Hum. Reprod. Advance Access published online on June 6, 2007
Human Reproduction, doi:10.1093/humrep/dem126
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Preserved fertility in a non-mosaic Klinefelter patient with a mutation in the fibroblast growth factor receptor 3 gene: Case Report
1 Department of Growth and Reproduction GR, Rigshospitalet Section 5064, University of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark 2 Department of Clinical Genetics, Rigshospitalet section 5064, University of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark
3 Correspondence address. Tel: +45-35455064; Fax: +45-35456054; E-mail: ajuul{at}rh.dk
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Abstract |
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Patients with Klinefelter syndrome (47,XXY) are characterized by eunuchoid body proportions, gynaecomastia, small firm testes and azoospermia. We describe a Klinefelter patient (non-mosaic 47,XXY karyotype) who was heterozygous for the classical 1138G > A mutation in the fibroblast growth factor receptor 3 (FGFR3) gene, which is a gain-of-function mutation resulting in achondroplasia. The patient had phenotypic characteristics of achondroplasia (e.g. short limbed dwarfism and frontal bossing). Testicular volume was 8 ml at 27 years of age and repeated semen samples showed sperm concentrations of 0.175 million/ml. Serum FSH levels were elevated (21.7 IU/l) compared to normal age-matched healthy male controls and patients with non-mosaic Klinefelter syndrome, and inhibin B levels were low-normal, in contrast to the usually undetectable inhibin B levels in adult Klinefelter patients. The patient fathered a child from a spontaneous pregnancy. The observed testicular size and function in our patient contrast the typical findings in classical Klinefelter syndrome. We speculate that the alteration of FGFR3 protein function in our Klinefelter patient alleviated the destruction of the seminiferous tubules and may suggest that the fibroblast growth factor family has a pleiotrophic function in human spermatogonia, which physiologically express FGFR3.
Key words: Klinefelter syndrome/achondroplasia/fibroblast growth factor receptor 3/fertility/testis
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Introduction |
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We describe a male patient with Klinefelter syndrome and achondroplasia due to a common fibroblast growth factor receptor 3 (FGFR3) mutation who had preserved fertility, and discuss the possible association between the fibroblast growth factor (FGF) family and spermatogenesis.
Patients with Klinefelter syndrome (47,XXY) are characterized by eunuchoid body proportions, gynaecomastia, small firm testes and azoospermia. The frequency is approximately 1 out of 600 newborn male infants (Bojesen et al., 2003
). The genotypic abnormality results from a meiotic non-disjunction event (maternal or paternal) resulting in a 47,XXY karyotype in the majority of patients. However, up to 3% of patients is mosaic 46,XY/47,XXY. Testicular biopsies from Klinefelter patients show mixed areas with Sertoli-cell-only tubules and sclerotic or hyalinized tubules as well as interstitial Leydig cell hyperplasia. However, scattered areas with focal spermatogenesis can be seen even in non-mosaic patients (Skakkebaek, 1969). Thus spontaneous pregnancies from fathers with non-mosaic Klinefelter syndrome are extremely rare, although such cases have been reported (Laron et al., 1982; Terzoli et al., 1992). In most cases of Klinefelter men who wish to father a child, the use of assisted reproductive techniques (testicular sperm extraction followed by ICSI) is required. Such techniques may have success rates of up to 50% (Schiff et al., 2005), suggesting that sperm retrieval and ICSI success in men with Klinefelter syndrome are comparable with other men with non-obstructive azoospermia.
Achondroplasia is the most frequent form of short-limb dwarfism occurring in 1:5000 to 1:40000 live births (Martinez-Frias et al., 1991). Affected individuals exhibit short stature caused by rhizomelic shortening of the limbs, characteristic faces with frontal bossing and mid-face hypoplasia, exaggerated lumbar lordosis, limitation of elbow extension, genu varum, and trident hand. Achondroplasia is caused by a mutation in the FGFR3 gene, which is located at 4p16.3 (Shiang et al., 1994). The most common mutation results in the substitution of an arginine residue for a glycine at position 380 of the mature protein, which is in the transmembrane domain of FGFR3. The effect of this mutation on male fertility has not been studied.
The rare combination of two distinct syndromes (47,XXY and achondroplasia) has previously been reported in three different cases (Sendrail et al., 1967; Kulakova and Ignat'ev, 1986; Sayli et al., 1994) in whom fertility was not described. In one of these reports a testicular biopsy described the typical histological pattern of dysgenetic Klinefelter testes (Sendrail et al., 1967), but it was not stated whether distinct areas within the testis with preserved spermatogenesis were present and no semen sample was collected.
