Heather Fawcett

Molecular analysis of mutations in DNA polymerase η in xeroderma pigmentosum-variant patients

Xeroderma pigmentosum variant (XP-V) cells are deficient in their ability to synthesize intact daughter DNA strands after UV irradiation. This deficiency results from mutations in the gene encoding DNA polymerase η, which is required for effecting translesion synthesis (TLS) past UV photoproducts. We have developed a simple cellular procedure to identify XP-V cell strains, and have subsequently analyzed the mutations in 21 patients with XP-V. The 16 mutations that we have identified fall into three categories. Many of them result in severe truncations of the protein and are effectively null alleles. However, we have also identified five missense mutations located in the conserved catalytic domain of the protein. Extracts of cells falling into these two categories are defective in the ability to carry out TLS past sites of DNA damage. Three mutations cause truncations at the C terminus such that the catalytic domains are intact, and extracts from these cells are able to carry out TLS. From our previous work, however, we anticipate that protein in these cells will not be localized in the nucleus nor will it be relocalized into replication foci during DNA replication. The spectrum of both missense and truncating mutations is markedly skewed toward the N-terminal half of the protein. Two of the missense mutations are predicted to affect the interaction with DNA, the others are likely to disrupt the three-dimensional structure of the protein. There is a wide variability in clinical features among patients, which is not obviously related to the site or type of mutation.

Mutation update for the CSB/ERCC6 and CSA/ERCC8 genes involved in Cockayne syndrome

Cockayne syndrome is an autosomal recessive multisystem disorder characterized principally by neurological and sensory impairment, cachectic dwarfism, and photosensitivity. This rare disease is linked to mutations in the CSB/ERCC6 and CSA/ERCC8 genes encoding proteins involved in the transcription‐coupled DNA repair pathway. The clinical spectrum of Cockayne syndrome encompasses a wide range of severity from severe prenatal forms to mild and late‐onset presentations. We have reviewed the 45 published mutations in CSA and CSB to date and we report 43 new mutations in these genes together with the corresponding clinical data. Among the 84 reported kindreds, 52 (62%) have mutations in the CSB gene. Many types of mutations are scattered along the whole coding sequence of both genes, but clusters of missense mutations can be recognized and highlight the role of particular motifs in the proteins. Genotype–phenotype correlation hypotheses are considered with regard to these new molecular and clinical data. Additional cases of molecular prenatal diagnosis are reported and the strategy for prenatal testing is discussed. Two web‐based locus‐specific databases have been created to list all identified variants and to allow the inclusion of future reports (www.umd.be/CSA/ and www.umd.be/CSB/). Hum Mutat 31:113–126, 2010.

Malfunction of nuclease ERCC1-XPF results in diverse clinical manifestations and causes Cockayne syndrome, xeroderma pigmentosum, and Fanconi anemia.

Cockayne syndrome (CS) is a genetic disorder characterized by developmental abnormalities and photodermatosis resulting from the lack of transcription-coupled nucleotide excision repair, which is responsible for the removal of photodamage from actively transcribed genes. To date, all identified causative mutations for CS have been in the two known CS-associated genes, ERCC8 (CSA) and ERCC6 (CSB). For the rare combined xeroderma pigmentosum (XP) and CS phenotype, all identified mutations are in three of the XP-associated genes, ERCC3 (XPB), ERCC2 (XPD), and ERCC5 (XPG). In a previous report, we identified several CS cases who did not have mutations in any of these genes. In this paper, we describe three CS individuals deficient in ERCC1 or ERCC4 (XPF). Remarkably, one of these individuals with XP complementation group F (XP-F) had clinical features of three different DNA-repair disorders--CS, XP, and Fanconi anemia (FA). Our results, together with those from Bogliolo et al., who describe XPF alterations resulting in FA alone, indicate a multifunctional role for XPF.

Genetic analysis of twenty-two patients with Cockayne syndrome.

Cockayne syndrome (CS) is an autosomal recessive disorder with dwarfism, mental retardation, sun sensitivity and a variety of other features. Cultured CS cells are hypersensitive to ultraviolet (UV) light, and following UV irradiation, CS cells are unable to restore RNA synthesis rates to normal levels. This has been attributed to a specific deficiency in CS cells in the ability to repair damage in actively transcribed regions of DNA at the rapid rate seen in normal cells. We have used the failure of recovery of RNA synthesis, following UV irradiation of CS cells, in a complementation test. Cells of different CS donors are fused. Restoration of normal RNA synthesis rates in UV irradiated heterodikaryons indicates that the donors are in different complementation groups, whereas a failure to effect this recovery implies that they are in the same group. In an analysis of cell strains from 22 CS donors from several countries and different racial groups, we have assigned five cell strains to the CS-A group and the remaining 17 to CS-B. No obvious racial, clinical or cellular distinctions could be made between individuals in the two groups. Our analysis will assist the identification of mutations in the recently clonedCSA andCSB genes and the study of structure-function relationships.

