Mutational analysis of the mature peptide region of inhibin genes in Indian women with ovarian failure

H. Dixit1, M. Deendayal2 and L. Singh1,3

1 Centre for Cellular and Molecular Biology, Uppal Road 2 Infertility Institute and Research Centre, Hyderabad, India

3 To whom correspondence should be addressed. Email: lalji{at}ccmb.res.in


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Clinically, premature ovarian failure (POF) is defined as unexplained amenorrhoea (>6 months) with a high FSH level (>40 IU) before the age of 40 years. POF is a heterogeneous genetic disease with unknown aetiology. Inhibin and activin regulate the FSH level by their opposing actions and thus have been considered as strong candidate genes in the aetiology of POF. METHODS: We have screened inhibin genes in patients with POF (n=80), primary amenorrhoea (n=33) and secondary amenorrhoea (n=4). RESULTS: INH{beta}B and INH{beta}A genes do not show any association with ovarian failure. We found the Ala257Thr missense mutation in INH{alpha} gene with high statistical significance in POF (nine out of 80, 11.2%) (Fisher's exact test, P=0.0005), primary amenorrhoea (three out of 33, 9.1%) (Fisher's exact test, P=0.014) and secondary amenorrhoea (two out of four, 50%) (Fisher's exact test, P=0.001) with complete absence of this mutation in controls (none out of 100). CONCLUSION: The INH{alpha} gene is a strong candidate gene for ovarian failure. Mutations in INH{beta}B and INH{beta}A genes are not associated with ovarian failure.

Key words: candidate gene/inhibin/mutation analysis/POF


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The loss of functional follicles occurring in women under the age of 40 years is defined as premature ovarian failure (POF). It occurs in ~1–2% of women (Coulam et al., 1986Go). Unexplained POF is clinically recognized as amenorrhoea (>6 months) with a low level of estrogen and raised level of FSH (>40 IU/l) before the age of 40. POF is now well documented as a heterogeneous disease, which can occur due to disturbance in any gene of the intra-ovarian as well as extra-ovarian pathways. This may lead to an elevation of the FSH level as well as poor or no response of follicles to gonadotrophins (Conway, 1997Go). Identification and population-based screening of candidate genes may provide a better understanding of the aetiology of POF. Abnormalities in the X chromosome have been reported in many idiopathic POF cases, suggesting the probable presence of a few candidate genes on the X chromosome, i.e. FMR1, DIA, ZFX, FMR2, the POF1 locus, the POF2 locus, the critical region, etc. (Zinn, 2001Go), but their functional role in ovarian dysfunction is not well defined. Their association studies are also not well supported in large-scale/sporadic POF cases (Mumm et al., 2001Go; Schlessinger et al., 2002Go).

Many association studies have been done with the key components of the hypothalamus–pituitary–ovarian axis. Very few mutations have been reported so far in gonadotrophins and the corresponding receptors in association with POF, except Ala189Val substitution in the extracellular domain of the FSH receptor (Aittomaki et al., 1995Go) in a Finnish population. The occurrence of this mutation was not found to be associated with POF in other populations (Kohek et al., 1998Go; Layman et al., 1998Go). FOXL2, a member of the winged helix/forkhead transcription factor family, is expressed predominantly in the human eyelid and ovary. Crisponi et al. (2001)Go reported mutations in the FOXL2 gene in families with the blepharophimosis/ptosis/epicanthus inversus (BPES) syndrome associated with eyelid abnormalities. In type 1 BPES, the females inherit ovarian failure in addition to the eyelid defect. In type 2 BPES, only the eyelid abnormalities are seen. Truncated FOXL2 is associated with BPES type 1, and expanded proteins with type 2 (Crisponi et al., 2001Go). Mutations in FOXL2 in patients with non-syndromic POF have been found (Harris et al., 2002Go), but these are not common.

Inhibin and activin are members of the transforming growth factor-{beta} (TGF-{beta}) superfamily and are dimers of three gene products: the INH{alpha} subunit, the INH{beta}A subunit and the INH{beta}B subunit, encoded by INH{alpha}, INH{beta}A and INH{beta}B genes, respectively. Inhibin and activin are dimeric glycoprotein hormones with one INH{alpha} subunit linked to either the INH{beta}A or INH{beta}B subunit by disulphide bonds, resulting in inhibin-A ({alpha}{beta}A) and inhibin-B ({alpha}{beta}B) respectively. Activins are dimers of {beta}-subunits only, forming activin A ({beta}A–{beta}A), activin-AB ({beta}A–{beta}B) and activin-B ({beta}B–{beta}B) (Ying, 1988Go).

