A novel mutation in the human thyroid peroxidase gene resulting in a total iodide organification defect.

In this study we describe a novel mutation of the thyroid peroxidase (TPO) gene that resulted in a total iodide organification defect. TPO activity and thyroxine formation in thyroglobulin in the thyroid gland of the patient were below the limits of detection. However, TPO mRNA was detectable at a similar size and concentration as compared with normal thyroid tissues when measured by Northern blot analysis. Sequence analysis of the TPO gene showed the presence of two mutations, a missense mutation in exon 7 and C insertion in exon 14. These mutations were heterozygous and located in different alleles. The latter mutation has already been reported as one of the mutations of the TPO gene resulting in total iodide organification defect. The former mutation was further analysed by mRNA transfection studies in which mutated mRNA was transfected to CHO-K1 cells by electroporation. The results of transfection studies showed that the cells transfected with mutated mRNA expressed similar size TPO molecules to those of cells transfected with wild-type mRNA but that they lacked TPO activity. The two mutations of the TPO gene resulting in the total iodide organification defect in the patient cosegregated from her parents.

Sequence analysis of the thyrotropin (TSH) receptor gene in congenital primary hypothyroidism associated with TSH unresponsiveness.

Congenital primary hypothyroidism due to thyrotropin (TSH) unresponsiveness is a very rare disorder and only a few cases have been documented previously. To elucidate whether structural abnormalities in the TSH receptor (TSHR) could be a primary underlying mechanism of this disorder, we analyzed nucleotide sequence of the entire coding region of the TSHR gene in three patients diagnosed with congenital primary hypothyroidism associated with TSH unresponsiveness. Diagnosis of TSH unresponsiveness was largely made based on the following criteria: (a) congenital primary hypothyroidism with autosomal recessive inheritance, (b) a nongoitrous thyroid gland in a normal position with low thyroidal radioactive iodine uptake, (c) normal in vitro TSH bioactivity or absent in vivo response to exogenous TSH, and (d) absence of thyroid autoantibodies. The TSHR cDNA was successfully obtained from RNA of peripheral mononuclear leukocytes with reverse transcription and polymerase chain reaction, and was sequenced directly. Comparison of these nucleotide sequences with the normal TSHR sequence revealed no difference in the predicted amino acid sequence with a heterozygous polymorphism in codon 601 in one patient, indicating absence of TSHR structural abnormalities in these patients. Our results indicate that congenital primary hypothyroidism associated with TSH unresponsiveness is unlikely to be due to mutations in the TSHR-structure gene.

Cytochrome C oxidase-deficient mitochondria in mitochondrial myopathy.

Electron microscopic cytochemistry was used to evaluate the behavior of cytochrome c oxidase (COX) in cultured skin fibroblasts from 4 patients with decreased COX activity (Leigh encephalopathy, fatal infantile COX deficiency). In patients with Leigh encephalopathy, all mitochondria reacted to COX staining either equivocally or negatively, indicating that all mitochondria were abnormal in these patients. In 1 patient with fatal infantile COX deficiency, intercellular heterogeneity of mitochondria was observed by COX staining. In another patient with fatal infantile COX deficiency, intracellular heterogeneity of mitochondria was observed. Patients with Leigh encephalopathy appeared to have a different type of mitochondrial COX deficiency than those with fatal infantile COX deficiency. Our result suggest that these 2 diseases may result from different genetic mechanisms.

Gonadal mixed germ cell tumor combined with a large hemangiomatous lesion in a patient with Turner's syndrome and 45,X/46,X, +mar karyotype.

In this report, we describe bilateral gonadal tumors with characteristic histopathological findings in a patient with Turner's syndrome who had 45,X/46,X, +mar mosaicism. The left gonad contained a gonadoblastoma and a remnant of streak gonad. The right gonad was entirely replaced by a 15x11x7-cm solid and cystic tumor, which was revealed to be a combination of a mixed germ cell tumor and a cavernous hemangiomatous lesion. The latter occupied approximately half of the entire tumor volume, and there was an incomplete boundary between it and the mixed germ cell tumor lesion. To our knowledge, this is the first reported case of Turner's syndrome with a combination of a mixed germ cell tumor and a hemangiomatous lesion in the gonad.

