Annalisa Nicoletti

Gonadoblastoma in Turner syndrome and Y-chromosome-derived material.

The identification of Y‐chromosome material is important in females with Ullrich–Turner syndrome (UTS) due to the risk of developing gonadoblastoma or other gonadal tumors. There is controversy regarding the frequency of the Y‐chromosome‐derived material and the occurrence of gonadoblastoma in these patients. The aim of our study was to evaluate a large number of patients with UTS, followed before and during the pubertal age for the prevalence of Y‐chromosome derived material, the occurrence of gonadoblastoma, and the incidence of possible neoplastic degeneration. An unselected series of 171 patients with UTS (1–34 years old), diagnosed cytogenetically, was studied for Y‐chromosome markers (SRY and Y‐centromeric DYZ3 repeats). The follow‐up was of 2–22 years; 101 of these patients were followed during pubertal age. Y‐chromosome material was found in 14 patients (8%): 12 of these were gonadectomized (2.8–25.9 years). A gonadoblastoma was detected in four patients under 16 years of age: in two, Y‐material was detected only at molecular analysis (at conventional cytogenetic analysis, one was included in the 45,X group and one in the X + mar group) and one had also an immature teratoma and an endodermal sinus carcinoma. The prevalence of gonadoblastoma in our series of gonadectomized UTS patients with Y‐positive material was of 33.3% (4/12). Our data suggest that the age of appearance and the possibility of malignant degeneration of gonadoblastoma can occur early in life. These patients, in particular those with 45,X or a marker chromosome may benefit from molecular screening to detect the presence of Y‐chromosome material; PCR is a rapid and inexpensive technique. At the moment, laparoscopy and preventive gonadectomy performed as soon as possible remain the procedures of choice for patients with UTS, when Y‐chromosome has been identified, as we are still unable to predict a future malignant evolution of gonadoblastoma.

Characterization of deletions at 9p affecting the candidate regions for sex reversal and deletion 9p syndrome by MLPA

The distal region on the short arm of chromosome 9 is of special interest for scientists interested in sex development as well as in the clinical phenotype of patients with the 9p deletion syndrome, characterized by mental retardation, trigonocephaly and other dysmorphic features. Specific genes responsible for different aspects of the phenotype have not been identified. Distal 9p deletions have also been reported in patients with 46,XY sex reversal, with or without 9p deletion syndrome. Within this region the strongest candidates for the gonadal dysgenesis phenotype are the DMRT genes; however, the genetic mechanism is not clear yet. Multiple ligation-dependent probe amplification represents a useful technique to evaluate submicroscopic interstitial or distal deletions that would help the definition of the minimal sex reversal region on 9p and could lead to the identification of gene(s) responsible of the 46,XY gonadal disorders of sex development (DSD). We designed a synthetic probe set that targets genes within the 9p23-9p24.3 region and analyzed a group of XY patients with impaired gonadal development. We characterized a deletion distal to the DMRT genes in a patient with isolated 46,XY gonadal DSD and narrowed down the breakpoint in a patient with a 46,XY del(9)(p23) karyotype with gonadal DSD and mild symptoms of 9p deletion syndrome. The results are compared with other patients described in the literature, and new aspects of sex reversal and the 9p deletion syndrome candidate regions are discussed.

Thyrotropin-Stimulating Hormone Receptor Gene Analysis in Pediatric Patients with Non-Autoimmune Subclinical Hypothyroidism

CONTEXT Mutations in TSH receptor (TSHR) are notoriously associated with congenital hypothyroidism as well as with non-autoimmune subclinical hypothyroidism, a mild form of TSH resistance that is not as well characterized by diagnostic procedures. OBJECTIVE The genetic analysis of the TSHR gene was performed to determine the prevalence of TSHR gene mutations in non-autoimmune subclinical hypothyroidism during the pediatric age. The new mutations were studied for genotypic-phenotypic correlation. PATIENTS Thirty-eight children (ages 0.5-18.0 yr) affected by non-autoimmune subclinical hypothyroidism diagnosed in our center (follow-up from 1 to 11.5 yr) and normal at neonatal screening were enrolled in the genetic study. In 11 cases, the relatives were included in the genetic analysis. RESULTS Eleven different mutations of the TSHR gene were identified in 11 of the 38 patients. Two are new: the nonsense mutation C31X and the missense mutation P68S, which shows a decrease in TSH binding capacity but not in biological activity. In all cases the carrier parent was identified. CONCLUSIONS To date, this study demonstrates the highest prevalence (29%) of TSHR gene mutations in children and adolescents with non-autoimmune subclinical hypothyroidism not selected by neonatal screening. Functional studies show that some mutations cause a slight inactivation of the TSHR. This reveals a possible limit of the in vitro study or of the knowledge of mechanisms involving TSHR, or that other candidate genes must be considered.

