SOX7 deficiency causes ventricular septal defects through its effects on endocardial-to-mesenchymal transition and the expression of Wnt4 and Bmp2.

SOX7 is a transcription factor-encoding gene located in a region on chromosome 8p23.1 that is recurrently deleted in individuals with ventricular septal defects (VSDs). We have previously shown that Sox7-/- embryos die of heart failure around E11.5. Here, we demonstrate that these embryos have hypocellular endocardial cushions with severely reduced numbers of mesenchymal cells. Ablation of Sox7 in the endocardium also resulted in hypocellular endocardial cushions, and we observed VSDs in rare E15.5 Sox7flox/-;Tie2-Cre and Sox7flox/flox;Tie2-Cre embryos that survived to E15.5. In atrioventricular explant studies, we showed that SOX7 deficiency leads to a severe reduction in endocardial-to-mesenchymal transition (EndMT). RNA-seq studies performed on E9.5 Sox7-/- heart tubes revealed severely reduced Wnt4 transcript levels. Wnt4 is expressed in the endocardium and promotes EndMT by acting in a paracrine manner to increase the expression of Bmp2 in the myocardium. Both WNT4 and BMP2 have been previously implicated in the development of VSDs in individuals with 46,XX sex reversal with dysgenesis of kidney, adrenals and lungs (SERKAL) syndrome and in individuals with short stature, facial dysmorphism and skeletal anomalies with or without cardiac anomalies 1 (SSFSC1) syndrome, respectively. We now show that Sox7 and Wnt4 interact genetically in the development of VSDs through their additive effects on endocardial cushion development with Sox7+/-;Wnt4+/- double heterozygous embryos having hypocellular endocardial cushions and perimembranous and muscular VSDs not seen in their Sox7+/- and Wnt4+/- littermates. These results provide additional evidence that SOX7, WNT4 and BMP2 function in the same pathway during mammalian septal development and that their deficiency can contribute to the development of VSDs in humans.

RERE deficiency contributes to the development of orofacial clefts in humans and mice.

Deletions of chromosome 1p36 are the most common telomeric deletions in humans and are associated with an increased risk of orofacial clefting. Deletion/phenotype mapping, combined with data from human and mouse studies, suggests the existence of multiple 1p36 genes associated with orofacial clefting including SKI, PRDM16, PAX7 and GRHL3. The arginine-glutamic acid dipeptide (RE) repeats gene (RERE) is located in the proximal critical region for 1p36 deletion syndrome and encodes a nuclear receptor co-regulator. Pathogenic RERE variants have been shown to cause neurodevelopmental disorder with or without anomalies of the brain, eye or heart (NEDBEH). Cleft lip has previously been described in one individual with NEDBEH. Here we report the first individual with NEDBEH to have a cleft palate. We confirm that RERE is broadly expressed in the palate during mouse embryonic development, and we demonstrate that the majority of RERE-deficient mouse embryos on C57BL/6 background have cleft palate. We go on to show that ablation of Rere in cranial neural crest (CNC) cells, mediated by a Wnt1-Cre, leads to delayed elevation of the palatal shelves and cleft palate and that proliferation of mesenchymal cells in the palatal shelves is significantly reduced in Rereflox/flox; Wnt1-Cre embryos. We conclude that loss of RERE function contributes to the development of orofacial clefts in individuals with proximal 1p36 deletions and NEDBEH and that RERE expression in CNC cells and their derivatives is required for normal palatal development.

Stromal Fat4 acts non-autonomously with Dchs1/2 to restrict the nephron progenitor pool.

Regulation of the balance between progenitor self-renewal and differentiation is crucial to development. In the mammalian kidney, reciprocal signalling between three lineages (stromal, mesenchymal and ureteric) ensures correct nephron progenitor self-renewal and differentiation. Loss of either the atypical cadherin FAT4 or its ligand Dachsous 1 (DCHS1) results in expansion of the mesenchymal nephron progenitor pool, called the condensing mesenchyme (CM). This has been proposed to be due to misregulation of the Hippo kinase pathway transcriptional co-activator YAP. Here, we use tissue-specific deletions to prove that FAT4 acts non-autonomously in the renal stroma to control nephron progenitors. We show that loss of Yap from the CM in Fat4-null mice does not reduce the expanded CM, indicating that FAT4 regulates the CM independently of YAP. Analysis of Six2(-/-);Fat4(-/-) double mutants demonstrates that excess progenitors in Fat4 mutants are dependent on Six2, a crucial regulator of nephron progenitor self-renewal. Electron microscopy reveals that cell organisation is disrupted in Fat4 mutants. Gene expression analysis demonstrates that the expression of Notch and FGF pathway components are altered in Fat4 mutants. Finally, we show that Dchs1, and its paralogue Dchs2, function in a partially redundant fashion to regulate the number of nephron progenitors. Our data support a model in which FAT4 in the stroma binds to DCHS1/2 in the mouse CM to restrict progenitor self-renewal.

