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From the Clinical Research Centers |
Department of Genetics (M.U., K.R.V., R.S.S.), and Center for Research on Reproduction and Women’s Health and Department of Obstetrics and Gynecology (L.-C.K., L.K.C., D.A.D., J.F.S.), University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; Division of Women’s Health (X.W., A.D.), Brigham and Women’s Hospital, Boston, Massachusetts 02115; Reproductive Endocrine Unit (A.S.), Massachusetts General Hospital, Boston, Massachusetts 02114; and Department of Obstetrics and Gynecology (R.S.L.), Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033
Address all correspondence and requests for reprints to: Margrit Urbanek, Department of Genetics, University of Pennsylvania, School of Medicine, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104-6145. E-mail: murbanek{at}mail.upenn.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Multiple biochemical pathways have been implicated in the pathogenesis
of PCOS (8). Candidate genes for PCOS selected from these
pathways include genes involved in: 1) steroid hormone biosynthesis or
metabolism; 2) gonadotropin action and regulation; 3) obesity and
energy regulation; and 4) insulin action (9). In an
initial analysis of 37 candidate genes belonging to these pathways, we
tested for linkage and association with PCOS or hyperandrogenemia (HA)
in data from 150 families (10). In the 39 affected sister
pairs from these families, the strongest evidence for linkage was
observed in the follistatin gene region. In this region, instead of the
expected identity by descent of 50%, identity by descent was elevated
(72%; 
2 = 12.97; nominal P =
3.2 x 10-4). These
findings remained statistically significant (P = 0.01) after
correction for multiple testing (33 independent gene regions tested).
The strongest evidence for a population association between a candidate
gene marker allele and PCOS was observed in the insulin receptor region
(D19S884; allele 5; 2 = 8.53), but these
findings were not statistically significant after correction for
multiple tests.
Follistatin was considered a candidate for the following reasons. It is an activin-binding protein that neutralizes the biological activity of activin in vivo and in vitro (11) and is expressed in multiple tissues, including the ovary, pituitary, adrenal cortex, and pancreas (12). Activin, a member of the transforming growth factor-ß superfamily, modulates the production of androgens by ovarian thecal cells, the development of ovarian follicles, and the secretion of FSH by the pituitary and insulin by pancreatic ß-cells (12, 13). Because follistatin inhibits the activity of activin, altered follistatin activity would be expected to affect follicular development, ovarian androgen production, pituitary FSH secretion, and insulin release. All these processes have been shown to be perturbed in PCOS (14, 15). Female transgenic mice that overexpress follistatin display reduced serum levels of FSH and arrested folliculogenesis (16).
Here, we describe variation in the follistatin gene and assess its relevance to the etiology of PCOS. Our analysis consisted of three parts: 1) we screened the follistatin gene for DNA sequence variants in 19 families with multiple affected daughters and in 31 unrelated PCOS women and 15 control women; 2) we tested a common variant in the follistatin gene for association with PCOS in 249 PCOS families; and 3) we examined follistatin messenger RNA (mRNA) expression levels in cultured fibroblast cells from 18 PCOS and 13 control women.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our earlier analysis of 37 candidate genes for PCOS, we
studied 150 nuclear families that included 189 affected daughters and
28 multiplex families (set 1). For the present follow-up study, we
analyzed 99 additional nuclear families (set 2). Of these families, 90
were of European descent, 5 of Caribbean or Mexican descent, 2 of
African-American descent, 1 of Asian Indian descent, and 1 of unknown
descent. Phenotypes were assigned as described by Legro et
al. (2) and Urbanek et al.
(10). Briefly, an index case was considered affected if
she had oligomenorrhea (
6 menses per year) and HA. Our operational
definition of HA was: a level of total testosterone or testosterone not
bound to sex hormone-binding globulin more than 2
SD above the control mean. As previously
described (2, 10), threshold levels for normal values were
established from analyses of 70 normal women. Coefficients of variation
within and between assays for total testosterone were estimated
as 6% and 10%, and for testosterone not bound to sex hormone-binding
globulin as 7% and 6%, respectively. Hyperprolactinemia and
nonclassical congenital adrenal hyperplasia were excluded by
appropriate tests (2). HA is found in a large fraction of
sisters of index cases and is the distinctive reproductive endocrine
phenotype in female relatives of our PCOS index cases 2 .
