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Hunter syndrome (mucopolysaccharidosis II [MPS II]), a deficiency of iduronate-2-sulfatase (IDS), causes an accumulation of glycosaminoglycans, giving rise to multiple systemic and CNS symptoms. The currently available therapies, idursulfase and idursulfase beta, are ineffective against the CNS symptoms because they cannot pass the blood-brain barrier (BBB). A novel IDS fused with anti-human transferrin receptor antibody (JR-141) has been shown to penetrate the BBB and ameliorate learning deficits in model mice. This first-in-human study evaluated the pharmacokinetics, safety, and potential efficacy of JR-141 in 14 patients with MPS II. In a dose-escalation study performed in two patients, JR-141 plasma concentrations were dose dependent and peaked at 3 hr after initiation of each infusion, and no or only mild adverse reactions were exhibited. In a subsequent 4-week evaluation at two dose levels, the plasma concentration profiles were similar between the first and final administration, indicating no drug accumulation. Levels of heparan sulfate (HS) and dermatan sulfate (DS) were suppressed in both plasma and urine and HS levels were significantly decreased in cerebrospinal fluid. Two patients experienced some amelioration of neurocognitive and motor symptoms. These results suggest that the drug successfully penetrates the BBB and could have CNS efficacy., Okuyama et al. report on a first-in-human study of iduronate-2-sulfatase fused with anti-human transferrin receptor antibody (JR-141) in 14 patients with mucopolysaccharidosis II to evaluate its pharmacokinetics, safety, and potential efficacy. The results show successful penetration of the compound across the blood-brain barrier, suggestive of its effect for neurodegeneration.

Mucopolysaccharidosis type II (MPS II or Hunter syndrome) is a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS), an enzyme that catabolizes glycosaminoglycans (GAGs) including heparan sulfate (HS) and dermatan sulfate (DS). GAG accumulation leads to severe neurological and somatic impairments. At present, the most common treatment for MPS II is intravenous enzyme replacement therapy; however, the inability of recombinant IDS to cross the blood-brain barrier (BBB) restricts therapeutic efficacy for neurological manifestations. We recently developed a BBB-penetrating IDS fusion protein, JR-141, and demonstrated its ability to reduce GAG accumulation in the brain of human transferrin receptor knock-in and Ids knock-out mice (TFRC-KI/Ids-KO), an animal model of MPS II, following intravenous administration. Given the impossibility of measuring GAG accumulation in the brains of human patients with MPS II, we hypothesized that GAG content in the cerebrospinal fluid (CSF) might serve as an indicator of brain GAG burden. To test this hypothesis, we optimized a high-sensitivity method for quantifying HS and DS in low-volume samples by combining acidic methanolysis and liquid chromatography-tandem mass spectrometry (LC/MS/MS). We employed this method to quantify HS and DS in samples from TFRC-KI/Ids-KO mice and revealed that HS but not DS accumulated in the central nerve system (CNS). Moreover, concentrations of HS in CSF correlated with those in brain. Finally, intravenous treatment with JR-141 reduced levels of HS in the CSF and brain in TFRC-KI/Ids-KO mice. These results suggest that CSF HS content may be a useful biomarker for evaluating the brain GAG accumulation and the therapeutic efficacy of drugs in patients with MPS II.

Background: Hunter syndrome (mucopolysaccharidosis II, MPS II) is an X-linked recessive lysosomal storage disease in which a deficiency of iduronate-2-sulfatase causes an accumulation of glycosaminoglycans, giving rise to multiple systemic as well as central nervous system (CNS) symptoms. The currently available enzyme replacement therapy with idursulfase is ineffective against the associated CNS symptoms, because the blood-brain barrier (BBB) prevents the enzyme from reaching the brain. A novel iduronate-2-sulfatase fused with anti-human transferrin receptor antibody (JR-141) has been shown to penetrate the BBB and ameliorate learning deficits in model mice. This first-in-human translational study evaluated the pharmacokinetics, safety and efficacy of JR-141 in patients with MPS II. Methods: In this phase 1/2 multicentre, open-label, randomised clinical trial, 15 patients with MPS II who were being treated with intravenous idursulfase were recruited in Japan, and treatment was switched to intravenous administration of JR-141. In Part 1 of the study, which was designed to evaluate the safety of JR-141, the dosage was escalated over a period of 4 weeks (from 0.01 to 2.0 mg/kg/week); in Part 2, a 4-week randomised evaluation of the pharmacokinetics, safety and exploratory efficacy of JR-141 (at 1.0 or 2.0 mg/kg/week) was made. Cerebrospinal fluid (CSF) concentrations of heparan sulfate (HS) and dermatan sulfate (DS) were compared at the start of JR-141 administration and after 3 weeks, and plasma and urine concentrations of HS and DS were compared at the start and after 4 weeks. Findings: Between 10th May, 2017 and 25th October, 2017, we recruited a total of 15 male patients from 9 investigational sites in Japan. Of these, 14 were enrolled after 1 was excluded as unsuitable. Two patients without intellectual disabilities were selected for the dose escalation study (Part 1) and were given 0*01, 0*1, 1.0, and 2*0 mg/kg/week of JR-141 intravenously; plasma concentrations of JR-141 were dose-dependent and peaked at 3 h after initiation of each infusion. One patient developed erythema and pyrexia, which abated quickly, and the other exhibited no adverse reactions. In Part 2, the remaining 12 patients were randomly allocated to JR-141 dosing groups of 1*0 or 2*0 mg/kg/week. The plasma concentration profiles of JR-141 did not differ between the first and final administration, indicating no drug accumulation. Plasma and urinary levels of HS and DS were both suppressed by JR-141, and HS concentrations in the CSF had significantly decreased after the last administration (by an average of 25*1% in patients taking 1*0 mg/kg/week, and 31*5% in those taking 2*0 mg/kg/week). Two patients showed some amelioration of neurocognitive and motor symptoms following the 4-week treatment. No drug-related serious adverse events were observed during the study period. Interpretation: Patients with Hunter syndrome who were treated with JR-141 showed significant reductions in neurotoxin levels in the CSF, suggesting the drug successfully penetrated the BBB. Furthermore, JR-141 was no less effective in treating systemic symptoms than conventional enzyme replacement therapy with idursulfase. If JR-141 reduces glycosaminoglycan accumulation in the CSF, it should also be effective in treating CNS symptoms. This will be investigated in future clinical trials. Clinical Trial Number: NCT03128593 Funding Statement: The trial was funded by JCR Pharmaceuticals. Declaration of Interests: Torayuki Okuyama reports research grants from Sanofi, JCR Pharmaceuticals, GC Pharma, Dainippon Sumitomo Pharma, Actelion, Alexion and Anges, along with honoraria from Sanofi, Dainippon Sumitomo Pharma, Actelion and Anges. Yoshikatsu Eto has conducted consultancy for JCR Pharmaceuticals, and he has been awarded grants and research support from Actelion, BioMarin Pharmaceutical Inc. and Sanofi; he has also received honoraria from Actelion, BioMarin Pharmaceutical Inc., Sanofi, Shire and Dainippon Sumitomo Pharma. Norio Sakai has conducted consultancy for JCR Pharmaceuticals, and he has been awarded grant/research support from Sanofi and Dainippon Sumitomo Pharma, and honoraria from Actelion, BioMarin Pharmaceutical Inc., Sanofi, Shire and Dainippon Sumitomo Pharma. Ethics Approval Statement: The study was conducted in 8 hospitals and specialised MPS centres in Japan, and was in compliance with the Declaration of Helsinki. The protocol and patient informed consent were reviewed and approved by the Institutional Review Board at each participating institution.

