|Ahead of print publication
Single-nucleotide polymorphisms in dendritic cell (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) gene of hepatocellular carcinoma patients from India
Manash Pratim Sarma1, Dipu Bharali2, Akan Das3, Minakshi Bhattacharjee4, Premashis Kar2
1 Department of Medicine, Maulana Azad Medical College, New Delhi; Department of Biotechnology, Assam Down Town University, Guwahati, Assam, India
2 Department of Medicine, Maulana Azad Medical College, New Delhi, India
3 Department of Bioengineering and Technology, Gauhati University-Institute of Science and Technology, Guwahati, Assam, India
4 Department of Biotechnology, Assam Down Town University, Guwahati, Assam, India
Manash Pratim Sarma,
Department of Biotechnology, Assam Down Town University, Guwahati, Assam
Source of Support: None, Conflict of Interest: None
Objective: Hepatocellular carcinoma (HCC) is one of the major causes of morbidity and mortality in the world. Numerous genomic and proteomic studies have been carried out across the globe to understand cancer biology related to HCC. Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is also known as cluster of differentiation 209. The current study was designed to investigate the association of mutation in DC-SIGN promoter region in HCC patients and healthy controls and to analyze the association of these mutations as a risk factor for HCC development from India.
Materials and Methods: total of 40 cases of HCC and 40 healthy controls without any underlying liver diseases were included in the study. A total of 5 ml of peripheral blood samples were collected, and genomic DNA was isolated using phenol–chloroform method. Polymerase chain reaction amplification was carried out for DC gene, and the amplicons were subjected to direct sequencing (Macrogen, Korea). Mutations were analyzed comparing these sequences with those published sequences from the database using bioinformatics software.
Results: A total of eight point mutations were observed in the HCC cases. The natures of mutation observed were deletion, transition, and transversion. All mutations were located in the 19th chromosome at nine different loci (51,079, 51,493, 51,561, 51,124, 51,125, 51,127, 51,169, 51,170, and 51,172).
Conclusion: Mutation in the promoter region of the DC-SIGN gene may be a possible risk factor for the development of HCC in India. The findings of the study reveal the possible role of these mutants with HCC, and future large-scale prospective studies will further validate the findings of the current study.
Keywords: Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin, hepatitis B virus, hepatocellular carcinoma, mutation, promoter region
|How to cite this URL:|
Sarma MP, Bharali D, Das A, Bhattacharjee M, Kar P. Single-nucleotide polymorphisms in dendritic cell (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) gene of hepatocellular carcinoma patients from India. J Can Res Ther [Epub ahead of print] [cited 2019 Oct 19]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=264227
| > Introduction|| |
Hepatocellular carcinoma (HCC) is one of the major causes of morbidity and mortality in the world (Perz, 2006). Hepatitis B virus (HBV) infection is the major risk factor associated with the development of HCC in regions with large populations such as China and southern Asia because of the high endemicity of the virus in these places (Perz et al., 2006). Association of gene mutation in HCC in comparison to asymptomatic carrier is well studied in India. The present study was designed with an aim to find the association and presence of mutation in DC-SIGN among HBV-associated HCC cases and compare that to healthy individuals from India.
Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is also known as cluster of differentiation 209 (CD209). DC-SIGN is a C-type lectin receptor present on the surface of both macrophages and dendritic cells (DCs) and has a high affinity for the ICAM3 molecule. DC-SIGN on macrophages recognizes and binds to mannose-type carbohydrates, a class of pathogen-associated molecular patterns commonly found on viruses, bacteria, and fungi. This binding interaction activates phagocytosis. On myeloid and preplasmacytoid DCs, DC-SIGN mediates DC rolling interactions with blood endothelium and activation of CD4+ T cells, as well as recognition of pathogen haptens. It binds various microorganisms by recognizing high-mannose-containing glycoproteins on their envelopes and especially functions as a receptor for several viruses such as human immunodeficiency virus (HIV) and hepatitis C.,, Binding to DC-SIGN can promote HIV and hepatitis C virus to infect T-cell from DCs., Thus, binding to DC-SIGN is an essential process for HIV infection. Besides functioning as an adhesion molecule, a recent study has also shown that DC-SIGN can initiate innate immunity by modulating Toll-like receptors although the detailed mechanism is not yet known. DC-SIGN together with other C-type lectins is involved in recognition of tumors by DCs. DC-SIGN is also a potential engineering target for DC-based cancer vaccine.
