New <i>NR5A1</i> mutations and phenotypic variations of gonadal dysgenesis

<div><p>Mutations in <i>NR5A1</i> have been reported as a frequent cause of 46,XY disorders of sex development (DSD) associated to a broad phenotypic spectrum ranging from infertility, ambiguous genitalia, anorchia to gonadal dygenesis and female genitalia. Here we present the clinical follow up of four 46,XY DSD patients with three novel heterozygous mutations in the <i>NR5A1</i> gene leading to a p.T40P missense mutation and a p.¹⁸DKVSG²²del nonframeshift deletion in the DNA-binding domain and a familiar p.Y211Tfs*83 frameshift mutation. Functional analysis of the missense and nonframeshift mutation revealed a deleterious character with loss of DNA-binding and transactivation capacity. Both, the mutations in the DNA-binding domain, as well as the familiar frameshift mutation are associated with highly variable endocrine values and phenotypic appearance. Phenotypes vary from males with spontaneous puberty, substantial testosterone production and possible fertility to females with and without Müllerian structures and primary amenorrhea. Exome sequencing of the sibling’s family revealed <i>TBX2</i> as a possible modifier of gonadal development in patients with <i>NR5A1</i> mutations.</p></div

Transactivation of the human <i>AMH</i>- and <i>STAR</i>-promoter.

<p>The transactivation capacity of mutant NR5A1-T40P and NR5A1-¹⁸DKVSG²²del were compared to wt-NR5A1 using human <i>AMH-</i> (A) and <i>STAR</i>-promoter (B) containing reporter genes. The empty pCMV-Myc vector represents background activity. Wild type (WT) activity was set 100%. RLU = relative luciferase activity. Error bars represent standard deviations, ***P = <0.001, t-test comparison of WT and mutant. C: Immunoblot detection of myc-tagged NR5A1 proteins. Equal loadings were verified by detection of total proteins using the TGX Stain Free system (BioRad). D: statistical analysis of 3 western blots of three different experiments showing an approximately equal protein accumulation of NR5A1-WT and mutant ¹⁸DKVSG²²del, while mutant T40P showed an 2–3 fold enhanced accumulation. Images were quantified and normalized to total protein using Image Lab 5.2.1 (BioRad).</p

Hormonal and clinical data of patients.

<p>Hormonal and clinical data of patients.</p

Electrophoretic mobility shift assay of NR5A1 mutants.

<p>A) DNA-binding capability of mutant NR5A1-T40P and NR5A1-¹⁸DKVSG²²del were compared to wt-NR5A1 using a SF-1 responsive element of the mouse <i>Amh</i> promoter. Nuclear extracts containing WT-SF1 shifted the labelled probe (arrow). A 200 fold excess of unlabelled competitor abolished the shifted signal, while a 200 fold excess of unlabelled mutated competitor rescues the shifted signal. Both mutant proteins have lost their DNA-binding capability. B) Aliquots of the nuclear extracts used in EMSA were separated by SDS gel electrophoresis, transferred to a nitrocellulose membrane and stained with Ponceau S and anti-myc antibodies to verify equal SF-1 protein concentrations.</p

Electropherogram of <i>NR5A1</i> mutations.

<p>Electropherogram of the heterozygous c.118A>C mutation in patient 1 (A), the nonframeshift deletion c.51_65delGGACAAGGTGTCCGG in patient 2 (B), the frame-shift deletion c.630_636delGTACGGC in patient 3 (C) and the pedigree of the family of patient 3 and 4 (D). Circles denote phenotypic females, squares denote phenotypic males. Filled squares and circles correspond to a DSD condition, dots to a carrier status.</p

Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome-4

<p><b>Copyright information:</b></p><p>Taken from "Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome"</p><p>http://www.biomedcentral.com/1471-2164/8/376</p><p>BMC Genomics 2007;8():376-376.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2212662.</p><p></p>ual transcripts are grouped by hierarchical cluster analysis and are displayed in rows while experiments are represented in columns. Expression values per gene are centered by the mean logred/green normalized ratio. Increasing red intensity corresponds to higher relative transcript levels compared to the mean expression level across all 15 array experiments. Increasing green intensity corresponds to relatively decreased transcript levels compared to the mean. On the right side, examples of individual genes (gene symbols according to S.O.U.R.C.E. []) discussed in the paper or falling into the biological processes and cellular pathways detected by PANTHER are displayed. Detailed data on figure 1 is available in additional files , , ,

Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome-2

<p><b>Copyright information:</b></p><p>Taken from "Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome"</p><p>http://www.biomedcentral.com/1471-2164/8/376</p><p>BMC Genomics 2007;8():376-376.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2212662.</p><p></p>on (B). In each of the two clusters A and B, the microarray experiments used to generate the underlying list of significant genes by SAM were removed before clustering resulting 57 remaining experiments in (A) and 58 in (B), respectively. The color code is the same as in Fig. 2. Based on the previous gene set, several highly virilized AIS 2 patients and normal scrotal fibroblasts were incorrectly classified female

Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome-3

<p><b>Copyright information:</b></p><p>Taken from "Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome"</p><p>http://www.biomedcentral.com/1471-2164/8/376</p><p>BMC Genomics 2007;8():376-376.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2212662.</p><p></p>normal scrotal cell lines. Semi-quantitative RT-PCR was performed on four samples of normal male scrotal fibroblasts (S4, S5, S8, S9), as well as on four samples of labia majora derived from CAIS patients (ARD402, ARD411, ARD682, ARD1097). Differences in expression levels between different cell lines were calculated according to the ΔΔ-CT method [19]. The y-axis represents the ratios of expression levels of CAIS labia majora fibroblasts divided by those from scrotal fibroblasts

Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome-0

<p><b>Copyright information:</b></p><p>Taken from "Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome"</p><p>http://www.biomedcentral.com/1471-2164/8/376</p><p>BMC Genomics 2007;8():376-376.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2212662.</p><p></p>ual transcripts are grouped by hierarchical cluster analysis and are displayed in rows while experiments are represented in columns. Expression values per gene are centered by the mean logred/green normalized ratio. Increasing red intensity corresponds to higher relative transcript levels compared to the mean expression level across all 15 array experiments. Increasing green intensity corresponds to relatively decreased transcript levels compared to the mean. On the right side, examples of individual genes (gene symbols according to S.O.U.R.C.E. []) discussed in the paper or falling into the biological processes and cellular pathways detected by PANTHER are displayed. Detailed data on figure 1 is available in additional files , , ,

Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome-1

<p><b>Copyright information:</b></p><p>Taken from "Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome"</p><p>http://www.biomedcentral.com/1471-2164/8/376</p><p>BMC Genomics 2007;8():376-376.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2212662.</p><p></p>interpretable data across the experiments whose expression levels were at least 2-fold different from the mean expression across all samples in at least 5 microarrays. (A) The cluster dendrogram demonstrating the degree of relatedness (Pearson correlation) between the expression patterns of the 259 genes in the cultured fibroblast samples. The length of the arms of the dendrogram reflects the degree of correlation between the samples. Samples are color coded to reflect the localization of the biopsy and the degree of external genital virilization according to a grading scheme developed by Sinnecker et al. [10]. The grey bar below indicates whether a sample was derived from dataset 1, 2 or 3. ''L.n.d.'' signifies that the biopsy localization was not accurately documented. Italics indicate a sample with a 46, XX karyotype. (B) Schematic depiction of the external genitalia phenotype of the cases from which the fibroblast cultures were derived using color coding that corresponds to the degree of genital ambiguity and the location of the biopsy. Color coding corresponds to the bar below the dendrogram in (A). (C) Cluster of AR-dependent transcripts that are highly expressed in the left ''male'' major branch of the cluster that are expressed at significantly lower levels in the ''female'' branch of the cluster on the right. TBX3, previously reported in ulnar mammary syndrome and IGF2, previously reported as being down-regulated in CAIS [20] are shown in this cluster. (D) Cluster of AR-dependent transcripts that are expressed at significantly lower levels in the lefthand ''male'' branch of the cluster and at higher levels in the righthand ''female'' branch. These include many extracellular matrix genes such as proteoglycan testican, versican, and fibrillin 1. Detailed data on figure 2 is available in additional files , , ,