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

Abstract

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 Rere^flox/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.

Introduction

Deletions of chromosome 1p36 are the most common telomeric deletions in humans (1). In addition to having neurocognitive problems, growth restriction, hearing loss, brain anomalies, congenital heart defects, cardiomyopathy and renal anomalies, approximately 5% of individuals with 1p36 deletion syndrome (MIM #607872) have orofacial clefts (1). Distal and proximal critical regions for 1p36 deletion syndrome have been defined (2). However, identifying the individual genes that cause or contribute to the development of individual phenotypes remains an important clinical and scientific research topic (2).

The arginine–glutamic acid dipeptide repeats gene (RERE; MIM #605226) is located in the proximal critical region for 1p36 deletion syndrome (3). Pathogenic, heterozygous variants in RERE have also been shown to cause neurodevelopmental disorder with or without anomalies of the brain, eye or heart (NEDBEH; MIM #616975) whose phenotypes closely resemble those seen in individuals with proximal 1p36 deletions (3,4). To date, one patient with NEDBEH has been described to have a cleft lip (Fregeau et al. 2016, Subject 7) (3). In contrast, cleft palate has not been previously associated with NEDBEH (3).

RERE encodes a widely expressed nuclear receptor co-regulator that positively regulates vitamin A/retinoic acid signaling in the developing embryo (5–7). In Xenopus laevis, decreases in retinoic acid signaling lead to a median cleft in the upper lip and primary palate (8). These observations led us to hypothesize that loss of RERE function may contribute to the development of cleft palate in individuals with 1p36 deletions and NEDBEH.

We have previously shown that on a mixed B6/129S6 background, mice bearing both a null allele (om) and a hypomorphic allele (eyes3) of Rere have a spectrum of malformations that is similar to that seen in humans with proximal 1p36 deletions and NEDBEH (3,9). These phenotypes include postnatal growth deficiency, brain hypoplasia, structural brain anomalies, microphthalmia, hearing loss, congenital heart defects and renal anomalies. Orofacial clefts were not documented in Rere^om/eyes3 mice in this mixed background. However, some defects, particularly congenital heart defects, are only seen on a pure B6 background, which is also associated with an increased level of embryonic/perinatal mortality (9).

Here we show that RERE is affected by a subset of 1p36 deletions associated with orofacial clefting and that orofacial clefting is seen in 9.2% (6/65) of individuals with isolated 1p36 deletions involving RERE. We also report an individual with a cleft palate and symptoms consistent with NEDBEH caused by a pathogenic, de novo variant in RERE. As further evidence for the role of RERE in palate development, we show that RERE is expressed in the developing mouse palate and that RERE-deficient mouse embryos on a C57BL/6 background have cleft palates. We go on to demonstrate that RERE plays a critical, cell-autonomous role in neural crest cells during palatal development and that mesenchymal cell proliferation in the palatal shelves is significantly reduced in Rere^flox/flox;Wnt1-Cre embryos.

Results

Deletion/phenotype mapping suggests the existence of multiple 1p36 genes associated with orofacial clefting

In a search of the literature, and local and public databases, we identified 26 individuals with isolated terminal and interstitial 1p36 deletions associated with orofacial clefting (Supplemental Material, Table S1). The smallest of the terminal deletions extended ~ 2.17 Mb into chromosome 1 (chr1:1–2,167,838) and was identified in an individual with cleft palate (DECIPHER 400898). This deletion has a partial overlap with an ~ 1.69 Mb interstitial deletion (chr1:1,786,789–3,472,907) in an individual with cleft palate. This suggests the existence of at least one, and possibly two orofacial clefting genes within the distal 1p36 critical region that has been defined as the region distal to marker D1S2870 (chr1:6,289,764–6,289,973; hg19) (2). Candidate genes that may contribute to the development of orofacial clefting in this region, based on mouse models, include SKI (chr1:2,160,134–2,241,652) and PRDM16 (chr1:2,985,742–3,355,185). Ski-null mice have midline facial clefting and PRDM16-deficient mice have cleft palate as a result of micrognathia and failed palate shelf elevation due to physical obstruction by the tongue (10,11).

