SEARCH

SEARCH BY CITATION

Keywords:

  • Digenic inheritance;
  • p.L173R

Abstract

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

ABSTRACT: Kallmann syndrome (KS) is characterized by the association of hypogonadotropic hypogonadism and anosmia or hyposmia. To date, 4 different genes have been identified as responsible for the presence of KS; however, in many cases no mutations have been found in any of these genes. Herein, we report the molecular findings regarding the analysis of fibroblast growth factor receptor 1 (FGFR1), prokineticin receptor 2 (PROKR2), and prokineticin (PROK2) in patients with KS. Twenty-four patients with KS were studied in whom mutations in KAL1 had been investigated previously. Polymerase chain reaction products from FGFR1, PROKR2, and PROK2 were sequenced and mutations were sought in the open reading frame of the 3 genes. Two patients presented a heterozygous T-to-G transversion in exon 2 (c.518T>G) of the PROKR2, which results in a leucine-to-arginine substitution at codon 173. Our results strengthen the hypothesis of possible digenic inheritance in some patients with KS. Likewise, our data extend previous reports demonstrating that PROKR2 plays a role in the etiology of this syndrome.

Kallmann syndrome (KS) is characterized by the association of hypogonadotropic hypogonadism and anosmia or hyposmia. It is also associated with additional features such as mirror movements, renal anomalies, cleft palate, and dental agenesis (Tsai and Gill, 2006). This disorder is caused by a neural migration arrest involving both the olfactory and the gonadotropin-releasing hormone (GnRH)-producing neurons (Hardelin et al, 1993). Migration of both olfactory axons and GnRH neurons is arrested within the meninges above the cribriform plate (Schwanzel-Fukuda et al, 1989; Matsumoto et al, 2006; Pitteloud et al, 2007b).

KS affects from 1 in 10 000 individuals, and although many cases occur sporadically, X-linked recessive, autosomal dominant, and autosomal recessive modes of inheritance were documented in several families (Dodé et al, 2003). However, afterwards it was demonstrated that this entity is heterogeneous and after studying a large number of patients, in whom no mutations were found in the KAL1 gene (mutations of the KAL1 gene are the cause of KS in approximately 10% of KS patients), it was suggested that these may more likely occur in autosomal genes (Maya-Nuñez et al, 1998a,b; Oliveira et al, 2001) and that the X-linked form of the disease accounts for the minority of the cases, because it has been documented that most affected subjects present mutations in autosomal genes (Dodé et al, 2003, 2006).

To date, 4 different genes have been identified as responsible for the presence of KS: KAL1 on Xp22.3 (NCBI GeneID 3730; OMIM No. 308700) (Franco et al, 1991; Legouis et al, 1991; Hardelin et al, 1992), fibroblast growth factor receptor 1 (FGFR1, also known as KAL2) on 8p11.2–12 (NCBI GeneID 2260; OMIM No. 136350) (Dodé et al, 2003), prokineticin receptor 2 (PROKR2) on 20p12.3 (NCBI GeneID 128674; OMIM No. 607123), and prokineticin (PROK2) on 13p21.1 (NCBI GeneID 60675; OMIM No. 607002) (Dodé et al, 2006).

Several heterozygous mutations of FGFR1 have been identified in approximately 10% of individuals with KS, including missense and nonsense mutations, splice site mutations, and intragenic insertions and deletions (Dodé et al, 2003; Sato et al, 2004; Albuisson et al, 2005; Pitteloud et al, 2006). Therefore, these mutations are believed to be loss-of-function mutations and this entity results from insufficient FGFR1-mediated signaling during embryonic development (Hebert et al, 2003; Dodé and Hardelin, 2004).

Recently, mutations in PROKR2 and its main ligand, PROK2, have been associated with KS (Dodé et al, 2006; Pitteloud et al, 2007b). It has been demonstrated that both genes are important in the development of the olfactory bulb and the reproductive system in mice (Ng et al, 2005; Matsumoto et al, 2006).

Herein, we report the molecular findings regarding the analysis of the FGFR1, PROKR2,and PROK2 genes in 24 patients with KS in whom mutations in the KAL1 gene had been previously sought.

