- Case Report
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Haploinsufficiencies of FOXF1, FOXC2 and FOXL1 genes originated from deleted 16q24.1q24.2 fragment related with alveolar capillary dysplasia with misalignment of pulmonary veins and lymphedema-distichiasis syndrome: relationship to phenotype
Molecular Cytogenetics volume 15, Article number: 48 (2022)
We describe a fetus with a 2.12-Mb terminal deleted fragment in 16q associated with alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) and lymphedema-distichiasis syndrome (LDS) and intend to provide a comprehensive prenatal management strategy for the fetuses with ACDMPV and LDS through reviewing other similar published studies.
The fetus presented a series of diverse structural malformations including congenital cardiovascular, genitourinary and gastro-intestinal anomalies in ultrasound at 23 + 5 weeks of gestation (GA). Amniocentesis was conducted for karyotype analysis and copy number variation sequencing (CNV-seq) after informed consent.
The fetal karyotype was 46,XX, however the result of CNV-seq showed an approximately 2.12-Mb deletion in 16q24.1q24.2 (85220000-87340000) × 1 indicating pathogenicity.
Genomic testing should be recommend as a first line diagnostic tool for suspected ACDMPV and/or LDS or other genetic syndromes for the fetuses with structural abnormalities in clinical practice.
ACDMPV (OMIM 265380) is a rare and deadly disorder characterized by severe respiratory distress and cyanosis with the incidence of 1/100,000 . In addition, about 50 to 75 percent of affected newborns have multiple-system abnormalities such as hypoplastic left heart syndrome (HLHS) and intestinal malrotation . In approximately 80–90% of ACDMPV cases, heterozygous single nucleotide variants (SNVs) or copy number variant (CNV) deletions involving forkhead box F1 (FOXF1, OMIM 601089) in chromosome 16q24.1 have been found [3, 4]. In this report, we describe a fetus featured by a series of diverse structural malformations. Meanwhile, CNV-seq revealed a deleted region in 16q24.1q24.2 related with ACDMPV  and LDS . Both ACDMPV and LDS (OMIM 153400) are rarely reported in adults simultaneously in practice because of nearly 100% mortality of the cases with ACDMPV in the newborn period . However, the severity of isolated LDS associated with pathogenetic forkhead box C2 (FOXC2, OMIM 602402) is variable and cannot be predicted, among which the majority have been found in late childhood or adolescence with classical lymphatic abnormalities  and the minority in fetuses with nuchal translucency thickness [9,10,11]. Furthermore, we compare the features of our fetus with the reported cases related with 16q24.1q24.2 microdeletion syndromes. We aim to provide a comprehensive prenatal management strategy for the fetuses with ACDMPV and LDS.
Materials and methods
A 28-year-old healthy multigravida woman resorted to prenatal diagnosis medical center of Xuzhou Central Hospital due to abnormal ultrasound results. She had no history of adverse pregnancy and drug usage, and the couple were non-consanguineous. The family has a healthy child. There were not family histories with any serious disorders. Prenatal ultrasound at 23 + 5 weeks of GA showed the following presentations of Fig. 1: (a) pulmonary artery (PA) dilatation; (b) complete atrioventricular septal defect (AVSD); (c) common atrioventricular valve (CAV), foramen ovale closure (FOC), atrial septal defect (ASD), ventricular septal defect (VSD) and right heart enlargement; (d) dilatation of the stomach, esophageal dilation (considering pyloric obstruction); (e) a hypodense mass in the upper pole of the left kidney on December 23, 2022. Amniotic fluid was collected for karyotype analysis and CNV-seq after informed consent. Although the fetal karyotype was 46,XX, the result of CNV-seq showed that there was an approximately 2.12-Mb pathogenetic deletion in 16q24.1q24.2 (85220000-87340000) × 1 (Fig. 2) which was confirmed to be de novo after CNV-seq results of the couple were verified. Finally after receiving sufficient genetic counseling, the couple provided informed consent and chose to terminate the pregnancy. This study was approved by Xuzhou Central Hospital Ethics Committee (No. XZXY-LK-20210812-019).
Chromosome analysis was performed on G-band metaphases from amniotic fluid sample according to the laboratory’s standard protocols.The following entire operation process of CNV-seq included extracting uncultured genomic DNA from the sample, constructing DNA libraries, massively sequencing in parallel and conducting the raw sequencing reads following the corresponding operating regulations . Finally, the results of data were assessed according to standards and guidelines of American College of Medical Genetics .
ACDMPV and LDS have been confirmed to be related with the deleted 16q24.1q24.2 fragment until now [5, 14]. In this case, CNV-seq detection showed a 2.12-Mb deleted region in 16q24.1q24.2 containing the following definite pathogenetic genes: FOXF1, FOXC2 and related regulatory genes including forkhead box L1 (FOXL1, OMIM 603252) 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 diagnosis of ACDMPV and LDS of the fetus was further defined. In addition to our fetus, Table 1 shows the other 10 cases with similar deleted fragment in the 16q24.1q24.2 region with complete information, and the sizes range from 0.9 to 3.5 Mb containing FOXF1, FOXL1 and FOXC2 genes, among which two fetuses were from de novo disease-causing variants of the above genes, four cases from maternal heredity, four cases from unknown origin, three females and seven males are enrolled from five literatures [3, 15,16,17,18]. And we present a figure visualizing the deleted regions of 11 cases harboring FOXF1, FOXC2, and FOXL1 according to different versions of the genome map from UCSC Genome Browser Home: (a) cases from C1 to C8 were plotted with HG18; (b) cases from 9 to 11 with HG19 (Fig. 3). As is shown, the deleted sizes of 16q24.1q24.2 fragment are not proportional to the severity of phenotypes, and both cardiac and renal anomalies are the two major manifestations during the fetal period, while the phenotypes of our fetus are the most serious, showing the changes of cardio-pulmonary structure such as PA dilatation, HLHS, complete AVSD, CAV, FOC, ASD, VSD; the upper pyloric obstruction manifestations; a hypodense mass in the left kidney. However, the prime symptoms of neonates after birth are featured by respiratory, gastro-intestinal and genitourinary manifestations. Moreover, the gestational ages of delivery range 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 h to 40 days. Therefore, early recognition of ACDMPV and LDS is essential in clinical practice.
