Discussion
Main Findings
In the present study, we found that abnormal CNVs are associated with several fetal structural malformations among stillborn fetuses. A pathogenic CNV deletion of 1q21.1 involving 46 genes (e.g.,CHD1L ) and a duplication of 21q22.13 involving 4 genes (SIM2 , CLDN14 , CHAF1B , HLCS ) were associated with a skeletal and cardiac defect, respectively.
Interpretation
In light of a previous large study that examined all fetuses,5 our findings of CNVs in gene-rich regions that are associated with fetal cardiac and skeletal anomalies in stillborn fetuses is noteworthy. Donnelly et. al. showed that chromosomal microarray analysis remained most informative in particularly identifying fetuses with cardiac and craniofacial anomalies, and most CNVs occurred in those with cardiac anomalies.5 In general, fetuses with anomalies in more than one system had higher frequency of abnormal CNVs compared with fetuses without anomalies (13.0%; p-value<0.001), suggesting that chromosomal microarray analysis identifies anomalies dependent on the type and number of organ systems involved.
Cardiac abnormalities are among the most common congenital malformations, associated with stillbirth13 and present in an estimated 0.8% of live births.8Skeletal defects involve a heterogeneous group of bone abnormalities resulting in abnormal growth and shape of the fetal skeleton.24 Skeletal defects in the first trimester are associated with nuchal translucent, and in lethal dysplasias, bone shortening may be obvious as early as 11 weeks’ gestation.24 Among fetuses with perinatal diagnosis of ventricular septal defects, most common cardiac defects, chromosomal microarray was particularly effective in identifying pathogenic CNVs.3 Thus, gene may potentially play a role in the etiology of fetal ventricular septal defects.
Deletion of 1q21.1 (4.0 Mb in size) and duplication of 21q22.13 (500 kb in size) CNVs were previously described as pathogenic abnormalities of stillbirth in the same study.4 However, these CNVs were not described in the context of fetal skeletal or cardiac defects. Expression analyses of CHD1L (chromodomain helicase DNA binding protein 1 like), among 46 genes implicated in 1q21.1 region in our study, showed that it promotes neuronal differentiation of human embryonic cells. This suggested that CHD1L plays an important role in the nervous system development.25 In addition, a pathogenic variant in CHD1L is also associated with short stature.26 In the adult population, distal variant CNVs of 1q21.1 were reported to be associated with cerebral and cognitive alterations.27 Furthermore, high prevalence of neurodevelopmental disorders are associated with CNVs at 1q21.28 Specifically, the CNV microdeletion and microduplication at 1q21 were enriched in patients with schizophrenia, intellectual disabilities and autism spectrum disorder.28-30 These studies highlight the pathophysiological mechanisms of CNVs in 1q21 in neurodevelopmental disorders.
Limitations and strengths
While our study has the potential to provide high-quality data of chromosomal abnormalities in fetuses suspected to have isolated cardiac and skeletal malformations, several limitations exist. First, microarray-based analyses may be limited to detect truly balanced rearrangements of chromosomes. The large chromosomal regions identified span several genes, and the Affy500K SNP Array is limited to detect genetic abnormalities at higher resolution, e.g., specific mutations in genes for clinical application. Up-to-date microarray platforms would be sensitive to detect CNVs at greater resolution (e.g., >150 kb), and with increased sequencing our knowledge of the pathogenicity of variants may change.31 Next-generation sequencing of cases will improve diagnostic yield of genes in fetal structural malformations.32 Further, due to lack of paternal DNA, we were not able to distinguish inherited from newly occurring CNVs in the placenta or fetus, which will be important future areas of investigation for identifying causative of genetic abnormalities. Finally, we have limited sample size to detect associations of CNVs with other fetal structural malformations. As resolution for determination of pathogenicity of CNVs is improved, CNVs that are thought to be benign may eventually be designated as pathogenic, increasing the sample size of stillborn fetuses identified as having abnormal CNVs in our study.
Several strengths of our study deserve mention. The population included in our study is geographically, ethnically and racially diverse, potentially ensuring the generalizability of our findings. Moreover, participants provided consent for a complete evaluation, including fetal standardized postmortem examination, placental pathological analysis, and maternal-fetal testing. These study design features provided careful phenotyping of stillbirth in our study, ensured accurate ascertainment of fetal structural malformations, and maximized the validity of the present analysis.
Conclusion
Among stillborn fetuses, specific CNVs involving several genes were associated with fetal structural malformations and warrant further investigation. These data improve genotype-phenotype databases, potentially informing more precise understanding of genetic etiologies of human developmental disorders.6 The data may also inform counseling and care surrounding pregnancies affected by fetal structural malformations at risk for stillbirth.