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Original Studies |
T) of the Human Follicle-Stimulating Hormone Receptor Gene in Four Populations Using Allele-Specific Hybridization and Time-Resolved Fluorometry1
Departments of Physiology (M.J., P.P., I.H.) and Biotechnology (C.N., A.I., K.P.), University of Turku, 20520 Turku; the Department of Clinical Genetics, Helsinki University Central Hospital (K.A.), 00029 Helsinki; and Folkhälson Institute of Genetics (A.d.I.C.), 00280 Helsinki, Finland; the Division of Endocrinology (T.T.), University Children’s Hospital, CH-8032 Zurich, Switzerland; the Department of Clinical Biochemistry (H.S.), Statens Serum Institute, 2300 Copenhagen, Denmark; the Department of Obstetrics and Gynecology, National University of Singapore (V.G.), 119074 Singapore; and the Comprehensive Cancer Center, Ohio State University (A.d.l.C.), Columbus, Ohio 43210
Address all correspondence and requests for reprints to: Prof. Ilpo T. Huhtaniemi, Department of Physiology, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland. E-mail: ilpo.huhtaniemi{at}utu.fi
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T) in the human FSH receptor (FSHR)
gene. In women, this mutation causes hypergonadotropic ovarian failure
with arrest of follicular maturation and infertility, whereas in men,
there is variable suppression of spermatogenesis, but no absolute
infertility. To determine whether the same FSHR mutation occurs in
other populations, its frequency was determined in Finland,
Switzerland, Denmark, and the Chinese population of Singapore. The
mutation was screened for using genomic DNA extracted from whole blood
or dried blood spots. Exon 7 of the FSHR gene was first amplified using
a pair of biotinylated primers. The PCR products were then immobilized
on streptavidin-coated microtitration wells and hybridized using short
allele-specific oligonucleotide probes labeled with europium.
Time-resolved fluorometry was used for europium signal detection. To
test the reliability of this method, 40 isolated DNA samples and 35
dried blood spot samples were blindly tested for the
566CT FSHR mutation. The analyses yielded identical
results with denaturing gradient gel electrophoresis and
allele-specific restriction enzyme digestion of the same samples, thus
demonstrating the reliability of the tested method. Automation of this
procedure allows the screening of large numbers of samples, which was
subsequently carried out to investigate the frequency of the
566CT mutation in the study populations. A total of 4981
samples from the above-mentioned 4 countries were analyzed. The
frequency of the 566CT mutation was 0.96% for all
Finnish samples (n = 1976), with a strong enrichment of the mutant
allele in the northeastern part of the country. Only 1 mutation carrier
was identified in the samples from Switzerland (n = 1162), whereas
none was found in samples from Denmark (n = 1094) and the
Singapore Chinese (n = 540). These results suggest that the
566CT mutation of the FSHR gene is enriched in Finland,
but is uncommon in other populations. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T point mutation in exon 7
of the FSH receptor (FSHR) gene, resulting in an Ala to Val change at
residue 189 of the extracellular ligand-binding domain of the FSHR, was
recently found in hypergonadotropic ovarian failure with normal female
karyotype (1). These studies were initiated in Finland with a
population-based investigation of women born between 1950–1976, and a
total of 75 females with ovarian failure and primary or early secondary
amenorrhea were identified (2). Of these, 22 women were found to have
the above-mentioned point mutation. The inactivation of FSH action
caused by this mutation resulted in the arrest of follicular maturation
and infertility in females (3). In 5 males homozygous for the same
mutation, variable suppression of spermatogenesis, but no azoospermia
or absolute infertility, was found (4). Detection of this mutation is
important because it explains the pathogenesis of hypergonadotropic
ovarian failure and suppressed spermatogenesis in this special group of
patients, thus facilitating the diagnosis of infertility and the choice
of therapy. Until the present study, nothing was known about the
occurrence of the 566CT FSHR mutation in other
populations.
Many hereditary diseases are caused by single point mutations, and
special methods are needed for their diagnosis, especially upon large
scale screening. Molecular analysis methods such as allele-specific
oligonucleotide hybridization (5), oligonucleotide ligation assay (6, 7), use of allele-specific oligonucleotide primers (8, 9), competitive
oligonucleotide priming (8), PCR amplification of specific alleles
(10), chemical mismatch analysis (11), sequencing (12), and
minisequencing (13) have been developed for this purpose. Our aim was
to develop an assay based on a sensitive nonradioactive detection
method with an easy to use assay format for large scale screening of
the inactivating FSHR gene point mutation (566CT) to
examine its allelic frequency in various populations.
