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The Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes (JSGIT) was established in July 1994 with the chief aim to improve the quality of therapy for type 1 diabetes in children, an entity far less common in Japan than in Europe. We proposed four initial research topics: (i) to determine the current status of medical care and glycemic control in Japanese children with type 1 diabetes mellitus; (ii) to standardize the measurement of hemoglobin A1c; (iii) to establish a registry of a large cohort of patients in order to enable prospective studies to improve the quality of therapy for children with type 1 diabetes in Japan; and (iv) to enable participants of the JSGIT to hold a workshop twice annually. We registered a total of 736 patients from 45 hospitals throughout Japan. Intervention via insulin treatment was instituted after 2 yr for those patients whose hemoglobin A1c level was more than 8.1%. The proportion of patients receiving multiple insulin injections increased after intervention; however, average hemoglobin A1c in females remained significantly higher than in males. We identified two forms of diabetes in Japanese children: a rapidly progressive form and a more slowly progressive form. There was a significantly higher prevalence of a family history of diabetes in first-degree relatives in the slowly progressive form. These preliminary findings are the result of the first collaborative study of childhood diabetes in Japan.

Background: The Beutler enzyme spot test is an effective assay for newborn mass screening of galactosemia, but it is qualitative and relies on visual interpretation. We describe a quantitative, instrumental modification of the assay. Methods: We modified the macroscopic visual Beutler enzyme spot test by adding extraction of blood components from filter paper, deproteinization with acetone-methanol, and quantification and recording by a fluorescent microplate reader and personal computer. All handling was performed in microplates. The measurement time was 90 min. Results: Fluorescence intensity (FI) of healthy controls correlated with hematocrit and galactose-1-phosphate uridyltransferase (GALT) activity. Patients with GALT deficiency were distinguished clearly from healthy subjects and heterozygous carriers by FI. FI decreased to 75% of the initial activity after storage at 25 °C for 3 days and to 40% after storage at 37 °C for 7 days. Screening of 46 742 newborns yielded 1 false-positive result (in a heterozygous carrier), 1 patient with glucose-6-phosphate dehydrogenase deficiency, and no apparent false negatives as judged by concurrent measurements of galactose and galactose-1-phosphate. Conclusions: The quantitative Beutler test can provide precise GALT activity in newborn mass screening, and can take into consideration the influence of high temperature and humidity, duration between sampling and testing, and anemia. This method is clinically useful, simple, automated, and highly reliable for newborn mass screening of galactosemia.

Galactokinase (GALK) deficiency is an autosomal recessive disorder, which causes cataract formation in children not maintained on a lactose-free diet. We characterized the human GALK gene by screening a Japanese genomic DNA phage library, and found that several nucleotides in the 5′-untranslated region and introns 1, 2, and 5 in our GALK genomic analysis differed from published data. A 20-bp tandem repeat was found in three places in intron 5, which were considered insertion sequences. We identified five novel mutations in seven unrelated Japanese patients with GALK deficiency. There were three missense mutations and two deletions. All three missense mutations (R256W, T344M, and G349S) occurred at CpG dinucleotides, and the T344M and G349S mutations occurred in the conserved region. The three missense mutations led to a drastic reduction in GALK activity when individual mutant cDNAs were expressed in a mammalian cell system. These findings indicated that these missense mutations caused GALK deficiency. The two deletions, of 410delG and 509–510delGT, occurred at the nucleotide repeats GGGGGG and GTGTGT, respectively, and resulted in in-frame nonsense codons at amino acids 163 and 201. These mutations arose by slipped strand mispairing. All five mutations occurred at hot spots in the CpG dinucleotide for missense mutations and in short direct repeats for deletions. These five mutations in Japanese have not yet been identified in Caucasians. We speculate that the origin of GALK mutations in Japanese is different from that in Caucasians.

Neopterin and biopterin concentrations were measured in cerebrospinal fluid (CSF) and urine samples from controls less than 1 year old. This is the first time for CSF reference data for controls less than 1 year old to be reported. The ratio of neopterin to biopterin in CSF 0-30 days (n = 48) of age in control samples was 0.65 +/- 0.31 (SD), which was far lower than that in urine over the same time period, 4.0 +/- 1.9 (SD), (n = 51). This finding is very important when diagnosing 6-pyruvoyltetrahydropterin synthase (PTPS) deficiency and peripheral form of PTPS deficiency in the neonatal period. Our CSF reference data for controls should be useful in the diagnosis of PTPS deficiency.

Two novel mutations of the beta-hexosaminidase alpha subunit gene were identified in Japanese patients with the infantile form of Tay-Sachs disease. One mutation was a one-base deletion at nt613C, which generated a stop codon at two codons downstream, in three unrelated patients. The other mutation was a one-base substitution of G-to-A at IVS 5, +1, which caused a splicing abnormality, in one patient. A missense mutation of R170W, which has already been reported in other ethnic groups, was also newly identified in one patient. In 1993, the most common mutation (IVS 5, -1G--T) in Japanese patients with Tay-Sachs disease was reported as the major mutation in Japan accounting for 80% of 56 mutant alleles from 28 unrelated patients. The deletion of nt613C was the second most common mutation, accounting for 5% of the mutant alleles. The previously reported mutation IVS 5, -1G--T and the nt613C deletion found in this study together accounted for 85% of the mutations causing Tay-Sachs disease among Japanese. Since these two mutations were located in or close to exon 6 and since they abolish Fok I (IVS 5, -1G--T) and Sfa NI (nt613C deletion) restriction sites, respectively, they were screened rapidly by single polymerase chain reaction followed by digestion with these enzymes.

