The prenatal diagnosis of FSD is important at the second trimester of gestation, however, it is still challenging due to diverse clinical and genetic heterogeneity of the disorder. For decades, ultrasound is widely used in the noninvasive detection of FSD. Pajkrt and Chitty (2019) found that FL and HL of the fetuses with skeletal dysplasia were 5% shorter than the normal value. Previous reports in China demonstrated that the diagnosis accuracy of continuous sequential follow-up ultrasound was over 80% and 87.2% in the second trimester of gestation (Wang et al., 2017). In this study, by ultrasound scanning, we found the HL and FL of the fetuses in the 24 cases was either lower or higher than the normal value except of case 10 (the woman with OI (osteogenesis imperfecta) before pregnancy). The BPD and HC of the fetus were normal in all the 24 cases. In one-year follow-up survey, we found the 8 infants (account for 33.3%) were normal in skeletal development after delivery when the HL and FL of these fetuses was 2SD-3SD lower than the mean value. Differently, when the HL and FL of the studied fetuses was 3SD less or 3SD more, only two newborns (8.33%) were normal. The results of this ultrasound-only dependent approach are inadequate for diagnosing the disorder and impossible to differentiate the complex types of FSD.
In recent years, NGS (WES and WGS, Whole exome sequencing and Whole gene sequencing) has been applied in the area of disease diagnosis (Han et al., 2020). The mutation detection tool needs to optimize since the detection rates of WES could be variable depending on many factors, such as the sample size, the analysis criteria, proband-only or trio WES, and so on (Chandler et al., 2018). More importantly, the complex of genetic variants was found to be associated with the diverse pathogenicity of FSD.
In case 4, The fetus carried the c.7842T>A (p. Ala2614Ala) variant in the FBN1 gene and the deletion of c.2135-3_2135-2delCA in OBSL1 gene. The mutation is synonymous mutation and the deletion is in the intron. The FBN1 gene variation (Newell et al., 2017) is reported to be associated with Weill Marchesani syndrome (clinical manifestations are short limb deformity, secondary glaucoma, short stature, etc.). The OBSL1 gene variation (Isik et al., 2021) is related to 3-M syndrome (clinical manifestations include severe intrauterine growth retardation, short stature, recessive spina bifida, compression deformation of long metaphysis). The inheritance patterns of Weill Marchesani syndrome and 3-M syndrome are both AR (autosomal recessive inheritance). The Sanger analysis indicated that the mutation was carried by the father and the deletion was carried by the mother (Fig. 1). The mother of case 4 had natural delivery by vaginal at 39+6weeks of gestation, and the newborn did not show any abnormality of bone development (up to 18-monthes old) at the end of the following-up survey.
In case 6, we detected the variant of c.700G>T (p.E234*) and the deletion of CDS4-5 in IMPAD1 gene for the fetus. The heterozygous deletion of CDS4-5 and the homozygous variant in gene IMPAD1 were inherited from their parents, as a compound heterozygote mutation (Fig. 2). IMPAD1-related chondrodysplasia is an autosomal recessive disease (Rosario et al., 2011). The pregnant women had cesarean section at 38 weeks of gestation in this case, and the newborn had typical short limb deformity and died within one month after delivery.
In the study, there were two women (case 9 and 10) diagnosed with osteogenesis imperfecta before pregnancy. We detected the variant of c.1118G>C (p. Gly373Ala) in COL1A2 gene for the fetus in case 9, and found the variant of c.178C>T (p. Arg60*) in GORAB gene for the fetus in case 10(Fig. 3, 4). The two variants related to osteogenesis imperfecta and inherited from their mother, but one genetic type is autosomal dominant (AD) (Augusciak-Duma et al., 2018) and the other is autosomal recessive (AR) (Yang et al., 2017). The fetus of case 9 died before delivery at 35 weeks of gestation, while the newborn of case 10 had no significant abnormality in bone development up to 2-years old in the following-up survey.
In the case 2, 5 and 8, we found the WNT1or COL1A1-related VUS in osteogenesis imperfecta, and DYNC2H1 and NEK1-related asphyxiative hypoplasia of thorax. Notably, the fetuses in these cases all had short lower limbs, which was determined after abortion, while the proteins encoded by the gene variants were predicted to be deleterious using the SIFT and Polyphen analysis. Our assumption includes: (1) the limited data depth which failed to achieve 100% coverage of the exon sequences, resulting in the related pathogenic gene variants was dismissed; (2) the unknown gene variation outside the exons, including non-coding region and intron mutation, possibly responsible for the pathogenicity; (3) other environmental factors that have not been found. Furthermore, we had negative findings by WES, and the 8 infants were normal in skeletal development after delivery in the rest 8 cases.