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Developmental Endocrinology Unit (A.P.A., E.B.T., E.M.F.C., B.B.M., A.C.L.), Laboratory of Hormone and Molecular Genetic Laboratory of Medical Investigation-42, Clinical Hospital, Medical School, Sao Paulo University, 05403-900 Sao Paulo, Brazil; Department of Internal Medicine (M.d.C., B.V.), Clinical Hospital, Medical School of Ribeirao Preto University of Sao Paulo, 14049-900 Ribeirao Preto, Brazil; and Department of Internal Medicine (M.T.M.B., H.M.G.), Clinical Hospital, State University of Campinas, Campinas, 13083-970 Sao Paulo, Brazil
Address all correspondence and requests for reprints to: Ana Claudia Latronico or Ana Paula Abreu, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, Disciplina de Endocrinologia e Metabologia, Avenue Dr. Eneas de Carvalho Aguiar, 155, 2 degree andar Bloco 6, 05403-900 Sao Paulo, SP, Brasil. E-mail: anacl{at}usp.br or apaulabreu{at}yahoo.com.br.
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Abstract |
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Objective: To investigate PROK2 and PROKR2 mutations in patients with hypogonadotropic hypogonadism (HH) associated or not with olfactory abnormalities.
Design: We studied 107 Brazilian 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 PCR followed by direct automatic sequencing.
Results: 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, p.L173R, and p.R268C missense mutations were identified in the heterozygous state in the HH patients and in their asymptomatic first-degree relatives. In addition, no mutations of FGFR1, KAL1, GnRHR, KiSS-1, or GPR54 were identified in these patients. Notably, the new nonsense mutation (p.Y140X) was identified in the homozygous state in an anosmic boy with micropenis, bilateral cryptorchidism, and high-arched palate. His asymptomatic parents were heterozygous for this severe defect.
Conclusion: We expanded the repertoire of PROK2 and PROKR2 mutations in patients with HH. In addition, we show that PROKR2 haploinsufficiency is not sufficient to cause Kallmann syndrome or normosmic HH, whereas homozygous loss-of-function mutations either in PROKR2 or PROK2 are sufficient to cause disease phenotype, in accordance with the Prokr2 and Prok2 knockout mouse models.
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Introduction |
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Kallmann syndrome is a heterogeneous genetic condition that is more prevalent in males. Although most of the cases appear to be sporadic, X-linked, autosomal dominant, and recessive transmissions have been reported (1, 4). Two distinct genes, Kallmann syndrome 1 gene (KAL1) and the fibroblast growth factor receptor 1 gene (FGFR1), have been implicated in the X chromosome-linked form and an autosomal dominant form of the disease, respectively (4). However, the molecular basis of Kallmann syndrome was elucidated in only 10–20% of the reported cases (4).
New promising candidate loci for Kallmann syndrome include genes with potential influence on GnRH neuron migration and on olfactory bulb morphogenesis. Matsumoto et al. (5) have shown that the activation of the prokineticin receptor-2 (PROKR2), a G protein-coupled receptor, is essential for the normal development of the olfactory bulbs and sexual maturation. Mice homozygous for null mutations in Prokr2 recapitulate the human phenotype of Kallmann syndrome, exhibiting severe atrophy of the reproductive system and hypoplastic olfactory bulb. A similar phenotype has been reported in mice lacking one of the Prokr2 ligands, prokineticin-2 (Prok2) (6).
Mutations in PROK2 and PROKR2 have been identified in patients with Kallmann syndrome and normosmic HH; most of these patients harbored missense mutations in the heterozygous state (7). Recently, homozygous loss-of-function mutations of PROK2 were described in families with HH, suggesting an autosomal recessive inheritance disorder (6, 8). In the present study, we investigated PROK2 and PROKR2 in a cohort of Brazilian patients with HH with or without olfactory abnormalities. In addition, we performed familial segregation analyses of PROKR2 defects associated with Kallmann syndrome to elucidate the mode of inheritance of this condition.
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Patients and Methods |
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Idiopathic HH was documented based on the following criteria: clinical signs of hypogonadism, prepubertal or low testosterone or estradiol levels for age, low or inappropriately normal gonadotropin levels, normal baseline and stimulated levels of the other anterior pituitary hormones, and normal hypothalamic-pituitary imaging. Olfactory tests (Smell Identification Test, Philadelphia, PA, or Alcohol Sniff Test, San Diego, CA) and magnetic resonance imaging of the hypothalamic-pituitary region, as well as of the olfactory bulbs and tracts, were evaluated in all patients (13). Body mass index (BMI: body weight in kilograms divided by the squared stature in meters) was assessed in all patients. Normal weight was defined as BMI from 20–24.9 kg/m2, overweight from 25–29.9 kg/m2, and obesity 30 kg/m2 or more.
One hundred adult individuals of both sexes with normal sexual development at the appropriate chronological age and no history of abnormal sense of smell were used as the control group.
