Patent application title: FIBROSIS SUSCEPTIBILITY GENE AND USES THEREOF
Inventors:
IPC8 Class: AC12Q168FI
USPC Class:
1 1
Class name:
Publication date: 2016-08-11
Patent application number: 20160230232
Abstract:
The present invention discloses the identification of a fibrosis
susceptibility gene locus, the CTGF gene locus, which can be used for
detecting predisposition to, diagnosis and prognosis of fibrosis as well
as for the screening of therapeutically active drugs. The invention
resides, in particular, in a method which comprises detecting in a sample
from the subject the presence of an alteration in the CTGF gene locus,
the presence of said alteration being indicative of the presence or
predisposition to fibrosis.Claims:
1.-14. (canceled)
15. A method of detecting one or more single nucleotide polymorphism (SNP) in the CTGF gene locus of a human subject, the method comprising: (a) obtaining a biological sample from the human subject; (b) hybridizing a probe to one or more target sequence that comprises the one or more single nucleotide polymorphism (SNP) in the CTGF gene locus; and (c) detecting the one or more single nucleotide polymorphism (SNP) in the sample, wherein the one or more target sequence comprises a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and combinations thereof.
16. The method of claim 15, wherein the one or more single nucleotide polymorphism (SNP) is selected from the group consisting of rs12527705 present in a sequence comprising SEQ ID NO: 1, rs12526196 present in a sequence comprising SEQ ID NO: 2, rs9399005 present in a sequence comprising SEQ ID NO: 3, rs6918698 present in a sequence comprising SEQ ID NO: 4, rs3037970 present in a sequence comprising SEQ ID NO: 5, rs1931002 present in a sequence comprising SEQ ID NO: 6, rs2151532 present in a sequence comprising SEQ ID NO: 7, and rs9402373 present in a sequence comprising SEQ ID NO: 8.
17. The method of claim 15, wherein the one or more single nucleotide polymorphism (SNP) is associated with an increased risk of hepatic fibrosis of the human subject.
18. The method of claim 17, wherein the hepatic fibrosis is caused by infection with a virus.
19. The method of claim 18, wherein the virus is hepatitis A virus.
20. The method of claim 18, wherein the virus is hepatitis B virus.
21. The method of claim 18, wherein the virus is hepatitis C virus.
22. The method of claim 17, wherein the hepatic fibrosis is caused by infection with Schistosoma.
23. The method of claim 22, wherein the Schistosoma is Schistosoma japonicum.
24. The method of claim 22, wherein the Schistosoma is Schistosoma mansoni.
25. A method of determining an increased risk of hepatic fibrosis in a human subject, the method comprising: (a) obtaining a biological sample from the human subject; (b) hybridizing a probe to one or more target sequence that comprises one or more single nucleotide polymorphism (SNP) of the CTGF gene locus; and (c) detecting the one or more single nucleotide polymorphism (SNP) in the sample, wherein the one or more target sequence comprises a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and combinations thereof.
26. The method of claim 25, wherein the one or more single nucleotide polymorphism (SNP) is selected from the group consisting of rs12527705 present in a sequence comprising SEQ ID NO: 1, rs12526196 present in a sequence comprising SEQ ID NO: 2, rs9399005 present in a sequence comprising SEQ ID NO: 3, rs6918698 present in a sequence comprising SEQ ID NO: 4, rs3037970 present in a sequence comprising SEQ ID NO: 5, rs1931002 present in a sequence comprising SEQ ID NO: 6, rs2151532 present in a sequence comprising SEQ ID NO: 7, and rs9402373 present in a sequence comprising SEQ ID NO: 8.
27. The method of claim 25, wherein the hepatic fibrosis is caused by infection with a virus.
28. The method of claim 27, wherein the virus is hepatitis A virus.
29. The method of claim 27, wherein the virus is hepatitis B virus.
30. The method of claim 27, wherein the virus is hepatitis C virus.
31. The method of claim 25, wherein the hepatic fibrosis is caused by infection with Schistosoma.
32. The method of claim 31, wherein the Schistosoma is Schistosoma japonicum.
33. The method of claim 31, wherein the Schistosoma is Schistosoma mansoni.
Description:
FIELD OF THE INVENTION
[0001] The present invention relates generally to the fields of genetics and medicine. The present invention discloses in particular the identification of a human fibrosis susceptibility gene, which can be used for the diagnosis or prognosis of fibrosis or for the detection of predisposition to fibrosis, occurring in hepatic diseases, in cirrhosis, cutaneous keloid, obesity and any fibrotic disease. The invention more particularly discloses certain alleles of the CTGF gene on chromosome 6 related to susceptibility to fibrosis and representing novel targets for the screening of therapeutically active drugs. The present invention relates more specifically to particular mutations in the CTGF gene and expression products, as well as to diagnostic tools and kits based on these mutations. The invention can be also used for the prevention and/or treatment of fibrosis occurring in all the fibrotic human diseases.
BACKGROUND OF THE INVENTION
[0002] Fibrosis is an excessive growth of fibrous connective tissue in an organ, any part, or tissue thereof, for example in a liver, any part or tissue thereof, especially in response to an injury. Abnormal fibrosis occurs in chronic hepatic inflammations of various aetiologies such as in Hepatitis Virus and Schistosome infections. It was shown previously that certain subjects infected by Schistosomes are slow fibrosers whereas others are rapid fibrosers and that this depends in part on a major gene located on Chr 6q22-q23 (Dessein et al., 1999; Mohamed-Ali et al., 1999).
[0003] Schistosomiasis is caused by helminths that develop in the vascular system of their hosts and lay eggs that are for some of them carried over to the liver where they trigger inflammation in the periportal space. Since worms live for years in their human host, chronic liver inflammation associated with much tissue destruction is common in infected subjects. Tissue repair requires the deposit of extracellular matrix protein (ECMP) in the damaged tissues that are later on turned over and replaced by normal hepatocytes. In some patients ECMP accumulate in the periportal space forming fibrosis deposits that reduce blood flow causing varicose veins, ascites. After months or years of chronic or repeated injury, fibrosis becomes permanent and irreversible. Subjects die of the consequences of fibrosis.
[0004] In South countries, it is estimated that 5 to 10% of the 350 millions of infected subjects may develop severe hepatic fibrosis. There is no good marker allowing to predict and follow hepatic fibrosis progression in Schistosome infected subjects.
[0005] Diagnosis of hepatic fibrosis is mostly based on liver biopsy, elastometry (Boursier et al., 2008; Macias et al., 2008) and ultrasound analysis (Richter et al., 2001; Lambertucci et al., 2004; King et al., 2003).
[0006] Biopsies are obtained via percutanous, transjugular, radiographically-guided fine-needle or laparoscopic route, depending upon the clinical setting. Histopathological examination enables the clinician to grade the severity of necroinflammation and stage the extent of fibrosis. The Metavir scoring system attributes a score to the stages of fibrosis on a 1-4 scale as follows: F0=no fibrosis, F1=portal fibrosis without septa, F2=portal fibrosis and few septae, F3=numerous septae without cirrhosis, F4=cirrhosis (Bedossa et al., 1996). Liver biopsy is an invasive and costly procedure, and samples only a small portion of the liver. Thus it cannot afford a global assessment of hepatic fibrosis, and is subject to sampling variation and inter- and intra-observer error. In addition, liver biopsy is associated with significant morbidity of 3% and a mortality rate of 0.03% (Garcia-Tsao et al., 1993). Potential complications include local hematoma, infection and pain related to the biopsy.
[0007] Noninvasive tests (i.e., serologic markers, elastometry, ultrasound analysis) are also used but are not yet ready for routine clinical use.
[0008] Panels of blood markers have been tested mostly in patients with chronic hepatitis C or cirrhosis due to viral hepatitis C. These studies revealed that serum markers can rule on or rule out fibrosis in approximately 35% of patients (Sebastiani et al., 2006). However, when looking at patients individually, these markers could not reliably differentiate between the various stages of fibrosis. A more recent study incorporated three panels of serum markers to devise an algorithmic approach that improved diagnostic accuracy (Parkes et al., 2006). The three panels evaluated were the APRI (aspartate transaminase to platelet ratio index), the Forns' index (platelets, gammaglutamyltranspeptidase, cholesterol) and the Fibrotest (GGT, haptoglobin, bilirubin, apolipoprotein A, alpha-2-macroglobulin). An algorithm consisting of the APRI followed by the Fibrotest boosted the diagnostic accuracy of fibrosis to above 90%. This group estimated that use of this algorithm could obviate the need for up to 50% of liver biopsies. However, the individual stages of fibrosis are not distinguishable using this algorithm. The limitation of these serum markers is the possibility of false positives when there is highly active hepatic inflammation.
