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Salt inducible kinase (SIK) 1, a member of the AMP-activated kinase (AMPK) family, is activated by the AMPK-activator LKB1 which phosphorylates SIK1 at Thr182. The activated SIK1 then auto-phosphorylates its Ser186 located at the +4 position of Thr182. The phospho-Ser186 is essential for sustained activity of SIK1, which is maintained by sequential phosphorylation at Ser186-Thr182 by glycogen synthase kinase (GSK)-3beta. Meanwhile, SIK1 represses the transcription factor cAMP-response element binding protein (CREB) by phosphorylating its co-activator transducer of regulated CREB activity (TORC). Recently, histone deacetylase (HDAC) 5 was identified as a new substrate of SIK1. Inhibition of SIK1 or AMPK results in the stimulation of glyconeogensis in the liver by enhancing dephosphorylation of TORC2 followed by up-regulation of peroxisome proliferator-activated receptor coactivator (PGC)-1alpha gene expression. However, expression of the PGC-1alpha gene has been found to be repressed in LKB1-defective muscle cells. Our findings show that the AMPK agonist 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR)-dependent expression of PGC-1alpha is diminished by inhibitors of GSK-3beta or SIKs in C2C12 myoblasts. Treatment with AICAR or the overexpression of SIK1 induces nuclear export of HDAC5 followed by the activation of myogenic transcription factor (MEF)-2C. The levels of phosphorylation at Thr182 and Ser186 of SIK1 in AICAR-treated C2C12 cells are elevated, and GSK-3beta enzyme purified from AICAR-treated cells shows enhanced phosphorylation activity of SIK1 in vitro. These observations suggest that GSK-3 beta and SIK1 may play important roles in the regulation of PGC-1alpha gene expression by inactivating HDAC5 followed by activation of MEF2C.

We identified two novel mutations of the aquaporin-2 (AQP2) gene in a sporadic Japanese patient diagnosed with an autosomal recessive nephrogenic diabetes insipidus (NDI). The patient, a Japanese boy, was referred to our clinic at the age of 5 months because of unexplained recurrent fever. He was diagnosed with NDI by clinical, biochemical and endocrine findings. Molecular analysis demonstrated that he was a compound heterozygote for two mutations. One mutation consisted of a two base deletion in exon 1 (197, 198 delCA). This deletion caused a frameshift in the open reading frame, resulting in a premature stop codon 186 bases downstream in exon 1. The second mutation was a G to A transition of the terminal exon splice site (1502-1G-->A). To date, several mutations in the AQP2 gene have been described, however no splicing mutation in the AQP2 gene has been identified. The deletion mutation described in this case study was inherited patemally and the splicing site mutation was inherited maternally, indicating an autosomal recessive inheritance. In the present case study, we identified two new mutations in the AQP-2 gene. Previous studies have shown that there is no hot spot for mutations in the AQP-2 gene, and thus genetic analysis for individual patients is helpful for genetic counseling and early diagnosis.

Gitelman syndrome is a renal disorder characterized by hypokalemia, hypomagnesemia, metabolic alkalosis and hypocalciuria due to the defective tubular reabsorption of magnesium and potassium. This disease is caused by mutations of thiazide-sensitive Na-Cl cotransporter (TSC) gene. Gitelman syndrome is usually distinguished from Bartter syndrome by the presence of both hypomagnesemia and hypocalciuria. However, a phenotypic overlap is sometimes observed. We encountered two sporadic Japanese patients with Gitelman syndrome and analyzed their TSC gene. These patients were diagnosed as Gitelman syndrome by the typical clinical findings and biochemical abnormalities, such as mild muscular weakness, periodic paralysis, tetany, metabolic alkalosis, hypomagnesemia and hypocalciuria. In patient 1, a novel two base deletion (del TG at nucleotide 731 and 732) in exon 5 and a two base deletion (del TT at nucleotide 2543 and 2544) in exon 21 previously reported in a Japanese patient were identified. The patient 2 had a missense mutation (L623P), that was also identified in Japanese patients, and a novel in-frame 18 base insertion in exon 6 as a heterozygous state. Family analysis of two patients confirmed an autosomal recessive inheritance. In conclusion, we add two new mutations of the TSC gene in Japanese patients with Gitelman syndrome. Because the differential diagnosis between Bartter syndrome and Gitelman syndrome is sometimes difficult, molecular analysis would be a useful diagnostic tool, particularly in unusual cases with phenotypic overlapping.

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.

The Wilms' tumor suppressor gene, WT1, plays an important role in the development of the urogenital system and also subsequent normal function of this system. Recently, the splice mutations in intron 9 of WT1 gene have been detected in Frasier syndrome, which is characterized by streak gonads, pseudohermaphroditism, slowly progressive nephropathy and frequent development of gonadoblastoma. Here to elucidate the molecular basis in a Japanese patient of Frasier syndrome, WT1 gene was analyzed by polymerase-chain-reaction (PCR) and direct sequencing. We identified the splice junction mutation in intron 9 of WT1, which is recognized as a mutation hot-spot in intron 9. This finding concludes that 1) the mutation in intron 9 might be the cause of Frasier syndrome, and 2) the mutation hot-spot in Japanese and Caucasian patients is similar.

To determine whether nonclassical steroid 21-hydroxylase deficiency in Japan has the same molecular basis as in western countries, we have characterized the mutations of the CYP21 gene in 7 Japanese patients with nonclassical (NC) steroid 21-hydroxylase deficiency. In the Japanese NC cases the P30L was present in one allele in 5 of the 7 patients and on both alleles in one patient. By contrast, the V281L mutation, which was present in about 60% of NC cases in western countries, was not identified in any patient. Among our 7 cases, 4 were detected through neonatal mass screening by a mild increase in serum 17-hydroxyprogesterone (without any symptoms of CAH) at birth, but the 2 cases who were diagnosed as adults were born before nationwide neonatal screening was instituted, so that the Japanese neonatal screening program does detect some cases of NC steroid 21-hydroxylase deficiency. We suggest that P30L mutation is more frequent in Japanese NC CAH than V281L and that the frequency of the mutations causing NC steroid 21-hydroxylase deficiency in Japan might be different from that in western countries.

A splicing junction mutation at nucleotide 656 (A-> G substitution, I2G) in the steroid 21-hydroxylase gene (CYP21) is the most frequently detected mutation in patients with the salt-wasting and simple-virilizing forms of steroid 21-hydroxylase deficiency (approximately 60%). In this disease, prenatal diagnosis and treatment to minimize the effects of excess androgen in affected females has been advocated. Therefore, to detect the I2G mutation rapidly, accurately, and without the use of radioisotope, we developed a modified polymerase chain reaction (PCR) with a mismatched 3' nucleotide primer to introduce a new restriction site upon PCR amplification of the mutant allele. This allowed the mutant allele to be identified readily by restriction enzyme digestion of the PCR product, and subsequently this PCR product was subjected to restriction enzyme digestion for diagnosis. Chorionic villus biopsy samples (CVS) were obtained at 10 to 11 weeks gestation from two females carrying fetuses at risk for steroid 21-hydroxylase deficiency. Prenatal diagnosis was successful in both cases. One affected female was treated with dexamethasone to term. In the other case, treatment was withdrawn at an early stage when testing revealed a normal fetus. The results demonstrate the rapid and accurate detection of the I2G mutation by this method, thereby indicating the feasibility of for prenatal diagnosis of the I2G mutation.