KR100643122B1 - Use of il10 haplotypes for predicting susceptibility to progression of hbv to hcc - Google Patents

Abstract

Interleukin-10 ( IL10 ) is a potent lymphoid cell produced by macrophages and monosite and T-cell lymphocyte replication and lymphoid cells that inhibit the secretion of inflammatory cytokines ( IL1, TNFA, TGFB IL6, IL8 and IL12 ). Th-2 cytokine. Genetic analysis of HBV-cohorts of well-documented clinical features for HBV infection revealed the development of hepatocellular carcinoma (HCC) in a dose-dependent form of IL10-ht2 , one of the IL10 haplotypes. It is very closely related to. The gene frequency of IL10-ht2 was very high in the HCC group, with HBV progression from chronic hapatitis (CH) to liver cirrhosis (LC) and liver cancer (HCC) among hepatitis B patients. Gene frequencies also increased significantly in the order of sensitivity to. In addition, survival analysis suggested that the onset age of liver cancer is promoted in patients with chronic hepatitis B with IL10-ht2 . The results of the limited category analysis also supported the strong association of IL10-ht2 with progression of liver cancer (HCC). It presents the fact that promoted the development of adjustment (up-regulate) the IL10 chronic hepatitis B (chronic hepatitis B) proceeds, in particular liver cancer (HCC) - IL10 to IL10 generated increases adjusted by the haplotype variant upward.

Haplotype, HBV, HCC, Interleukin-10 (IL10)

Description

Use of IL10 haplotypes to predict susceptibility to HV progression / HCC generation {USE OF IL10 HAPLOTYPES FOR PREDICTING SUSCEPTIBILITY TO PROGRESSION OF HBV TO HCC}

1 shows a cleavage map of human interleukin-10 (IL10) on chromosome 1q31-33.

Figure 2a shows the HCC generation age based on IL10-ht2 as a Kaplan-Meier survival curve: a) is-/-, b) is- / ht2 and c) is IL10-ht2 / ht2 .

2B is a curve for the dominant model comparing IL10 − / − with ht2 / ht2 or IL10 − / ht2 as the Kaplan-Meier survival curve.

2C is a curve for the recessive model comparing IL10 ht2 / ht2 to IL10 − / − or − / ht2 as a Kaplan-Meier survival curve.

3A shows the frequency of IL10-ht2 / ht2 or IL10 − / ht2 in three categorical analyzes of HBV progression in HbsAg positive patients (dominant model).

3B shows the frequency of IL10-ht2 / ht2 or IL10 − / ht2 in three categorical analyzes of HBV progression in HbsAg positive patients.

3C shows the frequency of IL10-ht2 / ht2 in three categorical analyzes of HBV progression in HbsAg positive patients.

4A and 4D show the frequency of IL10-ht2 / ht2 or IL10 − / ht2 in two defined categorical analyzes of HCC development in HbsAg positive patients (dominant model).

4B and 4E show the frequency of IL10 − / ht2 in two defined categorical analyzes for HCC development in HbsAg positive patients.

4C and 4F show the frequency of IL10-ht2 / ht2 in two defined categorical analyzes for HCC development in HbsAg positive patients.

Technical Field

The present invention relates to polymorphism of genes associated with disease. More specifically, the present invention measures polymorphism of the human IL10 gene which is dose-dependently proportional to sensitivity to HBV progression / HCC development and provides an association with sensitivity to HBV progression / HCC development.

Prior art

Viral hepatitis B infection is one of the major infectious diseases with more than 260 million chronic illnesses worldwide: 78% of these chronic diseases are Asian, 16% Africa, 3% South America, 3% Europe, Distributed in North America and Oceania. Hepatitis is chronic hepatitis (CH), cirrhosis (LC) and hepatocellular carcinoma (HCC), one of the 10 most common carcinomas (Ref. MA Kane, Soz Praventivmed 43 Suppl 1 , S24-6, S98-100 (1998)) Is a major risk factor. The link between HBV and HCC is now well established. Chronic carriers of HBV have a higher risk of developing HCC compared to non-infected people living in HBV endemic areas. [Ref. K. Huh, SY Choi, YS Whang, DS Lee, J Korean Med Sci 13 , 306- 10 (Jun, 1998); BS Blumberg, IARC Sci Publ , 243-61 (1984); And Merican et al. , J Gastroenterol Hepatol 15 , 1356-61 (Dec, 2000)]. Generally, HCC occurs 30 to 50 years after infection; As a result, HCC is more readily observed in individuals exposed to HBV prior to adulthood. Although chronic hepatitis B virus (HBV) infection can lead to serious liver disease, the risk of development depends on the group of carriers of HBV. Some carriers of HBV remain healthy (without developing) even after decades after infection. In light of this, the incidence of HBV infection is not determined by viral strains, but rather, allelic variants in the human genome appear to have more influence on the progression of viral hepatitis after infection. M. Thursz, Antiviral Res 52 , 113-6 (Nov, 2001)].

