Application Of Maternal Plasma DNA Sequencing In X-linked Disorder Screening

Objective: To develop a fetal sex determination method based on maternal plasmasequencing (MPS), assess its performance and potential use in X-linked disorder counseling.Methods:900cases of MPS data from a previous study were reviewed, in which100and800cases were used as training and validation set, respectively. The percentage of uniquelymapped sequencing reads on Y chromosome was calculated, and was used to classify maleand female cases based on cut-off value determined by ROC curve. Eight pregnant womenthat are carriers of Duchenne muscular dystrophy (DMD) mutations were recruited, whoseplasma were subjected to multiplex sequencing and fetal sex determination analysis. Toperform a comprehensive analysis for error cases of fetal sex determination, fetalchromosomal aneuploidies as well as clinical records were included, in which fetalchromosomal aneuploidies detection was established using Z-test with internal referencecontrol introduced.Results: In the training set, a sensitivity of96%and false positive rate of0%for male casesdetection were reached in our method. The blinded validation results showed421in423malecases and374in377female cases were successfully identified, revealing sensitivity andspecificity of99.53%and99.20%for fetal sex determination, at as early as12gestationalweeks. Fetal sex for all8DMD genetic counseling cases were correctly identified, whichwere confirmed by amniocentesis. Z-test with internal reference control method wasestablished, with sensitivity of100%for all three chromosome (Chr13, Chr18and Chr21) andspecificity of98.7%,100%and100%respectively. Fetal aneuploidies, gestational weeks,maternal age, down’s risk and pregnancy history factors were excluded in the five error casesof fetal sex determination.Conclusions: Based on MPS, high accuracy of noninvasive fetal sex determination can beachieved. This method can potentially be used for prenatal genetic counseling underappropriate ethical considerations.