Massive interstitial copy-neutral loss-of-heterozygosity as evidence for cancer being a disease of the DNA-damage response

BMC Medical Genomics, Jul 2015

Background The presence of loss-of-heterozygosity (LOH) mutations in cancer cell genomes is commonly encountered. Moreover, the occurrences of LOHs in tumor suppressor genes play important roles in oncogenesis. However, because the causative mechanisms underlying LOH mutations in cancer cells yet remain to be elucidated, enquiry into the nature of these mechanisms based on a comprehensive examination of the characteristics of LOHs in multiple types of cancers has become a necessity. Methods We performed next-generation sequencing on inter-Alu sequences of five different types of solid tumors and acute myeloid leukemias, employing the AluScan platform which entailed amplification of such sequences using multiple PCR primers based on the consensus sequences of Alu elements; as well as the whole genome sequences of a lung-to-liver metastatic cancer and a primary liver cancer. Paired-end sequencing reads were aligned to the reference human genome to identify major and minor alleles so that the partition of LOH products between homozygous-major vs. homozygous-minor alleles could be determined at single-base resolution. Strict filtering conditions were employed to avoid false positives. Measurements of LOH occurrences in copy number variation (CNV)-neutral regions were obtained through removal of CNV-associated LOHs. Results We found: (a) average occurrence of copy-neutral LOHs amounting to 6.9 % of heterologous loci in the various cancers; (b) the mainly interstitial nature of the LOHs; and (c) preference for formation of homozygous-major over homozygous-minor, and transitional over transversional, LOHs. Conclusions The characteristics of the cancer LOHs, observed in both AluScan and whole genome sequencings, point to the formation of LOHs through repair of double-strand breaks by interhomolog recombination, or gene conversion, as the consequence of a defective DNA-damage response, leading to a unified mechanism for generating the mutations required for oncogenesis as well as the progression of cancer cells.

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Massive interstitial copy-neutral loss-of-heterozygosity as evidence for cancer being a disease of the DNA-damage response

Gastric Massive interstitial copy-neutral loss-of- heterozygosity as evidence for cancer being a disease of the DNA-damage response Yogesh Kumar 0 2 Jianfeng Yang 0 2 Taobo Hu 0 2 Lei Chen 1 Zhi Xu 3 Lin Xu 7 Xiao-Xia Hu 6 Gusheng Tang 6 Jian-Min Wang 6 Yi Li 5 Wai-Sang Poon 5 Weiqing Wan 4 Liwei Zhang 4 Wai-Kin Mat 2 Frank W. Pun 2 Peggy Lee 2 Timothy H. Y. Cheong 2 Xiaofan Ding 2 Siu-Kin Ng 2 Shui-Ying Tsang 2 Jin-Fei Chen 3 Peng Zhang 8 Shao Li 8 Hong-Yang Wang 1 Hong Xue 2 0 Equal contributors 1 Eastern Hepatobiliary Surgery Institute, Second Military Medical University , Shanghai , China 2 Division of Life Science, Applied Genomics Centre and Centre for Statistical Science, Hong Kong University of Science and Technology , Clear Water Bay , Hong Kong 3 Department of Oncology, Nanjing First Hospital, and Collaborative Innovation Center for Cancer Personalized Medicine, Nanjing Medical University , Nanjing , China 4 Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University , 6 Tiantan Xili, Dongcheng District, Beijing 100050 , China 5 Department of Surgery, The Chinese University of Hong Kong , Hong Kong , China 6 Department of Hematology, Changhai Hospital, Second Military Medical University , Shanghai , China 7 Jiangsu Key Laboratory of Cancer Molecular Biology and Translational Medicine, Jiangsu Cancer Hospital , Nanjing , China 8 MOE Key Laboratory of Bioinformatics and Bioinformatics Division, TNLIST, and Department of Automation, Tsinghua University , Beijing 100084 , China Background: The presence of loss-of-heterozygosity (LOH) mutations in cancer cell genomes is commonly encountered. Moreover, the occurrences of LOHs in tumor suppressor genes play important roles in oncogenesis. However, because the causative mechanisms underlying LOH mutations in cancer cells yet remain to be elucidated, enquiry into the nature of these mechanisms based on a comprehensive examination of the characteristics of LOHs in multiple types of cancers has become a necessity. Methods: We performed next-generation sequencing on inter-Alu sequences of five different types of solid tumors and acute myeloid leukemias, employing the AluScan platform which entailed amplification of such sequences using multiple PCR primers based on the consensus sequences of Alu elements; as well as the whole genome sequences of a lung-to-liver metastatic cancer and a primary liver cancer. Paired-end sequencing reads were aligned to the reference human genome to identify major and minor alleles so that the partition of LOH products between homozygous-major vs. homozygous-minor alleles could be determined at single-base resolution. Strict filtering conditions were employed to avoid false positives. Measurements of LOH occurrences in copy number variation (CNV)-neutral regions were obtained through removal of CNV-associated LOHs. Results: We found: (a) average occurrence of copy-neutral LOHs amounting to 6.9 % of heterologous loci in the various cancers; (b) the mainly interstitial nature of the LOHs; and (c) preference for formation of homozygous-major over homozygous-minor, and transitional over transversional, LOHs. Conclusions: The characteristics of the cancer LOHs, observed in both AluScan and whole genome sequencings, point to the formation of LOHs through repair of double-strand breaks by interhomolog recombination, or gene conversion, as the consequence of a defective DNA-damage response, leading to a unified mechanism for generating the mutations required for oncogenesis as well as the progression of cancer cells. Copy number variation; Double strand break repair; Gain-of-heterozygosity; Gene conversion; Inter-homologous recombination; Loss-of-heterozygosity Background As a common feature of cancer cells, LOHs have been investigated by cytogenetics, fluorescence in situ hybridization, comparative genomic hybridization (CGH), array-CGH, and single nucleotide polymorphism (SNP)based microarrays [ 1–4 ]. With the application of nextgeneration sequencing, analysis of LOH in cancer can further be conducted at the level of single base resolution [ 5, 6 ]. However, owing to the importance of LOHs giving rise to loss of major alleles and inactivation of tumor suppressor genes, hitherto investigations of LOHs in cancers have been focused mainly on LOHs that yield homozygous-minor genotypes. Yet a comprehensive understanding of the properties and origins of LOHs in cancers requires analysis of all types of LOHs in multiple cancers. Accordingly, in the present study next-generation sequencing was applied to determine at single-base resolution the LOHs in the genomic sequences of various types of cancers, covering not only sequence regions that have undergone loss of heterozygosity but also single nucleotide changes where a heterozygous position has mutated to a homozygous one. Thirty tumor-control pairs of six different types of cancers including glioma (glioblastoma and astroglioma), acute myeloid leukemia, gastr (...truncated)


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Frank W. Pun, Gusheng Tang, Hong Xue, Hong-Yang Wang, Jian-Min Wang, Jianfeng Yang, Jin-Fei Chen, Lei Chen, Lin Xu, Liwei Zhang, Peggy Lee, Peng Zhang, Shao Li, Shui-Ying Tsang, Siu-Kin Ng, Taobo Hu, Timothy H. Y. Cheong, Wai-Kin Mat, Wai-Sang Poon, Weiqing Wan, Xiao-Xia Hu, Xiaofan Ding, Yi Li, Yogesh Kumar, Zhi Xu. Massive interstitial copy-neutral loss-of-heterozygosity as evidence for cancer being a disease of the DNA-damage response, BMC Medical Genomics, 2015,