Gonadotrophin therapy in Kallmann syndrome caused by heterozygous mutations of the gene for fibroblast growth factor receptor 1: report of three families:Case report

Naoko Sato1, Tomonobu Hasegawa2, Naoaki Hori2, Maki Fukami1, Yasunori Yoshimura3 and Tsutomu Ogata1,4

1Department of Endocrinology and Metabolism, National Research Institute for Child Health and Development, Tokyo 157-8535 and Departments of 2 Pediatrics and 3 Departments of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo 160-8582, Japan

4 To whom correspondence should be addressed. Email: tomogata{at}nch.go.jp


    Abstract
 Top
 Abstract
 Introduction
 Case reports
 Discussion
 Acknowledgements
 References
 
Gonadotrophin therapy (GT) is frequently used to induce fertility in Kallmann syndrome (KS). We studied the effects and the consequences of GT in autosomal dominant KS caused by heterozygous FGFR1 mutations. Three Japanese families were examined. In family A, an adult male received GT and had two sons. In family B, an adult female received GT and gave birth to dizygotic male and female twins. In family C, an adult female received GT and produced a son and a daughter. Direct sequencing was performed for FGFR1, and clinical assessment was carried out for KS features. The father and the elder son of family A had P745S mutation, the mother and the female twin of family B had G687R mutation, and the mother and the two children of family C had S107X mutation. KS phenotype was detected for the mutation-positive subjects, except for the elder son of family A who had apparently normal phenotype. GT in FGFR1 mutations is effective in acquiring fertility but has a risk of transmitting the mutation and the disease phenotype to the next generation.

Key words: fertility/FGFR1 mutation/genetic counselling/gonadotrophin therapy/Kallmann syndrome


    Introduction
 Top
 Abstract
 Introduction
 Case reports
 Discussion
 Acknowledgements
 References
 
Kallmann syndrome (KS) is a developmental disorder defined by the combination of hypogonadotrophic hypogonadism (HH) and olfactory dysfunction (Kallmann et al., 1944Go). This condition is genetically heterogeneous, with reports indicative of X-linked, autosomal dominant, and autosomal recessive transmissions (Dodé and Hardelin, 2004Go). To date, two genes have been identified for KS, i.e. KAL1 (Kallmann syndrome 1) on Xp22.3 (Franco et al., 1991Go; Legouis et al., 1991Go) and FGFR1 (fibroblast growth factor receptor 1, also known as KAL2) on 8p12 (Dodé et al., 2003Go). Hemizygous KAL1 deletions or mutations are responsible for the X-linked form, and frequently associated with renal agenesis and mirror movements, whereas heterozygous FGFR1 loss-of-function mutations are involved in one autosomal dominant form, and occasionally accompanied by cleft palate and dental agenesis as well as mirror movements (Dodé and Hardelin, 2004Go).

Gonadotrophin therapy is frequently used to induce fertility. In particular, this therapy has been reported to be effective in HH including KS (Buchter et al., 1998Go), because it can directly compensate for the cause of infertility. This therapeutic intervention, however, may transmit the parental mutation to the next generation. The possibility is especially important in an autosomal dominant form of HH such as KS caused by FGFR1 mutations, because the transmission of a mutant allele can reproduce the same disease in the next generation.

To our knowledge, however, while such an effect and a risk are well predicted for the gonadotrophin therapy, there has been no report describing the transmission of mutant allele and resultant disease phenotype after gonadotrophin therapy. Here, we report on the effects and the consequences of gonadotrophin therapy in three KS families with FGFR1 mutations.


    Case reports
 Top
 Abstract
 Introduction
 Case reports
 Discussion
 Acknowledgements
 References
 
Family A
Proband (I-1) was seen at 20 years of age because of lack of pubertal development, and was diagnosed as having typical KS on the basis of HH and anosmia (Table I). He had no other clinical features reported in KS. Subsequent clinical course is summarized in Figure 1. He was first placed on testosterone enanthate (TE) therapy (200 mg i.m. once per month) for 1 year to advance sexual development. He then received gonadotrophin therapy with FSH (150 IU i.m. twice per week) and hCG (5000 IU i.m. twice per week) to gain fertility. This therapy successfully increased testicular volume, serum testosterone value, and ejaculated semen volume and sperm count. After marriage at 23 years of age, the gonadotrophin therapy was interrupted a few times because of poor compliance, and the therapeutic status was well reflected by the testicular volume and the serum testosterone value. After gonadotrophin therapy for 13 consecutive months, a son was born after intrauterine insemination. Furthermore, after 3 years of TE therapy, he received gonadotrophin therapy again for 12 consecutive months, and a second son was born after natural conception. Thereafter, he was placed on TE therapy.


