Mechanisms in disorders of sex development

Sammanfattning: Is it a boy or a girl? This is usually the first question that parents have when their baby is born. Sometimes it is not possible to give an immediate answer. This is the case when the newborn presents ambiguous external genitalia and an immediate sex assignment is not possible. This situation represents the most typical and dramatic presentation of a disorder of sex development (DSD). Other DSDs can manifest later in life, for example in a girl with primary amenorrhea, who finds out that she has a 46,XY karyotype and will never be fertile. The overall aim of this thesis was to identify mechanisms in DSD, in order to better understand normal and atypical sex development, and furthermore to offer better diagnostics and genetic counselling to patients with DSD and their families. Congenital adrenal hyperplasia (CAH) due to CYP21A2 deficiency is the single most common cause of ambiguous external genitalia in the newborn. The wide spectrum of clinical manifestations ranges from prenatal virilisation in XX girls and salt-wasting in the neonatal period to precocious pubarche and late-onset hyperandrogenic symptoms during adulthood, depending on the CYP21A2 genotype. By in vitro expression of CYP21A2 we have evaluated the residual enzyme activities of four mutant enzymes that carry novel or rare missense mutations identified in patients with CAH. All mutants had a residual activity below 1%, and are thus associated with severe enzyme deficiency. Therefore, these mutations are predicted to cause classic CAH if found in trans with other severe mutations (Paper I). Mutations in the androgen receptor (AR) gene cause androgen insensitivity syndrome (AIS). Patients with completely female external genitalia are classified as having complete AIS (CAIS). However, some of these patients have signs of internal male genital differentiation due to missense mutations that show a low degree of residual function. We studied the expression of two isoforms of the AR in two CAIS patients in relation to the development of male internal genital structures. One patient had a mutation (L7fsX33) that affects only the full-length AR-B form of the AR, whereas the other had a nonsense mutation (Q733X) affecting both isoforms, as shown by Western blot analysis of proteins from gonadal and genital skin fibroblasts. No signs of Wolffian duct development were present in any of the patients, indicating that the AR-A form is not sufficient for Wolffian duct maintenance and differentiation (Paper II). A genome wide investigation by high resolution BAC array CGH (Comparative Genomic Hybridization) was used to identify gene dosage imbalances in 10 patients with female external genitalia due to XY gonadal dysgenesis (GD). We identified and characterised a 637 kb duplication at Xp21 containing DAX1 in a girl with isolated 46,XY GD (Paper III). We also identified another XY patient with isolated partial GD and ambiguous external genitalia, by MLPA (Multiplex Ligation Probe-dependent Amplification) analysis using a synthetic probe set that we designed to identify gene dosage imbalances for known genes involved in DSD (Paper IV). These reports describe the first duplications on Xp21.2 identified in patients with isolated GD because all previously described XY subjects with Xp21 duplications presented with GD as part of a more complex phenotype, including mental retardation and/or malformations. These data support DAX1 as a dosage sensitive gene responsible for GD and highlight the importance of considering DAX1 locus duplications in the evaluation of all cases of 46,XY GD. More recently, we identified an additional family with several members affected with XY GD, where a small DAX1 duplication is segregating through the female line. These data suggest that DAX1 duplications might be as common as SRY mutations causing 46,XY GD. A terminal 9p deletion, of a region already involved in DSD, was also identified by array-CGH, and confirmed by a MLPA probe set, designed to enable screening of loss of candidate DSD genes at 9p23.4. The identification of submicroscopic deletions at 9p24 is of help to understand the mechanisms that lead to GD in some patients with 9p deletions, and to narrow down the monosomy 9p syndrome candidate region (Paper V). By array-CGH we have also identified two novel chromosomal imbalances that are candidate regions for XY GD: a duplication of 3.7 Mb at chromosome band 12q21.31 and a duplication on chromosome 6 that extends from exon 5 to exon 12 of the SUPT3H gene. These regions will be the subjects of further investigations in order to identify new genes involved in gonadal development (Paper VI). The work of this thesis has led to the establishment of the genetic diagnosis in several patients with DSD, thus allowing not only a better genetic counselling but also, at least in some cases, a better patient management. Furthermore, our genetic diagnostic arsenal has been expanded, as we can offer sequencing of more genes and gene dosage investigations by MLPA.