Am. J. Hum. Genet. 51:1201-1212, 1992

Segregation and Manifestations of the mtDNA tRNA'YS A--'GO8344 of and Ragged-Red Fibers (MERRF) Syndrome Nils-Goran Larsson,* Mar H. TuliniusT Elisabeth Holme,* Anders Oldfors,1 Oluf Andersen,§ Jan Wahistrom, 11 and Jan Aasly# Departments of *Clinical Chemistry, tPediatrics, $Pathology, §, and IlClinical Genetics, University of Gothenburg, Gothenburg, Sweden; and #Department of Neurology, University of Trondheim, Trondheim, Norway

Summary We have studied the segregation and manifestations of the tRNALYS A-)'G(8344) mutation of mtDNA. Three unrelated patients with myoclonus epilepsy and ragged-red fibers (MERRF) syndrome were investigated, along with 30 of their maternal relatives. Mutated mtDNA was not always found in the offspring of women carrying the tRNALYS mutation. Four women had 10%-33% of mutated mtDNA in lymphocytes, and no mutated mtDNA was found in 7 of their 14 investigated children. The presence of mutated mtDNA was excluded at a level of 3:1,000. Five women had a proportion of 43%-73% mutated mtDNA in lymphocytes, and mutated mtDNA was found in all their 12 investigated children. This suggests that the risk for transmission of mutated mtDNA to the offspring increases if high levels are present in the mother and that, above a threshold level of 35%-40%, it is very likely that transmission will occur to all children. The three patients with MERRF syndrome had, in muscle, both 94%-96% mutated mtDNA and biochemical and histochemi- cal evidence of a respiratory-chain dysfunction. Four relatives had a proportion of 61%-92% mutated mtDNA in muscle, and biochemical measurements showed a normal respiratory-chain function in muscle in all cases. These findings suggest that >92% of mtDNA with the tRNALYS mutation in muscle is required to cause a respiratory-chain dysfunction that can be detected by biochemical methods. There was a positive correla- tion between the levels of mtDNA with the tRNALYS mutation in lymphocytes and the levels in muscle, in all nine investigated cases. The levels of mutated mtDNA were higher in muscle than in lymphocytes in all cases. In two of the patients with MERRF syndrome, muscle specimens were obtained at different times. In both cases, biochemical measurements revealed a deteriorating respiratory-chain function, and in one case a progressive increase in the amount of cytochrome c oxidase-deficient muscle fibers was found.

Introduction 1985; Wallace et al. 1988). mtDNA is exclusively ma- Myoclonus epilepsy and ragged-red fibers (MERRF) ternally inherited (Giles et al. 1980) and contains the syndrome is a maternally inherited progressive mito- for 13 subunits of the respiratory-chain enzyme chondrial encephalomyopathy characterized by myo- complexes, 22 transfer RNA genes, and 2 ribosomal clonic and tonic-clonic , , , RNA genes (Andersson et al. 1981). The non- muscle weakness, and mitochondrial abnormalities in Mendelian inheritance of MERRF syndrome was ex- (Fukuhara et al. 1980; Rosing et al. plained when a that changes a highly conserved adenine to a guanine at nucleotide 8344 in Received May 22, 1992; revision received August 14, 1992. the tRNALYS of mtDNA (tRNALYs mutation) was Address for correspondence and reprints: Nils-Goran Larsson, found (Shoffner et al. 1990; Yoneda et al. 1990). In Department of Clinical Chemistry, Sahigren's Hospital, S-413 45 Gothenburg, Sweden. vitro experiments with transmitochondrial lines i 1992 by The American Society of Human Genetics. All rights reserved. have demonstrated that the tRNALYS mutation causes 0002-9297/92/5106-0003$02.00 a mitochondrial protein synthesis defect (Chomyn et