Glycogen storage disease type Ib (GSD-Ib) is an inborn error of metabolism with autosomal recessive inheritance, caused by defects in microsomal transport of glucose-6-phosphate. Recently, Gerin et al isolated a human cDNA encoding a putative transporter homologous to bacterial transporters of hexose-6-phosphate, and identified two mutations in its gene in two patients with GSD-Ib (9). Independently, a linkage analysis mapped the GSD-Ib gene on chromosome 11q23 (10). It remains to be elucidated whether the two genes are identical or GSD-Ib is genetically heterogeneous. We first mapped the transporter gene on chromosome 11 by using a DNA panel of human/hamster hybridoma cells. The result suggested that the GSD-Ib genes identified by the two distinct approaches may be identical and GSD-Ib was allelic. We then studied four unrelated Japanese families with GSD-Ib, and found three novel mutations: a four-base deletion/two-base insertion, a point mutation within a consensus splicing donor site, and a missense mutation (W118R). The W118R mutation was found in 4 out of 8 mutant alleles, suggesting that it is prevalent among Japanese patients.

We determined the prevalence of antibodies to glutamic acid decarboxylase (anti-GAD) in Japanese diabetic patients. Anti-GAD were detected by RIP Anti-GAD Hoechst, which is a new sensitive radioimmunoassay (RIA) kit using purified pig brain GAD as the antigen. One thousand nine hundred Japanese patients were collected by the Study Group for Antibodies to GAD. The prevalence of anti-GAD in the subjects of this study was: 35.4% (326/921) in all patients with IDDM, 50.3% (96/191) in patients with IDDM less than 1-year duration, 4.3% (29/680) in NIDDM, 37.9% (39/103) in slowly progressive IDDM, 10.5% (4/38) in gestational diabetes mellitus, 0% (0/27) in impaired glucose tolerance, 4.8% (6/124) in the school children with glycosuria, 2.1% (1/47) in the relatives of IDDM and 5.0% (1/20) in neurological diseases without diabetes. The prevalence in normal subjects was 2.2% (7/323). Anti-GAD are frequently detected by the RIA kit in patients with IDDM of short duration and this assay may be useful for population screening for IDDM and for better understanding of its pathogenesis.

The influence of glycemic control on growth and on the development of complications in diabetic children was studied. The subjects of the study were 107 children with insulin-dependent diabetes mellitus (IDDM), who were enrolled in a Summer camp program for diabetic children in Kinki District, Japan from 1972 to 1990, and who had at least three determinations of HbA1 during the observation period. Many of the children had high mean levels of HbA1, regardless of age. The height and weight were below the standards for the respective ages in many children, indicating the retardation of growth. However, S.D. scores for height and weight and other physical indices were not related to the mean levels of HbA1. By contrast, the prevalence of diabetic retinopathy was related to an elevated mean level of HbA1, but that of albuminuria was not. Serum cholesterol levels were higher in children with higher mean levels of HbA1, but serum triglycerides appeared not to be related to glycemic control. The incidence of retinopathy during the observation period closely related to the degree of the mean levels of HbA1, but that of albuminuria did not.

Limb lengthening was performed on a patient with achondroplasia. The first operation was performed without growth hormone (GH) therapy, and the second operation was performed with GH therapy. The effect of GH on limb lengthening was studied. For the lengthening of limbs, the Orthofix device was used on the femur, and the Ilizarov method was used on the tibia. It took more time for the consolidation of the callus and severe narrowing of the femur was observed at the second limb lengthening. GH treatment was of no benefit for the consolidation of the callus in this patient.

1972年から1990年までの近畿小児糖尿病キャンプ参加者で, 3回以上HbA1値を測定した18歳未満のもの107名 (男児43名, 女児64名) について, コントロール状態と発育および合併症との関係について検討した。平均HbA1値は高値を示すものが多い, 年齢による差はみられなかった。発育状況は, 身長, 体重とも標準値を下回るものが多く, また身長, 体重のSD scoreも負領域で, 発育の遅延が示唆された。しかし, 平均HbA1値と身長, 体重のSD score, Kaup指数, Rohrer指数, PDWとの関係はみられなかった。糖尿病性網膜症の合併率は平均HbA1値が高くなるとともに有意に上昇した。しかし, タンパク尿はコントロールとは関係がなかった。一方, 血清コレステロ ール値は, 平均HbA1値が高くなるほど有意に上昇したが, 血清中性脂肪値との関係はみられなかった。観察期間中の新たな糖尿病性網膜症の発生率はコントロ一ル状態の悪い群ほど高く, その差は有意であった。

The mechanism of action of steroid hormones on skeletal growth is not understood in detail. We examined the interactions of steroid hormones and insulin-like growth factor-I (IGF-I) during DNA and sulfated proteoglycan synthesis in rabbit costal chondrocytes. Progesterone at 0.05 nM stimulated the incorporation of [3H]thymidine into DNA by 30% above the control level in confluent cultures, but neither testosterone nor 17 beta-estradiol stimulated DNA synthesis. None of the hormones affected [3H]thymidine incorporation stimulated by IGF-I when chondrocytes were incubated with one of the hormones and IGF-I simultaneously. In contrast, when confluent cultures were incubated with one of the sex steroids for 24 h before the addition of IGF-I, stimulation of DNA synthesis by the growth factor was enhanced about 45% above the control value by 0.5 nM progesterone, 50% by 0.5 nM testosterone, and 80% by 50 nM 17 beta-estradiol. The effects of IGF-I on proteoglycan synthesis, as judged by the incorporation of [35S]sulfate, were stimulated by treatment with progesterone or testosterone. Dexamethasone at physiological concentrations inhibited chondrocyte DNA synthesis in confluent cultures to 10% of the control level. At 50 nM, dexamethasone suppressed IGF-I induction of DNA synthesis by 60%. This suppression was greater when dexamethasone was added before IGF-I than when the additions were simultaneous. When chondrocytes were treated with hydrocortisone or dexamethasone for 24 h before the addition of IGF-I, the glucocorticoids synergistically accelerated proteoglycan synthesis mediated by IGF-I. These findings suggest that steroid hormones have priming effects on the biological action of IGF-I in cartilage metabolism.

