Petra Laspe

Cyclosporin A, but not everolimus, inhibits DNA repair mediated by calcineurin: implications for tumorigenesis under immunosuppression

Abstract:  Unlike other immunosuppressive drugs including everolimus, cyclosporin A causes a dramatic increase of UV‐induced skin cancer, a feature that is reminiscent of xeroderma pigmentosum (XP), where defective nucleotide excision repair (NER) of UV‐induced DNA damage results in cutaneous carcinogenesis. The molecular basis of the clinically important differential activities of cyclosporin A and everolimus is still unclear. We measured post‐UV cell survival of cyclosporin A‐ and everolimus‐treated human fibroblasts and lymphoblasts using a cell proliferation assay (MTT). The cellular NER capacity was assessed by host cell reactivation. Using an ELISA and specific antibodies, cyclobutane pyrimidine and pyrimidine‐6,4‐pyrimidone photoproduct removal from the cellular genome was measured. The effect of calcineurin on NER was investigated using a calcineurin A expression vector and specific RNAi. Cyclosporin A led to a dose dependent decrease in post‐UV cell survival, inhibited NER and blocked photoproduct removal. In contrast, none of these effects where seen in everolimus‐treated cells. Overexpression of calcineurin A resulted in increased NER and complemented the Cyclosporin A‐induced reduction of NER. Downregulation of calcineurin using RNAi inhibited NER comparable to cyclosporin A‐treatment. We conclude that cyclosporin A, but not everolimus, leads to an increased skin cancer risk via a calcineurin signalling‐dependent impairment of NER.

No association between three xeroderma pigmentosum group C and one group G gene polymorphisms and risk of cutaneous melanoma

Xeroderma pigmentosum (XP) patients exhibit a 1000-fold increased risk for developing skin cancers including malignant melanoma. We investigated the role of three variant alleles of the DNA repair gene XPC and one variant allele of the XPG gene in a hospital-based case–control study of 294 Caucasian patients from Germany with malignant melanoma and 375 healthy control individuals from the same area matched by sex. The polymorphisms G1580A (XPC exon 8; Arg492His), T1601C (XPC exon 8; Val499Ala), G2166A (XPC exon 10; Arg687Arg), and C3507G (XPG exon 15; Asp1104His) were not in linkage disequilibrium. The allele frequencies (cases: controls) were for 1580A 6.29%: 5.63%, for 1601C 79.08%: 78.28%, for 2166A 26.19%: 28.13%, and for 3507G 79.86%: 78.61%. We found no association of the homozygous 1580A, 1601C, 2166A, and 3507G genotypes with increased risks of melanoma: OR 1.254 (95% CI: 0.486–3.217), OR 1.108 (95% CI: 0.629–1.960), OR 0.817 (95% CI: 0.490–1.358), and OR 1.168 (95% CI: 0.670–2.044), respectively. Exploratory analyses of subgroups of melanoma patients compared to all controls indicated no association of these genotypes with increased risks for development of multiple primary melanomas (n=28), a negative family history for melanoma (n=277), melanomas in individuals with a low number of nevi (n=273), melanomas in individuals older than 55 years (n=142), and melanomas thicker than 1 mm (n=126).

Strict sun protection results in minimal skin changes in a patient with xeroderma pigmentosum and a novel c.2009delG mutation in XPD (ERCC2).

Abstract:  We examined the clinical, molecular and genetic features of a 16‐year‐old boy (XP2GO) with xeroderma pigmentosum (XP) and progressive neurological symptoms. The parents are not consanguineous. Increased sun sensitivity led to the diagnosis of XP at 2 years of age and a strict UV protection scheme was implemented. Besides recurrent conjunctivitis and bilateral pterygium, only mild freckling was present on his lips. He shows absent deep tendon reflexes, progressive sensorineural deafness and progressive mental retardation. MRI shows diffuse frontal cerebral atrophy and dilated ventricles. Symptoms of trichothiodystrophy (brittle hair with a tiger‐tail banding pattern on polarized microscopy) or Cockayne syndrome (cachectic dwarfism, cataracts, pigmentary retinopathy and spasticity) were absent. XP2GO fibroblasts showed reduced post‐UV cell survival (D37 = 3.8 J/m2), reduced nucleotide excision repair, reduced expression of XPD mRNA and an undetectable level of XPD protein. Mutational analysis of the XPD gene in XP2GO revealed two different mutations: a common p.Arg683Trp amino acid change (c.2047C>T) known to be associated with XP and a novel frameshift mutation c.2009delG (p.Gly670Alafs*39). The latter mutation potentially behaves as a null allele. While not preventing neurological degeneration, early diagnosis and rigorous sun protection can result in minimal skin disease without cancer in XP patients.

