222

hmrnal ~]the Neuroh)gic~z;~'~len,:e,~, 113 (it~J2) 222-22'4 , 1992Elsevier Science Publishers B.V. All rigbls i:esei~ed 11022-51(iX/92/~05.!t(i

JNS 03877

Correlation between clinical and molecular features in two MELAS families Andrea Martinuzzi ~, Luigi Bartolomei b, Rosalba Carrozzo ~, Marialuisa Mostacciuolo ", Costantino Carbonin b, Vito Toso b, Emma Ciafaloni d, Sara Shanske d, saivatore DiMauro d and Corrado Angelini ~' " Neuromuscular Center, Department of Neurology, and ;5

5~

227 TABLE 2 M I T O C H O N D R I A L E N Z Y M E ACTIVITIES IN M U S C L E O F PATIENTS For identification of each m e m b e r see fig. la and case report. Case

I

Family A ILl 11-3 II-5 III-1 III-3 Ill-5

160.5 141.6 155.0 135.8 97.0 131.1

Control SD

235.3 34.1

Family B I-2 11-1 1I-2 1I-4 1I-5 III-4 Ill-5 Control + SD

I +III

li

II + Ili

IV

5.9 4.7 5.6 4.5 3.8 4.6

6.0 4.2 4.0 4.4 2.2 (35%) 5.2

24.11 15.8 13.0 9.6 (61%) 8.0 (51%) 14.7

20.7 6.3

5.9 1.7

6.2 1.7

15.8 3.3

221.6 238.8 224.8 2411.9 249.1 115.4 (49%) 118.11 (511%)

21.4 30.9 (149%) 22.9 27.7 32.8 (158%) 1.8 (9%) 5.1 (25%)

5.3 5.6 4,5 4.3 4.3 5.2 5.6

6.1/ 7.3 6.1 4.9 4.3 1.4 (23%) 1.6 (26%)

16.5 16,1 19,3 20.8 17.7 11.8 (5%) 5.9 (37%)

235.3 34.1

20.7 6.3

5.9 1.7

6.2 1.7

15.8 3.3

(68%) (60%) (66%) (58%) 141%) (56%)

9.3 11.1 9.5 8.1 7.2 7.9

(45%) (54%) (46%) (39%) (35%) (38%)

I: complex I ( N A D H dehydrogenase), I + I I I : NADH-cytochrome c reductase, II: complex I1 (succinate dehydrogenase), I1 + lII: succinate-cytochrome c reductase, IV complex IV (cytochrome c oxidase). Values are expressed as n M / m i n / m g non collagen protein and corrected for citrate synthase activity.

The proportion of mutated mtDNA in muscle was higher than in blood in all cases (Table 1), but the ratio between proportion of mutated mtDNA in blood and in muscle varied in different patients. M u s c l e / b l o o d mutated mtDNA ratio was over 5 in asymptomatic members of family A, and below 3 in affected relatives; in family B, the ratio varied between 2.57 and 1.25 in both asymptomatic and symptomatic patients. Respiratory chain enzymes activities in muscle from patients and relatives of family A (Table 2A) showed low complex I (68-41% of normal control), and low complex III (54-35% of normal control). In addition, patient III-3 (the ocular myopathy patient), and her sister (lII-1) had low complex IV activity (51 and 61% of normal control, respectively). Complex II activity was normal in all tested samples. In family B, asymptomatic cases showed no alteration of respiratory chain enzymes. Patient III-4 and his sister showed very low complex I, IIl, and IV activities (Table 2B).

Discussion

The two families described in this study are among the largest MELAS pedigrees in which clinical, histochemical, biochemical, and molecular investigations have been performed. Although typical full MELAS

syndrome was seen only in two patients from family A and in one patient from family B, the same A to G transition at nt 3243 of mtDNA was demonstrated in all their maternal relatives. In all patients the mtDNA was heteroplasmic, and the proportion of wild versus mutated mtDNA varied greatly among different individuals and between skeletal muscle and blood in each case. The variation in the proportion of mutated mtDNA was reflected in different clinical phenotypes, but we could not detect a precise threshold for expression of the mutation, even after correction for age. This was especially true in family A, in which the asymptomatic case II-1 had a higher proportion of mutated mtDNA in muscle than her younger sister (II-3) and brother (1I-5) with full MELAS. Family A is also interesting because of the late onset of the full syndrome (over 40 years in both II-3 and II-5, and probably also in 1-1). All patients in the first reported series (Pavlakis et al. 1984), and most patients described thereafter (Van Hellenberg Hubar et al. 1991; Ciafaloni et al. 1992) experienced their first stroke-like episode before age 33 (mean 14.7 years). A few cases with late-onset stroke have been described (Lach et al. 1986; Byrne et al. 1988; Hammans et al. 1991), but they differ from ours in that myoclonus was a prominent feature in some (in one case myoclonus had been present for more than 30 years before the first stroke), and CPEO was present for years before the stroke

