Abstract
Six patients in two unrelated families from the eastern Arabian peninsula presented with childhood-onset progressive external ophthalmoplegia (PEO), mild facial and proximal limb weakness, and severe cardiomyopathy requiring cardiac transplantation. Muscle biopsies showed ragged-red and cytochrome c oxidase-negative fibers. The activities of several complexes in the electron-transport chain were decreased and Southern blot analysis showed multiple mtDNA deletions. The apparent autosomal-recessive inheritance and the association with cardiomyopathy distinguish this syndrome from autosomal-dominant PEO with multiple mtDNA deletions. The combination of autosomal-recessive PEO, cardiomyopathy, and multiple mtDNA deletions appears to be another disease due to a defect of communication between the nuclear and mitochondrial genomes.
Progressive external ophthalmoplegia (PEO) has been associated with several different mutations of mitochondrial DNA (mtDNA). Single deletions of mtDNA are typically seen in muscle from patients with Kearns-Sayre syndrome (KSS) or with isolated ocular myopathy. 1 Both conditions are sporadic and are characterized by massive mitochondrial proliferation, with ragged-red fibers (RRF) in the muscle biopsy specimen. Maternally inherited forms of PEO are associated with point mutations in mtDNA, most commonly with the A3243G mutation in the tRNA1eu(UUR) (the “MELAS mutation’) 2 and, less frequently, with other mutations in the same or other tRNA genes. 3 Patients with these mutations also show RRF on muscle biopsy.
A distinct form of familial PEO, transmitted by mendelian inheritance and characterized by the co-existence in muscle of multiple mtDNA deletions, initially described in an Italian family by Zeviani et al., 4 has been later reported in several pedigrees from various countries. 5–8 Zeviani et al. 4 and Zeviani and Tirantig suggested that this autosomal-dominant PEO (AD-PEO) is due to a mutation in a nuclear gene that controls mtDNA integrity, a first example of a group of disorders due to defects of communication between the nuclear and the mitochondrial genomes.
We now report two unrelated families from the eastern Arabian peninsula with an autosomal-recessive syndrome characterized by PEO, severe cardiomyopathy, RRF, and multiple mtDNA deletions, presumably representing another defect of intergenomic communication.
Materials and Methods
Patients
The pedigrees of the two families are represented in figure 1.
Figure 1. Pedigrees of the two families. Solid symbols indicate affected members (see text). Arrows indicate the propositi.
Family A is from the eastern area of the Arabian peninsula and the asymptomatic parents of our propositi were first cousins.
Patient IV-5 developed ptosis and limitation of eye movements at about 8 years of age. At age 11, exertional dyspnea, palpitations, and chest pain led to the diagnosis of congestive-dilative cardiomyopathy, and he died of intractable heart failure at the age of 15.
Examination at age 13 showed a thin young man (weight, 38 kg, height, 146 cm) with edema of both ankles. Neurologic examination showed bilateral ptosis, complete ophthalmoplegia, and mild weakness of facial, neck flexor, and proximal limb muscles. Fundus was normal and there was no deafness, ataxia, or sensory loss. Deep tendon reflexes were hypoactive and plantar reflexes were flexor. Echocardiography showed poor ventricular contraction and moderate dilation of all heart chambers. At age 12, ejection fraction was 26% (normal <60%).
Patient IV-4, the propositus, also developed ptosis and ophthalmoparesis at age 9, followed by chest pain, palpitations, and exercise intolerance, which were attributed to heart failure. ECG showed tachycardia with nonspecific conduction defect. Echocardiography showed severe dilated cardiomyopathy with poorly contracting right ventricle and tricuspid regurgitation. Ejection fraction was 20%. He was also found to be heterozygous for the sickle cell trait, with 29% hemoglobin-S.
At age 14, he received orthotopic cardiac transplantation. At age 15, he developed proximal limb weakness, premature fatigue, and growth retardation. Examination at age 22 showed bilateral ptosis, complete ophthalmoplegia, and moderate weakness of facial, neck, and proximal limb muscles (arms more than legs). Fundoscopy was normal, and there was no deafness, ataxia, or sensory loss. Deep tendon reflexes were hypoactive.
