Congenital Hypothyroidism with Thyroid in situ: A Case Report with NKX2-1 and DUOX2 Hypomorphic Variants.

INTRODUCTION: NK2 homeobox 1 (NKX2-1) encodes a transcription factor, NKX2-1, that is expressed in the thyroid gland, lung, and brain. Dual oxidase 2 (DUOX2) encodes an enzyme which generates hydrogen peroxide and is involved in the thyroid hormone synthesis. Cases of congenital hypothyroidism (CH) with dyshormonogenesis showing two or more genetic variants are increasingly reported. We describe the first case of transient dyshormonogenesis who had experimentally verified a loss-of-function NKX2-1 variant and DUOX2 variants. CASE PRESENTATION: The proband was a 15-year-old female patient with CH who was diagnosed in the frame of newborn screening for CH. She had a mildly elevated serum TSH level (14.56 mU/L), a low free thyroxine level (0.87 ng/dL), and a high thyroglobulin (Tg) level (>800 ng/mL). Ultrasonography revealed goiter. She was followed clinically without levothyroxine treatment and showed normal growth and development. She had slightly high Tg levels throughout the clinical course. Next-generation sequencing-based genetic analysis revealed that the patient was heterozygous for an NKX2-1 variant (p.Ile228Ser), a nonsense DUOX2 variant (p.[Lys530*;His678Arg]), and a functional DUOX2 polymorphism (p.His678Arg). NKX2-1 p.Ile228Ser showed about 50% reduced residual activity on the Tg promoter. CONCLUSION: A partial loss-of-function NKX2-1 variant with a monoallelic nonsense DUOX2 variant and a DUOX2 functional polymorphism can cause transient CH with high serum Tg levels.

Risk Factors of Coronary Artery Abnormalities and Resistance to Intravenous Immunoglobulin Plus Corticosteroid Therapy in Severe Kawasaki Disease: An Analysis of Post RAISE.

BACKGROUND: Coronary artery abnormalities (CAAs) still occur in patients with Kawasaki disease receiving intensified treatment with corticosteroids. We aimed to determine the risk factors of CAA development and resistance to intensified treatment in Post RAISE (Prospective Observational Study on Stratified Treatment With Immunoglobulin Plus Steroid Efficacy for Kawasaki Disease)-the largest prospective cohort of Kawasaki disease patients to date. METHODS: In Post RAISE, 2648 consecutive patients with Kawasaki disease were enrolled. The present study analyzed 724 patients predicted to be intravenous immunoglobulin (IVIG) nonresponders (Kobayashi score ≥5) who received intensified treatment consisting of IVIG plus prednisolone. The association between the baseline characteristics and CAA at 1 month after disease onset was examined. The association between the baseline characteristics and treatment resistance was also investigated. RESULTS: Maximum Z score at baseline ≥2.5 (odds ratio, 3.4 [95% CI, 1.5-7.8]), age at fever onset <1 year (odds ratio, 3.4 [95% CI, 1.6-7.4]), and nonresponsiveness to IVIG plus prednisolone treatment (odds ratio, 6.8 [95% CI, 3.3-14.0]) were independent predictors of CAA development. Nonresponsiveness to IVIG plus prednisolone was significantly associated with 8 baseline variables. Baseline total bilirubin (odds ratio, 1.4 [95% CI, 1.2-1.7]) was the only significant independent predictor other than the variables included in the Kobayashi score, enabling treatment resistance to be identified at diagnosis. The area under the ROC curve was 0.74 (95% CI, 0.69-0.79). At a cutoff point of 1.0, the sensitivity and specificity for predicting treatment resistance were 71% and 65%, respectively. CONCLUSIONS: In Post RAISE, younger age at fever onset, a larger maximum Z score at baseline, and nonresponsiveness to IVIG plus prednisolone were risk factors significantly associated with CAA development. Nonresponders were able to be identified at diagnosis based on the total bilirubin value. To prevent CAA, more intensified or adjunctive therapies using other agents, such as pulsed methylprednisolone, ciclosporin, infliximab, and Anakinra, should be considered for patients with these risk factors. Registration: URL: https://www.umin.ac.jp/ctr/; Unique identifier: UMIN000007133.

A novel screening method for pediatric urinary tract infection using ordinary diapers.

