Noriyuki Katsumata

Mastermind-like domain-containing 1 (MAMLD1 or CXorf6) transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence.

Although chromosome X open reading frame 6 (CXorf6) has been shown to be a causative gene for hypospadias, its molecular function remains unknown. To clarify this, we first examined CXorf6 protein structure, identifying homology to mastermind-like 2 (MAML2) protein, which functions as a co-activator in canonical Notch signaling. Transactivation analysis for wild-type CXorf6 protein by luciferase assays showed that CXorf6 significantly transactivated the promoter of a noncanonical Notch target gene hairy/enhancer of split 3 (Hes3) without demonstrable DNA-binding capacity. Transactivation analysis was also performed for the previously described three apparently pathologic nonsense mutations, indicating that E124X and Q197X proteins had no transactivation function, whereas R653X protein retained a nearly normal transactivation function. Subcellular localization analysis revealed that wild-type and R653X proteins co-localized with MAML2 protein in nuclear bodies, whereas E124X and Q197X proteins were incapable of localizing to nuclear bodies. Thus, further studies were performed for R653X, revealing the occurrence of nonsense mediated mRNA decay in vivo. Next, transient knockdown of CXorf6 was performed using small interfering RNA, showing reduced testosterone production in mouse Leydig tumor cells. Furthermore, steroidogenic factor 1 (SF1) protein bound to a specific sequence in the upstream of the CXorf6 coding region and exerted a transactivation activity. These results suggest that CXorf6 transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence, thereby providing the first clue to clarify the biological role of CXorf6. We designate CXorf6 as MAMLD1 (mastermind-like domain-containing 1) based on its characteristic structure.

SAMD9 mutations cause a novel multisystem disorder, MIRAGE syndrome, and are associated with loss of chromosome 7

Adrenal hypoplasia is a rare, life-threatening congenital disorder. Here we define a new form of syndromic adrenal hypoplasia, which we propose to term MIRAGE (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy) syndrome. By exome sequencing and follow-up studies, we identified 11 patients with adrenal hypoplasia and common extra-adrenal features harboring mutations in SAMD9. Expression of the wild-type SAMD9 protein, a facilitator of endosome fusion, caused mild growth restriction in cultured cells, whereas expression of mutants caused profound growth inhibition. Patient-derived fibroblasts had restricted growth, decreased plasma membrane EGFR expression, increased size of early endosomes, and intracellular accumulation of giant vesicles carrying a late endosome marker. Of interest, two patients developed myelodysplasitc syndrome (MDS) that was accompanied by loss of the chromosome 7 carrying the SAMD9 mutation. Considering the potent growth-restricting activity of the SAMD9 mutants, the loss of chromosome 7 presumably occurred as an adaptation to the growth-restricting condition.

A nationwide attempt to standardize growth hormone assays.

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.

New Compound Heterozygous Mutation in the CYP17 Gene in a 46,XY Girl with 17α-Hydroxylase/17,20-Lyase Deficiency

Background: 17α-Hydroxylase/17,20-lyase deficiency is caused by a defect of P450c17 which catalyzes both 17α-hydroxylase and 17,20-lyase reactions in adrenal glands and gonads. Results: In the present study, we analyzed the CYP17 gene in a Japanese patient with 17α-hydroxylase/17,20-lyase deficiency. The patient was a phenotypic girl and referred to us for right-sided inguinal hernia at the age of 4 years. Biopsy of the herniated gonad showed testicular tissue. The karyotype was 46,XY. At 6 years of age, hypertension was clearly recognized and the patient was diagnosed as having 17α-hydroxylase/17,20-lyase deficiency based on the clinical and laboratory findings. Analysis of the CYP17 gene revealed a compound heterozygous mutation. One mutation was an undescribed single nucleotide deletion at codon 247 in exon 4 (CTT to CT: 247delT) and the other was a missense mutation resulting in a substitution of His to Leu at codon 373 in exon 6 (CAC to CTC: H373L), which has been previously shown to abolish both 17α-hydroxylase and 17,20-lyase activities. The functional expression study of the 247delT mutant showed that this 247delT mutation completely eliminates both 17α-hydroxylase and 17,20-lyase activities. Conclusions: Together, these results indicate that the patient is a compound heterozygote for the mutation of the CYP17 gene (247delT and H373L) and that these mutations inactivate both 17α-hydroxylase and 17,20-lyase activities and give rise to clinically manifest 17α-hydroxylase/17,20-lyase deficiency.

