High-throughput splicing assays identify missense and silent splice-disruptive POU1F1 variants underlying pituitary hormone deficiency.

Pituitary hormone deficiency occurs in ∼1:4,000 live births. Approximately 3% of the cases are due to mutations in the alpha isoform of POU1F1, a pituitary-specific transcriptional activator. We found four separate heterozygous missense variants in unrelated individuals with hypopituitarism that were predicted to affect a minor isoform, POU1F1 beta, which can act as a transcriptional repressor. These variants retain repressor activity, but they shift splicing to favor the expression of the beta isoform, resulting in dominant-negative loss of function. Using a high-throughput splicing reporter assay, we tested 1,070 single-nucleotide variants in POU1F1. We identified 96 splice-disruptive variants, including 14 synonymous variants. In separate cohorts, we found two additional synonymous variants nominated by this screen that co-segregate with hypopituitarism. This study underlines the importance of evaluating the impact of variants on splicing and provides a catalog for interpretation of variants of unknown significance in POU1F1.

Renal Function Evolution and Hypoaldosteronism Risk After Unilateral Adrenalectomy for Primary Aldosteronism.

Few studies demonstrated a percentage decrease in the estimated glomerular filtration rate (eGFR) at a single time and the rate of hypoaldosteronism after adrenalectomy for primary aldosteronism (PA). Our aim was to investigate the evolution of renal function and the hypoaldosteronism risk after adrenalectomy for PA. Aldosterone, renin, eGFR, and electrolyte levels were determined before and at 1 week, 1, 3 and 6 months after unilateral adrenalectomy in 94 PA patients (40 men and 54 women). The main outcome was the postoperative eGFR decline using analysis of covariance with the preoperative eGFR as a covariate. eGFR decreased during first postoperative week compared to 3 months before surgery. During the first 6 months, eGFR remained stable at similar levels to the first week after surgery. Age (p=0.001), aldosterone levels (p=0.021) and eGFR 3 months before surgery (p+<+0.0001) had a significant correlation with eGFR during first postoperative week. High aldosterone levels at diagnosis were correlated with decline in renal function in the univariate model (p=0.033). In the multivariate analysis, aldosterone levels at diagnosis had a tendency to be an independent predictor of renal function after surgery (p=0.059). Postoperative biochemical hypoaldosteronism was diagnosed in 48% of the cases after adrenalectomy, but prolonged hyperkalemia occurred in only 4 cases (4.5%). Our findings showed a decrease of eGFR after unilateral adrenalectomy for PA. Additionally, aldosterone levels at diagnosis correlated with postoperative renal function. Postoperative biochemical hypoaldosteronism occurred in almost half of the patients, but prolonged hyperkalemia with fludrocortisone replacement was less frequent.

Genetic and clinical aspects of paediatric pheochromocytomas and paragangliomas.

OBJECTIVE: Few and conflicting reports have characterized the genetics of paediatric pheochromocytomas and paragangliomas (PPGLs). This study aimed to investigate the clinical and genetic features of Brazilian children with PPGL. PATIENTS AND METHODS: This study included 25 children (52% girls) with PPGL. The median age at diagnosis was 15 years (4-19). The median time of follow-up was 145 months. The genetic investigation was performed by Sanger DNA sequencing, multiplex ligation-dependent probe amplification and/or target next-generation sequencing panel. RESULTS: Of the 25 children with PPGL, 11 (44%), 4 (16%), 2 (8%), 1 (4%) and 7 (28%) had germline VHL pathogenic variants, SDHB, SDHD, RET and negative genetic investigation, respectively. Children with germline VHL missense pathogenic variants were younger than those with SDHB or SDHD genetic defects [median (range), 12 (4-16) vs. 15.5 (14-19) years; P = .027]. Moreover, 10 of 11 cases with VHL pathogenic variants had bilateral pheochromocytoma (six asynchronous and four synchronous). All children with germline SDHB pathogenic variants presented with abdominal paraganglioma (one of them malignant). The two cases with SDHD pathogenic variants presented with head and neck paraganglioma. Among the cases without a genetic diagnosis, 6 and 2 had pheochromocytoma and paraganglioma, respectively. Furthermore, metastatic PPGL was diagnosed in four (16%) of 25 PPGL. CONCLUSIONS: Most of the paediatric PPGL were hereditary and multifocal. The majority of the affected genes belong to pseudohypoxic cluster 1, with VHL being the most frequently mutated. Therefore, our findings impact surgical management and surveillance of children with PPGL.

