Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair.

Nucleotide excision repair (NER) promotes genomic integrity by removing bulky DNA adducts introduced by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the ss/ds DNA junction. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido-L-phenylalanine (4AZP or pAzF) was incorporated at Arg-158 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent XPA protein (XPACy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate XPACy3 and detail in vitro experimental conditions required to stably maintain the protein during biochemical and biophysical studies.

Predominant neurological phenotype in a Hungarian family with two novel mutations in the XPA gene-case series.

OBJECTIVE: The prevalence of xeroderma pigmentosum (XP) is quite low in Europe, which may result in a delay in determining the appropriate diagnosis. Furthermore, some subtypes of XP, including XPA, may manifest themselves with quite severe neurological symptoms in addition to the characteristic dermatological lesions. Accordingly, the aim of the current study is to highlight the predominant neurological aspects of XPA, as well as mild-to-moderate dermatological signs in a Hungarian family with 5 affected siblings. CASE REPORTS: The symptoms of the Caucasian male proband started to develop at 13-14 years of age with predominantly cerebellar, hippocampal, and brainstem alterations. His elder sister and three younger brothers all presented similar, but less expressed neurological signs. The diagnostic work-up, including clinical exome sequencing, revealed 2 novel compound heterozygous mutations (p.Gln146_Tyr148delinsHis, p.Arg258TyrfsTer5) in the XPA gene. Surprisingly, only mild-to-moderate dermatological alterations were observed, and less severe characteristic ophthalmological and auditory signs were detected. CONCLUSIONS: In summary, we present the first family with genetically confirmed XPA in the Central-Eastern region of Europe, clearly supporting the notion that disturbed function of the C-terminal region of the XPA protein contributes to the development of age-dependent neurologically predominant signs. This case series may help clinicians recognize this rare disorder.

Xeroderma Pigmentosa Group A (XPA), Nucleotide Excision Repair and Regulation by ATR in Response to Ultraviolet Irradiation.

The sensitivity of Xeroderma pigmentosa (XP) patients to sunlight has spurred the discovery and genetic and biochemical analysis of the eight XP gene products (XPA-XPG plus XPV) responsible for this disorder. These studies also have served to elucidate the nucleotide excision repair (NER) process, especially the critical role played by the XPA protein. More recent studies have shown that NER also involves numerous other proteins normally employed in DNA metabolism and cell cycle regulation. Central among these is ataxia telangiectasia and Rad3-related (ATR), a protein kinase involved in intracellular signaling in response to DNA damage, especially DNA damage-induced replicative stresses. This review summarizes recent findings on the interplay between ATR as a DNA damage signaling kinase and as a novel ligand for intrinsic cell death proteins to delay damage-induced apoptosis, and on ATR's regulation of XPA and the NER process for repair of UV-induced DNA adducts. ATR's regulatory role in the cytosolic-to-nuclear translocation of XPA will be discussed. In addition, recent findings elucidating a non-NER role for XPA in DNA metabolism and genome stabilization at ds-ssDNA junctions, as exemplified in prematurely aging progeroid cells, also will be reviewed.

Case Report: Identification of a Heterozygous XPA c.553C>T Mutation Causing Neurological Impairment in a Case of Xeroderma Pigmentosum Complementation Group A.

We aimed to determine if an adolescent patient presenting with neurological impairment has xeroderma pigmentosum (XP). For this purpose, whole-exome sequencing was performed to assess mutations in XP genes. Dermal fibroblasts were established from a skin biopsy and XPA expression determined by immunoblotting. Nucleotide excision repair (NER) capacity was measured by detection of unscheduled DNA synthesis (UDS) in UVC-irradiated patient fibroblasts. Genetic analysis revealed two recessive mutations in XPA, one known c.682C>T, p.Arg228Ter, and the other c.553C>T, p.Gln185Ter, only two cases were reported. XPA protein was virtually undetectable in lysates from patient-derived fibroblast. The patient had significantly lower UV-induced UDS (3.03 ± 1.95%, p < 0.0001) compared with healthy controls (C5RO = 100 ± 12.2; C1UMN = 118 ± 5.87), indicating significant NER impairment. In conclusion, measurement of NER capacity is beneficial for the diagnosis of XP and in understanding the functional impact of novel mutations in XP genes. Our findings highlight the importance of neurologists considering XP in their differential diagnosis when evaluating patients with atypical neurodegeneration.

Neurochemistry evaluated by MR spectroscopy in a patient with xeroderma pigmentosum group A.