Determination of serum growth hormone (GH) levels is mandatory for diagnosis of GH deficiency and excess. In the present study, we, the Study Committee for GH and Its Related Factors, The Foundation for Growth Science, Japan measured GH values in serum samples using all the commercially available kits in Japan. Significant discrepancies in the GH values were observed among the kits in spite of using the unified recombinant human GH-based standards. To deal with the discrepancies, we established a formula using a linear structural relationship model and were able to standardize the GH values. We propose to use the formula to diagnose GH deficiency and excess in Japan.

Background: Patients with Turner syndrome (TS) are prone to having metabolic abnormalities, such as obesity, dyslipidemia, hypertension, hyperinsulinemia and type 2 diabetes mellitus, resulting in increased risks of developing atherosclerotic diseases. Objective: To determine the effect of growth hormone (GH) therapy on serum cholesterol levels in prepubertal girls with TS enrolled in the Turner syndrome Research Collaboration (TRC) in Japan. Patients and methods: Eighty-one girls with TS were enrolled in the TRC, and their total cholesterol (TC) levels before GH therapy were compared with reported levels of healthy school-aged Japanese girls. TC levels after 1, 2 and 3 yr of GH treatment were available for 28 of the 81 patients with TS. GH was administered by daily subcutaneous injections, 6 or 7 times/wk, with a weekly dose of 0.35 mg/kg body weight. Results: Baseline TC levels revealed an age-related increase in TS that was in contrast to healthy girls showing unchanged levels. During GH therapy, TC decreased significantly after 1 yr of GH treatment and remained low thereafter. Conclusions: Girls with untreated TS showed an age-related increase in TC that was a striking contrast to healthy girls, who showed unchanged levels. GH therapy in girls with TS brought about a favorable change in TC that indicates the beneficial impact of GH on atherogenic risk.

Growth hormone (GH) therapy was approved in 1999 for only GH-deficient Turner syndrome (TS) in Japan. It was subsequently approved for all cases of TS regardless of GH secretory status since 1999. The dose of GH is 1.0 u (0.35 mg)/kg/wk at present, but it was 0.5 u (0.175 mg)/kg/wk before 1999. The adult height in patients with TS on the dose of 0.5 u/kg/wk was studied from the report on of Foundation for Growth Science in 2000. GH therapy was registered for 920 cases, and 258 cases reached adult height. The mean adult height was 145.7 cm. The adult height in patients with TS without GH therapy was reported to be 138 cm in Japan. Thus, the height gain by GH treatment was 7.7 cm. The mean age at the start of GH therapy was 12.0 yr old. The mean duration of GH therapy was 5.6 yr. The mean age at the start of estrogen therapy was 17.0 yr old. Patients in Japan were older at the start of GH and estrogen therapy than in the US and Europe at that time. The adult height and gain of height SD were not correlated with age at the start GH therapy in this study. This may be the result of the older age at the start of GH therapy and the low dose of the GH therapy. Patients are beginning to start GH therapy at a much earlier age and the dose has been doubled in Japan. We expect that the recent data concerning adult height in the patients with TS after GH therapy will improve better than this report.

Aim To investigate the final adult heights and pubertal growth patterns in Japanese patients with congenital hypothyroidism (CH) detected by neonatal screening. Methods A retrospective chart review was conducted of female patients >15 y of age (n = 18) and male patients >18 y of age (n = 9), who were detected by neonatal screening and kept on continuous thyroid hormone replacement therapy. Final height standard deviation scores (FHSDS) and target height standard deviation scores (THSDS) were determined. Parameters characterizing the pubertal growth process (such as age at onset of pubertal growth spurt and age at peak pubertal growth) were obtained from each patient's growth rate chart. Menarchial age was determined in each female patient by reviewing the medical record. The impact on FHSDS of the etiology of CH, the severity of CH, the time of initiation of therapy and the adequacy of treatment during the first year of life was assessed. Results All patients had received initial thyroid hormone treatment no later than 50 d of age, and had reached their final height. The mean FHSDS for female and male patients were +0.17 +/- 0.99 and -0.03 +/- 0.99, respectively. The mean FHSDS-THSDS for female and male patients was +0.09 +/- 0.77 and -0.19 +/- 0.53, respectively. No difference was seen in pubertal growth parameters for either gender compared with that of the reference population, except for a greater peak height velocity and pubertal height gain in male patients. The mean menarchial age was identical to that of the reference population. No significant relationship was found between the FHSDS and any of the factors investigated. Conclusion The adult height of patients with CH detected by neonatal screening was equivalent to that of the reference population and their target height. As long as early intervention and satisfactory management are ensured, severe CH does not appear to reduce final adult height.

