Application of real-time PCR of sex-independent insertion-deletion polymorphisms to determine fetal sex using cell-free fetal DNA from maternal plasma

Sherry Sze Yee Ho; Angela Barrett; Henna Thadani; Cecille Laureano Asibal; Evelyn Siew-Chuan Koay; Mahesh Choolani

doi:10.1515/cclm-2014-0881

Article

Application of real-time PCR of sex-independent insertion-deletion polymorphisms to determine fetal sex using cell-free fetal DNA from maternal plasma

Sherry Sze Yee Ho , Angela Barrett , Henna Thadani , Cecille Laureano Asibal , Evelyn Siew-Chuan Koay and Mahesh Choolani

Published/Copyright: January 10, 2015

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From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 53 Issue 8

Abstract

Background: Prenatal diagnosis of sex-linked disorders requires invasive procedures, carrying a risk of miscarriage of up to 1%. Cell-free fetal DNA (cffDNA) present in cell-free DNA (cfDNA) from maternal plasma offers a non-invasive source of fetal genetic material for analysis. Detection of Y-chromosome sequences in cfDNA indicates presence of a male fetus; in the absence of a Y-chromosome signal a female fetus is inferred. We aimed to validate the clinical utility of insertion-deletion polymorphisms (INDELs) to confirm presence of a female fetus using cffDNA.

Methods: Quantitative real-time PCR (qPCR) for the Y-chromosome-specific sequence, SRY, was performed on cfDNA from 82 samples at 6–39 gestational weeks. In samples without detectable SRY, qPCRs for eight INDELs were performed on maternal genomic DNA and cfDNA. Detection of paternally inherited fetal alleles in cfDNA negative for SRY confirmed a female fetus.

Results: Fetal sex was correctly determined in 77/82 (93.9%) cfDNA samples. SRY was detected in all 39 samples from male-bearing pregnancies, and none of the 43 female-bearing pregnancies (sensitivity and specificity of SRY qPCR is therefore 100%; 95% CI 91%–100%). Paternally inherited fetal alleles were detected in 38/43 samples with no SRY signal, confirming the presence of a female fetus (INDEL assay sensitivity is therefore 88.4%; 95% CI 74.1%–95.6%). Since paternally inherited fetal INDELs were not used in women bearing male fetuses, the specificity of INDELs cannot be calculated. Five cfDNA samples were negative for both SRY and INDELS.

Conclusions: We have validated a non-invasive prenatal test to confirm fetal sex as early as 6 gestational weeks using cffDNA from maternal plasma.

Keywords: ambiguous genitalia; amniocentesis; cell-free fetal DNA; complete androgen insensitivity; congenital adrenal hyperplasia; hemophilia; non-invasive prenatal diagnosis; sex-linked disorders

Corresponding author: Dr. Sherry Sze Yee Ho, Molecular Diagnosis Centre, Department of Laboratory Medicine, National University Hospital NUH Main Building Level 3 5 Lower Kent Ridge Road, Singapore, Phone: +65 119074 6772 4294, Fax: +65 6775 1757, E-mail: sherry_sy_ho@nuhs.edu.sg

Acknowledgments

We thank Shanice Tan, Kevin Kong, Kenneth Ee and Joey Koh from the Department of Life Science and Chemical Technology, Ngee Ann Polytechnic, Singapore for running the qPCR experiments.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Financial support: None declared.

Employment or leadership: None declared.

Honorarium: None declared.

Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Received: 2014-9-4

Accepted: 2014-12-10

Published Online: 2015-1-10

Published in Print: 2015-7-1

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Articles in the same Issue

https://doi.org/10.1515/cclm-2014-0881

Keywords for this article

ambiguous genitalia; amniocentesis; cell-free fetal DNA; complete androgen insensitivity; congenital adrenal hyperplasia; hemophilia; non-invasive prenatal diagnosis; sex-linked disorders