Inhibin and activin have opposing regulatory actions for modulation of the FSH level. Activin enhances FSH secretion while inhibin forms a negative feedback loop control of the FSH level. The presence of low serum inhibin levels in POF provides strong evidence of the involvement of inhibins and activins in the pathophysiology of POF (Petraglia et al., 1998Go). An elevated serum FSH level and low inhibin B level in the early follicular phase has also been reported to correlate with reproductive ageing (Shahara et al., 1998Go) and diminished ovarian reserve (MacNaughton et al., 1992Go), and this has been included in the clinical counselling report by the American Society for Reproductive Medicine Practice Committee (2002)Go. The first report of population screening for inhibin genes which revealed a strong association of the Ala257Thr missense mutation in the INH{alpha} gene with POF (Shelling et al., 2000Go) was supported further by the study of Marozzi et al. (2002)Go. Our study provides further strong support for the association of the Ala257Thr mutation in the INH{alpha} gene with ovarian failure.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patient information
A total of 117 non-familial cases of POF (n=80), primary amenorrhoea (n=33) and secondary amenorrhoea (n=4) were included in the study. Patients were recruited by the Infertility Institute and Research Centre (IIRC), Hyderabad. The diagnostic criteria for POF include at least 6 months of amenorrhoea before the age of 40 years, with a high FSH serum level (>40 IU/l). Primary amenorrhoea is defined as a condition with complete absence of menses or only induced menses. Secondary amenorrhoea is defined as a cessation of menses with previous history of menses before the age of 40. All patients were assessed clinically, with complete medical and gynaecological history including history of menses, age at menopause, serum FSH level (three times at 1 month intervals) and serum LH level with no history of autoimmune disease. Karyotyping with high-resolution GTG banding was carried out for all patients for cytogenetic anomalies. Patients with chromosomal abnormalities were excluded from the study. Controls (n=100) were recruited as normal females with a regular menstrual history and normal FSH level below the age of 40 years.

DNA extraction and karyotyping
A 5 ml aliquot of peripheral blood was collected in EDTA vacutainers for genomic DNA isolation and another 5 ml of peripheral blood was collected in heparin vacutainers for cytogenetic analysis. DNA was extracted using the Nucleon BACC2 DNA extraction kit (Amersham Pharmacia Biotech., NJ) according to the manufacturer's protocol. Chromosomal analysis was performed on phytohaemagglutinin (PHA)-stimulated peripheral lymphocyte cultures using standard cytogenetic methods (Benn and Perle, 1992Go; Gosden et al., 1992Go).

PCR
Primers were synthesized for amplification of the mature peptide region of each inhibin gene, i.e. for INH{alpha} (nucleotides 841–1242) using INH{alpha}F and INH{alpha}R primers, INH{beta}B (nucleotides 717–1061) using INH{beta}BF and INH{beta}BR primers, and INH{beta}A (nucleotides 1167–1528) using INH{beta}AF and INH{beta}AR primers (Shelling et al., 2000Go). PCR conditions for the mature peptide region of each inhibin gene were as described earlier (Shelling et al., 2000Go; Marozzi et al., 2002Go).

DNA sequencing
All PCR products were obtained from the above primers amplifying the mature peptide region of each inhibin gene. Sequencing was performed using the Big dye terminator sequencing protocol, supported by Applied Biosystems using an ABI prism 3700 DNA analyser.

RFLP analysis
The PCR products of the INH{alpha}1F and INH{alpha}1R primers were analysed by restriction fragment length polymorphism (RFLP) for the 769G->A mutation using BbvI restriction enzyme from NEB, which is a prototype of Bst71I described earlier (Shelling et al., 2000Go). This restriction digestion yields three fragments of 134, 85 and 25 bp in wild-type, four fragments of 159, 134, 85 and 25 bp in heterozygous mutants and and two fragments of 159 and 85 bp in homozygous mutant. The restriction digestion was performed using 1 x NEB2 buffer, 2 U of BbvI enzyme and 10 µl of PCR product, and sterile water to make a final volume of 15 µl. All reaction mixtures were incubated at 37°C for 16 h. The RFLP products were separated by 3.5% agarose gel electrophoresis.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In our study, we analysed POF (n=80), primary amenorrhoea (n=33), secondary amenorrhoea (n=4) cases and controls (n=100) for the mature peptide region of all three inhibin genes. The sequencing analysis of INH{alpha} showed a statistically significant prevalence of the 769G->A missense mutation in patients with ovarian disorder, females showed the normal sequence (Figure 1a). Eight out of 80 POF cases, three out of 33 primary amenorrhoea cases and two out of four secondary amenorrhoea cases were heterozygous for this mutation, while one POF case was homozygous. One primary amenorrhoea case showed a missense mutation 896A->C in the INH{beta}A gene (Figure 1b) which leads to the Gln299Pro amino acid change. One silent mutation 942C->T was found in the INH{beta}B gene in a POF case (Figure 1c). All the above mutations were confirmed by repeating the sequencing three times, including sequencing the amplification product in the reverse direction.