Human glutathione S-transferase A (GSTA) family genes are regulated by steroidogenic factor 1 (SF-1) and are involved in steroidogenesis

Steroidogenic factor 1 (SF‐1) is a master regulator for steroidogenesis. In this study, we identified novel SF‐1 target genes using a genome‐wide promoter tiling array and a DNA microarray. SF‐1 was found to regulate human glutathione S‐transferase A (GSTA) family genes (hGSTA1–hGSTA4), a superfamily of detoxification enzymes clustered on chromosome 6p12. All hGSTA genes were up‐regulated by transduction of SF‐1 into human mesenchymal stem cells, while knockdown of endogenous SF‐1 in H295R cells down‐regulated all hGSTA genes. Chromatin immunoprecipitation assays, however, revealed that SF‐1 bound directly to the promoters of hGSTA3 and weakly of hGSTA4. Chromosome conformation capture assays revealed that the coordinated expression of the genes was based on changes in higher‐order chromatin structure triggered by SF‐1, which enables the formation of long‐range interactions, at least between hGSTA1 and hGSTA3 gene promoters. In steroidogenesis, dehydrogenation of the 3‐hydroxy group and subsequent Δ5‐Δ4 isomerization are thought to be enzymatic properties of 3β‐hydroxysteroid dehydrogenase (3β‐HSD). Here, we demonstrated that, in steroidogenic cells, the hGSTA1 and hGSTA3 gene products catalyze Δ5‐Δ4 isomerization in a coordinated fashion with 3β‐HSD II to produce progesterone or Δ4‐androstenedione from their Δ5‐precursors. Thus, hGSTA1 and hGSTA3 gene products are new members of steroidogenesis working as Δ5‐Δ4 isomerases.—Matsumura, T., Imamichi, Y., Mizutani, T., Ju, Y., Yazawa, T., Kawabe, S., Kanno, M., Ayabe, T., Katsumata, N., Fukami, M., Inatani, M., Akagi, Y., Umezawa, A., Ogata, T., Miyamoto, K., Human glutathione S‐transferase A (GSTA) family genes are regulated by steroidogenic factor 1 (SF‐1) and are involved in steroidogenesis. FASEB J. 27, 3198–3208 (2013). www.fasebj.org

Novel CYP17A1 mutation in a Japanese patient with combined 17α-hydroxylase/17,20-lyase deficiency

Combined 17alpha-hydroxylase/17,20-lyase deficiency is caused by a defect of P450c17 that catalyzes both 17alpha-hydroxylase and 17,20-lyase reactions in adrenal glands and gonads. In the present study, we analyzed the CYP17A1 gene in a Japanese girl with 17alpha-hydroxylase/17,20-lyase deficiency. The patient was referred to us for clitoromegaly at the age of 3 years. The karyotype was 46,XY. The patient was diagnosed as having 17alpha-hydroxylase/17,20-lyase deficiency based on the clinical and laboratory findings. Analysis of the CYP17A1 gene revealed a compound heterozygous mutation. One mutation was a deletion of codon 53 or 54 encoding Phe (TTC) in exon 1 (DeltaF54) on a maternal allele, which has been previously shown to partially abolish both 17alpha-hydroxylase and 17,20-lyase activities. The other was a novel missense mutation resulting in a substitution of Asn (AAC) for His (CAC) at codon 373 in exon 6 (H373N) on a paternal allele. Functional expression study demonstrated that the H373N mutation almost completely eliminates enzymatic activity. Previous studies have demonstrated that replacement of histidine by leucine at position 373 causes complete loss of both 17alpha-hydroxylase and 17,20-lyase activities with a defect in heme binding due to a global alteration of P450c17 structure, indicating the importance of H373 for P450c17 structure and function. Together, these results indicate that the patient is a compound heterozygote for the DeltaF54 and H383N mutations and that these mutations inactivate both 17alpha-hydroxylase and 17,20-lyase activities and give rise to clinically manifest combined 17alpha-hydroxylase/17,20-lyase deficiency.