Subjects and Methods

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Patients

The study was approved by the Institute's Human Research Committee. Twenty-four unrelated male patients with proven diagnosis of KS were molecularly studied; in all cases the sequence of the open reading frame of the KAL1 gene had been studied previously, with the finding that 10 of 24 patients exhibited mutations in this gene (Maya-Nuñez et al, 1998b; Söderlund et al, 2002). All patients had a Mexican-mestizo ethnic origin and in all cases family history was negative for consanguinity. Patients ranged in age from 18 to 54 years. Anosmia or hyposmia detected by performing the olfactory test described by Rosen et al (1979) had been present since early childhood. During adolescence there was absence of or subnormal pubertal development. In all cases, the high-resolution G-banded karyotype was 46,XY. Clinical characteristics and molecular data of these patients had been previously published (Maya-Nuñez et al, 1998b; Söderlund et al, 2002).

Methods

Baseline plasma levels of luteinizing hormone, follicle-stimulating hormone, and testosterone were measured as described previously (Maya-Nuñez et al, 1998a).

Genomic DNA was isolated from blood leukocytes by standard techniques (Sambrook and Russell, 2001). DNA was amplified by polymerase chain reaction (PCR) in 50 μl of reaction mixture containing 300 ng of genomic DNA, 0.2 mM 2′-deoxynucleoside 5′-triphosphates, 2.0 U of thermostable DNA polymerase (New England BioLabs Inc, Beverly, Massachusetts), and 0.4 μM of each specific set of FGFR1, PROKR2, or PROK2 primers. The sequence of the primers, the sizes of the amplified products, and the PCR conditions have been described previously (Sato et al, 2004; Dodé et al, 2006). After amplification, PCR products were electrophoresed on 1.2% agarose gels and stained with ethidium bromide to verify the correct size of the expected fragments.

PCR products from the FGFR1, PROKR2, and PROK2 genes were purified by QIAEX II (QIAGEN GmbH, Hilden, Germany). DNA sequences (100 ng DNA template/reaction) of each PCR product were determined by cycle sequencing on an automated DNA sequencer ABI 377 (PE Biosystems, Foster City, California) using the DNA Sequencing Kit BigDye Terminator Cycle Sequencing Ready Reaction (PE Biosystems). Sequencing was performed following the protocol supplied by the manufacturer. Each mutation was confirmed in 3 independent PCR amplifications and sequencings.

Results

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

The sequence of the open reading frame of the FGFR1 and PROK2 genes was analyzed and no mutations were found in any of the patients studied. Direct sequencing of all exons of both genes demonstrated that no mutant alleles were present in any of the patients studied.

A genetic defect in the PROKR2 gene was found in 2 of the 24 patients after sequencing both exons. Patients 7 and 10 presented a heterozygous T-to-G transversion at nucleotide 518 (c.518T>G) in exon 2, which results in a leucine-to-arginine substitution at codon 173 (p.L173R) (Figure 1B and C); exon 1 showed no sequence variations. Two hundred normal male individuals (400 alleles) with the same ethnic origin as the patients did not harbor this mutation (p.L173R), being homozygous wild type.

image

Figure 1. . Partial sequence of the PROKR2 gene from a normal male control (A), from patient 7 (B), and from patient 10 (C). A heterozygous thymine-to-guanine transversion at nucleotide 518 (c.518T>G) in exon 2, which results in a leucine-to-arginine substitution at codon 173 (p.L173R), is observed.

Download figure to PowerPoint

In addition, Patient 7 presented a heterozygous G-to-A transition at nucleotide 1540 (c.1540G>A) in exon 11 of the KAL1 gene, which results in a glutamic acid—to-lysine substitution at codon 514 (p.E514K) (Figure 2).

image

Figure 2. . Partial sequence of the KAL1 gene from a normal male control (A) and from patient 7 (B). A heterozygous guanine-to-adenine transition at nucleotide 1540 (c.1540G>A) in exon 11, which results in a glutamic acid—to-lysine substitution at codon 514 (p.E514K), is observed.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

In the present study we analyzed the FGFR1, PROKR2, and PROK2 genes in 24 patients with KS, finding the same heterozygous mutation in PROKR2 in 2 unrelated patients (Patient 7, described previously by Maya-Nuñez et al, 1998b; and Patient 10, described previously by Söderlund et al, 2002).

