The mitochondrial encephalomyopathies are a diverse group of disorders that result from the struc-tural, biochemical or genetic derangement of mi-tochondria.1Mitochondrial encephalomyopathy,
lactic acidosis and stroke-like episodes (MELAS) syndrome is one of the mitochondrial disorders characterized by seizures, repeated stroke-like episodes with focal neurologic deficits, recurrent
A Follow-up Study in a Taiwanese Family
with Mitochondrial Myopathy,
Encephalopathy, Lactic Acidosis and
Stroke-like Episodes Syndrome
Jie-Yuan Li,1Rong-Hong Hsieh,2Nan-Jing Peng,3Ping-Hong Lai,4Cheng-Feng Lee,5
Yuk-Keung Lo,1Yau-Huei Wei5*
Background/Purpose: MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) syndrome is often associated with A3243G point mutation of mitochondrial DNA (mtDNA). We previously described a MELAS family characterized by harboring an additional ∼260 bp tandem dupli-cation in the D-loop and a novel C3093G point mutation in the 16S rRNA gene of mtDNA in the proband. We studied the clinical progression and fluctuation of mtDNA mutations in this Taiwanese MELAS family. Methods: We followed up the clinical course in all members of this family (1 proband, her mother and 3 sons) for 12 years. Mutations of mtDNA in serial muscle biopsies of the proband and blood samples and hair follicles taken at different time points from the members of this family were analyzed.
Results: The proband developed repeated stroke-like episodes, chronic intestinal pseudo-obstruction, polyneu-ropathy, progressive renal failure and dilated cardiomyopathy with heart failure. During the follow-up period, the mother and one of the siblings of the proband developed stroke-like episodes at age 62 and 12, respectively. There was no significant difference in the proportions of mtDNA with A3243G mutation among five serial muscle biopsies of the proband. In one carrier (I-2), the proportion of A3243G mutated mtDNA in blood cells was slightly increased with disease progression.
Conclusion: This study underlines the importance of early detection of extraneuromuscular symptoms in the members of a family with MELAS syndrome by adequate follow-up. The age of onset of stroke-like episode in MELAS syndrome may be as late as 62 years. We suggest that the manifestations of MELAS syndrome in this family might be associated with the additional ∼260 bp tandem duplication in the D-loop region and the coexistence of C3093G mutation in the 16S rRNA gene with the A3243G mutation of mtDNA. [J Formos Med Assoc 2007;106(7):528–536]
Key Words: follow-up study, MELAS, mitochondrial disease, mitochondrial DNA, mutation
©2007 Elsevier & Formosan Medical Association
. . . . 1Division of Neurology, and Departments of 3Nuclear Medicine and 4Radiology, Kaohsiung Veterans General Hospital, Kaohsiung and National Yang-Ming University, Taipei, 2School of Nutrition and Health Sciences, Taipei Medical University, and 5Department of Biochemistry and Molecular Biology, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan.
Received: November 30, 2006 Revised: December 28, 2006 Accepted: March 13, 2007
*Correspondence to: Professor Yau-Huei Wei, Department of Biochemistry and Molecular Biology,
School of Life Sciences, National Yang-Ming University, 155 Li-Nong Street, Section 2, Shih-Pai, Taipei 112, Taiwan.
migraine-like headache, vomiting, cognitive im-pairment with disease progression and ragged red fibers on muscle biopsy.2,3 Most patients with MELAS syndrome have been associated with the A3243G mutation in the tRNALeu(UUR) gene.4,5 However, recent studies showed that the clinical spectrum of the A3243G mutation is expanding and other genetic mutations may also occur in the affected tissues of patients with MELAS syn-drome.6–9These findings support the notion that MELAS syndrome is a genetically heterogeneous disease.
We described previously a unique MELAS family characterized by harboring the A3243G mutation and an additional ∼260 bp tandem du-plication in the D-loop region of mitochondrial DNA (mtDNA).10Soon we also identified a novel C3093G mtDNA mutation in the 16S rRNA gene of mtDNA in the proband and her mother in this MELAS family.11Besides the manifestations of en-cephalomyopathy, the proband presented with diabetes mellitus and hyperthyroidism. Since the proband harbored multiple mtDNA mutations, study of the progression of the clinical pheno-type of this family and the correlation between clinical manifestations and mtDNA mutations would be of particular interest.
