TT Mutations Research Articles

Clinical, histological, and molecular differences in melanoma due to different TERT promoter mutations subtypes. A retrospective cross-sectional study in 684 melanoma patients.

Differences in survival according to the pTERT mutation subtypes (-124C > T, -146C > T, and tandem -138_139CC > TT) have been observed. The present study aimed to describe the clinical as the histopathological and molecular cutaneous melanoma features according to the presence of the three most prevalent pTERT mutation subtypes (-124C > T, -146C > T, and tandem -138_139CC > TT). A retrospective cross-sectional study including 684 patients was designed, and a Partial Least-Squares Discriminant Analysis (PLS-DA) was performed. After the PSL-DA, it was observed that the tandem -138_139CC > TT subtype differs from the other subtypes. The model demonstrated that the -124C > T and the -138_139 CC > TT subtypes were associated with fast-growing melanomas (OR 0.5, CI 0.29-0.86, p = .012) and with Breslow >2 mm (OR 0.6, CI 0.37-0.97, p = .037), compared to the -146C > T mutation. Finally, the -124C > T appeared to be more associated with the presence of TILs (non-brisk) than the -146C > T (OR 0.6, CI 0.40-1.01, p = .05). These findings confirmed that the -124C > T and the tandem -138_139 CC > TT subtypes are both highly associated with the presence of features of aggressiveness; however, only the -124C > T was highly associated with TILs. This difference could explain the worse survival rate associated with the tandem -138_139CC > TT mutations.

Pulmonary squamous cell carcinoma and lymphoepithelial carcinoma - morphology, molecular characteristics and differential diagnosis.

Squamous cell carcinoma (SCC) comprises one of the major groups of non-small-cell carcinoma of the lung, and is subtyped into keratinising, non-keratinising and basaloid SCC. SCC can readily be diagnosed using histomorphology alone in keratinising SCC. Confirmatory immunohistochemical analyses should always be applied in non-keratinising and basaloid tumours to exclude differential diagnoses, most prominently adenocarcinoma and high-grade neuroendocrine carcinoma, which may have important therapeutic consequences. According to the World Health Organisation (WHO) classification 2015, the diagnosis of SCC can be rendered in resections of morphologically ambiguous tumours with squamous immunophenotype. In biopsies and cytology preparations in the same setting thecurrent guidelines propose a diagnosis of 'non-small-cell carcinoma, favour SCC' in TTF1-negative and p40-positive tumours to acknowledge a possible sampling bias and restrict extended immunohistochemical evaluation in order to preserve tissue formolecular testing. Most SCC feature a molecular 'tobacco-smoke signature' with enrichment in GG > TTmutations, in line with the strong epidemiological association of SCC with smoking. Targetable mutations are extremely rare but they do occur, in particular in younger and non- or light-smoking patients, warranting molecular investigations. Lymphoepithelial carcinoma (LEC) is a poorly differentiated SCC with a syncytial growth pattern and a usually prominent lymphoplasmacytic infiltrate and frequent Epstein-Barr virus (EBV) association. In this review, we describe the morphological and molecular characteristics of SCC and LEC and discuss the most pertinent differential diagnoses.

HOGA1 variants in Chinese patients with primary hyperoxaluria type 3: genetic features and genotype-phenotype relationships.

The aim of our study is to describe the genetic features and correlation between the genotype and phenotype of Chinese patients with primary hyperoxaluria type 3 (PH3). The genetic and clinical data of PH3 patients in our cohort were collected and analyzed retrospectively. All published studies of Chinese PH3 populations between January 2010 and November 2022 were searched and enrolled based on inclusive standards. A total of 60 Chinese PH3 patients (21 cases from our cohort and 39 cases from previous studies) were included. The mean age of onset was 1.62 ± 1.35 (range 0.4-7) years. A total of 29 different variants in the HOGA1 gene were found. The mutations were most commonly clustered in exons 1, 6, and 7. Among the genotypes, exon 6 skipping (c.834G > A and c.834_834 + 1GG > TT mutations) was the most common, followed by c.769T > G; the allele frequencies (AFs) were 48.76% and 12.40%, respectively. Patients homozygous for exon 6 skipping exhibited a median age of onset of 0.67 (0.58-1) years, which was significantly lower than that observed among heterozygotes and nonexon 6 skipping patients (p = 0.021). A total of 22.5% (9/40) of PH3 patients had a decreased estimated glomerular filtration rate, and one patient with homozygous exon 6 skipping developed end-stage renal disease. A hotspot mutation, potential hotspot mutation and genotype-phenotype correlation were found in Chinese PH3 patients. This study expands the mutational spectrum and contributes to the understanding of genotypic profiles of PH3, which may provide a potential diagnostic and therapeutic target.

