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A 16q deletion involving FOXF1 enhancer is associated to pulmonary capillary hemangiomatosis
© Dello Russo et al. 2015
Received: 4 July 2015
Accepted: 4 October 2015
Published: 13 October 2015
Pulmonary capillary hemangiomatosis (PCH) is an uncommon pulmonary disorder, with variable clinical features depending on which lung structure is affected, and it is usually linked to pulmonary arterial hypertension. Congenital PCH has been very rarely described and, so far, the only causative gene identified is EIF2AK4, which encodes for a translation initiation factor. However, not all PCH cases might carry a mutation in this gene.
We report the clinical and cytogenetic characterization of a patient (male, newborn, first child of healthy non-consanguineous parents) died after three days of life with severe neonatal pulmonary hypertension, due to diffuse capillary hemangiomatosis diagnosed post mortem.
Conventional karyotyping, Microarray-Based Comparative Genomic Hydridization (CGHa) and quantitative PCR were performed. CGHa revealed a heterozygous chromosome 16q23.3q24.1 interstitial deletion, spanning about 2.6 Mb and involving a FOXF1 gene enhancer. Quantitative PCR showed that the proband’s deletion was de novo. Microsatellite analysis demonstrate that the deletion occurred in the maternal chromosome 16.
FOXF1 loss of function mutation have been so far identified in alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV), a lung disease different from PCH. Our data suggest the hypothesis that disruption of the FOXF1 gene enhancer could be a genetic determinant of PCH. Moreover, our findings support the idea that FOXF1 is a paternally imprinted gene.
Pulmonary capillary hemangiomatosis (PCH) is a rare disorder that was first reported in 1978, with less than a few hundred nonrelated cases reported, so far . The PCH frequency within the general population is actually unknown [2, 3]. PCH anatomopathological features are pulmonary hypertension and excessive neovascularization characterized by capillary-sized blood vessels within the pulmonary interstitial tissue, vasculature, and airways [4, 5]. Clinically, PCH cases are quite variable; also because, this disease could mimic different lung diseases, including pulmonary veno-occlusive disease, idiopathic pulmonary arterial hypertension and atypical interstitial lung disease . Nowadays, the only genetic cause identified is a mutation in EIF2AK4 gene (Eukaryotic translation initiation factor 2 alpha kinase 4, in chromosome 15q15.1), which encodes for a translation factor . However, not all PCH cases might carry a mutation in this gene .
We report the clinical and genetic characterization of a newborn male presenting early severe pulmonary hypertension, due to a post-mortem diagnosis of PCH.
He carried a 2.6 Mb sized 16q23.3q24.1 deletion, as demonstrated by CGHa. He displayed no additional anomalies or malformations, and no familial occurrence.
The patient was the first child of nonrelated healthy parents with a negligible familial history. The patient was a full term (41 weeks) male infant, born by induced vaginal delivery to a 38-year old mother, whose pregnancy was uneventful. Birth weight, length and head circumference were 3340 gr, 50.2 cm and 34.0 cm, respectively. Apgar scores were 4-10-10. After birth, since the patient did not show spontaneous breathing and was hypotonic, he was stimulated and ventilated by ambu (FiO2: 0.4). Because he presented an increased O2-requirement after one hour of life, he was subjected to oxygenation by FiO2 40 %. Chest radiography showed widespread hypodiafania of the right hemithorax. Blood tests were negative for infection. Ultrasound revealed a normal brain anatomy, with mild hyperechoic periventricular left rear. Ultrasonography was compatible with wet lung disease. 21 h after birth, the infant developed a deteriorating respiratory distress and echocardiography documented a severe pulmonary hypertension in the absence of congenital structural abnormalities. He died at the third day of life from respiratory distress, pulmonary hypertension, heart failure and an extreme bradycardia. The autopsy demonstrated a lung parenchyma characterized by marked edema and stasis.
Materials and methods
Histology and immunohistochemistry
Tissue from autoptic lung samples were fixed in 4 % formalin for about 24 h. Fixed tissue was then paraffin embedded and 4 μm slides were stained with hematoxilin and eosin. Immunohistochemical stains for CD31 (1:200, clone JC70A, Dako, Denmark), CD34 (1:50, clone QBEND10, Dako, Denmark) and histochemical Masson’s trichrome stain were performed for vascular pattern observation.