[0009] Fibroscan is another innovative approach to staging hepatic fibrosis, which is based on elastography, which provides rapid measurement of mean hepatic tissue stiffness (Ziol et al., 2005). A probe is employed to transmit a vibration of low frequency and amplitude into the liver. This vibration wave triggers an elastic shear wave, whose velocity through the liver is directly proportional to tissuestiffness measured in kilopascals (kPa). Sensitivity of the Fibroscan technique ranged from 79 to 95%, and specificity from 78 to 95%, compared to the liver biopsy. However, the limitations of this technique are associated with attenuation of elastic waves in fluid or adipose tissue, which would impair assessment of fibrosis in patients. In addition, Fibroscan is an extremely expensive instrument.
[0010] However, no efficient method exists to prognose the fibrosis progression and the treatment efficiency.
[0011] A large number of molecules have been tested for treatment of hepatic fibrosis. For example, corticosteroids have been used to suppress hepatic inflammation in autoimmune and alcoholic hepatitis (Czaja et al., 2003). Ursodeoxycholic acid has been proven to increase survival in PBC patients by binding bile acids, and thus also decreasing hepatic inflammation (Poupon et al., 1997). Neutralizing inflammatory cytokines with specific receptor antagonists (TNFalpha, IL-1 receptor antagonists) and prostaglandin E have been tested in murine models, but not yet in humans (Bruck et al., 1997).
[0012] Another attractive target in curtailing hepatic fibrosis is the downregulation of hepatic stellate cell activation. Interferon gamma is used in combination with ribavirin for therapy of hepatitis C infection. It is postulated that the antifibrotic effects of the interferons may be partially related to downregulation of stellate cell activation. This mechanism could explain the improvement in fibrosis described in patients with viral hepatitis C who do not have a virologic response to interferon alpha (Poynard et al., 1998).
[0013] Trials of antioxidants (nacetylcysteine, alpha-tocopherol) are currently underway in humans. Angiotensin II receptors are upregulated in stellate cell activation, thus angiotensin converting enzyme inhibitors and angiotensin receptor blockers have demonstrated antifibrotic activity in vitro and in animals. This has yet to be replicated in humans (Jonsson et al., 2001). Promoting matrix degradation through matrix metalloproteinases is an antifibrotic strategy shown to be beneficial in a murine model (Iimuro et al., 2003). Specific apoptosis of hepatic stellate cells is another interesting theoretical idea, but has not yet been investigated (Gressner et al., 1998). Treatments aimed at reversing the fibrosis are usually too toxic for long-term use (i.e., corticosteroids, penicillamine) or have no proven efficacy (i.e., colchicine).
[0014] In conclusion, efficient and well-tolerated antifibrotic drugs are currently lacking and current treatment of fibrosis is limited to withdrawal of the noxious agent.
[0015] It has been previously reported that fibrosis development is markedly influenced by a major locus on Chr 6q23 (Dessein et al., 1999). It has been also proposed that CTGF gene contributes to increasing fibrosis by synergizing with various pro-fibrogenic growth factors (Leask et al., 2006) including PGDF, VEGF and the master fibrogenic molecule TGF-.beta.. More specifically CTGF acts as a TGF-.beta. downstream modulator (Leask et al., 2006; Leask et al., 2003). It increases TGF-.beta. binding to its receptor, interferes with the negative Smad-7 feedback loop on TGF-.beta. (Wahab et al., 2005); it inhibits receptor binding of the principal TGF-.beta. antagonist BPM-7 (Abreu et al., 2002). An important consequence of CTGF action on TGF-.beta. is the stimulation of the trans-differentiation of hepatic stellate cells and other parenchymal cells into ECMP producing myofibroblasts which is involved in progressive fibrotic process (Kalluri et al., 2003; Neilson et al., 2005). CTGF is also thought to increase ECMP networking trough its binding capacities to fibronectin domains on ECMP (Gressner et al., 2007; Yoshida et al., 2007). CTGF is produced by a variety of cells including hepatocytes (Kobayashi et al., 2005; Gressner et al., 2007), hepatic stellate cells, myofibroblasts and endothelial cells (Gressnet et al., 2008). An overexpression of CTGF transcripts have been reported in different tissues including liver (Rachfal et al., 2003) affected by fibrosis of different aetiological origin. Experimental work in rats has shown that inhibiting CTGF by siRNAs prevents or reduces tissue fibrosis (Li et al., 2006; George et al., 2007).
[0016] However, the genetic factors that control the human fibrosis susceptibility were not identified as well as their effect on fibrosis progression.
SUMMARY OF THE INVENTION
[0017] The purpose of the present invention is to provide a new genetic approach for fibrosis prognosis and treatment. The present invention now discloses the identification of a human fibrosis susceptibility gene locus, the CTGF gene locus (CCN2), which can be used for detecting predisposition to, diagnosis and prognosis of fibrosis, especially of hepatic fibrosis, as well as for the screening of therapeutically active drugs. The invention resides, in particular, in a method which comprises detecting in a sample from the subject the presence of an alteration in the CTGF gene locus (CCN2), the presence of said alteration being indicative of the presence or predisposition to fibrosis.
[0018] A particular object of this invention resides in an in vitro method of detecting predisposition to or diagnosis and/or prognosis of fibrosis occurring in a subject, the method comprising detecting the presence of an alteration in the CTGF gene or polypeptide in a sample from the subject, the presence of said alteration being indicative of the presence of fibrosis or the predisposition to fibrosis. A particular object of this invention resides in a method for assessment (prediction) of the progression of fibrosis.
[0019] In a preferred embodiment, said alteration is located within 20 kb, upstream the start codon of the CTGF gene and 20 kb, downstream the 3'UTR of the CTGF gene.
[0020] Preferably, the alteration lies in the surrounding sequences of 15.3 kb region, upstream the starting codon of the CTGF gene and 14.1 kb region, downstream the untranslated region (3'UTR).
[0021] In another preferred embodiment, said alteration is a mutation, an insertion or a deletion of one or more bases. In a more preferred embodiment, said alteration is one or several single nucleotide polymorphism(s) SNP(s) or a haplotype of SNPs associated with fibrosis. Preferably, said single nucleotide polymorphisms are SNPs flanking CTGF gene, which are allelic variants lying close to the CTGF gene.
[0022] The method of the invention allows for detection and prognosis of fibrosis which occurs in a human fibrotic disease selected from hepatic diseases fibrosis, cirrhosis, cutaneous keloid, hypertrophic scars and obesity. Especially, the hepatic fibrosis may be caused by hepatic A virus, hepatic B virus, hepatic C virus (HCV), Schistosoma japonicum (S. japonicum) or Schistosoma mansoni (S. mansoni) infection.
[0023] In a particular embodiment, the method comprises detecting the presence of SNP rs9402373 and/or rs6918698 in the CTGF gene locus in a biological sample of a subject infected with HCV, wherein the presence of an allele C at position rs9402373 and/or allele G at position rs6918698 is indicative of a risk of developing a hepatic fibrosis or of the development of a hepatic fibrosis, or of a poor prognostic of hepatic fibrosis in the subject.
[0024] Preferably, the alteration in the CTGF gene locus is determined by performing a selective hydridization assay, a sequencing assay, a microsequencing assay, and/or an allele-specific amplification assay.
[0025] In another aspect of the invention, said alteration in the CTGF gene is determined by restriction enzyme digestion, the detection of at least one said SNP being an indication of fibrosis.
[0026] This invention also relates to a method for selecting a therapeutic compound for a subject that has or is predisposed to develop fibrosis, said method comprising contacting a test compound with a CTGF polypeptide or gene or a fragment thereof and determining the ability of said test compound to enhance or reduce biological activity or function of a pathway related to the CTGF gene.
LEGEND TO THE FIGURES
[0027] FIG. 1: Correlation bins in the CTGF (=CCN2) gene locus in fishermen.
[0028] Thirty three markers were genotyped on 70 unrelated subjects as described in Materials and Methods. Correlations (r2 values) between SNPs were determined using Haploview software. The darkest colors indicate the strongest correlations. Correlation bins (r2>0.05) were as follows SNPs rs9321314, rs6940184, rs12523697, rs9493149 and rs12529636 were in bin 1; SNPs rs12527705 and rs12526196 in bin 2; SNPs rs9399005, rs6918698, and deletion/insertion (D/I) rs3037970 in bin 3; SNP rs2151532 and SNP rs1931002 in bin 4; SNPs rs9493150, rs928501, rs11966728, rs12198610 and a new insertion/deletion (132319925-/GAAA) were in bin 5; SNPs rs7747601, rs7768619 and rs9402373 in bin 6; SNPs rs12527379 and rs2095252 in bin 7.