1201 1202 Larsson et al. al. 1991). The tRNALYs mutation is found in most II-6, and 111-8) (fig. 1). For the other individuals, data MERRF patients, and there is always were obtained from interviews with relatives. The with a mixture of normal and mutated mtDNA. The clinical findings are summarized in table 1. proportion of mutated mtDNA varies between differ- Probands. -The first symptom was ataxia in the pro- ent individuals in the same family and even between bands of families A and B, with onset at the age of 0.5 different tissues in the same individual (Shoffner et al. and 5 years, respectively. The first symptoms in the 1990; Noer et al. 1991; Seibel et al. 1991; Zeviani et proband of family C were vision and hearing loss with al. 1991). Patients with MERRF syndrome have a onset at the age of 9 years. The dominating clinical high proportion of mutated mtDNA in muscle, and a findings in all three probands were photosensitive deficiency of the respiratory-chain enzyme complexes myoclonic, generalized tonic-clonic, and akinetic sei- I and IV is frequently found (Shoffner et al. 1990; zures, truncal and limb ataxia, and myoclonic jerks Seibel et al. 1991; Zeviani et al. 1991). Maternal rela- affecting the whole body. The onset ofmyoclonic jerks tives of MERRF patients are often unaffected or have and seizures was at the age of 7, 14, and 11 years in less severe encephalomyopathic manifestations. Large the probands of families A, B, and C, respectively. cervical have been reported in patients with The probands of families A and C also had dementia, MERRF syndrome and also in otherwise unaffected dysarthria, spastic paraparesis, optic , and maternal relatives of MERRF patients (Berkovic et al. sensorineural hearing loss. 1989, 1991; Holme et al., submitted). At the age of 10 years the proband offamily A could We have studied the segregation and manifestations still walk and run, but he had poor endurance. Muscle of the tRNALYS mutation. Three unrelated patients weakness was mainly noted in distal muscle groups. with MERRF syndrome and 30 maternal relatives of At the age of 13 years considerable progress of all two of these patients were studied. The correlation symptoms had occurred. He was almost completely between the levels of mtDNA with the tRNALYS muta- wheelchair bound and could only walk short distances tion and clinical manifestations and morphological with support. General muscle weakness and atrophy and biochemical findings in skeletal muscle was stud- were noticed in both the proximal and distal muscle ied in the three patients with MERRF syndrome and groups. At the age of 15 years he was wheelchair in four maternal relatives of two of these patients. In bound and had developed spastic paraparesis with two of the patients with MERRF syndrome, multiple both equinus posture of the feet and contractures ne- muscle biopsies during a 5- and a 3-year period were cessitating bilateral achillotenotomy. performed to study the progression of the . At the age of 21 years the proband of family B had severe fatigue, headache, and concentration diffi- culties. He had exercise-induced muscle pain and bi- Material and Methods lateral pes cavus but no . Muscle weakness was more prominent in proximal than in distal muscle Patients groups. The probands of families A-C had MERRF syn- At the age of 17 years the proband of family C had drome (fig. 1). Maternal relatives of the probands of muscle weakness mainly in proximal muscle groups, families A and B were investigated. Family C declined but he could walk long distances. At the age of 20 further investigation. The probands of families A and years he had severe muscular weakness and atrophy C have been described elsewhere (cases 13 and 8 in of proximal muscle groups, and he could walk only Tulinius et al. 1991a, 1991b). The three probands short distances. and case V-2 of family A were thoroughly investigated Family A. - Celiac disease was suspected in case V-2 according to a protocol described elsewhere (Tulinius at the age of 1 year, because of failure to thrive. He et al. 1 991a). Case IV-21 offamily A and II-5 and III-6 was treated with a gluten-free diet until the age of 4 of family B were extensively investigated to exclude years. He was on a normal diet during age 4-6 years, minor neurological, audiological, cardiological, and but a duodenal biopsy specimen at the age of 6 years ophthalmological signs. A clinical examination was showed subtotal villus atrophy. A gluten-free diet was performed in another 24 members of family A (cases again started and has been continued since. A control II-2, II-4-II-6,. III-1, III-1 1-III-17,9 III-21, III-23 , IV-7,~ duodenal biopsy specimen after 2 years of a gluten-free IV-9-IV-11, IV-13-IV-15, IV-19, and V-3-V-4) and diet showed normal villus structure. At 12 years of in another 5 members of family B (cases I-1, 11-2-II-3, age, case V-2 began to complain of exercise-induced Segregation and Manifestations of tRNALYS Mutation 1203

FamilXA

Family S Family C ///* 88% 777 74% 46%

1 2 11 1 2 /s90%

III

807 50% 597

Figure I Pedigrees ofthe three investigated families. The arrows indicate the probands. The mean values ofthe proportion ofmtDNA with the tRNALYs mutation in lymphocytes from the investigated individuals are shown. Levels below the detection limit (i.e., 0.3%) of the PCR assay are labeled by 0%. no and 0 = Clinically examined; E andO = carrier ofthe tRNALYs mutation; and * = MERRF syndrome. muscle pain and fatigue. At age 13 years he had minor classical migraine, a slight hearing loss, and decreased concentration difficulties and needed extra assistance vibration sensibility in the lower extremities. Case at school. The results of a neurological examination IV-9 had a slight ataxia in the lower extremities. Cases were normal. The proband's mother, case IV-21, had 11-4-II-6 experienced increasing mental deterioration multiple symmetric lipomas around the neck, with on- with loss of short-term that started in their set during her 20s. The results of investigations of 80s, compatible with senile dementia. The oldest in- the lipomas will be reported elsewhere (Holme et al., vestigated member offamily A, case 11-2, was healthy, submitted). Thorough clinical and laboratory investi- without any symptoms of senile dementia, at the age gations revealed no neuromuscular signs. The pro- of 90 years. band's maternal grandmother, case 111-21, had very Family B. -The proband's brother (case III-6) and large, disfiguring lipomas around the neck and shoul- mother (case 11-S) were healthy, and thorough clinical ders, with onset during her 40s. Case 111-20 was hyper- and laboratory investigations revealed no neuromus- active, clumsy, and slightly atactic. He died of pneu- cular signs. Case 11-3 had truncal and limb ataxia, monia in 1961, at the age of 8 years. Case 111-23 had proximal muscle weakness, muscle atrophy, and pes 1204 Larsson et al.

