Identification of mutations in the hepatocyte nuclear factor (HNF)-1 alpha gene in Japanese subjects with IDDM.

One form of maturity-onset diabetes of the young, MODY3, is characterized by a severe insulin secretory defect, compared with MODY2, a glucokinase-deficient diabetes. It has recently been shown that mutations of the gene encoding the transcription factor hepatocyte nuclear factor (HNF)-1 alpha cause MODY3. Because of the rapid progress to overt diabetes and the high prevalence of required insulin treatment in patients with MODY3, we screened the HNF-1 alpha gene for mutations in Japanese subjects with IDDM. Ten exons and flanking introns of the HNF-1 alpha gene in these subjects were amplified by polymerase chain reaction and direct sequencing of the products. Mutations were identified in three (5.5%) of the 55 unrelated subjects with IDDM. A missense mutation of R272H (replacement of Arg by His in codon 272) in the DNA binding domain of HNF-1 alpha was found in a subject who developed IDDM 1 year after diagnosis of NIDDM at 8 years of age. A frameshift mutation of P291 fsinsC (insertion of a C in a polyC tract around codon 291 for Pro), which would generate a mutant truncated protein of 340 amino acids, was found in a subject who started insulin treatment when hyperglycemia and ketonuria were noticed at 13 years of age. A missense mutation of R583G (replacement of Arg by Gly in codon 583) in the transactivation domain of HNF-1 alpha was found in a subject with sudden-onset IDDM at 20 years of age. None of these mutations were present in 100 nondiabetic subjects (200 normal chromosomes). These results indicate that the HNF-1 alpha gene defects could lead to the development of not only early-onset NIDDM but also IDDM, implicating the importance of subclassification of HNF-1 alpha-deficient IDDM from a classical type of autoimmune-based IDDM in Japanese.

Aberrant dynamics of histone deacetylation at the thyrotropin-releasing hormone gene in resistance to thyroid hormone.

Histone acetylation status influences transcriptional activity, and the mechanism of negative gene regulation by thyroid hormone remains unclear, although its impairment by a mutant thyroid hormone receptor (TR) is critical for resistance to thyroid hormone (RTH). We found a novel RTH mutant, F455S, that exhibited impaired repression of the TRH gene and had a strong dominant-negative effect on the gene. F455S strongly interacted with nuclear receptor corepressor (NCoR) and was hard to dissociate from it. To analyze the dynamics of histone acetylation status in vivo, we established cell lines stably expressing the TRH promoter and wild-type or F455S TR. Treatment with a histone deacetylase (HDAC) inhibitor completely abolished the repression of the gene by T3. The histones H3 and H4 at the TRH promoter were acetylated, and addition of T3 caused recruitment of HDACs 2 and 3 within 15 min, resulting in a transient deacetylation of the histone tails. TR and NCoR were located on the promoter, and T3 caused NCoR dissociation and steroid receptor coactivator-1 recruitment. In the presence of F455S, the histones were hyperacetylated, and HDAC recruitment and histone deacetylation were significantly impaired. This is the first report demonstrating the direct involvement of aberrant dynamics of chromatin modification in RTH.

Relationship between serum levels of methylguanidine and glycemic control in IDDM children.

OBJECTIVE: To examine the serum levels of methylguanidine in IDDM children and compare them with markers for glycemic control. Reports have indicated that active oxygen, which damages various tissues, increases in diabetes mellitus. The increase of active oxygen is one of the risk factors for diabetic complications. The synthesis of methylguanidine, a metabolic product of guanidine, is mainly regulated by active oxygen. RESEARCH DESIGN AND METHODS: Forty-eight children with IDDM (mean age 13.3 yr) and 17 age-matched nondiabetic control subjects were studied. Diabetic children were divided into a well-controlled group (HbA1c < 8%, n = 24) and a poorly controlled group (HbA1c > 8%, n = 24). Serum concentrations of methylguanidine were measured by enzymatic assay. RESULTS: Levels of methylguanidine in the poorly controlled group (1.31 +/- 0.08 microM) were significantly higher than those in both the well-controlled group (0.85 +/- 0.08 microM) and the control group (0.59 +/- 0.11 microM), respectively (P < 0.01). Methylguanidine levels showed a positive correlation with the levels of HbA1c (P < 0.01) or fructosamine (P < 0.01). No significant correlations were noted between methylguanidine levels and age, sex, duration of diabetes, or insulin dose. CONCLUSIONS: Our data indicate that the levels of methylguanidine in IDDM children might be affected by glycemic control and that the determination of serum methylguanidine levels could be a useful test for evaluating the state of diabetic control.