Tumor Necrosis Factor-alpha ( TNFA ) in Hepatitis C [Ref: LJ Yee, J. Tang, J. Herrera, RA Kaslow, DJ van Leeuwen, Genes Immun 1 , 386-90 (Aug, 2000); And HR Rosen et al. , Transplantation 68 , 1898-902 (Dec 27, 1999)], Transforming Growth Factor-beta ( TGFB ), angiotensinogen ( AT ) [Reference: EE Powell et al. , Hepatology 31 , 828-33 (Apr, 2000)], Human Leukocyte Antigen (HLA) [N. Kuzushita et al. , Hepatology 27 , 240-4 (Jan, 1998)], Transforming Growth Factor-alpha ( TGFA ) and Insulin-like Growth Factor ( IIF -II ) [Ref. : S. Tanaka, K. Takenaka, T. Matsumata, R. Mori, K]; N-Acetyltransferase ( NAT ) in hepatitis B [Reference: MC Yu, JM Yuan, S. Govindarajan, RK Ross, Can J Gastroenterol 14 , 703-9 (Sep, 2000)], vitamin D Receptor (Vitamin D Receptor; VDR ) [Ref . R. Bellamy et al. , J Infect Dis 179 , 721-4 (Mar, 1999), HLA 13 [Ref. A. Almarri, JR Batchelor, Lancet 344 , 1194-5 (Oct 29, 1994); In chronic liver disease, polymorphisms in genes that decode Aldehyde Dehydrogenase-2 ( ALDH2 ) were found to be involved in the progression of hepatitis.

A strong genetic predisposition to determine the outcome of hepatitis B virus (HBV) infection has been established by studies on twins [S. Shimbo et al. , Southeast Asian J Trop Med Public Health 28 , 500-6 (Sep, 1997). The likelihood of developing ongoing chronic hepatitis B was largely determined by the age of infection. Chronicization rate is approximately 90% for mainstream infections [Ref .: CE Stevens, RP Beasley, J. Tsui, WC Lee, N Engl J Med 292 , 771-4 (Apr 10, 1975)], 1-5 years old. 50% for [Ref .: RP Beasley et al. , J Infect Dis 146 , 198-204 (Aug, 1982); And in P. Coursaget et al. , J Med Virol 22 , 1-5 (May, 1987)], and 5% of adulthood infections [Ref . NC Tassopoulos et al. , Gastroenterology 92 , 1844-50 (Jun, 1987)]. In HBV endemic regions, including Korea, infections occur early in fetal or early childhood, which is the cause of high chronic HBV infection rates. In addition, the risk of HBV infection persists after age 5, but infection at this time does not materially contribute to high rates of chronic infection [Ref: MS Favero, JE Maynard, RT Leger, DR Graham, RE Dixon, Ann Intern Med 91 , 872-6 (Dec, 1979); And in C. Ferrari et al. , J Hepatol 31 Suppl 1 , 31-8 (1999). Prior to initiating HBV vaccination in 1983, most infections in Korea were caused by perinatal transmission [Ref . WK Chung et al. , J Infect Dis 151 , 280-6 (Feb, 1985)]. Perinatal infections are clinically distinct from later infections. Most newborns go through an asymptomatic carrier, despite high levels of HBV replication, because the immune response is still immature, and most adults get rid of HBV [Ref. M. Ruiz-Moreno, J. Hepatol 17 Suppl 3 , S64-6 (1993)].

HCC outbreaks are geographically different, most common in Asia and Africa, where HBV infection is endemic and infection usually occurs in early childhood [Ref: MC Yu, JM Yuan, S. Govindarajan, RK Ross, Can J Gastroenterol 14 , 703 -9 (Sep, 2000)]. Persistent HBV infection is considered an essential factor in HCC development. Thus, early vertical infections from mothers who are carriers are the most important cause of cirrhosis and HCC. The risk of progressive HCC has been demonstrated to be at least 100-fold higher in HbsAg carriers than in non-carriers [WHC Rajen Koshy, Hepatitis B Virus . 1st, Ed. (Imperial College Press, London, UK, 1998).

