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B. B. Mendonça, M. A. Zogno, B. L. Wajchenberg, D. Giannella-Neto and S. P. A. Toledo

Abstract. X-chromatin body (XCB) frequencies in oral mucosa were investigated in 14 female patients with the simple non-salt-losing form of congenital virilizing adrenal hyperplasia (CVAH) due to 'partial' 21-hydroxylase deficiency (CVAH-type I), using toluidine blue (TBS) and Feulgen's stains (FS).

They were found to be lower than normal controls by both TBS (12.6 ± 1.73 vs 18.38 ± 0.41) and FS (13.60 ± 2.16 vs 17.94 ± 0.76) methods. After 4–5 weeks on glucocorticoid therapy, XCB frequencies overlapped with those of normal controls using both TBS (16.54 ± 1.76) and FS (17.09 ± 1.98). Karyotypes performed in 8 of the 14 cases showed a normal female pattern.

Two possible interpretations are proposed: First, variations of XCB counts may reflect the average nuclear size of the cell populations studied. Second, high androgen levels found in untreated cases with CVAH-type I might lead to partial chromosome depiralization and increasing euchromatic areas. At present the first possibility appears more likely.

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M G Teles, E B Trarbach, S D Noel, G Guerra-Junior, A Jorge, D Beneduzzi, S D Bianco, A Mukherjee, M T Baptista, E M Costa, M De Castro, B B Mendonça, U B Kaiser and A C Latronico

Context

Loss-of-function mutations of the kisspeptin-1 receptor gene, KISS1R, have been identified in patients with normosmic isolated hypogonadotropic hypogonadism (nIHH).

Objective

To investigate KISS1R defects in patients with absent or delayed puberty.

Patients

We investigated KISS1R gene defects in a cohort of 99 Brazilian patients with nIHH or constitutional delay of puberty (CDP).

Methods

The entire coding region of KISS1R was amplified by PCR followed by automatic sequencing. In addition, screening for KISS1R exonic deletions was performed by multiplex ligation-dependent probe amplification.

Results

One novel homozygous KISS1R mutation was identified in two siblings with nIHH. This variant was an insertion/deletion (indel) mutation characterized by the deletion of three nucleotides (GCA) at position −2 to −4, and by the insertion of seven nucleotides (ACCGGCT) at the same position, within the 3′ splice acceptor site of intron 2 of KISS1R. The brothers who carried this KISS1R mutation had no clinical evidence of pubertal development at the ages of 14 and 20 years. Computational analysis of this indel mutation predicted the generation of an abnormal protein. In addition, a new heterozygous KISS1R variant (p.E252Q) was identified in a male patient with sporadic nIHH. However, in vitro studies of this variant did not demonstrate functional impairment. Only known polymorphisms were identified in patients with CDP.

Conclusion

Loss-of-function mutations of KISS1R represents a rare cause of nIHH, and was absent in patients with CDP. We have described a novel KISS1R homozygous splice acceptor site mutation in the familial form of nIHH.

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Daiane Beneduzzi, Anita K Iyer, Ericka Barbosa Trarbach, Acacio P Silveira-Neto, Letícia G Silveira, Cintia Tusset, Kathleen Yip, Berenice B Mendonça, Pamela L Mellon and Ana Claudia Latronico

Context

Necdin activates GNRH gene expression and is fundamental for the development, migration, and axonal extension of murine GNRH neurons. In humans, necdin plays a potential role in the hypogonadotropic hypogonadism phenotype in patients with Prader–Willi syndrome.

Aim

To investigate necdin gene (NDN) variants in patients with isolated hypogonadotropic hypogonadism (IHH).

Patients and methods

We studied 160 Brazilian patients with IHH, which includes 92 with Kallmann syndrome and 68 with normosmic IHH. Genomic DNA was extracted and the single NDN exon was amplified and sequenced. To measure GNRH transcriptional activity, luciferase reporter plasmids containing GNRH regulatory regions were transiently transfected into GT1-7 cells in the presence and absence of overexpressed wild-type or mutant necdin.

Results

A heterozygous variant of necdin, p.V318A, was identified in a 23-year-old male with Kallmann syndrome. The p.V318A was also present in affected aunt and his father and was absent in 100 Brazilian control subjects. Previous FGFR1 gene analysis revealed a missense mutation (p.P366L) in this family. Functional studies revealed a minor difference in the activation of GNRH transcription by mutant protein compared with wild type in that a significant impairment of the necdin protein activity threshold was observed.

