origins of heavy and light strand replication, respectively;PH and PL, heavyand light strand transcription promoters, respectively;ND 1~: NADH dehydrogenasesubunits; CO 1 III: cytochrome c oxidase subunits; Cyt b: cytochrome b; single capital letters indicate amino acid specific tRNA genes [1]. vular flow, a global dilatation and a small thrombus in the right ventricle. The cardiac status worsened, leading the patient to strict rest. Cardiac transplantation was decided and performed in October 1991. Eighteen months later, cardiac evolution and neurological status were stable. Histology and electron microscopy performed on the explanted heart showed mild mitochondrial abnormalities (Figs 2, 3), but histoenzymology and enzymological studies of mitochondrial respiratory chain complexes were normal. Southern blot analysis showed the presence of two populations of m t D N A , one normal (16.5 kb) and one partly deleted (about 12 kb; 25% of the total mtDNA). Using the PCR method, a fragment of 3.5 kb was amplified with the first combination of primers consistent with a 4.5 kb deletion. No fragment was amplified using the two other primer combinations, suggesting that the deleted m t D N A lost the positions 13832 and 15613. The deletion preserved Sac I (position

9643) and Xba I (position 10256) restriction sites but included Hind III (positions 11680 and 12570), EcoRI (position 12640), Ava II (positions 12629 and 13367) and Bam HI (position 14258) restriction sites. These results indicate that the 4.5 kb deletion extended from at least position 11680 to position 15613 with lower and upper limits of 10256 and 16142. The deleted region included the genes encoding for three subunits of complex I (ND4, ND5 and ND6), cytochrome b and four of five t R N A s (Fig. 4). DISCUSSION In the absence of retinal degeneration, our patient presented an incomplete form of KSS. A diagnosis of chronic progressive external ophthalmoplegia (CPEO) could have been proposed, but cardiac abnormalities are unusual in this syndrome. The pathological data with the presence of ragged-red fibres are consistent with

Cardiac Transplantation in KSS

the diagnosis of mitochondrial cytopathy, whatever the name of the syndrome. Mitochondrial cytopathies (MC) are due to a primary defect of mitochondrial oxidative phosphorylation [2] and have been associated with various mutations in mtDNA [3-6]. In our case, molecular analysis of the mtDNA demonstrated a heteroplasmic deletion in heart muscle consistent with KSS. Cardiac conduction abnormalities are common in KSS. They are usually asymptomatic (first degree heart block, bundle branch block) but in some cases with complete heart block, a permanent pace-maker can be necessary. Cardiac function is rarely reported to be severely impaired but electrocardiography can demonstrate non-specific ST segment or T wave abnormalities. A severe rapidly congestive cardiac failure has been rarely reported in KSS [7, 8]. But in other MC, a dilated cardiomyopathy can be the single manifestation of the disease. Ubiten or coenzyme Ql0 (CoQl0) administration increases mitochondrial succinate dehydrogenase activity and electron transfer in mitochondria [9]. Different trials have been performed in patients with various mitochondrial myopathies (which may or may not be due to a defect in CoQl0) [10-12]. In some patients a decrease of lactate level after effort has been demonstrated, but there was no clinical improvement. In cases with cardiac involvement, Ubiten 1-2 mg kg-~ could improve ECG abnormalities. Cardiac transplantation has been reported in only one case of KSS [8]. A 21-yr-old patient developed rapidly progressive congestive cardiac failure. Histopathological studies of excised heart showed degeneration of myocardiac fibres with perinuclear vacuolation pleomorphism and enlargement. Electron microscopy showed v a ~ ' i o u s mitochondrial abnormalities; mitochondria were increased in number and of varying size and shape, and had electron dense areas. Neither biochemical nor molecular studies were performed. In our case, two questions were raised before transplantation: (a) the neurological future of this patient? and (b) the mechanism of the cardiomyopathy? The neurological status (external ophthalmoplegia without muscular weakness in limbs) has been stable for several years and allowed a normal life. Cardiomyopathy in MC is thought to be due to mitochondrial abnormalities in heart muscle. Since mitochondrial