For genetic analysis, therefore, sisters of index cases were considered
affected if they had HA, whether or not they had oligomenorrhea.
Accordingly, we use "PCOS/HA" for the phenotype of the affected
sisters; this term applies to women with PCOS or with HA only (HA
without oligomenorrhea). Among the 75 sisters with the PCOS/HA
phenotype, 35 had PCOS, and 40 had HA only. Thus, among the 324
affected women in this study, 249 (index cases) plus 35 (sisters)
= 284 (88%) had PCOS. Sisters who had irregular menstrual cycles (but
normal androgen levels), were taking any confounding medications
(e.g. oral contraceptives or insulin-sensitizing drugs), or
were not of reproductive age were excluded from the analysis, as were
brothers.
Additional PCOS and control women
Additional studies were carried out in PCOS women and controls who fulfilled the diagnostic criteria outlined above but who were not part of our family study. Genomic DNA was screened by single-strand conformation polymorphism (SSCP) for follistatin gene variants (PCOS n = 31, control n = 15), and follistatin gene expression was examined in cultured skin fibroblasts (PCOS n = 18, control n = 13). The control women had regular menses and normal plasma androgen levels and were not taking any confounding medications (2). Fibroblast cultures were established from punch-skin biopsies (17).
Promoter sequencing
Genomic clone pHFG302–5.3, containing a 5.3-kb insert that
included the putative human follistatin promoter region and
transcription start site, was kindly provided by Dr. Shunichi
Shimasaki, University of California San Diego (18). The
insert was digested with HindIII and PstI and
subcloned into pBluescript II SK for sequencing. Three subclones were
obtained containing insert fragments of 2.6, 1.7, and 0.9 kb. Initial
sequencing was performed using specific primers derived from the
published 5' region of human follistatin complementary DNA (cDNA)
sequence and T3 or SK primers. We used internal primers,
generated from sequence data obtained from prior rounds of sequencing,
to complete the sequencing of the three subclones. Full-length
sequences of each of the three subclones were assembled by a
commercially available computer program (Sequencher 3.1.1, Gene
Codes Corporation, Ann Arbor MI). Final analyses revealed that the
0.9-kb insert subclone represented the most upstream fragment, and the
2.6-kb insert subclone represented the fragment most proximal to the
follistatin gene. The 2.6-kb fragment contains 1674 bp of sequence
upstream of the initiation codon (Fig. 1
), the translational start
site, the first exon, and 771 bp of the first
intron.
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The follistatin gene (Fig. 2) was
sequenced in 85 members of 19 multiplex families. Sequencing the gene
in multiple offspring in each family allowed us to confirm the
segregation of any putative sequence variants. Any biologically
relevant variants should be preferentially transmitted to the affected
daughters. Sixteen families were from set 1 (10), and 3
families were from set 2. We generated templates for the sequencing
reactions from genomic DNA by amplifying the follistatin gene in 3
fragments (Fig. 2B). Fragment 1 contained 850 bp of the sequence of the
putative proximal promoter, exon 1 (signal peptide), and 318 bp of
intronic sequence. Fragment 2 included exons 2–5 plus approximately
120 bp (intron A) and 130 bp (intron E) of flanking intronic sequences.
Fragment 3 contained exon 6 plus flanking sequences (120 bp of intron E
and 541 bp 3' of the follistatin gene). The introns between exons 1 and
2 and between exons 5 and 6 of the follistatin gene were not sequenced
completely. However, for both of these introns, sufficient DNA flanking
the exons (>100 bp) was sequenced to encompass elements that may
regulate splicing (19).
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) were taken
from the published sequence of the follistatin gene (18)
for the coding regions or were based on our own sequence of the
putative proximal promoter region. Each fragment was sequenced in both
directions using primers spaced approximately 500–600 bp apart (Fig. 2C
).