Deficiency of FRAS1-related extracellular matrix 1 (FREM1) causes congenital diaphragmatic hernia in humans and mice.

Congenital diaphragmatic hernia (CDH) is a common life-threatening birth defect. Recessive mutations in the FRAS1-related extracellular matrix 1 (FREM1) gene have been shown to cause bifid nose with or without anorectal and renal anomalies (BNAR) syndrome and Manitoba oculotrichoanal (MOTA) syndrome, but have not been previously implicated in the development of CDH. We have identified a female child with an isolated left-sided posterolateral CDH covered by a membranous sac who had no features suggestive of BNAR or MOTA syndromes. This child carries a maternally-inherited ~86 kb FREM1 deletion that affects the expression of FREM1's full-length transcripts and a paternally-inherited splice site mutation that causes activation of a cryptic splice site, leading to a shift in the reading frame and premature termination of all forms of the FREM1 protein. This suggests that recessive FREM1 mutations can cause isolated CDH in humans. Further evidence for the role of FREM1 in the development of CDH comes from an N-ethyl-N-nitrosourea -derived mouse strain, eyes2, which has a homozygous truncating mutation in Frem1. Frem1(eyes2) mice have eye defects, renal agenesis and develop retrosternal diaphragmatic hernias which are covered by a membranous sac. We confirmed that Frem1 is expressed in the anterior portion of the developing diaphragm and found that Frem1(eyes2) embryos had decreased levels of cell proliferation in their developing diaphragms when compared to wild-type embryos. We conclude that FREM1 plays a critical role in the development of the diaphragm and that FREM1 deficiency can cause CDH in both humans and mice.

Delineation of a less than 200 kb minimal deleted region for cardiac malformations on chromosome 7p22.

Cardiac malformations are commonly seen in individuals with terminal and interstitial deletions involving chromosome band 7p22. Although these malformations represent a significant cause of morbidity, the dosage-sensitive gene(s) that underlie these defects have yet to be identified. In this report, we describe a 16-month-old male with tetralogy of Fallot, bilateral second branchial arch remnants, and mild dysmorphic features. Array comparative genomic hybridization analysis revealed a less than 400 kb interstitial deletion on chromosome 7p22. The deletion was confirmed by real-time quantitative PCR and FISH analyses and was not detected in samples obtained from the child's parents. Molecular data from this de novo deletion, in combination with data from other isolated 7p deletions in the literature, can be used to define a less than 200 kb minimal deleted region for cardiac malformations on 7p22. This minimal deleted region spans all, or portions, of the coding regions of four known genes-MAD1L1, FTSJ2, NUDT1, and SNX8-and may include upstream regulatory elements of EIF3B. It is likely that one or more of these five genes, alone or in combination, plays an important, yet previously uncharacterized, role in cardiac development.

RERE deficiency leads to decreased expression of GATA4 and the development of ventricular septal defects.

Deletions of chromosome 1p36 are associated with a high incidence of congenital heart defects (CHDs). The arginine-glutamic acid dipeptide repeats gene (RERE) is located in a critical region for CHD on chromosome 1p36 and encodes a cardiac-expressed nuclear receptor co-regulator. Mutations affecting RERE cause atrial and ventricular septal defects (VSDs) in humans, and RERE-deficient mice also develop VSDs. During cardiac development, mesenchymal cells destined to form part of the atrioventricular (AV) septum are generated when endocardial cells in the AV canal undergo epithelial-to-mesenchymal transition (EMT) and migrate into the space between the endocardium and the myocardium. These newly generated mesenchymal cells then proliferate to fill the developing AV endocardial cushions. Here, we demonstrate that RERE-deficient mouse embryos have reduced numbers of mesenchymal cells in their AV endocardial cushions owing to decreased levels of EMT and mesenchymal cell proliferation. In the endocardium, RERE colocalizes with GATA4, a transcription factor required for normal levels of EMT and mesenchymal cell proliferation. Using a combination of in vivo and in vitro studies, we show that Rere and Gata4 interact genetically in the development of CHDs, RERE positively regulates transcription from the Gata4 promoter and GATA4 levels are reduced in the AV canals of RERE-deficient embryos. Tissue-specific ablation of Rere in the endocardium leads to hypocellularity of the AV endocardial cushions, defective EMT and VSDs, but does not result in decreased GATA4 expression. We conclude that RERE functions in the AV canal to positively regulate the expression of GATA4, and that deficiency of RERE leads to the development of VSDs through its effects on EMT and mesenchymal cell proliferation. However, the cell-autonomous role of RERE in promoting EMT in the endocardium must be mediated by its effects on the expression of proteins other than GATA4.This article has an associated First Person interview with the first author of the paper.

