Ikuma Fujiwara

Molecular Analysis Expands the Spectrum of Phenotypes Associated with GLI3 Mutations

A range of phenotypes including Greig cephalopolysyndactyly and Pallister‐Hall syndromes (GCPS, PHS) are caused by pathogenic mutation of the GLI3 gene. To characterize the clinical variability of GLI3 mutations, we present a subset of a cohort of 174 probands referred for GLI3 analysis. Eighty‐one probands with typical GCPS or PHS were previously reported, and we report the remaining 93 probands here. This includes 19 probands (12 mutations) who fulfilled clinical criteria for GCPS or PHS, 48 probands (16 mutations) with features of GCPS or PHS but who did not meet the clinical criteria (sub‐GCPS and sub‐PHS), 21 probands (6 mutations) with features of PHS or GCPS and oral‐facial‐digital syndrome, and 5 probands (1 mutation) with nonsyndromic polydactyly. These data support previously identified genotype–phenotype correlations and demonstrate a more variable degree of severity than previously recognized. The finding of GLI3 mutations in patients with features of oral–facial–digital syndrome supports the observation that GLI3 interacts with cilia. We conclude that the phenotypic spectrum of GLI3 mutations is broader than that encompassed by the clinical diagnostic criteria, but the genotype–phenotype correlation persists. Individuals with features of either GCPS or PHS should be screened for mutations in GLI3 even if they do not fulfill clinical criteria. Hum Mutat 31:1142–1154, 2010.

Molecular and clinical analysis of RAF1 in Noonan syndrome and related disorders: dephosphorylation of serine 259 as the essential mechanism for mutant activation†

Noonan syndrome (NS) and related disorders are autosomal dominant disorders characterized by heart defects, facial dysmorphism, ectodermal abnormalities, and mental retardation. The dysregulation of the RAS/MAPK pathway appears to be a common molecular pathogenesis of these disorders: mutations in PTPN11, KRAS, and SOS1 have been identified in patients with NS, those in KRAS, BRAF, MAP2K1, and MAP2K2 in patients with CFC syndrome, and those in HRAS mutations in Costello syndrome patients. Recently, mutations in RAF1 have been also identified in patients with NS and two patients with LEOPARD (multiple lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth, and sensorineural deafness) syndrome. In the current study, we identified eight RAF1 mutations in 18 of 119 patients with NS and related conditions without mutations in known genes. We summarized clinical manifestations in patients with RAF1 mutations as well as those in NS patients withPTPN11, SOS1, or KRAS mutations previously reported. Hypertrophic cardiomyopathy and short stature were found to be more frequently observed in patients with RAF1 mutations. Mutations in RAF1 were clustered in the conserved region 2 (CR2) domain, which carries an inhibitory phosphorylation site (serine at position 259; S259). Functional studies revealed that the RAF1 mutants located in the CR2 domain resulted in the decreased phosphorylation of S259, and that mutant RAF1 then dissociated from 14‐3‐3, leading to a partial ERK activation. Our results suggest that the dephosphorylation of S259 is the primary pathogenic mechanism in the activation of RAF1 mutants located in the CR2 domain as well as of downstream ERK. Hum Mutat 30:1–11, 2010.

PTHrP-independent hypercalcemia with increased proinflammatory cytokines and bone resorption in two children with CD19-negative precursor B acute lymphoblastic leukemia.