Particular Evolutionary Aspects in Children with Cystic Fibrosis

Abstract Background: Cystic fibrosis was considered a fatal condition years ago but, nowadays, it can have a favorable evolution if diagnosed and treated early. Objective: Screening clinical aspects of patients suspected for cystic fibrosis and later diagnosed by means of specific sweat and genetic tests. Methods: A retrospective descriptive study was performed on 13 patients during five years, between 2015-2020 from the Pediatric Clinic of the Emergency County Hospital in Constanta. Results: There were five patients present in ΔF508 homozygous form, which it seems to be the most frequent mutation, translated by the deletion of phenylalanine at position 508. Among the heterozygous patients, 2 patients aged 10 and 14 years have the ΔF508 del 2.3 mutation; a 10-year-old patient has the mutation ΔF508, 2183 AA>G; a 7-year-old patient has the ΔF508 R553X mutation; a 7-year-old patient has the N1303K, 394del TT mutation; a 1-year-old patient is heterozygous for the 394del TT and R347P mutations, a 3-year-old patient has the compound mutation ΔF508del and R334W, and finally, we find a 2-year-old patient heterozygous for the Δ508 mutation. Ten from 11 patients were diagnosed in the first year of life by performing the sweat test, and 2 patients based on the genetic test. In 30% of cases the onset manifestation were represented by diarrhea and growth failure, and 38% of cases had added respiratory manifestation. Singular manifestations such as respiratory disease, or respiratory disease accompanied by loss of appetite and growth failure, or chronic cough and steatorrhea followed by growth failure occurred in 8% each. If not properly diagnosed and treated, cystic fibrosis can put the patient’s life at risk. The analysis between an early diagnosis and the decrease of the incidence in pulmonary complications with pathogenic agents was statistically significant.

A Maternally Deposited Endosperm Cuticle Contributes to the Physiological Defects of transparent testa Seeds.

Mature dry seeds are highly resilient plant structures where the encapsulated embryo is kept protected and dormant to facilitate its ultimate dispersion. Seed viability is heavily dependent on the seed coat's capacity to shield living tissues from mechanical and oxidative stress. In Arabidopsis (Arabidopsis thaliana), the seed coat, also called the testa, arises after the differentiation of maternal ovular integuments during seed development. We recently described a thick cuticle tightly embedded in the mature seed's endosperm cell wall. We show here that it is produced by the maternal inner integument 1 layer and, remarkably, transferred to the developing endosperm. Arabidopsis transparent testa (tt) mutations cause maternally derived seed coat pigmentation defects. TT gene products encode proteins involved in flavonoid metabolism and regulators of seed coat development. tt mutants have abnormally high seed coat permeability, resulting in lower seed viability and dormancy. However, the biochemical basis of this high permeability is not fully understood. We show that the cuticles of developing tt mutant integuments have profound structural defects, which are associated with enhanced cuticle permeability. Genetic analysis indicates that a functional proanthocyanidin synthesis pathway is required to limit cuticle permeability, and our results suggest that proanthocyanidins could be intrinsic components of the cuticle. Together, these results show that the formation of a maternal cuticle is an intrinsic part of the normal integumental differentiation program leading to testa formation and is essential for the seed's physiological properties.

MTHFR Gene Polymorphism-Mutations and Air Pollution as Risk Factors for Breast Cancer: A Metaprediction Study.

BackgroundThe methylenetetrahydrofolate reductase gene (MTHFR) is one of the most investigated genes associated with breast cancer for its role in epigenetic pathways.ObjectivesThe objectives of this metaprediction study were to examine the polymorphism-mutation risk subtypes of MTHFR and air pollution as contributing factors for breast cancer.MethodsFor triangulation purposes in metapredictive analyses, we used a recursive partition tree, nonlinear association curve fit, and heat maps for data visualization, in addition to the conventional comparison procedure and pooled analyses.ResultsWe included 36,683 breast cancer cases and 40,689 controls across 82 studies for MTHFR 677 and 23,252 cases and 27,094 controls across 50 studies for MTHFR 1298. MTHFR 677 TT was a risk genotype for breast cancer (p = .0004) and in the East Asian subgroup (p = .005). On global maps, the most polymorphism-mutations on MTHFR 677 TT were found in the Middle East, Europe, Asia, and the Americas, whereas the most mutations on MTHFR 1298 CC were located in Europe and the Middle East for the control group. The geographic information system maps further revealed that MTHFR 677 TT mutations yielded a higher risk of breast cancer for Australia, East Asia, the Middle East, South Europe, Morocco, and the Americas and that MTHFR 1298 CC mutations yielded a higher risk in Asia, the Middle East, South Europe, and South America. Metapredictive analysis revealed that air pollution level was significantly associated with MTHFR 677 TT polymorphism-mutation genotype.DiscussionWe present the most comprehensive analyses to date of MTHFR polymorphism-mutations and breast cancer risk. Future nursing studies are needed to investigate the health impact on breast cancer of epigenetics and air pollution across populations.