Aim To determine the natural growth pattern of Japanese children with Down's syndrome. Methods Longitudinal height data of 85 patients (43 males, 42 females) from birth to final height were analyzed. Based on these data, semi-longitudinal standard growth curves and height velocity curves for Down's syndrome were drawn. Results The means +/- SD of final height of males and females with Down's syndrome were 153.2 +/- 5.6 and 141.9 +/- 4.2 cm, respectively. They were -3.0 SD and -2.8 SD for Japanese standards. Mean peak height velocities were 8.9 and 7.5 cm y(-1), and the ages at peak height velocity were 11.6 and 10.2 y for males and females, respectively. Conclusion The mean height of patients with Down's syndrome was around -2 SD for normal children before puberty. Their pubertal growth spurt starts about 1 y earlier and their peak height velocity was about 1.3-1.4 cm shorter than for normal children.

Duchenne muscular dystrophy (DMD) is an incurable genetic disease with early mortality that exhibits skeletal muscle weakness with chronic inflammation. Multipotent mesenchymal stromal cells (MSCs) could be a potential therapeutics because of their immunosuppressive properties and ability of differentiation to myogenic cells in situ. In the present study, we examined the strategies for effective cell transplantation to develop a novel approach for functional recovery of the skeletal muscles using a dog model of Duchenne muscular dystrophy.

The Growject® database on human GH treatment in Turner syndrome was analyzed in the Turner Syndrome Research Collaboration, and the relationships of the frequencies of spontaneous breast development and spontaneous menarche with karyotype and GH treatment were investigated. One hundred and three cases started GH treatment with 0.5 IU/kg/ week (0.5 IU group), and their dose was increased to 0.35 mg/kg/wk midway through the treatment course. Another 109 cases started GH at a dose of 0.35 mg/kg/wk (0.35 mg group). Spontaneous breast development was observed in 77 (36.3%) of the 212 patients, and spontaneous menarche occurred in 31 patients (14.6%). The frequency of spontaneous breast development was significantly lower in patients with the 45,X karyotype and significantly higher in patients with a structural abnormality of the second X chromosome. The frequency of spontaneous menarche was significantly higher in patients with mosaicism characterized by X monosomy and a cellular line with no structural abnormality of the X chromosome. No significant differences in frequencies of spontaneous breast development and spontaneous menarche were observed between the two dose groups, indicating that GH treatment does not increase the frequency of spontaneous puberty.

It is still in doubt whether the standard-dose growth hormone (GH) used in Japan (0.5 IU/kg/week, 0.167 mg/kg/week) for growth hormone deficiency is effective for achieving significant adult height improvement in non-growth hormone deficient (non-GHD) short children. We compared the growth of GH-treated non-GHD short children with that of untreated short children to examine the effect of standard-dose GH treatment on non-GHD short children. GH treatment with recombinant human growth hormone (rhGH) was started before the age of 11 yr in 64 boys and 76 girls with non-GHD short stature registered at the Foundation for Growth Science who have now reached their adult height. In 119 untreated boys and 127 untreated girls whose height standard deviation score (SDS) was below -2 SD at the age of 6 yr, height growth was followed until 17 yr. Height SDS was significantly lower before GH treatment in the GH-treated group than at the age of 6 yr in the untreated group, in both sexes. Adult height and adult height SDS were significantly greater in the untreated group than in the GH-treated group, in both sexes, although the change in height SDS did not differ significantly. Height SDS was significantly lower before GH treatment in the GH-treated group than at the age of 6 yr in the untreated group, so 57 boys and 57 girls whose height SDS at the age of 6 yr in the untreated group closely matched the height SDS before GH treatment in the GH-treated group were chosen for comparison. Height SDS did not differ significantly between the GH-treated group before GH treatment and the untreated group at the age of 6 yr, nor were there differences between these subgroups in adult height, adult height SDS, or height SDS change, in either sex. The effect of GH treatment is reported to be dose-dependent and doses over 0.23 mg/kg/week are reported to be necessary to improve adult height in non-GHD short children. Currently, the GH dose is fixed at 0.175 mg/kg/week in Japan, and we expected to find, and indeed concluded, that ordinary GH treatment in Japanese, non-GHD short children does not improve adult height.

In this study, we sent questionnaires to doctors treating severe short stature with severe GH deficiency (GHD) (height SDS (HtSDS) below -4 and all peak GH to provocative stimuli below 2 micro/L) (abbreviated as Severe Case), and obtained effective replies of 51 cases. The clinical characteristics, etiologies, and pathophysiology of these patients were examined. Among the 51 Severe Cases no consanguinity was observed, 44 were IGHD (24 males and 20 females), 3 were GH-1 gene deletion, 2 were Pit-1 gene mutation, and 2 were achondroplasia. HtSDS in these Severe Cases was already remarkably low at 12 (-3.0) and 24 months old (-3.9), while their birth weight and birth length were within normal ranges. Among 44 patients with IGHD, 12 were isolated GHD, and the remaining 32 were combined pituitary hormone deficiency (CPHD). Pituitary MRI was undergone in 25 idiopathic GHD, and abnormal findings (pituitary atrophy, interruption of stalk, and ectopic posterior lobe) were observed in 21 patients with CPHD. More than half of these patients had the history of breech delivery. Three patients with GH-1 gene mutation showed normal pituitary MRI, whereas one of two patients with Pit-1 mutation showed pituitary atrophy and narrowing of pituitary stalk. In conclusion, Severe Cases tended to have CPHD, and the incidence of Severe Case was only 0.6% of total IGHD. Although GHD due to genetic disorders is considered to be extremely rare (0.06% of total IGHD), the incidence reaches high levels (9.8%) among Severe Cases. Growth disorders in these Severe Cases seem to occur soon after delivery. Much earlier diagnosis and hGH treatment are desirable to attain better final height in the Severe Cases. GH-1 and Pit-1 gene analyses are crucial, when genetic abnormalities other than achondroplasia are suspected.

