Satoshi Narumi

Transcription Factor Mutations and Congenital Hypothyroidism: Systematic Genetic Screening of a Population-Based Cohort of Japanese Patients

CONTEXT Gene mutations of transcription factors that are predominantly expressed in the thyroid gland cause congenital hypothyroidism (CH). The prevalence of CH due to transcription factor mutations remains undetermined. OBJECTIVE This study was designed to define the prevalence of CH due to mutations of PAX8, NKX2-1 [encoding thyroid transcription factor (TTF)-1], FOXE1 (encoding TTF-2), and NKX2-5 among patients with permanent primary CH and in the general population in Japan. SUBJECTS AND METHODS We enrolled 102 CH patients that represent 353,000 newborns born in Kanagawa prefecture from October 1979 to June 2006. We sequenced PAX8, NKX2-1, FOXE1, and NKX2-5 using PCR-based methods. Additionally, deletion/duplication of PAX8, NKX2-1, and FOXE1 was screened by multiplex ligation-dependent probe amplification. Molecular functions of putative mutations were verified in vitro. RESULTS We identified a novel small duplication of PAX8 (p.K80_A84dup) in two half-sibling patients with thyroid hypoplasia. We also found a novel NKX2-1 variation (p.H60W) in a sporadic nonsyndromic CH patient. In vitro experiments showed that K80_A84dup PAX8 had impaired transactivation of the thyroglobulin promoter. H60W TTF-1 exhibited a comparable transactivating capacity with wild-type TTF-1, suggesting a benign variation. We estimate the prevalence of PAX8 mutations to be 2.0% (two in 102) among Japanese CH patients and one in 176,000 (two in 353,000) in the general Japanese population. CONCLUSIONS Using a population-based sample, we confirmed that a minor subset of CH patients has transcription factor mutations, but they are rare. In our cohort, PAX8 mutations were the leading cause of such a rare condition.

SAMD9 mutations cause a novel multisystem disorder, MIRAGE syndrome, and are associated with loss of chromosome 7

Adrenal hypoplasia is a rare, life-threatening congenital disorder. Here we define a new form of syndromic adrenal hypoplasia, which we propose to term MIRAGE (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy) syndrome. By exome sequencing and follow-up studies, we identified 11 patients with adrenal hypoplasia and common extra-adrenal features harboring mutations in SAMD9. Expression of the wild-type SAMD9 protein, a facilitator of endosome fusion, caused mild growth restriction in cultured cells, whereas expression of mutants caused profound growth inhibition. Patient-derived fibroblasts had restricted growth, decreased plasma membrane EGFR expression, increased size of early endosomes, and intracellular accumulation of giant vesicles carrying a late endosome marker. Of interest, two patients developed myelodysplasitc syndrome (MDS) that was accompanied by loss of the chromosome 7 carrying the SAMD9 mutation. Considering the potent growth-restricting activity of the SAMD9 mutants, the loss of chromosome 7 presumably occurred as an adaptation to the growth-restricting condition.

Molecular Basis of Thyroid Dyshormonogenesis: Genetic Screening in Population-Based Japanese Patients

