De novo EEF1A2 mutations in patients with characteristic facial features, intellectual disability, autistic behaviors and epilepsy.

Eukaryotic elongation factor 1, alpha-2 (eEF1A2) protein is involved in protein synthesis, suppression of apoptosis, and regulation of actin function and cytoskeletal structure. EEF1A2 gene is highly expressed in the central nervous system and Eef1a2 knockout mice show the neuronal degeneration. Until now, only one missense mutation (c.208G > A, p.Gly70Ser) in EEF1A2 has been reported in two independent patients with neurological disease. In this report, we described two patients with de novo mutations (c.754G > C, p.Asp252His and c.364G > A, p.Glu122Lys) in EEF1A2 found by whole-exome sequencing. Common clinical features are shared by all four individuals: severe intellectual disability, autistic behavior, absent speech, neonatal hypotonia, epilepsy and progressive microcephaly. Furthermore, the two patients share the similar characteristic facial features including a depressed nasal bridge, tented upper lip, everted lower lip and downturned corners of the mouth. These data strongly indicate that a new recognizable disorder is caused by EEF1A2 mutations.

Clinico-radiological features of subarachnoid hyperintensity on diffusion-weighted images in patients with meningitis.

AIM: To investigate the clinical and radiological features of meningitis with subarachnoid diffusion-weighted imaging (DWI) hyperintensity. MATERIALS AND METHODS: The clinical features, laboratory data, and radiological findings, including the number and distribution of subarachnoid DWI hyperintense lesions and other radiological abnormalities, of 18 patients seen at five institutions were evaluated. RESULTS: The patients consisted of eight males and 10 females, whose ages ranged from 4 months to 82 years (median 65 years). Causative organisms were bacteria in 15 patients, including Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus agalactiae, Staphylococcus aureus, Klebsiella pneumoniae, and Listeria monocytogenes. The remaining three were fungal meningitis caused by Cryptococcus neoformans. Subarachnoid DWI hyperintense lesions were multiple in 16 of the 18 cases (89%) and predominantly distributed around the frontal lobe in 16 of the 18 cases (89%). In addition to subarachnoid abnormality, subdural empyema, cerebral infarction, and intraventricular empyema were found in 50, 39, and 39%, respectively. Compared with paediatric patients, adult patients with bacterial meningitis tended to have poor prognoses (7/10 versus 1/5; p = 0.1). CONCLUSION: Both bacterial and fungal meningitis could cause subarachnoid hyperintensity on DWI, predominantly around the frontal lobe. This finding is often associated with poor prognosis in adult bacterial meningitis.

Postpuncture CSF leakage: a potential pitfall of radionuclide cisternography.

OBJECTIVE: We sought to evaluate radioisotope cisternography (RICG)-related postpuncture CSF leakage by MRI. METHODS: We conducted a prospective 3-day imaging study. Ten patients with orthostatic headache and other symptoms underwent pre-RICG brain and spinal MRI, magnetic resonance myelography (MRM), RICG, and post-RICG spinal MRI and MRM. For RICG, we used a 25-gauge pencil point spinal needle at the L3/4 or L4/5 level after which subjects took bed rest for 2.5 hours. RESULTS: On pre-RICG MRI and MRM, none of the 10 patients showed CSF leakage. However, 5 subjects (50%) showed epidural abnormalities suggesting CSF leakage on MRI after lumbar puncture for RICG. On RICG and subsequent MRM, 4 of the subjects showed definite findings of CSF leakage and 1 showed minimal leakage. CONCLUSIONS: RICG carries a risk of iatrogenic CSF leakage even with careful puncturing using a fine needle. This leakage produces abnormal RICG and MRM findings at the lumbosacral level. Therefore, abnormal RICG findings restricted to the lumbosacral level should be carefully interpreted when diagnosing SIH.

Expansion of the first PolyA tract of ARX causes infantile spasms and status dystonicus.

