Lutz Wünsch

Patients with disorders of sex development (DSD) at risk of gonadal tumour development: management based on laparoscopic biopsy and molecular diagnosis.

Study Type – Therapy (case series)

Partial deletion of DMRT1 causes 46,XY ovotesticular disorder of sexual development

OBJECTIVE Ovotesticular disorder of sexual development (DSD) is an unusual form of DSD, characterized by the coexistence of testicular and ovarian tissue in the same individual. In a subset of patients, ovotesticular DSD is caused by 46,XX/46,XY chimerism or mosaicism. To date, only a few monogenetic causes are known to be associated with XX and XY ovotesticular DSD. DESIGN AND METHODS Clinical, hormonal, and histopathological data, and results of high-resolution array-comparative genomic hybridization (CGH) were obtained from a female patient with 46,XY ovotesticular DSD with testicular tissue on one side and an ovary harboring germ cells on the other. Results obtained by array-CGH were confirmed by RT-quantitative PCR. RESULTS We detected a deletion of ∼35 kb affecting exons 3 and 4 of the DMRT1 gene in a female patient with 46,XY ovotesticular DSD. To the best of our knowledge, this is the smallest deletion affecting DMRT1 presented to this point in time. CONCLUSIONS We suggest that haploinsufficiency of DMRT1 is sufficient for both XY gonadal dysgenesis and XY ovotesticular DSD. Furthermore, array-CGH is a very useful tool in the molecular diagnosis of DSD.

Management of disorders of sex development

The medical term disorders of sex development (DSDs) is used to describe individuals with an atypical composition of chromosomal, gonadal and phenotypic sex, which leads to differences in the development of the urogenital tract and reproductive system. A variety of genetic factors have been identified that affect sex development during gonadal differentiation or in specific disorders associated with altered androgen biosynthesis or action. The diagnosis of DSDs in individuals and the subsequent management of patients and their families requires a targeted and structured approach, involving a multidisciplinary team with effective communication between the disciplines. This approach includes distinct clinical, imaging, laboratory and genetic evaluations of patients with DSDs. Although treatment of patients with DSDs can include endocrine and surgical options, many patients have concerns that arise from past incorrect treatments that were founded on the traditional binary concept of the sexes. To dispel these concerns, it is necessary to create centres of expertise for DSDs that include physicians, surgeons, psychologists and specialists in diagnostic procedures to manage patients and their families. Additionally, the inclusion of trained peer support in the multidisciplinary DSD team seems to be integral to the supportive management of patients with DSDs. Most importantly, dealing with DSDs requires acceptance of the fact that deviation from the traditional definitions of gender is not necessarily pathologic.

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

BackgroundTo better understand the molecular programs of normal and abnormal genital development, clear-cut definition of androgen-dependent gene expression patterns, without the influence of genotype (46, XX vs. 46, XY), is warranted. Previously, we have identified global gene expression profiles in genital-derived fibroblasts that differ between 46, XY males and 46, XY females with complete androgen insensitivity syndrome (CAIS) due to inactivating mutations of the androgen receptor (AR). While these differences could be due to cell autonomous changes in gene expression induced by androgen programming, recent work suggests they could also be influenced by the location from which the fibroblasts were harvested (topology). To minimize the influence of topology, we compared gene expression patterns of fibroblasts derived from identical urogenital anlagen: the scrotum in normally virilized 46, XY males and the labia majora from completely feminized 46, XY individuals with CAIS.Results612 transcripts representing 440 unique genes differed significantly in expression levels between scrotum and CAIS labia majora, suggesting the effects of androgen programming. While some genes coincided with those we had identified previously (TBX3, IGFBP5, EGFR, CSPG2), a significant number did not, implying that topology had influenced gene expression in our previous experiments. Supervised clustering of gene expression data derived from a large set of fibroblast cultures from individuals with partial AIS revealed that the new, topology controlled data set better classified the specimens.ConclusionInactivating mutations of the AR, in themselves, appear to induce lasting changes in gene expression in cultured fibroblasts, independent of topology and genotype. Genes identified are likely to be relevant candidates to decipher androgen-dependent normal and abnormal genital development.