Time-resolved fluorometry has been used in the signal detection of DNA
assays for the detection of mutations by allele-specific amplification
and allele-specific hybridization (14, 15, 16, 17, 18, 19, 20, 21). The first step of this
analysis is PCR using biotin end-labeled primers and capture of the
amplicons in streptavidin-coated microtitration wells. The two
different alleles are determined by hybridization of the immobilized
DNA using short allele-specific oligonucleotide probes (14-mer) labeled
with europium (Eu) (22, 23). In the present study, this technique was
used to amplify a 69-nucleotide region around the
566CT mutation of the FSHR gene using
biotinylated primers. Thereafter, short allele-specific oligonucleotide
probes were used in the hybridization reaction, quantified by
time-resolved fluorometry based on fluorescence emission. The assay
reliably classified the samples as homozygotes for the wild-type
sequence, carrier heterozygotes, or mutation homozygotes. This simple
single label hybridization assay is suitable for automation and
analysis of a large number of DNA samples to detect specific point
mutations. In the present study, this screening test was applied for
determination of the frequency of the inactivating
566CT mutation of the FSHR gene in four
populations.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 1094 blood spot samples were collected consecutively and anonymously from the routine newborn screening cards (PKU cards) used for phenylketonuria and TSH screening in Denmark. From the eastern area of Switzerland, 1162 blood spot samples were collected randomly for the routine newborn screening for metabolic and endocrinological diseases. Another 540 blood spot samples were collected in Singapore from subjects of Chinese ethnic origin. The 1528 samples from the northern and eastern areas of Finland were obtained through the Finnish Red Cross (Helsinki, Finland). The samples were collected from consecutive blood donors for viral antibody testing (hepatitis and human immunodeficiency virus) and were prepared for PCR as described previously (24). The 448 samples from the southwestern part of Finland were collected for newborn screening of the Finnish aspartylglucosaminuria mutation (13) and subjected to a rapid cell lysis procedure (25). Samples were collected at random from both female and male donors. Positive and negative control blood spot samples were prepared using 60 µL whole blood from subjects known to be homozygous or heterozygous for the FSHR mutation and from wild-type homozygotes. The samples were pipetted onto filter papers that were dried overnight in a fume hood and stored in plastic bags at 4 C until used. One 3-mm diameter blood spot disk from each filter paper card was punched out using a hand puncher. The blood disks were boiled in alkaline solution to liberate DNA (17). Forty samples of isolated DNA and 35 blood spot samples were studied to validate the methodology. The collection of samples and the study protocol were approved by the local institutional review boards.
Oligonucleotides
Primers (WO594, WO595), probes (WO552, WO553), and target DNAs
(WO548, WO549; Table 1
) were synthesized
using the PE Applied Biosystems model 392 DNA/ribonucleic
acid synthesizer (Perkin Elmer, Foster City, CA) and
phosphoramidate chemistry. All oligonucleotides were purified on 15%
polyacrylamide urea gel using standard methods (26). For
biotinylation of the PCR primers, an aliphatic amino group was
introduced at the 5'-end of the oligonucleotide using
5'-O-dimethoxytrityl-N4-(trifluoroacetylamidohexyl)-deoxycytidine-3'-O-(ß-cyanoethyldiisopropylamino)-phosphoramidate
(27). Twenty
5'-O-dimethoxytrityl-N4-(trifluoroacetylamidohexyl)-deoxycytidine-3'-O-(ß-cyanoethyldiisopropylamino)-phosphoramidates
were introduced at the 5'-end of the detection probe for labeling with
Eu chelate.
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The detection probes were labeled using Eu chelate W2014 (Wallac OY, Turku, Finland), which contains isothiocyanate as the reactive group. For this purpose, 50 µg oligonucleotide were dissolved in 440 µL sterile water, and the pH was adjusted to 10 with 1 mol/L Na2CO3. A 25-fold molar excess of the chelate per amino group was added (27), and the reaction mixture was incubated overnight at room temperature. After labeling, the probes were purified on a Sephadex DNA grade G-50 column (Pharmacia Biotech, Uppsala, Sweden) using 10 mmol/L Tris-HCl (pH 7.5), 50 µmol/L ethylenediamine tetraacetate, and 50 mmol/L NaCl as elution buffer. The final probe contained 8–11 Eu chelates/oligonucleotide molecule. The degree of labeling of the detection probes was measured according to the method of Dahlén et al. (15).