Aberrantly Expressed APOBEC3B Induces Mutations in Multiple Myeloma

Multiple myeloma (MM) is a malignant plasma cell tumor that arises secondarily from monoclonal gammopathy of uncertain significance (MGUS) due to accumulation of genetic abnormalities. Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) is a family of DNA cytosine deaminases that play critical roles in innate and acquired immunity. APOBEC proteins catalyze cytosine to uracil deamination and eventually induce C to T mutations in DNA. Recent genome-wide analysis revealed that APOBEC-induced signature DNA mutations accumulate during progression of MM and are associated with poor prognosis (Bolli et. al., Nat Commun. 2014). We previously found that one of the APOBEC3 proteins, APOBEC3B (A3B), induces genomic DNA mutations in a human cell culture model (Shinohara et. al., Sci Rep. 2012) and hypothesized that A3B might induce DNA mutations during onset and progression of MGUS and MM.A deletion polymorphism that removes the entire A3B gene is reported to affect breast cancer prognosis, therefore we first investigated the frequency of A3B deletion allele in 88 Japanese MM/MGUS patients and in healthy controls. 41 patients (46.6%) showed wild type, 42 patients (47.7%) heterozygous, and 5 patients (5.7%) had homozygous deletion allele. We could not detect a significant difference between MM/MGUS patients and healthy controls, suggesting that A3B may not contribute to disease initiation. In contrast, real-time PCR analysis revealed a considerably high expression of A3B mRNA in 23 bone marrow specimens from MM/MGUS patients, except for two samples with homozygous deletion allele. We sorted myeloma cells using anti-CD138 beads and the enriched myeloma cell samples exhibited even higher A3B mRNA expression in 9 MM/MGUS patients (relative quantity to peripheral blood cells [PBMC]: range 1.06 to 55.14, median 7.67).Next, we investigated the APOBEC3 family protein expression profile in six myeloma cell lines: U266, RPMI8226, SKMM1, AMO1, OPM2 and THK72. As expected, all of these cell lines demonstrated remarkably high levels of A3B expression by real-time PCR analysis (relative quantity to PBMC: range 1.22 to 489.4, median 62.47). Western blot analysis using an anti-A3B antibody that we newly generated also confirmed the high A3B protein expression in these myeloma cell lines. In addition, fluorescent immunostaining analysis confirmed high expression of A3B protein localized in the nucleus. Because previous studies showed that A3B expression is activated via the NF-kB signaling pathway, we investigated the regulation of A3B transcription in myeloma cells by p65 knockdown through shRNA lentivirus, resulting in suppression of A3B at protein level.To investigate the mutagenic potential of aberrantly expressed A3B in myeloma cell lines, we evaluated somatic mutations in genomic DNA by differential DNA denaturation PCR (3D-PCR) using stable A3B knock-down cells by shRNA lentivirus simultaneously transduced with a fluorescent gene, mCherry. Interestingly, we detected that scramble shRNA-transduced cells lost fluorescence much faster than those transduced with shRNA against A3B. 3D-PCR amplified mCherry fragments more efficiently at a lower denaturation temperature, indicating that these fragments include more AT-rich sequences. We cloned these DNA fragments and sequenced about 4000 bp in total, and detected prominently more TC > TT mutations in the mCherry sequences from control shRNA-transduced cells (C to T mutation, 3.001%) than in those from A3B shRNA-tranduced cells (C to T mutation, 0.0024%). Additionally, the amplified mCherry sequences from scramble shRNA-transduced cells contained various deletions which may be repaired by microhomology-mediated end joining. Since DNA double strand breaks (DSB) are constitutively activated in myeloma cells, we also examined whether aberrantly expressed A3B can induce DSBs. A3B knockdown in RPMI8226 decreased gH2AX signals by fluorescent immunostaining and Western blot analysis, suggesting that A3B induces DSBs.The present study clearly shows that myeloma cells express aberrantly high levels of A3B and suggests that A3B might play crucial roles in clonal evolution or genomic instability of MM/MGUS albeit A3B may not contribute to initiation of MGUS/MM. Whether A3B accelerates chemoresistance or disease progression remains to be elucidated, however, A3B might be a potential therapeutic target in MM/MGUS. DisclosuresIida:Janssen Pharmaceuticals: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Takaori-Kondo:Mochida Pharmaceutical: Research Funding; Takeda Pharmaceutical: Research Funding; Pfizer: Research Funding; Kyowa Kirin: Research Funding; Astellas Pharma: Research Funding; Eisai: Research Funding; Alexion Pharmaceuticals: Research Funding; Chugai Pharmaceutical: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Shionogi: Research Funding; Toyama Chemical: Research Funding; Cognano: Research Funding.

