Clinical, functional, and genetic analysis of NER defective patients and characterization of five novel XPG mutations

Abstract Xeroderma pigmentosum (XP), Trichothiodystrophy (TTD), and Cockayne Syndrome (CS) are rare (incidence ~1 to 1 million) recessively inherited genetic diseases arising from genetic defects in the nucleotide excision repair (NER) which is responsible for the removal of UV-induced DNA lesions. Increased UV sensitivity is a common symptom, whereas only XP patients exhibit freckling within sun-exposed skin and a more than 1000-fold increased skin cancer susceptibility. Beyond that, a high phenotypic heterogeneity results in at least seven overlapping phenotypes: XP, XP plus neurological abnormalities, TTD, CS, XP/TTD complex, XP/CS complex, and COFSS (Cerebro-Oculo-Facio-Skeletal Syndrome). Additionally, different mutations affecting the same gene may result in different phenotypes depending on their localization. In this by far largest analysis of 23 NER defective patients in Germany 12 XP-C, eight XP-D, and three XP-G patients were assessed by molecular-genetic characterization of their corresponding fibroblast cells and correlation with their clinical course of disease. Neurological symptoms were absent in all but one of the XP-C patients. Of the XP-D patients, generally phenotypically more variable, five patients exhibited the XP phenotype, two patients the TTD, and one patient the XP/CS complex phenotype. Two of the three XP-G patients exhibited a XP/CS complex phenotype. All patients’ fibroblasts showed an increased UV sensitivity and a decreased NER capacity compared to wild type fibroblasts. Co-transfection of plasmids expressing XPC, XPD, or XPG cDNA increased relative NER capacity in XP-C, -D, and –G cells, respectively, thereby confirming patients’ complementation groups. The mRNA expression of the mutated genes was determined compared to the mean expression level of nine wild type fibroblast cell cultures set to 100 %. XPC mRNA expression levels were significantly decreased (range 9.5 % – 25.7 %; p< 0.001, Student’s T-test) in all but one XP-C patients’ fibroblasts (274.1 %), whereas XPD and XPG mRNA expression in the corresponding patients’ cells ranged nearly within the SEM of wild type cells. Mutational analysis revealed all XP-C patients being homozygous and identified four novel XPC mutations: p.A116YfsX4 (1/12), p.R475EfsX18 (1/12), p.G723SfsX44 (1/12), and p.I812del (1/12) which is a unique novel mutation resulting in an unusually elevated XPC mRNA expression. The novel XPD mutation, p.D681H (2/8), was identified in patients carrying the TTD-causing mutation p.R112H on the other allele. One patient exhibited TTD- and the other one CS-like symptoms indicating that dominance of the alleles is probably differently influenced by other factors such as epigenetic effects or SNPs. Five novel XPG mutations were identified. Four mutations, p.Q150X with p.L778P and p.E727X with p.W814S, were found in a compound heterozygous and one, p.G805R, in a homozygous state. Correlation of missense mutations with a XP/CS phenotype was rather unexpected. Usually missense mutations impairing NER result in XP, whereas truncating mutations impairing NER and transcription result in XP/CS. Allele-specific complementation analysis of these five novel mutations identified only p.L778P and p.W814S retaining some residual repair activity. In line with the XP/CS phenotypes, even the missense mutations failed to interact with the transcription factor IIH subunits XPD and cdk7 in co-immunoprecipitation assays probably resulting in destabilized TFIIH. Immunofluorescence techniques revealed a mutation-specific effect on early XP protein recruitment to localized photodamage and a delayed redistribution in vivo. In summary, in very rare diseases, novel XPC, XPD, and XPG mutations were identified. Comprehensive analysis of five novel XPG mutations identified the first single amino acids crucial for interaction with TFIIH.