CONTEXT Inborn errors of thyroid hormone biosynthesis are collectively referred to as thyroid dyshormonogenesis (DH). Seven genes have been implicated in DH, including the dual oxidase 2 gene (DUOX2), the thyroglobulin gene (TG), and the thyroid peroxidase gene (TPO). OBJECTIVE We aimed to define the prevalence and phenotypic spectrum of DH with single gene mutations. SUBJECTS AND METHODS A population-based cohort of 102 patients with permanent congenital hypothyroidism was enrolled. Fourteen were diagnosed as DH and were analyzed for the seven causative genes including DUOX2, TG, and TPO. Several common mutations were screened in the remaining 88 patients. Pathogenicity of single amino acid mutations was verified in vitro. RESULTS We identified four, five, and two patients with seemingly biallelic mutations in DUOX2, TG, and TPO, respectively. We also found two patients having one heterozygous DUOX2 mutation and one uncommon single-nucleotide polymorphism (SNP) p.H678R (rs57659670, allele frequency 0.035) and another two patients with homozygous p.H678R. Expression experiments and RT-PCR revealed that p.H678R is a functional SNP with theoretical 40% loss of function, supporting a role of p.H678R in the onset of DH. As for clinical phenotypes, patients with inactive DUOX2 alleles (mutations and/or p.H678R) showed characteristic time-dependent improvement of thyroid function and morphology. All three evaluated patients had a negative result in the perchlorate test. CONCLUSIONS Mutations (or a functional SNP) in DUOX2, TG, or TPO were observed in 93% (95% confidence interval = 70-99%) of DH patients. Inactive DUOX2 alleles cause a broader phenotypic spectrum than currently accepted.

Identification and Functional Characterization of Two Novel NPR2 Mutations in Japanese Patients With Short Stature

CONTEXT C-type natriuretic peptide-natriuretic peptide receptor B (NPR-B) signaling is critical for endochondral ossification, which is responsible for longitudinal growth in limbs and vertebrae. Biallelic NPR2 mutations cause acromesomelic dysplasia, type Maroteaux, which is bone dysplasia characterized by severe short stature and short limbs. A monoallelic NPR2 mutation has been suggested to mildly impair long bone growth. OBJECTIVE The goal of this study was to identify and characterize NPR2 mutations among Japanese patients with short stature. SUBJECTS AND METHODS We enrolled 101 unrelated Japanese patients with short stature. NPR2 and NPPC were sequenced, and the identified variants were characterized in vitro. RESULTS In two subjects, we identified two novel heterozygous NPR2 mutations (R110C and Q417E) causing a loss of C-type natriuretic peptide-dependent cGMP generation capacities and having dominant-negative effects. R110C was defective in trafficking from the endoplasmic reticulum to the Golgi apparatus. In contrast, Q417E showed clear cell surface expression. CONCLUSIONS We identified heterozygous NPR2 mutations in 2% of Japanese patients with short stature. Our in vitro findings indicate that NPR2 mutations have a dominant negative effect, and their dominant-negative mechanisms vary corresponding to the molecular pathogenesis of the mutations.

Various types of LRP5 mutations in four patients with osteoporosis- pseudoglioma syndrome: Identification of a 7.2-kb microdeletion using oligonucleotide tiling microarray

Osteoporosis‐pseudoglioma syndrome (OPS; OMIM 259770) is an autosomal‐recessive genetic disorder characterized by severe osteoporosis and visual disturbance from childhood. Biallelic mutations in the low‐density lipoprotein receptor‐related protein 5 gene (LRP5) have been frequently detected, while a subset of patients had only one or no detectable mutation. We report on the clinical and molecular findings of four unrelated Japanese patients with the syndrome. The four patients had typical skeletal and ocular phenotypes of OPS, namely severe juvenile osteoporosis and early‐onset visual disturbance, with or without mental retardation. We undertook standard PCR‐based sequencing for LRP5 and found four missense mutations (p.L145F, p.T244M, p.P382L, and p.T552M), one nonsense mutation (p.R1534X), and one splice site mutation (c.1584+1G>A) among four OPS patients. Although three patients had two heterozygous mutations, one had only one heterozygous splice site mutation. In this patient, RT‐PCR from lymphocytic RNA demonstrated splice error resulting in 63‐bp insertion between exons 7 and 8. Furthermore, the patient was found to have only mutated RT‐PCR fragment, implying that a seemingly normal allele did not express LRP5 mRNA. We then conducted custom‐ designed oligonucleotide tiling microarray analyses targeted to a 600‐kb genome region harboring LRP5 and discovered a 7.2‐kb microdeletion encompassing exons 22 and 23 of LRP5. We found various types of LRP5 mutations, including an exon‐level deletion that is undetectable by standard PCR‐based mutation screening. Oligonucleotide tiling microarray seems to be a powerful tool in identifying cryptic structural mutations.