Abstract 4510: KIT melanomas are developmentally racially biased

Abstract KIT is a critical pathway for melanocytic homeostasis and it is often down-regulated in cutaneous melanoma. However, KIT activation plays a critical role in a subpopulation of melanomas especially those in acral locations and melanomas on chronically sun damaged skin. We analyzed exome and RNAseq data from The Cancer Genome Atlas (TCGA) Network (321 cutaneous melanomas in the data set) to identify patterns unique to KIT activation. The melanomas were divided based on known mutually exclusive mutations (BRAF, NRAS and KIT) vs those without (WT). We compared RNA data between each group. KIT group had significantly higher expression of KIT than BRAF, NRAS and WT (p<0.01, FDR<0.01) and isoform 2 expression was higher than 1. KIT melanomas were found to overexpress Wnt pathway, pigmentation and genes involved epithelial-mesenchymal transition. From the exome data, germline SNPs with incidence in ethnic groups (Caucasian, Asian and African) and more likely to be present in a specific melanoma groups were selected. Comparing the 3 ethnicity groups across the 4 tumor groups Asian SNPs were significantly enhanced in KIT tumors, African in WT and Caucasian in BRAF and NRAS melanomas. We evaluated the tumors for the primary type of genetic damage. The three signatures that appeared most divergent were CC>TT for UV, (G/A)C(G)>(G/A)T(G) for deamination, and G>T for oxidative damage. UV and deamination appeared inversely proportional, while oxidative damage appeared to be independent of those other two features. KIT and WT had a greater% of non UV high deamination tumoral environments. A fraction of these tumors also had very high oxidative signatures. We then compared 2 KIT subgroups UV vs non UV (absence of CC>TT mutations) for germline ethnicity differences. Asian SNPs were highly increased in non UV subgroup whereas Caucasian SNPs were in UV subgroup. Further, we divided KIT non UV subgroup into deamination and oxidative damage subgroups and compared ethnicity differences. Deamination was significantly higher in Asians whereas oxidative damage was higher in Caucasians. A similar analysis was done to the WT group, where African SNPs were significantly increased in the non UV subgroup and were primarily associated with oxidative damage. This data implies that KIT mutant melanomas develop in a unique genetic and mutational environments and makes this an ideal system to in which study racial disparities at the molecular level. Citation Format: Margaret I. Sanchez, James M. Grichnik. KIT melanomas are developmentally racially biased. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4510.

A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer

Human deoxycytidine deaminase APOBEC3A (Apo3A) acts as an HIV-1 restriction factor in cells of myeloid lineage yet functions separately as a potent mutator for genomic DNA. Apo3A activity and C motif deamination specificity exhibit a striking dependence on pH that reflects these two distinct biological processes. Upon infection of macrophages, HIV-1 induces the formation of autophagosomes, and requires autophagosomes for replication, whereas inhibiting lysosomal fusion indicative of late stage autophagy. Here we show that Apo3A has optimal activity and a strict 5'-YYCR motif specificity in the pH 5.8-6.1 range, characteristic of enclosed autophagosomal membrane compartments, and reflective of the mutation pattern of HIV-1. In contrast to the high activity and narrow specificity of Apo3A at acid pH, a 13-30-fold reduction in specific activity is accompanied by relaxed C deamination specificity at pH 7.4-8. Notably, Apo3A is also expressed in keratinocytes, and is up-regulated in skin lesions. At pH 7.9, we show that Apo3A generates transcription-dependent CC → TT tandem mutations on the non-transcribed strand, a hallmark signature of skin cancer. The biochemical data taken in conjunction with the biological up-regulation of Apo3A in skin lesions suggests that enzyme-catalyzed deaminations at adjacent C sites followed by normal replication generating CC → TT mutations provides an alternative molecular basis for the initiation events in skin cancer in contrast to well established pathways in which CC dimers formed in response to UV radiation either undergo nonenzymatic spontaneous deaminations or aberrant replication.

