the clinical diagnosis and molecular genetics of kearns

Introduction

Kearns-Sayre syndrome (KSS; OMIM #530000) is a rare, sporadic disorder representing a heterogeneous group of metabolic diseases known as mitochondrial encephalomyopathies. The clinical picture of KSS consists of a classic triad of symptoms namely: onset of the disease before 20 years of age; progressive external ophthalmoplegia with ptosis; and pigmentary degeneration of the retina. The disease is also manifested by many secondary abnormalities including cardiac conduction defects, muscle weakness, neurological abnormalities (neural deafness, cerebellar ataxia, mental retardation, dementia, convulsions and neuropathy) and several endocrine disorders (diabetes mellitus, hypoparathyroidism, thyroiditis, hypogonadism and short stature) (Table 1) (1-3). The true incidence is unknown, and no racial prevalence study has been reported. KSS affects both sexes equally and is characterized by progression of multisystemic clinical features leading to premature death in most cases. Mitochondrial DNA (mtDNA) rearrangements lie at the molecular background of this disease. The disease is usually dominated by the involvement of skeletal muscles and the nervous system because of mitochondrial dysfunction (hence the term “mitochondrial encephalomyopathy”). In most cases a variety of deletions and/or duplications in mtDNA are found, affecting genes encoding respiratory chain proteins (4-7). These rearrangements impair oxidative phosphorylation and the energy metabolism of mitochondria and lead to dysfunction of many tissues, especially those with high energy demand, such as muscles and brain. The characteristic histological manifestations of damaged mitochondria are the ragged red fibres seen in modified Gomori

The Clinical Diagnosis and Molecular Genetics of Kearns-Sayre Syndrome: a Complex Mitochondrial

EncephalomyopathyJarosław Maceluch, PhD, Marek Niedziela, MD, PhD

Department of Pediatric Endocrinology and Diabetes, Poznan University of Medical Sciences, Poznan, Poland

Corresponding author: Dr. Marek Niedziela, Department of Pediatric Endocrinology and Diabetes, Poznan University of Medical Sciences, Szpitalna Street 27/33, 60-572 Poznan, Poland, Tel: +48 61 849 1481, Fax: +48 61 848 0291, Home Address: Palacza Street 122F/8, 60-278 Poznan, Poland, Tel: +48 61 662 13 24, e-mail: [email protected]

REFERENCE N0 206 IS NOT MENTIONED IN THE TEXT PLEASE ADD

Abstract

F rom the first description by Kearns and Sayre in 1958, this syndrome has been diagnosed in several hundred patients. However, the labile character

of its clinical manifestations makes diagnosis difficult and delayed. Only recently, some thirty years from the first diagnosis, have we recognized mitochondrial DNA rearrangements as the molecular basis of the disease. This has lead to increasing interest in the contribution which mtDNA deletions make to Kearns-Sayre Syndrome (KSS) and other disorders. Although the true prevalence of this syndrome in the general population is unknown, a basic awareness of the KSS phenotype, as well as of the essential elements of patient evaluation is important for appropriate patient management. Although methods of assessing patients for mtDNA rearrangements are well developed, ambiguity in patient diagnosis often remains even after detailed, multisystem testing. Advances in our understanding of the genetic background and the tissue specific effects of mtDNA deletions, in addition to resolving the inheritance pattern, will also increase our ability to diagnose, manage and counsel patients with this disorder.

Ref: Ped. Endocrinol. Rev. 2006;2:??-??

Key words: Kearns-Sayre Syndrome; KSS; Mitochondrial DNA; Deletions; Duplications; Encephalomyopathy; Inheritance

Pediatric Endocrinology Reviews (PER) n Volume 4 n No. 2 n December 200622

trichrome stained muscle biopsies. On Electron Microscope (EM) images accumulations of abnormal mitochondria can be seen beneath the cell surface. Another characteristic laboratory finding in KSS is an elevated protein level (above 100 mg/dl) in the cerebrospinal fluid. The mutated mtDNA coexists with normal molecules (heteroplasmy) and the proportion of mutated to normal mtDNA has an influence on both the occurrence and severity of clinical symptoms. To date over 100 different deletions have been identified in human mtDNA [see MITOMAP database (8)], the most common is 4977 base pairs in length and spans between nucleotides 8469 and 13447. Deletion breakpoints are often flanked by direct repeats and, in most cases, they occur spontaneously. There is no effective treatment of KSS and the complicated character of its clinical features makes the correct diagnosis of this disease extremely difficult.

Clinical manifestations of Kearns-Sayre syndrome

In 1958 Kearns and Sayre first described a specific multisystem disorder consisting of chronic progressive external ophthalmoplegia, retinitis pigmentosa and atrioventricular heart block (9). Despite its rare occurrence, significant progress has been made since then in delineating the natural history and complications of KSS, a prototype for mitochondrial genetic syndromes. Although the specific clinical phenotype of KSS associated with mtDNA deletion is now well known, there are still problems in determining the correct diagnosis, especially in non-classical cases, because of the significant overlap in clinical manifestations with other mitochondrial cytopathies (10). Diagnosis is now based on the clinical picture and the results of laboratory tests, inseparably including the genetic analysis of mitochondrial DNA. While genetic

counseling is the primary focus of care, studies have shown the benefit of appropriate medical and surgical treatment.

Children with Kearns-Sayre syndrome usually appear normal at birth. Males and females are affected equally and although some patients have different, transient systemic abnormalities and metabolic disorders, the early development is normal. Ptosis is usually the first sign of the disease (Figure 1A); the child may be observed using their brow muscles to elevate the eyelids. Ptosis is followed within a few years by progressive external ophthalmoplegia (PEO) (11). PEO usually begins after the age of five. External ophthalmoplegia is a relatively common feature of patients with different mitochondrial encephalomyopathies (12). This weakness affects all the ocular muscles equally with respect to the pupils, which appear fully functional. Atypical retinitis pigmentosa, with a “salt and pepper-like” appearance, is another ocular characteristic of KSS (Figure 1B) (13). Bone-spicule formation in the retina is uncommon, and as pigmentary epithelial change is not confined to the posterior pole, these changes differ from those seen in standard retinitis pigmentosa (14,15). However, pigmentary changes indicate that the retinal pigment epithelium is affected and this can lead to blindness (14). Unusual ophthalmic presentations of KSS in early childhood include congenital glaucoma and corneal dystrophy (16-18). Histopathologically pigmentary epithelial atrophy, with overlying photoreceptor degeneration, occurs at first

PtosisOphthalmicOphthalmoplegiaPigmentary retinopathy

CardiomyopathyCardiacDysrhythmiaConduction defectsEncephalomyopathyNeurologicalSensorineural deafnessCognitive impairmentCerebellar ataxiaSeizuresNystagmusProximal myopathyMuscularMuscle weaknessExercise intoleranceDiabetes mellitusEndocrineHypoparathyroidismHypogonadismShort statureThyroid abnormalitiesAdrenal insufficiencyDelayed puberty

Table 1. The clinical manifestations of Kearns-Sayre syndrome

23Pediatric Endocrinology Reviews (PER) n Volume 4 n No. 2 n December 2006

Clinical and Molecular Review of KSS

and reduced phagocytosis of photoreceptor debris can be noted. Macrophages containing the outer segments of the photoreceptors may be observed within the affected pigmentary epithelium, but peripheral photoreceptors are relatively spared (19,20).