Table I Clinical Features of Patients with MERRF Syndrome and of Their Relatives Agea Hearing Pes Case (years) Myoclonus Dementia Ataxia Spasticity Loss Cavus Family A: II-2 ...... 90 11-4 ...... 86 11-5 ...... 85 11-6 ...... 83 III-1. 60 III-11. 65 III-12 ...... 64 111-13 ...... 62 III-14 ...... 59 III-15 ...... 44 III-16 ...... 60 III-17 ...... 59 III-21 ...... 59 III-23 ...... 48 IV-7 ...... 42 IV-9 ...... 29 IV-10 ...... 28 IV-11 ...... 24 + IV-13 ...... 35 IV-14 ...... 32 IV-15 ...... 31 IV-19 ...... 37 IV-21 ...... 37 Proband 16 V-2 ...... 13 V-3 ...... 5 V-4 ...... 3 Family B: I-1 ...... 70 11-2 ...... 46 11-3 ...... 44 +_ II-5 ...... 42 11-6 ...... 38 +_ Proband 23 +_ 111-6 ...... 20 III-8 ...... 8 Family C: + Proband 23 + + + + + +_ NOTE. -A plus sign ( + ) indicates that the clinical feature was present, and a minus sign ( - ) indicates that the clinical feature was not present. a Age at last investigation.