Urinary tract infection (UTI) is one of the most common bacterial infections in children. The symptoms of UTI in young children are nonspecific, therefore urine should be examined whenever UTI cannot be ruled out. In clinical settings, however, collecting urine from children who are not toilet trained is sometimes difficult, presenting a challenge in UTI management. Here, we developed a "diaper UTI test", which enables the quick detection of pyuria in ordinary diapers, and investigated its sensitivity and specificity in a clinical study. The diaper UTI test is based on a leukocyte esterase reaction. Reagent was prepared in liquid form so that it can be absorbed by disposable diapers, where it will produce a violet color in the presence of pyuria. For the clinical study, we enrolled children younger than 3 years with potential UTI who underwent bladder catheterization for urine culture and urinalysis. Of the 65 children included, 21 were diagnosed with UTI. The sensitivity and specificity of the diaper UTI test were 90.5% (95% CI 69.6-98.8) and 93.2% (95% CI 81.3-98.6), respectively. Because of its convenience and good sensitivity, the diaper UTI test may be useful in the screening of pediatric UTI.

Pubertal and Adult Testicular Functions in Nonclassic Lipoid Congenital Adrenal Hyperplasia: A Case Series and Review.

Lipoid congenital adrenal hyperplasia (LCAH) is caused by mutations in STAR and characterized by a defect in steroidogenesis and lipid droplet accumulation in steroidogenic cells. Patients with 46,XY and classic LCAH will typically present with female-type external genitalia. However, those with nonclassic LCAH will have masculinized external genitalia. The rarity of the nonclassic form has precluded the clarification of the long-term outcomes of testicular function in nonclassic LCAH. We report the cases of three adult males with nonclassic LCAH in whom primary adrenal insufficiency had been diagnosed at 5 days, 4 years, and 5 years of age. All exhibited complete male external genitalia and had completed pubertal development without androgen replacement. The endocrinological data showed preserved gonadal function in patients 1 and 2 and hypergonadotropic hypogonadism in patient 3. Semen analyses showed normozoospermia in patient 1 and mild oligozoospermia in patient 2. Electron microscopic analysis of a testicular biopsy specimen from patient 2 at 13 years of age revealed prominent lipid accumulation in the cytosol of Leydig cells. Patients 1 and 2 shared the same compound heterozygous mutations in STAR (p.Glu258* and p.Arg272Cys). Patient 3 possessed a heterozygous dominant-negative mutation in STAR (p.Gly22_Leu59del). A functional assay of a variant STAR-Arg272Cys determined the residual activity as 35% of the wild-type STAR. The results from the present case series and a review of four previously reported adult cases indicate that testosterone synthesis can be preserved in most males with nonclassic LCAH to complete pubertal development and induce germ cell maturation despite lipid accumulation in the Leydig cells.

Effects of pre- and post-pubertal dihydrotestosterone treatment on penile length in 5α-reductase type 2 deficiency.

Steroid 5α-reductase type 2 deficiency (5αRD2) is a congenital disorder of sex development caused by impairment of conversion from testosterone (T) to 5α-dihydrotestosterone (DHT). DHT deficiency leads to various degrees of undervirilized external genitalia including micropenis, primarily correlated with mutations of the SRD5A2 gene that encodes 5α-reductase type 2. Four Japanese boys with isolated micropenis were diagnosed as 5αRD2 by elevated ratios of serum T/DHT, and decreased ratios of urinary 5α/5ß-reduced steroid metabolites. Genetic analyses for SRD5A2 identified that the four patients shared a hypomorphic mutation R227Q that has a residual activity related to the mild-form of 5αRD2. For prepubertal micropenis, DHT was transdermally applied to the four patients at the ages of 4-11 year, increasing a median of stretched penile lengths (SPLs) from 2.6 cm (-2.5 SD) to 4.4 cm (-0.2 SD). Nevertheless, the post-pubertal penile growth was apparently retarded, despite normal levels of T secreted from well-developed testes. The second course of DHT treatment underwent at ages of 12-18 year, but unable to normalize SPLs at a range of 6.0 to 7.0 cm (-3.4 to -2.4 SD). The prostate volumes of two patients were variable at 8.1 and 21 cm3, and a sperm cell count of one patient was normal as young adult. DHT treatment contributes to development of the penis and prostate, which are favorable for the potential fertility of 5αRD2 adults. Meanwhile, the retarded penile growth and a risk of prostate overgrowth may complicate the post-pubertal management with DHT for 5αRD2 males.

Efficacy and safety of intravenous immunoglobulin plus prednisolone therapy in patients with Kawasaki disease (Post RAISE): a multicentre, prospective cohort study.