[0029] Sequencing CCN2 (3.2 kb) and 15.3 kb upstream the starting codon and 14.1 kb in the 3'UTR region was done on 8 cases and 2 controls. It revealed 61 SNPs among whose 50 had been described before.
[0030] Since the inventors were looking for a gene with major effects they focused the study on 53 SNPs with a MAF>20%. 33 SNPs with a MAF>20% were genotyped on 70 fishermen. Correlations (r2 values) between SNPs were determined using Haploview software. Bottom figure shows r.sup.2 values .times.100. The darkest colors indicate the strongest correlations. Above this representation, a physical map with the relative position of each marker is also provided. These markers are grouped in seven correlation (r2=0.5) bins (I to VII).
[0031] SNPs rs9321314, rs6940184, rs12523697, rs9493149 and rs12529636 were in bin 1; SNPs rs12527705 and rs12526196 in bin 2; SNPs rs9399005, rs6918698 and D/I rs3037970 in bin 3; SNP rs2151532 and SNP rs1931002 in bin 4; SNPs rs9493150, rs928501, rs11966728, rs12198610 and a new insertion/deletion (132319925-/GAAA) were in bin 5; SNPs rs7747601, rs7768619 and rs9402373 in bin 6; SNPs rs12527379 and rs2095252 in bin 7.
[0032] Twenty two SNPs were further genotyped in the whole fisherman sample (201 controls and 99 cases). The results of the univariate analysis are shown on the top of FIG. 1 and in Tables 1 and 4. SNPs associated with hepatic fibrosis were SNP rs12527705 (p=0.02, OR=2.3) and SNP rs12526196 (p=0.02, or=2.2) in bin II, SNP rs9399005 (p=0.02, OR=2.2), SNP rs6918698 (p=0.02, OR=2) and D/I rs3037970 (p=0.003, OR=2.6) in bin III, SNP rs1931002 (p=0.004, OR=2.3) and rs2151532 (p=0.02, OR=1.98) in bin IV and SNP rs9402373 (p=0.015, OR=2) in bin VI. The analysis was adjusted on gender (p<0.001) exposure (fishing years: p<0.001, born on boat: p<0.01) and number of treatments (p<0.01).
[0033] In the present analysis, D/I rs3037970 excluded rs6918698 in a multivariate analysis testing simultaneously both SNPs. Likewise SNP rs1931002 excluded SNP rs2151532; SNP rs1256196 excluded SNP rs12527705 and SNP rs9399005. This indicated that SNPs or deletions rs12526196, rs30337970, rs1931002, rs9402373 had the strongest association with hepatic fibrosis (HF). When all four SNPs were tested in the same regression model (lower part Table 4), SNPs rs12526196 (p=0.007, OR=3), rs9402373 (p=0.002, OR=2.8) and rs1931002 (p=0.002, OR=2.8) showed independent associated with HF. An haplotype 1002C, 6196T, present in 53.9% controls and 67.5 cases was associated with HF (p<0.005). The inventors also tested a phenotype that associated advanced HF with evidence of portal hypertension. They found (154 controls and 151 cases) rs9402373 (p=0.005, OR=2.6) and rs3037970 (p=0.05, OR=2) were associated with that phenotype, SNP rs1256196 showed a trend for an association with this more severe disease phenotype (p=0.12). Gender (p=0.001) entered the model as covariate. This suggested that both rs9402373 and rs3037970 could have a more important contribution to severe disease.
[0034] FIG. 2A: Electrophoretic mobility shift assay reveals allele specific binding of nuclear factors with SNPs rs12526196 and rs9402373.
[0035] EMSA were performed according to Materials and Methods using nuclear extracts of a stimulated human hepatocyte cell line (HEPG2). The 12526196T allele bound nuclear factors (complex 1) with a higher affinity than the C allele. SNP rs9402373C allele bound nuclear factors (complex 2) that were not bound by the G allele. No allele specific binding was observed with SNP rs12527705 and SNP rs1931002.
[0036] FIG. 2B: Competitive electrophoretic mobility shift assay for rs9402373 polymorphism. Competitive reactions were done with 100 or 200 fmol of unbiotinylated rs9402373G and C probe. Competitive reactions performed with unbiotinylated rs9402373C probe at concentration (200 fmol) but not with the unbiotinylated rs9402373G has competed the binding of the biotinylated rs9402373C probe.
[0037] FIG. 3 shows the meta analysis of the association of SNP rs9402373 with hepatic fibrosis in HCV or Schistosome infected subjects.
DETAILED DESCRIPTION OF THE INVENTION
[0038] This invention identifies the most critical steps in fibrosis development and provides valuable genetic markers to predict disease progression in fibrosis, especially in hepatic fibrosis.
[0039] Early detection of fibrosis and regular monitoring of fibrosis, would allow for initiation of anti-fibrotic therapies capable of halting and even reversing this process. This would in turn prevent progression to human fibrosis disease, for example hepatic fibrosis or hepatic cirrhosis, and the morbidity and mortality this condition entails. The development of these various early fibrosis detection techniques bodes well for the future care of patients with liver disease.
[0040] The inventors have now identified the major gene associated with human fibrosis. They have shown that fibrosis in two Chinese, one Sudanese and one Brazilian cohort infected with Schistosoma japonicum and with Schistosoma mansoni respectively is markedly dependent on allelic variants lying in the CTGF gene. Two of these variants affect nuclear factor binding.
[0041] Various nucleic acid samples from individuals with fibrosis were submitted to a particular analysis process. Four populations with hepatic fibrosis were examined, inter alia a population of fishermen of the Dong Ting lake in central China infected with Schistosoma japonicum, a population of farmers living in Hunan province also infected with Schistosoma japonicum.
[0042] Two additional populations infected with Schistosoma mansoni were also included (a Sudanese and a Brazilian population). This analysis process led to the identification of particular single nucleotide polymorphism(s) SNP(s) in said populations that are overrepresented in subjects having hepatic fibrosis.
[0043] The inventors first examined fibrosis in Fishermen of the Dong Ting lake in central China near Yueyang city. Analysis of the various factors that influence the development of fibrosis in the fishermen population has shown that gender, exposure to infected waters and anti-schistosome treatments with Praziquantel were significantly associated with the risk of fibrosis. The testing of several covariates has shown that the "the number of years fishing" and "being born on a fishing boat" were the best covariates to measure exposure. The "treatment" covariate is the number of Praziquantel treatments over the last twenty years.
[0044] Using these covariates, the inventors tested on a population sample (N=300) whether SNPs (three per gene) in 12 candidate genes (two genes in the 6q23 region (IFNGR1, CTGF) and ten genes out of this region) were associated with the risk of fibrosis. One SNP rs9399005 near the CTGF gene yield a suggestive association (p=0.02) with fibrosis.
[0045] Then, the inventors sequenced the 3.2 kb of the CTGF gene and 15.3 kb upstream the starting codon and 14.1 kb in the 3'UTR region in 8 cases and 2 controls (fisherman population). 61 SNPs were recorded, among whose 50 had been described before. Fifty three SNPs (53 SNPs) had a MAF>20%. Since the inventors were looking for a gene with major effects, they selected SNPs with a MAF>20%. 33 out of 53 SNPs were genotyped on 70 fishermen.
[0046] They found seven correlation bins (r2=0.5), using data from the Hap Map project, that included 5, 2, 3, 2, 5, 3, 2 SNPs in bins I to VII respectively (FIG. 1). Finally, 22 SNPs or genetic variations were genotyped for further analysis. These 22 were added to the three SNPs tested in the early steps.
[0047] On these 25 SNPs or genetic variations (22+3) of the CTGF gene locus, 8 genetic variations were identified as being correlated to hepatic fibrosis in human subjects (rs12527705, rs12526196, rs9399005, rs6918698, rs3037970, 1931002, rs2151532 and rs9402373). Thus, a particular object of this invention resides in a method of detecting predisposition to and/or prognosis of fibrosis, the method comprising detecting the presence of one or several SNPs or alteration selected in the group consisting of rs12527705, rs12526196, rs9399005, rs6918698, rs3037970, 1931002, rs2151532 and rs9402373.
[0048] Among these SNPs, C allele of rs9402373 or T allele of rs12526196 specifically binds nuclear factors SRY, Lyf-1, CdxA, CP2, Ik-2, or Nkx-2.
[0049] SNPs rs6918698 and rs9402373 were shown to be associated with hepatitis fibrosis in HCV infected subjects. In particular, allele C of rs9402373 was shown to be deleterious, i.e. it is strongly correlated with severe hepatic fibrosis.
[0050] Although the experimental data gathered by the inventors did not allow to confirm association of certain alleles, depending on the tested population, the present invention is not limited to the particular SNPs that were found significantly correlated with fibrosis in all tested populations. Indeed, several reasons could account for the failure in confirming significant correlation in some populations, including an insufficient cohort, the incomplete assessment of confounding variables, a lower frequency of the SNPs in said populations, etc.