The CNV-seq result of our fetus indicated a 2.12-Mb deleted fragment in 16q24.1q24.2 (Fig. 2) including the FOX family of transcription factors (FOXF1, FOXL1 and FOXC2), FENDRR, and FOXF1 corresponding enhancer region. The FOX transcription factors play critical roles in the process of cellular proliferation, differentiation [19, 20]. FOXF1 involves in development of pulmonary alveoli, capillaries and embryonic development of organs associated with airways, gastrointestinal tract and urinary tract in diverse-type cells including capillary endothelial cells, fibroblasts, and peribronchial smooth muscle cells [21, 22]. In epithelial cells of the peripheral lung mesenchyme, sonic hedgehog (SHH) signaling pathway mediated by FOXF1 is one of the key pathways regulating formation. Moreover, the interactions between FOXF1-SHH and semaphorins-neuropilin or vascular endothelial growth factor/vascular endothelial growth factor receptor 2 (VEGF/VEGFR2) signaling may result in structural abnormalities of multiple systems, especially the lung, cardiovascular, gastrointestinal and urinary systems . Hence, the haploinsufficiency of FOXF1 gene is related with manifestations of lung, gastrointestinal and urinary tracts such as HLHS, duodenal atresia and distal ureteral dilatation [5, 16, 22] because of point disease-causing variant of FOXF1 or CNV deletions overlapping FOXF1 or the change of its upstream regulatory region located ~ 270 kb upstream to FOXF1 gene (chr16:86178434-86238313, hg19) . In addition, the genetic effects of FOXF1 gene inactivation have been confirmed in FOXF1-deficient mice with severe alveolarization and angiogenesis defects, stenosis of esophageal and tracheal, lung repair defects, et al. [16, 23]. In our case, the fetus presenting similar multi-system clinical manifestations may be associated with the haploinsufficiency of FOXF1.
The deleted fragment in our fetus includes the other three genes—FOXC2, FOXL1 and FENDRR. FOXC2 is the key gene of LDS characterized by lymphedema of the limbs and double rows of eyelashes [14, 24], which is essential for lymphatic valve maintenance by regulating lymphatic endothelial cells junctional integrity and cellular quiescence . FOXC2 pathogenetic variant has been identified in cases with LDS to impair transcriptional activity and cell proliferation  through VEGF-C/VEGFR3 signaling pathway commonly correlated with primary lymphedema, lymphatic valve formation and other lymphatic malformations . The FOXC2-inactiviation mice exhibited lymphatic abnormalities, VSD, interrupted aortic arch, et al. [28, 29]. In this report, although the characteristic phenotypes associated with LDS may be atypical in the fetal stage, CNV-seq detection confirms the diagnosis of LDS. Therefore, genetic detection should be recommended as a first-line diagnostic tool for the fetuses with suspected ACDMPV and/or LDS early during the fetal period . In addition, the disease-causing variant of FOXL1 gene is mainly related with gastrointestinal manifestations, as has been confirmed in mice with FOXL1 gene knocked out . 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 . Therefore, we speculate that the present phenotypes of our fetus resulted from the deleted 16q24.1q24.2 fragment including FOXF1, FOXC2, FOXL1 and FENDRR, and the severity might derive from the integration of multiple genes disease-causing variants of the above four genes. Our fetus has been confirmed with ACDMPV 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 genetic testing should now be recommend as a first-line diagnostic tool for suspected ACDMPV and/or LDS or other genetic syndromes for the fetuses with structural abnormalities in clinical practice, which may switch traditional histological examination of ACDMPV especially during the fetal period.
Availability of data and materials
The data and materials in the current study were available from the corresponding author upon reasonable request.
Alveolar capillary dysplasia with misalignment of pulmonary veins
Atrial septal defect
Atrioventricular septal defect
Common atrioventricular valve
Copy number variation sequencing
- FENDRR :
FOXF1 Adjacent non-coding developmental regulatory RNA
Foramen ovale closure
- FOXC2 :
Forkhead box C2
- FOXF1 :
Forkhead box F1
- FOXL1 :
Forkhead box L1
Ventricular septal defect
Vascular endothelial growth factor/vascular endothelial growth factor receptor 2
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We would like to thank the couple and the staff for participating in this study.
The work was supported by the science and technology bureau project of Xuzhou (No. KC21189), Jiangsu Province Maternal and Child Health Research Project (No. F201942) and the general project of Development Fund of Xuzhou Medical University (No. XYFM2020014).
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All examinations were approved by the ethical standards of the responsible committee. The pregnant woman provided written informed consent for the study.
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Wang, X., Guo, L., Zhang, B. et al. Haploinsufficiencies of FOXF1, FOXC2 and FOXL1 genes originated from deleted 16q24.1q24.2 fragment related with alveolar capillary dysplasia with misalignment of pulmonary veins and lymphedema-distichiasis syndrome: relationship to phenotype. Mol Cytogenet 15, 48 (2022). https://doi.org/10.1186/s13039-022-00627-9
- Haploinsufficiencies of FOXF1
- FOXC2 and FOXL1 genes
- Multiple-system structural malformations
- Prenatal diagnosis