The heterozygous mutation c.518T>G p.L173R found is identical to the one described by Dodé et al (2006) in 2 familial cases and 4 sporadic cases with KS. This mutation leads to a nonconservative amino acid substitution, changing a highly conserved residue (Dodé et al, 2006). PROKR2 belongs to the family of G protein—coupled receptors (Lin et al, 2002) and the p.L173R mutation is located within the fourth transmembrane domain of the PROKR2 protein (Dodé et al, 2006). Taking into consideration that our 2 mutated individuals, who belong to the same ethnic group, share the same mutation with 2 familial cases and 4 sporadic cases from Caucasian populations, the finding might indicate the presence of a hot spot in this region of the PROKR2 gene.

As has been indicated by Dodé et al (2006), in the absence of functional testing, one cannot be sure that the p.L173R mutation found in KS individuals is causative of the disease. However, this mutation was not identified in 400 alleles studied by us, nor in 500 other alleles analyzed at this particular region by Dodé et al (2006), making the possibility of polymorphism very unlikely; if this is really a polymorphism, it has a very low frequency and to date has not been demonstrated.

Interestingly, Dodé et al (2006) reported that 1 of the heterozygous patients for the p.L173R mutation in PROKR2 (sporadic case) also carried a point mutation in exon 8 of the KAL1 gene, and that this mutation modified the first amino acid residue of the linker between the second and third fibronectinlike type III repeats. Likewise, we found that patient 7, heterozygous for the same mutation in PROKR2, also carried a missense mutation in KAL1 (p.E514K); however, although this mutation was located in exon 11, it is located within the region encoding the third fibronectin type III—like repeat of the KAL protein (Maya-Nuñez et al, 1998b). Although E514K is a nonneutral polymorphism (rs28937309), and functional studies have demonstrated that it does not affect synthesis, processing, or protein release, Robertson et al (2001) demonstrated that E514K increases anosmin-1 binding to an extracellular target, critically affecting the function of anosmin-1. Likewise, Cariboni et al (2004) demonstrated that 514K proved unable to induce migration of immortalized GnRH neurons, and that variant induces an almost complete loss of this function. The presence of the polymorphism and the mutation in KAL1 and PROKR2 in the same patient constitute susceptibility factor(s) for the development of Kallmann syndrome.

To date, only 2 cases of possible digenic inheritance have been reported in KS; 1 case presented mutations in KAL1 and PROKR2 genes (Dodé et al, 2006) and the other case in FGFR1 and nasal embryonic luteinizing hormone—releasing hormone factor (NELF) genes (Pitteloud et al, 2007a). Our report constitutes the third such case. KS appears to follow the pattern of other disorders that were initially thought to be monogenic, but have been proven to be attributed to more than 1 gene defect (Badano and Katsanis, 2002; Carlton et al, 2003). Dodé et al (2006) proposed that the KAL1 gene product anosmin-1, which has been reported to enhance fibroblast growth factor signaling through FGFR1 (Gonzalez-Martinez et al, 2004), could also play a role in prokineticin signaling through PROKR2. Therefore, both mutant proteins may act at different levels of the same intracellular pathway and may contribute to its progressive dysfunction until a critical threshold is reached, yielding the pathological entity (Pitteloud et al, 2007a). Alternatively, both proteins may participate in a multiprotein complex that becomes progressively compromised by the additional mutations (Badano and Katsanis, 2002). On this basis, we postulate that the presence of both mutations in the KAL1 and PROKR2 genes contributes in an important fashion to the etiology of the KS. Unfortunately, the family members of the probands did not accept to participate in the study proof to prove the p.L173R of PROKR2 segregates with E514K of KAL1; however, data of several studies showed that digenic mutations could be de novo (Muntoni et al, 2006; Tosch et al, 2006; Gao et al, 2007).