Familial congenital hypopituitarism with central diabetes insipidus.

Congenital hypopituitarism (CH) presenting with central diabetes insipidus is typically associated with midline facial deformities or ophthalmological abnormalities. We present three brothers with CH and central diabetes insipidus not associated with any of these predisposing conditions. All three subjects presented with clinical features typical for CH (neonatal hypoglycemia, short stature, protruding forehead, and microgenitalia). All had hypoplastic genitalia indicating in utero gonadotropin deficiency, and all had complete GH deficiency. One represented low levels of thyroid hormones and TSH, indicating central hypothyroidism. Water deprivation examination in two of the brothers demonstrated complete arginine vasopressin deficiency in one and partial deficiency in the other. Magnetic resonance imaging indicated absence of the pituitary stalk, severe hypoplastic anterior pituitary in all three brothers, and absence of any posterior pituitary gland in two of the three. The other sibling had an ectopic posterior pituitary. This first report of familial CH with central diabetes insipidus may represent a previously unknown midline anomaly and provide new insights into the genetic control of pituitary and hypothalamic development.

A new compound heterozygous mutation (W17X, 436 + 5G --> T) in the cytochrome P450c17 gene causes 17 alpha-hydroxylase/17,20-lyase deficiency.

A genetic disorder in cytochrome P450c17 results in 17 alpha-hydroxylase/17,20-lyase deficiency. In the present study, a Japanese patient with 17 alpha-hydroxylase/17,20-lyase deficiency underwent molecular analysis. The patient presented with complete female genitalia with a 46,XY karyotype, absent pubertal development, and hypertension. the exons and exon-intron boundaries of P450c17 genetic region were amplified and sequenced. DNA sequencing revealed a compound heterozygous mutation. One allele showed a G to A transition corresponding to a premature termination codon at tryptophane in codon 17 (W17X). The other allele showed a G to T substitution at the fifth nucleotide from the splice donor site in intron 2 (436 + 5G --> T). W17X was found in one allele of the father, and 436 + 5G --> T was found in one allele of the mother. A previous report presented a patient with 17 alpha-hydroxylase/17,20-lyase deficiency who was homozygous for W17X. However, the present case is a novel 436 + 5G --> T mutation. Reverse transcription-PCR analysis using total ribonucleic acid isolated from the testes of the patient revealed that an intron 2 donor site mutation caused abnormal splicing, such that exon 2 was spliced with intron 2. Skipping the exon alters the translational reading frame of exon 3 and introduces a premature termination codon. In semiquantitative analysis, the majority of the transcript for 436 + 5G --> T skips exon 2. The present findings indicate that in this patient, 17 alpha-hydroxylase/17,20-lyase deficiency was caused by the compound heterozygous mutation of exon and splice site mutation in cytochrome P450c17 gene.

Detection of skewed X-inactivation in two female carriers of vasopressin type 2 receptor gene mutation.

