Haplotype-Based Noninvasive Prenatal Testing For Duchenne Muscular Dystrophy And Becker's Muscular Dystrophy

ObjectiveTo explore the accuracy and feasibility of the noninvasive prenatal testing for Duchenne muscular Dystrophy(DMD)and Becker's Muscular Dystrophy(BMD)using Proband-assisted haplotype phasing(PAHP).Methods 1.A total of 96 families had a high risk to get a DMD/BMD baby were collected from Prenatal Diagnosis Center in the Third Affiliated Hospital of Guangzhou Medical University from 2015 to 2017.2.The inclusion and exclusion criterias were set.17 families with no evidence of NIPT contraindications were included in The NIPT study and the genetic diagnosis of their probands who can provide blood samples were also clear.3.Blood samples from probands,pregnant women,and their husbands were collected for NIPT.The gDNA samples of them were captured and sequenced in the target area.The haplotype information linked to the pathogenic sites was obtained.Then,with the help of the haplotypes of the husband and wife,it is possible to infer whether the fetal haplotypes obtained from the parents in the dystrophin gene region are consistent with the probands by analyzing and counting the snp sites available in the plasma data.The male fetuses carrying the same haplotype as the proband were affected while the female fetuses were carriers and the rest were normal fetuses.4.NIPT results were compared with the gold standard for Dystrophin gene analysis to verify the accuracy of NIPT.Results 1.The mean maternal age of 17 pregnant women participating in the NIPT study was 32.05±4.91 years-old.The mean gestational week of NIPT blood collection was 15+1w,and the minimum week was 11+1w.15 probands were Dystrophin gene deletion mutations,and 2 probands were Dystrophin gene point mutations.2.The average Sequencing Depth for gDNA samples and plasma cf-DNA samples was 142.64X±46.04X(78.25X-311.74X)and 256.60X±61.35X(279.38X-479.31X),respectively.The average capture efficiency,coverage and repetition rate at 20 X sequencing depth were 51.6%±8.62%(29.73%-74.6%),98.83%±0.92%(94.37%-99.72%)and 29.73%±6.09%(14.94%-45.15%).The results showed that the average sequencing error rate was 0.57%(0.21%-2.16%), and the average fetal concentration was 9.80%(3.61%-16.97%).3.Based on the average sequencing depth and the coverage rate of the sequencing depth ?4X in the Y chromosome target region of the plasma sample,the families F-03?F-06?F-07?F-10?F-11?F-13?F-14?F-15?F-16 were inferred to be male and other to be female.The results were consistent with the results of sex diagnosis tested by fetal amniotic fluid/villi samples.4.NIPT found that 5 families had recombination events within the Dystrophin gene,including F-04?F-09?F-10?F-11?F-13.The mutation site is far from the recombination site.5.Among the 17 families,3 fetuses were diagnosed as patients and 4 fetuses were diagnosed as carriers.The results were consistent with Dystrophin gene detection diagnosed by villus/amniotic fluid and there were no false and positives results.6.The follow-up results suggest that 3 pregnant women with NIPT-inferred DMD/BMD fetuses was terminated after diagnosis.The remaining 14 pregnant women continued to be pregnant till full-term delivery.ConclusionThe NIPT based on Proband-assisted haplotype phasing is convenient and can avoid interventional procedures.It is potential to apply to prenatal detection for DMD/BMD of high detection accuracy.