Am. J. Hum. Genet. 51:457-468, 1992
A New Disease-related Mutation for Mitochondrial Encephalopathy Lactic Acidosis and Strokelike Episodes (MELAS) Syndrome Affects the ND4 Subunit of the Respiratory Complex I P. Lertrit,* A. S. Noer,* M. J. B. Jean-Francois,t R. Kapsa,* X. Dennett,$ D. Thyagarajan,t K. Lethlean,§ E. Byrnet and S. Marzuki* *Department of Biochemistry and Center for Molecular Biology and Medicine, Monash University, Clayton, Victoria; TDepartment of Neurology, St. Vincent's Hospital, Fitzroy, Victoria; $State Neuropathology Services, University of Melbourne, Parkville, Victoria; and §Department of Neurology, Prince Henry Hospital, Sydney
Summary The molecular lesions in two patients exhibiting classical clinical manifestations of MELAS (mitochondrial encephalopathy, lactic acidosis, and strokelike episodes) syndrome have been investigated. A recently reported disease-related A-OG base substitution at nt 3243 of the mtDNA, in the DHU loop of tRNAL U, was detected by restriction-enzyme analysis of the relevant PCR-amplified segment of the mtDNA of one patient but was not observed, by either restriction-enzyme analysis or nucleotide sequencing, in the other. To define the molecular lesion in the patient who does not have the A-OG base substitution at nt 3243, the total mitochondrial genome of the patient has been sequenced. An A- G base substitution at nt 11084, leading to a Thr-to-Ala amino acid replacement in the ND4 subunit of the respiratory complex I, is suggested to be a disease-related mutation.
Introduction
The importance of mtDNA mutations as the underlying molecular lesions in a wide range of human disorders has become apparent in recent years. The mtDNA, which is maternally inherited (Case and Wallace 1981; Egger and Wilson 1983), has been shown to have a mutation rate which is 10 times higher than that of the average nuclear genes (Brown et al. 1979; Giles et al. 1980). Two types of disease-related mutations have been observed: (a) large multigene deletions of 1-8 kb, observed in the CPEO (chronic progressive external ophthalmoplegia) syndrome (e.g., see Lestienne and Ponsot 1988; Zeviani et al. 1988; Moraes
Received November 4, 1991; final revision received May 22, 1992. Address for correspondence and reprints: Dr. S. Marzuki, Department of Biochemistry, Monash University, Clayton, Victoria 3168, Australia. i 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/92/5103-0002$02.00
et al. 1989), Pearson-marrow syndrome (Pearson et al. 1979; Rotig et al. 1990), and idiopathic cardiomyopathy (Ozawa et al. 1990), and (b) single base substitutions such as the nt 11778 G-INA base substitution observed in the ND4 gene in LHON (Leber hereditary optic neuropathy) (Wallace et al. 1988) and the nt 8344 A-GOG base substitution in the tRNALYS gene in the MERRF (myoclonic epilepsy with ragged-red fiber) syndrome (Shoffner et al. 1990; Yoneda et al. 1990; Noer et al. 1991). In recent years, evidence has accumulated indicating the heterogeneity of the molecular lesions which underlie distinct clinical syndromes associated with mitochondrial respiratory-chain dysfunction. Thus, in European studies, only 50% of patients with LHON have been shown to carry, in the ND4 coding region, the nt 11778 GN-*A mutation initially reported to be a causal mutation (Holt et al. 1989; Vilkki et al. 1989), and several alternative mutations have been suggested (Howell et al. 1991; Huoponen et al. 1991). It is significant that only 75% of MELAS (mitochondrial encephalopathy, lactic acidosis, and strokelike 457
458