Genetics, clinical features and outcomes of non-syndromic pituitary gigantism: experience of a single center from Sao Paulo, Brazil.

PURPOSE: Non-syndromic pituitary gigantism (PG) is a very rare disease. Aryl hydrocarbon receptor-interacting protein (AIP) and G protein-coupled receptor 101 (GPR101) genetic abnormalities represent important etiologic causes of PG and may account for up to 40% of these cases. Here, we aimed to characterize the clinical and molecular findings and long-term outcomes in 18 patients (15 males, three females) with PG followed at a single tertiary center in Sao Paulo, Brazil. METHODS: Genetic testing for AIP and GPR101 were performed by DNA sequencing, droplet digital PCR and array comparative genomic hybridization (aCGH). RESULTS: Pathogenic variants in the AIP gene were detected in 25% of patients, including a novel variant in splicing regulatory sequences which was present in a sporadic male case. X-LAG due to GPR101 microduplication was diagnosed in two female patients (12.5%). Of interest, these patients had symptoms onset by age 5 and 9 years old and diagnosis at 5 and 15 years, respectively. X-LAG, but not AIP, patients had a significantly lower age of symptoms onset and diagnosis and a higher height Z-score when compared to non-X-LAG. No other differences in clinical features and/or treatment outcomes were observed among PG based on their genetic background. CONCLUSION: We characterize the clinical and molecular findings and long-term outcome of the largest single-center PG cohort described so far.

Evaluation of SHOX defects in the era of next-generation sequencing.

Short stature homeobox (SHOX) haploinsufficiency is a frequent cause of short stature. Despite advances in sequencing technologies, the identification of SHOX mutations continues to be performed using standard methods, including multiplex ligation-dependent probe amplification (MLPA) followed by Sanger sequencing. We designed a targeted panel of genes associated with growth impairment, including SHOX genomic and enhancer regions, to improve the resolution of next-generation sequencing for SHOX analysis. We used two software packages, CONTRA and Nexus Copy Number, in addition to visual analysis to investigate the presence of copy number variants (CNVs). We evaluated 15 patients with previously known SHOX defects, including point mutations, deletions and a duplication, and 77 patients with idiopathic short stature (ISS). The panel was able to confirm all known defects in the validation analysis. During the prospective evaluation, we identified two new partial SHOX deletions (one detected only by visual analysis), including an intragenic deletion not detected by MLPA. Additionally, we were able to determine the breakpoints in four cases. Our results show that the designed panel can be used for the molecular investigation of patients with ISS, and it may even detect CNVs in SHOX and its enhancers, which may be present in a significant fraction of patients.

A 46,XX testicular disorder of sex development caused by a Wilms' tumour Factor-1 (WT1) pathogenic variant.

Molecular diagnosis is rarely established in 46,XX testicular (T) disorder of sex development (DSD) individuals with atypical genitalia. The Wilms' tumour factor-1 (WT1) gene is involved in early gonadal development in both sexes. Classically, WT1 deleterious variants are associated with 46,XY disorders of sex development (DSD) because of gonadal dysgenesis. We report a novel frameshift WT1 variant identified in an SRY-negative 46,XX testicular DSD girl born with atypical genitalia. Target massively parallel sequencing involving DSD-related genes identified a novel heterozygous WT1 c.1453_1456del; p.Arg485Glyfs*14 variant located in the fourth zinc finger of the protein which is absent in the population databases. Segregation analysis and microsatellite analysis confirmed the de novo status of the variant that is predicted to be deleterious by in silico tools and to increase WT1 target activation in crystallographic model. This novel and predicted activating frameshift WT1 variant leading to the 46,XX testicular DSD phenotype includes the fourth zinc-finger DNA-binding domain defects in the genetic aetiology of 46,XX DSD.