The role of DC-SIGN in viral infection indicates that it is involved in the initial stages of the HIV infection, as the HIVgp120 molecule causes cointernalization of the DC-SIGN molecule and HIV particle (virion). The DC then migrates to the cognate lymphoid organ, whereupon recycling of the DC-SIGN/HIV virion complex to the cell periphery facilitates HIV infection of CD4+ T-cells by interaction between DC-SIGN and ICAM-3.
DC-SIGN encoded by CD209 on chromosome 19p13.3 is expressed on subsets of DCs and alveolar macrophages,,, and functions both as a cell adhesion receptor and as a pathogen recognition receptor. Acting as a pathogen uptake receptor, DC-SIGN could mediate interactions with a plethora of pathogens including bacteria such as Helicobacter pylori;, viruses such as HIV-1, Ebola,, Cytomegalovirus, hepatitis-C virus,, dengue virus,,, and SARS-coronavirus; and parasites such as Leishmania pifanoi. Several studies have recently reported on the role of DC-SIGN promoter variants in the susceptibility to or pathogenesis of various infectious diseases, such as dengue fever,, tuberculosis,,,, acquired immune deficiency syndrome,,,, and celiac disease. Desprès et al. reported that the G allele of the variant DC-SIGN-336 was associated with strong protection against dengue fever. In addition, several previous reports suggested that variants in the DC-SIGN promoter conferred protection against tuberculosis., However, whether DC-SIGN promoter variants have effects on susceptibility to NPC is still unknown, and so far, no study has reported on the variants in the DC-SIGN promoter in Cantonese population. DC-SIGN is a 44 kDa type II transmembrane protein. Amino acid sequence analysis confirmed that DC-SIGN is composed of 404 amino acids organized into three distinct domains. It has been shown that some pathogens target DC-SIGN to escape lysosomal degradation in early infection and result in chronic infection. DC-SIGN is encoded by the CD 209 gene which is located within the 19p13.2-3 chromosomal region. Several studies, have reported the role of DC-SIGN promoter region polymorphism in the susceptibility to or prognosis of various infectious diseases. The most common mutation in DC-SIGN promoter region is located in positions −139 and −336 as observed in HIV.
It has also been shown that −336T exerts high protection against parenteral HIV-1. About tuberculosis, research has shown that −336T also decreased the risk of developing tuberculosis. HBV, a kind of lentivirus, is an important factor of chronic hepatitis. Humoral transmission is the main pathway of HBV infection that makes HBV to have opportunity to interact with DCs. Some researchers have shown that HBV could be detected on or within DCs. In addition, HBV preS1 and preS2 carried N-glycosylation. These suggested that a DC-specific receptor such as DC-SIGN may be involved in the pathogenesis of HBV infection. In light of this, the hypothesis that some mutations in DC-SIGN promoter region are related to HBV is plausible. On this basis, the study is designed with an aim to analyze the DC-SIGN promoter region polymorphism and to investigate whether there is characteristic mutation within DC-SIGN promoter region in a cohort of HCC cases infected by HBV in India.
| > Materials and Methods|| |
A total of 40 cases of HCC in India and 40 cases of healthy controls without any liver diseases were included in the study. The study includes cases from three locations, namely, Lok Nayak Hospital, New Delhi, Rajaji Government Hospital, Madurai, and Gauhati Medical College with different cultural and linguistic ethnicity. The study was approved by the ethical committee of all the three hospitals and the study confronted to the ethical committee guidelines of EASL Helsinki 1975. All the study participants provided written informed consent before the enrollment in the study.
Sample collection/human DNA extraction
Five-milliliter peripheral whole blood was collected using sterile vacutainer. Blood was processed and subjected to genomic DNA isolation using the standard protocol of DNA isolation from whole blood under aseptic condition.
