Catherine Pienkowski

Identification and functional analysis of a new WNT4 gene mutation among 28 adolescent girls with primary amenorrhea and Müllerian duct abnormalities: A french collaborative study

CONTEXT Müllerian duct development depends on gene and hormone interactions. Female Wnt4-knockout mice lack müllerian ducts and are virilized due to the inappropriate expression of the enzymes required for androgen production (normally repressed in female ovary). The WNT4 mutation was recently reported to be associated with failure of müllerian duct formation and virilization in two 46, XX women. OBJECTIVES This collaborative work was designed to determine whether the WNT4 mutation could be identified in a group of adolescent girls with Mayer-Rokitansky-Küster-Hauser syndrome. RESULTS We analyzed 28 DNA samples from adolescent girls with primary amenorrhea and failure of müllerian duct formation by direct sequencing and identified a new L12P mutation within exon 1 of the WNT4 gene. The substitution of leucine by proline is crucial for the conformation of the expressed protein. This amino acid substitution is unlikely to be a polymorphism because it was not found in 100 DNAs from control subjects. Functional analysis revealed that the mutation induces significantly increased expression of the enzymes involved in androgen biosynthesis (3beta-hydroxysteroid dehydrogenase and 17alpha-hydroxylase). It is interesting to note that the adolescent carrying the mutation was referred to our clinic for primary amenorrhea and hyperandrogenism (severe acne and plasma testosterone: 1.8 vs. 1.2 nmol/liter in controls). She also presented with uterine hypoplasia and follicle depletion. CONCLUSIONS We suggest that in adolescent girls with primary amenorrhea, müllerian duct abnormalities, and hyperandrogenism, a WNT4 mutation should be sought. Moreover, our data confirm that WNT4 is involved in the regulation of müllerian duct development and ovarian androgen biosynthesis. WNT4 may also contribute to human follicle development and/or maintenance.

Ovarian torsion. Management and ovarian prognosis: a report of 45 cases

BACKGROUND/PURPOSE Ovarian torsion in childhood and adolescence is a rare entity. Traditionally, treatment is oophorectomy. The aim of this study was to evaluate ovarian outcome and to propose a decision-making protocol for suspected ovarian torsion. METHODS Between January 1986 and December 2007, 45 ovarian torsion cases in 40 girls were operated on. In all the cases, when the ovary was preserved, patients were clinically and ultrasonographically followed up for several months. RESULTS Median age was 11 years. Median delay between the first symptoms and surgical procedure was 3 days. There was a statistical difference (P = .0003) between the mean of the largest diameter of twisted normal ovary and the mean of the largest diameter of twisted diseased ovary. Underlying pathology was benign in 22 cases and low-grade malignancy in 2 (one grade II immature teratoma and one steroid cell tumor). Conservative management was performed in 26 cases. At follow-up, 17 ovaries were follicular, 7 being black-bluish during surgery. CONCLUSIONS Conservative approach after detorsion of black-bluish ovaries is safe and effective in children. Although very unlikely, the fear of missing malignancy must incite to proceed with caution and can lead, when the size of the twisted ovary is greater than 75 mm, to prefer laparotomy to laparoscopy.

Auxological and Endocrine Evolution of 28 Children with Prader-Willi Syndrome: Effect of GH Therapy in 14 Children

We report on the auxological and endocrine evolution of 28 patients presenting with Prader-Willi syndrome. Half of them received growth hormone (GH) therapy (group 2). The spontaneous auxological evolution was analyzed in the two groups from 2 to 8 years; the mean SDS for height remained stable (–0.6 ± 0.6) in group 1 and decreased (from –2.0 ± 0.9 to –2.7 ± 0.6) in group 2. Magnetic resonance imaging showed marked pituitary hypoplasia in the two groups. In group 2, the mean GH peak after two provocative tests was 3.8 ± 2.4 µg/l, the mean SDS values for insulin-like growth factor I levels were –2.0 ± 1.5 (range from –0.5 to –5.0). The mean duration of GH treatment was 3.6 ± 2.9 (range 1–9.3) years. 14 children completed 1 year of treatment. The two groups had opposite evolutions in ΔSDS for height (–0.8 ± 0.8 vs. +1.1 ± 0.8), for growth velocity (–1.9 ± 2.2 vs. +2.9 ± 2.7), and for Z score of the body mass index (+0.37 ± 1.3 vs. –0.14 ± 0.76; group 1 vs. group 2). This retrospective study shows that, in children with Prader-Willi syndrome and true GH deficiency, long-term GH therapy is effective in increasing growth velocity and in maintaining body mass index.

