Uncommon nucleotide excision repair phenotypes revealed by targeted high-throughput sequencing

Abstract

BackgroundDeficient nucleotide excision repair (NER) activity causes a variety of autosomal recessive diseases including xeroderma pigmentosum (XP) a disorder which pre-disposes to skin cancer, and the severe multisystem condition known as Cockayne syndrome (CS). In view of the clinical overlap between NER-related disorders, as well as the existence of multiple phenotypes and the numerous genes involved, we developed a new diagnostic approach based on the enrichment of 16 NER-related genes by multiplex amplification coupled with next-generation sequencing (NGS).MethodsOur test cohort consisted of 11 DNA samples, all with known mutations and/or non pathogenic SNPs in two of the tested genes. We then used the same technique to analyse samples from a prospective cohort of 40 patients. Multiplex amplification and sequencing were performed using AmpliSeq protocol on the Ion Torrent PGM (Life Technologies).ResultsWe identified causative mutations in 17 out of the 40 patients (43 %). Four patients showed biallelic mutations in the ERCC6(CSB) gene, five in the ERCC8(CSA) gene: most of them had classical CS features but some had very mild and incomplete phenotypes. A small cohort of 4 unrelated classic XP patients from the Basque country (Northern Spain) revealed a common splicing mutation in POLH (XP-variant), demonstrating a new founder effect in this population. Interestingly, our results also found ERCC2(XPD), ERCC3(XPB) or ERCC5(XPG) mutations in two cases of UV-sensitive syndrome and in two cases with mixed XP/CS phenotypes.ConclusionsOur study confirms that NGS is an efficient technique for the analysis of NER-related disorders on a molecular level. It is particularly useful for phenotypes with combined features or unusually mild symptoms. Targeted NGS used in conjunction with DNA repair functional tests and precise clinical evaluation permits rapid and cost-effective diagnosis in patients with NER-defects.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-016-0408-0) contains supplementary material, which is available to authorized users.

Defective transcription of ATF3 responsive genes, a marker for Cockayne Syndrome

“…Gene expression was uploaded at GEO: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE87540). Sample preparation and NGS sequencing of XP genes were described in the same way as in 26 . All primer sequences are available upon request.…”

Prenatal diagnosis of cerebro‐oculo‐facio‐skeletal syndrome: Report of three fetuses and review of the literature

“…After genetic counseling, a molecular analysis was performed on clinical basis on fetal DNA by high throughput sequencing of a custom panel of 104 genes involved in cortical malformations, including lissencephaly, without identifying any pathogenic or potentially pathogenic variant. According to the organization of the laboratory, a panel of 17 genes involved in NER was also captured (Calmels et al, ) in the same assay and sequencing revealed heterozygous nonsense variants in ERCC5 (NM_000123.3): c.1034T>A, p.(Leu345*) in exon 8 and c.2038C>T, p.(Gln680*) in exon 9. Targeted Sanger sequencing of ERCC5 exons 8 and 9 on the fetus and his parents confirmed the compound heterozygosity (variants in exon 8 and 9 inherited from the mother and the father, respectively).…”

“…An ERCC8/CSA defective cell line, an ERCC4/XPF defective cell line and a normal cell line were used as controls. Recovery of RNA synthesis (RRS) and unscheduled DNA synthesis (UDS) were performed and evaluated as previously described (Calmels et al, ). Fetal amniocytes showed a decreased RRS level similarly to the CSA defective cell line, and a decreased UDS level similarly to the XPF defective cell line, indicating a decreased efficiency of the TC‐NER and the GG‐NER, respectively (Supporting Information Figure S2).…”

Heterogeneity and overlaps in nucleotide excision repair disorders

“…Both XP‐B and XP‐D cohorts include pure XP cases with minimal neurological abnormalities as well as combined XP/CS presentations of varying severity. Two couples of young siblings with a suspected clinical diagnosis of UVSS have been also recently reported to carry alterations in either XPB or XPD . Diagnosis is however difficult in young patients because skin cancers may develop later in life.…”

“…Circles flanking the alterations indicate the associated clinical phenotypes: XP (close blue), CS or COFS (close red), TTD (close green), FA (close light blue), XFE (yellow), XP/CS (open red), XP/TTD (open green), XP with HD‐like (open black), XP/CS/FA (open light blue). The symbol § refers to patients recently reported with suspected UVSS for whom, because of young age, long‐term follow‐up is needed to confirm clinical diagnosis. In XPD, the asterisks denote the identical combination of mutated alleles (p.[R112H];[D681H]) exceptionally found in two patients with TTD and XP/CS .…”