A novel pathogenic variant in fibroblast growth factor 23 outside the furin-recognizing RXXR motif in an autosomal dominant hypophosphatemic rickets patient.

INTRODUCTION: Autosomal dominant hypophosphatemic rickets (ADHR) is caused by pathogenic variants in the fibroblast growth factor 23 (FGF23) gene, which plays a key role in the regulation of phosphorus metabolism. FGF23 has the RXXR motif recognized by furin, leading to cleavage between R179 and S180 and thereby inactivating the protein's function. Previously reported variants in FGF23 causing ADHR occurred only affecting residues R176 or R179, which are located in the RXXR motif, leading to impaired cleavage. Impairment of protein cleavage increases bioactive FGF23 levels, subsequently resulting in the development of ADHR. CASE PRESENTATION: A 13-year-old boy with ADHR with the appearance of rickets on bone radiographs as well as documented hypophosphatemia was found to have a novel S180I variant in the FGF23 gene. Unlike previously reported pathogenic variants, this novel variant was located outside the RXXR motif. Subsequently, western blotting showed that the S180I mutant was resistant to proteolysis than the wild-type, similar to pathogenic variants model mutant (R176Q/R179Q). CONCLUSION: The novel variant in FGF23 presented herein, found in a patient with ADHR, is the first pathogenic variant found outside the typical furin recognition sequence. It exhibits proteolysis resistance due to impaired cleavage.

Development of multi-drug resistance to anticancer drugs in HepG2 cells due to MRP2 upregulation on exposure to menthol.

BACKGROUND: Menthol exerts relaxing, antibacterial, and anti-inflammatory activities, and is marketed as a functional food and therapeutic drug. AIM: In the present study, the effects of menthol on the expression of multidrug resistance associated protein 2 (MRP2) and its association with the cytotoxicity of epirubicin (EPI) and cisplatin (CIS) were examined using HepG2 cells. METHODS: The expression levels of target genes were examined by real-time PCR. The intracellular concentration of incorporated EPI was measured by high-performance liquid chromatography. Cell viability was evaluated by MTT analysis. RESULTS: The expression of MRP2 mRNA was increased by exposing HepG2 cells to menthol for 24 hr. Consistent with a previous report suggesting an inverse correlation between MRP2 and Akt behavior, increased expression of MRP2 was also observed on suppression of the Akt function. Intracellular accumulation of EPI was significantly decreased by exposure of HepG2 cells to menthol, and a significant decrease in the intracellular concentration of EPI remaining was observed in HepG2 cells exposed to menthol. The decreased intracellular accumulation of EPI was significantly suppressed by treatment with MK-571, but not verapamil. Both EPI and CIS exerted cytocidal effects on HepG2 cells, but the decrease in cell viability was significantly attenuated by 24-hr menthol pre-exposure. CONCLUSION: These results demonstrate that menthol causes hepatocellular carcinoma to acquire resistance to anticancer drugs such as EPI and CIS by MRP2 induction.

Differences in Transport Characteristics and Cytotoxicity of Epirubicin and Doxorubicin in HepG2 and A549 Cells.

BACKGROUND/AIM: Epirubicin (EPI), an epimer of doxorubicin (DOX), and DOX are anthracycline agents with broad-spectrum antitumor activity. The aim of the present study was to elucidate the transport characteristics of EPI and DOX in human hepatocellular carcinoma HepG2 cells and human non-small cell lung cancer A549 cells, and to examine the relationship of intracellular drug accumulation with their cytotoxic effects. MATERIALS AND METHODS: Intracellular concentrations of EPI and DOX were measured using high-performance liquid chromatography (HPLC). Expression level of targeted genes was analyzed by real-time quantitative PCR. Cell viability was evaluated using the MTT assay. RESULTS: Similar to DOX, EPI was taken up into HepG2 and A549 cells by organic cation transporter 6 and passive diffusion; however, the efficiency of saturable and non-saturable uptake of EPI was greater than that of DOX in both cell types. EPI served as a substrate of P-glycoprotein and multidrug associated protein (MRP) 1 and MRP2 similarly to DOX, but the efflux efficiency of each transporter was markedly different between EPI and DOX. The intracellular accumulation of EPI was significantly greater than that of DOX in all cells, and the accumulated level reflected the cytotoxic effects of these drugs. However, the intracellular drug amount did not correspond to the degree of cytotoxicity when compared between HepG2 and A549 cells, which can be explained by the higher expression of Bcl-xl in A549 cells. CONCLUSION: This study suggested that the transport characteristics are markedly different between EPI and DOX in HepG2 and A549 cells, and that intracellular accumulation is the predominant factor affecting the cytotoxicity of EPI and DOX in individual cells.