Biallelic and monoallelic ESR2 variants associated with 46,XY disorders of sex development.

PURPOSE: Disorders or differences of sex development (DSDs) are rare congenital conditions characterized by atypical sex development. Despite advances in genomic technologies, the molecular cause remains unknown in 50% of cases. METHODS: Homozygosity mapping and whole-exome sequencing revealed an ESR2 variant in an individual with syndromic 46,XY DSD. Additional cases with 46,XY DSD underwent whole-exome sequencing and targeted next-generation sequencing of ESR2. Functional characterization of the identified variants included luciferase assays and protein structure analysis. Gonadal ESR2 expression was assessed in human embryonic data sets and immunostaining of estrogen receptor-ß (ER-ß) was performed in an 8-week-old human male embryo. RESULTS: We identified a homozygous ESR2 variant, c.541_543del p.(Asn181del), located in the highly conserved DNA-binding domain of ER-ß, in an individual with syndromic 46,XY DSD. Two additional heterozygous missense variants, c.251G>T p.(Gly84Val) and c.1277T>G p.(Leu426Arg), located in the N-terminus and the ligand-binding domain of ER-ß, were found in unrelated, nonsyndromic 46,XY DSD cases. Significantly increased transcriptional activation and an impact on protein conformation were shown for the p.(Asn181del) and p.(Leu426Arg) variants. Testicular ESR2 expression was previously documented and ER-ß immunostaining was positive in the developing intestine and eyes. CONCLUSION: Our study supports a role for ESR2 as a novel candidate gene for 46,XY DSD.

Androgen receptor mRNA analysis from whole blood: a low-cost strategy for detection of androgen receptor gene splicing defects.

Androgen insensitivity syndrome (AIS) is caused by defects in the androgen receptor (AR) gene and is the most common aetiology of 46,XY disorders of sex development. Allelic variants in the AR gene are found in 90% of complete AIS (CAIS), but in only 28% to 50% of cases of partial AIS. Even a single nucleic acid change can disrupt splicing sites or splicing regulatory sequences, resulting in inadequate exon and intron recognition, ultimately leading to an aberrant transcript. Therefore, we tested the feasibility of conducting AR cDNA analysis from whole blood and from gonadal tissue in a patient with CAIS due to AR synonymous mutation (c.1530C > T, p.Ser510Ser; NM_000044.3), which led to an aberrant splicing site causing deletion of 92 nucleotides resulting in a very short transcript. AR cDNA sequencing was similar in the whole blood and in the gonadal tissue, with similar evidence of a consequent altered AR transcript. We propose that analysis of AR RNA extracted from whole blood with AR DNA sequencing can help to improve the frequency of molecular diagnosis, particularly for partial AIS.

Pathogenic copy number variants in patients with congenital hypopituitarism associated with complex phenotypes.

