Congenital diaphragmatic hernia: focus on abnormal muscle formation.
BACKGROUND
CDH is a major birth defect, characterized by high mortality. How the initial defective mesenchymal substructures affects muscle malformation is unclear. Defects of genes involved in diaphragmatic development, such as friend-of-GATA2 (Fog2), may play an important role in its pathogenesis. We investigated the expression of Fog2 and proteins of myogenesis in a series of CDH and in diaphragms at different fetal ages, in order to clarify the role of muscular components during diaphragmatic development in cases with CDH.


METHODS
Specimen were obtained from seven diaphragms of CDH cases undergoing surgery, 3 entire diaphragms from non repaired CDH, 5 control diaphragms at different gestational ages (16, 17, 22, 32, and 40g.w.), and 3 biopsy samples of normal voluntary muscle. The thickness of diaphragms at the edge of the defect in CDH and in developing diaphragms was measured. All samples were processed for HE staining and immunohistochemistry. Immunohistochemical expression of MyoD, Myf4, Pax7, Mib1 and Fog2 was evaluated.


RESULTS
Mean thickness at the edge of the defect was 4.14mm. Contralateral hemi-diaphragm in 3 autopsies and in controls at 32 and 40weeks measured 2.25mm; histology showed a higher density of desmin-positive muscular cells at the edge of defect. CDH displayed scattered Myf4-positive cells (range 0%-10%, mean 2.4%), numerous Pax7-positive cells (range 0%-24%, mean 12.1%) and less than 1% Mib1-positive cells. Controls showed a reduction of positive cell with the progression of gestational age for Myf4 (30% at 16 weeks, 20% at 17 weeks, 5% at 22 weeks, 1% at 32 and 40 weeks), Pax7 (85% at 16 weeks and 17 weeks, 35% at 22 weeks, 11% at 32 weeks) and Mib1 (20% at 16 weeks, 8% at 17 weeks, 7% at 22weeks, 2% at 32 weeks). Fog-2 was diffusely positive in mesenchymal, mesothelial and muscular cells, in diaphragms from 16 to 22 weeks, decreasing to 20% of positive muscular cells in 32-week diaphragm. In CDH only mesothelial and mesenchymal cells were positive. Stem cell markers were negative in cases and controls.


CONCLUSIONS
CDH shows a thick muscular border, with high number of mature muscle cells and significant increase of quiescent satellite cells (PAX7+, Mib1-). Abnormal architecture may affect the normal process of myogenesis and thus signaling and cell-cell interactions of myocytes. The expression of Fog2 in mesothelial and mesenchymal cells in CDH demonstrates the absence of a genetic defect involving Fog2 in our cases. Being Fog2 expressed in muscle cells at early stage supports the hypothesis that the altered diaphragmatic genesis may undermine also the muscular component instead of the only mesenchymal one.
Medicine Department DIMED, Pathology Unit, University of Padua.
R
R
Alaggio
Pediatric Surgery, University of Padua.
P
P
Midrio
Pediatric Surgery, University of Padua. Electronic address: albertosgro@gmail.com.
A
A
Sgrò
Pediatric Surgery, University of Padua.
G
G
Piovan
Medicine Department DIMED, Pathology Unit, University of Padua.
V
V
Guzzardo
Section of Anatomy, Dept. Exp. Med. & Biochem. Sci., University of Perugia.
R
R
Donato
Section of Anatomy, Dept. Exp. Med. & Biochem. Sci., University of Perugia.
G
G
Sorci
Neonatal Intensive Care Unit, University of Padua.
P
P
Lago
Pediatric Surgery, University of Padua.
P G
PG
Gamba
D003964:Diaphragm / Q000002:abnormalities*; D005260:Female; D065630:Hernias, Diaphragmatic, Congenital / Q000175:diagnosis*; D006801:Humans; D007150:Immunohistochemistry; D007231:Infant, Newborn; D008297:Male; D024510:Muscle Development; D011446:Prospective Studies
CDH; Development; Diaphragm; Muscle