Early growth hormone treatment accelerates delayed onset of puberty in patients with growth hormone deficiency.

We investigated whether growth hormone (GH) treatment could accelerate the onset of puberty in patients with isolated GH deficiency (GHD). Of the 135 boys and 89 girls who started GH treatment before the onset of puberty and were followed up at Tanaka Growth Clinic, 83 boys and 51 girls who started GH treatment sufficiently earlier than the average age at onset of puberty of GHD patients (<10 years vs. 11.7 years for boys; <9.5 years vs. 11.4 years for girls) were analyzed. Age at onset of puberty significantly positively correlated to age at the start of GH treatment (boys: r = 0.427, p < 0.0001; girls: r = 0.302, p < 0.05). When the subjects were divided into two groups each: for boys, Groups A (n = 45) and B (n = 39), treatment was started at age <8 and 8 to <10 years, respectively; for girls, Groups A (n = 26) and B (n = 21), treatment was started at age <7 and 7 to <9.5 years, respectively, age at the onset of puberty was significantly lower in Groups A than in Groups B by the Mann-Whitney U test (boys: p < 0.01; girls: p < 0.05) and Kaplan-Meier log-rank test (boys: p < 0.01; girls: p < 0.05). These results indicate that GH treatment accelerates the delayed onset of puberty in patients with GHD. Heights at the onset of puberty in Groups A and B were not significantly different, suggesting that early treatment does not increase adult height.

DNA Methylation Status of SHOX-Flanking CpG Islands in Healthy Individuals and Short Stature Patients with Pseudoautosomal Copy Number Variations.

SHOX resides in the short arm pseudoautosomal region (PAR1) of the sex chromosomes and escapes X inactivation. SHOX haploinsufficiency underlies idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). A substantial percentage of cases with SHOX haploinsufficiency arise from pseudoautosomal copy number variations (CNVs) involving putative enhancer regions of SHOX. Our previous study using peripheral blood samples showed that some CpG dinucleotides adjacent to SHOX exon 1 were hypomethylated in a healthy woman and methylated in a woman with gross X chromosomal rearrangements. However, it remains unknown whether submicroscopic pseudoautosomal CNVs cause aberrant DNA methylation of SHOX-flanking CpG islands. In this study, we examined the DNA methylation status of SHOX-flanking CpG islands in 50 healthy individuals and 10 ISS/LWD patients with pseudoautosomal CNVs. In silico analysis detected 3 CpG islands within the 20-kb region from the translation start site of SHOX. Pyrosequencing and bisulfite sequencing of genomic DNA samples revealed that these CpG islands were barely methylated in peripheral blood cells and cultured chondrocytes of healthy individuals, as well as in peripheral blood cells of ISS/LWD patients with pseudoautosomal CNVs. These results, in conjunction with our previous findings, indicate that the DNA methylation status of SHOX-flanking CpG islands can be affected by gross X-chromosomal abnormalities, but not by submicroscopic CNVs in PAR1. Such CNVs likely disturb SHOX expression through DNA methylation-independent mechanisms, which need to be determined in future studies.

Systematic molecular analyses of SHOX in Japanese patients with idiopathic short stature and Leri-Weill dyschondrosteosis.

The etiology of idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD) in European patients is known to include SHOX mutations and copy-number variations (CNVs) involving SHOX and/or the highly evolutionarily conserved non-coding DNA elements (CNEs) flanking the gene. However, the frequency and types of SHOX abnormalities in non-European patients and the clinical importance of mutations in the CNEs remains to be clarified. Here, we performed systematic molecular analyses of SHOX for 328 Japanese patients with ISS or LWD. SHOX abnormalities accounted for 3.8% of ISS and 50% of LWD cases. CNVs around SHOX were identified in 16 cases, although the ~47 kb deletion frequently reported in European patients was absent in our cases. Probably damaging mutations and benign/silent substitutions were detected in four cases, respectively. Although CNE-linked substitutions were detected in 15 cases, most of them affected poorly conserved nucleotides and were shared by unaffected individuals. These results suggest that the frequency and mutation spectrum of SHOX abnormalities are comparable between Asian and European patients, with the exception of a European-specific downstream deletion. Furthermore, this study highlights the clinical importance and genetic heterogeneity of the SHOX-flanking CNVs, and indicates a limited clinical significance of point mutations in the CNEs.