BACKGROUND: ARX is a paired-type homeobox gene located on the X chromosome that contains five exons with four polyalanine (PolyA) tracts, a homeodomain, and a conserved C-terminal aristaless domain. Studies in humans have demonstrated remarkable pleiotropy: malformation phenotypes are associated with protein truncation mutations and missense mutations in the homeobox; nonmalformation phenotypes, including X-linked infantile spasms (ISS), are associated with missense mutations outside of the homeobox and expansion of the PolyA tracts. OBJECTIVE: To investigate the role of ARX, we performed mutation analysis in 115 boys with cryptogenic ISS. This included two pairs of brothers. RESULTS: We found an expansion of the trinucleotide repeat that codes for the first PolyA tract from 10 to 17 GCG repeats (c.333_334ins[GCG]7) in six boys (5.2%) ages 2 to 14, from four families, including the two pairs of brothers. In addition to ISS, all six boys had severe mental retardation and generalized dystonia that appeared around the age of 6 months and worsened, eventually leading to stable severe quadriplegic dyskinesia within age 2 years. Three children experienced recurrent, life-threatening status dystonicus. In four children brain MRI showed multiple small foci of abnormal cavitation on T1 and increased signal intensity on T2 in the putamina, possibly reflecting progressive multifocal loss of tissue. CONCLUSION: The phenotype of infantile spasms with severe dyskinetic quadriparesis increases the number of human disorders that result from the pathologic expansion of single alanine repeats. ARX gene testing should be considered in boys with infantile spasms and dyskinetic cerebral palsy in the absence of a consistent perinatal history.

Are animal models useful for understanding the pathophysiology of lysosomal storage disease?

UNLABELLED: Spontaneously occurring genetic lysosomal storage diseases are as rare in other mammalian species as in man. However, the advent of gene targeting technology has revolutionized the state of animal models of genetic diseases. Nearly all lysosomal storage diseases known in man have been duplicated in the mouse. The technology now allows, not only complete inactivation of endogenous genes, but also the introduction of essentially any type of mutation. These animal models can overcome many of the limitations inherent in studies of human patients--rarity of the disease, extremely complex genetic background and logistical and ethical constraints in the design and execution of experiments with human subjects. For example, genetic manipulations of germ cells or cross-breeding experiments between two mutants are readily feasible with animal models. Two major areas of the utility of animal models are the clarification of the pathophysiology/pathogenetic mechanism of disease and the exploration of therapeutic approaches. Examples of experiments using animal models of lysosomal storage disease are presented, primarily from studies undertaken in our own laboratory. CONCLUSION: Animal models have proved invaluable in extending our knowledge of the lysosomal storage diseases and exploring potential therapies.

FDG-PET findings in sclerosing hemangioma of the lung: a case report.

We report a case of sclerosing hemangioma of the lung that showed an intermediately increased accumulation of 18F-fluorodeoxyglucose (FDG) on positron emission tomography (PET). We suggest that FDG-PET may be useful for considering a lesion as benign or low-grade malignant.

Psychosine is as potent an inducer of cell death as C6-ceramide in cultured fibroblasts and in MOCH-1 cells.

Cytotoxic capacity of psychosine (galactosylsphingosine) was evaluated in comparison with C6-ceramide in cultured fibroblasts and the glia-derived MOCH-1 cells that have characteristics of myelinating cells (1). Psychosine caused cytotoxic cell death and DNA fragmentation at concentrations similar to C6-ceramide and MOCH-1 cells were substantially more sensitive to their cytotoxic effects than fibroblasts. In this system, pretreatment with GM1-ganglioside failed to protect the cells from the deleterious effects of these compounds. These findings are consistent with the hypothesis that psychosine is the cytotoxic metabolite that causes apoptotic death of the oligodendrocyte in globoid cell leukodystrophy (Krabbe disease). They further suggest that the protective capacity of GM1-ganglioside is unlikely to be the explanation for the paradoxical improvement of the phenotype of globoid cell leukodystrophy in the mouse simultaneously deficient in two lysosomal beta-galactosidases, galactosylceramidase and GM1-ganglioside beta-galactosidase.

A mutation in the saposin A domain of the sphingolipid activator protein (prosaposin) gene results in a late-onset, chronic form of globoid cell leukodystrophy in the mouse.

