SUN-084 A Quantitative-PCR Based Rapid and Cost-Effective Diagnostic Method for Turner Syndrome and Its Variants

Turner’s syndrome (TS) is a common aneuploidy diagnosed by peripheral blood karyotyping in patients. Karyotyping involves manual labour, time and costs. Quantitative real-time PCR (qPCR) is a rapid molecular diagnostic test for TS that yields results of upto 48 samples within two hours at low cost with reasonable accuracy. To assess the sensitivity and specificity of qPCR in the rapid diagnosis of TS, genomic DNA was isolated and estimated from peripheral blood 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 normal males(46,XY). qPCR was done using 96 well plates, fast real-time PCR. 4 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 ΔΔ CT method was used for calculation of the ‘X gene dose’ with respect to the housekeeping gene and X genes from normal females. Differences of doses of the four X-chromosomal primers in different karyotypes were analysed. ROC curves were plotted to determine cut-offs to discriminate the different karyotypes of TS from normal females. qPCR could distinguish classical TS from normal females with >95% sensitivity and specificity. SHOX gene primer was the best to diagnose TS of all karyotypes combined and also classical TS(XO) from normal females. qPCR could also identify non-classical TS with >92% sensitivity and specificity,the best primer being ARSE, for detecting both mosaics and isochromosomes. The cut-offs determined from our study corroborates with past similar studies.(1,2) qPCR using an appropriate panel of primers on the short and long arms of X chromosomes can be a rapid and cheaper alternative to karyotyping to diagnose TS of different karyotypes. The choice of primers should be guided by the need for a more sensitive or specific test depending on the clinical scenario. If used as a neonatal screening test, SHOX should be the best primer. For diagnostic purposes, when the pre-test probability is low, a more specific primer like SHOX would be more appropriate. However, when the pre-test probability is high, a sensitive primer for ruling out TS like VAMP7 is better. In case there is a high pre test probability of the patient having a non-classic TS rather than classic TS, ARSE should be used. This is the first study to show good sensitivity of qPCR in detecting non-classic TS of different karyotypes in addition to classic TS. References: 1. Ibarra-Ramírez M, Martínez-de-Villarreal LE. Clinical and genetic aspects of Turner’s syndrome. Medicina universitaria. 2016 Jan 1;18(70):42-8. 2 .Rocha MN et al. Applicability of real-time PCR methodology in the neonatal detection of Turner syndrome. Hormone and metabolic research. 2010 Aug;42(09):677-81.