Rare pseudoautosomal copy-number variations involving SHOX and/or its flanking regions in individuals with and without short stature.

Pseudoautosomal region 1 (PAR1) contains SHOX, in addition to seven highly conserved non-coding DNA elements (CNEs) with cis-regulatory activity. Microdeletions involving SHOX exons 1-6a and/or the CNEs result in idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). Here, we report six rare copy-number variations (CNVs) in PAR1 identified through copy-number analyzes of 245 ISS/LWD patients and 15 unaffected individuals. The six CNVs consisted of three microduplications encompassing SHOX and some of the CNEs, two microduplications in the SHOX 3'-region affecting one or four of the downstream CNEs, and a microdeletion involving SHOX exon 6b and its neighboring CNE. The amplified DNA fragments of two SHOX-containing duplications were detected at chromosomal regions adjacent to the original positions. The breakpoints of a SHOX-containing duplication resided within Alu repeats. A microduplication encompassing four downstream CNEs was identified in an unaffected father-daughter pair, whereas the other five CNVs were detected in ISS patients. These results suggest that microduplications involving SHOX cause ISS by disrupting the cis-regulatory machinery of this gene and that at least some of microduplications in PAR1 arise from Alu-mediated non-allelic homologous recombination. The pathogenicity of other rare PAR1-linked CNVs, such as CNE-containing microduplications and exon 6b-flanking microdeletions, merits further investigation.

Mutation spectrum and phenotypic variation in nine patients with SOX2 abnormalities.

Multiple mutations in SOX2 have been identified in patients with ocular anomalies and/or pituitary dysfunction. Here, we identified SOX2 abnormalities in nine patients. The molecular defects included one missense, one nonsense and four frameshift mutations, and three submicroscopic deletions involving SOX2. Three of the six mutations and all deletions were hitherto unreported. The breakpoints determined in one deletion were located within Alu repeats and accompanied by an overlap of 11 bp. Three of the six mutations encoded SOX2 proteins that lacked in vitro transactivation activity for the HESX1 promoter, whereas the remaining three generated proteins with ∼15-∼20% of transactivation activity. All cases manifested ocular anomalies of various severities, together with several complications including arachnoid cyst and hamartoma. There was no apparent correlation between the residual activity and clinical severity. The results indicate that molecular defects in SOX2 are highly variable and include Alu repeat-mediated genomic rearrangements. Our data provide further evidence for wide phenotypic variation of SOX2 abnormalities and the lack of genotype-phenotype correlation in patients carrying SOX2 lesions.

Acroscyphodysplasia as a phenotypic variation of pseudohypoparathyroidism and acrodysostosis type 2.

Acroscyphodysplasia (OMIM250215) is a distinctive form of metaphyseal dysplasia characterized by the distal femoral and proximal tibial epiphyses embedded in cup-shaped, large metaphyses known as metaphyseal scypho ("scypho" = cup) deformity. It is also associated with severe growth retardation and brachydactyly. The underlying molecular mechanism of acroscyphodysplasia has not yet been elucidated, although scypho-deformity of the knee has been reported in three patients with acrodysostosis due to a mutation in the PDE4D gene. We report on the clinical, radiological, and molecular findings of five female patients with acroscyphodysplasia; two were diagnosed as pseudohypoparathyroidism (PHP) or Albright hereditary osteodystropy, and the other three as acrodysostosis. They all had radiological findings consistent with severe metaphyseal scypho-deformity and brachydactyly. Heterozygous mutations were identified in the PHP patients consisting of one novel (p.Q19X) and one recurrent (p.R231C) mutation of the GNAS gene, as well as, in the acrodysostosis patients consisting of two novel mutations (p.T224I and p.I333T) of the PDE4D gene. We conclude that metaphyseal acroscyphodysplasia is a phenotypic variation of PHP or acrodysostosis caused by either a GNAS or PDE4D mutation, respectively.

