CINTIA TUSSET
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- Mutational analysis of TAC3 and TACR3 genes in patients with idiopathic central pubertal disorders(2012) TUSSET, Cintia; NOEL, Sekoni D.; TRARBACH, Ericka B.; SILVEIRA, Leticia F. G.; JORGE, Alexander A. L.; BRITO, Vinicius N.; CUKIER, Priscila; SEMINARA, Stephanie B.; MENDONCA, Berenice B. de; KAISER, Ursula B.; LATRONICO, Ana ClaudiaObjective: To investigate the presence of variants in the TAC3 and TACR3 genes, which encode NKB and its receptor (NK3R), respectively, in a large cohort of patients with idiopathic central pubertal disorders. Subjects and methods: Two hundred and thirty seven patients were studied: 114 with central precocious puberty (CPP), 73 with normosmic isolated hypogonadotropic hypogonadism (IHH), and 50 with constitutional delay of growth and puberty (CDGP). The control group consisted of 150 Brazilian individuals with normal pubertal development. Genomic DNA was extracted from peripheral blood and the entire coding region of both TAC3 and TACR3 genes were amplified and automatically sequenced. Results: We identified one variant (p.A63P) in NKB and four variants, p.G18D, p.L58L (c.172C > T), p.W275* and p.A449S in NK3R, which were absent in the control group. The p.A63P variant was identified in a girl with CPP, and p.A449S in a girl with CDGP. The known p.G18D, p.L58L, and p.W275* variants were identified in three unrelated males with normosmic IHH. Conclusion: Rare variants in the TAC3 and TACR3 genes were identified in patients with central pubertal disorders. Loss-of-function variants of TACR3 were associated with the normosmic IHH phenotype.
article 8 Citação(ões) na Scopus Clinical and molecular aspects of congenital isolated hypogonadotropic hypogonadism(2011) TUSSET, Cintia; TRARBACH, Ericka B.; SILVEIRA, Leticia Ferreira Gontijo; BENEDUZZI, Daiane; MONTENEGRO, Luciana; LATRONICO, Ana ClaudiaCongenital isolated hypogonadotropic hypogonadism (IHH) is characterized by partial or complete lack of pubertal development due to defects in migration, synthesis, secretion or action of gonadotropin-releasing hormone (GnRH). Laboratory diagnosis is based on the presence of low levels of sex steroids, associated with low or inappropriately normal levels of pituitary gonadotropins (LH and FSH). Secretion of other pituitary hormones is normal, as well magnetic resonance imaging of the hypothalamohypophyseal tract, which shows absence of an anatomical defects. When IHH is associated with olfactory abnormalities (anosmia or hyposmia), it characterizes Kallmann syndrome. A growing list of genes is involved in the etiology of IHH, suggesting the heterogeneity and complexity of the genetic bases of this condition. Defects in olfactory and GnRH neuron migration are the etiopathogenic basis of Kallmann syndrome. Mutations in KAL1, FGFR1/FGF8, PROK2/PROKR2, NELF, CHD7, HS6ST1 and WDR11 are associated with defects in neuronal migration, leading to Kallmann syndrome. Notably, defects in FGFR1, FGF8, PROKR2, CHD7 and WDR11 are also associated with IHH, without olfactory abnormalities (normosmic IHH), although in a lower frequency. Mutations in KISS1R, TAC3/TACR3 and GNRH1/GNRHR are described exclusively in patients with normosmic IHH. In this paper, we reviewed the clinical, hormonal and genetic aspects of IHH. Arq Bras Endocrinol Metab. 2011;55(8):501-11- Absence of Functional LIN28B Mutations in a Large Cohort of Patients with Idiopathic Central Precocious Puberty(2012) SILVEIRA-NETO, Acacio P.; LEAL, Leticia Ferro; EMERMAN, Amy B.; HENDERSON, Katherine D.; PISKOUNOVA, Elena; HENDERSON, Brian E.; GREGORY, Richard I.; SILVEIRA, Leticia F. Gontijo; HIRSCHHORN, Joel N.; NGUYEN, Thutrang T.; BENEDUZZI, Daiane; TUSSET, Cintia; REIS, Ana Claudia S.; BRITO, Vinicius N.; MENDONCA, Berenice B.; PALMERT, Mark R.; ANTONINI, Sonir R.; LATRONICO, Ana ClaudiaAim: To investigate LIN28B gene variants in children with idiopathic central precocious puberty (CPP). Patients and Methods: We studied 178 Brazilian children with CPP (171 girls, 16.8% familial cases). A large multiethnic group (1,599 subjects; Multiethnic Cohort, MEC) was used as control. DNA analysis and biochemical in vitro studies were performed. Results: A heterozygous LIN28B variant, p. H199R, was identified in a girl who developed CPP at 5.2 years. This variant was absent in 310 Brazilian control individuals, but it was found in the same allele frequency in women from the MEC cohort, independent of the age of menarche. Functional studies revealed that when ectopically expressed in cells, the mutant protein was capable of binding pre-let-7 microRNA and inhibiting let-7 expression to the same extent as wild-type Lin28B protein. Other rare LIN28B variants (p.P173P, c.198+32_33delCT, g.9575731A>C and c.-11C>T) were identified in CPP patients and controls. Therefore, no functional mutation was identified. Conclusion: In vitro studies revealed that the rare LIN28B p.H199R variant identified in a girl with CPP does not affect the Lin28B function in the regulation of let-7 expression. Although LIN28B SNPs were associated with normal pubertal timing, rare variations in this gene do not seem to be commonly involved in the molecular pathogenesis of CPP.
