Improved interpretation of aneuploidy screening QF PCR using relevant allele ratio ranges
Quantitative fluorescence polymerase chain reaction (QF PCR) is an alternative approach to FISH for rapid aneuploidy screening of selected chromosomes. Using this method, samples from uncultured amniocytes and CVS tissue can be readily analysed for the most common chromosomal aneuploidies found in prenatal specimens (chromosomes 13, 18, 21, X and Y). QF PCR amplifies chromosome specific short tandem repeats (STRs) to reveal polymorphic patterns that indicate the chromosomal balance in the sample.
A twenty five marker, three-reaction QF PCR assay was developed during 2003. Since launching the QF PCR screen in 2004, our lab has analysed over 1700 samples. Five percent of these samples had abnormal chromosome profiles, including 67 cases of trisomy 13, 18 or 21, 15 sex chromosome aneuploidies and eleven triploidies.
The basis for QF PCR is a quantitative assessment of the peak areas of two alleles at a heterozygous locus. Homozygous loci do not contribute information, except on the X chromosome where 45,X and 47,XXY genotypes are to be excluded, and tri-allelic loci are also indicative of trisomy. The best practice guidelines state that the range for allele ratios should not lie outside of 0.8-1.4, with values of less than 0.65 or more than 1.8 defining the trisomic range. However, data obtained during our clinical service showed some STR loci with a wider allele ratio range of 0.8 to 1.8 in normal samples. To help interpret this wide range, discreet analysis of all allele pair ratios was used. Using this approach, we observed that while the average ratio of some allele pairs at some loci was high, (e.g. 1.7) the standard deviation for any pair was low (± 0.09). This analysis revealed the presence of subsets of allele ratios within the greater allele ratio set. The possible reason for the anomalous amplifications of these specific alleles is being further investigated. This analysis resulted in a robust test with improved interpretation for rapid and accurate aneuploid screening.