Development of a bioassay system for human growth hormone determination with close correlation to immunoassay.

Serum growth hormone (GH) level is measured largely through immunoassays in clinical practice. However, a few cases with bioinactive and immunoreactive GH have also been reported. We describe here a new bioassay system for GH determination using the BaF/GM cell line, which proliferates in a dose-dependent manner on hGH addition; cell proliferation was blocked by anti-hGH antibody. This bioassay had the lowest detection limit (∼0.02 ng/ml) reported thus far and the highest specificity for GH. The bioassay results were compared with those of an immunoradiometric assay across 163 patient samples in various endocrine states. A close correlation (the ratio of bioactivity/immunoreactivity was 1.04 ± 0.33, mean ± SD) was observed between bioactivity and immunoreactivity in these samples. The newly developed system is a specific, sensitive, easy, and fast bioassay system for GH determination; we consider it useful for evaluating GH bioactivity in various endocrine states.

Glycated albumin but not HbA1c reflects glycaemic control in patients with neonatal diabetes mellitus.

AIMS/HYPOTHESIS: It is difficult to use HbA(1c) as an indicator of glycaemic control in patients with neonatal diabetes mellitus (NDM) because of high levels of fetal haemoglobin (HbF) remaining in the blood. In this study, glycated albumin (GA), which is not affected by HbF, and HbA(1c) were compared to evaluate whether they reflect glycaemic control in patients with NDM. METHODS: This study included five patients with NDM. Age at diagnosis was 38 ± 20 days. Insulin therapy was started in all patients, and levels of GA, HbA(1c) and HbF were measured monthly for 6 months. One-month average preprandial plasma glucose (aPPG) was calculated using self-monitoring of blood glucose. RESULTS: Plasma glucose and GA were elevated (29.7 ± 13.1 mmol/l [n = 5] and 33.3 ± 6.9% [n = 3], respectively) but HbA(1c) was within normal limits (5.4 ± 2.6% [35.5 ± 4.9 mmol/mol]; n = 4) at diagnosis. With diabetes treatment, aPPG (r = -0.565, p = 0.002), GA (r = -0.552, p = 0.003) and HbF (r = -0.855, p < 0.0001) decreased with age, whereas HbA(1c) increased (r = 0.449, p = 0.004). GA was strongly positively correlated with aPPG (r = 0.784, p < 0.0001), while HbA(1c) showed no correlation with aPPG (r = 0.221, p = 0.257) and was significantly inversely correlated with HbF (r = -0.539, p = 0.004). CONCLUSIONS/INTERPRETATION: GA is a useful indicator of glycaemic control in patients with NDM, whereas HbA(1c) is influenced by age-related changes in HbF and does not accurately reflect glycaemic control.

Measurement of glycated hemoglobin and glycated albumin in umbilical cord: evaluation of the glycemic control indicators in neonates.

OBJECTIVE: As neonatal blood contains a high proportion of fetal hemoglobin (HbF), it is difficult to use high-performance liquid chromatography (HPLC) method, latex-immunoturbidimetry (LA) method and enzymatic methods, which determine hemoglobin A(1C) (HbA(1C)) in order to provide the glycemic control indicators of neonates. In this study, we evaluated glycated hemoglobin (GHb) and glycated albumin (GA) as appropriate indicators of the glycemic control in the neonatal period. STUDY DESIGN: Umbilical cord blood samples collected during delivery were subjected to measurements of GHb (HPLC methods using two different instruments, LA method, enzymatic method and affinity method) and serum GA. RESULT: HbA(1C) levels determined by the HPLC method, the LA method and the enzymatic method were as low as <3.0% in all the cases. Although GHb determined by the affinity method was 3.6 ± 0.2%, this method may not measure accurately the values of glycated HbF plus glycated HbA. Serum GA was 9.4 ± 1.1%. CONCLUSION: We speculate that serum GA, but not GHb, could be used as glycemic control indicators in neonates.

Partial paternal uniparental disomy of chromosome 6 in monozygotic twins with transient neonatal diabetes mellitus and macroglossia.