DEFINITIONS
[0051] Within the context of this invention, "fibrosis" designates all types of human fibrosis occurring in all the fibrotic human diseases, for example in hepatic diseases, cirrhosis, cutaneous keloid, hypertrophic scars, sclerodermia, obesity and any fibrotic disease. Within the context of this invention, "hepatic fibrosis" or "HF" designates all types of fibrosis occurring in a liver, tissue thereof or any part of tissue thereof. Hepatic fibrosis occurs especially in response to an injury. Hepatic fibrosis can be the common response to chronic liver injury, ultimately leading to cirrhosis and its complications, portal hypertension, liver failure, and hepatocellular carcinoma. Hepatic fibrosis is overly exuberant wound healing in which excessive connective tissue builds up in the liver. The extracellular matrix is either overproduced, degraded deficiently, or both. The trigger is chronic injury, especially if there is an inflammatory component. Various types of chronic liver injury can cause fibrosis, such as chemical fibrosis (CCl.sub.4), bacterial (i.e., brucellosis), parasitic (i.e., bilharziosis/schistosomiasis caused by Schistosoma species; or echinococcosis infections) or viral (i.e., hepatitis caused by hepatic A virus (HAV), hepatic B virus (HBC) or hepatic C virus (HCV) infections).
[0052] Within the context of this invention, "cutaneous keloid" is an excessive growth of scar tissue on the skin. More particularly, keloids and hypertrophic scars (HSc) are dermal fibroproliferative disorders unique to humans that occur following trauma, inflammation, surgery, burns and sometimes spontaneously. These are characterized by excessive deposition of collagen in the dermis and the subcutaneous tissues. Contrary to the fine line scar characteristics of normal wound repair, the exuberant scarring of keloid and HSc results typically in disfigurement, contractures, pruritis and pain. Keloids occur in individuals with a familial disposition among the Blacks, Hispanics and Orientals. Unlike HSc, the keloid scars enlarge and extend beyond the margins of the original wound and rarely regress. These disorders represent aberrations in the fundamental processes of wound healing, which include cell migration and proliferation, inflammation, increased synthesis and secretion of cytokines and extra cellular matrix (ECM) proteins and remodelling of the newly synthesized matrix. Biologically, keloids are fibrotic tissue characterized by a collection of atypical fibroblasts with excessive deposition of extracellular matrix components, especially collagen, fibronectin, elastin, and proteoglycans. Generally, keloids contain relatively acellular centers and thick, abundant collagen bundles that form nodules in the deep dermal portion of the lesion. The release and activation of growth factors during the inflammatory phase of healing are pre-requisites for the scar processes, including angiogenesis, reepithelialization, recruitment and proliferation of fibroblasts and matrix deposition. Then, abnormal production of activity of the regulating cytokine including CTGF, could contribute to the development of keloids.
[0053] Within the context of this invention, "the CTGF gene locus" (Connection Tissue Growth Factor), also called CCN2 gene locus, designates all sequences or products in a cell or organism, including CTGF coding sequences, CTGF non-coding sequences (e.g., introns), CTGF regulatory sequences controlling transcription and/or translation (e.g., promoter, enhancer, terminator, etc.), all corresponding expression products, such as CTGF RNAs (e.g., mRNAs) and CTGF polypeptides (e.g., a pre-protein and a mature protein); as well as surrounding sequences of 20 kb region, preferably 15.3 kb region, upstream the starting codon of the CTGF gene and 20 kb region, preferably 14.1 kb region, downstream the untranslated region (3'UTR). For example, the CTGF locus comprises surrounding sequences comprising the 61 SNPs identified by sequencing (FIG. 1) and in particular 8 SNPs of Table 1. However in a particular embodiment most alterations are not in the promoter sequence.
[0054] Within the context of the present invention, the term "prognosis" includes the detection, monitoring, dosing, comparison, etc., at various stages, including early, pre-symptomatic stages, and late stages, in adults, children and pre-birth. Prognosis typically includes the assessment (prediction) of the progression of fibrosis and the characterization of a subject to define most appropriate treatment (pharmaco-genetics), etc. The present invention provides prognostic methods to determine the speed of the progression of fibrosis or an associated disorder resulting from a mutation or a polymorphism in the CTGF gene locus. Prognosis, which analyzes and predicts response to a treatment or drug, or side effects to a treatment or drug, aims at determining whether an individual should be treated with a particular treatment drug. For example, if the prognosis indicates a likelihood that an individual will respond positively to treatment with a particular drug, the drug may be administered to the individual. Conversely, if the prognostic indicates that an individual is likely to respond negatively to treatment with a particular drug, an alternative course of treatment may be prescribed. A negative response may be defined as either the absence of an efficacious response or the presence of toxic side effects. Clinical drug trials represent another application for the CTGF gene locus SNPs. One or more CTGF SNPs indicative of response to a drug or to side effects to a drug may be identified using the methods described above. Thereafter, potential participants in clinical trials of such an agent may be screened to identify those individuals most likely to respond favourably to the drug and exclude those likely to experience side effects. In that way, the effectiveness of drug treatment may be measured in individuals who respond positively to the drug, without lowering the measurement as a result of the inclusion of individuals who are unlikely to respond positively in the study and without risking undesirable safety problems.
Alterations
[0055] The alteration may be determined at the level of the CTGF DNA, RNA or polypeptide. Optionally, the detection is performed by sequencing all or part of the CTGF gene locus or by selective hybridization or amplification of all or part of the CTGF gene locus. More preferably a CTGF gene locus specific amplification is carried out before the alteration identification step. An alteration in the CTGF gene locus may be any form of mutation(s), deletion(s), rearrangement(s) and/or insertions in the coding and/or non-coding region of the locus, alone or in various combination(s). Mutations more specifically include point mutations. Deletions may encompass any region of two or more residues in a coding or non-coding portion of the gene locus, such as from two residues up to the entire gene or locus. Typical deletions affect smaller regions, such as domains (introns) or repeated sequences or fragments of less than about 50 consecutive base pairs, although larger deletions may occur as well. Insertions may encompass the addition of one or several residues in a coding or non-coding portion of the gene locus. Insertions may typically comprise an addition of between 1 and 50 base pairs in the gene locus. Rearrangement includes inversion of sequences. The CTGF gene locus alteration may result in the creation of stop codons, frameshift mutations, amino acid substitutions, particular RNA splicing or processing, product instability, truncated polypeptide production, etc. The alteration may result in the production of a CTGF polypeptide with altered function, stability, targeting or structure. The alteration may also cause a reduction in protein expression or, alternatively, an increase in said production.
[0056] In a preferred embodiment, said alteration is a mutation, an insertion or a deletion of one or more bases. In a particular embodiment of the method according to the present invention, the alteration in the CTGF gene locus is selected from a point mutation, a deletion and an insertion in the CTGF gene or corresponding expression product, more preferably a point mutation and a deletion. The alteration may be determined at the level of the CTGF DNA, RNA or polypeptide.
[0057] A particular object of this invention is a method of detecting predisposition to and/or prognosis of fibrosis, the method comprising detecting the presence of one or several alterations or SNPs selected in the group consisting of rs9402373, rs1931002, rs12526196, rs1257379, rs12527705, rs9399005, rs6918698, rs3037970, rs2151532, rs9321314, rs9321315, rs6910279, rs6940184, rs12523697, rs9493149, rs12529636, rs6917644, rs9493150, rs928501, rs11966728, rs7747601, rs12198610, 132319925 D/I, rs9483364, rs2095252, rs6926879, rs34118837, rs1931003, rs12191459, rs12206863, rs7768619, rs10872386 and rs2327184.
[0058] The 132319925 D/I (deletion/insertion) polymorphism is a new polymorphism that was never described before. To be able to identify this polymorphism the inventors gave as name the position according to the coordinate system and D/I to indicate that this polymorphism induces a deletion or an insertion. This polymorphism can be also written 132319925-/GAAA.
[0059] Preferably, said alteration(s) or SNP(s) associated with fibrosis is(are) selected from the group consisting of rs12527705, rs12526196, rs9399005, rs6918698, rs3037970, rs1931002, rs2151532 and rs9402373. More preferably, said rs9402373C and rs12526196T alleles preferentially bind nuclear factors.
[0060] These alterations/SNPs are reported in the following Table 1.