cavus. Case II-6 had pes cavus, but she was otherwise ents. This study was approved by the Ethics Commit- healthy. tee of the Medical Faculty at Gothenburg University. Family C. -The proband's brother, case II-1, had Muscle Biopsies generalized tonic-clonic seizures with onset at 20 years of age. He has not been examined by us. Muscle specimens for mtDNA analysis, biochemis- All investigations were performed after informed try, and morphology were obtained from the quadri- consent was obtained from the patients or their par- ceps muscle by open biopsy according to a method Segregation and Manifestations of tRNALYs Mutation 1205 described elsewhere (Tulinius et al. 1991a). The last PCR reactions of each sample. Quantitation with the biopsy specimen of the proband of family A was ob- PCR-based assay of Zeviani et al. (1991) leads to an tained from the gastrocnemius muscle during achillo- underestimation of the proportion of mutated mtDNA, tenotomy. All skeletal muscle biopsies were performed because of the formation of heteroduplexes during under local anesthesia, except for the biopsies in the PCR (Tanno et al. 1991). A standard curve was ob- proband offamily A and the first biopsy in the proband tained by analyzing seven of the samples (with dif- of family C, where general anesthesia was used. ferent proportions of mutated mtDNA), with radio- labeled primer added only in the last cycle of mtDNA Analysis amplification, according to the procedure of Tanno et DNA was isolated from muscle, lymphocytes, and al. (1991) (fig. 2). This standard curve was used to fibroblasts and was analyzed by Southern blotting ac- correct for the heteroduplex formation. PCR reactions cording to a method described elsewhere (Larsson et were performed by using the same conditions as de- al. 1990). The asymmetric primer method with prim- scribed above, but without labeled primers. One extra ers corresponding to nucleotides (nt) 8161-8180 cycle was then performed after the addition ofradiola- (NG25; 50 pmol) of the heavy strand and to nt 8537- beled primer NG25 (50 pmol), unlabeled primer 8556 (NG24; 4 pmol) of the light strand of mtDNA NG29 (50 pmol), and Taq DNA polymerase (2.5 U). was used to generate single-stranded DNA for direct The radiolabeled PCR fragments were then analyzed sequencing with a primer corresponding to nt 8411- as described above. 8430 (NG28) ofthe light strand ofmtDNA, according The detection limit, with PCR, of mtDNA with the to a method described elsewhere (Larsson et al. 1991). tRNALYs mutation was determined by analyzing serial The PCR-based assay of Zeviani et al. (1991) was dilutions of muscle DNA with mainly mutated used to analyze the proportion of mtDNA with the mtDNA in normal muscle DNA. Muscle DNA from tRNALYS mutation, in various tissues and individuals. the third biopsy specimen from the proband of family A primer corresponding to nt 8345-8390 (NG29) of A (5 ng, 4 ng, 3 ng, 2 ng, 1 ng, 0.5 ng, 0.4 ng, 0.3 the light strand of mtDNA with the normal AG dinu- ng, 0.2 ng, and 0.1 ng) was mixed with muscle DNA cleotide at nt 8352 and 8353 replaced by a CC dinucle- (100 ng) from a healthy individual and was analyzed otide was synthesized. This primer creates a BglI re- striction-enzyme site around nt 8344 when mtDNA with the tRNALYs mutation is amplified. The primer 1.0 NG25 was end-labeled with 32P by using standard c.Ce methods (Ausubel et al. 1989). PCR with the primers :z c3 NG29 and radiolabeled NG25 was performed under 4--) the same conditions as described elsewhere (Larsson et E al. 1991). Approximately 0.1-0.2 of the resulting a, gg 4-i 230-bp radiolabeled PCR fragment was digested over- 4-, night with 27 units of BglI and electrophoresed in .5 6% acrylamide gels. Kodak X-OMAT S films were 4-)m exposed, without intensifying screens, for 8 h-1 wk 8 at room temperature. Two radiolabeled fragments- 0 one of 230 bp, corresponding to normal mtDNA, and .r-f the other of 184 bp, corresponding to mtDNA with 4-J the tRNALYs mutation-were detected. Autoradiograms C- were scanned according to a method described else- LL- where (Larsson et al. 1990). The mean proportion 0 .5 1.0 of mtDNA with the tRNALYs mutation in the muscle Fraction of mutated mtDNA, B specimens from the three probands and case V-2 of A was obtained after 10 different Figure 2 Standard curve to correct for heteroduplex forma- family analyzing tion. Seven samples with different fractions of mtDNA with the PCR reactions of each sample. The mean proportion tRNALYS mutation were analyzed by a PCR-based assay in which of mtDNA with the tRNALYS mutation in the other radiolabeled primer was added in the first cycle (A) or only in the samples was obtained after analyzing three different last cycle (B) of amplification. 1206 Larsson et al. with PCR as described above. The point mutation at A nt 3243 that is associated with mitochondrial myopa- 1 2 3 4 5 6 7 8 9 10 thy, encephalopathy, lactic acidosis, and strokelike epi- sodes (MELAS) syndrome (Goto et al. 1990) and the point mutation at nt 8993 that is associated with reti- nitis pigmentosa/ataxia syndrome (Holt et al. 1990) were analyzed with Southern blotting after cleavage with ApaI and AvaI, respectively. Morphological Examination of Muscle Histological, enzyme-histochemical, and ultrastruc- B tural analyses of skeletal muscle were performed ac- 1 2 cording to methods described elsewhere (Tulinius et al. 1991a). Biochemistry Polarographic measurements of the respiratory- chain function were performed on isolated muscle mi- tochondria from fresh muscle tissue, essentially ac- cording to the method described by Scholte et al. Figure 3 Analysis of the proportion of mtDNA with the (1981), with modifications described by Larsson et al. tRNALYs mutation, by the PCR-based assay. The upper arrow indi- (1991). Spectrophotometric measurements were done cates the 230-bp fragment corresponding to normal mtDNA, and the lower arrow indicates the 184-bp fragment corresponding to on freeze-thawed mitochondrial preparations ac- mtDNA with the tRNALYs mutation. A, Analysis of the fraction of cording to methods described elsewhere (Tulinius et mtDNA with the tRNALYs mutation, in muscle obtained from the al. 1991a). proband of family A (case V-1) at the ages of 10, 13, and 15 years The first biopsy specimen from the probands offam- (lanes 1-3, respectively), case V-2 of family A (lane 4), case IV-21 ilies A and C was analyzed before 1986. The method of family A (lane 5), the proband of family C (case 11-2) at the ages of 17 and 20 years (lanes 6 and 7, respectively), the proband of for isolation of mitochondria was less efficient than family B (case 111-5) (lane 8), case III-6 of family B (lane 9), and case the currently used method, and the activity ofcomplex II-5 of family B (lane 10). B, Analysis of serial dilutions of muscle IV was measured in the presence of 60 jmol of DNA with mainly mutated mtDNA in normal muscle DNA. Muscle N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD)/ DNA from the third muscle biopsy specimen of the proband of liter, instead of 500 jmol/liter, which is the concen- family A (0.1 ng, 0.2 ng, 0.3 ng, 0.4 ng, 0.5 ng, 1 ng, 2 ng, 3 ng, 4 ng, and 5 ng) was mixed with muscle DNA (100 ng) from a healthy tration used at present (Tulinius et al. 1991a). No individual and was analyzed with PCR (lanes 1-10, respectively). spectrophotometric measurements were performed before 1986. A, even after prolonged exposure of the autoradio- Results grams. The proportion of mutated mtDNA in muscle is shown in table 2. The proportions of mutated mtDNA Analysis mtDNA of lymphocytes, muscle, and fibroblasts in Sequencing of muscle mtDNA from the three pro- nine investigated cases are shown in table 3. bands showed the adenine-to-guanine transition mu- A 1:100 proportion of mutated mtDNA was de- tation at nt 8344. A PCR-based assay was used to tected after overnight exposures of the autoradio- quantitate the proportion ofmtDNA with the tRNALYS grams, when serial dilutions ofmuscle DNA with 95% mutation (fig. 3A), and the values were corrected for mutated mtDNA in normal muscle DNA were ana- heteroduplex formation (fig. 2). In figure 1 the individ- lyzed (fig. 3B). A 3:1,000 proportion of mutated uals of families A-C who carry the tRNALYs mutation mtDNA was detected after prolonged exposures ofthe are indicated by blackened symbols, and the propor- autoradiograms. tions of mutated mtDNA in lymphocytes of all investi- The point associated with MELAS syn- gated cases are given. No mutated mtDNA was de- drome and retinitis pigmentosa/ataxia syndrome were tected in cases II-2, III-1, III-1 1-111-1 2, III-15, III-16- not present in any tissue from the probands. No III-17, III-23, IV-7, IV-13 TIV-15, and IV-19 offamily mtDNA deletions were found. Segregation and Manifestations of tRNALYS Mutation 1207