Methylenetetrahydrofolate Reductase Gene 677CT Polymorphism and Isolated Congenital Heart Disease in a Mexican Population

Introduction and objectives The frequency of the 677C>T mutation in the methylenetetrahydrofolate reductase gene in Mexico is one of the highest worldwide. Some studies have shown that both the homozygous state of this mutation and a high homocysteine concentration are associated with congenital heart disease. The aim of this study was to determine whether this association exists in the Mexican population. Methods Genotypes were analyzed in 60 patients with congenital heart disease and in their mothers, and the levels of homocysteine were determined in the latter group. The genotypes were compared with those of a control group (n=62) and of their mothers. All the possible mother-child genotype combinations were also compared. Results There were no significant differences in allele or genotype frequencies between the patients with congenital heart disease and the controls or their respective mothers ( P>.05). Although no significant differences were observed when the homocysteine concentrations in the presence of the CC or the TT genotype were compared, a clear trend was observed ( P=.0621). We found no significant differences in homocysteine concentrations in relation to folic acid intake. The study cases and controls did not differ in terms of the possible combinations of mother-child genotypes. Conclusions The frequencies obtained were consistent with those reported for Mexico. No significant differences were found between groups. Nor did we find any association between TT mutations in both the mother and child and hyperhomocysteinemia. There was no evidence of an association between any of the mother-child genotype combinations and congenital heart disease. Similar studies with larger numbers of patients are required to confirm or refute some of the trends observed in this report.

Polimorfismo 677CT del gen de la metilentetradihidrofolato reductasa y cardiopatías congénitas aisladas en población mexicana

Introduction and objectives The frequency of the 677C>T mutation in the methylenetetrahydrofolate reductase gene in Mexico is one of the highest worldwide. Some studies have shown that both the homozygous state of this mutation and a high homocysteine concentration are associated with congenital heart disease. The aim of this study was to determine whether this association exists in the Mexican population. Methods Genotypes were analyzed in 60 patients with congenital heart disease and in their mothers, and the levels of homocysteine were determined in the latter group. The genotypes were compared with those of a control group (n=62) and of their mothers. All the possible mother-child genotype combinations were also compared. Results There were no significant differences in allele or genotype frequencies between the patients with congenital heart disease and the controls or their respective mothers ( P>.05). Although no significant differences were observed when the homocysteine concentrations in the presence of the CC or the TT genotype were compared, a clear trend was observed ( P=.0621). We found no significant differences in homocysteine concentrations in relation to folic acid intake. The study cases and controls did not differ in terms of the possible combinations of mother-child genotypes. Conclusions The frequencies obtained were consistent with those reported for Mexico. No significant differences were found between groups. Nor did we find any association between TT mutations in both the mother and child and hyperhomocysteinemia. There was no evidence of an association between any of the mother-child genotype combinations and congenital heart disease. Similar studies with larger numbers of patients are required to confirm or refute some of the trends observed in this report. Full English text available from: www.revespcardiol.org

Acceleration of 5-Methylcytosine Deamination in Cyclobutane Dimers by G and Its Implications for UV-Induced C-to-T Mutation Hotspots

Sunlight-induced C→T mutation hotspots occur most frequently at methylated CpG sites in tumor suppressor genes and are thought to arise from translesion synthesis past deaminated cyclobutane pyrimidine dimers (CPDs). While it is known that methylation enhances CPD formation in sunlight, little is known about the effect of methylation and sequence context on the deamination of 5-methylcytosine ( mC) and its contribution to mutagenesis at these hotspots. Using an enzymatic method, we have determined the yields and deamination rates of C and mC in CPDs and find that the frequency of UVB-induced CPDs correlates with the oxidation potential of the flanking bases. We also found that the deamination of T mC and mCT CPDs is about 25-fold faster when flanked by G's than by A's, C's or T's in duplex DNA and appears to involve catalysis by the O6 group of guanine. In contrast, the first deamination of either C or mC in AC mCG with a flanking G was much slower ( t 1/2 > 250 h) and rate limiting, while the second deamination was much faster. The observation that C mCG dimers deaminate very slowly but at the same time correlate with C→T mutation hotspots suggests that their repair must be slow enough to allow sufficient time for deamination. There are, however, a greater number of single C→T mutations than CC→TT mutations at C mCG sites even though the second deamination is very fast, which could reflect faster repair of doubly deaminated dimers.

