Target Enrichment Technology Identifies Genetic Loci Of Nasopharyngeal Carcinoma Within6p21.3and Preliminary Study On Possible Functions

BackgroudNasopharyngeal carcinoma （NPC） is a malignant tumor that arises in the epithelial lining of the nasopharynx. This neoplasm has remarkable ethnic and geographic distribution, with a high prevalence in southern China （e.g. Guangdong, Guangxi, Hunan）, HongKong, Taiwan and Singapore. The annual incidence rate reaches10-50cases per100,000people in the endemic regions. NPC is rare in northern China and Caucasians. Intermediate incidence rates among Chinese people who have migrated abroad and more than10%of NPC patients have a familiar predisposition, suggesting that genetic and environmental factors play important roles in NPC. In addition, studies have shown a male preponderance over the female with a ratio of about2-3:1. Taken together, these findings indicate that NPC is likely to have a complex etiology involving genetic, viral and environmental factors.The MHC region located at6p21.3（-4Mb） occupies0.13%of the human genome （3×109bp）, but contains-0.5%（>150） of the-32000known protein coding genes. Many of the MHC gene products are ligands, receptors, interacting proteins, signaling factors and transcription regulators involved in the inflammatory response, antigen processing and presentation as part of the adaptive immune response, and interactions with NK cells and cytokines as part of the innate immune responses. The human MHC, regarded as the human leukocyte antigen （HLA）, is the most complex system regulating specific immune response and hereditary susceptibility in people. It is characterized by extensive LD and high polymorphisms, which have been reported to associate with many diseases, e.g. rheumatoid arthritis （RA）, osteoarthritis （OA）, Kawasaki disease （KD）, Ankylosing Spondylitis （AS）.Since1974, many studies have indicated that some specific human leukocyte antigen （HLA） haplotypes and genes within the HLA complex are potentially associated with NPC. Our previous meta-analysis showed that populations in geographical areas with higher risk of developing NPC display HLA distribution patterns different and opposite from areas of low incidence. HLA complex is undoubtedly playing an important role in NPC predisposition, either by playing a functional role in modulating an innate and adaptive immune response against EBV, or as a marker of an unrelated predisposition locus in close linkage. Recently two independent research groups conducted GWA studies in Taiwanese and southern Chinese respectively and consistently identified HLA as a major susceptibility locus in addition to some new loci.With the rapid development of next generation sequencing, some new ideas have been provided in terms of pathogenesis, prevention, diagnosis and the treatment of diseases. The exon region of genes contains protein coding messages covering a majority of functional variation associated with various phenotypes. Because the exome occupies～1%of the human genome, it is easy, economic and high-efficient to sequence the exome. Therefore, exome sequencing is becoming a new powerful strategy to discover causative genes and susceptibility gene for monogenic disorders, complex diseases and cancers. With the SureSelect Target Enrichment System, many genomic areas of interest can be sequenced with an efficient process that reduces costs and allows more samples to be analyzed. Meanwhile, reducing the amount of DNA being interrogated can allow investigators to perform their experiments with more statistically relevant numbers of samples. ObjectiveAs the MHC region is the major susceptibility locus and very complex, we used SureSelect Target Enrichment System to sequence the exonic and regulatory regions of genes within MHC in order to explore the functional SNPs. This may provide some new clues for further elucidating the NPC susceptibility and pathogenesis.Contents and methods1. The SureSelect Target Enrichment System and next-generation sequencing technology was used to identify potentially associated SNPs within MHC.（1） Agilent eArray, a free web-based design tool, was used to create and order SureSelect target enrichment oligo sets.（2） The genomic DNA from40patients was captured according to the specification, and then associated captured libraries were sequenced by Illumina Solexa.（3） The above results were aligned with the data of40Northern Chinese controls from the1000Genomes Project, and found the potentially associated SNPs for the following replication study.2. The potentially associated SNPs were confirmed by Sequenom MassArray System.（1） In the preliminary stage, we selected and genotyped potentially associated SNPs in310patients and310controls.（2） In the second stage, significant SNPs were examined in another independent study population including768cases and759controls.（3） A joint association analysis comibined samples from stage one and two was performed to search for SNPs associated with NPC susceptibility in southern Chinese.3. Preliminary studies on the possible function of one of the susceptibility SNPs.（1） The SNP possible function was predicted by bioinformatics tools （FastSNP）.（2） The genomic DNA from NPC cell lines and NPC and NP tissues was extracted, and genotyped by Sequenom MassArray System and sequencing.（3） The gene mRNA expression was comparatively measured by real-time PCR in NPC cell lines, NPC tissues and noncancerous nasopharyngeal tissues with different genotypes. The gene protein expression was comparatively measured by western blot in NPC tissues with different genotypes.Results1. The SureSelect Target Enrichment System and next-generation sequencing technology was used to identify potentially associated SNPs within MHC.9983SNPs were found in the exonic and regulatory regions of genes within MHC using target capturing and sequencing.122SNPs of them were significantly different between NPC and controls. They were located in the genes such as HLA-A, HLA-B, HLA-DQA1/DQB1, PSORS1C1, etc..2. The potentially associated SNPs were confirmed by Sequenom MassArray System.In the stage one,122potentially associated SNPs were designed to be genotyped. After quality controls, we conducted association analysis.9significant SNPs were significant and selected for the second stage of study. The joint association analysis of combined two stages" s data, showed that chr6₃0280350（novel）, rs1265053, rsl610696, chr6₃1190425（novel） and rs17604492were statistically significant; their P values were6.35E-14,2.61E-15,0.006398,0.006398,0.009038,0.0107respectively and their OR values were1.902（1.605-2.254）,0.6056（0.5347-0.686）,0.664（0.4938-0.8928）,0.662（0.4341-0.8911）,0.7386（0.5849-0.9328） respectively. These SNPs are located in the exonic and regulational regions of TRIM26, C6orf15, HLA-G, PSORS1C1and NOTCH4respectively.3. The TRIM26chr6₃0280350（A/T, novel） polymorphism may affect the mRNA and protein expression level of TRIM26.（1）. Using a bioinformatics tool, FastSNP （http://fastsnp.ibms.sinica.edu.tw/pages/input novel.jsp）, we predicted the possible functions of chr6₃0280350. There were different transcription factor binding sites between A site and T site. SRY, MZF1, STATx and C/EBP can bind to A site, only MZF1link to T site.（2） All NPC cell lines were TT type except C666-1（AT） for chr6₃0280350. Subsequently, qRT-PCR showed that the expression of TRIM26was decreased in C666-1compared to other NPC cell lines with TT type （F=34.007, P<0.001）. （3） By genotyping NPC and NP tissues, we prepared9AA/AT-samples and25TT-samples for the detection of TRIM26expression. In consistent with the data from NPC cell lines, the expression level of TRIM26was lower in AA/AT samples than TT samples （t=-3.25, P=0.003）.（4） Western blot showed the down-regulated protein expression level of TRIM26in AA/AT type samples compared to TT type samples.Conclusions1. The SureSelect Target Enrichment System is feasible in capturing the MHC region. About122potentially associated SNPs could be caught for the further research.2. Through two-stage case-control analysis, we identified five new susceptibility loci, TRIM26（chr6₃0280350）, C6orf15（rs1265053）, HLA-G （rs161096）, PSORS1C1（chr6₃1190425） and NOTCH4（rs17604492）.3. The SNP chr6₃0280350with A allele may reduce the gene expression of TRIM26and hereby increase the susceptibility of NPC.