episodes) syndrome (Pavlakis et al. 1984) patients examined to date showed the nt 3243 A-GG mutation, in the tRNALeU, recently suggested to be the diseaserelated mutation for this syndrome (Goto et al. 1990; Kobayashi et al. 1990; Tanaka et al. 1991). MELAS is characterized clinically by short stature; episodic vomiting; seizures and recurrent cerebral insults resembling strokes causing hemiparesis, hemianopia, or cortical blindness; and lactic acidosis. The maternal mode of inheritance observed in some families, as well as the sporadic nature of its expression in others, suggested that this mitochondrial disorder is the result of mutation(s) in the mtDNA. The lack of nt 3243 A--G base substitution in a significant proportion of cases raises the possibility that several mutations may lead to the clinical phenotype. Information with regard to the correlation between mutation and biochemical and clinical phenotype is not only of medical relevance with regard to the pathophysiology of mitochondrial respiratory disorders but also is of fundamental significance to our understanding of the mitochondrial development and genetics in humans. We have thus examined two patients of Caucasian origin who exhibited the classical features of MELAS. Our results show that one of the patients exhibited the base substitution at nt 3243 but that the other does not have this mutation. Nucleotide sequencing analysis of the mtDNA suggests that the underlying mutation in this patient is an A---G base substitution, at nt 11084 in the mtDNA, which causes an amino acid change from Thr to Ala in the ND4 subunit of the respiratory complex I. It is of interest that two very distinct mutations, one in the tRNALeU and the other affecting the ND4 subunit of the respiratory complex I, should lead to the same clinical phenotype. Patients and Methods Patients Patient SVR86-1, her mother, and a sister were studied clinically at St. Vincent's Hospital in Melbourne. Patient SVR90-8 and her mother were studied at Prince of Wales Hospital in Sydney. Muscle biopsies were obtained from the patients, their relatives, and
several normal and disease controls after informed consent had been granted, as described elsewhere (Byrne and Trounce 1985). Isolation of DNA
DNA was isolated from skeletal muscle biopsy samples. Tissue homogenates were prepared by adding
Lertrit et al. 0.3 M Tris buffer pH 8.0 containing 0.1 M NaCI, 0.2 M sucrose, and 0.01 M EDTA to the muscle sample material (2 ml/ 100 mg tissue) and then homogenizing the tissue in a glass hand tissue homogenizer until no pieces were visible. SDS and proteinase K were then added to a final concentration of 0.1% and 200 gg/ml, respectively, and the suspensions were incubated at 370C overnight. After the addition of 8 M potassium acetate to a final concentration of 1.5 M, the suspensions were held on ice for 1 h. DNA was then extracted by phenol/chloroform, precipitated in ethanol according to standard procedures (Davis et al. 1986), and dissolved in 10 mM Tris-HCI buffer pH 8.0 containing 1 mM EDTA. Restriction-Enzyme Analysis
The segment of interest in the mtDNA was first amplified by PCR prior to restriction-enzyme analysis. PCR was performed in an Innovonics "Gene Machine" by using 2.5 units of recombinant Taq DNA polymerase (Promega) (Saiki et al. 1988). The reaction mixture (final volume 100 pl) consisted of the amplification buffer (10 mM Tris-HCl pH 8.3 containing 50 mM KCI and 0.01% [w/v] gelatin), 1.25 mM MgC12, 200 gM of each dNTP, 80 pmol of each synthetic oligonucleotide primer, and 5-10 ng of DNA template. The reaction was carried out for 30 cycles of denaturation (60 sat 950C), annealing (90 sat 560C), and polymerization (150 s at 720C). For the analysis of the nt 3243 A oG MELASassociated mutation (Goto et al. 1990; Kobayashi et al. 1990; Tanaka et al. 1991), the primer sets were B1F (corresponding to nt 2826-2849 of the light strand) and ND2H (corresponding to nt 5459-5482 of the heavy strand); nucleotide numbering is according to the Cambridge sequence (Anderson et al. 1981), and details of primers are as previously published (Marzuki et al. 1990). The PCR-amplified products, extracted by phenol/ chloroform and purified using GenecleanT (Bio 101; La Jolla), were digested with the appropriate restriction endonuclease at 370C overnight. The restriction fragments were then electrophoretically separated on a 1% or 2.5% agarose gel as indicated, stained with ethidium bromide at a final concentration of 5 ig/ml, and were photographed under UV light. DNA Sequencing Analysis
