Molecular Human Reproduction, Vol. 8, No. 10, 887-892,
October 2002
© 2002 European Society of Human Reproduction and Embryology
Reproductive endocrinology |
Functional study of a recombinant form of human LHß-subunit variant carrying the Gly102Ser mutation found in Asian populations
1 Department of Physiology, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland, 2 Department of Clinical Biochemistry, Statens Serum Institute, DK-2300 Copenhagen, Denmark and 3 Department of Biological Sciences, Florida International University, Miami, FL 33199, USA
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Abstract |
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Genetic variants of human LH caused by amino acid replacements in the ß-subunit have been demonstrated to affect reproductive function. Occurrence of a G1502A substitution in the LHß gene leading to Gly102Ser replacement of the LHß protein has been found to be associated with infertility in the Singapore Chinese population. In the present study, a search for this LHß allele from 383 DNA samples from different continents, using a PCR-based strategy, demonstrated its total absence in these populations. Functional properties of the variant (V) (Gly102Ser substitution) LHß subunit were assessed using a recombinant (r) form of V-LH produced in HEK293 cells, in comparison with wild-type (WT) LH or hCG. The synthesized V-LH was purified by a single step of immunoaffinity chromatography, and it had a molecular weight of 30 kDa as determined by SDS–PAGE. The affinities of the WT-hCG and rV-LH in mouse Leydig tumour (mLT-1) cell LH receptor binding were similar, with Kd values of 0.140 ± 0.03 and 0.156 ± 0.01 nmol/l respectively. Likewise, the effects of WT- and V-rLH preparations on mLT-1 cell cAMP and progesterone production were concentration-dependent and with similar biopotencies. In addition, HEK293 cells expressing the human LH receptor documented similar dose-dependent increases in inositol phosphate production by the two rLH forms. In conclusion, these findings demonstrate that Gly102Ser mutation of the LHß gene does not affect receptor binding and bioactivity of the hormone, when tested in vitro.
functional study/luteinizing hormone/variant
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Introduction |
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LH is a heterodimeric protein belonging to a family of glycoprotein hormones that also includes FSH, thyroid-stimulating hormone and hCG. All these hormones share a similar structure consisting of a common
-subunit and a specific ß-subunit. The peptide dimers bind to their hormone-specific receptors and trigger the G-protein-mediated cascade of signal transduction, leading to cellular responses (Pierce and Parsons, 1981
; Gharib et al., 1990). The receptors for glycoprotein hormones mainly transduce their signal by activating adenylate cyclase, but there is also activation of other signalling pathways, including inositol phosphate (IP) turnover, calcium and chloride fluxes, and mitogen-activated protein kinase pathways (Tena-Sempere and Huhtaniemi, 1999).
Normal development and function of the mammalian reproductive system are dependent on complex hormonal interactions, where LH and FSH have essential roles in both sexes. Due to their critical role in reproduction, the gonadotrophin subunit genes are highly conserved and exhibit very rare mutations (Themmen and Huhtaniemi, 2000). The only exception known so far is the common variant of LH (cV-LH) with two amino acid replacements, Trp8Arg and Ile15Thr, which has world-wide occurrence in various ethnic populations (Pettersson et al., 1992, 1994; Nilsson et al., 1997, 1998). In the original report on cV-LH from Japan, homozygous individuals for the variant LHß allele exhibited menstrual irregularities and infertility (Furui et al., 1994). In subsequent studies, heterozygous women from a predominately Jewish population from Boston, USA with cV-LH were reported to have problems with fertility (Cramer et al., 2000). On the other hand, in both Singapore Chinese men and women, no association was found between infertility and cV-LH (Ramanujan et al., 1999, 2000).