OBJECTIVES: The aetiology of congenital hypopituitarism (CH) is unknown in most patients. Rare copy number variants (CNVs) have been implicated as the cause of genetic syndromes with previously unknown aetiology. Our aim was to study the presence of CNVs and their pathogenicity in patients with idiopathic CH associated with complex phenotypes. DESIGN AND PATIENTS: We selected 39 patients with syndromic CH for array-based comparative genomic hybridization (aCGH). Patients with pathogenic CNVs were also evaluated by whole exome sequencing. RESULTS: Twenty rare CNVs were detected in 19 patients. Among the identified rare CNVs, six were classified as benign, eleven as variants of uncertain clinical significance (VUS) and four as pathogenic. The three patients with pathogenic CNVs had combined pituitary hormone deficiencies, and the associated complex phenotypes were intellectual disabilities: trichorhinophalangeal type I syndrome (TRPS1) and developmental delay/intellectual disability with cardiac malformation, respectively. Patient one has a de novo 1.6-Mb deletion located at chromosome 3q13.31q13.32, which overlaps with the region of the 3q13.31 deletion syndrome. Patient two has a 10.5-Mb de novo deletion at 8q23.1q24.11, encompassing the TRPS1 gene; his phenotype is compatible with TRPS1. Patient three carries a chromosome translocation t(2p24.3;4q35.1) resulting in two terminal alterations: a 2p25.3p24.3 duplication of 14.7 Mb and a 4-Mb deletion at 4q35.1q35.2. CONCLUSIONS: Copy number variants explained the phenotype in 8% of patients with hypopituitarism and additional complex phenotypes. This suggests that chromosomal alterations are an important contributor to syndromic hypopituitarism.

Long-term outcomes and molecular analysis of a large cohort of patients with 46,XY disorder of sex development due to partial gonadal dysgenesis.

BACKGROUND: Follow-up data on patients with 46,XY partial gonadal dysgenesis (PGD) until adulthood are scarce, making information on prognosis difficult. OBJECTIVE: To analyse the long-term outcomes of patients with 46,XY PGD regarding testosterone production, germ cell tumour risk, genotype and psychosexual adaptation. METHODS: A retrospective longitudinal study of 33 patients (20 assigned male and 13 patients assigned female at birth). Molecular diagnosis was performed by Sanger sequencing or by targeted massively parallel sequencing of 63 genes related to disorders of sex development (DSDs). RESULTS: Age at first and last visit ranged from 0.1 to 43 and from 17 to 53 years, respectively. Spontaneous puberty was observed in 57% of the patients. During follow-up, six of them had a gonadectomy (four due to female gender, and two because of a gonadal tumour). At last evaluation, five of six patients had adult male testosterone levels (median 16.7 nmol/L, range 15.3-21.7 nmol/L) and elevated LH and FSH levels. Germ cell tumours were found in two postpubertal patients (one with an abdominal gonad and one patient with Frasier syndrome). Molecular diagnosis was possible in 11 patients (33%). NR5A1 variants were the most prevalent molecular defects (n = 6), and four of five patients harbouring them developed spontaneous puberty. Gender change was observed in four patients, two from each sex assignment group; all patients reported satisfaction with their gender at final evaluation. Sexual intercourse was reported by 81% of both gender and 82% of them reported satisfaction with their sexual lives. CONCLUSION: Spontaneous puberty was observed in 57% of the patients with 46,XY PGD, being NR5A1 defects the most prevalent ones among all the patients and in those with spontaneous puberty. Gender change due to gender dysphoria was reported by 12% of the patients. All the patients reported satisfaction with their final gender, and most of them with their sexual life.

Long-Term Outcomes of Patients with Central Precocious Puberty due to Hypothalamic Hamartoma after GnRHa Treatment: Anthropometric, Metabolic, and Reproductive Aspects.

