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Genotyping common FSHR polymorphisms based on competitive amplification of differentially melting amplicons (CADMA).

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Abstract

Purpose

To provide an improved platform for simple, reliable, and cost-effective genotyping.

Background

Modern fertility treatments are becoming increasingly individualized in an attempt to optimise the follicular response and reproductive outcome, following controlled ovarian stimulation. As the field of pharmacogenetics evolve, genetic biomarkers such as polymorphisms of the follicle stimulating hormone receptor (FSHR) may be included as a predictive tool for individualized fertility treatment. However, the currently available genotyping methods are expensive, time-consuming or have a limited analytical sensitivity. Here, we present a novel version of “competitive amplification of differentially melting amplicons” (CADMA), providing an improved platform for simple, reliable, and cost-effective genotyping.

Methods

Two CADMA based assays were designed for the two common polymorphisms of the FSHR gene: rs6165 (c.919A > G, p. Thr307Ala, FSHR 307) and rs6166 (c.2039A > G, p. Asn680Ser, FSHR 680). To evaluate the reliability of the new CADMA-based assays, the genotyping results were compared with two conventional PCR based genotyping methods; allele-specific PCR (AS-PCR) and Sanger sequencing.

Results

The genotype frequencies for both polymorphisms were 35 % (TT), 42 % (CT), and 23 % (CC), respectively. A 100 % accordance was observed between the CADMA-based genotyping results and sequencing results, whereas 5 discrepancies were observed between the AS-PCR results and the CADMA-based genotyping results. Comparing the CADMA-based assays to (AS-PCR) and Sanger sequencing, the CADMA based assays showed an improved analytical sensitivity and a wider applicability.

Conclusions

The new assays provide a reliable, fast and user-friendly genotyping method facilitating a wider implication in clinical practise.

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Acknowledgments

The authors would like to acknowledge Yvonne Giwercman from Molecular Genetic Reproductive Medicine, Department of Clinical Sciences, Faculty of Medicine, Lund University, Malmö, Sweden, for providing reference DNA of known genotypes, as well as AS-PCR assays the FSHR polymorphisms rs6165 and rs6166.

Source of funding

This work was supported by the faculty of Health, University of Aarhus, and by ReproHigh, an Interregional EU project.

Conflicts of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Biomedicine, University of Aarhus, Aarhus, Denmark

    Tanni Borgbo & Lise Lotte Hansen

  2. Laboratory of Reproductive Biology, Rigshospitalet, Blegdamsvej 9, DK-2100, Copenhagen, Denmark

    Tanni Borgbo & Claus Yding Andersen

  3. Department of Hematology, Rigshospitalet, Copenhagen, Denmark

    Lasse Sommer Kristensen

  4. Molecular Genetic Reproductive Medicine, Department of Clinical Sciences, Lund University, Malmö, Sweden

    Ida Lindgren

Authors
  1. Tanni Borgbo
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  2. Lasse Sommer Kristensen
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  3. Ida Lindgren
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  4. Claus Yding Andersen
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  5. Lise Lotte Hansen
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Corresponding author

Correspondence to Tanni Borgbo.

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Fig. S1

False-positive amplification using AS-PCR. We encountered recurrent problems with uneven amplification (left panel) and false-positive amplification (mid and right panel), when genotyping with AS-PCR. Each assay consists of two PCR runs, one for each genotype, containing a PCR control fragment (CF; upper band) and a genotype specific fragment (GF; lower band). The presence of a lower band marks the genotype. As the two polymorphisms are in linkage disequilibrium, the genotype of FSHR 307 should match the genotype of FSHR 680. (GIF 333 kb)

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Borgbo, T., Sommer Kristensen, L., Lindgren, I. et al. Genotyping common FSHR polymorphisms based on competitive amplification of differentially melting amplicons (CADMA).. J Assist Reprod Genet 31, 1427–1436 (2014). https://doi.org/10.1007/s10815-014-0329-6

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  • DOI: https://doi.org/10.1007/s10815-014-0329-6

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