PMC:2694971 / 17718-25885 JSONTXT

{"project":"2_test","denotations":[{"id":"19500772-12114478-2052321","span":{"begin":2391,"end":2393},"obj":"12114478"},{"id":"19500772-11499682-2052321","span":{"begin":2391,"end":2393},"obj":"11499682"},{"id":"19500772-12114478-2052322","span":{"begin":2486,"end":2488},"obj":"12114478"},{"id":"19500772-9409679-2052323","span":{"begin":2725,"end":2727},"obj":"9409679"},{"id":"19500772-15664398-2052323","span":{"begin":2725,"end":2727},"obj":"15664398"},{"id":"19500772-12648490-2052324","span":{"begin":2975,"end":2977},"obj":"12648490"},{"id":"19500772-16469829-2052324","span":{"begin":2975,"end":2977},"obj":"16469829"},{"id":"19500772-11238010-2052325","span":{"begin":4408,"end":4410},"obj":"11238010"},{"id":"19500772-11493558-2052325","span":{"begin":4408,"end":4410},"obj":"11493558"},{"id":"19500772-11437453-2052325","span":{"begin":4408,"end":4410},"obj":"11437453"},{"id":"19500772-11437453-2052326","span":{"begin":5032,"end":5034},"obj":"11437453"},{"id":"19500772-11313147-2052327","span":{"begin":5395,"end":5397},"obj":"11313147"},{"id":"19500772-18055845-2052328","span":{"begin":5475,"end":5477},"obj":"18055845"},{"id":"19500772-10756340-2052329","span":{"begin":5783,"end":5785},"obj":"10756340"},{"id":"19500772-17881493-2052330","span":{"begin":5994,"end":5996},"obj":"17881493"},{"id":"19500772-17881493-2052331","span":{"begin":6053,"end":6055},"obj":"17881493"},{"id":"19500772-15229177-2052331","span":{"begin":6053,"end":6055},"obj":"15229177"},{"id":"19500772-11493558-2052332","span":{"begin":6184,"end":6186},"obj":"11493558"},{"id":"19500772-11437453-2052332","span":{"begin":6184,"end":6186},"obj":"11437453"},{"id":"19500772-8626802-2052332","span":{"begin":6184,"end":6186},"obj":"8626802"},{"id":"19500772-11943666-2052332","span":{"begin":6184,"end":6186},"obj":"11943666"},{"id":"19500772-14607778-2052332","span":{"begin":6184,"end":6186},"obj":"14607778"},{"id":"19500772-19014668-2052333","span":{"begin":6643,"end":6645},"obj":"19014668"},{"id":"19500772-19014668-2052334","span":{"begin":6977,"end":6979},"obj":"19014668"},{"id":"19500772-16299066-2052335","span":{"begin":7203,"end":7205},"obj":"16299066"}],"text":"Discussion\nOur report of deletions and point mutations within the FOX transcription factor gene cluster at 16q24.1 highlights the differing phenotypic consequences of gene alterations in cis. The patients described in this report can be classified into three groups on the basis of genotype and phenotype. Patients in group 1 have deletions that encompass all three FOX genes in the 16q24.1 cluster—FOXF1, FOXC2, and FOXL1—as well as MTHFSD (genomic deletions). Group 2 patients have deletions spanning either none (patients D9 and D10) or some (patients D6 and D7; genic deletions) but not all of the genes in the FOX cluster. Group 3 patients comprise those with inactivating point mutations in FOXF1. All group 1 and group 3 patients with histologic evaluation had ACD/MPV, but there were consistent differences in the spectrum of associated malformations between the two groups. All six group 1 patients had cardiovascular malformations. These ranged in severity from patent ductus arteriosus (PDA) (D1, D3, D5, and D8) through interrupted aortic arch (D5) and tetralogy of Fallot (D3) to reduced left ventricular size (D5) and hypoplastic left heart (D2 and D4). Just one of the four patients in group 3 (M1) had a cardiac malformation, a partial AV canal defect, together with PDA. Group 1 patients had gastrointestinal atresias (esophageal in D1, duodenal and anal in D3), whereas group 3 patients had intestinal malrotation or congenital short bowel. As expected, patients in group 2 were less readily classifiable. Patients with deletions, either upstream of (D9 and D10) or encompassing (D6) FOXF1, had ACD/MPV; one patient (D7) with a downstream deletion encompassing just FOXC2 and FOXL1 did not. Likewise, malformations occurring in this group were of group 1 type, such as hypoplastic left heart and imperforate anus, and group 3 type, such as intestinal malrotation. Additional malformations not seen in either group 1 or group 3 occurred in this group: possible abdominal heterotaxy in patient D6, Chiari malformation and meningeal cysts in patient D7.\nWe propose that ACD/MPV results from haploinsufficiency of FOXF1 and that the differences in the associated malformations between groups 1 and 3 relate to haploinsufficiency for FOXC2 and FOXL1. Point mutations in FOXC2, probably leading to its haploinsufficiency, are responsible for lymphoedema-distichiasis syndrome;21,22 a small proportion of these patients also have congenital heart defects (6.8% in one series,21 including tetralogy of Fallot and ventricular septal defect but not hypoplastic left heart). Mice with targeted inactivation of the orthologous Foxc2 have interrupted aortic arch, as well as defects of the skull and vertebral column.23,24 Haploinsufficiency of FOXL1 in humans has not previously been described. Targeted disruption of Foxl1 in mice is associated with aberrant epithelial cell positioning in the small intestine, hyperplasia of the gastric mucosa and retarded growth.25–27 MTHFSD is a methylene tetrahydrofolate synthase domain-containing gene of unknown function.