The broader phenotypic spectrum of congenital caudal abnormalities associated with mutations in the caudal type homeobox 2 gene

Abstract The caudal type homeobox 2 (CDX2) gene encodes a developmental regulator involved in caudal body patterning. Only three pathogenic variants in human CDX2 have been described, in patients with persistent cloaca, sirenomelia and/or renal and anogenital malformations. We identified five patients with de novo or inherited pathogenic variants in CDX2 with clinical phenotypes that partially overlap with previous cases, that is, imperforate anus and renal, urogenital and limb abnormalities. However, additional clinical features were seen including vertebral agenesis and we describe considerable phenotypic variability, even in unrelated patients with the same recurrent p.(Arg237His) variant. We propose CDX2 variants as rare genetic cause for a multiple congenital anomaly syndrome that can include features of caudal regression syndrome and VACTERL. A causative role is further substantiated by the relationship between CDX2 and other proteins encoded by genes that were previously linked to caudal abnormalities in humans, for example, TBXT (sacral agenesis and other vertebral segmentation defects) and CDX1 (anorectal malformations). Our findings confirm the essential role of CDX2 in caudal morphogenesis and formation of cloacal derivatives in humans, which to date has only been well characterized in animals.


| INTRODUCTION
Caudal type homeobox (cdx) genes encode transcriptional regulators that have a broad role in early mesodermal fate decisions and development of the body plan. [1][2][3] For example, they regulate axial extension, as well as anteroposterior patterning in embryogenesis. 4 The human genome contains three known cdx genes, that is, CDX1, CDX2 (also known as CDX3), and CDX4, which are ParaHox genes of the HOXL subclass. 5,6 The developmental role of CDX2 has been extensively studied in animal model systems. Its role in human development and disease remains less understood, although ectopic activation of the gene is involved in the development of some cancers. 7 Furthermore the gene is involved in human caudal morphogenesis.
Only three pathogenic germline CDX2 variants have been described in humans. 8,9 De novo CDX2 variants have been reported in two individuals with persistent cloaca. Inherited CDX2 variants were identified in two families with extremely variable phenotypes that ranged from imperforate anus, renal agenesis and urogenital malformations to the most severe form of caudal abnormality sirenomelia, a malformation sequence characterized by fused legs and visceral abnormalities. 9 Here, we describe five additional patients with pathogenic variants in the CDX2 gene. We show that the associated phenotypic spectrum is broad and occasionally extends beyond caudal abnormalities. These findings highlight the pivotal role of the CDX2 gene in the development of the uro-recto-genital tract, vertebrae, and the limbs in humans.

| MATERIALS AND METHODS
Whole exome sequencing was performed as described previously 10 using DNA isolated according to standard procedures from blood, chorion villi or skin biopsies. Exome capture was done using the Agilent SureSelect XT Human All Exon kit (Agilent, Santa Clara, CA; patient 1 and 4), the Agilent Sureselect Clinical Research Exome (CRE) Capture kit (patient 2) or the Nimblegen SeqCap_ EZ_Exome_v3 kit (Roche Nimblegen, Pleasanton, CA; patient 3).
Exome libraries were sequenced on an Illumina HiSeq instrument (Illumina, San Diego, CA). Sequence reads were aligned to the hg19 reference genome using BWA version 0.5.9-r16 or Novoalign version 3. A mean coverage was obtained of 111x (patient 1), 56.5x (patient 2), 174x (patient 3) and 121x (patient 4), with at least 99.3%, 96.5%, 98.6%, and 96% of exome nucleotides covered by at least 10 sequence reads respectively. Variants were subsequently called by the GATK unified genotyper, version 3.2-2 or higher version and annotated using custom diagnostic annotation pipelines as described previously 10,11 or by Cartagenia software (Agilent Technologies). Variants were filtered using a frequency of <1% in dbSNP and the Genome Aggregation Database (gnomAD). Data were subsequently filtered for homozygous, compound heterozygous variants or X-linked inheritance modes, and for the de novo inheritance in parent-offspring trio data. CDX2 gene variants were reported by our laboratories in the routine diagnostic genetic work-up of the patients involved in this study.
The patients in this study were recruited via matching submissions for the CDX2 gene to the Genematcher website. 12 Description of the patients' clinical phenotype was done by the consulting Clinical Geneticists as part of the routine genetic work-up according to standard procedures for this medical profession. Parents were investigated either in a whole exome sequencing trio analysis, 10 Table 1 gives details of the genotypic and phenotypic findings in the five patients compared with patients described in the literature. 8,9 Figure 1 is a schematic presentation of the CDX2 variants described here and previously.

