Novel FOXF1 mutations in sporadic and familial cases of alveolar capillary dysplasia with misaligned pulmonary veins imply a role for its DNA binding domain

Abstract

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare and lethal developmental disorder of the lung defined by a constellation of characteristic histopathological features. Nonpulmonary anomalies involving organs of gastrointestinal, cardiovascular, and genitourinary systems have been identified in approximately 80% of patients with ACD/MPV. We have collected DNA and pathological samples from more than 90 infants with ACD/MPV and their family members. Since the publication of our initial report of four point mutations and 10 deletions, we have identified an additional 38 novel nonsynonymous mutations of FOXF1 (nine nonsense, seven frameshift, one inframe deletion, 20 missense, and one no stop). This report represents an up to date list of all known FOXF1 mutations to the best of our knowledge. Majority of the cases are sporadic. We report four familial cases of which three show maternal inheritance, consistent with paternal imprinting of the gene. Twenty five mutations (60%) are located within the putative DNA-binding domain, indicating its plausible role in FOXF1 function. Five mutations map to the second exon. We identified two additional genic and eight genomic deletions upstream to FOXF1. These results corroborate and extend our previous observations and further establish involvement of FOXF1 in ACD/MPV and lung organogenesis.

Figure 1

Sections of formalin fixed paraffin embedded lung tissue obtained either at autopsy (Cases 1 and 2) or diagnostic lung biopsy (Case 7) are examined stained with hematoxylin and eosin for the constellation of changes seen in ACD/MPV. These changes are often confirmed with a variety of additional stains including Movat pentachrome, trichrome and elastic stains and immunostains for pulmonary epithelium (CK7), the microvasculature (CD31) and vascular smooth muscle (Smooth Muscle Actin) (not shown). These changes include increased medial smooth muscle in small pulmonary arteries (black arrows), malposition of pulmonary veins (white arrows) adjacent to small pulmonary arteries in their normal parabronchiolar location (B identified bronchioles), and lobular underdevelopment seen in all panels. Other changes include paucity of alveolar capillaries and abnormal capillary size and location, seen in panels “a” and “b” as centrally located thin-walled dilated and congested alveolar wall vessels. Images are from Case 1 (a), Case 2 (b), and Case 7 (c); all H&E stain.

Figure 2

Genomic and genic deletions identified in the 16q24.1 region in patients with ACD/MPV. Patients D1–D6 and D8–D10 have been reported in Stankiewicz et al 2009 and the remainder except 94.3 and 104.3 have been reported in Szafranski et al (2013). Note that many of the deletions are flanked by Alu repetitive segments. SDR represents the smallest deletion overlap defining the cis-regulatory region mapping 250 kb upstream of FOXF1 (Szafranski et al 2012). The numbers with decimals refer to patients with ACD/MPV in our cohort.

Figure 3

A: Schematic representation of FOXF1 at 16q24.1. The upper segment depicts the genomic arrangement of FOXF1 with two exons in the gene shown by open boxes. The non-coding part of the gene is shown in shaded boxes. The numbers underneath show the beginning and end of the coding sequences and the exon intron junctions. The lower segment depicts the protein with the relative location of the DNA binding “Forkhead box” domain (DBD) and, the Cell-type specific activation domain in exon 1 and the general activation domain in exon 2. The numbers underneath indicate the position of amino acids for the domains. B: Positional representation of all reported and novel mutations in FOXF1. The green solid lines and green shaded boxes represent the first exon while the red solid line and the red shaded box represent the second exon. The first exon contains the DNA binding domain and the cell-type specific activation domain and the second exon contains the general activation domain. The locations of the mutations are shown with arrows. Golden arrows indicate nonsense mutations, purple, blue and green arrows indicate missense mutations, frameshift and indel mutations and no-stop mutations, respectively. The details of the mutations are shown above respective arrows. The black arrow indicates the nonsynonymous SNP. C: Evolutionary conservation of the amino acid sequence of the DNA binding domain of FOXF1 among vertebrates and conservation of the mutated amino acids within other forkhead family proteins. The identical amino acids are shown in blue while conservative changes are shown in burgundy and non-conservative changes shown in green. Only missense mutations are shown. The resulting amino acids after mutations are shown between the two panels. Although two of the missense mutations in unrelated patients involve the same amino acids the resulting changed amino acids are different in each case. The resulting amino acids are shown one above the other.