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Figure 1. (a) Electropherogram showing heterozygous and homozygous 769G->A transitions in the INH{alpha} gene compared with the normal sequence. (b) Electropherogram showing the heterozygous 896C->A transversion in the INH{beta}A gene. (c) Electropherogram showing the heterozygous 942C->T transition in the INH{beta}B gene.

 
The presence of the 769G->A mutation was confirmed further by RFLP using the BbvI restriction enzyme (Figure 2), which showed the same pattern of digestion as reported earlier (Shelling et al., 2000Go). The mutation summary and clinical details of the patients tested positive for mutations are shown in Tables I and II, respectively.



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Figure 2. RFLP profile of the INH{alpha}1 product with BbvI restriction enzyme digestion. Lane 1, the 50 bp marker; lane 2, undigested PCR product; lanes 3 and 4, wild-type; lanes 5 and 6, the heterozygous mutant; lane 7, the homozygous mutant (as described by Shelling et al., 2000Go).

 

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Table I. Summary of mutation analysis of inhibin genes

 

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Table II. Clinical details of the patients who were positive for mutations

 
Our sequencing results further confirm the prevalence of the 769G->A mutation in Indian women with sporadic POF (nine out of 80, 11.2%) (Fisher's exact test, P=0.0005), sporadic primary amenorrhoea (three out of 33, 9.1%) (Fisher's exact test, P=0.014), secondary amenorrhoea (two out of four, 50%) (Fisher's exact test, P=0.001) and its absence in controls (none out of 100, 0%).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Presently, POF is widely accepted as a heterogeneous disorder, which may involve a large number of genes, and, furthermore, mutation(s) in any of the genes may provide the answer to its aetiology. To date, there is no such gene known which can be accepted as a genetic marker for POF except a promising involvement of the INH{alpha} gene in the world population.

Inhibin production is restricted to granulosa cells. Activins are produced mainly by granulosa cells but are also produced in a variety of tissues including the anterior pituitary (Drummond et al., 1996Go; Findlay et al., 2001Go). Inhibins exert their inhibitory effect either by interfering with the activin-induced signalling pathway (Lebrun and Vale, 1997Go) or by competition of full-length INH{alpha} precursor with FSH for binding to the FSH receptor (Schneyer et al., 1991Go). Inhibin complexed with its co-receptor betaglycan antagonizes activin signalling by competing for the activin receptor A type-II and activin receptor B type-II with equal binding affinity. This complex does not stimulate phosphorylation of ALK4 type-I receptor and subsequent intracellular Smad-dependent signalling which is imperative for FSH production by gonadotrophins (Bernard et al., 2002Go). Inhibin binds to betaglycan via its {alpha}-subunit (Lewis et al., 2000Go; Esparza-Lopez et al., 2001Go), thus a mutation in the {alpha}-chain would probably impair inhibin binding to betaglycan as well as its antagonistic role in activin signalling. A deficiency in inhibin function or secretion allows activin to elevate the FSH level, which concurs with reproductive ageing (Welt et al., 1999Go) and rapid exhaustion of the follicular reserve during the menopausal transition (Richardson et al., 1987Go). Increased activity of activin produced by pre-ovulatory follicles has been shown to repress the growth of neighbouring follicles (Mizunuma et al., 1999Go).