The only mutation in the LHß-subunit gene with a profound functional effect at the protein level was identified in a male patient with a homozygous Gln54Arg substitution (Weiss et al., 1992). This single amino acid alteration causes a change in the ß-subunit structure so that the incorrectly folded hormone is unable to bind to its receptor. Interestingly, only the heterozygous male members in the family were affected with infertility, while the heterozygous females were normally fertile (Weiss et al., 1992). In addition, two mutations causing amino acid replacements in the LHß subunit have been detected (Roy et al., 1996; Jiang et al., 2002). Both of these seem to be restricted to certain populations. A G52A mutation in exon 2, resulting in Thr–3Ala amino acid substitution in the signal peptide of the LHß subunit, was found in three heterozygous individuals out of 100 DNA samples from Rwanda (Jiang et al., 2002). Functional studies of the recombinant (r) form of LH carrying the Thr–3Ala variant site revealed that the mutation did not interfere with signal peptide cleavage nor the efficacy of heterodimerization. However, some differences were detected between the wild-type (WT) and mutant LH in signal transduction function, with the potential to cause phenotypic effects.
The Gly102Ser mutation, the topic of the current study, was first found in the Singapore Chinese population (Roy et al., 1996). Subsequently, this variant was identified in three additional Asian populations, yet its highest allelic frequency (0.018) was in the Singapore Chinese (Ramanujam et al., 1998). This rare mutation has been found exclusively in infertility or subfertility patients, both male and female (Ramanujam et al., 1999, 2000).
Previously, different recombinant forms of LH, WT and variants (Arg8/Thr15 and Ala–3) have been produced in our laboratory (Jiang et al., 2002; Manna et al., 2002). The functional properties of the recombinant hormones have been ascertained. Thus, these constructs are valuable tools in characterizing the functional effects of the LH mutations. In this study, we have screened the Gly102Ser mutation in several phylogenetically distinct populations representing three continents. In addition, we have produced a recombinant form of LH carrying the Gly102Ser variant (V-LH). Functional studies were performed with rV-LH to examine its effect on signal transduction and to identify potential differences between the WT- and V-LH signalling mechanisms, to explain the phenotypic consequences that were found to be associated in Singapore with the Gly102Ser mutation (Ramanujam et al., 1999, 2000).
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Materials and methods |
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Subjects
A total of 383 genomic DNA samples were collected anonymously and randomly from apparently healthy donors screened for infectious, metabolic or endocrinological diseases, or collected for anthropological studies in populations from Finland (n = 60), Bengali/North-East India (n = 78), Denmark (n = 145) and Rwanda (n = 100). Appropriate local guidelines for the ethics of sample collection were obeyed.
Mutation analysis
The Gly102Ser mutation was screened for using PCR amplification followed by restriction enzyme digestion. After DNA amplification of a 826 bp fragment of nucleotides (nt) 243–1068 [nt numbering according to Fiddes and Talmadge (Fiddes and Talmadge, 1984)] using thermostabile DNA polymerase (Dynazyme II; Finnzymes OY, Espoo, Finland), the fragment was digested using restriction enzyme EcoO 109 I (New England Biolabs, Inc., Beverly, MA, USA). According to the published sequence (Fiddes and Talmadge, 1984), the WT fragment was expected to generate 707 and 119 bp fragments, while the variant amplicon was expected to remain uncut. Against expectations, the WT fragment was cut into 447, 260 and 119 bp fragments. The unexpected restriction site for EcoO 109 I lead to digestion of the 707 bp fragment into 447 and 260 bp fragments. Sequencing of intron 2 of the LH ß-gene showed a nucleotide change A
G at position 689 compared with the published sequence, creating an extra restriction site and explaining the result (Jiang et al., 2002). Thus, the correct size fragments after EcoO 109 I digestion were 447, 260 and 119 nt for WT-LH and 447 and 379 nt for V-LH.
Mutagenesis
To study the functional characteristics of the Gly102Ser variant, rLH carrying the mutation was produced by mutagenesis. Incorporation of the Gly102Ser mutation GGTAGT into exon 3 of the LHß gene was performed using the QuickChange Site-Directed Mutagenesis Kit according to the manufacturer’s specifications (Stratagene Cloning Systems, La Jolla, CA, USA). The full length WT-LHß gene (1511 bp) was inserted into the eukaryotic expression vector pM2, downstream of the Harvey murine sarcoma virus long terminal repeat, as previously described (Suganuma et al., 1996). The expression vector was kindly provided by Dr N.Suganuma (Department of Obstetrics and Gynecology, Nagoya University School of Medicine, Nagoya, Japan). The nucleotide change was confirmed by automated sequencing (Perkin-Elmer Life Sciences, Boston, MA, USA) and plasmid purification was performed using Nucleobond cartridges (Nucleobond AX, Macherey-Nagel, Düren, Germany).