Transient neonatal diabetes mellitus (TNDM) usually develops within the first few weeks of life and resolves at a median age of 3 months. In most of the cases, TNDM is caused by the over-expression of a paternally expressed imprinted PLAGL1 locus on chromosome 6q24. The most frequent manifestation other than TNDM is intrauterine growth retardation (IUGR), and in some cases macroglossia. We investigated monozygotic twins who had macroglossia without IUGR. Both of the twins developed insulin-dependent hyperglycemia within the first week of life, which subsequently resolved. DNA profiling with polymerase chain reaction amplification was performed for polymorphic microsatellite markers of chromosome 6. The six informative markers, located between 6p24 and 6q15, showed normal biparental inheritance. However, the six distal informative markers, located between 6q23.2 and the 6q telomeric region, showed the absence of a maternal allele and the presence of a single paternal allele. The monosomy of the 6q telomeric region was not confirmed by chromosome banding showing 46, XX. These findings provide further evidence that partial paternal uniparental disomy of chromosome 6 (pUPD6) causes TNDM. The phenotypes other than diabetes observed in patients with partial pUPD6 may differ from those observed in patients with complete pUPD6.

A novel mutation (V101A) of the LHX4 gene in a Japanese patient with combined pituitary hormone deficiency.

OBJECTIVE: LHX4, a LIM-homeodomain transcription factor, is required for development of the pituitary and nervous system. Several mutations of the LHX4 gene have been identified in patients with combined pituitary hormone deficiency (CPHD). The objective of the study was to clarify the molecular basis of a Japanese patient of CPHD with a small anterior pituitary and an ectopic posterior pituitary. METHODS: Genomic DNA was extracted from blood samples of the patient. Exons and exon-intron junctions of the LHX4 gene were amplified and sequenced. An expression vector of the mutant LHX4 protein was constructed and its function was analyzed in vitro. RESULTS: A novel missense mutation (V101A) was identified. IN VITRO transfection studies demonstrated that V101A mutant LHX4 was unable to activate the POU1F1 and FSHbeta subunit gene promoter, indicating a loss of function mutation. CONCLUSION: Our results identify a novel loss of function mutation of the LHX4 gene in a Japanese patient with CPHD.

High serum levels of procollagen type III N-terminal amino peptide in patients with congenital heart disease.

OBJECTIVE: The serum concentration of aminoterminal procollagen type III (PIIIP) is considered a useful marker of tissue fibrogenesis. The present study tested the hypothesis that: serum PIIIP levels are elevated in patients with congenital heart disease (CHD) and abnormal haemodynamic loading and/or hypoxaemia; PIIIP levels are associated with the severity of haemodynamic load or hypoxaemia, both of which enhance myocardial fibrosis. METHODS AND RESULTS: Serum PIIIP levels were measured in five groups of CHD patients (42 patients with ventricular septal defect (VSD), 26 with coarctation of the aorta (COA, n = 19) or aortic stenosis (AS, n = 7), 36 with atrial septal defect (ASD), 39 with pulmonary stenosis (PS) and 20 with tetralogy of Fallot (TOF)). PIIIP levels of CHD patients were significantly higher than those of 42 control subjects (p<0.05, each). Serum PIIIP levels increased in parallel with increased ventricular volume load in VSD and ASD, and with the severity of PS. In TOF patients, PIIIP levels correlated negatively with arterial oxygen saturation. Treatment with an angiotensin-converting enzyme inhibitor (ACEI) was associated with low levels of PIIIP in COA/AS patients despite the existing haemodynamic load. CONCLUSION: The increased serum PIIIP levels in proportion to the severity of ventricular load or cyanosis suggest enhanced myocardial synthesis of collagen type III in patients with CHD. Suppression of the PIIIP level by ACEI suggests the involvement of the renin-angiotensin-aldosterone system in myocardial fibrosis. These data provide the basis for the development of new diagnostic and therapeutic strategies in patients with CHD.

Skewed X chromosome inactivation failed to explain the normal phenotype of a carrier female with MECP2 mutation resulting in Rett syndrome.

Mutations in the X-linked MECP2 gene cause Rett syndrome, a neurodevelopmental disorder that exclusively affects girls. Females with the MECP2 mutations exhibit a broad spectrum of clinical presentations ranging from classical Rett syndrome to asymptomatic carriers, which can be explained by differences in X chromosome inactivation (XCI). Here, we report a family with a girl with Rett syndrome in whom a novel missense mutation in the MECP2 gene was transmitted through the maternal germ line. The carrier mother was asymptomatic and presented non-random XCI in the peripheral blood cells, which resulted in the X chromosome harboring the mutant allele that was predominantly active. Thus, the presence of non-random XCI in the peripheral blood cells did not provide an explanation for the normal phenotype of the carrier mother. This result suggests that mechanisms other than XCI may contribute to the phenotypic heterogeneity associated with MECP2 mutations.

Heterozygous mutation in the cholesterol side chain cleavage enzyme (p450scc) gene in a patient with 46,XY sex reversal and adrenal insufficiency.