Changes in somatomedin activity in anorexia nervosa.

Somatomedin (SM) activity, GH, T3 and T4 were investigated in 6 girls with anorexia nervosa during hospitalization and at outpatient clinic. On admission, serum T3 (27-62 ng/dl) and SM activity (0.24-0.55 U/ml) were low in all cases, while basal GH was extremely high in 2 cases. A significant negative correlation was found between SM activity and basal GH during the course of treatment (r = -0.61, p less than 0.02). The change in SM activity was related to that of the serum T3 level and a significant positive correlation was found between SM activity and serum T3 (r = 0.80, p less than 0.001). These data suggest that decreased SM activity may suppress the inhibitory effect of SM on GH release and may raise the basal GH level. SM activity is one of the indicators of the nutritional condition in anorexia nervosa as well as the serum T3 concentration.

[Longitudinal studies on gonadotropin levels in patients with Turner's syndrome and patients with prepubertal castration].

Basal and LH-RH induced plasma FSH and LH levels were determined longitudinally in 41 patients aged 4 to 22 years with Turner's syndrome and in 4 male patients with prepubertal castration. In 12 patients with Turner's syndrome over 18 yrs of age without pubertal change, basal and LH-RH induced FSH levels studied at age 11--22 yrs were all significantly increased over normal levels. However, some of these patients had normal basal and LH-RH induced LH levels. In 5 patients with mosaic Turner's syndrome with spontaneous puberty, basal and LH-RH induced FSH and LH levels studied at age 6--12 yrs were always within the normal range for age-matched controls. In 10 patients studied at age 11--18 yrs, basal and LH-RH induced FSH levels were also strikingly increased over normal levels except for one patient. This patient had normal basal FSH and LH levels and serum estradiol level was increased from 49 to 199 pg/ml after HMG test. In 14 patients aged 4--10 years, nine patients had elevated basal FSH levels and abnormally high responses to LH-RH. The remaining 5 patients had normal basal FSH levels, and 3 of them also had normal FSH responses to LH-RH. The data on the 5 patients studied again at the age of greater than 13 yrs rose to high levels in adult castrated ranges. In 24 patients aged 4 to 17 years, 23 patients were thought to have no ovarian function, and one was thought to have spontaneous puberty. In 4 male patients with prepubertal castration, basal and LH-RH induced FSH levels were increased over normal levels after 11 yrs of age. However, basal LH levels in some patients were within the normal range for age-matched controls after 12 yrs of age. From these results, we conclude that basal and LH-RH induced FSH levels may provide definitive evidence of absent ovaries or testes in patients over 11 yrs of age with primary hypogonadism.

Quantitation of urinary gonadotropins in normal children.

A simple and improved method for the quantification of urinary LH and FSH was developed. Urinary gonadotropin concentrations were determined by polyclonal double antibody RIA after ammonium sulfate extraction. Urinary LH and FSH concentrated by ammonium sulfate were coeluted with an iodinated LH and FSH tracer. Gel chromatography of the urine revealed that the majority of immunoreactive LH and FSH were eluted coincident with 125I-LH and 125I-FSH. Good correlation was observed between urinary gonadotropin/creatinine ratios in first morning voided and full 24-h urine collections. Age-dependent changes in urinary LH excretion were significant in normal boys and girls 6-17 y of age. Urinary FSH excretion in these children did not change in an age-dependent fashion.

Monthly urinary LH and FSH secretory patterns in normal children and patients with sexual disorders.

Urinary gonadotropin concentrations were determined by polyclonal double antibody RIA after ammonium sulfate extraction. Good correlation was observed between urinary gonadotropin/creatinine ratios in first morning voided and full 24-h urine collections. Using consecutive 30-d first morning voided urine specimens from normal children and from patients with sexual disorders, we have studied the monthly patterns of nighttime gonadotropin secretion. In normal prepubertal girls, the levels of urinary LH were low with few variations and those of urinary FSH were higher with episodic fluctuations. In early pubertal girls, the levels of urinary LH increased with striking, rhythmic fluctuations. The same changes were seen in urinary FSH. A single big surge of urinary gonadotropins was observed in postmenarcheal girls. In normal boys, the secretory patterns of urinary gonadotropins were similar to those of normal girls, but varied less. In patients with idiopathic precocious puberty, the patterns of urinary gonadotropins were similar to those of normal subjects matched for sexual stage. The measurement of 30-d first morning voided urinary gonadotropins can provide a simple and physiologic test of gonadotropin function in children.