Deletions or mutations in the gene encoding the basolateral chloride channel CLC-Kb (CLCNKB) cause classic Bartter syndrome (MIM 602023), which is characterized by hypokalemic metabolic alkalosis, hyperreninemic hyperaldosteronism and hypercalciura. These patients are usually diagnosed during infancy or childhood due to failure to thrive and growth retardation. The purpose of this study was to investigate the underlying mutations in Japanese patients with classic Bartter syndrome. Seven Japanese patients from seven different families diagnosed as having classic Bartter syndrome were studied. Analysis of CLCNKB demonstrated a large deletion in two patients, a partial deletion in one patient and two mutations (DeltaL130 in exon 4 and W610X in exon 16) in the remaining four patients. DeltaL130 is a novel mutation, but W610X was previously reported in three unrelated Japanese patients. Six out of the seven patients were diagnosed due to typical characteristics of classic Bartter syndrome such as failure to thrive and poor weight gain however, one patient was asymptomatic with mild hypokalemia. In conclusion, we identified a novel mutation of the CLCNKB gene, DeltaL130. We did not determine whether the W610X mutation in our patients was from a common ancestor or if this mutation is frequent in Japan.

Aim: To evaluate the usefulness of pancreatic ultrasonography in diagnosing Shwachman-Bodian-Diamond syndrome (SBDS). Methods: Diagnostic methods in two infants with SBDS were retrospectively reviewed. Results: Patient 1 first presented with hepatic steatosis in the early infantile period, and later developed myelodysplastic syndrome. Patient 2 presented with frequent infections and elevated liver function tests without any haematological alterations. Both patients were considered to have impaired exocrine pancreatic function. Abdominal ultrasonography (US) obtained at 9 mo for patient 1 and at 10 mo for patient 2 showed diffuse high echogenicity, which raised suspicions of SBDS. SBDS gene analysis confirmed SBDS in both patients. Conclusion: Pancreatic US is sensitive in detecting pathological change in SBDS patients. It should be applied to the diagnostic approach for patients who show only minor signs of SBDS.

We evaluated the effect of leuprorelin treatment on adult height (AH) and followed recovery of reproductive function in 63 girls and 13 boys with central precocious puberty (CPP). Mean treatment durations were 3.8 ± 2.0 and 4.1 ± 2.5 yr, and posttreatment follow-up durations were 3.5 ± 1.3 and 2.6 ± 1.1 yr for girls and boys, respectively. AH was 154.5 ± 5.7 cm for girls, and 89.5% of girls reached AH within their target height range. For boys, AH was 163.2 ± 13.0 cm, and 90.9% reached target height range. It appeared that the Bayley-Pinneau method, modified for Japanese children, using a table for advanced bone age (BA), overestimated AH in CPP; and this method, using a table for average BA and projected height for BA, was suitable for prediction of AH in CPP. Menarche or remenarche occurred in 96.8% of girls at the age of 13.1 ± 1.5 yr. Of 11 girls who contributed urine samples, all seven idiopathic and two organic cases were considered to have ovulation. Serum testosterone levels reached normal adult level in all boys. In conclusion, long-term leuprorelin treatment for children with CPP improved AH and had no adverse effects on recovery of reproductive function.

We report on molecular and clinical findings in 10 Japanese patients (four males and six females) from eight families (two pairs of siblings and six isolated cases) with Antley-Bixler syndrome accompanied by abnormal genitalia and/or impaired steroidogenesis. Direct sequencing was performed for all the 15 exons of cytochrome P450 oxidoreductase gene (POR), showing two missense mutations (R457H and Y578C), a 24-bp deletion mutation resulting in loss of nine amino acids and creation of one amino acid (L612_W620delinsR), a single bp insertion mutation leading to frameshift (I444fsX449), and a silent mutation (G5G). R457H has previously been shown to be a pathologic mutation, and computerized modeling analyses indicated that the 15A>G for G5G could disturb an exonic splicing enhancer motif, and the remaining three mutations should affect protein conformations. Six patients were compound heterozygotes, and three patients were R457H homozygotes; no mutation was identified on one allele of the remaining one patient. Clinical findings included various degrees of skeletal features, such as brachycephaly, radiohumeral synostosis, and digital joint contractures in patients of both sexes, normal-to-poor masculinization during fetal and pubertal periods in male patients, virilization during fetal life and poor pubertal development without worsening of virilization in female patients, and relatively large height gain and delayed bone age from the pubertal period in patients of both sexes, together with maternal virilization during pregnancy. Blood cholesterol was grossly normal, and endocrine studies revealed defective CYP17A1 and CYP21A2 activities. The results suggest that Antley-Bixler syndrome with abnormal genitalia and/or impaired steroidogenesis is caused by POR mutations, and that clinical features are variable and primarily explained by impaired activities of POR-dependent CYP51A1, CYP17A1, CYP21A2, and CYP19A1.

The Growth Hormone (GH) and Its Related Factors Study Committee of the Foundation for Growth Science, Japan, has been conducting a quality control study for 15 years to improve the equality of diagnosis of GH deficiency. It found that the greatest differences in measured GH values were due to the different potencies of the kit standards, which were primarily adjusted to WHO standards for human GH of pituitary origin. With the collaboration of kit makers and the Study Group of Hypothalamo-Pituitary Disorders of the Ministry of Health, Labor and Welfare, all GH kits in Japan have begun using the same recombinant human GH standard since April 2005. As a result the diagnostic cut-off peak GH has changed from 10 to 6 ng/ml.