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Table I. Clinical features in FGFR1 mutation positive patients

 


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Figure 1. Summary of family A. (Upper panel) Clinical course of the proband. This patient received testosterone enanthate (TE, 200 mg i.m. once per month) therapy or gonadotrophin therapy consisting of FSH (150 IU i.m. twice per week) and hCG (5000 IU i.m. twice per week). Semen was obtained after 5–7 days of abstinence. PL=penile length; PH=pubic hair; TV=testicular volume; T=testosterone. (Lower panel) Left: Pedigree of family A. N.E.=not examined; WT=wildtype. Middle: Electrochromatogram showing a heterozygous mutation (2233C->T, P745S) at exon 17 in the father (I-1). The same mutation was also identified in the elder son (II-1). Right: BsaI digestion analysis for the mutation. The 2233C->T mutation should disrupt a BsaI site and, consistent with this, BsaI digestion of the 563 bp PCR products encompassing exon 17 produced two fragments (382 and 181 bp) in the younger son (II-2) and a control subject (C) and three fragments (563, 382 and 181 bp) in the father (I-1) and the elder son (II-1). The 563 bp fragment is specific to the mutation. M=molecular weight marker.

 
Clinical assessment of the elder son (II-1) at 6 years of age indicated apparently normal phenotype, although it was uncertain whether he could have sufficient pubertal development (Table I). The younger son also had normal phenotype at 6 months of age.

Family B
Proband (I-2), who married at 25 years of age, was referred to us because of primary amenorrhoea, and was found to have typical KS with HH and anosmia (Table I). She had no other phenotype described in KS. She was treated with conjugated estrogen (CE) (0.625 mg per os once per day) for the first 6 months and received cyclic HRT consisting of CE on days 1–14, CE plus medroxyprogesterone acetate (5 mg per os once per day) on days 15–21, and no drug on days 22–28 for the next 6 months. At that time, she had sufficient pubertal development (breast, Tanner stage 4; pubic hair, Tanner stage 3) and withdrawal bleeding. She hoped to have a child, and received gonadotrophin therapy with a regimen shown in Figure 2. At the second cycle of this regimen, she became pregnant, and delivered dizygotic twins by a Caesarean section at 35 weeks of gestation. Subsequently, gonadotrophin treatment was discontinued and HRT was re-started.



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Figure 2. Summary of families B and C. (Upper panel) Regimen of the gonadotrophin therapy employed in the mothers of families B and C. hMG was started from the fourth day of withdrawal bleeding, and was continued for 12–16 days until a follicle(s) of ≥17 mm in diameter was delineated by transvaginal ultrasounds. A single dose of a large amount of hCG was then given, followed by intermittent hCG injections (twice per week) for 14 days. (Middle panel) Left: Pedigree of family B. N.E.=not examined; WT=wildtype. Right: Electrochromatogram showing a heterozygous mutation (2059G->A, G687R) at exon 16 in the mother (I-2). The same mutation was also identified in the female twin (II-2). This mutation was indicated by the direct sequencing, and confirmed by the sequencing of the subcloned normal and mutant alleles. (Lower panel) Left: Pedigree of family C. WT=wildtype. Middle: Electrochromatogram showing a heterozygous mutation (320C->A, S107X) at exon 3 in the son (II-1). The same mutation was also identified in the mother (I-2) and the sister (II-2). Right: BfaI digestion analysis for the mutation. The 320C->A mutation should create a BfaI site, and BfaI digestion of the 434 bp PCR products encompassing exon 3 produced two fragments (350 and 84 bp) in the father (I-1) and a control subject (C) due to a naturally occurring BfaI site, and four fragments (350, 278, 84 and 72 bp) in the mother (I-2), the son (II-1), and the daughter (II-2) because of the mutation specific BfaI site cutting the 350 bp fragment into 278 and 72 bp fragments t; in this figure, the 350 and 278 bp bands only are shown, and the 278 bp fragment is specific to the mutation. M=molecular weight marker.

 
Clinical evaluation of the female twin (II-2) at 3 months of age revealed olfactory bulb aplasia indicative of KS and good FSH response to GnRH stimulation (Table I), while it was uncertain whether she would continue to have olfactory dysfunction only phenotype or show both olfactory dysfunction and HH in later life. Other features reported in KS were undetected in the female twin. The twin male had left cryptorchidism, but a GnRH test yielded good responses of LH (8.1->19.3 IU/l) and FSH (4.6->10.5 IU/l) and brain magnetic resonance imaging delineated normal olfactory bulbs and sulci.