Conclusion

A rare variant of necdin (p.V318A) was described in a family with Kallmann syndrome associated with a FGFR1 mutation. Familial segregation and in vitro analysis suggested that this non-synonymous variant did not have a direct causative role in the hypogonadism phenotype. NDN mutations are not a frequent cause of congenital IHH.

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Luciana R Montenegro, Andrea C Leal, Debora C Coutinho, Helena P L Valassi, Mirian Y Nishi, Ivo J P Arnhold, Berenice B Mendonca and Alexander A L Jorge

Background

Hypomethylation of the paternal imprinting center region 1 (ICR1) is the most frequent molecular cause of Silver–Russell syndrome (SRS). Clinical evidence suggests that patients with this epimutation have mild IGF1 insensitivity.

Objective

To assess in vitro IGF1 action in fibroblast culture from a patient with SRS and IGF1 insensitivity.

Methods

Fibroblast cultures from one patient with SRS due to ICR1 demethylation and controls were established. The SRS patient has severe growth failure, elevated IGF1 level, and poor growth rate during human recombinant GH treatment. IGF1 action was assessed by cell proliferation, AKT, and p42/44-MAPK phosphorylation. Gene expression was determined by real-time PCR.

Results

Despite normal IGF1R sequence and expression, fibroblast proliferation induced by IGF1 was 50% lower in SRS fibroblasts in comparison with controls. IGF1 and insulin promoted a p42/44-MAPK activation in SRS fibroblasts 40 and 36%, respectively, lower than that in control fibroblasts. On the other hand, p42/44-MAPK activation induced by EGF stimulation was only slightly reduced (75% in SRS fibroblasts in comparison with control), suggesting a general impairment in MAPK pathway with a greater impairment of the stimulation induced by insulin and IGF1 than by EGF. A PCR array analysis disclosed a defect in MAPK pathway characterized by an increase in DUSP4 and MEF2C gene expressions in patient fibroblasts.

Conclusion

A post-receptor IGF1 insensitivity was characterized in one patient with SRS and ICR1 hypomethylation. Although based on one unique severely affected patient, these results raise an intriguing mechanism to explain the postnatal growth impairment observed in SRS patients that needs confirmation in larger cohorts.

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Luiz Roberto Salgado, Maria Candida B Villares Fragoso, Mirta Knoepfelmacher, Marcio Carlos Machado, Sorahia Domenice, Maria Adelaide Albergaria Pereira and Berenice Bilharinho de Mendonça

Objective: Ectopic ACTH syndrome (EAS) occurs in about 5–10% of all patients with ACTH-dependent hypercortisolism with most of them caused by intrathoracic neoplasms. It may be associated with overt malignancies or with occult and indolent tumors. We assessed the accuracy of dynamic tests, inferior petrosal sinus sampling (IPSS) using desmopressin, and imaging in the work-up diagnosis of EAS.

Design and subjects: Tumor markers, imaging, and outcome data from 25 patients (13F/12M) aged 18–72 years. High dexamethasone suppression test (HDDST), desmopressin test, GHRP-6 test, corticotropin-releasing hormone (CRH) test, IPSS, computed tomography (CT), magnetic resonance imaging (MRI), and 111In-pentetreotide scintigraphy were revised.

Results: In 5 out of 20 patients HDDST was positive. In 13 patients who underwent desmopressin test, ACTH- and cortisol-positive responses were seen in six and five patients respectively. GHRP-6 test was positive in two out of three cases. Two patients underwent CRH test with negative response. In the seven patients submitted to IPSS using desmopressin in six of them, none had ACTH gradients. CT was positive in 15 out of 21 patients and MRI in 8 out of 17 cases. 111In-pentetreotide scintigraphy was positive in three out of five patients. Fourteen patients had intrathoracic tumors, five had pheochromocytomas, three had pancreatic tumors, one had a glomic tumor, and had three occult tumors. Six out of 11 patients with metastasis died and 3 others without metastasis died.

Conclusions: IPSS with desmopressin was helpful for differential diagnosis. Patients initially harboring occult carcinoids may also exhibit severe hypercortisolism and those harboring tymic carcinoids had poor prognoses when compared with bronchial carcinoids and pheocromocytomas.

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V R V Yance, J A M Marcondes, M P Rocha, C R G Barcellos, W S Dantas, A F A Avila, R H Baroni, F M Carvalho, S A Y Hayashida, B B Mendonca and S Domenice

Background

The presence of virilizing signs associated with high serum androgen levels in postmenopausal women is rare. Virilizing ovarian tumors (VOTs) and ovarian stromal hyperthecosis (OH) are the most common etiologies in virilized postmenopausal women. The differential diagnosis between these two conditions is often difficult.

