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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

Abstract

Objective

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.

Methods

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.

Results

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.

Conclusion

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.

Background

ACDMPV (OMIM 265380) is a rare and deadly disorder characterized by severe respiratory distress and cyanosis with the incidence of 1/100,000 [1]. In addition, about 50 to 75 percent of affected newborns have multiple-system abnormalities such as hypoplastic left heart syndrome (HLHS) and intestinal malrotation [2]. 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 [5] and LDS [6]. 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 [7]. 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 [8] 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

Case presentation

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).

Fig. 1
figure 1

Fetal ultrasound at 23 + 5 weeks gestation showed a pulmonary artery (PA) dilatation with an internal diameter of about 4.5 mm; b complete atrioventricular septal defect manifestation during diastole; c common atrioventricular valve (red arrow), foramen ovale closure (yellow arrow), atrial septal defect with the width of 2.2 mm (green arrow), ventricular septal defect with the width of 2.6 mm (white arrow) and right heart enlargement manifestations during systole; d dilatation of the stomach measuring about 32 × 13 mm and esophageal dilation with the widest internal diameter of 9 mm (considering pyloric obstruction); e a 11 × 7.5 mm hypodense mass in the upper pole of the left kidney. Abbreviation: ESO, esophagus; LA, left atrium; LK left kidney; LV, left ventricle; PA, pulmonary artery; RA, right atrium; RV, right ventricle; STO, stomach

Fig. 2
figure 2

The CNV-seq result of fetus showed a 2.12-Mb deletion in 16q24.1q24.2 (85220000-87340000)

Methods

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 [12]. Finally, the results of data were assessed according to standards and guidelines of American College of Medical Genetics [13].

Discussion

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.

Table 1 Features of patients with 16q24.1q24.2 deletion harboring FOXF1, FOXL1 and FOXC2
Fig. 3
figure 3

Schematic representation of the genomic region harboring FOXF1, FOXC2, and FOXL1 showed the extent and primary gene content of the regions deleted in 11 cases, 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

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 [22]. 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) [4]. 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 [25]. FOXC2 pathogenetic variant has been identified in cases with LDS to impair transcriptional activity and cell proliferation [26] through VEGF-C/VEGFR3 signaling pathway commonly correlated with primary lymphedema, lymphatic valve formation and other lymphatic malformations [27]. 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 [30]. 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 [31]. 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 [32]. 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.

Abbreviations

ACDMPV:

Alveolar capillary dysplasia with misalignment of pulmonary veins

ASD:

Atrial septal defect

AVSD:

Atrioventricular septal defect

CAV:

Common atrioventricular valve

CNV-seq:

Copy number variation sequencing

FENDRR :

FOXF1 Adjacent non-coding developmental regulatory RNA

FOC:

Foramen ovale closure

FOX:

Forkhead-box

FOXC2 :

Forkhead box C2

FOXF1 :

Forkhead box F1

FOXL1 :

Forkhead box L1

GA:

Gestation

LDS:

Lymphedema-Distichiasis syndrome

PA:

Pulmonary artery

SHH:

Sonic hedgehog

VSD:

Ventricular septal defect

VEGF/VEGFR2:

Vascular endothelial growth factor/vascular endothelial growth factor receptor 2

References

  1. Al-Hathlol K, Phillips S, Seshia MK, et al. Alveolar capillary dysplasia. Report of a case of prolonged life without extracorporeal membrane oxygenation (ECMO) and review of the literature. Early Hum Dev. 2000;57:85–94.

    Article  CAS  PubMed  Google Scholar 

  2. Nogee LM. Interstitial lung disease in newborns. Semin Fetal Neonatal Med. 2017;22(4):227–33.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Kozłowska Z, Owsiańska Z, Wroblewska JP, et al. Genotype-phenotype correlation in two Polish neonates with alveolar capillary dysplasia. BMC Pediatr. 2020;20(1):320.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Szafranski P, Gambin T, Dharmadhikari AV, et al. Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins. Hum Genet. 2016;135(5):569–86.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Bourque DK, Fonseca IC, Staines A, et al. Alveolar capillary dysplasia with misalignment of the pulmonary veins and hypoplastic left heart sequence caused by an in frame deletion within FOXF1. Am J Med Genet A. 2019;179(7):1325–9.