IL10, on the other hand, is a potent Th-2 cytokine produced by lymphoid cells that inhibit macrophage and monosite and T-cell lymphocyte replication and secretion of inflammatory cytokines (IL1, TNFA, TGFB IL6, IL8 and IL12). [Reference: L. Meda et al. , J Neuroimmunol 93 , 45-52 (Jan 1, 1999); And in A. Eigler et al. , J Leukoc Biol 68 , 97-103 (Jul, 2000)]. IL10 has also been shown to regulate neutrophil infiltration, hepatocyte proliferation and hepatic fibrosis induced by carbon tetrachloride in mice [H. Louis et al. , Hepatology 28 , 1607-15 (Dec, 1998). IL10 also normalized serum ALT levels, improved liver histology, and resulted in hepatic fibrosis reduction in a large number of patients treated [Ref: DR Nelson, GY Lauwers, JY Lau, GL Davis, Gastroenterology 118 , 655-60 (Apr, 2000)]. Increasing serum IL10 levels in patients with acute liver disease could predict improved outcomes in these patients [Ref . E. Yumoto et al. , J Gastroenterol Hepatol 17 , 285-94 (Mar, 2002)]. Hepatitis C virus (HCV) increased IL10 production by human CD4 + T cells (H9) for a long time. [Reference: O. Delpuech, DB Buffello-Le Guillou, E. Rubinstein, C. Feray, MA Petit, Eur Cytokine Netw 12 , 69-77 (Mar, 2001). It is reported that one of the promoter polymorphisms of IL10 can be used to predict the response of chronic hepatitis C to Interferon-alpha (IFNA). [Reference: CJ Edwards-Smith et al. , Hepatology 30 , 526-30 (Aug, 1999).

In addition, IL10, initially designated as cytokine synthesis inhibitory factor (CSIF), is a pleiotropic cytokine that exhibits immunosuppressive or immunostimulatory effects on various types of cells. In various reports, variants in the IL10 promoter region showed functional differences and reported functional analysis of IL10 promoter variants. Nuclear -binding activity by IL10-592 A> C polymorphism [Ref .: HD Shin et al. , Proc Natl Acad Sci USA 97 , 14467-72 (Dec 19, 2000)], serum total IgE levels [reference: K. Hobbs, J. Negri, M. Klinnert, LJ Rosenwasser, L. Borish, Am J Respir Crit Care Med 158 , 1958-62 (Dec, 1998)] and IL10 levels [reference: J. Zhang et al. , Zhonghua Yi Xue Za Zhi 82 , 114-8 (Jan 25, 2002); And LJ Rosenwasser, L. Borish, Am J Respir Crit Care Med 156 , S152-5 (Oct, 1997)], and similarly, different levels of IL10 by IL10-1082 A> G polymorphism [Ref. DM Turner et al. , Eur J Immunogenet 24 , 1-8 (Feb, 1997)], transcriptional activity [Ref . LE Rees et al. , Cell Mol Life Sci 59 , 560-9 (Mar, 2002)] and serum total IgE levels [Reference: K. Hobbs, J. Negri, M. Klinnert, LJ Rosenwasser, L. Borish, Am J Respir Crit Care Med 158 , 1958-62 (Dec, 1998). In addition, other levels of IL10 [Me Helminen, S. Kilpinen, M. Virta, M. Hurme, J Infect Dis 184 , 777-80 (Sep 15, 2001)] and transcriptional activity [Ref. E. Crawley] et al. , Arthritis Rheum 42 , 1101-8 (Jun, 1999)] were mediated by IL10 promoter haplotype.