- New genetic factors implicated in human GnRH-dependent precocious puberty: The role of kisspeptin system(2011) TELES, Milena Gurgel; SILVEIRA, Leticia Ferreira Gontijo; TUSSET, Cintia; LATRONICO, Ana ClaudiaHuman puberty is triggered by the reemergence of GnRH pulsatile secretion with progressive activation of the gonadal function. A number of genes have been identified in the complex regulatory neuroendocrine network that controls puberty initiation. KISS1 and KISS1R genes, which encode kisspeptin and its cognate receptor, respectively, are considered crucial factors for acquisition of normal reproductive function. Recently, rare missense mutations and single nucleotide polymorphisms (SNPs) of the kisspeptin system were associated with puberty onset. Two gain-of-function mutations of the KISS1 and KISS1R genes were implicated in the pathogenesis of GnRH-dependent precocious puberty, previously considered idiopathic. These discoveries have yielded significant insights into the physiology and pathophysiology of this important life transition time. Here, we review the current molecular defects that are implicated in human GnRH-dependent precocious puberty.
- Combined use of multiplex ligation-dependent probe amplification and automatic sequencing for identification of KAL1 defects in patients with Kallmann syndrome(2013) MONTENEGRO, Luciana Ribeiro; SILVEIRA, Leticia F. G.; TUSSET, Cintia; CASTRO, Margaret de; VERSIANI, Beatriz R.; LATRONICO, Ana Claudia; MENDONCA, Berenice Bilharinho; TRARBACH, Ericka B.Objective: To investigate the role of KAL1 abnormalities in Brazilian patients with Kallmann syndrome. Design: In vitro experiments. Setting: Academic medical center. Patient(s): One hundred fifteen Brazilian patients (98 men) with Kallmann syndrome. Intervention(s): Peripheral blood leukocytes were used to obtain DNA. Main Outcome Measure(s): Direct sequencing and multiplex ligation-dependent probe amplification were used to identify KAL1 abnormalities. Result(s): We identified four KAL1 mutations (p.Met1?, p.Ala33Glyfs, p.Arg257*, and p.Trp462*) and two multiple exon deletions (exons 1-2 and 3-14) in six new male patients. Overall, 17 KAL1 defects (14.8%) were identified in the entire cohort of patients with Kallmann syndrome, including previously studied cases. KAL1-mutated patients presented with a more severe reproductive and nonreproductive phenotype (synkinesia, renal malformations, cryptorchidism, and anatomic olfactory abnormalities) in comparison with patients without KAL1 mutations. Intragenic deletions were one of the most often encountered defects (29.4%). These deletions can be missed by polymerase chain reaction (PCR) due to Yq11.2 KAL1 pseudogene (KALP) spurious amplification. Conclusion(s): These results indicate that intragenic multiexon deletions are one of the most frequent KAL1 abnormalities, which can be more accurately detected by multiplex ligation-dependent probe amplification. In addition, KAL1 sequencing results should be interpreted with caution, and stringency conditions of the PCR reaction should be adjusted to avoid pseudogene amplification. (C) 2013 by American Society for Reproductive Medicine.
- Mutational analysis of the necdin gene in patients with congenital isolated hypogonadotropic hypogonadism(2011) BENEDUZZI, Daiane; IYER, Anita K.; TRARBACH, Ericka Barbosa; SILVEIRA-NETO, Acacio P.; SILVEIRA, Leticia G.; TUSSET, Cintia; YIP, Kathleen; MENDONCA, Berenice B.; MELLON, Pamela L.; LATRONICO, Ana ClaudiaContext: 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.
- TACR3 mutations disrupt NK3R function through distinct mechanisms in GnRH-deficient patients(2014) NOEL, Sekoni D.; ABREU, Ana Paula; XU, Shuyun; MUYIDE, Titilayo; GIANETTI, Elena; TUSSET, Cintia; CARROLL, Jessica; LATRONICO, Ana Claudia; SEMINARA, Stephanie B.; CARROLL, Rona S.; KAISER, Ursula B.Neurokinin B (NKB) and its G-protein-coupled receptor, NK3R, have been implicated in the neuroendocrine control of GnRH release; however, little is known about the structure-function relationship of this ligand-receptor pair. Moreover, loss-of-function NK3R mutations cause GnRH deficiency in humans. Using missense mutations in NK3R we previously identified in patients with GnRH deficiency, we demonstrate that Y256H and Y315C NK3R mutations in the fifth and sixth transmembrane domains (TM5 and TM6), resulted in reduced whole-cell (79.3 +/- 7.2%) or plasma membrane (67.3 +/- 7.3%) levels, respectively, compared with wild-type (WT) NK3R, with near complete loss of inositol phosphate (IP) signaling, implicating these domains in receptor trafficking, processing, and/or stability. We further demonstrate in a FRET-based assay that R295S NK3R, in the third intracellular loop (IL3), bound NKB but impaired dissociation of G(q)-protein subunits from the receptor compared with WT NK3R, which showed a 10.0 +/- 1.3% reduction in FRET ratios following ligand binding, indicating activation of G(q)-protein signaling. Interestingly, R295S NK3R, identified in the heterozygous state in a GnRH-deficient patient, also interfered with dissociation of G proteins and IP signaling from wild-type NK3R, indicative of dominant-negative effects. Collectively, our data illustrate roles for TM5 and TM6 in NK3R trafficking and ligand binding and for IL3 in NK3R signaling.-Noel, S. D., Abreu, A. P., Xu, S., Muyide, T., Gianetti, E., Tusset, C., Carroll, J., Latronico, A. C., Seminara, S. B., Carroll, R. S., Kaiser, U. B. TACR3 mutations disrupt NK3R function through distinct mechanisms in GnRH-deficient patients.