Novel FOXF1 Deep Intronic Deletion Causes Lethal Lung Developmental Disorder, Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins

Abstract

Haploinsufficiency of FOXF1 causes an autosomal dominant neonatally lethal lung disorder, Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV). We identified novel 0.8-kb deletion within the 1.4-kb intron of FOXF1 in a deceased newborn diagnosed with ACDMPV. The deletion arose de novo on the maternal copy of the chromosome 16, and did not affect FOXF1 minigene splicing tested in lung fibroblasts. However, FOXF1 transcript level in the ACDMPV peripheral lung tissue was reduced by almost 40%. We found that, in an in vitro reporter assay, the FOXF1 intron exhibited moderate transcriptional enhancer activity, correlating with the presence of binding sites for expression regulators CTCF and CEBPB, whereas its truncated copy, that lost major CTCF and CEBPB binding sites, inhibited the FOXF1 promoter. Our data further emphasize the importance of testing the non-protein coding regions of the genome currently not covered by diagnostic chromosomal microarray analyses or whole exome sequencing.

Two deletions overlapping a distant FOXF1 enhancer unravel the role of lncRNA LINC01081 in etiology of alveolar capillary dysplasia with misalignment of pulmonary veins

“…DNA was extracted from peripheral blood, and RNA was extracted from FFPE ACDMPV lung tissue, frozen normal lung tissue, and normal human fetal lung fibroblasts MRC-5 and IMR-90 (ATCC) as described [Szafranski et al 2013a,b]. PCR products were directly sequenced by the Sanger method.…”

“…Heterozygous point mutations or genomic deletions of FOXF1 (MIM 601089) have been reported in most patients with ACDMPV [Stankiewicz et al, 2009; Sen et al, 2013a,b; Szafranski et al, 2013a]. Recently, we have defined a ~ 75 kb differentially methylated and evolutionarily conserved cis -regulatory region mapping to a protein-coding gene desert ~ 257 kb upstream to FOXF1 and functioning as its tissue-specific enhancer [Szafranski et al, 2013b].…”

“…Recently, we have defined a ~ 75 kb differentially methylated and evolutionarily conserved cis -regulatory region mapping to a protein-coding gene desert ~ 257 kb upstream to FOXF1 and functioning as its tissue-specific enhancer [Szafranski et al, 2013b]. This region harbors, among others, genes for fetal lung-enriched long non-protein coding RNAs (lncRNAs), transcription factor GLI2-binding sites, and the differentially-methylated CpG island, likely contributing to the paternal genomic imprinting of the FOXF1 in the human lungs [Sen et al, 2013a,b; Szafranski et al, 2013a]. …”

ROBO1 deletion as a novel germline alteration in breast and colorectal cancer patients

“…However, some examples showing intronic alterations associated with human conditions have been described [28][29][30][31]. In most cases, pathogenic intronic deletions lead to abnormal RNA splicing via the creation/removal of splice sites minimizing intron size [28][29][30][31]. In cancer, deep intronic mutations had been related to hereditary retinoblastoma and melanoma [32,33].…”

“…According to Kearney et al [26], small CNVs that affect only intronic regions have no apparent effect on gene function. However, some examples showing intronic alterations associated with human conditions have been described [28][29][30][31]. In most cases, pathogenic intronic deletions lead to abnormal RNA splicing via the creation/removal of splice sites minimizing intron size [28][29][30][31].…”