Eiichi Kinoshita

The role of fibroblast growth factor 23 for hypophosphatemia and abnormal regulation of vitamin D metabolism in patients with McCune-Albright syndrome.

McCune–Albright syndrome (MAS) is sometimes complicated by hypophosphatemia and abnormally low levels of 1,25(OH)2D in the presence of hypophosphatemia. Recently, fibroblast growth factor 23 (FGF-23) was reported as a phosphaturic and a causal factor of abnormal vitamin D metabolism. This abnormal phosphate and vitamin D metabolism is well known to be found in oncogenic and X-linked hypophosphatemia. We furthermore reported increased circulating plasma FGF-23 levels in patients with oncogenic and X-linked hypophosphatemia. To determine whether FGF-23 may be involved in the pathogenesis of MAS, we measured plasma FGF-23 levels in six MAS patients. As a control for hypophosphatemia, we also investigated the plasma FGF-23 levels in two patients with hereditary hypophosphatemic rickets with hypercalciuria (HHRH). We also investigated the correlation of plasma FGF-23 levels with serum phosphate and 1,25(OH)2D levels after short-term pamidronate therapy in three MAS patients. Plasma FGF-23 levels were significantly increased in patients with MAS compared to normal controls, whereas they were not increased in HHRH patients. Serum phosphate levels of the MAS patients were significantly lower than those observed in normal controls. Plasma FGF-23 levels showed significant negative correlation with serum phosphate concentrations. In three MAS patients, pamidronate therapy decreased plasma FGF-23 levels, which showed significant negative correlation with serum 1,25(OH)2D concentrations. These data suggested that FGF-23 is a possible causal factor for hypophosphatemia and abnormal vitamin D metabolism in MAS.

A Mutational Hot Spot in the Prop-1 Gene in Russian Children with Combined Pituitary Hormone Deficiency

Combined pituitary hormone deficiency (CPHD), including growth hormone (GH), prolactin (Prl) and thyroid-stimulating hormone (TSH) in children is now considered a heterogeneous syndrome. Recent findings on expression of mouse pituitary-specific homeodomain factors demonstrate dependence of adenopituitary ontogeny on interactive expression of these factors, suggesting their involvement in etiology of CPHD. Prophet of Pit-1 (Prop-1) gene, a novel pituitary-specific homeodomain factor, was analyzed in 14 Russian children with CPHD, in whom Pit-1 gene was intact. We found a mutational hot spot in three patients from two families in homeodomain part of the second exon of Prop-1 gene. The common 2- base pair deletion (GA296) in the homozygous state resulted in a Serine to Stop codon (S109X) substitution and generated a truncated Prop-1 protein. Parents were phenotypically normal and heterozygous for GA296 deletion, indicating an autosomal recessive inheritance. These results demonstrate a novel type of Prop-1 gene mutation as one of the causes of CPHD in Russian patients.

Rarity of PIT1 involvement in children from Russia with combined pituitary hormone deficiency

To ascertain the molecular background of combined pituitary hormone deficiency, screening for mutations in the pituitary-specific transcription factor (Pit-1/GHF-1) gene (PIT1) was performed on a cohort of 15 children from Russia with combined growth hormone (GH)/prolactin (Prl)/thyroid-stimulating hormone (TSH) deficiency. The group of patients, suspected of PIT1 mutations, consisted of four familial cases (seven patients) and eight sporadic cases. All had complete GH deficiency and complete or partial Prl and TSH deficiency. Direct sequencing of all six exons of PIT1 and its promoter region showed a C to T transition mutation at codon 14 of exon 1 in a 3 8/12-year-old girl. This novel PIT1 mutation results in a proline to leucine substitution (P14L). The patient was heterozygous for mutant and normal alleles. The heterozygous P14L mutation was also present in her mother as well as in her maternal aunt and grandmother, all of whom were phenotypically normal. There was no mutation in the fathers DNA, suggesting the need for reevaluation of genomic imprinting. In other children of our series, no mutation in PIT1 or in its promotor region was identified. This is the first report on the analysis of PIT1 and its promoter region in Russian children with GH/Prl/TSH deficiency. However, as the involvement of PIT1 mutation is rare in Russia, the other negative cases need to be analyzed for another candidate gene responsible for combined GH/Pr/TSH deficiency.

