Two approaches for calculating female fetal DNA fraction in noninvasive prenatal testing based on size analysis of maternal DNA fragments

The concentration of cell-free fetal DNA fragments should be detected before noninvasive prenatal testing(NIPT).The fetal DNA molecules have significant clinical potential in determining the overall performance of NIPT and clinical interpretation.It is important to measure fetal DNA fraction before NIPT.However,there is still little research on how to calculate the concentration of female fetuses.Two estimation approaches were proposed to calculate fetal DNA fraction,including the fragments size-based approach,aneuploid-based approach,which are all approaches based on chromosome segments.Based on high-throughput sequencing data,two approaches to calculate the DNA fraction of male fetuses were tested and obtained the experiment values,which were close to the actual values.The correlation coefficient of fragments size-based approach was 0.9243(P<0.0001)and the aneuploid-based approach reached 0.9339(P<0.0001).We calculated the concentration of female fetuses and obtained remarkable experimental results.We came up with two approaches for calculating the fetal DNA fraction of female fetuses.It provides an important theoretical basis for the detection of female fetal concentration in future clinical diagnosis.

Noninvasive Prenatal Testing for Fetal XXY Aneuploidies Among Pregnancies in Beijing of China

Objective:To evaluate the screening performance of noninvasive prenatal testing(NIPT)based on high-throughput massively parallel sequencing technology for the fetal XXY aneuploidies among pregnancies in Beijing of China.Methods:The study enrolled 26913 consecutive pregnancies,20-50 years old,who attended the Peking Union Medical College Hospital,Beijing,China,for prenatal screening from January 1,2016 to December 31,2019.Cell-free DNA was extracted from maternal peripheral blood to have a high-throughput massively parallel sequencing procedure.Cases with high-risk of fetal XXY were suggested to take invasive prenatal diagnosis(IPD)for confirmation.Maternal DNA sequencing was performed,if necessary,to find other potential factors that may lead to high-risk results of XXY by NIPT.Results:Among a cohort of 26913 pregnant women,34 were high-risk for fetal XXY,among which 30 accepted IPD while 4 declined.In those who accepted IPD,19 cases were confirmed fetal XXY by chromosome karyotyping analysis while 11 were verified as false positive.Among the 19 confirmed fetal XXY cases,14 elected pregnancy termination.For all the 34 high-risk cases,two were verified maternal sex chromosome aneuploidy.The calculated detection rate,positive predictive value,and false-positive rate of NIPT for fetal XXY in this cohort was 100.00%(19/19),63.33%(19/30),and 0.04%(11/26890),respectively.And the percentage of pregnancy termination was 73.68%(14/19).Conclusion:NIPT could be used as a potential method for fetal XXY screening,although the accuracy needs to be improved.As NIPT is not diagnostic,IPD is strongly recommended for those with high-risk results.For cases with discordance between NIPT and fetal karyotyping,maternal DNA sequencing would help to identify the cause of false-positive/false-negative results.

Reproductive management through integration of PGD and MPS-based noninvasive prenatal screening/diagnosis for a family with GJB2-associated hearing impairment

A couple with a proband child of GJB2 （encoding the gap junction protein connexin 26）-associated hearing impairment and a previous pregnancy miscarriage sought for a reproductive solution to bear a healthy child. Our study aimed to develop a cus- tomized preconception-to-neonate care trajectory to fulfill this clinical demand by integrating preimplantation genetic diagno- sis （PGD）, noninvasive prenatal testing （NIPT）, and noninvasive prenatal diagnosis （N1PD） into the strategy. Auditory and ge- netic diagnosis of the proband child was carried out to identify the disease causative mutations. The couple then received in-vitro-fertilization treatment, and eight embryos were obtained for day 5 biopsy. PGD was performed by short-tandem-repeat linkage analysis and Sanger sequencing of GJB2 gene. Transfer of a GJB2c.235delC heterozygous embryo resulted in a sin- gleton pregnancy. At the 13th week of gestation, genomic DNA （gDNA） from the trio family and cell-free DNA （cfDNA） from maternal plasma were obtained for assessment of fetal chromosomal aneuploidy and GJB2 mutations. NIPT and NIPD showed the absence of chromosomal aneuploidy and GJB2-associated disease in the fetus, which was later confirmed by inva- sire procedures and postnatal genetic/auditory diagnosis. This strategy successfully prevented the transmission of hearing im- pairment in the newborn, thus providing a valuable experience in reproductive management of similar cases and potentially other monogenic disorders.

Prenatal Testing or Screening?

Over the past 50 years,the scope and extent of prenatal diagnosis and screening for genetic disorders have improved geometrically.There has been a pendulum like swing from testing to screening back and forth as new technologies emerge.The concurrent developments of cell free fetal DNA analysis of maternal blood has dramatically changed patient’s choices towards screening.However,with the use of array comparative genomic hybridization of fetal DNA that requires diagnostic procedures(Chorionic villus sampling and amniocentesis),much more extensive diagnosis can be obtained.Until noninvasive methods can replicate what can be done with diagnostic procedures there still will be a"price to be paid"for opting for the non-invasive methods.