Truncating mutations in the last exon of NOTCH3 cause lateral meningocele syndrome

Abstract

Lateral meningocele syndrome (LMS, OMIM%130720), also known as Lehman syndrome, is a very rare skeletal disorder with facial anomalies, hypotonia and meningocele-related neurologic dysfunction. The characteristic lateral meningoceles represent the severe end of the dural ectasia spectrum and are typically most severe in the lower spine. Facial features of LMS include hypertelorism and telecanthus, high arched eyebrows, ptosis, midfacial hypoplasia, micrognathia, high and narrow palate, low-set ears and a hypotonic appearance. Hyperextensibility, hernias and scoliosis reflect a connective tissue abnormality, and aortic dilation, a high-pitched nasal voice, wormian bones and osteolysis may be present. Lateral meningocele syndrome has phenotypic overlap with Hajdu-Cheney syndrome. We performed exome resequencing in five unrelated individuals with LMS and identified heterozygous truncating NOTCH3 mutations. In an additional unrelated individual Sanger sequencing revealed a deleterious variant in the same exon 33. In total, five novel de novo NOTCH3 mutations were identified in six unrelated patients. One had a 26 bp deletion (c.6461_6486del, p.G2154fsTer78), two carried the same single base pair insertion (c.6692_93insC, p.P2231fsTer11), and three individuals had a nonsense point mutation at c.6247A > T (pK2083*), c.6663C > G (p.Y2221*) or c.6732C > A, (p.Y2244*). All mutations cluster into the last coding exon, resulting in premature termination of the protein and truncation of the negative regulatory proline-glutamate-serine-threonine rich PEST domain. Our results suggest that mutant mRNA products escape nonsense mediated decay. The truncated NOTCH3 may cause gain-of-function through decreased clearance of the active intracellular product, resembling NOTCH2 mutations in the clinically related Hajdu-Cheney syndrome and contrasting the NOTCH3 missense mutations causing CADASIL.

Keywords: Hajdu-Cheney syndrome; Lehman syndrome; NOTCH3; PEST domain; dural ectasia; lateral meningocele syndrome.

FIG. 1

Photographs of Individual 1 at age 24 years showing face and head (A and B) with arched eye brows, ptosis, flat midface, thin upper lip, low-set and posteriorly angulated ears and low posterior hairline, and hands (C and D) with short and wide distal 2nd and 3rd fingers (pseudo-clubbing). Individual 20 ‘s facial features at age 13 years (E) are similar to those at age 4 years (F and G) with coarse and curly hair, tall forehead, high arched brows, ptosis, midfacial hypoplasia, long flat philtrum and thin upper lip, micrognathia and low-set ears. Facial photographs (H and I) of Individual 26 at age 6 years, showing a high forehead, shallow supraorbital ridges with arched eye brows, ptosis, flat midface, thin and tented upper lip, low-set and posteriorly angulated ears, low posterior hairline and a submandibular scar with keloid formation.

FIG. 2

Sagittal T2-weighted MR images of the spine from Individual 7 at age 8 years showing numerous lateral meningoceles increasing in size from the thoracic (A) to the lumbar spine (B); from Individual 26 showing numerous lateral meningoceles increasing in size from the high thoracic (C) to the lumbar region (D); and similar findings seen on MR images obtained at age 5 years from Individual 28 (E, F).

FIG. 3

Photographs of Individual 28 at age 12 months (A) and 5 2/12 years (B–D), showing dolichocephaly, high forehead, shallow supraorbital ridges with high arched eyebrows, telecanthus and ptosis, a short nasal tip, thin upper lip, midfacial hypoplasia, low-set and posteriorly angulated ears and coarse hair.

FIG. 4

Diagram of NOTCH3 mutations in lateral meningocele syndrome, not to scale. (A) Structural organization of the NOTCH3 receptor: NOTCH3 is expressed on the cell surface as a heterodimer composed of a large extracellular domain non-covalently linked to the intracellular domain. The extracellular domain contains 34 epidermal growth factor (EGF-like) repeats and 3 cysteine-rich repeats (LNR). The intracellular domain includes a RAM23 domain; nuclear localizing signals (NLS); and ankyrin repeats (ANK) for protein protein interaction. At the carboxyl terminus, the PEST domain is a region rich in proline (P), glutamine (E), serine (S) and threonine (T) residues used for protein degradation. A close up of exon 33's structure indicates the location of the 5 different de novo DNA variants identified in this study. Panel (B) shows the sequencing chromatogram for each of the six individuals. All sequences are presented 5′ -> 3′. Double sequences indicate the frameshift mutations in patient 1, 7, and 15. Asterisk (*) indicates a common SNP (rs 1044009) identified in Individual 15.

FIG. 5

Diagram of normal and abnormal NOTCH signaling. Binding of a Notch ligand to the membrane bound NOTCH receptor triggers proteolytic cleavage (indicated by red arrows) and release of the Notch intracellular domain (NICD). NICD translocates to the nucleus, where it binds to specific transcription factors (not shown). This activated complex directs the transcription of target genes. In wildtype NOTCH3, the PEST sequence (red box) serves as a signal for protein degradation by the proteasome machinery and controls the intracellular half-life of NICD. In mutant NOTCH3, truncation of the C-terminal PEST domain presumably impairs proteasome degradation of NICD (Stop sign), resulting in an extended half-life of NICD and prolonged signaling (gain-of-function effect).