Family C
Proband (II-1) was previously reported as a sporadic case with KS resulting from a heterozygous S107X mutation of FGFR1 at 11 years of age (Sato et al., 2004Go), and subsequent familial study disclosed an autosomal dominant form of KS. In brief, the proband was referred to us because of HH (peak LH value of 0.2 IU/l and FSH value of 0.1 IU/l in a GnRH test) and anosmia at 10 8/12 years of age. At 13 0/12 years of age, he exhibited no pubertal development, and clinical studies confirmed HH and olfactory dysfunction (Table I). To induce pubertal development, he was treated with TE with a gradual dosage increase from 25 mg to 200 mg i.m. per month. At 15 years of age, he manifested good pubertal development (genitalia, Tanner stage 4; pubic hair, Tanner stage 3), although his testes remained ~1 ml in volume. He had no other KS features.

His younger sister (II-2) also came to us because of no pubertal development at 12 4/12 years of age. Endocrine results were suggestive of mild HH, while her olfactory function was apparently normal. She was placed on CE therapy, to induce pubertal development. She exhibited no other KS phenotype.

The mother (I-2) had a history of delayed puberty, oligomenorrhoea and hyposmia, although she had good pubertal development spontaneously. During the clinical examinations of the daughter, she confessed that she was seen at a local hospital because of infertility for 2 years after marriage at 25 years of age. Subsequently, her menses occurred at a frequency of six to eight times per year, and the measurement of basal body temperature indicated probable ovulation in roughly one-third of the cycles. She received gonadotrophin therapy with a regimen shown in Figure 2, under a provisional diagnosis of KS. At the second cycle of this regimen, she became pregnant and delivered the son (II-1) at 39 weeks of gestation. Three years later, she received the gonadotrophin therapy again and, consequently, she became pregnant in the third cycle of this regimen and gave birth to the daughter (II-2) at 40 weeks of gestation. Clinical assessment at 43 years of age, in conjunction with the history, indicated mild KS without other associated features (Table I). At that time, she still had oligomenorrhoea with a frequency of three or four times per year, although it was uncertain whether the cycles were associated with ovulation.

Molecular studies
This study has been approved by the Institutional Review Board Committees at National Center for Child Health and Development and Keio University Hospital. After obtaining written informed consent, leukocyte genomic DNA was amplified by PCR for all the coding region of FGFR1 (exons 2–18), and the PCR products were subjected to direct sequencing from both directions on a CEQ 8000 autosequencer (Beckman Coulter, Fullerton, CA, USA). The primer sequences and the PCR conditions have been described previously (Sato et al., 2004Go). To confirm heterozygous mutations, the corresponding PCR products were digested with appropriate restriction enzyme, or they were subcloned with TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA, USA) and normal and mutant alleles were sequenced separately. For controls, leukocyte genomic DNA samples from 50 normal subjects were similarly analysed with permission.

Heterozygous FGFR1 mutations were identified in families A–C. In family A, the proband (I-1) had a missense mutation at exon 17 (2233C->T, P745S), and this mutation was present in the elder son (II-1) and absent in the younger son (II-2) (Figure 1). In family B, the proband (I-2) had a missense mutation at exon 16 (2059G->A, G687R), and this mutation was present in the female twin (II-2) and absent in the male twin (II-1) (Figure 2). In family C, a nonsense mutation at exon 3 (320C->A, S107X) was shared by the mother (I-2), the son (II-1), and the daughter (II-2) (Figure 2). These mutations were absent from 100 chromosomes of the 50 control subjects.


    Discussion
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 Abstract
 Introduction
 Case reports
 Discussion
 Acknowledgements
 References
 
Three different heterozygous FGFR1 mutations were identified in families A–C. S107X in the hinge region between the first and the second immunoglobulin-like domains would be an amorphic mutation, because it should result in the production of a severely truncated protein missing most of the functional domains of FGFR1 protein (Groth and Lardelli, 2002Go). For G687R and P745S, both mutations were found in KS patients and absent from the 100 chromosomes of the control subjects. Furthermore, G867R and P745S have not been reported as a polymorphism in Japanese Single Nucleotide Polymorphism Database (http://www.snp.ims.u-tokyo.ac.jp/), National Center for Biotechnology Information (NCBI) Database (http://www.ncbi.nlm.nih.gov/), and Ensembl Genome Browser Database (http://www.ensembl.org/), and the G867 and the P745 residues are conserved across various species including mouse, chicken and frog (NCBI and Ensembl Databases). Since G867R and P745S exist at the tyrosine kinase domain 2 (TK2) in the intracellular part of the receptor, they may affect the TK activity required for the FGFR1 signalling (Groth and Lardelli, 2002Go). In addition, several missense mutations, including P745S in a sporadic Japanese male patient with KS (Sato et al., 2004Go), have been identified in TK2 (Dodé and Hardelin, 2004Go); this may provide further support for the pathogenic effects of missense mutation in TK2. Thus, although functional studies have not been performed, the three mutations would be responsible for the development of KS in families A–C.