Most cases of congenital nephrogenic diabetes insipidus (NDI) are inherited in an X-linked manner, which is due to the mutations of the vasopressin type 2 receptor (V2R) gene. However, recent reports have presented female NDI patients with heterozygote V2R gene mutations. The mechanism of inheritance was thought to be skewed X-inactivation. We present a family with congenital NDI. Three male members were diagnosed with NDI, and examination of their V2R gene revealed a G inserted at nucleotide 804 of the open reading frame. Three female individuals display different degrees of symptoms of NDI, and all of them possess both the normal and abnormal genes. The X-inactivation patterns of the female members were investigated via the detection of methylated trinucleotide repeat in the human androgen receptor gene. The grandmother showed extremely skewed methylation of one X chromosome, and the mother revealed moderately skewed methylation. The daughter of the grandmother's sister, who has no symptoms of NDI, showed random methylation. The highly skewed X-inactivation pattern of the grandmother suggests that her NDI phenotype is caused by dominant methylation of the normal allele of V2R gene.

Identification of novel human GH-1 gene polymorphisms that are associated with growth hormone secretion and height.

Height, which is partially determined by GH secretion, is genetically influenced. The purpose of this study was to identify polymorphisms in the GH-1 gene, which are associated with altered GH production. The subjects included prepubertal short children with GH insufficiency without gross pituitary abnormalities (n = 43), short children with normal GH secretion (n = 46), and normal adults (n = 294). A polymorphism in intron 4 (P-1, A or T at base 1663) was identified. Two additional polymorphic sites (P-2, T or G at base 218, and P-3, G or T at base 439) in the promoter region of the GH-1 gene were also identified and matched with the P-1 polymorphism (A or T, respectively) in more than 90% of the subjects. P-1, P-2, and P-3 were considered to be associated with GH production, and the results of P-2 are explained as a representative in this abstract. For example, the allele frequency of T at P-2 in prepubertal short children with GH insufficiency without gross pituitary abnormalities (58.1%) was significantly different from that in short children with normal GH secretion and normal adults (37.0% and 43.5%, respectively; P < 0.001). Furthermore, significant differences were observed in maximal GH peaks in provocative tests (11.1 vs. 18.2 ng/mL, P = 0.006), insulin-like growth factor I SD scores (SDS) (-2.4 vs. -0.8, P < 0.0001), and height (Ht) SDS (-3.7 vs. -3.0, P = 0/001) in children with the T/T or G/G genotypes at P-2, respectively. In the entire study group, significant differences in insulin-like growth factor SDS (T/T, -0.9; G/G, -0.2; P = 0.0009) and Ht SDS (T/T, -1.0; G/G, -0.4; P = 0.022) were observed between the T/T and G/G genotypes at P-2. These data indicate that GH secretion is partially determined by polymorphisms in the GH-1 gene, which explain some of the variations in GH secretion and Ht.

A novel point mutation (R243Q) in exon 7 of the c-erbA beta thyroid hormone receptor gene in a family with resistance to thyroid hormone.

Resistance to thyroid hormone (RTH) is characterized by variable tissue hyporesponsiveness to thyroid hormones. Recently, a large number of different point mutations have been identified in the c-erbA beta thyroid hormone receptor (TR beta) in subjects with RTH. We describe a Japanese family with RTH with a novel point mutation in exon 7 of the TR beta gene. A single nucleotide substitution, guanine for adenine, was identified at the second position of codon 243 located in the hinge domain between the ligand binding and DNA binding domains in one of the two alleles of the proband and his mother, resulting in the substitution of the normal arginine (CGG) with a glutamine (CAG). Except for one family, point mutations so far described in RTH are clustered at exons 8-10 of the TR beta gene. This report presents a novel mutation in the characteristic portion in exon 7 of the TR beta.

Novel inactivating missense mutations in the thyrotropin receptor gene in Japanese children with resistance to thyrotropin.

