Sachiko Kitanaka

Inactivating Mutations in the 25-Hydroxyvitamin D3 1α-Hydroxylase Gene in Patients with Pseudovitamin D–Deficiency Rickets

BACKGROUND Pseudovitamin D-deficiency rickets is characterized by the early onset of rickets with hypocalcemia and is thought to be caused by a deficit in renal 25-hydroxyvitamin D3 1alpha-hydroxylase, the key enzyme for the synthesis of 1alpha,25-dihydroxyvitamin D3. METHODS We cloned human 25-hydroxyvitamin D3 1alpha-hydroxylase complementary DNA (cDNA) using a mouse 1alpha-hydroxylase cDNA fragment as a probe. Its genomic structure was determined, and its chromosomal location was mapped by fluorescence in situ hybridization. We then identified mutations in the 1alpha-hydroxylase gene in four unrelated patients with pseudovitamin D-deficiency rickets by DNA-sequence analysis. Both the normal and the mutant 1alpha-hydroxylase proteins were expressed in COS-1 cells and were assayed for 1alpha-hydroxylase activity. RESULTS The gene for 25-hydroxyvitamin D3 1alpha-hydroxylase was mapped to chromosome 12q13.3, which had previously been reported to be the locus for pseudovitamin D-deficiency rickets by linkage analysis. Four different homozygous missense mutations were detected in this gene in the four patients with pseudovitamin D-deficiency rickets. The unaffected parents and one sibling tested were heterozygous for the mutations. Functional analysis of the mutant 1alpha-hydroxylase protein revealed that all four mutations abolished 1alpha-hydroxylase activity. CONCLUSIONS Inactivating mutations in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene are a cause of pseudovitamin D-deficiency rickets.

Positive and Negative Regulations of the Renal 25-Hydroxyvitamin D3 1α-Hydroxylase Gene by Parathyroid Hormone, Calcitonin, and 1α,25(OH)2D3 in Intact Animals1

Reflecting the prime role of 1α,25(OH)2D3 in calcium homeostasis, the activity of 25-hydroxyvitamin D3 1α-hydroxylase, a key enzyme for 1α,25(OH)2D3 biosynthesis, is tightly regulated by 1α,25(OH)2D3, PTH and calcitonin. Its significant activity is found in kidney, though the enzymatic activity is also reported in extra-renal tissues. In the present study, we found that the 1α-hydroxylase gene abundantly expresses in kidney, and at low levels in other tissues and in some cell lines. Positive and negative regulations of 1α-hydroxylase gene by PTH, calcitonin, or 1α,25(OH)2D3 were observed at transcriptional levels in kidneys of animals and in a mouse proximal tubule cell line. Moreover, the protein kinase A inhibitor abrogated the PTH-mediated positive regulation. In mice lacking the vitamin D receptor, the 1α-hydroxylase gene expression was overinduced, and the inducible effect of either PTH or calcitonin, but not the repression by 1α,25(OH)2D3, was evident. Thus, vitamin D receptor is essential for the n...

Selective Interaction of Vitamin D Receptor with Transcriptional Coactivators by a Vitamin D Analog

ABSTRACT The nuclear vitamin D receptor (VDR) is a member of a nuclear receptor superfamily and acts as a ligand-dependent transcription factor. A family of cotranscriptional activators (SRC-1, TIF2, and AIB-1) interacts with and activates the transactivation function of nuclear receptors in a ligand-dependent way. We examined interaction of VDR with these coactivators that was induced by several vitamin D analogs, since they exert differential subsets of the biological action of vitamin D through unknown mechanisms. Unlike other vitamin D analogs tested, OCT (22-oxa-1α,25-dihydroxyvitamin D3) induced interaction of VDR with TIF2 but not with SRC-1 or AIB-1. Consistent with these interactions, only TIF2 was able to potentiate the transactivation function of VDR bound to OCT. Thus, the present findings suggest that the structure of VDR is altered in a vitamin D analog-specific way, resulting in selective interactions of VDR with coactivators. Such selective interaction of coactivators with VDR may specify the array of biological actions of a vitamin D analog like OCT, possibly through activating a particular set of target gene promoters.