Association studies of multiple IL10 polymorphisms for various diseases have been conducted to date. Resistance to AIDS, HCV infection and antiviral treatment [reference: PG Vidigal, JJ Germer, NN Zein, J Hepatol 36 , 271-7 (Feb, 2002); LJ Yee et al. , Hepatology 33 , 708-12 (Mar, 2001); And CJ Edwards-Smith et al. , Hepatology 30 , 526-30 (Aug, 1999)], advanced alcoholic liver disease [Ref .: J. Grove, AK Daly, MF Bassendine, E. Gilvarry, CP Day, Gut 46 , 540-5 (Apr, 2000) ], Gastrointestinal cancer [Reference: MS Wu et al. , J Infect Dis 185 , 106-9 (Jan 1, 2002)], cervical cancer [Reference: GA Stanczuk et al. , Int J Cancer 94 , 792-4 (Dec 15, 2001)], multiple myeloma [Ref . C. Zheng et al. , Int J Cancer 95 , 184-8 (May 20, 2001)], malignant melanoma [Reference: WM Howell, SJ Turner, AC Bateman, JM Theaker, Genes Immun 2 , 25-31 (Feb, 2001)] , Systemic Lupus Erythematosus [Reference: W. Gibson et al. , J Immunol 166 , 3915-22 (Mar 15, 2001); And in M. Lazarus et al. , J Rheumatol 24 , 2314-7 (Dec, 1997). ( 58, 59 )], inflammatory bowel disease [Ref. K. Koss, J. Satsangi, GC Fanning, KI Welsh, DP Jewell, Genes Immun 1 , 185 -90 (Feb, 2000), Schizophrenia [Reference: LB Chiavetto et al. , Biol Psychiatry 51 , 480-4 (Mar 15, 2002)], endometriosis [Ref . J. Kitawaki et al. , Am J Reprod Immunol 47 , 12-8 (Jan, 2002)], longevity [Ref . D. Lio et al. , Genes Immun 3 , 30-3 (Feb, 2002)], EBV infection [Ref .: ME Helminen, S. Kilpinen, M. Virta, M. Hurme, J Infect Dis 184 , 777-80 (Sep 15, 2001) ], Primary Sjogren's syndrome [Reference: J. Hulkkonen et al. , Arthritis Rheum 44 , 176-9 (Jan, 2001)], extended oligoarthritis [Ref . E. Crawley et al. , Arthritis Rheum 42 , 1101-8 (Jun, 1999), rheumatoid arthritis [Reference: AH Hajeer et al. , Scand J Rheumatol 27 , 142-5 (1998)] and atopy / asthma [LJ Rosenwasser, L. Borish, Am J Respir Crit Care Med 156 , S152-5 (Oct, 1997)]. Was observed.

Genetic diversity between individuals results in differences between individuals in the toxicity and efficacy of the drug. Therefore, considering the effects on the genetic diversity of such pharmaceutically important proteins in the early stages of drug development is an important factor that can reduce the risk of drug development failure. One criterion for measuring genetic diversity among individuals is the "haplotype." Haplotype refers to the combination of polymorphisms in each gene sequence within a single population, which is known to provide more accurate and reliable information about genetic diversity than individual polymorphisms (Ulbrecht M et al., 2000 Am J Respir Crit Care Med 161 : 469-74).

The present invention aims to discover polymorphisms of genes associated with HBV progression / HCC development. In particular, in an effort to find additional polymorphisms in the genes involved in HBV progression / HCC development, the present inventors have identified a single nucleotide polymorphism in the interleukin-10 (IL10) gene as a potential candidate gene for HBV. Nucleotide polymorphisms (SNPs) and IL10 haplotypes were examined closely.

The present invention also aims to provide a method for predicting HBV progression / HCC generation by measuring IL10 haplotype, which is dose-dependently proportional to susceptibility to HBV progression / HCC development.

The present invention also provides a kit for diagnosing and predicting susceptibility to HBV progression / HCC development, which includes a probe specifically binding to IL10 haplotype.

In the present invention, the distribution of SNP genotype and haplotype in chronic hepatitis and HCC patients was compared by logistic regression analysis. In addition, Cox proportional hazards model [Ref . DR Cox, JR Stat. Soc. B34 , 187-202 (1972); And PD Allison, pp. 155-197 (1995)] to analyze differences in progression to HCC. In addition, several defined category comparisons were performed.

