Editors: Norio Fujiki, M.D. & Darryl R.J. Macer, Ph.D.
Ikuya Nonaka and Yu-ichi Goto
Departments of Laboratory Medicine and Child Neurology, National Center Hospital for Nervous, Mental and Muscular Disorders, NCNP, Kodaira, Tokyo 187, Japan
1. Mitochondrial (mt) DNA
A mitochondria contains several copies of its own DNA, which is circular, consisting of 16,569 base pairs. Different to the nuclear DNA, there are no introns but only exons. The mtDNA encodes 2 rRNA, 22 tRNA, and 13 subunits of enzymes including 3 subunits of cytochrome c oxidase and 7 of complex I (Fig. 1). All mitochondria in human beings originate from the mother, because the mitochondria-rich sperm tail is cleaved off at fertilization, and only the mitochondrial free head penetrates the egg, while mitochondria in the ova itself remain. Since the mtDNAs in our body are all from our mother, most mitochondrial diseases due to mtDNA mutations are inherited through a maternal trait. This maternal inheritance is quite a rare mode of transmission, and therefore little is known about its the importance and about the problems it raises for genetic counseling of the maternally inherited diseases.
2. MtDNA mutations and diseases
Mitochondrial diseases (encephalomyopathies) have been classified into three major forms based on the clinical characteristics, these are chronic progressive external ophthalmoplegia (CPEO), mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) and myoclonus epilepsy associated with ragged-red fibres (MERRF) (Table 1)(2). Recently it has become clear that each disease has disease specific mutations associated with it; large scale deletions in CPEO, and different respective point mutations in MELAS and MERRF. Here we will focus discussion of the former type, MELAS, the most frequently occurring children's mitochondrial disease.
3. Clinical and pathological findings in MELAS
In 1984, Pavlakis et al. (1) first described MELAS characterized by clinically by repeated "stroke-like" episodes mostly beginning in children from 5 to 15 years of age. The episodes include episodic headache and vomiting, convulsions, transient hemiplegia, hemianopsia, and occasional total cortical blindness. With repeated attacks, patients developed progressive mental retardation, paralysis, emaciation, infection, and cardiac insufficiency leading to death.
On brain CT and MRI, there are focal ischemic lesions, predominantly in the occipital lobe(s) which accounts for the frequent ocular symptoms of hemianopsia and cortical blindness. The ischemic lesions are not necessarily identical to the regions supplied by major arteries. Unlike infarctions due to thrombosis seen in adults, the ischemic changes are usually transient with rapid recovery. As we might suppose from the above clinical findings, blood vessel abnormality is the major pathogenetic mechanism in MELAS (3, 4). Electron microscopic examination of autopsied brain and biopsied muscle specimens, reveals that abnormal mitochondria are accumulated in the smooth muscle cells of the arteries (Fig. 2). Accordingly, there is systemic blood vessel involvement (angiopathy) in MELAS which probably results in the ischemic changes in the central nervous system.
Figure 1:Human mitochondrial DNA and disease specific mutations.
Figure 2: Note an increased number of enlarged mitochondria in smooth muscle cells (SM) in an intra-muscular arteriole from a patient with MELAS. The endothelial cells (E) are less affected. Lumen (L). X6,000.
Genetic analyses of MELAS families have shown evidence of maternal inheritance. Goto et al. sequenced the mtDNA of a family with this maternal inheritance and found an A-to-G substitution in the tRNA-Leu (UUR) coding region at the nucleotide pair (np) 3243 (3243 mutation)(5). Since this 3243 mutation was seen in 80% of MELAS patients, but in none of the normal controls, it is now regarded as a disease specific one. Furthermore, this mutation is also detectable in DNA extracted from blood, which is helpful in confirming clinically a diagnosis of MELAS.
Goto et al. further analyzed the mtDNA sequences of the remaining 20% of patients and found a T-to-C mutation in the same tRNA coding region at np 3271 (3271 mutation) in 10% of patients (6). Therefore approximately 90% of patients with MELAS had either a 3243 or 3271 mutation, and the remaining 10% had unidentified mutations.