    CAS  PubMed  Google Scholar 

  6. Tavian D, Missaglia S, Maltese PE, et al. FOXC2 disease-mutations identified in lymphedema-distichiasis patients cause both loss and gain of protein function. Oncotarget. 2016;7(34):54228–39.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Slot E, Edel G, Cutz E, et al. Alveolar capillary dysplasia with misalignment of the pulmonary veins: clinical, histological, and genetic aspects. Pulm Circ. 2018;8(3):1–8.

    Article  CAS  Google Scholar 

  8. Mansour S, Brice GW, Jeffery S, Mortimer P, et al. Lymphedema-Distichiasis syndrome. In: Adam MP, Everman DB, Mirzaa GM, et al., editors. GeneReviews. Seattle, WA: University of Washington; 2005.

    Google Scholar 

  9. Jones GE, Richmond AK, Navti O, et al. Renal anomalies and lymphedema distichiasis syndrome. A rare association? Am J Med Genet A. 2017;173(8):2251–6.

    Article  CAS  PubMed  Google Scholar 

  10. Liu Y, Ding J, Peng Y, et al. Genetic variant analysis of a pedigree affected with lymphedema-distichiasis syndrome. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2020;37(4):434–7.

    PubMed  Google Scholar 

  11. Hu G, Liu B, Chen M, Qian Y, Dong M. Genetic analysis and clinical phenotype of a family with lymphedema-distichiasis syndrome. Zhejiang Da Xue Xue Bao Yi Xue Ban. 2020;49(5):581–5.

    PubMed  PubMed Central  Google Scholar 

  12. Wang J, Chen L, Zhou C, et al. Prospective chromosome analysis of 3429 amniocentesis samples in China using copy number variation sequencing. Am J Obstet Gynecol. 2018;219(287): e281.

    Google Scholar 

  13. Riggs ER, Andersen EF, Cherry AM, et al. Technical standards for the interpretation and reporting of constitutional copy-number variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics(ACMG) and the Clinical Genome Resource (ClinGen). Genet Med. 2020;22:245–57.

    Article  PubMed  Google Scholar 

  14. Michelson M, Lidzbarsky G, Nishri D, et al. Microdeletion of 16q24.1-q24.2-A unique etiology of Lymphedema-Distichiasis syndrome and neurodevelopmental disorder. Am J Med Genet A. 2022;188(7):1990–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Yu S, Shao L, Kilbride H, Zwick DL. Haploinsufficiencies of FOXF1 and FOXC2 genes associated with lethal alveolar capillary dysplasia and congenital heart disease. Am J Med Genet A. 2010;152A(5):1257–62.

    Article  CAS  PubMed  Google Scholar 

  16. Stankiewicz P, Sen P, Bhatt SS, et al. Genomic and genic deletions of the FOX gene cluster on 16q24.1 and inactivating mutations of FOXF1 cause alveolar capillary dysplasia and other malformations. Am J Hum Genet. 2009;84(6):780–91.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zufferey F, Martinet D, Osterheld MC, et al. 16q24.1 microdeletion in a premature newborn: usefulness of array-based comparative genomic hybridization in persistent pulmonary hypertension of the newborn. Pediatr Crit Care Med. 2011;12(6):e427–32.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Garabedian MJ, Wallerstein D, Medina N, Byrne J, Wallerstein RJ. Prenatal diagnosis of cystic hygroma related to a deletion of 16q24.1 with haploinsufficiency of FOXF1 and FOXC2 genes. Case Rep Genet. 2012;2012:490408.

    PubMed  PubMed Central  Google Scholar 

  19. Perl AK, Whitsett JA. Molecular mechanisms controlling lung morphogenesis. Clin Genet. 1999;56(1):14–27.

    Article  CAS  PubMed  Google Scholar 

  20. Costa RH, Kalinichenko VV, Lim L. Transcription factors in mouse lung development and function. Am J Physiol Lung Cell Mol Physiol. 2001;280(5):L823–38.