Hypomorphic PCNA mutation underlies a human DNA repair disorder

Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNAs interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration.

Deep phenotyping of 89 xeroderma pigmentosum patients reveals unexpected heterogeneity dependent on the precise molecular defect.

Significance Xeroderma pigmentosum (XP) is a genetic disorder caused by defective repair of DNA damage. Affected patients are mutated in one of eight genes and develop skin pigmentation changes, skin cancers, ocular surface abnormalities, and, in some cases, acute sunburn and neurodegeneration. The XP proteins are involved in different steps in the repair of DNA damage. Examination of 89 patients, the largest reported cohort under long-term follow-up, by the same multidisciplinary team of clinicians and scientists has revealed unexpected clinical heterogeneity dependent on the affected gene and the exact mutation. Our findings provide new insights into the mechanisms of carcinogenesis, ocular surface disease, and neurodegeneration, as well as providing improved clinical management and more definitive prognostic predictions. Xeroderma pigmentosum (XP) is a rare DNA repair disorder characterized by increased susceptibility to UV radiation (UVR)-induced skin pigmentation, skin cancers, ocular surface disease, and, in some patients, sunburn and neurological degeneration. Genetically, it is assigned to eight complementation groups (XP-A to -G and variant). For the last 5 y, the UK national multidisciplinary XP service has provided follow-up for 89 XP patients, representing most of the XP patients in the United Kingdom. Causative mutations, DNA repair levels, and more than 60 clinical variables relating to dermatology, ophthalmology, and neurology have been measured, using scoring systems to categorize disease severity. This deep phenotyping has revealed unanticipated heterogeneity of clinical features, between and within complementation groups. Skin cancer is most common in XP-C, XP-E, and XP-V patients, previously considered to be the milder groups based on cellular analyses. These patients have normal sunburn reactions and are therefore diagnosed later and are less likely to adhere to UVR protection. XP-C patients are specifically hypersensitive to ocular damage, and XP-F and XP-G patients appear to be much less susceptible to skin cancer than other XP groups. Within XP groups, different mutations confer susceptibility or resistance to neurological damage. Our findings on this large cohort of XP patients under long-term follow-up reveal that XP is more heterogeneous than has previously been appreciated. Our data now enable provision of personalized prognostic information and management advice for each XP patient, as well as providing new insights into the functions of the XP proteins.

A novel mutation in the XPA gene associated with unusually mild clinical features in a patient who developed a spindle cell melanoma

Background  Xeroderma pigmentosum (XP) is an autosomal recessive disorder of, in most cases, defective nucleotide excision repair (NER) of ultraviolet radiation (UV)‐ and chemical‐induced DNA damage. The condition is characterized by an increased sensitivity of the skin to UV radiation, with early development of pigmentary changes and premalignant lesions in sun‐exposed areas of the skin, signs of photoageing and a greatly increased incidence from a young age of skin tumours including melanoma. Approximately 20% of patients with XP show neurological abnormalities of varying severity due to primary neuronal degeneration. Genetic analysis by somatic cell hybridization has led to the identification in the NER‐defective form of XP of seven complementation groups, designated XP‐A to XP‐G. These complementation groups correspond to different proteins involved in the NER process. XP‐A classically includes some of the most severely affected patients.

Patients with xeroderma pigmentosum complementation groups C, E and V do not have abnormal sunburn reactions

Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder of DNA repair. It is divided into eight complementation groups: XP‐A to XP‐G (classical XP) and XP variant (XP‐V). Severe and prolonged sunburn reactions on minimal sun exposure have been considered a cardinal feature of classical XP. However, it has recently become clear that not all patients have abnormal sunburn reactions.