The cardiac manifestations dominate the later clinical picture of KSS (21). The heart is a highly ATP-dependant organ and mitochondria constitute about one-third of the total cytoplasmic volume of cardiomyocytes. It has long been speculated that inadequate energy production may be an important factor contributing to heart failure. Clinical manifestations of cardiac disease occur in more than half (57%) of the patients with KSS. These conditions include syncopal attacks, congestive heart failure and cardiac arrest (22). Those patients with KSS who have ventricular conduction defects are also shown to have an accelerated and unpredictable rate of progression to complete AV block, with an associated mortality rate of 20% in one study (23). The cardiac pathology in KSS typically involves the distal bundle of His, bundle branches and infranodal conductions (24-26). Electrophysiological investigations have shown an increase in the H-V interval at rest that lengthens further upon atrial pacing (27). The ECG change typically found in KSS is PR

interval prolongation preceding 2nd or 3rd degree AV block. Intracardiac electrophysiological studies of patients with KSS have shown that primary abnormalities are concentrated in the AV node-His-Purkinje system with shortened atrial-His conduction, together with prolonged H-V intervals (27). At the cellular level, the mitochondria in patients with KSS have an abnormal structure and excessive augmentation. These, along with a loss of myofibrils in both skeletal and heart muscle cells, are the classical pathological features of the disease (28). There is poor correlation between ultrastructural abnormalities of the myocytes and clinical heart disease other than conduction abnormalities, which are observed as electrocardiographic changes (23). Recent advances in mitochondrial cytopathies have suggested that in the subgroup of mitochondrial encephalomyopathies, mtDNA deletions within the cardiomyocytes cause dilated cardiomyopathy, a known complication of KSS (29). The deletion of mtDNA at the “common deletion” site found in about one third of patients with KSS is thought to cause the cardiac conduction defect (5,30). Several studies have demonstrated that the same deletion of mtDNA is present in skeletal muscle and in myocardial tissues (31-33). The percentage of deleted mitochondrial genome in the heart muscle is reported to be between 15% and 40% (30,32). In addition to mtDNA deletions, the proportion of mtDNA duplications has been shown to be higher in patients with KSS, particularly in heart tissue (34).

Further organ involvement, particularly that of the CNS, is common in Kearns-Sayre syndrome (35). Tissues with high energy demands such as skeletal muscle, the CNS and the retina, appear to be more severely affected and the clinical symptoms depend on the extent of different organ affection (36). Since brain energetics depends heavily on oxidative metabolism, the CNS is particularly susceptible to mitochondrial dysfunction (37,38). Furthermore, different brain regions seem to have different tolerance thresholds for metabolic dysfunction (39). The mtDNA rearrangements in KSS which occur in mitochondrial Oxidative Phosphorylation (OXPHOS) dysfunction cause many neurological disturbances. The most common neurological symptom is cognitive impairment which ranges from mildly delayed development to severe mental retardation (40). Muscle hypotonia, ataxia, dystonic movements, and myoclonias are common. Sensorineural hearing loss is a clinically relevant and treatable symptom (41,42). Nystagmus and dementia can develop, and spongiform degeneration may be seen in the cerebral cortex white matter, basal ganglia and brain stem (43). These lesions may affect the cranial nuclei, including the oculomotor nuclei. Brain stem lesions in the medulla may account for respiratory distress and the tendency for episodic coma in KSS patients (44,45). Moreover, intracranial calcifications are common (46). The CSF protein content is usually elevated (over 100 mg/dl) and may rise with time, with values exceeding 200 mg/dl having been reported (20). MRI scans of the brains of KSS patients reveal widespread abnormalities, which are probably responsible for the different neurological symptoms (40,47). Cerebral and

Figures 1A and 1B: Ophthalmological symptoms of KSS. A – Both eye ptosis in a 14-years-old girl with KSS. B – Atypical retinitis pigmentosa, with a “salt and pepper-like” appearance, revealed in the fundus of the right eye of the same patient.



cerebellar atrophy are common findings. On T2-weighted spin-echo images, the patients show high-signal lesions bilaterally in the subcortical white matter, thalamus, and brain stem (Figure 2). The white matter lesions can extend into the deep cerebral white matter and can also affect the cerebellum. The combination of bilateral high-signal lesions in the globus pallidus with high-signal foci in subcortical cerebral white matter is characteristic of KSS. Diffuse vacuolation of white matter has also been stressed as a characteristic feature in some histopathological studies (19). MRI abnormalities have been known to increase in parallel with neurologic progression of KSS, although there is little correlation between specific neurological deficits and any particular MRI finding (48).

Symptoms of CNS dysfunction, such as cognitive impairment or dementia, have also been described in mitochondrial encephalomyopathies. Considering the widespread physical abnormalities the data on functional aspects of regional cerebral energy dysfunction is limited and conflicting (49-52). There is evidence for cerebral metabolic impairment, even in patients without obvious CNS symptoms (53). In contrast to extensive research into the morphological and metabolic

aspects of brain affection, comparatively little is known regarding neuropsychological capabilities in mitochondrial disease patients. Some reports showed equivocal results with respect to both general intellectual capabilities and focal cognitive function (54-56). A recent study of a well-defined group of patients with KSS or CPEO (chronic progressive external ophthalmoplegia) examined the range and extent of putative cognitive dysfunction using a comprehensive neuropsychological test battery and a group of healthy controls (57). The neuropsychological testing in this study did not reveal general intellectual deterioration, but provided evidence of specific focal deficits, suggesting particular impairment of visuospatial perception associated with the parieto-occipital lobes and executive deficits associated with the prefrontal cortex.

Progressive muscle weakness and exercise intolerance appear as common features, and cause many inconveniences to the patients. The muscular weakness affects the facial, pharyngeal, trunk and shoulder muscles in particular, leading to dysarthria and dysphagia in many patients (58), some of whom may even become malnourished as a result. The characteristic appearance of muscle fibres in biopsy specimens of KSS patients is also very informative. Once cellular energy production falls below a certain threshold in cells with perturbed oxidative phosphorylation, a compensatory proliferation of all mitochondria, including affected ones, occurs. Thus, a typical finding in muscle fibres is an increased number of atypical mitochondria which stain red with a modified Gomori trichrome stain, and hence are referred to as “ragged-red fibres” (RRF). A deficiency of cytochrome-c oxidase (COX) can often be shown by measurement of the enzyme activity in homogenates of affected muscles. Single COX-negative fibres, usually corresponding to ragged-red fibres, can be demonstrated histochemically on muscle biopsy specimens (Figure 3).

Mitochondrial disorders like KSS are often believed to be rare conditions seen only in children with severe mental retardation and multi-system failure. These classical cases

Figure 2. Patient with Kearns–Sayre syndrome. Axial T2 weighted image demonstrates diffuse high signal intensity regions within the corona radiata and subcortical white matter. Reprinted from: Molecular Genetics and Metabolism; Lerman-Sagie T, Leshinsky-Silver E, Watemberg N, Luckman Y, Lev D. White matter involvement in mitochondrial diseases; Copyright 2005;84:127-136 with permission from Elsevier.

Figure 3. Serial cross-sections of muscle from a patient with the Kearns–Sayre syndrome, showing increased mitochondrial activity in ragged-red fibers on staining with succinate dehydrogenase (asterisks in panel A; x120) and the absence of activity on staining with cytochrome c oxidase (asterisks in panel B; x120). Reprinted from: DiMauro S, Schon EA. Mitochondrial respiratory-chain diseases. N Engl J Med 2003;348:2656-2668. Copyright © 2003 Massachusetts Medical Society. All rights reserved.



are in reality only a part of the phenotype spectrum of these disorders and primary involvement of a single system, especially endocrine disease, in other way clinically normal children is quite common. This is especially true early in the course of mitochondrial disease. Although endocrine abnormalities are common in mitochondrial disorders, in the majority of patients they are masked by encephalomyopathy. Consequently, the correct etiological diagnosis is often missed when the disease first manifests as an endocrinopathy with normal neuromuscular function and no particular family history. Several cases of KSS in which various endocrinopathies were the presenting feature have been reported, including diabetes (59), adrenal insufficiency (59-61) and growth hormone deficiency (62,63). Tetany and other symptoms secondary to hypoparathyroidism induced hypocalcemia can also be the first symptoms in cases with developing KSS (3,64). The most common endocrine organs affected in KSS are: the pituitary gland (leading to hypopituitarism, growth retardation, thyroid and gonadal dysfunction); the thyroid gland (65,66) (hypo- and hyperthyroidism, Hashimoto thyroiditis); the parathyroid gland (64,67-69) (hypo- or hyperparathyroidism); pancreas (mitochondrial diabetes mellitus); the adrenal gland (60,61,70,71) (insufficiency with hypoaldosteronism, hyponatriaemia and Addison disease); or the gonads (72) (delayed puberty and hypogonadism). Symptoms of hypopituitarism or hypothyroidism may overlap with symptoms of skeletal muscle manifestations such as fatigue, general weakness, slowing or hypotonia. Short stature is observed in about 38% of KSS cases, and diabetes mellitus in 20% (3). Skin and renal involvement are rare in KSS (73-75). Delayed bone age and osteoporosis are also seen in KSS patients (72).Osteoporosis in those cases can result from sex steroid deficiency, immobilization in later stages of the disease, but also from growth hormone deficiency. In some specific cases of KSS, where the first and predominant symptoms of the disease were various endocrine disorders, such as diabetes mellitus and adrenal insufficiency, a specific 7.4 kb deletion in mtDNA was found (59). It has been proposed that this second most common deletion of mtDNA in KSS, should be considered as one of the candidate causes for phenotypically uncommon cases of endocrinopathies.