Incidence and Risk Factors for Adrenal Crisis in Pediatric-onset Adrenal Insufficiency: A Prospective Study.

CONTEXT: Adrenal crisis (AC) is a life-threatening complication that occurs during follow-up of patients with adrenal insufficiency (AI). No prospective study has thoroughly investigated AC in children with primary and secondary AI. OBJECTIVE: This work aimed to determine the incidence and risk factors for AC in patients with pediatric-onset AI. METHODS: This multicenter, prospective cohort study conducted in Japan enrolled patients diagnosed with AI at age ≤15 years. The incidence of AC was calculated as events per person-year (PY), and risk factors for AC were assessed using Poisson regression multivariable analysis. RESULTS: The study population comprised 349 patients (164 male, 185 female) with a total follow-up of 961 PY. The median age at enrollment was 14.3 years (interquartile range [IQR] 8.5-21.2 years), and the median follow-up was 2.8 years (IQR 2.2-3.3 years). Of these patients, 213 (61%) had primary AI and 136 (39%) had secondary AI. Forty-one AC events occurred in 31 patients during the study period. The calculated incidence of AC was 4.27 per 100 PY (95% CI, 3.15-5.75). Poisson regression analysis identified younger age at enrollment (relative risk [RR] 0.93; 95% CI, 0.89-0.97) and increased number of infections (RR 1.17; 95% CI, 1.07-1.27) as significant risk factors. Female sex (RR 0.99; 95% CI, 0.53-1.86), primary AI (RR 0.65; 95% CI, 0.30-1.41), or equivalent dosage of hydrocortisone per square meter of body area (RR 1.02; 95% CI, 0.96-1.08) was not a significant risk factor. CONCLUSION: A substantial proportion of patients with pediatric-onset AI experience AC. Younger age and an increased number of infections are independent risk factors for developing AC in these patients.

HLA-class II and class I genotypes among Japanese children with Type 1A diabetes and their families.

OBJECTIVE: To determine the HLA-DRB1, DQB1, DPB1, A, C, and B genotypes among Japanese children with autoimmune type 1 diabetes. METHODS: Four hundred and thirty patients who were GADAb and/or IA-2Ab-positive (Type 1A) were recruited from 37 medical centers as part of a nationwide multicenter collaborative study. DNA samples from 83 siblings of the children with Type 1A diabetes and 149 parent-child trios were also analyzed. A case-control study and a transmission disequilibrium test (TDT) were then performed. RESULTS: The susceptible and protective DRB1 and DQB1 alleles and haplotypes were confirmed. DPB1 alleles unique to the Japanese population and those common to multiple ethnic groups were also present. A linkage disequilibrium (LD) analysis showed both susceptible and protective haplotypes. The TDT did not reveal any alleles that were transmitted preferentially from the mother or father to children with Type 1A. Homozygosity for DRB1-09:01-DQB1-03:03 and heterozygosity for DRB1-04:05-DQB1-04:01 and DRB1-08:02-DQB1-03:02 were associated with an extremely high risk of Type 1A. A comparison of children with Type 1A and their parents and siblings suggested a dose effect of susceptible DRB1-DQB1 haplotypes and an effect of protective alleles on immunological pathogenesis. DRB1-09:01 appeared to be strongly associated with an early onset in preschool children with Type 1A diabetes. CONCLUSIONS: This study demonstrated the characteristic association of HLA-class II and class I genes with Type 1A diabetes among Japanese children. A TDT did not reveal the genomic imprinting of HLA-class II and class I genes in Type 1A diabetes.

Usefulness of insulin detemir in Japanese children with type 1 diabetes.

