Teruhisa Miike

Unstable expansion of CAG repeat in hereditary dentatorubral–pallidoluysian atrophy (DRPLA)

Hereditary dentatorubral–pallidoluysian atrophy (DRPLA) is an autosomal dominant neurologic disorder characterized by variable combinations of myoclonus, epilepsy, cerebellar ataxia, choreoathetosis and dementia. By specifically searching published brain cDNA sequences for the presence of CAG repeats we identified unstable expansion of a CAG in a gene on chromosome 12 in all the 22 DRPLA patients examined. A good correlation between the size of the CAG repeat expansion and the ages of disease onset is found in this group. Patients with earlier onset tended to have a phenotype of progressive myoclonus epilepsy and larger expansions. We propose that the wide variety of clinical manifestations of DRPLA can now be explained by the variable unstable expansion of the CAG repeat.

Atrophy of the cerebellum and brainstem in dentatorubral pallidoluysian atrophy. Influence of CAG repeat size on MRI findings.

To elucidate how the size of the expanded CAG repeat of the gene for dentatorubral pallidoluysian atrophy (DRPLA) and other factors affect the atrophy of the brainstem and cerebellum, and the appearance of high-intensity signals on T2-weighted MRI of the cerebral white matter of patients with DRPLA, we quantitatively analyzed the MRI findings of 26 patients with DRPLA, the diagnosis of which was confirmed by molecular analysis of the DRPLA gene. When we classified the patients into two groups based on the size of the expanded CAG repeat of the DRPLA gene (group 1, number of CAG repeat units≥66; group 2, number of CAG repeat units ≤65), we found strong inverse correlations between the age at MRI and the areas of midsagittal structures of the cerebellum and brainstem in group 1 but not in group 2. Multiple regression analysis, however, revealed that both the patients age at MRI and the size of the expanded CAG repeat correlated with the areas of midsagittal structures. Involvement of the cerebral white matter as detected on T2-weighted images was observed more frequently in patients belonging to group 2 than in group 1 patients. Furthermore it was demonstrated that high-intensity signals can be detected on T2-weighted images of the cerebral white matter of patients with a largely expanded CAG repeat (group 1) in their thirties. These results suggest that patient age as well as the size of the expanded CAG repeat are related to the degree of atrophy of the brainstem and cerebellum, and the white matter changes in patients with DRPLA.

Immunohistochemical dystrophin reaction in synaptic regions

To investigate the functional significance of dystrophin, we studied various tissues of control and mdx mice, and rats immunohistochemically, using anti-dystrophin antibodies. In control animals, we observed the immunohistochemical localization of dystrophin in synaptic regions such as neuromuscular junctions, the cornea and outer plexiform layer of the retina, the taste buds and neurons in the brain, as well as on the surface membrane of skeletal, cardiac and smooth muscle fibers. In mdx mice, dystrophin was absent from the surface membrane of muscle fibers and the outer plexiform layer of the retina. These results suggest that dystrophin plays an important physiological and/or structural role in the conduction system.

Possible systemic smooth muscle layer dysfunction due to a deficiency of dystrophin in Duchenne muscular dystrophy

The localization of dystrophin was examined immunohistochemically in various tissues from mice and rats as well as from biopsied human muscle specimens, using polyclonal antibodies against dystrophin. Although dystrophin was completely absent in biopsied muscle specimens from 3 male DMD patients, it was faintly observed in the surface membrane of almost all muscle fibers examined in a female DMD patient. In all controls, human and animal, a strong dystrophin reaction was observed in the surface membrane of intrafusal muscle fibers and at the neuromuscular junctions, rather than in the surface membrane of skeletal and cardiac muscle fibers. In addition, dystrophin was clearly localized in the surface membrane of smooth muscle fibers in the viscera, bronchial system, ureter, and tunica media of blood vessels, including arteries and veins, in all examined animal tissues. In mdx mice, dystrophin was absent in almost all muscle and smooth muscle fibers in various tissues and blood vessels. These results suggested a possible systemic dysfunction of smooth muscle layers, especially those of blood vessels, as well as skeletal muscle fibers, due to a deficiency of dystrophin in Duchenne muscular dystrophy.

A school refusal case with biological rhythm disturbance and melatonin therapy

An 18-year-old male high school student with school refusal and circadian rhythm disturbance is reported. At 17 years of age, he was unable to attend school because of a reversal of the daily rhythm and a moderate depressive feeling. Other circadian rhythms, including deep body temperature (DBT), and plasma melatonin, cortisol and beta-endorphin, also showed quite different or abnormal curves compared with those in normal controls. He was treated with methyl B12 and melatonin, which normalized the circadian rhythm, i.e. it became entrained to a 24-h period, and the DBT and hormonal rhythms became closer to normal patterns. These results suggest that desynchronization of the biorhythms, particularly the circadian rhythm, may be one of the important causes of school refusal in Japan, and melatonin and methyl B12 might be useful for treatment of the condition.

