ESPE Abstracts

Background: Turner syndrome (TS) is a common aneuploidy diagnosed by peripheral-blood-cell (PBC) karyotyping. Karyotyping is costly, time-staking and subject to manual errors. Quantitative real-time PCR (qPCR) is a cheap molecular diagnostic test which can detect several samples of TS within hours.

Objective: To assess the performance of qPCR in detecting TS and variants.

Methods: Genomic DNA was isolated from 50 TS patients (45, XO: 23, XO/XX mosaics : 10, Isochromosome-Xq: 12, XO/XY mosaics : 5), 25 normal females (46, XX) and 5 males (46, XY). qPCR was done using 96-well plates, fast real-time PCR. Four primers were used – two on Xp [SHOX and ARSE] and two on Xq [VAMP7 and XIST]. Autosomal gene HBB was used as housekeeping-gene. The ΔΔCT method was used for calculation of the ‘X gene-dose’ with respect to housekeeping gene and X-genes from karyotype-normal (46, XX) females. ROC curves were plotted to determine cut-offs for the genes.

Results: Using a criterion for either SHOX < 0.752 or ARSE < 0.885, all TS cases could be detected (Table 1). TS cases thus diagnosed should undergo qPCR for VAMP7 dose and a value > 0.723 would indicate presence of isochromosome-Xq. For non-isochromosome cases, SHOX < 0.511 indicates classic TS (45, X) and SHOX > 0.511 means 45, X/46, XX mosaic. A qualitative PCR for the SRY on all diagnosed TS cases could detect XO/XY mosaics. Next, we analysed samples of nine girls with phenotypic diagnosis of TS but karyotype from 30-PBC being 46, XX in six and 46, XY in three. Using our novel approach, we found that the qPCR-diagnosis for 5 girls having 46, XX on 30-PBC karyotyping was 45, XO/46, X,iXq (n = 2) or 45,XO/46,XX (n = 3) and in all the three girls with 46,XY, the qPCR-diagnosis was 45,XO/46,XY. We did a 100 cell karyotyping for these nine samples and found that the qPCR based diagnosis matches with 100 cell karyotype result in 8 out of the 9 cases, Cohen’s κ statistic being 0.72 indicating substantial agreement.

Conclusion: Our qPCR based method involving multiple X-primers can be used for rapid and cost-effective diagnosis of all TS variants, including cases with dubious results on 30-PBC karyotyping.