Cyclosporin A inhibits nucleotide excision repair via downregulation of the xeroderma pigmentosum group A and G proteins, which is mediated by calcineurin inhibition.

Abstract:  Cyclosporin A (CsA) inhibits nucleotide excision repair (NER) in human cells, a process that contributes to the skin cancer proneness in organ transplant patients. We investigated the mechanisms of CsA‐induced NER reduction by assessing all xeroderma pigmentosum (XP) genes (XPA‐XPG). Western blot analyses revealed that XPA and XPG protein expression was reduced in normal human GM00637 fibroblasts exposed to 0.1 and 0.5 μm CsA. Interestingly, the CsA treatment reduced XPG, but not XPA, mRNA expression. Calcineurin knockdown in GM00637 fibroblasts using RNAi led to similar results suggesting that calcineurin‐dependent signalling is involved in XPA and XPG protein regulation. CsA‐induced reduction in NER could be complemented by the overexpression of either XPA or XPG protein. Likewise, XPA‐deficient fibroblasts with stable overexpression of XPA (XP2OS‐pCAH19WS) did not show the inhibitory effect of CsA on NER. In contrast, XPC‐deficient fibroblasts overexpressing XPC showed CsA‐reduced NER. Our data indicate that the CsA‐induced inhibition of NER is a result of downregulation of XPA and XPG protein in a calcineurin‐dependent manner.

Molecular genetic analysis of 16 XP‐C patients from Germany: environmental factors predominately contribute to phenotype variations

Patients belonging to xeroderma pigmentosum (XP) complementation group C comprise one‐third of all XP patients. Only four major reports compiled larger groups of XP‐C patients from southern Europe (12 pts), North America (16 pts) and Africa (14 and 56 pts) as well as their genetic background (46 XPC mutations). We identified 16 XP‐C patients from Germany. Interestingly, only five patients exhibited severe sun sensitivity. The mean age of XP diagnosis was 9.4 years, and the median age of the first skin cancer was 7 years. Neurological symptoms were absent in all but two patients. Primary fibroblasts from all 16 patients showed reduced post‐UV cell survival (mean: 50% vs 93% in normal cells) and reduced reactivation of an UV‐treated luciferase reporter gene (mean: 6.4% vs 30.7% in normal cells). XPC mRNA expression was also greatly reduced compared with normal cells (mean: 14.3%; range 8.3–25.7%) except in XP47MA (274.1%). All patients carried homozygous XPC mutations. Four mutations have been described previously: c.1747_1748delTG (found in 4/16), c.567 C>T (4/16), c.1839 C>T (1/16) and a complex insertion/deletion mutation in exon 9 (1/16). The novel frameshift mutations c.446_447delAG (2/16), c.1525insA (1/16) and c.2271delC (1/16) lead to truncated XPC proteins as does the novel nonsense mutation c.843C>T (1/16). XP47MA carries an interesting mutation (c.2538_2540delATC; p.Ile812del) resulting in an in‐frame single amino acid deletion. This mutation results in a classical XP phenotype, a non‐functional XPC protein, but elevated XPC mRNA expression. Our study indicates that extrinsic factors may contribute to XP‐C symptom severity due to nonsense‐mediated message decay.

Eight novel mutations and consequences on mRNA and protein level in pyruvate kinase-deficient patients with nonspherocytic hemolytic anemia.

Pyruvate kinase (PK) deficiency (PKD) is an autosomal recessive disorder with the typical manifestation of nonspherocytic hemolytic anemia. We analyzed the mutant enzymes of 10 unrelated patients with PKD, whose symptoms ranged from a mild, chronic hemolytic anemia to a severe anemia, by sequence analysis for the presence of alterations in the PKLR gene. In all cases the patients were shown to be compound heterozygous. Eight novel mutations were identified: 458T→C (Ile153Thr), 656T→C (Ile219Thr), 877G→A (Asp293Asn), 991G→A (Asp331Asn), 1055C→A (Ala352Asp), 1483G→A (Ala495Thr), 1649A→T (Asp550Val), and 183‐184ins16bp. This 16 bp duplication produces a frameshift and subsequent stop codon resulting in a drastically reduced mRNA level, and probably in an unstable gene product. Surprisingly, the existence of M2‐type PK could be demonstrated in the patients red blood cells. The study of different polymorphic sites revealed, with one exception, a strict linkage of the 1705C, 1738T, IVS5(+51)T, T(10) polymorphisms and the presence of 14 ATT repeats in intron 11. Our analyses show the consequences of a distorted structure on enzyme function and we discuss the correlations between the mutations identified and the parameters indicative for enzyme function. Hum Mutat 15:261–272, 2000.