Of the 24 patients studied, 1 had a mutation in KAL1 as well as a mutation in PROKR2; another individual presented a mutation in PROKR2 alone; and 9 patients had mutations in the KAL1 gene. Thirteen patients did not present mutations in any of the genes studied, indicating that molecular defects in those individuals could be present in the untranslated regulatory regions of KAL1, FGFR1, PROKR2, or PROK2. Besides, defects in other gene(s) different from the ones studied could explain the existence of the disorder, because to date only approximately 30% of KS patients bear the presence of a mutation that explains the syndrome.

In conclusion, our results strengthen the hypothesis of possible digenic inheritance in some patients with KS (Dodé et al, 2006; Pitteloud et al, 2007a). Likewise, our data extend previous reports demonstrating that PROKR2 plays a role in the etiology of this syndrome.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

We thank Laura Márquez, MSc, from the Laboratorio de Biología Molecular del Instituto de Biología, UNAM, for her technical assistance.

References

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
  • Albuisson J., Pecheux C., Carel JC, Lacombe D., Leheup B., Lapuzina P., Bouchard P., Legius E., Matthijs G., Wasniewska M., Delpech M., Young J., Hardelin JP, Dodé C.. Kallmann syndrome: 14 novel mutations in KAL1 and FGFR1(KAL2). Hum Mutat. 2005;25: 9899.
    Direct Link:
  • Badano JL, Katsanis N.. Beyond Mendel: an evolving view of human genetic disease transmission. Nat Rev Genet. 2002;3: 779789.
  • Cariboni A., Pimpinelli F., Colamarino S., Zaninetti R., Piccolella M., Rumio C., Piva F., Rugarli EI, Maggi R.. The product of X-linked Kallmann's syndrome gene (KAL1) affects the migratory activity of gonadotropin-releasing hormone (GnRH)-producing neurons. Hum Mol Genet. 2004;13: 27812791.
  • Carlton VE, Harris BZ, Puffenberger EG, Batta AK, Knisely AS, Robinson DL, Strauss KA, Shneider BL, Lim WA, Salen G., Morton DH, Bull LN. Complex inheritance of familial hypercholanemia with associated mutations in TJP2 and BAAT. Nat Genet. 2003;34: 9196.
  • Dodé C., Hardelin JP. Kallmann syndrome: fibroblast growth factor signaling insufficiency? J Mol Med. 2004;82: 725734.
  • Dodé C., Levilliers J., Dupont JM, de Paepe A., le Du N., Soussi-Yanicostas N., Coimbra RS, Delmaghani S., Compain-Nouaille S., Baverel F., Pêcheux C., le Tessier D., Cruaud C., Delpech M., Speleman F., Vermeulen S., Amalfitano A., Bachelot Y., Bouchard P., Cabrol S., Carel JC, de Waal H Delemarre-Van, Goulet-Salmon B., Kottler ML, Richard O., Sanchez-Franco F., Saura R., Young J., Petit C., Hardelin JP. Loss-of-function mutations in FGFR1 cause autosomal dominant Kallmann syndrome. Nat Genet. 2003;3: 463465.
  • Dodé C., Teixeira L., Levilliers J., Fouveaut C., Bouchard P., Kottler ML, Lespinasse J., Lienhardt-Roussie A., Mathieu M., Moerman A., Morgan G., Murat A., Toublanc JE, Wolczynski S., Delpech M., Petit C., Young J., Hardelin JP. Kallmann syndrome: mutations in the genes encoding prokineticin-2 and prokineticin receptor-2. PLoS Genet. 2006;2: 16481652.
  • Franco B., Guioli S., Pragliola A., Incerti B., Bardoni B., Tonlorenzi R., Carrozzo R., Maestrini E., Pieretti M., Taillon-Miller P., Brown CJ, Willard HF, Lawrence CH, Persico MG, Camerino G., Ballabio A.. A gene deleted in Kallmann's syndrome shares homology with neural cell adhesion and axonal path-finding molecules. Nature. 1991;353: 529536.