There has been no consistent view about the cross-species reactivity of islet cell antibodies (ICA), and no report about their target antigens in the pancreatic islet cells of different species. Therefore, we examined the cross-reactivity of human ICA against rodent pancreatic islet cells, and found at least two types of ICA, one having a comparatively strong cross-reactivity and the other lacking it. Furthermore, using human as well as some rodent pancreatic tissues that had been modified chemically, we came to suspect that the target antigens of ICA were sialic acid residues of glycolipids. Therefore, we suggest that there are at least two types of ICA recognizing sialic acid residues of glycolipids, one reacting with antigen(s) commonly present in human and rat islets, and the other with antigen(s) only present in human islets.

We identified three mutations in four Japanese patients with central type 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency. One missense mutation was a C-to-T transition, resulting in the substitution of Pro by Ser at codon 87 (P87S) in exon 5. Another missense mutation was a G-to-A transition, resulting in the substitution of Asp by Asn at codon 96 (D96N) in exon 5. A splicing mutation was found by skipping of exon 4 on PTPS mRNA analysis, and a G-to-A transition at the third base of codon 81 (E81E) and at the terminal base in exon 4 were detected on genomic PTPS DNA analysis. The E81E mutation affected the splice donor site of exon 4 and caused the splicing error. In COS cell expression analysis, the P87S and D96N mutant constructs revealed, respectively, 52% and 10% of wild-type activity. Patients with P87S/P87S (52%/52% in-vitro PTPS activity) exhibited 0.11 and 0 microU/g hemoglobin [Hb] in erythrocyte PTPS activity (wild-type control: 11-29 microU/gHb) erythrocyte PTPS activity, and the patient with P87S/D96N mutations (52%/10%) had 0.97 microU/gHb in PTPS erythrocyte activity. The PTPS erythrocyte activity did not coincide with the in-vitro PTPS activity based on patient genotype.

It is not clear yet when pancreatic islet cells begin to express antigens for islet cell antibodies (ICA). Therefore, we studied whether human ICA-positive sera, crossreacting with adult rat islet cells, react with fetal and neonatal rat islet cells immunohistologically. The ICA did not react with fetal islet cells. On the other hand, neonatal islet cells over 2-3 weeks of age expressed the reactivities almost similar to those of the adult. It was suspected that pancreatic islet cells started to express their antigens to induce ICA gradually after birth.

We characterized phenylalanine hydroxylase (PAH) genotypes of Japanese patients with phenylketonuria (PKU) and hyperphenylalaninemia (HPA). PKU and HPA mutations in 41 Japanese patients were identified by denaturing gradient gel electrophoresis and direct sequencing, followed by restriction fragment length polymorphism analysis to find a large deletion involving exons 5 and 6. Of 82 mutant alleles, 76 (92%) were genotyped showing 21 mutations. The major mutations were R413P (30.5%), R243Q (7.3%), R241 C (7.3%), IVS4nt-1 (7.3%), T2781 (7.3%), E6nt-96A-->g (6.1%), Y356X (4.9%), R111X (3.7%), and 442-706delE5/6 (2.4%). Eight new mutations (L52 S, delS70, S70P, Y77X, IVS3nt-1, A132 V, W187 C, and C265Y) and a polymorphism of IVS10nt-14 were detected. In vitro PAH activities of mutant PAH cDNA constructs were determined by a COS cell expression system. Six mutations, viz., R408Q, L52 S, R241 C, S70P, V388 M, and R243Q, had 55%, 27%, 25%, 20%, 16% and 10% of the in vitro PAH activity of normal constructs, respectively. The mean pretreatment phenylalanine concentration (0.83+/-0.21 mmol/l) of patients carrying the R408Q, R241 C, or L52 S mutation and a null mutation was significantly lower (P