BACKGROUND: Hypothalamic hamartoma (HH) represents the commonest cause of organic central precocious puberty (CPP). Follow-up of these patients in adulthood is scarce. OBJECTIVE: To describe the anthropometric, metabolic, and reproductive parameters of patients with CPP due to HH before and after treatment with gonadotropin-releasing hormone analog (GnRHa). METHODS: We performed a retrospective and cross-sectional study in a single tertiary center including 14 patients (7 females) with CPP due to HH. RESULTS: The mean duration of GnRHa treatment was 7.7 ± 2.4 years in boys and 7.9 ± 2.1 years in girls. GnRHa treatment was interrupted at the mean chronological age (CA) of 12.1 ± 1.1 years in boys and 10.7 ± 0.5 years in girls. At the last visit, the mean CA of the male and female patients was 21.5 ± 3.2 and 24 ± 3.9 years, respectively. Eleven of the 14 patients reached normal final height (FH) (standard deviation score -0.6 ± 0.9 for males and -0.6 ± 0.5 for females), all of them within the target height (TH) range. The remaining 3 patients had predicted height within the TH range. The mean body mass index and the percentage of body fat mass was significantly higher in females, with a higher prevalence of metabolic disorders. All patients presented normal gonadal function in adulthood, and 3 males fathered a child. CONCLUSION: All patients with CPP due to HH reached normal FH or near-FH. A higher prevalence of overweight/obesity and hypercholesterolemia was observed in the female patients. Finally, no reproductive disorder was identified in both sexes, indicating that HH per se has no deleterious effect on the gonadotropic axis in adulthood.

Central Precocious Puberty Caused by a Heterozygous Deletion in the MKRN3 Promoter Region.

CONTEXT: Loss-of-function mutations in the coding region of MKRN3, a maternally imprinted gene at chromosome 15q11.2, are a common cause of familial central precocious puberty (CPP). Whether MKRN3 alterations in regulatory regions can cause CPP has not been explored to date. We aimed to investigate potential pathogenic variants in the promoter region of MKRN3 in patients with idiopathic CPP. PATIENTS/METHODS: A cohort of 110 patients with idiopathic CPP was studied. Family history of precocious sexual development was present in 25%. Mutations in the coding region of MKRN3 were excluded in all patients. Genomic DNA was extracted from peripheral blood leukocytes, and 1,100 nucleotides (nt) of the 5'-regulatory region of MKRN3 were amplified and sequenced. Luciferase assays were performed in GT1-7 cells transiently transfected with plasmids containing mutated and wild-type MKRN3 promoter. RESULTS: We identified a rare heterozygous 4-nt deletion (c.-150_-147delTCAG; -38 to -41 nt upstream to the transcription start site) in the proximal promoter region of MKRN3 in a girl with CPP. In silico analysis predicted that this deletion would lead to the loss of a binding site for a downstream res-ponsive element antagonist modulator (DREAM), a potential transcription factor for MKRN3 and GNRH1 expression. Luciferase assays demonstrated a significant reduction of MKRN3 promoter activity in transfected cells with a c.-150_- 147delTCAG construct plasmid in both homozygous and heterozygous states when compared with cells transfected with the corresponding wild-type MKRN3 promoter region. CONCLUSION: A rare genetic alteration in the regulatory region of MKRN3 causes CPP.

Neonatal 17-hydroxyprogesterone levels adjusted according to age at sample collection and birthweight improve the efficacy of congenital adrenal hyperplasia newborn screening.

INTRODUCTION: The primary concern related to congenital adrenal hyperplasia (CAH) newborn screening (NBS) is the high rate of false-positive results (FPR) associated with prematurity; false-negative results (FNR) can also occur due to precocious sample collection. OBJECTIVE: To determine the neonatal 17-hydroxyprogesterone (N17OHP) normal range in newborns in Sao Paulo using different references according to age and birthweight (BW) and to establish the optimal NBS cut-off levels. METHODS: Neonatal 17-hydroxyprogesterone levels from 271 810 newborns (NBs) according to sample collection time (G1: 48-<72 h and G2: ≥72 h) and BW (≤1500 g, 1501-2000 g, 2001-2500 and >2500 g) were evaluated. N17OHP was measured by an fluoroimmunoassay, and serum 17OHP was measured by liquid chromatography-mass spectrometry. Affected and asymptomatic NBs with persistently increased 17OHP levels were submitted to CYP21A2-sequencing. RESULTS: Neonatal 17-hydroxyprogesterone levels in G1 were lower than G2 in all BW groups (P < 0·001). The FPR rate in G1/G2 was 0·2% using the 99·8th and 0·5% using the 99·5th percentile. The 99·8th percentile N17OHP value was the best cut-off for distinguishing between unaffected and affected NBs. Forty-four salt wasters, and five simple virilisers were diagnosed; N17OHP levels ranged from 93·3 to 2209·8 nmol/l, and no affected neonates with FNR were identified. The positive predictive value in G1 and G2 using the 99·8th percentile was 5·6% and 14·1%, respectively, and 2·3% and 7%, respectively, using the 99·5th percentile. Molecular tests identified two NBs with the nonclassical form among the 29 FPR. CONCLUSION: Neonatal 17-hydroxyprogesterone levels adjusted to sample collection age and birthweight reduced the FPR, and the use of N17OHP values based upon the 99·8th percentile improved the NBS efficacy.

