Hypertension is a characteristic complication of X-linked hypophosphatemia.

X-linked hypophosphatemia (XLH) is a group of rare disorders caused by defective proximal tubular reabsorption of phosphate. Mutations in the PHEX gene are responsible for the majority of cases. There are very few reports of long-term complications of XLH other than skeletal and dental diseases. The aim of this study was to identify the phenotypic presentation of XLH during adulthood including complications other than skeletal and dental diseases. The clinical and biochemical phenotype of 22 adult patients with a PHEX gene mutation were examined retrospectively from their medical records. 6 patients had hypertension. The average age of hypertension onset was 29.0 years. Secondary hyperparathyroidism preceded the development of hypertension in 5 patients. 1 patient developed tertiary hyperparathyroidism. 15 patients had nephrocalcinosis. 2 patients had chronic renal dysfunction. Patients with hypertension had a significantly lower eGFR (p=0.010) compared to patients without hypertension. No significant difference was found in any other parameters. To examine the genotype-phenotype correlation, 10 adult males were chosen for analysis. No significant genotype-phenotype correlation analysis was revealed in any of the complications. However, there was a possibility that the age at nephrocalcinosis onset was younger in the non-missense mutation group than in the missense mutation group (p=0.063). This study corroborated the view that early-onset hypertension could be one of the characteristic complications seen in XLH patients. Considering the limited number of our patients, further study is necessary to address a potential cause of hypertension. XLH patients require careful lifelong treatment.

Free T3 to free T4 ratio less than 2.0 suggests low T3 syndrome rather than central hypothyroidism from the age of two to eighteen years.

No serological cut-off exists to separate low T3 syndrome (LT) and central hypothyroidism (CH). The objective of this retrospective study was to propose such a cut-off. The first participant group comprised 52 patients from the age of six to twenty years. This group consisted of patients of 36 anorexia nervosa with LT and 16 CH. The second participant groups comprised 229 patients of all the same range of ages at the same hospital and included LT (n = 58) and CH (n = 4) patients, respectively. The third group of participants comprised 125 LT and 27 CH patients at the same hospital at all ages less than eighteen years. The last group of participants comprised 10 CH patients from the other two hospitals. The main outcome measure was fT3/fT4 ratio (pg/mL, ng/dL respectively). This ratio in the first group was significantly different (p < 0.05) between LT and CH. When the cut off value of fT3/fT4 was set as 2.0, the sensitivity of the LT and CH patients in the second group was 62% and 100%, respectively. This cut-off value of 2.0 was useful for distinguishing LT from CH only above the age of two years, as shown in the third group. The fT3/fT4 in 10 subjects with CH in the last group, aged 2 to 7 years, ranged from 2.55 to 7.71. In conclusion, fT3/fT4 less than 2.0 suggests LT rather than CH for patients from the age of two to eighteen years.

Lower body weight and BMI at birth were associated with early adiposity rebound in 21-hydroxylase deficiency patients.

21-hydroxylase deficiency (21-OHD) is the most common type of congenital adrenal hyperplasia. In addition to the clinical problems caused by adrenal insufficiency and androgen excess, a risk for obesity and metabolic syndrome during young adulthood is a major ramification of the disease. Although glucocorticoid therapy is very likely to be one of the contributory factors, the precise causes of the metabolic status of adult 21-OHD patients remain to be clarified. Previously we reported that 21-OHD patients developed early onset AR, a condition which might create a risk for obesity and metabolic syndrome in adulthood. In order to elucidate the association between the onset of AR and factors during the fetal period to early infancy, we conducted a retrospective longitudinal analysis of 29 21-OHD patients (male: 14 cases, female: 15 cases, salt wasting type: 16, simple virilizing type: 13), who were identified by newborn screening and followed up at least until the age 10 years. Body size at birth, lower body weight, and lower BMI were found to precipitate the timing of AR. On the other hand, no significant association was observed between the timing of AR and sex, gestational age, treatment regimen (including cumulative dose of HDC), and disease severity (the type of the disease, the value of DHEA-S and 17-OHP). There are two points to consider: first, in 21-OHD patients treated with glucocorticoid substitution therapy, the risk for early AR cannot be reduced by adjusting the dose of glucocorticoid; second, fetal factors might affect the metabolic status of 21-OHD patients.