BACKGROUND: The RAISE study showed that additional prednisolone improved coronary artery outcomes in patients with Kawasaki disease at high risk of intravenous immunoglobulin (IVIG) resistance. However, no studies have been done to test the steroid regimen used in the RAISE study. We therefore aimed to verify the efficacy and safety of primary IVIG plus prednisolone. METHODS: We did a multicentre, prospective cohort study at 34 hospitals in Japan. We included patients diagnosed with Kawasaki disease according to the Japanese diagnostic criteria, and excluded those who were treated at other hospitals before being transferred to a participating hospital. Patients who were febrile at diagnosis received primary IVIG (2 g/kg per 24 h) and oral aspirin (30 mg/kg per day) until the fever resolved, followed by oral aspirin (5 mg/kg per day) for 2 months after Kawasaki disease onset. We stratified patients using the Kobayashi score into predicted IVIG non-responders (Kobayashi score ≥5) or predicted IVIG responders (Kobayashi score <5). For predicted non-responders, each hospital independently decided whether to add prednisolone (intravenous injection of 2 mg/kg per day for 5 days) to the primary IVIG treatment, according to their respective treatment policy, and we further divided these patients based on the primary treatment received. The primary endpoint was the incidence of coronary artery abnormalities determined by two-dimensional echocardiography at 1 month after the primary treatment in predicted non-responders treated with primary IVIG plus prednisolone. Coronary artery abnormalities were defined according to the criteria of the Japanese Ministry of Health and Welfare and of the American Heart Association (AHA). This study is registered with the University Hospital Medical Information Network Clinical Trials Registry, number UMIN000007133. FINDINGS: From July 1, 2012, to June 30, 2015, we enrolled 2628 patients with Kawasaki disease, of whom 724 (27·6%) were predicted IVIG non-responders who received IVIG plus prednisolone as primary treatment. 132 (18·2%) of 724 patients did not respond to primary treatment. Among patients with complete data, coronary artery abnormalities were present in 40 (incidence rate 5·9%, 95% CI 4·3-8·0) of 676 patients according to the AHA criteria or in 26 (3·8%, 2·5-5·6) of 677 patients according to the Japanese criteria. Serious adverse events were reported in 12 (1·7%) of 724 patients treated with primary IVIG plus prednisolone; two of these patients had hypertension and bacteraemia that was probably related to prednisolone. One patient died possibly due to severe inflammation from the Kawasaki disease itself. INTERPRETATION: Primary IVIG plus prednisolone therapy in this study had an effect similar to that seen in the RAISE study in reducing the non-response rate and decreasing the incidence of coronary artery abnormalities. A primary IVIG and prednisolone combination therapy might prevent coronary artery abnormalities and contribute to lowering medical costs. FUNDING: Tokyo Metropolitan Government Hospitals and the Japan Kawasaki Disease Research Center.

A novel mutation in the C-propeptide of COL2A1 causes atypical spondyloepiphyseal dysplasia congenita.

Spondyloepiphyseal dysplasia congenita (SEDC, OMIM #183900) is one of the type II collagenopathies caused by a heterozygous mutation in the COL2A1 gene. Although typical SEDC shows delay of pubic bone ossification on radiographs, atypical SEDC exists without this finding. We identified an atypical SEDC patient with a novel missense mutation in the C-propeptide region of COL2A1. This case suggests that a COL2A1 C-propeptide mutation can cause atypical SEDC.

Human Chorionic Gonadotropin Stimulation Test in Prepubertal Children with Micropenis Can Accurately Predict Leydig Cell Function in Pubertal or Postpubertal Adolescents.

BACKGROUND/AIM: To evaluate the accuracy of the human chorionic gonadotropin (hCG) stimulation test in children with micropenis in predicting later Leydig cell function. METHODS: We conducted a retrospective investigation of testosterone response to a 3-day hCG test (3,000 IU/m2/day) in prepuberty to indicate the need for hormone replacement therapy (HRT) in adolescence. RESULTS: Fifty Japanese boys (range, 0.8-15.4 years of age; median, 8.9) with micropenis were enrolled. Thirty-four spontaneously developed puberty and preserved the ability of testosterone production (group 1), while 16 did not develop any pubertal signs without HRT (group 2). Serum testosterone levels after the hCG test (post-hCG T) in group 2 (range, <0.05-1.1 ng/ml; median, 0.24) were significantly lower than in group 1 (range, 0.5-8.7 ng/ml; median, 2.4; p < 0.0001). Based on true positives who required continuous HRT, the area under the receiver-operating characteristics curve for post-hCG T was 0.983 [95% confidence interval (CI), 0.90-1.00]. The post-hCG T cut-off level corresponding to the Youden index was 1.1 ng/ml (95% CI, 1.0-1.1), with a sensitivity of 100.0% (95% CI, 79.4-100.0) and a specificity of 94.1% (95% CI, 80.3-99.3). CONCLUSIONS: The hCG test in prepubertal children with micropenis can be useful for predicting Leydig cell function in pubertal or postpubertal adolescents. The post-hCG T cut-off level of 1.1 ng/ml is recommended to screen for those who will likely require HRT for pubertal development.

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.

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.