We describe Japanese siblings with resistance to thyrotropin (TSH) who are compound heterozygotes for two novel mutations in the TSH receptor gene. The affected siblings had increased serum TSH, normal serum thyroid hormones, and normal positioned but slightly hypoplastic thyroid glands. The mutated paternal allele has the substitution of His (CAC) in place of Arg (CGC) at codon 450 (R450H) of the TSH receptor. The mutated maternal allele has the substitution of Ser (AGT) in place of Gly (GGT) at codon 498 (G498S) of the TSH receptor. COS-7 cells transfected with the R450H mutant exhibited a slightly decreased TSH binding and a slightly decreased cyclic adenosine monophosphate (cAMP) response to TSH, whereas cells transfected with the G498S mutant exhibited a markedly decreased TSH binding and a markedly decreased cAMP response to TSH. Flow immunocytofluorometry analysis demonstrated that the G498S mutant resulted in extremely low expression at the cell surface as compared with the wild type receptor and the R450H mutant, in spite of a normal intracellular synthesis. The present cases are the first Japanese patients with TSH resistance in whom mutations in the TSH receptor gene have been identified. These novel mutations may contribute to understanding of the struc-ture-function relationship of the TSH receptor.

A novel point mutation of thyroid hormone receptor beta gene in a family with resistance to thyroid hormone.

Resistance to thyroid hormone (RTH) is characterized by variable tissue hyporesponsiveness to thyroid hormone caused by mutations of thyroid hormone receptor beta (TRbeta) gene. We found a novel point mutation of the TRbeta gene in a family (F123) with RTH, a transition of a guanine to adenine at nucleotide 1215, which replaced the normal Met-310 with Ile. This substitution was found in only one allele of affected family members. In vitro transcription and translation of this mutant TRbeta demonstrated a 12-fold reduction of the affinity for triiodothyronine (T3) compared with the wild type TRbeta. Thyroid function tests were similar to a previously reported RTH family (F99) who had a different mutation in the same codon (Thr 310).

Detection of point mutation for human growth hormone in patients with anti-pituitary antibody positive type 1 diabetes mellitus.

We investigated genetic mutations in the coding region of the human growth hormone (hGH) gene in anti-pituitary antibody (APA)-positive patients with type 1 diabetes mellitus (n = 6) or autoimmune thyroid diseases (n = 10) and in APA-negative, healthy controls (n = 10). A point mutation in the hGH gene was discovered in two patients with type 1 diabetes mellitus. No mutations were found in the hGH gene in control subjects, patients with autoimmune thyroid diseases (Hashimoto's thyroiditits, Graves' disease) or in the remaining four patients with type 1 diabetes mellitus. The mutation was located in the coding region for the second amino acid in the N-terminal region of hGH. This point mutation was identified in codon 2 in exon 2 of the hGH gene. We successfully developed an allele-specific amplification method for detecting this mutation using the polymerase chain reaction.

Clinical case seminar in pediatric thyroid disease.

Pediatric thyroid diseases cover a large spectrum of congenital and acquired forms, ranging from congenital primary or central hypothyroidism, autoimmune thyroid disease, iodine deficiency, rare genetic defects of thyroid hormone action, metabolism and cell membrane transport to benign nodules and malignant tumors. The previous 15 papers of the textbook Paediatric Thyroidology gave a systematic overview of the current knowledge and guidelines on all these diseases. In this final paper, the authors collected a series of patient histories from their clinics illustrating frequently encountered clinical problems and providing key learning points and references to each case. Although not fully comprehensive, it aims at providing relevant clinical knowledge on thyroid diseases of the neonate, the child, and the adolescent.

Transport of triiodothyronine by erythrocytes from premature and term infants.

To analyze hypothyroxinemia in low birth weight (LBW) infants, the initial uptake of T3 into erythrocytes from term and premature infants was measured. Thirty-eight infants and 18 prepubertal children as controls were subjected in this study. The infants were divided into group 1 (premature infants; below 2,500 g, n = 12) and group 2 (term infants; 2,501-3,500 g, n = 26). Infants with LBW for gestational age were excluded from this study. Vmax for group 1 (mean +/- SE, 6.4 +/- 0.5 pmol/min/10(8) cells), having low thyroid hormone levels, was significantly less than in group 2 (9.0 +/- 0.7 pmol/min/10(8) cells; p < 0.05). Mean Vmax for both groups exceeded normal controls (3.9 +/- 0.3 pmol/min/10(8) cells; p < 0.01). Km in the perinatal period varied widely and was essentially the same as in the normal controls. These findings indicate that T3 uptake by erythrocytes of newborns may possibly be high regardless of maturation stage and blood levels of thyroid hormones may be reflected not by change in the affinity of the transport system but by that in the number of membrane carriers.

