Mol. Hum. Reprod. Advance Access originally published online on August 12, 2005
Molecular Human Reproduction 2005 11(8):601-605; doi:10.1093/molehr/gah198
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Human FSHß subunit gene is highly conserved
1Department of Physiology, Institute of Biomedicine, University of Turku, Kiinamyllynkatu, Turku, Finland, 2Department of Clinical Biochemistry, Statens Serum Institute, Copenhagen, Denmark and 3Institute of Reproductive and Developmental Biology, Imperial College London, Hammersmith Campus, London, UK
4 To whom correspondence should be addressed at: Institute of Reproductive and Developmental Biology, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 ONN, UK. E-mail: ilpo.huhtaniemi{at}imperial.ac.uk
5 Present address: Department of Paediatrics, Glostrup University Hospital, Glostrup, Denmark
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Abstract |
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FSH is a pituitary gonadotropin that along with LH plays a key role in the regulation of gonadal function. The gonadotropic hormones are composed of two subunits, the common
subunit and the hormone-specific ß subunit, which determines the binding to specific receptors and induction of biological response. Unlike the LHß gene, shown in earlier studies to harbour several amino acid-altering polymorphisms and mutations, information about the eventual sequence variation of the human FSHß subunit is not available. In this study, we made sequence analysis and comparison of polymorphisms found in FSHß in two Caucasian populations, the Finns and the Danes. It was found that FSHß subunit is highly conserved in these populations. Compared with the published sequences, only three silent polymorphisms were detected in the coding regions of the gene, and the promoter sequence was completely identical with the reported sequence. Two of the polymorphisms found were novel, one in the Finnish and one in the Danish population. The results of the sequence analysis show that the human FSHß gene is highly conserved and amino acid changing mutations are apparently extremely rare, at least in the samples collected randomly from control populations. This may be due to the crucial role of normal FSH function in the regulation of fertility. Key words: FSHß/gene/polymorphism/population study
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Introduction |
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FSH is a member of the family of glycoprotein hormones together with LH, HCG and thyroid-stimulating hormone (TSH). The gonadotropins LH and FSH are secreted from the anterior pituitary, and they have essential roles in the regulation of development and function of the reproductive system. These hormones share a heterodimeric structure where the common
subunit is non-covalently associated with a unique hormone-specific ß subunit (Pierce and Parsons, 1981
). Only the heterodimer can bind to the hormone-specific receptor and evoke signal transduction and hormonal stimulation. LH and HCG are structurally similar and use the same LH/HCG receptor for signal transduction, but FSH binds only to the specific FSH receptor (Simoni et al., 1997).
The biological function of FSH has two structural requirements: subunit heterodimerization and specific receptor binding. The common subunit is shared by all glycoprotein hormones, and it forms heterodimers with the hormone-specific ß subunits. The ß subunit enables the correct interaction with the binding sites in the extracellular domain of membrane-bound receptor (Simoni et al., 1997; Fan and Hendrickson, 2005). Theoretically, mutations in these subunits could have different consequences depending on the affected subunit and specific location of the mutation. Inactivating mutations in the common subunit would have widespread effects starting in early development because of disturbed HCG function that would probably seriously compromise pregnancy. Subsequently, an affected individual would be hypogonadal and hypothyroid. Probably for this reason, no germ-line mutations of the subunit have been discovered. Only one patient has been reported with a somatic subunit mutation in undifferentiated carcinoma tissue of the femoral region, but even in this case the mutated subunit co-existed with the normal subunit allele (Nishimura et al., 1986). Perhaps for the same reason, functionally significant mutations in the HCGß subunit have not been discovered. An amino acid altering point mutation V79M with 4.2% frequency was reported about 5 years ago from American mid-west (Miller-Lindholm et al., 1999), but the existence of this polymorphism could not be verified when analysed in 580 DNA samples from four European populations (Jiang et al., 2004).