BACKGROUND Antley-Bixler syndrome (ABS) is characterized mainly by abnormal skeletal morphogenesis such as craniosynostosis and radiohumeral synostosis, and by ambiguous genitalia in some cases. The mechanisms resulting in these deformities have not been determined. METHODS The adrenal and gonadal function of three Japanese ABS patients were evaluated. Patient 1 (17-year-old-male) had bilateral cryptoorchidism, delayed puberty and symptoms of glucocorticoid deficiency. Patient 2 (14-year-old male) and patient 3 (4-year-old female) presented with emaciation. Additionally, patient 3 had partial labial fusion and common urogenital sinus. In each patient, blood sampling for steroid analysis before and after rapid adrenocorticotropic hormone (ACTH) stimulation was carried out. Additionally, urinary steroids were quantified. Molecular analysis of CYP17 and CYP21A2 were also performed. RESULTS All patients showed elevated basal 17alpha-deoxysteroid levels. Although the 17alpha-deoxysteroid levels further increased after rapid ACTH stimulation, 17alpha-hydroxysteroids including cortisol did not respond, suggesting impaired 17alpha-hydroxylation. Patient 1 and patient 2 showed low adrenal androgen blood levels both before and after rapid ACTH stimulation. Patient 3 showed lower than normal excretions of urinary androgens. Additionally, a prolonged ACTH stimulation in patient 3 failed to elicit significant increase of adrenal androgens. These findings suggested impaired 17,20-lyase activity. In contrast to attenuated 17alpha-hydroxycorticosteroids, notably cortisol, elevated 17alpha-hydroxyprogesterone (17OHP) levels were observed, not only in pubertal patients (1 and 2) but also in prepubertal patient 3, indicating impaired 21-hydroxylation. This assumption was supported by increased urinary 21-deoxycortisol metabolite excretion in patients 2 and 3. With the exception of a heterozygous mutation of CYP17 in one of the patients, other mutations of this gene or CYP21A2 were identified in any of the patients. CONCLUSION Combined decreased 17alpha-hydroxylation, 17,20-lyase activity and 21-hydroxylation was detected in three ABS patients. Considering that the enzymes responsible are all cytochrome P450 enzymes and that another cytochrome P450 enzyme, lanosterol 14alpha-demethylase, has recently been shown to be impaired in an ABS patient, we speculate that dysfunction of a system which commonly regulates cytochrome P 450 activity may be responsible for the ABS phenotype.

Background: Duchenne muscular dystrophy (DMD) is a congenital disease causing progressive deterioration of skeletal and cardiac muscles because of mutations in the dystrophin gene. Supplementation of dystrophin using rAAV is effective to improve pathogenesis of animal models of DMD. However, we have previously reported that local injection of rAAV2 or rAAV8 to canine skeletal muscles without immunosuppression resulted in insufficient transgene expression with potent immune responses. Here we used DMD dog (CXMDJ) to investigate three strategies of inducing immune tolerance to the rAAV vector and transgene expression with rAAV9-microdystrophin (rAAV-µDys).Methods: For fetal transduction, we tried two methods to induce immune tolerance against rAAV and microdystrophin. First, direct injection of rAAV into amniotic fluid at embryonic day 35 (oral ingestion of rAAV). Second, pregnant CXMDJ heterozygote with embryonic day 30 fetuses was injected with rAAV by intravenous injection (trans-placental rAAV transduction). Furthermore, for postnatal transduction, we tried mesenchymal stem cells (MSCs) pretreatment with rAAV transduction. Bone-marrow derived MSCs and rAAV9-Luciferase or rAAV9-µDys were intramuscularly or intravenously injected into the normal or CXMDJ dog at 8 weeks old. Seven days after injection, MSCs were systemically injected again. At 8 days after 1st injection, rAAV9-Luciferase or rAAV9-µDys was intramuscularly or intravenously injected into the same dog. To examine the immune response against rAAV, purified canine peripheral leukocytes were exposed to rAAV9 for 4 hours, and then IFN-γ expression was analyzed using qRT-PCR. Skeletal muscles of the rAAV-Luc or rAAV-µDys injected animals were sampled by biopsy for expression analysis at 4 weeks after rAAV injection.Results: Following the fetal transduction, expression of IFN-γ in the purified peripheral blood leukocytes after the rAAV exposure were not induced in both of the rAAV oral ingestion and trans-placental transduced dogs, suggesting the successful induction of immune tolerance against rAAV. rAAV-derived microdystrophin expression were confirmed by immunohistochemistry in the transduced affected dogs from additional rAAV injection in both methods. In normal or CXMDJ puppy, administration of rAAV-Luc or rAAV-µDys following MSCs treatment resulted in higher expression of transgene, compared to the rAAV transduction alone. Expression of IFNγ in the purified peripheral blood leukocytes after the rAAV exposure were not enhanced in the rAAV with MSCs, suggesting the immune suppressive effects of the MSCs.Conclusion: Our results demonstrate that induction of immune tolerance against rAAV and/or transgene can be achieved both by fetal or postnatal rAAV injections. These strategies would be effective approach to analyze the expression and function of transgene in vivo. These findings also support the future feasibilities of rAAV-mediated protein supplementation strategies.

In order to estimate the mortality in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21OHD) diagnosed after the introduction of a newborn screening program in Japan, a questionnaire was sent to all members of the Japanese Society for Pediatric Endocrinology. Three hundred and eighty two out of 960 questionnaires were available for the analysis. Twelve deaths were reported. Two died in an early neonatal period, and 10 patients were on maintenance therapy. This suggests that mortality is estimated to be one in 25-80 21OHD patients on maintenance therapy in childhood. It was speculated that cortisol deficiency somewhat contributed to their deaths. Of note, 4 died during a gastrointestinal infection. The need to increase hydrocortisone doses, and the importance of parenteral hydrocortisone administration during stress or illnesses should be repeatedly informed to carers and their physicians.