Hormone assays
Serum LH, FSH, testosterone, and estradiol levels were measured by fluorometric assays (Delfia, Wallac, Inc., Turku, Finland) in the majority of the studied patients. The coefficient of variation was 5% or less for all assays. The lower limits of detection were 0.6 IU/liter for LH, 1.0 IU/liter for FSH, 13 pg/ml (47 pmol/liter) for estradiol, and 19 ng/dl (0.6 nmol/liter) for testosterone. Serum LH and FSH were measured at –15, 0, 15, 30, 45, and 60 min after the iv administration of 100 µg GnRH. The results were compared with previously reported normal values established in our population (14).
DNA analysis
Genomic DNA was extracted from peripheral blood leukocytes using standard procedures. The entire coding regions of PROK2 (GenBank accession number M021935) and PROKR2 (NM_144773) were amplified by PCR and automatically sequenced. The four exons of PROK2 were amplified using primers and conditions previously described (7). The PCR amplification of the two exons of the PROKR2 was performed using the following primers: 1 foward 5'-TGAAAGAGCAGAAGGTCTGGA-3' and 1 reverse 5'-TCCTCATTTAGGCTCTGACAGG-3', 2 foward 5'-GATTCACTGTGCCACTGCTT-3' and 2 reverse 5'-CCAGTACTCAGAGCATCACCC-3'. The PCR amplifications were performed in 25-µl reaction mixtures containing 200–500 ng genomic DNA, 0.2 mM dNTPs, 1.5 mM MgCl2, 0.6 pmol each primer, 1x PCR buffer, and 1 U Go Taq DNA polymerase (Promega, Madison, WI). Amplification was performed for 35 cycles. The PCR products were electrophoresed on 1.0% agarose gel, stained with ethidium bromide, and photographed.
The PCR products of both PROK2 and PROKR2 were pretreated with an enzymatic combination of exonuclease I and shrimp alkaline phosphatase (U.S. Biochemical Corp., Cleveland, OH) and directly sequenced using the BigDye terminator cycle sequencing ready reaction kit (PE Applied Biosystems, Foster City, CA) in an ABI PRISM 310 automatic sequencer (PerkinElmer Cetus, Shelton, CT). All mutations identified were confirmed in three independent PCR products and by sequencing both DNA strands.
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Results |
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We identified a homozygous T insertion between nucleotides 297 and 298 (c.297_298insT) within exon 4 of PROK2, leading to a previously described p.G100fsX121 frameshift mutation in two brothers with Kallmann syndrome (Table 1
, patients 1 and 2) (7). Their parents were apparently nonconsanguineous. The proband was a 23-yr-old male with gynecomastia, micropenis, and anosmia. Hormonal profile revealed low testosterone levels and prepubertal basal and GnRH-stimulated gonadotropins. His brother also had anosmia and failed to go through puberty. Their asymptomatic parents were not available for molecular studies.
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). PROKR2 analysis
We identified four different defects, including one nonsense and three missense mutations, in six unrelated patients with HH (patients 4–9, Table 1). Two of these mutations, c.238C
T (p.R80C) and c.420CG (p.Y140X), are new PROKR2 alterations. The p.R80C mutation, located in the first intracellular loop of the receptor, was identified in the heterozygous state in a 21-yr-old anosmic female patient with primary amenorrhea and lack of spontaneous breast development. Her basal and GnRH-stimulated gonadotropin levels were at normal pubertal range, but associated with basal undetectable estradiol levels (patient 4, Table 1). Her mother and an 18-yr-old sister carried the same mutation in the heterozygous state (Fig. 1), and both had normal pubertal development and olfactory tests.
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). Consanguinity was denied. His basal gonadotropin and testosterone levels were at the prepubertal range. His parents were normosmic and carried the same mutation in the heterozygous state (Fig. 1).
One adult female patient (patient 6, Table 1), who carried the p.L173R mutation in the heterozygous state, had anosmia, primary amenorrhea, and lack of breast development. Subsequently, she developed metabolic syndrome characterized by obesity, type II diabetes mellitus, chronic arterial hypertension, and hypertriglyceridemia. Her mother did not have the mutation (Fig. 1). This mutation was also identified in an anosmic and obese 52-yr-old patient who referred absence of pubertal development until 25 yr of age (patient 7, Table 1).
The p.R268C mutation was identified in the heterozygous state in a 15-yr-old boy with anosmia, micropenis, and cryptorchidism. His basal gonadotropin and testosterone levels were at the prepubertal range (patient 8, Table 1). His asymptomatic father was also heterozygous for the mutation (Fig. 1). This mutation was also identified in a 42-yr-old male patient with normosmic HH (patient 9, Table 1). His testosterone levels were in the prepubertal range with normal basal gonadotropin levels measured by RIA. A GnRH stimulation test revealed prepubertal gonadotropin response.
None of the patients who harbored PROK2 or PROKR2 defects had mutations in KAL1, FGFR1, GnRHR, GPR54, or KiSS-1.