Mutation and Gene Copy Number Analyses of Six Pituitary Transcription Factor Genes in 71 Patients with Combined Pituitary Hormone Deficiency: Identification of a Single Patient with LHX4 Deletion

CONTEXT Mutations of multiple transcription factor genes involved in pituitary development have been identified in a minor portion of patients with combined pituitary hormone deficiency (CPHD). However, copy number aberrations involving such genes have been poorly investigated in patients with CPHD. OBJECTIVE We aimed to report the results of mutation and gene copy number analyses in patients with CPHD. SUBJECTS AND METHODS Seventy-one Japanese patients with CPHD were examined for mutations and gene copy number aberrations affecting POU1F1, PROP1, HESX1, LHX3, LHX4, and SOX3 by PCR-direct sequencing and multiplex ligation-dependent probe amplification. When a deletion was indicated, it was further studied by fluorescence in situ hybridization, oligoarray comparative genomic hybridization, and serial sequencing for long PCR products encompassing the deletion junction. RESULTS We identified a de novo heterozygous 522,009-bp deletion involving LHX4 in a patient with CPHD (GH, TSH, PRL, LH, and FSH deficiencies), anterior pituitary hypoplasia, ectopic posterior pituitary, and underdeveloped sella turcica. We also identified five novel heterozygous missense substitutions (p.V201I and p.H387P in LHX4, p.T63M and p.A322T in LHX3, and p.V53L in SOX3) that were assessed as rare variants by sequencing analyses for control subjects and available parents and by functional studies and in silico analyses. CONCLUSIONS The results imply the rarity of abnormalities affecting the six genes in patients with CPHD and the significance of the gene copy number analysis in such patients.

Mutations in intron 3 of GH-1 gene associated with isolated GH deficiency type II in three Japanese families

Isolated GH deficiency (IGHD) type II is a disorder inherited in an autosomal dominant manner. Three mutations at the donor splice site of intron 3 of the GH‐I gene have been identified in five families. In this report, we describe a novel mutation also at the donor splice site of intron 3 in patients with IGHD type II.

Persistent hepatitis associated with chronic active Epstein-Barr virus infection.

A previously healthy boy developed persistent hepatitis without fever or lymphoproliferative disorder. Although serologic tests were not indicative, Epstein-Barr virus (EBV) genome and transcripts were detected from the liver tissue, and real time PCR detected extremely high levels of EBV viremia. EBV infection should be included in the differential diagnoses of hepatitis of unknown etiology, even with unremarkable serologic data.

Pit-1 and hypopituitarism

The story of Pit-1 and hypopituitarism in humans provides an excellent example of pleiotrophism or multiple phenotypic effects resulting from a single genetic alteration. It shows how defects in this single gene cause the absence o f several pituitary hormones. Three recent articles reviewed here provide examples of different mutations in this homeobox gene encoding a transcriptional activation protein that is vital to the embryologic development, survival, and differentiated function of somatotropes, lactotropes, and thyrotropes.

Two novel and one recurrent PTPN11 mutations in LEOPARD syndrome.