Methods
Patients
The clinical symptoms of the proband of this fam-ily with MELAS syndrome have been described previously.10 She (patient II-2, Figure 1) had a history of hyperthyroidism and received subtotal thyroidectomy at age 26. She presented with dis-orientation, psychiatric symptoms and epileptic seizures at age 30. MELAS syndrome was diagnosed based on the findings of multiple infarcts on brain computed tomography (CT), elevated cerebral spinal fluid lactate and ragged red fibers (RRF) on muscle biopsy. Endocrinologic studies disclosed diabetes mellitus and hyperthyroidism.10Analysis of mtDNA showed an AG transition at nucleo-tide position (np) 3243 (A3243G mutation) in the
tRNALeu(UUR)gene and an additional ∼260 bp tan-dem duplication in the D-loop region of mtDNA in the three generations of the family.
In this family, subjects I-2, II-4, III-1, III-2 and III-3 were all asymptomatic carriers at the time of the first stage of diagnosis. We have been con-ducting follow-up studies on all of these family members except II-4 for 12 years. Investigations included physical examination, blood biochem-istry, urinalysis, EKG, EEG, and ophthalmologic, otologic and cardiologic examinations.
Medical imaging study
Serial single photon emission computed tomog-raphy (SPECT) and magnetic resonance imaging (MRI) scans and two positron emission tomog-raphy (PET) scans of the brain were performed in all maternal relatives of the proband and chrono-logic changes were observed. A brain SPECT scan was performed 60 minutes after intravenous in-jection of 99mTc-HMPAO on a dual-head gamma camera (Multispect II; Siemens, New York, NY, USA) equipped with low-energy, high-resolution collimators. Imaging was performed parallel to the canthomeatal plane, starting below the level of the cerebellum and extending to the vertex. Brain MRI was performed on the 1.5-T superconducting
1 Family pedigree 1 2 2 I II III 3 1 2 3 4
Figure 1. Family pedigree of the patients with MELAS syn-drome. The proband is indicated by an arrow. Solid symbols show the members with full-blown MELAS syndrome. Stripped symbols indicate oligosymptomatic relatives. Open symbols show healthy relatives.
system (GE Signa MRI; GE Healthcare) with a cir-culatory polarizing head coil. The imaging stud-ies included axial T1-weighted spin-echo (500/30/2 repetition time/echo time/excitations) and T2-weighted fast spin-echo (4000/100/2) with echo train length 8. Sections (5-mm thick) with 2.5-mm interslice gaps, 24-cm field of view, and 256× 192 matrix were used for all scans. A PET scan-ner (PC4096-15WB; Scanditronix Co., Uppsala, Sweden) with eight ring detectors was used for the study. The system produces 15 contiguous slices, each in the thickness of 7 mm, and has a transaxial resolution of 5.6 mm full-width at half maximum in the center of the field of view. PET brain imaging was performed in the fasting state 45 minutes after intravenous injection of 18F-fluorodeoxyglucose. Transmission scanning for attenuation correction was conducted prior to the emission study. Images were reconstructed by standard filtered back projection using a Hann filter with a filter width of 4.2 mm for 128× 128 images.
Mitochondrial DNA mutation analysis
Muscle biopsies of the proband, done serially from June 1993 to January 1997, were analyzed for change in the proportion of mutated mtDNA with the progression of disease. Blood samples and hair follicles were collected annually from June 1994 to February 1997. Total DNA was extracted from skeletal muscle, blood leukocytes and hair follicles. The target mtDNA fragment was amplified in a 100µL reaction mixture containing 2–5 ng DNA, 200µM of the dNTPs, 20 pmol of each primer, 1.5 units of Taq DNA polymerase (Perkin-Elmer/Cetus, Norwalk, CT, USA), 50 mM KCl, 1.5 mM MgCl2 and 10 mM Tris-HCl (pH 8.3). We performed polymerase chain reaction (PCR) with 1 minute of denaturation at 94°C, 1 minute of annealing at 56°C, and 1 minute of extension at 72°C for 30 cycles and then added 0.5µCi of α-[33P]-dATP in the last cycle in a Perkin-Elmer/Cetus DNA ther-mal cycler. The region encompassing np 3058– 3343 of mtDNA was amplified by using a pair of primers, L3058 (np 3058–3092, 5’-TACGT-GATCTGAGTTCAGACCGGAGTAATCCAGAT-3’)
and H3343 (np 3343–3324, 5’-TGGGTACAAT-GAGGAGTAGG-3’), of human mtDNA. The
∼260 bp tandem duplication and A3243G and C3093G mutations of mtDNA were analyzed as described previously.10,11 The amplified DNA fragments were digested with either Apa I (for the A3243G mutation) or Dpn II (for the C3093G mutation) and the restricted DNA fragments were separated by electrophoresis on a 1.5% agarose gel or 3% Nusieve plus 1% agarose gel at 100 volts for 45 minutes. The relative proportion of the mutated mtDNA was determined by scanning densitometry and autoradiography of a Kodak X-ray film after exposure to the DNA bands in the agarose gel.
