573: A non-invasive prenatal test for Alpha Thalassemia Hb Barts and Hb Constant Spring

Abstract

Hb Barts and Hb Constant Spring are the two most frequent severe forms of alpha thalassemia. The UNITYTM Carrier screen is a Lab Developed Test that performs reflex alpha thalassemia NIPT when the mother is determined to be an alpha-thalassemia carrier. This study analyzes the ability of the UNITYTM NIPT to exclude inheritance of Hb Barts and detect maternal inheritance of Hb Constant Spring. The UNITYTM Alpha Thalassemia NIPT was performed on simulated cell-free DNA samples. The assay is a multiplex PCR reaction that employs multiple technologies to evaluate the fetal genotype. Polymorphic alleles contained within the SEA deletion region are assayed to exclude inheritance of double deletions and therefore Hb Barts. Quantitative counting templates are used to accurately measure the number of DNA molecules present in the reaction. Relative mutation dosage is performed to determine the maternal and paternal inheritance of the Constant Spring allele. Gap-PCR is used to detect paternal inheritance of an SEA deletion. A likelihood ratio is used to combine these measurements and determine the Hb Constant Spring status of the fetus. Simulated cell-free DNA from pregnant mothers was prepared by shearing and combining genomic DNA samples. Hb Barts exclusion was tested using SEA carrier DNA mixed with healthy or Hb Barts DNA. Hb Barts was excluded from all healthy DNA samples and was not excluded from all affected samples (n=96). Maternal inheritance of the Constant Spring variant was tested on Constant Spring carrier DNA mixed with healthy SEA carrier DNA. All calls were correct at 5%, 10% and 20% fetal fractions, two 5% samples resulted in no-calls (n=12). These results analytically demonstrate the ability of the UNITYTM alpha thalassemia NIPT to identify healthy fetuses in carrier mothers. The UNITYTM alpha thalassemia carrier screen with reflex NIPT will enable detection of both maternal carrier status and fetal disease risk from a single blood tube. This removes the need for paternal DNA and decreases the turn around time to identify high-risk fetuses.View Large Image Figure ViewerDownload Hi-res image Download (PPT)