Four unrelated Japanese patients with infantile Sandhoff disease (beta-hexosaminidase beta-subunit deficiency) have been studied for the molecular basis of their severe phenotype. Two patients had complex base substitutions; one patient was homoallelic for a triple mutation (P417L, K121R, and S255R) and the other was a compound heterozygote of a double (P417L and K121R) mutation and the triple mutation. K121R is known to be a functional polymorphism, while P417L (exon 11, +8 C--T) generates predominantly an abnormally spliced mRNA at base +112 of exon 11 and has been described in two patients with a juvenile form of the disease. The mild phenotype is attributed to the presence of a small amount of normally spliced mRNA. S255R is a novel mutation without prior description in the literature. An expression study of the normally spliced cDNA with the double and the triple mutations gave about 70% and 30% of normal activity, respectively. This finding suggests that S255R further reduces the catalytic activity of the already below-threshold amount of normally spliced mRNA and accounts for the more severe phenotype in our patients. In the other two patients, a novel disease-causing base transition was found within intron 10, away from the intron/exon junction (-17 a--g). This mutation caused abnormal 3' splicing at position -37 of intron 10, and no normally spliced product was detectable upon RT-PCR analysis. We noted an unusually low splice site score (61.8) for the exon 10/intron 11 junction and suspected that this might be partially responsible for the aberrant splicing in these mutations. To test this hypothesis, we constructed four chimeric cDNAs all with an additional intron 10 inserted and evaluated their splicing efficiency. They, respectively, had the normal sequence, P417L (exon 11, +8 C--T), the intronic mutation (-17 a--g), and the intronic mutation with an artificially engineered intron 10/exon 11 junction of a higher splice site score (85.1). Of the total transcripts, 67% and 32% were correctly spliced in the normal chimeric construct and P417L, respectively, while no normally spliced product was generated either in the chimeric construct with -17 a--g or in that with a high splice site score. The sequence around the adenosine -17 residue upstream of the normal acceptor splice site in this report, UGCAAU (-21 to -16), matches the consensus branchpoint sequence YNYRAY (Y, pyrimidine; R, purine; N, any base) reported in the literature. The mutation in this study is most likely to abolish lariat formation because the artificial site of the high splice site score did not improve splicing efficiency.

An outbreak of Escherichia coli O157:H7 infection occurred in July 1996 in Sakai City. About 5000 children were infected, 122 of whom developed hemolytic uremic syndrome (HUS). In this outbreak, almost all patients were administrated some type of antibiotics. The effects of antibiotics on E. coli O157 associated hemorrhagic colitis (HC) have been controversial. In this study, we focused on the effects of antibiotics on development of HUS in the HUS in the Sakai outbreak. We retrospectively determined the antibiotics administrated within three days after the onset of HC, clinical courses, and laboratory data of 301 patients who were hospitalized and identified as Escherichia coli O157 infection by stool culture, from results of questionnaires sent by the Osaka Prefecture Medical Association to hospitals in Osaka Prefecture. The antibiotics used could be identified for 216 patients. The incidence of HUS among these patients was 11.6%. They were divided into 19 groups based on the type of antibiotics administrated. The incidence of HUS in the new quinolone (3.7%) group was low, but was high in the intravenous cephalosporin (18.2%) group. The differences in the incidence of HUS among the 19 antibiotic groups was significant (p < 0.05) on analysis of covariance which eliminated the contributions of variables including age, sex and laboratory data. These findings indicate that the suitable antibiotics can prevent the development of E. coli O157-associated HUS.

The present study was carried out to elucidate whether renal hemodynamic changes are associated with angiotensin converting enzyme (ACE) gene polymorphism in patients with insulin dependent diabetes mellitus (IDDM). We studied 32 Japanese patients with IDDM (aged 15 +/- 3 years in mean +/- SD) without renal failure or retinopathy. Renal hemodynamics were examined by duplex Doppler sonography and arterial resistance index was calculated. ACE genotypes were determined by polymerase chain reaction amplification. Resistance index (RI) of arcuate arteries in IDDM patients with DD genotype was significantly elevated, being 0.64 +/- 0.04, 0.66 +/- 0.05, and 0.71 +/- 0.05 for II, ID and DD genotype groups, respectively (II vs. DD, p0.02). In patients with DD genotype with normoalbuminuria (n = 27), it was also significantly elevated in DD genotype patients (II vs. DD, p0.02). In addition, multiple regression analysis with a forward elimination procedure showed that only the ACE genotype was associated with RI of arcuate arteries (R2 = 0.24, p0.01) among the parameters of sex, age, IDDM duration, body mass index, HbA1c, plasma glucose levels, serum levels of total cholesterol and creatinine, urinary albumin excretion index, mean blood pressure and ACE genotype. The present study demonstrated that renal arterial resistance is elevated in IDDM patients with DD genotype. ACE gene polymorphism which could be linked to intrarenal circulatory disturbance may be associated with the initiation and progression of diabetic nephropathy.

Succinyl-CoA:3-ketoacid CoA-transferase (SCOT; EC 2.8.3.5) deficiency (McKusick 245050) is a rare inherited metabolic disease. This enzyme is a key enzyme for utilization in peripheral tissues of ketone bodies that are produced by the liver. The absence of SCOT activity blocks peripheral ketone body utilization and causes recurrent attacks of severe ketoacidosis beginning in the neonatal or infantile period. Five cases of SCOT deficiency have been reported (Tildon and Cornblath 1972; Spence et al 1973; Middleton et al 1987; Saudubray et al 1987; Perez-Cerda et al 1992). The prognosis of these patients seems to parallel the severity of the SCOT deficiency. Here we report a new case of SCOT deficiency in Japan. We also report SCOT activity in the lymphocytes of the patient and his family.

We characterized two novel mutations of the galactose-1-phosphate uridyltransferase (GALT) gene intwo Japanese patients with GALT deficiency and identified N314D and R333W mutations, previouslyfound in Caucasians. One novel missense mutation was an G-to-A transition in exon 8, resulting in thesubstitution of arginine by histidine at the codon 231 (R231H). GALT activity of the R231H mutantconstruct was reduced to 15% of normal controls in a COS cell expression system. The other was asplicing mutation, an A-to-G transition at the 38th nucleotide in exon 3 (318AG), resulting in a38-bp deletion in the GALT cDNA by activating a cryptic splice acceptor site. In seven Japanesefamilies (14 alleles for classic form and one allele for Duarte variant) with GALT deficiency, the R231H and 318AG mutations were found only on both alleles of the proband. The N314D and R333W mutations were found on one allele each. The Q188R was prevalent in the United States butnot in Japanese patients. The N314D mutation was associated with the Duarte variant in Japanesepersons, as well as in the United States. We speculate that classic galactosemia mutations appear todiffer between Japanese and Caucasian patients. Our limited data set on galactosemia mutations in Japanese suggests that the N314D GALT mutation encoding the Duarte variant arose before Asianand Caucasian people diverged and that classic galactosemia mutations arose and/or accumulated afterthe divergence of Asian and Caucasian populations. © 1995 Wiley-Liss, Inc.

