Masaki Takagi

A Novel Mutation in LEPRE1 That Eliminates Only the KDEL ER- Retrieval Sequence Causes Non-Lethal Osteogenesis Imperfecta

Prolyl 3-hydroxylase 1 (P3H1), encoded by the LEPRE1 gene, forms a molecular complex with cartilage-associated protein (CRTAP) and cyclophilin B (encoded by PPIB) in the endoplasmic reticulum (ER). This complex is responsible for one step in collagen post-translational modification, the prolyl 3-hydroxylation of specific proline residues, specifically α1(I) Pro986. P3H1 provides the enzymatic activity of the complex and has a Lys-Asp-Glu-Leu (KDEL) ER-retrieval sequence at the carboxyl terminus. Loss of function mutations in LEPRE1 lead to the Pro986 residue remaining unmodified and lead to slow folding and excessive helical post-translational modification of type I collagen, which is seen in both dominant and recessive osteogenesis imperfecta (OI). Here, we present the case of siblings with non-lethal OI due to novel compound heterozygous mutations in LEPRE1 (c.484delG and c.2155dupC). The results of RNA analysis and real-time PCR suggest that mRNA with c.2155dupC escapes from nonsense-mediated RNA decay. Without the KDEL ER- retrieval sequence, the product of the c.2155dupC variant cannot be retained in the ER. This is the first report of a mutation in LEPRE1 that eliminates only the KDEL ER-retrieval sequence, whereas other functional domains remain intact. Our study shows, for the first time, that the KDEL ER- retrieval sequence is essential for P3H1 functionality and that a defect in KDEL is sufficient for disease onset.

Gradual Loss of ACTH Due to a Novel Mutation in LHX4: Comprehensive Mutation Screening in Japanese Patients with Congenital Hypopituitarism

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.

A recurrent mutation in the 5′-UTR of IFITM5 causes osteogenesis imperfecta type V

A Recurrent Mutation in the 50-UTR of IFITM5 Causes Osteogenesis Imperfecta Type V Masaki Takagi, Shuhei Sato, Keiichi Hara, Chihiro Tani, Osamu Miyazaki, Gen Nishimura, and Tomonobu Hasegawa* Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan Department of Endocrinology and Metabolism, Tokyo Metropolitan Children’s Medical Center, Tokyo, Japan Perinatal Medical Center of Aomori Prefectural Central Hospital, Aomori, Japan Department of Pediatrics, Kure Medical Center, Hiroshima, Japan Department of Radiology, Hiroshima City Hospital, Hiroshima, Japan Department of Radiology, National Center for Child Health and Development, Tokyo, Japan Department of Radiology, Tokyo Metropolitan Children’s Medical Center, Tokyo, Japan

A family of pseudohypoparathyroidism type Ia with an 850-kb submicroscopic deletion encompassing the whole GNAS locus†

A Family of Pseudohypoparathyroidism Type Ia With an 850-kb Submicroscopic Deletion Encompassing the Whole GNAS Locus Toshikatsu Mitsui, Keisuke Nagasaki, Masaki Takagi, Satoshi Narumi, Tomohiro Ishii, and Tomonobu Hasegawa* Department of Pediatrics, Keio University School of Medicine, Tokyo Japan Division of Pediatrics, Department of Homeostatic Regulation and Development, Niigata University Graduate School of Medicine and Dental Sciences, Niigata, Japan

Heterozygous C-propeptide mutations in COL1A1: Osteogenesis imperfecta type IIC and dense bone variant†‡

Osteogenesis imperfecta type IIC (OI IIC) is a rare variant of lethal OI that has been considered to be an autosomal recessive trait. Twisted, slender long bones with dense metaphyseal margins and normal vertebral bodies in OI IIC contrast with crumpled, thick long bones and multiple vertebral compression fractures in OI IIA. Here, we report on two sporadic patients with classical OI IIC and a pair of siblings, with features of OI IIC but less distortion of the tubular bones (OI dense bone variant). One case with OI IIC and the sibs had novel heterozygous mutations in the C‐propeptide region of COL1A1, while the second patient with clear‐cut OI IIC had no mutation in this region. Histological examination in the two sporadic cases showed a network of broad, interconnected cartilaginous trabeculae with thin osseous seams in the metaphyses. These changes differed from the narrow and short metaphyseal trabeculae found in other lethal or severe cases of OI. Our experience sheds light on the genetics and etiology of OI IIC and on its phenotypic spectrum.

A novel mutation in SOX3 polyalanine tract: a case of kabuki syndrome with combined pituitary hormone deficiency harboring double mutations in MLL2 and SOX3

IntroductionBoth duplications encompassing SOX3 and loss-of function mutations in SOX3 have been reported in a minor portion of X-linked isolated growth hormone deficiency (GHD) or combined pituitary hormone deficiency (CPHD) patients with or without mental retardation.Patients and methodsWe report a Japanese male patient with molecularly confirmed Kabuki syndrome who was found to have CPHD. We analyzed all coding exons and flanking introns of currently known nine genes responsible for CPHD by PCR-based sequencing.ResultsIn this CPHD patient, we identified a novel hemizygous 21-base pair deletion, resulting in the loss of 7 alanine residues from polyalanine (PA) tracts of SOX3. The clinically and endocrinologically normal mother of the patient carried the same deletion in a heterozygous manner. In vitro experiments showed that the del 7A SOX3 had increased transactivation of the HESX1 promoter.ConclusionOur study provides additional evidence that deletion in PA tracts of SOX3 is associated with hypopituitarism. Female carriers of SOX3 PA tract deletions will show a broad phenotypic spectrum, ranging from clinically normal to CPHD.

