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Abstract The objectives of this study were the biostratigraphic and paleobiogeographic interpretations of the calcareous nannofossil records of the Romualdo Formation (Aptian-Albian) in the south-central portion of the Araripe Basin. The methods used to process the samples were decanting, smear slide, centrifugation, and ultra-thin section confection. The taxonomic and biostratigraphic interpretations were carried out through qualitative and quantitative investigations. A total of 11 genera and 17 species of calcareous nannofossils, namely, Biscutum constans, Biscutum sp., Braarudosphaera africana, Braarudosphaera sp., Calculites sp., Discorhabdus ignotus, Hayesites albiensis, Hayesites sp., Nannoconus bucheri, Nannoconus sp., Retecapsa surirella, Rhagodiscus sp., Thoracosphaera sp., Watznaueria barnesiae, Watznaueria sp., Zeughrabdotus erectus, and Zeughrabdotus noeliae were identified. The integrated analysis of three outcrops suggests two distinct marine phases: the first in the Aptian, recognized in two outcrops, and during the Albian age. The genera Hayesites and Nannoconus suggest a strong Tethyan affinity. Differently, other calcareous nannofossils such as Watznaueria, Thoracosphaera, and Braarudosphaera recorded here are considered more resistant, cosmopolitan, and/or opportunistic species. Biscutum and Zeughrabdotus are sensitive calcareous nannofossils because they are more susceptible to dissolution and cannot be surely used to define this paleobiogeography and biostratigraphic range.

Abstract Background Schimke immuno-osseous dysplasia (SIOD) is a multisystemic disorder caused by biallelic mutations in the SWI/SNF-related matrix-associated actin-dependent regulator of chromatin, subfamily A-like 1 (SMARCAL1) gene. Changes in gene expression underlie the arteriosclerosis and T-cell immunodeficiency of SIOD; therefore, we hypothesized that SMARCAL1 deficiency causes the focal segmental glomerulosclerosis (FSGS) of SIOD by altering renal gene expression. We tested this hypothesis by gene expression analysis of an SIOD patient kidney and verified these findings through immunofluorescent analysis in additional SIOD patients and a genetic interaction analysis in Drosophila. Results We found increased expression of components and targets of the Wnt and Notch signaling pathways in the SIOD patient kidney, increased levels of unphosphorylated β-catenin and Notch1 intracellular domain in the glomeruli of most SIOD patient kidneys, and genetic interaction between the Drosophila SMARCAL1 homologue Marcal1 and genes of the Wnt and Notch signaling pathways. Conclusions We conclude that increased Wnt and Notch activity result from SMARCAL1 deficiency and, as established causes of FSGS, contribute to the renal disease of most SIOD patients. This further clarifies the pathogenesis of SIOD and will hopefully direct potential therapeutic approaches for SIOD patients.

Mutations in transcription factors genes, which are well regulated spatially and temporally in the pituitary gland, result in congenital hypopituitarism (CH) in humans. The prevalence of CH attributable to transcription factor mutations appears to be rare and varies among populations.This study aimed to define the prevalence of CH in terms of nine CH-associated genes among Japanese patients. We enrolled 91 Japanese CH patients for DNA sequencing of POU1F1, PROP1, HESX1, LHX3, LHX4, SOX2, SOX3, OTX2, and GLI2. Additionally, gene copy numbers for POU1F1, PROP1, HESX1, LHX3, and LHX4 were examined by multiplex ligation-dependent probe amplification. The gene regulatory properties of mutant LHX4 proteins were characterized in vitro. We identified two novel heterozygous LHX4 mutations, namely c.249-1G>A, p.V75I, and one common POU1F1 mutation, p.R271W. The patient harboring the c.249-1G>A mutation exhibited isolated growth hormone deficiency at diagnosis and a gradual loss of ACTH, whereas the patient with the p.V75I mutation exhibited multiple pituitary hormone deficiency. In vitro experiments showed that both LHX4 mutations were associated with an impairment of the transactivation capacities of POU1F1 andαGSU, without any dominant-negative effects. The total mutation prevalence in Japanese CH patients was 3.3%. This study is the first to describe, a gradual loss of ACTH in a patient carrying an LHX4 mutation. Careful monitoring of hypothalamic-pituitary -adrenal function is recommended for CH patients with LHX4 mutations.