Four isolated interstitial 1p36 deletions associated with orofacial clefting did not include either of these genes. The first two are large deletions (chr1:3,768,946–18,563,553 and chr1:4,795,388–17,364,849; hg19) that encompass the proximal critical region for 1p36 (chr1: 8,395,179–11,362,893; hg19). This proximal critical region includes RERE (chr1:8,412,463–8,877,698; hg19).

The third interstitial deletion (chr1:11,816,673–19,201,956; hg19) partially overlaps the first two but does not overlap the proximal critical region for 1p36 and does not include RERE. Variants in PAX7 (chr1:18,957,500–19,075,360; hg19), a gene located in this region, have been found to be associated with an increased risk of orofacial clefting (12,13).

The fourth interstitial deletion (chr1:23,689,659–25,570,112; hg19) has no overlap with any of the other deletions associated with orofacial clefting. GRHL3 (chr1:24,645,812–24 ,690,972; hg19) is located in this region. Autosomal dominant variants in this gene cause van der Woude syndrome 2 (MIM #606713) and contribute to the development of nonsyndromic cleft palate (14,15). A subset of Grhl3-null mice also develops cleft palate (14).

RERE is deleted in a subset of isolated 1p36 deletions associated with orofacial clefting

In a search of the literature and local databases, we identified 65 isolated terminal and interstitial 1p36 deletions that affected RERE. Of these, six (9.2%) were associated with various types of orofacial clefting (Supplemental Material, Table S2) including submucosal cleft palate (n = 2), cleft palate (n = 1) and cleft lip and palate (n = 3).

Identification of a pathogenic RERE variant in an individual with cleft palate

Pathogenic variants in RERE have been shown to cause most of the medical problems associated with deletions of the 1p36 proximal critical region (3,4). Although one individual with NEDBEH has been described to have a cleft lip (Fregeau et al., 2016, Subject 7), cleft palate has not been previously described in association with NEDBEH. However, we have recently identified a 6-year, 2-month old White male of Polish descent (Subject 1) who carries a pathogenic, de novo c.4313_4318dupTCCACC, p.Leu1438_His1439dup [NM_012102.3] variant in RERE. This variant has been previously described in three individuals with NEDBEH (Fregeau et al., 2016, Subject 2 and Jordan et al., Subjects 6 and 7) (3,4).

Figure 1

RERE is expressed in the palate at E13.5 and loss of RERE function causes cleft palate. (A) Coronal sections of the palate were prepared from wild-type mouse embryos at E13.5 and stained with anti-RERE antibodies. RERE was expressed in the palatal shelves and bend region of the palate at E13.5. (B and C) High-power views of regions indicated by the white arrow (B) or yellow arrow (C) in panel A. Representative images were selected from three independent experiments. (D–I) Coronal sections of the anterior (D, E), middle (F, G) and posterior (H, I) regions of the palate were obtained from wild-type and Rere^om/eyes3 embryos at E15.5 and H&E stained. At this stage, the palatal shelves of wild-type embryos have fused at the midline (D, F and H). In contrast, the palatal shelves of Rere^om/eyes3 embryos were not fused (E, G and I). Representative images were selected from four independent littermates of each genotype. NS, nasal septum; PS, palatal shelf; T, tongue.

His family history was negative for orofacial clefting and other major medical problems. The pregnancy was complicated by polyhydramnios and maternal bleeding, and he was born prematurely at 33 weeks of gestation. At birth, he was found to have a cleft hard and soft palate. Other findings included atrial and ventricular septal defects and ambiguous genitalia with undescended testicles and chordae. He was also noted to be hypotonic and required nasogastric feeding. A neonatal cranial ultrasound revealed ventriculomegaly and brain calcifications.