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.

Nonclassic TSH resistance: TSHR mutation carriers with discrepantly high thyroidal iodine uptake

CONTEXT Inactivating mutations in the TSH receptor gene (TSHR) cause TSH resistance. Most patients with TSH resistance have low to normal thyroidal radioiodine uptake (RAIU), which is consistent with the physiological knowledge that TSH stimulates iodine uptake. To date, only one TSHR mutation-carrying family with discrepantly high RAIU has been reported. OBJECTIVE We aimed to test whether TSHR mutation carriers with high RAIU are observed in a cohort of Japanese patients with congenital hypothyroidism (CH). SUBJECTS AND METHODS Twenty-four Japanese CH patients with high RAIU were screened for TSHR mutations. The capacities of mutant TSHR to activate Gs- and Gq-coupled signaling pathways were evaluated in vitro. RESULTS Two patients were found to have biallelic TSHR mutations: p.[T145I]+[R450H] in one and p.[R450H]+[I661fs] in the other. The two subjects had permanent CH with slightly high RAIU (41.8 and 43.0%, reference 8-40) but did not have goiter. One had a slightly high perchlorate discharge rate (10%, reference <10). Expression experiments revealed that T145I-TSHR retained partial ability to transduce both Gs- and Gq-coupled pathways, whereas I661fs-TSHR could transduce neither of them. R450H-TSHR had partial ability to transduce Gs-coupled signaling but had abrogated ability to transduce Gq-coupled signaling, indicating that coupling to Gq was dominantly affected. CONCLUSIONS We show that 8% of Japanese CH patients with high RAIU (two in 24) has inactivating TSHR mutations. Expression of this apparently discrepant phenotype, which we term nonclassic TSH resistance, is presumably associated with the characteristic signaling property of the mutant TSHR, namely the Gq-dominant coupling defect.

Quantitative and sensitive detection of GNAS mutations causing mccune-albright syndrome with next generation sequencing.

Somatic activating GNAS mutations cause McCune-Albright syndrome (MAS). Owing to low mutation abundance, mutant-specific enrichment procedures, such as the peptide nucleic acid (PNA) method, are required to detect mutations in peripheral blood. Next generation sequencing (NGS) can analyze millions of PCR amplicons independently, thus it is expected to detect low-abundance GNAS mutations quantitatively. In the present study, we aimed to develop an NGS-based method to detect low-abundance somatic GNAS mutations. PCR amplicons encompassing exons 8 and 9 of GNAS, in which most activating mutations occur, were sequenced on the MiSeq instrument. As expected, our NGS-based method could sequence the GNAS locus with very high read depth (approximately 100,000) and low error rate. A serial dilution study with use of cloned mutant and wildtype DNA samples showed a linear correlation between dilution and measured mutation abundance, indicating the reliability of quantification of the mutation. Using the serially diluted samples, the detection limits of three mutation detection methods (the PNA method, NGS, and combinatory use of PNA and NGS [PNA-NGS]) were determined. The lowest detectable mutation abundance was 1% for the PNA method, 0.03% for NGS and 0.01% for PNA-NGS. Finally, we analyzed 16 MAS patient-derived leukocytic DNA samples with the three methods, and compared the mutation detection rate of them. Mutation detection rate of the PNA method, NGS and PNA-NGS in 16 patient-derived peripheral blood samples were 56%, 63% and 75%, respectively. In conclusion, NGS can detect somatic activating GNAS mutations quantitatively and sensitively from peripheral blood samples. At present, the PNA-NGS method is likely the most sensitive method to detect low-abundance GNAS mutation.

Functional characterization of four novel PAX8 mutations causing congenital hypothyroidism: new evidence for haploinsufficiency as a disease mechanism

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 early-truncating 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.

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