The gonadotrophin therapy successfully induced fertility in both male and female cases. This is consistent with the previous finding that the gonadotrophin therapy is effective in acquiring fertility in most patients with HH including KS (Aharoni et al., 1989Go; Buchter et al., 1998Go; Kitahara et al., 1998Go). In this context, FGFR1 mutation-positive patients with one normal allele might actually respond better to the gonadotrophin therapy, because some of them can have normal reproductive function (Dodé et al., 2003Go). It should be pointed out, however, that not all disorders with HH respond to gonadotrophin therapy. It is known that male patients with mutant DAX1 (DSS–AHC critical region on the X chromosome, gene 1) do not respond to the gonadotrophin therapy, although they have HH (Seminara et al., 1999Go). This would primarily be due to the presence of primary as well as secondary testicular dysfunction in such patients (Seminara et al., 1999Go). Indeed, DAX1 is expressed in gonads (Sertoli and interstitial cells) as well as adrenals, in addition to hypothalamus and pituitary (Ikeda et al., 2001Go).

The gonadotrophin therapy resulted in the transmission of the mutant allele to the next generation. Furthermore, consistent with the dominant effect of FGFR1 mutations, mutation-positive children in families B and C showed clinically discernible KS phenotype. For the elder son of family A, it is uncertain whether he remains as an apparently non-manifesting carrier or exhibits KS phenotype such as defective pubertal development at a later age. Indeed, phenotypic spectrum is known to be variable in FGFR1 mutation-positive patients of both sexes, ranging from typical KS phenotype to apparently normal phenotype with intact olfactory and reproductive functions (Dodé et al., 2003Go). Taken together, the results actually demonstrate that gonadotrophin therapy in FGFR1 mutation-positive patients risks transmitting the mutation to the next generation and causing the KS phenotype in most, if not all, mutation-positive patients.

In addition to the effects and consequences of the gonadotrophin therapy, clinical variability in mutation-positive patients appears to be noteworthy (Table I). Several factors would be involved in the phenotypic diversity. First, there should be an influence of ascertainment bias. Indeed, the probands (I-1 in family A, I-2 in family B, and II-1 in family C) had typical KS phenotype, whereas other patients had mild or apparently normal phenotype. Second, sex difference may contribute to the phenotypic variability. In this regard, FGFR1 and anosmin-1 encoded by KALI are likely to interact in FGF signalling involved in the development of olfactory bulbs (the target regions in KS) (Dodé and Hardelin, 2004Go), and the local concentration of anosmin-1 should be higher in females than in males, because KALI partially escapes X-inactivation (Franco et al., 1991Go; Legouis et al., 1991Go). This would provide better FGF signalling in females than in males with heterozygous FGFR1 mutations, mitigating KS phenotype in affected females. Indeed, the phenotype was milder in the affected females than in the affected male in family C with the same mutation. In addition, the sex difference in the KALI dosage would explain why apparently the non-X linked form of KS is more prevalent in males than in females (Dodé and Hardelin, 2004Go). Third, the type of mutations and other genetic and environmental factors would also be relevant to the clinical diversity. KS phenotype should be more severe in patients with amorphic mutations than in those with hypomorphic mutations, and haploinsufficiency of genes involved in human development frequently is known to show a wide range of penetrance and expressivity, probably depending on other genetic and environmental factors (Fisher and Scambler, 1994Go). Such complexity would explain the lack of straightforward genotype–phenotype correlations in affected individuals.

In summary, the results indicate that gonadotrophin therapy in FGFR1 mutations is effective in acquiring fertility but has a risk of transmitting the mutation and the disease phenotype to the next generation. Although the occurrence of such events is quite expected, this point should be considered in the application of gonadotrophin therapy and relevant genetic counselling.


    Acknowledgements
 Top
 Abstract
 Introduction
 Case reports
 Discussion
 Acknowledgements
 References
 
This work was supported by a grant for Child Health and Development (14C-1) and a grant for Research on Children and Families from the Ministry of Health, Labor, and Welfare, by a Pfizer Fund for Growth and Development Research, and by a Grant-in-Aid for Scientific Research on Priority Areas (16086215).


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 Introduction
 Case reports
 Discussion
 Acknowledgements
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Submitted on December 26, 2004; resubmitted on February 24, 2005; accepted on April 7, 2005.





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