| DISCUSSION
Our findings indicate that variants in CDX2 are a rare genetic cause for congenital abnormalities affecting the development of the anus, the renal and urogenital system, the vertebrae and/or the limbs in varying sequences and severity. We postulate that CDX2 abnormalities cause a highly diverse and variable clinical phenotype, which shows overlap with VACTERL, that is, renal, vertebral and limb malformations and cardiac features (see Table 1). A consistent feature is uro-recto-genital malformation, with imperforate anus being the most frequent. The CDX2-associated clinical phenotype overlaps with caudal regression syndrome, which encompasses a range of congenital defects. 15 We propose that caudal regression syndrome, sirenomelia and persistent cloaca are part of a variable phenotypic spectrum that may also include VACTERL-like features. A common pathogenesis for these malformations has been proposed [16][17][18] and our findings may link these conditions genetically, although larger cohort studies are needed to further substantiate this.
Animal models have defined the role for CDX2 orthologues in caudal morphogenesis. The Drosophila caudal protein for example, is required for formation of posterior structures [19][20][21] and in other arthropods the CDX2 orthologue is also required for posterior axis elongation 22 . In Amphioxus the cdx gene is essential for gut, anus and tail patterning. 23 In the mouse cdx2 is essential for anteroposterior patterning of embryonal axis and morphogenesis of cloacal structures. [24][25][26][27] Strikingly, Cdx2 heterozygous conditional mutant mice show a variable phenotype that can include an imperforate anus, sirenomelia, posterior vertebral truncations, and bladder anomalies, 25,26,28 which is similar to the human clinical phenotype (Table 1).
CDX2 together with transcription factor T Brachyury (TBXT) coactivates a regulatory network of target genes during posterior axial elongation and both proteins instruct the "trunk to tail" transition in mice. 29  F I G U R E 1 Schematic representation of the functional domains of the CDX2 proteins and the variants described in literature 8,9 and in this study (underlined). The Figure is based on CDX2 protein reference sequence NP_001256. Amino acid positions are indicated as numbers below the protein domains. The poly-alanine ("Poly A"), poly-glutamine ("Poly Q"), and poly-proline ("Poly P") stretches in the protein are indicated above the domains [Colour figure can be viewed at wileyonlinelibrary.com] functions in posterior axis elongation in mice 27 and have strong coexpression during anorectal morphogenesis in human embryos. 34 A mutation in the HOXL gene HOXD13 has been linked to VACTERL. 35 We are unable to link the type of CDX2 variant to the severity or diversity of the phenotype. The recurrent pathogenic missense variant in the HOX domain of the protein, p.(Arg237His), that was found in three unrelated families exhibits remarkable variability in phenotypic expression. This ranges from persistent cloaca, 8 sirenomelia and renal/ urogenital anomalies in offspring of mildly affected mothers with imperforate anus 9 and Müllerian abnormalities in patient 4, with a solitary kidney in her mildly affected father. Patient 2 has a missense variant in the activation domain, while the other variants either concern nonsense or frameshift variants or missense variants in the HOX domain. Remarkably, patient 2 only had radial abnormalities, which are often seen in VACTERL-like phenotypes, but caudal morphogenesis defects were absent. It remains unclear however whether this is due to location of the CDX2 variant because the number of CDX2 patients currently is too small for a thorough genotype-phenotype analysis. Another limitation of our study is the fact that we did not perform functional or animal studies that may further define the pathogenic mechanisms causing the phenotype and may explain its variability.
The reason for the observed phenotypic diversity therefore remains unclear but may be related to (epi)genetic modifiers of the phenotype or, teratogenic environmental or maternal factors, as postulated. 18,36,37 Differences in control of homeostasis of retinoic acid (RA) may possibly be involved as well. CDX2 indirectly inhibits RA by upregulating CYP26A1, a cytochrome that catabolizes RA. Loss of CDX2 function therefore leads to prolonged RA bioactivity which impairs axial mesoderm ontogenesis. 25 Interestingly, RA exposure of heterozygous conditional mutant mice resulted in the development of sirenomelia, underscoring the molecular interplay between CDX2 and RA signaling. 25 In conclusion, our findings confirm that CDX2 gene variants should be considered as a rare cause of vertebral, urogenital, limb, and/or anal anomalies.