Serum lactate was normal at rest (1.8 mM/l; normal, up to 2.2), but increased excessively after mild exercise (7.1 mM/l). Serum CK varied between 320 and 850 IU/l (normal, <195). CSF protein and lactate were normal. EEG, evoked potentials, and brain MRI were normal.
ECG and echocardiography showed moderate biventricular dysfunction of the dilated type.
Patient IV-3 developed ophthalmoparesis at age 8, followed by exercise intolerance, mild proximal weakness, and occasional palpitations. At age 21, his weight is 52 kg and his height 156 cm. He has no ptosis, but complete ophthalmoplegia, and mild weakness of facial, neck, and proximal limb muscles. Fundus is normal, and there is no ataxia, deafness, or evidence of peripheral neuropathy. He has no evidence of heart failure or valve disease. Ejection fraction is 48%. Holter monitoring shows occasional paroxysmal atrial tachycardia, controlled with medication.
Family B is from Bahrain, an island-state in the Persian Gulf. The parents were not consanguineous.
Patient II-9, the propositus, was evaluated at age 17 because of ophthalmoplegia, weight loss, and exercise intolerance. He was born after normal pregnancy and delivery and had normal early development. Droopy eyelids and limitations of eye movements were first noticed at 8 years of age. His school performance was excellent, but he could never keep up with his peers in physical activities because of weakness and poor stamina. Examination at age 17 revealed a thin (39 kg) young man with obvious wasting of all shoulder girdle muscles. There was bilateral ptosis, complete ophthalmoplegia, and moderate weakness of facial muscles (he could not whistle or puff up his cheeks). There was generalized weakness of both axial and limb muscles, which was especially pronounced in proximal arm muscles. Fundoscopic examination was normal and there was no hearing loss measured by audiometry. Deep tendon reflexes were hypoactive and plantar reflexes were flexor. There was no ataxia and no evidence of cerebellar dysfunction. Sensory examination was normal.
Normal blood tests included: lactate and pyruvate, ammonia, amino acids, immunoglobulins, anti-acetylcholine receptor antibodies, and thyroid function tests. CSF protein and lactate were also normal. Serum CK was 580 U/l (normal 24 to 195). EMG showed myopathic motor units. Motor and sensory nerve conduction velocities were normal. Brain MRI was normal.
ECG showed left anterior fascicular block and left ventricular hypertrophy. Echocardiography disclosed increased thickness of the left ventricular wall, reduced contractility, and decreased ventricular filling; ejection fraction was around 48% (normal, <60%). Cardiac catheterization suggested severe diastolic dysfunction of both ventricles with global hypokinesia of the left ventricle. Cardiac output was reduced to 3.7 l/min (normal, 6.0). Coronary angiography was normal.
He was readmitted 4 months later with marked dyspnea at rest, dependent edema, hepatomegaly, and ascites. Deterioration of left ventricular function was confirmed by echocardiography and radionucleotide angiography. Ejection fraction had dropped to 25%. The heart failure was resistant to conventional therapy and he died in the hospital while awaiting heart transplantation.
Patient II-10. This older sister of the propositus also developed ptosis and ophthalmoplegia around age 9, accompanied by facial and proximal limb weakness. Around age 12, she developed exertional dyspnea, tachycardia, and dependent edema. She died of heart failure at the age of 18.
Patient II-7. The clinical picture in this younger sister of the propositus was virtually identical to that of her affected siblings: ptosis and ophthalmoplegia started in childhood, followed by weakness and by rapidly progressive heart failure. She died at age 13 after a prolonged hospitalization in the coronary care unit.
Both parents and four siblings were healthy and there was no history of PEO or cardiac disease in previous generations.
