Nozomi Shinohara

Novel missense mutation (Leu466Arg) of the DAX1 gene in a patient with X-linked congenital adrenal hypoplasia.

We identified a DAX1 missense mutation, a substitution of arginine for leucine at codon 466 (Leu466Arg), in an infant with X-linked congenital adrenal hypoplasia (AHC). A heterozygous substitution, Leu466Arg, was also identified in his mother and sister. Since leucine at position 466 is well conserved among other orphan nuclear hormone receptor superfamilies and Leu466Arg was not detected among 50 normal Japanese control individuals, the mutation is most likely responsible for X-linked AHC. It is interesting to note that Leu466Arg among all mutations ever reported is located at the most C-terminal region of the DAX-1 protein. Most mutations identified previously were located in the C-terminal presumptive ligand binding domain. Hence, the C-terminal end of the DAX-1 protein may play an important role in the biological function, such as in normal adrenal embryogenesis.

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.

Near-normal linear growth in the setting of markedly reduced growth hormone and IGF-1. A case report.

A 14.2-year-old prepubertal boy diagnosed with complete-type growth hormone deficiency and tertiary hypothyroidism, keeps growing in the height range between –1 and –2 SD. He has been treated with levothyroxine only. To understand the growth mechanism of this boy, we analyzed the serum growth hormone (GH) with a radioimmunoassay (RIA), serum GH bioactivity with Nb2 and erythroid progenitor cell bioassays, and growth hormone-binding protein (GHBP) with a ligand-mediated immunofunctional assay (LIFA). In addition, IGF-1 and free IGF-1 were analyzed by immunoradiometric assay (IRMA) and insulin-like growth factor-binding protein-3 (IGFBP-3) by Western immunoblot. Peak GH-RIA responses to insulin, arginine and GH-releasing factor, and nocturnal GH secretion, were low (0.5–2.3 ng/ml); bioactive GH was low (0.313 ng/ml), and GHBP was elevated (84 ng/ml). The serum levels of IGF-1 and free IGF-1 were continuously low, 17.1–39.3 and 0.17–0.26 ng/ml, respectively. Moreover, serum IGFBP-3 levels were low (1.68– 1.39 mg/l) and IGFBP-3 protease activity was negative. Prolactin and insulin were in the normal range. The result of the assay for growth-promoting activity showed that the patient’s serum stimulated normal erythroid progenitor cells twice as potently as did healthy thin adult control serum. These results suggest that GH and IGF-1 are not indispensable for maintaining physical growth in this boy. Thus, it appears that circulating GH and IGF-1 are not mandatory requirements for maintaining normal physical growth, and other, as yet uncharacterized, pathways or growth factors might be sufficiently compensatory under certain conditions.

Predominance of the mutation at 1138 of the cDNA for the fibroblast growth factor receptor 3 in Japanese patients with achondroplasia

SummaryFibroblast growth factor receptor 3 (FGFR3) has recently been identified as a putative gene for achondroplasia. Since a guanine to adenine mutation at 1138 of the cDNA for FGFR3 had been identified in most of the patients in Western population, we examined 13 Japanese patients to see if they also share the same mutation. Specific endonuclease digestion of the amplified coding sequence for the transmembrane domain of the FGFR3 revealed that the 12 patients have the G to A change at 1138, while the other had the G to C substitution at the same point, both of which result in G380A substitution. As far as we studied, the homogeneity of the point mutation at 1138 is also authentic to Japanese patient as well as Western patients.

A NEW POINT MUTATION OF SRY GENE IN TWO SISTERS WITH 46 XY GONADAL DYSGENESIS

The sex determining region of Y(SRY) is required for the male sex determination. Recently several mutations of SRY gene have been identified in 46XY gonadal dysgenesis(GD). All mutations reported so far are located within the putative DNA binding motif known as HMG box domain. We investigated SRY gene of four sporadic cases and two sisters in one family with 46XY GD by polymerase chain reaction and single strand conformation polymorphism and subsequent DNA direct sequencing. Four sporadic cases did not show any mutations in SRY gene, while two sisters in one family shared the same one base mutation T to A, which exists out of the putative DNA binding motif region of SRY gene. By this mutation, a codon TTG(Leucine) changes a stop codon TAG. Generation of this stop codon would be expected to make a truncated nonfunctional SRY gene products, and affect DNA binding activity, resulting in 46,XY GD.From these results it is concluded that in addition to the mutations in HMG box domain of SRY gene new mutation reported here cause 46 XY GD. Furthermore, this new mutation in this family will shed light on disclosing the mechanism of genetic transmission in the familiar cases of 46 XY GD.

IGFs AND IGF BINDING PROTEINS IN HUMAN CORD SERA: RELATIONSHIP TO INTRAUTEINE GROWTH

The IGF autocrine/paracrine system is believed to play a major role in the regulation of human fetal growth. We have examined the ontogeny of IGF-I, IGF-II, IGFBP-1, 2, 3 concentrations in fetal development throughout gestation using cord sera from 97 normal newborns between 26 and 42 weeks. We also compared these variables with those from 18 SFD and 9 LFD newborns.IGF-I and IGF-II were measured by RIA and ELISA after acid-ethanol extraction. IGFBP-1, 2, 3 were measured by ELISA newly deveoloped.In relation to gestitional age and birth weight, IGF-I and IGFBP-3 had positive correlation and IGFBP-1 had negative correlation. IGF-II and IGFBP-2 did not show any correlation. In cord sera from SFD newborns the decreased IGF-I and IGFBP-3 levels and the increased IGFBP-1 levels were observed. In contrast, in LFD cord sera these variables were not significantly different from those of normals.These results imply that these IGF-related peptides play significant role in human fetal growth by positive and negative regulatory mechanism. In contrast IGF-II and IGFBP-2 do not play a role in fetal growth during late stage of fetal development.