A Novel Mutation in OTX2 Causes Combined Pituitary Hormone Deficiency, Bilateral Microphthalmia, and Agenesis of the Left Internal Carotid Artery.

BACKGROUND: Mutations in OTX2 cause hypopituitarism, ranging from isolated growth hormone deficiency to combined pituitary hormone deficiency (CPHD), which are commonly detected in association with severe eye abnormalities, including anophthalmia or microphthalmia. Pituitary phenotypes of OTX2 mutation carriers are highly variable; however, ACTH deficiency during the neonatal period is not common in previous reports. OBJECTIVE: We report a novel missense OTX2 (R89P) mutation in a CPHD patient with severe hypoglycemia in the neonatal period due to ACTH deficiency, bilateral microphthalmia, and agenesis of the left internal carotid artery (ICA). RESULTS: We identified a novel heterozygous mutation in OTX2 (c.266G>C, p.R89P). R89P OTX2 showed markedly reduced transcriptional activity of HESX1 and POU1F1 reporters compared with wild-type OTX2. A dominant negative effect was noted only in the transcription analysis with POU1F1 promoter. Electrophoretic mobility shift assay experiments showed that R89P OTX2 abrogated DNA-binding ability. CONCLUSION: OTX2 mutations can cause ACTH deficiency in the neonatal period. Our study also shows that OTX2 mutations are associated with agenesis of the ICA. To the best of our knowledge, this is the first report of a transcription factor gene mutation, which was identified due to agenesis of the ICA of a patient with CPHD. This study extends our understanding of the phenotypic features, molecular mechanism, and developmental course associated with mutations in OTX2.

Nck influences preosteoblastic/osteoblastic migration and bone mass.

Migration of the cells in osteoblastic lineage, including preosteoblasts and osteoblasts, has been postulated to influence bone formation. However, the molecular bases that link preosteoblastic/osteoblastic cell migration and bone formation are incompletely understood. Nck (noncatalytic region of tyrosine kinase; collectively referred to Nck1 and Nck2) is a member of the signaling adaptors that regulate cell migration and cytoskeletal structures, but its function in cells in the osteoblastic lineage is not known. Therefore, we examined the role of Nck in migration of these cells. Nck is expressed in preosteoblasts/osteoblasts, and its knockdown suppresses migration as well as cell spreading and attachment to substrates. In contrast, Nck1 overexpression enhances spreading and increases migration and attachment. As for signaling, Nck double knockdown suppresses migration toward IGF1 (insulin-like growth factor 1). In these cells, Nck1 binds to IRS-1 (insulin receptor substrate 1) based on immunoprecipitation experiments using anti-Nck and anti-IRS-1 antibodies. In vivo, Nck knockdown suppresses enlargement of the pellet of DiI-labeled preosteoblasts/osteoblasts placed in the calvarial defects. Genetic experiments indicate that conditional double deletion of both Nck1 and Nck2 specifically in osteoblasts causes osteopenia. In these mice, Nck double deficiency suppresses the levels of bone-formation parameters such as bone formation rate in vivo. Interestingly, bone-resorption parameters are not affected. Finally, Nck deficiency suppresses repair of bone injury after bone marrow ablation. These results reveal that Nck regulates preosteoblastic/osteoblastic migration and bone mass.

A 45,X/46,XY DSD (Disorder of Sexual Development) case with an extremely uneven distribution of 46,XY cells between lymphocytes and gonads.