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Case story |
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The patient was born at term after an uneventful pregnancy with a birth weight of 3000 gram and birth length 52 cm. He was clinically diagnosed with achrondroplasia shortly after birth. Apart from the typical abnormal growth pattern his childhood was uneventful, and puberty occurred spontaneously, albeit with a slight delay. The patient was heterozygous for the classical c.1138G > A mutation resulting in an arginine to glycine substitution at position 380. At the age of 17 years he was diagnosed with non-mosaic Klinefelter syndrome during chromosomal analysis of the family, which was carried out because of mental retardation of his sister. Karyotype was established on 30 metaphases and revealed 47,XXY in all studied peripheral blood leucocytes. He attended normal school, was educated in economy/business and serves a regular job. He has had no physical complaints from back and joints during childhood and adolescence. He obtained a final height of 135.1 cm, which corresponds to –7.0 SD compared to Danish references (Andersen et al., 1982
), with typical achondroplastic body proportions (sitting height/height ratio 0.66).
Reproductive history
He had slightly delayed puberty, and at the age of 14 he was in genital stage 2 according to Tanner et al. (1966) (both testes 6 ml by orchidometry (Zachmann et al., 1974)). Puberty progressed (Table 1) and at adult age his testes remained at a size of 8 ml with a normal texture upon palpation. Scrotal ultrasound revealed a normal sonographic pattern of the testes (echo-score 2) (Lenz et al., 1993). Reproductive hormones are shown in Table 1. He married at 26 years of age to a woman with achondroplasia and the same FGFR3 mutation as our patient (c.1138G > A) confirmed by DNA testing. She became pregnant naturally and following genetic counselling they decided to have the child. Chorion villus analysis demonstrated that the child was affected having one allele with the c.1138G > A mutation. At 37 weeks of gestation his wife delivered a boy (by Caesarian section) with the typical features of achondroplasia. Genetic analyses revealed that the c.1138G > A allele was inherited from his mother.
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Laboratory findings
Serum concentrations of reproductive hormones in the patient were determined by published immunoassays , and compared to levels in normal age-matched healthy male controls (n = 706) (Andersson et al., 1997
, 2004a,b) and patients with non-mosaic Klinefelter syndrome (n = 31) (Aksglaede et al., 2007) using the same assays. The data are shown in Table 1. The presence of an additional X chromosome in the patient was verified by the detection of the XIST gene transcript (Brown et al., 1991; Penny et al., 1996). The analysis was performed by RT–PCR analysis of RNA isolated from blood cells, according to the method of Kleinheinz and Schulze (1994).
The patient delivered three semen samples, which were examined according to World Health Organization (WHO) criteria (WHO, 1999) (Table 2). Fluorescence in situ hybridization (FISH) analysis using X and Y probes (a gift from DAKO Cytomation, Glostrup, Denmark) were performed on cells from the semen sample (Fig. 1). The FISH procedure was performed as previously described (Hoei-Hansen et al., 2006), with a small modification involving a stronger proteinase K pretreatment, required to increase a signal from condensed sperm DNA. All spermatozoa in this analysis were apparently haploid, showing either X or Y signal (Fig. 1A). One semen sample was used to purify DNA for mutational analyses. FGFR3 mutation was confirmed in DNA isolated from sperm.
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To test whether FGFR3 is expressed at the protein level in the human testis, we performed immunohistochemical staining in five different paraffin-embedded anonymous specimens from our tissue archives. The samples contained preserved tissue with complete spermatogenesis in orchidectomy specimens previously removed from patients with testicular germ cell cancer. Immunohistochemical staining with a monoclonal mouse-anti-human FGFR3 antibody (Santa Cruz Biotechnology, S. Cruz, USA) was performed according to a standard indirect immunoperoxidase protocol with a microwave heating pre-treatment, essentially as previously described for other antibodies (Rajpert-De Meyts et al., 1999
; Hoei-Hansen et al., 2006). A sample of human skin was used as a positive control, and for a negative control the procedure was performed on serial sections with a dilution buffer used instead of the primary antibody. All samples showed specific staining of spermatogonia (Fig. 1B). |
Discussion |
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We present an interesting case of preserved fertility in a patient with non-mosaic Klinefelter syndrome and a mutation in the FGFR3 gene, which causes achondroplasia. Both testes were much larger (8 ml) compared to what is usually seen in Klinefelter patients (2–4 ml), and repeated semen analyses revealed ongoing spermatogenesis with approximately 0.175 x 106 spermatozoa per ml. The patient fathered a son following a spontaneous pregnancy.