Example

Example 1. Logistic Regression Analysis

We screened for SNPs in IL10 for 1,237 patients infected with chronic hepatitis B, including HBV carriers without any signs, namely chronic hepatitis (CH), cirrhosis (LC) and hepatocellular carcinoma (HCC). The interleukin-10 ( IL10 ) gene on the 1q31-32 chromosome contained five exons (total size ~ 10 Kb). Four monobasic variants in the IL10 gene (FIG. 1): three upstream of the promoter region (AG transition at -1082 position from transcription initiation site, TC transition at -819 position, and AC transition at -592 position And TC transfer at +117 calculated from the first nucleotide in the 5'UTR was genotyped by a single nucleotide expansion method. Sequences for amplification and genotyping for IL10 -1082 A> G, IL10-819 T> C, IL10 -592 A> C, and IL10 +117 T> C IL10 are each expressed as translocation 5'-ccaactggctccccttaccttctac-3 ' Number: 1), inversion 5'-caggattccatggaggctgg-3 '(SEQ ID NO: 2), extension 5'-actttcctcttacctatccctacttcccc-3' (SEQ ID NO: 3); Potential 5′-gggtgaggaaaccaaattctcag-3 ′ (SEQ ID NO: 4), inversion 5′-ggtagtgctcaccatgacccc-3 ′ (SEQ ID NO: 5), extension 5′-gtacccttgtacaggtgatgtaa-3 ′ (SEQ ID NO: 6); Potential 5'-aggtggaaacatgtgcctgag-3 '(SEQ ID NO: 7), inversion 5'-ctcaaagttcccaagcagcc-3' (SEQ ID NO: 8), extension 5'-ttccattttactttccagagactggcttcctacag-3 '(SEQ ID NO: 9); Potential 5'-atagctgacccagcccctt-3 '(SEQ ID NO: 10), inversion 5'-aaatcgttcacagagaagctcagt-3' (SEQ ID NO: 11), extended 5'-gctcagtaaataaatagaaatgggggttgaggtatcagaggtaataaatattctat-3 '(SEQ ID NO: 12). 0.15 unit of Taq DNA polymerase (Applied Biosystems, Foster City, Calif.), 50 ng of genome DNA, 250 μM of dNTPs and 1.25 pmol of each primer were mixed and PCR was performed using the method provided by the manufacturer. PCR amplification was performed on an ABI Prizm GeneAmp PCR 9700 system (Applied Biosystems, Foster City, Calif.). To complete the PCR reaction for primer extension, 1 unit of SAP (Amersham Life Sciences, Cleveland, OH) and 2 units of Exo I (Amersham Life Sciences, Cleveland, OH) were added to the PCR product. The mixture was incubated at 37 ^° C. for 1 hour and then treated at 72 ^° C. for 15 minutes to inactivate the enzyme. Primer extension reaction was used SNaPshot ddNTP Primer Extension Kit (Applied Biosystems, Foster City, CA). To complete the primer extension reaction, SAP 1 unit was added to the reaction mixture, the mixture was incubated at 37 ^° C. for 1 hour and then placed at 72 ^° C. for 15 minutes to inactivate the enzyme. DNA samples containing the expanded product and standard solutions of Genescan 120 Liz size were mixed in Hi-Di formamide (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. The mixture was incubated at 95 ^° C. for 5 minutes, then placed on ice for 5 minutes and subjected to electrophoresis using an ABI Prism 3100 Genetic Analyzer. Results were analyzed using ABI Prism GeneScan and Genotyper (Applied Biosystems, Foster City, Calif.).

All four SNPs were either complete (| D '| = 1 and d ² ≠ 1) or absolute linkage disequilibrium (| D' | = 1 and d ² = 1). In Koreans (population number: n = 1,237), the recessive frequencies of alleles at each mononucleotide site were 0.07, 0.30, 0.30 and 0.03, respectively. Four haplotypes were identified without problems with paging due to LDs between SNPs (FIG. 1). Further analysis was performed with three SNPs ( IL10-1082A> G, -592A> C and + 117T> C) and two haplotypes ( Haplotype 2 and Haplotype 4), which were IL10-819 T> C and IL10-592 A> C are in absolute LD and haplotypes 1 and 3 are in IL10-592 A> C and IL10 + 117 T> C , respectively. It was because it is equivalent (FIG. 1). Exon, SNP and short tandem repeat (STR) polymorphisms are shown in FIG.

All four of the analytical models (co-dominant, dominant and recessive models for the control, rare alleles) used logistic regression analysis using age and gender as secondary variables to show the alternative effects of the variables. Association analysis of IL10 SNPs with HCC development is summarized in Table 1.

In Table 1, i) HCC development of HBV includes HBsAg positive and patients who had LC prior to progression to HCC, ii) HBsAg positive patients without CH, LC and / or HCC. IL10-819 was not analyzed because it is present in IL10-592 and absolute LD ( d ² = 1). Since haplotypes 1 and 3 are equivalent to IL10-592 and IL10 + 117 , respectively, haplotype analysis was performed only for ht2 and ht4. IL10-1082 , IL10 + 117 and ht4 recessive models were not analyzed due to their low frequency.

Statistical analysis. The logistic regression model was used to measure age and sex as auxiliary variables, while the probability (95% confidence interval) and corresponding P value for each SNP site and haplotype were used for the measurement. Common alleles were used as control genotypes for heterozygotes and homozygotes of unusual alleles. P values of the co-dominant, dominant and recessive models are also shown. As expected, age and gender showed a high association with the development of HCC ( P <0.0001). All patients included in the study were HBsAg positive (chronic hepatitis). The mean age (± SD) of the CH, LC and HCC groups was 46.9 (± 10.4), 49.9 (± 9.1) and 55.8 (± 8.8), respectively.