More girls with phenylketonuria (PKU) enter childbearing ages, and most such women are mentally normal, having been born since newborn screening was initiated in the 1970s and treated from early infancy with a low phenylalanine (Phe) diet. Women with PKU not treated prior to conception can have a pregnancy that results in serious fetal damage1. Maternal PKU as a cause of mental retardation and birth defects is a new phenomenon. There will be an increased need for specific therapies in maternal PKU. Low Phe diet is essential for the treatment of maternal PKU. It should be started before pregnancy and it is necessary to maintain their plasma Phe levels around 5 mg/dl throughout their pregnancy2. However they are usually controlled around 10 mg/dl because of the difficulty of the diet therapy. We made an animal model of maternal PKU by the intravenous injection of Phe to pregnant guinea-pigs, and examined plasma, liver and brain Phe levels in their fetuses after an intravenous administration of 6R-5,6,7,8-tetrahydrobiopterin (R-BH4) to the mothers.

Tetrahydrobiopterin (BH4) synthase deficiency has a high incidence of low birth weight,1 and some of them had a mild mental retardation in spite of their early treatment2. In this study we performed an intravenous loading of 2,4-diamino-6-hydroxypyrimidine (DAHP) with a small amount of BHU, and successfully made a model of fetal BH4 deficiency. We investigated the possibility of the fetal therapy of BH4 deficiency in this model by measurements of phenylalanine(Phe), tyrosine(Tyr), BH4, dopamine and catecholamines.

Tetrahydrobiopterin (BH4) has been used for the therapy of BH4-deficient patients. However, BH4 is poorly absorbed from the intestine. The plasma level of biopterin that was reached after oral administration of BH4 to a BH4-deficient patient was reported to be 1–2% of the levels reached after either intravenous or subcutaneous administration1. This poor absorption from the intestine needs a large amount of BH4 for the oral therapy, leading to high costs.

The frequency and distribution of eight mutations (R111X, IVS4nt-1, Y204C, R243Q, IVS7nt-2, W326X, Y356X, and R413P) in the phenylalanine hydroxylase gene of Orientals in Japan and Korea were examined by allele-specific oligonucleotide hybridization. The mutant alleles comprised 54 and 55% of the phenylketonuria (PKU) chromosomes examined in 36 patients in Japan and 10 patients in Korea, respectively. The spectrum of PKU mutations in Japan was similar to that in China, particularly in northern China, but different from that in Korea. The IVS4nt-1 mutation had a high frequency in Korea and southern China, due to the result of the founder effect and genetic drift. The R413P mutation, which may have originated in the regions surrounding the Baikal, expanded to northern China and Japan. We did not find Caucasian mutations in the Japanese or Korean PKU chromosomes. Thus, PKU mutations occurred after racial divergence between Caucasians and Mongoloids, and there were different founding populations for PKU in the two populations.

Purpose: Several noniiivasive imaging methods can detect epileptic ioci. We compared tnagnetic source imaging (MSI) of epilcptic spikes with the results of other imaging mcthoda, including positron emission tomography (PET), single photon emission CT (SPECT), and MRI, in order to determine characteristics of each. Methods: We studied 9 patients, age ranging from 1–23 years. Etiologies included acute encephalopathy, tubcrous sclerosis, pcrisylvian syndrome, and cerebral infarct in I patient each, unknown in the rest. Eight exhibited bca/ization-related epilepsy (LRE) and the remaining iut syndrome (LGS). Informed cnsent was obtained from the patients or their parents. Thc magnctocncephalograins (MEG) were measured in 21 magnetically shielded room using a layingtype whole-cortex MEG with 160-channel first-order gradiometers. We estimated the equivalent current dipoles (ECD) of spikes in the simultaneously recorded EEG. MRI scanning was performed at 5 m m thickness in 3 directions including the markers for superimposition with MEG. IXF-FDG PET was performed with a Headtome IV in 4 paticnts and 123Tc ECD SPECT with a Toshiba GCA9300 in 8 patients. We visually evaluated these images. Results: We could estimate the ECD in 8 patients as having I or multiple locations. All 4 patients examined by PET cxhibitcd 1 or more hypometabolic arcas on PET. SPECT studies revealed hypoperfusion areas in 3 patients m d hyperperfusion areas in 2 patients. One id the patients with hypcrpcrfusion was on ictal study. Structural ahnormalities were presented on MRI in 3 patients. Casc I had advcrsivc seizures with turning to the lcft and exhibited a broad right frontotemporal hypometabolic area on PET. Her ECDs wcrc cstimatcd to originate from the right prcmotor area, in agreement with a hypoperfusion spot on ictal SPECT. Case 2 had epilepsy as a residuum of past cnccphalopathy and had complcx partial seizures (CPS) with rapid generalim tion. The foci detected on MSI, PET, and SPECT did not agree. Casc3 had CPS and anterotemporal hypomctabolism on PET. ECDs were focused in the lateral mterotemporal area. Case 4 had tuberous xlc- rosis and a past history of status cpilcpticus. MRI revealed cortical tubers and white matter lesions. Cortical tubers exhibited hypoperlu- sion on SPECT. However, ECDs werc cstimatcd to originate from the right occipital hypoperfusion area and not thc cortical tubcrs. C was suspected to have idiopathic LRE and exhibited no focus on PET and SPECT. ECDs were estimated to originate from the left frontal cortex. Case 6 had pcrisylvian syndrome with LGS. Bilaterally abnormal cortex exhibited hyperperfusion on SPECT, and ECDs wcrc citimated to originate lrom this cortex. Casc 7 had CPS and left temporal sporadic spikes on previous EEGs. The findings of SPECT and PET studies were normal, and ECDs could not be estimated because there was no spike in the MEG study. Case 8 had right leg pain and frontal slow wave bursts on the EEG. PET rcvcaled right frontal and left parietal hypometabolism, and ECDs were estimated to originate from the left parietal area. Case 9 had an old inraretion in the lcft occipital lobe and CPS. SPECT revealed perfusion defect in the infarction and right frontal hypoperfusion. ECDs were estimated to originatc from the margin of the infrarction. Conclusions: Interictal MEG studics can dctcct irritable zones of epileptic foci, hut their findings sometimes do not accurately reveal the epileptogcnic zone. However the majority of our cstimatcd ECDs agreed with the ictal symptomatology and the foci detected by PET and SPECT. The estimated area of ECDs was usually smaller than hypometabolic areas on PET and hypoperfusion area on SPECT. Multimodal interictal studies with noninvasive technologics will enablc more enable detection of epileptic foci.