Hypercalcemia in childhood acute lymphoblastic leukemia (ALL) is rare and occasionally associated with parathyroid hormone‐related protein (PTHrP). However, the pathogenesis of PTHrP‐independent hypercalcemia remains unclear. We report two children with precursor B ALL who had marked hypercalcemia (15.8 and 16.6 mg/dl, respectively) and disseminated osteolysis. Serum tumor necrosis factor‐α (TNF‐α) and IL‐6 were markedly elevated, whereas 1,25(OH)2 vitamin D3, intact PTH and PTHrP were decreased or undetected. Analysis of urinary deoxypyridinoline (DPY) or bone biopsy of the osteolytic lesion showed an increased bone resorption, and administration of bisphosphonate improved the hypercalcemia. Patients had ALL with immunophenotype positive for CD10, CD34, and HLA‐DR but negative for CD19 and obtained remission with chemotherapy. These findings suggest that increased osteoclastic bone resorption via stimulation with TNF‐α and IL‐6 may be mechanism causing PTHrP‐independent hypercalcemia in some patients with precursor B ALL lacking CD19 expression. Pediatr Blood Cancer 2007;49:990–993.

Abnormal Regulation of Renal 25-Hydroxyvitamin D-1α-Hydroxylase Activity in X-Linked Hypophosphatemia: A Translational or Post-Translational Defect†

The hyp mouse exhibits abnormal metabolic/hormonal regulation of renal 25(OH)D‐1α‐hydroxylase activity. Whether this results from aberrant transcriptional regulation of the 1α‐hydroxylase gene, CYP27B1, remains unknown. To investigate this possibility, we compared phosphate and parathyroid hormone effects on renal proximal convoluted tubule and thyrocalcitonin effects on proximal straight tubule enzyme activity and mRNA expression in normal and hyp mice. We assayed 25(OH)D‐1α‐hydroxylase activity by measuring 1,25(OH)2D production and mRNA by ribonuclease protection. Phosphate‐depleted mice exhibited a 3‐fold increment of 25(OH)D‐1α‐hydroxylase activity compared with normals, whereas hyp mice displayed no enhanced enzyme function. Phosphate‐depleted mice concurrently displayed a 2‐fold increase in mRNA transcripts; in contrast, despite failure to alter enzyme activity, hyp mice exhibited a similar increment in mRNA transcripts. Parathyroid hormone stimulation of normal mice increased 25(OH)D‐1α‐hydroxylase activity 10‐fold, while eliciting only a 2‐fold increment in hyp mouse enzyme function. This disparity occurred despite increments of mRNA transcripts to comparable levels (22.2 ± 3.5‐ vs. 19.9 ± 1.8‐fold). The dissociation between phosphate‐ and parathyroid hormone‐mediated transcriptional activity and protein function was not universal. Thus, thyrocalcitonin stimulation of normal and hyp mice resulted in comparable enhancement of mRNA transcripts and enzyme activity. These observations indicate that abnormal regulation of vitamin D metabolism in hyp mice occurs in the proximal convoluted tubule and results, not from aberrant transcriptional regulation, but from a defect in translational or post‐translational activity.

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.

Mutations in PIGL in a patient with Mabry syndrome

Mabry syndrome, hyperphosphatasia mental retardation syndrome (HPMRS), is an autosomal recessive disease characterized by increased serum levels of alkaline phosphatase (ALP), severe developmental delay, intellectual disability, and seizures. Recent studies have revealed mutations in PIGV, PIGW, PIGO, PGAP2, and PGAP3 (genes that encode molecules of the glycosylphosphatidylinositol (GPI)‐anchor biosynthesis pathway) in patients with HPMRS. We performed whole‐exome sequencing of a patient with severe intellectual disability, distinctive facial appearance, fragile nails, and persistent increased serum levels of ALP. The result revealed a compound heterozygote with a 13‐bp deletion in exon 1 (c.36_48del) and a two‐base deletion in exon 2 (c.254_255del) in phosphatidylinositol glycan anchor, class L (PIGL) that caused frameshifts resulting in premature terminations. The 13‐bp deletion was inherited from the father, and the two‐base deletion was inherited from the mother. Expressing c.36_48del or c.254_255del cDNA with an HA‐tag at the C‐ or N‐terminus in PIGL‐deficient CHO cells only partially restored the surface expression of GPI‐anchored proteins (GPI‐APs). Nonsynonymous changes or frameshift mutations in PIGL have been identified in patients with CHIME syndrome, a rare autosomal recessive disorder characterized by colobomas, congenital heart defects, early onset migratory ichthyosiform dermatosis, intellectual disability, and ear abnormalities. Our patient did not have colobomas, congenital heart defects, or early onset migratory ichthyosiform dermatosis and hence was diagnosed with HPMRS, and not CHIME syndrome. These results suggest that frameshift mutations that result in premature termination in PIGL cause a phenotype that is consistent with HPMRS.

