Use of uracil-DNA glycosylase enzyme to reduce DNA-related artifacts from formalin-fixed and paraffin-embedded tissues in diagnostic routine

Background: Detection of somatic mutations is a mandatory practice for therapeutic definition in precision oncology. However, somatic mutation detection protocols use DNA from formalin-fixed and paraffin-embedded (FFPE) tumor tissues, which can result in detection of nonreproducible sequence artifacts, especially C:G > T:A transitions, in DNA. In recent studies, DNA pretreatment with uracil DNA glycosylase (UDG), an enzyme involved in base excision repair, significantly reduced the number of DNA artifacts after mutation detection by next-generation sequencing (NGS) and other methods, without affecting the capacity to detect real mutations. This study aimed to evaluate the effects of UDG enzymatic pretreatment in reducing the number of DNA sequencing artifacts from FFPE tumor samples, to improve the accuracy of genetic testing in the molecular diagnostic routine. Methods: We selected 12 FFPE tumor samples (10 melanoma, 1 lung, and 1 colorectal tumor sample) with different storage times. We compared sequencing results of a 16-hotspot gene panel of NGS libraries prepared with UDG-treated and untreated samples. Results: All UDG-treated samples showed large reductions in the total number of transitions (medium reduction of 80%) and the transition/transversion ratio (medium reduction of 75%). In addition, most sequence artifacts presented a low variant allele frequency (VAF < 10%) which are eliminated with UDG treatment. Conclusion: Including UDG enzymatic treatment before multiplex amplification in the NGS workflow significantly decreased the number of artifactual variants detected in FFPE samples. Thus, including this additional step in the current methodology should improve the rate of true mutation detection in the molecular diagnostic routine.

Uracil-DNA glycosylase-treated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination

Here, we describe a uracil-DNA glycosylase (UNG)-treated reverse transcription loop-mediated isothermal amplification (uRT-LAMP) for the visual detection of all subtypes of avian influenza A virus (AIV). The uRT-LAMP assay can prevent unwanted amplification by carryover contamination of the previously amplified DNA, although the detection limit of the uRT-LAMP assay is 10-fold lower than that of the RT-LAMP without a UNG treatment. To the best of our knowledge, this is the first successful application of deoxyuridine triphosphate/UNG strategy in RT-LAMP for AIV detection, and the assay can be applied for the rapid, and reliable diagnosis of AIVs, even in contaminated samples.

Frequently ABL kinase domain G:C→A:T mutation and uracil DNA glycosylase abnormal expression in TKI-resistant acute lymphoblastic leukemia of Chinese population / 中国实验血液学杂志

Most Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) patients often show rapid recurrence and development of ABL kinase domain (KD) mutation after tyrosine kinase inhibitor (TKI) treatment. To further investigate the mechanism of Ph(+) ALL fast relapse after TKI treatment, ABL KD mutation in 35 Chinese Ph(+) ALL with TKI resistance was detected by direct sequencing. The results showed that 77.1% (27/35) Ph(+) ALL patients with TKI resistance had ABL KD mutation and 55.6% (15/27) Ph(+) ALL patients with ABL KD mutation had T315I. Interestingly, 77.8% (21/27) Ph(+)ALL showed ABL mutation G: C→A:T, including T315I, E255K and E459K. Furthermore, all the Ph(+) ALL patients with two or more ABL KD mutations collaborated with complex chromosome abnormality and all the TKI-resistant Ph(+) ALL patients, whose karyotype progressed from simple t (9;22) into complex, developed ABL KD mutation. Moreover, the expression level of uracil-DNA glycosylase UNG2, which inhibits G:C→A:T transition in genomic DNA, decreased in Ph(+) ALL with TKI-resistance compared to that in newly diagnosis Ph(+) ALL. It is concluded that there is a high frequent ABL KD G:C→A:T mutation and a high genomic instability in Chinese TKI-resistant Ph(+) ALL. In addition, the decreased UNG2 expression in TKI-resistant Ph(+) ALL probably contributes to their high rate of ABL KD G:C→A:T mutation.