The diagnosis of pseudohypoparathyroidism type 1a (PHP1a) is challenging, because both the osteodystrophy, such as brachydactyly and round face, and the symptomatic hypocalcemia usually develop beyond infancy. Although ectopic calcification may be an early sign of PHP1a, there are no systematic reviews regarding the time of its appearance. We here report on two PHP1a patients who presented with subcutaneous calcification in neonatal period.

The objectives of this study were the biostratigraphic and paleobiogeographic interpretations of the calcareous nannofossil records of the Romualdo Formation (Aptian-Albian) in the south-central portion of the Araripe Basin. The methods used to process the samples were decanting, smear slide, centrifugation, and ultra-thin section confection. The taxonomic and biostratigraphic interpretations were carried out through qualitative and quantitative investigations. A total of 11 genera and 17 species of calcareous nannofossils, namely, Biscutum constans, Biscutum sp., Braarudosphaera africana, Braarudosphaera sp., Calculites sp., Discorhabdus ignotus, Hayesites albiensis, Hayesites sp., Nannoconus bucheri, Nannoconus sp., Retecapsa surirella, Rhagodiscus sp., Thoracosphaera sp., Watznaueria barnesiae, Watznaueria sp., Zeughrabdotus erectus, and Zeughrabdotus noeliae were identified. The integrated analysis of three outcrops suggests two distinct marine phases: the first in the Aptian, recognized in two outcrops, and during the Albian age. The genera Hayesites and Nannoconus suggest a strong Tethyan affinity. Differently, other calcareous nannofossils such as Watznaueria, Thoracosphaera, and Braarudosphaera recorded here are considered more resistant, cosmopolitan, and/or opportunistic species. Biscutum and Zeughrabdotus are sensitive calcareous nannofossils because they are more susceptible to dissolution and cannot be surely used to define this paleobiogeography and biostratigraphic range. [ABSTRACT FROM AUTHOR]

SOX9, a transcription factor, is expressed in the undifferentiated XX and XY gonads. SRY induces significant upregulation of SOX9 expression in XY gonads. Loss‐of‐function SOX9 variants cause testicular dysgenesis in 46,XY patients, while duplication of the total gene or the upstream regulatory region results in testicular development in 46,XX patients. However, gain‐of‐function (GoF) SOX9 variants have not been reported previously. We report the case of a 16‐year‐old female patient with a 46,XX karyotype who had masculinized external genitalia and unilateral ovotestis. Next‐generation sequencing‐based genetic screening for disorders of sex development led to the identification of a novel SOX9 variant (p.Glu50Lys), transmitted from the phenotypically normal father. Expression analysis showed that E50K‐SOX9 enhanced transactivation of the luciferase reporter containing the testis enhancer sequence core element compared with that containing the wildtype‐SOX9. This GoF activity was not observed in the luciferase reporter containing Amh, the gene for anti‐Müllerian hormone. We genetically engineered female mice (Sox9E50K/E50K), and they showed no abnormalities in the external genitalia or ovaries. In conclusion, a novel SOX9 variant with a promoter‐specific GoF activity was identified in vitro; however, the disease phenotype was not recapitulated by the mouse model. At present, the association between the GoF SOX9 variant and the ovotestis phenotype remains unclear. Future studies are needed to verify the possible association. [ABSTRACT FROM AUTHOR]

