Discussion
By studying the WES test results of 79 pregnant women from the third affiliated hospital of Zhengzhou University from August 2018 to April 2020, the overall detection rate of skeletal abnormalities was 31.6%. This is higher than the previously reported detection rate (15.4%(Lord, et al., 2019) and 24%(Petrovski, et al., 2019)). This may be due to our stricter requirements for the ultrasound test results of the enrolled cases. Interestingly, we find that for different types of skeletal abnormalities, the detection rate through WES varies greatly. Among them, WES has a higher detection rate for short limbs. Especially when FL<-4.0SD or HL<-4.0SD, the detection rate can rise to 41.6%. However, when the fetal has short limbs with other bone abnormal phenotypes, the detection rate will be higher. For example, when the limbs are short with bone curved, the WES detection rate can reach 100%; when the limbs are short with nasal bone dysplasia, WES detection rate can reach 80%. On the contrary, if the phenotype of short limbs is not detected by ultrasound testing and only other skeletal abnormal phenotypes exists, such as only fetal hand or foot deformities, the WES detection rate will be very low, only 11.1% . It may be that achondroplasia is the cause for the most of skeletal abnormalities (Pauli, 2019), which is also the most common form of inherited disproportionate short stature(Waller, et al., 2008). In our study, through ultrasound,46 of 79 fetal skeletal abnormalities had clinical manifestations of short limbs, accounting for 58.2%, consistent with previous reports(Georgoulis, et al., 2011). This shows that the diagnosis of achondroplasia is the key to the diagnosis of fetal skeletal abnormalities.
Through our research, we find that for the diagnosis of fetalFGFR3 -related achondroplasia, the WES detection rate is highly correlated with the results of ultrasound testing of the fetal limb shortness and severity. Through our research, we find that the diagnosis of fetal FGFR3 -related achondroplasia(Wynn, et al., 2007) accounts for the highest proportion of all skeletal abnormalities, reaching 28%. Almost all the ultrasound test results of fetal achondroplasia have been detected with FL<-4.0SD or HL<-4.0SD, suggesting that FL<-4.0SD or HL<-4.0SD is the most critical basis for the diagnosis of achondroplasia by fetal ultrasound testing. At the same time, the detection of macrocephaly through ultrasound is also an important evidence for the diagnosis of fetal achondroplasia. Among the 7 cases ofFGFR3 -related achondroplasia, 6 cases were caused by FGFR3gene c.1138G>A mutation. FGFR3 gene c.1138G>A mutation is the most common mutation inFGFR3 -related achondroplasia, which accounts for more than 99% of all FGFR3 -related achondroplasia together with FGFR3gene c.1138G>C mutation(Foldynova-Trantirkova, et al., 2012; Xue, et al., 2014). At this point, our research is consistent with these reports. In addition, we have also detected a case of FGFR3gene c.1620C>A mutation through WES, which is related to hypochondroplasia(Bellus, et al., 1995). FGFR3 gene c.1620C>A mutation has been reported extensively(Deutz-Terlouw, et al., 1998) and is considered to account for 50%-76% of FGFR3-related hypochondroplasia(Tarja, et al., 2012). Therefore, FGFR3 gene c.1138G>A mutation and c.1620C>A mutation may be the two most common causes of skeletal abnormalities in the fetal period and require special clinical attention.
Osteogenesis imperfecta caused by the COL1A1 gene is also a major cause of fetal skeletal dysplasia. The main feature of Osteogenesis imperfecta is multiple fractures usually caused by minor trauma(W, et al., 1985; Willing, et al., 1990). This may be manifested by the limbs being curved by ultrasound testing during the prenatal period. In our study, osteogenesis imperfecta due to COL1A1 gene mutation was detected in the presence of limbs slightly curved through ultrasound in the fetal period, which suggests that the presence of this phenotype during the fetal period may be highly correlated with COL1A1-related osteogenesis imperfecta. In addition, COL1A1 gene c.896G>A mutation and c.1301G>A mutation found in our research have not been reported in other studies before , which broadens the clinical understanding of this gene mutation. COL1A1gene c.3235G>A mutation, according to previous reports, shows that it has a highly variable phenotype in the family, and family members with this gene mutation can only show signs of disease without fractures(D., et al., 2012; Kaneko, et al., 2011). This may explain why in our study, the husband of the pregnant woman also has the heterozygous mutation of the gene but the phenotype was not abnormal.
In our study, 2 cases of cleidocranial dysplasia related to RUNX2gene mutation were detected, which were c.931_946del mutation and c.568C>T mutation. The main clinical features of cleidocranial dysplasia include persistently open skull sutures with bulging calvaria, hypoplasia or aplasia of the clavicles permitting abnormal facility in apposing the shoulders, wide pubic symphysis, short middle phalanx of the fifth fingers, dental anomalies, and often vertebral malformation(Pan, et al., 2017). It has been pointed out in previous reports that cleidocranial dysplasia can also be related to the phenotype of nasal bone loss(Pan, et al., 2017), which is consistent with our case study. In our study, some other genetic variations or chromosomal aneuploidies related to the nasal bone loss phenotype are also found, such as the ARSE gene c.331C>T variation and trisomy 21. ARSE gene mutation is related to X-linked recessive chondrodysplasia punctata, which is manifested as nasal dysplasia and distal phalanx dysplasia(Brunetti-Pierri, et al., 2003), which is consistent with the phenotype observed during fetal ultrasound testing. Trisomy 21 is the most frequent form of mental retardation caused by a microscopically demonstrable chromosomal aberration, is characterized by well-defined and distinctive phenotypic features and natural history(Zhu, et al., 2013). It has been reported that nasal bone dysplasia is a common detected phenotype of fetal trisomy 21(De Jong Pleij, et al., 2012), which is consistent with the phenotype of a trisomy 21 case found in our study. Therefore, in the diagnosis of fetal skeletal abnormalities, nasal bone dysplasia may be another typical indication of ultrasound abnormality in addition to short limbs.
In our study, some gene mutations related to skeletal abnormalities related to hand and foot abnormalities are also detected. For example,SCN4A gene c.4361G>A mutation(Gay, et al., 2010),NEB gene c.1569+5G>A and c.2278C>T compound heterozygous mutation(Lehtokari, et al., 2014), FGFR2gene c.755C>G mutation(Miraoui, et al., 2010). Most of the diseases related to these gene mutations mainly affect the function of the muscular system as the main cause(Coen A. C. Ottenheijm, 2009; Matthews, et al., 2011), and their detection rate in fetal skeletal abnormalities is low (only FGFR2 gene c.755C>G mutation), but their detection can play an important guiding role in clinical diagnosis and treatment.