1 School of Clinical Medical Sciences, University of Newcastle NE2 4HH, UK, 2 Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Medical School, Chicago, IL 60611, USA, 3 Centre for Human Growth and Maturation, Department of Medicine and Institute of Child Health, University College London, London WC1N 1EH, UK and 4 Department of Physiology, University of Turku, Turku, FIN-20520, Finland
5 To whom correspondence should be addressed at: Department of Paediatrics, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP, UK. e-mail: t.d.cheetham{at}ncl.ac.uk
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Abstract |
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Key words: FSH/FSH receptor/infertility/ovary/puberty
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Introduction |
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The G protein-coupled FSHR receptor consists of seven transmembrane domains and an amino-terminal extracellular hormone binding region (Simoni et al., 1997; Themmen and Huhtaniemi, 2000
). The gene encoding FSHR is located on the short arm of chromosome 2 in humans and consists of 10 exons spanning 54 kB of genomic DNA. The first 9 exons of this gene encode the extracellular domain of the FSHR. Exon 10 encodes the transmembrane and intracellular domains, as well as the proximal portion of the extracellular domain.
The first inactivating mutation of the FSHR gene was described in individuals from several Finnish relatives who presented with poorly developed secondary sexual characteristics, primary amenorrhoea and recessively inherited hypergonadotrophic ovarian failure (Aittomäki et al., 1995, 1996). Functional studies demonstrated that this Ala189Val mutation results in a functionally inactive FSHR, consistent with the severe phenotype reported in affected patients (Aittomäki et al., 1995
). More recently, three patients with compound heterozygous mutations in the FSHR have been described. Two patients (harbouring the mutations Asp224Val/Leu601Val and Ala189Val/Ala419Thr) had normal secondary sexual characteristics and primary amenorrhoea (Touraine et al., 1999
; Doherty et al., 2002
). The other patient (Ile160Thr/Arg573Cys) had a milder phenotype consisting of normal secondary sexual development and secondary amenorrhoea (Beau et al., 1998
). In this report, we describe a novel complete loss-of-function mutation in the FSHR in an adolescent girl with hypergonadotrophic ovarian failure and primary amenorrhoea.
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Materials and methods |
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Microsatellite analysis
Microsatellite analysis was performed using fluorescently labelled primers to amplify highly polymorphic regions around the FSHR locus (2p1125.3). PCR products were electrophoresed using an ABI373 sequencer and results were analysed using genescan and genotyper software (Applied Biosystems).
Chromosome painting
Whole chromosome painting by fluorescence in-situ hybridization (FISH) was performed on fixed metaphase chromosome preparations from family members using chromosome 2 specific probes (Quest Diagnostics, Nichols Institute, San Juan Capistrano, CA, USA).
Construction of hFSHR expression vectors
Wild-type (WT) and mutant (Pro348Arg) FSHR expression vectors were created for use in functional studies. The wild-type vector was constructed by cloning hFSHR cDNA directly into a pSVL expression vector (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The mutant vector was constructed by site-directed mutagenesis, using an overlapping PCR strategy with wild-type hFSHR cDNA as a template. This mutant cDNA fragment was ligated into the full-length hFSHR and transferred into a pSVL expression vector. The presence of the mutation was confirmed by sequencing.
Functional studies
Cell culture experiments were performed using tsa201 cells, a human embryonic kidney line. These cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 µg/ml streptomycin in a 5% CO2 atmosphere at 37°C.
Intracellular cAMP assays
Cells were transiently transfected with 500 ng of either negative control (empty vector), wild-type FSHR construct (WT) or mutant construct (Pro348Arg) by calcium phosphate DNA precipitation. After 48 h, cells were treated with 0.2 mmol/l 3-isobutyl-1- methyl-xanthine (IBMX) and varying concentrations of human FSH (0400 IU/l, IRP-68/140; SigmaAldrich, S Louis, MO, USA) for 30 min. Media were removed, cells were snap-frozen on dry ice, and 750 µl cold 0.1 mol/l HCl was added to each well. Cell lysates were centrifuged (16 000 g) for 10 min at 4°C and 10 µl of each supernatant was removed and neutralized with an equal volume of 150 mmol/l TrisHCl (pH 8.0). Cyclic AMP concentrations were measured by radioimmunoassay (Biomedical Technologies Inc., Stoughton, MA, USA) according to the manufacturers instructions. Representative data for triplicate transfections (mean ± SEM) are shown.
Transient gene expression studies using a cAMP-responsive luciferase reporter
Cells were transiently transfected with 50 ng pSVL expression vector (empty vector, WT or Pro348Arg) and 20 ng pA3846luc (cAMP-responsive) reporter. After 48 h, cells were treated with 0.2 mmol/l IBMX and varying concentrations of human FSH (0400 IU/l) for 6 h. Cell extracts were prepared and luciferase assays performed. Representative data for triplicate transfections (mean ± SEM) are shown.
Hormone binding studies
Transfection
Monkey kidney COS-7 cells were transiently transfected with expression vectors containing either wild type or mutated FSHR, or the vector only using FuGENE 6 Transfection Reagent (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturers instructions. Co-transfections with a luciferase-expressing vector were done to control the transfection efficiency.
