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Doctoral Thesis
DOI
https://doi.org/10.11606/T.5.2011.tde-18022011-134611
Document
Author
Full name
Ana Paula de Abreu e Silva
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2011
Supervisor
Committee
Xavier, Ana Claudia Latrônico (President)
Abucham Filho, Júlio Zaki
Arnhold, Ivo Jorge Prado
Costa, Elaine Maria Frade
Guerra Júnior, Gil
Title in Portuguese
Mutações inativadoras dos genes PROK2 e PROKR2 em pacientes com hipogonadismo hipogonadotrófico isolado
Keywords in Portuguese
Hormônio liberador de gonadotropina
Mutações
Neurogênese do bulbo olfatório
Procineticina 2
Receptor tipo 2 da procineticina
Síndrome de Kallmann hipogonadismo
Abstract in Portuguese
O sistema da procineticina desempenha um papel importante na migração dos neurônios secretores de GnRH e na neurogênese do bulbo olfatório. Camundongos com ablação dos genes que codificam a procineticina 2 (PROK2) e seu receptor (PROKR2) apresentaram fenótipos semelhantes ao da síndrome de Kallmann descrita em humanos. Mutações inativadoras nos genes PROK2 e PROKR2 foram identificadas em pacientes com hipogonadismo hipogonadotrófico isolado. Com base nestes achados, investigamos a presença de alterações estruturais nos genes PROK2 e PROKR2 em 107 pacientes brasileiros (63 com síndrome de Kallmann e 47 com hipogonadismo hipogonadotrófico isolado normósmico). Cem indivíduos brasileiros que relataram desenvolvimento puberal normal foram utilizados como grupo controle. As regiões codificadoras dos genes PROK2 e PROKR2 foram amplificadas utilizando-se oligonucleotídeos intrônicos específicos, seguida de purificação enzimática e sequenciamento automático. Duas mutações no gene PROK2 foram identificadas: a mutação p.G100fsX121 em homozigose presente em dois irmãos com síndrome de Kallmann; e a mutação p.I55fsX56 em heterozigose identiificada em um paciente com HHIn. Quatro mutações foram identificadas no gene PROKR2 (p.R80C, p.Y140X, p.L173R e p.R268C) em cinco pacientes com síndrome de Kallmann e um paciente com HHIn. Essas mutações não foram encontradas no grupo controle. As mutações do tipo missense, p.R80C, p.L173R e p.R268C foram identificadas em heterozigose. Mutações nos genes FGFR1, GnRHR, KiSS-1 e GPR54 foram excluídas nesses pacientes. O paciente portador da mutação p.R268C do PROKR2 apresentou deleção dos exons 1 e 2 do gene KAL1. Adicionalmente, as mutações p.R80C e p.R268C foram identificadas em heterozigose em parentes de primeiro grau assintomáticos dos casos índices. A nova mutação p.Y140X do PROKR2, única alteração em homozigose, foi identificada em um paciente com micropênis, criptorquidia bilateral, anosmia e palato ogival. Os pais deste paciente eram portadores da mutação p.Y140X em heterozigose e relataram desenvolvimento puberal normal e ausência de anormalidades olfatórias. Estudos in vitro da nova mutação p.R80C localizada na primeira alça intracelular demonstraram que o acúmulo de fofatidil-inositol (IP), assim como a ativação da via da MAPK foram significativamente afetadas em células transfectadas com o receptor mutado em relação ao receptor selvagem, indicando que a mutação p.R80C determina uma menor atividade do receptor. Avaliação da expressão por Western blot mostrou uma diminuição na expressão do receptor mutado R80C e uma maior expressão de receptores imaturos. Esses achados sugeriram o papel crítico da arginina localizada na posição 80 na atividade normal do receptor. Em conclusão, expandimos o repertório de mutações deletérias nos genes PROK2 e PROKR2 em pacientes com hipogonadismo hipogonadotrófico isolado. A haploinsuficiência do PROKR2 não foi suficiente para causar síndrome de Kallmann ou HHIn, entretanto mutações inativadoras em homozigose nos genes PROK2 e PROKR2 foram responsáveis pelo fenótipo reprodutivo e olfatório anormal, em concordância com os estudos prévios de ablação gênica em modelos animais. Arginina localizada na posição 80 do PROKR2 desempenha um papel crucial na adequada maturação do receptor
Title in English
PROK2 and PROKR2 inactivating mutations in patients with idiopathic hypogonadotropic hypogonadism
Keywords in English
Kallmann syndrome hypogonadism
Mutations
Neurogenesis of olfactory bulb
Prokineticin 2
Prokineticin receptor 2
Abstract in English
