Katsuaki Motomura

Cytochrome P450 oxidoreductase deficiency: identification and characterization of biallelic mutations and genotype-phenotype correlations in 35 Japanese patients.

CONTEXT Cytochrome P450 oxidoreductase (POR) deficiency is a rare autosomal recessive disorder characterized by skeletal dysplasia, adrenal dysfunction, disorders of sex development (DSD), and maternal virilization during pregnancy. Although multiple studies have been performed for this condition, several matters remain to be clarified, including the presence of manifesting heterozygosity and the underlying factors for clinical variability. OBJECTIVE The objective of the study was to examine such unresolved matters by detailed molecular studies and genotype-phenotype correlations. PATIENTS Thirty-five Japanese patients with POR deficiency participated in the study. RESULTS Mutation analysis revealed homozygosity for R457H in cases 1-14 (group A), compound heterozygosity for R457H and one apparently null mutation in cases 15-28 (group B), and other combinations of mutations in cases 29-35 (group C). In particular, FISH and RT-PCR sequencing analyses revealed an intragenic microdeletion in one apparent R457H homozygote, transcription failure of apparently normal alleles in three R457H heterozygotes, and nonsense mediated mRNA decay in two frameshift mutation-positive cases examined. Genotype-phenotype correlations indicated that skeletal features were definitely more severe, and adrenal dysfunction, 46,XY DSD, and pubertal failure were somewhat more severe in group B than group A, whereas 46,XX DSD and maternal virilization during pregnancy were similar between two groups. Notable findings also included the contrast between infrequent occurrence of 46,XY DSD and invariable occurrence of 46,XX DSD and pubertal growth pattern in group A mimicking that of aromatase deficiency. CONCLUSIONS The results argue against the heterozygote manifestation and suggest that the residual POR activity reflected by the R457H dosage constitutes the underlying factor for clinical variability in some features but not other features, probably due to the simplicity and complexity of POR-dependent metabolic pathways relevant to each phenotype.

Expression of parathyroid hormone-related protein in rat articular cartilage

Expression and localization of parathyroid hormone-related protein (PTHrP) in rat articular cartilage during fetal and postnatal periods were investigated by immunohistochemistry and in situ hybridization. PHTrP displayed distinct distribution and intensity of staining at different ages. In fetal (18-day-old) and young (3-week-old) rats, articular chondrocytes expressed abundant PTHrP throughout the entire thickness of cartilage. In contrast, in 60-week-old rats, PTHrP was expressed in a few articular chondrocytes of superficial and middle layers. Regulation of PTHrP and PTH/PTHrP receptor mRNA was also studied in cultured rat articular chondrocytes. Northern blot analysis revealed that both transforming growth factor-β (TGF-β), an important stimulator for chondrocyte proliferation and differentiation, and 10% fetal bovine serum (FBS) stimulated the expression of PTHrP mRNA with down-regulation of its receptor mRNA. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) down-regulated the expression of receptor without changes of PTHrP mRNA level. These results suggest that the changes in abundance and localization of PTHrP and its receptor may be directly involved in the cell growth and differentiation of articular cartilage.

Mutation and Gene Copy Number Analyses of Six Pituitary Transcription Factor Genes in 71 Patients with Combined Pituitary Hormone Deficiency: Identification of a Single Patient with LHX4 Deletion

CONTEXT Mutations of multiple transcription factor genes involved in pituitary development have been identified in a minor portion of patients with combined pituitary hormone deficiency (CPHD). However, copy number aberrations involving such genes have been poorly investigated in patients with CPHD. OBJECTIVE We aimed to report the results of mutation and gene copy number analyses in patients with CPHD. SUBJECTS AND METHODS Seventy-one Japanese patients with CPHD were examined for mutations and gene copy number aberrations affecting POU1F1, PROP1, HESX1, LHX3, LHX4, and SOX3 by PCR-direct sequencing and multiplex ligation-dependent probe amplification. When a deletion was indicated, it was further studied by fluorescence in situ hybridization, oligoarray comparative genomic hybridization, and serial sequencing for long PCR products encompassing the deletion junction. RESULTS We identified a de novo heterozygous 522,009-bp deletion involving LHX4 in a patient with CPHD (GH, TSH, PRL, LH, and FSH deficiencies), anterior pituitary hypoplasia, ectopic posterior pituitary, and underdeveloped sella turcica. We also identified five novel heterozygous missense substitutions (p.V201I and p.H387P in LHX4, p.T63M and p.A322T in LHX3, and p.V53L in SOX3) that were assessed as rare variants by sequencing analyses for control subjects and available parents and by functional studies and in silico analyses. CONCLUSIONS The results imply the rarity of abnormalities affecting the six genes in patients with CPHD and the significance of the gene copy number analysis in such patients.

MECHANISMS OF THYROID HORMONE ACTION: Implications for the Clinical Manifestation of Thyrotoxicosis

Serum thyroid hormone concentrations alone do not explain the variability and severity of the range of symptoms observed in thyrotoxic patients. Despite gaps in our understanding of the links between the clinical manifestations of thyrotoxicosis and the underlying mechanisms, much has been learned. A limited number of markers directly reflect T3 action. The future elucidation of T3 targets that mediate these effects should ultimately lead to additional clinical markers of tissue-specific T3 action. The availability of such tests should allow for more specific treatment of individual patients.