In contrast to common and HCGß subunits, several mutations and polymorphisms have been found in the LHß subunit (Huhtaniemi, 2003). Five sequence variations of LHß are known today. A Q54R mutation was found in one Caucasian male with normal intrauterine masculinization but lack of spontaneous puberty. The mutation leads to altered hormone structure so that the formed LH /ß heterodimer is unable to bind to the LH receptor (Weiss et al., 1992). Another mutation, G36D, was recently reported in another male with very similar phenotype (Valdes-Socin et al., 2004). Two mutations, A-3T and G102S in the LHß subunit, have been found only in single populations from Africa (Rwanda) and Asia (Singapore Chinese), respectively. The effects of the latter alterations on LH function are minor or nonexisting, although the G102S mutation has been reported to be associated with infertility in both sexes in Asian populations (Liao et al., 1998; Ramanujam et al., 1999; Jiang et al., 2002; Lamminen et al., 2002). In addition to these rare and probably population-specific polymorphisms, a common LH variant (V-LH), due to two-linked amino acid alterations, W8R/I15N, has been found in numerous populations worldwide with a variable allelic frequency (Nilsson et al., 1997). V-LH has increased in vitro bioactivity but shortened circulating half-life, and it has been associated with several clinical conditions with potentially altered gonadotropin function (Manna et al., 2002; Huhtaniemi, 2003, 2004).
With regard to the FSHß subunit, four inactivating mutations, either altering single amino acids or deleting nucleotides leading to premature stop codons and truncated FSH ß-subunit protein, have been reported in four female and three male patients (Matthews et al., 1993; Layman et al., 1997, 2002; Lindstedt et al., 1998; Phillip et al., 1998). The phenotypic characteristics caused by these mutations in both sexes are severe, resulting in absent or incomplete pubertal development and infertility.
Although some FSHß mutations have been found in patients with reproductive problems, the variability and conservation of the FSHß gene sequence remains unknown. In this study, we wanted to determine the frequency of polymorphisms in the FSHß gene and in its promoter in randomly selected, apparently healthy individuals of two Caucasian populations, the Finns and the Danes. The Finns represent a population with a Uralic, more precisely Finno–Ugric language and have a unique genetic heritage moulded by isolation from other populations, geographical location and strong internal migration movement in the 1500s (Rosser et al., 2000; Norio, 2003a,b). The Danish population belongs to the Indo-European, Germanic language-speaking populations and have a strongly diverse genetic Y-chromosomal haplogroup structure from the Finns (Rosser et al., 2000). Systematic comparison of genetic differences between these two populations and published sequences can give information about the frequency of polymorphisms and mutations occurring in the human FSHß gene.
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Materials and methods |
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Subjects
Two hundred DNA samples were collected anonymously and randomly from apparently healthy donors in populations from Finland (n = 101) and Denmark (n = 99). Appropriate local guidelines for the ethics of sample collection and handling were obeyed.
Sequence analysis
The study was carried out by sequencing PCR-amplified DNA of 50 Finnish and 50 Danish samples, except for fragment 1 (see Table I) which was sequenced on 15 Finnish and 10 Danish samples. The primer sequences for amplifications are listed in Table I, and the locations of the primers in the FSHß gene structure are shown in Figure 1. Fragment 1 covers the promoter region 489 bp upstream of exon 1, and fragment 2 covers exon 1, the preceding TATA box and part of the non-coding region flanking exon 1. Fragments 3 and 4 cover exon 2 and translated part of exon 3, respectively. Intronic sequences included into the amplified fragments were sequenced in 20–50 nucleotide regions flanking the exons. The numbering of the exons is according to Jameson et al. (1988).
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, signal peptide;
PCR products (Dynazyme II, Finnzymes OY, Espoo, Finland) were run on 1% agarose gels, purified using the GFXTM PCR DNA and Gel Band Purification Kit (Amersham Biosciences, Buckinghamshire, UK) and sequenced using the BigDyeTerminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequencing was made with AbiPrism 377 DNA Sequencer and AbiPrism 310 DNA Analyzer (Applied Biosystems).