Biotinylation of oligonucleotides
For biotinylation, 50-µg aliquots of oligonucleotides (WO594, WO595) were dissolved in 40 µL water, and the pH was adjusted to 10 by adding 1 mol/L Na2CO3. Biotin-N-hydroxysuccinimide-ester (Sigma Chemical Co., St. Louis, MO) dissolved in N,N-dimethylformamide was added to the reaction to yield a 50-fold molar excess of active biotin per amino group. After overnight incubation at room temperature, free biotin was removed from the reaction mixture by using a NAP-5 and, subsequently, a NAP-10 column (Pharmacia Biotech). The biotinylation was verified using high performance liquid chromatography and a reverse phase column PEP RPC 5/5 (Pharmacia).
PCR amplification
A 69-bp fragment of exon 7 of the FSHR gene including nucleotide
566 was amplified using a pair of biotinylated primers. PCR
amplification was performed in a PTC-200 Peltier Thermal Cycler
(MJ Research, Inc., Watertown, MA) or Techne PHC-2
Dri-Block Cycle (Techne, Cambridge, Duxford, Cambridge, UK) using a
program of 95 C for 1 min, 56 C for 2 min, and 72 C for 2 min for 35
cycles. About 100 ng isolated DNA or an unspecified amount of liberated
DNA from the blood spots in a volume of 20 µL supernatant, or a 3-mm
diameter blood spot sample, were added to the reaction mixture. The
final amplification reaction (50 µL) contained 10 mmol/L Tris-HCl (pH
8.8), 1.5 mmol/L MgCl2 for DNA samples/5 mmol/L
MgCl2 for blood spot samples, 300 µmol/L of each
deoxy-NTP, 50 mmol/L KCl, and 0.1% Triton X-100. The Taq
DNA polymerase (DyNAZyme II, Finnzymes OY, Espoo, Finland) was added
after the initial denaturation step using 1 U/reaction. PCR primers
(Table 1
) were used at 0.1 µmol/L concentrations. Water instead of
DNA was used as a control in each reaction to detect possible
contamination. A sequence-verified DNA sample from a mutation
homozygote, heterozygote, and wild-type homozygote were amplified in
each PCR run as controls.
The Delfia oligonucleotide hybridization test for the FSHR
566CT mutation
The assay flow chart is described in Fig. 1. The analysis of the amplified DNA with
time-resolved fluorescence was performed in two separate,
allele-specific hybridization reactions in microtitration wells coated
with streptavidin (SA). The Delfia assay buffer (Wallac) was added in a
volume of 50 µL, and 5 µL of the PCR amplification product or
oligonucleotide standard (10 µL) were added in triplicate and
incubated in SA-coated microtitration wells (Wallac). The wells were
incubated at room temperature with shaking in a Delfia plateshaker
(Wallac) for 1 h to capture the amplified DNA onto the surface of
the microtitration wells. After collection, the well strips were washed
three times at room temperature with Delfia wash solution (Wallac) in
an automated Delfia platewasher (1296-024, Wallac). A 100-µL volume
of 50 mmol/L NaOH was added per well to denature the bound PCR product
during an additional shaking for 5 min at room temperature. After
denaturation, the wells were briefly washed with Delfia wash solution.
The subsequent hybridization was performed in 100 µL of the
hybridization buffer (Delfia assay buffer with 1 mol/L NaCl and 0.1%
Tween-20) containing 1 ng of the probe labeled with Eu chelate. One
Eu-labeled probe (wild-type probe) was complementary to the wild-type
DNA sequence and the other (mutant probe) to the mutated sequence. The
hybridization was carried out for 3 h at room temperature. After
hybridization, the wells were washed at 35 C using Delfia wash
solution, and unbound probes were removed. For detection of the bound
Eu-labeled probes, 200 µL Delfia enhancement solution (Wallac) were
added to each microtitration well and shaken for 30 min at room
temperature. Time-resolved fluorescence was measured in a fluorometer
(ARCUS 1230 Fluorometer, Wallac). A signal ratio was first calculated
from the amount of fluorescence detected for the normal (NP) and mutant
(MP) probes after subtraction of the background signal, and then
modified to logarithmic transformation because the signal ratio (NP/MP)
did not display normal distribution.
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T mutation
To validate the methodology and to confirm the positive samples
obtained during the screening, both RFLP and DGGE were used as control
methods. For RFLP, genomic DNA was amplified by PCR as described above,
and 20 µL of the PCR product were digested with 20 U BsaMI
(Promega Corp., Madison, WI) and electrophoresed through a
12% nondenaturing polyacrylamide gel. DNA was visualized with ethidium
bromide (1). DGGE was used to detect the 566CT
mutation as described in our earlier studies (1).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All of the different sample materials, i.e. isolated DNA, blood cell lysates, supernatants of the blood spots, and dried blood spots, were suitable for the screening test. For practical reasons, cell lysates and dried blood spot samples were used in this study. The direct use of blood spot disks (3 mm) in PCR amplification with 5 mmol/L MgCl2 reduces the number of experimental steps and would be preferable for large scale screening studies.