Table 2 Summary of Investigations Performed on Skeletal Muscle from Probands and Family Members RESPIRATORY RATEa AGE AT PROPORTION OF FIBERS LACKING BioPsY MUTATED mtDNA Complex IY Complex IVd RAGGED-RED COX ACTIVITY CASE (years) (%) (% of mean) (% of mean) FIBERSb (%) Family A: Probande 10 10 94 12 69 + 10 Proband 13 94 12 27 + 40 Probandf 15 15 95 19 30 + 60 - V-2 13 ...... 92 94 90 10 IV-21 37 37 80 107 88 - <1 III-23 48...... <.3 ND ND - 0 11-6 ...... 83 14 ND ND - <1 Family B: Proband 21 96 47 76 + 5 111-6 .20 20 61 107 106 - 0 II-5 4...... 42 73 136 109 + 1 Family C: Probande ... 17 95 50 88 + <1 Proband 20 96 36 74 + <1 a Absolute values are given in table 4. ND = not done. b A plus sign ( + ) indicates that ragged-red fibers were present, and a minus sign (-) indicates that ragged-red fibers were not present. c Polarographic measurements, with pyruvate and malate. d Polarographic measurements, with ascorbate and TMPD. Measurements were performed before 1986, with a less efficient isolation procedure for muscle mitochondria and with a lower concentra- tion of TMPD (see Material and Methods). f Muscle biopsy specimen was obtained from the gastrocnemius muscle. (All other muscle biopsy specimens were obtained from the quadriceps muscle.) Examination Muscle Morphological of dria. Apart from the typical ragged-red fibers, many The three probands and case TI-S of family B had other fibers also showed accumulation of mitochon- ragged-red fibers, cytochrome c oxidase (COX)-defi- dria, as revealed by high succinate dehydrogenase ac- cient fibers, and ultrastructurally abnormal mitochon- tivity. Case V-2 of family A had no ragged-red fibers, Table 3 Proportion of mtDNA with tRNALYS Mutation, in Different Tissues