Copy number variations in 375 patients with oesophageal atresia and/or tracheoesophageal fistula.

Oesophageal atresia (OA) with or without tracheoesophageal fistula (TOF) are rare anatomical congenital malformations whose cause is unknown in over 90% of patients. A genetic background is suggested, and among the reported genetic defects are copy number variations (CNVs). We hypothesized that CNVs contribute to OA/TOF development. Quantifying their prevalence could aid in genetic diagnosis and clinical care strategies. Therefore, we profiled 375 patients in a combined Dutch, American and German cohort via genomic microarray and compared the CNV profiles with their unaffected parents and published control cohorts. We identified 167 rare CNVs containing genes (frequency<0.0005 in our in-house cohort). Eight rare CNVs - in six patients - were de novo, including one CNV previously associated with oesophageal disease. (hg19 chr7:g.(143820444_143839360)_(159119486_159138663)del) 1.55% of isolated OA/TOF patients and 1.62% of patients with additional congenital anomalies had de novo CNVs. Furthermore, three (15q13.3, 16p13.3 and 22q11.2) susceptibility loci were identified based on their overlap with known OA/TOF-associated CNV syndromes and overlap with loci in published CNV association case-control studies in developmental delay. Our study suggests that CNVs contribute to OA/TOF development. In addition to the identified likely deleterious de novo CNVs, we detected 167 rare CNVs. Although not directly disease-causing, these CNVs might be of interest, as they can act as a modifier in a multiple hit model, or as the second hit in a recessive condition.

1p36 deletion syndrome: an update.

Deletions of chromosome 1p36 affect approximately 1 in 5,000 newborns and are the most common terminal deletions in humans. Medical problems commonly caused by terminal deletions of 1p36 include developmental delay, intellectual disability, seizures, vision problems, hearing loss, short stature, distinctive facial features, brain anomalies, orofacial clefting, congenital heart defects, cardiomyopathy, and renal anomalies. Although 1p36 deletion syndrome is considered clinically recognizable, there is significant phenotypic variation among affected individuals. This variation is due, at least in part, to the genetic heterogeneity seen in 1p36 deletions which include terminal and interstitial deletions of varying lengths located throughout the 30 Mb of DNA that comprise chromosome 1p36. Array-based copy number variant analysis can easily identify genomic regions of 1p36 that are deleted in an affected individual. However, predicting the phenotype of an individual based solely on the location and extent of their 1p36 deletion remains a challenge since most of the genes that contribute to 1p36-related phenotypes have yet to be identified. In addition, haploinsufficiency of more than one gene may contribute to some phenotypes. In this article, we review recent successes in the effort to map and identify the genes and genomic regions that contribute to specific 1p36-related phenotypes. In particular, we highlight evidence implicating MMP23B, GABRD, SKI, PRDM16, KCNAB2, RERE, UBE4B, CASZ1, PDPN, SPEN, ECE1, HSPG2, and LUZP1 in various 1p36 deletion phenotypes.

Identification of critical regions and candidate genes for cardiovascular malformations and cardiomyopathy associated with deletions of chromosome 1p36.

Cardiovascular malformations and cardiomyopathy are among the most common phenotypes caused by deletions of chromosome 1p36 which affect approximately 1 in 5000 newborns. Although these cardiac-related abnormalities are a significant source of morbidity and mortality associated with 1p36 deletions, most of the individual genes that contribute to these conditions have yet to be identified. In this paper, we use a combination of clinical and molecular cytogenetic data to define five critical regions for cardiovascular malformations and two critical regions for cardiomyopathy on chromosome 1p36. Positional candidate genes which may contribute to the development of cardiovascular malformations associated with 1p36 deletions include DVL1, SKI, RERE, PDPN, SPEN, CLCNKA, ECE1, HSPG2, LUZP1, and WASF2. Similarly, haploinsufficiency of PRDM16-a gene which was recently shown to be sufficient to cause the left ventricular noncompaction-SKI, PRKCZ, RERE, UBE4B and MASP2 may contribute to the development of cardiomyopathy. When treating individuals with 1p36 deletions, or providing prognostic information to their families, physicians should take into account that 1p36 deletions which overlie these cardiac critical regions may portend to cardiovascular complications. Since several of these cardiac critical regions contain more than one positional candidate gene-and large terminal and interstitial 1p36 deletions often overlap more than one cardiac critical region-it is likely that haploinsufficiency of two or more genes contributes to the cardiac phenotypes associated with many 1p36 deletions.