Optimization of the PCR amplification
The PCR was optimized for the amount of DNA, volume of supernatant (5–40 µL), amount of Taq DNA polymerase, concentration of MgCl2 (1.5–5 mmol/L), and number of PCR cycles (30, 35, or 40 cycles; data not shown). Optimal DNA amplification and maximal hybridization signal were obtained with 100 ng isolated DNA, 5 µL blood cell lysate, and 20 µL supernatant from a blood spot, using 1 U Taq DNA polymerase/PCR reaction, added after the initial denaturation step. The optimum PCR program was as follows: 95 C for 1 min, 56 C for 2 min, and 72 C for 2 min, for 35 cycles. The concentration of 1.5 mmol/L MgCl2 was optimal for isolated DNA samples, and that of 5 mmol/L was optimal for the blood spot samples.
Optimization of oligonucleotide hybridization
The fragment of the FSHR gene containing the 566CT
mutation in exon 7 was amplified using a pair of biotinylated primers.
Synthetic biotinylated target oligonucleotides (Table 1) for the
wild-type and mutated FSHR gene were used to optimize the hybridization
assay. The kinetic properties of the hybridization were studied for
both capture and hybridization procedures. The highest signal was
reached during 1-h capture reaction with shaking and a 3-h
hybridization without shaking at room temperature. The amounts of
hybridization probes (0.1, 0.5, 1, 1.5, 2, 5, and 10 ng/reaction) were
tested by monitoring the signal to noise ratio (hybridization
signal/unspecific signal) with a fixed amount of the synthetic target
(1 x 1010 molecules/well). The optimal signal ratio
was obtained at 1 ng/well. The optimal hybridization temperature was
studied at the range of 4–50 C, and the optimum occurred at room
temperature. When the washing temperature was tested at 25–55 C, the
best signal to cross-reaction ratio was obtained at 35 C.
Assay validation
A total of 40 DNA samples and 35 dried blood spot samples were
initially tested blindly to assess the accuracy of the hybridization
assay. The results were clear cut after subtraction of the background
signal from the original fluorescence. When the logarithmic signal
ratio was calculated from the amount of fluorescence detected for the
normal (NP) and mutant probes (MP), three different categories were
clearly distinguished. The logarithmic signal ratio (NP/MP) was able to
discriminate the three possible genotypes, namely wild-type
homozygotes, heterozygotes, and mutation homozygotes. A log NP/MP ratio
above 1 corresponded to a wild-type homozygote, a ratio between 1–0
corresponded to a heterozygote, and a ratio below 0 corresponded to a
mutant homozygote (Fig. 2). The test
results of the 75 samples were in full agreement with earlier analyses
of the same samples using DGGE and allele-specific RFLP. The technical
details were described in our early studies (1, 4). There were 18
homozygotes, 27 carriers for the 566CT
mutation, and 30 wild-type homozygotes among the samples tested. During
the population screenings (see below), we continued carrying out
control analyses of the positive samples with RFLP and DGGE. We
detected some false positive results in the samples from Denmark and
Singapore Chinese, analyzed from blood spots (see
Discussion). These samples were initially considered to
represent mutation carriers, but were subsequently classified as
wild-type homozygotes on the basis of the control analysis. No false
negative results were observed during the assay validation.
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Samples from four populations were analyzed to determine the
frequency of the 566CT transition in exon 7 of the FSHR
gene (Table 2). The results showed that
the frequency of this mutation was clearly higher in the Finnish
population than in samples received from Denmark, Switzerland, and
Singapore Chinese. Excluding Finland, there was only one mutation
carrier among the 2796 samples studied. This carrier was identified in
a sample from Switzerland. In Finland, the geographic distribution of
the 566CT mutation was highly uneven (Fig. 3). Of the 19 mutation carriers, only one
was detected in the samples collected from the southwestern part of the
country, whereas the others were all identified in samples collected
from the northeastern regions. The birth places of the 30 obligatory
mutation carriers (parents of tested patients) identified to date show
the same geographical distribution (Fig. 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T) of the FSHR gene was shown to be the
cause of hypergonadotropic ovarian failure in a proportion of Finnish
patients (1, 2). Subsequent phenotypic studies in female and male
homozygotes demonstrated ovarian failure with ovaries shown to contain
oocytes but with arrest of follicular development in females (3),
whereas the males showed variable disturbance of spermatogenesis
without azoospermia (4). The present study was initiated to study the
carrier frequency of this mutation in Finland and other countries.