5. The 3243 mutation produces heterogeneous symptoms
As described above, we analyzed the mtDNA mutations of patients with typical MELAS symptoms and found 80% of them had the 3243 mutation. On the other hand, when we examined members of the families of MELAS patients, they had a wide variety of symptoms from asymptomatic through mildly symptomatic patients easily fatigued and having short stature, to typical MELAS patients with stroke-like episodes. Recently, the 3243 mutation has been described as present in familial diabetes mellitus and in patients with idiopathic cardiomyopathy.
6. Why does the 3243 mutation produce heterogeneous symptoms?
As discussed above, patients with the 3243 mutation show heterogeneous symptoms including diabetes mellitus, muscle weakness and fatigue, and central nervous system symptoms of stroke-like episodes, mental retardation and psychiatric problems. This heterogeneity can be explained by the presence of normal and mutant mitochondrial genomes coexisting in a heteroplasmic state, but differing in their relative populations from tissue to tissue and from individual to individual even in the same family. This heteroplasmic distribution of normal and mutant mtDNA is seen not only in MELAS but also in all other mitochondrial diseases, including CPEO and MERRF, with the exception of a portion of patients with Leber hereditary optic neuropathy.
When the mutant mtDNA increases over a certain threshold in a given cell, all mitochondria in the cell lose their function. In an in vitro study, those cultured cells containing the 3243 mutation in over 94% of the total mtDNA lose all mitochondrial function (7), suggesting that the threshold level is about 90% for the 3243 mutation.
We and Dr. Ohama et al. have proposed that the pathogenetic mechanism for the stroke-like episode is angiopathy, from the morphologic finding of an increased number of mitochondria in the arteriolar walls. The abnormal blood vessels were proven to contain an increased amount of mutant mtDNA by an in situ hybridization study and by the PCR amplification method after their direct dissection.
7. Is there any relationship between the amount of mutant mtDNA and disease severity?
If patients with a larger amount of mtDNA in blood or muscle samples show severer clinical symptoms, we can predict the prognosis of the disease from the amount of mutant mtDNA. All muscle biopsies from patients with the typical MELAS symptoms contained at least 50% of mtDNA with the 3243 mutation, but over half the patients who had high populations of mutant mtDNA did not show typical symptoms, i.e. there was no close relationship between the amount of mutant mtDNA in muscles and clinical phenotypes (8). In some autopsied patients, the relative populations of wild and mutant mtDNA differed significantly from organ to organ. Since the major symptoms in MELAS are related to the central nervous system, it's quite understandable that the prognosis and disease severity can not be predicted from the amount of mutant mtDNA in the peripheral tissues such as blood and skeletal muscle.
An important fact seen in all mitochondrial diseases is that the population of wild and mutant mtDNA in blood differs from that in muscle. In some instances, mutated mtDNA is detectable by muscle biopsy but not in blood samples. Accordingly it's hard to determine whether patients or their family members have mutant mtDNA from blood samples. Although DNA analysis on muscle sample is more reliable than blood, it is impossible to decide whether a family member is a carrier of the disease even by this method.
The discovery of mtDNA mutations in various mitochondrial diseases is now aiding the performance and confirmation of clinical diagnoses. However, it is not necessarily helpful in predicting the prognosis, or in genetic counseling, for the following reasons:
a) Although all patients with the 3243 mutation have more than 50% of mutant mtDNA in their muscle, patients with the same population of mutant DNA are not necessarily symptomatic. The prognosis of the disease cannot be predicted from the amount of mutant mtDNA in blood sample.
b) The disease becomes worse with generation in most of families, though there are some exceptional instances. A mother with the mutation sometimes has a healthy child who has no DNA mutation in their blood.
c) The population of wild and mutant mtDNA in blood differs from that in muscle. A negative result does not necessarily mean no mutation is present in other organs. Therefore it's hard to determine carrier status from analysis of blood and muscle samples.
d) The population of wild and mutant mtDNA changes with time and progression of the disease. Prenatal or early diagnosis has no meanings for prediction of the onset or prognosis of the disease.