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SSCP analysis of the follistatin gene
Each exon including the exon/intron boundaries of the
follistatin gene was amplified by PCR from the genomic DNA with
specific primers (Table 2). PCR
was performed in a reaction vol of 25 µL containing 10 mM
Tris-Cl (pH 8.3), 1.5 mM MgCl2, 10%
dimethylsulfoxide, 0.2 mM dNTP, 0.1 µg genomic DNA, 50
pM of each primer, 0.01 µCi/µL
[
-32P]deoxycycidine triphosphate, and 1 U
Taq DNA polymerase (Perkin-Elmer Corp., Foster
City, CA). PCR was initiated by denaturation at 94 C for 5 min,
followed by 30 cycles of amplification (94 C, 30 sec; 62 C, 30 sec; 72
C, 30–60. sec) and a final extension of 7 min at 72 C. PCR fragments
of exons 2, 5, and 6, which were too large for SSCP analysis, were
digested before electrophoresis. The PCR fragments from exons 2 and 5
were digested with PstI and HaeIII, respectively,
whereas the PCR fragment of exon 6 was digested with HaeIII,
HindIII, and XbaI. One microliter of sample was
mixed with 3 µL of single-stranded DNA loading buffer (80%
formamide, 1 mM EDTA, 10 mM
NaOH, 0.01% Bromophenol blue, and 0.01% xylene cyanol), incubated for
5 min at 90 C, chilled on ice, and electrophoresed on a 5%
polyacrylamide gel, with or without 10% glycerol, at 40 W for 3–4 h
at 4 C, or at 10 W overnight at room temperature (only for gels with
10% glycerol). X-ray film was exposed to the dried gel at -80 C. PCR
fragments from 4 subjects showing SSCP were purified in agarose (1.2%)
and sequenced.
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The short tandem repeat polymorphism markers (D5S474, D5S822,
and D5S623) were genotyped as described by Urbanek et al.
(10) using fluorescently labeled primers and a 377 DNA
Sequencer, and the GeneScan Analysis and Genotyper programs
(PE Applied Biosystems). The single nucleotide
polymorphism (SNP) in exon 6 was genotyped using SSCP. For each PCR
reaction, 45 ng of genomic DNA was amplified in a total vol of 8 µL
in the presence of 200 µM dNTPs (Amersham Pharmacia Biotech, Piscataway, NJ), 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 1.5
mM MgCl2, 0.36 U AmpliTaq
polymerase (Roche Molecular Biochemicals,
Branchburg, NJ), 2.5 µCi [-33P]
deoxycycidine triphosphate, and 0.5 µM of
forward primer (5'-CACACATGGGCTGCTGCTTTTTGC-3') and 0.5
µM of reverse primer
(5'-TGTAGTCCTGGTCTTCATCTTCCTC-3'). Samples were electrophoresed
overnight at room temperature at 25 W on a MDE gel (FMC BioProducts,
Rockland, ME).
Statistical analysis
We tested for linkage disequilibrium (presence of both linkage and association) between specific alleles at the candidate gene markers and PCOS/HA using the transmission/disequilibrium test (TDT) (20). The TDT assesses transmission of marker alleles from heterozygous parents to affected offspring. If a particular allele is transmitted in this situation with a frequency greater than 50%, there is evidence that the disease is linked to the marker locus (and associated with that marker allele). Transmission with frequency less than 50% implies a so-called protective effect, associated with the marker allele. DNA samples could not be obtained from 8 parents in the 99 set-2 families. Genotypes for missing parents were reconstructed using genotypes of unaffected sibs or those with unknown phenotype. None of these sibs were included in the statistical analysis. When one parent was missing, the available parent’s genotype was used only if the inheritance could be determined unambiguously and without bias in affected individuals (21).