    Article  CAS  PubMed  Google Scholar 

  21. Mahlapuu M, Pelto-Huikko M, Aitola M, Enerbäck S, Carlsson P. FREAC-1 contains a cell-type-specific transcriptional activation domain and is expressed in epithelial-mesenchymal interfaces. Dev Biol. 1998;202(2):183–95.

    Article  CAS  PubMed  Google Scholar 

  22. Karolak JA, Gambin T, Szafranski P, et al. Perturbation of semaphorin and VEGF signaling in ACDMPV lungs due to FOXF1 deficiency. Respir Res. 2021;22(1):212.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kalinichenko VV, Lim L, Stolz DB, et al. Defects in pulmonary vasculature and perinatal lung hemorrhage in mice heterozygous null for the Forkhead Box f1 transcription factor. Dev Biol. 2001;235(2):489–506.

    Article  CAS  PubMed  Google Scholar 

  24. Brice G. Analysis of the phenotypic abnormalities in lymphoedema-distichiasis syndrome in 74 patients with FOXC2 mutations or linkage to 16q24. J Med Genet. 2002;39(7):478–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Sabine A, Bovay E, Demir CS, et al. FOXC2 and fluid shear stress stabilize postnatal lymphatic vasculature. J Clin Investig. 2015;125(10):3861–77.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Tavian D, Missaglia S, Michelini S, et al. FOXC2 disease mutations identified in lymphedema distichiasis patients impair transcriptional activity and cell proliferation. Int J Mol Sci. 2020;21(14):5112.

    Article  CAS  PubMed Central  Google Scholar 

  27. Norden PR, Sabine A, Wang Y, et al. Shear stimulation of FOXC1 and FOXC2 differentially regulates cytoskeletal activity during lymphatic valve maturation. Elife. 2020;9: e53814.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Kriederman BM, Myloyde TL, Witte MH, et al. FOXC2 haploinsufficient mice are a model for human autosomal dominant lymphedema-distichiasis syndrome. Hum Mol Genet. 2003;12(10):1179–85.

    Article  CAS  PubMed  Google Scholar 

  29. Kanzaki-Kato N, Tamakoshi T, Fu Y, et al. Roles of forkhead transcription factor Foxc2 (MFH-1) and endothelin receptor A in cardiovascular morphogenesis. Cardiovasc Res. 2005;65(3):711–8.

    Article  CAS  PubMed  Google Scholar 

  30. Callan EA, Geddes G, Konduri GG, et al. A case of alveolar capillary dysplasia with misalignment of the pulmonary veins (ACD/MPV): the importance of early genetic testing. J Neonatol. 2021;35(1):38–41.

    Article  Google Scholar 

  31. Fukuda K, Yoshida H, Sato T, et al. Mesenchymal expression of Foxl1, a winged helix transcriptional factor, regulates generation and maintenance of gut-associated lymphoid organs. Dev Biol. 2003;255(2):278–89.

    Article  CAS  PubMed  Google Scholar 

  32. Szafranski P, Gambin T, Karolak JA, Popek E, Stankiewicz P. Lung-specific distant enhancer cis regulates expression of FOXF1 and lncRNA FENDRR. Hum Mutat. 2021;42(6):694–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We would like to thank the couple and the staff for participating in this study.

Funding

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).

Author information

Authors and Affiliations

Authors

Contributions

JZ conceived the project. JS designed the molecular approach. BZ, JW, JZ and ML collaborated in the molecular analyses. HT and YS participated in the recruitment, clinical information acquisition of the patient and her families. XW and LG wrote the clinical description and discussion. XW, LG and JZ designed and wrote the first draft with molecular aspects. All authors included modifications and suggestions to the initial version. All authors read and approved the final version of the manuscript.

Corresponding authors

Correspondence to Jingfang Zhai or Min Liu.

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Ethics approval and consent to participate

All examinations were approved by the ethical standards of the responsible committee. The pregnant woman provided written informed consent for the study.

Consent for publication

Written informed consent for publication and the fetal clinical details were obtained from the couple.

Competing interests

No interests.

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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

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Keywords

  • ACDMPV
  • LDS
  • Haploinsufficiencies of FOXF1
  • FOXC2 and FOXL1 genes
  • Multiple-system structural malformations
  • Prenatal diagnosis