Polymerase chain reaction amplification
Purified DNA was subjected to polymerase chain reaction (PCR) amplification using specifically designed BLAST primer both forward and reverse and maintaining standard PCR conditions. The reaction mixture contained PCR Master Mix at 25 mm, forward primer at 1.0 ml, reverse primer at 1.0 ml, distilled water at 19.0, and genomic DNA at 4.0 microgram for a 50 μL reaction. The thermocycler condition was used with a slight modification over the already published literature. It involved an initial denaturation for 5 min, 30 cycles of repetition (involving 95 for 45 s, 55 for 45 s, and 72 for 45 s), and final extension of 10 min at 72°. The amplified product was subjected to agarose gel electrophoresis. PCR amplification is followed by direct sequencing (Macrogen, Korea) along with comparing those sequences with the published sequences of DC from database.
Genotyping of dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin promoter variants
A region approximately 1041 bp upstream of the ATG start codon that includes the promoter region was amplified using the following primers: 5-”GCAGTCTTGGTTCCTTGGAG-3” for forward primer 1 and 5-CTTGCAGTGCCTCCTCAGT-3' for reverse primer 1; 5'-TGCTGCTGTCCTCATTTTTG-3' for forward primer 2 and 5'-GCATACAGAAACCCCGTTG-3' for reverse primer 2. Primer 1 delimits the promoter region between nt −602 and 28 and amplifies a 630 bp fragment [Figure 1]. Primer 2 delimits the promoter region between nt −404 and nt −1041 and amplifies a 638 bp fragment. PCR amplification was performed in a volume of 20 μL as follows: 15.85 μL ddH2O, 2.0 μL 10× reaction buffer (with Mg2+), 0.5 μL 4× dNTP (10 mmol L-1), 0.2 μL of each primer (20 μM), 1.25 U Taq DNA polymerase, and 1 μL genomic DNA (20 ng). Touchdown PCR was performed in the model 9700 Gene Amp PCR system (Applied Biosystems, Foster City, CA, USA) with the following conditions: one initial denaturation of 95°C for 5 min, and then, 5 cycles of 94°C for 30 s, 61°C for 30 s (−0.5°C every cycle), and 72°C for 45 s; then, 32 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s, followed by one elongation step at 72°C for 10 min. The amplified products were analyzed by 1.5% agarose gel electrophoresis followed by ethidium bromide staining. PCR amplification was performed using the same conditions for primer 1 and primer 2 as described above. PCR products were recovered and further purified. Sequencing reactions were performed using PCR primers, and all nucleotide sequences were obtained using the automated sequencer (ABI), Macrogen, Korea. Sequence alignment and SNP search were inspected using DNASTAR analysis programs (DNASTAR, Madison, WI, USA) using the nucleic acid sequences from GenBank at National Center for Biotechnology Information as the prototype sequence (GenBank accession: KF990501 to KF990529). The mutations were detected by ClustalW alignment, and few representative pictures were captured as screen view [Figure 2]a, [Figure 2]b, [Figure 2]c, [Figure 2]d, [Figure 2]e.
|Figure 1: Gel photograph depicting 630 bp band size of dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin promoter region of gene fragment. M: ø X 174 Hae III digested 100 bp DNA ladder. Lane 1, 3, 6, 7: Cases and Controls. Lane 2, 4, 5: Blank|
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|Figure 2: (a-e) Nucleotide alignment showing mutation by ClustalW alignment and captured as print screen view|
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| > Results|| |
A total of eight point mutations were observed in the HCC cases. The nature of mutation observed was deletion, transition, and transversion. All mutations were located in the 19th chromosome at nine different loci (51079, 51493, 51561, 51124, 51125, 51127, 51169, 51170, and 51172) [Table 1]. However, none of the sequences from healthy control harbored any of the above mutations.
|Table 1: Types and positions of mutation in dendritic cell gene of hepatocellular carcinoma cases from India|
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| > Discussion|| |
Mutation in the DC-SIGN promoter gene has been associated with ovarian and lung cancer, and −336C mutation has been suspected as a genetic risk factor for developing chronic hepatitis B in a study conducted by Chen et al., 2011. However, studies on HCC are scanty and first of its kind from India.
| > Conclusion|| |
Mutation such as 51079, 51493, 51561, 51124, 51125, 51127, 51169, 51170, and 51172 in the DC-SIGN promoter region may be a possible risk factor for the development of HCC in Indian patients.
