Warsaw breakage syndrome: Further clinical and genetic delineation

Abstract

Warsaw breakage syndrome (WBS) is a recently recognized DDX11-related rare cohesinopathy, characterized by severe prenatal and postnatal growth restriction, microcephaly, developmental delay, cochlear anomalies, and sensorineural hearing loss. Only seven cases have been reported in the English literature, and thus the information on the phenotype and genotype of this interesting condition is limited. We provide clinical and molecular information on five additional unrelated patients carrying novel bi-allelic variants in the DDX11 gene, identified via whole exome sequencing. One of the variants was found to be a novel Saudi founder variant. All identified variants were classified as pathogenic or likely pathogenic except for one that was initially classified as a variant of unknown significance (VOUS) (p.Arg378Pro). Functional characterization of this VOUS using heterologous expression of wild type and mutant DDX11 revealed a marked effect on protein stability, thus confirming pathogenicity of this variant. The phenotypic data of the seven WBS reported patients were compared to our patients for further phenotypic delineation. Although all the reported patients had cochlear hypoplasia, one patient also had posterior labyrinthine anomaly. We conclude that while the cardinal clinical features in WBS (microcephaly, growth retardation, and cochlear anomalies) are almost universally present, the breakage phenotype is highly variable and can be absent in some cases. This report further expands the knowledge of the phenotypic and molecular features of WBS.

Keywords: DDX11; Warsaw breakage syndrome; cochlear anomalies; cohesinopathy; exome sequencing; growth restriction; hearing loss; microcephaly.

Fig. 1:. CT and MRI of the inner ear in order of the degree of cochlear hypoplasia. Patient 1 (a-d), patient 3 (e-h), patient 2 (i-l) and patient 4 (m-p).

Axial CT image (a) demonstrates lateral tapering (arrow) of the basal turn at the junction of the ductus reuniens. Axial CT (b) and axial T2W (c) images demonstrate a cystic cochlea (arrows) and a persistent anlage of the lateral semicircular canal (open arrows). Sagittal T2W image (d) reveals 4 nerves in the internal auditory canal. The cochlear nerve (arrow) is present, but smaller than the other nerves. Axial CT (e) demonstrates a hypoplastic basal turn and cochlea (arrow). Axial CT (f) demonstrates an amorphous cystic cochlea (arrow) and a hypoplastic cochlear nerve canal (black arrow). Axial CT image (g) demonstrates a persistent anlage of the posterior semicircular canal (open arrow), lacking a bone island. Axial CT (h) reveals a truncated superior semicircular canal with only the anterior limb (arrow). The apex and posterior limb are absent. Axial CT image (i) reveals a tiny cochlea (arrow). Axial CT image (j) reveals a normal bone island within the lateral semicircular canal (open arrow). Axial T2W (k) image shows a tiny cochlea (arrow). Sagittal T2W image (l) reveals only 2 nerves (facial and vestibular nerves) (arrow) in the internal auditory canal. The cochlear nerve is absent and the internal auditory canal is small. Axial CT (m) reveals absent basal turn of the cochlea (*), while axial CT (n) demonstrates a hypoplastic cochlea (black arrow). 3D surface reconstruction (o) also confirms cochlear hypoplasia (arrow) and absent basal turn, while lack of cochlear nerve (white arrow) is shown on axial T2W MRI (l). The semicircular canals and vestibule are normal.

Fig. 2:. MRI findings on patient 1, 2 and 5.

Patient 1 (a/a¹), Patient 2 (b/b¹) MRI images obtained at 8 months of age. Patient 5 (c/c1) MRI obtained at 15 months of age. All 3 patients show similar microcephaly with simplified gyri and age appropriate myelin maturation. There is metric breaking in all, consistent with early suture closure. Sagittal T1W image (a) of patient 1 demonstrates a normal corpus callous, brainstem and vermis. Axial T2W image (a¹) shows normal ventricular size. Sagittal T2W image (b) in patient 2 shows similar findings. Axial T2W image (b¹) in patient 2 shows mild ventricular and pericerebral CSF prominence (correlate with HC in each). Sagittal T1W image (c) in patient 5 reveals very mild thinning of the corpus callosum, small cerebellar vermis and mildly prominent 4^th ventricle. Axial T1W image (c¹) in patient 5 demonstrates normal lateral and third ventricular size and pericerebral CSF spaces.

Fig. 3:. Facial features of individuals with WBS.