Objective

To evaluate the contribution of clinical features, hormonal profiles and radiological studies to the differential diagnosis of VOT and OH.

Design

A retrospective study.

Setting

A tertiary center.

Main outcome measures

Clinical data, hormonal status (T, E2, LH and FSH), pelvic images (transvaginal sonography and MRI) and anatomopathology were reviewed.

Patients

Thirty-four postmenopausal women with a diagnosis of VOT (13 women) and OH (21 women) were evaluated retrospectively.

Results

Clinical signs of hyperandrogenism were more prevalent in the VOT group than the OH group. Although the VOT group showed higher T and E2 levels and lower gonadotropin levels than the OH group, a great overlap occurred among the hormone levels. A pelvic MRI provided an accurate differentiation of these two conditions.

Conclusion

In this group of patients, the main features contributing to the differential diagnosis of VOT and OH were serum levels of testosterone and gonadotropins and the presence of an ovarian nodule identified on the MRI. Although the association of clinical, hormonal and radiological features contributes to the differential diagnosis of these two conditions, histopathological analysis remains the gold standard for the diagnosis of ovarian hyperandrogenism in postmenopausal women.

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Patricia N Pugliese-Pires, Jean-Philippe Fortin, Thais Arthur, Ana Claudia Latronico, Berenice B Mendonca, Sandra Mara F Villares, Ivo J P Arnhold, Alan S Kopin and Alexander A L Jorge

Background

A limited number of mutations in the GH secretagogue receptor gene (GHSR) have been described in patients with short stature.

Objective

To analyze GHSR in idiopathic short stature (ISS) children including a subgroup of constitutional delay of growth and puberty (CDGP) patients.

Subjects and methods

The GHSR coding region was directly sequenced in 96 independent patients with ISS, 31 of them with CDGP, in 150 adults, and in 197 children with normal stature. The pharmacological consequences of GHSR non-synonymous variations were established using in vitro cell-based assays.

Results

Five different heterozygous point variations in GHSR were identified (c.−6 G>C, c.251G>T (p.Ser84Ile), c.505G>A (p.Ala169Thr), c.545 T>C (p.Val182Ala), and c.1072G>A (p.Ala358Thr)), all in patients with CDGP. Neither these allelic variants nor any other mutations were found in 694 alleles from controls. Functional studies revealed that two of these variations (p.Ser84Ile and p.Val182Ala) result in a decrease in basal activity that was in part explained by a reduction in cell surface expression. The p.Ser84Ile mutation was also associated with a defect in ghrelin potency. These mutations were identified in two female patients with CDGP (at the age of 13 years, their height SDS were −2.4 and −2.3). Both patients had normal progression of puberty and reached normal adult height (height SDS of −0.7 and −1.4) without treatment.

Conclusion

This is the first report of GHSR mutations in patients with CDGP. Our data raise the intriguing possibility that abnormalities in ghrelin receptor function may influence the phenotype of individuals with CDGP.

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Daniel F de Carvalho, Mirela C Miranda, Larissa G Gomes, Guiomar Madureira, José A M Marcondes, Ana Elisa C Billerbeck, Andresa S Rodrigues, Paula F Presti, Hilton Kuperman, Durval Damiani, Berenice B Mendonca and Tania A S S Bachega

Background

Most congenital adrenal hyperplasia (CAH) patients carry CYP21A2 mutations derived from conversion events involving the pseudogene, and the remaining carry new mutations.

Objective

To review causal mutations and genotype–phenotype correlation in 480 Brazilian patients.

Methods

DNA was extracted from 158 salt-wasters (SWs), 116 simple virilizing (SV), and 206 nonclassical (NC) patients. Fourteen point mutations were screened by allele-specific PCR, large rearrangements by Southern blotting/MLPA, and sequencing was performed in those with incomplete genotype. The gene founder effect was analyzed by microsatellite studies. Patients were divided into six genotypes (Null; A: <2%; B: 3–7%; C: >20% of residual enzymatic activity (EA); D: unknown EA; E: incomplete genotype).

Results

Targeted methodologies defined genotype in 87.6% of classical and in 80% of NC patients and the addition of sequencing in 100 and 83.5%, respectively. The most frequent mutations were p.V281L (26.6% of alleles), IVS2-13A/C>G (21.1%), and p.I172N (7.5%); seven rare mutations and one novel mutation (p.E351V) were identified. Gene founder effect was observed in all but one (p.W19X) mutation. Null, A, B, and C genotypes correlated with SW (88%), SW (70%), SV (98%), and NC forms (100%), respectively. In group D, the p.E351V mutation correlated with classical form and group E comprised exclusively NC-patients. ACTH-stimulated 17OHP level of 44.3ng/mL was the best cutoff to identify NC-patients carrying severe mutations.