Hereditary vitamin D-resistant rickets: a report of four cases with two novel variants in the VDR gene and successful use of intermittent intravenous calcium via a peripheral route.

Background Hereditary vitamin D-resistant rickets (HVDRR) is caused by vitamin D receptor (VDR) defects. Patients with HVDRR do not respond to standard doses of calcitriol and oral calcium (Ca) treatment and need to be treated with intravenous Ca (IV-Ca) via a central route. However, central catheter-related complications can cause significant morbidity. Case presentation Four unrelated patients with HVDRR presenting with rickets and alopecia totalis were administered intermittent IV-Ca treatment (2-5 times/week) through a peripheral route. No complications such as infection, extravasation or arrhythmias were detected upon peripheral infusion. Peripheral 1-22 months' duration of IV-Ca normalized parathyroid hormone (PTH) and alkaline phosphatase (ALP) in all patients, after which, oral Ca of 200-400 mg/kg/day and calcitriol of 0.5 µg/kg/day were sufficient to maintain normal PTH levels. Molecular studies on the VDR gene showed a previously reported homozygous c.454C > T (p.Q152*) pathogenic variant in two patients. Two novel homozygous variants in the other two patients were detected: (1) c.756-2A > G, which affects the splice acceptor site, and (2) c.66dupG (p.I23Dfs*20) variant leading to a frameshift that results in a premature stop codon. Conclusions Peripheral IV-Ca treatment is an effective and practical alternative treatment mode that provides dramatic clinical benefit in patients with HVDRR.

Functional analyses of a novel missense and other mutations of the vitamin D receptor in association with alopecia.

Hereditary 1,25-dihydroxyvitamin D-resistant rickets (HVDRR) is a rare disorder, caused by bialellic mutations of the vitamin D receptor (VDR) gene, sometimes associated with alopecia. The aim of this study is to elucidate the mechanism of functional disruption of a novel mutation, detected in a patient with HVDRR, comparing to other mutations with or without alopecia. The patient was a 2-year-old girl with alopecia, who was clinically diagnosed as HVDRR. Genetic analysis revealed a novel homozygous mutation, S360P, located in ligand binding domain (LBD). The mutation was predicted as not disease causing by Polyphen2 and SIFT. But the transcriptional activity of S360P was disrupted as well as other reported mutations, Q152X (located in the hinge lesion), and R274L, H305Q (located in LBD). Following assays revealed no ligand binding affinity, no interaction with cofactors or RXR and no functioning of nuclear localization signals. Our results provide an additional evidence for the previous findings suggesting that DNA binding by the VDR/RXR heterodimer is essential for the function of the VDR in hair development. In conclusion, we identified a novel missense mutation of VDR causing HVDRR with alopecia. Functional analyses revealed that the single amino acid substitution could disrupt the function of the protein.

Vitamin D-Deficient Rickets in Japan.

Objectives: Our study aimed to clarify the trend of vitamin D deficiency and rickets diagnosed in the past 10 years. Methods: This observational study used health insurance claims data from 2005 to 2014. The number of beneficiaries for 2005 and 2014 were 91 617 and 365 800, respectively. We included children aged 0 to 15 years diagnosed with vitamin D deficiency or vitamin D-deficient rickets; those with congenital/secondary rickets and low-birth-weight infants were excluded. We analyzed the number of patients and the temporal trend of these diseases in Japan. Results: The annual number of patients from 2005 to 2008 was <5. The number of patients in 2009 and 2014 were 3.88 (95% confidence interval = 1.77-7.37) and 12.30/100 000 (95% confidence interval = 8.97-16.46), respectively. Conclusions: Diagnosed cases of vitamin D deficiency and vitamin D-deficient rickets have apparently increased in Japan. Further studies to identify the trend, cause, and prevention of vitamin D-deficient rickets are necessary.