The thyroid gland dysfunctions are quite common in the course of Kearns-Sayre Syndrome and other mitochondrial encephalomyopathies (3). In most cases thyroid abnormalities present as hypo or hyperthyroidism, but there are also cases of Hashimoto thyroiditis reported (65). In the latter, predisposed by mitochondrial deletion, anti-thyroid antibodies may have interfered with mitochondrial cerebral function, causing Hashimoto encephalopathy and facilitating ophthalmoplegia – it may be therefore important to study anti-thyroid antibodies in every case of KSS (65). But the spectrum of mtDNA deletions associated with thyroid dysfunction does not restrict only for mitochondrial encephalomyopathies. Point mutations

and large scale rearrangements of mtDNA are found in most thyroid diseases, including tumors and cancers (76). Large scale deletions in mtDNA are quite prevalent in healthy and diseased thyroid. However, the proportion of aberrant mtDNA molecules accounts for a very small part of total mtDNA and does not seem to correlate with the pathology of thyroid tumors. Common deletion is most abundant in Hurtle cell tumors, yet it also occurs in other thyroid diseases as well as in normal tissue. The principal difference between the common deletion and other deleted mtDNA molecules is that the former does not depend on the relative mtDNA content in the tissue, whereas in a subset of thyroid tumors, such as radiation-associated papillary carcinomas and follicular adenomas, there is a strong correlation between mtDNA levels and prevalence of large-scale deletions. Relative mtDNA levels by themselves are elevated in most thyroid tumors in comparison to normal thyroid tissue. Distinct differential distribution and prevalence of mutational mtDNA burden in normal tissue and thyroid lesions suggest the implication of altered mtDNA in thyroid diseases, especially in cancer (76).

Hypoparathyroidism is a frequent symptom in KSS patients with multiple endocrine abnormalities. It can be demonstrated by low concentrations of PTH, hypocalcaemia, hypomagnesemia or hyperphosphatemia and may be combined with coexisting renal failure. Different types of mutations were described in KSS patients presenting with hypoparathyroidism: single deletions (64,77), multiple deletions (78), coexisting deletion and duplication (69,79), and a point mutation in tRNALeu

gene (67). In the cases where the existence of duplications was confirmed biochemically, higher proportions of duplicated mtDNA in white blood cells were demonstrated (79-81). Regardless of the mtDNA defect, all patients reported had an early onset of symptoms with a multisystemic involvement, and in only three cases was hypoparathyroidism the initial manifestation (69,78,82). The conclusion is that in these cases, the presence of a duplication/deletion of mtDNA may be more frequently associated with endocrinopathies, as suggested by Poulton et al (83). The frequency of the duplications in these cases could be higher than previously suspected because most of the KSS cases with multiple endocrine involvements reported earlier were not properly investigated for the presence of this rearrangement.

Abnormalities of the adrenal axis have been reported in several patients with KSS, but are not routinely investigated. Some patients with elevated plasma levels of renin and aldosterone (84-86) and some with salt craving of unknown pathogenesis (87,88) have been reported. Low basal morning cortisol levels with normal responses to corticotropin releasing hormone was reported (63). One child was found to have partial growth hormone and ACTH deficiencies (89). Two cases with KSS were reported to have low urinary 17-ketosteroid excretion, but clinical signs of Addison disease were not mentioned (90). First report of non-autoimmune Addison



disease in KSS showed in 1998, and the authors stressed that early recognition of adrenal insufficiency is crucial to prevent mortality from this cause (61). Primary adrenal insufficiency as a presenting feature of mitochondrial disorder were described only a few years ago (59,60). Formerly, only North and colleagues reported an 18-month-old girl with a respiratory chain defect and a usual phenotype characterized by neonatal onset of chronic lactic acidosis, lipid storage myopathy, bilateral cataracts, and, starting at 7 months of age, primary adrenal insufficiency. The molecular genetic defect in this case however, was not defined (71). Given the fact that correct cause is not established in a number of cases with adrenal insufficiency, these reports may provide a clue to the potential etiology in these patients as an impaired mitochondrial ATP production in adrenal gland resulting in a defect in the secretory capacity of adrenocortical cells.

Although growth disturbance is a common feature in mitochondrial encephalomyopathies (22,59,91), information regarding endocrine evaluation and the clinical effectiveness of GH therapy is limited (62,92,93), mainly because deficiency of growth hormone only accounts for a part of these cases. Normal GH responses following insulin-induced hypoglycemia and following arginine have been reported in most cases (3,63,94). In patients suffering from mitochondrial encephalomyopathies such as KSS, normal or nearly normal serum IGF-1 levels were reported, suggesting that their hypothalamic-pituitary-somatomedin axis is mostly not affected. GH treatment-induced increase in growth velocity in these patients is usually limited to the first years of the therapy, as generally found in short non-GH-deficient children treated with GH. However, no catch-up growth is observed in mitochondrial encephalomyopathy patients. GH treatment of patients suffering from mitochondrial disorders, such as KSS/CPEO, may be ineffective or transient (95), indicating that short stature in these cases may be caused by disease-related insufficient protein substrate support rather than by GH deficiency. Growth stimulation induced by GH therapy increases energy demand of the every cell in the patient’s body. Deficient oxidative phosphorylation productivity of mutated mtDNA containing mitochondria cannot cope with the needs of increased energy production and protein synthesis. As an effect, no significant or short lasting results of GH administration are observed. In some cases GH therapy may even lead to a worsening of other clinical symptoms and progression of the disease, due to elevated cellular energy demands. Increased mitochondrial activity in the presence of heteroplasmic mtDNA deletion may lead to the lowering of the threshold level when deficient mitochondria fail and, in consequence, to the occurrence of the disease symptoms in new tissues and organs. Increased production of reactive oxygen molecules provoked by OXPHOS stress may also contribute to the failure of cellular energy production. The hypothesis of other factors, such as insufficient caloric intake

and feeding difficulties, being responsible for the resistance of mitochondrial cytopathy patients to GH treatment can also be considered. But these difficulties occur rarely in mitochondrial cytopathies, so their influence on commonly found growth disturbances can only be marginal. The other important issue is that the majority of KSS patients continue to experience weight loss and weakness. Human GH has been reported to have an important role in regulating protein metabolism and lean body mass in GH-deficient adults (96). Therefore, treatment with GH could promote protein synthesis and reduce wasting in some patients, but care should be taken during GH therapy to avoid the possible adverse effects. The quality of life for some patients could be at least temporarily improved by GH treatment, but other patients can suffer from severe multi-system failures. KSS patients with growth disorders can undergo GH therapy, but they should be carefully monitored for effectiveness of GH stimulation, and when there is no significant improvement in growth velocity or new, or more severe disease symptoms appear, the GH therapy should be terminated.

Dysfunction of the pituitary-gonadal axis is also common (about 20% of cases) and affects both sexes equally. Females present with late menarche, primary or secondary amenorrhoea or delayed secondary sexual attributes development. Dynamic endocrine testing indicates that the pituitary is responsive to GnRH and the defect therefore resides at the hypothalamic level (3). In one case, a girl with KSS and diabetes also presented with delayed puberty, primary amenorrhea, a low FSH, and normal LH, suggesting a latent hypothalamic hypogonadism. Boys presenting with similar symptoms were also reported (97).