Sphingolipid activator proteins (saposins A, B, C and D) are small homologous glycoproteins derived from a common precursor protein (prosaposin) encoded by a single gene. They are required for in vivo degradation of sphingolipids with short carbohydrate chains. Six cysteines and one glycosylation site are strictly conserved in all four saposins. Total deficiency of all saposins and specific deficiency of saposin B or C are known among human patients. A mouse model of total saposin deficiency closely mimics the human disease. However, no specific saposin A or D deficiency is known. We introduced an amino acid substitution (C106F) into the saposin A domain by the Cre/loxP system which eliminated one of the three conserved disulfide bonds. Saposin A(-/-) mice developed slowly progressive hind leg paralysis with clinical onset at approximately 2.5 months and survival up to 5 months. Tremors and shaking, prominent in other myelin mutants, were not obvious until the terminal stage. Pathology and analytical biochemistry were qualitatively identical to, but generally much milder than, that seen in the typical infantile globoid cell leukodystrophy (GLD) in man (Krabbe disease) and in several other mammalian species, due to genetic deficiency of lysosomal galactosylceramidase (GALC) (EC 3.2.1.46). Thus, saposin A is indispensable for in vivo degradation of galactosylceramide by GALC. It should now be recognized that, in addition to GALC deficiency, genetic saposin A deficiency could also cause chronic GLD. Genetic saposin A deficiency might be anticipated among human patients with undiagnosed late-onset chronic leukodystrophy without GALC deficiency.

Simple and low-cost tele-nuclear medicine conference system with the e-mail protocol.

PURPOSE: Because of the recent innovative growth in computer technology, digital imaging, and the Internet, we can take advantage of these facilities for education and clinical work in nuclear medicine. We developed a tele-nuclear medicine conference system with electronic mail (e-mail) on the Internet. METHODS: Twenty-one physicians (20 radiologists, 1 neurologist), 6 technologists and 2 medical students in six university hospitals (Japan 5, Canada 1), 5 local hospitals in Japan participated in this project. We used digital still cameras (330 k pixels) equipped with a floppy disk drive and 10 x optical zoom to digitize images with JPEG compression (640 x 480 matrix). The images were attached to e-mail messages (containing a brief description of each case). The mail was sent simultaneously to all members on the mailing list. Scintigram and SPECT images as well as other radiological images were sent by e-mail. Reply mails about each case were sent to all members via the mailing list. RESULTS: During a period of 6 months, 18 cases (tumor/infection: 7, bone: 6, cardiovascular: 1, neurology; 3, endocrine: 1) with 144 e-mails (average 5.6/case) were submitted to the conference. The average period of discussion was 15.6 days. The number of attached images was 1 to 9 (average, 4.2/e-mails). JPEG compression rate was 1/10 to 1/20. The quality of the images was good enough for discussion. Some cases required additional images for further discussion. CONCLUSION: Our tele-nuclear medicine conference with an electronic mailing list and digital camera was simple and low-cost. The conference system was useful for education and clinical work.

Paradoxical influence of acid beta-galactosidase gene dosage on phenotype of the twitcher mouse (genetic galactosylceramidase deficiency).

We have cross-bred twitcher mice (galactosylceramidase deficiency) and acid beta-galactosidase knockout mice (G(M1) gangliosidosis) and found that the acid beta-galactosidase gene dosage exerts an unexpected and paradoxical influence on the twitcher phenotype. Twitcher mice with an additional complete deficiency of acid beta-galactosidase have the mildest phenotype with the longest lifespan and nearly rescued CNS pathology. In contrast, twitcher mice with a single functional acid beta-galactosidase gene have the most severe disease with the shortest lifespan, despite the fact that G(M1) gangliosidosis carrier mice with an otherwise normal genetic background are phenotypically normal. A significant proportion of these galc(-/-), bgal(+/-) mice clinically develop additional extreme hyper-reactivity and generalized seizures not seen in any other genotypes. Consistent with the clinical seizures, widespread neuronal degeneration is present in the galc(-/-), bgal(+/-) mice, most prominently in the CA3 region of the hippocampus. The double knockout mice show a massive accumulation of lactosylceramide in all tissues. The brain inexplicably contains only a half-normal amount of galactosylceramide, which may account for the mild clinical and pathological phenotype. On the other hand, brain psychosine level is increased in all twitcher mice, but galc(-/-), bgal(+/-) mice show a significantly higher level than other genotypes. The reduced galactosylceramide in the brain of the double knockout mice and the significantly higher psychosine in the brain of the galc(-/-), bgal(+/-) mice cannot readily be explained from the genotypes of these mice. These observations are contrary to the expected outcome of Mendelian autosomal recessive single gene disorders and may also be interpreted as that the acid beta-galactosidase gene functions as a modifier gene for the phenotypic expression of genetic galactosylceramidase deficiency.