Novel frem1-related mouse phenotypes and evidence of genetic interactions with gata4 and slit3.

The FRAS1-related extracellular matrix 1 (FREM1) gene encodes an extracellular matrix protein that plays a critical role in the development of multiple organ systems. In humans, recessive mutations in FREM1 cause eye defects, congenital diaphragmatic hernia, renal anomalies and anorectal malformations including anteriorly placed anus. A similar constellation of findings-microphthalmia, cryptophthalmos, congenital diaphragmatic hernia, renal agenesis and rectal prolapse-have been described in FREM1-deficient mice. In this paper, we identify a homozygous Frem1 missense mutation (c.1687A>T, p.Ile563Phe) in an N-ethyl-N-nitrosourea (ENU)-derived mouse strain, crf11, with microphthalmia, cryptophthalmos, renal agenesis and rectal prolapse. This mutation affects a highly conserved residue in FREM1's third CSPG domain. The p.Ile563Phe change is predicted to be deleterious and to cause decreased FREM1 protein stability. The crf11 allele also fails to complement the previously described eyes2 allele of Frem1 (p.Lys826*) providing further evidence that the crf11 phenotype is due to changes affecting Frem1 function. We then use mice bearing the crf11 and eyes2 alleles to identify lung lobulation defects and decreased anogenital distance in males as novel phenotypes associated with FREM1 deficiency in mice. Due to phenotypic overlaps between FREM1-deficient mice and mice that are deficient for the retinoic acid-responsive transcription factor GATA4 and the extracellular matrix protein SLIT3, we also perform experiments to look for in vivo genetic interactions between the genes that encode these proteins. These experiments reveal that Frem1 interacts genetically with Gata4 in the development of lung lobulation defects and with Slit3 in the development of renal agenesis. These results demonstrate that FREM1-deficient mice faithfully recapitulate many of the phenotypes seen in individuals with FREM1 deficiency and that variations in GATA4 and SLIT3 expression modulate some FREM1-related phenotypes in mice.

An allelic series of mice reveals a role for RERE in the development of multiple organs affected in chromosome 1p36 deletions.

Individuals with terminal and interstitial deletions of chromosome 1p36 have a spectrum of defects that includes eye anomalies, postnatal growth deficiency, structural brain anomalies, seizures, cognitive impairment, delayed motor development, behavior problems, hearing loss, cardiovascular malformations, cardiomyopathy, and renal anomalies. The proximal 1p36 genes that contribute to these defects have not been clearly delineated. The arginine-glutamic acid dipeptide (RE) repeats gene (RERE) is located in this region and encodes a nuclear receptor coregulator that plays a critical role in embryonic development as a positive regulator of retinoic acid signaling. Rere-null mice die of cardiac failure between E9.5 and E11.5. This limits their usefulness in studying the role of RERE in the latter stages of development and into adulthood. To overcome this limitation, we created an allelic series of RERE-deficient mice using an Rere-null allele, om, and a novel hypomorphic Rere allele, eyes3 (c.578T>C, p.Val193Ala), which we identified in an N-ethyl-N-nitrosourea (ENU)-based screen for autosomal recessive phenotypes. Analyses of these mice revealed microphthalmia, postnatal growth deficiency, brain hypoplasia, decreased numbers of neuronal nuclear antigen (NeuN)-positive hippocampal neurons, hearing loss, cardiovascular malformations-aortic arch anomalies, double outlet right ventricle, and transposition of the great arteries, and perimembranous ventricular septal defects-spontaneous development of cardiac fibrosis and renal agenesis. These findings suggest that RERE plays a critical role in the development and function of multiple organs including the eye, brain, inner ear, heart and kidney. It follows that haploinsufficiency of RERE may contribute-alone or in conjunction with other genetic, environmental, or stochastic factors-to the development of many of the phenotypes seen in individuals with terminal and interstitial deletions that include the proximal region of chromosome 1p36.