Endocrine phenotype of 91 probands screened for 9 genes.

<p>Endocrine phenotype of 91 probands screened for 9 genes.</p

Transactivation assays of R84X and V75I LHX4 using <i>POU1F1</i>(<i>PIT1</i>) and<i>αGSU</i> reporter.

<p><i>A and B</i>: COS7 cells were cotransfected with the pRL-CMV internal control vector, indicated amount (nanograms) of the effector plasmids, and the <i>POU1F1</i>(A) or<i>αGSU</i> (B) reporter. The data are the mean ± s.e.m. of at least three independent experiments performed in triplicate transfections. The white, black, red, and blue bars indicate the data of the empty expression vectors, expression vectors with wild type (WT) LHX4, expression vectors with R84X LHX4, and V75I LHX4, respectively. R84X LHX4 exhibited markedly reduced transactivation, whereas V75I LHX4 retained partial activity. The two mutants did not exhibit any dominant negative effect. The data are mean ± SEM of at least three independent experiments performed in triplicate transfections. <i>C and D</i>: GH3 cells were cotransfected with the pRL-CMV internal control vector, indicated amount (nanograms) of the effector plasmids, and the <i>POU1F1</i>(C) or<i>αGSU</i> (D) reporter.</p

Endocrinological findings in Propositus of pedigree 2.

<p>The conversion factors to the SI unit are as follows: GH 1.0 (μg/liter), TSH 1.0 (mIU/liter), LH 1.0 (IU/liter), FSH 1.0 (IU/liter), testosterone, 0.035 (nmol/liter), prolactin 1.0 (μg/liter), ACTH 0.22 (pmol/liter), cortisol 27.59 (nmol/liter), IGF-I 0.131 (nmol/liter), free T4 12.87 (pmol/liter), and free T3, 1.54 (pmol/liter).</p>a<p>Reference data of pre-pubertal Japanese boys (younger than 10 years) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Ito1" target="_blank">[22]</a></p>b<p>Reference data of UK children (younger than 10 years) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Crofton2" target="_blank">[23]</a></p>c<p>Reference data of Japanese boys (younger than 1 years old) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Fujieda1" target="_blank">[24]</a></p>d<p>Reference data of Japanese boys (7-9 years old) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Fujieda1" target="_blank">[24]</a></p

Endocrinological findings in Propositus of pedigree 1.

<p>The conversion factors to the SI unit are as follows: GH 1.0 (µg/liter), LH 1.0 (IU/liter), FSH 1.0 (IU/liter), TSH 1.0 (mIU/liter), prolactin 1.0 (µg/liter), ACTH 0.22 (pmol/liter), cortisol 27.59 (nmol/liter), IGF-I 0.131 (nmol/liter), free T4 12.87 (pmol/liter), free T3, 1.54 (pmol/liter), and estradiol 3.671 (pmol/liter).</p>a<p>Reference data of pre-pubertal Japanese girls <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Ito1" target="_blank">[22]</a></p>b<p>Reference data of pubertal (Tanner 2–3) Japanese girls <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Ito1" target="_blank">[22]</a></p>c<p>Reference data of UK children (younger than 10 years) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Crofton2" target="_blank">[23]</a></p>d<p>Reference data of UK children (older than 10 years) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Crofton2" target="_blank">[23]</a></p>e<p>Reference data of Japanese girls (5–7 years old) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Fujieda1" target="_blank">[24]</a></p>f<p>Reference data of Japanese girls (15–17 years old) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Fujieda1" target="_blank">[24]</a></p>g<p>Reference data of Japanese girls (15 years old) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046008#pone.0046008-Japan1" target="_blank">[25]</a></p

Results of MR scans of probands screened for 9 genes.

<p>Results of MR scans of probands screened for 9 genes.</p

Functional characterization of two mutant LHX4.

<p><i>A</i>, Protein expression level of myc-tagged WT and two LHX4 mutants was assessed by western blot using a monoclonal anti-myc antibody. The expression of V75I LHX4 was comparable to that of WT, whereas R84X LHX4 was not detected. Tubulin was used as a control. <i>B</i>, Subcellular localization analysis. For subcellular localization analyses, we visualized and photographed COS7 cells transfected with GFP-tagged LHX4 using a Leica TCS-SP5 laser scanning confocal microscope, after mounting the cells in Vectashield-DAPI solution. The WT and V75I LHX4 are localized to the nucleus. <i>C</i>, EMSA experiments. WT LHX4 showed specific binding to the elements, which was competed by excess amount of (200 times) cold competitors. The V75I LHX4, which has an intact HD, bound with similar or slightly high efficiency to the WT LHX4.</p

The pedigrees of the affected families.

