論文

Introduction: Non-autoimmune hyperthyroidism (NAH) is a rare genetic disorder caused by germline-activating variants in the TSH receptor (TSHR) gene. While most NAH-related TSHR variants are located in the seven-transmembrane domain (7TMD), three variants (p.Leu267Phe, p.Ser281Asn, and p.Asn406Ser) have been identified in the extracellular domain (ECD), with p.Leu267Phe previously not showing constitutively active in vitro. Methods: We searched for TSHR variants in a Japanese family with NAH using PCR-based direct sequencing. We created three ECD variants (p.Leu267Phe, p.Ser281Asn, and p.Asn406Ser) and a series of variants in which Leu267 was mutated to 18 amino acids other than Leu and Phe. Based on the cryo-electron microscopic structures, we evaluated the structure-function relationship of TSHR. We compared their cAMP-producing capacities to WT-TSHR in the luciferase activity assay using HEK293 cells. Western blot and fluorescence immunostaining were performed using HA-tagged TSHR vectors to compare Leu267Phe-TSHR with WT-TSHR. Results: A heterozygous TSHR variant (p.Leu267Phe) was identified. Comparison of the cryo-electron microscopy structures of the activated and inactivated TSHRs revealed a significant change in the ECD structure around Leu267. The ligand-independent cAMP-producing capacities compared to WT-TSHR were 238 ± 20% (mean ± SEM) for Leu267Phe. Of all 19 possible variants created through systematic mutagenesis, only Leu267Phe-TSHR and Leu267Tyr-TSHR exhibited significantly higher ligand-independent cAMP-producing capacities. Immunoblotting and fluorescence immunostaining showed that the Leu267Phe variant did not affect protein expression levels and intracellular localization of TSHR. Discussion/Conclusion: Leu267Phe-TSHR causes NAH. Substituting Leu267 to aromatic amino acids may shift the equilibrium of the TSHR state toward activation.

Pediatric brain tumors, the most devastating cancers affecting children, are believed to originate from neural stem/progenitor cells in developing brain. In precise timing and specific regions during the brain development, chromatin deregulation plays crucial roles in redirecting normal neuronal differentiation pathways toward tumorigenesis. Indeed, epigenomic abnormalities are thought to be more important for brain tumor formation especially in children than adults, as pediatric brain tumors generally exhibit fewer genetic mutations compared to adult brain tumors. Given the small number of mutations, targeting such limited alterations involved in cancer epigenomes is expected to be more effective in pediatric brain tumors. The mechanisms of cancer epigenomes include mutation or dysregulation of chromatin remodelers, histone modifiers, histones themselves, and DNA methylation enzymes. Furthermore, genomic rearrangements and/or higher-order chromatin topology also contribute to these epigenomic mechanisms. These mechanisms are commonly observed in various types of pediatric brain tumors. However, alterations in chromatin regulatory factors differ across tumor types, reflecting the unique epigenetic landscapes shaped by their tumor origins. Accordingly, clarifying their functional similarities and differences across tumor types could offer valuable insights for finding new therapeutic strategies. Thus, this review article focuses on elucidating how pediatric brain tumors arise from epigenomic deregulation and what epigenetic molecules or mechanisms could serve as therapeutic targets.

Biallelic mutations of SLC26A4 (encoding pendrin) cause Pendred syndrome (PS), an autosomal recessive genetic disorder with deafness and goiter. The mechanism underlying the development of the goiter is unknown. Here, we report clinical and molecular findings of a patient with PS. This 27-year-old woman was born to nonconsanguineous healthy parents. She was seen at our hospital due to hearing loss at age 3 years, and subsequently developed goiter at age 10 years. From age 15 years, her thyroid gland showed progressive enlargement accompanied by elevation of serum thyroglobulin reaching 10-fold the normal amount. Thyroidal iodine uptake was also increased during goiter progression ((123)I uptake at 24 hr: 20.2% at age 17 years; 69.4% at age 24 years; reference, 8-40), while serum thyrotropin (TSH) levels and iodine organification (examined by the per-chrolate or thiocyanate discharge test) remained normal. We sequenced SLC26A4 using standard PCR-based technique, and found one novel (p.T537P) and one recurrent (p.H723R) mutations in a compound heterozygous state. Expression experiments using COS-7 cells showed that the two mutants were entrapped in the endoplasmic reticulum and were poorly localized at the plasma membrane. In summary, a molecularly confirmed PS patient showed goiter progression accompanied by elevated serum thyroglobulin and increased thyroidal iodine uptake, but normal serum TSH levels and normal iodine organification. This implies that some pendrin mutations may involve direct stimulation of thyroid cell proliferation with no TSH hyperstimulation and no iodine organification defect. (C) 2010 Wiley-Liss, Inc.

Sotos syndrome (SoS, OMIM #117550) is an overgrowth syndrome. Deletions or intragenic mutations of the NSD1 , which is located at chromosome 5q35, are responsible for more than 75% of SoS. Conventionally, neonatal hypoglycemia was reported briefly as one of the infrequent symptoms of SoS. However, Matsuo et al. published a report describing five patients with SoS who presented with transient hyperinsulinemic hypoglycemia (HIH) in the neonatal period. We report on an additional patient of SoS, who presented transient HIH in the neonatal period. All of this patient and previous patients have microdeletions at the 5q35 chromosome. Therefore, we examined the following three in considering the possibility that other factor than NSD1 caused HIH. 1) This patient had no mutation of four currently known HIH related genes, ABCC8, KCNJ11, GLUD1, and GCK. 2) He had no further deletion than commonly observed region encompassing NSD1 by comparative genomic hybridization to DNA microarrays. 3) He had no mutation in the 5q35 region in the non-deleted chromosome using exsome sequence analysis. In conclusion, our patient supported that HIH could be one of the characteristic symptoms of SoS in the neonatal period, and could be useful for early diagnosis.