There are several observations suggesting that mtDNA deletions and dysfunction of the respiratory chain may be involved in the pathogenesis of diabetes mellitus (DM). First, diabetes mellitus is one of the clinical manifestations of KSS (3,97-99). Complex mtDNA rearrangements (tandem duplications) have been described in patients with KSS, suffering from diabetes (7,100), two other patients of mitochondrial disorders reported with duplications were also diabetic (74,101). In fact, between 0.5 and 1% of all diabetics harbour a causative mtDNA mutation (102). Second, direct evidence for mtDNA involvement in diabetes has been found in pedigrees with maternally transmitted diabetes mellitus and deafness (103,104). The family described by Ballinger et al. consisted of an affected mother, six affected children and an affected maternal grandmother. DM in this family was associated with a maternally inherited complex mtDNA rearrangements (coexisting deletion duplication in mtDNA involving identical breakpoint junctions). Third, it is more common to inherit diabetes from an affected mother than from an affected father, suggesting maternal inheritance of predisposing factors 105. Therefore mtDNA dysfunction may play a role in the pathogeneicity of diabetes mellitus and



large scale rearrangements of mtDNA in KSS patients may be those predisposing factors. It has been demonstrated that mtDNA and intact respiratory function are necessary for glucose-stimulated insulin secretion in an insulin-producing cell line studied in vitro (106). This suggests that mtDNA mutations and other causes of impaired respiratory chain function may lead to reduced insulin secretion and subsequent development of diabetes. Diabetes mellitus in KSS is usually non-insulin dependent in its origin, but the patients progressively become insulin-deficient and, finally, almost all need insulin for their metabolic control (107). Non-insulin-requiring diabetes in KSS may be demonstrable only on glucose tolerance testing, sometimes developing after glucocorticoid therapy for neurological symptoms or, as reported in one case, for nephrotic syndrome (108). Diabetic patients with mtDNA mutations are in general islet cell antibody - (ICA) and glutamic acid decarboxylase antibody - (GADA) negative (109), although a few cases with low titres of ICA have been described (110,111). Insulin dependent diabetes mellitus (IDDM) may first appear with ketoacidosis (112) and no insulin resistance is described in KSS (113). Insulin therapy can even be stopped for a considerable time and given again for a second episode of diabetic coma (113). In a 10 year follow-up study of one patient with KSS and DM, a marked worsening of clinical symptoms was observed, but not those of the diabetes that was well controlled by once-a-day insulin therapy (114). Normal insulin receptors have been found in KSS patients without diabetes mellitus (115). Autopsies in some cases of KSS with DM have shown extensive fatty infiltration, or severe atrophy, of the pancreas (82,115). The mechanism of DM in KSS is unknown. As mitochondrial oxidative phosphorylation and ATP production is impaired in KSS (117), what is also true for mitochondrial diabetes associated with deafness (118), is the possibility that energy deficiency leads to pancreatic involvement, either in the form of a limited beta cell reserve or impaired insulin secretion (119). Abnormal function of the rearranged mtDNA can have influence on both development and function of pancreatic islet cells since glucose-stimulated insulin secretion is energy dependent. However, persistent insulin secretion was observed many years after the onset of the disease. On the other hand, increased oxidative stress may also be present leading to premature ageing of pancreatic beta cells and concomitant decompensation of pancreatic function (120). Other immune, genetic and environmental factors may also contribute.

In conclusion, it is highly probable that mtDNA rearrangements in mitochondrial disorders like KSS and their effect in reduced ATP production lead to impaired hormone production in endocrine organs, to no release of hormones at all, or to a decrease in the number of endocrine cells themselves. It is important to analyze every endocrine abnormality in detail in each case and to study how the mtDNA dysfunction influences the pathophysiology of the disorder. It is equally important to

treat these associated disorders what will significantly improve the patient’s quality of life in this complex syndrome.

Genetic Background of the Disease

One of the characteristic features that distinguish mitochondria from other cellular organelles is the possession of their own DNA (mtDNA). The mitochondrial genome is a 16569 base pair, double-stranded, circular molecule (Figure 4A) (121). It encodes 13 peptides of the respiratory chain, and also two rRNAs and 22 tRNAs which are required for the expression of this genome. All the other enzymes and factors which are necessary for mitochondrial DNA transcription, translation and replication, are encoded by the nuclear genome, translated in the cytoplasm and then imported into the mitochondrion (122). All 13 mtDNA encoded polypeptides are components of the respiratory chain/oxidative phosphorylation (OXPHOS) system which is located in the inner mitochondrial membrane (Figure 4B). Somatic cells usually contain many copies of the mitochondrial genome in each organelle and up to several hundred mitochondria per cell. It is important to appreciate that all respiratory chain complexes contain protein subunits encoded by the cell’s nucleus. Complex I contains the largest number of mitochondrially encoded proteins, i.e. seven polypeptide chains, and only complex II is encoded entirely by the nuclear DNA. One important difference between mitochondrial and nuclear DNA is that the genes encoded by mtDNA are contiguous with each other without introns and that translation of the mitochondrial mRNAs occurs on the mitochondrial ribosomes. Therefore, in order to be able to evaluate the possible significance of mitochondrial DNA mutations, it is important to be aware of the differences between nuclear and mitochondrial genetics (58,123). In humans the inheritance of mitochondrial DNA is exclusively maternal. Beginning from the zygote all the cells of an organism are generated through mitotic cell division. Mitochondria within the cells replicate autonomously and are passed on from generation to generation via the cytoplasm of the dividing cell. Thus segregation of mtDNA occurs at random and, in the case of an mtDNA mutation, may result in varying amounts of mutated DNA in different cells of the body. The term for the state in which more than one sequence variant of mtDNA, i.e. normal and deleted, co-exist in the same cell, or even in the same mitochondrium, is heteroplasmy. Heteroplasmy is a feature of mtDNA diseases where homoplasmy for severe pathogenic mutants may be lethal. One consequence of heteroplasmy is that the amount of mutated mtDNA determines the probability of expression of mitochondrial disease and influences the clinical symptoms of affected individuals. In addition, tissue specific threshold effects need to be taken into account as they may substantially influence the expression of the clinical phenotype (124). The threshold for the disease is lower in tissues that are highly dependent on oxidative metabolism,



such as brain, heart, skeletal muscle, retina, renal tubules, and endocrine glands. These tissues will therefore be especially vulnerable to the effects of pathogenic mutations in mtDNA. Mitochondria are not partitioned equally to daughter cells during cell division which can result in the non-uniform distribution of mutated mtDNA in these cells. This can result in an individual having completely different amounts of normal and mutant mtDNA in different tissues and indeed in the same

tissue. Moreover, mtDNA undergoes continuous replication, even in non-dividing cells (125). In patients with mitochondrial disease this may lead to a change in the level of heteroplasmy in non-dividing tissues, such as skeletal muscle and nerve, if the mutant and wild-type mtDNA replicate at different rates which may contribute to the late onset of symptoms found in some patients. Furthermore, the mutational rate of mtDNA is 10 to 20 times higher than that of nuclear DNA (126,127). Reasons for this greater rate are the lack of protective histones of mitochondrial DNA, inefficiency of the mitochondrial DNA repair system, the lack of intrones and the physical proximity of the mitochondrial genome to the respiratory chain where relatively high amounts of reactive oxygen species (ROS) are physiologically produced (128,129). The mitochondrial ageing theory claims that ROS, generated as by-products of the OXPHOS activity, react with and mutate mtDNA leading to impaired function of the respiratory chain which, in turn, is thought to further promote the generation of free radicals (130). Tissue specific gene expression and the specific energy demands of different tissues need to be taken into account when considering the possible pathogenicity of a mitochondrial mutation (131). Another complexity of mitochondrial disorders and their genetic basis is that a mitochondrial genotype changes throughout the life of an individual. The mitochondrial genome acquires somatic mutations during the normal life span. With age a wide spectrum of mtDNA rearrangements accumulate in postmitotic tissues such as the brain and skeletal muscle, which correlates with the marked age-related decrease in OXPHOS capacity (132-134). The rate of accumulation may be much faster in certain disease states [such as in the brain in Alzheimer’s disease (135,136) and in muscles in myocardial ischaemia (137) and inflammatory muscle disease (138,139)]. The mean level of these mutations in individual tissues is usually low (<1%) in comparison with pathogenic mtDNA mutations (132,133). Single cell studies have shown that the mutations may clonally accumulate to high levels in ageing human tissues leading to mitochondrial dysfunction (140) but the importance of these mutations, and their role in ageing and neurodegenerative diseases, remains to be determined.