Other molecular findings
One known intronic PROK2 polymorphism (IVS3 + 14GA) and three known coding-synonymous PROKR2 polymorphisms (c.465CT, c.525CG, and c.585GC) were identified in patients with Kallmann and normosmic HH and normal controls at similar frequencies to those described in the Ensembl database.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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In the current study, we identified two known frameshift mutations (p.G100fsX121 and p.I55fsX56) in PROK2 and four nonsynonymous mutations (p.R80C; p.Y140X; p.L173R and p.R268C) in PROKR2 from a cohort of 107 Brazilian patients with sporadic and familial HH. The p.R80C and p.Y140X mutations had not been previously reported. These PROKR2 mutations were not identified in 200 alleles from control individuals. All identified mutations were located at highly conserved amino acids across species, suggesting a critical role of these residues in the normal PROK2 signaling.
The homozygous p.G100fsX121 mutation of PROK2 was identified in two Brazilian brothers with Kallmann syndrome, whereas the heterozygous p.I55fsX56 mutation was identified in a normosmic HH patient. These two PROK2 mutations have been previously described in American and French studies (6, 8). Interestingly, the p.I55fsX56 mutation was previously described in the homozygous state in three Portuguese siblings, two brothers with Kallmann syndrome and their sister with normosmic HH (6). Another asymptomatic brother was a carrier of this mutation in the heterozygous state. In vitro analysis of this truncated protein revealed lack of bioactivity in a CHO cell line that stably expressed PROKR2 (6).
The three heterozygous missense mutations of PROKR2 (p.R80C, p.L173R, and p.R268C) were identified in four patients with Kallmann syndrome. The heterozygous p.R268C mutation was also identified in a normosmic male patient with HH. Two of these mutations (p.L173R and p.R268C) had been previously described (7). Remarkably, three first-degree relatives of two patients with Kallmann syndrome also carried the p.R80C and p.R268C mutations of PROKR2 in the heterozygous state (Fig. 1). These asymptomatic carriers advocate that PROKR2 haploinsufficiency is not sufficient to cause Kallmann syndrome phenotype. Potential cryptic mutations within the regulatory or intronic regions affecting the other allele of PROKR2 gene could, however, result in a complete loss of function of the protein in affected individuals.
Two families with a phenotypic variability harboring FGFR1 missense mutations associated with NELF or GnRHR mutations have recently been described (16). Similarly, Dodé et al. (7) described a patient with Kallmann syndrome who carried a heterozygous p.L173R mutation in PROKR2 and a missense mutation in KAL1. Therefore, digenic models could account for some of the phenotypic heterogeneity seen in Kallmann syndrome and normosmic HH (7, 16). Nevertheless, previous analyses of FGFR1, KAL1, GnRHR, GPR5, or KiSS-1 in the Brazilian patients did not reveal any additional abnormalities (9, 10, 11, 12, 17).
Interestingly, a novel homozygous nonsense PROKR2 mutation (p.Y140X), located in the third transmembrane helix of the receptor was identified in an anosmic boy with micropenis, bilateral cryptorchidism and high arched palate. This p.Y140X mutation probably results in a PROKR2 with complete loss of function through the generation of an aberrant transcript that can be unstable or encodes for a truncated protein, lacking the carboxyl-terminal domain. The parents of this boy, who harbored the same nonsense mutation in the heterozygous state, had had normal pubertal development, spontaneous fertility, and no olfactory abnormalities. This further supports the idea that PROKR2 haploinsufficiency is not sufficient to cause disease phenotype.
Body weight and reproduction have been linked for decades, but the neuroendocrine circuits that serve as convergence points between these two systems are not completely identified (18). Here, we observed that three of the five Kallmann syndrome patients with PROKR2 mutations were obese and one 50-yr-old female patient who carried the heterozygous p.L173R mutation developed metabolic syndrome. Remarkably, Dodé et al. (7) reported a patient with Kallmann syndrome carrying the heterozygous p.R73C mutation of PROK2, who presented marked obesity and suffered from a severe sleep disorder. Indeed, the association between the PROK/PROKR2 system and obesity has been previously demonstrated in rodents (19). The injection of a prokineticin homolog into the arcuate nucleus of rat brains, a region with high PROKR2 expression, leads to the suppression of diurnal, nocturnal, deprivation-induced and neuropeptide Y-stimulated feeding, indicating that the activation of this system results in an anorexigenic response (19).
We expanded the repertoire of mutations of PROK2 and PROKR2 in patients with HH with or without olfactory dysfunction. In addition, the presence of heterozygous deleterious mutations involving PROKR2 in asymptomatic first-degree relatives indicates that the haploinsufficiency of these genes is not sufficient to cause HH phenotype. In contrast, the presence of biallelic deleterious mutations is apparently sufficient to cause Kallmann syndrome or normosmic HH.
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online August 5, 2008
Abbreviations: BMI, Body mass index; HH, hypogonadotropic hypogonadism; PROKR2, prokineticin receptor-2.
Received May 2, 2008.
Accepted July 28, 2008.
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