In an initial analysis, recessive alleles of IL10-592A> C (C alleles) suggested a weak association with HCC development from chronic hepatitis B infection ( P = 0.009-0.08; Table 1). On the other hand, all other sites showed no association at all. IL10-592 C allele has a significant effect on the development of HCC from chronic hepatitis (OR = 1.37-1.57). Compared to the homozygotes for the common genotypes (A / A genotypes), heterozygotes and homozygotes for the C allele of IL10-592A> C showed similar effects on HCC development (OR = 1.41, P = 0.05) OR = 1.37, P = 0.02). Individual genotype groups in the co-dominant model showed significantly more significant associations (OR = 1.37, P = 0.009) than the other two association models (dominant and recessive models) (Table 1).

Example 2. Age-dependent Survival Model for HCC

In the age- dependent survival model for HCC, IL10-1082A> G showed a weak protective effect (RH = 0.70-0.68 , P = 0.05-0.04) in the co-dominant and dominant models, while IL10-592A> C was all 3 The two analytical models each showed a weak sensitivity effect ( IL10-592A>C; RH = 1.22-1.39 , P = 0.03-0.11) (Table 2 below).

Table 2 . Cox relative hazards analysis of the age of HCC development with respect to single nucleotide polymorphisms (SNPs) and haplotypes in the IL10 gene

location n / event Co-dominance dominant zeal χ ² RH P χ ² RH P χ ² RH P IL10-1082 1,159 / 294 3.97 0.70 0.05 4.24 0.68 0.04 0.00 0.97 0.98 IL10-592 1,100 / 277 4.52 1.22 0.03 3.29 1.25 0.07 2.58 1.39 0.11 IL10 + 117 1,100 / 274 0.86 0.78 0.35 0.69 0.80 0.41 . . . ht2 (A-C-C-T) 1,073 / 271 12.57 1.43 0.0004 7.77 1.41 0.005 10.79 2.13 0.001 ht4 (G-C-C-T) 1,073 / 271 1.92 0.69 0.17 1.91 0.69 0.17 . . .

Statistical analysis. Cox models were used to measure the relative risk, P value for each SNP site and haplotype, with gender adjustment. In areas where HBV is endemic, for example, most patients in Korea have been vertically infected with HBV and progressed to HCC after a persistent chronic disease phase. Here we used the HCC development age variable under the assumption of vertical or perinatal infection. ht2 results are shown as Kaplan-Meier survival curves (FIG. 2). The IL10-ht4 recessive model was not analyzed because of its low frequency.

In a further analysis, four haplotypes were identified from the IL10 gene. Only two haplotypes [ IL10-Haplotype2 (ACCT) and IL10-Haplotype4 (GCCT) ; Abbreviated IL10-ht2 and IL10-ht4 , respectively, for statistical analysis, since the other two of them ( IL10-ht1 and IL10-ht3 ) are equivalent to single SNPs.

IL10-ht2 is relatively common in Koreans (freq. = 0.23, n = 1,639), and also in Caucasians (0.28-0.00) and Africans (0.28-0.00) [Ref . HD Shin et al. Proc Natl Acad Sci USA 97 , 14467-72 (Dec 19, 2000); And in C. Donger et al. , Eur J Clin Invest 31 , 9-14 (Jan, 2001). IL10-ht4, on the other hand, had a very low frequency (freq. = 0.04).

IL10-ht2- related SNPs are IL10-1082 A> G and IL10-592A> C (absolute LD with IL10819T> C ), which are quantitative in IL10 transcription and / or expression in which several previous reports have been mediated by alleles. Of particular interest in demonstrating the difference [Reference: CJ Edwards-Smith et al. , Hepatology 30 , 526-30 (Aug, 1999); DMTurner et al. , Eur J Immunogenet 24 , 1-8 (Feb, 1997); E. Crawley et al. , Arthritis Rheum 42 , 1101-8 (Jun, 1999); E. Crawley, D. Isenberg, P. Woo, R. Kay, Arthritis Rheum 42 , 590-3 (Mar, 1999); S. Lim, E. Crawley, P. Woo, PJ Barnes, Lancet 352 , 113 (Jul 11, 1998); SB Cohen, JB Crawley, MC Kahan, M. Feldmann, BM Foxwell, Immunology 92 , 1-5 (Sep, 1997); K. Hobbs, J. Negri, M. Klinnert, LJ Rosenwasser, L. Borish, Am J Respir Crit Care Med 158 , 1958-62 (Dec, 1998); And LJ Rosenwasser, L. Borish, Am J Respir Crit Care Med 156 , S152-5 (Oct, 1997).