Functional and molecular genetic analyses of nine newly identified XPD-deficient patients reveal a novel mutation resulting in TTD as well as in XP/CS complex phenotypes

The xeroderma pigmentosum (XP) group D protein is involved in nucleotide excision repair (NER) as well as in basal transcription. Determined by the type of XPD mutation, six different clinical entities have been distinguished: XP, XP with neurological symptoms, trichothiodystrophy (TTD), XP⁄TTD complex, XP⁄Cockayne syndrome (CS) complex or the cerebro‐oculo‐facio‐skeletal syndrome (COFS). We identified nine new XPD‐deficient patients. Their fibroblasts showed reduced post‐UV cell survival, reduced NER capacity, normal XPD mRNA expression and partly reduced XPD protein expression. Six patients exhibited a XP phenotype in accordance with established XP‐causing mutations (c.2079G>A, p.R683Q; c.2078G>T, p.R683W; c.1833G>T, p.R601L; c.1878G>C, p.R616P; c.1878G>A, p.R616Q). One TTD patient was homozygous for the known TTD‐causing mutation p.R722W (c.2195C>T). Two patients were compound heterozygous for a TTD‐causing mutation (c.366G>A, p.R112H) and a novel p.D681H (c.2072G>C) amino acid exchange, but exhibited different TTD and XP/CS complex phenotypes, respectively. Interestingly, the XP/CS patients cells exhibited a reduced but well detectable XPD protein expression compared with hardly detectable XPD expression of the TTD patients cells. Same mutations with different clinical outcomes in NER‐defective patients demonstrate the complexity of phenotype–genotype correlations, for example relating to additional genetic variations (parental consanguinity), different allelic expression due to SNPs or differences in the methylation status.

An unusual mutation in the XPG gene leads to an internal in-frame deletion and a XP/CS complex phenotype.

DEAR EDITOR, The nucleotide excision repair (NER) pathway repairs ultraviolet (UV)-induced photoproducts. According to the respective mutated genes (XPA–XPG) and a variant form with a defect in translesion synthesis (Pol H), seven xeroderma pigmentosum (XP) complementation groups (XP-A to XP-G) have been identified. Patients belonging to XP complementation groups B, D, F and G can exhibit XP symptoms, including photosensitivity, freckling and a high increase in the risk of skin cancer, combined with Cockayne syndrome (CS) symptoms, which include photosensitivity, neurological abnormalities and failure to thrive, but no preponderance for skin cancer (XP/CS complex phenotype). This indicates a role of the respective proteins in DNA repair, as well as in basal transcription. The human XPG/ERCC5 gene (OMIM: 278780) encodes an 1186-amino acid protein and is located on chromosome 13q32 3-q33 1. During NER, XPG performs the 30 incision of the damage-containing strand and stabilizes the basal transcription factor IIH (TFIIH) by two interaction

A novel mutation in the XPA gene results in two truncated protein variants and leads to a severe XP/neurological symptoms phenotype.

The nucleotide excision repair (NER) pathway repairs UV‐induced DNA lesions in an accurate fashion and prevents UV‐irradiated areas of the skin from tumour formation. The XPA protein plays a major role in DNA damage demarcation as well as stabilization of other NER factors and was found to be defective in xeroderma pigmentosum (XP) complementation group A patients.

Photosensitive form of trichothiodystrophy associated with a novel mutation in the XPD gene

Department of Dermatology, Venereology and Allergology, University Medical Center, Georg August University, G€ ottingen, Germany. Clinic for Dermatology and Venereology, University Medical Center Rostock, Rostock, Germany. DWI – Leibniz Institute for Interactive Materials, Aachen, Germany. Interdisciplinary Pediatric Center for Children with Developmental Disabilities and Severe Chronic Disorders, University Medical Center, Georg August University, G€ ottingen, Germany. Lower Saxony Institute of Occupational Dermatology, University Medical Center G€ ottingen and University of Osnabr€ uck, Osnabr€ uck, Germany.