BACKGROUND: This multicenter observational study was conducted to investigate the efficacy and safety of insulin detemir (detemir) for diabetes management in Japanese children and adolescents. METHODS: Data from the Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes database were analyzed. Ninety children (32 boys, 58 girls; mean age, 11.9 ± 3.8 years) who transferred from a neutral protamine Hagedorn insulin or insulin glargine basal-bolus regimen to detemir basal-bolus therapy and who were observed for at least 12 months were identified. Clinical data obtained at 0, 3, 6, and 12 months were analyzed to determine the type of bolus insulin used, number and timing of detemir injections, detemir dose as a proportion of the total insulin dose, hemoglobin A1c (HbA1c), fasting blood glucose (FBG) and frequency of severe hypoglycemia. RESULTS: Twelve months after switching to detemir, the detemir dose represented 39.8% of the total insulin dose, and 37.8% of patients were being treated with twice-daily injections. HbA1c and FBG were significantly reduced from baseline at 3 and 6 months but not at 12 months. Considering the seasonal HbA1c variation in the Japanese population, a separate analysis was performed using data for 65 children (21 boys, 44 girls; mean age, 11.6 ± 2.9 years) who switched to detemir during the winter. Subset analysis showed significant HbA1c reductions from baseline at all specified times. The incidence of severe hypoglycemia during detemir treatment was 4.4 episodes per 100 patient-years. CONCLUSIONS: Detemir is an effective and safe basal insulin for diabetes management in Japanese children and adolescents.

The Boy:Girl Ratio of Children Diagnosed with Growth Hormone Deficiency-Induced Short Stature Is Associated with the Boy:Girl Ratio of Children Visiting Short Stature Clinics.

BACKGROUND: About twice as many boys as girls undergo growth hormone (GH) therapy in GH deficiency (GHD). However, this sex difference may not correctly reflect a real incidence. OBJECTIVES: We analyzed the evidence of a selection bias whereby more boys seek treatment at short stature clinics. SUBJECTS AND METHODS: The present study included 3,902 children who visited 17 short stature clinics with a height SD score of -2 SD or less. The percentage of children who underwent the GH stimulation test was compared between boys and girls, as was the percentage of children ultimately diagnosed with GHD. RESULTS: The children comprised 2,390 boys (61.3%) and 1,512 girls (38.7%), with a boy:girl ratio of 1.58:1. The percentage of children who underwent the GH stimulation test did not differ between boys (45.7%) and girls (49.8%). Among the children who underwent the GH stimulation test, the percentage diagnosed with GHD did not differ significantly between boys (22.0%) and girls (20.1%). The boy:girl ratio of children diagnosed with GHD was 1.59:1. CONCLUSIONS: The boy:girl ratio of children with short stature (1.58:1) did not differ significantly from that of children diagnosed with GHD (1.59:1). These results indicate that the predominance of boys in GHD does not reflect a real incidence, but rather a selection bias whereby a higher proportion of boys with short stature seek treatment at clinics. This difference arises because parents are more concerned about boys' height, and because boys reach adult height at an older age.

Liver transplantation for an infant with neonatal intrahepatic cholestasis caused by citrin deficiency using heterozygote living donor.

NICCD is an autosomal recessive genetic disorder, characterized by cholestasis, coagulopathy, hypoglycemia, fatty liver and multiple amino acidemia. NICCD develops in the neonatal/infantile period and has been reported as a "naturally curable" disease within one yr of life. Recently, we experienced an infantile NICCD who developed progressive liver failure, and required subsequent LT using a heterozygote living donor at eight months of age. Diagnosis of NICCD was established before transplantation, and donor evaluation included mutation in the SLC25A13 gene for exclusion of individuals with citrin deficiency citrullinemia. LDLT, from blood type identical mother using a left lateral segment graft, was performed without serious complication. Plasma amino acid concentration was normalized rapidly, and the patient was discharged 30 days after transplant. During one yr follow up, the recipient has been doing well without additional medication for NICCD. NICCD should be considered in the differential diagnosis as a cause of neonatal/infantile cholestatic disease. LT using a heterozygote living donor is an effective alternative in countries where a deceased donor is not available.

Increasing secular trends in height and obesity in children with type 1 diabetes: JSGIT cohort.