Cytochrome P450scc, the mitochondrial cholesterol side chain cleavage enzyme, is the only enzyme that catalyzes the conversion of cholesterol to pregnenolone and, thus, is required for the biosynthesis of all steroid hormones. Congenital lipoid adrenal hyperplasia is a severe disorder of steroidogenesis in which cholesterol accumulates within steroidogenic cells and the synthesis of all adrenal and gonadal steroids is impaired, hormonally suggesting a disorder in P450scc. However, congenital lipoid adrenal hyperplasia is caused by mutations in the steroidogenic acute regulatory protein StAR; it has been thought that P450scc mutations are incompatible with human term gestation, because P450scc is needed for placental biosynthesis of progesterone, which is required to maintain pregnancy. In studying patients with congenital lipoid adrenal hyperplasia, we identified an individual with normal StAR and SF-1 genes and a heterozygous mutation in P450scc. The mutation was found in multiple cell types, but neither parent carried the mutation, suggesting it arose de novo during meiosis, before fertilization. The patient was atypical for congenital lipoid adrenal hyperplasia, having survived for 4 yr without hormonal replacement before experiencing life-threatening adrenal insufficiency. The P450scc mutation, an in-frame insertion of Gly and Asp between Asp271 and Val272, was inserted into a catalytically active fusion protein of the P450scc system (H2N-P450scc-Adrenodoxin Reductase-Adrenodoxin-COOH), completely inactivating enzymatic activity. Cotransfection of wild-type and mutant vectors showed that the mutation did not exert a dominant negative effect. Because P450scc is normally a slow and inefficient enzyme, we propose that P450scc haploinsufficiency results in subnormal responses to ACTH, so that recurrent ACTH stimulation leads to a slow accumulation of adrenal cholesterol, eventually causing cellular damage. Thus, although homozygous absence of P450scc should be incompatible with term gestation, haploinsufficiency of P450scc causes a late-onset form of congenital lipoid adrenal hyperplasia that can be explained by the same two-hit model that has been validated for congenital lipoid adrenal hyperplasia caused by StAR deficiency.

46, XY pure gonadal dysgenesis: a case with Graves' disease and exceptionally tall stature.

A case of 46, XY pure gonadal dysgenesis with very tall stature was investigated. The 24-year-old, phenotypically female patient consulted our clinic because of linear growth persisting into adulthood. The patient was found to have no mutation or deletion of a sex-determining region of the Y chromosome, and also was found to have Graves' disease. Growth was arrested with height remaining at 187 cm after normalization of the thyroid function by treatment with an antithyroid agent, although follow-up to monitor growth was limited to 3 months. In some cases of gonadal dysgenesis, then, Graves' disease may contribute to an abnormally tall stature.

Two mutations of the Gsalpha gene in two Japanese patients with sporadic pseudohypoparathyroidism type Ia.

Pseudohypoparathyroidism Ia (PHP-Ia), is an inherited disease with clinical hypoparathyroidism caused by parathyroid hormone resistance (PTH), and shows the phenotype of Albright hereditary osteodystrophy (AHO), including short stature, obesity, round face, brachydactyly, and subcutaneous ossification. This disease is caused by mutation that inactivates the alpha-subunit of Gs, the stimulatory regulator of adenylyl cyclase. Here, a novel frameshift mutation (delG at codon 88) in exon 4, and a missense mutation (R231H) in exon 9 of the Gsalpha gene were identified in two Japanese patients with sporadic PHP-Ia. Deletion of a G in exon 4 at codon 88 in the first patient produced a premature stop codon, resulting in the truncated protein. The second patient had a previously reported R231H mutation. Because this amino acid is located in a region, switch 2, that is thought to interact with the betagamma subunit of Gsalpha protein, this mutation may impair Gs protein function. We report here one novel Gsalpha mutation, and note that mutations in Japanese patients with PHP-Ia are probably heterogeneous.

RIP 140 modulates transcription of the steroidogenic acute regulatory protein gene through interactions with both SF-1 and DAX-1.