The polymorphism in exon 3, Y76 TAT
TAC (Liao et al., 1998), was screened by using PCR amplification followed by restriction enzyme digestion. Amplified fragment 4 was digested using restriction enzyme AccI (New England Biolabs, Beverly, MA, USA) according to the manufacturer’s recommendations.
Nomenclature for the sequence variations
Nomenclature for the sequence variations follows the international recommendations (Dunnen and Antonarakis, 2001).
Statistical analysis
The statistical analyses of frequencies of the detected polymorphisms were conducted by chi-square test (StatView software, SAS Institute, Cary, NC, USA). P < 0.05 was considered statistically significant.
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Results |
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Sequence analysis revealed that the FSHß promotor and translated regions are highly conserved in the populations studied. Compared with the published FSHß sequence (Jameson et al., 1988
) and Celera sequence (Celera Discovery System, Applied Biosystems), only three polymorphisms were found in the translated part of exon 3 of the gene (Table II).
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All of the mutations found were silent and did not thus cause amino acid changes in the coded peptide. One of the mutations Y76 TATTAC has been reported earlier [Liao et al., 1998; National Center for Biotechnology Information, SNP databank (NCBI/SNP) 2005], but the two others are novel findings. The frequencies of the novel silent polymorphisms are statistically significantly different in the Finnish and the Danish populations. The frequency of heterozygous carriers of K58 AAGAAA polymorphism is 16% in the Danish and 0% in the Finns populations (P < 0.0001), and the heterozygote frequency of K104 AAGAAA is 8 and 0% in the Finns and the Danes, respectively (P = 0.0039). In addition to these, two intronic changes were observed. The first of these is an inversion 49–52inv4 after exon 2. The inversion was found in all samples studied, and because the Celera sequence has GGCC in this position, it is most probably a mistake in the original published sequence. The second one is a heterozygous intronic nucleotide change at IIS + 33C > T. The allelic frequencies for allele T are 43 and 59% for the Finnish and the Danish populations, respectively (P = 0.0236).
The silent mutation in codon 76, TATTAC, was screened in a larger number of samples in Danish (n = 99) and Finnish (n = 101) populations by restriction enzyme analysis. In the Asian populations, the frequency of the silent mutation was determined earlier by Liao et al. (1998). The C nucleotide seems to be more common in the Nordic populations, whereas in the Asian populations, T is the primary nucleotide. Both the Finnish and the Danish populations have the genotype frequencies in Hardy–Weinberg equilibrium, so the observed frequencies of heterozygotes do not differ significantly from those expected, the P values being 0.944 and 0.915, respectively. The allele frequencies are listed in Table III. Statistically significant (P < 0.05) differences were found in pair-wise comparisons of genotype frequencies between the Nordic populations and all three Asian populations.
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The FSHß promoter was sequenced on 15 Finns and 10 Danish samples. The sequence spanned 430 bp upstream of exon 1 (sequence according to Jameson et al., 1988). No sequence variation was observed when compared with the published sequence.
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Discussion |
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FSH is a pituitary gonadotropin that together with LH plays a key role in the regulation of gonadal function. FSH stimulates ovarian folliculogenesis up to the antral stage and is essential for Sertoli cell proliferation and maintenance of sperm quality in the testis (Layman and McDonough, 2000
). Mutations in the FSHß gene have been found to induce infertility in men and women. The reported mutations have been inactivating (Matthews et al., 1993; Layman et al., 1997, 2002; Lindstedt et al., 1998; Phillip et al., 1998) or neutral (Liao et al., 1998) in nature. The number of reported mutations is very small; three leading to amino acid changes, one 2 bp deletion and one silent mutation. The reason for the low frequency of FSHß mutations may be their expected effect on reproduction, which eliminated them rapidly from the gene pool.
The rarity of the reported silent mutations and polymorphisms in FSHß is interesting. In addition to the reported mutations and complete FSHß sequence in the Celera Sequence Bank, 14 SNPs are listed in the NCBI/SNP databank. Four of these SNPs are located in the coding region of FSHß gene. Interestingly, the situation is different with LHß gene whose coding sequence has been studied systematically and reported both in the Celera Sequence Bank and in a population study from Singapore (Roy et al., 1996). Altogether nine mutations have been found in the LHß gene, six of which cause amino acid changes and three are silent. In the NCBI/SNP databank, 28 sequence variations have been reported in the gene region of LHß (NCBI/SNP databank).