Molecular analysis of brazilian patients with combined pituitary hormone deficiency and orthotopic posterior pituitary lobe reveals eight different PROP1 alterations with three novel mutations.

BACKGROUND: Mutations in PROP1, HESX1 and LHX3 are associated with combined pituitary hormone deficiency (CPHD) and orthotopic posterior pituitary lobe (OPP). OBJECTIVE: To identify mutations in PROP1, HESX1 and LHX3 in a large cohort of patients with CPHD and OPP (35 Brazilian, two Argentinian). DESIGN AND METHODS: We studied 23 index patients with CPHD and OPP (six familial and 17 sporadic) as well as 14 relatives. PROP1 was sequenced by the Sanger method in all except one sporadic case studied using a candidate gene panel. Multiplex ligation-dependent probe amplification (MLPA) was applied to one familial case in whom PROP1 failed to amplify by PCR. In the 13 patients without PROP1 mutations, HESX1 and LHX3 were sequenced by the Sanger method. RESULTS: We identified PROP1 mutations in 10 index cases. Three mutations were novel: one affecting the initiation codon (c.1A>G) and two affecting splicing sites, c.109+1G>A and c.342+1G>C. The known mutations, c.150delA (p.Arg53Aspfs*112), c.218G>A (p.Arg73His), c.263T>C (p.Phe88Ser) and c.301_302delAG (p.Leu102Cysfs*8), were also detected. MLPA confirmed complete PROP1 deletion in one family. We did not identify HESX1 and LHX3 mutations by Sanger. CONCLUSION: PROP1 mutations are a prevalent cause of congenital CPHD with OPP, and therefore, PROP1 sequencing must be the first step of molecular investigation in patients with CPHD and OPP, especially in populations with a high frequency of PROP1 mutations. In the absence of mutations, massively parallel sequencing is a promising approach. The high prevalence and diversity of PROP1 mutations is associated with the ethnic background of this cohort.

High Frequency of MKRN3 Mutations in Male Central Precocious Puberty Previously Classified as Idiopathic.

BACKGROUND/AIMS: Recently, loss-of-function mutations in the MKRN3 gene have been implicated in the etiology of familial central precocious puberty (CPP) in both sexes. We aimed to analyze the frequency of MKRN3 mutations in boys with CPP and to compare the clinical and hormonal features of boys with and without MKRN3 mutations. METHODS: This was a retrospective review of clinical, hormonal and genetic features of 20 male patients with idiopathic CPP evaluated at an academic medical center. The entire coding regions of MKRN3, KISS1 and KISS1R genes were sequenced. RESULTS: We studied 20 boys from 17 families with CPP. All of them had normal brain magnetic resonance imaging. Eight boys from 5 families harbored four distinct heterozygous MKRN3 mutations predicted to be deleterious for protein function, p.Ala162Glyfs*14, p.Arg213Glyfs*73, p.Arg328Cys and p.Arg365Ser. One boy carried a previously described KISS1-activating mutation (p.Pro74Ser). The frequency of MKRN3 mutations among these boys with idiopathic CPP was significantly higher than previously reported female data (40 vs. 6.4%, respectively, p < 0.001). Boys with MKRN3 mutations had typical clinical and hormonal features of CPP. Notably, they had later pubertal onset than boys without MKRN3 abnormalities (median age 8.2 vs. 7.0 years, respectively, p = 0.033). CONCLUSION: We demonstrated a high frequency of MKRN3 mutations in boys with CPP, previously classified as idiopathic, suggesting the importance of genetic analysis in this group. The boys with CPP due to MKRN3 mutations had classical features of CPP, but with puberty initiation at a borderline age.