In 45,X/46,XY DSDs, the proportion of the two cell lineages is uneven in different organs and tissues, and 45,X and 46,XY cells can be found throughout the body. The gonadal development of 45,X/46,XY patients depends on the population of 46,XY cells in the gonads and the clinical features are variable. We had a 45,X/46,XY DSD patient whose 46,XY population in peripheral blood was extremely low, less than 0.2%, and was not detected by FISH analysis. However, the patient showed bilateral testicular development and more than 50% of the cells in the gonads had the 46,XY karyotype. This case suggests that a drastically imbalanced distribution could occur in 45,X/46,XY DSD cases.

Hypophosphatemic rickets developed after treatment with etidronate disodium in a patient with generalized arterial calcification in infancy.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) was originally reported as a responsible gene for generalized arterial calcification in infancy (GACI). Though the prognosis of GACI patients is poor because of myocardial infarction and heart failure in relation to medial calcification of the coronary arteries, some patients rescued by bisphosphonate treatment have been reported. Recently, ENPP1 is also reported as responsible for autosomal recessive hypophosphatemic rickets type 2. We show here a boy with homozygous ENPP1 mutations diagnosed as having GACI in early infancy. After the diagnosis, he was treated with etidronate disodium (EHDP) in combination with antihypertensive drugs. The calcification of major arteries was diminished and disappeared by the age of eight months. He also showed mild hypophosphatemia (2.6-3.7 mg/dl) from the age of one year. After the treatment with EHDP for five years, he showed genu valgum with hypophosphatemia (2.6 mg/dl). He was diagnosed as having hypophosphatemic rickets at the age of seven years. The findings that hyper-mineralization of the arteries and hypo-mineralization of the bone observed in the same patient are noteworthy. ENPP1 could be regarded as a controller of the calcification of the whole body at least in part.

Urinary calcium to creatinine ratio: a potential marker of secondary hyperparathyroidism in patients with vitamin D-dependent rickets type 1A.

Patients with vitamin D-dependent rickets type 1A (VDDR1A) are usually treated with alfacalcidol, an analog of vitamin D. Around puberty, an increased dose of alfacalcidol is recommended for these patients to avoid hypocalcemia and secondary hyperparathyroidism. However, no indicators of secondary hyperparathyroidism except for PTH are presently known. The aim of this study is to evaluate whether urinary calcium to creatinine ratio (U-Ca/Cr) is useful as a biomarker of secondary hyperparathyroidism in VDDR1A patients in order to determine the proper dose of alfacalcidol. Two brothers with VDDR1A were recruited who had null mutations of CYP27B1 which encodes 1-alpha-hydroxylase of vitamin D. We investigated the relationship between U-Ca/Cr and intact-PTH around puberty when the brothers showed hypocalcemia with secondary hyperparathyroidism. The results were compared to those of five patients with vitamin D deficiency (VDD). As a result, high intact-PTH levels were observed when U-Ca/Cr decreased to less than 0.1 (mg/mg) in both VDDR1A brothers. This relationship was also observed in the VDD patients. However, it is necessary to take into account body calcium status, either in depletion or in excess, to accurately evaluate the relationship between U-Ca/Cr and secondary hyperparathyroidism. First, low U-Ca/Cr was detected in situations with calcium depletion without hyperparathyroidism in the VDDR1A patients. Second, high U-Ca/Cr with hyperparathyroidism could be detected theoretically in a condition of excess calcium supply. In conclusion, a U-Ca/Cr ratio of less than 0.1 (mg/mg) in VDDR1A patients is useful to accurately evaluate calcium depletion and secondary hyperparathyroidism.

[Updates on rickets and osteomalacia: pathogenesis and pathophysiology of rickets].

Rickets is a condition of inadequate mineralization of osteoid and cartilage at the growing ends of bones in children. In this brief review, we first explained the regulation of serum Ca and P concentrations to understand Rickets. Second, four types of sub-division of Rickets are presented ; 1) Vitamin D dysfunction-related, 2) Phosphate deficiency-related, 3) both 1) and 2) -related, 4) others. Finally, as most common entities, diagnosis and treatment in vitamin D deficiency and inherited hypophosphatemic Rickets/Osteomalacia are described. Over production of Klotho and inactivating mutations of FAM20c are explained as recent etiologies of non-hypercaluciuric inherited hypophosphatemic Rickets/Osteomalacia.