TABLE-US-00001 TABLE 1 Fibrosis-associated alterations in the CTGF gene locus Nucleotide position in genomic sequence of Alteration/SNP Sequence chromosome reference Polymorphism reference 132304944 rs12527705 A/T SEQ ID NO: 1 132305169 rs12526196 C/T SEQ ID NO: 2 132310657 rs9399005 C/T SEQ ID NO: 3 132314950 rs6918698 C/G SEQ ID NO: 4 132316891 rs3037970 --/TAAAA (D/I) SEQ ID NO: 5 132320175 rs1931002 A/G SEQ ID NO: 6 132316423 rs2151532 C/T SEQ ID NO: 7 132319124 rs9402373 C/G SEQ ID NO: 8 132321533 rs12527379 A/G SEQ ID NO: 9 132294634 rs9321314 A/G SEQ ID NO: 10 132294969 rs9321315 A/T SEQ ID NO: 11 132295757 rs6910279 A/G SEQ ID NO: 12 132299137 rs6940184 G/T SEQ ID NO: 13 132299717 rs12523697 C/T SEQ ID NO: 14 132300452 rs9493149 A/G SEQ ID NO: 15 132301414 rs12529636 A/G SEQ ID NO: 16 132307358 rs6917644 A/G SEQ ID NO: 17 132315684 rs9493150 C/G SEQ ID NO: 18 132317130 rs928501 A/C SEQ ID NO: 19 132318298 rs11966728 C/T SEQ ID NO: 20 132318597 rs7747601 A/G SEQ ID NO: 21 132318713 rs12198610 A/G SEQ ID NO: 22 132323491 rs9483364 A/G SEQ ID NO: 23 132325281 rs2095252 A/G SEQ ID NO: 24 132304054 rs6926879 A/C SEQ ID NO: 25 132307157 rs1931003 A/G SEQ ID NO: 26 132309432 rs12191459 C/T SEQ ID NO: 27 132310735 rs12206863 A/C SEQ ID NO: 28 132319026 rs7768619 C/T SEQ ID NO: 29 132323557 rs10872386 C/T SEQ ID NO: 30 132324717 rs2327184 A/G SEQ ID NO: 31 132304021 rs34118837 C/G SEQ ID NO: 32 132319925 -- (promoteur 5) --/GAAA (D/I)
[0061] Alterations in the CTGF gene may be detected by determining the presence of an altered CTGF RNA expression. Altered RNA expression includes the presence of an altered RNA sequence, the presence of an altered RNA splicing or processing, the presence of an altered quantity of RNA, etc. These may be detected by various techniques known in the art, including by sequencing all or part of the CTGF RNA or by selective hybridisation or selective amplification of all or part of said RNA, for instance.
[0062] In a further variant, the method comprises detecting the presence of an altered CTGF polypeptide expression. Altered CTGF polypeptide expression includes the presence of an altered polypeptide sequence, the presence of an altered quantity of CTGF polypeptide, the presence of an altered tissue distribution, etc. These may be detected by various techniques known in the art, including by sequencing and/or binding to specific ligands (such as antibodies), for instance.
[0063] As indicated above, various techniques known in the art may be used to detect or quantify altered CTGF gene or RNA expression or sequence, including sequencing, hybridisation, amplification and/or binding to specific ligands (such as antibodies). Other suitable methods include allele-specific oligonucleotide (ASO), allele-specific amplification, Southern blot (for DNAs), Northern blot (for RNAs), single-stranded conformation analysis (SSCA), PFGE, fluorescent in situ hybridization (FISH), gel migration, clamped denaturing gel electrophoresis, heteroduplex analysis, RNase protection, chemical mismatch cleavage, ELISA, radio-immunoassays (RIA) and immuno-enzymatic assays (IEMA). Some of these approaches (e.g., SSCA and CGGE) are based on a change in electrophoretic mobility of the nucleic acids, as a result of the presence of an altered sequence. According to these techniques, the altered sequence is visualized by a shift in mobility on gels. The fragments may then be sequenced to confirm the alteration. Some others are based on specific hybridization between nucleic acids from the subject and a probe specific for wild-type or altered CTGF gene or RNA. The probe may be in suspension or immobilized on a substrate. The probe is typically labelled to facilitate detection of hybrids. Some of these approaches are particularly suited for assessing a polypeptide sequence or expression level, such as Northern blot, ELISA and RIA. These latter require the use of a ligand specific for the polypeptide, more preferably of a specific antibody.
[0064] In a preferred embodiment, the method comprises detecting the presence of an altered CTGF gene expression profile in a sample from the subject. As indicated above, this can be accomplished more preferably by sequencing, selective hybridisation and/or selective amplification of nucleic acids present in said sample.
Sequencing
[0065] Sequencing can be carried out using techniques well known in the art, using automatic sequencers. The sequencing may be performed on the complete CTGF gene locus or, more preferably, on specific domains thereof, typically those known or suspected to carry deleterious mutations or other alterations.
Amplification
[0066] Amplification is based on the formation of specific hybrids between complementary nucleic acid sequences that serve to initiate nucleic acid reproduction. Amplification may be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). These techniques can be performed using commercially available reagents and protocols. Preferred techniques use allele-specific PCR or PCR-SSCP. Amplification usually requires the use of specific nucleic acid primers, to initiate the reaction. Nucleic acid primers useful for amplifying sequences from the CTGF gene locus are able to specifically hybridize with a portion of the CTGF gene locus that flank a target region of said locus, said target region being altered in certain subjects having fibrosis or associated disorders. Examples of such target regions are provided in Table 2. Another particular object of this invention resides in a nucleic acid primer useful for amplifying sequences from the CTGF gene or locus including surrounding regions. Such primers are preferably complementary to, and hybridize specifically to nucleic acid sequences in the CTGF gene locus. Particular primers are able to specifically hybridize with a portion of the CTGF gene locus that flank a target region of said locus, said target region being altered in certain subjects having fibrosis or associated disorders. Primers that can be used to amplify CTGF target region comprising SNPs as identified in Table 2 may be designed based on their sequence or on the genomic sequence of CTGF.
[0067] The invention also relates to a nucleic acid primer, said primer being complementary to and hybridizing specifically to a portion of a CTGF gene locus coding sequence (e.g., gene or RNA) altered in certain subjects having fibrosis or associated disorders. In this regard, particular primers of this invention are specific for altered sequences in a CTGF gene locus or RNA. By using such primers, the detection of an amplification product indicates the presence of an alteration in the CTGF gene locus. In contrast, the absence of amplification product indicates that the specific alteration is not present in the sample. The invention also concerns the use of a nucleic acid primer or a pair of nucleic acid primers as described above in a method of detecting the presence of or predisposition to fibrosis or an associated disorder in a subject or in a method of assessing the response of a subject to a treatment of fibrosis or an associated disorder.
Selective Hybridization
[0068] Hybridization detection methods are based on the formation of specific hybrids between complementary nucleic acid sequences that serve to detect nucleic acid sequence alteration(s). A particular detection technique involves the use of a nucleic acid probe specific for wild-type or altered CTGF gene or RNA, followed by the detection of the presence of a hybrid. The probe may be in suspension or immobilized on a substrate or support (as in nucleic acid array or chips technologies). The probe is typically labeled to facilitate detection of hybrids. In this regard, a particular embodiment of this invention comprises contacting the sample from the subject with a nucleic acid probe specific for an altered CTGF gene locus, and assessing the formation of an hybrid. In a particular preferred embodiment, the method comprises contacting simultaneously the sample with a set of probes that are specific, respectively, for wild type CTGF gene locus and for various altered forms thereof. In this embodiment, it is possible to detect directly the presence of various forms of alterations in the CTGF gene locus in the sample. Also, various samples from various subjects may be treated in parallel. Within the context of this invention, a probe refers to a polynucleotide sequence which is complementary to and capable of specific hybridization with a (target portion of a) CTGF gene or RNA, and which is suitable for detecting polynucleotide polymorphisms associated with CTGF alleles which predispose to or are associated with fibrosis. Probes are preferably perfectly complementary to the CTGF gene, RNA, or target portion thereof. Probes typically comprise single-stranded nucleic acids of between 8 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. It should be understood that longer probes may be used as well. A preferred probe of this invention is a single stranded nucleic acid molecule of between 8 to 500 nucleotides in length, which can specifically hybridize to a region of a CTGF gene locus or RNA that carries an alteration.
[0069] The method of the invention employs a nucleic acid probe specific for an altered (e.g., a mutated) CTGF gene or RNA, i.e., a nucleic acid probe that specifically hybridizes to said altered CTGF gene or RNA and essentially does not hybridize to a CTGF gene or RNA lacking said alteration. Specificity indicates that hybridization to the target sequence generates a specific signal which can be distinguished from the signal generated through non-specific hybridization. Perfectly complementary sequences are preferred to design probes according to this invention. It should be understood, however, that certain mismatch may be tolerated, as long as the specific signal may be distinguished from non-specific hybridization.