Morphology
Muscle samples from the two propositi, patient IV-4 (family A) and patient II-9 (family B), were frozen in isopentane cooled in liquid nitrogen, stored in liquid nitrogen, and shipped in dry ice. Frozen serial sections, 8 km thick, were utilized for the demonstration of succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) activities, as described. 10 Additional serial sections were stained with hematoxylin and eosin (H-E), Nile red, and the Gomori trichrome. 11
Biochemistry
Mitochondria1 enzyme activities were measured in crude muscle extracts from both propositi and from patient IV-3 (family A) as described. 12
Molecular genetic analyses
Total DNA extraction from muscle, preparation of mtDNA probes, and Southern blot analysis were performed as described. 13 Briefly, about 5 kg of total DNA are digested with the restriction endonuclease Puu II or Bam HI to linearize the mtDNA, and are run on a 0.8% agarose gel, blotted onto a nylon membrane, then hybridized with full-length human mtDNA labeled by the random primer method 14 in the presence of 32P-dATP.
Mapping of mtDNA deletions was performed by a combination of Southern blot, PCR, and direct sequencing.
Southern blot mapping. Total DNA, digested with Puu II and processed by electrophoresis as described above, was blotted onto two nylon membranes, and hybridized to a set of probes corresponding to different mtDNA regions. The fragments used as probes were obtained by polymerase chain reaction (PCR) amplification of mtDNA extracted from normal human muscle, using the following oligonucleotides (Cambridge sequence 15): Probe 1, forward nt126-150, backward nt1753–1776; Probe 2, nt3115–3134 and nt4521–4542; Probe 3, nt7433–7468 and nt8228–8321; Probe 4, nt8274-8305 and nt9931–9950; Probe 5, nt9007–9036 and nt10240–10269; Probe 6, nt10353–10377 and nt12392–12412. The fragments were purified by Gene-Clean (Bio 101) and labeled as described above. Before each hybridization, the membranes were stripped by boiling for 15 to 20 min and exposed at −70°C to an x-ray film (Kodak-XAR) to verify the absence of any residual radioactive signal from the previous hybridization.
PCR amplification To confirm the presence of the deletions, we performed PCR amplification by the “primer shifting” method, 16 which excludes nonspecific PCR amplification and facilitates localization of the deleted regions. We performed seven amplifications, with the following pair of primers (forward; backward): (1) nt7433–7468, and nt16033–16060; (2) nt7955–7979, and nt16033-16060; (3) nt8274-8305, and nt15574-15600; (4) nt8274-8305, and nt13692-13720; (5) nt8274-8305, and nt12966-12995; (6) nt9007-9036, and nt15574-15600; (7) nt9744-9767, and nt162-184. PCR conditions were: 94 °C X 1, 60 or 65°C X2, 72 °C Xl′ 25 cycles.
Direct sequencing. We performed sequencing analysis of the major bands obtained with primer sets 4 and 6 to localize the deletion breakpoints. The bands were extracted from low-melting-temperature gel, purified by Geneclean, and sequenced using the “ds DNA cycle sequencing kit” (Gibco BRL), according to the manufacturer's instructions. Sequencing products were processed by electrophoresis through a 6% polyacrylamide/7M urea gel (19:1 acrylamide:bis-acrylamide). The gel was vacuum-dried for 1 hour and exposed to XAR-Kodak film for 24 hours at room temperature.
Results
Morphology
Muscle biopsy specimens from the two propositi (IV-4 of family A and II-9 of family B) showed RRF with Gomori trichrome and SDH stains, indicating mitochondrial proliferation (figure 2A). In both patients, the COX reaction showed a mosaic of COX-positive and COX-negative fibers. While most RRF were COX-negative, many more COX-negative fibers did not show histochemical evidence of mitochondrial proliferation (figure 2B). The overall proportion of COX-negative fibers was 17.3% in patient II-9 and 30% in patient IV-4.