High Frequency Induction of CC to TT Tandem Mutations in DNA Repair‐proficient Mammalian Cells

The CC to TT tandem mutation is induced by UV radiation exposure, though at relatively low frequencies when compared with the more commonly induced C to T mutation. Induction of the tandem mutation by UV is enhanced in mammalian cells with certain genetic deficiencies; however, conditions have not been described in which the frequency of this mutation is enhanced in DNA repair-proficient mammalian cells. For this study, an integrated construct that detects C to T and CC to TT mutations at a single codon in mouse Aprt was used to examine UVB mutagenesis under various conditions. Oxidative stress, in the form of intracellular hydrogen peroxide, increased the frequency of UVB-induced CC to TT mutations. Surprisingly, exposure of the cells to two antioxidants (N-acetylcysteine and trolox), either alone or in combination, also enhanced UVB induction of CC to TT tandem mutations. These results demonstrate, for the first time, that the frequency of UVB-induced CC to TT tandem mutations can be enhanced dramatically in DNA repair-proficient mammalian cells, and suggest that the enhancing effect does not require direct damage to DNA.

Mutation spectrum in UVB‐exposed skin epidermis of Xpa‐knockout mice: Frequent recovery of triplet mutations

Knockout mutations in both alleles of the Xpa gene give rise to a complete deficiency in nucleotide excision repair (NER) in mammalian cells. We used transgenic mice harboring the lambda-phage-based lacZ mutational reporter gene to study the effect of Xpa null mutation (Xpa(-/-)) on damage induction, repair, and mutagenesis in mouse skin epidermis after UVB irradiation. UVB induced equal amounts of cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (64PPs) in mouse skin epidermis of Xpa(-/-) and wild-type mice. Neither photolesion was removed in the Xpa(-/-) epidermis by 12 hr after irradiation whereas removal of 64PPs was observed in the epidermis of wild-type mice. Irradiation with 200 and 300 J/m(2) UVB increased the lacZ mutant frequency in the epidermis of Xpa(-/-) mice, but the induced mutant frequencies were not significantly different from those previously determined for wild-type mice. One-hundred lacZ mutants isolated from the UVB-exposed epidermis of Xpa(-/-) mice were analyzed and compared with mutant sequences previously determined for irradiated wild-type mice. The distribution of the mutations along the lacZ transgene and the preferred dipyrimidine context of the UV-specific mutations were similar in mutants from the Xpa(-/-) and wild-type mice. The spectra of the mutations in the two genotypes were both highly UV-specific and similar in a dominance of C --> T transitions at dipyrimidine sites; however, Xpa(-/-) mice had a higher frequency than wild-type mice of two-base tandem substitutions, including CC --> TT mutations, three-base tandem mutations and double base substitutions that were separated by one unchanged base in a three-base sequence (alternating mutations). These tandem/alternating mutations included a remarkably large number of triplet mutations, a recently reported, novel type of UV-specific mutation, characterized by multiple base substitutions or frameshifts within a three-nucleotide sequence containing a dipyrimidine. We conclude that the triplet mutation is a UV-specific mutation that preferably occurs in NER-deficient genetic backgrounds.

Frequent recovery of triplet mutations in UVB-exposed skin epidermis of Xpc-knockout mice

Mutations of the Xpc gene cause a deficiency in global genome repair, a subpathway of nucleotide excision repair (NER), in mammalian cells. We used transgenic mice harboring the λ-phage-based lacZ mutational reporter gene to study the effect of an Xpc null mutation ( Xpc −/− ) on damage induction, repair and mutagenesis in mouse skin epidermis after UVB irradiation. UVB induced equal amounts of cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (64PPs) in mouse skin epidermis of Xpc −/− and wild-type mice. CPDs were not significantly removed in either of the mouse genotypes by 12 h after irradiation, whereas removal of 64PPs was observed in the wild-type. Irradiation with 300 and 400 J/m 2 UVB increased the lacZ mutant frequency in the Xpc −/− epidermis to at least twice as high as in the wild-type. Ninety-nine lacZ mutants isolated from the UVB-exposed epidermis of Xpc −/− mice were analyzed and compared with mutant sequences from irradiated wild-type mice. The spectra of the mutations in the two genotypes were both highly UV-specific and similar in the dominance of C → T transitions at dipyrimidine sites; however, Xpc −/− mice had a higher frequency of two-base tandem substitutions, including CC → TT mutations, three-base tandem substitutions and double base substitutions that were separated by one unchanged base in a three-base sequence (alternating mutations). These tandem/alternating mutations included a remarkably large number of triplet mutations, a recently reported, novel type of UV-specific mutation, characterized by multiple base substitutions or frameshifts within a three-nucleotide sequence containing a dipyrimidine. We concluded that the triplet mutation is a UV-specific mutation that preferably occurs in NER deficient genetic backgrounds.