In HCC incidence analysis in patients with chronic HBV infection, IL10-ht2 showed a dose-dependent effect: the heterozygote (OR = 1.42, P = 0.05) had a higher risk than the homozygotes (OR = 1.91, P = 0.0001). Indicated. In the co-dominant, dominant and recessive models, IL10-ht2 shows the highest dominance in the co-dominant model (OR = 1.65, P = 0.0002) and the highest risk in the recessive model (OR = 3.08, P = 0.0005) By showing a very significant sensitivity effect on the generation of HCC. The role of IL10-ht2 in the age of HCC development among 1,073 HBV patients was analyzed by Cox relative risk model (Table 2) and demonstrated by Kaplan-Meier survival curve (FIG. 2). In FIG. 2, it was evident that there was a very significant difference in the promotion of HCC development among chronic HBV patients with IL10-ht2 allele. Homozygous IL10-ht2 / ht2 (line c) shows significantly different HCC incidence compared to heterozygote- / ht2 (line b; RH = 1.85, P = 0.01) and wild type (line a; RH = 2.35, P = 0.0004) Indicated. And the heterozygote- / ht2 also showed increased HCC incidence compared to the-/-genotype (RH = 1.31, P = 0.04) (FIG. 2A). Homozygous IL10-ht2 / ht2 and heterozygous-/ ht2 showed the same risk orientation and the effect was shown in a dose-dependent manner: RH = 2.35 (P = 0.0004) and for homozygous IL10-ht2 / ht2 RH = 1.31 for heterozygous- / ht2 (P = 0.04). The dominant and recessive models for Cox relative risk analysis were also demonstrated (FIGS. 2B and C). Although the two analyzes showed significant differences, the recessive model for IL10-ht2 was much stronger and more pronounced than the dominant model. While all patients with homozygotes for IL10-ht2 (FIG. 2C, line a) progressed to HCC before age 65.7, progression to HCC at only the same age among patients without IL10-ht2 was only 57.9%. . This comprehensive analysis demonstrated that IL10-ht2 has a dose-dependent effect on age-dependent HCC development.

Example 3 Disease Category Analysis

3 categories analyzed

The dependence of the IL10-ht2 containing genotype on rapid progression to LC and HCC was evident in defined disease categorical analysis (FIGS. 3A-3C), which made it possible to include susceptibility groups for LC.

In order of susceptibility to HBV progression (LC and HCC), they were divided into three categories: I. Patients who did not progress to LC and HCC up to cutoff age; II. Group of patients who progressed LC (without HCC) before cutoff age; And III. Patients who progressed to HCC before cutoff age. Patients who progressed to HCC without LC were excluded to eliminate the complexity of HBV progression other than the CH → LC → HCC pathway. Considering the high incidence of LC and HCC, 50, 55 and 60 years were chosen as cutoff ages. Although it was not possible to collect the exact onset age from all patients (because most of the patients have a history of LCs), we chose a group that had already advanced to LC before the cutoff age (category II), Regardless of age of progression, a susceptibility group for LC generation from CH was introduced. The incidence of susceptible IL10-ht2 containing genotypes in three categories of HBV progression increased proportionally with sensitivity to HBV progression (LC and HCC) at all cutoff ages (FIG. 3A; P = 0.005-0.00008, dominant model). ). In addition, individual genotype frequencies of homozygotes and heterozygotes were compared (FIG. 3B and C). The numbers listed above in the bar graphs represent the total number of patients in each category and denoted Mantel-Hasenszel Chi-Square (MHC) P values. The frequency of homozygotes for IL10-ht2 showed a significantly increased form (Fig. 3c, P = 0.05-0.003), whereas for heterozygotes, only slightly increased form (Fig. 3b, P = 0.04-0.44) . Among patients who did not progress to LC or HCC before age 55 (healthy carriers), the homozygote, IL10-ht2 / ht2, did not appear at all (FIG. 3C). The categorical analysis of FIGS. 3A-3C also shows that IL10-ht2 acts gene number dependently on the progression of HBV.

2 categories analysis

Analysis of other HCC developmental categories by cutoff age demonstrated the effect of IL10-ht2 on HCC development. The classification consists of two groups; I) Patients who progressed to HCC before the cutoff age, and II) Patients who did not progress to HCC even beyond the cutoff age. The same cutoff age was chosen as in FIGS. 3A-3C. In this comparison, although the effect of the heterozygotes was not significant, homozygotes for IL10-ht2 showed very different frequencies in the two defined classes. The incidence of IL10-ht2 / ht2 among HCC patients was much higher in patients without HCC at all cutoff ages (OR = 5.59-23.6, P = 0.00003-0.0009) (FIGS. 4A-4C).