Figure 2. Serial frozen muscle sections from patient IV-4 (family A) stained for succinate dehydrogenase (SDH) (A) and cytochrome c oxidase (COX) (B) activities. The SDH stain reveals one ragged-red fiber (RRF) (star), and the COX stain shows seuera1 COX-negative fibers (arrows), including the RRF (star). Magnification X 180, before 22.5% reduction.
Biochemistry
In muscle extracts from both propositi and from patient IV-3 (family A), the activities of all electron transport chain complexes containing mtDNA-encoded subunits (complexes I, III, and IV) were moderately to markedly decreased (table). These results would be even more striking if activities were referred to citrate synthase, a matrix enzyme encoded by nuclear DNA and reflecting mitochondrial “volume”.
Table Mitochondria1 enzymes in muscle biopsies from patients with AR-PEO and multiple mtDNA deletions
Molecular genetics
Southern blot analysis of muscle DNA from the two propositi showed the 16.5-kb band corresponding to normal mtDNA and several additional smaller bands ranging from approximately 11.5 kb to 7.5 kb (figure 3). Both pattern and intensity of the abnormal bands were similar but not identical in the two patients. PCR amplification of selected mtDNA fragments documented that the most abundant abnormal band in each patient corresponded to the “common deletion” of 4.977 kb. 17 This was confirmed by direct sequencing.
Figure 3. Southern blot analysis of muscle mtDNA in patient II-9 of family B (P1), patient IV-4 of family A (P2), and a control (C). Total human mtDNA was used as a probe in lanes 1 to 3; in addition to the 16.6-kb band corresponding to normal mtDNA, several additional bands are seen in P1 and P2, corresponding to deleted mtDNA molecules. Probe 6 (see Materials and Methods) was used in lanes 4 to 6: this probe encompasses the genes for tRNAArg, ND3, ND4L, ND4, tRNASer(AGY), tRNA1eu(CUN), and part of the ND5 gene. Only the normal 16.5-kb band is evident, suggesting that these genes were deleted. Probe 2 (see Materials and Methods) was used in lanes 7 to 9: this probe comprises the genes for 16s rRNA, tRNA1eu(UUR), ND1, tRNAIle, tRNAGln, tRNAf-Met, and part of the ND2 gene. The reappearance of the pattern seen in lanes 1 to 3 suggests that these genes were spared by the deletions.
Discussion
Six patients from two unrelated families from the eastern region of the Arabian peninsula presented a novel syndrome characterized by childhood-onset PEO, facial and proximal limb weakness, and fatal cardiomyopathy requiring cardiac transplantation. Muscle histochemistry in the two propositi showed RRF, most of which were COX-negative by histochemistry, but also numerous scattered non-ragged-red COX-negative fibers. Biochemical analysis of muscle from the two propositi and an affected sibling showed partial defects of respiratory chain enzymes, involving all complexes containing mtDNA-encoded subunits, and sparing SDH and citrate synthase, which are exclusively encoded by the nuclear genome.
The combination of isolated COX-negative fibers and multiple respiratory chain defects suggested an alteration of mtDNA, which was confirmed by molecular genetic analysis. Southern blot of mtDNA showed multiple smaller bands in addition to the normal mtDNA band of 16.5 kb. The presence of multiple mtDNA deletions in muscle was confirmed by PCR analysis. The breakpoints of the deletions were defined by a combination of PCR and direct sequencing. Most of the deletions were contained within an arc of mtDNA spanning the region from the COX II gene to the cytochrome b gene and sparing the origins of heavy and light strand replication. Mitochondrial genomes from the two propositi harbored similar, though not identical, deletions.
The association of PEO and multiple mtDNA deletions with a mendelian pattern of inheritance makes this syndrome very similar to the autosomal-dominant form of chronic progressive ophthalmoplegia (AD-CPEO) described by Zeviani et al., 4 Zeviani, 5 and Servidei et a1. 6 in Italian families and by Suomalainen et al. 7 in a Finnish family. In these patients, PEO typically started in the third decade, was accompanied by proximal limb weakness and wasting, and the course was slowly progressive. In the Italian patients, hearing loss, peripheral neuropathy, cataracts, tremor, and ataxia were frequently, but not invariably, present. In the Finnish family, depression was the most important accompanying sign. All patients with AD-CPEO had RRF on muscle biopsy, and mild elevations of serum lactate were reported in the Italian patients. Cardiomyopathy was not a feature and ECGs were normal in most patients. 6,7 Although excessive numbers of mitochondria, some containing abnormal inclusions and distorted cristae, were found by electron microscopy in the myocardium of the Finnish patient, the activities of respiratory chain enzymes were normal.
