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CASE REPORT |
Neuroendocrine Unit, Division of Endocrinology, Department of Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Pedro de Toledo 910, 04039002 São Paulo, Brazil
(Correspondence should be addressed to J Abucham; Email: julioabucham{at}uol.com.br)
Abstract
Kallmann syndrome (KS) describes the association of isolated hypogonadotropic hypogonadism with hypo/anosmia. A few KS patients may reverse hypogonadism after testosterone withdrawal, a variant known as reversible KS. Herein, we describe the first mutation in KAL1 in a patient with reversible KS and review the literature. The proband was first seen at 22 years complaining of anosmia and lack of puberty. His brother had puberty at 30 years and a maternal granduncle had anosmia and delayed puberty. On physical examination, he was P2G1, testes were 3 ml and bone age was 14 years. During 20 years of irregular testosterone replacement, he developed secondary sexual characteristics and testicular enlargement. At the age of 41 years, after stopping testosterone replacement for 5 months, his testes were 15 ml, serum testosterone, LH, and FSH responses to GnRH were normal, and his wife was pregnant. The molecular study revealed a cytosine insertion in exon 2 of KAL1, generating a frameshift at codon 75 and a premature stop at codon 85. The expected gene product is a truncated peptide with 85 of the 610 amino acids present in the wild-type protein. Fourteen cases of reversible KS have been described but the genotype was only studied in a single case showing a heterozygous fibroblast growth factor receptor type 1 (FGFR1) mutation. Considering the low prevalence of mutations in KAL1 or FGFR1 in KS, it is possible that these genotypes are more prevalent in reversible KS than in other KS patients, but additional studies are necessary to confirm this hypothesis.
Introduction
Kallmann syndrome (KS) describes the association of isolated hypogonadotropic hypogonadism (HH) with hypo/anosmia. The association of hypogonadism and anosmia was first described in 1856 by Maestre de San Juan in an autopsy report of a man with small penis, infantile testes, no pubic hair, and absence of olfactory bulbs, who was known to lack the sense of smell (1). In 1944, Kallmann recognized the genetic basis of this condition in three families, and thereafter this association has been known as Kallmann syndrome (2). Hypogonadism in one form of KS was later shown to be due to deficient gonadotropin-releasing hormone (GnRH) secretion caused by defective migration of GnRH neurons which depend on the guidance of olfactoterminal nerve axons to reach the hypothalamus (3–5).
Kallmann syndrome can be sporadic or familial and affects more males than females (6). Familial cases display different modes of inheritance: X-linked, autosomal dominant and, more rarely, autosomal recessive inheritance (7). So far, inactivating mutations in two distinct genes have been implicated in this condition, KAL1 (Xp22.3) and FGFR1 (8p11p12) (8–11). KAL1 mutations are responsible for X-linked KS (XKS) and fibroblast growth factor receptor type 1 (FGFR1) mutations underlie one form of autosomal dominant KS (AKS), but mutations in these two genes account for only 20–25% of KS cases (10, 12–14).
KAL1 encodes anosmin1, a secreted glycoprotein expressed in various extracellular matrices, and FGFR1 encodes FGFR1, a member of the receptor tyrosine kinase superfamily that binds fibroblast growth factor 2 (FGF2) and other FGF ligands. Anosmin1 and FGFR1 are both expressed during organogenesis of the olfactory-GnRH system where they regulate neuronal migration and axon elongation and branching (15–17). Mutations in these genes lead to defective olfactory tract formation and are likely to account for associated defects in other developing tissues observed in KS patients, such as renal agenesia, synkinesia and cleft lip, palate, and dental agenesis (8, 13, 14, 18, 19). A defective migration of GnRH neurons to their final destination in the hypothalamic anterior septopreoptic area has been documented in a 19-week human fetus with a KAL1 deletion, but a similar defect has not been investigated in KS patients with FGFR1 mutations (5).
In addition to the classical phenotypes, other variants of KS have been frequently observed among individuals with the same mutation within a family, including normal and mild phenotypes (e.g. isolated anosmia, delayed puberty without anosmia). Both KAL1 and FGFR1 genotypes have been shown to underlie this same phenotypic variability (13, 14, 20–23). Interestingly, a few KS patients have been reported to sustain improved gonadal function, including fertility, after testosterone withdrawal, a variant known as reversible KS (24–31). The only previous molecular study of a patient with reversible KS has shown a heterozygous FGFR1 mutation (32). In the present study, we describe a male patient with reversible KS showing a mutation in KAL1 and review all reversible KS found in the literature.
