A non-coding insertional mutation of Grhl2 causes gene over-expression and multiple structural anomalies including cleft palate, spina bifida and encephalocele

Abstract

Orofacial clefts, including cleft lip and palate (CL/P) and neural tube defects (NTDs) are among the most common congenital anomalies, but knowledge of the genetic basis of these conditions remains incomplete. The extent to which genetic risk factors are shared between CL/P, NTDs and related anomalies is also unclear. While identification of causative genes has largely focused on coding and loss of function mutations, it is hypothesized that regulatory mutations account for a portion of the unidentified heritability. We found that excess expression of Grainyhead-like 2 (Grhl2) causes not only spinal NTDs in Axial defects (Axd) mice but also multiple additional defects affecting the cranial region. These include orofacial clefts comprising midline cleft lip and palate and abnormalities of the craniofacial bones and frontal and/or basal encephalocele, in which brain tissue herniates through the cranium or into the nasal cavity. To investigate the causative mutation in the Grhl2Axd strain, whole genome sequencing identified an approximately 4 kb LTR retrotransposon insertion that disrupts the non-coding regulatory region, lying approximately 300 base pairs upstream of the 5' UTR. This insertion also lies within a predicted long non-coding RNA, oriented on the reverse strand, which like Grhl2 is over-expressed in Axd (Grhl2Axd) homozygous mutant embryos. Initial analysis of the GRHL2 upstream region in individuals with NTDs or cleft palate revealed rare or novel variants in a small number of cases. We hypothesize that mutations affecting the regulation of GRHL2 may contribute to craniofacial anomalies and NTDs in humans.

Figure 1

Midline cleft lip and cleft palate in Grhl2 over-expressing fetuses. Frontal view of snout in wild-type (A) and Grhl2^Axd/Axd (B) fetus at E18.5 reveals a distinct midline cleft in the homozygous mutant (asterisk in B). (C–E) View of the lip and palate (lower jaw removed) at E18.5. In comparison with wild-type (C), a midline gap is clearly present in Grhl2^Axd/Axd mutants (asterisks in D,E), with cleft of the primary palate evident in a proportion of embryos (arrow in E). (F) Grhl2 mRNA is significantly more abundant in Grhl2^Axd/Axd embryos than wild-type at E9.5 (^*P < 0.05, t-test). (G,H) At E11.5, an abnormal midline groove was detectable between the medial nasal prominences of Grhl2^Axd/Axd embryos (arrow in H). Scale bar represents 50 μm (A–E) and 100 μm (G–H).

Figure 2

Abnormal nasal septum and cleft palate. (A–H) Toluidine blue staining of coronal sections showed abnormal splitting of the nasal septum at anterior and medial levels in Grhl2^Axd/AXd fetuses at E17.5. (I,J) Hematoxylin and eosin–stained sections also revealed splitting of the nasal septum in Grhl2^Axd/Axd (arrows in C,E,G) and the presence of ectopic tissue in the midline (arrow in J). Some fetuses showed failure of fusion of the nasal septum with the primary palate (^* in F,J), with small unfused palatal shelves (F,H). Scale bar represents 250 μm.

Figure 3

Grhl2 over-expression causes anterior and basal encephalocele. (A–I) Examination of fetuses at E18.5 (A–C) and E17.5 (D–I) showed the presence of unilateral bulges in Grhl2^Axd/Axd fetuses that were not present in wild-type (arrow in B,C,H,I) and showed signs of hemorrhage (arrow in H,I). Arrowhead indicates midline cleft lip in I. (J,K) Hematoxylin and eosin–stained coronal sections at the level of the lesion showed protrusion of skin-covered brain tissue (K), with apparent lateral displacement of the frontal bone (arrow in K). (L–O) Coronal sections stained with toluidine blue (L,M) or hematoxylin and eosin (N,O) of E17.5 fetuses without anterior encephalocele revealed the presence of abnormal ventral protrusion of brain tissue, at the level of the olfactory bulbs (arrow in M,O). This basal encephalocele corresponded with apparent deflection of the palatine bone (arrowhead in M). Scale bar represents 1 mm.

Figure 4

Viscerocranium and frontal bone abnormalities in Grhl2^Axd/Axd fetuses. (A–D) Alcian blue and Alizarin red staining of cartilage and bone was used to stain skeletal elements in the head of wild-type (A) and Grhl2^Axd/Axd (B–D) fetuses. All Grhl2^Axd/Axd fetuses showed a midline cleft lip, and additional anomalies included frontal encephalocele (C″). Images in A″-D″ show enlarged views of the boxed area in A–D. Progress of frontal bone ossification appears to be prevented at the site of encephalocele (white arrow in C″), compared with fetuses without encephalocele (white arrow in A″, B″). Additional abnormalities were noted in the anterior portion of the vomer bone (white arrowheads), posterior portion of the palatine process of the premaxilla (yellow arrowheads), premaxillary bone (red arrowheads) and sometimes in the palatine process of the palatine bone. (E,F) μCT analysis of E18.5 fetuses confirmed the presence of abnormal midline spacing of palatine bone (white arrowheads; E,F show three orientations of the same fetuses). bo, basioccipital; bs, basisphenoid, p, palatine bone; ppmx, palatine process of maxilla; pppl, palatine process of palatine; pppx, palatal process of premaxilla; pm, premaxilla; vm, vomer. Scale bar represents 1 mm (A–D and A′–D′), 250 μm (A″–D″).

Figure 5

Characterization of the Axd mutation. (A) Schematic diagram of the region of mouse chromosome 15, which contains the promoter and exon of Grhl2 and Gm16136 lncRNA, with WGS coverage in wild-type (n = 2) and Grhl2^Axzd/Axd (n = 2) samples. Loss of coverage in the Grhl2^Axd/Axd samples is indicated (lines on coverage map) with the corresponding region that was amplified by genomic PCR. Orientation of the inserted IAP transposable element in Grhl2^Axd is indicated (red arrow). (B,C) Genomic PCR, using primer pairs flanking the region with loss of sequence coverage in mutant embryos, generates products of expected size using wild-type DNA as template but not using Grhl2^Axd/Axd DNA (B), whereas a product of larger size (~6 kb) was amplified using long-range PCR (C) in mutant but not wild-type DNA. (D,E) RT-PCR using primer pairs that amplify Gm16136 and (D) flank the insertion site, or (E) include a primer within the inserted sequence, shows that the lncRNA is expressed and has increased size owing to transcription of the inserted sequence in Grhl2^Axd/Axd embryos. (F,G) Quantitative real-time RT-PCR showed that Gm16136 has increased expression in Grhl2^Axd/Axd embryos compared with wild-type. The primer pairs amplify isoform (F) 204 or (G) isoforms 202 and 204 (Supplementary Material, Fig. S4).