Tissue-specific transcription profiles of sex steroid biosynthesis enzymes and the androgen receptor

Abstract17β-hydroxysteroid dehydrogenase (17β-HSD) and 5α-reductase isoenzymes play a crucial role in the formation and metabolism of sex steroids. Not only the key androgens testosterone and dihydrotestosterone but also their precursors are potent activators of the androgen receptor and are, therefore, likely to act as determinants of male sexual differentiation and maturation in a differentially regulated way. The aim of the present study was to relatively quantify the expression of the mRNA of 17β-HSD isoenzymes, namely, type 1, 2, 3, 4, 5, 7, and 10, together with the 5α-reductase type 1 and 2, and the androgen receptor in normal human males and females. RNA was isolated from peripheral blood cells of both sexes and from genital skin fibroblasts (GSFs) of two different localizations (foreskin and scrotal skin) obtained from phenotypically normal males. mRNA expression was semi-quantified by quantitative reverse-transcriptase polymerase chain reaction with the LightCycler Instrument (Roche). The examined enzymes show statistically significant differences in their transcription pattern between the blood and the GSF RNA samples. Within the GSF samples, there are also significant variations between the two examined localizations in the transcription of 17β-HSD type 1, 2, 4, and 5 as well as for the androgen receptor. We found large interindividual variation of enzyme transcription patterns in all investigated tissues. In peripheral blood cells, no sex-specific differences were seen. We conclude that sex steroid enzymes are expressed not only in genital primary target tissues but also in peripheral blood. The expression in different target tissues may contribute to both the individual sexual and tissue-specific phenotype in humans.

Selective down-regulation of the α6-integrin subunit in melanocytes by UVB light

Abstract:  In vivo, melanocytes bind to laminin (LM) molecules of the basement membrane (BM) via the integrins α3β1 and α6β1, and they adhere to neighbouring keratinocytes via E‐cadherin. Only few studies have addressed the impact of ultraviolet (UV) light on the interaction of melanocytes with their microenvironment. In this report, we examined the influence of UVB irradiation on the expression of the most important melanocyte‐adhesion molecules (E‐, N‐cadherin, α2‐, α3‐, α5‐, α6‐, αV‐, β1‐, β3‐integrins and ICAM‐1) in vitro by flow cytometry. We were able to demonstrate that the α6‐integrin subunit is selectively and reversibly down‐regulated by UVB in a dwzm 150ose‐dependent manner. In comparison, keratinocytes lacked UVB‐inducible alterations in the expression of α6‐integrin. In the presence of LM‐1, the UVB‐induced down‐regulation of α6‐integrin in melanocytes was significantly reduced. Moreover, LM‐1 increased the resistance of melanocytes to UVB‐induced cell death, as measured by annexinV‐binding analysis. This effect was reversed by preincubation with an α6‐integrin‐blocking antibody. By immunofluorescence, we could demonstrate that UVB leads to a dose‐dependent internalization of α6‐integrin, providing an obvious explanation for the down‐regulation on the outer cell surface observed by flow cytometry. We suggest that adhesion to LM‐1 through α6‐integrin represents a protective mechanism for melanocytes to withstand UVB damage. Through α6‐integrin internalization, sunburns might alter the interaction between melanocytes and the BM, resulting in apoptosis induced by loss of anchorage (anoikis). Repeated sunburns may then lead to the selection of a population of melanocytes which are capable of anchorage‐independent survival, culminating in solar nevogenesis and melanoma development.

Requirements for a multicentric multidisciplinary registry on patients with disorders of sex development.

Disorders of Sexual Development (DSDs) are a group of rare to very rare congenital anomalies of the genito-urinary tract of genetic and endocrine causes. Recently, an international database I-DSD was successfully implemented to register patients with DSD and to provide the basis for epidemiologic, genetic, and clinical research. This tool needs to be adjusted and supplemented with additional modules in order to better assess the anatomical basis of DSD as well as to monitor risk factors such as gonadal histology. A proposal for the additional information to be obtained is discussed.