Physiological activation of the prokineticin pathway has a critical role in olfactory bulb morphogenesis and GnRH secretion. Knock-out mice for genes that encode prokineticin 2 (PROK2) and the prokineticin receptor 2 (PROKR2) exhibited a phenotype similar to the Kallmann syndrome (KS). Inactivating mutations in PROK2 and PROKR2 have been identified in patients with isolated hypogonadotropic hypogonadism. Based on these findings, we investigated the presence of inactivating mutations of the genes PROK2 and PROKR2 in Brazilian patients with isolated hypogonadotropic hypogonadism associated or not with olfactory abnormalities and performed in vitro studies of the new identified mutations. We studied 107 patients with HH (63 with Kallmann syndrome and 44 with normosmic HH) and 100 control individuals. The coding regions of PROK2 and PROKR2 were amplified by polymerase chain reaction followed by enzymatic purification and direct automatic sequencing. In PROK2, two known frameshift mutations were identified. Two brothers with Kallmann syndrome harbored the homozygous p.G100fsX121 mutation, whereas one male with normosmic HH harbored the heterozygous p.I55fsX56 mutation. In PROKR2, four distinct mutations (p.R80C, p.Y140X, p.L173R and p.R268C) were identified in five patients with Kallmann syndrome and in one patient with normosmic HH. These mutations were not found in the control group. The p.R80C and p.R268C missense mutations were identified in heterozygous state in the HH patients and in their asymptomatic first-degree relatives. The p.L173R was also identified in heterozygous state. In addition, no mutations of FGFR1, GnRHR, KiSS-1 or GPR54 were identified in these patients. The patient with the PROKR2 mutation p.R268C also has a deletion of the exon 1 and 2 in the gene KAL1. Notably, the new nonsense mutation (p.Y140X) was identified in homozygous state in an anosmic boy with micropenis, bilateral cryptorchidism and high-arched palate. His asymptomatic parents were heterozygous for this severe defect. In vitro studies of the new mutation, p.R80C, were performed in order to access the mechanism by which this mutation could affect the activity of the PROKR2. In vitro studies showed that the amount of fofatidil-inositol (PI) and the activation of MAPK were significantly lower in cells transfected with the R80C mutant receptor than in cells transfected with the wild receptor, indicating that this variant is a loss-of-function mutation. Binding studies and Western blot showed a reduction in the expression levels of the receptor in the plasma membrane and in whole cell, respectively. Additionally, Western blot analysis of R80C PROKR2 revealed an additional smaller molecular weight band that represents the presence of immature unglycosylated receptors. The arginine 80 in ICL1 is important for post-translational processing of PROKR2. In conclusion, we expanded the repertoire of PROK2 and PROKR2 mutations in patients with HH and showed that PROKR2 haploinsufficiency is not sufficient to cause Kallmann syndrome or normosmic HH, whereas homozygous loss-of-function mutations either in PROK2 or PROKR2 are sufficient to cause disease phenotype, in accordance with the Prokr2 and Prok2 knockout mouse models. In vitro studies suggested that the arginine located at position 80 of the receptor seems to play an important role in the receptor function
 
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Publishing Date
2011-02-22
 
WARNING: The material described below relates to works resulting from this thesis or dissertation. The contents of these works are the author's responsibility.
  • Abreu, A.P.S., et al. Evidence of the importance of the first intracellular loop of prokineticin receptor 2 in receptor function [doi:10.1210/me.2012-1102]. Molecular Endocrinology (Baltimore, Md.) [online], 2012, vol. 26, p. 1417-1427.
  • Abreu, A.P.S., et al. Loss-of-function mutations in the genes encoding prokineticin-2 or prokineticin receptor-2 cause autosomal recessive kallmann syndorme [doi:10.1210/jc.2008-0958]. The Journal of Clinical Endocrinology and Metabolism [online], 2008, vol. 93, p. 4113-4118.
  • Abreu, A.P.S., et al. The role of prokineticins in the pathogenesis of hypogonadotropic hypogonadism [doi:10.1159/000308880]. Neuroendocrinology (Basel) [online], 2010, vol. 91, p. 283-290.
  • Avbelj Stefanija, M., et al. An ancient founder mutation in PROKR2 impairs human reproduction [doi:10.1093/hmg/dds264]. Human Molecular Genetics [online], 2012, vol. 21, p. 4314-4324.
  • Abreu, A.P.S., et al. A PROKR2 Mutant Exerts Dominant Negative Effects on Wild-Type PROKR2: New Insights into the Effects of PROKR2 Heterozygous Mutations. In The Endocrine Society 94th Annual Meeting, Houston, 2012. Programa do Congresso.Houston, 2012. Abstract.
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