Case report
A 22-year-old male presented at our outpatient pituitary clinic due to absent pubertal development. He also had anosmia and neurosensorial hearing impairment but no mirror movements or midline defects. His family history included a mentally retarded brother who entered puberty at the age of 30 years and one maternal granduncle with delayed puberty and anosmia. On physical examination, his weight was 55 kg, height was 163 cm, and arm span was 163 cm. Tanner pubertal stage was P2G1, testes were 3 ml each, and axillary hair was scarce (33). Bone age was 14 years. Serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were low and total testosterone was in the peripubertal range. Sexual chromatin in the cells of the oral mucosa was negative. He was diagnosed with KS, a luteinizing hormone-releasing hormone (LHRH) stimulation test was indicated, but he refused the test.
Testosterone replacement (Durateston, Organon do Brasil, São Paulo, Brazil; 250 mg i.m. each 4 weeks) was soon started, but his adherence to treatment and follow-up visits were irregular. He was seen again at the ages of 26 and 28 years, with increased pubic, facial and body hair, and penis and testicular enlargement. At the age of 37 years, he complained of decreased libido in the week preceding each testosterone injection, and his total testosterone, measured 4 weeks after the injection, was low (54 ng/dl), and a shorter interval between injections was suggested. He was seen again 4 years later, complaining of reduced libido after discontinuing testosterone for the last 5 months. He also reported that his wife was pregnant (first trimester). He was fully virilized, his testicular volume was 15 ml each, total testosterone was normal (453 ng/dl) and serum LH and FSH were both normal before and after i.v. administration of 100 g LHRH (LH peak, 16.8 mIU/ml; FSH peak, 13.1 mIU/ml).
The patient was kept off testosterone replacement. Eight months after the last testosterone injection, he complained of decreased libido, lack of energy, weight loss (3.5 kg), and his total testosterone was in the low normal range (288 ng/dl). Bone densitometry revealed osteopenia at the lumbar spine (T-score 1.8). Prolactin, thyroid-stimulating hormone, FT4, and cortisol serum levels were normal. A summary of clinical and hormonal features of the patient is shown in Table 1
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After the patient consent, DNA was extracted from peripheral lymphocytes of the patient using a Qiagen Midi Kit (Qiagen), following manufacturer’s protocol. Exons 1–14 of KAL1 and exons 1–18 of FGFR1 were amplified by PCR. One hundred nanograms of human genomic DNA were used as template in a 100 l PCR mixture containing 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM deoxy-NTPs, 2.5 U Taq polymerase (PCR Reagent System, Life Technologies), and 0.1 M upstream and downstream specific primers. The sequence of the PCR primers and the PCR thermal cycling program used were based on previous publications (8, 12, 19). PCR products were analyzed in 1.8% agarose gels and purified using a PCR Product Purification Kit (Life Technologies). Direct sequencing of the PCR products was carried out in both directions, using the ABI Prism Big Dye terminator cycle sequencing ready reaction version 2.0 (Applied Biosystems, Foster City, CA, USA) in an ABI Prism 3100 DNA Sequencer (Perkin-Elmer Applied Biosystems).
As shown in Fig. 1, direct sequencing of the PCR products revealed a cytosine insertion in exon 2 of KAL1, causing a frameshift at codon 75 and a premature stop at codon 85. The expected gene product is a truncated peptide with only 85 of the 610 amino acids present in the wild-type protein. This insertion was confirmed by sequencing the products of two different PCRs. Sequencing of the exon 2 of KAL1 under the same conditions using DNA from a normal male as template revealed no abnormalities. No mutation was found in FGFR1.
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Cases of reversible Kallmann syndrome were searched in Pubmed (US National Library of Medicine) using the search terms ‘Kallmann’, ‘reversible Kallmann’, ‘delayed puberty’, and ‘variant Kallmann syndrome’. The diagnosis of reversible KS included all patients, who became fertile without gonadotropin or GnRH therapy and/or who showed improved testosterone secretion after discontinuing gonadotropin, GnRH, or testosterone replacement.
As shown in Table 2, 14 unrelated patients with reversible KS, including our patient, have been described in the medical literature. All patients presented delayed puberty associated with anosmia. In four, additional features of KS were described: neurosensorial hearing loss (n = 2), facial asymmetry (n = 2), thoracic asymmetry (n = 1), and palatine cleft (n = 1). Five patients had other family members with KS and one family presented a high rate of consanguinity (26). The diagnosis of KS in these patients was established between 15 and 31 years of age. Ten patients were treated exclusively with testosterone replacement, three were also given human chorionic gonadotropin to induce fertility, and a single one received FSH additionally. Testosterone replacement was discontinued in seven patients by their own judgment and in three patients for inclusion in studies of gonadotropin secretion in KS.
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Discussion
In addition to the two classical diagnostic criteria for KS (hypogonadotropic hypogonadism and anosmia), our patient had neurosensorial hearing loss, which may also be present in KS, and a positive family history of delayed puberty in a brother and a maternal grand-uncle. Although his compliance with testosterone replacement therapy was variable, he achieved full virilization. The possibility that his hypogonadism had reversed was raised by his account of his wife’s pregnancy, but a DNA paternity test was not performed. Reversal of his hypogonadal state was confirmed by testicular enlargement, from 3 to 15 ml, over nearly 20 years of irregular testosterone without gonadotropin replacement, and by a normal serum testosterone level long after testosterone replacement had been discontinued.