DAX-1 Gene Mutations and Deletions in Japanese Patients with Adrenal Hypoplasia Congenita and Hypogonadotropic Hypogonadism

Abnormality of the DAX-1 gene accounts for many instances of congenital adrenal hypoplasia. In the present study, we performed molecular genetic analysis of DAX-1 in 4 unrelated Japanese patients with adrenal hypoplasia congenita and hypogonadotropic hypogonadism. A double-point mutation for V126M and W171X was identified in 1 family and a complex de novo insertion-deletion mutation was identified in a second. The DAX-1 gene was entirely deleted in a 3rd patient as well as in a 4th with the additional feature of glycerol kinase deficiency.

Increased proliferative activity of osteoblasts in congenital hemifacial hypertrophy

Congenital hemifacial hypertrophy is expressed in facial asymmetry resulting from unilateral overgrowth. We examined proliferative activity of primary cultured osteoblasts taken from the hemifacial hypertrophic side of an 18-year-old woman compared with the normal side. Both fetal calf serum and basic fibroblast growth factor stimulated osteoblast DNA synthesis, and cultured osteoblast proliferation was markedly increased in the hypertrophic side. Neutralizing antibody against basic fibroblast growth factor was effective to partially block fetal calf serum-induced DNA synthesis. These results suggest that fibroblast growth factor and its receptor signal transduction axis may be selectively involved in affected osteoblasts, leading to progression of congenital hemifacial hypertrophy.

Impaired induction of heat shock protein implicated in decreased thermotolerance in a temperature-sensitive multinucleated cell line.

Abstract Cells of a temperature-sensitive mutant line (tsFT101) derived from a mouse mammary carcinoma cell line (FM3A) become multinucleated at a non-permissive temperature of 39°C because of disturbed cytokinesis. To explore how this relates to thermotolerance, we examined the proliferative activity of, and heat shock protein (HSP) expression in, FM3A and tsFT101 cells cultured at 37°C and 39°C after heat shock pretreatment (15 min exposure at 45°C). FM3A cells developed thermotolerance when cultured at both 37°C and 39°C, but whereas tsFT101 cells developed thermotolerance at 37°C, this was markedly reduced at 39°C. Western blot analysis showed similar degrees of expression of constitutive HSP70 (HSP73) in FM3A and tsFT101 cells after heat shock pretreatment at both 37°C and 39°C. However, expression of inducible HSP70 (HSP72) was reduced in tsFT101 cells at 39°C compared to 37°C and to FM3A cells at both 37°C and 39°C. Heat shock pretreatment activated DNA binding of heat shock transcription factor (HSF) in FM3A cells at 37°C and 39°C, but only at 37°C in tsFT101 cells. These results indicate that (1) multinucleation caused by disturbed cytokinesis increases temperature sensitivity, (2) HSP70 is critical for the development of thermotolerance in both FM3A and tsFT101 cells, and (3) decreased expression of inducible HSP70 parallels deficient development of thermotolerance in tsFT101 cells cultured at a non-permissive temperature.

A Japanese patient with a mild Lenz–Majewski syndrome

AbstractWe report on a sclerosing bone dysplasia, associated with cutis laxa, enamel dysplasia, and mental retardation. The patient was a 17-year-old Japanese boy of normal height and muscular build. Cutis laxa with prominent veins in the scalp and abdominal wall and delayed eruption of permanent teeth attracted the attention of clinicians in infancy and adolescence, respectively. The clinical manifestations included a progeroid facial appearance with prognathism, wrinkled skin, and interdigital webbing. The intelligence quotient was estimated at 60. Enamel dysplasia was histologically confirmed. Skeletal changes included calvarial hyperostosis, sclerosis of the skull base, an enlarged, sclerotic mandible, broad clavicles and ribs, and diaphyseal undermodeling of the tubular bones. Metaepiphyseal sclerosis or longitudinal striation was found in the long bones. Metaphyseal equivalents of the axial skeleton showed dense osteosclerosis. These clinical and radiological manifestations overlapped with those of Lenz-Majewski syndrome. Unlike the classical phenotype of the disorder, however, he did not show brachymesophalangy with proximal symphalangism or growth failure. The present case may be considered to fall in the mildest end in the phenotypic continuum of Lenz-Majewski syndrome, suggesting that the clinical spectrum of the disorder may be broader than currently thought.

Profilin gene expression and regulation in a temperature-sensitive breast cancer cell line: tsFT101

Abstract The temperature-sensitive mutant cells (tsFT101) derived from a mouse mammary carcinoma cell line, FM3A, become multinucleated at a non-permissive temperature of 39°C. To further understand the molecular mechanism of such cytokinetic disturbance, we examined the expression of profilin, the main regulator of the transition of globular actin (G-actin) to filamentous actin (F-actin). RT-PCR analysis of mouse profilin cDNA from tsFT101 showed a point mutation (177 A → G) which was a wobble mutation causing no change in the encoded amino acid. The expression level of profilin mRNA was, however, diminished in cultured tsFT101 cells under non-permissive temperatures compared with wild-type FM3A cells in association with multinucleation. A stable transfection of profilin cDNA expression vector to tsFT101 cells prevented multinuclear cell formation when cultured at 39°C. In contrast, antisense profilin cDNA expression vector did not alter multinuclear cell formation. The primary cause of the cytokinetic disturbance of tsFT101 cells may be due to the diminished level of profilin gene expression.

Molecular Chaperone and Folding: Lessons from Heat Shock Protein Research

Regulation of heat shock protein (HSP) is a subject that has generated much literature over the years. It is beyond the scope of this review to consider in detail the interrelation between HSPs and various peptides; instead, current knowledge on HSP and its related peptides is highlighted with special references. The controversy surrounding the mechanism of newly synthesized protein-chaperon and folding is discussed.