In vivo function of VDR in gene expression-VDR knock-out mice

Vitamin D exerts many biological actions through nuclear vitamin D receptor (VDR)-mediated gene expression. The transactivation function of VDR is activated by binding 1alpha,25-dihydroxyvitamin D3[1alpha,25(OH)2D3], an active form of vitamin D. Conversion from 25(OH)D3 is finely regulated in kidney by 25(OH)D3 1alpha-hydroxylase[25(OH)D 1alpha-hydroxylase], keeping serum levels of 1alpha,25(OH)2D3 constant. Deficiency of vitamin D and mutations in the genes like VDR (type II genetic rickets) are known to cause rickets like lowered serum calcium, alopecia and impaired bone formation. However, the molecular basis of vitamin D VDR system in the vitamin D action in intact animals remained to be established. In addition, the 1alpha-hydroxylase gene from any species had not yet been cloned, irrespective of its biological significance and putative link to the type I genetic rickets. We generated VDR-deficient mice (VDR KO mice). VDR KO mice grew up normally until weaning, but after weaning they developed abnormality like the type II rickets patients. These results demonstrated indispensability of vitamin D-VDR system in mineral and bone metabolism only in post-weaning life. Using a newly developed cloning system, we cloned the cDNA encoding a novel P450 enzyme, mouse and human 1alpha-hydroxylase. The study in VDR KO mice demonstrated the function of liganded VDR in the negative feed-back regulation of 1alpha,25(OH)2D3 production. Finally, from the analysis of type I rickets patients, we found missense genetic mutations in 1alpha-hydroxylase, leading to the conclusion that this gene is responsible for the type I rickets.

MLL2 and KDM6A mutations in patients with Kabuki syndrome

Kabuki syndrome is a congenital anomaly syndrome characterized by developmental delay, intellectual disability, specific facial features including long palpebral fissures and ectropion of the lateral third of the lower eyelids, prominent digit pads, and skeletal and visceral abnormalities. Mutations in MLL2 and KDM6A cause Kabuki syndrome. We screened 81 individuals with Kabuki syndrome for mutations in these genes by conventional methods (n = 58) and/or targeted resequencing (n = 45) or whole exome sequencing (n = 5). We identified a mutation in MLL2 or KDM6A in 50 (61.7%) and 5 (6.2%) cases, respectively. Thirty‐five MLL2 mutations and two KDM6A mutations were novel. Non‐protein truncating‐type MLL2 mutations were mainly located around functional domains, while truncating‐type mutations were scattered through the entire coding region. The facial features of patients in the MLL2 truncating‐type mutation group were typical based on those of the 10 originally reported patients with Kabuki syndrome; those of the other groups were less typical. High arched eyebrows, short fifth finger, and hypotonia in infancy were more frequent in the MLL2 mutation group than in the KDM6A mutation group. Short stature and postnatal growth retardation were observed in all individuals with KDM6A mutations, but in only half of the group with MLL2 mutations.

Molecular Genetics of Vitamin D- Dependent Hereditary Rickets

Vitamin D exerts a wide variety of biological actions. The active form of vitamin D, 1α,25(OH)₂D₃, is biosynthesized from cholesterol. The final, critical step in this biosynthesis is conversion from 25-hydroxyvitamin D₃ to 1α,25(OH)₂D₃ by the enzyme 25-hydroxyvitamin D₃ 1α-hydroxylase(CYP27B1)[1α(OH)ase]. 1α,25(OH)₂D₃ transcriptionally controls the expression of a particular set of target genes mediated through nuclear vitamin D receptor(VDR) acting as a ligand-inducible factor. Two types of vitamin D-dependent hereditary rickets (VDDR) are known to be caused by mutations in the 1α(OH)ase and VDR genes. The 1α(OH)ase gene is responsible for VDDR type I, and VDR for type II. Both of the diseases display an autosomal-recessive trait, but clinical features and response to administrated 1α,25(OH)₂D₃ are distinct. The phenotypes of the gene KO mice deficient of 1α(OH)ase and VDR exhibited the clinical abnormalities observed in the VDDR patients.