Immunoreactive somatomedin C/insulin-like growth factor I in urine from normal subjects, pituitary dwarfs, and acromegalics.

Using antibodies to somatomedin C/insulin-like growth factor I (SmC) produced in rabbits using the recombinant hormone, we have developed a radioimmunoassay for SmC. Gel-chromatography of urine revealed that the vast majority of immunoreactive SmC was eluted coincident with 125I-SmC and a small portion eluted with fractions having a mol. wt. range of 30,000-40,000. The SmC concentration in urine was determined by radioimmunoassay after ammonium sulfate extraction. Values did not ordinarily exceed 1 ng/ml. When the values from normal subjects were expressed as ng/mg creatinine, high levels were observed in the neonatal period. These values fell rapidly in infancy, declined more gradually in childhood, were slightly elevated at early puberty, and were lowest in adulthood. Urine SmC concentrations in 15 pituitary dwarfs were lower than the averages obtained from agematched control subjects, and six of them showed abnormally low values. Three patients with active acromegaly had high SmC values in urine. In conclusion, 1) SmC, mainly of monomeric form, was immunologically detected in urine. 2) Radioimmunoassay for urine SmC revealed that values varied considerably with age in normal subjects and were partially dependent on the human growth hormone status. However, the full meaning of the findings remains to be elucidated.

Prolonged activation of hypothalamo-pituitary-ovarian axis during early infancy in female patients with salt-losing 21-hydroxylase deficiency.

We observed prolonged genital bleeding during the first 2-3 months after treatment in five of 13 female patients with salt-losing 21-hydroxylase deficiency. Their relatively low concentrations of serum follicle-stimulating hormone and luteinizing hormone before therapy increased rapidly to high levels which were maintained for 1-3 wk and then decreased. The duration of these relatively high levels after therapy was longer in the patients with genital bleeding than those without. Before therapy, there was no release of serum follicle-stimulating hormone and luteinizing hormone following the administration of synthetic luteinizing hormone-releasing hormone in two patients; 1 month after therapy, the response to luteinizing hormone-releasing hormone increased significantly. Serum estradiol increased above 300 pg/ml in four patients with genital bleeding but was less than 175 pg/ml in three patients without bleeding. The etiology of genital bleeding in these female patients may be more prolonged activation of the hypothalamo-pituitary-ovarian axis and a greater increase in the responsiveness of internal genitalia to gonadotropins and sex hormones, perhaps induced by prolonged exposure to excessive adrenal steroids starting before birth.

Monthly urinary gonadotropin and ovarian hormone excretory patterns in normal girls and female patients with idiopathic precocious puberty.

To identify the developmental changes in monthly urinary gonadotropin and ovarian hormone excretion, consecutive 30-d first morning void urinary specimens were collected from 36 normal girls, one normal woman, and 15 female patients with idiopathic precocious puberty. Of these children, three normal girls and three patients with precocious puberty volunteered to collect these specimens on 2-3 occasions over a time interval of 0.5-3.2 y. When sampled, six were early prepubertal, nine late prepubertal, eight early pubertal, eight mid-pubertal, and eight late pubertal normal girls, and six were early pubertal and 14 mid-pubertal patients with precocious puberty. The mean level of monthly urinary LH, FSH, and total estrogen excretions increased with pubertal maturation. In prepuberty, the mean LH level was lower than the mean FSH level, and neither showed significant episodic fluctuations. In early puberty, mean FSH levels increased with remarkable fluctuations, and mean LH levels were low with few variations in the course of a month. At the onset of puberty, gonadotropin excretory patterns underwent specific changes, showing at the same time periodically and every other day fluctuating patterns. Urinary total estrogen and pregnanediol excretion fluctuated independently from these periodic variations in urinary gonadotropins. These patterns were observed in six out of 16 patterns in normal pubertal girls and 10 out of 20 patterns in precocious puberty. Once the urine LH level exceeded the urine FSH level, however, these periodic variations disappeared. The cycle of a normal postmenarcheal girl aged 14 y showed a pattern similar to that of a normal adult. In patients with precocious puberty, the hormonal patterns were similar to those of sexual stage-matched normal girls.