Figure 2

Cleft palate in Rere^flox/flox;Wnt1-Cre embryos at E15.5. (A–F) Coronal sections from the anterior (A, B), middle (C, D) and posterior (E, F) regions of the developing palate were obtained from Rere^flox/flox;Wnt1-Cre embryos and control littermate embryos at E15.5. Sections were stained with H&E. In Rere^flox/flox;Wnt1-Cre embryos, the palatal shelves were elevated but failed to meet in the midline (B, D and F). Representative images were selected from five independent littermates of each genotype. NS, nasal septum; PS, palatal shelf.

Figure 3

Delayed palatal shelf elevation in Rere^flox/flox;Wnt1-Cre embryos. (A–F) Coronal sections were obtained from Rere^flox/flox;Wnt1-Cre embryos and control embryos at E13.5 and stained with H&E. Vertical growth of the palatal shelves was comparable between Rere^flox/flox;Wnt1-Cre embryos and their control littermate embryos at E13.5 in all regions of the palate. (G–L) H&E-stained coronal sections prepared from Rere^flox/flox;Wnt1-Cre embryos and control littermate embryos at E14.5. The palatal shelves of Rere^flox/flox;Wnt1-Cre embryos remained in vertical position (H, J and L), whereas the palatal shelves were horizontally elevated in littermate control embryos at E14.5 (G, I and K). Representative images from the anterior, middle and posterior regions of developing palate were selected from four independent littermates of each genotype. PS, palatal shelf; T, tongue.

Figure 4

Proliferative activity in the medial half of the palatal shelves of Rere^flox/flox;Wnt1-Cre embryos is significantly reduced. (A and B) Coronal sections obtained from Rere^flox/flox;Wnt1-Cre embryos and control embryos at E13.5 and stained with anti-pHH3 antibodies. White dashed lines divide the palatal shelves into medial halves (yellow arrows) and lateral halves (red arrows). (C) Mitotic cells in medial halves or lateral halves of the palatal shelves were counted and normalized by area. The number of mitotic cells in medial halves of the palatal shelves from Rere^flox/flox;Wnt1-Cre embryos was significantly less than the number from control littermate embryos at E13.5. ^*P < 0.03. Quantification was performed using eight slides containing at least three sections from three independent littermates of each genotype. Data represent the mean ± SD. Student’s unpaired two-tailed t-test was used to determine P-values.

At 18 months of age he was able to sit independently. He started walking at 4 years of age, and he was nonverbal at 6 years of age. Hence, he was given a diagnosis of global developmental delay. He was also noted to have some autistic traits and has been diagnosed with moderate intellectual disability. He has hypermetropic astigmatism and bilateral mild/moderate conductive hearing loss. He is currently fed through a gastrostomy tube.

At 6 years of age his height and weight were at the ninth centile and his occipital frontal circumference (OFC) was between the second and ninth centile. His neurological examination was normal, but a brain magnetic resonance imaging study revealed bilateral mild ventricular dilation and nonspecific patchy white matter high signal. Dysmorphic features include proptosis, hypertelorism, a flat mid-face, a small upturned nose with a flat nasal bridge, dysplastic, low-set, posteriorly rotated ears, a very small mouth with significant micrognathia and wide-spaced nipples. He had overlapping fingers on both hands and tended to hold his fists in a clenched position.

RERE is expressed in the developing mouse palate

To determine if RERE deficiency was likely to predispose individuals to development of cleft palate, we examined the expression pattern of RERE in the palatal shelves of embryonic mice by immunohistochemistry. We found RERE-positive cells in both the palatal mesenchyme and epithelium of palatal shelves at embryonic day (E)13.5 (Fig. 1A and B). In addition, RERE was expressed in the bend region between the palatal shelf and the cranial base at E13.5 (Fig. 1A and C).

