Analysis of FOXF1 and the FOX gene cluster in patients with VACTERL association

Abstract

VACTERL association, a relatively common condition with an incidence of approximately 1 in 20,000 –35,000 births, is a non-random association of birth defects that includes vertebral defects (V), anal atresia (A), cardiac defects (C), tracheo-esophageal fistula (TE), renal anomalies (R) and limb malformations (L). Although the etiology is unknown in the majority of patients, there is evidence that it is causally heterogeneous. Several studies have shown evidence for inheritance in VACTERL, implying a role for genetic loci. Recently, patients with component features of VACTERL and a lethal developmental pulmonary disorder, alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV), were found to harbor deletions or mutations affecting FOXF1 and the FOX gene cluster on chromosome 16q24. We investigated this gene through direct sequencing and high-density SNP microarray in 12 patients with VACTERL association but without ACD/MPV. Our mutational analysis of FOXF1 showed normal sequences and no genomic imbalances affecting the FOX gene cluster on chromosome 16q24 in the studied patients. Possible explanations for these results include the etiologic and clinical heterogeneity of VACTERL association, the possibility that mutations affecting this gene may occur only in more severely affected individuals, and insufficient study sample size.

Introduction

VACTERL is an acronym used to describe the non-random clustering of congenital anomalies: vertebral defects (V), anal atresia (A), cardiac defects (C) tracheo-esophageal fistula (TE), renal anomalies (R) and limb malformations (L). It is often termed an association in order to emphasize that these malformations appear together more often than would be expected by chance [2], [13]. To diagnose the condition, most clinicians look for the presence of three constituent features of VACTERL association, without evidence of an alternate, overlapping diagnosis [17]. Diagnosis is difficult due to the number of disorders that have overlapping features with VACTERL, including Feingold syndrome, CHARGE syndrome, 22q11.2 deletion syndrome, Townes–Brocks syndrome, Pallister–Hall syndrome, Holt–Oram syndrome, Fanconi anemia, and Baller–Gerold syndrome [16].

VACTERL is likely a defect of blastogenesis, with an estimated incidence of 1 in 20,000 to 1 in 35,000 births [2], [11], [13]. Although relatively common, the causes of VACTERL have yet to be elucidated in the majority of patients. VACTERL is thought to largely occur sporadically, though there is evidence for inheritance in at least a subset of patients [17], suggesting that genetic factors may play a key role. A number of studies give evidence of heterogeneous etiologies [2], [7], [13], [17].

Although few large studies looking for genetic or other causes of VACTERL have been performed to date, due to relatively few familial cases and the heterogeneity, scattered case reports describe possible genetic causes. Thus far, patients with VACTERL association have been found to have mutations in HOXD13 [4], ZIC3 [20], PTEN [12], and mitochondrial genes [3]; additionally, mutations and deletions involving FOXF1 and the FOX gene cluster [15], [19], [21] have been identified in patients with VACTERL association features as well as a specific pulmonary phenotype (see below). The pathogenicity of each of these mutations as they relate to VACTERL association is not uniformly clear. However, animal models of the FOXF1 gene and related signaling pathways have provided clues as to its pathogenicity, making FOXF1 an intriguing candidate gene for VACTERL association.

Recently, Stankiewicz et al. (2009) identified overlapping microdeletions in 16q24.1q24.2 in seven patients with component features of VACTERL association, namely vertebral anomalies, gastrointestinal atresia, TE fistula, and cardiac malformations. In addition, these patients also had alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV), a rare and lethal developmental pulmonary disorder [19]. These deletions included the FOX transcription factor gene cluster at 16q24.1q24.2. All but one of these deletions contained FOXF1, a gene that plays a pivotal role in the development of the lung and foregut [10]. Interestingly, the one patient with the deletion not involving FOXF1 had a distinct phenotype with less overlap with VACTERL association in terms of the type of congenital malformations. Similar clinical and genetic findings have been reported by Yu et al (2009), who reported a patient with a 1.37 Mb deletion of chromosome 16q24.1q24.2. In addition to ACD/MPV, this patient had genitourinary, cardiac, and intestinal anomalies [21].

Stankiewicz et al (2009) further identified four unrelated patients with ACD/MPV who had heterozygous mutations in FOXF1. In addition to ACD/MPV, all of these patients had associated malformations, namely cardiac, intestinal, and urinary tract malformations [19]. Specifically, one patient had a cardiac malformation, with a partial atrio-ventricular canal defect and a patent ductus arteriosus. Three patients had gastrointestinal malformations, which included intestinal malrotation, annular pancreas, duodenal stenosis, congenital short bowel, omphalocele, and a Meckel’s diverticulum. Anomalies affecting the urinary tract were present in three patients, and were described as including hydronephrosis, hydroureter, bladder dilatation, and obstructive renal dysplasia [19]. As the pattern of these malformations is similar to those seen in patients with VACTERL association, these human studies provide compelling evidence for the possible role of FOXF1 in the pathogenesis of VACTERL.

The pivotal role of FOXF1 in the development of the lung and foregut has support from mouse models, where haploinsufficiency of Foxf1 causes a variable phenotype including lung immaturity and hypoplasia, fusion of the right lung lobes, narrowing of the esophagus and trachea, esophageal atresia, and TE fistula. Of note, similar malformations have been found in Sonic hedgehog (Shh) mouse mutants [8], [10]. Further evidence for the connection between these two genes has been shown in mice, where Foxf1 expression was induced by exogenous Shh in the lung and gut [9], [10]. In addition, expression patterns of Foxf1 mirror the basal levels of Shh expression [10]. The reproduction of similar VACTERL-type anomalies in Shh (−/−) and Foxf1 (+/−) mouse mutants implies that these genes share a common pathway whose aberration may contribute to the VACTERL phenotype [8], [9], [10].

If the pathway linking SHH and FOXF1 is thus involved in VACTERL association, the logical question is at what point in that pathway might genetic changes result in the type of anomalies observed in patients with VACTERL association? Several factors hint that it is not likely that this pathway would be altered at the level of SHH itself. Not only do loss-of-function mutations in SHH cause holoprosencephaly in humans, but multiple studies have shown negative mutation analysis of the SHH gene in patients with VACTERL association features without any signs of holoprosencephaly [1], [6], [14]. These studies provide a basis for further investigations aimed at downstream targets of the SHH pathway, such as FOXF1.

In summary, due to the involvement of SHH signaling in gut development, as well as the role of its downstream effectors, including FOXF1, the demonstration of FOXF1 as a SHH target, the presence of FOXF1 mutations and deletions in patients with features of VACTERL association, and the VACTERL association-type phenotype present in mice heterozygous for Foxf1 mutations, we carried out a mutational analysis of the candidate gene FOXF1 and a copy number analysis of the 16q24.1q24.2 region containing the FOX gene cluster in a cohort of 12 patients with VACTERL association but without clear evidence of the pulmonary condition observed in previous patients with mutations affecting this gene, with the hypothesis that mutations in FOXF1 could result in a variable, and milder phenotype than in the patients originally described.