[0070] Particular examples of such probes are nucleic acid sequences complementary to a target portion of the genomic region including the CTGF gene locus or RNA carrying a point mutation as listed in Table 1 above. More particularly, the probes can comprise a sequence selected from the group consisting of SEQ ID NO 1 to 8 or a fragment thereof comprising the SNP or a complementary sequence thereof.
[0071] The sequence of the probes can be derived from the sequences of the CTGF gene and RNA as provided in the present application. Nucleotide substitutions may be performed, as well as chemical modifications of the probe. Such chemical modifications may be accomplished to increase the stability of hybrids (e.g., intercalating groups) or to label the probe. Typical examples of labels include, without limitation, radioactivity, fluorescence, luminescence, enzymatic labelling, etc. The invention also concerns the use of a nucleic acid probe as described above in a method of detecting the presence of or predisposition to fibrosis or an associated disorder in a subject or in a method of assessing the response of a subject to a treatment of fibrosis or an associated disorder.
Specific Ligand Binding
[0072] As indicated above, alteration in the CTGF gene locus may also be detected by screening for alteration(s) in CTGF polypeptide sequence or expression levels. In this regard, contacting the sample with a ligand specific for a CTGF polypeptide and determining the formation of a complex is also described. Different types of ligands may be used, such as specific antibodies. In a specific embodiment, the sample is contacted with an antibody specific for a CTGF polypeptide and the formation of an immune complex is determined. Various methods for detecting an immune complex can be used, such as ELISA, radio-immunoassays (RIA) and immuno-enzymatic assays (IEMA). Within the context of this invention, an antibody designates a polyclonal antibody, a monoclonal antibody, as well as fragments or derivatives thereof having substantially the same antigen specificity. Fragments include Fab, Fab'2, CDR regions, etc. Derivatives include single-chain antibodies, humanized antibodies, poly-functional antibodies, etc. An antibody specific for a CTGF polypeptide designates an antibody that selectively binds a CTGF polypeptide, i.e., an antibody raised against a CTGF polypeptide or an epitope-containing fragment thereof. Although non-specific binding towards other antigens may occur, binding to the target CTGF polypeptide occurs with a higher affinity and can be reliably discriminated from non-specific binding.
[0073] It is also disclosed a diagnostic kit comprising products and reagents for detecting in a sample from a subject the presence of an alteration in the CTGF gene locus or polypeptide, in the CTGF gene or polypeptide expression, and/or in CTGF activity. Said diagnostic kit comprises any primer, any pair of primers, any nucleic acid probe and/or any ligand, preferably antibody, described in the present invention. Said diagnostic kit can further comprise reagents and/or protocols for performing a hybridization, amplification or antigen-antibody immune reaction.
Linkage Disequilibirum
[0074] Once a first SNP has been identified in a genomic region of interest, more particularly in CTGF gene locus, other additional SNPs in linkage disequilibrium with this first SNP can be identified. Indeed, any SNP in linkage disequilibrium with a first SNP associated with fibrosis or an associated disorder will be associated with this trait. Therefore, once the association has been demonstrated between a given SNP and fibrosis, the discovery of additional SNPs associated with this trait can be of great interest in order to increase the density of SNPs in this particular region. Identification of additional SNPs in linkage disequilibrium with a given SNP involves: (a) amplifying a fragment from the genomic region comprising or surrounding a first SNP from a plurality of individuals; (b) identifying of second SNP in the genomic region harboring or surrounding said first SNP; (c) conducting a linkage disequilibrium analysis between said first SNP and second SNP; and (d) selecting said second SNP as being in linkage disequilibrium with said first marker. Sub-combinations comprising steps (b) and (c) are also contemplated. These SNPs in linkage disequilibrium can also be used in the methods according to the present invention, and more particularly in the diagnostic methods according to the present invention.
Causal Mutation
[0075] Mutations in the CTGF gene locus which are responsible for fibrosis may be identified by comparing the sequences of the CTGF gene locus from patients presenting fibrosis or an associated disorder and control individuals. Based on the identified association of SNPs of CTGF, the identified locus can be scanned for mutations. In a preferred embodiment, functional regions such as exons and splice sites, promoters and other regulatory regions of the CTGF gene locus are scanned for mutations. Preferably, patients presenting fibrosis carry the mutation shown to be associated with fibrosis and control individuals do not carry the mutation or allele associated with fibrosis. It might also be possible that patients presenting fibrosis carry the mutation shown to be associated with fibrosis with a higher frequency than control individuals. The method used to detect such mutations generally comprises the following steps: amplification of a region of the CTGF gene locus comprising a SNP or a group of SNPs associated with fibrosis from DNA samples of the CTGF gene locus from patients presenting fibrosis and control individuals; sequencing of the amplified region; comparison of DNA sequences of the CTGF gene from patients presenting fibrosis or an associated disorder and control individuals; determination of mutations specific to patients presenting fibrosis.
Drug Screening
[0076] New methods for the screening of drug candidates or leads are also described. These methods include binding assays and/or functional assays, and may be performed in vitro, in cell systems, in animals, etc. A particular object of this invention resides in a method of selecting biologically active compounds, said method comprising contacting in vitro a test compound with a CTGF gene or polypeptide according to the present invention and determining the ability of said test compound to bind said CTGF gene or polypeptide. Binding to said gene or polypeptide provides an indication as to the ability of the compound to modulate the activity of said target, and thus to affect a pathway leading to fibrosis in a subject. In a preferred embodiment, the method comprises contacting in vitro a test compound with a CTGF polypeptide or a fragment thereof according to the present invention and determining the ability of said test compound to bind said CTGF polypeptide or fragment. The fragment preferably comprises a binding site of the CTGF polypeptide. Preferably, said CTGF gene or polypeptide or a fragment thereof is an altered or mutated CTGF gene or polypeptide or a fragment thereof comprising the alteration or mutation. A particular object of this invention resides in a method of selecting compounds active on fibrosis, said method comprising contacting in vitro a test compound with a CTGF polypeptide according to the present invention or binding site-containing fragment thereof and determining the ability of said test compound to bind said CTGF polypeptide or fragment thereof. Preferably, said CTGF polypeptide or a fragment thereof is an altered or mutated CTGF polypeptide or a fragment thereof comprising the alteration or mutation. The method for the screening of drug candidates comprises contacting a recombinant host cell expressing a CTGF polypeptide according to the present invention with a test compound, and determining the ability of said test compound to bind said CTGF and to modulate the activity of CTGF polypeptide. Preferably, said CTGF polypeptide or a fragment thereof is an altered or mutated CTGF polypeptide or a fragment thereof comprising the alteration or mutation. The determination of binding may be performed by various techniques, such as by labelling of the test compound, by competition with a labelled reference ligand, etc. The method of selecting biologically active compounds also comprises contacting in vitro a test compound with a CTGF polypeptide and determining the ability of said test compound to modulate the activity of said CTGF polypeptide. Preferably, said CTGF polypeptide or a fragment thereof is an altered or mutated CTGF polypeptide or a fragment thereof comprising the alteration or mutation.
[0077] The method of selecting biologically active compounds for a subject that has or is predisposed to develop fibrosis, also comprises contacting in vitro a test compound with a CTGF gene according to the present invention and determining the ability of said test compound to modulate the expression of said CTGF gene. Preferably, said CTGF gene or a fragment thereof is an altered or mutated CTGF gene or a fragment thereof comprising the alteration or mutation.
[0078] The method of screening, selecting or identifying active compounds, particularly compounds active on fibrosis, also comprises contacting a test compound with a recombinant host cell comprising a reporter construct, said reporter construct comprising a reporter gene under the control of a CTGF gene promoter, and selecting the test compounds that modulate (e.g. activate or inhibit) expression of the reporter gene. Preferably, said CTGF gene promoter or a fragment thereof is an altered or mutated CTGF gene promoter or a fragment thereof comprising the alteration or mutation.
[0079] The above screening assays may be performed in any suitable device, such as plates, tubes, dishes, flasks, etc. Typically, the assay is performed in multi-wells plates. Several test compounds can be assayed in parallel. Furthermore, the test compound may be of various origin, nature and composition. It may be any organic or inorganic substance, such as a lipid, peptide, polypeptide, nucleic acid, small molecule, etc., in isolated or in mixture with other substances. The compounds may be all or part of a combinatorial library of products, for instance.
Pharmaceutical Compositions, Therapy
[0080] It is also described a pharmaceutical composition comprising (i) a CTGF polypeptide or a fragment thereof, a nucleic acid encoding a CTGF polypeptide or a fragment thereof, a vector or a recombinant host cell as described above and (ii) a pharmaceutically acceptable carrier or vehicle. The invention also relates to a method of treating or preventing fibrosis or an associated disorder in a subject, the method comprising administering to said subject a functional (e.g., wild-type) CTGF polypeptide or a nucleic acid encoding the CTGF gene locus. It is also described a method of treating or preventing fibrosis in a subject, the method comprising administering to said subject a compound that modulates, preferably that activates or mimics, expression or activity of a CTGF gene locus or protein according to the present invention. Said compound can be an agonist or an antagonist of CTGF, an antisense or a RNAi of CTGF, an antibody or a fragment or a derivative thereof specific to a CTGF polypeptide. In a particular embodiment of the method, the modulation is an inhibition. In another particular embodiment of the method, the modulation is an activation.