Pathogenic mtDNA mutations of different types are found in about one in 8000 individuals (141,142). The common cause for Kearns-Sayre syndrome is mitochondrial DNA rearrangements – either deletions or duplications (6,7,81). These mtDNA rearrangements were the first pathogenic mtDNA mutations discovered (6). In one study the prevalence of single, large scale deletions was calculated to be 1.6/100 000 in the adult Finnish population (143). Large scale deletions remove a part of the mtDNA molecule, approximately 9% to 50% of the mitochondrial genome, commonly removing mitochondrial genes encoding OXPHOS subunits and tRNA genes, but rarely removing rRNA genes (144). Deletions of mtDNA have at least two different consequences at different levels of mitochondrial protein synthesis. Missing protein genes

Figures 4A and 4B. Human mitochondrial DNA map (A), and schematic organization of the respiratory chain in the inner mitochondrial membrane (B). The mitochondrial genes encoding protein subunits of particular complexes are shaded in picture A in the same pattern as complexes in picture B.A. The arrows indicate the direction of translation of particular genes. The tRNA genes are denoted with a single letter amino-acid code. The approximate position of the common, 4977-bp deletion is marked. Abbreviations: ND1-6: NADH dehydrogenase (complex I); CYTb: cytochrome B (complex III); COI-III: cytochrome C oxidase (complex IV); ATP6, 8: ATP synthase (complex V); rRNA: ribosomal RNA genes. D-loop: displacement loop (regulatory region); OH, OL: origins of replication of the heavy and light strands, respectively.B. The respiratory chain is composed of five enzyme complexes (complexes I-V), coenzyme Q (CoQ) and cytochrome c (Cyt C). Electrons from oxidized NADH and succinate are transferred through coenzyme Q, complex III, cytochrome c and complex IV to molecular oxygen (O2), which is reduced to water (H2O). Protons (H+) are pumped out of the mitochondrial matrix by complexes I, III and IV. The proton gradient formed across the inner mitochondrial membrane is used by ATP synthase (complex V) to synthesize ATP.



cause a defined enzyme deficiency, due to a defect at the level of transcription. The resulting impairment at this level of mitochondrial function depends on the influence of the deleted protein on the total function of the mitochondrium. Missing tRNA, however, causes dysfunction due to a defect at the level of translation. This translation defect necessarily affects all mitochondrial encoded proteins and should have a higher impact on the impairment of mitochondrial function than deletions of protein coding genes (145,146). The deleted mtDNA exists in a heteroplasmic manner within the cell and the level of heteroplasmy affects the onset and severity of the disease in different tissues. There is evidence in vitrothat reduced mitochondrial protein synthesis and biochemical dysfunction result when this proportion is greater than 50-60% of the total mtDNA (147-149). The level of heteroplasmy can change during the life-span. There is some evidence that the proportion of deleted mtDNA in muscle increases with time, along with the amount of ragged-red fibres (150,151). It has been suggested that any increase in the deleted fraction results from a replicative advantage conferred by shorter length, as most deletions preserve mtDNA replication origins and rRNA genes, thus leaving the deleted molecule replication competent. On the other hand, some tissues may have a different capability of selecting against mutant mtDNA, which may reflect the different replicative behavior of deleted and duplicated mtDNA in different tissues. In non-dividing tissues, i.e. muscle and brain, there is unlikely to be selection against deleted mtDNA as compared, for example, with rapidly dividing cells in bone marrow, spleen and testis (19). Deletions are prevalent in the postmitotic tissues of KSS patients but are present only in trace amounts in blood (5,152). The most frequently identified deletion, the so-called “common deletion”, involves 4977 nucleotides in positions 8469 – 13447, and is usually flanked by small, 13 bp direct repeats of the mtDNA sequence (152-155). The deleted mtDNA segment contains all, or part of, the genes encoding for polypeptides for complex I, one for complex IV, two for complex V and five tRNA genes. Approximately one-third to one-half of patients with KSS and CPEO have been reported to harbour the “common deletion” (5) and, interestingly, this deletion made up 63% of all the deletions in the tissues of a patient with multiple mtDNA deletions (156). The presence of direct tandem repeat elements flanking the deletion breakpoints has been proposed as a genetic criterion to classify mtDNA single deletions (157). The mechanism by which single deletions are produced is not well understood and several explanations have been proposed, namely intramolecular recombination via an unequal crossing over mechanism (152,157), a “slip replication” model arising from the erroneous annealing of H and L strands of mtDNA (154), an “illegitimate elongation” model (158), the “pyrimidine content” hypothesis involving DNA polymerase gamma (159), or a concept involving topoisomerase II (160). However, mechanisms of slipped-replication or illegitimate

recombination via direct repeats, are considered to be the most probable cause of human mtDNA deletions.

It is not known for certain whether or not deletions of mtDNA at different levels of heteroplasmy correlate with altered patterns of mitochondrial enzymes (161), nor how these altered enzyme patterns influence the functional properties of mitochondria (162,163). There are reports that patients with larger deletions have an earlier onset of the disease (164,165). Another study reported that patients who manifest non-neuromuscular multisystemic disorders at a very young age usually harbour mutant mtDNA with novel or rare deletions (166). Generally it is assumed that only severe enzyme defects are detectable as OXPHOS dysfunction, since the maximum activity of most single enzymes is higher than the maximum fluxes through the metabolic system of mitochondria (162,167). This is the metabolic reason for threshold in mitochondrial diseases. It was demonstrated that in patients with single deletions at a heteroplasmy level of about 50%, the amount of mitochondria within the cells was nearly doubled (168). Therefore, even considerably reduced levels of enzyme activity can be compensated by increased amounts of mitochondria. It may be speculated that increasing the amount of mitochondria in diseased skeletal muscles could be a strategy to minimize the functional consequences of deletions. In another investigation, an approximately ninefold amplification of mtDNA in muscle was detected (169). This amplification was probably the reason for the normal levels of respiratory chain enzyme activity and mild clinical course of the disease found in the patients in this study, despite a high mutant load (92%). Other reports also indicate a synchronous increase in the levels of deleted and normal mtDNA in the course of KSS (170).

Structurally more complex mtDNA duplications produce an mtDNA molecule that is larger than the normal mtDNA and contains two tandemly arranged mtDNA molecules – one normal, with a full length 16.6 kb mtDNA molecule coupled to a deleted mtDNA molecule (81,100). Duplications of the mitochondrial genome can cause symptoms similar to those with deletions, but not so severe (171,172). Deleted mtDNAs are thought to be pathogenic, largely as a result of loss of tRNAs, but this does not apply to duplicated molecules. It has been suggested that translation products of the duplication-deletion junction impair mitochondrial function (81). Duplicated mtDNAs, although they may not be pathogenic in and of themselves, could still have pathogenic consequences if they are recombination intermediates that could give rise to deleted mtDNAs, which are pathogenic (81,173). This concept is supported by a decrease in the proportion of duplicated molecules throughout life concomitant with an increase in the proportion of deleted mtDNA (101). This is particularly relevant to those disorders in which duplicated mtDNAs, but not the corresponding deleted mtDNAs, have been detected, such as maternally inherited diabetes and deafness (101,103,174).