We found normal testosterone and LH levels and normal LH/T ratio in our index patient, which contrasts the impaired Leydig cell function usually found in non-mosaic 47,XXY patients (Aksglaede et al., 2007). FSH levels were elevated, but we found detectable inhibin B levels (71 and 23 pg/ml) in our patient, which also contrasts the usually undetectable inhibin B levels found in adult Klinefelter patients (Tomasi et al., 2003). We found haploid spermatozoa in the ejaculate of our patient by FISH technique, confirming previous studies of Klinefelter sperm. Typically, the spermatozoon of a non-mosaic Klinefelter patient carries only one sex chromosome, but the risk of disomy may be slightly increased in apparently non-mosaic patients: 3–4% disomy (XX or XY) as demonstrated by FISH (for review see Sarrate et al., 2005). A single study analysed sperm chromosomes from a 46,XY/47,XXY male and found a significantly increased incidence (0.9%) of hyperhaploid gonosomal 24,XY sets, with a lack of the expected corresponding gonosomal hypohaploidies, and a normal rate of autosomal non-disjunctions. This could suggest that 47,XXY cells are able to go through meiosis and to form spermatozoa (Skakkebaek et al., 1969; Cozzi et al., 1994), although more recent studies postulate that these cells cannot enter the meiotic process (Mroz et al., 1999; Blanco et al., 2001) in agreement with our present findings.
The initial cord formation in mice is strongly dependent on FGF9, which binds to FGFR3 protein. Knockout mice studies demonstrated that in the absence of FGF9, gonadal development is directed along the female pathway of differentiation. This sex reversal occurred in the majority of XY mice, although a few animals did display male traits in the form of hypoplastic testes (Colvin et al., 2001). The FGFR3 gene is expressed in testicular cords of the urogenital complex at embryonic day 14.5 of mice. At postnatal day 14, all cell types of the seminiferous tubules showed abundant FGFR3 staining. By postnatal day 25, expression was localized to the spermatogonia and spermatocytes, while in the adult mice FGFR3 expression was restricted to the spermatogonia (Willerton et al., 2004). A study of human expression pattern of FGFRs in the adult testis, demonstrated that FGFR2 was expressed in the cytoplasm of spermatogonia, while FGFR3 was present in nuclei of all germ cells except elongated spermatids (Steger et al., 1998). However, the nuclear localization of the latter casts doubt on the quality of the antibody used in this study. To investigate this further, we examined the pattern of expression of FGFR3 in the adult human testis by immunohistochemistry using a commercially available antibody. We established that in the adult testis, FGFR3 was present exclusively in spermatogonia, predominantly in the cell membrane and to a lesser extent in the cytoplasm (Fig. 1B). A very similar immunohistochemical pattern of expression of FGFR3 obtained with a different antibody (CAB004231) is also presented online in the publicly available Human Protein Atlas (Persson et al., 2006). A detailed analysis of the developmental expression pattern of FGFR3 and its ligands in a larger series of samples will be published separately.
In our patient, the FGFR3 mutation (1138G > A) was present both in peripheral blood leucocytes and in DNA isolated from sperm. This suggests that the mutation is in fact present in the spermatogenic epithelium of our patient. The typical FGFR3 mutation found in our patient is a gain-of-function mutation, which in bone causes premature ossification and closure of sutures, whereas in the testis the activation of the FGFR may stimulate spermatogonial proliferation, and/or prevent apoptosis, giving germ cells a selective advantage. Such a mechanism was first proposed by the group of Wilkie and coworkers (Goriely et al., 2003; Wilkie, 2005).
The preserved fertility in our patient suggests that the increased signalling through the FGFR3 may stimulate the proliferation of spermatogonia and alleviate, at least partially, the destruction of the seminiferous tubules usually observed in Klinefelter syndrome.
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Acknowledgements |
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The authors wish to thank Inger D. Garn and Lene Andersen for excellent technical assistance, and DakoCytomation for providing FISH probes. This work was supported in part by a grant from the Svend Andersen Foundation.
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References |
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Submitted on December 18, 2006; resubmitted on March 22, 2007; accepted on April 17, 2007.
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