MEAN OF WITH THE IN AGE (range) mtDNA tRNALYsMUTATION, CASE (years) Muscle Lymphocytes Fibroblasts Family A: Proband ...... 10 94 (92-96) Proband ...... 13 94 (92-96) 43(41-45) Proband ...... 15 95 (93-97) 88 (86-89) 41(39-43) V-2 ...... 13 92 (90-93) 77 (74-79) 66(66-66) IV-21 ...... 37 80 (78-83) 72 (70-73) 62(62-62) III-23 ...... 58 <.3 <.3 <.3 11-6 ...... 83 14 (12-15) 10 (7-12) 7(5-8) Family B: Proband ...... 21 96 (94-97) 80 (77-81) 54(51-59) 111-6 ...... 20 61(59-63) 50 (50-51) 48 (47-50) 11-5 ...... 42 73 (72-75) 66 (63-69) 50(49-50) Family C: Proband ...... 17 95 (94-97) 91(89-93) Proband ...... 20 96 (92-97) 90 (88-91) 1208 Larsson et al. but 10% of the fibers were devoid of COX activity. In Biochemistry cases IV-21 and II-6 of family A, occasional fibers Evaluation ofthe respiratory-chain function in fresh were COX deficient. mitochondria revealed decreased oxidation rates in Three different muscle specimens were obtained the presence ofcomplex I-dependent substrates (pyru- from the proband of family A (case V-1) during a vate + malate and glutamate + malate) in all biopsy S-year period. The first biopsy showed absence of specimens from the three probands (tables 2 and 4). COX in 10% of the muscle activity fibers. The second The proband of family A had a profound deficiency in and third showed absence of COX biopsy activity in all three biopsy specimens, whereas the deficiency was 40% and 60% of the muscle fibers. Many additional less pronounced in the other probands. The proband fibers showed weak COX in activity relation to the of family C had a more pronounced deficiency at the amount of as mitochondria, determined by succinate age of 20 years than at the age of 17 years. In the in consecutive dehydrogenase activity sections. proband of family A, polarographic measurements re- In the two muscle obtained specimens 3 years apart vealed a slightly decreased complex IV activity (ascor- from the of proband family C (case II-2), <1% of the bate + TMPD) at 10 years of age. A severely decreased fibers lacked COX activity. Many additional fibers activity was found at the ages 13 and 15 years. In the showed weak COX activity in relation to the amount proband of family C, polarographic measurements at of mitochondria. The morphological findings are sum- age 17 years showed a complex IV activity well within marized in table 2. the normal range, but at 20 years the activity was at

Table 4 Polarographic Measurements of Respiratory-Chain Function in Isolated Muscle Mitochondria from Probands and Family Members Ascorbate + Ascorbate + Isolated Pyruvate + Glutamate + Succinate + TMPD TMPD Age at Mitochondria Malate Malate Rotenone (60 ipmol/liter) (500 imol/liter) Biopsy (mg protein/ (nmol O/min/ (nmol O/min/ (nmol O/min/ (nmol O/min/ (nmol O/min/ Case (years) g muscle) mg protein) mg protein) mg protein) mg protein) mg protein) Family A: Probanda ...... 10 .83 14 17 30 40 Proband ...... 13 7.51 13 16 54 79 Probandb ...... 15 2.49 20 23 62 88 V-2 ...... 13 4.91 101 109 136 261 IV-21 ...... 37 3.10 115 131 125 256 Family B: Proband ...... 21 3.05 50 61 147 219 III-6 ...... 20 3.17 115 139 157 307 II-5 ...... 42 2.59 146 140 158 315 Family C: Probanda ...... 17 .87 57 60 84 51 Proband ...... 20 3.55 39 39 116 214 Controls (n = 6):c Mean ...... 35 2.76 107 114 122 290 Range ...... 21-63 1.26-6.28 95-118 95-128 98-151 214-339 Controls (n = 4)a'd Mean...... 15 114 116 88 58 Range ...... 9-20 75-152 73-163 54-125 45-68 a Measurements were performed before 1986, with a less efficient isolation procedure for muscle mitochondria and with a lower concentra- tion of TMPD (see Material and Methods). Spectrophotometric measurements were not done. b Muscle biopsy specimen was obtained from the gastrocnemius muscle. (All other muscle biopsy specimens were obtained from the quadriceps muscle.) ' Controls are the same as in the study by Larsson et al. (1991). d Controls are the same as control group A in the study by Tulinius et al. (1991). Segregation and Manifestations of tRNALYs Mutation 1209 the lower limit of the normal range. The proband of of family A and cases I-1 and 11-5-11-6 of family B) family B had an activity at the lower limit ofthe normal had 43%-73% mutated mtDNA in lymphocytes, and range. The respiratory rates in specimens from the mutated mtDNA was detected in all 12 oftheir investi- other cases were normal. gated children. This suggests that the risk for transmis- In the second and third biopsy specimens from the sion of mutated mtDNA to the offspring increases if proband of family A, the COX activity in disrupted high levels ofmutated mtDNA are present in the mother mitochondria, as measured by spectrophotometry, and that, above a certain threshold level of - 35%- was <10% of the normal mean. In the probands of 40%, it is very likely that transmission to all children families B and C, the COX activity was reduced to will occur. We found widely varying levels of mtDNA - 30% ofthe normal mean (table 5). The results ofthe with the tRNAALYS mutation (range <0.3%-88%) in polarographic and spectrophotometric measurements lymphocytes from the 27 investigated individuals of of respiratory-chain function in muscle are summa- family A (fig. 1). The proband's maternal great-grand- rized in tables 4 and 5. mother (case 11-6) had 7%-14% mutated mtDNA in fibroblasts, lymphocytes, and muscle. No mutated mtDNA was found in fibroblasts, lymphocytes, and Discussion muscle from the great-grandmother's son (case III-23), We have found that mutated mtDNA is not always whereas her daughter (case 111-21) had 73% mutated detected in the offspring of women carrying the tRNALYS mtDNA in lymphocytes. Cases 111-13 and III-14 had mutation. Four women (cases 11-4, 11-6, 111-13, and 28% and 14% mutated mtDNA in lymphocytes, re- III-14 of family A) had 10%-33% mutated mtDNA spectively, and no mutated mtDNA was found in three in lymphocytes, and no mutated mtDNA was found of their children (cases IV-7, IV-13, and IV-15), in 7 of their 14 investigated children. The presence of whereas high levels, 49%-72%, were found in an- mutated mtDNA was excluded at a level of <3:1 ,000, other three of their children (cases IV-9, IV-10, and which is, for the PCR assay, the detection limit of IV-14). This shows that, although a mother has a mutated mtDNA. Five women (cases 111-21 and IV-21 lowproportion ofmutated mtDNA, a high proportion