Results
Table 1 presents the clinical manifestations and paraclinical data of the proband and her mother and three children. During the follow-up period, the proband suffered from repeated stroke-like episodes and progressive gait disturbance. Serial brain SPECT revealed focal hyperperfusion during the stroke-like episode (Figure 2A) and diffuse hypoperfusion at the late stage (Figure 2B) of the disease in the proband. Brain MRI showed multiple infarcts and brain atrophy (Figure 2C). Episodes of acute ileus (intestinal pseudo-obstruction) were noted on plain abdominal X-ray (Figure 2D). Poly-neuropathy and progressive renal failure with proteinuria also developed subsequently. Thyroid function test indicated sick euthyroid state. She then developed congestive heart failure with acute pulmonary edema and died of heart failure at age 35.
Her mother (I-2) had been noted to have severe sensorineural hearing impairment since the age of 56. At age 62, she was sent to our emergency room due to episodes of abdominal fullness and generalized tonic-clonic seizure. Brain MRI re-vealed acute cerebral infarcts in the left temporo-parieto-occipital areas. Intestinal pseudo-obstruction, chronic renal failure, recurrent stroke-like epi-sodes and cortical blindness developed later. She
Table 1. Clinical manifestations and paraclinical findings of the members in the MELAS family examined in this study
Family member I-2 II-2 III-1 III-2 III-3
Age (yr) at diagnosis 53 30 10 7 4
Sex F F M M M
Clinical manifestation
At diagnosis None Headache, episodic None None Lactic acidemia
vomiting, lactic acidosis, stroke-like episodes, NIDDM, hyperthyroidism
During follow-up SNHL, stroke-like Brain atrophy with Occasional Mild memory Mental retardation, episodes, seizure, dementia, dilated headache, impairment, mild limb ataxia,
myopathy, cardiomyopathy, nausea, occasional recurrent headache
vomiting, intestinal severe myopathy, vomiting, headache and vomiting, pseudo-obstruction, intestinal anorexia and and blurred stroke-like
chronic pseudo-obstruction, blurred vision vision episodes,
nephropathy polyneuropathy, seizures, SNHL,
chronic nephropathy myopathy
EEG
At diagnosis Normal Generalized slowing Normal Normal Normal
During follow-up Focal slowing of No change Normal Normal Focal spike over
cerebral activity over left posterior T-P
left T-P-O regions areas and
background slowing SPECT
At diagnosis Normal Focal hyperperfusion Normal Normal Normal
during the stroke-like episodes
During follow-up NA Diffuse hypoperfusion Normal Normal Hypoperfusion over
bil. occipital areas MRI
At diagnosis Normal Old and new focal Normal Normal Normal
infarcts
During follow-up Multiple infarcts Multiple infarcts, Normal Normal Initially, high T2
brain atrophy signal lesions in
bil. PVWM, mild cerebral and Ceb atrophy; Later, multiple infarcts PET (during follow-up)
Regional glucose Normal (before NA Normal Normal Bil. F, bil. Ceb,
metabolic deficit stroke-like right Th (before
episodes) stroke-like
episodes) and generalized areas at later stage NIDDM = non-insulin dependent diabetes mellitus; SNHL = sensorineural hearing loss; NA = not available; bil. = bilateral; PVWM = periventricular white matter; Ceb = cerebellum; Th = thalamus; F = frontal area; T-P-O = temporo-parieto-occipital area.
died at home from an unknown cause at the age of 64.