We are grateful to University to all members of PCR Hepatitis Laboratory, Department of Medicine, MAMC, New Delhi, India.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Khoo US, Chan KY, Chan VS, Lin CL. DC-SIGN and L-SIGN: The SIGNs for infection. J Mol Med (Berl) 2008;86:861-74.
McGreal EP, Miller JL, Gordon S. Ligand recognition by antigen-presenting cell C-type lectin receptors. Curr Opin Immunol 2005;17:18-24.
Lozach PY, Burleigh L, Staropoli I, Amara A. The C type lectins DC-SIGN and L-SIGN: Receptors for viral glycoproteins. Methods Mol Biol 2007;379:51-68.
Lozach PY, Amara A, Bartosch B, Virelizier JL, Arenzana-Seisdedos F, Cosset FL, et al.
C-type lectins L-SIGN and DC-SIGN capture and transmit infectious hepatitis C virus pseudotype particles. J Biol Chem 2004;279:32035-45.
Geijtenbeek TB, Kwon DS, Torensma R, van Vliet SJ, van Duijnhoven GC, Middel J, et al.
DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 2000;100:587-97.
Wu L, Martin TD, Vazeux R, Unutmaz D, KewalRamani VN. Functional evaluation of DC-SIGN monoclonal antibodies reveals DC-SIGN interactions with ICAM-3 do not promote human immunodeficiency virus type 1 transmission. J Virol 2002;76:5905-14.
den Dunnen J, Gringhuis SI, Geijtenbeek TB. Innate signaling by the C-type lectin DC-SIGN dictates immune responses. Cancer Immunol Immunother 2009;58:1149-57.
Aarnoudse CA, Garcia Vallejo JJ, Saeland E, van Kooyk Y. Recognition of tumor glycans by antigen-presenting cells. Curr Opin Immunol 2006;18:105-11.
Soilleux EJ, Barten R, Trowsdale J. DC-SIGN; a related gene, DC-SIGNR; and CD23 form a cluster on 19p13. J Immunol 2000;165:2937-42.
Tailleux L, Pham-Thi N, Bergeron-Lafaurie A, Herrmann JL, Charles P, Schwartz O, et al.
DC-SIGN induction in alveolar macrophages defines privileged target host cells for mycobacteria in patients with tuberculosis. PLoS Med 2005;2:e381.
Tailleux L, Schwartz O, Herrmann JL, Pivert E, Jackson M, Amara A, et al.
DC-SIGN is the major mycobacterium tuberculosis receptor on human dendritic cells. J Exp Med 2003;197:121-7.
Geijtenbeek TB, Torensma R, van Vliet SJ, van Duijnhoven GC, Adema GJ, van Kooyk Y, et al.
Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses. Cell 2000;100:575-85.
Soilleux EJ, Morris LS, Leslie G, Chehimi J, Luo Q, Levroney E, et al.
Constitutive and induced expression of DC-SIGN on dendritic cell and macrophage subpopulations in situ
and in vitro
. J Leukoc Biol 2002;71:445-57.
Geijtenbeek TB, van Vliet SJ, Engering A, t Hart BA, van Kooyk Y. Self- and nonself-recognition by C-type lectins on dendritic cells. Annu Rev Immunol 2004;22:33-54.
Bergman MP, Engering A, Smits HH, van Vliet SJ, van Bodegraven AA, Wirth HP, et al. Helicobacter pylori
modulates the T helper cell 1/T helper cell 2 balance through phase-variable interaction between lipopolysaccharide and DC-SIGN. J Exp Med 2004;200:979-90.
Alvarez CP, Lasala F, Carrillo J, Muñiz O, Corbí AL, Delgado R. C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola virus in cis and in trans. J Virol 2002;76:6841-4.
Simmons G, Reeves JD, Grogan CC, Vandenberghe LH, Baribaud F, Whitbeck JC, et al.