In addition, limited analyzes limited to HCC patients (number of patients; n = 211) showed that the age of HCC development was similar to the susceptibility effects of IL10-ht2 , but this was not statistically significant (FIGS. 4D-4F). .

In conclusion, IL10-ht2 shows a strong association between HCC incidence (Table 1) and HCC age (Table 2 and FIG. 2) in a gene number dependent method among chronic hepatitis B patients. Patients containing IL10-ht2 (particularly homozygotes) have a much higher risk compared to patients without IL10-ht2 . The promotion of HCC development in patients with IL10-ht2 homozygotes or heterozygotes is also number-dependent (co-dominant). In addition, defined category analysis confirms the facilitating impact on HBV progression (FIG. 3) and HCC development (FIG. 4). The mechanism of susceptibility effects of IL10-ht2 may be the result of modified IL10 production, which plays an important role in HCC progression.

IL10 polymorphism has been identified by the inventors to play an important role in HBV progression / HCC development. In particular, the effect of IL10 haplotype on HBV progression susceptibility has been clarified that the effect of polymorphic sites on IL10 is a result of multiple genotypes of multiple SNPs rather than a single polymorphism. As such, HCC promotion mediated by IL10 promoter haplotype (IL10-ht2) in chronic hepatitis B patients provides diagnostic information about the risk of HCC development and targeted immunotherapeutic methods of IL10 cytokines. It can mimic or enhance natural inhibitory forces to prevent fatal progression to HCC.

<110> SNP Genetics, Inc.          SHIN, hyoung doo          LEE, hyo suk <120> USE OF IL10 HAPLOTYPES FOR PREDICTING SUSCEPTIBILITY TO          PROGRESSION OF HBV TO HCC <130> PM022503 <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer for IL10 SNP genotyping for IL10-1082 A> G <400> 1 ccaactggct ccccttacct tctac 25 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for IL10 SNP genotyping for IL10-1082 A> G <400> 2 caggattcca tggaggctgg 20 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer for IL10 SNP genotyping for IL10-1082 A> G <400> 3 actttcctct tacctatccc tacttcccc 29 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer for IL10 SNP genotyping for IL10-819 T> C <400> 4 gggtgaggaa accaaattct cag 23 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer for IL10 SNP genotyping for IL10-819 T> C <400> 5 ggtagtgctc accatgaccc c 21 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer for IL10 SNP genotyping for IL10-819 T> C <400> 6 gtacccttgt acaggtgatg taa 23 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer for IL10 SNP genotyping for IL10-592 A> C <400> 7 aggtggaaac atgtgcctga g 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for IL10 SNP genotyping for IL10-592 A> C <400> 8 ctcaaagttc ccaagcagcc 20 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer for IL10 SNP genotyping for IL10-592 A> C <400> 9 ttccatttta ctttccagag actggcttcc tacag 35 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer for IL10 SNP genotyping for IL10 + 117 T> C <400> 10 atagctgacc cagcccctt 19 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer for IL10 SNP genotyping for IL10 +117 T> C <400> 11 aaatcgttca cagagaagct cagt 24 <210> 12 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> primer for IL10 SNP genotyping for IL10 + 117 T> C <400> 12 gctcagtaaa taaatagaaa tgggggttga ggtatcagag gtaataaata ttctat 56

Claims (12)

delete delete delete delete delete delete delete delete IL10 haplotype IL-ht2 for predicting susceptibility to HBV progression or HCC development. 10. The method of claim 9, wherein the IL10 haplotype IL-ht2 is A at nucleotide position -1082 of the promoter of the IL10 gene, C at nucleotide position -819 and C at nucleotide position -592, and T at nucleotide position +117 of the IL10 gene. IL10 haplotype, characterized in that consisting of IL-ht2. 11. IL10 haplotype IL-ht2 according to claim 9 or 10, characterized in that IL10 haplotype IL-ht2 is dose-dependently proportional to sensitivity to HBV progression or HCC development. The method according to claim 9 or 10, wherein the IL10 haplotype IL-ht2 is SEQ ID NO: 1 to SEQ ID NO: 3, SEQ ID NO: 4 to SEQ ID NO: 6, SEQ ID NO: 7 to SEQ ID NO: 9 and SEQ ID NO: : IL10 haplotype IL-ht2, which is detected by PCR using a primer set consisting of 10 to SEQ ID NO: 12.

Images