Using a next-generation sequencing strategy, we identified a novel KAL1 missense mutation (p.His568Gln) in a patient with combined pituitary hormone deficiency, right microphthalmia, right renal aplasia and severe developmental delay. Our findings will provide additional evidence that KAL1 mutations are associated with hypopituitarism, in addition to luteinizing hormone, and follicle-stimulating hormone deficiencies, and improve our understanding of the phenotypic features and developmental course associated with KAL1 mutations.

Pseudohypoaldosteronism type II (PHA II) is a renal tubular disease that causes hyperkalemia, hypertension, and metabolic acidosis. Mutations in four genes (WNK4, WNK1, KLHL3, and CUL3) are known to cause PHA II. We report a patient with PHA II carrying a KLHL3 mutation, who also had congenital hypopituitarism. The patient, a 3-yr-old boy, experienced loss of consciousness at age 10 mo. He exhibited growth failure, hypertension, hyperkalemia, and metabolic acidosis. We diagnosed him as having PHA II because he had low plasma renin activity with normal plasma aldosterone level and a low transtubular potassium gradient. Further investigations revealed defective secretion of GH and gonadotropins and anterior pituitary gland hypoplasia. Genetic analyses revealed a previously known heterozygous KLHL3 mutation (p.Leu387Pro), but no mutation was detected in 27 genes associated with congenital hypopituitarism. He was treated with sodium restriction and recombinant human GH, which normalized growth velocity. This is the first report of a molecularly confirmed patient with PHA II complicated by congenital hypopituitarism. We speculate that both GH deficiency and metabolic acidosis contributed to growth failure. Endocrinological investigations will help to individualize the treatment of patients with PHA II presenting with growth failure.

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.

Background: Recent reports have indicated that loss-of-function mutations in the immunoglobulin superfamily member 1 gene (IGSF1, OMIM 300888) cause congenital central hypothyroidism with macroorchidism. Methods: We conducted a next-generation sequencing-based comprehensive mutation screening for pituitary hormone deficiencies to elucidate molecular mechanisms other than anatomical abnormalities of the pituitary that might be responsible for multiple anterior hormone deficiency in a male patient who originally visited our institute complaining of short stature. He was born large for gestational age (4,370 g, + 3.0 SD) after an obstructed labour. Endocrinological evaluation revealed growth hormone and thyroid-stimulating hormone deficiency. Magnetic resonance imaging showed a discontinuity of the pituitary stalk with an ectopic posterior lobe and a hypoplastic anterior lobe, likely explaining multiple anterior pituitary hormone deficiency. Result: We identified a novel hemizygous IGSF1 mutation (c.1137_1138delCA, p.Asn380Glnfs*6) in the patient. In reviewing the literature, we noticed that all reported Japanese male IGSF1 mutation carriers were born larger than mean standards for gestational age (mean birth weight SD score of +2.0, 95% confidence interval 1.0-3.0). Conclusion: This case suggests that more attention should be paid to intrauterine growth and birth history when patients are suspected of having an IGSF1 mutation. (C) 2015 S. Karger AG, Basel

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.

Context: Defects in MKRN3, DLK1, KISS1, and KISS1R and some disorders, such as Temple syndrome (TS14), cause central precocious puberty (CPP). Recently, pathogenic variants (PVs) in MECP2 have been reported to be associated with CPP. Objective: We aimed to clarify the contribution of (epi)genetic abnormalities to CPP and clinical and hormonal features in each etiology. Methods: We conducted targeted sequencing for MKRN3, DLK1, MECP2, KISS1, and KISS1R and methylation analysis for screening of imprinting disorders such as TS14 associated with CPP in 90 patients with CPP (no history of brain injuries and negative brain magnetic resonance imaging) and collected their clinical and laboratory data. We measured serum DLK1 levels in 3 patients with TS14 and serum MKRN3 levels in 2 patients with MKRN3 genetic defects, together with some etiology-unknown patients with CPP and controls. Results: We detected 8 patients with TS14 (6, epimutation; 1, mosaic maternal uniparental disomy chromosome 14; 1, microdeletion) and 3 patients with MKRN3 genetic defects (1, PV; 1, 13-bp deletion in the 5′-untranslated region [5′-UTR]; 1, microdeletion) with family histories of paternal early puberty. There were no patients with PVs identified in MECP2, KISS1, or KISS1R. We confirmed low serum MKRN3 level in the patient with a deletion in 5′-UTR. The median height at initial evaluation of TS14 patients was lower than that of all patients. Six patients with TS14 were born small for gestational age (SGA). Conclusion: (Epi)genetic causes were identified in 12.2% of patients with CPP at our center. For patients with CPP born SGA or together with family histories of paternal early puberty, (epi)genetic testing for TS14 and MKRN3 genetic defects should be considered.