BACKGROUND: Recently, anthropometric indices in children with type 1 diabetes mellitus (T1DM) have begun to change. OBJECTIVE: To examine secular trends in patients' anthropometric indices. SUBJECTS: Japanese children with T1DM from the 1995, 2000, 2008 and 2013 cohorts of The Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes. METHODS: We analysed serum haemoglobin A1c (HbA1c) levels, the incidence of severe hypoglycaemic events, the types and doses of insulin, height standard deviation scores (SDS), body mass index (BMI) percentiles compared with healthy Japanese children and obesity prevalence over time. We also stratified the patients according to glycaemic control levels of <58 mmol/mol (optimal), 58-75 mmol/mol (suboptimal) and ≥75 mmol/mol (high-risk). RESULTS: Data for 513-978 patients from each of the cohorts were analysed. The incidence of severe hypoglycaemic events decreased over time (from 21 to 4.8/100 patient-years), while the proportion of insulin analogue doses increased (14.6% to 98.6%). In addition, patient height SDS (-0.22 to +0.17), BMI percentile (52.1 to 58.7) and obesity prevalence (2.1% to 5.1%) increased. Height SDS increased in all of the glycaemic control subgroups, while BMI percentile and obesity prevalence increased in the suboptimal and high-risk groups. CONCLUSIONS: Since 1995, the average height of children with T1DM has increased in parallel with increasing insulin doses. Clinicians should be aware of increased BMI in these patients and the associated risk of developing cardiovascular disease in the future.

Retrospective study of the renal function using estimated glomerular filtration rate and congenital anomalies of the kidney-urinary tract in pediatric Turner syndrome.

Although Turner syndrome (TS) is frequently associated with congenital anomalies of the kidney-urinary tract (CAKUT), which is a major cause of pediatric chronic kidney disease, renal function in TS is usually considered normal. The present study aimed to analyze the frequency of renal dysfunction and CAKUT in pediatric patients with TS. Our study included 122 patients with TS between the ages of 2 and 18 years from 30 hospitals across Japan. Clinical data related to renal function and CAKUT were retrospectively collected. The estimated glomerular filtration rate (eGFR) was calculated using the serum creatinine-based formula recommended by the Japanese Society for Pediatric Nephrology. An eGFR <90 mL/min/1.73 m2 for two consecutive years was defined as renal dysfunction. Fifteen (13.5%) of 122 patients had CAKUT, and four patients had renal dysfunction (3.2%, 95% confidence interval: 0%-6.7%). Three of the four did not have CAKUT. Of the CAKUT manifestations, horseshoe kidney, renal hypodysplasia, and multicystic dysplastic kidney were seen in nine, two, and one patient, respectively. Eight of the nine patients with horseshoe kidney had a normal renal function; however, the remaining patient with renal hypodysplasia had renal dysfunction. A small percentage of patients with pediatric TS may had an eGFR below 90 mL/min/1.73 m2 which was not necessarily associated with CAKUT.

A case report: primary extragonadal yolk sac tumor of penile shaft in a 2-year-old child.

A 2-year-old boy, who had the chief complaints of penile swelling and pain, was brought to the hospital by his mother. Penile contusion/trauma was suspected and he was admitted the same day to undergo emergency surgery to eliminate hematoma. The surgery revealed that the origin of the bleeding was not trauma but a tumor lesion of the penile shaft. It was histopathologically identified as a yolk sac tumor and no tumorous lesions were found except that in the penis. Therefore the patient was diagnosed as definitely having a yolk sac tumor originating in the penis. The patient received four cycles of cisplatin, etoposide and bleomycin treatment as adjuvant chemotherapy. Although it was impossible to completely resect the tumor, cisplatin, etoposide and bleomycin chemotherapy was effective and a complete response was achieved. We plan to carefully monitor the patient in the future.

(Epi)genetic defects of MKRN3 are rare in Asian patients with central precocious puberty.

We sequenced MKRN3, the major causative gene of central precocious puberty in Western countries, in 24 Japanese or Chinese patients and examined the DNA methylation and copy-number statuses of this gene in 19 patients. We identified no (epi)genetic defects except for one previously reported mutation. These results, together with reports from Korea, indicate that MKRN3 defects are rare in Asian populations. The ethnic differences likely reflect Western country-specific founder mutations and the rarity of de novo mutations.