Our study demonstrated the strong association of the 769G->A mutation in the coding region of the INH{alpha} gene in Indian women with sporadic POF, which agrees with two earlier studies (Shelling et al., 2000Go; Marozzi et al., 2002Go). Marozzi et al. (2002)Go found this mutation in 25% of patients with primary amenorrhoea (three out of 12, 25%) (Fisher's exact test, P<0.001) which is a significantly higher frequency than we have found in our population (three out of 33, 9.1%) (Fisher' exact test, P=0.014). The significance of this difference is unknown, but could be due to the investigation of different ethnic groups. All patients with the mutation were below the age of 25 years at the time of clinical diagnosis, except for one secondary amenorrhoea case (Figure 3). The POF patients carrying the 769G->A mutation attained menopause between the age of 18 and 24 years, with a mean age at menopause of 21 years. The POF patients without mutation presented with a wider range of age at menopause, from between the ages of 15 and 36 years, with a mean age at menopause at 27 years. Therefore, the presence of the 769G->A mutation is associated with an earlier age of menopause, as has been found previously (Shelling et al., 2000Go).



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Figure 3. Graph showing the distribution of POF patients with the 769G->A mutation in the INH{alpha} gene and POF patients without this mutation against the age of menopause.

 
The patient who was homozygous for the 769G->A mutation attained menopause at 23 years, with a surprisingly high FSH (100, 88 and 85 IU/l) and LH (90 IU/l) levels. This is the first patient identified in the literature to be homozygous for the 769G->A mutation. It is interesting to note that she does not have a particularly severe phenotype, such as primary amenorhoea. Shelling et al. (2000)Go hypothesized that the Ala257Thr mutation is sufficient to impair the binding affinity of inhibin for its receptors, and leads to the subsequent inability to regulate the FSH level by negative feedback. The increased level of FSH leads to rapid depletion of follicles, resulting in ovarian failure. Therefore, in the homozygous patient, it is plausible that the presence of relatively higher levels of FSH and LH, as compared with patients with the heterozygous mutation, is a reflection of the increased loss of negative feedback of INH{alpha} on FSH in the homozygous state. Since a few POF patients without this mutation (8.5%) also had FSH levels (>100 IU/l) as high as the homozygous mutant for the 769G->A mutation, this is a strong indication that other candidate genes also have a role in the aetiology of ovarian failure, and these need to be identified.

It is interesting to note that Matzuk et al. (1992)Go generated INH{alpha} knockout mice and showed that inhibin-deficient mice developed gonadal tumours followed by cancer cachexia such as wasting syndrome, hepatocellular necrosis around the central vein, parietal cell depletion and mucosal atrophy. Inhibin-deficient mice, if gonadectomized at an early age, do not develop a wasting syndrome but develop adrenal cortex tumours (Matzuk et al., 1994Go). These studies indicate that inhibin acts as a gonadal and adrenal tumour suppressor. The 769G->A mutation in the INH{alpha} gene perhaps does not completely inactivate the functional activity of inhibin and does not show such a severe phenotypic outcome as seen in the knockout mice.

Mutations in INH{beta}A and INH{beta}B did not show any association with ovarian failure in our study. One patient with primary amenorrhoea was found to have an 896A->C missense mutation (Gln299Pro) in the INH{beta}A gene. She had a Turner's phenotype, which included shortening of the fourth metatarsal, and her ears were low set, but she was found to have a normal karyotype. The glutamine at position 299 and its surrounding amino acids are highly conserved in cow, pig, horse, sheep, rat and mouse sequences (Figure 4). It is difficult to determine what the role of the non-conservative Gln299Pro mutation might be in ovarian failure, as it is found in the propeptide region, which would not lead to a change in the structure of the mature peptide chain.



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Figure 4. Comparison of the human INH{beta}A protein sequence with the cow, horse, mouse, sheep, rat, pig and chicken INH{beta}A sequences. Conservation of sequences around the glutamine (arrowhead) at position 299, which is altered to proline in one primary amenorrhoea case.

 
Our study, in combination with the two earlier studies, strongly suggests that the 769G->A mutation in the INH{alpha} gene is a significant genetic marker for the diagnosis of POF and probably associated with early onset of POF. The presence of this mutation can be diagnosed by RFLP using Bst71I or BbvI restriction enzymes in clinical laboratories. This genetic test may help in the early diagnosis of POF, prior to the complete depletion of follicles, and may offer opportunities for patients to plan their families earlier.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We are grateful to Ms V.V.Padmalatha and Ms M.K.Kanakavalli for providing the chromosomal diagnosis, Ms D.V.Jayanthi of the Infertility Institute and Research Centre, Secunderabad, for her help in patient recruitment, and Mrs Lakshmi Rao for her constant support during the study. The study was supported by a grant from the Indian Council of Medical Research, New Delhi. H.D. was supported by fellowship from the Council of Scientific and Industrial Research (CSIR), Government of India.


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Submitted on January 8, 2004; accepted on May 6, 2004.