Unlike deletions, the mtDNA duplications in patients with KSS are usually present in moderate amounts in the blood (74,100,101). These duplications may represent a distinctive feature of KSS that is absent from individuals with CPEO or PS (83,173). Alternatively, because patients with CPEO may have some, but not all, of the elements of KSS it has been suggested that CPEO and KSS describe different degrees of severity of the same disease (175). Deletion dimers (two joined deleted molecules) also occur and, theoretically, they should have the same effects on mitochondrial protein synthesis as standard deletions (81). Approximately 80% of KSS patients and 50% of CPEO patients harbour mtDNA rearrangements (5,36). The mtDNA mutations usually arise spontaneously very early during embryonic development (176), although it is probable that duplications are maternally transmitted (74,101,177). An important factor to consider in the analysis of KSS cases is the possibility that mtDNA point mutations in mitochondrial tRNA genes coexist in the same fashion as large scale mtDNA rearrangements (178). The existence of multiple mtDNA deletions, together with an autosomal inheritance, is characteristic of an underlying nuclear mutation, which is typical for CPEO (179,180). It is also possible that patients with KSS symptoms, but without identified mtDNA deletions, may have unidentified mutations in either their nuclear or mitochondrial OXPHOS associated genes.

Mitochondrial DNA deletions are also commonly found in two other clinical forms of encephalomyopathy, namely chronic progressive external ophthalmoplegia (CPEO), which primarily affects the ocular muscles, and Pearson’s syndrome (PS). PS is a fatal disorder which begins in early infancy and is characterized by bone-marrow involvement with anemia, leucopenia and thrombocytopenia, plus hepatic and exocrine pancreatic dysfunction. Pearson’s syndrome patients require frequent transfusions and most of them die before the age of 3 years. Virtually all patients with KSS harbor single mtDNA deletions that are detectable in muscle and other tissues by Southern blot analysis (4,5). Approximately 50% of patients with CPEO and ragged-red fibers have single deletions, detectable in muscles but not in other tissues (5,181). In PS, a disorder mainly affecting the hematopoietic system, mtDNA deletions are abundant in white blood cells (182,183). In fact, it is considered that PS, KSS and CPEO are all different clinical manifestations of the same disorder caused by mtDNA rearrangements. Patients with Pearson’s syndrome who survive the first critical period may develop the features of Kearns-Sayre syndrome in later childhood, suggesting that these two disorders represent different phenotypes of the same genetic defect. The mode of initial presentation probably depends on the amount of deleted mtDNA present and its distribution between tissues (184). The mildest variant is CPEO, in which clinical symptoms develop during adulthood and are limited mostly to the ocular system. A high prevalence of mtDNA deletions in blood cells will lead to PS in infancy. If the patient survives, mtDNA rearrangements may disappear from the blood

but the persistence of the mutation in other tissues results in the development of KSS. The blood content of deleted mtDNA is critical in these cases and it appears that this tissue mutant load is susceptible to drastic changes during the life span. There are conflicting reports of either an increase (151) or of a reduction in (184,185) mutated mtDNA levels in the hematopoietic system in PS, but in many reported cases the percentage of deleted mtDNA in blood cells ranged from 80% to 90% (183,186). A fully functional respiratory chain was observed in B lymphoblastoid cell lines from PS, despite the presence of 60% of deleted mtDNA (187). No more than 80% of mtDNA was deleted in all the reported cases of KSS with deletions in blood cells. These lines of evidence suggest that 20% of normal mtDNA may be sufficient for normal mitochondrial function in blood cells, although the possibility of a higher demand during the critical neonatal period cannot be excluded.

The majority of single large-scale deletions of mtDNA is sporadic and is therefore believed to be the result of the clonal amplification of a single mutational event, occurring in the maternal oocyte or early during the development of the embryo (153,188). On the other hand, in many cases of multiple rearrangements autosomal recessive or dominant trait of inheritance occurs. Since mitochondria depend on numerous nuclear encoded factors for its integrity and replication, mutations in these factors affect mtDNA directly, either quantitatively or qualitatively, and cause diseases that are inherited as mendelian traits (189). These forms of mitochondrial diseases are also called disorders of nuclear-mitochondrial intergenomic signaling (190). A quantitative alteration appears as abnormal reduction in the number of mtDNA molecules, down even to 35% of the normal mtDNA level – this abnormality is called mtDNA depletion syndrome (191,192). A qualitative alteration manifests itself by multiple deletions, in contrast to the single mtDNA deletions occurring spontaneously in KSS and other mitochondrial disorders (180,193,194). Both quantitative and qualitative defects may result from impairment of the integrity of the mitochondrial genome. Such impairment can be direct (e.g., affecting proteins required for the replication and maintenance of mtDNA) or indirect (e.g. affecting proteins required for proper maintenance of nucleotide polls in mitochondria). During the last few years several nuclear genes were showed to influence mtDNA structure, replication and stability. Autosomal dominant progressive external ophthalmoplegia (adPEO) is a mendelian disorder characterized by the accumulation of multiple deletions of mtDNA in patients’ tissues (179). This disease shares many clinical features with KSS (progressive muscle weakness, ophthalmoplegia, ptosis, cardiomyopathy, hypogonadism and RRFs). Most of the adPEO families carry heterozygous mutations in one of three genes: ANT1, encoding the muscle-heart-specific mitochondrial adenine nucleotide translocator, which is responsible for transporting ATP across the inner mitochondrial membrane in exchange for ADP (180); Twinkle, encoding a putative mtDNA helicase that



may be involved in mitochondrial DNA replication (193); and POLG1, encoding the catalytic subunit of the mtDNA-specific polymerase gamma (194). Mutations in Twinkle and ANT1 are not common; Twinkle mutations have been found in 15% of adPEO families and ANT1 mutations have been found in 11% of adPEO patients (193,195). The frequency of POLG1 mutations remains to be determined. All these three genes are strongly involved in maintenance of mtDNA replication and integrity. Mutations in both POLG1 alleles were also found in autosomal recessive PEO sibships with multiple affected members and in apparently sporadic cases (196). The exact mechanism of how mutations in POLG1 lead to the mtDNA deletions is unknown. It was shown that a prevalent mutation in POLG1 gene, the Y955C mutation, dramatically reduces the enzyme’s binding affinity for nucleoside triphosphates in vitro and also the accuracy for base pair substitutions (197), so it may provoke error prone mtDNA replication in vivo and subsequent rearrangements of the whole mtDNA genome. Another disease in this series is mitochondrial neuro-gastro-intestinal encephalomyopathy (MNGIE) clinically characterized by ophthalmoparesis, peripheral neuropathy, leucoencephalopathy, gastro-intestinal syndromes (recurrent nausea, vomiting or diarrhea) with intestinal dysmotility and histologically abnormal mitochondria in muscle including RRF (198). Mutations in the TP gene, encoding thymidine phosphorylase, causing the loss of enzyme function, are associated with MNGIE (199). TP is an important factor involved in the control and maintenance of the pyrimidine nucleoside pool of the cell. Defects of TP are thought to produce an excess of thymidine, resulting in the imbalance of dNTP pools that can ultimately affect both the rate and fidelity of mtDNA replication. This is reflected by a molecular phenotype of MNGIE, which is characterized by both multiple deletions and partial depletion of muscle mtDNA (200). The role of nucleotides is reinforced by the pathogenicity of the ANT1 mutations and by recent findings that mutations in mitochondrial thymidine kinase and deoxyguanosine kinase are associated with the myopathic and hepatocerebral forms of mtDNA depletion (201,202). Knowledge of these mutations makes prenatal diagnosis feasible for some families and may offer new approaches to therapeutic intervention [e.g., lowering blood thymidine concentrations in patients with MNGIE (203)].

Diagnosing KSS

As mitochondrial cytopathies are multisystemic disorders, a comprehensive clinical investigation must be carried out in order to arrive at a correct diagnosis. In the case of Kearns-Sayre syndrome the discovery of classical symptoms, such as progressive external ophthalmoplegia, pigmentary retinopathy, cardiac conduction defects and muscle weakness, together with accompanying neurological abnormalities and a variety of endocrine disorders, provide the clinician with a definite diagnosis. In non-classical cases more advanced and detailed examinations have to be undertaken in order to obtain the final answer and here the biochemical and molecular genetic tests help to confirm the clinical diagnosis. The family history is also important to obtain the whole view of the disease and its inheritance pattern (Figure 5). MtDNA disease is often only considered after many other diagnoses have been excluded.