Gonadotrophin therapy in Kallmann syndrome caused by heterozygous mutations of the gene for fibroblast growth factor receptor 1: report of three families: case report.

Gonadotrophin therapy (GT) is frequently used to induce fertility in Kallmann syndrome (KS). We studied the effects and the consequences of GT in autosomal dominant KS caused by heterozygous FGFR1 mutations. Three Japanese families were examined. In family A, an adult male received GT and had two sons. In family B, an adult female received GT and gave birth to dizygotic male and female twins. In family C, an adult female received GT and produced a son and a daughter. Direct sequencing was performed for FGFR1, and clinical assessment was carried out for KS features. The father and the elder son of family A had P745S mutation, the mother and the female twin of family B had G687R mutation, and the mother and the two children of family C had S107X mutation. KS phenotype was detected for the mutation-positive subjects, except for the elder son of family A who had apparently normal phenotype. GT in FGFR1 mutations is effective in acquiring fertility but has a risk of transmitting the mutation and the disease phenotype to the next generation.

Clinical assessment and mutation analysis of Kallmann syndrome 1 (KAL1) and fibroblast growth factor receptor 1 (FGFR1, or KAL2) in five families and 18 sporadic patients.

We report on the clinical and molecular findings in 25 males and three females with Kallmann syndrome (KS) aged 10-53 yr. Ten males were from five families, and the remaining 15 males and three females were apparently sporadic cases. Molecular studies were performed for Kallmann syndrome 1 (KAL1) and fibroblast growth factor receptor 1 (FGFR1, also known as KAL2) by sequence analysis for all the coding exons, by PCR-based deletion analysis, and by fluorescence in situ hybridization (FISH) analysis, showing six novel and two recurrent intragenic KAL1 mutations in seven familial and four sporadic male cases and two novel intragenic FGFR1 mutations in two sporadic male cases. In addition, submicroscopic deletions at Xp22.3 involving VCX-A, STS, KAL1, and OA1 were identified in three familial cases and one sporadic male case affected by a contiguous gene syndrome. Clinical assessment in the 15 males with KAL1 mutations showed normal and borderline olfactory function in two males and right-side dominant renal lesion in seven males, in addition to variable degrees of hypogonadotropic hypogonadism (HH) in all the 15 males and olfactory dysfunction in 13 males. The two males with FGFR1 mutations had HH and anosmia and lacked other features. Clinical features in the remaining 11 cases with no demonstrable KAL1 or FGFR1 mutations included right renal aplasia in one female, cleft palate in one male, cleft palate and perceptive deafness in one male, and dental agenesis and perceptive deafness in one male, in addition to a variable extent of HH and olfactory dysfunction. The results suggest the following: 1) KAL1 mutations might be more prevalent in the Japanese patients than previously estimated in the Caucasian patients and can be associated with apparently normal olfactory function; 2) FGFR1 mutations account for approximately 10% of KS patients, as previously reported in the Caucasian patients, and can result in HH and olfactory dysfunction-only phenotype; and 3) renal aplasia, which is characteristic of KAL1 mutations, and cleft palate and dental agenesis, which are characteristic of FGFR1 mutations, can occur in patients without KAL1 and FGFR1 mutations.

A case of impairment of mitochondrial fatty acid beta-oxidation.

We describe a patient with impairment of mitochondrial fatty acid P-oxidation. A Japanese baby boy was delivered in the 38th week of gestation by emergency cesarean section due to fetal asphyxia. His birth weight was 1,985 g (<10th percentile), length 44.8 cm (<10th percentile), and head circumference 31.0 cm (10th percentile). His Apgar scores were 3 and 5 at 1 min and 5 min, respectively. Blood glucose was 12 mg/dl at 1 hour after birth, requiring glucose administration. On day 1 his serum CK was 20,780 IU/l, which was thought to be due to asphyxia. His serum CK levels gradually began to decrease. At 3 months of age, he sucked poorly, had poor body weight gain, and muscle hypotonia was observed. On day 117 his general condition was impaired, and marked hepatomegaly was observed. The blood glucose level was 43 mg/dl. The patient's urine was negative for ketone bodies. His serum triglyceride level was 3,670 mg/dl. Abdominal CT scan revealed a fatty liver. Serum levels of acyl carnitine from very-long chain fatty acid increased. On day 118 he died due to ventricular fibrillation. On necropsy, massive lipid deposition was observed in the liver, cardiac muscle, kidney, skeletal muscle, and intestinal mucosa. The ratio of very-long chain acyl-CoA dehydrogenase (VLCAD) activity for C16/C8 fatty acid was 0.50 (normal control 1.29), suggesting abnormal VLCAD. He was diagnosed as having impairment of mitochondrial fatty acid beta-oxidation, presumably due to the VLCAD deficiency.