The serum lactate level had been high in pa-tient III-3 since the first diagnosis when he was about 4 years old. He presented with mental re-tardation, recurrent headache, nausea and vom-iting at age 6. Brain MRI at age 9 disclosed high signal lesions in bilateral periventricular regions and mild cerebral and cerebellar atrophy on T2-weighted images. PET showed reduced cerebral metabolic rate of glucose in bilateral frontal cor-tex and cerebellum, and in the right thalamus. At age 12, he was noted to develop severe headache, seizure and right hemianopsia. Brain MRI revealed cerebral infarcts in the left parietal and occipital regions. At age 16, a PET scan revealed generalized severe hypometabolism in the brain.
There were no ophthalmoplegia, heart con-duction block or abnormal findings of oph-thalmoscopy or echocardiography in all family members during the follow-up period except that audiometric measurement showed moder-ate to severe hearing loss in III-3. None of these
family members developed diabetes mellitus or hyperthyroidism.
Mitochondrial DNA mutation analysis
There was no significant difference in the propor-tions of A3243G mutated mtDNA among four serial muscle biopsies of the proband (Table 2). Quantitative PCR analysis showed that the propor-tion of mtDNA with the C3093G mutapropor-tion was approximately 51% in the muscle of the proband. The levels of C3093G mutant mtDNA in blood cells and hair follicles were 9% and 22% in the pro-band and 4% and 10% in the patient I-2, respec-tively (Table 3). However, the C3093G mutation was not found in the blood cells and hair follicles of the three sons of the proband. The C3093G mu-tation of mtDNA was not found in the blood cells or hair follicles of 23 normal healthy subjects.
The distribution of the A3243G and C3093G mutant mtDNAs in the blood cells and hair folli-cles of all maternal relatives and muscle biopsy of the proband are summarized in Table 3. The proportion of mtDNA with “+A3243G+C3093G”
A B
C D
Figure 2. Brain single photon emission computed tomography of the proband in the MELAS family: (A) decreased radioactivity in the left occipital area and
intense uptake in the right frontal region during the stroke-like episode; (B) diffuse hypoperfusion at the late stage. (C) Brain T2-weighted magnetic resonance
imaging shows multiple infarcts and diffuse brain atrophy. (D) Severe intestinal pseudo-obstruction on abdominal X-ray.
genotype of the proband was higher than those of the other three genotypes in muscle. In the mus-cle of the proband, numus-cleotide sequence analysis showed that the PCR-amplified mtDNA fragment harbored both point mutations, which is consis-tent with the findings of PCR-RFLP analysis.
Discussion
This family presented many features common to previously described MELAS pedigrees.5,12,13 Three members of the family (I-2, II-2, III-3) had the full-blown syndrome and the other
Table 2. Fluctuation of the proportions of the A3243G mutant mtDNA in various tissues of the MELAS family members*
Sample number† I-2 II-2 III-1 III-2 III-3
B H B H M B H B H B H 1 4.6a 33.2a 41.5a 52.8a 79.6a 51.5a 45.5a 45.5a 43.5a 65.8a,b 74.2a 2 4.7a 33.3a 20.2b 54.3a 71.4b,d 47.9a 44.7a 42.9a 43.9a 65.4a,b 77.1a 3 6.6a,b 31.6a 41.0a 51.8a 74.7a,c 47.0a 43.6a 44.7a 45.4a 67.3a 77.5a 4 10.5b,c 31.6a 35.3a 54.8a 69.3d 38.1b 43.3a 38.6b 45.1a 54.2b,c 74.1a 5 13.5c NA 41.4a NA 73.6b,c 49.9a NA 45.1a NA 50.5c NA
*Statistical difference between the data of each tissue of any two of the five samples was determined by non-parametric independent analysis: presence of the same superscripted letter indicates no significant difference (p> 0.05) between the two samples under comparison, and absence of similar super-scripted letters represents a significant difference (p< 0.05) between the two samples under pair-wise comparison; †sample numbers 1 to 5 indicate the
mtDNAs of blood cells, hair follicles and muscle biopsies of the five family members (I-2, II-2, III-1, III-2 and III-3) collected at different time points. B= blood; H = hair follicles; M = muscle; NA = not available.