LEOPARDsyndrome (LS) is a congenital developmental disorder named by the acronym of multiple lentigines, electrocardiographic conductionabnormalities, ocularhypertelorism, pulmonary stenosis, abnormalities of genitalia, retardation of growth, and sensorineural deafness [Gorlin et al., 1969]. This condition is also often associated with other features such as cardiovascular anomalies, hypertrophic cardiomyopathy (HCM), and mental retardation. These clinical features in LS overlap with those in Noonan syndrome (NS). Recently, it has been shown that both NS and LS are caused by heterozygous missense mutations of the gene for protein-tyrosine phosphatase, nonreceptor type 11 (PTPN11) [Tartaglia et al., 2001; Digilio et al., 2002]. PTPN11 encodes cytoplasmic tyrosinephosphatasewith two tandemlyarranged Src homology 2 (SH2) domains (N-SH2 and C-SH2) on the Nterminal side and a protein-tyrosine phosphatase (PTP) domain on the C-terminal side, and plays a critical role in regulating the responses of eukaryotic cells to multiple extracellular signals in various tissues. Since the missense mutations cause aminoacid substitutions in and around the broad N-SH2/PTP interaction surface, they have been suggested to affect the intramolecular N-SH2/PTP binding in the absence of a phosphopeptide, leading to a gain-of-function effect with an excessive phosphatase activity [Tartaglia et al., 2001]. To date, while variousmutations have been found inNS [Tartaglia et al., 2001, 2002], only three mutations have been identified in LS [Digilio et al., 2002; Legius et al., 2002; Conti et al., 2003; Sarkozy et al., 2003]. Here, we report on two novel and one recurrent PTPN11 mutations identified in LS patients. Six Japanese patients (five males and one female) with LS aged 8.3–26.0 years were studied (Table I). All cases had a normal karyotype. Case 5 had hypochondroplasia resulting fromthe commonAsn540Lysmutation ofFGFR3 [Ramaswami et al., 1998]. The mother of case 4 also had LS phenotype and deceased of cardiac failure at 34 years of age. The remaining cases were sporadic. All the cases 1–6 showed multiple lentigines and characteristic face with hypertelorism, and had at least three of the cardinal features outlined by Varon et al. [1976]. Short stature (< 2.0 SD) was present in five cases. Cardiovascular lesions included HCM in three cases, and pulmonary valve stenosis, patent ductus arteriosus, and electrocardiographic conduction abnormality in single different cases. Deafness was exhibited by five cases, cryptorchidism by two of the five male cases, and mental retardation by five cases. After obtaining informed consent, leukocyte genomic DNA was amplified by PCR for all the 15 exons and its flanking introns of PTPN11, and was subjected to direct sequencing from both directions on a CEQ 8000 autosequencer (Beckman Coulter, Fullerton, CA). The primer sequences and the PCR conditions have beendescribed previously [Kosaki et al., 2002]. To confirm mutations, three independent PCR products were similarly examined. Two novel heterozygous missense mutations, Ala461Thr and Gly464Ala were identified in single different cases, together with a recurrent heterozygous mutation, Tyr279Cys, in two cases (Table I and Fig. 1). These mutations were absent

Rapid detection of a point mutation in thyroid-stimulating hormoneβ-subunit gene causing congenital isolated thyroid-stimulating hormone deficiency

SummaryPrevious study showed that congenital isolated TSH deficiency in Japan is resulted exclusively from a G-A transition at nucleotide 145 in exone 2 of the TSHβ-subunit gene. All reported cases were from the inbred in Shikoku Island. We describe here a 10-year-old boy with hereditary TSH deficiency in the same area. The patient was born with a weight of 3,225 g to non-consanguineous parents. Evaluation at age 2 months revealed typical manifestations of cretinism without goiter. Serum T4, T3, and TSH values were 2.53 μg/dl, 107 ng/dl, and 0.5 μU/ml, respectively. A TRH stimulation test showed no increment of serum TSH value. Other anterior pituitary hormone levels were all within the normal range. Two oligonucleotide primers T1a and T1b were synthesized according to the sequence data. Amplified 169 bp nucleotides in exon 2 of the TSHβ gene with this primer set were digested with MaeI. Both the phenotypically normal brother and normal controls showed only the 169 bp fragment, whereas the proband showed 140 and 29 bp fragments and both parents showed three fragments; 169, 140, and 29 bp. These results were consistent with the point mutation of TSHβ gene in Japanese patients with congenital isolated TSH deficiency. Our PCR method with MaeI digestion contributes to the rapid detection of the homozygous patient and the heterozygous carrier.

Pubertal Changes in Testicular 3β-Hydroxysteroid Dehydrogenase Activity in a Male with Classical 3β-Hydroxysteroid Dehydrogenase Deficiency Showing Spontaneous Secondary Sexual Maturation

Males with classical 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency manifest appropriate secondary sexual maturation with an elevation in serum testosterone levels at pubertal age. To define the origin of serum testosterone, we evaluated a male patient with classical 3 beta-HSD who showed pubertal development. High values of testosterone and a ratio of delta(5) to delta(4) steroids in the spermatic vein indicated direct production of considerable amounts of testosterone and a persistent defect of 3 beta-HSD activity in the gonad. Immunohistochemical analysis showed distinct immunoreactivity in the Leydig cells of the patient. The patient was homozygous for a nonsense mutation in the type-II 3 beta-HSD gene. We propose that gonadal type-I 3 beta-HSD could be expressed by gonadotropin stimulation at pubertal age, and delta(4)-steroid precursors would convert to testosterone.