UNLABELLED The optimal strategy in neonatal screening for congenital hypothyroidism is still a subject of controversy. In Kanagawa Prefecture in Japan, simultaneous thyroid-stimulating hormone (TSH) and T4/fT4 determination has been used, while the results of our program may provide valuable information. Cumulative findings were analysed to determine the type and frequency of thyroid disorders in infants detected by simultaneous TSH and T4/fT4 determination, and the TSH and T4/fT4 screening strategy was validated. A total of 1284130 neonates were screened between October 1979 and September 1997 and infants followed because of low T4/fT4 without elevated TSH (T4 < 51.5 nmol/L or fT4 < 9 pmol/L and TSH < 15 mU/L) were retrospectively analysed. The first survey was carried out within 6 mo of birth and the second in 1998; 258 infants were diagnosed with congenital hypothyroidism at the first medical evaluation, 15 of them with hypothalamo-pituitary hypothyroidism. However, in the second survey, only 8 children were confirmed as having hypothalamo-pituitary hypothyroidism, therefore the incidence detected by the present strategy was 1/160516. Of 8 children with hypothalamo-pituitary hypothyroidism, mental retardation was prevented in 3 owing to early treatment. CONCLUSIONS Simultaneous measurement of TSH and T4/fT4 is a useful strategy for detecting hypothalamo-pituitary hypothyroidism, but more studies are needed to show the cost-benefits of using this strategy.

We report 4 female patients and 1 male patient with a 22q11.2 deletion and Graves' disease diagnosed at age 27 months, 7, 10, 17, and 16 years, respectively. The clinical presentations were typical for hyperthyroidism, but 1 female infant had seizures in addition to symptoms of hyperthyroidism. All patients had elevated serum levels of thyroid hormones in association with suppressed thyroid-stimulating hormone levels. From these observations, we suggest that Graves' disease may be a part of the clinical spectrum associated with the 22q11.2 deletion syndrome.

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/Purpose: Congenital absence of the portal vein (CAPV) is a subtype of congenital portosystemic shunt, which can cause a broad spectrum of clinical manifestations. The authors report on 4 patients with CAPV including a boy with CAPV-associated encephalopathy, which was resolved effectively by liver transplantation (LT). Methods: The case records of 4 pediatric patients with CAPV who were referred to the author's institution between 1984 and 1999 were reviewed. Results: The patients (3 boys and 1 girl) ranged in age at diagnosis from 0.8 to 14 years. Two patients had growth retardation or disturbed consciousness, and the other 2 had no specific manifestations. Not only high serum levels of bile acids, ammonia, and transaminases but also low plasma levels of branched-chain amino acids were common laboratory findings. The absent portal vein was replaced by a large portosystemic shunt, which connected the splanchnic vein to the inferior vena cava or the left renal vein. Two patients survived without any symptoms, but 1 with growth retardation died of hepatic failure. The other with encephalopathy did not respond to medical therapy and underwent LT, which resolved symptoms and metabolic disorders effectively. Conclusions: Patients with CAPV do not always have a good prognosis. They should be followed up with careful observation of their symptoms, hepatic function, and metabolic abnormalities. LT might be indicated for patients with symptomatic CAPV unresponsive to medical therapy. J Pediatr Surg 36:1026-1031. Copyright © 2001 by W.B. Saunders Company.

A gradual loss of anterior pituitary hormones is suspected in patients treated with irradiation due to brain tumors. Development of growth hormone deficiency (GHD) with age has been documented in patients with idiopathic GHD. A gradual loss of adrenocorticotropic hormone (ACTH) secretion has been also shown in a patient with severe GHD and an invisible pituitary stalk on magnetic resonance imaging (MRI). The purpose of this longitudinal and cross-sectional study was to evaluate the gradual loss of growth hormone (GH) and ACTH in a homogeneous group of patients with hypopituitarism. Twenty-eight patients (23 males, 5 females) from four hospitals were diagnosed as having prenatal or perinatal-onset hypothalamic hypopituitarism. They had an abnormal pituitary stalk on MRI (invisible in 18 patients, thin in 10 patients) without any other organic disease of the brain. Each patient had GHD upon initial evaluation. Height (n=20) was analyzed as standard deviation score (SDS). Longitudinal (n=8) and cross-sectional (n=28) GH secretion capacity was evaluated by GH peaks, in response to insulin tolerance test (ITT) and growth hormone releasing factor test (GRF test). Longitudinal (n=10) and cross-sectional (n=28) ACTH secretion capacity was evaluated by cortisol peaks in response to ITT. Height SDS decreased each year in all the untreated patients after birth. GH peaks decreased gradually with age. Longitudinal data showed decreased GH peaks with age in seven out of eight patients using ITT and in all four patients using GRF tests. Cortisol peaks also decreased gradually together with signs and symptoms for adrenal deficiency such as general fatigue. Cortisol peaks of less than 414 nmol/L (15 microg/dl) in response to ITT were seen in 24% of the tests before age 10 and 56% before age 25. In conclusion, GHD and ACTH deficiency developed gradually in patients with prenatal or perinatal-onset hypothalamic hypopituitarism who had invisible or thin pituitary stalks examined by MRI.

X-linked hypophosphatemic rickets (XLH) is an X-linked dominant disorder characterized by renal phosphate wasting, abnormal vitamin D metabolism, and defects of bone mineralization. The phosphate-regulating gene on the X-chromosome (PHEX) that is defective in XLH has been cloned, and its location identified at Xp22.1. It has been recognized to be homologous to certain endopeptidases. So far, a variety of PHEX mutations have been identified mainly in European and North American patients with XLH. To analyze the molecular basis of four unrelated Japanese families with XLH, we determined the nucleotide sequence of the PHEX gene of affected members. We detected a new nonsense mutation (R198X) in exon 5, a new 3 nucleotides insertion mutation in exon 12 and a new missense mutation (L160R) in exon 5 as well as a previously reported nonsense mutation in exon 8 (R291X). These results suggest that: 1) PHEX gene mutations are responsible for XLH in Japanese patients, and 2) PHEX gene mutations are heterogeneous in the Japanese population similarly to other ethnic populations.