Table 3. Proportions of the three mutant mtDNAs in different tissues of the five members of the MELAS family
Mitochondrial DNA mutation Tissues Proportion of mutant mtDNA
I-2 II-2 III-1 III-2 III-3
A3243G mutation Muscle NA 82.0 NA NA NA
Blood 8.0 35.0 49.0 49.0 72.0
Hair follicles 36.0 55.0 45.0 47.0 83.0
C3093G mutation Muscle NA 51.0 NA NA NA
Blood 4.0 9.0 ND ND ND
Hair follicles 10.0 22.0 ND ND ND
260 bp tandem duplication Muscle NA 38.0 NA NA NA
Blood 0.8 1.6 0.8 0.4 1.6 Hair follicles 0.4 0.8 0.8 0.8 0.4 +A3243G+C3093G Blood ND ND NA NA NA +A3243G−C3093G Blood 8.7 32.7 NA NA NA −A3243G+C3093G Blood 4.6 7.4 NA NA NA −A3243G−C3093G Blood 86.9 60.5 NA NA NA
+A3243G+C3093G Hair follicles ND ND NA NA NA
+A3243G−C3093G Hair follicles 35.1 47.8 NA NA NA
−A3243G+C3093G Hair follicles 9.7 24.8 NA NA NA
−A3243G−C3093G Hair follicles 55.2 27.4 NA NA NA
+A3243G+C3093G Muscle NA 41.8 NA NA NA
+A3243G−C3093G Muscle NA 32.2 NA NA NA
−A3243G+C3093G Muscle NA 10.2 NA NA NA
−A3243G−C3093G Muscle NA 15.8 NA NA NA
oligosymptomatic members (III-1, III-2) had rel-atively mild features of the syndrome. During the follow-up period, the proband developed pro-gressive dementia, generalized muscle atrophy, polyneuropathy, episodic subacute ileus, progres-sive renal failure and dilated cardiomyopathy with congestive heart failure. Patient III-3 developed re-current headache and repeated stroke-like episodes at the age of 12 and patient I-2 showed these symptoms at the age of 62. Although diagnostic criteria dictate that stroke-like episodes occur be-fore age 40 in MELAS, it usually occurs bebe-fore the age of 30.12To the best of our knowledge, late onset of the stroke-like episode at age 62, as seen in this patient, has never been reported before in patients with MELAS syndrome.
Apart from the brain and muscle, several ex-traneuromuscular organs were involved in the family examined in this study. Many symptoms of multisystemic involvement such as intestinal pseudo-obstruction, chronic nephropathy, dilated cardiomyopathy and sensorineural hearing im-pairment developed gradually with disease progres-sion. These findings are consistent with previous reports.14–18Mitochondrial encephalomyopathy is a multisystemic disorder mainly involving skele-tal muscle and the central nervous system, and may also involve cardiac muscle, kidney and gas-trointestinal tract.1,2,19Mitochondrial defect can affect virtually all organ systems.13 Our findings suggest that attention should be paid to the devel-opment of systemic manifestations and symptoms that can be alleviated by earlier treatment.
The endocrinologic abnormalities in the pro-band, including diabetes mellitus and hyper-thyroidism have been described previously.10,11 However, these symptoms were not seen in other family members. Although A3243G mutation of mtDNA has been associated with diabetes,9,19,20 hyperthyroidism is rarely seen in MELAS patients. Thyroid function test of the proband revealed sick euthyroid state at the later stage, which may be due to previous subtotal subthyroidectomy. Whether the hyperthyroidism observed in the proband is related to MELAS syndrome needs further investigation.
