Recent insights into the structure, function, and regulation of the eukaryotic transfer RNA splicing endonuclease complex.

The splicing of transfer RNA (tRNA) introns is a critical step of tRNA maturation, for intron-containing tRNAs. In eukaryotes, tRNA splicing is a multi-step process that relies on several RNA processing enzymes to facilitate intron removal and exon ligation. Splicing is initiated by the tRNA splicing endonuclease (TSEN) complex which catalyzes the excision of the intron through its two nuclease subunits. Mutations in all four subunits of the TSEN complex are linked to a family of neurodegenerative and neurodevelopmental diseases known as pontocerebellar hypoplasia (PCH). Recent studies provide molecular insights into the structure, function, and regulation of the eukaryotic TSEN complex and are beginning to illuminate how mutations in the TSEN complex lead to neurodegenerative disease. Using new advancements in the prediction of protein structure, we created a three-dimensional model of the human TSEN complex. We review functions of the TSEN complex beyond tRNA splicing by highlighting recently identified substrates of the eukaryotic TSEN complex and discuss mechanisms for the regulation of tRNA splicing, by enzymes that modify cleaved tRNA exons and introns. Finally, we review recent biochemical and animal models that have worked to address the mechanisms that drive PCH and synthesize these studies with previous studies to try to better understand PCH pathogenesis. This article is categorized under: RNA Processing > tRNA Processing RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition.

Acid-labile subunit (ALS) measurements in children.

Almost all of the serum IGFs are found in a ternary complex composed of IGF, IGFBP-3 and acid-labile subunit (ALS). It was reported that ALS levels were age- and sex-dependent. In the present study we measured serum ALS levels in 264 normal children (145 boys and 119 girls) aged from 5 days to 16 years, and 15 patients with growth hormone deficiency (GHD) aged from 11 months to 13 years. Serum ALS levels increased during childhood, and reached peak values in mid to late puberty. ALS levels reached their highest levels 2 years earlier in girls than in boys. Serum ALS levels were significantly correlated with serum IGF-I levels and IGFBP-3 levels. Serum ALS levels were below -2SD in 6 out of 7 children with complete GHD (CGHD), while serum ALS levels were below -2SD in 1 out of 8 patients with partial GHD (PGHD). These results indicate that serum ALS levels are regulated by GH, and that the measurement of ALS is useful for the diagnosis of CGHD in children.

Bone mineral density in Turner syndrome: relation to GH treatment and estrogen treatment.

The bone mineral density (BMD) of the second metacarpal bone of the left hand was measured in 57 patients with Turner syndrome by the digital image processing (DIP) method to study the relations between the treatment regimen and their bone mineral density. BMD SD score in the patients who had started the GH treatment before 10 years old was within +/-2SD of the standard before 14 years, but the score decreased to below -2SD after 14 years. In the patients who had started GH treatment after 10 years old, BMD score were significantly lower than -2SD, although there was tendency to increased. In the patients who had estrogen after 15 years old, BMD did not increase with GH alone and slowly increased after estrogen replacement. In the other two patients who had started sex steroid hormone replacement treatment before 15 years old, BMD maintained +/-2SD. In patients who received combined GH and LH-RH analog treatment, their BMD score did not increase during LH-RH analog treatment. It slowly increased but was still below -3SD after stop of LH-RH analog and start of estrogen treatment. In Turner syndrome, GH may play a role in maintaining prepubertal BMD levels [4], and estrogen plays an important role in pubertal BMD increment. It is recommended that estrogen treatment is started before 15 years of age for maintenance of normal BMD level.

Analysis of the FGFR3 gene in Japanese patients with achondroplasia and hypochondroplasia.

It has been reported that mutations in the FGFR3 gene cause autosomal dominant forms of dwarfism, achondroplasia (ACH) and hypochondroplasia (HCH). In the present study, we analyzed the FGFR3 gene in 26 Japanese patients with ACH and 14 with HCH. Genomic DNAs of the patients were isolated from whole blood. For the ACH patients, a 164-bp fragment of the FGFR3 gene that spans the entire transmembrane domain was amplified by polymerase chain reaction (PCR), and the PCR products were analyzed by direct sequencing of the PCR products and by digestion of the PCR products with restriction enzymes. For the HCH patients, a 206-bp fragment of the FGFR3 gene which encodes a part of the TK1 domain was amplified, and the PCR products were directly sequenced. The heterozygous G380R mutations were identified in all of the 26 ACH patients, whereas the heterozygous N540K mutations were identified in 8 out of 14 HCH patients. These results were consistent with previous reports from abroad.

