Identification and Characterization of a Novel Recurrent ERCC6 Variant in Patients with a Severe Form of Cockayne Syndrome B

Abstract

Cockayne syndrome (CS) is a rare disease caused by mutations in ERCC6/CSB or ERCC8/CSA. We report here the clinical, genetic, and functional analyses of three unrelated patients mutated in ERCC6/CSB with a severe phenotype. After clinical examination, two patients were investigated via next generation sequencing, targeting seventeen Nucleotide Excision Repair (NER) genes. All three patients harbored a novel, c.3156dup, homozygous mutation located in exon 18 of ERCC6/CSB that affects the C-terminal region of the protein. Sanger sequencing confirmed the mutation and the parental segregation in the three families, and Western blots showed a lack of the full-length protein. NER functional impairment was shown by reduced recovery of RNA synthesis with proficient unscheduled DNA synthesis after UV-C radiations in patient-derived fibroblasts. Despite sharing the same mutation, the clinical spectrum was heterogeneous among the three patients, and only two patients displayed clinical photosensitivity. This novel ERCC6 variant in Tunisian patients suggests a founder effect and has implications for setting-up prenatal diagnosis/genetic counselling in North Africa, where this disease is largely undiagnosed. This study reveals one of the rare cases of CS clinical heterogeneity despite the same mutation. Moreover, the occurrence of an identical homozygous mutation, which either results in clinical photosensitivity or does not, strongly suggests that this classic CS symptom relies on multiple factors.

Keywords: Cockayne syndrome; DNA repair disorder; ERCC6; accelerated aging; neurodegeneration.

Figure 1

Pedigree of three unrelated Tunisian families. (A) pedigree of the CS10 family (B) pedigree of the CS12 family (C) pedigree of the CS14 family. The studied proband is indicated with an arrow.

Figure 2

MRI images of patient CS10. (A) Axial T1-weighted image, (B,C) axial T2-weighted images, and (D,E) axial FLAIR-weighted images, showing isointensity of periventricular white matter on T1 and hyperintensity on T2. FLAIR suggestive of hypomyelinating leukodystrophy (red arrows). (F) CT scan shows lenticular calcifications (green arrow).

Figure 3

Genetic analysis. (A) Electropherogram showing the c.3156dup mutation at homozygous state in CS12, at a heterozygous state in the mother CS12-P, compared to a wild-type sample. (B) Schematic representation of the domains in the CSB protein (upper panel) and the novel variant (lower panel). The inverted red triangle represents the frameshift mutation and the red arrow the stop codon 8 aa downstream.

Figure 4

Western blot of CSB. (A,C) α-CSB rabbit polyclonal Abcam. (B,D) α-CSB rabbit polyclonal Bethyl. (A,B): the full-length form of the CSB protein is absent from CS10 and CS14 fibroblasts whereas it is present in a healthy control. (C,D): larger-sized Western blots at a lower exposure than in (A,B); arrows indicate the expected position of the CSB-piggyBac fusion protein (which is more abundant than the full-length CSB); hatched rectangles correspond to the part of the blot shown in (A,B). Lower panels: immunoblot of the loading control GAPDH.

Figure 5

Response to UV radiation in fibroblasts. CS10 and CS14 fibroblasts compared to multiple CS, a XPF, and a healthy control. (A) RRS 24h after UV irradiation expressed in percentage of recovery after 5-EU incorporation, showing the defect of RNA Synthesis after UV exposure in CS fibroblasts. (B) UDS expressed in arbitrary units (a.u.) of 5-EdU fluorescence intensity. Differently from XP, CS patients show normal levels of unscheduled DNA synthesis.