Adjuvant radiotherapy for the primary treatment of adrenocortical carcinoma: Are we offering the best?

PURPOSE: To evaluate the role of ARDT after surgical resection of ACC. MATERIALS AND METHODS: Records of patients from our institutional ACC database were retrospectively assessed. A paired comparison analysis was used to evaluate the oncological outcomes between patients treated with surgery followed by ARDT or surgery only (control). The endpoints were LRFS, RFS, and OS. A systematic review of the literature and meta-analysis was also performed to evaluate local recurrence of ACC when ARDT was used. RESULTS: Ten patients were included in each Group. The median follow-up times were 32 months and 35 months for the ARDT and control Groups, respectively. The results for LRFS (p=0.11), RFS (p=0.92), and OS (p=0.47) were similar among subsets. The mean time to present with local recurrence was significantly longer in the ARDT group compared with the control Group (419±206 days vs. 181±86 days, respectively; p=0.03). ARDT was well tolerated by the patients; there were no reports of late toxicity. The meta-analysis, which included four retrospective series, revealed that ARDT had a protective effect on LRFS (HR=0.4; CI=0.17-0.94). CONCLUSIONS: ARDT may reduce the chance and prolong the time to ACC local recurrence. However, there were no benefits for disease recurrence control or overall survival for patients who underwent this complementary therapy.

Central precocious puberty caused by mutations in the imprinted gene MKRN3.

BACKGROUND: The onset of puberty is first detected as an increase in pulsatile secretion of gonadotropin-releasing hormone (GnRH). Early activation of the hypothalamic-pituitary-gonadal axis results in central precocious puberty. The timing of pubertal development is driven in part by genetic factors, but only a few, rare molecular defects associated with central precocious puberty have been identified. METHODS: We performed whole-exome sequencing in 40 members of 15 families with central precocious puberty. Candidate variants were confirmed with Sanger sequencing. We also performed quantitative real-time polymerase-chain-reaction assays to determine levels of messenger RNA (mRNA) in the hypothalami of mice at different ages. RESULTS: We identified four novel heterozygous mutations in MKRN3, the gene encoding makorin RING-finger protein 3, in 5 of the 15 families; both sexes were affected. The mutations included three frameshift mutations, predicted to encode truncated proteins, and one missense mutation, predicted to disrupt protein function. MKRN3 is a paternally expressed, imprinted gene located in the Prader-Willi syndrome critical region (chromosome 15q11-q13). All affected persons inherited the mutations from their fathers, a finding that indicates perfect segregation with the mode of inheritance expected for an imprinted gene. Levels of Mkrn3 mRNA were high in the arcuate nucleus of prepubertal mice, decreased immediately before puberty, and remained low after puberty. CONCLUSIONS: Deficiency of MKRN3 causes central precocious puberty in humans. (Funded by the National Institutes of Health and others.).

Quality of life of patients with 46,XX and 46,XY disorders of sex development.