Hyperalaninemia hyperpyruvicemia and lactic acidosis due to pyruvate carboxylase deficiency of the liver; treatment with thiamine and lipoic acid.

A 16-month-old female infant with severe mental and motor retardation, clinically diagnosed as Leigh's encephalomyelopathy, forms the basis of this study. This infant was found to have lactic acidosis, low cerebrospinal fluid glucose, hyperalaninemia, and increased levels of urine lactate, pyruvate and alanine. These laboratory studies suggested an inborn error in gluconeogenesis. Further investigation revealed a low level of hepatic pyruvate carboxylase activity. The patient's elder sister who also had mental and motor deterioration was then also found to have an elevated blood lactate. These two siblings clinically and biochemically showed improvement with treatment consisting of thiamine and lipoic acid.

Neonatal hypophosphatasia with elevated serum parathyroid hormone.

Two cases of neonatal hypophosphatasia are described. In case 1, hypercalcemia developed at 2 1/2 months of age and continued until death at 10 1/2 months of age. Serum calcium levels decreased transiently in response to phosphate supplementation, prednisolone, and calcitonin. Significantly elevated levels of PTH were detected at 2 1/2 months of age. At autopsy, no parathyroid glands were found. In case 2, hypercalcemia was not detected in his course. Elevated level of serum PTH was recorded on the 17th day of life. A post-mortem examination revealed the presence of one normal parathyroid gland.

Measurement of the skin urocanic acid content in normal and histidinemic infants.

The urocanic acid content of the skin was measured photometrically in a large number of normal and histidinemic infants. A very high content was demonstrated in the normal newborn infants, followed by a rapid decrease throughout early infancy. In contrast, 36 measurements in 17 infants with histidinemia revealed a much lower content even in their newborn periods. Thus, the quantification of skin urocanic acid was considered to be simple and useful for confirming the diagnosis of histidinemia, especially in a neonatal mass-screening program.

[Somatomedin (bioassay) response to long-term growth hormone treatment of idiopathic pituitary dwarfism].

Serum somatomedin (SM) activity by bioassay was measured in 46 idiopathic pituitary dwarfs before and during long-term hGH treatment. The mean (+/- SD) pretreatment SM activity was 0.38 +/- 0.17 U/ml. The mean (+/- SD) pretreatment annual growth rate was 3.4 +/- 1.0 cm/year. None of the patients had a SM activity within the normal range of age-matched controls. The mean SM activity 1 day post GH was 1.11 +/- 0.28 U/ml. Neither the mean SM activity nor the growth rate during the entire treatment period up to 8 years decreased with the duration of the treatment, always being more than 1.0 U/ml and 5.0 cm/year respectively. With respect to normal data for chronological and bone age separately, 43.3% and 60% of the SM activity in the patients during hGH treatment attained the levels of age-matched normal controls. The increase in SM activity in the patients with treatment was not related to their growth rate. SM activity measured by bioassay is useful for evaluating clinical response to hGH therapy.

Hunter's syndrome. An ultrastructural study of an autopsy case.

An autopsy case of a 10-year, 8-month-old boy with Hunter's syndrome is reported with emphasis on the ultrastructural findings of almost all the organs, except the brain. Intracytoplasmic inclusion bodies were observed in various organs as follows: nerve cells and glia in the spinal cord, hepatocytes and Kupffer cells in the liver, sinusoidal endothelium of the spleen, proximal tubules, podocytes and epithelium of Bowman's capsule of the kidney, interstitial fibroblast-like cells among cardiac muscle bundles, cardiac valves and aorta, exocrine and endocrine cells of the pancreas, adrenocortical cells, follicular epithelial cells of the thyroid. Leydig cells of the testis, chondrocytes, fibroblasts and endothelium of capillaries throughout the body. Three types of inclusion bodies were morphologically distinguishable. Type 1: clear vacuole, Type 2: zebra body, Type 3: clear vacuole with a lipid-like lamellar structure. The clear vacuole (Type 1) was thought to represent an accumulation of glycosaminoglycans, and the zebra body (Type 2), probably ganglioside. The type 3 inclusion body might be an intermediate and mixed form of the type 1 and type 2 inclusions. Histochemical study also suggested that the type 3 inclusion body contained glycosaminoglycan and a type of lipid.