RNA isolation and RT-PCR
Total RNA was isolated from cultured fibroblast cells with the SV Total RNA Isolation System (Promega Corp., Madison, WI). The protocol provided by the manufacturer was followed. The RNA samples were kept in ribonuclease-free water at -80 C. For RT-PCR, first-strand cDNA was synthesized from the total RNA samples with the First-Strand cDNA Synthesis Kit from Amersham Pharmacia Biotech). Briefly, 0.5 µg total RNA was incubated in 8 µL ribonuclease-free water for 10 min at 65 C and then quickly chilled in an ice-water bath. The sample was supplemented with 1 µL NotI-d(T)18 primer (1 µg/µL), 1 µL dithiothreitol (0.2 M), and 5 µL Bulk First-Strand cDNA Reaction Mix provided by the kit and incubated for 1 h at 37 C. PCR was performed in a 25-µL reaction consisting of 1 µL of the first-strand cDNA, 0.2 mM dNTP, 10 mM Tris-Cl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 30 pmol each of the follistatin forward (5'-GAACTGCGGCTCCGTCAAGCGAAGA-3') and reverse (5'-ATTACAGGTCACACAGTAGGCATTAT-3') primers, and 2 U AmpliTaq DNA polymerase for 10 cycles of amplification with 1 min at 94 C, 40 sec at 62 C, and 30 sec at 72 C, after denaturation for 3 min at 94 C. The actin forward (5'-AGCCATGTACGTTGCTATCCAGGCTG-3') and reverse (5'-CAGCGGAACGCTCATTGCCAATGGT-3') primers were then added, and the PCR was performed for 20 additional cycles. After a final extension for 7 min at 72 C, PCR products were separated on a 2% agarose gel and visualized by ethidium bromide staining. The intensity of the DNA bands was determined by a laser densitometer (MultiAnalytic System, Bio-Rad Laboratories, Inc., Hercules, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We determined the sequence of the putative proximal promoter from
a clone containing the follistatin gene (pHFG302–5.3). The human, pig,
and rat promoter sequences are shown in Fig. 1 (22, 23).
The human sequence is very similar to the corresponding sequence in the
rat and the pig promoters (89% and 93% identity, respectively, in the
500 bp directly upstream of the translational initiation codon) (Fig. 1). There are multiple transcription factor-binding sites in the rat
that are conserved in the human sequence (see Fig. 1), and two out of
three potential TATA boxes in the rat are conserved in the human
sequence (22). In the rat promoter, the 455 bp proximal to
the translational initiation codon were sufficient for basal
transcription and contain elements responsive to forskolin and
12-O-tetradecanoylphorbol 13-acetate (22). Because of
the high sequence similarity between the human and rat sequences, this
region in humans most likely also contains the key elements driving
follistatin gene expression. There is a dinucleotide repeat located
approximately 600 bp upstream of the ATG. In the rat, this sequence
contains 34 continuous CA repeats; but, in the human, there are only 15
CA repeats, and they are interrupted by nonrepeat sequences. There was
evidence for only slight variation (1 variant in 30 alleles among 30
unrelated chromosomes genotyped) at the human repeat (data not
shown).
For the putative promoter region and the genomic region encompassing exons 1–5, we numbered the nucleotides, based on the published genomic sequence by Shimasaki et al. (18) (GenBank accession nos. M19480 and J03771). The A in the translational initiation codon was designated +1, with upstream sequences being negative. Because the sequence for the exon 6 region is not contiguous with the fragment containing exons 1–5 described in Shimasaki et al., we numbered the nucleotides in the region containing exon 6 according to Shimasaki’s genomic sequence of the fragment containing exon 6 (GenBank accession nos. M19481 and J03771), but we added an x to the nucleotide number to distinguish them from the sequences of exons 1–5.
We sequenced the entire coding region, flanking intronic sequences, and
500 bp of the proximal putative promoter (Fig. 2) in 85 individuals
belonging to 19 multiplex PCOS families. These families contained 43
PCOS/HA offspring. We identified 20 variants at 17 positions (Table 3). For 15 variants, transmission of the
variant allele from parent to offspring could be documented. Five
variants were seen only in parents but in more than one family. Most of
the variants are rare; 13 of 20 variants occur at a frequency of less
than 5% of parental chromosomes. Three of the variants are located in
exons [nucleotide 17 in the signal peptide (exon 1); nucleotide 3368
in exon 3; nucleotide 343x in exon 6]. The A-to-C change at
nucleotide 17 results in a change of a histidine (CAC) to proline (CCC)
residue. This variant occurred in three individuals in one family. At
this point, its functional relevance is not known; however, because of
its low frequency, we do not expect it to play a major role in the
etiology of PCOS. The G-to-A change at nucleotide 3368 in exon 3
results in a conservative change of an arginine (CGG) to glutamine
(CAG) residue. This variant occurred in three individuals in a single
family and is not likely to cause a functional change in the
follistatin protein. The exon 6 variant (T-to-A change) is located 78
nucleotides downstream of the termination codon in the 3' untranslated
region of exon 6 and, therefore, is not translated (18).