SRD5A2 gene analysis in an Italian population of under-masculinized 46,XY subjects

Objective  The differential diagnosis of male under‐masculinization, including a wide spectrum of phenotypes and a heterogeneous genetic basis, is crucial for the correct management of the patients. To characterize an Italian population of under‐masculinized males, we performed the molecular analysis of the SRD5A2 gene (2p23), encoding the 5α‐reductase‐2 enzyme that converts testosterone (T) to dihydrotestosterone (DHT), and is required for full masculinization of the male foetus.

17β-Hydroxysteroid dehydrogenase-3 deficiency: From pregnancy to adolescence

Objective: Aim of this study is to report on basal clinical phenotype and follow up after diagnosis, of patients with 17β-hydroxysteroid-dehydrogenase type 3 (17β-HSD3) deficiency in Italy. Setting: Pediatric Endocrine Departments, University Hospitals. Patients: The cases of 5 Italian subjects affected by 17β-HSD3 deficiency are presented in this study. Interventions: Laboratory and genetic assessment. Gonadectomy and female sex assignment (4 patients) or GnRH analog therapy to regress puberty and gender identity disorder (1 patient). Results: Presentation lasted from pregnancy (pre-natal diagnosis of a 46,XY fetus with female external genitalia) to infancy (inguinal hernia containing testes/clitoromegaly) and adolescence (virilisation). All subjects but one (subject 1, Central-Northern Italy) were from small areas of Southern Italy. Endocrine data (baseline and/or stimulated testosterone/A4-androstenedione ratio) were informative. Two girls were homozygous for 17β-HSD3 gene mutations (G289S/G289S; R80W/R80W), while the others were compound heterozygous (IVS325+4 A>T/A203V; L212Q/M235V; R80W/A235E). Four patients were confirmed as females and were well-adjusted with assigned sex; gender identity disorder improved during treatment with GnRH analog in the last subject. Conclusions: 17βHSD3 deficiency may present from pregnancy to puberty for different clinical issues. Albeit testosterone/A4-androstenedione ratio represents the most accurate endocrine marker to diagnose the disorder, hCG-stimulation is mandatory in pre-puberty. Molecular analysis of 17β-HSD3 gene should be performed to confirm the diagnosis. Temporary GnRH analog treatment may regress gender identity disorder and provide time to confirm or change the birth sex assignment. Female individuals seems to be compliant with their sex, providing that virilisation does not occur. In Italy, the disorder seems to be more prevalent in the Southern regions and shows genetic heterogeneity.

A sequence variation in 3’UTR of CYP21A2 gene correlates with a mild form of congenital adrenal hyperplasia

Background: Congenital adrenal hyperplasia (CAH) is mainly caused by the deficiency of the 21-hydroxylase enzyme coded by the CYP21A2 gene. However, some alleles in the non-classical form (NC-CAH) remain without identified mutations, suggesting the involvement of regulatory regions. Aim: Our objective was to study an allele carrying the variant *13 G>A in the 3’UTR of the CYP21A2 gene identified in some patients with a mild form of NC-CAH in order to verify the possible implication of this variation with the phenotype observed. Subjects and methods: Among all the subjects in whom the CYP21A2 gene was analyzed, 14 patients and 7 relatives heterozygous or homozygous for the *13 G>A substitution in 3’UTR were selected. Sequencing of DNA, genotyping, multiplex ligation-dependent probe amplification (MLPA), in vitro studies and bioinformatic analysis were performed. Results: The haplotype of the *13 G>A allele was identical in all the subjects with a monomodular structure composed by one C4A gene and one CYP21A2 gene without a second module with the CYP21A1P pseudogene. No other concomitant mutations were found in the region extending from 3 kb in the promoter and encompassing the polyadenylation signal. Both bioinformatic analysis and in vitro studies predicted an alteration of the RNA folding and expression, but no miRNA target sequences were found in this region. Conclusions: The identification of a substitution in the 3’UTR of the gene associated with a mild form of NC-CAH suggests the importance of analyzing the CYP21A2 untranslated regions to better characterize and treat this subgroup of patients.

Three novel AMH gene mutations in a patient with persistent mullerian duct syndrome and normal AMH serum dosage.

Background:Persistentmüllerian duct syndrome (PMDS) is characterized by the presence of uterus, fallopian tubes and the upper part of the vagina in 46,XY patients with perfectly virilized external genitalia. It is mostly caused by mutations of the AMH or AMH type 2 receptor (AMHR2) gene. The AMH serum level is very often low or undetectable in the AMH gene defect and normal in the AMHR2 gene defect. Aim: We investigate an Italian patient, genotypically and phenotypically male, observed at 1 month of age for a right inguinal hernia that at surgery showed the presence of both testes in the same hernial sac and uterus and fallopian tubes in the abdomen. Results: Genetic tests first showed the absence of the common 27-bp deletion in the AMHR2 gene, then the presence of three new sequence variations in the AMH geneleading to the following variants: the p.A405P carried by the paternal allele; the p.G326V plus the p.V508A carried by the maternal allele. Conclusions: The determination of serum AMH, performed after the genetic analysis, showed a normal level for age, suggesting that these mutations may affect the function and the bioactivity of the hormone and not the secretion rate.