Clinical practice guidelines for congenital hyperinsulinism.

Congenital hyperinsulinism is a rare condition, and following recent advances in diagnosis and treatment, it was considered necessary to formulate evidence-based clinical practice guidelines reflecting the most recent progress, to guide the practice of neonatologists, pediatric endocrinologists, general pediatricians, and pediatric surgeons. These guidelines cover a range of aspects, including general features of congenital hyperinsulinism, diagnostic criteria and tools for diagnosis, first- and second-line medical treatment, criteria for and details of surgical treatment, and future perspectives. These guidelines were generated as a collaborative effort between The Japanese Society for Pediatric Endocrinology and The Japanese Society of Pediatric Surgeons, and followed the official procedures of guideline generation to identify important clinical questions, perform a systematic literature review (April 2016), assess the evidence level of each paper, formulate the guidelines, and obtain public comments.

Cytochrome P450 oxidoreductase gene mutations and Antley-Bixler syndrome with abnormal genitalia and/or impaired steroidogenesis: molecular and clinical studies in 10 patients.

We report on molecular and clinical findings in 10 Japanese patients (four males and six females) from eight families (two pairs of siblings and six isolated cases) with Antley-Bixler syndrome accompanied by abnormal genitalia and/or impaired steroidogenesis. Direct sequencing was performed for all the 15 exons of cytochrome P450 oxidoreductase gene (POR), showing two missense mutations (R457H and Y578C), a 24-bp deletion mutation resulting in loss of nine amino acids and creation of one amino acid (L612_W620delinsR), a single bp insertion mutation leading to frameshift (I444fsX449), and a silent mutation (G5G). R457H has previously been shown to be a pathologic mutation, and computerized modeling analyses indicated that the 15A>G for G5G could disturb an exonic splicing enhancer motif, and the remaining three mutations should affect protein conformations. Six patients were compound heterozygotes, and three patients were R457H homozygotes; no mutation was identified on one allele of the remaining one patient. Clinical findings included various degrees of skeletal features, such as brachycephaly, radiohumeral synostosis, and digital joint contractures in patients of both sexes, normal-to-poor masculinization during fetal and pubertal periods in male patients, virilization during fetal life and poor pubertal development without worsening of virilization in female patients, and relatively large height gain and delayed bone age from the pubertal period in patients of both sexes, together with maternal virilization during pregnancy. Blood cholesterol was grossly normal, and endocrine studies revealed defective CYP17A1 and CYP21A2 activities. The results suggest that Antley-Bixler syndrome with abnormal genitalia and/or impaired steroidogenesis is caused by POR mutations, and that clinical features are variable and primarily explained by impaired activities of POR-dependent CYP51A1, CYP17A1, CYP21A2, and CYP19A1.

SLC25A13 gene analysis in citrin deficiency: sixteen novel mutations in East Asian patients, and the mutation distribution in a large pediatric cohort in China.

BACKGROUND: The human SLC25A13 gene encodes citrin, the liver-type mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), and SLC25A13 mutations cause citrin deficiency (CD), a disease entity that encompasses different age-dependant clinical phenotypes such as Adult-onset Citrullinemia Type II (CTLN2) and Neonatal Intrahepatic Cholestasis caused by Citrin Deficiency (NICCD). The analyses of SLC25A13 gene and its protein/mRNA products remain reliable tools for the definitive diagnoses of CD patients, and so far, the SLC25A13 mutation spectrum in Chinese CD patients has not been well-characterized yet. METHODS AND RESULTS: By means of direct DNA sequencing, cDNA cloning and SNP analyses, 16 novel pathogenic mutations, including 9 missense, 4 nonsense, 1 splice-site, 1 deletion and 1 large transposal insertion IVS4ins6kb (GenBank accession number KF425758), were identified in CTLN2 or NICCD patients from China, Japan and Malaysia, respectively, making the SLC25A13 variations worldwide reach the total number of 81. A large NICCD cohort of 116 Chinese cases was also established, and the 4 high-frequency mutations contributed a much larger proportion of the mutated alleles in the patients from south China than in those from the north (χ(2) = 14.93, P<0.01), with the latitude of 30°N as the geographic dividing line in mainland China. CONCLUSIONS: This paper further enriched the SLC25A13 variation spectrum worldwide, and formed a substantial contribution to the in-depth understanding of the genotypic feature of Chinese CD patients.

