The Frequency of an Inactivating Point Mutation (C3T) of the Human Follicle-Stimulating Hormone Receptor Gene in Four Populations Using Allele-Specific Hybridization and Time-Resolved Fluorometry*

We have described previously in the Finnish population an inactivating point mutation (C3T) 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 C3T 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 C3T mutation in the study populations. A total of 4981 samples from the above-mentioned 4 countries were analyzed. The frequency of the C3T mutation was 0.96% for all Finnish samples (n 5 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 5 1162), whereas none was found in samples from Denmark (n 5 1094) and the Singapore Chinese (n 5 540). These results suggest that the C3T mutation of the FSHR gene is enriched in Finland, but is uncommon in other populations. (J Clin Endocrinol Metab 83: 4338–4343, 1998) A 566 C3T 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 populationbased 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 C3T 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 allelespecific 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 forReceived June 10, 1998. Revision received August 14, 1998. Accepted August 26, 1998. 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: [email protected]. * This work was supported by research grants from the Academy of Finland and the Sigrid Jusélius Foundation. 0021-972X/98/$03.00/0 Vol. 83, No. 12 Journal of Clinical Endocrinology and Metabolism Printed in U.S.A. Copyright © 1998 by The Endocrine Society