Disorders of sex development (DSD) result from abnormalities in the complex process of sex determination and differentiation. An important consideration to guide the assignment of social sex in newborns with ambiguous genitalia is the quality of life (QoL) of these patients in adulthood. The rarity of most DSD conditions makes it difficult to conduct a long-term follow-up of affected patients through adulthood. This review of papers on the QoL of DSD patients evaluated in developing and developed countries by qualitative and quantitative instruments revealed a large spectrum of QoL, ranging from very poor to similar to, or even better than, the normal population. A more adequate QoL was found in patients from tertiary centres, indicating that the medical care of DSD patients should be multidisciplinary and carried out by specialized teams.

Quality of life in a large cohort of adult Brazilian patients with 46,XX and 46,XY disorders of sex development from a single tertiary centre.

OBJECTIVE: Few studies have focused on the quality of life (QoL) of patients with disorders of sex development (DSD). Our aim was to evaluate QoL in DSD patients with defined diagnoses followed until adulthood in a single tertiary centre. PATIENTS AND METHODS: Adult patients with DSD (56 patients with 46,XX DSD - 49 with female social sex and 7 with male social sex as well as 88 patients with 46,XY DSD - 54 with female social sex and 34 with male social sex). MEASUREMENTS: QoL using WHOQOL-Bref questionnaire. RESULTS: Both patients with 46,XX DSD and patients with 46,XY DSD had similar QoL scores on the WHOQOL-Bref, comparable to the scores of the Brazilian general population. The chronological age at the start of treatment was negatively and significantly associated with general QoL score. Patients with male social sex DSD had better scores on the psychological domain than patients with female social sex DSD, as found in the Brazilian general population. In addition, among the 46,XY DSD group, the male social sex patients had better QoL compared with the female social sex patients. There was a positive and significant correlation between sexual performance and general QoL, although it explained only 4% of the variability of the general QoL score. The most influencing variables were general health, positive feelings and spirituality, religion and personal beliefs, each of them contributing with 18% of the variability of the general QoL score. CONCLUSION: Our large cohort of adult patients with DSD, which was followed by a multidisciplinary team in a single tertiary centre, had good QoL in adulthood; in addition, late treatment compromised the QoL of patients with DSD, whereas sexual performance has little influence on QoL.

Expression of LIN28 and its regulatory microRNAs in adult adrenocortical cancer.

OBJECTIVE: LIN28 control cells reprogramming and pluripotency mainly through miRNA regulation and has been overexpressed in many advanced cancers. In this study, we evaluated the prognostic role of LIN28 and its regulatory miRNAs in a large cohort of adrenocortical tumours (ACTs). PATIENTS AND METHODS: LIN28 protein expression was assessed in 266 adults ACTs (78 adenomas and 188 carcinomas) from Brazil and Germany. LIN28A and LIN28B gene expression was analysed in 59 ACTs (31 adenomas and 28 carcinomas) and copy number variation in 39 ACTs. In addition, we determined the expression of let-7 family, mir-9, mir-30 and mir-125 in 28 carcinomas. RESULTS: LIN28A gene was overexpressed in aggressive ACCs when compared with adenomas and nonaggressive ACCs, but no LIN28A copy number variation was found in ACTs. Unexpectedly, weak LIN28 protein expression was significantly associated with reduced disease-free survival in ACC patients (P = 0·01), but for overall survival only a trend was detectable (P = 0·117). In the multivariate analysis, only Ki67 index ≥10% (HR 4·6, P = 0·000) and weak LIN28 protein expression (HR 2·0, P = 0·03) were independent predictors of recurrence in ACC patients. Interestingly, mir-9 expression, a negative LIN28A/B regulator, was significantly higher in aggressive than in nonaggressive ACCs [2076 (from 36 to 9307) vs 133·4 (from 2·4 to 5193); P = 0·011] and was highly associated with reduced overall (P = 0·01) and disease-free survival (P = 0·01). However, mir-9 prognostic role should be further evaluated in a larger cohort. CONCLUSION: Weak LIN28 protein expression was associated with recurrence in ACCs. Additionally, overexpression of mir-9, a negative LIN28A regulator, was associated with poor outcome.