Growth and metabolic disturbances in a patient with total parenteral nutrition: a case of hypercalciuric hypercalcemia.

Hypercalciuria is a common side effect during total parenteral nutrition (TPN). We report a patient with long-term TPN, who demonstrated hypercalciuria, hypercalcemia and growth retardation. The patient is a six-year-old Japanese girl with Hirschsprung disease (jejunal agangliosis). Jejunostomy was performed at one-month old and since then her nutrition has depended mostly on TPN. When she was 3 years old, continuous TPN was switched to cyclic TPN (on TPN for 11 hrs and off TPN for 13 hrs). The urinary calcium level has been elevated (Ca/Cre ratio, 1.0) since 3 months of age, whereas serum calcium levels stayed within normal range for a while. The serum calcium levels started to elevate to 12 to approximately 13 mg/dl when she was 3 years and 8 months old. She showed growth retardation (height SD score was -4.2SD when she was 5 years and 8 months old) and deteriorated renal tubular function with renal glycosuria, elevated beta 2-microglobulin (beta2-MG) and N-acetyl-beta-D-glucosaminidase. She was referred to our division for the investigation and treatment of growth disturbance and Ca metabolism. Her bone age was delayed (BA/CA 0.62) and serum IGF-I level was decreased but her GH response to provocation test was normal. Bilateral nephrocalcinosis was revealed by renal echogram and CT scan. By reducing calcium content in TPN solution, the serum and urinary calcium levels could be maintained within normal range and her renal function and growth velocity was improved.

Mechanism for the development of ovarian cysts in patients with congenital lipoid adrenal hyperplasia.

OBJECTIVE: Although ovarian cysts commonly occur in patients with congenital lipoid adrenal hyperplasia (CLAH), the mechanism of development remains to be determined. To clarify the pathogenesis of the ovarian cysts, endocrinological examinations were performed in patients with CLAH. METHODS: The subjects were three Japanese CLAH patients. Basal body temperature, serum and urinary gonadotropin levels, serum and/or urinary ovarian hormones and mutations of the steroidogenic acute regulatory protein (StAR) gene were examined. RESULTS: The basal body temperature was not biphasic in any patient. Basal LH levels were high in all CLAH patients and markedly responded to LH-releasing hormone in two patients. Urinary gonadotropin analysis revealed repetitive LH surges in the menstrual cycles of the CLAH patients. No increase in the urinary pregnanediol suggested anovulation in all patients, and bilateral ovarian cysts were found in two of the subjects. Examination of the StAR gene revealed a frameshift mutation 840delA at codon 238, a nonsense mutation Q258X at codon 258, a homozygotic mutation at Q258X, and a compound heterozygotic mutation with 251insG and Q258X. CONCLUSIONS: We concluded that the development of ovarian cysts may be derived from continued anovulation in CLAH patients. Elevated LH levels may be explained by increased sensitivity of the anterior pituitary to circulating estrogen.

Genetic and endocrinological evaluations of three 46,XX patients with congenital lipoid adrenal hyperplasia previously reported as having presented spontaneous puberty.

Congenital lipoid adrenal hyperplasia (CLAH) is an autosomal recessive disorder characterized by impaired synthesis of adrenal and gonadal steroids. It was demonstrated that loss-of-function mutations in the steroidogenic acute regulatory protein (StAR) gene cause CLAH and that 46,XX patients with CLAH develop spontaneous puberty. We had reported that three 46,XX patients with CLAH had presented spontaneous puberty and one of the patients had developed life-threatening ovarian cysts, before the etiology of CLAH had been clarified. In the present study, we analyzed their StAR gene and demonstrated mutations. Endocrinological examinations of the patients revealed that serum LH and FSH levels and their responses to the LHRH stimulation were not exaggerated before the onset of puberty. Serum LH levels and its response to LHRH were increased during puberty, whereas serum FSH levels remained within the normal range. Serum estradiol increased after the administration of human menopausal gonadotropins in the pubertal patient, suggesting that the ovary might have another system than StAR to facilitate cholesterol transport into the mitochondria. Although the patients had menstrual cycles, they remained anovulatory, and the resultant increased secretion of LH was speculated to be responsible for the development of ovarian cysts.

Japanese familial patients with male-limited precocious puberty.