Multimodal Approach to Monitoring and Investigating Cone Structure and Function in an Inherited Macular Dystrophy

PURPOSE To examine a female subject, her father, and a brother harboring a missense mutation of the retinitis pigmentosa 1-like 1 (RP1L1) gene, over 2 years of follow-up. DESIGN Observational case series. METHODS setting: Fondazione G.B. Bietti IRCCS, Rome, Italy. STUDY POPULATION RP1L1 family members and controls. MAIN OUTCOME MEASURES Images of the cone mosaic acquired with an adaptive optics retinal camera, spectral-domain optical coherence tomography (SD OCT), and full-field and multifocal electroretinography (mfERG). RESULTS In the proband, best-corrected visual acuity (≤0.7 logMAR) was stable in both eyes during follow-up, though analysis of adaptive optics images showed decreased cone density in the central 9 degrees from the fovea with respect to controls (P < .05) and cone density loss in the parafoveal area (2 degrees; <12%-16%) during follow-up. Texture analysis of SD OCT images identified abnormalities of the ellipsoid zone in the central 7 degrees, while mfERG response amplitudes were reduced only in the central 5 degrees relative to controls. In the probands father, who had 0.0 logMAR visual acuity, significant cone loss was found in the central 7 degrees from the fovea (P < .05); abnormal SD OCT and mfERG values with respect to controls were found in corresponding retinal areas. No defects in the cone structure and function were found in the probands brother, who had 0.0 logMAR visual acuity. CONCLUSIONS Occult macular dystrophy was diagnosed based on genetic and multimodal ophthalmic findings. The quantitative assessment of photoreceptor survival or loss, based on analysis of adaptive optics retinal images, was valuable to monitor disease progression at a cellular level.

Osteoporosis-pseudoglioma syndrome: Report of two cases and a manifesting carrier

ABSTRACT Background: Osteoporosis-pseudoglioma syndrome is a very rare disease mainly characterized by severe eye abnormalities and osteoporosis but also causing a broader range of clinical features. The syndrome is associated with homozygous or compound heterozygous variations in the LRP5 gene. In this report, we describe two children with a severe early-onset form of familial exudative vitreoretinopathy associated with skeletal abnormalities. Materials and methods: Two probands (4 and 7 years of age respectively) and their parents were assessed by genetic analysis and comprehensive ophthalmic examination. Results: In both probands, the diagnosis of osteoporosis-pseudoglioma syndrome was confirmed by detection of three new pathogenic LRP5 variants: p.(Asp379Asn), found in the homozygous state in one proband, and p.(Asp203Ala) in the compound heterozygous state with p.(Cys612Valfs*25) in the other. The clinical and genetic study was extended to their parents, confirming that heterozygous carriers may also have incomplete clinical manifestation of this syndrome. Conclusions: To our knowledge, these are the first two cases of the syndrome described in Italy. Genetic testing proved to be fundamental for definition of the syndrome and confirms the importance of early detection of LRP5 variants for management of systemic features of the disease in patients and carrier relatives.

Cystoscopic diagnosis of polypoid cystitis in two pet rabbits

CASE DESCRIPTION AS-year-old male Dwarf rabbit and 4-year-old female Mini-Rex rabbit were evaluated because of anorexia and urine scalding of the perineum. CLINICAL FINDINGS Abdominal radiography revealed a diffuse increase in the opacity of the urinary bladder attributable to urinary sludge. In 1 rabbit, abdominal ultrasonography revealed several mass-like lesions protruding from the mucosal surface into the lumen of the urinary bladder. Rabbits were anesthetized, and cystoscopy was performed with a rigid 2.7-mm, 30° endoscope. Histologic analysis of tissue samples obtained through the cystoscope operating channel revealed findings consistent with polypoid cystitis. TREATMENT AND OUTCOME To remove the urinary sludge from each rabbit, the urinary bladder was filled with sterile saline (0.9% NaCl) solution and emptied with a gentle massage several times until the ejected fluid was transparent. Rabbits were treated with NSAIDs, antimicrobials (chosen following microbial culture of urine and antimicrobial susceptibility testing), bathing of the perineum, and a low-calcium diet. The male rabbit died of unrelated causes 18 months later; postmortem examination findings confirmed the polypoid cystitis. The female rabbit remained disease free through to last follow-up (12 months after initial evaluation). CLINICAL RELEVANCE This was the first report of polypoid cystitis in pet rabbits. Although ultrasonographic findings supported this diagnosis, a definitive diagnosis was achieved through cystoscopy and lesion biopsy. Treatments administered were intended to reduce the potential sources of irritation. Research is needed to investigate the effectiveness of the applied interventions and the association between excessive urinary calcium excretion and polyploid cystitis in rabbits.