The strategy employed for the sequencing of mtDNA has been reported elsewhere (Marzuki et al. 1991; Noer et al. 1991). The entire mitochondrial
Mutant ND4 Subunit in MELAS genome was amplified in six overlapping segments by PCR. The six synthetic oligonucleotide primer pairs used for this purpose correspond to the following locations on the human mitochondrial genome (subscripts refer to the heavy [H] or light [L] strands): AL, 1590415930; AH, 2569-2546; BL, 2322-2345; BH, 54825459; CL, 5223-5246; CH, 9247-9224; EL, 83168345; EH, 11942-11918; FL, 11580-11603; FH, 13200-13177; GL, 12367-12390; and GH, 1654016515. Details of the primers have been previously published (Marzuki et al. 1990). The PCR products were sequenced by the dideoxy termination procedure (Sanger et al. 1977), either directly after further asymmetrical amplification (fragment G) or after cloning into the pUCi 9 vector (Marzuki et al. 1991; Noer et al. 1991). For fragments sequenced after cloning, at least three independent clones (in many cases five clones) were analyzed. Results Case Study
SVR86-I.-This subject was a 19-year-old female tertiary student presented to the hospital with a 6-mo history of intermittent migraine headaches and a 6-d history of severe headaches and increasing drowsiness. A history was obtained of bilateral sensorineural hearing loss which had progressed during childhood and of bilateral cataracts which had required surgery when the patient was age 8 years. Several grand mal seizures had occurred 1 year before presentation. Over a short period, the patient developed two further strokelike episodes which failed to conform to a given vascular territory. The patient was extremely thin, with reduced muscle mass. Resting serum lactate levels were found to be elevated in four separate determinations: 5.7, 6.0, 5.5, and 6.0 mmol/liter (normal range
A New Disease-related Mutation for Mitochondrial Encephalopathy Lactic Acidosis and Strokelike Episodes (MELAS) Syndrome Affects the ND4 Subunit of the Respiratory Complex I P. Lertrit,* A. S. Noer,* M. J. B. Jean-Francois,t R. Kapsa,* X. Dennett,$ D. Thyagarajan,t K. Lethlean,§ E. Byrnet and S. Marzuki* *Department of Biochemistry and Center for Molecular Biology and Medicine, Monash University, Clayton, Victoria; TDepartment of Neurology, St. Vincent's Hospital, Fitzroy, Victoria; $State Neuropathology Services, University of Melbourne, Parkville, Victoria; and §Department of Neurology, Prince Henry Hospital, Sydney
Summary The molecular lesions in two patients exhibiting classical clinical manifestations of MELAS (mitochondrial encephalopathy, lactic acidosis, and strokelike episodes) syndrome have been investigated. A recently reported disease-related A-OG base substitution at nt 3243 of the mtDNA, in the DHU loop of tRNAL U, was detected by restriction-enzyme analysis of the relevant PCR-amplified segment of the mtDNA of one patient but was not observed, by either restriction-enzyme analysis or nucleotide sequencing, in the other. To define the molecular lesion in the patient who does not have the A-OG base substitution at nt 3243, the total mitochondrial genome of the patient has been sequenced. An A- G base substitution at nt 11084, leading to a Thr-to-Ala amino acid replacement in the ND4 subunit of the respiratory complex I, is suggested to be a disease-related mutation.