The activities of 14 lysosomal enzymes in chorionic villi at gestational ages of 6-12 weeks were assayed. Arylsulphatases A and B, alpha-glucosidase and beta-glucuronidase activities increased with advancing gestational age. When compared with the activity in cultured amniotic fluid cells, arylsulphatase A, beta-galactosidase, alpha-glucosidase, heparan N-sulphatase, alpha-L-iduronidase, alpha-mannosidase, neuraminidase, and sphingomyelinase showed significant differences. All except beta-glucuronidase showed lower activity in chorionic villi than in cultured amniotic fluid cells. Prenatal diagnosis using chorionic villi was possible except for alpha-L-iduronidase. Storage at -20 degrees C up to 42 days did not significantly affect activity. The results emphasize the importance of using fresh or frozen age-matched control tissue for diagnosis.

OBJECTIVE To determine whether hemodynamic changes in retinal arteries precede clinical manifestations of diabetic retinopathy and to examine the effects of control of hyperglycemia on retinal artery blood flow. RESEARCH DESIGN AND METHODS We assessed blood flow in bilateral central retinal arteries in 50 insulin-dependent diabetes mellitus (IDDM) patients without retinopathy and 20 sex- and age-matched control subjects using duplex Doppler sonography. We determined the peak systolic velocity (PSV), end-diastolic velocity (EDV), time-averaged velocity (TAV), resistance index (RI), and pulsatility index (PI). RESULTS PSV, EDV, and TAV were significantly lower in IDDM patients than in control subjects (P < 0.05, P < 0.01, and P < 0.01, respectively). The RI was significantly higher in IDDM patients than in control subjects (P < 0.01) and was significantly correlated with plasma levels of glucose in IDDM patients (r = 0.0.310, P = 0.0248). Multiple regression analysis identified the plasma levels of glucose as a significant determination of RI in IDDM patients. After 14 days of intensive insulin therapy in 7 IDDM patients, the RI and plasma levels of glucose showed significant decreases (P = 0.018, P = 0.001, respectively). CONCLUSIONS Our results showed that changes in retinal hemodynamics were present before the clinical detection of overt diabetic retinopathy and suggest that the presence of short-term hyperglycemia partly contributes to impaired retinal circulation.

Serum immunoglobulin (IgG, A, M) levels were measured in 123 children with Type I diabetes in order to clarify the relationship between immunoglobulin levels and islet cell antibodies (ICA) or the duration of illness and to investigate the incidence of selective IgA deficiency in children with Type I diabetes in Japan.IgA levels in Type I childhood diabetics were significantly higher than in normal children; however, IgG and IgM levels in Type I childhood diabetics were similar to those in normal children. There was no correlation between immunoglobulin (IgG, A, M) levels and the duration of illness. Immunoglobulin levels in patients with ICA were about the same in those without ICA. IgM levels in ICA-positive patients with a duration of less than one year were significantly higher in ICA-negative patients. Two out of 123 children (1.6%) with Type I diabetes had selective IgA deficiency.From these results, it was suggested that high IgM levels in ICA-positive diabetic children with short duration might show recent viral infection and that the incidence of IgA deficiency in Japanese children with Type I diabetes was slightly lower than in Caucasians.

In order to prevent vertical transmission of hepatitis B virus (HBV), yeast recombinant HB vaccine at a dose of either 5 mcg or 10 mcg was administered to 185 infants born to mothers who were positive for HBs antigen (HBsAg). All of them developed antibody to HBsAg (anti-HBs) after three vaccinations. Generally, the cutaff index (COI) of anti-HBs by radioimmunoassay (RIA) was higher in the 10 mcg dosage group than in the 5 mcg dosage group. The geometric mean titer (GMT) of anti-HBs, as measured by quantitative RIA, in the former group was 2.4 times that in the latter one month after the third vaccination. The incidence of clinical reactions was only 4% in a total of 561 injections, and none of the reactiom were severe. It is conduded that recombinant HB vaccine is safe and has excellent immunogenicity for infants requiring prevention of HBV vertical transmission. It is also suggested that 10 mcg doses of the recombinant HB vaccine can provide more solid protection to high-risk infants without serious adverse reactions. (Acta Paediutr Jpn 1989; 31: 180–185)