We report a mother and two daughters with partial Xp monosomy. Clinical assessment for Turner phenotype revealed that the three females manifested low-normal to mild short stature (-1.6 to approximately -2.3 SD) and variable degrees of skeletal features, such as cubitus valgus, short 4th matacarpals, and Madelung deformity, but no soft tissue or visceral anomalies or gonadal dysfunction. Cytogenetic studies for lymphocytes showed that the karyotype was 45,X[3]/46,X,del(X)(p21.1)[27] in the mother and non-mosaic 46,X,del(X)(p21.1) in the two daughters. Fluorescence in situ hybridization and microsatellite analyses for 19 loci/regions on the X chromosome demonstrated that the del(Xp) chromosome was missing SHOX and had the breakpoint between DMD and CYBB. The results are consistent with the recently proposed notion that haploinsufficiency of SHOX results in not only short stature, but also Turner skeletal features in association with maturational effects of gonadal estrogens. The lack of soft tissue or visceral anomalies suggests the presence of the putative lymphogenic gene on the del(Xp) chromosome; the preservation of ovarian function appears to be compatible with meiotic pairing failure being relatively mild.

This study reports on Y chromosomal genotypes of three patients with gonadoblastoma and one patient with gonadoblastoma and mixed germ cell tumor. Molecular analysis for 35 Y chromosomal loci was performed for DNA samples taken from peripheral leukocytes and lymphoblastoid cell lines, showing that the four patients shared the region between DYS267 at interval 4A and DYF50S1 at interval 6D, with the exception of the region around DYS202 at interval 5K. In the patient with gonadoblastoma and mixed germ cell tumor, Y chromosomal material was preserved in the gonadoblastoma but was lost from the mixed germ cell tumor. The results, in conjunction with previous reports, suggest that GBY (gonadoblastoma locus on the Y chromosome) may be located to a roughly 5-Mb pericentromeric region between DYS267 at interval 4A and DYS270 at interval 5A. The presence of Y chromosomal material in gonadoblastoma is consistent with GBY being involved in the development of gonadoblastoma, and the absence of Y chromosomal material in mixed germ cell tumor would be explained as a consequence of Y chromosomal loss from rapidly proliferating gonadal cancer cells.

A case of an 8-year-old Japanese boy with a testicular large cell calcifying Sertoli cell tumor (LCCSCT) is presented. This report appears to be the first Japanese case of LCCSCT. The patient presented with left testicular swelling and gynecomastia. His family history was not contributory; however, his past history was remarkable for a benign myxoid tumor in the nasal cavity, which was removed at the age of 2 months. After removal of the testicular tumor, the gynecomastia disappeared gradually and no recurrence or metastasis developed during a 15 month follow-up period. Although the tumor was initially interpreted as a Leydig cell tumor, a review of the slides after the patient’s past history of nasal myxoid tumor was revealed led us to the diagnosis of LCCSCT. An accurate diagnosis of LCCSCT is crucial because this tumor is occasionally associated with Carney complex, which can comprise various pathological conditions, including cardiac myxoma, that may be life- threatening. Myxoma of Carney complex has been described to occur in the heart, skin, oral cavity and breast in a wide age range, but there have been no reports referring to nasal myxoid tumor associated with Carney complex.

To know the effect of the age factor on growth response to growth hormone (GH) therapy in patients less than three years of age with complete idiopathic growth hormone deficiency (GHD). One hundred thirty-seven prepubertal children with complete idiopathic GHD from the database of the International Growth Cooperative Study (ICGS) of Japan for more than two years were analyzed. The patients were divided into four groups according to age at the start of GH therapy; group 1, nine years. In group 1 the mean birth weight SDS of -0.17 was not the lowest. Patients in group 1 had the lowest HSDS (-4.53) at the start of the therapy (P

We report the case of a 17-year-old boy with delayed puberty, who presented a complexity of clinical problems. An analysis of steroid hormones led to a diagnosis of 17alpha-hydroxylase/17,20-lyase deficiency (17OHD). Unlike typical cases of 17OHD, however, the patient had pubertal development without medical intervention. In addition, he never exhibited the symptoms of mineralocorticoid excess, showing instead the symptoms of glucocorticoid deficiency, including fatigability, emaciation, and weight-loss induced by minor infection. He also had dysmorphic features, which comprised marfanoid habitus, arachnodactyly and putative craniosynostosis. The combination of these malformations substantially resembled that of Shprintzen-Goldberg syndrome. Direct sequencing of the CYPl7 gene did not reveal any significant aberrations in the exons or exon-intron boundaries. We speculate that the association of partial combined 17OHD with the Shprintzen-Goldberg phenotype in the present patient may result from an aberration of a hitherto unknown gene that controls both steroid hormone synthesis and skeletal development.

Duchenne muscular dystrophy (DMD) is a congenital disease causing progressive deterioration of skeletal and cardiac muscles because of mutations in the dystrophin gene. Supplementation of dystrophin using rAAV is effective to improve pathogenesis of animal models of DMD. However, we have previously reported that local injection of rAAV2 or rAAV8 to canine

The phenotypes of chromosomal 22q11.2 microdeletion are quite variable among individuals and hypoparathyroidism (HP) constitutes a definite portion of the clinical spectrum. For the correct diagnosis and pertinent follow up of the HP children due to del22q11.2, we tried to delineate the clinical characteristics of such patients. By employing fluorescence in situ hybridization (FISH) to all the patients diagnosed as HP in our clinic, ten possessed the 22q11.2 microdeletion. Among them, the incidence of cardiac defect (5/10), recurrent infection (1/10) and cleft palate (1/10) was modest. Additionally, seven of them had been diagnosed as HP during the infantile period, when their facial abnormality and intellectual problem had not become evident. Notably, two patients were complicated by Graves disease, while the association of idiopathic thrombocytopenic purpura was also observed in two girls.HP due to del22q11.2 may be misdiagnosed as idiopathic, especially in an infant who lacks apparent complications like cardiac anomaly. They should be closely followed up for auto-immune complications.