Familial male-limited precocious puberty (FMPP) is a rare disease caused by constitutively activating mutations in the luteinizing hormone receptor (LH-R) gene. In the present study, we analyzed the LH-R gene in members of a Japanese FMPP family. Two males of the family were affected and had a heterozygous M398T mutation; one patient developed pubertal signs as early as 2 years of age, and the other at 6 years of age. Both patients had elevated serum testosterone levels and prepubertal gonadotropin secretions. The father of the latter patient carried the M398T mutation, but lacked history of precocious puberty. Thus, phenotypic differences were observed in the three males with the same LH-R mutation belonging to the same family. In summary, we have described a Japanese family with FMPP.

A Japanese family with autosomal dominant growth hormone deficiency.

UNLABELLED: We report a 1-year-old Japanese boy and his father with isolated growth hormone deficiency II. In both cases, a G-->A transition of the first base of the donor splice site of intron 3 of the growth hormone-1 gene was detected. All unaffected family members were homozygous normal. CONCLUSION: This is the fourth reported case of autosomal isolated growth hormone deficiency II with a G-->A transition. The CG dinucleotide at the exon 3-intron 3 junction of the growth hormone-1 gene appears to be a hot spot for point mutations.

[Childhood diabetes mellitus].

Long-term check of 68 type 1 diabetes patients (1978-1998); 23 under age 19, mean age 13.4 +/- 4.1 (group A) and 45 above age 20, mean age 26.7 +/- 4.9 (group B) were analyzed. 1) Mean onset age were 5.5 +/- 3.9 years old in group A whereas 7.8 +/- 3.5 in B. 2) In all type 1 diabetes high prevalences of positive HLA-DQ region gene and ICA and/or GADA were found. About 11% of all these patients showed the clinical pattern of slowly progressive variants. 3) Treatment modalities in individuals were successfully selected out of various combinations of insulin and multiple daily injection (MDI) styles. 4) Late complication prevalences were none in A and very little in B (8.9% background and none proliferative retinopathy, 2.2% nephropathy and 2.2% hypertension). 5) Five married females were able to have 7 normal babies. Sixteen type 2 diabetes patients, including one MODY 2, were successfully treated with combinations of sulfonylureas/alphaglucosidase inhibitors/insulin.

When and how to combine growth hormone with a luteinizing hormone-releasing hormone analogue.

The effect of combined treatment with growth hormone (GH) and a luteinizing hormone-releasing hormone (LHRH) analogue, or GH alone, on pubertal height gain was assessed in an uncontrolled study in 15 boys and 10 girls with GH deficiency (GHD). Seven boys and six girls were treated with GH alone (group 1), and eight boys and four girls were treated with a combination of GH and an LHRH analogue during puberty (group 2). Mean ages (+/- SD) at the start of GH treatment and at the onset of puberty were significantly lower in group 2 (8.0 +/- 3.3 years and 11.2 +/- 0.8 years, respectively, in boys, and 6.3 +/- 1.6 years and 10.8 +/- 0.7 years in girls) than in group 1 (12.8 +/- 1.9 years and 13.7 +/- 1.4 years in boys, and 11.2 +/- 1.0 years and 12.5 +/- 1.2 years in girls). Height at the onset of puberty was less in group 2 than in group 1, but the difference was significant only for the boys. Combination treatment was started at a mean age of 11.7 +/- 1.2 years in boys and 11.5 +/- 1.0 years in girls. The duration of the combination treatment was 5.1 +/- 1.5 years in boys and 2.3 +/- 0.7 years in girls. The duration of the period between the onset of puberty and the end of GH treatment was significantly longer in group 2 (6.8 +/- 1.2 years in boys and 5.5 +/- 1.0 years in girls) than in group 1 (4.3 +/- 1.6 years in boys and 3.6 +/- 1.4 years in girls). The pubertal height gain was also significantly greater in group 2 (36.7 +/- 6.5 cm in boys and 29.0 +/- 8.3 cm in girls) than in group 1 (21.9 +/- 4.1 cm in boys and 18.6 +/- 4.1 cm in girls). Final height was significantly greater in group 2 than in group 1 in boys. Although there was no significant difference in final height between groups in the girls, the change in height SDS from the start of GH treatment until final height was significantly greater in group 2 (2.7 +/- 1.6 in boys and 4.5 +/- 0.5 SD in girls) than in group 1 (1.0 +/- 0.8 in boys and 1.8 +/- 0.9 SD in girls), in both boys and girls. In conclusion, it appears that combination of an LHRH analogue and GH may increase the pubertal height gain and the final height of children with GHD. The improvement is attributed to the prolongation of the treatment period, permitting slow bone maturation, and to the maintenance of height velocity. This combination treatment appears to be more effective in boys than girls. To fully assess this therapeutic approach, prospective controlled studies are needed.

