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The HLA-DO molecule is a non-classical class II heterodimer composed of alpha and beta chains. We have previously recognized that all eight of the allelic variations of the HLA-DOA gene represent non-synonymous amino acid substitution. In the present study, to analyze genetic polymorphism and allelic variation of the HLA-DOB gene which may affect the efficiency of class II restricted antigen presentation thereby being involved in the susceptibility of HLA associated diseases, we conducted direct DNA sequencing of HLA-DOB in 36 HLA class II homozygous typing cells and identified six new allelic variations (DOB*0101101, *0101102, *01012, *01022, *0104101 and *0104102) including five single nucleotide polymorphisms with only one amino acid substitution. Furthermore, strong linkage disequilibrium was detected between DOB*01022 and DRB1*1502 only, with no linkage disequilibrium between the DOA and the DOB genes. The HLA-DOB gene has been identified in other mammals, and their nucleotide sequences are well conserved. These facts suggest that limited polymorphism in the DOB gene is profitable to execute their unique function as a co chaperone and so strong selective pressure is operating to prevent generic variation against the DOB molecule interacting with the DM molecule and thus maintaining the specified immunological function of regulating antigen presentation.

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.

The HLA-DO molecule, a heterodimer consisting of two novel members of the class II gene family, DOA and DOB, has recently been suggested to function as an important modulator in the HLA class II restricted antigen presentation pathway by interaction with the HLA-DM molecule. In this study, we have analyzed genetic polymorphism and allelic variation of the HLA-DOA gene in 37 HLA class II homozygous typing cells using the direct DNA sequencing technique. As a result, we recognized at least eight allelic variations, DOA*01011, *0101201, *0101202, *0101203, *01013, *0101401, *0101402 and *01015. None of them, however, result in amino acid substitution. The HLA-DOA gene has been identified in other mammals as well, and the nucleotide sequences were well conserved among these species. These results suggest that the DOA molecule has undergone strong selective pressure to preserve functional structure and conformation required for interaction with the DM molecule, preventing non-synonymous amino acid substituiton Note.

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.

The serologically defined HLA-DR53 antigen is associated with HLA-DR4, -DR7, and -DR9 antigens, and these haplotypes contain two functional genes, DRB1 and DRB4, and two pseudogenes, DRB7 and DRB8. The DRB4 gene encodes the DR53 antigen, and has been officially recognized to contain three allelic variants (DRB4*0101, 0102, and 0103). In this study, we have established the polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) method for DRB4 genotyping and analyzed genetic polymorphism of the DRB4 gene in Japanese population. DRB4*0101, DRB4*0102, and DRB4*0103 could be observed at the frequencies of 0.5%, 1.1% and 32.7%, respectively. The same DRB1 allele does not necessarily share an identical DRB4 allele. Further, a tight linkage disequilibrium was found between DRB4*0I02 and DRB1*0401 in Japanese population, whereas DRB1*0401 was associated with DRB4*0101 or *0103 in Caucasian population. These findings reveal extensive diversity of the HLA-DRB1 and -DRB4 haplotypes and may have important implications for HLA-disease associations and donor selection in unrelated transplantation.

In the HLA-D region, one of the class II genes, DMA and DMB have been identified between the DQ and DP genes, and four allelic polymorphisms in each of the DMA (DMA*0101 approximately 0104) and DMB (DMB*0101 approximately 0104) genes have been so far recognized. Several recent studies suggested that the DM molecule is required for class II antigen presentation pathway especially by promoting the binding of antigenic peptides to the classical HLA class II molecule. In this study, we have analyzed genetic polymorphism and allelic variation of the DMB gene in a Japanese population by the direct DNA sequencing technique and also by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, and could recognize DMB*0101 (49.3%), DMB*0102 (23.2%), DMB*0103 (23.2%), and DMB*0104 (0.4%). Further, a new DMB allele, DMB*0105 characterized by the presence of Val and Ile at two polymorphic sites, codons 144 and 179, respectively was identified. Strong linkage disequilibria were found between DMB*0101 and DRB1*0101, DPB1*0402 and DRB1*1502, and also between DMB*0103 and DRB1*1501 and DQB1*0602. HLA-DMB genotyping using the PCR-RFLP method established here will provide accurate evaluation of the effects of sequence allelism in the DMB gene on the HLA class II disease associations.

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.

HLA is an important etiologic genetic factor in Type I diabetes and specific HLA-class II genes are closely related to the onset of the disease. Many differences in the patterns of susceptible and resistant DRB1, DQA1, and DQB1 genes have been observed among various ethnic groups. We have previously shown that DRB1*0405, DRB1*0901 and DQA1*0301-DQB1*0302 were the major susceptible alleles or haplotype to Type I diabetes while DR-DQ haplotype studies suggested the important role of DR and DQ alleles in susceptibility and resistance in Japanese patients. Based on the analysis of 90 Japanese patients with childhood onset Type I diabetes and 136 unrelated healthy Japanese controls by polymerase chain reaction-restriction fragment polymorphism method (PCR-RFLP), we report here the association of Type I diabetes with DPB1*0201 (relative risk = 2.29; Pc = 0.027) in this population. Comparison of linkage disequilibrium patterns between patients and controls showed that the significantly high prevalence of DPB1*0201 among patients cannot be attributed simply to linkage disequilibrium with susceptible DRB1 alleles and DQA1-DQB1 haplotypes. Our results suggest that in addition to alleles at the DRB1, DQA1, DQB1 loci, polymorphism at DPB1 locus also influences the risk of Type I diabetes.

We identified 14 mutations in 15 Japanese subjects from 13 families with galactose-1-phosphate uridyltransferase (GALT) deficiency using denaturing gradient gel electrophoresis (DGGE) and direct sequence analysis. These mutations accounted for 22 (96%) of 23 mutant alleles in 15 Japanese subjects. The mutational spectrum included nine missense mutations (M142V, G179D, A199T, R231H, W249R, N314D, P325L, R333Q, and R333W), two deletions (L275fsdelT and Q317fsdelC), a nonsense mutation (W249X), and two splicing mutations (V85-N97fsdel38bp and IVS4nt+1). Ten of the 14 mutations have not been reported in Caucasians. Differences in frequency and spectrum of GALT mutations suggest that the mutations may have occurred after racial divergence of Caucasians and Asians. The Duarte variant in Japanese was associated with the N314D mutation, g.1105GC, g.1323GA, and g.1391GA (SacI -) polymorphisms, as in Caucasians. The Duarte variant may have occurred before racial divergence, and was an ancient mutation. In vitro GALT activities of nine missense mutations were determined by a COS cell expression system, and indicated between 1.3% and 35% of wild-type control. Patients with R333Q (29% in vitro GALT activity) or A199T (35%) showed mild clinical phenotypes, i.e. no ovarian failure or neurological deterioration. Genotype determination is useful for predicting biochemical and clinical phenotypes in classic galactosaemia, and can be of further help in managing patients with this disorder.

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.

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.

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.

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.

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.

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)

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.

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.

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.

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.

It has been reported that the serum level of uric acid is elevated in Down's syndrome. The present study was undertaken to investigate whether the regulation mechanism of uric acid biosynthesis by hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and adenine phosphoribosyltransferase (APRT) is impaired in Down's syndrome. The activities of HGPRT and APRT in erythrocytes were determined in 14 cases of Down's syndrome and 10 cases of healthy children. There was no significant difference in activities of both enzymes between Down's syndrome and controls. These findings indicate that the activity of HGPRT or APRT may not be controlled by chromosome 21.