Serum insulin-like growth factor I (somatomedin-C) level in normal subjects from infancy to adulthood, pituitary dwarfs and normal variant short children.

Serum levels of IGF-I were radioimmunoassayed after acid ethanol extraction in 1075 normal subjects from infants through young adults, and the normal range for each age was established. The mean value for infants which was relatively low increased gradually with age, and rose sharply after that reaching the peak levels at mid adolescence, then it decreased slowly to the young adult levels. Significantly higher mean values were observed in females at the age of 9, 10, 11 and 12 years. Each of 23 cases with pituitary dwarfism exhibited a lower concentration than the lower limit of the bone age matched normal range. All of the 59 normal variant short children except three showed normal values, but the values were distributed over the lower side of the range.

The application of microspectrocytofluorometric measurement of Feulgen nuclear DNA content in experimental tumors of rat submandibular gland, 2. The correlation between proliferative ability and nuclear DNA content of tumor tissue in autotransplanted areas.

We have reported that there is a difference in the variation of the nuclear DNA content of tumor cells among cases of squamous cell carcinoma induced by 9,10-dimethyl-1,2-benzanthracene (DMBA) in the rat submandibular gland. In the present investigation, the relationship between the nuclear DNA content of tumor cells in autotransplanted sections collected from primary lesions of DMBA-induced tumors and their proliferative ability in the subfascial area of the rat abdomen was examined. As a result of autotransplantation, proliferation in the autotransplanted area was observed in 6 of 14 (42.8%) cases of autotransplantation. Five of these had a keratinizing squamous cell carcinoma while the remaining one had a sarcomatoid tumor. The histological type of the tumor of the proliferative lesion in the transplanted area was very similar to that of the tumor tissues in the primary lesion or the transplanted section collected from the primary lesions. In the proliferative group, marked variation of the nuclear DNA content was observed in the tumor cells of the transplanted section. The proliferative index (PI) was high for these tumor cells in this group. There was no variation in the nuclear DNA content in the tumor cells of the nonproliferative group, and the PI was also low. These results were considered to suggest that there was a correlation between the nuclear DNA content of these experimental tumor cell and their proliferative ability in the autotransplanted area. Therefore, the determination of nuclear DNA content by this method can be used as an objective index of the proliferative ability of tumor tissue.

Serum somatomedin activity assayed by the enhancement of proteoglycan sulphation using chick embryo chondrocytes in normal children at various ages and children of short stature.

It was possible to obtain a sufficient amount of homogeneous chondrocytes from 13-day old chick embryo-sterna in an 8-day suspension culture. Serum somatomedin (SM) activity was measured on the basis of ability of serum to stimulate labelled sulphate uptake in these chondrocytes. Somatomedin A and multiplication stimulating activity (MSA) stimulated the sulphation in this assay. Serum SM activity in term cord sera and newborn sera was lower than that in normal adults. Serum SM activity in early infants increased rapidly and attained the levels of that in normal adults within 1 month following birth. Serum SM activity in infants (1 month - 2 years) and pubertal children (10-16 years), when the annual height increment is greatest, was higher than in normal adults. Serum SM activity in children with constituted dwarfism aged 6-10 years and over 10 years was normal and lower, respectively, compared to that in age-matched controls. Pituitary dwarfs had markedly low SM activity compared to age-matched controls. Diagnostic evaluation of serum SM activity in children requires comparison with age-matched controls. Assay for serum SM activity may provide a useful and simple procedure fo differentiating the cause of growth disorders from growth hormone deficiency.