Analysis of skin cancer risk factors in immunosuppressed renal transplant patients shows high levels of UV-specific tandem CC to TT mutations of the p53 gene

Immunosuppressed renal transplant recipients (RTRs) are predisposed to non-melanoma skin cancers (NMSCs), predominantly squamous cell carcinomas (SCCs). We have analyzed skin lesions from RTRs with aggressive tumors for p53 gene modifications, the presence of Human Papillomas Virus (HPV) DNA in relation to the p53 codon 72 genotype and polymorphisms of the XPD repair gene. We detected 24 p53 mutations in 15/25 (60%) NMSCs, 1 deletion and 23 base substitutions, the majority (78%) being UV-specific C to T transitions at bipyrimidine sites. Importantly, 35% (6/17) are tandem mutations, including 4 UV signature CC to TT transitions possibly linked to modulated DNA repair caused by the immunosuppressive drug cyclosporin A (CsA). We found 8 p53 mutations in 7/17 (41%) precancerous actinic keratosis (AK), suggesting that p53 mutations are early events in RTR skin carcinogenesis. Immunohistochemical analysis shows a good correlation between p53 accumulation and mutations. HPV DNA was detected in 78% of skin lesions (60% Basal Cell Carcinomas, 82%AK and 79% SCCs). Thus, immunosuppression has increased the risk of infections by HPVs, predominantly epidermodysplasia verruciformis, speculated to play a role in skin cancer development. No association is found between HPV status and p53 mutation. Moreover, p53 codon 72 or frequencies of three XPD genotypes of RTRs are comparable with control populations. The p53 mutation spectrum, presenting a high level of CC to TT mutations, shows that the UV component of sunlight is the major risk factor and modulated DNA repair by immunosuppressive drug treatment may be significant in the skin carcinogenesis of RTRs.

Spontaneous mutation in Big Blue mice from fetus to old age: tissue-specific time courses of mutation frequency but similar mutation types.

Transgenic mouse mutation detection systems permit rapid determination of the frequency and type of mutations allowing direct examination of mutational markers for aging, neurodegeneration, and cancer. The Big Blue transgenic mouse mutation detection system was used to determine the frequency and nature of spontaneous mutations versus age in multiple tissue types. Nuclear DNA was extracted from whole fetus at 13.5 days postcoitus (dpc) and from six tissues postbirth (cerebellum, forebrain, thymus, liver, adipose tissue, and male germline) of Big Blue transgenic mice at four ages: 10 days and at 3, 10, and 25 months postbirth. Forty million total plaque-forming units (pfu) were screened. The time course of mutation frequency with age had a significantly different shape in different tissues (P < 10(-6)). By 13.5 dpc, the whole fetus mutation frequency had already started increasing from the theoretical zero at conception to a value that was about one-half the mid-adulthood (3-10 months) average. From 10 days to 3 months, mutation frequency increased significantly in liver (P = 0.007) and showed an increasing trend in cerebellum, forebrain, and thymus. From 3 to 10 months, there was no significant change in mutation frequency in any tissue examined. From 10 to 25 months, the mutation frequency increased significantly in liver (P < 10(-6)) and adipose tissue (P = 0.002), but not in the other tissues examined (cerebellum, forebrain, and male germline). It is of interest that the mutation frequency in the male germline is consistently the lowest, remaining essentially unchanged in old age. The spectrum of mutation types was unaltered with age, tissue type and gender, although, as previously reported, tandem GG-->TT mutations are tissue specific and show significant increases with age and certain hotspots (Buettner VL et al. [1999]: Environ Mol Mutagen 33:320-324; Hill KA et al. [2003]: Mutat Res 534:173-186). The spectrum of mutation types was generally the same for all tissue types, despite the tissue-specific increases in mutation frequency with age. These data provide a useful reference for future studies of endogenous and exogenous mutagenesis.