Context: PAX8 is a transcription factor required for thyroid development, and its mutation causes congenital hypothyroidism (CH). More than 20 experimentally verified loss-of-function PAX8 mutations have been described, and all but one were located in the DNA-binding paired domain.Objective: We report the identification and functional characterization of 3 novel truncating PAX8 mutations located outside the paired domain.Methods: Three CH probands, diagnosed in the frame of newborn screening, had thyroid hypoplasia and were treated with levothyroxine. Next-generation sequencing-based mutation screening was performed. Functionality of the identified mutations were verified with Western blotting, intracellular localization assays, and transactivation assays with use of HeLa cells. Luciferase complementation assays were used to evaluate the effect of mutations on the interaction between PAX8 and its partner, NKX2-1.Results: Each proband had novel truncating PAX8 mutations that were I160Sfs*52, Q213Efs*27, and F342Rfs*85. Western blotting showed destabilization of the I160fs-PAX8 protein. Q213fs-PAX8 and F342fs-PAX8 showed normal protein expression levels and normal nuclear localization, but showed loss of transactivation of the luciferase reporter. By luciferase complementation assays, we showed that PAX8-NKX2-1 interaction was defective in Q213fs-PAX8. We also characterized the recombinant PAX8 proteins, and found that the protein sequence corresponding to exon 10 (363-400 aa residues) was essential for the PAX8-NKX2-1 interaction.Conclusions: Clinical and molecular findings of 3 novel truncating PAX8 mutations located outside the paired domain were reported. Experiments using cultured cells and recombinant proteins showed that the C-terminal portion (ie, 363-400 aa) of PAX8 is required for the PAX8-NKX2-1 interaction. [ABSTRACT FROM AUTHOR]

Objective: Biallelic TSHR mutations cause congenital hypothyroidism (CH). Serum TSH levels of monoallelic mutation carriers range from normal to mildly elevated, and thus the size of its effect remains unclear. The objectives were to examine the association between monoallelic TSHR mutations and positivity at newborn screening (NBS) for CH, and to test whether the association was modified by another genetic factor. Subjects and methods: We enrolled 395 patients that had a positive result in NBS and sequenced TSHR. Monoallelic TSHR mutation carriers were further sequenced for DUOX2. Molecular functions of the mutations were verified in vitro. The frequency of the mutations in the study subjects was compared with a theoretical value in the Japanese general population. Odds ratio (OR) for NBS positivity associated with the mutation was calculated. Using Bayes' theorem, we estimated a posterior probability of NBS positivity given the mutation. Results: Twenty-six monoallelic TSHR mutation carriers were found. Four out of the 26 also had a monoallelic DUOX2 mutation (double heterozygotes). The frequencies of monoallelic TSHR mutation carriers (6.6%) and double heterozygotes (1.0%) were significantly higher than those in the general population (0.58% and 0.0087%, respectively). OR for NBS positivity of having a monoallelic TSHR mutation or being a double heterozygote was 12.0 or 117.9, respectively. Posterior probability of NBS positivity was 0.38% in monoallelic TSHR mutation carriers and 3.8% in double heterozygotes. Conclusions: Monoallelic TSHR mutations are significantly associated with NBS positivity, and the association is further strengthened by the coexistence of monoallelic DUOX2 mutations. [ABSTRACT FROM AUTHOR]

Partial lipodystrophy (PD), a condition similar to metabolic syndrome without obesity, is one of the late complications of hematopoietic stem cell transplantation (HSCT) performed during childhood. We aimed to investigate the prevalence and risk factors of PD. A cross-sectional survey was performed in a children's hospital, targeting patients treated for a malignancy or hematological disorder, and who were disease-free for > 24 mo. PD was defined as gluteal lipoatrophy and lipohypertrophy of the cheeks or neck associated with diabetes and/or fatty liver disease. In total, 65 patients were enrolled. Six patients (9.2%) were judged to have PD, all of whom had received 10-14 Gy total body irradiation. Compared with the patients without PD, patients with PD were older at investigation (P < 0.01), had a longer elapsed time following HSCT (P < 0.01), had more frequent disease recurrence (P < 0.05), and were more likely to have undergone multiple HSCT (P < 0.05). In addition, they had higher blood pressure and showed higher levels of low-density lipoprotein-cholesterol and triglycerides, whereas their adiponectin levels were significantly lower. In conclusion, a large number of patients developed PD following HSCT, with unfavorable metabolic profiles at a later age, especially when they experienced a complex disease course. [ABSTRACT FROM AUTHOR]