[0081] It is also disclosed the use of a functional CTGF polypeptide, a nucleic acid encoding the CTGF gene locus, or a compound that modulates expression or activity of a CTGF gene or protein according to the present invention, in the manufacture of a pharmaceutical composition for treating or preventing fibrosis in a subject. Said compound can be an agonist or an antagonist of CTGF, an antisense or an RNAi of CTGF, an antibody or a fragment or a derivative thereof specific to a CTGF polypeptide. In a particular embodiment of the method, the modulation is an inhibition. In another particular embodiment of the method, the modulation is an activation.
[0082] The present invention demonstrates the correlation between fibrosis and the CTGF gene locus. The invention thus provides a novel target of therapeutic intervention. Various approaches can be contemplated to restore or modulate the CTGF activity or function in a subject, particularly those carrying an altered CTGF gene locus. Supplying wild-type function to such subjects is expected to suppress phenotypic expression of fibrosis in a pathological cell or organism. The supply of such function can be accomplished through gene or protein therapy, or by administering compounds that modulate or mimic CTGF polypeptide activity (e.g., agonists as identified in the above screening assays).
[0083] The wild-type CTGF gene or a functional part thereof may be introduced into the cells of the subject in need thereof using a vector as described above. The vector may be a viral vector or a plasmid. The gene may also be introduced as naked DNA. The gene may be provided so as to integrate into the genome of the recipient host' cells, or to remain extra-chromosomal. Integration may occur randomly or at precisely defined sites, such as through homologous recombination. In particular, a functional copy of the CTGF gene may be inserted in replacement of an altered version in a cell, through homologous recombination. Further techniques include gene gun, liposome-mediated transfection, cationic lipid-mediated transfection, etc. Gene therapy may be accomplished by direct gene injection, or by administering ex vivo prepared genetically modified cells expressing a functional CTGF polypeptide.
[0084] Other molecules with CTGF activity (e.g., peptides, drugs, CTGF agonists, or organic compounds) may also be used to restore functional CTGF activity in a subject or to suppress the deleterious phenotype in a cell. Restoration of functional CTGF gene locus function in a cell may be used to prevent the development of fibrosis or to reduce progression of said disease.
[0085] Further aspects and advantages of the present invention will be disclosed in the following experimental section, which should be regarded as illustrative and not limiting the scope of the present application.
Materials and Methods
Statistical Analysis
[0086] Multivariate logistic regression was used to analyse the relationship between the probability of an individual developing fibrosis and genetic variants including the main covariates known to affect disease progression in subjects infected with schistosomes. The statistical SPSS software (version 10.0) was used for this analysis. Age and gender were tested in the regression models and kept when they showed an association (p<0.05) with disease. Since the cohorts were matched for gender and age, these covariates had little effects on the association between genetic variants and disease. The number of Praziquantel treatments, infection with HBV and exposure to infection were included in the regression models when these covariates could be evaluated accurately as in the Chinese fishermen (exposure, number of treatments) or in the Chinese farmers (HBV infection, place of birth).
DNA Extraction
[0087] Aliquots of 5 to 15 ml of blood were collected on sodium citrate and kept at -20.degree. C. DNA was extracted using the standard salting out method (Sambrook et al., 1989). Some subjects refused bleeding. In this case, buccal cell samples were collected using foam-tipped applicators and applied to indicating FTA1 cards following the protocol described by Whatman (http://www.whatman.co.uk/). Place the indicating FTA1 card on a clean, dry, flat surface. Label the FTA1 card with a unique identifying name or number. Remove one foam-tipped applicator from the protective packaging. Holding the plastic handle of the applicator, place the foam tip in the mouth and rub one side of the foam tip on the inside of the cheek for 30 s. Repeat using the opposite side of the foam tip for the other cheek. Run the foam tip along the gum-line and fold of the cheek and under the tongue, soaking up as much saliva as possible. Remove the applicator from the mouth. Carefully lift the paper cover of the indicating FTA1 card and press the flat and circular foam applicator tip with the sample area. Without lifting the foam tip from the card. Squeeze the tip using a side to side motion (908 in each direction) three times to completely saturate the sample area. The sample area will turn white upon transfer of the sample. Position the FTA1 card for drying on a clean surface. Allow the card to dry at least 1 h at room temperature. Afterwards the punches are washed three times in 200 .mu.l of FTA purification buffer and in TE buffer successfully.
DNA Amplification
[0088] All the DNA purified from FTA card were pre amplified before genotyping. Polymerase chain reactions (whole genome amplifications) were conducted in 50 .mu.l reactions containing one punch of biological sample (FTA1-bound buccal cell DNA) or 100 ng of genomic DNA, 1.5 OD of 15-base totally degenerate random primer (Genetix, Paris, France), 200 mM dNTPs, 5 mM MgCl.sub.2, 5 ml of 10.times.PCR buffer and 0.5 unit of high fidelity Taq DNA polymerase (BIOTAQ DNA Polymerase, Bioline London, England). Samples were amplified in a multiblock thermocycler as follows: a pre-denaturation step of 3 min at 94.degree. C., 50 cycles consisting of 1 min at 94.degree. C., 2 min at 37.degree. C., 1 min of ramp (37-55.degree. C.), and 4 min at 55.degree. C. Final extension step of 5 min at 72.degree. C.
Sequencing
[0089] Purified PCR products were sequenced using ABI Prism BigDye Terminator cycle sequencing system (PE Applied Biosystems, Foster City, U.S.A.) on ABI Prism automatic sequencer. Sequencing reactions were performed on both strands Sequencing by GATC biotech (GATC, Marseille France). The sequencing primers are described in Table 2. Sequencing CCN2 (3.2 kb) and 15.3 kb upstream the starting codon and 14.1 kb in the 3'UTR region in eight cases and two controls revealed 61 SNPs (FIG. 1) among whose 50 had been described before.
Polymorphism Genotyping by PCR with Specific TaqMan Probes
[0090] Allelic discrimination was assessed using TaqMan probe assays (Applied Biosystems, Lafayette USA). Each reaction contained 12.5 ng of genomic DNA, TaqMan Universal PCR Master Mix (Applied Biosystems, Lafayette USA), 900 nM of each primer and 200 nM of each fluorescently-labelled hybridisation probe in a total volume of 5 .mu.l. RT-PCR was conducted in an ABI Prism Sequence Detection System 7900 (Applied Biosystems, Lafayette USA) using the following conditions: 50.degree. C. for 2 min, 95.degree. C. for 10 min and 40 cycles of amplification (95.degree. C. denaturation for 15 s, 60.degree. C. annealing/extension for 1 min). The following SNPs or alterations were genotyped by this methodology: rs9321314, rs9321315, rs6910279, rs6940184, rs12523697, rs9493149 rs12529636, rs12527705, rs12526196, rs6917644, rs9399005, rs6918698, rs9493150, rs2151532, rs3037970, rs928501, rs11966728, rs7747601, rs12198610, rs9402373, 132319925 D/I, rs1931002, rs12527379, rs9483364, rs2095252.
Polymorphism Genotyping by PCR and Restriction Enzyme Digestion
[0091] Polymorphisms (rs6926879, rs34118837, rs1931003, 12191459, rs12206863, 7768619, rs10872386 and rs2327184) were genotyped by restriction enzyme analysis under standard conditions described by the enzyme manufacturers (Euromedex, Mundolsheim, France or New England Biolabs, Beverly, USA). Polymerase chain reaction (PCR) amplifications were carried out on a robocycler gradient 96 (Stratagene, La Jolla, U.S.A.) according to standard protocol. Each digestion was resolved on either agarose or acrylamide gels, stained with ethidium bromide and visualized by UV.Primers are described in Table 2.
Nuclear Extract Preparation
[0092] Nuclear extracts were prepared from human hepatocyte cell line (HEPG2) stimulated for one hour with dexamethasone (1 mM) since hepatocytes produce CTGF in hepatic fibrosis (HF) Kobayashi et al., 2005, Gressner et al., 2007) together with hepatic stellate cells, and endothelial cells and myo-fibroblasts (Gressner et al., 2008). The extracts were prepared with the nuclear and cytoplasmic extraction reagents from Pierce (NE-PER; Pierce, Rockford, Ill., USA).
