FIGURE LEGENDS
Figure 1. A. Nucleotide Excision Repair (NER) Pathway and brain imaging of patient INE4CC. Global Genome NER (left) and Transcription Coupled NER (right) converge. Representative Xeroderma Pigmentosum and Cockayne Syndrome patients shown. Photo Credit: Child with xeroderma pigmentosum in Rukum, Nepal by Lax and Child with Cockayne Syndrome by Eric Bixel / Creative Commons BY 4.0. 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.ERCC6GAPDH expression. B . Whole cell extracts were prepared from the aforementioned cells and CSB protein levels were detected by immunoblotting. ⍺Tubulin served as a loading control.C. Measurement of UV-induced unscheduled DNA synthesis (UDS) in dermal fibroblast lines (C5RO: unaffected donor; XP51RO: patient with a mutation in XPF affecting NER; the patient in this study: INE4CC). Cells were UV-C- or sham-irradiated and afterwards cells were 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. Measurement of RNA synthesis in fibroblasts following UV irradiation. Expression of the house-keeping genesDHFR and GAPDH was measured after UV-C irradiation of cells from C5RO (normal control), XP51RO (XPF mutation with a diagnosis of XFE progeroid syndrome), CS20LO (CS caused by a mutation inERCC1 ), and the patient in this study (INE4CC). Expression was measured at baseline (no UV) and at 6 and 24 hr post-irradiation and normalized to the amount of 18s rRNA. The results were plotted as the ratio of expression in irradiated vs. sham-irradiated cells. qPCR reactions were performed in triplicate for five independent experiments. Values represent mean ± SD, ns 0.05, **p <0.01, ***p <0.001, ****p <0.0001 measured by unpaired two-tailed Student’s t test or one-way ANOVA with Tukey’s test.