Table 5 Spectrophotometric Measurements of Respiratory-Chain Enzyme Activities in Isolated Muscle Mitochondria from Probands and Family Members NADH Succinate Ferricyanide Cytochrome c Cytochrome c Citrate Age at Reductase Reductase Oxidase Synthase Biopsy (imol/min (nmol/min (rate constant [k]/ (jimol/min Case (years) mg protein) mg protein) min mg protein) mg protein) Family A: Proband ...... 13 1.16 114 .521 2.32 Probanda ...... 15 2.69 201 .979 2.58 V-2 ...... 13 4.83 367 10.8 2.19 IV-21 ...... 37 5.88 276 9.71 2.07 Family B: Proband ...... 21 2.60 194 3.05 2.69 III-6 ...... 20 7.12 476 15.4 2.33 11-5 ...... 42 7.15 423 14.6 2.60 Family C: Proband ...... 20 2.84 279 2.89 2.76 Controls (n = 6) b Mean ...... 35 6.39 263 10.0 2.03 Range ...... 21-63 3.08-8.50 207-309 7.7-11.3 1.26-2.54 a Muscle biopsy specimen was obtained from the gastrocnemius muscle. (All other muscle biopsy specimens were obtained from the quadriceps muscle.) b Controls are the same as in the study by Larsson et al. (1991). 1210 Larsson et al.