\nThe lack of intestinal atresia in group 3 suggests that either FOXC2 or FOXL1 may also contribute to this phenotype in humans, despite the fact that, in mice, haploinsufficiency for Foxf1 alone is associated with esophageal atresia. It will be fascinating to determine whether deletions or inactivating mutations contribute to malformations reported here when they occur in isolation, in particular for hypoplastic left heart syndrome and intestinal malrotation, for which no gene has yet been causally implicated. The presence of overlapping deletions upstream of the FOX cluster in two patients (D9 and D10) with ACD/MPV strongly suggests that critical regulatory sequences are harbored within the minimal interval, and future work will focus on the identification of any evolutionarily conserved elements within this interval and the elucidation of their functional role.\nAlthough the abnormal lung phenotype of Foxf1+/− mice has some overlap with that of ACD/MPV in humans with alveolarization abnormalities, paucity of alveolar capillaries and pulmonary lymphangiectasia being common to both, there is no evidence of MPV, the defining histologic feature of ACD/MPV, in the mutant mice. Two different mouse models showed that Foxf1−/− embryos die by 8.5 days postcoitum (dpc) from defects in extraembryonic mesoderm development.18,19,28 Interestingly, in one model, half of the newborn Foxf1+/− mice had reduced (20% of wild-type) levels of pulmonary Foxf1, died from pulmonary hemorrhage, and showed severe defects in alveolarization and vasculogenesis (lung immaturity, hypoplasia) with fusion of the right lung lobes and lobar arteries, as well as an abnormal gall bladder, narrowing of the esophagus and trachea, esophageal atresia with tracheo-esophageal fistula, and defects in liver and lung repair. The other half of the Foxf1+/− mice had normal Foxf1 levels and normal lung morphology, interpreted as a “compensation” for the alveolarization defect.28 We suggest that the occurrence of these two distinct populations of mice is consistent with imprinting of Foxf1, in which the expression level of Foxf1 in Foxf1+/− mice is dependent upon the parent of origin of the normal Foxf1 allele. Supporting this notion, the expression from the Foxf1 promoter has been shown to be regulated by tissue-specific methylation29 and FOXF1 was computationally predicted to be paternally imprinted in humans.30 However, the facts that all six de novo deletions that we studied for the parental origin arose on a maternal chromosome and one mutation of FOXF1 (in patient M4) occurred on a paternal chromosome, as well as the observation of a relatively normal phenotype associated with paternal uniparental disomy 16,31 argue against FOXF1 imprinting in humans; nevertheless, a complex tissue-specific genomic imprinting cannot be excluded.\nFoxf1 has been shown to be downregulated in Shh−/− mice, probably through Gli proteins,32 and activated by bone morphogenetic protein 4 (BMP4).32,33 In Foxf1−/+ mice, a diminished expression of Vegfa, Vegfr2, Bmp-4, Tbx, Lklf, Fgf-10, Gli3, and Notch-2 has been found.19,28,34–36 Some of these genes, critical for lung development, may be mutated in patients with ACD/MPV without detectable abnormalities in FOXF1.\nDNA sequence analysis of the deletion breakpoints in nine patients (D1–D9) revealed that in six patients (D1, D3–D6, and D8), both breakpoints map in the portions of Alu elements showing perfect microhomology (eight to 43 base pairs) (Table 3), suggesting replication error MMBIR/FoSTeS as a mechanism of their formation.37 Alternatively, these deletions might have occurred via nonallelic homologous recombination (NAHR). In patients D2, D7, and D9, the presence of short microhomologies (D2 and D7) and AGC insertion (D9) at the junction fragments indicate that these deletions probably arose via nonhomologous end joining or the MMBIR/FoSTeS mechanism.37\nOur data may contribute to a better understanding of the pathogenesis of malformations seen in patients with the VACTERL (vertebral, anal, cardiac, tracheo-esophageal, renal and limb malformations) association (MIM 192350).38 Cardiac malformations, esophageal and anal atresia, renal and vertebral/axial malformations, and a single umbilical artery appear to be common to both VACTERL association and 16q24.1 microdeletion. However, although lung malformations have been reported occasionally in cases of VACTERL association, ACD/MPV has not been described in this context. We suggest that microdeletions at 16q24.1 should be considered in cases of apparent VACTERL association for which there is a severe respiratory distress and/or neonatal lethality. We propose that patients with intestinal atresia, isolated diastasis recti, or umbilical hernia should be screened for mutations and deletions in FOXL1. Given that FOXF1 maps only ∼3.7 Mb from 16qter, we also suggest that a balanced rearrangement involving 16qter (e.g., submicroscopic subtelomeric reciprocal translocation) should be excluded in parents with an ACD/MPV-affected child from whom no material is available for testing."}