Coregulators have been suggested to act as a bridging apparatus between nuclear receptors and the transcriptional machinery. The orphan receptor SF-1 plays a role in controlling the basal and cAMP-stimulated expression of the human steroidogenic acute regulatory protein gene. DAX-1 is the gene responsible for X-linked adrenal hypoplasia congenita and blocks steroid biosynthesis by impairing the expression of steroidogenic acute regulatory protein. In the present study we examined the role of coregulators in the actions of SF-1 and DAX-1 on the human steroidogenic acute regulatory protein promoter. We found that the coregulator RIP 140 interacts with SF-1 in the yeast two-hybrid system. Glutathione-S-transferase pull-down assays and coimmunoprecipitations confirmed the interaction between RIP 140 and SF-1. RIP 140 was also shown to interact with DAX-1. When an RIP 140 expression vector was introduced into Y-1 cells, basal and cAMP-stimulated human steroidogenic acute regulatory protein promoter activities decreased. The inhibitory effect of RIP 140 on human steroidogenic acute regulatory protein promoter activity was dependent upon the presence of SF-1. The cAMP response of an SF-1 response element was inhibited by both RIP 140 and DAX-1 expression vectors at low concentrations of plasmids. We conclude that RIP 140 binds to the orphan nuclear receptor SF-1 and DAX-1 and modulates their actions on the human steroidogenic acute regulatory protein promoter.

Compound heterozygous mutations in the gamma subunit gene of ENaC (1627delG and 1570-1G-->A) in one sporadic Japanese patient with a systemic form of pseudohypoaldosteronism type 1.

The systemic form of pseudohypoaldosteronism type 1 (PHA1) is a rare autosomal recessive disorder with salt-wasting, hyperkalemia, metabolic acidosis, and multiorgan aldosterone unresponsiveness. Recently, this form of PHA1 was found to be caused by the loss-of-function mutations in the gene of each subunit (alpha, beta, and gamma) of the epithelial sodium channel (ENaC). To investigate the molecular basis of one sporadic Japanese patient with a systemic form of PHA1, we determined the nucleotide sequence of the genes of every subunit of ENaC of this patient. The patient was found to be a compound heterozygote for one base deletion in exon 12 (1627delG) in combination with 1570-1-->GA substitution at the 5' splice acceptor site of intron 11 in the gamma subunit gene of ENaC. The 1627delG mutation altered a reading frame, resulting in a premature stop codon in exon 12. Messenger RNA from the allele harboring the splice site mutation was not identified by RT-PCR. In conclusion, two novel mutations in the gamma subunit gene of ENaC caused systemic PHA1 in the sporadic Japanese patient. Identification of the molecular basis of PHA1 is helpful for early diagnosis and understanding the pathophysiology of the disease.

Three novel PHEX gene mutations in Japanese patients with X-linked hypophosphatemic rickets.

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.

Gonadotropin-releasing hormone analog therapy failed to improve predicted final height in two children with central precocious puberty and microcephalus.

Long-acting gonadotropin-releasing hormone (GnRH) analog treatment for central precocious puberty (CPP) suppresses excessive bone maturation by inhibiting the pituitary-gonadal axis, and usually assures favorable results for growth potential. Recently, we encountered two children with CPP and microcephalus in whom GnRH analog therapy arrested pubertal development, but could not suppress bone age maturation effectively. Eventually, their final height deteriorated rather than improved. The reason why these two cases did not respond to GnRH analog therapy remains unknown. However, microcephalus and minor cerebral anomalies may have some links to deterioration of final height. Our cases suggest that careful evaluation will be required especially for CPP with microcephalus throughout treatment with GnRH analog.

A novel missense mutation in the HMG box region of the SRY gene in a Japanese patient with an XY sex reversal.

The sex-determining region of the Y chromosome, the SRY gene, located on the short arm of the Y chromosome, is appreciated as one of the genes that is responsible for directing the process of sex differentiation. To date, 34 different mutations, including 29 missense and nonsense mutations in the SRY gene, have been described in XY female patients. We investigated the molecular basis of the sex reversal in one Japanese XY female patient by determining the nucleotide sequence of the SRY gene, using polymerase chain reaction and direct sequencing. We identified a novel mutation, of the substitution of Tyr for Asn at nucleotide position 87 (N87Y). This Asn residue is located within the DNA-binding high-mobility-group (HMG) motif, which is considered to be the main functional domain of the SRY protein. Further, this amino acid, Asn, is a conserved residue among mammalian SRY genes. These findings indicate that this amino acid substitution may be responsible for the sex reversal in this patient.

Establishment and characterization of a culture system for enzymatically released rat dental pulp cells.