<p><i>A–C</i>, Pedigrees of families 1–3. Arrow indicates the propositus. ND: not determined.</p

Endocrinological findings (baseline) in Family members of pedigree 1.

a<p>Follicular phase</p

Individual Variation of the Genetic Response to Bisphenol A in Human Foreskin Fibroblast Cells Derived from Cryptorchidism and Hypospadias Patients

<div><h3>Background/Purpose</h3><p>We hypothesized that polymorphic differences among individuals might cause variations in the effect that environmental endocrine disruptors (EEDs) have on male genital malformations (MGMs). In this study, individual variation in the genetic response to low-dose bisphenol A (BPA) was investigated in human foreskin fibroblast cells (hFFCs) derived from child cryptorchidism (CO) and hypospadias (HS) patients.</p> <h3>Methodology/Principal Findings</h3><p>hFFCs were collected from control children without MGMs (<em>n</em> = 5) and child CO and HS patients (<em>n</em> = 8 and 21, respectively). BPA exposure (10 nM) was found to inhibit matrix metalloproteinase-11 (<em>MMP11</em>) expression in the HS group (0.74-fold, <em>P</em> = 0.0034) but not in the control group (0.93-fold, <em>P</em> = 0.84) and CO group (0.94-fold, <em>P</em> = 0.70). Significantly lower levels of <em>MMP11</em> expression were observed in the HS group compared with the control group (0.80-fold, <em>P</em> = 0.0088) and CO group (0.79-fold, <em>P</em> = 0.039) in response to 10 nM BPA. The effect of single-nucleotide polymorphism <em>rs5000770</em> (G>A), located within the aryl hydrocarbon receptor nuclear translocator 2 (<em>ARNT2</em>) locus, on individual sensitivity to low-dose BPA was investigated in the HS group. A significant difference in neurotensin receptor 1 (<em>NTSR1</em>) expression in response to 10 nM BPA was observed between AA and AG/GG groups (<em>n</em> = 6 and 15, respectively. <em>P</em> = 0.031). However, no significant difference in <em>ARNT2</em> expression was observed (<em>P</em> = 0.18).</p> <h3>Conclusions/Significance</h3><p>This study advances our understanding of the specificity of low-dose BPA effects on human reproductive health. Our results suggest that genetic variability among individuals affects susceptibility to the effects of EEDs exposure as a potential cause of HS.</p> </div

Identification of Novel Low-Dose Bisphenol A Targets in Human Foreskin Fibroblast Cells Derived from Hypospadias Patients

<div><h3>Background/Purpose</h3><p>The effect of low-dose bisphenol A (BPA) exposure on human reproductive health is still controversial. To better understand the molecular basis of the effect of BPA on human reproductive health, a genome-wide screen was performed using human foreskin fibroblast cells (hFFCs) derived from child hypospadias (HS) patients to identify novel targets of low-dose BPA exposure.</p> <h3>Methodology/Principal Findings</h3><p>Gene expression profiles of hFFCs were measured after exposure to 10 nM BPA, 0.01 nM 17β-estradiol (E2) or 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 h. Differentially expressed genes were identified using an unpaired Student's t test with <em>P</em> value cut off at 0.05 and fold change of more than 1.2. These genes were selected for network generation and pathway analysis using Ingenuity Pathways Analysis, Pathway Express and KegArray. Seventy-one genes (42 downregulated and 29 upregulated) were identified as significantly differentially expressed in response to BPA, among which 43 genes were found to be affected exclusively by BPA compared with E2 and TCDD. Of particular interest, real-time PCR analysis revealed that the expression of matrix metallopeptidase 11 (MMP11), a well-known effector of development and normal physiology, was found to be inhibited by BPA (0.47-fold and 0.37-fold at 10 nM and 100 nM, respectively). Furthermore, study of hFFCs derived from HS and cryptorchidism (CO) patients (<em>n</em> = 23 and 11, respectively) indicated that MMP11 expression was significantly lower in the HS group than in the CO group (0.25-fold, <em>P</em> = 0.0027).</p> <h3>Conclusions/Significance</h3><p>This present study suggests that an involvement of BPA in the etiology of HS might be associated with the downregulation of MMP11. Further study to elucidate the function of the novel target genes identified in this study during genital tubercle development might increase our knowledge of the effects of low-dose BPA exposure on human reproductive health.</p> </div