DNA was extracted from proband’s blood and parents’ buccal smear samples using Gentra Puregene Blood Kit (Qiagen, Germantown, MD). DNA from frozen lung autopsy samples from five previously reported ACDMPV cases (pts 60.4, 64.5, 179.3, 180.3, and 205.3) was extracted using DNeasy Blood and Tissue Kit (Qiagen). RNA was isolated from pts 99.3 [9] and 217.3 FFPE lung tissues using Quick-RNA FFPE Kit (Zymo Res., Irvine, CA).

Array CGH analysis was done using customized chr16q24.1 region-specific 4 × 180 K oligonucleotide microarray (Agilent, Santa Clara, CA) according to manufacturers’ protocol.

Targeted next-generation sequencing (NGS) was done by Invitae (San Francisco, CA) using Illumina technology with ≥ 50 × depth. CNVs were called using an in-house algorithm that determines CNV at each target.

CNV deletion junction was amplified by long-range PCR using LA Taq DNApol (Takara Bio., Madison, WI) and primer pair 5′-GACCCTGATCTTGCATGTTCCTCGT-3′/5′-GAAGAATCGCCATCCCAGGTCAACG-3′, directly Sanger sequenced, and aligned with the human genome sequence GRCh37/hg19 using BLAT tool in the UCSC Genome Browser (https://genome.ucsc.edu).

Parental origin of the CNV deletion was determined using informative SNPs amplified for sequencing from the patient’s region of hemizygosity on chr16q24.1 and from corresponding region of parents’ chr16 with a primer pair 5′-TAACCAGAACTCCTCCCTGCCTGAG-3′ / 5′-AAAGCACCTGTTGATGGACTCTGGT-3′.

To determine the epigenetic characteristics of the described portion of Unit 2 of the FOXF1 enhancer, we analyzed the methylation status of cytosine and adenine within the DMRs, hemizygous in ACDMPV patients with paternal (pts 179.3, 180.3 and 205.3) or maternal (pts 60.4 and 64.5) CNV deletions of the enhancer, leaving FOXF1 intact (Additional file 2: Fig. S2). Genomic DNA (500 ng) was treated overnight at 37 °C with 5 units of HhaI or MboI (NEB, Ipswich, MA) in 25 µl of the rCutSmart buffer (NEB). The activity of HhaI is blocked by cytosine methylation (5mC) in CpG context, whereas the activity of MboI is blocked by methylation at 6N of adenine (m6A) in ApT context or cytosine in CpGs if they partially overlap with the MboI site. Following the treatment with restrictases, DMR1 and DMR2, both of which contain a single HhaII and MboI recognition site, were PCR amplified from 50 ng of nuclease treated or untreated DNA in a volume of 25 µl, using primer pairs listed in Additional file 8: Table S1, and LA Taq DNApol, applying 25 amplification cycles. In addition, the randomly selected 0.2-kb region, not recognized by HhaII or MboI, was simultaneously amplified as an internal control. The PCR products were resolved on EtBr-agarose gels and semi-quantified from gel images using ImageJ software (https://imagej.nih.gov). For comparison of methylation frequencies at maternal and paternal allele of DMRs, the ratios of the intensities of the DMR DNA band to the internal control band were used.

Transcript levels of FOXF1 and its downstream target TMEM100 [10] were measured by RT-qPCR. RNA samples were converted to cDNA using SuperScript III First-Strand Synthesis System (Invitrogen, Waltham, MA). TaqMan gene expression assays were obtained from Applied Biosystems (Foster City, CA). RT-qPCRs were performed on BioRad CFX Connect Real-Time System using TaqMan Universal PCR Master Mix (Applied Biosystems). For relative quantification of the transcripts/cDNA, the comparative ∆∆Ct method was used. FOXF1 and TMEM100 transcript (cDNA) levels were normalized to that of GAPDH, and showed as a fold change of an average FOXF1 or TMEM100 transcript (cDNA) level from two normal lung autopsy samples.

Immunostaining for TMEM100 (1:50, Sigma HPA055936) on cases 99.3 and 217.3 was performed on the Ventana BenchMark Ultra stainer after CC1 antigen retrieval on FFPE 5-µm sections of the lung. After pretreatment on the stainer, slides for FOXF1 immunohistochemistry (1:100, AF4798, R&D Systems) were blocked for one hour at room temperature using 5% normal donkey serum (Jackson Immuno Research Laboratories) in PBS containing 0.3% Triton X-100 and then incubated with primary antibody diluted in blocking buffer overnight at 4 °C. They were visualized with DAB peroxidase substrate after incubation with donkey anti-goat HRP at a dilution of 1:200 in blocking buffer for 1 h at room temperature. Images were captured with a digital camera mounted on a Nikon Eclipse 80i microscope using NIS-Elements Advanced Research Software v4.60.