ACD/MPV and LDS have been confirmed to be related with the deleted 16q24.1-q24.2 fragment until now [5, 8]. In this case, CNV-seq detection showed a 2.12-Mb deleted region in 16q24.1q24.2 containing two definite pathogenic genes: FOXF1, FOXC2 and related regulatory genes including FOXL1 and FOXF1 adjacent non-coding developmental regulatory RNA (FENDRR). Combined with the abnormal results of multi-system malformations of the fetus such as congenital cardiac, lung, genitourinary and gastro-intestinal anomalies, the diagnoses of ACD/MPV and LDS of the fetus were further defined. In addition to our fetus, table 1 shows another 10 patients with a different fragment deletion in the 16q24.1q24.2 region and complete information, and the fragment sizes range from 0.9 Mb to 3.5 Mb, containing FOXF1, FOXL1 and FOXC2 genes, among which, two fetuses were from de novo mutation, four patients from maternal heredity, four patients from unknown origin, three females and seven males are enrolled from five literatures [3, 9–12]. As is shown, the deleted sizes of 16q24.1q24.2 fragment are not proportional to phenotype severity and both the cardiac and renal anomalies are the two major symptoms during the fetal period, while the phenotypes of our fetus are the most serious, showing the changes of cardio-pulmonary structure such as pulmonary artery dilatation, HLHS, complete AVSD, CAV, FOC, ASD, VSD; the upper pyloric obstruction manifestations; a hypodense mass in the left kidney. However, after birth the prime symptoms of neonates are featured as respiratory, gastro-intestinal and genitourinary manifestations. Moreover, the gestational ages of delivery ranged from 22 to 39 + 1 weeks, among which three couples opted to terminate the pregnancies at second trimester of pregnancy and all of them died of respiratory diseases and their lifespans ranged from 16 hours to 40 days. Therefore, early recognition of ACD/MPV and LDS is essential in clinical practice.
The CNV-seq result of our fetus indicated an approximately 2.12-Mb deletion in 16q24.1-q24.2 (85220000–87340000) (Fig. 2) including the FOX family of transcription factors (FOXF1, FOXL1 and FOXC2), FENDRR FOXF1 and their corresponding deletion of enhancer region. The FOX transcription factors play critical roles in the process of cellular proliferation, differentiation [13, 14]. FOXF1, which is expressed in diverse-type cells including capillary endothelial cells, fibroblasts, and peribronchial smooth muscle cells, involves in development of pulmonary alveoli, capillaries and embryonic development of organs associated with airways, gastrointestinal tract and urinary tract [15–16]. The haploinsufficiency of FOXF1 has been confirmed to lead to manifestations of lung, gastrointestinal and urinary tracts [5, 10, 16] because of FOXF1 point mutations or CNV deletions overlapping FOXF1 or the change of its upstream regulatory region located ~ 270 kb upstream to FOXF1 gene (chr16:86178434–86238313;hg38) [4]. Hence, our fetus with hemizygous FOXF1 gene has similar clinical and pathological changes in the organs of cardiovascular, lung, gastrointestinal and urinary tracts. In addition, the haploinsufficiency of FOXC2 gene or associated with FOXL1 might contributed to congenital heart disease in our fetus [10]. The mutation of FOXL1 gene are mainly related to gastrointestinal manifestations, as has been confirmed in mice with FOXL1 gene knocked out [17]. Furthermore, FENDRR gene expression has been verified to be regulated both in cis and in trans by FOXF1, indicating that FENDRR involves in FOXF1-linked diseases including ACDMPV [18]. In addition, the mutation of FOXL1 gene mainly accouts for gastrointestinal manifestation. Therefore, the phenotypes of our fetus resulted from the deleted 16q24.1-q24.2 fragment including FOXF1, FOXL1, FOXC2, FENDRR and the severity may derive from the integration of multiple genes mutations.
LDS is another autosomal dominant disorder characterized by lymphedema of the limbs and double rows of eyelashes and FOXC2 is the key gene of LDS [8, 19]. FOXC2 is essential for lymphatic valve maintenance by regulating lymphatic endothelial cells junctional integrity and cellular quiescence [20]. FOXC2 mutation has been identified in patients with LDS to impair transcriptional activity and cell proliferation [21] through the VEGF-C/VEGFR3 signaling pathway commonly correlated with primary lymphedema, lymphatic valve formation and other lymphatic malformations [22]. And FOXC1 and FOXC2 have been verified to negatively regulate increased Ras/ERK signaling during embryonic development and lead to lymphangiogenesis obstruction which suppress formation of hyperplastic lymphatic vessels [22]. The clinical diagnosis of LDS is established in clinical symptoms and the identification of a heterozygous FOXC2 variant by molecular genetic testing [23]. In this report, the characteristic phenotypes associated with LDS may be atypical due to the fetal stage, however, the pathogenic variant by molecular genetic testing confirms the diagnosis of LDS. Therefore, genetic detection should be recommended as a first-line diagnostic tool for the fetuses with suspected ACD/MPV and / or LDS early during the fetal period [24]. Although clinical manifestations of the individuals in the table 1 are very different, there are no one who presented ACD/MPV and LDS simultaneously. Our fetus has been confirmed with ACD/MPV and LDS through CNV-seq detection.
In conclusion, this case supports the value of antenatal CNV-seq detection in multiple congenital abnormalities of the fetus. And molecular genetic testing should now be recommend as a first line diagnostic tool for suspected ACD/MPV and / or LDS or other genetic syndromes for the fetuses with structural abnormalities in clinical practice, which may switch traditional histological examination of ACD/ MPV especially during the fetal period.