A Novel Mutation in SOX2 Causes Hypogonadotropic Hypogonadism with Mild Ocular Malformation

Background: Heterozygous SOX2 mutations have been reported to cause isolated hypogonadotropic hypogonadism (HH) in addition to ocular and brain abnormalities. Objective: We report a novel missense SOX2 (Y110C) mutation in an HH patient with mild ocular malformation. Patients: The 20-year-old male was referred because of typical signs of complete hypogonadism, with small intrascrotal testes (2 ml), no pubic hair (P1), and a micropenis. Hormone assays revealed very low plasma testosterone levels and very low levels of plasma gonadotropin. He was found to have retinal detachment in his right eye and surgery was performed at the age of 14 years. Results: Using a next-generation sequencing strategy, we identified a novel heterozygous SOX2 mutation, c.329A>G (p.Y110C). Y110C SOX2 had reduced transactivation and no dominant negative effect. Subcellular localization revealed no significant difference between wild-type and mutant SOX2. EMSA experiments showed that the Y110C SOX2 abrogated DNA-binding ability. Conclusion: The Y110C mutation affects a critical residue in the SOX2 protein. This study extends our understanding of the phenotypic features, molecular mechanism, and developmental course associated with mutations in SOX2. When multiple genes need to be analyzed for mutations simultaneously, targeted sequence analysis of interesting genomic regions is an attractive approach.

Novel compound heterozygous mutations of the growth hormone-releasing hormone receptor gene in a case of isolated growth hormone deficiency

OBJECTIVE To elucidate the pathogenesis of isolated growth hormone (GH) deficiency in a Japanese girl without consanguinity. DESIGN A 2-year-old girl of height 77.2 cm (-3.0 SD for Japanese girls) was found to have an insulin-like growth factor (IGF)-1 level of 7 ng/mL and IGF binding protein-3 (IGFBP-3) level of 0.41 μg/mL. GH responded modestly to a series of pharmacological stimulants, increasing to 2.81 ng/mL with insulin-induced hypoglycemia, 3.78 ng/mL with arginine, and 3.93 with GH-releasing hormone (GHRH). Following direct sequencing of the GHRH receptor (GHRHR) gene, evaluation by the luciferase reporter assay, immunofluorescence study, and in vitro splicing assay with minigene constructs was conducted. RESULTS Novel compound heterozygous GHRHR gene mutations were identified in the patient. A p.G136V substitution elicited no luciferase activity increment in response to GHRH stimulation, with normal membranous expression. Splicing assay demonstrated that the IVS2+3a>g mutation would lead to aberrant splicing. CONCLUSIONS A case of isolated GH deficiency due to novel GHRHR gene mutations was identified.

A 2.0 Mb microdeletion in proximal chromosome 14q12, involving regulatory elements of FOXG1, with the coding region of FOXG1 being unaffected, results in severe developmental delay, microcephaly, and hypoplasia of the corpus callosum

We identified 2.0 Mb of a novel deletion on chromosome 14q12, involving 8 genes and putative regulatory elements of FOXG1 by array CGH in a patient with severe growth and psychomotor retardation, hypotonia, microcephaly, dysmorphic face, and hypoplasia of the corpus callosum. Case of a submicroscopic 14q12 deletion, involving regulatory elements of FOXG1, with the coding region of FOXG1 being unaffected, is extremely rare. Using fibroblast cell line established from the patient, we showed that the expression level of FOXG1 in our patient was decreased. Our finding provides additional evidence that not only over-dosage of FOXG1 as previously mentioned, under-dosage of FOXG1 is also associated with phenotype, overlapping between congenital variant of Rett syndrome with FOXG1 mutations and 14q12 microdeletion, not including the coding region of FOXG1. Though the gene dosage of FOXG1 appears to be critical for the normal development of brain, the complex mechanism of its regulation of gene expression remains to be elucidated.

A case of Rabson-Mendenhall syndrome with a novel mutation in the tyrosine kinase domain of the insulin receptor gene complicated by medullary sponge kidney

Abstract Rabson-Mendenhall syndrome (RMS) is a genetic disorder characterized by severe insulin resistance and somatic characteristics. Recombinant insulin-like growth factor 1 (r-IGF-1) is used to treat RMS, as the IGF-1 and insulin receptors share homology. However, the effect of r-IGF-1 varies in patients and it is difficult to manage metabolic status appropriately in r-IGF-1 resistant cases. We report a Japanese boy with RMS who showed resistance to r-IGF-1 therapy and a novel mutation in the insulin receptor in the tyrosine kinase domain. Mutations in this region disturb tyrosine kinase catalytic activity in IGF-1 receptors as a result of dominant negative effects. We consider this mutation to be the cause of resistance to r-IGF-1. The patient also exhibited radiographical features of medullary sponge kidney and had severe nephrocalcinosis and hypokalemia, indicating Bartter syndrome. However, analysis revealed no mutations in the responsible genes and the etiology of the renal abnormalities therefore remains unknown.