Conclusions

We identified a good genotype–phenotype correlation in CAH, providing useful data regarding prediction of disease’s severity; moreover, we suggest that ACTH-stimulated 17OHP levels could predict carrier status for severe mutations. Sequencing is essential to optimize molecular diagnosis in Brazilian CAH patients.

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Maria Candida B V Fragoso, Madson Queiroz Almeida, Tania L Mazzuco, Beatriz M P Mariani, Luciana P Brito, Talita Cardoso Gonçalves, Guilherme A Alencar, Lorena de O Lima, Andre M Faria, Isabelle Bourdeau, Antonio M Lucon, Daniel S Freire, Ana Claudia Latronico, Berenice B Mendonca, Andre Lacroix and Antonio M Lerario

Background

A recent microarray study identified a set of genes whose combined expression patterns were predictive of poor outcome in a cohort of adult adrenocortical tumors (ACTs). The difference between the expression values measured by qRT-PCR of DLGAP5 and PINK1 genes was the best molecular predictor of recurrence and malignancy. Among the adrenocortical carcinomas, the combined expression of BUB1B and PINK1 genes was the most reliable predictor of overall survival. The prognostic and molecular heterogeneity of ACTs raises the need to study the applicability of these molecular markers in other cohorts.

Objective

To validate the combined expression of BUB1B, DLGAP5, and PINK1 as outcome predictor in ACTs from a Brazilian cohort of adult and pediatric patients.

Patients and methods

BUB1B, DLGAP5, and PINK1 expression was assessed by quantitative PCR in 53 ACTs from 52 patients – 24 pediatric and 28 adults (one pediatric patient presented a bilateral asynchronous ACT).

Results

DLGAP5–PINK1 and BUB1B–PINK1 were strong predictors of disease-free survival and overall survival, respectively, among adult patients with ACT. In the pediatric cohort, these molecular predictors were only marginally associated with disease-free survival but not with overall survival.

Conclusion

This study confirms the prognostic value of the combined expression of BUB1B, DLGAP5, and PINK1 genes in a Brazilian group of adult ACTs. Among pediatric ACTs, other molecular predictors of outcome are required.

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F P Paranhos-Neto, L Vieira Neto, M Madeira, A B Moraes, L M C Mendonça, I C B Lima, C L R Chagas, D A Lira, J F Spitz, J A M Guimarães, M E L Duarte and M L F Farias

Introduction

The role of vitamin D on bone microarchitecture and fragility is not clear.

Objective

To investigate whether vitamin D deficiency (25(OH)D <20 ng/mL) increases cortical bone loss and the severity of fractures.

Design

Cross-sectional study of 287 elderly women with at least one prevalent low-impact fracture.

Methods

Biochemistry, X-rays to identify vertebral fractures (VFs) and to confirm non-vertebral fractures (NonVFs), and high-resolution peripheral quantitative computed tomography (HR-pQCT) to evaluate bone microstructure.

Results

Serum 25(OH)D levels were associated with body mass index (BMI: r = −0.161, P = 0.006), PTH (r = −0.165; P = 0.005), CTX (r = −0.119; P = 0.043) and vBMD at cortical bone (Dcomp: r = 0.132; P = 0.033) and entire bone (D100: r = 0.162 P = 0.009) at the distal radius, but not at the tibia. Age and PTH levels were potential confounding variables, but in the multiple linear regressions only BMI (95% CI: 0.11–4.16; P < 0.01), 25(OH)D (95% CI: −0.007 to 1.70; P = 0.05) and CTX (95% CI: −149.04 to 21.80; P < 0.01) predicted Dcomp, while BMI (95% CI: 1.13–4.18; P < 0.01) and 25(OH)D (95% CI: 0.24–1.52; P < 0.01) predicted D100. NonVFs predominated in patients with 25(OH)D <20 ng/mL (P = 0.013). Logistic regression analysis showed a decrease in the likelihood of presenting grade 2–3 VFs/NonVFs for every increase in 25(OH)D (OR = 0.962, 95% CI: 0.940–0.984; P = 0.001), BMI (OR = 0.932, 95% CI: 0.885–0.981; P = 0.007) and D100 at radius (OR = 0.994, 95% CI: 0.990–0.998; P = 0.005).

Conclusion

In elderly patients with prevalent fractures, vitamin D deficiency was associated with cortical bone loss and severity of fractures.