There are some reports of long-term studies of serial imaging in MELAS patients.21,22 In the proband, brain CT/MRI showed progression of infarcts. The infarction did not conform to defi-nite vascular territories and tended to fluctuate. Focal hyperperfusion on SPECT was observed dur-ing acute stroke and diffuse hypoperfusion durdur-ing follow-up. This is consistent with the findings of previous studies.22,23 EEG and brain SPECT did not reveal any abnormalities in asymptomatic car-riers before the development of stroke-like episodes during follow-up. MRI of the brain in patient III-3 before the development of stroke-like episodes showed hyperintense lesions on T2-weighted im-ages in bilateral periventricular regions and mild cerebral and cerebellar atrophy. These MRI find-ings are similar to those reported by previous studies.21,24 The initial PET finding was abnor-mal when subject III-3 was clinically asympto-matic, which suggests that PET is a sensitive tool to evaluate the cerebral metabolism in MELAS patients. Sano et al25also reported a patient with MELAS syndrome, in whom PET study demon-strated that the regional metabolic rate of oxygen was remarkably decreased, while regional meta-bolic rate of glucose was not decreased. We be-lieve that the regional glucose metabolic deficit is truly pathologic and significant. To determine whether these abnormalities are related to stroke-like episodes in MELAS, further study including measurement of oxygen consumption is needed. Disease progression in patients with mitochon-drial disorders is thought to be related to the pro-portion of mutant mtDNA. Larsson et al26found an increase with time of the mutated mtDNA in muscle in two cases and this increase paralleled the progression of the disease. Another study reported that a decline in the level of A3243G mutant mtDNA is associated with clinical and histo-chemical improvement.27The results obtained in this study (Table 2) do not show significant change in the proportion of mutated mtDNA in the mus-cle tissues of the proband during the progression of the disease. This suggests that this pathogenic mutation of mtDNA was distributed in a stable manner in affected tissues since the early stage
of embryonic development. In patient I-2, the proportion of A3243G mutated mtDNA in blood cells slightly increased during disease progres-sion. This finding is consistent with the report of a previous study.26
The results compiled in Table 3 show that the proportion of the A3243G mutant mtDNA of the blood cells and hair follicles of subjects III-1, III-2 and III-3 were very high or even higher than those of the proband. However, C3093G mutant mtDNA could not be detected in the blood cells and hair follicles of the three matrilineal members of this family. It is apparent that the C3093G mutant mtDNA was completely selected out in just one generation.
The mode of segregation and transmission of mtDNA with the C3093G mutation in this family suggest that the C3093G mutant mtDNA is segregated with tissue specificity and with rapid and extreme genetic shift in one generation of this family. The mechanisms of fluctuation or fixa-tion of the mutant mtDNA in the maternal line-ages remain unclear. These findings suggest that the C3093G mutation of mtDNA first occurred at low abundance in the proband’s mother in this family and that the proband inherited a higher proportion of the C3093G mutant mtDNA from her mother and widely spread in the muscle tissue and manifested more severe clinical symptoms. Although more extensive studies are required be-fore a solid conclusion can be made, our findings suggest that the C3093G mutant mtDNA had been more rapidly amplified in the affected tissues of the proband of this MELAS family and was then actively and completely selected out in her three sons. The origin of the triple mutations in the mtDNA of the proband in this family could not be traced. Whether the A3243G mutation predis-poses to the other two mutations in the mito-chondrial genome remains unclear. There are some previous reports of coexistence of two mutations in patients with mitochondrial diseases. Ohno et al28 found a MELAS and Kearns-Sayre syndrome co-mutation in a patient with myopathy and au-toimmune polyendocrinopathy. They suggested that the A3243G mutation probably predisposed
to the large-scale mtDNA deletion. Bidooki et al29 documented a patient with mitochondrial encep-halopathy who harbored two mutations at differ-ent tRNA genes in mtDNA. The contribution of the two mutations to the phenotypic expression was not determined. Arenas et al30also reported a double mutation in the tRNALysgene, which was associated with myoclonus epilepsy with ragged red fibers (MERRF) syndrome.
In conclusion, this family is rather interesting because the proband and her maternal relatives harbored several pathologic mutations in mtDNA at widely varied levels. Many pathologic mani-festations of the affected organs developed grad-ually during the follow-up study. The very late onset of stroke-like episodes in patient I-2 is ex-tremely rare for MELAS syndrome. The manifes-tations of MELAS syndrome in this family might be associated with the additional ∼260 bp tandem duplication in the D-loop region and the coexis-tence of C3093G mutation in the 16S rRNA gene of mtDNA.
Acknowledgments
This work was supported by grants NSC94-2320-B-010-052 and NSC94-2321-B-010-YC from the National Science Council and in part by the grant “Aim for the Top University Plan” from the Ministry of Education, Executive Yuan, Taiwan.
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