A simple baseline investigation for the diagnosis of KSS is the determination of lactate and pyruvate levels in blood. As there is a demonstrable increase in blood lactate at rest and/or during exercise testing in approximately 50% of patients with KSS, it is wise to include a bicycle ergometer test in the investigation. Other clinical investigations such as an EMG (which may show a myogenic pattern), or tests for elevated CSF lactate and protein levels are also helpful. Proton magnetic resonance spectroscopy is a useful tool to demonstrate increased lactate levels in the damaged white brain matter of KSS patients and to support mitochondrial respiratory chain insufficiency as the underlying cause. The discovery of a doublet at 1.33 ppm is characteristic of the presence of lactate (204). Other neuroradiological examinations, such as

Figure 5. Proposed pathway for patient evaluation in the diagnosis of KSS.



MRI or CT, may visualize some characteristic brain changes. CT may reveal cortical and white matter atrophy, low density in the cerebral and cerebellar white matter and variable low density or calcifications in the basal ganglia, thalamus and/or cerebral hemispheres (46,205). The serum creatine kinase may be raised, but is often normal even when the patient has a proximal myopathy (10). Urinary organic and amino acids may also be abnormal (178). Every patient with seizures or cognitive decline should have an electroencephalogram (EEG) which may show evidence of seizure activity, or diffuse slow waves suggestive of a subacute encephalomyopathy.

Only three clinical features are significantly associated with an mtDNA mutation: progressive external ophthalmoplegia, myopathy and pigmentary retinopathy. The only investigation that provides specific evidence of an underlying mtDNA mutation is histochemical staining of muscle biopsy specimens (296,207) and therefore a muscle biopsy is always recommended for histological examination and to provide material for molecular genetic testing. Atypical mitochondria containing paracrystalline inclusions may sometimes be revealed by electron microscopy, even early in the course of the disease. In addition to the verification of ragged-red fibres, other histochemical and immunological investigations of the muscle tissue are helpful. These include staining for cytochrome-c oxidase (COX) and succinate dehydrogenase (SDH) and immunohistochemical investigations, using antibodies generated against the individual subunits of the respiratory chain complexes. Muscle tissue provides the material for both biochemical investigation of the respiratory chain and for genetic testing. DNA isolated from blood is frequently used for genetic testing but, because of the continuous turnover of mitochondria in this tissue, the mutation levels may be low. It is therefore preferable, when looking for deletions and duplications, to use muscle tissue. It should be noted that, owing to the different distribution of mutated DNA in different tissues, a negative result does not exclude an mtDNA mutation.

It is essential to assess cardiac function by ECG and echocardiography and to carry out an oral glucose tolerance test because of the high prevalence of both cardiac complications and diabetes in patients with mtDNA disease. If the diagnosis of KSS is confirmed a regular cardiological follow-up should be carried out so that any cardiac arrhythmias which may develop can be treated at an early stage.

Perspectives for the Patient

No curative treatment is currently available for patients with Kearns-Sayre syndrome (or indeed for those with other mitochondrial cytopathies). Therapy must therefore consist of the prevention and treatment of the typical symptoms and complications associated with the disease (e.g., insulin therapy in the cases with DM or hormone substitution in case

of other endocrine disturbances). Of great significance to the clinical management are the cardiovascular manifestations. Patients with KSS should be routinely and regularly evaluated for AV conduction disturbances. Those with ophthalmoplegia and retinitis, but without a confirmed diagnosis of KSS, should be followed carefully for heart block, because this typically develops after the ophthalmologic manifestations. Because of the genetic basis of the disease, screening, which should include a routine ECG, should be performed for family members of affected patients, although the yield may be low in otherwise asymptomatic individuals. It is important to realize that prophylactic pacemaker therapy is advisable in KSS patients because of the potential progression of the heart conduction abnormalities and the high risk of sudden death. The fact that mitochondrial OXPHOS function in KSS changes before manifestations become clinically evident underscores the need for electrocardiographic surveillance to determine the optimal timing for pacemaker implantation (27). A recent study (208) illustrates the need to pace KSS patients with ECG changes indicative of conduction defects. This study, carried out between 1976 and 1999 on a substantial cohort of 67 patients, found that, over a 10 year period, patients had a 32% likelihood of sustaining cardiac conduction defects, a 12% likelihood of having a pacemaker implanted, and a 5% likelihood of sudden death. The authors therefore concluded that, without pacing, patients with KSS had a high risk of sudden cardiac death. This study supports the practice of pacemaker implantation in patients with KSS based on ECG changes. The 2002 ACC/AHA (American College of Cardiology/American Heart Association) guidelines also recommend that pacing should be considered in patients with neuromuscular diseases with AV block, such as those with KSS, with or without other symptoms, because there may be an unpredictable progression of AV conduction disease (209).

Further management includes the use of bicarbonate and dialysis to correct episodes of severe lactic acidosis. Endocrine function should be investigated and the necessary corrective measures initiated. Blood glucose levels should be checked regularly. Caution should be exercised in the administration of anticonvulsants and anesthetics, as severe systemic complications may occur (210,211). Surgical correction of ptosis is possible, but the benefits are usually transient. A moderate degree of exercise has been shown to improve exercise tolerance and muscle metabolism, and this should be recommended to patients with mtDNA disease (212). A ketogenic diet may also bring some benefit in restoring proper mitochondrial function, providing energy source through the pathway omitting the deficient enzyme complexes in the energy production chain. Ketogenic treatment may even lead to a reduction in the deleted mtDNA fraction, as has been shown in vitro (213). Specific regeneration of mtDNA mutation-affected muscle tissue from satellite muscle cells has



also been tried, in an attempt to restore a wild-type muscle phenotype (214,215).

In addition to the procedures mentioned above, various methods of medical treatment for KSS have been employed in an attempt to moderate the progression of the neurological disease process. These mainly consist of nutritional supplements, such as coenzyme Q10 and ubiquinone (216). Coenzyme Q10 is thought to function as an electron carrier between flavoproteins and the application of this supplementary therapy should compensate for reduced levels of Q10 in the mitochondria of KSS patients. This has been shown to accompany clinical improvement in both neurological function and electrocardiographic abnormalities (217-219). Other studies have shown a transient effect of such supplements with a decrease in lactate levels within brain lesions (220). Some studies report an improvement of AV-block with this type of therapy, but others report no changes in the electrocardiogram and echocardiogram, or in clinical function (221,222). Antioxidants, electron-transfer mediators, enzyme cofactors and calcium blocking agents have also been tried in patients with mitochondrial encephalomyopathies (223). Other agents utilized in the treatment of patients with respiratory chain defects include thiamine, riboflavin, biotin, ascorbic acid, vitamin K and vitamin E, all of which have been used with some success in a small number of patients with complex II deficiency. The therapeutic effectiveness of all these dietary supplements still needs to be proven (224), but at least in some cases they have been useful (225). Succinate, a respiratory chain substrate which is coupled to the respiratory chain via complex II, has been used in patients with complex I deficiency and L-carnitine has been used in those patients with a secondary carnitine deficiency. Further therapeutic possibilities include the use of creatine (Cr), based on its potential neuroprotective and antioxidant effects (226). However, trials of Cr therapy in patients with mitochondrial cytopathies yielded controversial results regarding clinical and ergogenic treatment effects (227-230). Overall, the usefulness of these treatment strategies in the clinical course of the disease is not certain but, because they are relatively free of side effects, there is a rationale for their use in individual cases.

