The role of FREM2 and FRAS1 in the development of congenital diaphragmatic hernia

Abstract

Congenital diaphragmatic hernia (CDH) has been reported twice in individuals with a clinical diagnosis of Fraser syndrome, a genetic disorder that can be caused by recessive mutations affecting FREM2 and FRAS1. In the extracellular matrix, FREM2 and FRAS1 form a self-stabilizing complex with FREM1, a protein whose deficiency causes sac CDH in humans and mice. By sequencing FREM2 and FRAS1 in a CDH cohort, and searching online databases, we identified five individuals who carried recessive or double heterozygous, putatively deleterious variants in these genes which may represent susceptibility alleles. Three of these alleles were significantly enriched in our CDH cohort compared with ethnically matched controls. We subsequently demonstrated that 8% of Frem2ne/ne and 1% of Fras1Q1263*/Q1263* mice develop the same type of anterior sac CDH seen in FREM1-deficient mice. We went on to show that development of sac hernias in FREM1-deficient mice is preceded by failure of anterior mesothelial fold progression resulting in the persistence of an amuscular, poorly vascularized anterior diaphragm that is abnormally adherent to the underlying liver. Herniation occurs in the perinatal period when the expanding liver protrudes through this amuscular region of the anterior diaphragm that is juxtaposed to areas of muscular diaphragm. Based on these data, we conclude that deficiency of FREM2, and possibly FRAS1, are associated with an increased risk of developing CDH and that loss of the FREM1/FREM2/FRAS1 complex, or its function, leads to anterior sac CDH development through its effects on mesothelial fold progression.

Figure 1.

Deficiency of FREM2 and FRAS1 cause anterior sac hernias in mice. (A) A retrosternal diaphragmatic hernia in a FREM2-deficient Frem2^ne/ne mouse as viewed from the thorax. The herniated viscera are covered by a membranous sac (yellow outline). (B) A retrosternal diaphragmatic hernia (yellow arrow) in a Frem2^ne/ne mouse as viewed from the abdomen. (C, D) H&E-stained coronal sections through the hernial sac of a Frem2^ne/ne mouse reveal herniated liver tissue (Lv) and the gallbladder (G, red arrow) surrounded by a thin membranous sac. There is a rapid transition from the muscularized diaphragm (*) to a thin amuscular sac (blue arrow). (E) A retrosternal diaphragmatic hernia in a FRAS1-deficient Fras1^{Q1263*/Q1263*} mouse as viewed from the thorax. The herniated viscera are covered by a membranous sac (yellow outline). (F) The same retrosternal diaphragmatic hernia (yellow arrow) shown in (E) but viewed from the abdomen after reduction of the gallbladder and a pedunculated liver mass (Lv). D, diaphragm; St, sternum.

Figure 2.

Morphogenetic abnormalities associated with anterior sac hernia development in FREM1-deficient mice. (A, B) By E16.5 wild-type embryos on a mixed B6Brd/129S6 background showed complete muscularization of the anterior diaphragm. In contrast, Frem1^eyes2/eyes2 embryos on an inbred B6Brd/129S6 background have a persistent region of amuscular diaphragm directly behind the sternum. Dashed yellow lines mark the boundary between amuscular and muscular regions of the diaphragm. (C, D) At P3 and P4, 45% (5/11) of Frem1^eyes2/eyes2 embryos on an inbred B6Brd/129S6 background had a persistent region of amuscular diaphragm directly behind the sternum (yellow arrow in panel C) and 27% (3/11) had frank herniation with a sac. The hernia contents have been reduced in the embryo pictured in (D) leaving a hernia sac (green arrow) and a circular defect in the diaphragm (yellow arrow) that is completely surrounded by muscular diaphragm. (E, F) Coronal sections through a wild-type E16.5 embryo on a mixed B6Brd/129S6 background, reveal complete muscularization of the anterior diaphragm (dashed red lines). In these embryos, the liver was attached to the anterior diaphragm only by the falciform ligament (red arrow). In contrast, the muscular components of the diaphragm (dashed red lines) fail to meet in the midline in a subset of E16.5 Frem1^eyes2/eyes2 embryos on an inbred B6Brd/129S6 background. In these embryos, the liver is also abnormally attached to the anterior diaphragm (red arrows). (G–J) Whole mount immunohistochemistry using a PECAM1 antibody, reveals a network of blood vessels surrounding the central tendon in wild-type E15.5 embryos on a mixed B6Brd/129S6 background (yellow dashed lines). In contrast, a poorly vascularized region of the anterior diaphragm (yellow dashed lines) was identified in 3/5 (60%) of Frem1^eyes2/eyes2 embryos on an inbred B6Brd/129S6 background at E15.5.