Functional characterization of LMX1B mutations associated with nail-patella syndrome

Nail-patella syndrome (NPS) is an autosomal dominant disease characterized by dysplastic nails, absent or hypoplastic patellae, elbow dysplasia, and nephropathy. Recently, it was shown that NPS is the result of heterozygous mutations in the LIM-homeodomain gene, LMX1B. Subsequently, many mutations of the LMX1B gene have been reported in NPS patients. However, functional analyses of the mutant proteins have been performed in only a few mutations. Furthermore, the mechanisms of dominant inheritance in humans have not been established. In the present study, we analyzed the LMX1B gene in three Japanese patients with NPS and identified two novel mutations, 6 nucleotide deletion (Δ246Ν 247Q) and V242L. These two mutations are located in the homeodomain of LMX1B. Functional analyses of the LMX1B mutants revealed that these mutants had diminished transcriptional activity and had lost DNA binding ability. Furthermore, we demonstrated that each mutant did not manifest a dominant-negative effect on the transcriptional activity of wild-type LMX1B. These results suggested that NPS is caused by loss-of-function mutations of LMX1B, and haploinsufficiency of LMX1B should be the predominant pathogenesis of NPS in humans.

A novel compound heterozygous mutation in the thyroglobulin gene resulting in congenital goitrous hypothyroidism with high serum triiodothyronine levels

AbstractThyroglobulin abnormality is a rare cause of congenital hypothyroidism and only a limited number of mutations in the thyroglobulin gene have been reported. We analyzed the thyroglobulin gene in a patient with congenital goitrous hypothyroidism. This girl was identified with hyperthyrotropinemia in a neonatal mass-screening test. The patient had goiter, and her body weight gain was poor. Distal femoral epiphysis was absent on roentgenography. Her serum thyroxine level was low; however, her triiodothyronine level was high. Autoantibodies against triiodothyronine, thyroid peroxidase, and thyroglobulin were all negative. Her serum thyroglobulin level was undetectable. The thyroglobulin gene from the genomic DNA of the patient was analyzed by direct sequencing. Two novel heterozygous missense mutations, Cys1897Tyr (exon 31) and Arg2336Gln (exon 40), were found in the patient. The former mutation was derived from her mother, suggesting a compound heterozygous state. Normal triiodothyronine and low thyroxine concentrations are often observed in patients with thyroglobulin gene mutations. We considered that some patients with thyroglobulin abnormality might have high triiodothyronine levels. In cases of congenital goitrous hypothyroidism with normal-to-high triiodothyronine levels and low serum thyroglobulin levels, thyroglobulin abnormality should be considered.

Citrin deficiency presenting with ketotic hypoglycaemia and hepatomegaly in childhood.