Urinary laminin P1 as an index of glycemic control in children with insulin-dependent diabetes mellitus.

OBJECTIVE: To evaluate the clinical meanings of urinary laminin P1, urinary laminin P1 concentrations in children with insulin-dependent diabetes mellitus (IDDM) were measured and compared with urinary levels of albumin, kappa light chain (kappa-LC), alpha 1-microglobulin (alpha 1-MG), beta 2-microglobulin (beta 2-MG), and n-acetyl-b-D-glucosaminidase (NAG). RESEARCH DESIGN AND METHODS: Forty-six children with IDDM and 31 age-matched controls were studied. The first urine in the morning was collected for three days and stored at -20 degrees C until assayed. The mean values were used for the study. Radioimmunoassay (RIA) was used to measure of laminin P1, kappa-LC, alpha 1-MG, and beta 2-MG. NAG and haemoglobin Aic (HbA1c) concentrations were measured by a colorimetric method and high-performance liquid chromatography, respectively. RESULTS: Urinary laminin P1 levels in IDDM were 54.2 + 3.4 (SE) mU/microM.Cr, significantly higher than those in control subjects (40.7 +/- 2.3 mU/microM.Cr, p < 0.01). In 10 out of 46 patients (21.7 percent), the values were higher than the mean +/- 2 SD of controls. Urinary albumin, kappa-LC, alpha 1-MG, beta 2-MG, and NAG in IDDM were higher than those in control subjects (p < 0.01). There were significant correlations between urinary laminin P1 levels and urinary albumin, kappa-LC, NAG, alpha 1-MG, and beta 2-MG. A significant correlation was also shown between urinary laminin P1 and HbA1c concentrations (p < 0.01). CONCLUSIONS: From the above results, we conclude that urinary laminin P1 concentrations could be one of the clinical indexes for glycemic control and damage of the glomerulus in IDDM.

Allelic variations of the D2 dopamine receptor gene in children with idiopathic short stature.

The D2 dopamine receptor (DRD2) plays a major role in growth hormone (GH) secretion. Recent reports indicate that Taq I A DRD2 gene alleles (A1 and A2) are related to the function of DRD2. Idiopathic short stature (ISS) is defined as short stature without accompanying malnutrition, chronic disease, and endocrinological disorders. However, some reports suggest that ISS is associated with a mild disturbance of GH secretion. In this study, we examined the notion that allelic variants of the DRD2 are associated with ISS. We studied 55 children with ISS aged 8.4 (SD 2.9) years; (group I) and 104 age-matched children of normal stature (group II). Informed consent was obtained from each child's parent or guardian. Genomic DNAs were extracted from peripheral mononuclear cells and amplified by polymerase chain reaction (PCR). The PCR products were digested by Taq1 and resolved by electrophoresis. The frequency of the A1 allel was significantly higher in group I (0.42) than in group II (0.26). The insulin-like growth factor (IGF)-I ratio (the ratio of the individual level to the normal mean value according to age at our laboratory center) was significantly lower in group I than in group II. When group I was subdivided into group A (with the A1 allele) and group B (with only the A2 allele), group A had a significantly lower peak GH response to the l-dopa test, lower levels of IGF-I, and retarded bone maturation. These findings indicate that polymorphism of the DRD2 gene may be one genetic factor that affects body height in childhood, acting through the hypothalamus (GH-releasing hormone)--pituitary (GH)--IGF-I axis.

Sequence analysis of thyroid transcription factor-1 gene reveals absence of mutations in patients with thyroid dysgenesis but presence of polymorphisms in the 5' flanking region and intron.

