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
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.
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.
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.
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.
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
A limited number of mutations in the GH secretagogue receptor gene (GHSR) have been described in patients with short stature.
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.
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.
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.
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
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.
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).
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.
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.
Lorena Guimaraes Lima Amato, Luciana Ribeiro Montenegro, Antonio Marcondes Lerario, Alexander Augusto Lima Jorge, Gil Guerra Junior, Caroline Schnoll, Alessandra Covallero Renck, Ericka Barbosa Trarbach, Elaine Maria Frade Costa, Berenice Bilharinho Mendonca, Ana Claudia Latronico and Leticia Ferreira Gontijo Silveira
Congenital hypogonadotropic hypogonadism (CHH) is a rare condition caused by GnRH deficiency. Several genes have been associated with the pathogenesis of CHH, but most cases still remain without a molecular diagnosis. The advent of next-generation sequencing (NGS) has allowed the simultaneous genotyping of several regions, faster, making possible the extension of the genetic knowledge of CHH.
Genetic characterization of a large cohort of Brazilian CHH patients.
Design and patients
A cohort of 130 unrelated patients (91 males, 39 females) with CHH (75 normosmic CHH, 55 Kallmann syndrome) was studied using a panel containing 36 CHH-associated genes.
Potential pathogenic or probably pathogenic variants were identified in 43 (33%) CHH patients. The genes ANOS1, FGFR1 and GNRHR were the most frequently affected. A novel homozygous splice site mutation was identified in the GNRH1 gene and a deletion of the entire coding sequence was identified in SOX10. Deleterious variants in the IGSF10 gene were identified in two patients with reversible normosmic CHH. Notably, 6.9% of the patients had rare variants in more than one gene. Rare variants were also identified in SPRY4, IL17RD, FGF17, IGSF1 and FLRT3 genes.
This is a large study of the molecular genetics of CHH providing new genetic findings for this complex and heterogeneous genetic condition. NGS has been shown to be a fast, reliable and effective tool in the molecular diagnosis of congenital CHH and being able to targeting clinical genetic testing in the future.