Subacute thyroiditis in children: Patient report and review of the literature

Subacute thyroiditis is extremely rare during childhood. We treated a 10 year-old prepubertal girl who presented with typical clinical features of subacute thyroiditis. Considering this case and previous reports in children, we characterized the clinical features of this disorder as it occurs in childhood. In contrast to the predominance of subacute thyroiditis in adults, the incidence of subacute thyroiditis is lower than that of acute suppurative thyroiditis in children. This may cause difficulty with differential diagnosis, particularly when leukocytosis is present or the lesion is localized to the left lobe. Ultrasonography can be helpful in differentiation.

Hypercalcemia due to all-trans retinoic acid therapy for acute promyelocytic leukemia : A case report of effective treatment with bisphosphonate

of vitamin A, has been introduced to the therapy of acute promyelocytic leukemia (APL).1 Most cases of APL show t (15;17) (q22;q21) chromosomal translocation, which produces PML/retinoic acid receptor α (RARα) fusion gene. The maturation of APL cells is blocked at the promyelocyte stage of myeloid differentiation, and ATRA can induce terminal differentiation and apotosis of leukemic cells.2 A very high rate of complete remission (CR) by use of ATRA for APL has been reported, whereas several side-effects including hypercalcemia have been reported to be associated with systemic ATRA therapy.1,3 In this study, we reported an 11-year-old boy with severe hypercalcemia caused by ATRA therapy for APL. Interestingly, bisphosphonate was effective to control ATRA-induced hypercalcemia in this patient.

Safety and efficacy of treatment with asfotase alfa in patients with hypophosphatasia: Results from a Japanese clinical trial

Hypophosphatasia (HPP) is a rare skeletal disease characterized by hypomineralization and low alkaline phosphatase activity. Asfotase alfa (AA) has been recently developed to treat HPP complications. This study evaluated its safety and efficacy in Japan.

Effects of RXR Agonists on Cell Proliferation/Apoptosis and ACTH Secretion/Pomc Expression.

Various retinoid X receptor (RXR) agonists have recently been developed, and some of them have shown anti-tumor effects both in vivo and in vitro. However, there has been no report showing the effects of RXR agonists on Cushing’s disease, which is caused by excessive ACTH secretion in a corticotroph tumor of the pituitary gland. Therefore, we examined the effects of synthetic RXR pan-agonists HX630 and PA024 on the proliferation, apoptosis, ACTH secretion, and pro-opiomelanocortin (Pomc) gene expression of murine pituitary corticotroph tumor AtT20 cells. We demonstrated that both RXR agonists induced apoptosis dose-dependently in AtT20 cells, and inhibited their proliferation at their higher doses. Microarray analysis identified a significant gene network associated with caspase 3 induced by high dose HX630. On the other hand, HX630, but not PA024, inhibited Pomc transcription, Pomc mRNA expression, and ACTH secretion dose-dependently. Furthermore, we provide new evidence that HX630 negatively regulates the Pomc promoter activity at the transcriptional level due to the suppression of the transcription factor Nur77 and Nurr1 mRNA expression and the reduction of Nur77/Nurr1 heterodimer recruiting to the Pomc promoter region. We also demonstrated that the HX630-mediated suppression of the Pomc gene expression was exerted via RXRα. Furthermore, HX630 inhibited tumor growth and decreased Pomc mRNA expression in corticotroph tumor cells in female nude mice in vivo. Thus, these results indicate that RXR agonists, especially HX630, could be a new therapeutic candidate for Cushing’s disease.