CSA mutations

We have identified 32 pathogenic genetic variants in CSA, of which 25 have not been reported previously. Six were missense mutations, all but one previously unreported, and one a small in-frame indel. Missense mutations are indicated below the CSA linear structure (figure 1A) with the previously unreported alterations indicated in bold. CSA comprises a seven-bladed WD40 propeller attached to the DDB1 protein via a helix-loop-helix motif at the N-terminus.12 All the newly identified missense mutations affect residues that are conserved among CSA orthologs and are located in WD40 repeats. Together with previously reported mutations, indicated above the CSA linear structure, there is a particularly high concentration of missense mutations around aa 200 (4 mutations within 12 aa) and aa 270 (3 mutations within 7 aa). All the 13 missense mutations are located in the blades of the beta propeller structure, with 8 of them clustered in blades 4 and 5 (figure 1B), and are likely to disrupt the structure of the protein.12

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Figure 1

Mutation distribution in CSA. (A) Distribution of missense mutations across the CSA protein. Mutations identified in this study are indicated below the protein with new mutations indicated in bold. Other previously identified missense mutations are indicated above the protein; 1–7 indicate the seven WD40 domains of the CSA protein according to the reference sequence NP_000073.1. (B) Missense mutations associated with different CS phenotypes mapped onto the three-dimensional CSA protein structure (RCSB PDB, DOI: 10.2210/pdb4a11/pdb11 12). Yellow, UVSS; orange, CS type I; violet, CS type II; dark grey, CS type III. (C) Distribution of truncation mutations. Mutations have been grouped in intervals of 40 aa and columns represent the number of mutations for each group. The interval 0–40 includes mutations resulting in no transcript (asterisks). Black: new mutations identified in this study; grey: previously reported mutations also present in this study; white: other previously reported mutations.

All the missense mutations are predicted to be pathogenic using Polyphen, MutPred2 and SIFT (see online supplemen tary table 1). We have confirmed their defective function by transducing a CS-A cell line, CS9LO, with virus containing the mutant cDNA (figure 2A, B). Wild-type CSA almost completely restored RRS to the recipient UV-irradiated CS9LO cell line. In contrast, when the cells were transduced with any of the six mutant cDNAs, RRS remained close to the level of the untransduced cells (figure 2A). The infection efficiency was similar for all transductions (figure 2B).

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Figure 2

Lack of complementation with CSA and CSB mutations. Wild-type and various mutant ERCC8/CSA (A, B) or ERCC6/CSB (C, D) cDNAs were ectopically expressed by recombinant lentivirus infection in fibroblasts derived from a patient with CS-A, CS9LO or patient with CS-B, CS10LO, respectively. (A, C) Recovery of RNA synthesis activities were detected 12 hours after UV irradiation (filled bars, 12 J/m² UVC irradiation; open bars, no UV irradiation), and the value was normalised to activity measurement in non-irradiated cells. (B, D) Viral infection efficiency was confirmed by immunofluorescent staining of V5-tagged wild-type and mutant CSA or CSB proteins, and calculated as the number of Alexa 488-positive cells using a semi-automatic VTI system. w/o, without virus infection; w.t., wild type. Results from at least three independent experiments. Error bars indicate SD.

We have identified 19 protein-truncating genetic variants, of which 14 are new, that include frameshift, splicing and premature stop mutations. They are predicted to result in 18 distinct truncated proteins (12 new), because we found that the Thr134Leufs*13 truncation (due to exon 5 deletion, ie, r.400_481del) is caused by two distinct mutations affecting either the splice acceptor site of intron 4 (c.400-2A>G in CS261ST) or the splice donor site of intron 5 (c.479C>T in CS133NY). In addition, two mutations resulting in large in-frame deletions (p.Val282_Gln347del, Val282_Glu374del and p.Val27_Arg92del) are respectively predicted from c.966C>A and a previously unreported rearrangement involving part of intron 2 and exon 3, which results in a transcript lacking exons 2 and 3 (patient CS1LE). Interestingly, the genomic mutation c.966C>A (in exon 10), previously described as resulting in a single, full-length normal-spliced transcript (r.966c>a, p.Tyr322*),39 was shown to generate also two abnormally spliced transcripts carrying the deletion of exons 10 and 11 (r.844_1122del; p.Val282_Glu374del in CS9IAF) and/or the deletion of exon 10 (r.844_1041del; p.Val282_Glu347del in CS5IAF and CS9IAF). Indeed, this mutation is predicted to alter an exonic splicing enhancer (ESE) site, and potentially alter the splicing as indicated by the bioinformatics tool HSF.

Finally, at the genomic level we identified three new CSA genetic variants located in splice donor sites (c.481+1G>C, c.1041+1G>T and c.1122+1 delG) most probably affecting splicing. The resulting transcripts could not be identified because of the unavailability of RNA samples. However, HSF analysis indicated potential splicing alterations for the three mutations as well as the activation of an intronic cryptic donor site for the latest two.