DC-SIGN and DC-SIGNR bind Ebola glycoproteins and enhance infection of macrophages and endothelial cells. Virology 2003;305:115-23.
Halary F, Amara A, Lortat-Jacob H, Messerle M, Delaunay T, Houlès C, et al.
Human cytomegalovirus binding to DC-SIGN is required for dendritic cell infection and target cell trans-infection. Immunity 2002;17:653-64.
Pöhlmann S, Zhang J, Baribaud F, Chen Z, Leslie GJ, Lin G, et al.
Hepatitis C virus glycoproteins interact with DC-SIGN and DC-SIGNR. J Virol 2003;77:4070-80.
Desprès P, Sakuntabhai A, Julier C. A variant in the CD209 (DC-SIGN) promoter is associated with severity of dengue disease. Med Sci (Paris) 2005;21:905-6.
Marzi A, Gramberg T, Simmons G, Möller P, Rennekamp AJ, Krumbiegel M, et al.
DC-SIGN and DC-SIGNR interact with the glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome coronavirus. J Virol 2004;78:12090-5.
Colmenares M, Puig-Kröger A, Pello OM, Corbí AL, Rivas L. Dendritic cell (DC)-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN, CD209), a C-type surface lectin in human DCs, is a receptor for Leishmania amastigotes
. J Biol Chem 2002;277:36766-9.
Tassaneetrithep B, Burgess TH, Granelli-Piperno A, Trumpfheller C, Finke J, Sun W, et al.
DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells. J Exp Med 2003;197:823-9.
Vannberg FO, Chapman SJ, Khor CC, Tosh K, Floyd S, Jackson-Sillah D, et al.
CD209 genetic polymorphism and tuberculosis disease. PLoS One 2008;3:e1388.
Barreiro LB, Neyrolles O, Babb CL, Tailleux L, Quach H, McElreavey K, et al.
Promoter variation in the DC-SIGN-encoding gene CD209 is associated with tuberculosis. PLoS Med 2006;3:e20.
Koizumi Y, Kageyama S, Fujiyama Y, Miyashita M, Lwembe R, Ogino K, et al.
RANTES-28G delays and DC-SIGN – 139C enhances AIDS progression in HIV type 1-infected Japanese hemophiliacs. AIDS Res Hum Retroviruses 2007;23:713-9.
Martin MP, Lederman MM, Hutcheson HB, Goedert JJ, Nelson GW, van Kooyk Y, et al.
Association of DC-SIGN promoter polymorphism with increased risk for parenteral, but not mucosal, acquisition of human immunodeficiency virus type 1 infection. J Virol 2004;78:14053-6.
Geijtenbeek TB, van Kooyk Y. DC-SIGN: A novel HIV receptor on DCs that mediates HIV-1 transmission. Curr Top Microbiol Immunol 2003;276:31-54.
Núñez C, Rueda B, Martínez A, Maluenda C, Polanco I, López-Nevot MA, et al.
A functional variant in the CD209 promoter is associated with DQ2-negative celiac disease in the Spanish population. World J Gastroenterol 2006;12:4397-400.
Sakuntabhai A, Turbpaiboon C, Casadémont I, Chuansumrit A, Lowhnoo T, Kajaste-Rudnitski A, et al.
A variant in the CD209 promoter is associated with severity of dengue disease. Nat Genet 2005;37:507-13.
Xu YF, Liu WL, Dong JQ, Liu WS, Feng QS, Chen LZ, et al.
Sequencing of DC-SIGN promoter indicates an association between promoter variation and risk of nasopharyngeal carcinoma in cantonese. BMC Med Genet 2010;11:161.
Chen L, Li C, Meng X, Zhu P. Single-nucleotide polymorphisms in dendritic cell -specific intercellular adhesion molecule 3-grabbing nonintegrin gene of hepatocellular carcinoma patients from India. J Cent South Univ Med Sci 2011;36:1052-8.
Gelao L, Criscitiello C, Esposito A, De Laurentiis M, Fumagalli L, Locatelli MA, et al
. Dendritic cell-based vaccines: clinical applications in breast cancer. Immunotherapy 2014;6:349-60.
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