Biochemistry and neuropathology of mice doubly deficient in synthesis and degradation of galactosylceramide.

We have generated mice doubly deficient in both synthesis and degradation of galactosylceramide by cross-breeding twitcher mice and galactosylceramide synthase (UDP-galactose:ceramide galactosyltransferase, CGT) knockout mice. The prediction that the phenotype of the doubly deficient mice should be the same as the cgt -/- mice, since the degrading enzyme should not be necessary if the substrate is not synthesized, proved to be only partially correct. In early stages of the disease, the doubly deficient mice (galc -/-, cgt -/-) were essentially indistinguishable from the cgt -/- mice. However, the doubly deficient mice had a much shorter life span than cgt -/- mice. Both galactosylceramide and galactosylsphingosine (psychosine), were undetectable in the brain of the cgt -/- and the doubly deficient mice. The characteristic twitcher pathology was never seen in the galc -/-, cgt -/- mice. However, after 43 days, neuronal pathology was observed in the brainstem and spinal cord. This late neuronal pathology has not been seen in the CGT knockout mice but has been described in some long surviving bone marrow-transplanted twitcher mice. Furthermore, the motor segment of the trigeminal nerve of the galc -/-, cgt -/- mice showed severe degeneration not seen in either twitcher or CGT knockout mice. Thus, the galc -/-, cgt -/- mice, while primarily showing the cgt -/- phenotype as predicted, develop late pathology that is seen only in twitcher mouse and also a unique pathology in the trigeminal nerve. These observations indicate that the functional relationship between galactosylceramidase and galactosylceramide synthase is complex.

Twitcher mice with only a single active galactosylceramide synthase gene exhibit clearly detectable but therapeutically minor phenotypic improvements.

Cross-breeding of mouse mutants, each defective in either synthesis (CGT knockout) or degradation (twitcher) of galactosylceramide, generates hybrids with a genotype of galc -/-, cgt +/-, in addition to doubly deficient mice. They are ideally suited to test the potential usefulness of limiting synthesis of the substrate as a treatment of genetic disorders due to degradative enzyme defects. The rate of accretion of galactosylceramide in the brain of CGT knockout carrier mice (cgt +/-) is approximately two-thirds of the normal, suggesting a gene-level compensation for the reduced gene dosage. Phenotype of twitcher mice with a single dose of normal cgt gene was indeed milder with statistical significance, albeit only slightly. Compared among 10 paired littermates, the difference in the life span was 7+/-3.9 days (S.D.) and the difference in the maximum attained body weight was 1.9+/-1.2 g (S.D.). Neuropathologists were able to distinguish blindly galc -/-, cgt +/- mice from galc -/-, cgt +/+ mice. The brain psychosine level in galc -/-, cgt +/- mice was also approximately two-thirds of the galc -/-, cgt +/+ mice. These observations indicate that reduction of galactosylceramide synthesis to two-thirds of the normal level results in minor but clearly detectable phenotypic improvements. Because of the detrimental consequences of drastic reduction in galactosylceramide synthesis that may be required for pragmatically meaningful improvements, this approach by itself is unlikely to be useful as the sole treatment but may be helpful as a supplement to other therapies.

