Birth defects bring serious psychological and financial burden to families and have become a crucial social problem. It is estimated that genetic factors contribute solely or collaboratively to about 80% of the occurrence of birth defects. Therefore, genetic studies can provide precise molecular targets for clinical screening, diagnosis and treatment[20]. In this study, we detected two novel missense mutations harbored in TREX1 gene (c.294dupA and c.296_299dupGTTT) in a AGS fetus, which were inherited from normal father and mother separately. During the PGT-M treatment, one out of three embryos were confirmed as euploid as well as homozygous state of TREX1 gene through CNV testing which was verified through Sanger sequencing and SNP haplotyping analysis. After transferring the selected embryo, a health fetus was born.
PGT has developed by leaps and bounds since 1989s[21]. It prevents not only invasive prenatal test which may constitute a finite risk to the fetus but also the risk of bearing an affected fetus and termination of pregnancy that may impose psychological and physical burdens on the pregnant woman. However, due to the DNA amount of biopsy cell is very limited, direct genetic tests through Sanger sequencing based on PCR has the risk of allele dropout(ADO), Which is one of the most important causes of the misdiagnoses. Here, besides direct detection by Sanger sequencing, we also used NGS-based SNP haplotyping to determine ADO. And SNP haplotyping results verified the Sanger sequencing.
Chromosomal mosaicism is at high prevalence in early developmental stage of embryos, which is much higher than reported in the prenatal and postnatal cytogenetic literature[22]. Mitotic errors following fertilization have been characterized as the mechanism of chromosomal mosaicism[23]. Nowadays, Chromosomal mosaicism is a potential factor of misdiagnosis and has been a raising problem in PGT[24]. Firstly, a single trophectoderm biopsy of a few cells couldn’t represent the entire embryo. Besides, amplification bias in WGA, sample DNA biological variability, sequencing methods and bioinformatic approaches may all influence detection limits, leads to false positives. Thirdly, embryo mosaicism may self-correct aneuploidy downstream[25, 26]. Recent studies provided evidence that mosaic embryos can develop into healthy euploid newborns[27, 28, 29]. Although compared with euploid embryo transfer, Several studies have confirmed mosaic embryo transfer is associated with a lower rate of live birth, increasing biochemical miscarriage[30, 31, 32]. Considering the possible risks of transferring mosaic embryos, we eventually selected an euploid carrier embryo E2 to transfer. In this study, two of three embryos were mosaicism, rate of mosaic seems more frequent than reported. A possible explanation might be that using laser pulse in biopsy improperly increased cell damage which influenced the sequencing results, leading to an overdiagnosis of normal embryos as mosaics. Furthermore, embryo E1 is a complex mosaic blastocyst involved with several chromosomes. Recently, a retrospective study showed that 61.6% of the complex mosaic were diagnosed as euploidy in re-biopsy[33]. Therefore, those complex mosaicism like E1 could be selected for re-biopsy to avoids waste of potential available embryos.
We reported here our experience of applying PGT for a couple carrying different mutations in TREX1 which led to the pregnancy termination of an affected fetus with AGS. According the age of onset, two clinical presentations of AGS could be delineated: early-onset neonatal form and later-onset presentation. The former highly reminiscent of congenital infection seen particularly with TREX1 mutations[34]. In our study, the proband 30 weeks’ ultrasound scan showed microcephaly and multiple intracranial calcifications which was initially misdiagnosed as acquired in utero viral infection. TREX1 playing an important role in processing or clearing anomalous DNA structures, failure of which results in the triggering of an abnormal innate immune response, therefore AGS is sometimes mistaken as the sequelae of congenital infection[35]. Genetics detection has obvious importance in distinguishing AGS from common perinatal infections.
In conclusion, the pedigrees suffering from AGS was validated harboring pathogenic TREX1 mutations through Trio-WES. To the best of our knowledge, this is the first report that we identified a novel compound mutation in the TREX1 gene. Our study combined haplotype analysis based on targeted capture sequencing and invasive prenatal detection to effectively block the transmission of genetic defects, resulting in the birth of a healthy baby. This approach was proven to be practicable and effective and could be expanded to other monogenic diseases.