Nck1 deficiency accelerates unloading-induced bone loss.

Mechanical stress is an important signal to determine the levels of bone mass. Unloading-induced osteoporosis is a critical issue in bed-ridden patients and astronauts. Many molecules have been suggested to be involved in sensing mechanical stress in bone, though the mechanisms involved in this phenomenon are not fully understood. Nck1 is an adaptor protein known to mediate signaling from plasma membrane-activated receptors to cytosolic effectors regulating actin cytoskeleton remodeling. Nck1 has also been implicated in cellular responses to endoplasmic reticulum stress. In vitro, in case of cell stress the actin cytoskeleton is disrupted and in such cases Nck1 has been reported to enter the nucleus of the cells to mediate the nuclear actin polymerization. However, the role of Nck1 in vivo during the bone response to mechanical stimuli is unknown. The purpose of this study is to examine the role of Nck1 in unloading-induced bone loss in vivo. Sciatic and femoral nerve resection was conducted. Neurectomy-based unloading enhanced Nck1 gene expression in bone about twofold. Using the Nck1 deficient mice and control Nck1+/+, effects of neurectomy-based unloading on bone structure were examined. Unloading reduced bone volume in wild type mice by 30% whereas the levels in bone loss were exacerbated to 50% in Nck1 deficient mice due to neurectomy after 4 weeks. These data demonstrate that Nck1 gene deficiency accelerates the mechanical unloading-induced bone loss suggesting Nck1 to be a crucial molecule in mechanical stress mediated regulation in bone metabolism.

Longitudinal analysis of growth and body composition of Japanese 21-OHD patients in childhood.

Substitution therapy of glucocorticoid is a major part of the treatment for 21-OHD (21-hydroxylase deficiency). However, the therapy causes two major adverse effects, impairment of linear growth and obesity, so that collecting precise growth data is essential for optimizing the therapy. We longitudinally evaluated the linear growth and the body composition of Japanese 21-OHD patients during childhood. For the present study, we chose 16 patients (eight of each sex) who were diagnosed during the newborn period, and continuously observed them in our institute until they were at least 15 years old. All patients were treated according to the guidelines from The Japanese Society for Pediatric Endocrinology. The final height standard deviation score (Ht-SDS) of all the patients was -1.18 ± 0.85 SD, and no significant differences were observed between males and females or between the simple virilizing form and the salt wasting form. As previously reported, in spite of nearly normal height at the onset of puberty, the pubertal height gains were severely impaired, resulting in reduced final heights. Body composition of the patients was evaluated with BMI-SDS. Our longitudinal data showed that BMI was increased up to +1.23 SD in males and up to +1.75 SD in females, and that adiposity rebound was precipitated. Our study should alert physicians to the risk of metabolic syndrome and provide a framework for further studies of metabolic syndrome in 21-OHD patients.

[Bone fracture and the healing mechanisms. Molecular bases of fracture healing].

Fracture healing is a process comprising of a local bleeding followed by inflammation and differentiation of mesenchymal cells that lead to formation of soft extracellular matrix tissue, cartilage and bone. This pathway includes endo-chondral bone formation and in part intra-membranous bone formation. During this process several sets of cytokines are involved in the regulation of the progress in fracture healing. This paper reviews the molecules involved in fracture healing.

ANA deficiency enhances bone morphogenetic protein-induced ectopic bone formation via transcriptional events.