Background: Schimke immuno-osseous dysplasia (SIOD) is a multisystemic disorder caused by biallelic mutations in the SWI/SNF-related matrix-associated actin-dependent regulator of chromatin, subfamily A-like 1 (SMARCAL1) gene. Changes in gene expression underlie the arteriosclerosis and T-cell immunodeficiency of SIOD; therefore, we hypothesized that SMARCAL1 deficiency causes the focal segmental glomerulosclerosis (FSGS) of SIOD by altering renal gene expression. We tested this hypothesis by gene expression analysis of an SIOD patient kidney and verified these findings through immunofluorescent analysis in additional SIOD patients and a genetic interaction analysis in Drosophila. Results: We found increased expression of components and targets of the Wnt and Notch signaling pathways in the SIOD patient kidney, increased levels of unphosphorylated β-catenin and Notch1 intracellular domain in the glomeruli of most SIOD patient kidneys, and genetic interaction between the Drosophila SMARCAL1 homologue Marcal1 and genes of the Wnt and Notch signaling pathways. Conclusions: We conclude that increased Wnt and Notch activity result from SMARCAL1 deficiency and, as established causes of FSGS, contribute to the renal disease of most SIOD patients. This further clarifies the pathogenesis of SIOD and will hopefully direct potential therapeutic approaches for SIOD patients. [ABSTRACT FROM AUTHOR]

We report a Japanese pedigree with familial primary hyperparathyroidism due to a CDC73 mutation. To our knowledge, this is the first report of cinacalcet as a treatment for CDC73- related primary hyperparathyroidism. The proband had severe psychomotor retardation and received laryngotracheal separation surgery. At 19 yr of age, he developed acute pancreatitis. Hypercalcemia (12.2-13.8 mg/dL), elevated levels of intact PTH (86-160 pg/mL), and a tumor detected upon neck ultrasonography led to the diagnosis of primary hyperparathyroidism. Family history and biochemical examinations revealed that three family members (the proband's mother, elder brother, and maternal grandfather) had primary hyperparathyroidism. We identified a novel heterozygous mutation, c.240delT, p.Glu81Lysfs*28, in the CDC73 gene in three affected family members, excluding the proband's elder brother who refused genetic testing. Parathyroidectomy for the proband was considered as high-risk, because the tumor was located close to the tracheostomy orifice. After receiving approval from the institutional review board and obtaining the consent, we initiated cinacalcet treatment. At 22 yr of age, treatment with 100 mg of cinacalcet m aintained serum calcium levels below 11.0 mg/dL with no apparent side effects. Our report presents the potential efficacy of cinacalcet as a treatment for CDC73-related primary hyperparathyroidism, in particularly inoperative cases. [ABSTRACT FROM AUTHOR]

Background: The ratio of serum free triiodothyronine (FT3) to free thyroxine (FT4) has been shown to be constant in healthy adults. However, this ratio has been found to be decreased in athyreotic adult patients on levothyroxine (L-T4) supplementation. In order to better evaluate thyroid-related pathologies in children as well as to establish a reference range, we investigated the FT3/FT4 ratio in a pediatric population. Furthermore, we evaluated this ratio in children with congenital hypothyroidism as well as those with central hypothyroidism. Methods: A reference range for the FT3/FT4 ratio was obtained from 129 Japanese children (3-17 y) with idiopathic short stature who were designated as the 'Control' group. Patients with congenital hypothyroidism due to athyreosis or severe thyroid hypoplasia (designated as 'A/Hypoplasia'), as well as patients with central hypothyroidism ('Central'), were recruited from the institutional database. For each group, the mean FT3/FT4 ratio was obtained. Results: In the Control group, the FT3/FT4 ratio was 3.03 ± 0.38 10-2 pg/ng (mean ± standard deviation) with no age or gender differences. A/Hypoplasia patients showed a significantly decreased mean FT3/FT4 ratio (2.17 ± 0.33, P < 0.001) compared to Control patients, with decreased FT3 and elevated FT4 levels. The Central group also showed a significantly decreased FT3/FT4 ratio (2.55 ± 0.45, P < 0.001) compared to the Control group, with decreased FT3 and equivalent FT4 levels. Conclusions: The FT3/FT4 ratio appears to be constant between the ages of 3-17 y. Children on L-T4 due to congenital thyroid a/hypoplasia or central hypothyroidism have a decreased FT3/FT4 ratio compared to short normal children. [ABSTRACT FROM AUTHOR]