As pharmacological therapy has proven to be of limited value, researchers have instead explored the possibility of gene therapy. One such approach has involved the expression of a wild-type copy of the mutated mitochondrial gene in the nucleus and targeting of this cytoplasmically synthesized protein to mitochondria (231,232). Another attempt included the selective destruction of mutant mtDNA through importation of a restriction enzyme into mitochondria (233). The more obvious approach of direct transfection of affected mitochondria with wild-type mitochondrial genes has also been attempted (234). However, the exogenous DNA was neither replicated nor transcribed and currently no practical

method of transfecting mammalian mitochondria exists. One improvement in this area involving the import of lacking tRNA into diseased mitochondria has recently been described (235). One of the most promising approaches to gene therapy involves attempts to alter the level of heteroplasmy by either selectively inhibiting the replication of, or destroying, the mutant DNA (236,237). In selected, isolated myopathy cases, reduction of heteroplasmic mutant load was obtained by controlled muscle fibre damage and regeneration by mutation-free satellite cells, using myotoxic drugs (238). Such strategies are based on the fact that a large amount of the mutant mtDNA is required before the effect of the mutation becomes phenotypically apparent. Effective reduction of the population of mutant DNA, should lead to the propagation of wild-type DNA, resulting in a normal phenotype. Additionally, recent in vitro work has shown that sequence specific peptide nucleic acids (PNA) used as antisense probes can selectively inhibit the replication of mutated mtDNA (239).

Inheritance of mtDNA Deletions

The majority of KSS cases are sporadic although, more rarely familial cases, which can be maternally inherited, do occur (177,240-242). Although mtDNA deletions themselves are not transmitted (181,243), mtDNA duplications may be transmitted from mother to offspring (74,101). The duplications themselves are not pathogenic (172) but they do predispose to deletion formation (81). It should be stressed that mtDNA duplications are rare, but women harbouring mtDNA duplication may have an affected child who could also harbour a pathogenic mtDNA deletion (244). This mechanism might provide an explanation for the apparent transmission of mtDNA deletions in some human pedigrees but, in at least one instance the maternally transmitted species appeared to be a deleted mtDNA molecule (242). It should also be realized that what may appear to be a maternal inheritance could simply reflect low levels of age-related mtDNA deletions in the mother. Several children of women with single mtDNA deletions were clinically normal and had no detectable deletions (181,243,245). A recent attempt to define and understand the recurrence risk for mtDNA deletion disorders showed that the incidence of such a disease does not increase with maternal age and, also that unaffected mothers are unlikely to have more than one affected child (246). Conversely, affected women were previously thought to have a negligible risk of having clinically affected offspring but the actual risk in this case study was found to be, on average, about one in 24 births. It is not clear why the inheritance pattern of mtDNA deletions and point mutations is so different, given that both types of mutation cause the same biochemical defect in OXPHOS activity. It is possible that mtDNA deletions are embryonic



lethal and that all cases of apparent deletion transmission really occur because of intermolecular recombination of mitochondrial genomes with the formation of duplications as an intermediary stage (247). The recombination events may occur only in the germ line and, as a result the duplications, may not be detected in the blood or muscle of the mother. Additionally, recent report of paternal transmission of mtDNA in skeletal muscle (but not in other tissues) in a patient with mitochondrial myopathy gives us an important warning that maternal inheritance of mtDNA is not an absolute rule, and in some specific cases other possibilities of the origin of the disease should be considered. Of course it does not negate the primacy of maternal inheritance of mtDNA in particular mitochondrial encephalomyopathies.

Evidence supporting the idea that pathogenic deletions associated with the ontogeny of sporadic KSS and CPEO can be transmitted in the female germ line has come from the demonstration that the “common deletion”, which is present in high amounts in many sporadic cases of KSS, can be detected in human oocytes (188,248). Another study showed that both oocytes and embryos harbour a KSS deletion in their mtDNAs, but the percentage of this mutation is significantly reduced after fertilization (249). There was no correlation between the mtDNA deletion in human gametes and patient age, suggesting that this deletion is not a marker for reproductive senescence. It may be that deleted mtDNA cannot be transferred from the oocyte to the implanted embryo, because of the postulated presence of a “genetic bottleneck”, limiting the number of transmitted mtDNA (244,250). This theory argues that, during early germline development of oocytes, there is a transient reduction/amplification event with respect to the total number of mtDNA causing only a small proportion of the available mitochondrial genomes to propagate to the following generations. This could lead to high proportions of mutated mtDNA in a very small number of oocytes and therefore to the elimination of mutated mtDNA in the offspring of the next generation. According to this hypothesis, the expected frequency and percentage of mutated mtDNA in human embryos should be low or possibly absent, when compared with oocytes. Although there have been recent advances in our understanding of this phenomenon (251,252), it is unlikely that the simple mathematical models proposed will be of any practical use in the clinic (253). The “bottleneck” hypothesis may describe the real events occurring in vivo, but the fine details need to be determined and reliable genetic counseling will only be possible when we have a much more complete understanding of the processes involved in the inheritance, segregation and expression of mtDNA defects.

It is extremely important to determine whether positive or negative selection of heteroplasmy exists according to transmission of mtDNA mutations in human pedigrees. If there is positive selection, and a tendency to increase mutational load with subsequent generations, this would lead to the

earlier onset of a more severe disease phenotype. By contrast, a tendency to decrease mutational load (negative selection) might simply lead to the loss of a deleterious mutation from the population without clinical effects (252). Both of these possibilities would have important implications for the counseling of women who harbour pathogenic mtDNA mutations in their germ line. Thousands of families have now been given a diagnosis of mtDNA disease, and they regularly ask about the risk of transmitting an mtDNA defect to their offspring.

The complexities of mtDNA segregation cause difficulties with prenatal diagnosis (251). A major component of the variance in mtDNA heteroplasmy levels between individuals seems to arise by the time oocytes are mature. If so, prenatal diagnosis, by sampling the embryo prior to implantation, would seem a logical strategy, although it would not be one that is readily available in many places. On the other hand, the level of mutated mtDNA in a chorionic villus sample (CVS) may be different from the level in the fetus. It is already known that the level of mutant mtDNA is not distributed evenly in most patients with mtDNA disease and this differential segregation probably occurs at the later stages of development. Even subtle variations in tissue mutation load may lead to a profound variation in the phenotype, and sampling of a single cell or chorionic villus may not reflect this load in clinically relevant organs such as the brain. In isolated cases, the level of mutant mtDNA has been uniformly distributed throughout the fetus, which suggests that a chorionic villus sampling may give useful information (254,255). Furthermore, sampling oocytes (e.g. after diagnostic superovulation) for mutated mtDNA levels might be useful in any effort to advise individual women about the likelihood of bearing an affected fetus (256). However, at present we know little about how the level of mutant mtDNA might change during development and it will take some time before we know whether the mutation load in the CVS provides clinically useful information.

Conclusions

In this review we have outlined the complicated clinical and genetic picture of Kearns-Sayre syndrome, a classical disease of mitochondrial origin. Although we have gained much information about the clinical course and genetic background of KSS, since the first description of this syndrome almost 50 years ago, much still remains to be resolved. The variable, multisystemic manifestation of KSS impedes the correct clinical diagnosis, which is critical for the early clinical treatment of the disease. Many investigators have explored the widespread spectrum of clinical manifestations of KSS, and it would appear that we know almost everything about the physical side of them. However, as no curative therapy has been discovered we still can do no more for our patients than attempt to treat the symptoms and complications associated with the disease.



The molecular causes of the disease, lying in rearrangements of mitochondrial DNA, are now well established. However the complex and uncertain inheritance pattern of mtDNA rearrangements makes the genetic counseling of the growing number of women affected by KSS a demanding but vital task. We must therefore focus on preventing the spread of mtDNA deletions, and therefore precise presymptomatic diagnostic methods must be found (257). Recent work has started to unravel the complexities of mtDNA tissue segregation and this should, hopefully, allow us to refine the counseling we can give our patients in the future. There is also the pressing need to discover the role of mtDNA mutations in human ageing and neurodegenerative disorders such as Alzheimer’s or Parkinson’s disease (258-261). So the coming years should see even greater interest in the wide spectrum of molecular and clinical mechanisms of mitochondrial DNA rearrangements.

Acknowledgements

We would like to thank Prof. Geoffrey Shaw for his critical reading of the manuscript and helpful suggestions. This work was financially supported by Research Grant nr 502-01-11004118-06037 from Poznan University of Medical Sciences to M. Niedziela and Grant nr 2 P05A 070 30 from Ministry of Education and Science to J. Maceluch.

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