RERE-deficient embryos have cleft palate

Fusion of the medial nasal processes and the bilateral maxillary processes forms the primary palate and the upper lip. The secondary palate arises through outgrowth and merging of the bilateral maxillary processes. In mice, palatogenesis is initiated from the oral side of the medial nasal processes and the maxillary processes around E11.5 (16,17). Vertical outgrowth of the bilateral maxillary processes leads to development of the palatal shelves in the oral cavity by E13.5. The palatal shelves elevate into a horizontal position by E14.5 and begin to grow toward the midline. After the palatal shelves contact in the midline around E15.5, they fuse to form the secondary palate.

To determine if RERE deficiency can cause cleft palate in mice, we looked for failure of palatal shelf fusion in Rere^om/eyes3 embryos on a B6 background. On this background, Rere^om/eyes3 pups are unrecoverable at P0, but they can be generated at Mendelian ratios at E15.5 (Supplemental Material, Table S3). At this stage, the palatal shelves of wild-type embryos were fused in the midline to form the secondary palate (Fig. 1D, F and H). In contrast, the palate shelves of their Rere^om/eyes3 littermates failed to contact each other in midline at E15.5 resulting in a palatal cleft (Fig. 1E, G and I). This palatal cleft was present in the midline from the anterior region to the posterior region in Rere^om/eyes3 embryos (Fig. 1E, G and I). On a B6 background, cleft palate was detected in 80% (4/5) of Rere^om/eyes3 embryos at E15.5. These data suggest that RERE deficiency causes cleft palate in mice.

Tissue-specific ablation of Rere in cranial neural crest cells causes cleft palate in mice

Cranial neural crest (CNC) cells are pluripotent cells that are derived from the lateral ridges of the neural plate and compose the palatal mesenchyme during the early stages of palatogenesis (18). Because of their pluripotency, CNC cells can differentiate into a variety of cell types including osteoblasts, chondrocytes and fibroblasts that contribute to the development of bony and nonbony craniofacial tissues. To determine if RERE deficiency in CNC cells and their derivatives causes cleft palate, we selectively ablated Rere in these cells using a Wnt-1 Cre in combination with a floxed Rere allele (18,19). Rere^flox/flox;Wnt-1 Cre pups are unrecoverable at P0, but they can be generated at Mendelian ratios at E15.5 (Supplemental Material, Table S4). We found that Rere^flox/flox;Wnt-1 Cre embryos developed cleft palates that were similar to those seen in Rere^om/eyes3 embryos at E15.5 (Fig. 2B, D and F). Specifically, the bilateral palatal shelves were not fused in the midline in Rere^flox/flox;Wnt1-Cre embryos, whereas the bilateral palatal shelves were fused in control embryos. As in RERE-deficient embryos, the cleft palates seen in Rere^flox/flox;Wnt1-Cre embryos occurred along the anterior–posterior axis in (Fig. 2B, D and F). These data suggest that tissue-specific ablation of Rere in CNC cells causes cleft palate in mice.

To determine if tissue-specific depletion of RERE in CNC cells has an effect on outgrowth and/or elevation of the palatal shelves, we performed hematoxylin and eosin (H&E) staining on sections that were prepared from Rere^flox/flox;Wnt-1 Cre embryos and control littermates at E13.5 and E14.5. Vertical outgrowth of the palatal shelves in Rere^flox/flox;Wnt-1 Cre embryos was comparable with that of wild-type littermates at E13.5 (Fig. 3A–F). However, the size of the palatal shelves in the anterior region of Rere^flox/flox;Wnt-1 Cre embryos was smaller than that of control embryos at E13.5 (Fig. 3A and B). At E14.5, palatal shelves were horizontally elevated in wild-type littermates (Fig. 3G, I and K). However, palatal shelves were not elevated in Rere^flox/flox;Wnt1-Cre embryos at same stage (Fig. 3H, J and L). Even though the palatal shelves of Rere^flox/flox;Wnt-1 Cre embryos were not fused in midline, the palatal shelves were elevated into horizontal position at E15.5 (Fig. 2). These data suggest that tissue-specific ablation of Rere in CNC cells results in delayed horizontal elevation of the palatal shelves and failure of palatal shelf fusion at E15.5.