CSB mutations

In CSB, we identified 73 pathogenic genetic variants of which 46 were previously unreported. Ten of the mutations were missense, 9 in-frame deletions (2 small and 7 large), 5 null mutations leading to unexpressed transcripts and the rest being truncations resulting from stop (16), frameshifts (20) or splice mutations (13). The 10 missense mutations are indicated in figure 3A, below the CSB linear structure, with the 6 new mutations indicated in bold. Other previously reported missense mutations are shown above the CSB linear structure.

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Figure 3

Mutation distribution in CSB. (A) Distribution of missense mutations across the CSB protein. Mutations identified in this study are indicated below the protein with new mutations indicated in bold. Other missense mutations reported as pathogenic are indicated above the protein, with those now classified as polymorphic variants in parenthesis. Different domains of the protein are indicated: A, acidic domain; N, nuclear localisation domain; I, IA, II–VI, helicase-like domains; U, ubiquitin-binding domain. (B) Distribution of truncation mutations. Mutations have been grouped in intervals of 100 aa and columns represent the number of mutations number for each group. The interval 0–100 includes mutations resulting in no transcript (asterisks). Black: new mutations identified in this study; grey: previously reported mutations also present in this study; white: other previously reported mutations.

All missense mutations are predicted to be pathogenic (see online supplementary table 1). We have confirmed the defective function of several of them by transducing a CS-B cell line, CS10LO, with virus containing the mutant cDNA (figure 2C, D). Similar to the CSA data, wild-type CSB almost completely restored RRS to the recipient UV-irradiated CS10LO cell line, whereas with the six mutant cDNAs tested, RRS remained close to the level of the untransduced cells (figure 2C). The infection efficiency was similar for all transductions (figure 2D).

The distribution of CSB truncation mutations is presented in figure 3B (lower panel). Some general conclusions may be drawn from the present in-depth investigation together with previous studies (data up to 2010 reviewed by Laugel et al,39 and since 2011 listed in online supplementary table 2). With two exceptions, all of the CSB missense mutations are located either in or very close to the seven helicase domains, in particular in domains I and III (four mutations each) and domains IV–VI (9 mutations within 110 amino acids), emphasising the crucial role of these domains in CSB function. These helicase domains are involved in the DNA-dependent ATPase activity of the protein and confirm that this activity is vital for preventing the features of CS. The N-terminal and C-terminal extensions are likely to be much more amenable to genetic variants that do not affect function, despite the demonstration that the C-terminal part of the protein is essential for a normal cellular response to UV irradiation.13 16 This may imply that the structure of the C-terminal ubiquitin—binding domain needs to be intact, but the precise amino acid sequence is less crucial. Again, as might be anticipated, the truncation mutations are spread rather evenly across the protein (figure 3B).

Interestingly, the c.1834C>T and c.2143G>T result in p.Arg612* and p.Gly715*, respectively, and their transcript was detected only in homozygous or hemizygous patients. This transcript must be poorly expressed, presumably because of nonsense-mediated decay, because in compound heterozygotes only the transcript resulting from the second allele was detected.

Furthermore, several genetic variants affect ERCC6/CSB splicing giving rise to either truncations or in-frame deletions. In particular, 10 mutations map at the canonical splice sites of different exons, four are located inside introns (c.1993-5A>G; c.2599-26A>G, c.1685+6T>G, c.543+4 delA) and three inside exons (c.466C>T, c.526C>T and c.2092_2093insG). All the exonic changes are likely to alter the splicing by creating novel exon-splicing enhancer (ESE in the case of c.466C>T) or exon-splicing silencer (ESS for c.526C>T and c.2092_2093insG) sites according to HSF prediction.

Recurring pathogenic genetic variants

Mutations found in several patients are indicative of either founder effects or mutation hotspots. Although haplotype analysis would be required to distinguish definitively between these two alternatives, as a first approximation, we assume that if the pathogenic genetic variant is only found in a relatively limited geographical location, it is more likely to be a founder effect. In ERCC8/CSA, we found a complex rearrangement involving exon 4 in 8 Japanese patients, also previously reported in four Japanese patients by Ren et al,40 strongly indicating a founder mutation. Moreover, the c.966C>A mutation found in two patients of our cohort (CS9IAF, CS5IAF) was also previously described in three cases (CS2IAF, CS886VI/CS887VI).39 Since all the patients are of Arabic origin, although from different countries (Israel or Lebanon), this is also most likely a founder mutation.

