Genomic and genic deletions of the FOX gene cluster on 16q24.1 and inactivating mutations of FOXF1 cause alveolar capillary dysplasia and other malformations

Abstract

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare, neonatally lethal developmental disorder of the lung with defining histologic abnormalities typically associated with multiple congenital anomalies (MCA). Using array CGH analysis, we have identified six overlapping microdeletions encompassing the FOX transcription factor gene cluster in chromosome 16q24.1q24.2 in patients with ACD/MPV and MCA. Subsequently, we have identified four different heterozygous mutations (frameshift, nonsense, and no-stop) in the candidate FOXF1 gene in unrelated patients with sporadic ACD/MPV and MCA. Custom-designed, high-resolution microarray analysis of additional ACD/MPV samples revealed one microdeletion harboring FOXF1 and two distinct microdeletions upstream of FOXF1, implicating a position effect. DNA sequence analysis revealed that in six of nine deletions, both breakpoints occurred in the portions of Alu elements showing eight to 43 base pairs of perfect microhomology, suggesting replication error Microhomology-Mediated Break-Induced Replication (MMBIR)/Fork Stalling and Template Switching (FoSTeS) as a mechanism of their formation. In contrast to the association of point mutations in FOXF1 with bowel malrotation, microdeletions of FOXF1 were associated with hypoplastic left heart syndrome and gastrointestinal atresias, probably due to haploinsufficiency for the neighboring FOXC2 and FOXL1 genes. These differences reveal the phenotypic consequences of gene alterations in cis.

Figure 1

Summary of the Results in Patients with Microdeletions in 16q24.1q24.2 (A) Schematic representation of the genomic region harboring the FOX transcription gene cluster, FOXF1, FOXC2, and FOXL1, showing the extent and gene content of the regions deleted in seven patients analyzed by array CGH (D1–D7). A non-ACD/MPV patient, D7, with a deletion of FOXC2 and FOXL1 and an intact FOXF1, is also shown. (B) Microdeletions identified via a custom designed 16q24-specific high-resolution 4×44K Agilent array in three patients with ACD/MPV (D8–D10). Note that the microdeletion in subject D8 contains FOXF1, whereas those in patients D9 and D10 are located upstream of FOXF1, indicating a position effect on this gene. Group 1 patients are indicated in red, group 2 in blue (see main text for explanation). In six out of nine deletions analyzed, breakpoints map within Alu repetitive elements, suggesting MMBIR/FoSTeS or NAHR mechanisms of formation. (C) Targeted array CGH plot obtained with V7.2 OLIGO (Agilent 105K) in patient D3, showing a deletion in 16q24.1 (red mark designated by arrow). (D) Metaphase chromosomes of patient D4 after FISH with a 16q24-specific BAC clone, RP11-542M13 (red), and a control chromosome 16 alfa satellite (Vysis) probe (green), showing absence of signal (arrow) on one chromosome 16, consistent with a heterozygous deletion. (E and F) NimbleGen whole-genome oligonucleotide array CGH profiles for subjects D3 (E) and D4 (F). (G) The ∼524 kb deletion in patient D9, located ∼52 kb upstream of FOXF1, was detected via a custom designed 16q24-specific high-resolution 4×44K Agilent array. Green dots shifted to the left between 84.43–84.95 represent the deleted segment.

Figure 2

Histopathology Studies Histopathology studies show that all deletion and mutation patients have characteristic changes of ACD/MPV, with medial hyperplasia of small pulmonary arteries, abnormally positioned pulmonary veins adjacent to membranous and terminal bronchioles and coursing with small pulmonary arteries (misalignment), lobular underdevelopment, and deficient numbers of normally positioned airspace wall capillaries with abnormal enlarged and centrally placed thin-walled vascular channels in airspace walls. The deletion cases often show marked airspace enlargement, thinner airspace walls, and pulmonary lymphangiectasis; these findings are uncommon in mutation cases. Hematoxylin and eosin, initial magnification 25x. (A) Patient D3 with FOXF1 deletion. Note two small pulmonary artery branches with moderately thickened smooth muscle (a), an adjacent malpositioned congested pulmonary vein (v), and a dilated lymphatic channel (l) all located adjacent to a dilated terminal bronchiole (b). In the inset, this same abnormal vascular configuration is seen adjacent to a membranous bronchiole and the lobular parenchyma is formed of markedly enlarged and simplified airspaces with thinner walls, compared to mutation cases. (B) Patient D9 with deletion upstream of FOXF1. Small pulmonary arteries (a) have strikingly thickened medial smooth muscle and only pin-point lumens; they share a common connective tissue sheath with pulmonary vein branches (v) that are neither dilated nor congested and both are adjacent to a membranous bronchiole (b). Normally positioned capillaries are not seen in airspace walls, although there are more centrally placed congested thin-walled vascular channels. A prominent lymphatic (l) is adjacent to the artery/vein combination. Airspaces appear prominently enlarged. (C) Patient M2 with frameshift mutation in exon 1. Multiple thick-walled small pulmonary arteries (a) and dilated congested malpositioned pulmonary veins (v) often share the same connective tissue sheath adjacent to a small bronchiole. The lobular parenchyma is formed of enlarged, simplified and poorly subdivided airspaces; however airspace enlargement is less dramatic than in the deletion cases. (D) Patient M3 with frameshift mutation in exon 2. Thick-walled small pulmonary arteries (a) and adjacent dilated and congested veins (v) are located next to a terminal bronchiole (b). The lobular parenchyma is underdeveloped with simplified and poorly subdivided airspaces separated by thickened septa in which there are dilated and congested central vascular channels, but only rare capillaries.

Figure 3

Genomic and Protein Structure of FOXF1 and DNA Sequencing Chromatograms Genomic and protein structure of FOXF1 and DNA sequencing chromatograms showing four mutations in the coding sequence of FOXF1 identified in patients with ACD/MPV. (A) A nonsense mutation c.150C > A; p.Y50X in exon 1 in patient M1. (B) A frameshift mutation c.775 dupT; p.Y259Lfs11X in exon 1 in subject M2. (C) A frameshift mutation c.956_957 delTT; p.F319CfsX66 in exon 2 in patient M3. (D) A no-stop mutation c.1063T > C; p.X355RextX74 in exon 2 in individual M4. The base numbering for the FOXF1 gene refers to A of the second ATG start codon as position 1 (nucleotide 119 of GenBank accession number NM_001451.2).