Proliferation of mesenchymal cells in the palatal shelves of Rere^flox/flox;Wnt-1 Cre embryos was significantly reduced

It has been proposed that reduced proliferation of medial mesenchymal cells could lead to both a failure of shelf elevation and a reduction in the horizontal outgrowth of the palatal shelves (17,20). To investigate if proliferation is affected in the palatal shelves of Rere^flox/flox;Wnt1-Cre embryos, mitotic cells in the lateral halves and medial (oral) halves of the palatal shelves were counted at E13.5 and normalized by area (Fig. 4). Proliferation of cells in the lateral halves of the Rere^flox/flox;Wnt1-Cre embryos was comparable with that of littermate controls at E13.5 (Fig. 4C). However, proliferative activity in the medial halves of the palatal shelves was significantly reduced in Rere^flox/flox;Wnt1-Cre embryos compared with that of littermate control embryos at E13.5 (Fig. 4A–C). These data suggest that RERE is required to regulate proliferation of palatal cells in the medial halves of the palatal shelves.

Discussion

Approximately 5% of individuals with 1p36 deletion syndrome have orofacial clefts (1). Deletion/phenotype mapping suggests that haploinsufficiency of multiple genes on chromosome 1p36 is likely to predispose individuals to the development of orofacial clefts (Supplemental Material, Table S1). The role of SKI, PRDM16, PAX7 and GRHL3 in this process is supported by mouse models and/or human studies (10–15). Pathogenic variants in RERE have been shown to cause NEDBEH, and haploinsufficiency of RERE is likely to contribute to most, if not all, of the phenotypes associated with deletion of the 1p36 proximal critical region (3,4). Although approximately 9% of individuals with 1p36 deletions that include RERE have some form of orofacial clefting—submucosal cleft palate, cleft palate and cleft lip and palate (Supplemental Material, Table S2)—the only type of orofacial clefting that has been previously associated with NEDBEH has been cleft lip that was documented in one individual (Fregeau et al., 2016, Subject 7) (3).

Here we have described the first individual with NEDBEH who has a cleft palate. The diagnosis of NEDBEH in this individual is strongly supported by their carrying a de novo c.4313_4318dupTCCACC, p.Leu1438_His1439dup [NM_012102.3] variant in RERE that has previously been described in three individuals with NEDBEH (3,4). To date, 19 other individuals with NEDBEH have been described. Hence, orofacial clefting can be estimated to effect ~10% (2/20) of individuals with NEDBEH, making is a relatively low penetrance phenotype.

As has been the case for other orofacial cleft-related genes on 1p36, mouse models provide additional evidence for the role of RERE in palatal development. Specifically, cleft palate was detected in 80% (4/5) of Rere^om/eyes3 embryos at E15.5 on a B6 background (Fig. 1). This leads us to conclude that decreased expression of RERE contributes to the development of cleft palate in individuals with proximal 1p36 deletions and NEDBEH, and that RERE deficiency also causes cleft palate in mice. On the basis of the incomplete penetrance seen in both humans and mice, we assume that other genetic, epigenetic, environmental and/or stochastic factors play a role in modulating the effect of RERE deficiency during palatal development.

Development of the secondary palate is initiated with outgrowth of the medial (oral) side of the paired maxillary prominences in the mouth (16,17). The outgrowth of these processes expands vertically to form the palatal shelves flanking the tongue. The palatal shelves consist of the mesenchymal cells derived from the neural crest and are covered by a thin layer of the epithelial cells (19). We have shown that RERE is expressed in palatal epithelial cells as well as in the palatal mesenchymal cells (Fig. 1) and that tissue-specific deletion of Rere in the CNC cells that form the palatal mesenchyme leads to a reduction of mesenchymal cell proliferation in the medial aspect of the palatal shelves (Fig. 4), retarded elevation of the palatal shelves (Fig. 3) and the development of cleft palate in Rere^flox/flox;Wnt1-Cre embryos (Fig. 2). We conclude that RERE expression in CNC-derived cells is required for normal palatal development in mice.