MRI of a female patient with xeroderma pigmentosum group A (XP-A) showed progressive cerebral atrophy, but no disease-specific lesion. MR spectroscopy with short TE sequences in the bilateral white matter revealed decreased N-acetyl aspartate (neuro-axonal marker) and increased myo-inositol (astroglial marker) with a normal concentration of choline (membrane marker), which are compatible with the neuropathology of XP-A, consisting of a reduced number of neurons, and fibrillary astrogliosis with preservation of myelinated fibers. MR spectroscopy reveals neurochemical derangement in XP-A, which cannot be observed on conventional MRI, and will be useful to monitor the neurochemical derangements of XP-A.

Meta-analysis on the association between xeroderma pigmentosum Group A A23G polymorphism and esophageal cancer in a Chinese population.

AIM OF THE STUDY: Several studies have evaluated the correlation between xeroderma pigmentosum Group A (XPA) A23G polymorphism (rs 1800975) and esophageal cancer in Chinese people. However, the results are inconsistent. To assess the effects of XPA A23G variants on the risk for development of esophageal cancer in the Chinese population, a meta-analysis was performed. MATERIALS AND METHODS: Studies were identified using PubMed and Chinese databases through December 2015. The associations were assessed with pooled odds ratios (ORs) and 95% confidence intervals (95% CIs). RESULTS: This meta-analysis identified seven studies including 1514 esophageal cancer cases and 2120 controls. In the overall analysis, no significant association between XPA A23G polymorphism and esophageal cancer was found in the Chinese population. In the subgroup analyses by geographic area(s) and source of controls, significant results were only found in studies with hospital-based controls (GG vs. AA: OR = 0.42, 95% CI = 0.28-0.62; GG vs. AA + AG: OR = 0.55, 95% CI = 0.39-0.78; GG + AG vs. AA: OR = 0.54, 95% CI = 0.40-0.72; G vs. A: OR = 0.61, 95% CI = 0.50-0.75). CONCLUSIONS: This meta-analysis suggested that XPA A23G gene polymorphism may be one low-penetrant risk factor for esophageal cancer in Chinese individuals.

XPA A23G polymorphism and risk of digestive system cancers: a meta-analysis.

BACKGROUND: Several studies have reported an association between the A23G polymorphism (rs 1800975) in the xeroderma pigmentosum group A (XPA) gene and risk of digestive system cancers. However, the results are inconsistent. In this study, we performed a meta-analysis to assess the association between XPA A23G polymorphism and the risk of digestive system cancers. METHODS: Relevant studies were identified using the PubMed, Web of Science, China National Knowledge Infrastructure, WanFang, and VIP databases up to August 30, 2014. The pooled odds ratio (OR) with a 95% confidence interval (CI) was calculated using the fixed or random effects model. RESULTS: A total of 18 case-control studies from 16 publications with 4,170 patients and 6,929 controls were included. Overall, no significant association was found between XPA A23G polymorphism and the risk of digestive system cancers (dominant model: GA + AA versus GG, OR 0.89, 95% CI 0.74-1.08; recessive model: AA versus GA + GG, OR 0.94, 95% CI 0.74-1.20; GA versus GG, OR 0.89, 95% CI 0.77-1.03; and AA versus GG, OR 0.87, 95% CI 0.64-1.19). When the analysis was stratified by ethnicity, similar results were observed among Asians and Caucasians in all genetic models. In stratified analysis based on tumor type, we also failed to detect any association between XPA A23G polymorphism and the risk of esophageal, gastric, or colorectal cancers. CONCLUSION: This meta-analysis indicates that the XPA A23G polymorphism is not associated with a risk of digestive system cancers.

Contribution of defective mitophagy to the neurodegeneration in DNA repair-deficient disorders.

DNA repair is a prerequisite for life as we know it, and defects in DNA repair lead to accelerated aging. Xeroderma pigmentosum group A (XPA) is a classic DNA repair-deficient disorder with patients displaying sun sensitivity and cancer susceptibility. XPA patients also exhibit neurodegeneration, leading to cerebellar atrophy, neuropathy, and hearing loss, through a mechanism that has remained elusive. Using in silico, in vitro, and in vivo studies, we discovered defective mitophagy in XPA due to PARP1 hyperactivation and NAD(+) (and thus, SIRT1) depletion. This leads to mitochondrial membrane hyper-polarization, PINK1 cleavage and defective mitophagy. This study underscores the importance of mitophagy in promoting a healthy pool of mitochondria and in preventing neurodegeneration and premature aging.

Progressive dysautonomia in two patients with xeroderma pigmentosum group A.

