Prenatal Diagnosis of Trisomy 21, 18 and 13 by Quantitative Pyrosequencing of Segmental Duplications

Abstract

Chromosomal aberration mostly occurs in chromosome 21, 18 and 13, with an incidence approximately 1 out of 160 live births in humans, therefore making prenatal diagnosis necessary in clinics. Current methods have drawbacks such as time consuming, high cost, complicated operations and low sensitivity. In this paper, a novel method for rapid and accurate prenatal diagnosis of aneuploidy is proposed based on pyrosequencing, which quantitatively detects the peak height ratio of different bases of segmental duplication. A direct PCR approach was undertaken where a small volume of amniotic fluid was used as the starting material without DNA extraction. Single-stranded DNA was prepared from PCR products and subsequently analyzed using pyrosequencing. The peak height ratio between target and reference chromosome of 2.2 for euploid and 3:2 for a trisomy fetus were used as reference. The reference intervals and z scores were calculated for discrimination of aneuploidy. A total of 132 samples were collected, within trisomy 21 (n = 11), trisomy 18 (n = 3), trisomy 13 (n = 2), and unaffected controls (n = 116). A set of six segmental duplications were chosen for analysis. This method had consistent results with karyotyping analysis, a correct diagnosis with 100% sensitivity and 99.9% specificity.

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