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phenotype and genotype are normal in the transplanted heart muscle, no cardiomyopathy can appear during subsequent generations of mitotically active cells. These two arguments led us to allow cardiac transplantation in this patient. Mitochondrial cytopathies have been correlated with various mtDNA mutations [13]. Kearns-Sayre syndrome is caused by a variety of heteroplasmic mtDNA deletion spanning up to 8 kb [3-6]. These deletions can be different in size and do not localize to a single region of the mitochondrial genome. There are no correlations between the site of the deletion, the biochemical abnormalities of respiratory chain enzymes and the clinical severity of KSS. Such deletions have been described and found in the myocardium of patients with isolated dilated cardiomyopathy [14]. In our case, the heteroplasmic deletion encompasses genes encoding for the subunits 4, 5 and 6 of complex I and for cytochrome b of the mitochondrial respiratory chain. Twenty-five percent of deleted mtDNA may not be sufficient to induce a defect in respiratory chain complex activities, since no biochemical abnormality was found in the myocardium. Thus, one can argue about the direct relation between genotype, biochemical and clinical phenotypes. REFERENCES 1. Heddi A, Lestienne P, Wallace D C, Stepien G. Mitochondrial DNA expression in mitochondrial myopathies and coordinated expression of nuclear genes involved in ATP production. J Biol Chem 1993; 268: 1215(~12163. 2. Karpati G, Arnold D, Matthews P, Carpenter S, Andermann F, Shoubridge E. Correlative multidisciplinary approach to the study of mitochondrial encephalomyopathies. Rev Neurol (Paris) 1991; 147:455-46 I. 3. Holt I J, Harding A E, Morgan Hughes J A. Deletions of mitochondrial DNA in patients with mitochondrial myopathies. Nature 1988; 337: 717-719. 4. Lestienne P, Ponsot G. Kearns-Sayre syndrome with muscle mitochondrial DNA deletion. Lancet 1988; 16: 885. 5. Zeviani M, Moraes C T, DiMauro S, et al. Deletions of mitochondrial DNA in Kearns-Sayre syndrome. Neurology 1988; 38: 1339-1346. 6. Moraes C T, DiMauro S, Zeviani M, et al. Mitoehondrial DNA deletions in progressive external ophthalmoplegia and Kearns-Sayre syndrome. N Engl J M e d 1989; 320: 1293-1299. 7. Picard R, Manigand G, Said G, Chauvin J P, Benit Ch. Myocardiopathie non obstructive primitive et ophtalmopl6gie externe progressive. Sere H @ Paris 1974; 50: 627~532. 8. Channer K S, Channer J L, Campbell M J, Russel Rees J. Cardiomyopathy in the Kearns-Sayre syndrome. Br Heart J 1988; 59: 486-490. 9. Schoffner J M, Wallace C. Oxidative phosphorylation diseases. Disorders of two genomes. In: Harris H,

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Hirschborn K, eds. Advances in Human Genetics. New York: Plenum, 1990; Chapter 5, 267-330. 10. Bresolin N, Bet L, Binda A, et al. Clinical and biochemical correlations in mitochondrial myopathies treated with coenzyme QI0. Neurology 1988; 38:892 899. 11. Ogasahara S, Nishikawa Y, Yorifuji S, et al. Treatment of Kearns-Sayre syndrome with coenzyme QI0. Neurology 1986; 36: 45-53. 12. Scarlato G, Bresolin N, Moroni I, et al. Multicenter

trial with ubidecarenone: treatment of 44 patients with mitochondrial myopathies. Rev Neurol (Paris) 1991; 174: 542-548. 13. Tritschler H S, Medori R. Mitochondrial DNA alterations as a source of human disorders. Neurology 1993; 43: 28~288. 14. Suomalainen A, Leinonen H, Peltonen L, Paetau A, Majander A, Somer H. Inherited idiopathic dilated cardiomyopathy with multiple deletions of mitochondrial DNA. Lancet 1992; 340: 1319-1320.