Molecular analysis of WNT4 gene in four adolescent girls with mullerian duct abnormality and hyperandrogenism (atypical Mayer-Rokitansky-Küster-Hauser syndrome)

In a collaborative study, we investigated four 46,XX adolescent girls with Mayer-Rokitansky-Küster-Hauser syndrome and hyperandrogenism. Molecular analysis of the WNT4 gene permitted us to identify a new mutation (p.A233T). Functional studies revealed partial repression of steroidogenic enzymes (normal repression of HSD3B2) contrasting with the abnormal reexpression of CYP17A1 enzyme in the OVCAR3 cell line. This fourth new WNT4 mutation confirms that this signaling molecule is involved in mullerian development and androgen biosynthesis repression in the ovary. Interestingly, this mutant partially lacks the capability to repress ovarian steroidogenic enzymes, with abnormal expression of 17α- hydroxylase.

Long-term evolution of endocrine disorders and effect of GH therapy in 35 patients with pituitary stalk interruption syndrome

We report long-term evolution of endocrine functions and the results of GH treatment in 35 patients (26 male and 9 female) with pituitary stalk interruption. At diagnosis, mean chronological age was 4.8 ± 2.7 years, mean SDS for height –3.1 ± 0.8 with a bone age retardation of 2.3 ± 1.3 years and a mean SDS for growth velocity of –0.5 ± 1.1; 80% presented complete GH deficiency (GHD) and 20% partial GHD; thyroid deficiency was present in 47.1% of children with complete GHD but absent in all partial GHD. Diagnosis was made during the first months of life in only 2 patients while 23% presented with severe neonatal distress; neonatal signs were only observed in the group with pituitary height below 2 mm (45.7% of patients). GHD was isolated in 40.6% of patients below 10 years while multiple hormone deficiencies was consistent at completion of growth in all patients. Height gain was significantly higher in patients who started GH treatment before 4 years (p = 0.002). GH treatment is very effective: in 13 patients, final height was –0.4 ± 1.0, total height gain 3.2 ± 1.2 and distance to target height –0.3 ± 1.6 SDS.

Aberrant expression of ovary determining gene FOXL2 in the testis and juvenile granulosa cell tumor in children.

PURPOSE FOXL2 is the earliest known marker of ovarian differentiation in mammals. It is involved in ovarian somatic cell differentiation and further follicle maintenance. FOXL2 is not implicated in determination of the male gonad and it is absent in the testis. We investigated whether the rare JGCTT (juvenile granulose cell tumor of the testis), named for its histological similarity to ovarian tumor, could be the first illustration of aberrant expression of this ovary determining gene in the human testis. MATERIALS AND METHODS Between 1990 and 2004, 3 boys with JGCTT were reported from the TGM95 database of the French Society for Childhood Cancer and from 8 pediatric endocrinology centers. Orchiectomy was performed in these patients. Immunohistochemistry of FOXL2, and co-immunofluorescence of FOXL2 and SOX9 were performed on tumor sections. RESULTS Testicular tumor cells showed aberrant expression of FOXL2, which resembled normal ovarian granulosa cells. The localization of FOXL2 expression was nuclear without any cytoplasmic sequestration, suggesting that FOXL2 had biological activity. Conversely SOX9, which is present in the nucleus of normal testicular cells, was sequestered in the cytoplasm of granulosa tumor cells or markedly under expressed in the nuclei. In this case of residual SOX9 nuclear expression the expression of FOXL2 and SOX9 was mutually exclusive. CONCLUSIONS To our knowledge we report the first human model of aberrant intratesticular expression of an ovary determining gene along with the extinction of SOX9 and the transdifferentiation of a testicular cell into a granulosa tumor cell.

Complete androgen insensitivity syndrome is frequently due to premature stop codons in exon 1 of the androgen receptor gene: an international collaborative report of 13 new mutations.

OBJECTIVE To confirm the clinical diagnosis of complete androgen insensitivity syndrome (CAIS) by molecular genetic analysis and to determine the prevalence of exon 1 mutations in the androgen receptor (AR) transactivation defects of a large series of CAIS patients. DESIGN International retrospective study. SETTING University Hospital of Montpellier, Department of Hormonology. PATIENT(S) 105 patients with normal female external genitalia, bilateral intra-abdominal or inguinal testis, normal breast development, absent or sparse pubic hair, normal or high endogenous testosterone production, hypoplastic or absent wolffian structures, and 46,XY karyotype. INTERVENTION(S) Sequencing of the AR gene. MAIN OUTCOME MEASURE(S) Prevalence of AR exon 1 mutations. RESULT(S) Over a 10-year period (1997 to 2007), we identified 78 AR gene mutations in 105 patients with CAIS; 21 of them were located in exon 1, and 13 of these were new mutations. We report 13 new mutations in the AR gene. All but one were stop codons, and the last was a splicing abnormality. CONCLUSION(S) The finding that 27% of the mutations in our cohort were localized in exon 1 versus 15% in previous works justifies the sequencing of this exon in patients with CAIS.