XRCC4 deficiency in human subjects causes a marked neurological phenotype but no overt immunodeficiency

BACKGROUND Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair mechanism in human cells. The final rejoining step requires DNA ligase IV (LIG4) together with the partner proteins X-ray repair cross-complementing protein 4 (XRCC4) and XRCC4-like factor. Patients with mutations in genes encoding LIG4, XRCC4-like factor, or the other NHEJ proteins DNA-dependent protein kinase catalytic subunit and Artemis are DSB repair defective and immunodeficient because of the requirement for NHEJ during V(D)J recombination. OBJECTIVE We found a patient displaying microcephaly and progressive ataxia but a normal immune response. We sought to determine pathogenic mutations and to describe the molecular pathogenesis of the patient. METHODS We performed next-generation exome sequencing. We evaluated the DSB repair activities and V(D)J recombination capacity of the patients cells, as well as performing a standard blood immunologic characterization. RESULTS We identified causal mutations in the XRCC4 gene. The patients cells are radiosensitive and display the most severe DSB repair defect we have encountered using patient-derived cell lines. In marked contrast, a V(D)J recombination plasmid assay revealed that the patients cells did not display the junction abnormalities that are characteristic of other NHEJ-defective cell lines. The mutant protein can interact efficiently with LIG4 and functions normally in in vitro assays and when transiently expressed in vivo. However, the mutation makes the protein unstable, and it undergoes proteasome-mediated degradation. CONCLUSION Our findings reveal a novel separation of impact phenotype: there is a pronounced DSB repair defect and marked clinical neurological manifestation but no clinical immunodeficiency.

A Distinct Genotype of XP Complementation Group A: Surprisingly Mild Phenotype Highly Prevalent in Northern India/Pakistan/Afghanistan.

Xeroderma pigmentosum (XP) is a rare inherited disorder of DNA repair. Affected individuals cannot repair ultraviolet radiation (UVR)einduced DNA damage, resulting in an increased skin cancer risk (Bradford et al., 2011), severe sunburn in approximately 50% of patients (Sethi et al., 2013), and progressive neurodegeneration in approximately 30% (Kraemer et al., 1987; Totonchy et al., 2013). XP can result from defects in any of eight genes (XPAeXPG and POLH). XPAeXPG are involved in nucleotide excision repair (NER) of DNA damage (Cleaver et al., 2009). Xeroderma pigmentosum complementation group A (XP-A) patients usually have a severe phenotype, with exaggerated sunburn and early onset of progressive neurodegeneration, which results in death, usually in the second or third decade (Anttinen et al., 2008). XPA protein is required for damage verification in the NER pathway. More than 20 different mutations have been identified in the XPA gene (States et al., 1998; Takahashi et al., 2010). Many of the reported cases come from Japan because of a founder mutation (c.390-1G>C) carried by 1% of the Japanese population (Hirai et al., 2006; Satokata et al., 1990). This mutation results in abnormal splicing of mRNA and subsequent production of truncated, nonfunctioning XPA protein and the typically severe clinical phenotype. Although a diagnosis of XP-A has usually been associated with a poor prognosis, a number of XP-A patients undergoing long-term follow-up at the UK National XP Clinic have a surprisingly mild phenotype. To examine this finding further, a detailed genotype-phenotype study in this cohort was conducted. Neurological analysis included audiometry, nerve conduction studies, brain magnetic resonance imaging and neuropsychometric evaluations. Informed written consent was obtained from all patients. The study was performed in accordance with protocols approved by the Research Ethics Committee of Guy’s and St. Thomas’ Hospitals NHS Foundation Trust, London (reference 12/LO/0325). Nineteen of 90 patients being studied at the UK National XP clinic were assigned to complementation group A (Table 1). Twelve of these patients, from eight consanguineous families, displayed a mild XP-A phenotype with no ocular surface disease, delayed onset or lack of skin cancer, and normal neurological and neuropsychometric evaluations (Figure 1aeh). Mean age at assessment was 32 years (range 6e79 years) and mean age at clinical diagnosis was 26 years (range 4e46 years), significantly higher than in the more severely affected XP-A group of patients, who showed progressive neurodegeneration presenting as developmental delay and cognitive impairment, sensorineural hearing loss, microcephaly, neuropathy, and cerebellar signs (Table 1). Remarkably, one of the patients, XP1CB, is aged 79 years without any XP-related neurological problems. He spent the first 30 years of his life in India working mostly outdoors and was only diagnosed clinically at age 46 years. These 12 patients all were homozygous for the mutation c.555þ8A>G, which previously was reported by Sidwell et al. (2006) in a 61-year-old Punjabi woman with no neurological problems. All 12 patients included in this study, as well as the case described by Sidwell et al., originate from a 950-km stretch of land