FSH binding assay
Recombinant human FSH (rhFSH; Organon, Oss, The Netherlands) was radioiodinated with Na[125I]iodine (IMS 300; Amersham Biosciences UK Ltd, Little Chalfont, UK) using a lactoperoxidase method (Karonen et al., 1975) to specific activity of 3800 cpm/ng. The transfected COS-7 cells were cultured in 9 cm diameter cell culture plates for 48 h, washed with phosphate-buffered saline and scraped into Dulbeccos PBS containing 0.1% bovine serum albumin (BSA; SigmaAldrich). Triplicate aliquots of cell suspensions, containing 2x105 cells, were incubated in the presence of increasing amounts of radio-iodinated rhFSH (from 3.3 to 131.6 ng) in a total volume of 300 µl. For non-specific binding, 2.5 IU of rhFSH was used at each hormone concentration. Cells were incubated overnight at room temperature, and the radioactivity of the cell pellets was counted in a
-spectrometer.
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Results |
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Microsatellite analysis
Microsatellite analysis showed that the proband is heterozygous for nine out of fourteen polymorphic markers spanning 2p (Table II). Segmental paternal uniparental isodisomy involving the D2S123 marker, a known region of chromosomal instability, was excluded as a cause of the presence of only C1043G mutant sequence in the proband. The microsatellite analysis also excludes the possibility that the proband inherited a large deletion within chromosome 2p from the mother.
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Functional studies
Functional studies demonstrated a dose-dependent increase in luciferase activity and cAMP production with the WT FSHR in response to FSH stimulation (Figure 3). In contrast, the Pro348Arg mutant FSHR showed complete loss of activity at all FSH concentrations.
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Discussion |
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Only a limited number of FSHR mutations have been reported to date (Figure 5). The best characterized of these is the Ala189Val (A189V) mutation that is found in the Finnish population (Aittomäki et al.; 1995, 1996; Tapanainen et al., 1997
; Rannikko et al., 2002
). Similar to our observations with the Pro348Arg (P348R) substitution, the Ala189Val mutation causes complete loss of receptor function, resulting in severe clinical features such as impaired pubertal development, primary amenorrhoea, low estradiol and elevated FSH in homozygous females. It is likely, therefore, that these cases represent the most extreme end of the phenotypic spectrum associated with FSHR mutations in women.
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A surprising feature of this pedigree is the absence of a mutation in the mother. The apparent homozygous genotype in the proband would suggest the inheritance of the same mutant allele (C1043G) from the mother and the father. Although the father was heterozygous for this change, the mother appeared to have wild-type sequence. A similar inheritance pattern was found for an SNP in a 3' region of genomic DNA located 1718 nucleotides from the exon 10 mutation. This inheritance pattern could be explained by one of three genetic mechanisms: paternal uniparental isodisomy (UPD), gene conversion, or deletion of part of the maternal allele.
Several cases of UPD involving chromosome 2 have been described, but these are usually whole chromosome changes that involve maternally inherited material in all cases (Kotzot, 1999). The presence of heterozygosity for several markers in the proband would exclude this. More recently, a case of maternal segmental UPD has been described that involves a region of
100 000 base pairs of chromosome 2p and includes the marker D2S123 (Stratakis et al., 2001
). This marker lies in the vicinity of the FSHR locus [GeneMap 99 (GB4), National Center for Biotechnology Information] and represents a region of high chromosomal recombination and potential instability. However, microsatellite analysis of our pedigree using this marker (D2S123) excludes paternal segmental UPD in this region as the cause of the genotype in the proband. It remains possible that a more limited region of paternal segmental UPD or somatic gene conversion has occurred (involving the FSHR exon 10 locus and region 3' to it), in which the mutant allele (from the father) undergoes genetic exchange with the wild-type allele (from the mother). The phenomenon of gene conversion is becoming increasingly recognized in humans (Jonkman et al., 1997
; Merke et al., 1999
).
The presence of heterozygous markers in the proband at positions D2S123 (cen) and D2S2227 (tel) exclude the presence of a very large deletion of a maternal allele involving the FSHR locus. Similarly, chromosome painting failed to detect any significant deletions or rearrangements. It remains possible that the proband harbours a small deletion (spontaneous or inherited from the mother) of this region of chromosome 2 that was not detected by our analysis. Indeed, a combined mutation/deletion has been reported in a patient with an inactivating LH receptor mutation previously (Laue et al., 1996). Given the inheritance pattern of the SNP in the 3' region, such a deletion would have to extend beyond the 3' boundary of exon 10 of the FSH receptor locus.
Although FSHR mutations appear to be rare in most populations (Layman et al., 1998; Jiang et al., 1998
; Conway et al., 1999
), this report confirms that FSHR mutations can be found in patients who present with spontaneous (non-familial) delayed puberty and primary amenorrhoea. The clinical and molecular features of these patients further our understanding of genotypephenotype correlations in this disorder and the role of the FSHR in the development and function of the ovary.
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Acknowledgements |
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References |
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Submitted on June 15, 2002; accepted on October 9, 2002.