Novel DHCR7 mutation in a case of Smith-Lemli-Opitz syndrome showing 46,XY disorder of sex development.

Smith-Lemli-Opitz syndrome is an autosomal recessive disease caused by mutations in 7-dehydrocholesterol reductase (DHCR7), which is rarely observed in Japan. We report a Japanese case with 46,XY disorder of sex development and Y-shaped 2-3 toe syndactyly. DHCR7 gene analysis revealed compound heterozygous mutations including the novel mutation H442R. Early diagnosis led to starting cholesterol treatment at an early age.

Detection of Hereditary 1,25-Hydroxyvitamin D-Resistant Rickets Caused by Uniparental Disomy of Chromosome 12 Using Genome-Wide Single Nucleotide Polymorphism Array.

CONTEXT: Hereditary 1,25-dihydroxyvitamin D-resistant rickets (HVDRR) is an autosomal recessive disease caused by biallelic mutations in the vitamin D receptor (VDR) gene. No patients have been reported with uniparental disomy (UPD). OBJECTIVE: Using genome-wide single nucleotide polymorphism (SNP) array to confirm whether HVDRR was caused by UPD of chromosome 12. MATERIALS AND METHODS: A 2-year-old girl with alopecia and short stature and without any family history of consanguinity was diagnosed with HVDRR by typical laboratory data findings and clinical features of rickets. Sequence analysis of VDR was performed, and the origin of the homozygous mutation was investigated by target SNP sequencing, short tandem repeat analysis, and genome-wide SNP array. RESULTS: The patient had a homozygous p.Arg73Ter nonsense mutation. Her mother was heterozygous for the mutation, but her father was negative. We excluded gross deletion of the father's allele or paternal discordance. Genome-wide SNP array of the family (the patient and her parents) showed complete maternal isodisomy of chromosome 12. She was successfully treated with high-dose oral calcium. CONCLUSIONS: This is the first report of HVDRR caused by UPD, and the third case of complete UPD of chromosome 12, in the published literature. Genome-wide SNP array was useful for detecting isodisomy and the parental origin of the allele. Comprehensive examination of the homozygous state is essential for accurate genetic counseling of recurrence risk and appropriate monitoring for other chromosome 12 related disorders. Furthermore, oral calcium therapy was effective as an initial treatment for rickets in this instance.

Hereditary 1,25-dihydroxyvitamin D-resistant rickets (HVDRR) caused by a VDR mutation: A novel mechanism of dominant inheritance.

Hereditary 1,25-dihydroxyvitamin D-resistant rickets (HVDRR) is caused by mutations in the VDR gene, and its inheritance is autosomal recessive. In this report, we aimed to confirm whether HVDRR is occasionally inherited as a dominant trait. An 18-month-old Japanese boy was evaluated for short stature and bowlegs. His father had been treated for rickets during childhood, and his paternal grandfather had bowlegs. We diagnosed him with HVDRR based on laboratory data and radiographic evidence of rickets. Sequence analyses of VDR were performed, and the functional consequences of the detected mutations were analyzed for transcriptional activity, ligand binding, and interaction with the retinoid X receptor, cofactors, and the vitamin D response element (VDRE). A novel mutation (Q400LfsX7) and a reported variant (R370H) were identified in the patient. Heterozygous Q400LfsX7 was detected in his father, and heterozygous R370H was detected in his healthy mother. Functional studies revealed that the transcriptional activity of Q400LfsX7-VDR was markedly disturbed. The mutant had a dominant-negative effect on wild-type-VDR, and the ligand binding affinity of Q400LfsX7-VDR was completely impaired. Interestingly, Q400LfsX7-VDR had a strong interaction with corepressor NCoR and could interact with VDRE without the ligand. R370H-VDR was functionally similar to wild-type-VDR. In conclusion, we found a dominant-negative mutant of VDR causing dominantly inherited HVDRR through a constitutive corepressor interaction, a mechanism similar to that in dominantly inherited thyroid hormone receptor mutations. Our report together with a reported pedigree suggested a distinct inheritance of HVDRR and enriched our understanding of VDR abnormalities.