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
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Abstract |
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Key words: candidate gene/inhibin/mutation analysis/POF
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Introduction |
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Many association studies have been done with the key components of the hypothalamuspituitaryovarian 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., 1995) in a Finnish population. The occurrence of this mutation was not found to be associated with POF in other populations (Kohek et al., 1998
; Layman et al., 1998
). FOXL2, a member of the winged helix/forkhead transcription factor family, is expressed predominantly in the human eyelid and ovary. Crisponi et al. (2001)
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., 2001
). Mutations in FOXL2 in patients with non-syndromic POF have been found (Harris et al., 2002
), but these are not common.
Inhibin and activin are members of the transforming growth factor- (TGF-
) superfamily and are dimers of three gene products: the INH
subunit, the INH
A subunit and the INH
B subunit, encoded by INH
, INH
A and INH
B genes, respectively. Inhibin and activin are dimeric glycoprotein hormones with one INH
subunit linked to either the INH
A or INH
B subunit by disulphide bonds, resulting in inhibin-A (
A) and inhibin-B (
B) respectively. Activins are dimers of
-subunits only, forming activin A (
A
A), activin-AB (
A
B) and activin-B (
B
B) (Ying, 1988
).
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., 1998). 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., 1998
) and diminished ovarian reserve (MacNaughton et al., 1992
), and this has been included in the clinical counselling report by the American Society for Reproductive Medicine Practice Committee (2002)
. The first report of population screening for inhibin genes which revealed a strong association of the Ala257Thr missense mutation in the INH
gene with POF (Shelling et al., 2000
) was supported further by the study of Marozzi et al. (2002)
. Our study provides further strong support for the association of the Ala257Thr mutation in the INH
gene with ovarian failure.
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Materials and methods |
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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, 1992; Gosden et al., 1992
).
PCR
Primers were synthesized for amplification of the mature peptide region of each inhibin gene, i.e. for INH (nucleotides 8411242) using INH
F and INH
R primers, INH
B (nucleotides 7171061) using INH
BF and INH
BR primers, and INH
A (nucleotides 11671528) using INH
AF and INH
AR primers (Shelling et al., 2000
). PCR conditions for the mature peptide region of each inhibin gene were as described earlier (Shelling et al., 2000
; Marozzi et al., 2002
).
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 INH1F and INH
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., 2000
). 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.
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Results |
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Discussion |
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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., 1996; Findlay et al., 2001
). Inhibins exert their inhibitory effect either by interfering with the activin-induced signalling pathway (Lebrun and Vale, 1997
) or by competition of full-length INH
precursor with FSH for binding to the FSH receptor (Schneyer et al., 1991
). 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., 2002
). Inhibin binds to betaglycan via its
-subunit (Lewis et al., 2000
; Esparza-Lopez et al., 2001
), thus a mutation in the
-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., 1999
) and rapid exhaustion of the follicular reserve during the menopausal transition (Richardson et al., 1987
). Increased activity of activin produced by pre-ovulatory follicles has been shown to repress the growth of neighbouring follicles (Mizunuma et al., 1999
).
Our study demonstrated the strong association of the 769GA mutation in the coding region of the INH
gene in Indian women with sporadic POF, which agrees with two earlier studies (Shelling et al., 2000
; Marozzi et al., 2002
). Marozzi et al. (2002)
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., 2000
).
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It is interesting to note that Matzuk et al. (1992) generated INH
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., 1994
). These studies indicate that inhibin acts as a gonadal and adrenal tumour suppressor. The 769G
A mutation in the INH
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 INHA and INH
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
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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Acknowledgements |
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References |
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Submitted on January 8, 2004; accepted on May 6, 2004.