We made a complete sequence study of the diversity of the FSHß gene in two Caucasian populations. For this purpose, we sequenced the proximal part of the promoter and the entire translated regions of FSHß on randomly selected and apparently healthy 50 Finnish and 50 Danish individuals. The result of the study was surprising: only three silent polymorphisms, two of which were novel, were revealed (Table II). It thus seems that the FSHß gene is highly conserved in humans. However, it must be remembered that although the samples were collected randomly, they represent individuals with normal health status. The result might be different if the samples had been collected from patients with reproduction problems. Also, the sample number and methodology to detect mutations must be considered. We used the most informative method to study DNA by sequencing amplified and purified DNA fragments. The amplified fragments were short to improve the fidelity of the polymerase used. The number of samples is always a compromise between economical and scientific needs. The number of 50 samples can be considered reasonable to draw primary conclusions about sequence variation. More samples would have been screened had any amino acid changing mutations been detected.
With regard to the populations chosen, the Finns represent an isolated gene pool with enriched gene differences compared with the ‘general’ Caucasian population (Norio, 2003a,b). The Danish sample set represents another Caucasian population with a more varied gene pool. The comparison of genetic variants between these two populations can provide information on both the general variation in FSHß and on variation that exists between populations. The conclusion drawn from this study is that some variation exists at the population level. The proof for this is that silent mutations K58 and K104 show population-specific occurrence. The Y76 polymorphism has similar but not identical distribution in the two Nordic populations. The difference between the Nordic and Asian populations is marked, to the extent that allele C considered polymorphic in the Asian populations, as the more frequent allele in the two Nordic populations.
The functional consequences of silent mutations is thought to be minor to nonexisting. However, Tong et al. (2000) found that the Y76T>C polymorphism may have association with polycystic ovarian syndrome (PCOS) especially in obese women. Although the Y76T>C polymorphism does not cause amino acid change, it may interact with other mutations and lead to a harmful cumulative effect on FSH action. These other mutations may occur in the regulatory regions of FSHß. It may also be in linkage with a more severe mutation in a neighbouring gene. Because no such data exist on other mutations, the reason for association of the T76T>C polymorphism with higher PCOS frequency in obese Chinese women remains unclear. The high frequency in the Nordic countries may refer to the fact that the polymorphism is harmless as such, but other factors, probably population-specific ones, may associate with Y76T>C polymorphism to PCOS in Asian populations.
In conclusion, according to this sequence analysis study, the human FSHß gene is less variable when compared with the LHß gene that has several functional and silent mutations (Huhtaniemi, 2003). Even silent polymorphisms are rare in FSHß. The low variability of this gene could be explained by the crucial role of FSH in reproduction and could therefore be effectively excluded from the gene pool. All the reported homozygous FSHß gene mutations cause infertility in both sexes (Rabinowitz et al., 1979; Matthews et al., 1993; Layman et al., 1997). Also, in females, complete FSH receptor inactivation leads to hampered pubertal development and infertility (Aittomäki et al., 1995). However, in males carrying inactivating FSH-receptor mutations, spermatogenesis is seriously reduced but not totally abolished because of compromised FSH action (Tapanainen et al., 1997). Normally FSH is needed in both females and males for reproduction, but for functionally abnormal FSH or FSH receptor, other factors affecting the complex mechanism of spermatogenesis can compensate for the lack of functional FSH receptor (Tapanainen et al., 1997). Uncompromised FSH action is thus necessary for female fertility but can be partly compensated for by other factors in males. The important role of FSH may explain the low variability and strict intolerance of genetic variants also detected in this study.
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Acknowledgements |
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We thank professor M.-L. Savontaus for kindly providing us with the Finnish DNA samples. The study was supported by grant from the Academy of Finland.
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References |
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Submitted on June 2, 2005; accepted on June 5, 2005.
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