Exon 6 is alternatively spliced to produce two transcripts. One
transcript retains the exon 6 variant site, whereas the other
transcript does not. Thus, the functional relevance of the exon 6
variant is unclear. This variant has been previously described by
Shimasaki et al. (18).
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All 6 exons of the follistatin gene and their intron/exon
boundaries were screened for variants by SSCP. Only 1 sequence
polymorphism was detected in the coding region and the intron/exon
boundaries of the follistatin gene of 31 PCOS and 15 control women. The
only sequence variant (T vs. A) was found at nucleotide
343x in exon 6 of the follistatin gene of both PCOS and control women
(see above). The other variants (Table 3) found by sequencing were not
observed in these samples using SSCP.
TDT
We tested for association between PCOS/HA and the alleles of 4
markers in, or closely linked to, the follistatin gene: 3 short tandem
repeat polymorphism markers (D5S474, D5S623, and D5S822) and the SNP in
exon 6 (see Fig. 3). The 249 families
(set 1 and set 2) contained 324 affected individuals. The results of
the TDT analysis are shown in Fig. 3. The 2 markers with the largest
2 values were the exon 6 variant (allele 1,
2 = 5.00, P = 0.025) and D5S623 (allele
11, 2 = 4.26, P = 0.039). The
number of transmissions with allele 11 of D5S623 was too small (n
= 19) for reliable evaluation. Although the number of transmissions of
allele 1 of the exon 6 variant (n = 245) was substantial, the
2 value does not remain significant at the
0.05 level after correction for testing 22 alleles (P > 0.5).
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Total RNA was extracted from 18 PCOS and 13 control fibroblast
cell lines. Follistatin mRNA was detected by RT-PCR with
follistatin-specific primers in
all cell lines (Fig. 4). To compare the
expression level of follistatin between PCOS and control women, the
band intensity of follistatin RT-PCR products was normalized with that
of ß-actin mRNA, which served as an internal control. No substantial
difference in follistatin expression between PCOS and control women was
found in duplicate experiments; the ratio of follistatin to actin, in
arbitrary densitometry units, was 1.1 ± 0.1 for PCOS and 1.0
± 0.1 for controls.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Extensive sequencing of the follistatin gene identified variants at 17 sites, but none of these seem to be likely etiological agents in PCOS. No common variants were detected in the coding regions of follistatin. The exon 6 variant, which is not translated, and other closely linked polymorphic sites outside the follistatin gene, were tested by the TDT for association and linkage. Even at exon 6, where the strongest evidence was found, the findings are, at most, marginally significant; and they are not significant after correction for multiple testing. Although this finding reduces support for the follistatin gene, it is not, by itself, conclusive. The magnitude of the TDT depends on the degree of linkage disequilibrium between the allele being tested and the putative functional variant, which, in turn, depends strongly on the population history of both the tested allele and the putative functional variant. It follows that the absence of a TDT effect at a given site strongly implies that the tested variant is not itself responsible for the phenotype, but absence of a TDT effect cannot be used to exclude an entire gene or region.
It therefore remains possible, in principle, that a major causal element is present in or near the follistatin gene but was missed because it is located in a region that was not sequenced (one of the introns, more distal promoter, or other control regions). This possibility would be consistent with the original observation in affected sib pairs, that suggested linkage (10). However, in the additional affected sib pairs that we have studied since the original report (10), there is no further evidence for linkage with follistatin.
The results of the expression analysis show that there are no significant differences in the mRNA levels of follistatin in cultured fibroblast cells from PCOS and control women. This implies that there are no common sequence variants that result in global changes in either transcription rates or mRNA stability of follistatin in PCOS. It is still possible that there are tissue-specific differences in mRNA level or protein stability that affect follistatin levels in PCOS. However, in support of our observations, others have also found no difference in follistatin mRNA levels in developing follicles of normal women and women with PCOS (A. Schneyer, unpublished results).
Among the various components of this study, the only evidence supporting a role for follistatin was the slightly elevated TDT for the exon 6 variant. The sequence and mRNA analysis provided no evidence implicating the follistatin gene, and the TDT results for nearby markers were not significant. The present study suggests that, if variation at follistatin or a nearby site makes a contribution to PCOS, it is most likely quite modest.
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Acknowledgments |
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Footnotes |
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Received May 2, 2000.
Revised July 26, 2000.
Accepted August 23, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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