Introduction
The importance of mtDNA mutations as the underlying molecular lesions in a wide range of human disorders has become apparent in recent years. The mtDNA, which is maternally inherited (Case and Wallace 1981; Egger and Wilson 1983), has been shown to have a mutation rate which is 10 times higher than that of the average nuclear genes (Brown et al. 1979; Giles et al. 1980). Two types of disease-related mutations have been observed: (a) large multigene deletions of 1-8 kb, observed in the CPEO (chronic progressive external ophthalmoplegia) syndrome (e.g., see Lestienne and Ponsot 1988; Zeviani et al. 1988; Moraes
Received November 4, 1991; final revision received May 22, 1992. Address for correspondence and reprints: Dr. S. Marzuki, Department of Biochemistry, Monash University, Clayton, Victoria 3168, Australia. i 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/92/5103-0002$02.00
et al. 1989), Pearson-marrow syndrome (Pearson et al. 1979; Rotig et al. 1990), and idiopathic cardiomyopathy (Ozawa et al. 1990), and (b) single base substitutions such as the nt 11778 G-INA base substitution observed in the ND4 gene in LHON (Leber hereditary optic neuropathy) (Wallace et al. 1988) and the nt 8344 A-GOG base substitution in the tRNALYS gene in the MERRF (myoclonic epilepsy with ragged-red fiber) syndrome (Shoffner et al. 1990; Yoneda et al. 1990; Noer et al. 1991). In recent years, evidence has accumulated indicating the heterogeneity of the molecular lesions which underlie distinct clinical syndromes associated with mitochondrial respiratory-chain dysfunction. Thus, in European studies, only 50% of patients with LHON have been shown to carry, in the ND4 coding region, the nt 11778 GN-*A mutation initially reported to be a causal mutation (Holt et al. 1989; Vilkki et al. 1989), and several alternative mutations have been suggested (Howell et al. 1991; Huoponen et al. 1991). It is significant that only 75% of MELAS (mitochondrial encephalopathy, lactic acidosis, and strokelike 457
458
episodes) syndrome (Pavlakis et al. 1984) patients examined to date showed the nt 3243 A-GG mutation, in the tRNALeU, recently suggested to be the diseaserelated mutation for this syndrome (Goto et al. 1990; Kobayashi et al. 1990; Tanaka et al. 1991). MELAS is characterized clinically by short stature; episodic vomiting; seizures and recurrent cerebral insults resembling strokes causing hemiparesis, hemianopia, or cortical blindness; and lactic acidosis. The maternal mode of inheritance observed in some families, as well as the sporadic nature of its expression in others, suggested that this mitochondrial disorder is the result of mutation(s) in the mtDNA. The lack of nt 3243 A--G base substitution in a significant proportion of cases raises the possibility that several mutations may lead to the clinical phenotype. Information with regard to the correlation between mutation and biochemical and clinical phenotype is not only of medical relevance with regard to the pathophysiology of mitochondrial respiratory disorders but also is of fundamental significance to our understanding of the mitochondrial development and genetics in humans. We have thus examined two patients of Caucasian origin who exhibited the classical features of MELAS. Our results show that one of the patients exhibited the base substitution at nt 3243 but that the other does not have this mutation. Nucleotide sequencing analysis of the mtDNA suggests that the underlying mutation in this patient is an A---G base substitution, at nt 11084 in the mtDNA, which causes an amino acid change from Thr to Ala in the ND4 subunit of the respiratory complex I. It is of interest that two very distinct mutations, one in the tRNALeU and the other affecting the ND4 subunit of the respiratory complex I, should lead to the same clinical phenotype. Patients and Methods Patients Patient SVR86-1, her mother, and a sister were studied clinically at St. Vincent's Hospital in Melbourne. Patient SVR90-8 and her mother were studied at Prince of Wales Hospital in Sydney. Muscle biopsies were obtained from the patients, their relatives, and
several normal and disease controls after informed consent had been granted, as described elsewhere (Byrne and Trounce 1985). Isolation of DNA