In a nationwide cross-sectional study of Japanese type-1 diabetics, whose disease had manifested itself before the age of 18, we found (1) the long-term outcome of childhood diabetes to be very poor, (2) the prevalence of microvascular complications to be stronly related to the age of the patients and the duration of diabetes, and (3) the prevalence of retinopathy to be dependent on the degree of diabetic control. Of 90 diabetics born before 1955, 7 had died, 71 had retinopathy of which 36 were proliferative, 47 had cataracts, 12 were blind, 44 had nephropathy, 42 had neuropathy, and 18 had hypertension. Retinopathy was present in 1.8% under the age of 12, in 8.3% from the age of 13 to 16, in 22.1% between the age of 17 to 20, and in 52.5% over the age of 20. A comparison between age-matched subgroups with and without retinopathy showed that the percentages of patients who were cooperative, with good control and without hyperlipidemia were higher in the group having no retinopathy than in the other.

Summary We describe a case with transient neonatal hyperphenylalaninemia, possibly due to immature biosynthesis of 7, 8-dihydrobiopterin (BH2). In the neonatal period his blood phenylalanine levels were high and the patterns of urinary pterins were similar to those found in defective synthesis of BH2, which results in a type of hyperphenylalaninemia. But the laboratory data became almost normal after three months. Transient hyperphenylalaninemia has generally been thought to be caused by the delayed maturation of phenylalanine hydroxylase. But it is suggested that some cases with transient neonatal hyperphenylalaninemia are due to immaturity of BH2 cofactor biosynthesis rather than that of phenylalanine hydroxylase.

Sprague-Dawley rats were given L-phenylalanine intraperitoneally from the 8th to 11th day of pregnancy. The hearts of the fetuses were examined on the 21st day of pregnancy. We found that the group given the higher doses of L-phenylalanine had significantly more heart defects than the group given the lower dose and the controls. Ventricular septal defect was found in 80 % of the fetuses with congenital deformities of the heart. [14C] Leucine uptake by the embryos was significantly lower in the group loaded with L-phenylalanine than in the controls. The activity of thymidine kinase, one of the rate-limiting enzymes in DNA synthesis, was significantly lower in the brain and heart tissues of the young rats loaded with L-phenylalanine than in the controls. Thus, when blood levels of phenylalanine are high in early pregnancy, an amino acid imbalance in the embryo occurs during fetal organogenesis. Also, thymidine kinase decreases, which may hinder DNA synthesis. Both of these mechanisms seem to be involved in the higher incidence of fetal heart malformations in maternal phenylketonuria.

Plasma concentrations of fructosamine, an indicator of glycated plasma proteins, were measured in non-diabetic children and children with insulin-dependent diabetes mellitus (IDDM) to see if they also correlate with glycemic control in children as well as in adults. Non-diabetic children aged less than 4 yr had significantly lower plasma fructosamine than non-diabetic children aged 4 or more. Total plasma protein in these children was slightly lower or close to that of older children. There was no difference in fructosamine between non-diabetic children aged 4 or more and healthy adult subjects. Plasma fructosamine in children with IDDM was twofold that of age-matched controls. In children with IDDM, correlations between fructosamine and HbAI (r = 0.799) or HbAIc (r = 0.841) were high. The measurement of plasma fructosamine, which is practical in children because of the small sample volume needed and no influence of HbF, is useful in the management of children with IDDM.

Pregnant rats were loaded with L-phenylalanine, and the distributions of [14C]leucine and [14C]urea into fetal plasma and tissues were examined. Uptake of [14C]leucine into the supernatant and protein fractions of fetal plasma and tissues was low in the rats loaded with phenylalanine. In contrast, [14C]urea was distributed identically in both groups, indicating that maternal hyperphenylalaninemia did not affect blood flow across the placenta. Administration of phenylalanine and p-chlorophenylalanine produced amino acid imbalance in fetal tissues. Along with these changes, polysomes of the affected fetal heart and brain disaggregated without changes in the ribonuclease activity. These results indicate that high phenylalanine levels in maternal plasma disturb the active transport of amino acids across the placenta, causing an amino acid imbalance and disaggregation of polysomes in fetal heart and brain. These changes may contribute to the congenital heart disease and mental retardation of maternal phenylketonuria.

Copper-binding proteins in the tissues from the patients with Menkes' kinky hair disease were examined by gel filtration on a Sephadex G-75 column. In the kidney, major part of copper was found to bind to low molecular weight protein, which corresponded chromatographically to metallothionein. This copper-binding protein contained a large amount of copper and a small amount of zinc. Cu: Zn ratio of this protein was different from that of metallothionein found in the fetal liver. In the liver of the same patient, however, there was no increase of copper bound to this protein.

A patient with a new variant of multiple sulphatase deficiency (MSDv) is reported. Unlike the usual type, onset was late and progress was slow. The phenotypic changes were those usually seen in multiple sulphatase deficiency but much milder. Cytoplasmic accumulations were found in skin fibroblasts, and urinary mucopolysaccharides and sulphatides were high. Arylsulphatases A, B and C (ASA, B and C), heparan N-sulphatase sulphoiduronate sulphatase, and N-acetylgalactosamine 6-sulphatase all had low activity in lymphocytes and cultured skin fibroblasts. Complementation for ASA activity was found in hybrids between MSDv and metachromatic leukodystrophy (MLD) as well as between multiple sulphatase deficiency (MSD) and MLD. Complementation for ASC activity was also seen in hybrids between MSDv and X-linked ichthyosis (XLI), and between MSD and XLI. However, neither ASA nor ASC activity increased in hybrid cells of MSDv and MSD. These results suggested that the mutations of MSDv and of MSD were allelic, although of different phenotypes.