Production and purification of rV-LH
Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)-Ham’s F-12 medium (1:1; Life Technologies, Rockville, MD, USA), supplemented with 10% heat-inactivated fetal calf serum (FCS) (Autogen BioClear, Wiltshire, UK) containing 100 mg/l gentamycin (Biological Industries, Beit Haemek, Israel) in a humidified atmosphere of air containing 5% CO2 at 37°C. Transfections were carried out at 65–75% confluency of the cells by LipofectAMINE (Life Technologies). Several ratios were originally used, but the best transfection efficiency was reached with a 1:1 ratio of the -subunit gene construct (in eukaryotic expression vector pM2, received from Dr Suganuma) to the variant ß-subunit construct created by mutagenesis.
Following 48 h of transfection, the level of rLH was determined from the media by an immunofluorometric assay (DELFIA LH Spec Kit; Wallac OY, Turku, Finland). For creation of clonal lines, the cells producing high levels of rLH were chosen, trypsinized and split into selection medium containing 800 mg/l geneticin (G418; Sigma Chemical Co, St Louis, MO, USA). After 3–4 weeks, the resistant colonies for geneticin were selected, grown and screened for LH production. The clonal lines were first cultured in 15 cm diameter culture plates in medium containing 250 mg/l geneticin and later transferred to culture flasks (Nunclon Delta TripleFlask; Nalge Nunc International, Rochester, NY, USA) where the cells were cultured to 70–80% confluency. Before collection of the media for hormone purification, the cells were cultured for 2 days in serum-free medium.
The collected media were concentrated using the PelliconTM-2 ultrafiltration system (Millipore Corporation, Bedford, MA, USA). The concentrated medium was partly lyophilized and further concentrated and equilibrated with buffer A (10 mmol/l sodium phosphate containing 0.6% saline, pH 7.2) using centrifugal concentrators (Centriprep-10; Amicon Inc, Beverly, MA, USA). The purification was performed by immunoaffinity chromatography using cyanogen bromide-activated Sepharose 4 Fast Flow matrix (Amersham-Pharmacia Biotech, Uppsala, Sweden). The coupling of monoclonal anti-human (h)LH for intact LH dimer (code 5303; Medix Biochemica, Kauniainen, Finland) was conducted at 4°C for 12–16 h, after which the non-reacted groups were blocked with 1.0 mol/l glycine. The anti-LH tagged sepharose was packed into 8 cm bed height to high resolution 10/10 columns (Amersham). Sepharose was washed four times using high and low pH buffers. The concentrated medium was loaded onto the sepharose column and allowed to bind to the matrix for 4–6 h at 4°C. Bound LH was eluted with 10 mmol/l sodium phosphate, 0.6% saline, pH 7.2, containing 3.0 mol/l sodium iodide as a chaotropic agent. The immunoreactive peak fractions were pooled, dialysed against sterile distilled H2O, lyophilized and stored at –70°C.
As a control for the V-LH function, rWT-LH was used. For the cAMP and progesterone production experiments and for receptor binding, highly purified rWT-LH and rhCG respectively were used (kindly provided by Organon Pharmaceuticals Ltd, Oss, The Netherlands). For IP production, rWT-LH prepared previously in our laboratory was used (Manna et al., 2002).