DNA was isolated from skeletal muscle biopsy samples. Tissue homogenates were prepared by adding
Lertrit et al. 0.3 M Tris buffer pH 8.0 containing 0.1 M NaCI, 0.2 M sucrose, and 0.01 M EDTA to the muscle sample material (2 ml/ 100 mg tissue) and then homogenizing the tissue in a glass hand tissue homogenizer until no pieces were visible. SDS and proteinase K were then added to a final concentration of 0.1% and 200 gg/ml, respectively, and the suspensions were incubated at 370C overnight. After the addition of 8 M potassium acetate to a final concentration of 1.5 M, the suspensions were held on ice for 1 h. DNA was then extracted by phenol/chloroform, precipitated in ethanol according to standard procedures (Davis et al. 1986), and dissolved in 10 mM Tris-HCI buffer pH 8.0 containing 1 mM EDTA. Restriction-Enzyme Analysis
The segment of interest in the mtDNA was first amplified by PCR prior to restriction-enzyme analysis. PCR was performed in an Innovonics "Gene Machine" by using 2.5 units of recombinant Taq DNA polymerase (Promega) (Saiki et al. 1988). The reaction mixture (final volume 100 pl) consisted of the amplification buffer (10 mM Tris-HCl pH 8.3 containing 50 mM KCI and 0.01% [w/v] gelatin), 1.25 mM MgC12, 200 gM of each dNTP, 80 pmol of each synthetic oligonucleotide primer, and 5-10 ng of DNA template. The reaction was carried out for 30 cycles of denaturation (60 sat 950C), annealing (90 sat 560C), and polymerization (150 s at 720C). For the analysis of the nt 3243 A oG MELASassociated mutation (Goto et al. 1990; Kobayashi et al. 1990; Tanaka et al. 1991), the primer sets were B1F (corresponding to nt 2826-2849 of the light strand) and ND2H (corresponding to nt 5459-5482 of the heavy strand); nucleotide numbering is according to the Cambridge sequence (Anderson et al. 1981), and details of primers are as previously published (Marzuki et al. 1990). The PCR-amplified products, extracted by phenol/ chloroform and purified using GenecleanT (Bio 101; La Jolla), were digested with the appropriate restriction endonuclease at 370C overnight. The restriction fragments were then electrophoretically separated on a 1% or 2.5% agarose gel as indicated, stained with ethidium bromide at a final concentration of 5 ig/ml, and were photographed under UV light. DNA Sequencing Analysis
The strategy employed for the sequencing of mtDNA has been reported elsewhere (Marzuki et al. 1991; Noer et al. 1991). The entire mitochondrial
Mutant ND4 Subunit in MELAS genome was amplified in six overlapping segments by PCR. The six synthetic oligonucleotide primer pairs used for this purpose correspond to the following locations on the human mitochondrial genome (subscripts refer to the heavy [H] or light [L] strands): AL, 1590415930; AH, 2569-2546; BL, 2322-2345; BH, 54825459; CL, 5223-5246; CH, 9247-9224; EL, 83168345; EH, 11942-11918; FL, 11580-11603; FH, 13200-13177; GL, 12367-12390; and GH, 1654016515. Details of the primers have been previously published (Marzuki et al. 1990). The PCR products were sequenced by the dideoxy termination procedure (Sanger et al. 1977), either directly after further asymmetrical amplification (fragment G) or after cloning into the pUCi 9 vector (Marzuki et al. 1991; Noer et al. 1991). For fragments sequenced after cloning, at least three independent clones (in many cases five clones) were analyzed. Results Case Study
SVR86-I.-This subject was a 19-year-old female tertiary student presented to the hospital with a 6-mo history of intermittent migraine headaches and a 6-d history of severe headaches and increasing drowsiness. A history was obtained of bilateral sensorineural hearing loss which had progressed during childhood and of bilateral cataracts which had required surgery when the patient was age 8 years. Several grand mal seizures had occurred 1 year before presentation. Over a short period, the patient developed two further strokelike episodes which failed to conform to a given vascular territory. The patient was extremely thin, with reduced muscle mass. Resting serum lactate levels were found to be elevated in four separate determinations: 5.7, 6.0, 5.5, and 6.0 mmol/liter (normal range
