Ending a diagnostic odyssey: Moving from exome to genome to identify cockayne syndrome

Abstract

Background: Cockayne syndrome (CS) is a rare autosomal recessive disorder characterized by growth failure and multisystemic degeneration. Excision repair cross-complementation group 6 (ERCC6 OMIM: *609413) is the gene most frequently mutated in CS.

Methods: A child with pre and postnatal growth failure and progressive neurologic deterioration with multisystem involvement, and with nondiagnostic whole-exome sequencing, was screened for causal variants with whole-genome sequencing (WGS).

Results: WGS identified biallelic ERCC6 variants, including a previously unreported intronic variant. Pathogenicity of these variants was established by demonstrating reduced levels of ERCC6 mRNA and protein expression, normal unscheduled DNA synthesis, and impaired recovery of RNA synthesis in patient fibroblasts following UV-irradiation.

Conclusion: The study confirms the pathogenicity of a previously undescribed upstream intronic variant, highlighting the power of genome sequencing to identify noncoding variants. In addition, this report provides evidence for the utility of a combination approach of genome sequencing plus functional studies to provide diagnosis in a child for whom a lengthy diagnostic odyssey, including exome sequencing, was previously unrevealing.

Keywords: ERCC6; DNA repair; cockayne syndrome; whole-genome sequencing.

FIGURE 1

(A) Nucleotide Excision Repair (NER) Pathway and brain imaging of patient INE4CC. Global Genome NER (left) and Transcription Coupled NER (right) converge. (B) Brain MRI and CT Scan: CT Scan age 5 (a and b) demonstrates diffuse cerebral and cerebellar atrophy. There is dense calcification of the bilateral globi pallidi and parieto‐occipital and left frontal (not shown) cortical calcification. MRI Scan age 6. T1 Images (c and d) show diffuse supra and infratentorial volume loss evidenced by ventriculomegaly, widened cortical sulci, diffuse thinning of the corpus callosum and inferior vermian and brainstem hypoplasia. There was an abnormal basal ganglia signal including mild T2 hyperintensity (not shown) T1 shortening and susceptibility artifact of the lenticular nuclei consistent with mineralization

FIGURE 2

Functional consequences of biallelic ERCC6 variants in INE4CC cells. (a) ERCC6 mRNA was quantified in patient (INE4CC) and control (C5RO) dermal fibroblasts by qRT‐PCR in triplicate using the ∆∆Ct method and normalized to GAPDH expression. (b) CSB protein levels were detected by immunoblotting of whole‐cell extracts with ⍺Tubulin as control. (c) Measurement of UV‐induced unscheduled DNA synthesis (UDS) in dermal fibroblast lines (C5RO: unaffected control; XP51RO: patient with ERCC4 mutation affecting NER; the patient in this study: INE4CC). Cells were UV‐C‐ or sham‐irradiated and then incubated with the thymidine analog EdU to allow time for DNA repair. Alexa Fluor 647 was conjugated to EdU incorporated into the nuclear genome before cell fixation, DAPI staining, and flow cytometric quantification of Alexa Fluor 647 intensity in G₁ cells. UDS was measured in triplicate for each sample and normalized to the control samples. (d) Recovery of RNA synthesis in fibroblasts following UV irradiation. Expression of the house‐keeping genes DHFR and GAPDH was measured after UV‐C irradiation of cells from C5RO (normal control), XP51RO (ERCC4 mutation with a diagnosis of XFE progeroid syndrome), CS20LO (CS caused by mutations in ERCC1), and the patient in this study (INE4CC). Expression was measured at baseline (no UV) and at 6 and 24 hr postirradiation and normalized to quantity of 18s rRNA. Results were plotted as the ratio of expression in irradiated versus sham‐irradiated cells. qPCR reactions were performed in triplicate for five independent experiments. Values represent mean ± SD, ns p ≥ 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared using unpaired two‐tailed Student's t test or one‐way ANOVA with Tukey's test