The Foundation for Growth Science has been controlling the use of GH by its registration system, which includes a scoring system for the eligibility for GH treatment according to the diagnostic criteria for GH deficiency (GHD) established by the Study Group for Hypothalamo-pituitary Disorder of the Ministry of Health and Welfare. Until 1995, 28,876 patients with GHD (19,432 boys and 9,444 girls) had been registered as eligible for GH treatment. The number of patients registered in a year increased gradually till 1990 due to the unlimited hGH supply by recombinant techniques and the change in the criteria for GH treatment and the number registered became stable after 1990. The frequency of GH-treated patients is calculated to be 55.2/100,000 persons (72.2/100,000 in boys and 37.1/100,000 in girls) in patients born between 1960 and 1990. The highest frequency was 148.4/100,000 persons (191.7/ 100,000 boys and 103.7/100,000 girls) in 1981, when 2,278 patients (1,508 boys and 770 girls) were born. Eligibility for GH treatment is assessed according to the scoring system which is basically dependent on peak GH values in provocation tests so that standardization of GH values measured with the various commercial GH kits is required to avoid inequality of patients' access to the treatment. In samples obtained by GRF test in 10 normal volunteers, hGH was measured with seven human GH (hGH) kits at a laboratory center. Since the RIA value has been used historically for the diagnosis of GHD, the mean of two RIA measurements was selected as the basis for the standardization procedure and the linear regression formula was used for each hGH kit. After the freely available supply of hGH obtained by recombinant DNA techniques, the role of the Foundation for Growth Science has changed to avoid hGH abuse. Even with this regulation, the frequency of registered patients may indicate a tendency to GH overuse.

The optimal timing for discontinuing treatment with a long-acting gonadotropin-releasing hormone (GnRH) analog (TAP-144SR) was investigated in patients with central precocious puberty (CPP). Thirty-five girls with CPP (21 with idiopathic disease and 14 with organic disease) were treated with the analog for 3 to 5 years. No significant differences were seen between the idiopathic and the organic CPP in the suppressive effect of bone maturation. Advancement of bone maturation was noticeably suppressed during the period between bone ages (BA) of 11.0 and 11.9. The height standard deviation score (Ht-SDS) for BA was consistently improved from 10 to 11.5 years of BA, and patients reached peak Ht-SDS at a BA of 11.5 years. The deltaHt-SDS (annual change in Ht-SDS) was noticeably decreased at BA over 12 years in spite of prolongation of the treatment. In the eight patients who have reached final height, the average Ht-SDS was -0.49 at end of the treatment (BA 11.7 years) and the final Ht-SDS was - 1.1 SD, respectively. Predicted adult height at the end of the treatment was significantly higher than the actual final height (P

We report two sporadic cases of a hitherto undescribed skeletal dysplasia with short stature and characteristic facies. The present patients, a 6-year-old girl and a 15-year-old boy, were almost equally affected. Craniofacial anomalies included a sloping forehead, bitemporal bulging, sparse medial eyebrows, a prominent nasal bridge, hypertelorism, proptosis, a beaked nose, hypoplastic alae nasi and a pointed chin. Shallow orbits, short anterior cranial fossae and bitemporal bossing found on skull radiograph corresponded with the facial dysmorphism. Thickening of the dorsum sellae was another hallmark in the skull. Skeletal survey revealed mild osteopenia, interpediculate narrowing of the lumbar spine with short neural arches and, most important, osteopathia striata of the long tubular bones. There was no sclerosis of the craniofacial bones. The clinical and radiological findings in the present patients were overall inconsistent with those of previously known skeletal dysplasias and congenital malformation syndromes, which possess osteopathia striata as a cardinal feature.The unique clinical and radiological constellation of our patients constitutes a hitherto unknown bone dysplasia.

The objective of this study was to discuss the etiology of cerebral infarction in three cases of congenital adrenal hyperplasia (CAH) based on detailed individual case reports. Patients consisted of three children, two female, one male, 3y, 2y 6mon., and 3y 6mon, with CAH who developed cerebral infarction. The possibility of cerebral infarction following hypoglycemia due to adrenal insufficiency can be considered but the evidence is not conclusive. The combination of cerebral infarction, a rare condition among children, and CAH might not be accidental but suggests the possibility that CAH itself or treatment with glucocorticoid and/or mineralocorticoid or an inadequate treatment under stressful condition may be the cause of the cerebral infarction.

The final heights of the patients treated with GH at our hospital were analysed. The mean final heights were 163.1±5.6 cm and 151.0±4.4 cm for boys and girls, respectively; 30% of the patients were shorter than -2SD. The mean final heights of the patients with gonadotropin deficiency (boys 165.8±5.4 cm; girls 154.3±3.3 cm) were greater than those of the patients without gonadotropin deficiency (boys 160.9±4.9 cm; girls 148.6±3.6 cm). The mean final height SDS of the patients with complete GH deficiency (maximum peak GH level at provocation tests ≤5 ng/ml) was significantly greater than that of the patients with partial GH deficiency (maximum peak GH >5 and ≤10 ng/ml) or non-endocrine short stature (maximum peak GH levels >10 ng/ml). When the subjects were restricted to those with complete GH deficiency and with gonadotropin deficiency, there was a significant negative correlation between final height SDS and age at the start of GH treatment.

A total of 1840 children and adolescents treated with cyproterone acetate (CPA) to block gonadal function, as a treatment for precocious puberty, short stature and other disorders, were registered to survey for the risk of developing hepatic tumors. Patients responding to follow-up numbered 1552 (85%). The cumulative dose and duration of CPA therapy for boys and girls were 110.4g and 2.6 years, and 122.9 g and 2.8 years, respectively. Among the 1552 patients, five hepatoma cases were found. Four underwent successful surgery and remain alive and well to date. Two of the 5 cases had been given more than 500g, the other 3 more than 1000 g, of CPA. Three had also been given androgens before CPA administration. Although further follow-up is necessary to monitor for the development of adenoma and hepatoma, the risk of developing these tumors among patients to whom limited doses of CPA were administered appears to be negligible. J Epidemiol, 1997 ; 7 : 173-178.