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| Author(s): |
Ana Paula de Abreu e Silva
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD) |
| Defense date: | 2011-01-14 |
| Examining board members: |
Ana Claudia Latrônico Xavier;
Júlio Zaki Abucham Filho;
Ivo Jorge Prado Arnhold;
Elaine Maria Frade Costa;
Gil Guerra Júnior
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| Advisor: | Ana Claudia Latrônico Xavier |
| Abstract | |
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 (AU) | |