Cytogenetics and molecular genetics
Blood samples of proband and parents were obtained after a signed informed consent. Parents informed consent was also given to analyse their own genomes. We explained that analysis of the parents would be useful to better understand the proband results. When CGHa is performed, obtaining blood samples from parents is part of our standard care.
Conventional high resolution karyotyping (GTG banding) was performed on proband blood lymphocytes following conventional procedures.
For CGHa analysis genomic DNA was isolated from an EDTA peripheral blood sample using QIAamp Blood Midi Kit according to the manufacturer procedure (Qiagen, Hilden, Germany). Molecular karyotyping was performed in accordance with the manufacturer procedure with an 180,000-oligonucleotide microarray (Sure Print G3 Human CGH Microarray Kit 180 k, Agilent Technologies, Santa Clara, CA, USA). The genomic sample was labelled and hybridized according with the Agilent Enzymatic Labelling protocol. We used a male DNA Coriell GM10851 (Coriell Institute, Camden, NJ, USA) as a normal reference. CGH Agilent Genomic Workbench Lite Edition 184.108.40.206 software and UCSC hg19 assembly were used to analyze the results. The presence of a copy number variation was defined by the presence of an abnormal log2 ratio for at least three contiguous oligonucleotides. The presence of the deletion in our patient was confirmed by quantitative PCR using 7300 Real Time PCR System (Applied BioSystems, Foster City, CA, USA) (data not shown). Primers inside OSGIN1, EMC8, ZDHHC7 genes were employed.
L17941, Forward: 5′- 6FAM-CTGGGTACTCTTCTGTGACA-3′;
L29692, Forward: 5′-6FAM-TGTGTGTCTTCTGGGGGAGT-3′;
The amplified products were analyzed by capillary electrophoresis (3500 IDX sequencer, Life Technologies).
Standard karyotyping showed no abnormalities (46,XY).
By quantitative PCR, our patient parents were evaluated: both subjects presented no abnormalities, thus suggesting the de novo origin of the deletion.
The PCH histopathologic lungs features show a crowded and congested alveolar capillary bed without pulmonary venous misalignment and lymphatic alteration. The alveolar capillary expansion is usually associated with intraalveolar hemorrhage [4, 5]. Based upon our microscopical findings, our patient suffers of PCH with structural abnormalities in preacinar and intra-acinar pulmonary arteries, consistent with the morphologic characteristics of persistent pulmonary hypertension of the newborn. The radial-alveolar counts were reduced, reflective of decreased alveolarization.
PCH is considered an underestimated pathology, because it may mimic idiopathic pulmonary arterial hypertension, pulmonary veno-occlusive disease, atypical interstitial lung disease, misaligment of lung vessel and alveolar capillary dysplasia or congenital pulmonary lymphangectasia [2, 11–13]. Based on familial occurrence, it is possible that congenital forms of this disease may have a genetic determination. Best et al.  have shown the involvement of EIF2AK4 gene mutations in PCH pathogenesis. However, it is likely that EIF2AK4 mutations do not account for all cases of PCH .
In our case, CGHa revealed a chromosome 16q23.3-q24.1 deletion that disrupts the distant FOXF1 transcriptional enhancer, which maps about 257 kb upstream to the FOXF1 gene. In this enhancer are located two lncRNAs: LINC01081 and LINC01082 . It has been demonstrated that in vitro abolition of LINC01081 by siRNA, reduces FOXF1 expression . Thus, our data could suggest that the disruption of the FOXF1 gene transcriptional enhancer induces cell proliferation and migration. This is consistent with other findings indicating FOXF1 as an oncosuppressor gene [15, 16]. The analysis of fusion progeny between mesenchymal stem cells and lung cancer cells has recently demonstrated that FOXF1 significantly reduced the growth rate and expression levels of proteins regulating the cell cycle . Accordingly, previous investigations have proposed PCH to be a lung endothelial neoplasia . Based upon the relevance of LINC01081 on FOXF1 expression regulation, our data could suggest that the disruption of this long non-coding RNA can lead to architectural changes in pulmonary vessels, resulting in neonatal-onset PCH.
This study has been performed in accordance with the Helsinki declaration. Written informed consent was obtained from the parents of the patient for publication of this case report. A copy of the written consent is available for review by the Editor of this journal.
This work has been funded by the Interreg SIGN (Slovenian Italian Genetic Network) project. We thank Catia Mio for the critical review of the manuscript.
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