As RERE functions as a transcription factor to modulate the expression of its target genes (5,21), it is likely that it regulates one or more signaling pathways during palatal development. Palatal shelf outgrowth is controlled by sonic hedgehog, fibroblast growth factor 10 and bone morphogenic protein signaling pathways (19,21). In X. laevis, decreases in retinoic acid signaling also leads to the development of cleft palate (8). Future studies will be aimed at identifying how RERE deficiency affects these signaling pathways.

Materials and Methods

Mice

All experiments using mouse models were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The associated protocols were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine (Animal Welfare Assurance #A3832-01).

Wild-type C57BL/6 embryos were used to define the expression pattern of RERE. The generation of the Rere om, null and eyes3 alleles was described previously (5,9). Experiments involving mice bearing these alleles were conducted on a C57BL/6 background.

The generation of the Rere flox allele was described previously (18). Briefly, the second coding exon of Rere, the same exon that is skipped in the om null allele, was flanked by loxP sites. Rere^flox/flox mice were generated in expected numbers in heterozygous crosses, were viable and fertile, and had no discernable abnormal phenotypes. In preparation for experiments on a C57BL/6 background, the B6/129S6 mice bearing the Rere flox allele were backcrossed for at least six generations onto the C57BL/6 background.

Histology

Embryos were harvested and fixed with Buffered Formalde-Fresh solution (Fisher Scientific, Pittsburgh, PA) for 1 day at 4°C. After washing with phosphate-buffered saline solution (PBS), tissues were dehydrated in ethanol and embedded in paraffin. Paraffin-embedded tissue blocks were sectioned at 6 μm with an RM2155 microtome (Fisher Scientific).

Coronal sections of embryos were stained with H&E for histomorphological analyses. For histomorphological analyses, images of H&E-stained sections corresponding to various regions of the developing palate were acquired using a Zeiss Axioplan microscope equipped with an AxioCam digital camera and imaging system (Carl Zeiss Microscopy GmbH, Gena, Germany).

For immunohistochemical analyses, tissue sections were deparaffinized and blocked with 1× PBS containing 1% bovine serum albumin (BSA) and 5% normal donkey serum for 1 h at room temperature. Sections were then incubated with anti-RERE (sc-98 415, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-Phospho-Histone H-3 (pHH3) (#9664, 1:200; Cell Signaling Technology) antibodies diluted in the same blocking solution (1% BSA and 5% normal donkey serum in 1× PBS) overnight at 4°C. After washing with 1× PBS, the sections were incubated with biotin conjugated anti-rabbit IgG or biotin conjugated anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA).

The immunoreactivity of each antibody was visualized using either a 3,3′-diaminobenzidine (DAB) substrate kit (Vector Laboratories, Burlingame, CA) or a tyramide signal amplification (TSA) kit (Invitrogen, Grand Island, NY) containing Alexa Fluor 488 dye or Alexa Fluor 568 dye for fluorescent labeling per manufacturer’s instructions. Images were acquired on a Zeiss Axioplan microscope equipped with an AxioCam digital camera and imaging system (Carl Zeiss Microscopy GmbH, Gena, Germany). For RERE expression studies, two slides, each containing at least three sections, were analyzed from each of three E15.5 embryos.

To quantify the mitotic cells in the developing palate, cells labeled with anti-pHH3 antibodies were counted and normalized to the area of the palate shelves on each section using Image J software (NIH, https://imagej.nih.gov/ij/). For statistical analysis, Student’s unpaired two-tailed t-test was used. The standard deviation of the mean [±standard deviation (SD)] was plotted as an error bar in the associated graphs.

Acknowledgements

The authors would like to thank Subject 1 and his family for their participation in this research study.

Conflict of Interest Statement. None declared.