(A1-2) patient 1 at 2 years of age showing flat supraorbital ridges, depressed nasal bridge, broad nasal tip, overhanging columella, normal philtrum, prominent upper lip, retrognathia and small cupped ears.; (B1-2) patient 2 at 2 years of age with a short forehead, triangular and small face, arched eyebrows, deep-set eyes, flat nasal bridge, short prominent nose with overhanging columella, short philtrum, full lips and retrognathia.; (C1-2) patient 3 at 6 years of age. Note short forehead, small face, deep-set eyes, epicanthic folds, droopy eyelids, depressed nasal bridge, overhanging columella, short philtrum, full lips, and retrognathia.; (D1-2) patient 5 at 5 years of age (D1) and at 6 years of age (D2) with a triangular small face, receding short forehead, prominent eyes, beaked nose, narrow nostrils, overhanging columella, short philtrum, thin lips and retrognathia, posteriorly rotated ears with marked prominence of antihelix and small and attached earlobes with prominent targus.

Fig.4:. Lower limbs of individuals with WBS.

(A) patient 2 at 2 years of age with pes planus, clinodactyly of the left 5^th toe. The 5^th toe overlaps the 4^th toe, bilaterally. On the right, the 4^th toe overlaps the 3^rd toe. The 5^th toe was proximally inserted, and there was 2/3 toes partial syndactyly, bilaterally. (B) patient 3 at 6 years of age with bilateral clinodactyly of the 5^th toe and hypoplastic toe nails. On the right, the left 5^th and 4^th toes were proximally inserted and there was 2/3 toes partial syndactyly. (C) patient 5 at 5 years of age with bilateral clinodactyly of the 3^rd, 4^th, and 5^th toes and the 2^nd toe overlaps the 3^rd toe.

Fig. 5:. WBS-linked Arg-378-Pro variant in DDX11.

(A) Location of disease-linked DDX11-R378P variant with respect to conserved helicase core motifs and Fe-S cluster. Conserved helicase motifs are shown in red and Fe-S motif is highlighted in yellow. Previously published variants in DDX11 shown in black and the novel variants from this series shown in blue. (B) Sequence alignment (Muscle Multiple Sequence Alignment) of region spanning R378 residue of DDX11 and related Fe-S containing DNA helicases XPD, FANCJ, and RTEL-1. (C) Coomassie-stained SDS-PAGE gel to analyze FLAG-M2 resin affinity-purified FLAG-tagged DDX11-WT and DDX11-R378P recombinant proteins expressed in 293T cells. E-elution. The intact full-length DDX11-R378P protein was hardly detectable in the eluted fraction compared to the full-length DDX11-WT protein. (D) Western blot analysis and quantification of DDX11 protein samples. Panel A. Sup- the supernatant obtained after centrifugation of cell lysates; Elution-Proteins obtained after 3X FLAG peptide elution in the final stage of purification. The quantification shows ~5-fold greater in wild-type DDX11 protein compared to the R378P DDX11 protein. (E) Western blot analysis of whole cell lysate protein from HeLa cells expressing recombinant DDX11-WT or DDX11-R378P. Actin serves as a loading control.

Fig.6:. Predicted and apparent destabilization of DDX11 protein by R378P mutation.

(A) Alignment of secondary structure of DDX11 based on Thermoplasma acidophillum (Ta) XPD as a template using Phre2 protein fold recognition server. Region of secondary structure spanning R378 amino acid (in Block) is shown. (B) Predicted modeled structure of DDX11 protein. Human DDX11 protein was modeled by Phyre2 protein fold recognition server. Structure is based on homology modeling using TaXPD (Kuper et al., 2012). Arg-378 with side chain (ball and stick) is shown and indicated in edged red surface. (C) Effect of Proteasome inhibitor MG132 on the expression of DDX11 proteins. 293T cells transfected with DDX11-WT, DDX11-R378P#1 (R378P#1), or DDX11-R378P#2 (R378P#2) were either treated with MG132 (10 μM) or DMSO used as a not treated (NT) for 14 h. Cell lysates were prepared and resolved by SDS-PAGE, followed by Western blot detection with FLAG antibody. Actin was used as a loading control.

Fig.7:. C-banding of metaphase chromosomes in patients 1 and 2. Short arrows show chromosome morphology suggestive of PCD; long arrows show chromosomes with PCS

(A) Representative metaphase from untreated (0 dose) culture of patient 1; (B) Thymidine-synchronized culture of patient 2.