Proximal promoter of the cytochrome P450 oxidoreductase gene: identification of microdeletions involving the untranslated exon 1 and critical function of the SP1 binding sites.

CONTEXT: POR (cytochrome P450 oxidoreductase) is a ubiquitously expressed gene encoding an electron donor to all microsomal P450 enzymes and several non-P450 enzymes. POR mutations cause an autosomal recessive disorder characterized by skeletal dysplasia, adrenal dysfunction, and disorders of sex development. Although recent studies have indicated the presence of a CpG-rich region characteristic of housekeeping genes around the untranslated exon 1 (exon 1U) and a tropic effect of thyroid hormone on POR expression via thyroid hormone receptor-ß, detailed regulatory mechanisms for the POR expression remain to be clarified. OBJECTIVE: Our objective was to report a pivotal element of the proximal promoter of POR. RESULTS: We first studied three patients (cases 1-3) with POR deficiency due to compound heterozygosity with an p.R457H mutation and transcription failure of an apparently normal allele, by oligoarray comparative genomic hybridization and serial direct sequencing of the deletion fusion points. Consequently, a 2,487-bp microdeletion involving exon 1U was identified in case 1 and an identical 49,604-bp deletion involving exon 1U and exon 1 was found in cases 2 and 3. We next analyzed the 2,487-bp region commonly deleted in cases 1-3 by in silico analysis, DNA binding analysis, luciferase assays, and methylation analysis. The results showed a critical function of the evolutionally conserved SP1 binding sites just upstream of exon 1U, especially the binding site at the position -26/-17, in the transcription of POR. CONCLUSIONS: The results suggest that the SP1 binding sites constitute an essential element of the POR proximal promoter.

Aromatase excess syndrome: identification of cryptic duplications and deletions leading to gain of function of CYP19A1 and assessment of phenotypic determinants.

CONTEXT: Aromatase excess syndrome (AEXS) is a rare autosomal dominant disorder characterized by gynecomastia. Although cryptic inversions leading to abnormal fusions between CYP19A1 encoding aromatase and its neighboring genes have been identified in a few patients, the molecular basis remains largely unknown. OBJECTIVE: The objective of the study was to examine the genetic causes and phenotypic determinants in AEXS. PATIENTS: Eighteen affected males from six families participated in the study. RESULTS: We identified three types of heterozygous genomic rearrangements, i.e. a 79,156-bp tandem duplication involving seven of 11 noncoding CYP19A1 exons 1, a 211,631-bp deletion involving exons 2-43 of DMXL2 and exons 5-10 of GLDN, and a 165,901-bp deletion involving exons 2-43 of DMXL2. The duplicated exon 1 functioned as transcription start sites, and the two types of deletions produced the same chimeric mRNA consisting of DMXL2 exon 1 and CYP19A1 coding exons. The DMXL2 exon 1 harbored a translation start codon, and the DMXL2/CYP19A1 chimeric mRNA was identified in only 2-5% of CYP19A1-positive transcripts. This was in contrast to the inversion-mediated chimeric mRNA that had no coding sequence on the fused exon 1 and accounted for greater than 80% of CYP19A1-positive transcripts. CYP19A1 was expressed in a limited number of tissues, whereas its neighboring genes involved in the chimeric mRNA formation were expressed widely. CONCLUSIONS: This study provides novel mechanisms leading to gain of function of CYP19A1. Furthermore, it appears that clinical severity of AEXS is primarily determined by the tissue expression pattern of relevant genes and by the structural property of promoter-associated exons of chimeric mRNA.