Congenital hypothyroidism is caused by several mechanisms. The most common cause worldwide is iodine deficiency, but in iodine-sufficient regions thyroid dysgenesis is the most common cause of congenital hypothyroidism. In the present study we analyzed the thyroid transcription factor-1 (TTF-1) gene in patients with congenital hypothyroidism due to thyroid dysgenesis: three patients with athyrosis, five with ectopy, and one with hypoplasia. Genomic DNA was isolated from peripheral leukocytes, and the TTF-1 gene, including a 5' flanking region, two exons and one intron was amplified by polymerase chain reaction (PCR) with 4 pairs of primers. The PCR products were directly sequenced by the Dye Terminator Cycle Sequencing method. We could not find any mutations specific for the thyroid dysgenesis in the 5' flanking region, two exons and one intron in the TTF-1 gene, but two heterozygous nucleotide substitutions were detected in the intron: a G to A transition at nucleotide 469 (G469A) and a C to A transversion at nucleotide 866 (C866A). The same nucleotide changes were detected in some normal subjects. Allelic frequencies of the polymorphisms G469A and C866A were 23% and 10%, respectively. Another normal polymorphism in the 5' flanking region was a G to T transversion at nucleotide -845 from the transcription start site (G-845T). The allelic frequency of the polymorphism G-845T was 28%. We also found 12 polymorphisms in the 5' flanking region, two in the intron and one in the 3' untranslated region. These polymorphisms were detected in 100% chromosomes. These results suggest that congenital hypothyroidism associated with thyroid dysgenesis is unlikely to be caused by mutations in the TTF-1 gene in which, however, were detected normal polymorphisms in the 5' flanking region, intron and 3' untranslated region.

Role of serum leptin in the regulation of weight gain in early infancy.

We investigated the changes in the serum leptin concentration from birth to 30 days of age in order to determine the effects of leptin in early infancy. Twenty-seven Japanese term infants (12 boys and 15 girls) were examined. The serum leptin concentration at 3-6 days of age was significantly lower than that in cord blood (p < 0.0001) and was significantly higher at 30 days than at 3-6 days (p < 0.0001). The serum leptin concentration did not differ with gender or nutrition. The body weight gain and the magnitude of leptin rise that occurred between 3-6 and 30 days of age showed a significant positive correlation (r = 0.79; p < 0.0001). These changes in leptin levels suggest that leptin plays an important role in the regulation of weight gain in early infancy.

Growth hormone and gonadotropin-releasing hormone analog therapy in haploinsufficiency of SHOX.

We report on GH (0.5 IU or 0.17 mg/kg/week) and GnRH analog (GnRHa, 60 microg/kg, every 4 weeks) therapy in SHOX haploinsufficiency. Case 1 was a 46,XY boy with microdeletion of the Y chromosomal pseudoautosomal region. At 7 years of age, he exhibited short stature (-3.9 SD) with a reduced growth rate (3.8 cm/year), short 4th metacarpals, and mild Madelung deformity. GH therapy resulted in a marked increase in height velocity (10.7 cm/year in the first year). Case 2 was a 46,XX girl with a heterozygous nonsense mutation of SHOX (C674T). At 6 years of age, she presented with short stature (-3.3 SD) with a low height velocity (4.0 cm/year). GH therapy caused a moderate increase in height velocity (6.6 cm/year in the first year and 6.0 cm/year in the second year) before puberty. Because of breast development, she received GnRHa from 9 8/12 years of age. At 10 10/12 years of age, she had mild shortening and borderline curvature of radius. Case 3 was a girl with a 46,X,der(X)t(X;2)(p22.3;p21) karyotype. She was treated with GH from 6 to 14 years of age, and also with GnRHa from 12 to 15 years of age. Her height remained around mean -4 SD, with no discernible alteration of height velocity. At 17 years of age, she had short stature (-4.1 SD), bilateral cubitus valgus, Madelung deformity, and full breast development. The results suggest that GH therapy may have variable statural effects in SHOX haploinsufficiency as in most disorders including Turner syndrome, and that GnRHa therapy after pubertal entry may be insufficient to prevent the development of skeletal lesions such as Madelung deformity.