Screening of MAMLD1 Mutations in 70 Children with 46,XY DSD: Identification and Functional Analysis of Two New Mutations

More than 50% of children with severe 46,XY disorders of sex development (DSD) do not have a definitive etiological diagnosis. Besides gonadal dysgenesis, defects in androgen biosynthesis, and abnormalities in androgen sensitivity, the Mastermind-like domain containing 1 (MAMLD1) gene, which was identified as critical for the development of male genitalia, may be implicated. The present study investigated whether MAMLD1 is implicated in cases of severe 46,XY DSD and whether routine sequencing of MAMLD1 should be performed in these patients. Seventy children with severe non-syndromic 46,XY DSD of unknown etiology were studied. One hundred and fifty healthy individuals were included as controls. Direct sequencing of the MAMLD1, AR, SRD5A2 and NR5A1 genes was performed. The transactivation function of the variant MAMLD1 proteins was quantified by the luciferase method. Two new mutations were identified: p.S143X (c.428C>A) in a patient with scrotal hypospadias with microphallus and p.P384L (c.1151C>T) in a patient with penile hypospadias with microphallus. The in vitro functional study confirmed no residual transactivating function of the p.S143X mutant and a significantly reduced transactivation function of the p.P384L protein (p = 0.0032). The p.P359S, p.N662S and p.H347Q variants are also reported with particularly high frequency of the p.359T- p.662G haplotype in the DSD patients. Severe undervirilization in XY newborns can reveal mutations of MAMLD1. MAMLD1 should be routinely sequenced in these patients with otherwise normal AR, SRD5A2 and NR5A1genes.

Ovarian Masses in Adolescent Girls

Ovarian masses are the most frequent gynecological pathology seen in adolescent girls. Functional or organic tumors of the ovary are usually benign and the incidence rises with age. Most cysts are functional and adnexal torsion is the main complication, but a malignant etiology must nevertheless always be eliminated. The clinical presentation is quite variable. Ultrasonography is the investigation of choice: the sonogram will reveal a strictly fluid, benign functional cyst, suggest an adnexal torsion, and provide evidence of a heterogeneous mass. Emergency surgery is indicated only in the case of suspected ovarian torsion, in order to perform detorsion. In all other cases, serum tumor marker measurements will orient the diagnosis and MRI is an essential complement to imaging of tumors with heterogeneous solid components. Surgery and histopathological examination then determine the stage and the benign or malignant nature of the mass. Ovarian tumors are classified by the World Health Organization based on the cell of origin into epithelial tumors, germ cell tumors and sex cord-stromal tumors. Surgery should always follow oncological standards and be as conservative as possible to preserve future fertility.

Ovarian germ cell tumors in children. Management, survival and ovarian prognosis. A report of 75 cases.

BACKGROUND/PURPOSE The aims of this study were to evaluate survival and ovarian prognosis in patients treated for ovarian germ cell tumor (OGCT) and to propose a decision-making protocol. METHODS Charts of girls operated on for OGCT from 1976 up to 2009 were reviewed retrospectively. Tumor characteristics were assessed by tumor markers, imaging, and pathology. RESULTS Charts were available in 71 children presenting 75 OGCT. Tumors were benign in 58 cases and malignant in 17 cases. The average of the largest diameter of benign OGCT was significantly lower than that of malignant OGCT (76.5 +/- 49 mm versus 169 +/- 54 mm, P < .0001). Ovarian-sparing tumorectomy was carried out in 27 benign OGCT; 23 (85%) preserved ovaries were follicular. Malignant OGCTs were managed according to the protocols of the French Society for Pediatric Oncology. Bilateral oophorectomy had to be performed in 2 children. One patient presented a recurrence and 1 died. CONCLUSIONS In our series, both benign and malignant OGCTs have a good prognosis. A 75-mm cutoff size is proposed as an important criterion to preoperatively differentiate between benign and malignant tumors. In benign OGCT, ovarian-sparing tumorectomy leads to preserve ovaries in approximately 85% of cases, and in malignant OGCT, high survival rate has been obtained.