Receptor ligand binding assays of rLHs
Highly purified hCG (CR-127, NIDDK) was radio-iodinated with Na[125I]-iodide (IMS 300; Amersham), using a solid phase lactoperoxidase method (Karonen et al., 1975). The binding studies were performed using two different methods. Method 1: incubating 2.5x105 intact mouse Leydig tumour (mLT-1) cells with [125I]-iodo-hCG (
100 000 cpm/100 µl) either in the absence (total binding) or presence (non-specific binding) of either 50 IU of unlabelled hCG or increasing amounts of rV-LH or hCG at room temperature for 16–18 h. Method 2: incubating 2.5x105 intact mLT-1 cells with increasing amounts of [125I]-iodo-hCG either in the absence (total binding) or presence (non-specific binding) of either 50 IU of unlabelled hCG or rV-LH at room temperature for 16–18 h. The incubation was stopped by placing the tubes on ice and adding 3 ml of ice-cold Dulbecco’s phosphate-buffered saline with 0.1% bovine serum albumin (BSA). After 30 min centrifugation (1850 g) at 4°C, the supernatants were discarded and the radioactivity of the cell pellets was determined in a 
-counter (1260 Multigamma II; AGG Wallac, Turku, Finland). The specific binding was calculated by subtracting the non-specific from total binding, and the data were converted into binding-inhibition curves and Scatchard plots.
Signal transduction studies
cAMP and progesterone production after LH stimulation
mLT-1 cells were cultured in Waymouth’s Growth Medium (Life Technologies) supplemented with 9% heat-inactivated horse serum, 4.5% heat-inactivated FCS and 50 mg/l gentamycin. mLT-1 cells were trypsinized and plated on 24-well plates (Greiner, Frickenhausen, Germany) at a density of 80 000 cells/1.9 cm2/well 18–24 h prior to stimulation. The cells were exposed for 2 h (cAMP) and 6 h (progesterone) to doses of 0–1000 µg/l of rLH. Cyclic AMP and progesterone levels from collected media were measured by radioimmunoassays as previously described (Harper and Brooker, 1975; Vuorento et al., 1989).
IP production after LH stimulation
HEK293 cells were transfected with the pcDNAneo-hLHR plasmid containing the human luteinizing hormone receptor (hLHR) cDNA (Manna et al., 2002). After 24 h, the cells were trypsinized and plated at a density of 5x105 cells/9.4 cm2/well into 6-well plates. After 12–16 h, the cells were labelled using 2 µCi/ml [3H]-inositol (NEN Life Sciences Products Inc., Boston, MA, USA) in DMEM containing 2–5% FCS. After 24–30 h of labelling, the cells were washed and prestimulated for 10–15 min in serum-free DMEM with 13 mmol/l LiCl, 58 mmol/l NaCl and 0.1% BSA 1 ml/well. After prestimulation, increasing doses of rLH, WT and V were added to the medium and the stimulation was continued up to 60 min. The reaction was stopped by transferring the plates onto ice and adding stopping buffer (10% perchloric acid, 0.5 mmol/l EDTA). The cells and media combined were centrifuged and the supernatant containing IP was neutralized with 1.5 mol/l KOH in 60 mmol/l HEPES buffer. The level of IP was determined in analytical grade 1 anion exchange columns (BioRad, Hercules, CA, USA) and radioactivity was counted in a liquid scintillation counter (1215 RackBeta II; LKB Wallac, Turku, Finland) as demonstrated previously (Tena-Sempere et al., 1999).
Data analysis
The data are presented as mean ± SEM. The statistical analyses were conducted by Student–Newman–Keuls test (SigmaPlot Version 2.0, SSPS Science, Chicago, USA). P < 0.05 was considered statistically significant.
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Results |
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Screening of the Gly102 Ser mutation
Because the Gly102Ser LHß mutation was found to have a possible correlation with infertility in the Chinese population from Singapore (Liao et al., 1998
; Ramanujam et al., 1999, 2000), we considered it important to study its prevalence in other ethnic groups. We screened DNA samples of 205 Caucasians from Northern Europe (60 Finns, 145 Danes), 100 East-African Hutus (Rwandan) and 78 Caucasians from Asia (Bengali). The screening was performed by RFLP analysis after PCR amplification of a 826 bp fragment. A WT amplicon harboured two cutting sites resulting in fragments of 447, 260 and 119 bp, while the Gly102Ser, GA mutation abolished the restriction site at nucleotide 1502, thus leading to a 447 and 379 bp profile (Figure 1). As a positive control for the screening, a known heterozygous sample from Singapore carrying the mutation was used (provided kindly by Drs Roy and Liao). Except for the positive control sample, the Gly102Ser mutation was not found in any of the individuals studied.