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.

High-performance liquid chromatographic determination of lipoamidase (lipoyl-X hydrolase) activity with a novel substrate, lipoyl-6-aminoquinoline

An HPLC lipoamidase (lipoyl-X hydrolase) assay method has been developed, which uses a novel fluorescent substrate, lipoyl-6-aminoquinoline (LAQ). LAQ is synthesized from lipoic acid and 6-aminoquinoline (AQ) through lipoyl chloride as an intermediate and is conveniently purified by washing with chloroform-methanol. Mechanistic studies on the time-course, the dependence on enzyme and substrate concentrations were performed by using LAQ and a model enzyme (milk lipoamidase). Moreover, this method was successfully applied to the direct determination of the lipoamidase (LAQ hydrolase) activity in samples of human liver, milk, stools and porcine serum. Using this novel synthetic lipoyl substrate, we demonstrated that LAQ hydrolase was present in some specific tissues; LAQ hydrolase was solely present in the grey matter and not in the white matter in the human cerebrum. Furthermore, LAQ hydrolase activity was shown to increase in human liver cancer. Thus, this enzyme assay method is expected to be applicable to the tissue distribution study and also to the basic research on human diseases such as cancer.

Serum protein determination by high-performance gel-permeation chromatography

A general high-performance gel-permeation chromatography (HPGPC) method was developed to determine protein in human serum with improved sensitivity and speed. The optimum UV wavelength for protein detection was found to be 210 nm, by comparing the protein values obtained by varying the UV wavelength of the HPLC detection system with the protein values obtained from spectrophotometric protein assays, i.e., the bicinchoninic acid (BCA) method and the biuret method. The analysis time was less than 1 min. Since this HPGPC serum protein assay method is simple and rapid, it is expected to be particularly well adapted for use in clinical laboratories.

A novel frameshift mutation 840delA and a novel polymorphism D203A in the steroidogenic acute regulatory protein gene in a Japanese patient with congenital lipoid adrenal hyperplasia

Congenital lipoid adrenal hyperplasia (CLAH) is an autosomalrecessive disorder characterized by impaired production of allsteroids including glucocorticoids, mineralocorticoids and sexsteroids. It has recently been reported that mutations in thesteroidogenic acute regulatory protein (StAR) gene cause CLAH. We analyzed the StAR gene in a Japanese patient with CLAH. The patient was revealed to be a compound heterozygote bearing a nonsense mutation Q258X, changing codon 258 (CAG) encoding Gln to the stop codon TAG, and a novel frameshift mutation 840delA resulting from deletion of one of the three adenosines normally present in codon 238 (AAA), thus leading to a frameshift after codon 237 (Thr) in the StAR gene. The patient was also revealed to be homozygous for a novel missense point mutation D203A, changing codon 203 (GAC) encoding Asp to GCC encoding Ala in the StAR gene. To elucidate the significance of the D203A mutation, we analyzed the StAR gene sequence in twenty normal subjects, and found that all of them were homozygous for the D203A mutation, indicating that the D203A mutation is an innocent polymorphism. In conclusion, we have identified a novel frameshift mutation 840delA which seems to cause 840delA and the first polymorphism D203A in the human StAR gene. Hum Mutat 11:331, 1998.