Chronic fatigue syndrome in childhood

Chronic fatigue occurring in previously healthy children and adolescents is one of the most vexing problems encountered by pediatric practitioners. We report three cases, 11, 12 and 13-year-old children, with chronic fatigue syndrome (CFS). They initially developed a low grade fever and generalized fatigue, followed by sleep disturbance and psychosomatic symptoms, and their performance ability deteriorated. They were diagnosed as having CFS on the basis of criteria. To investigate the brain function in CFS patients, we examined the regional cerebral blood flow by single-photon emission-computed tomography (SPECT) with 111 MBq [123I]-iodoamphetamine (123I-IMP) or xenon-computed tomography (Xe-CT), and brain metabolic levels by MR spectroscopy (MRS). Blood flow, expressed as the corticocerebellar ratio (CCR), in the left temporal and occipital lobes was markedly lower in cases 2 and 3 than that in healthy subjects reported by another investigator. In case 1, however, blood flow in the left basal ganglia and thalamus was markedly higher than in healthy subjects. The MR spectroscopy (MRS) study revealed remarkable elevation of the choline/creatine ratio in the patients with CFS. None of our patients exhibited evidence of focal structural abnormalities on MRI. These findings suggest that the various clinical symptoms in CFS patients may be closely related to an abnormal brain function.

Dystrophin immunostaining and freeze-fracture studies of muscles of patients with early stage amyotrophic lateral sclerosis and Duchenne muscular dystrophy

We used polyclonal antibodies against dystrophin for the immunohistochemical localization of this protein in human skeletal muscle. Dystrophin was localized in the sarcolemma of the myofibers in 8 infantile and 11 adult normal control muscles and in 10 early stage patient muscles with amyotrophic lateral sclerosis (ALS). The protein was absent or markedly decreased in 8 early stage patients with Duchenne muscular dystrophy (DMD). Moreover the densities of sarcolemmal plasma membrane assemblies, orthogonal arrays and their pits were estimated by freeze-fracture electron microscopy studies in the same number of muscle samples in each disease and control case. The group median densities of orthogonal arrays and their pits in the ALS group and adult control group were 4.8 with a midrange of 1.1-13.5 (25-75%) and 7.5 with a midrange of 2.3-12.9, respectively (P greater than 0.1, Wilcoxon rank-sum test), whereas those of the DMD group and child control group were 0 with a midrange of 0-1.1 and 10.8 with a midrange of 5.4-16.7 respectively (P less than 0.01). The skeletal muscles of mdx mice and their controls were also investigated by the same techniques. In mdx mice, the absence or marked deficiency of dystrophin was also noted; however, the decrease of orthogonal arrays was not as severe as in DMD, which might relate to the milder clinical features in mdx mice as compared with those in DMD.

Immunohistochemical study of calpain and its endogenous inhibitor in the skeletal muscle of muscular dystrophy.

A calcium-dependent proteinase (calpain) has been suggested to play an important role in muscle degradation in Duchenne muscular dystrophy (DMD). In immunohistochemical studies, calpain and its endogenous inhibitor (calpastatin) were located exclusively in the cytoplasm in normal human muscles. The intensity of the staining was stronger in type 1 than in type 2 fibers. Quantitative immunohistochemical study showed an increase of calpain in biopsied muscles from the patients with DMD and Becker muscular dystrophy. Abnormal increases in calpain and calpastatin were demonstrated mainly in atrophic fibers, whereas necrotic fibers showed moderate or weak immunoreactions for the enzymes. Opaque fibers and hypertrophic fibers were negative. Not all dystrophin-deficient muscle fibers necessarily showed a strong reaction for calpain. We suggest that calpain may play an important role in muscle fiber degradation, especially in the early stage of muscle degradation in muscular dystrophy.

Learning and memorization impairment in childhood chronic fatigue syndrome manifesting as school phobia in Japan

For the last 15 years, we have tried to understand the pathophysiology of childhood chronic fatigue syndrome (CCFS) in Japan. In this condition, two major symptoms are important: easy fatigability and disturbed learning and memorization. In CCFS patients we clinically evaluated autonomic nervous system function, circadian rhythm of hormonal secretion (melatonin, cortisol and 3-endorphin), core body temperature, and sleep-wake pattern. Most patients showed autonomic nervous system dysfunction and circadian rhythm disturbances, similar to those observed in jet lag. Radiological imaging studies (SPECT, Xe-CT, and MRS) revealed decreased blood flow in the frontal and thalamic areas, and accumulation of choline in the frontal lobe. We analyzed the relationship between the laboratory data and clinical symptoms in CCFS.

Disturbed circadian core body temperature rhythm and sleep disturbance in school refusal children and adolescents.

We examined the circadian rhythm of core body temperature (CBT) in 22 school refusal patients, ages between 12 and 18 years, who did not have any physical or psychiatric disorders, but had indefinite complaints, and were suspected to have a circadian rhythm disturbance. To obtain normal data for analysis, CBT in 9 healthy age-matched school attendants who did not have any sleep, psychiatric, or medical disturbance were monitored. Circadian variation of CBT in school refusal patients did not present a clear rhythm, and appearance time of their lowest CBT was markedly delayed compared to healthy subjects. Amplitude of circadian CBT changes, fitted to a cosinor curve by the least square method, was significantly smaller in school refusals than in healthy subjects. These findings suggest that in school refusal patients who do not have physical and psychiatric disorders, clinical psychosomatic symptoms (e.g., fatigue and memory disturbance) and school refusal could be closely related to the desynchronization of their biorhythms, particularly the circadian rhythm of body temperature and sleep-wake rhythm.