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Production and purification of rV-LH
HEK293 cells were transfected with both the LH
- and the V-LHß-subunit genes within the pM2 expression vector plasmids containing the bacterial neomycin resistance gene. The selection of clonal cell lines for large-scale LH production was based on resistance to the antibiotic G-418 and on high LH production. The amount of LH in cell culture medium was monitored constantly using immunofluorometric assay. Routinely, a total of 7 l of medium was collected and concentrated. The purification of rV-LH was performed by immunoaffinity using the coupling of rV-LH to an anti-LH monoclonal antibody for an intact LH dimer. The purity and molecular size of rV-LH (20–50 ng of V-LH) from crude medium and immunoaffinity purified medium was determined by SDS–PAGE (not shown). The major band representing V-LH in purified medium was 30 kDa in size. Removal of impurities and unwanted proteins diminished the amount of rV-LH. However, the specific activity of the purified protein was increased 17.6-fold; in crude medium the specific activity was 26 IU/mg, while in the immunoaffinity purified preparation the specific activity was 460 IU/mg. Thus, the purified rV-LH demonstrated high biological activity and potency of signal transduction (see below). Because the HEK293 cells used do not express the endogenous gonadotrophin subunit genes, the LH activity detected in the culture media was encoded by the transfected plasmid, and sequencing verified that it carried the Gly102Ser LHß mutation.
LH receptor binding studies
To define the characteristics of specific V-LH and WT-LH binding to LH receptors, ligand binding studies were performed in mLT-1 cells using [125I]iodide-hCG as a tracer. No significant differences were observed in binding of rV-LH or hCG. The equilibrium dissociation constants (Kd) calculated from two Scatchard plots were 0.140 ± 0.03 nmol/l for hCG and 0.156 ± 0.01 nmol/l for V-LH, and the maximum binding values were 7.3 ± 1 and 6.3 ± 1.5 pmol/l respectively (mean ± range of two individual measurements). These data demonstrate that the mutational change Gly102Ser did not markedly affect the affinity or capacity of LH binding to the LH receptor, although some variance between the intercept of the hormone preparations was seen (Figure 2). The slightly, but not significantly, lower bioactivity of V-LH as compared with the rWT hormones may be due to its lower stability in the incubations.
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Effects of rLH on mLT-1 cell cAMP and progesterone production
The in-vitro biological activity of rV-LH was compared with that of rWT-LH produced by Organon, by determining the cAMP and progesterone responses of mLT-1 cells. The data presented in Figure 3
show dose-dependent increases in the levels of cAMP (Figure 3A) and progesterone (Figure 3B) with increasing doses of the two rLHs (0–500 µg/l). The experiment was repeated three times and the data showed that the maximum responses in terms of cAMP were 134 ± 24-fold for WT-LH and 121 ± 28-fold for V-LH, and in terms of progesterone, 707 ± 197- and 734 ± 123-fold respectively. No statistically significant differences were found in these responses between the two LH preparations. Neither did the ED50 values obtained with the three experiments exhibit significant differences between the rWT-LH and rV-LH either in cAMP (ED50 for WT-LH 6.8 ± 3.1 µg/l, for V-LH 4.1 ± 0.6 µg/l) or progesterone (ED50 for WT-LH 0.40 ± 0.03 µg/l, for V-LH 0.51 ± 0.18 µg/l) responses.