228 episodes in others. No myoclonus or C P E O was ever evident in our cases, who, apart from age, exhibited all the clinical hallmarks of MELAS. There were two main phenotypes in family A: a group of patients (cases I-1, 11-3, 1I-5, II1-5) were short, deaf since their twenties, had few R R F , and developed (or are at risk for developing) MELAS in their 40s; a second group (cases II-1, llI-1 and 111-31 presented no clinical CNS involvement, had higher stature, abundant RRF. Patient III-3 had the unusal feature of CPEO, but otherwise shared the same characteristics as the second group, represented by her mother and sister. She had the highest resting serum lactate, the highest proportion of R R F in her muscle, the most severe defect in respiratopy chain enzyme activity, and the highest proportion of mutated m t D N A in muscle. All these data are in good agreement with her severe muscle involvement. Although she also had the highest proportion of mutated m t D N A in blood, her clinical presentation was mild compared to her uncles with full MELAS. However, she was still young, especially considering the late-onset of stroke-like episodes in her family. Even more surprising is her sister (III-1), who is completely asymptomatic in spite of the significant muscle enzymatic defect and the relevant proportion of mutated m t D N A . The clinical follow-up of these patients, and of patient Ill-5, who also has a very high proportion of mutated genomes in muscle, will be of particular interest. A common feature in patients of both groups were the E E G abnormalities, which were found in 4 of 6 patients, although in the asymptomatic cases they were non-specific. In contrast to family A, in family B there was good correlation between phenotype and molecular abnormality in blood, suggesting that muscle may not be the best indicator of mutated m t D N A distribution in other tissues, especially the central nervous tissue. In this family there is a substantial increase of the proportion of mutated m t D N A in both muscle and blood from generation to generation (actually no mutated m t D N A could be detected in the blood of case I-2), and this is accompanied by increasingly severe signs of mitochondrial dysfunction. Genetic anticipation is described in some autosomal disorders (e.g., myotonic dystrophy; Harley et al. 1992), and a similar situation has been observed in other families with m t D N A defects (Driscoil et al. 1987; Rosing et al. 1985; Wallace et al. 1988; Lott et al. 1990). In our cases, the increasing clinical severity is paralleled by an increase in the number of the mutated genomes. A similar occurrence in Leber's hereditary optic neuropathy ( L H O N ) pedigrees (Lott et al. 1990) was explained on the basis of random meiotic segregation. A characteristic of our M E L A S families is the variation of clinical presentation among members of the

same family. This clinical heterogeneity can he partly explained in terms of variable proportion and distribution of mutated mtDNA. AnOther possible explanation is the coexistence of more than one mutation in a single mtDNA. By PCR or Sou:thern analysis we detected only the nt3243 mutation, but other mulations could have been missed. If nlultiple deleterious mutalions coexisted in the same mtDNA, the phenotype could result from the additive effect of the various mutations. Multiple mutation in conserved sitcs have been described in MELAS (Tanaka et al. 1091; Ozawa ct al. 1991) and L H O N (Johns and Berman 199t), but their significance is not clear, t;iven the strictly materhal inheritance of mammalian m t D N A it is unlikeb that different members of the same maternal line would have different m t D N A mutations. However the fixation of a new mutation in mammalian m t D N A within ~me generation has been shown m be possible (Koehter et al. 1991). A third possibilily is that a nuclear gent modulates the effect of the m t D N A mutation. The respirato~ ~ competence of transformant cells obtained by fusion of various mitochondrial donors with Rho" cells (King and Attardi 19891 is influenced b), the specific interactions between nuclear and mitochondrial genomes. Liability to develop optic atrophy in L H O N was shown to be linked to the DXS7 locus at proximal Xp (Vilkki et al. 19911. An X-chromo~ontai effect can thus explain the male predominance typical of LHON. According to some authors (Van tlallcnmerg Hubar et al. 1991), MELAS also appears (o show a male predominance, and m our families males wcrc more severely affected. Linkage analysis in our families with various X chromosome markers is no~ underway to test this hypothesis. In conclusion, our studies t l) confirm tile clinical variability of MELAS syndrontc, (2) show thal the phenotypic variability is reflected at the molecular level, (31 demonstrate the need to extend clinical, morphological, biochemical, and molecular investigations to the asymptomatic relatives of index cases, (4) underlies the importance of studying each family separately, since the g e n o t y p e / p h e n o t y p c correlation cannot bc extended to different pedigrees Acknowledgements This work was supported in part by the Associazione Italian a per la Promozionc dellc Ricerche Ncurologichc (ARIN) toA.M, and by CNR grant to ('.A. Wethank DJ.F Sanson of the Division of Neurology, Thicnc tt~spital, for referring to us case 11-3 of lamily A.

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Correlation between clinical and molecular features in two MELAS families.

We describe the clinical, morphological, biochemical presentation in two MELAS families, and correlate it with the distribution and proportion of mito...
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