The regulatory effect of growth hormone on growth hormone-binding protein in human serum.

To study the acute and long-term effect of GH on its down-stream axis, we measured serum GH-binding protein (GHBP) by ligand-mediated immunofunctional assay. Diurnal changes in serum GHBP were determined by every 20-min sampling for 24 h in four normal short children. There were small or negligible fluctuations in serum GHBP levels, and no correlation with GH pulses was observed. The short-term effect of GH on GHBP levels was assessed in eight GH-deficient children by daily administration of GH (0.1 U/kg) for 10 days. Serum GHBP levels did not significantly change, but IGF-I levels increased significantly. We also studied the long-term effect of GH administration on GHBP levels in seventeen patients with GH deficiency over six months. In twelve out of 17 patients, serum GHBP levels showed an increase when compared to the levels before treatment. In these patients, BMI was not largely changed during treatment, while it tended to decrease in patients whose GHBP levels did not increase. In conclusion, endogenous pulsatile GH secretion and short-term exogenous GH administration had no effect on serum GHBP levels. On the other hand, long-term GH administration increased GHBP levels in 70% of patients with GH deficiency. Changes in BMI may partly be attributed to this change. The direct long-term regulatory effect of GH on GHBP should be further studied.

Nutrition related hormonal changes in obese children.

Children with simple obesity (SO) show increased linear growth with normal or high serum insulin-like growth factor-I (IGF-I) levels during prepubertal period, despite low GH secretion. We measured IGF-I, IGFBP-1, GHBP and other factors to clarify the hormonal relation between the nutrition and the linear growth in SO and compared these factors with children with normal short stature (NS). Subjects were 23 SO and 19 NS children, and their height standard deviation (SD) scores were 0.7 +/- 0.2 SD and -3.4 +/- 0.3 SD (mean +/- SEM) (P < 0.01), respectively. Oral glucose tolerance test (OGTT) was performed in all the subjects and GH-releasing factor (GRF) test was also performed in 13 of SO and 17 of NS. The peak levels of GH in the GRF test were significantly lower in SO than in NS (12.8 +/- 1.7 vs. 39.8 +/- 6.9 ng/ml) and showed a significantly positive correlation with sigma IGFBP-1 (r = 0.63, P < 0.01). Serum GHBP level and IGF-I level were significantly higher in SO than in NS on pubertal stage matching. There was a positive correlation between GHBP and sigma insulin during OGTT (r = 0.75, P < 0.01). When the sum of the values during OGTT was expressed as sigma, sigma insulin, sigma C-peptide and sigma glucose were significantly higher in SO than in NS on pubertal stage matching. Basal and sigma IGFBP-1 were significantly lower in SO than in NS, but IGFBP-3 levels showed no significant difference between the two groups either in prepuberty or midpuberty. In conclusion, it can be hypothesized that the overnutrition causes hyperinsulinemia which increases GH receptor and IGF-I secretion despite low GH secretion. Hyperinsulinemia also may increase free IGF-I by lowering IGFBP-1. These two mechanism are supposed to be the nutrition related hormonal changes in SO and can explain the growth of SO. In addition, the increased free IGF-I may contribute the decreased GH secretion due to negative feedback in SO.

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. Mutations in brief no. 117. Online.

Congenital lipoid adrenal hyperplasia (CLAH) is an autosomalrecessive disorder characterized by impaired production of all steroids including glucocorticoids, mineralocorticoids and sexsteriods. It has recently been reported that mutations in the steriodogenic 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 framshift 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.

A new compound heterozygous mutation in the 11 beta-hydroxysteroid dehydrogenase type 2 gene in a case of apparent mineralocorticoid excess.