Site-Specific Mutagenesis in Escherichia coli by Bulky Exocyclic Amino-Substituted Guanine and Adenine Derivatives in Double-Stranded or Gapped Plasmids / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: 7-Bromomethylbenz[alpha]anthracene is a known mutagen and carcinogen. The mutagenic potency of its two major DNA adducts, i.e., N2-(benz[alpha]anthracen-7-ylmethyl)-2'-deoxyguanosine (b[alpha]a2G) and N6-(benz[alpha]anthracen-7-ylmethyl)-2'-deoxyadenosine (b[alpha]a6A), as well as the simpler benzylated analogs, N2-benzyl-2'-deoxyguanosine (bn2G) and N6-benzyl-2'-deoxyadenosine (bn6A), were determined in E. coli. MATERIALS AND METHODS: Double-stranded and gapped plasmid vectors were used to determine the mutagenicity of b[alpha]a2G, b[alpha]a6A, bn2G and bn6A in E. coli. The four, suitably protected, bulky exocyclic amino-substituted adducts were incorporated into 16-base oligodeoxyribonucleotides, in place of normal guanine or adenine residues, which form part of the ATG initiation codon for the lacZ' alpha-complementation gene. The site-specifically modified oligodeoxyribonucleotides were then incorporated into double-stranded plasmids, which contained uracil residues in the complementary strand in the vicinity of the initiation codon. The uracil residues lead to the creation of a gap in the complementary strand due to the actions of E. coli uracil-DNA glycosylase and AP endonuclease. Following the transfection of these plasmid vectors into E. coli strain GP102, a lacZ alpha complementing version of the parent strain AB1157, their propensity to induce mutation was investigated. RESULTS: The percentages of mutant colonies produced by the four modified nucleosides, in both the double-stranded and gapped plasmid vectors, were not significantly different from those produced by the unmodified plasmids. The mutagenicities of the b[alpha]a2G and b[alpha]a6A were extremely low, and a totally unexpected result, whereas, those of the bn2G and bn6A were undetectable. CONCLUSION: In this E. coli site-specific mutagenesis system, these bulky aralkylated adducts exhibited no significant mutagenicities, either with or without SOS induction.

Cloning of human uracil N-glycosylase and its detection in cancer tissues by quantitative RT-PCR / 生物工程学报

The uracil in DNA comes from either the misincorporation of dUTP in place of dTTP or deamination of cytosine. In the latter case, it can result in a GC to AT transition mutation if the uracil is not removed before DNA replication. Base excision repair (BER) is a major pathway for removing DNA lesions arising from endogenous processes as well as those induced by exposure to exogenous chemicals or irradiation. BER is initiated by DNA glycosylases that excise aberrant bases from DNA by cleavage of the N-glycosidic bond linking to the base of its deoxyribose sugar. Uracil N-glycosylase (UNG) is the enzyme responsible for the first step in the BER pathway that specifically removes uracil from DNA. The UNG gene undergoes both temporal and spatial regulation mainly at the level of transcription. Normally cancer cells undergo over-proliferation and up-regulate their UNG during tumorigenesis. In this study we examine the correlation between UNG level and carcinogenesis, and explore the possibility of using UNG as a marker for cancer diagnosis. Human UNG gene was amplified from the total RNA of the human choriocarcinoma cell line, JEG-3, by RT-PCR. After purification, the 942bp full-length UNG cDNA coding sequence was digested with EcoR I and Sal I, and cloned into the digested pET-21 to construct a recombinant vector, pUNG. The UNG protein was expressed under the control of T7 promoter in E. coli BL21 (DE3) cells induced with IPTG. After ultrasonic treatment, the cell lysate and precipitate were analyzed by SDS-PAGE and a 39kD band was detected. The plasmid was serially diluted at appropriate concentrations and employed as standards in the subsequent quantification. Total RNAs were extracted from 18 pairs of clinical samples, each pair contains a sample of esophageal squamous cell carcinoma (ESCC) tissue and its surrounding normal esophageal epithelia. The copy numbers of UNG mRNA in these RNA samples were determined by real-time quantitative RT-PCR using a Lightcycler (Roche). UNG was present in 13 cases of ESCC (13/18, n = 18) but absent in all of the normal tissues. The results indicated that there was a correlation between high level of UNG expression and the carcinogenesis of ESCC.