For the detection of amplified target sequences, several methods are
available. In large scale screening studies, automation combined with
technical reliability, specificity, and nonradioactive signal detection
methods are required. To achieve large scale population screening of
the 566CT mutation, we used a single label,
allele-specific hybridization assay with time-resolved fluorometry as
the signal detection system. In the initial validation of this method,
75 test samples were genotypically studied by this method, and the
results were confirmed by RFLP analysis and/or DGGE. There were no
discrepancies among the results obtained by the three methods, and the
results of the tested method showed clearly different values of
logarithmic NP/MP ratio for each of the three genotypes. However, to
confirm the study results, we also reanalyzed all positive samples
detected in population screening. In this control testing, 8 false
positive samples were identified. They had all been analyzed from blood
spots and interpreted as carriers, although they were shown to be
wild-type homozygotes upon control analysis (RFLP). This would have led
to false interpretation of the carrier frequency if the positive
results had not been additionally tested. In contrast, no false
negative interpretations were made when studying either mutation
homozygotes or heterozygotes. Therefore, the single label hybridization
assay with time-resolved fluorometry can be considered a reliable
screening method for mutations. Positive findings, however, have to be
verified using an alternative method, such as RFLP, DGGE, or
sequencing. The hybridization assay in combination with control
analysis, as used in the present study, proved to be an effective
method with high through-put in screening large numbers of samples for
the FSH receptor 566CT point mutation.
On the population level, the results showed a carrier frequency of
0.96% for the mutation considering all samples from Finland, whereas
its frequency in other populations was very low or could not be
defined. This is in agreement with the findings in some other
disease-causing genes that are enriched in Finland and underlie some of
the 30 inherited diseases that are common in Finland but are uncommon
elsewhere (28). This pattern is caused by a combination of profound
founder effects followed by genetic drift in a population expanding
while isolated (29). Instead, inbreeding, as known consanguineous
marriages, is considered relatively uncommon in Scandinavian and
Central European populations. In several of these disorders, this is
also displayed as an enrichment of specific mutations in geographically
defined subpopulations inside the country (30). This typical feature is
also displayed by the 566CT mutation, which
together with some other disorders, such as congenital chloride
diarrhea (31), shows a typical northeastern geographical enrichment.
The fact that a specific mutation is enriched in Finland does not,
however, exclude its presence in other populations. On the contrary, it
is known that in some disorders, such as progressive myoclonus
epilepsy, common mutations are shared with other populations (32).
Based on the present results, however, it seems that the
566CT mutation of FSHR is uncommon in other countries.
The same is suggested by the recent study by Layman et al.,
who were not able to find this mutation in 35 patients with premature
ovarian failure (33). Therefore, it is most likely that in other
populations, different mutations will be identified in the FSHR and
other genes. Indeed, another heterozygous inactivating genomic point
mutation was serendipitiously identified by Gromoll et al.
(34) in a healthy female of German origin. However, no homozygotes for
this mutation have been detected to date (35). This indicates that
other inactivating mutations in the FSHR gene are possible, although
not yet identified. Compared with the number of mutations identified to
date in the LH receptor gene (36, 37), the number of FSHR mutations is
surprisingly low and not readily explicable. On the one hand, the
obligatory infertility that the homozygous mutation causes in females
should contribute to a low incidence in the genetic pool, unless
heterozygosity for it has a selective advantage. On the other hand, the
mild male phenotype of the homozygotes, with no absolute infertility,
may contribute to preservation of the mutation. Nevertheless, it is
likely that new FSHR mutations will be found in the future in other
populations.
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Acknowledgments |
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Footnotes |
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Received June 10, 1998.
Revised August 14, 1998.
Accepted August 26, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-antitrypsin deficiency: application of an improved
method for amplifying mutated gene sequences. Lab Invest. 59:403–408.[Medline]
1-antitrypsin deficiency. Clin Chem. 39:1626–1631.[Abstract]
F508 in the
cystic fibrosis gene using allele-specific PCR primers and
time-resolved fluorometry. PCR Methods Appl. 2:157–162.[Medline]
F508 mutation analyses in newborn screening for
cystic fibrosis: an anonymous pilot study in Denmark. Screening. 2:1–11.
ß]-labeled oligo DNA hybridization
probes. Nucleos Nucleot. 7:655–659.
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