Apparent mineralocorticoid excess (AME) characterized by early-onset hypertension and hypokalemia is due to congenital deficiency of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). Two isoforms of human 11 beta HSD are known, and the type 2 isoform (11 beta HSD2) has been recently shown to be responsible for AME. In this study we have analyzed the 11 beta HSD2 gene of a Japanese patient with AME. PCR amplification and subsequent nucleotide sequencing of the 11 beta HSD2 gene from the patient and his family members revealed that the patient has a compound heterozygous mutation of this gene. In 1 allele, an undescribed single nucleotide transition in codon 208 in exon 3 resulted in a substitution of arginine to histidine (CGC to CAC: R208H). In the other allele, a deletion of 3 nucleotides in codons 337-338 in exon 5 resulted in a substitution of arginine to histidine and a deletion of tyrosine residue (CGCTAT to CAT: R337H, delta Y338), which has been previously shown to abolish 11 beta HSD2 enzyme activity. A chloramphenicol acetyltransferase assay-based expression study involving the mineralocorticoid receptor indicated that the novel R208H mutation eliminates the enzymatic activity of 11 beta HSD2. From the genetic analysis of 50 healthy subjects, the novel R208H mutation was unlikely to be due to polymorphism. Together, these results indicate that this patient is a compound heterozygote for the mutation in the 11 beta HSD2 gene (R208H and R337H, delta Y338) and that these mutations inactivate the 11 beta HSD2 function and give rise to clinically manifest AME.

[Sexual precocity].

Sexual precocity results from both GnRH-dependent and GnRH-independent mechanisms. The GnRH-dependent forms of precocious puberty can be treated effectively with long-acting agonist analogues of GnRH. However, for some children who have a poor growth rate during the analogue therapy, an additional growth hormone therapy should be considered to get them near to their normal final height. The analogue treatment could be continued until a bone age of 13 or more. The GnRH-independent forms of precocious puberty have unique mechanisms and unknown pathophysiology. For instance, even though it is known that testotoxicosis and McCune Albright syndrome are caused by the molecular mechanism disorders, further elucidation of their etiologic basis of sexual precocity is needed. Thus being, in treating the GnRH-independent forms they should be assessed for each particular disorder.

Follow-up study of children with precocious puberty treated with cyproterone acetate. Ad hoc Committee for CPA.

A total of 1840 children and adolescents treated with cyproterone acetate (CPA) to block gonadal function, as a treatment for precocious puberty, short stature and other disorders, were registered to survey for the risk of developing hepatic tumors. Patients responding to follow-up numbered 1552 (85%). The cumulative dose and duration of CPA therapy for boys and girls were 110.4g and 2.6 years, and 122.9 g and 2.8 years, respectively. Among the 1552 patients, five hepatoma cases were found. Four underwent successful surgery and remain alive and well to date. Two of the 5 cases had been given more than 500g, the other 3 more than 1000 g, of CPA. Three had also been given androgens before CPA administration. Although further follow-up is necessary to monitor for the development of adenoma and hepatoma, the risk of developing these tumors among patients to whom limited doses of CPA were administered appears to be negligible.

Homozygous Q258X mutation in the steroidogenic acute regulatory gene in a Japanese patient with congenital lipoid adrenal hyperplasia.

Congenital lipoid adrenal hyperplasia (CLAH) is an autosomal recessive disorder characterized by impaired synthesis of all adrenal and gonadal steroid hormones. It has recently been reported that mutations in the steroidogenic acute regulatory protein (StAR) gene cause CLAH. We analyzed the nucleotide sequences of exon 7 of the StAR gene in a Japanese CLAH patient with a karyotype of 47,XYY, and her parents. The patient was homozygous for a nonsense mutation Q258X, which changed codon 258 (CAG) encoding Gln to the stop codon TAG, and the her parents were heterozygous for the Q258X mutation. Since the Q258X mutation destroys a MvaI site normally present in the StAR gene sequence, we confirmed the Q258X mutation by means of the restriction endonuclease MvaI digestion of the PCR products. Endocrinological examinations of the parents revealed normal responses of adrenal steroid hormones to exogenous adrenocorticotropin administration, confirming the failure to detect the heterozygous carriers of CLAH by hormonal evaluation.

Urinary biotinidase and alanine excretion in patients with insulin-dependent diabetes mellitus.

Twenty-four-hour urine specimens from 21 juvenile insulin-dependent diabetics and 10 healthy controls were compared with respect to biotinidase activity and alanine content. Urinary biotinidase activity was analysed by a newly developed high-performance liquid chromatography (HPLC) method. It was found that the excretion of biotinidase in urine was elevated in diabetics (7.02 mU/d; p < 0.005) as compared with controls (not detectable). Alanine excretion was also found to increase (p < 0.01) in diabetics. Biotinidase excretion in diabetics was correlated with alanine excretion (rS = 0.667; p < 0.01), but not with protein, albumin or N-acetyl-beta-glucosaminidase excretion. The simultaneous elevation of urinary biotinidase and alanine excretion in juvenile diabetics suggests that changes in kidney metabolism arise in the early stages of diabetes.