Ectopic bone formation after joint replacement or brain injury in humans is a serious complication that causes immobility of joints and severe pain. However, mechanisms underlying such ectopic bone formation are not fully understood. Bone morphogenetic protein (BMPs) are defined as inducers of ectopic bone formation, and they are regulated by several types of inhibitors. ANA is an antiproliferative molecule that belongs to Tob/BTG family, but its activity in bone metabolism has not been known. Here, we examined the role of ANA on ectopic bone formation activity of BMP. In ANA-deficient and wild-type mice, BMP2 was implanted to induce ectopic bone formation in muscle. ANA deficiency increased mass of newly formed bone in vivo compared with wild-type based on 3D-muCT analyses. ANA mRNA was expressed in bone in vivo as well as in osteoblastic cells in vitro. Such ANA mRNA levels were increased by BMP2 treatment in MC3T3-E1 osteoblastic cells. Overexpression of ANA suppressed BMP-induced expression of luciferase reporter gene linked to BMP response elements in these cells. Conversely, ANA mRNA knockdown by small interference RNA enhanced the BMP-dependent BMP response element reporter expression. It also enhanced BMP-induced osteoblastic differentiation in muscle-derived C2C12 cells. Immunoprecipitation assay indicated that ANA interacts with Smad8. Thus, ANA is a suppressor of ectopic bone formation induced by BMP, and this inhibitory ANA activity is a part of the negative feedback regulation of BMP function.

Analysis of the hypothalamus in a case of X-linked lissencephaly with abnormal genitalia (XLAG).

X-linked lissencephaly with abnormal genitalia (XLAG) is characterized by lissencephaly, absent corpus callosum and ambiguous genitalia. We examined hypothalamic dysfunctions in a XLAG case with a novel mutation of the ARX gene, and performed immunohistochemical evaluation of the diencephalons in autopsy brain. A 1-year-old boy showed intractable epilepsy, persistent diarrhea and disturbed temperature regulation. This case had abnormalities in circadian rhythms and pituitary hormone reserve test. He died of pneumonia. The globus pallidus and subthalamic nucleus was not identified, and the putamen and thalamus were dysplasic. The suprachiasmatic nucleus was absent. A few neurons immunoreactive for vasopressin seemed to form the ectopic supraoptic-like nucleus. The diencephalons were disturbed differently in each sub-region, and the changes may be related to various hypothalamic dysfunctions.

Small cell carcinoma of the uterine cervix metastasizing to the bone marrow: a case report.

We report a case of small cell carcinoma (SmCC) of the uterine cervix that metastasized to the bone marrow. A 60-year-old woman with stage IIB SmCC of the cervix was treated with three courses of neoadjuvant chemotherapy followed by radical hysterectomy. Because of the presence of a large residual tumor, the patient underwent postoperative adjuvant chemotherapy. Two months after the last course of chemotherapy, severe pancytopenia developed, and erythroblastic cells were found in the peripheral blood. The hematological disorder was shown to be secondary to bone marrow metastasis, and no other metastases were found. The patient died of the disease 8 months after the initial diagnosis. This case suggests that SmCC of the cervix can metastasize to bone marrow, that such metastasis can occur in isolation and lead to severe pancytopenia, influencing the clinical course of the disease.

Liquid chromatography-tandem mass spectrometric method for determination of salivary 17alpha-hydroxyprogesterone: a noninvasive tool for evaluating efficacy of hormone replacement therapy in congenital adrenal hyperplasia.

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS-MS) method for the quantification of 17alpha-hydroxyprogesterone (17OHP) in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata-X cartridge, derivatized with a highly proton-affinitive reagent, 2-hydrazinopyridine, and subjected to LC-MS-MS. Quantification was based on the selected reaction monitoring, and deuterated 17OHP was used as the internal standard. This method allowed the reproducible and accurate quantification of the salivary 17OHP using a 200-mul sample, and the limit of quantitation was 5.0 pg/ml. The developed method was applied to clinical studies. A linear relationship was found to be positive (r(2)=0.975) between the blood 17OHP level and the salivary 17OHP level measured using the proposed method. The result from the salivary 17OHP measurement in patients with congenital adrenal hyperplasia demonstrated that the proposed method is very useful for monitoring of the therapeutic efficacy during hormone replacement therapy.