Nine CSB/ERCC6 pathogenic genetic variants occur in three or more patients (table 3). The most common of these multiple occurrences are c.2203C>T, c.2167C>T and c.466C>T, respectively found in 12, 11 and 7 patients. Whereas c.2167C>T and c.466C>T are found almost exclusively in the UK patients and may result from founder effects, c.2203C>T is found in individuals from several different countries and likely results from independent mutations. Interestingly, the C>T mutations in table 3 that are more likely to result from a founder mutation are at CpA sites, whereas those more likely to result from independent mutations are at CpG sites. CpG sites are known to be mutational hotspots in the human genome.41

Relationship to clinical features

No obvious genotype-phenotype correlation was identified in the patients with CS-A reported in previous investigations (45 cases from 33 families). With the present study, we have expanded the cohort of patients with CS-A by describing 39 new cases, the majority of which are homozygotes. Focusing on the homozygous patients with CS-A (33 from 24 families in the literature and 30 from 30 families in our cohort, excluding the 9 Japanese cases with a recurrent mutation), missense mutations appear to be more frequently associated with mild phenotypes than protein-truncating mutations. The observation that the missense alteration p.Trp361Cys, which interferes with transcription-coupled NER but not with the oxidative stress response, is associated with UVSS, a rare disorder characterised only by cutaneous photosensitivity,20 strongly supports the notion that the severity of the clinical features is related to the effects of the mutation on the additional roles of CSA outside transcription-coupled NER, which include oxidative damage response, mitochondrial function maintenance and ribosomal DNA transcription.

Previous analysis of mutations in patients with CS-B (51 homozygous cases from 29 families/kindreds and 37 compound heterozygotes from 32 families) have not identified any clear correlation between the site or the nature of the mutations with the type and severity of the clinical features,42 43 although some more subtle relationships have been suggested. Several years ago, Horibata et al suggested that CSB truncations generating no functional protein resulted in the mild phenotype of UVSS, whereas more C-terminal truncations might generate inactive protein that could interfere with other processes, thereby resulting in more severe phenotypes.19 Weiner and colleagues showed that the human ERCC6/CSB gene contains a PiggyBac transposon insertion in intron 544 45 (see table 2 and figure 3). They showed that translation of ERCC6/CSB resulted in bona fide CSB protein, but also a CSB-PiggyBac fusion protein. Truncation mutations upstream of intron 5 would generate neither protein, whereas those downstream would generate only the CSB-PiggyBac fusion, which was proposed to have deleterious effects.44 We have analysed the severity of the clinical features in our patient cohort to see if they are in accord with these suggestions. In eight patients homozygous for truncations in the first five exons, six could be categorised as type I, and one as type II. No information is available for one patient. In contrast, in 28 patients homozygous for truncations downstream of exon 5, the numbers assigned to types I, II and III are 5, 12 and 3, respectively. There thus appears to be a tendency to more severe phenotypes (type II) associated with downstream truncations, although this does not seem to be an absolute correlation. Patients with truncations upstream of the Piggy-Bac insertion but severe clinical features have been reported previously.46 Furthermore, of the four patients homozygous for the mutation Asp1355Valfs*32, two were classified as type I and two as type III (see table 2). Altogether, these observations indicate that other factors, apart from the site of mutation, contribute to the severity of the pathological phenotype.

In an earlier analysis, Laugel suggested that type II features were more prevalent in patients with CS-B than in patients with CS-A.6 This is supported by our current data. The distributions for those patients for whom we have clinical data for types I, II and III are 67%, 21% and 12.5% for CS-A (21 patients) and 35%, 56% and 10% for CS-B (60 patients), respectively. The individual clinical features for which we have information are summarised in table 4, where they are also compared, where possible, with data from a recent analysis of 102 CS individuals by Wilson et al.4 Within our own cohort, there are few differences between patients with CS-A and CS-B, with the possible exceptions of cataracts, low birth weight and microphthalmia, which are more prevalent in patients with CS-B. The incidence of several features appears to be higher in our cohort than in that studied by Wilson et al (see table 4). Two possible explanations for this are: (1) they could represent genuine differences between the two cohorts; (2) the analytical clinical criteria may differ between the two studies. Of the patients subjected to molecular analysis in4 the ratio of CS-B to CS-A cases is very similar to that reported here.

In a recent survey of patients with CS in Japan, nearly all of them (41/47) were categorised clinically as type I.3 Unfortunately, this survey did not include molecular analyses. However, our data strongly suggest that there is a ERCC8/CSA founder mutation in Japanese patients with CS. We may extrapolate this to suggest that many of the patients analysed in the survey by Kubota et al are likely also to have carried this founder mutation. As mentioned above, patients with CS-A are more likely to fall into the type I category. The features of the 41 Japanese patients with type I CS are also included in table 4. Deafness, photosensitivity and retinal degeneration appear to be higher in the Japanese cohort. This may be partially explained by the average age of the Japanese patients (17.5 years), which appears to be significantly higher than in our cohort. Deafness and retinal degeneration are progressive and therefore more likely to occur in older patients.

As also reported in earlier studies, clinical photosensitivity was found in the majority of our patients, even those with skin types IV and V on the Fitzpatrick Skin Type Scale (see tables 1, 2 and 4). Nevertheless, as in other reports,47 we found no skin cancers in any of our patients. This may be explained by a recent finding that CS fibroblasts are not hypermutable by UV radiation.48

In conclusion, our analyses show that the human mutation spectrum of the CS genes is not yet saturated, but missense mutations are largely confined to a few relatively short regions. There are no definitive correlations between genotype and phenotype, but truncation mutations C-terminal to the PiggyBac insertion in ERCC6/CSB are more likely to confer a severe clinical phenotype than mutations N-terminal to this insertion or mutations in ERCC8/CSA.