  • Gao YQ, Danciger M., Ozgul RK, Gribanova Y., Jacobson S., Farber DB. Association of the Asn306ser variant of the SP4 transcription factor and an intronic variant in the beta-subunit of transducin with digenic disease. Mol Vis. 2007;13: 287292.
  • Gonzalez-Martinez D., Kim SH, Hu Y., Guimond S., Schofield J., Winyard P., Vannelli GB, Turnbull J., Bouloux PM. Anosmin-1 modulates fibroblast growth factor receptor 1 signaling in human gonadotropin-releasing hormone olfactory neuroblasts through a heparan sulfate-dependent mechanism. J Neurosci. 2004;24: 1038410392.
  • Hardelin JP, Levilliers J., Blanchard S., Carel JC, Leutenegger M., Pinard-Bertelletto JP, Bouloux P., Petit C.. Heterogeneity in the mutations responsible for × chromosome-linked Kallmann syndrome. Hum Mol Genet. 1993;2: 373377.
  • Hardelin JP, Levilliers J., Del Castillo I., Cohen-Salmon M., Legouis R., Blanchard S., Compain S., Bouloux P., Kirk J., Moraine C., Chaussain J-L, Weissenbach J., Petit C.. X chromosome-linked Kallmann syndrome: stop mutations validate the candidate gene. Proc Natl Acad Sci U S A. 1992;89: 81908194.
  • Hebert JM, Lin M., Partanen J., Rossant J., McConnell SK. FGF signaling through FGFR1 is required for olfactory bulb morphogenesis. Development. 2003;130: 11011111.
  • Legouis R., Hardelin JP, Levilliers J., Claverie JM, Compain S., Wunderle V., Millasseau P., le Paslier D., Cohen D., Caterina D., Bougueleret L., de Waal H Delemarre-Van, Lutfalla G., Weissenbach J., Petit CH. The candidate gene for the X-linked Kallmann syndrome encodes a protein related to adhesion molecules. Cell. 1991;67: 423435.
  • Lin DC, Bullock CM, Ehlert FJ, Chen JL, Tian H., Zhou QY. Identification and molecular characterization of two closely related G protein-coupled receptors activated by prokineticins/endocrine gland vascular endothelial growth factor. J Biol Chem. 2002;277: 1927619280.
  • Matsumoto S., Yamazaki C., Masumoto KH, Nagano M., Naito M., Soga T., Hiyama H., Matsumoto M., Takasaki J., Kamohara M., Matsuo A., Ishii H., Kobori M., Katoh M., Matsushime H., Furuichi K., Shigeyoshi Y.. Abnormal development of the olfactory bulb and reproductive system in mice lacking prokineticin receptor PKR2. Proc Natl Acad Sci U S A. 2006;103: 41404145.
  • Maya-Nuñez G., Cuevas-Covarrubias S., Zenteno JC, Ulloa-Aguirre A., Kofman-Alfaro S., Méndez JP. Contiguous gene syndrome due to deletion of the first three exons of the kallmann gene and complete deletion of the steroid sulphatase gene. Clin Endocrinol. 1998a;48: 713718.
  • Maya-Nuñez G., Zenteno JC, Ulloa-Aguirre A., Kofman-Alfaro S., Mendez JP. A recurrent missense mutation in the KAL gene in patients with X-linked Kallmann's syndrome. J Clin Endocrinol Metab. 1998b;83: 16501653.
  • Muntoni F., Bonne G., Goldfarb LG, Mercuri E., Piercy RJ, Burke M., Yaou RB, Richard P., Récan D., Shatunov A., Sewry CA, Brown SC. Disease severity in dominant Emery Dreifuss is increased by mutations in both emerin and desmin proteins. Brain. 2006;129: 12601268.
  • Ng KL, Li JD, Cheng MY, Leslie FM, Lee AG, Zhou QY. Dependence of olfactory bulb neurogenesis on prokineticin 2 signaling. Science. 2005;308: 19231927.