To establish a cell culture system that reflects the dentin formation in dental pulp tissue, we used dental pulp cells enzymatically isolated from rat incisor teeth. During the 20-day culture period, the cells exhibited various phenotypes of the odontoblast differentiation process, from the immature stage to the terminal mineralization stage. The cells began to form the mineralized nodules from day 10, and the nodules became larger by day 20. Alkaline phosphatase (ALP)-positive cells surrounded the mineralized nodules. The ALP activity in the cell layers was maximal on day 5, and gradually decreased to day 20. The calcium content in the cell layers was very low by day 10, and significantly increased from day 15. Sulfated glycosamino-glycans (GAGs) contained in the cell layers increased by day 15, but the content then decreased by day 20. The dental pulp cells produced a small amount of osteocalcin that was released into the culture medium by day 10, and the amount secreted increased markedly from day 15. The expression of osteocalcin and parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor mRNA was evident as early as day 5, and the expression of each gradually increased with the number of days in culture. Dentin matrix protein (Dmp1) mRNA gene transcripts were identified by use of the reverse transcription polymerase chain reaction (RT-PCR) in the cells throughout the culture period. The present results demonstrate that this culture system is useful for studying the differentiation process of the odontoblast-like cells.

A novel missense mutation of mineralocorticoid receptor gene in one Japanese family with a renal form of pseudohypoaldosteronism type 1.

Pseudohypoaldosteronism type 1 (PHA1) is a rare condition characterized by neonatal salt loss with dehydration, hypotension, hyperkalemia, and metabolic acidosis, despite elevated plasma aldosterone levels and PRA. Two modes of inheritance of PHA1 have been described: an autosomal dominant form and an autosomal recessive form. An autosomal recessive form manifests severe life-long salt wasting resulting from multiple mineralocorticoid target tissue such as sweat, salivary glands, the colonic epithelium, and lung. Contrary, an autosomal dominant PHA1 manifests milder salt wasting that gradually improves with advancing age. Recently, in one sporadic and four dominant cases, four different mutations including two frame shift mutations, two premature termination codons, and one splice site mutation in the mineralocorticoid receptor (MR) gene were identified. We studied the molecular mechanisms of one Japanese family with a renal form of PHA1. PCR and direct sequencing of the MR gene identified a heterozygous point mutation changing codon 924 Leu (CTG) to CCG (Pro) (L924P) in all affected members. COS-1 cells were transfected with expression vectors for either wild type or the mutant MR-L924P receptors, together with the reporter plasmid (glucocorticoid response element tk-CAT). Aldosterone increased CAT activity in cells expressing wild-type receptor, but had no effect in cells expressing the mutant receptors. These results suggest that mineralocorticoid resistance in this family is due to a missense mutation in the MR gene. To our knowledge, this is the first case of the missense mutation of the MR gene in renal PHA1.

Pharmacokinetics and metabolic effects of high-dose growth hormone administration in healthy adult men.

To evaluate pharmacokinetics of growth hormone (GH) and its effects on IGF-I, glucose, insulin, nonesterified fatty acid (NEFA) and triglyceride (TG), fifteen Japanese healthy adult male volunteers (20-27 years old) were studied. The subjects were divided into three groups, and received with a single s.c. injection of 0.075, 0.15 and 0.30 IU/kg of GH, respectively. The subjects assigned to receive 0.30 IU/kg were administered for additional 6 days. After a single administration of GH, Cmax and AUC of GH were increased in a dose-dependent manner. There was a significant positive correlation between the AUC and the T1/2 (r=0.516, P<0.05). Total body clearance was significantly greater in 0.075 IU/kg group than the other groups and showed a significant negative correlation with Cmax (r=-0.694, P<0.005) and AUC (r=-0.723, P<0.005). After a single administration of each dose, serum IGF-I concentrations were increased gradually. In the repeated administered group (0.30 IU/kg), IGF-I concentrations almost reached a plateau at a significantly high level four days after the start of administration and remained at a high level (786-405.4 ng/ml) until day 8. There was no significant difference in diurnal change of blood glucose and serum insulin after a single administration of GH among three groups. In the 0.3 U/kg group, there was no significant difference in diurnal change of blood glucose between day 1 and day 7, but serum insulin level was significantly higher in day 7 than in day 1 (P<0.01). Serum concentrations of NEFA were increased over time after administration in all subjects administered once or repeatedly. TG concentrations showed no changes after single administration of each dose level, but were significantly increased on day 7 in the subjects repeatedly treated with 0.30 IU/kg/day. This effect is speculated to be caused by high dose GH treatment. The above findings demonstrated that higher GH dose significantly influences on carbohydrate and lipid metabolism. It remains necessary to elucidate what kinds of effects of the long-lasting increased levels of insulin and triglyceride, even if reversible, would have on glucose and lipid metabolism.