When Kan-baku-taiso-to (TJ-72) (0.1~0.25 g/kg/day) was given to 12 children with breath-holding spells (BHS) of the cyanotic type, the attacks were lessened starting in the second week of administration. There were no side effects even when administration was continued for more than one year. We think that the drug should be given primarily for BHS when the frequency of severe attacks is high and also to patients who did not respond to other medication.

Tetrahydrobiopterin (BH4) deficiency has been described as a form of hyperphenylalaninaemia in which severe neurological symptoms develop despite early treatment with low phenylalanine diet. In recent years it has become apparent that biopterin deficiency may be caused by a defect either of dihydropteridine reductase (DHPR, EC 1.6.99.10) or dihydrobiopterin synthetase (DHBS) (Niederwieser et al., 1979). Since it was proposed that treatment with precursors of the neurotransmitters involved could prevent neurological deterioration if started within the first months after birth (Curtius et al., 1979), screening of all neonates with hyperphenylalaninaemia for biopterin disorders, and a non-invasive reliable method for the diagnosis of two types of BH4 deficiency are needed urgently. Assessment of pterin derivatives in biological fluids, mostly in urine, by high performance liquid chromatography (HPLC) is proposed as a reliable diagnostic method and Crithidia fasciculata bioassay is also a very sensitive method of measuring biopterin activity. Thus normal values of pterin derivatives during the neonatal period are needed. Nevertheless, few reports on a small number of neonates have so far been found (Niederwieser et al., 1980). In this study we describe normal values of pterin derivatives in urine and biopterin activity in serum, and their age-related change in early neonates, young infants, children and adults.

In 6 cases of high risk pregnancies for Tay-Sachs disease, prenatal diagnosis was successfully carried out by examining hexosaminidase A activity in the supernatant of amniotic fluid and the uncultured and cultured amniotic fluid cells. In 5 out of 6 cases, the activity of hexosaminidase A was found to be within normal or heterozygous levels. They continued their pregnancies and were delivered of healthy children. In the remaining 1 case, the activity was undetected. Her pregnancy was terminated at the 23rd week of gestation. There was no activity of hexosamindase A in the brain and liver from the aborted fetus. A moderate increase in GM2 ganglioside was found in the brain. Electron-microscopic findings revealed membranous cytoplasmic bodies in the spinal ganglion cells as well as in the nerve cells of the retina. These biochemical and histological findings of Tay-Sachs fetus suggest that the disease proceeds early in fetal period. It was found that the cultured amniotic fluid cells was the most reliable material for the prenatal diagnosis of Tay-Sachs disease, because the values of hexoxaminidase A in the cultured cells were well in accord with those in serum from the consequently bord children. Hexosaminidase pattern in the supernatant of amniotic fluid on DEAE-cellulose columnchromatogram showed a distinct difference between homozygote, heterozygote and normal. This procedure also may be useful for prenatal diagnosis of Tay-Sachs disease.

Microcomputers have been used in our hospital for the past five years to summarize data from patients and to remind us to carry out routine tests. Recently, we have shown computer data on the screen to patients; this helps us to explain and discuss the monitoring of their condition with them. Unlike some other systems, general data as well as data on self-monitored blood glucose levels are recorded in our computer system. The data consist of five categories: personal profile, treatment, etiological research, indices of control and data for checking complications. The analysis and evaluation of our system are done by the modes of display, statistical calculations, early detection of complications and graphics. The system is compatible with input from a keyboard, optical character readers and interface to a glucose meter with a memory.

Summary In a summer camp for 47 diabetic children in Kinki district, Japan, in 1984, the relationship between hypoglycemic symptoms and blood glucose levels by self-monitoring was analyzed. During the 7-day camp, self-monitoring of blood glucose (SMBG) was carried out 599 times in total, 12.7 times per camper. SMBG due to hypoglycemic complaints amounted to 371. 154 measurements out of 371 indicated blood glucose levels under 80 mg/dl, but 78 monitorings were found to be over 200 mg/dl. Fatigue or weakness were the most frequent hypoglycemic symptoms, as was hunger sensation, each reaching approximately 40% in frequency. In most complaints of tremor, the blood glucose level was critically low. Prompt measurement of blood glucose is indeed necessary to properly treat diabetic children with ‘hypoglycemic’ symptoms.

Multiple sulphatase deficiency (MSD) and metachromatic leukodystrophy (MLD) are both characterized by a deficiency of arylsulphatase A (ARS A) activity, although they are inherited as separate autosomal recessive traits. However, it has been found that the immunologically active substance with anti-ARS A antibody is present in quite normal levels in MLD and in smaller quantities in MSD fibroblasts (Fiddler, 1979). Indirect immunofluorescence staining with anti-ARS A antibody displayed a coarse granular and diffuse distribution of ARS A or cross-reacting material (CRM) in the normal control and MLD fibroblasts, whereas very weak fluorescence staining was observed in MSD fibroblasts proportional to the decrease in the ARS A activity observed in the lysate enzyme assay. These results suggest that ARS A deficiency in MLD cells is due to an enzymatically deficient ARS A molecule, which is immunologically cross-reactive with anti-normal ARS A antibody. ARS A deficiency in MSD cells appears to be due to a reduced amount of normal ARS A.