Novel Insights into 46,XY Disorders of Sex Development due to NR5A1 Gene Mutation

The differential diagnosis of 46,XY disorders of sex development (DSD) is based on the distinction between forms of gonadal dysgenesis and disorders of androgen biosynthesis and action. However, clinical and endocrine evaluations are often not conclusive. Here, we describe an adolescent female with hirsutism and hyperandrogenization at puberty. Her karyotype was 46,XY, and clinical investigation demonstrated clitoromegaly, but no uterine remnants were detected. Histology of the gonads revealed a testicular structure with a Sertoli-cell-only pattern. Endocrine evaluation showed hypergonadotropic hypogonadism, and the Sertoli cell markers inhibin B and anti-Müllerian hormone were also low. Several molecular genetic studies were initiated. While analyses of the androgen receptor gene, the SRD5A2 gene and HSD17B3 gene were uninformative, a novel p.L230R mutation was found in the NR5A1 gene. A mutant construct proved a severe dysfunction of this variant in functional analysis after recreation and transfection into HeLa cells. We conclude that the NR5A1 p.L230R mutation most likely leads to a spatial and time-dependent Leydig cell and Sertoli cell dysfunction during development not causing the classical gonadal dysgenesis phenotype. This case demonstrates that the current classification should be updated to encompass the overlapping phenotypes of some genetic conditions within 46,XY DSD.

New NR5A1 mutations and phenotypic variations of gonadal dysgenesis

Mutations in NR5A1 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 NR5A1 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 TBX2 as a possible modifier of gonadal development in patients with NR5A1 mutations.

Perineal ultrasound offers useful information in girls with congenital adrenal hyperplasia

A variable spectrum of urogenital malformations exists in girls with congenital adrenal hyperplasia (CAH). The vagina may enter the urethra at a variable level, and relations to the sphincter complex vary accordingly. Furthermore, an enlarged clitoris and variations in the bladder sphincter anatomy can be found. Endoscopy, genitography or magnetic resonance imaging (MRI) are commonly used for the assessment of these anomalies, and to provide information for counselling and treatment. When surgery is planned, introitoplasty cosmetical reduction of the clitoris and labioplasty are discussed with the families; introitoplasty is the most demanding aspect. In order to plan the most appropriate surgical approach, the entrance level of the vagina into the urethra and its relation to the bladder sphincter must be known. Thus, imaging has an important role in CAH. The imaging techniques mentioned above require sedation, anaesthesia or involve ionizing radiation of the gonads and, thus, are relatively invasive. It would therefore be highly desirable to have a minimally invasive and accurate technique that provides images of the individual anatomic situation. The present paper describes experience with perineal ultrasound in the initial imaging evaluation of girls with CAH. Ultrasound findings were compared to the results of endoscopy that was performed before surgery. From 2006 to 2012, 11 girls had perineal ultrasound and endoscopy. Measurements of clinical relevance for introitoplasty were: the length of the urogenital sinus, the distance to the vaginal opening into the urogenital sinus, and the length of the bladder neck. This retrospective analysis showed that the entrance point of the vagina into the urogenital sinus could be identified in 10 of 11 girls. In some cases, the correlation of endoscopic and ultrasound data showed a correlation between endoscopic and sonographic findings. The length of the bladder neck and the length of the urogenital sinus could be measured by ultrasound in 10 of 11 girls, and were subsequently confirmed by endoscopy. This showed, for the first time, that perineal ultrasound could provide the information required for surgical correction of the urogenital sinus anomaly in CAH. Advantages of these techniques are the minimal invasiveness and wide availability. Because long-term problems are not uncommon, perineal ultrasound may also be of value during follow-up. Widespread use of this technique has the potential to reduce costs and morbidity associated with endoscopy and genitography.