The real prevalence of reversible KS is unknown, but it is probably higher than the 5% found in a series of 76 KS patients without performing routine reassessment of gonadal function whilst off testosterone replacement therapy (28). In practice, special attention should be paid to testicular volume, both at diagnosis and during follow-up of KS patients, since testicular enlargement indicates increased gonadotropin secretion. In addition, periodical interruption of testosterone replacement and reassessment of gonadal function in KS patients may be desirable in order to avoid unnecessary hormone replacement and inappropriate reproductive prognosis.
The molecular study of our patient showed a novel mutation in KAL1, the gene responsible for XKS, and no mutations in FGFR1, the gene responsible for an AKS. This KAL1 mutation predicts a prematurely truncated protein that lacks all functional domains of anosmin1: the N-terminal cysteine-rich region, the whey acidic protein (WAP)-like four disulfide core motifs, the four tandem fibronectin type III (FnIII)-like repeats homologous to neural cell adhesion molecules with heparan sulfate (HS)-binding activity, and the histidine-rich C-terminus. In vitro studies have shown that the WAP domain influences axon targeting and that the FnIII domains are essential for this function and also for axon branching (15, 17). In vitro, both FGFR1 and anosmin1 require HS for their cooperation within a multimeric FGFR1–FGF2–HS–anosmin1 complex leading to functional and specific activation of FGFR1 signaling through the MAPK pathway. Accordingly, anosmin1 can be viewed as an FGFR1-specific modulatory coligand that interacts with the FGFR1–FGF2 complex to amplify intracellular downstream signaling (16).
In our patient, a severely dysfunctional protein was predicted from his KAL1 mutation. However, in the absence of anosmin1, FGF2 activation of the FGFR1 receptor is more likely to be decreased than abolished (16). In the patient with reversible KS with a heterozygous FGFR1 mutation previously described, intracellular signaling was also likely decreased, but not abolished, which can be explained by his FGFR1 haploinsufficiency (32). The reversal of the hypogonadism in KS indicates that at least a population of GnRH neurons have successfully migrated to the hypothalamus and established functional connections with other neurons as well as with the vessels in the median eminence. Theoretically, variations in the amount of neurons effectively reaching the hypothalamus and establishing adequate connections could explain the broad spectrum of gonadal function in KS, including severe hypogonadism, delayed puberty, and reversible hypogonadism.
The possibility that patients with reversible KS represent an extreme degree of pubertal delay that would eventually enter puberty if left untreated cannot be ruled out. Delayed puberty with or without anosmia is known to occur in other family members of KS patients sharing the same mutation (6, 23, 32). On the other hand, testosterone replacement therapy could play a role in reversible KS. Precocious puberty can be triggered by increased androgen levels as observed in patients with congenital adrenal hyperplasia, androgen-secreting tumors, and testotoxicosis.
In conclusion, the present mutation represents the first mutation in KAL1, the gene responsible for X-linked KS, in a patient presenting reversible KS. This finding, together with the previous report of a heterozygous FGFR1 mutation, indicates that the reversible KS phenotype is not restricted to mutations in a single gene. Considering the low prevalence (~10%) of mutations in each of these two genes in familial KS, it is rather intriguing that they have already been found in the only patients with reversible KS with molecular studies reported so far. It is tempting to speculate that mutations in KAL1 and FGFR1 may be more prevalent among patients with reversible KS than in other KS patients, but molecular studies in a larger number of patients are necessary to confirm this hypothesis.
Acknowledgements
We are grateful to the staff of the Molecular Endocrinology Laboratory, particularly to Teresa S Kasamatsu, for assistance in the experiments, and to Gustavo S Guimarães and Gilberto K Furuzawa for their helpful discussions. This work was supported by a FAPESP research grant 04/011625 (TCV) and CNPq grant 133493/20 032 (RSR).
References
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  A. A. Sinisi, R. Asci, G. Bellastella, L. Maione, D. Esposito, A. Elefante, A. De Bellis, A. Bellastella, and A. Iolascon Homozygous mutation in the prokineticin-receptor2 gene (Val274Asp) presenting as reversible Kallmann syndrome and persistent oligozoospermia: Case Report Hum. Reprod., July 1, 2008; (2008) den247v1. [Abstract] [Full Text] [PDF]
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 T. Raivio, J. Falardeau, A. Dwyer, R. Quinton, F. J. Hayes, V. A. Hughes, L. W. Cole, S. H. Pearce, H. Lee, P. Boepple, et al. Reversal of Idiopathic Hypogonadotropic Hypogonadism N. Engl. J. Med., August 30, 2007; 357(9): 863 - 873. [Abstract] [Full Text] [PDF]
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