Somatic mitochondrial mutations in melanoma resection specimens

As epidemiological data suggest ultraviolet (UV) radiation to be the major environmental factor for the development of melanoma, we screened this malignancy for UV-specific C right curved arrow T and CC right curved arrow TT DNA mutations as described in squamous and basal cell carcinomas of the skin. Mitochondrial (mt) DNA was addressed which is known to be highly more susceptible for mutations than nuclear DNA. In the mt genome we chose part of the non-coding displacement-loop (D-loop) containing two hypermutable (C)n tracts especially prone for C right curved arrow T and CC right curved arrow TT changes. A total of 69 melanoma resection specimens was investigated by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) electrophoresis, followed by DNA cloning and sequencing. We detected alterations of the mt D-loop fragment in 4/34 (12%) melanoma primary tumours and in 7/35 (20%) cutaneous and subcutaneous metastases which was no statistically higher proportion upon Fisher's exact test (p=0.17). Among these 11 positive cases, 9 exhibited alterations in the (C)n microsatellite sequences indicating microsatellite instability (MSI) of mtDNA (C)n tracts. Besides 7 insertions and 2 deletions of nucleotides, 11 mutations occurred including only 4 UV-specific C right curved arrow T mutations in a nodular melanoma of the skin and in two subcutaneous metastases. CC right curved arrow TT mutations were not detected in any tissue sample. As (C)n tract alterations occurred in 2/9 primary melanomas with subsequent metastasis, whereas all 20 non-metastasizing cutaneous melanomas were not affected, these somatic changes may reflect genomic imbalance during tumour progression and may be useful for the assessment of patients' prognosis.

Deamination of 5-Methylcytosines within Cyclobutane Pyrimidine Dimers Is an Important Component of UVB Mutagenesis

UVB mutagenesis is characterized by an abundance of C --> T and 5-methylcytosine --> T transitions at dipyrimidine sequences. It is not known how these mutations might arise. One hypothesis is that UV-induced mutations occur only after deamination of the cytosine or 5-methylcytosine within the pyrimidine dimer. It is not clear how methylation of cytosines at the 5-position influences deamination and how this affects mutagenesis. We have now conducted experiments with a CpG-methylated supF shuttle vector that was irradiated with UVB and then incubated at 37 degrees C to allow time for deamination before passage through a human cell line to establish mutations. This led to a significantly increased frequency of CC --> TT mutations and of transition mutations at 5'-PymCG-3' sequences. A spectrum of deaminated cis-syn cyclobutane pyrimidine dimers in the supF gene was determined using the mismatch glycosylase activities of MBD4 protein in combination with ligation-mediated PCR. Methylation at the C-5 position promoted the deamination of cytosines within cis-syn cyclobutane pyrimidine dimers, and these two events combined led to a significantly increased frequency of UVB-induced transition mutations at 5'-PymCG-3' sequences. Under these conditions, the majority of all supF mutations were transition mutations at 5'-PymCG-3', and they clustered at several mutational hot spots. Exactly these types of mutations are frequently observed in the p53 gene of nonmelanoma skin tumors. This particular mutagenic pathway may become prevalent under conditions of inefficient DNA repair and slow proliferation of cells in the human epidermis.

Oxidative DNA damage induced by copper and hydrogen peroxide promotes CG-->TT tandem mutations at methylated CpG dinucleotides in nucleotide excision repair-deficient cells.

Oxidative DNA damage may play an important role in human disease including cancer. Previously, mutational spectra have been determined using systems that include transition metal ions and hydrogen peroxide (H2O2). G-->T transversions and C-->T transitions were the most common mutations observed including some CC-->TT tandem mutations. C-->T transition mutations at methylated CpG dinucleotides are the most common mutations in human genetic diseases. It has been hypothesized that oxidative stress may increase the frequency of mutations at methylated CpG sequences. Here we have used a CpG-methylated shuttle vector to derive mutational spectra of copper/H2O2-induced DNA damage upon passage of the shuttle vector through human fibroblasts. We find that copper/H2O2 treatment produces higher numbers of CpG transition mutations when the CpGs are methylated but does not create clear C-->T hotspots at these sites. More strikingly, we observed that this treatment produces a substantial frequency of mutations that were mCG-->TT tandem mutations. Six of seven tandem mutations were of this type. mCG-->TT mutations (6/63 = 10% of all mutations) were observed only in nucleotide excision repair-deficient (XP-A) cells but were not found in repair-proficient cells. The data suggest that this novel type of mutation may be produced by vicinal or cross-linked base damage involving 5-methylcytosine and a neighboring guanine, which is repaired by nucleotide excision repair. We suggest that the underlying oxidative lesions could be responsible for the progressive neurodegeneration seen in XP-A individuals.