Ceramide accumulation is associated with increased apoptotic cell death in cultured fibroblasts of sphingolipid activator protein-deficient mouse but not in fibroblasts of patients with Farber disease.

Ceramide is recognized as an intracellular mediator of cell growth, differentiation and apoptosis. Tumour necrosis factor, anti-fas antibody, radiation and anticancer drugs such as actinomycin D are known to induce apoptosis in several cell types through generation of ceramide by activation of the sphingomyelinase pathway or ceramide synthetase. In this study, we examined the occurrence of apoptosis in fibroblasts from patients with Farber disease and from sphingolipid activator protein-deficient (sap -/-) mouse. These cells accumulate ceramide as the result of genetic deficiency of acid ceramidase and the ceramidase activator (sap-D), respectively. Amounts of ceramide in fibroblasts from Farber patients and in fibroblasts from sap -/- mouse were increased 2.9-fold and 2.8-fold, respectively, over the level of controls. Despite the similar degree of ceramide accumulation, cells exhibiting apoptotic features were increased only in fibroblasts from the sap -/- mouse but not those from the Farber patients. Thymidine uptake of Farber fibroblasts was normal while that of sap -/- mouse fibroblasts was twice normal, consistent with the apparently normal growth and the different rates of apoptotic cell death in these two cell lines. These data suggest that intralysosomal accumulation of ceramide due to defective acid ceramidase or its activator may not play an important role as a mediator of apoptosis. The increased apoptosis in the cultured fibroblasts from the sap -/- mouse may be caused by mechanisms other than the ceramide accumulation. Although more frequent than normal, significant apoptotic cell death was not observed in sap -/- mouse brain in vivo.

[A case of Sanfilippo syndrome type C: long-term clinical course and treatment].

We reported a Japanese girl with the Sanfilippo syndrome type C. She was born to healthy parents married consanguineously. She began to deteriorate and became disoriented at the age of 6 year and 8 month. She also developed sleep problems and dysphagia. Physical examination revealed short stature, slightly coarse facial features, contracture of the PIP joints and hypertrophy of the tonsils. There was neither hepatomegaly nor corneal clouding. Laboratory examination demonstrated an increase in urinary excretion of glycosaminoglycan. Electrophoresis of the urinary glycosaminoglycans indicated that heparan sulfate was the predominant component. Enzymatic assay using her skin fibroblasts demonstrated a complete deficiency of acetyl-CoA: a-glucosaminide N-acetyltransferase activity. Low dose erythromycin alleviated hypertrophy of her tonsils, thereby improving dysphagia.

[Automatic calculation of left ventricular volume and ejection fraction from gated myocardial perfusion SPECT--basic evaluation using phantom].

We evaluated accuracy of Quantitative Gated SPECT Program that enabled calculation of the left ventricular (LV) volume and ejection fraction by automatically tracing the contour of the cardiac surface. Cardiac phantoms filled with 99mTc-solution were used. Data acquisition was made by 180-degree projection in L type and 360-degree projection in opposed type. Automatic calculation could be done in all processes, which required 3-4 minutes. Reproducibility was sufficient. The adequate cut off value of a prefilter was 0.45. At this value LV volume was 93% of the actual volume in L type acquisition and 95.9% in opposed type acquisition. The LV volume obtained in L type was smaller than that obtained in opposed type (p < 0.05). The tracing of the defects was fair, on the cardiac phantoms with all of 90-degree defects and 180-degree defects of the septal and lateral wall. The LV volume was estimated to be larger on the phantom with 180-degree defect of the anterior wall, and to be smaller on the phantom of 180-degree defect of the inferoposterior wall. Because tracing was deviated anteriorly at the defects. In the patients with similar conditions to 180-degree defect of the anterior wall or inferoposterior wall, the LV volume should be carefully evaluated.

Evidence for active acetylcholine metabolism in human amniotic epithelial cells: applicable to intracerebral allografting for neurologic disease.