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Influence of rLH on IP production
To study further the signal transduction after stimulation with rWT-LH and rV-LH, the IP production was monitored. HEK293 cells were transfected with a plasmid construct carrying the human LH receptor cDNA. Cells were incubated with [3H]-inositol and stimulated with increasing concentrations of rLH, and dose-dependent IP production was observed. The fold increase in IP production with V-LH compared with WT-LH did not demonstrate significant differences at any of the LH doses when the results of two independent experiments with duplicate samples were performed (Figure 4
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Discussion |
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Mutations in gonadotrophin subunit genes are rare. For example, only four genetic variants, Gln54Arg, Trp8Arg/Ile15Thr, Gly102Ser and Ala–3Thr, have been detected in the LHß subunit (Weiss et al., 1992
; Furui et al., 1994; Pettersson et al., 1994; Roy et al., 1996; Jiang et al., 2002). The LHß variants differ from each other in several aspects, including their distribution among the human population, frequency of occurrence and functional properties. The most common and also the most extensively studied is the common variant, cV-LH, with two amino acid replacements Trp8Arg and Ile15Thr. The allelic frequency of this variant varies extensively between different ethnic groups, and its distribution shows that the cV-LHß allele is a universally occurring polymorphism, and thus different from the other LH variants so far detected, which have a more restricted distribution. The wide occurrence of this allele in populations with different evolutionary histories, and the wide frequency variability may imply that this polymorphism represents an ancient form of LH and it may have offered an advantage in certain environments during human evolution (Lamminen and Huhtaniemi, 2001).
Studies on the biochemical properties of cV-LH have revealed increased bioactivity in vitro based on its higher potency upon signal transduction, but a shorter half-life in circulation compared with WT-LH (Haavisto et al., 1995; Manna et al., 2002). Still, the phenotypic effect of cV-LH remains somewhat unclear. In large population studies, the effect of cV-LH on several conditions related to LH action, such as polycystic ovarian syndrome, breast cancer, recurrent spontaneous abortions, frailty of old age and progression of puberty, have been found to be on the borderline of statistical significance (Rajkhova et al., 1995; Raivio et al., 1996; Tulppala et al., 1998; Tapanainen et al., 1999; Van den Beld et al., 1999; Akhmedkhanov et al., 2000; Cramer et al., 2000; I. Huhtaniemi et al., unpublished data). However, both homozygosity and heterozygosity for the cV-LH allele have shown a significant association with infertility or subfertility in some populations (Furui et al., 1994; Takahashi et al., 1998, 1999; Cramer et al., 2000).
Compared with cV-LH, the Gln54Arg mutation has contrasting features. The proband was a 17-year-old Caucasian man with delayed puberty, and infertility was repeatedly found in his maternal uncles, yet all the female relatives studied were fertile (Weiss et al., 1992). Although the LH of the patient was immunologically active, in-vitro bioassays demonstrated that it was totally devoid of bioactivity due to its inability to bind to the LH receptor. An AG missense mutation was found in codon 54 of this LHß variant, causing a Gln to Arg substitution. Functional studies confirmed the inactivating effect of the mutation.
Two other variants of the LHß subunit exhibit more restricted distribution and may be population-specific according to the screenings of various ethnic groups. A G52A mutation in exon 2, resulting in a Thr–3Ala amino acid substitution in the LHß signal peptide, was found upon screening for LHß gene polymorphisms in random population samples (Jiang et al., 2002). The variant was found in only three heterozygous individuals out of 100 DNA samples from Rwanda. This may indicate an African origin for the polymorphism and possibly its limited existence in populations of this continent. Protein sequencing of a recombinant form of this LH variant revealed normal signal peptide cleavage. Neither was the efficacy of heterodimerization of LH markedly influenced. However, an increase in IP production and a minor decrease in the LH-stimulated cAMP response were observed when signal transduction was compared between WT-LH and the mutant LH. The phenotypic consequences of this variant are unknown, but extensive functional studies of the recombinant hormone variant Thr–3Ala suggest that this mutation may have some influence on fertility.
