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Letters to the Editor |
1 Departamento de Genómica Estructural Neocodex, Sevilla, Spain
2 Servicio de Ginecología y Obstetricia Hospital Universitario, Virgen de las Nieves, Granada, Spain
3 Servicio de Ginecología y Obstetricia Hospital San Juan de Alicante, Alicante, Spain
4 CEOGA Clínica de Ginecología, Lugo, Spain
5 Servicio de Ginecología Clínica Sanatorio Bilbaíno, Bilbao, Spain
6 Clínica Diatros Gavá, Barcelona, Spain
aAddress correspondence to this author at: Departamento de Genómica Estructural. Neocodex. C/ Charles Darwin no.6, Acc. A. Parque Tecnológico Isla de la Cartuja, 41092-Sevilla, Spain. Fax 34-955-047325; e-mail aruiz{at}neocodex.es.
To the Editor:
Germline mutations in different genes are associated with premature ovarian failure (POF, OMIM 311360), defined as premature menopause with amenorrhea occurring before the age of 40 years along with increased gonadotropin concentrations [follicle-stimulating hormone (FSH) >40 IU/L]. A new candidate gene, bone morphogenetic protein 15 (BMP15) has been investigated in POF. In a family affected by hypergonadotropic ovarian failure, a mutation in the pro-region of the BMP15 gene (Y235C) was found in 2 affected sisters (1), and 3 linked markers within the BMP15 gene (–673C>T, –9C>G and IVS1 + 905A>G) are associated with high follicle production in women undergoing recombinant FSH stimulation (2).
Several heterozygous variations affecting the pro-region and mature peptide of the BMP15 gene have been identified in women with POF (3)(4)(5), but A180T was the only variant found in all reported studies, occurring with relatively high frequency in POF women (8 of 502, 1.6%) but not at all in control groups.
To clarify the role of the A180T allele in early menopause and ovarian failure, we used a pyrosequencing protocol (Biotage) to evaluate the A180T variant. This technology allows an easy 96-well typing format. The selected primers for pyrosequencing analysis and PCR conditions were forward: 5'-ACC GCC ATC ATC TCC AAC TAA-3', reverse: 5'-biotine-CCT GTG TCC CTT GTT ATT CCA-3', and sequencing: 5'-AAC CTT CCC TGA TGT CT-3'. We used reextraction and conventional resequencing of a random set of samples (10%) with the CEQ Dye Terminator Cycle Sequencing Quick Start Kit (Beckman) to assure the quality of our genotyping protocol (see Figure 1 in the Data Supplement that accompanies the online version of this letter at http://www.clinchem.org/content/vol53/issue4).
We investigated the presence of the A180T allele and also reproductive, gynecological, and other important traits in 1157 white Spanish postmenopausal women divided into 3 clinical groups. Group A included women whose age at natural menopause was in the normal range (
40 years, n = 852), group B included women with secondary amenorrhea (SA; age at natural menopause <40 years, n = 46), and group C included women with menopause induced by surgery for treatment of benign or malignant gynecological pathologies (n = 249). The observed mean (SD) ages at menopause and menarche [48.4 (5.0) and 12.9 (1.56) years, respectively] were strictly concordant with others previously reported. Written informed consent was obtained from all study participants, and the study protocol was approved by the referral center Ethics Committees and Neocodex.
Genotyping revealed the A180T variant only in 7 unrelated individuals, suggesting a variant allele frequency of 0.3% in the Spanish population. The clinical data and related morbidity of the 7 women who were heterozygous carriers of A180T variant are reported in Table 1
. Among these women, 1 patient had menopause caused by a surgical intervention for endometriosis (group C). Only 1 patient displayed the SA phenotype (group B). The other 5 women had proven fertility and a natural menopause occurring at >40 years (group A).
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To analyze relevant variables involved in age at menopause, such as age at menarche, reproductive period, pregnancies, and body mass index, we further studied patients with a natural menopause (n = 898) by dividing them into 2 genotypic groups (presence/absence of A180T variant). Our results indicated that the groups did not differ in any clinical variable studied (P >0.13). Nevertheless, women carrying the A180T variant seemed to have a lower mean (SD) age at menopause [45.3 (5.6) vs 48.5 (5.0)], and they had a shorter reproductive period [32.8 (5.4) vs 35.6 (5.1) years], although because of the small number of individuals, this finding was not statistically significant.
Research to date has detected the A180T variant in only 10 women. Of these, 5 of 166 were Italian women with POF (3); 1 of 133 and 2 of 60 Indian women with POF or primary amenorrhea, respectively (4); and 2 of 203 European women with POF (5). Furthermore, no control individuals analyzed to date have been carriers the A180T allele (n = 462, merged sample size). In contrast with these observations, we detected the A180T allele in 5 women with natural menopause occurring at >40 years of age. Our results indicate that the A180T allele is a rare variant that is not sufficient to generate SA in humans. That the allele has not been found in control groups to date does not exclude the possible presence of this rare variant in unaffected individuals, because the number of controls used in studies to date may have been insufficient to detect very low frequency alleles. However, our data also support the finding that genetic variation in the BMP15 gene may contribute to variation in age at menopause in a complex manner, but this interpretation should be viewed with caution.
To elucidate the role of BMP15 in ovulation rate, POF, and SA, exhaustive analysis of this gene in independent large populations would be required.
Acknowledgments
We are deeply grateful to the postmenopausal women who participated in this study. We are very grateful to Eva Molero, Antonio Gónzalez, and Rocío Pascual for their collaboration during this work. The authors F.J. Moron, M.E. Saez, and A. Ruiz, have declared that conflicts of interest exist. Some of the work described here is subject to patent filings for diagnostics purposes. Neocodex has been partially funded by the Ministerio de Educación y Ciencia of Spain (PTQ2003-0546, PTQ2003-0549, PTQ2003-0783) and the European Commission (Gendisrupt project: QLK4-CT-2002-02403).
Grant/funding support: None declared.
Financial disclosures: None declared.
References
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