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Identification of low-frequency variants of UGT1A3 associated with bladder cancer risk by next-generation sequencing.

ABSTRACT: Although genome-wide association studies (GWASs) have successfully revealed many common risk variants for bladder cancer, the heritability is still largely unexplained. We hypothesized that low-frequency variants involved in bladder cancer risk could reveal the unexplained heritability. Next-generation sequencing of 113 patients and 118 controls was conducted on 81 genes/regions of known bladder cancer GWAS loci. A two-stage validation comprising 3,350 cases and 4,005 controls was performed to evaluate the effects of low-frequency variants on bladder cancer risk. Biological experiments and techniques, including electrophoretic mobility shift assays, CRISPR/Cas9, RNA-Seq, and bioinformatics approaches, were performed to assess the potential functions of low-frequency variants. The low-frequency variant rs28898617 was located in the first exon of UGT1A3 and was significantly associated with increased bladder cancer risk (odds ratio = 1.50, P = 3.10 × 10^-6). Intriguingly, rs28898617 was only observed in the Asian population, but monomorphism was observed in the European population. The risk-associated G allele of rs28898617 increased UGT1A3 expression, facilitated UGT1A3 transcriptional activity, and enhanced the binding activity. In addition, UGT1A3 deletion significantly inhibited the proliferation, invasion, and migration of bladder cancer cells and xenograft tumor growth. Mechanistically, UGT1A3 induced LAMC2 expression by binding CBP and promoting histone acetylation, which remarkably promoted the progression of bladder cancer. This is the first targeted sequencing study to reveal that the novel low-frequency variant rs28898617 and its associated gene UGT1A3 are involved in bladder cancer development, providing new insights into the genetic architecture of bladder cancer.

SUBMITTER: Zheng R

PROVIDER: S-EPMC8016664 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:COVID-19 is caused by SARS-CoV-2, several virulent variants of which have emerged since 2019. More than 529 million people have been infected, and at least 6 million have died. Our aim was to develop a fast, accurate, low-cost method for detecting and identifying newly emerging variants of concern (VOCs) that could pose a global threat. The 341-bp DNA sequence of a specific region of the SARS-CoV-2's spike protein was amplified by a one-step PCR on RNA samples from 46 patients. The product was sequenced using next-generation sequencing (NGS). DNA sequences from seven genomes, the original Wuhan isolate and six different representative variants obtained from the GISAID website, were used as references. Complete whole-genome sequences from local isolates were also obtained from the GISAID website, and their RNA was used for comparison. We used an amplicon-based NGS method (termed VOC-NGS) for genotyping and successfully identified all 46 samples. Fifteen (32.6%) were like the original isolate. Twenty-seven were VOCs: nine (19.5%) Alpha, eight (19%) Delta, six (14%) Beta, and four (8.7%) Omicron. Two were variants of interest (VOI): one (2%) Kappa and one (2%) Zeta. Two samples were mixtures of two variants, one of Alpha and Beta and one of Alpha and Delta. The Spearman correlation between whole-genome sequencing (WGS) and VOC-NGS was significant (P < 0.001) with perfect agreement (Kappa = 0.916) for 36/38 (94.7%) samples with VOC-NGS detecting all the known VOCs. Genotyping by VOC-NGS enables rapid screening of high-throughput clinical samples that includes the identification of VOCs and mixtures of variants, at lower cost than WGS. IMPORTANCE The manuscript described SARS-Cov-2 genotyping by VOC-NGS, which presents an ideal balance of accuracy, rapidity, and cost for detecting and globally tracking VOCs and some VOI of SARS-CoV-2. A large number of clinical samples can be tested together. Rapid introduction of new mutations at a specific site of the spike protein necessitates efficient strain detection and identification to enable choice of treatment and the application of vaccination, as well as planning public health policy.

Project description:Next-generation DNA sequencing is opening new avenues for genetic association studies in common diseases that, like deep vein thrombosis (DVT), have a strong genetic predisposition still largely unexplained by currently identified risk variants. In order to develop sequencing and analytical pipelines for the application of next-generation sequencing to complex diseases, we conducted a pilot study sequencing the coding area of 186 hemostatic/proinflammatory genes in 10 Italian cases of idiopathic DVT and 12 healthy controls.A molecular-barcoding strategy was used to multiplex DNA target capture and sequencing, while retaining individual sequence information. Genomic libraries with barcode sequence-tags were pooled (in pools of 8 or 16 samples) and enriched for target DNA sequences. Sequencing was performed on ABI SOLiD-4 platforms. We produced > 12 gigabases of raw sequence data to sequence at high coverage (average: 42X) the 700-kilobase target area in 22 individuals. A total of 1876 high-quality genetic variants were identified (1778 single nucleotide substitutions and 98 insertions/deletions). Annotation on databases of genetic variation and human disease mutations revealed several novel, potentially deleterious mutations. We tested 576 common variants in a case-control association analysis, carrying the top-5 associations over to replication in up to 719 DVT cases and 719 controls. We also conducted an analysis of the burden of nonsynonymous variants in coagulation factor and anticoagulant genes. We found an excess of rare missense mutations in anticoagulant genes in DVT cases compared to controls and an association for a missense polymorphism of FGA (rs6050; p = 1.9 × 10(-5), OR 1.45; 95% CI, 1.22-1.72; after replication in > 1400 individuals).We implemented a barcode-based strategy to efficiently multiplex sequencing of hundreds of candidate genes in several individuals. In the relatively small dataset of our pilot study we were able to identify bona fide associations with DVT. Our study illustrates the potential of next-generation sequencing for the discovery of genetic variation predisposing to complex diseases.

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Identification of low-frequency variants of UGT1A3 associated with bladder cancer risk by next-generation sequencing.

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