Results

Initial Array CGH Screening
Using BAC clone and oligonucleotide array CGH, we identified overlapping microdeletions in 16q24.1q24.2, ranging in size from ∼100 kb to ∼3.5 Mb, in seven patients referred for high-resolution genome analysis (Figures 1A and 1C, Table 1). The first of these patients (D1) was ascertained through our study of the genetic basis of esophageal atresia, for which, to date, 80 syndromic cases have been studied. The remainder were ascertained by queries of databases of patients referred for high-resolution genome analysis for a variety of reasons. Parental samples were available in six out of seven patients (not D2). One deletion (D7) was inherited from a phenotypically abnormal parent; the remaining five were de novo, supporting their likely pathogenic effect. No deletion CNVs in this genomic region were found, either in the Database of Genomic Variants or in the BCM and Signature Genomics databases of over 30,000 patients studied by array CGH. Five of the seven deletions (D2–D5 and D7) were independently verified by FISH analysis (Figure 1D). High-resolution oligonucleotide array CGH (385K or 2.1M NimbleGen) confirmed the deletions and enabled breakpoint characterization in all seven cases (Figures 1E and 1F).
The deletions centered around the FOX transcription factor gene cluster at 16q24.1. All but one harbored FOXF1, a gene with a role in lung and foregut development ascertained on the basis of previous studies of mice.18–20 Patient D7 had an ∼131 kb deletion encompassing FOXC2 and FOXL1 but not FOXF1.

Clinical Characterization
Of the six patients with chromosomal deletions harboring FOXF1 (D1–D6), five (D1 and D3–D6) died from pulmonary insufficiency in the first two months of life, and the mother of a sixth (D2) underwent elective termination of pregnancy at 22 weeks. Three patients (D1, D3, and D4) had ACD/MPV documented by histopathological examination (Figure 2A). Two other patients (D5 and D6) had severe respiratory distress, raising the possibility of ACD/MPV, but no postmortem examination was performed. Cardiac malformations were present in five of the six patients; two patients (D2 and D4) had a hypoplastic left heart, and a third (D5) had a small left ventricle. One patient (D1) manifested esophageal atresia with tracheo-esophageal fistula, whereas duodenal and anal atresias were present in another patient (D3). Five patients (D2–D6) had abnormalities consistent with urinary tract obstructive lesions, ranging from uretero-pelvocaliectasis to severe hydronephrosis. The phenotype in patient D7 was clearly distinct from that in the other six patients. This patient did not have respiratory insufficiency in the neonatal period and was still alive at the age of 3 years, having presented with atrial septal defect, bilaterally dilated and tortuous ureters with left hydronephrosis, diastasis recti, and developmental delay (Figure 2A and Table 1). The deletion was inherited from the patient's mother, whose sole recognized phenotypic abnormality was the presence of an umbilical hernia.

Mutation Analysis of the FOXF1 Gene
Two lines of evidence suggested that FOXF1 haploinsufficiency might be responsible for ACD/MPV in our initial cohort. First, patients with deletions harboring FOXF1 (D1–D6) had a severe respiratory phenotype (confirmed histologically as ACD/MPV in three cases), whereas a patient with a deletion harboring FOXC2 and FOXL1 but not FOXF1, patient D7, did not. Second, mice haploinsufficient for Foxf1 have abnormal alveolar development, notwithstanding the fact that the histological changes are not identical to those of ACD/MPV.18,19 We therefore sequenced FOXF1 in a cohort of 18 patients with ACD/MPV and other malformations.10 We identified four de novo heterozygous mutations in the coding sequence of FOXF1 in four unrelated patients with sporadic ACD/MPV. Patient M1 had a nonsense mutation (c.150C→A; p.Y50X) in exon 1; patient M2 had a frameshift mutation (c.775dupT; p.Y259Lfs11X) in exon 1; patient M3 had a frameshift mutation (c.956_957delTT; p.F319CfsX66) in exon 2 adding 29 amino acids to the protein, as predicted by conceptual translation; and patient M4 had a T→C substitution in the first base of the stop codon, a no-stop mutation (c.1063T→C; p.X355RextX74) adding 73 amino acids to the protein, as predicted by conceptual translation (Figures 3A–3D). All four patients had associated malformations, including a partial atrioventricular canal defect (1/4 cases), patent ductus arteriosus (1/4), bowel malrotation (3/4), a congenital short bowel (1/4), an annular pancreas (1/4), and urinary tract malformations (3/4) (Table 2).

Custom-Designed Array CGH
We hypothesized that some of the cases of ACD/MPV in the cohort might be due to whole-gene deletions, as was seen for patients D1–D6. We therefore designed a 16q24 region-specific high-resolution 44K oligonucleotide microarray and used it to screen the 14 mutation-negative patients from our ACD cohort. This analysis revealed an ∼1.8 Mb microdeletion harboring FOXF1 in patient D8. In addition, two microdeletions, one ∼524 kb (patient D9) and one ∼145 kb (patient D10) in size, located ∼52 kb and ∼259 kb upstream of FOXF1, respectively, were detected (Figures 1B and 1G, Table 1). All three rearrangements arose de novo.

Histopathology
Of 14 patients, ten had lung tissue available for histologic review. All ten showed the constellation of changes required for the histologic diagnosis of ACD/MPV (Figure 2). Many also showed other superimposed changes; consequences of respiratory failure and therapeutic support, terminal events, postmortem changes, and focal inflammation (rarely). However, these did not obscure the diagnostic hallmarks of ACD/MPV. Pulmonary lymphangiectasis was evident in all analyzed deletion cases (D1, D3, D4, and D8–D10), as well as in one (M2) of four examined patients with mutations. In the patients with lung biopsy but without autopsy (D9 and D10), information related to other malformations was based on clinical and/or imaging observations without pathologic confirmation; for those with neither lung biopsy nor autopsy (D2, D5, and D6), lung histology remains unknown and information related to other malformations is based on clinical and/or imaging observations only. No pulmonary tissue is available from patient D7, and that patient remains alive at three years of age, without respiratory symptomatology.

DNA Sequence Analysis
DNA sequence analysis enabled breakpoint sequencing in patients D1–D9. This confirmed the deletions and revealed that in patients D1, D3–D6, and D8, both breakpoints occurred in the portions of Alu elements showing eight to 43 base pairs (bp) of perfect microhomology. In patients D2 and D7, microhomologies of 4 and 3 bp, respectively, were found, and in patient D9, a 3 bp AGC insertion was identified at the junction fragments (Table 3).

Parental Origin of Deletions and Mutation
We investigated, when possible, the parent of origin, and we showed by microsatellite polymorphism and SNP studies that the deletions in patients D1, D3, D4, D8, D9, and D10 are maternal in origin. In patient M4, DNA sequencing of the cloned PCR product revealed that the no-stop mutation c.1063T→C was associated with the C allele inherited from the patient's father, whereas the G allele was associated with the wild allele, demonstrating that the mutation occurred on the paternal chromosome (Table S1).