Despite the clinical and molecular genetic similarities, there are a few unique features that distinguish the present syndrome from AD-CPEO. The ophthalmoplegia presented earlier in life, and the clinical picture was dominated by intractable cardiomyopathy, which caused death before age 20 in three of the six patients and made cardiac transplantation necessary in one; in addition, the Arabian patients had no clinical or electrophysiologic evidence of central or peripheral nervous system involvement. Another major distinctive feature was the mode of inheritance: involvement of several siblings of both sexes, parental consanguinity in one family, and lack of involvement in previous generations strongly suggests autosomal recessive rather than dominant inheritance in our families. That both families originated from the eastern region of the Arabian peninsula further suggests the possibility of a “founder effect” for this syndrome.
Multiple mtDNA deletions in muscle occur in a variety of conditions, including sporadic inclusion body myositis, 18 mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), 19,20 and late-onset mitochondrial myopathy, 21 as well as in an assortment of individual cases with diverse symptoms. 8,22–27 The pathogenic role of the multiple deletions in these disorders is questionable, especially in older patients, in whom they may be a consequence of normal aging. 28 Kawashima et a1. 8 described a condition that closely resembled the syndrome of our patients. A Japanese man had PEO; weakness of facial, respiratory, and proximal limb muscles; and neurosensory hearing loss, He had an affected sister and there was parental consanguinity, suggesting autosomal-recessive inheritance. However, the onset of PEO was around age 30 and there was no cardiopathy. 8 Autosomal recessive transmission was also evident in another Japanese family in which two brothers suffered from ptosis, limb weakness, optic atrophy, and sensory neuropathy 29 and had RRF and multiple deletions on muscle biopsies. 22 However, there was minimal limitation of extraocular movements, onset of weakness was late, and the heart was not affected. Abundant mtDNA multiple deletions were also detected in heart and muscle from a mother and son with dilated cardiomyopathy. 30 However, neither patient had PEO, and inheritance appeared to be autosomal dominant or, less likely, maternal.
When multiple mtDNA deletions are abundant enough in muscle to be detected by Southern blot analysis in young adult patients with PEO and RRF, and the trait is clearly transmitted by mendelian inheritance, as in the families with AD-CPEO and in the Arabian families with autosomal-recessive CPEO (AR-CPEO) and cardiopathy, the multiple rearrangements of the mitochondrial genome may be a direct consequence of mutations in nuclear genes. 4,7,9 Such mutations could somehow facilitate an intrinsic propensity of mtDNA to undergo rearrangements (such as those occurring with age), or it could affect factors involved in the recognition and elimination of spontaneously occurring rearrangements. 9 Linkage analysis is being conducted to identify the gene or genes implicated in AD-CPEO, and the gene responsible for AD-CPEO in the Finnish family has been assigned to chromosome 10q23.-24.3. 31 We are presently carrying out linkage analysis in the two Arabian families in the hope of mapping the presumably single gene associated with AR-CPEO and cardiopathy.
Irrespective of further molecular genetic analysis, this novel and severe syndrome should be included in the differential diagnosis of the familial progressive ophthalmoplegias.
Footnotes
-
Supported by National Institutes of Health grants NS 11766, HD 32062, and K08-NS01617, and by a grant from the Muscular Dystrophy Association. Dr. Santorelli is supported by a fellowship from Telethon Italia.
Received July 3, 1995. Accepted in final form July 13, 1995.
- Copyright 1996 by the American Academy of Neurology
References
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