may be found in her offspring. Once a high proportion 61%-80% of mutated mtDNA in muscle. Polaro- of mutated mtDNA is established in a female, it will graphic and spectrophotometric measurements re- probably be maintained in subsequent generations, as vealed normal respiratory-chain function in all four demonstrated by the offspring of case I-1 of family B cases. This suggests that a proportion of >92% of and by the offspring of case 111-21 of family A. This mtDNA with the tRNALYS mutation has to be present suggestion is also supported by the findings in two to cause, in muscle, a respiratory-chain dysfunction other MERRF families, where high levels of mtDNA that can be detected by biochemical methods. These with the tRNALYS mutation were found in all investi- findings support the suggestion of Shoffner et al. gated maternally related individuals (Shoffner et al. (1990) that a small amount of normal tRNALYS is 1990; Seibel et al. 1991). sufficient to maintain a normal respiratory-chain func- Investigations in Holstein cows have demonstrated tion. COX-deficient muscle fibers were found in the that the proportion of mutated mtDNA can vary con- probands, in cases V-2 and IV-21 of family A, and in siderably between animals ofthe same sibship (Ashley case 11-5 of family B (but not in case III-6 of family B, et al. 1989). Vilkki et al. (1990) reported that the the case with the lowest amount of mutated mtDNA). levels of mutated mtDNA varied between 0% and The occurrence of COX-deficient muscle fibers in >95% in different individuals of a family with Leber Kearns-Sayre syndrome is caused by low levels of nor- hereditary optic neuropathy and the NADH-dehy- mal mtDNA in certain muscle fibers with accumula- drogenase subunit 4 gene point mutation at nt 11778 tion of deleted mtDNA (Mita et al. 1989; Oldfors et of mtDNA. This indicates that the widely varying lev- al. 1992). The COX-deficient muscle fibers found in els ofmutated mtDNA in individuals with the tRNALYS asymptomatic carriers of the tRNALYS mutation may mutation are not specific for the inheritance of this be caused by such an uneven distribution and lack of mutation but, rather, reflect the general principles of normal mtDNA in certain muscle fibers. inheritance of heteroplasmic mtDNA mutations. Multiple symmetrical lipomas of the neck were However, homoplasmy for the tRNALYs mutation has found in the mother (case IV-21) (Holme et al., sub- never been observed, most likely because this is not mitted) and maternal grandmother (case III-21) of the compatible with life. It has been suggested that the proband of family A. Lipomas have been reported in widely varying levels of mutated mtDNA in different other families with MERRF syndrome (Berkovic et al. individuals of the same sibship may be explained by 1989, 1991) and may represent a manifestation of the a founder effect where only a proportion of the thou- tRNALYS mutation. sands of mitochondria in the oocyte are transmitted In the oldest living generation of family A, three of to the fetus (Ashley et al. 1989). The founder effect can four individuals (cases II-4-11-6) were carriers of the also explain why a certain threshold level of mutated tRNALYs mutation, and they all had senile dementia. mtDNA in the mother is required for transmission to The oldest individual of family A (case 11-2) had no all children, since high levels of mutated mtDNA will detectable levels of mutated mtDNA, and she had no increase the probability that all mitochondria in the signs ofdementia. Dementia is very common in people oocyte contain mutated mtDNA. >80 years of age, and the correlation between the The three probands all fulfilled the clinical and mor- tRNALYs mutation and dementia in this family may phological criteria for MERRF syndrome (Fukuhara just be fortuitous. It has been suggested that mtDNA et al. 1980), although the age at onset and the rate of mutations are ofimportance in aging and degenerative progression varied considerably. The probands had a disease (Miquel and Fleming 1986; Wallace 1992). 94%-96% proportion of mutated mtDNA in muscle, Further studies are needed to clarify whether senile and there was biochemical and morphological evi- dementia is more common in otherwise asymptomatic dence for a respiratory-chain dysfunction. Thorough individuals carrying the tRNALYS mutation than in the investigations were performed in four maternal rela- general population. tives (cases IV-21 and V-2 of family A and cases II-5 There was a clear correlation between the levels and III-6 of family B) of two of the probands. A of mutated mtDNA in lymphocytes and the levels in brother (case V-2) of the proband in family A had muscle. The levels were higher in muscle in all nine exercise-induced muscle pain. In the other three cases, investigated cases (table 2). Higher levels of mutated extensive investigations revealed no neuromuscular mtDNA in muscle than in were also found in involvement. Case V-2 of family A had 92% of mu- three patients investigated by Seibel et al. (1991). De- tated mtDNA in muscle, and the other three cases had termination of the proportion of mutated mtDNA in Segregation and Manifestations of tRNALYS Mutation 1211 blood can possibly be used to predict both the minimal mtDNA proportion increases with time. We have pre- level of mutated mtDNA in muscle and, thus, the risk viously reported that such an increase occurs in Kearns- for development of MERRF syndrome. The constant Sayre syndrome (Larsson et al. 1990). However, as finding of higher levels of mutated mtDNA in muscle demonstrated by our and other cases of MERRF syn- may be due to selective increase of mitochondria with drome, the proportion of mutated mtDNA needed to deficient respiratory-chain function as suggested by cause disease in muscle is >92%, and the range avail- morphological findings. Another possibility is that able for progressive increase is thus very limited. We clones of cells with high levels of mutated mtDNA in did obtain increasing mean values of the proportion the bone marrow may have a growth disadvantage of mutated mtDNA in muscle from the probands of and may be selected out. families A and C when 10 determinations ofeach sam- The underlying cause of the progressive nature of ple were made, but the differences were not significant. MERRF syndrome is unclear. We followed the pro- The precision ofthe PCR method was too low to ascer- bands of families A and C for 7 and 8 years, respec- tain a difference of a few percent. Longitudinal studies tively, and performed multiple muscle biopsies over 5 that start at an early age in presymptomatic cases, who and 3 years, respectively. There was a clear clinical may have a lower proportion of mutated mtDNA in progression of neuromuscular symptoms in both pa- muscle, are needed to clarify whether the progression tients, accompanied by biochemical evidence ofdeteri- in MERRF syndrome is due to a gene-dosage effect. orating respiratory-chain function. In the proband of family A there was also an increase in the amount of COX-deficient muscle fibers. Lombes et al. (1989) Acknowledgments described a patient with premature fatigue and muscle This study was supported by grants from the Swedish cramps during childhood who developed myoclonic Medical Research Council, projects 585, 3921, and 7122, jerks and progressive gait ataxia from age 20 years. by the Sven Jerring Foundation, and by the First of May- This case may be similar to case V-2 of family A, who flower Annual Campaign. had exercise-induced muscle pain with onset at age 12 years. It is possible that our case also will develop a severe myopathy and MERRF syndrome. 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