Ketotic hypoglycaemia and hepatomegaly during childhood may be one of the symptoms of citrin deficiency. Adult-onset type II citrullinaemia (CTLN2) is characterised by late-onset attacks of hyperammonaemia often leading to rapid death [4]. CTLN2 is caused by mutations in the SLC25A13gene, encoding citrin, a Ca-stimulated aspartate glutamate carrier [2]. SLC25A13gene mutations also cause neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) [3, 4, 5,6]. The clinical features of NICCD usually disappear by 12 months of age, and some subjects develop symptoms of CTLN2 decades later [4,5]; however, the signs and symptoms during childhood are not well recognised. Our patient, a girl, had abnormal results on neonatal mass screening: hyperphenylalaninaemia at 5 days of age, and hypergalactosaemia with hypermethioninaemia at 17 days of age. Poor weight gain and hepatomegaly were noted. Biochemical data at 41 days of age revealed mild cholestasis, aminoacidaemia including a high citrulline level, but normal phenylalanine and methionine levels (Table 1). Galactosaemia was excluded by enzyme assay. Her weight gain and biochemical data improved by 6 months of age. At the ages of 1 year and 9 months and 2 years and 4 months, she presented with pallor and drowsiness in the morning after taking no meal since the evening before. Laboratory investigations revealed marked hypoglycaemia, lactic acidosis, and ketonuria (Table 1). During an oral glucose tolerance test, the blood lactate level decreased from 37.7 mg/dl to 10.6 mg/dl. A glucagon test showed no rise in blood glucose both in the fasting and postprandial state. Treatment with nocturnal uncooked cornstarch improved blood lactate levels and hepatomegaly. Hypoglycaemia recurred three times following failure to take cornstarch. Psychomotor development and laboratory data were normal at 4 years of age (Table 1). Her younger sister had normal neonatal mass screening results. She had a mild episode of hypoglycaemia (40 mg/dl) at 1 year and 10 months of age, and hepatomegaly was noticed. She had a slightly elevated citrulline level (Table 1). Both the index case and her sister liked beans and peanuts. Enzyme assay or DNA analysis for hepatic glycogen storage diseases were all normal. Citrin deficiency was suspected because of the abnormal neonatal mass screening [3]. Analysis of the SLC25A13gene was performed as described [6] with informed consent of the parents, and compound heterozygous mutations for 851del4 and IVS11+1G>A were found in the patient and her sister, indicating that they were suffering from citrin deficiency. The parents were heterozygous carriers. We considered that hypoglycaemia and hepatomegaly might be symptoms of citrin deficiency during childhood, from the co-segregation of the mutation and from data of a similar patient (M. Hasegawa, personal communication). Deficiency of citrin may block the malate aspartate shuttle, leading to increased cytosolic NADH/NAD, which inhibits gluconeogenesis from reduced substrates. This, together with the difficulty of conversion of alanine to glucose, may cause hypoglycaemia [4]. In addition, impaired fatty acid b-oxidaM. Hachisu Æ S. Noma Tokyo Metropolitan Hachioji Children’s Hospital, Tokyo, Japan

Validity of a self‐administered diet history questionnaire for estimating vitamin D intakes of Japanese pregnant women

Maternal vitamin D status is important for fetal development and the prevention of pregnancy complications. Mothers require both sufficient intakes and skin production of this vitamin. We investigated the validity and test-retest reliability of a self-administered diet history questionnaire (DHQ) to establish a method of assessing vitamin D intakes of Japanese pregnant women, using a serum marker. A total of 245 healthy pregnant women in the second trimester, who were not taking vitamin D supplements, were recruited at a university hospital in Tokyo between June 2010 and July 2011. Serum 25-hydroxyvitamin D [25(OH)D] concentrations were measured as an indicator of vitamin D status. To assess the test-retest reliability of the DHQ, 58 pregnant women completed it twice within a 4-5-week interval. Significant positive correlations between intakes and serum concentrations of vitamin D were found (r = 0.266 for daily intakes and r = 0.249 for energy-adjusted intakes). In the winter investigation in which the serum 25(OH)D concentrations were less likely to be affected by sunlight exposure, the correlation coefficients were 0.304 for both daily and energy-adjusted intakes. After excluding participants with pregnancy-associated nausea, the coefficients increased. The intraclass correlation coefficient between vitamin D intakes estimated from the two-time DHQ was 0.638. The DHQ provides an acceptable validity and reliability of the vitamin D intake of Japanese pregnant women. However, the data of women with nausea should be interpreted with caution. We believe that the DHQ is a useful questionnaire to grasp and improve vitamin D intakes during pregnancy.