  • Oliveira LM, Seminara SB, Beranova M., Hayes FJ, Valkenburgh SB, Schipani E., Costa EM, Latronico AC, Crowley WF Jr, Vallejo M.. The importance of autosomal genes in Kallmann syndrome: genotype-phenotype correlations and neuroendocrine characteristics. J Clin Endocrinol Metab. 2001;86: 15321538.
  • Pitteloud N., Meysing A., Quinton R., Acierno JS Jr, Dwyer AA, Plummer L., Fliers E., Boepple P., Hayes F., Seminara S., Hughes VA, Ma J., Bouloux P., Mohammadi M., Crowley WF Jr. Mutations in fibroblast growth factor receptor 1cause Kallmann syndrome with a wide spectrum of reproductive phenotypes. Mol Cell Endocrinol. 2006;254–255: 6069.
  • Pitteloud N., Quinton R., Pearce S., Raivio T., Acierno J., Dwyer A., Plummer L., Hughes V., Seminara S., Cheng YZ, Li WP, Maccoll G., Eliseenkova AV, Olsen SK, Ibrahimi OA, Hayes FJ, Boepple P., Hall JE, Bouloux P., Mohammadi M., Crowley W.. Digenic mutations account for variable phenotypes in idiopathic hypogonadotropic hypogonadism. J Clin Invest. 2007a;117: 457463.
  • Pitteloud N., Zhang C., Pignatelli D., Li JD, Raivio T., Cole LW, Plummer L., Jacobson-Dickman EE, Mellon PL, Zhou QY, Crowley WF Jr. Loss-of-function mutation in the prokineticin 2 gene causes Kallmann syndrome and normosmic idiopathic hypogonadotropic hypogonadism. Proc Natl Acad Sci U S A. 2007b;104: 1744717452.
  • Robertson A., MacColl GS, Nash JA, Boehm MK, Perkins SJ, Bouloux PM. Molecular modelling and experimental studies of mutation and cell-adhesion sites in the fibronectin type III and whey acidic protein domains of human anosmin-1. Biochem J. 2001;357: 647659.
  • Rosen SW, Gann P., Rogol AD. Congenital anosmia: detection thresholds for seven odorant classes in hypogonadal and eugonadal patients. Ann Otol Rhinol Laryngol. 1979;88: 288292.
  • Sambrook J., Russell D.. Preparation and analysis of eukaryotic genomic DNA. In: Nolan C., ed. Molecular Cloning: A Laboratory Manual. New York, NY: Cold Spring Harbor Laboratory Press; 2001; 6.16.31.
  • Sato N., Katsumata N., Kagami M., Hasegawa T., Hori N., Kawakita S., Minowada S., Shimotsuka A., Shishiba Y., Yokozawa M., Yasuda T., Nagasaki K., Hasegawa D., Hasegawa Y., Tachibana K., Naiki Y., Horikawa R., Tanaka T., Ogata T.. Clinical assessment and mutation analysis of Kallmann syndrome 1 (KAL1) and fibroblast growth factor receptor 1 (FGFR1, or KAL2) in five families and 18sporadic patients. J Clin Endocrinol Metab. 2004;89: 10791088.
  • Schwanzel-Fukuda M., Bick D., Pfaff DW. Luteinizing hormone-releasing hormone (LHRH)-expressing cells do not migrate normally in an inherited hypogonadal (Kallmann) syndrome. Brain Res Mol Brain Res. 1989;6: 311326.
  • Söderlund D., Canto P., Mendez JP. Identification of three novel mutations in the KAL1 gene in patients with Kallmann syndrome. J Clin Endocrinol Metab. 2002;87: 25892592.
  • Tosch V., Rohde HM, Tronchère H., Zanoteli E., Monroy N., Kretz C., Dondaine N., Payrastre B., Mandel JL, Laporte J.. A novel PtdIns3P and PtdIns(3,5)P2 phosphatase with an inactivating variant in centronuclear myopathy. Hum Mol Genet. 2006;15: 30983106.
  • Tsai P-S, Gill JC. Mechanisms of disease: insights into X-linked and autosomal-dominant Kallmann syndrome. Nat Clin Pract Endocrinol Metab. 2006;3: 160171.
Footnotes
  1. Supported by the Instituto Mexicano del Seguro Social, México, grant 2005/1/I/173.