Recent reports have indicated the role of the prokineticin receptor 2 gene (PROKR2) in the etiology of congenital hypopituitarism, including septo-optic dysplasia and Kallmann syndrome. In the present study, using next-generation targeted sequencing, we identified a novel heterozygous PROKR2 variant (c.742C>T; p.R248W) in a female patient who had combined pituitary hormone deficiency (CPHD), morning glory syndrome and a severely malformed pituitary gland. No other mutation was present in 27 genes related to hypogonadotropic hypogonadism, pituitary hormone deficiency and optic nerve malformation. The substituted amino acid was located on the third intracellular loop of the PROKR2 protein, which is a G protein-coupled receptor. Computational analyses with two programs (SIFT and PolyPhen-2) showed that the substitution was deleterious to PROKR2 function. The P.R248W mutation was transmitted from the patient's mother, who had a slightly delayed menarche. Collectively, we provide further genetic evidence linking heterozygous PROKR2 mutations and the development of CPHD. [ABSTRACT FROM AUTHOR]

Background: The prevalence of congenital hypopituitarism (CH) attributable to known transcription factor mutations appears to be rare and other causative genes for CH remain to be identified. Due to the sporadic occurrence of CH, de novo chromosomal rearrangements could be one of the molecular mechanisms participating in its etiology, especially in syndromic cases. Objective: To identify the role of copy number variations (CNVs) in the etiology of CH and to identify novel genes implicated in CH. Subjects and methods: We enrolled 88 (syndromic: 30; non-syndromic: 58) Japanese CH patients. We performed an array comparative genomic hybridization screening in the 30 syndromic CH patients. For all the 88 patients, we analyzed PAX6 by PCR-based sequencing. Results: We identified one heterozygous 310-kb deletion of the PAX6 enhancer region in one patient showing isolated GH deficiency (IGHD), cleft palate, and optic disc cupping. We also identified one heterozygous 6.5-Mb deletion encompassing OTX2 in a patient with bilateral anophthalmia and multiple pituitary hormone deficiency. We identified a novel PAX6 mutation, namely p.N116S in one non-syndromic CH patient showing IGHD. The p.N116S PAX6 was associated with an impairment of the transactivation capacities of the PAX6-binding elements. Conclusions This study showed that heterozygous PAX6 mutations are associated with CH patients. PAX6 mutations may be associated with diverse clinical features ranging from severely impaired ocular and pituitary development to apparently normal phenotype. Overall, this study identified causative CNVs with a possible role in the etiology of CH in <10% of syndromic CH patients. [ABSTRACT FROM AUTHOR]

Oral sodium phosphate formulations indicated for hypophosphatemia are commercially available worldwide. In Japan, however, many medical institutes have used hospital dispensary or foreign over-the-counter formulations because no such medication with an indication covered by the health insurance system is domestically available. To address this problem, we initiated the development of Phosribbon®. The present study evaluated the efficacy and safety of Phosribbon® in 16 patients with hereditary hypophosphatemic rickets. The optimal dosage and an administration pattern were also investigated. Administration of the agent resulted in an increase in the level of serum phosphorus in all patients, which implied that the employed dosage was appropriate. The dosage and administration pattern were adjusted based on comprehensive considerations, including changes in clinical laboratory values such as serum phosphorus, alkaline phosphatase and intact PTH, the dosage of a concomitantly administered activated vitamin D formulation and characteristics of individual patients. Adverse drug reactions were observed in 2 patients, neither of which were serious or necessitated therapy dose reduction or discontinuation. We conclude that Phosribbon® is a safe and effective treatment for patients with hypophosphatemic rickets and that dose adjustment in this therapy can be guided by the results of regular clinical examination and renal ultrasonography. [ABSTRACT FROM AUTHOR]

Renal coloboma syndrome is an autosomal dominant condition characterized by renal lesions and optic nerve abnormalities. We report an 11-yr-old Japanese girl with familial renal coloboma syndrome, who also had Graves' disease. Four affected family members had a previously reported heterozygous mutation (c.76dupG, p.Val26Glyfs*28) in the PAX2 gene. We hypothesized that PAX2 mutations may increase the risk of autoimmune diseases through alterations of human B-defensin 1 expression. [ABSTRACT FROM AUTHOR]