The GA transition in nucleotide 1502 was found by single strand conformational polymorphism analysis in the Chinese population of Singapore (Roy et al., 1996). Subsequently, the mutation was screened in three populations from South-East Asia, but it was found only in the Chinese population of Singapore with an allelic frequency of 0.018 (Ramanujam et al., 1998). The mutation leads to a Gly102Ser replacement in exon 3 of the LHß subunit gene. Amino acid Gly102 is highly conserved among mammalian species, and therefore a change in this position may cause a conformational change and affect the normal function of LH (Liao et al., 1998). Indeed, this mutation has been found in both females and males with reproductive problems (Liao et al., 1998; Ramanujam et al., 1999, 2000), and was first identified in two infertile women with endometriosis. However, 50 infertile women either with menstrual disorders, polycystic ovarian syndrome and endometriosis or idiopathic infertility and 212 healthy fertile controls lacked this variant (Liao et al., 1998). In another study of Singapore Chinese women, 7/176 (4%) patients with menstrual disorders were found to be heterozygous for the Gly102Ser mutation, while 200 normal controls were WT homozygotes (Ramanujam et al., 1999). In the Singapore Chinese male population, the mutation was found at a frequency of 5/145 (3.4%) in infertile males, but was absent in 200 healthy controls (Ramanujam et al., 2000). Interestingly, in men the variant LH was associated with varicocele. It was suggested that the Gly102SerLH variant together with varicocele may cause infertility (Ramanujam et al., 2000). On the other hand, it has been reported that LH concentrations are higher in infertile patients with varicocele than in males with normal fertility (Freire and Nahoum, 1981). In the Singaporean study, the LH concentrations were normal in four patients and low in one patient harbouring the Gly102Ser replacement. Therefore, the possible connection between this LH mutation and varicocele-associated infertility remains open.
We have screened for the Gly102Ser mutation in random samples representing populations from three continents. No positive samples for the mutation were found (Jiang et al., 2002; this study). Therefore, based on a small-scale population study of Finnish, Danish, Bengali and Rwandan samples, it seems that the occurrence of this mutation may be population-specific and possibly restricted to Eastern Asian populations, and even more specifically to the Singapore Chinese. However, in the present study the Gly102Ser mutation was screened in random samples for which the status of infertility/fertility was unknown. To study the population specificity of this mutation, it would be necessary to screen samples of infertile donors from populations other than the Singapore Chinese.
Although mutations in the LHß-subunit gene are very rare, the study of their biological actions is important in elucidating normal and abnormal functions of LH. The biological activity and function of the hormones can be reliably studied in vitro and in vivo using recombinant molecules carrying either WT or mutated structures of LH (Suganuma et al., 1996; Jiang et al., 2002; Manna et al., 2002). In the present study, we produced a recombinant form of LH with the Gly102Ser replacement in exon 3 of the ß-subunit. The biological activity was evaluated using dose-dependent stimulation of mLT-1 cells with WT-LH and V-LH and measuring the cAMP, progesterone and IP responses, as well as binding to the LH receptor. Although some variance was observed between the hormones studied, the differences can be explained by the experimental variance. Thus, no marked differences were found in any of the parameters studied, suggesting similar ligand binding and signal transduction characteristics among the two hormones. Therefore, according to our experiments, there seems be no obvious functional defect in signal transduction caused by the Gly102Ser mutation that could cause infertility in people harbouring this mutation. However, it must be remembered that this study only examines a small part of the complex signalling pathways in vitro, and neither the regulation of expression of the LHß allele nor the in-vivo behaviour of the variant hormone were studied. Therefore, more extensive experiments may reveal functional differences.
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Acknowledgements |
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We would like to thank Drs M.Poutanen and P.Ryhänen (Department of Physiology, University of Turku) and C.Nilsson (Department of Biotechnology, University of Turku) for advice and help during this study. Professor M.-L.Savontaus is acknowledged for kindly providing us with the Finnish DNA samples. The technical assistance of Ms T.Laiho and Ms R.Kytömaa is gratefully acknowledged. H.Peuravuori (Department of Pathology, University of Turku) is thanked for assistance in the lyophilization of rLH. We thank Dr N.Suganuma (Department of Obstetrics and Gynecology, Nagoya University School of Medicine, Nagoya, Japan) for the expression vectors pM2LH
and pM2LHß. Drs A.C.Roy and W.X.Liao (Department of Obstetrics and Gynecology, Faculty of Medicine, National University of Singapore) are thanked for donating the positive DNA sample for the Gly102Ser mutation screening. This study was supported by grants from the Academy of Finland, and partially by an NIH grant to R.J.H. |
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4 To whom correspondence should be addressed. E-mail: ilpo.huhtaniemi{at}utu.fi
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References |
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Submitted on April 10, 2002; accepted on July 22, 2002.
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