BACKGROUND: Xeroderma pigmentosum group A (XPA) is a rare autosomal-recessive disorder caused by a defect in nucleotide excision repair. Progressive dysautonomia in patients with XPA is rarely described. PATIENTS: Two juvenile male patients with XPA suffered from dysphagia, sleep interruption, and dysuria from the age of 10 to 19 years, successively. These autonomic symptoms might have been caused by progressive descending degeneration of cranial nerves IX and X and the sacral parasympathetic nerve, including Onuf's nucleus. One patient died from sudden cardiopulmonary arrest during postural change and tracheal suction. RESULTS: Heart rate variability analyses of these patients revealed parasympathetic dysautonomia, based on decreased high-frequency values. CONCLUSIONS: The insidiously progressive dysautonomia in these two patients with XPA suggested progressive descending degeneration extending from the medulla oblongata to the sacral spinal cord, which is an ominous sign of end-stage disease and a risk factor of sudden death attributable to XPA.

Mutational spectrum of Xeroderma pigmentosum group A in Egyptian patients.

Xeroderma pigmentosum (XP) is a rare autosomal recessive hereditary disease characterized by hyperphotosensitivity, DNA repair defects and a predisposition to skin cancers. The most frequently occurring type worldwide is the XP group A (XPA). There is a close relationship between the clinical features that ranged from severe to mild form and the mutational site in XPA gene. The aim of this study is to carry out the mutational analysis in Egyptian patients with XP-A. This study was carried out on four unrelated Egyptian XP-A families. Clinical features were examined and direct sequencing of the coding region of XPA gene was performed in patients and their parents. Direct sequencing of the whole coding region of the XPA gene revealed the identification of two homozygous nonsense mutations: (c.553C >T; p.(Gln185)) and (c.331G>T; p.(Glu111)), which create premature, stop codon and a homodeletion (c.374delC: p.Thr125Ilefs 15) that leads to frameshift and premature translation termination. We report the identification of one novel XPA gene mutation and two known mutations in four unrelated Egyptian families with Xermoderma pigmentosum. All explored patients presented severe neurological abnormalities and have mutations located in the DNA binding domain. This report gives insight on the mutation spectrum of XP-A in Egypt. This would provide a valuable tool for early diagnosis of this severe disease.

Slow accumulation of mutations in Xpc-/- mice upon induction of oxidative stress.

XPC is one of the key DNA damage recognition proteins in the global genome repair route of the nucleotide excision repair (NER) pathway. Previously, we demonstrated that NER-deficient mouse models Xpa(-/-) and Xpc(-/-) exhibit a divergent spontaneous tumor spectrum and proposed that XPC might be functionally involved in the defense against oxidative DNA damage. Others have mechanistically dissected several functionalities of XPC to oxidative DNA damage sensitivity using in vitro studies. XPC has been linked to regulation of base excision repair (BER) activity, redox homeostasis and recruitment of ATM and ATR to damage sites, thereby possibly regulating cell cycle checkpoints and apoptosis. XPC has additionally been implicated in recognition of bulky (e.g. cyclopurines) and non-bulky DNA damage (8-oxodG). However, the ultimate contribution of the XPC functionality in vivo in the oxidative DNA damage response and subsequent mutagenesis process remains unclear. Our study indicates that Xpc(-/-) mice, in contrary to Xpa(-/-) and wild type mice, have an increased mutational load upon induction of oxidative stress and that mutations arise in a slowly accumulative fashion. The effect of non-functional XPC in vivo upon oxidative stress exposure appears to have implications in mutagenesis, which can contribute to the carcinogenesis process. The levels and rate of mutagenesis upon oxidative stress correlate with previous findings that lung tumors in Xpc(-/-) mice overall arise late in the lifespan and that the incidence of internal tumors in XP-C patients is relatively low in comparison to skin cancer incidence.

De Sanctis-Cacchione Syndrome in a female infant - Case report / Sindrome de De Sanctis-Cacchione em lactente do sexo feminino - Relato de caso

The De Sanctis-Cacchione Syndrome is the rarest and most severe kind of xeroderma pigmentosum, characterized by microcephaly, hypogonadism, neurological disorders, mental and growth retardation, with very few cases published. The clinical findings compatible with De Sanctis-Cacchione Syndrome and the therapeutic approach used to treat a one year and nine months old child, with previous diagnosis of xeroderma pigmentosum, are reported.

A síndrome de de Sanctis-Cacchione é a forma mais rara e grave do xeroderma pigmentoso e é caracterizada por microcefalia, hipogonadismo, alterações neurológicas e retardo mental e de crescimento, com poucos casos publicados. Relatam-se os achados clínicos compatíveis com essa síndrome e a terapêutica instituída em uma lactente de um ano e nove meses, com diagnóstico prévio de xeroderma pigmentoso.