Introduction Classic Bartter syndrome is a salt-wasting tubulopathy caused by mutations in the CLCNKB (chloride channel Kb) gene. Although growth hormone deficiency has been suggested as a cause for persistent growth failure in patients with classic Bartter syndrome, in our opinion the diagnoses of growth hormone deficiency has been unconvincing in some reports. Moreover, Gitelman syndrome seems to have been confused with Bartter syndrome in some cases in the literature. In the present work, we describe a new case with CLCNKB gene mutations and review the reported cases of classic Bartter syndrome associated with growth hormone deficiency. Case presentation Our patient was a Japanese boy diagnosed as having classic Bartter syndrome at eight months of age. The diagnosis of Bartter syndrome was confirmed by CLCNKB gene analysis, which revealed compound heterozygous mutations with deletion of exons 1 to 3 (derived from his mother) and ΔL130 (derived from his father). His medical therapy consisted of potassium (K), sodium chloride, spironolactone, and anti-inflammatory agents; this regime was started at eight months of age. Our patient was very short (131.1cm, -4.9 standard deviation) at 14.3 years and showed profoundly impaired growth hormone responses to pharmacological stimulants: 0.15μg/L to insulin-induced hypoglycemia and 0.39μg/L to arginine. His growth response to growth hormone therapy was excellent. Conclusions The present case strengthens the association between classic Bartter syndrome and growth hormone deficiency. We propose that growth hormone status should be considered while treating children with classic Bartter syndrome. [ABSTRACT FROM AUTHOR]

Background: Individuals carrying a heterozygous inactivating PAX8 mutation are affected by congenital hypothyroidism (CH), although heterozygous Pax8 knockout mice are not. It has remained unclear whether CH in PAX8 mutation carriers is caused by haploinsufficiency or a dominant negative mechanism. Objective: To report clinical and molecular findings of four novel PAX8 mutations, including one earlytruncating frameshift mutation. Subjects and methods: Four probands were CH patients. Two had family history of congenital or childhood hypothyroidism. Three probands were diagnosed in the frame of newborn screening for CH, while one had a negative result in screening but was diagnosed subsequently. Three had thyroid hypoplasia and one had a slightly small thyroid with low echogenicity. For these probands and their family members, we sequenced PAX8 using a standard PCR-based method. Pathogenicity of identified mutations was verified in vitro. Results: We found four novel heterozygous PAX8 mutations in the four probands: L16P, F20S, D46SfsX24, and R133Q. Family studies showed four additional mutation carriers, who were confirmed to have high serum TSH levels. Expression experiments revealed that three mutations (L16P, F20S, and R133Q) had defects in target DNA binding, while D46fs had protein instability that was rescued by the proteasome inhibitor MG132. All four mutations had reduced transactivation on the thyroglobulin promoter, supporting that they were inactivating mutations. Conclusion: D46fs is the first PAX8 mutation with confirmed protein instability. Our clinical and in vitro findings together suggest that pure PAX8 haploinsufficiency can cause CH in humans. [ABSTRACT FROM AUTHOR]

Background: Arteriosclerosis and emphysema develop in individuals with Schimke immuno-osseous dysplasia (SIOD), a multisystem disorder caused by biallelic mutations in SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1). However, the mechanism by which the vascular and pulmonary disease arises in SIOD remains unknown. Methods: We reviewed the records of 65 patients with SMARCAL1 mutations. Molecular and immunohistochemicalanalyses were conducted on autopsy tissue from 4 SIOD patients. Results: Thirty-two of 63 patients had signs of arteriosclerosis and 3 of 51 had signs of emphysema. The arteriosclerosis was characterized by intimal and medial hyperplasia, smooth muscle cell hyperplasia and fragmented and disorganized elastin fibers, and the pulmonary disease was characterized by panlobular enlargement of air spaces. Consistent with a cell autonomous disorder, SMARCAL1 was expressed in arterial and lung tissue, and both the aorta and lung of SIOD patients had reduced expression of elastin and alterations in the expression of regulators of elastin gene expression. Conclusions: This first comprehensive study of the vascular and pulmonary complications of SIOD shows that these commonly cause morbidity and mortality and might arise from impaired elastogenesis. Additionally, the effect of SMARCAL1 deficiency on elastin expression provides a model for understanding other features of SIOD. [ABSTRACT FROM AUTHOR]