Human amniotic epithelial (HAE) cells have been used for allotransplantation in patients with lysosomal storage disease due to lack of expression of HLA antigens. Previously, we have reported the expression of differentiation markers for both neural stem cells, and neuron and glial cells. In the present study, we investigated the presence of choline acetyltransferase (ChAT) and acetylcholine (ACh) in HAE cells using different experimental approaches. Cultured HAE cells showed strong immunoreactivity against ChAT antibody. ChAT activity in primary cells was 24.9 +/- 8.5 pmol/mg protein/h. Using HPLC with electrochemical detection, ACh was detected in both cell incubation media and cell pellets indicating that these cells synthesize and release ACh in a time-dependent manner. Additional confirmation of this hypothesis was gained from the data obtained from RT-PCR and Western blot analyses which revealed the expression of ChAT mRNA and ChAT protein, respectively, in HAE cells. Results of the present study suggest that HAE cells can possibly be applied for intracerebral allografting to treat neurologic diseases in which cholinergic neurons are damaged.

Characterization of human amniotic epithelial cells transformed with origin-defective SV40 T-antigen gene.

This paper describes characteristics of human amniotic epithelial cells (AEC) transfected with a gene of origin-defective simian virus (SV) 40 large T-antigen (pMTIOD). Normal AEC before transfection with pMTIOD exhibited only low proliferative potential under our culture conditions. On the other hand, AEC cells transfected with pMTIOD exhibited greater proliferative potentials. Flow cytometry and immunohistochemistry analyses showed that both the primary and the transfected AEC did not express appreciable levels of class II antigens. However, the expression of class I antigen of the transfected AEC cells was slightly increased. The cells obtained in this experiment have the ability to induce tumors in severely combined immunodeficiency mice. This finding suggests that established AEC line can be used as a tool to investigate possible expression of the desired gene in human AEC and the gene products, however, was not suitable as a gene carrier to the recipient. Further experiments will be required to establish AEC as a transgene carrier for somatic cell gene therapy.

Epileptic seizures and event-related potentials (P300) in childhood partial epilepsies.

To clarify the relationship between cognitive function and clinical seizures, auditory event-related potentials (P300) were examined in 72 patients (185 trials) with partial epilepsy. Twenty-six patients (67 trials) had idiopathic partial epilepsies (IPE), and 46 (118 trials) symptomatic or cryptogenic partial epilepsies (SPE). In this study, to rule out the effects of epileptogenesis and other factors, we only examined patients with partial epilepsies undergoing carbamazepine (CBZ) monotherapy at doses of less than 16 mg/kg/day. The results were: 1) the mean age-corrected P300 latency in the patients with SPE (394 +/- 38 msec) was significantly prolonged compared with that in the patients with IPE (378 +/- 28). 2) The prolongation of the P300 latency had no relationship to the seizure frequency, seizure type or seizure duration. 3) In both epileptic groups, there was no significant correlation between the seizure-free period and the age-corrected P300 latency. Our results suggest that the effect of clinical seizures on the cognitive function may be relatively little, and that the cognitive dysfunction in partial epilepsies may mainly originate from epileptogenesis or other factors.

Event-related potentials (P300) and EEG activity in childhood partial epilepsy.

To clarify the relationship between the cognitive function and EEG activity, auditory event-related potentials (P300) were examined in 72 patients with partial epilepsy. Twenty-six patients (67 trials) had idiopathic partial epilepsies (IPE), and 46 (118 trials) symptomatic or cryptogenic partial epilepsies (SPE). For this study, patients undergoing carbamazepine monotherapy with a dose of less than 16 mg/kg/day were selected to rule out the effects of anti-epileptic drugs. The results were as follows: (1) The P300 latency tended to be prolonged in association with the EEG slowing in both epileptic groups. (2) There was no clear relationship between the frequency of paroxysmal discharges and the P300 latency. (3) The P300 latency was slightly prolonged in the patients with temporal foci compared with that in ones with extra-temporal foci. (4) There was no significant relationship between the generalization of focal paroxysmal discharges and the P300 latency. These results suggested that the influence of EEG abnormalities (particularly paroxysmal discharges) on the P300 latency is relatively little, and the cognitive dysfunction in partial epilepsies mainly originates from other factors such as the epileptogenic lesion itself.