Electrophoretic Mobility Shift Assay (EMSA)
[0093] Complementary single-stranded oligonucleotides were commercially synthesized to span approximately 10 bp on either side of the variant nucleotide, as follows:
TABLE-US-00002 (SEQ ID NO: 33) rs9402373C GCTCTCAAAACTAAGCCCAACTC (SEQ ID NO: 34) rs9402373G GAGTTGGGCTTAGTTTTGAGAGC (SEQ ID NO: 35) rs12527705A GTAATATAGAAGATGGGTCTA (SEQ ID NO: 36) rs12527705T GTAATATAGATGATGGGTCTA (SEQ ID NO: 37) rs12526196C GAATATACAACGAATATGGGC (SEQ ID NO: 38) rs12526196T GAATATACAATGAATATGGGC (SEQ ID NO: 39) rs1931002A TGGATGATTCAAACAACTTGG (SEQ ID NO: 40) rs1931002G TGGATGATTCGAACAACTTGG
[0094] Complementary strands were annealed by placing reactions (oligonucleotide sens and oligonucleotide antisens) in boiling water for 10 min and allowing to cool to room temperature. Binding reactions were set up with LightShift Chemiluminescent EMSA Kit (Pierce, Rockford, Ill., USA). Aliquots of 20 fmol of complementary DNA were incubated at room temperature for 20 min with 4 mg of nuclear extract in 10 mM Tris, 50 mM KCl, 1 mM DTT, 2.5% glycerol, 5 mM MgCl.sub.2, 50 ng/.mu.l poly d(I-C), 0.05% NP-40, pH 7.5. Then reactions were loaded onto an 8% non-denaturing polyacrylamide gel and run for 150 min at 110V. Free DNA and DNA/protein complexes were transferred to nylon N+ membrane by capillary action. Binding was detected according to manufacturer's instructions (Pierce, Rockford, Ill., USA).
TABLE-US-00003 TABLE 2 Primers used to prepare templates for sequencing PCR Position according to the product Primer sequences (forward and reverse) coordinate system. size AAGGGGCAAGAGACCAAAGT (SEQ ID NO: 41) 132325212-132325194 831 bp CACCCTGCCATTTCATAGAAC (SEQ ID NO: 42) 132324384-132324404 ACCAGGGTGATGGTGCTAAA (SEQ ID NO: 43) 132324557-132324538 783 bp AATGACCATGAAAGGGCTTG (SEQ ID NO: 44) 132323775-132323794 TGCCCCCATATGTACAGAAA (SEQ ID NO: 45) 132323861-132323842 844 bp GGGACATTTTGCAAGGGTTA (SEQ ID NO: 46) 132323018-132323037 TTGCTAGAAAAAGGCTGTCAA (SEQ ID NO: 47) 132323290-132323270 827 bp CAGCCCAATATTCCACCAAG (SEQ ID NO: 48) 132322464-132322483 GCCAAGTTTATTTTGGCAGGT (SEQ ID NO: 49) 132322614-132322593 826 bp TTGGTTCTTCTTGATTGTGGTTT (SEQ ID NO: 50) 132321788-132321810 GCAAAGCAACATTGGTTCAA (SEQ ID NO: 51) 132321921-132321912 844 bp GGTATGCTTTGGGAGGCTTA (SEQ ID NO: 52) 132321088-132321107 TCTCAGCCGAAACAAGACTG (SEQ ID NO: 53) 132321195-132321176 756 bp TTTAGCGGGAACGCTTCTTA (SEQ ID NO: 54) 132320440-132320459 ACCGTGAAAGGGCTTTGTAA (SEQ ID NO: 55) 132320556-132320537 808 bp TGCTCAGCTTATTTTTGTCACC (SEQ ID NO: 56) 132319749-132319770 CCCTAACTCTCCCCATCCTC (SEQ ID NO: 57) 132319847-132319828 823 bp ATGTGCAGCTCAAGGAGACA (SEQ ID NO: 58) 132319044-132319025 TTGATTTTCTCTGAGCTCTCACC (SEQ ID NO: 59) 132319238-132319216 812 bp TAGGAATGAGCCTGGTGGTC (SEQ ID NO: 60) 132318427-132318446 GGTGTCCCCAAATCACACAT (SEQ ID NO: 61) 132318639-132318620 838 bp TATGGGCATCTGGACAGTGA (SEQ ID NO: 62) 132317802-132317821 ACGCCCTCTCCTTAACCTTC (SEQ ID NO: 63) 132318031-132318012 838 bp AGCCCCTTTGATTGACAGC (SEQ ID NO: 64) 132317194-132317212 GTTAATTCCTCATTTTACCACGA (SEQ ID NO: 65) 132317353-132317331 647 bp AGTCCCTCGAAGTCTCAAAAA (SEQ ID NO:66) 132316706-132316726 GCACAGAAACTTTTTCTTTCCTG (SEQ ID NO: 67) 132316835-132316813 625 bp AAGGGTAGGGATGTGCAGTG (SEQ ID NO: 68) 132316211-132316230 CAGGCATGAAGATGGTGGTA (SEQ ID NO: 69) 132316364-132316345 679 bp GGTGCAAAGATCGGCTTTAG (SEQ ID NO: 70) 132315687-132315706 TCTTCTTGGCTATTTTTCAACAGA (SEQ ID NO: 71) 132315803-132315780 739 bp GGGGTTCACCTAGGAGCATT (SEQ ID NO: 72) 132315065-132315084 CAAGACAAACCAAATCCAATCC (SEQ ID NO: 73) 132315178-132315157 706 bp TATCCTCTTCGCACCACTCC (SEQ ID NO: 74) 132314474-132314493 GTGGACAGAACAGGGCAAAC (SEQ ID NO: 75) 132314570-132314551 637 bp GCTTACCCGGCTGCAGAG (SEQ ID NO: 76) 132313934-132313951 ACAGCCCCGAGACGACAG (SEQ ID NO: 77) 132314127-132314110 635 bp GCAGCAGCTGGAGAAAGAAA (SEQ ID NO: 78) 132313593-132313512 AAAAAGAAACCGCTCGGACT (SEQ ID NO: 79) 132313535-1323135016 602 bp CCAGGCAGTTGGCTCTAATC (SEQ ID NO: 80) 132312934-132312953 TCAGGGTCGTGATTCTCTCC (SEQ ID NO: 81) 132313102-132313083 746 bp TGGAGATTTTGGGAGTACGG (SEQ ID NO: 82) 132312357-132312376 TCAAACTTCCTCCCCTCAAA (SEQ ID NO: 83) 132312529-132312510 704 bp TGCTCCTAAAGCCACACCTT (SEQ ID NO: 84) 132311826-132311845 GGAAAAGATTCCCACCCAAT (SEQ ID NO: 85) 132311978-1323119459 659 bp CCATTTCATGCTTTGAACGA (SEQ ID NO: 86) 132311320-132311339 TTGTGTGTGTGTGTGTGTGTGT (SEQ ID NO: 87) 132311497-132311476 806 bp AATGCCAAAAGGACCAAGTG (SEQ ID NO: 88) 132310673-132310692 CAGACTTGCAGGCATACACA (SEQ ID NO: 89) 132310821-132310802 844 bp TGGTTTGTGGTGTGGACAGT (SEQ ID NO: 90) 132309978-132309997 CCACATGAGGATAGCTGAGGA (SEQ ID NO: 91) 132310141-132310121 794 bp GCTTTGTTGGAGTGCTAGGC (SEQ ID NO: 92) 132309348-132309367 CCAAATCATCAACCACCGTA (SEQ ID NO: 93) 132309544-132309525 828 bp TTTCTGTAACCTGGCTTATTTCA (SEQ ID NO: 94) 132308717-132308739 TCTGCACCTCCATGTTCATT (SEQ ID NO: 95) 132308929-132308910 845 bp TTTGTAGGGATTTGGCTTCA (SEQ ID NO: 96) 132308085-132308104 ATTGAGTGGGTCAGGGACAA (SEQ ID NO: 97) 132308323-132308304 806 bp CCCTTTTCTTTAAACTCCAGCA (SEQ ID NO: 98) 132307518-132307539 TGGACAGGCAAAGAAAATCC (SEQ ID NO: 99) 132307722-132307703 833 bp TGCAAATAAACAGGCAGAAATG (SEQ ID NO: 100) 132306889-132306911 GCCCTTGCTGAGGAACCTAC (SEQ ID NO: 101) 132307075-132307056 846 bp ACACATTTCTTGCCCCAGAC (SEQ ID NO:102) 132306230-132306250 TCCCCCTACATCTGCCTACA (SEQ ID NO: 103) 132306391-132306072 839 bp AGCCTGTCTTCTGAGGCACT (SEQ ID NO: 104) 132305563-132305572 GGGGGACAGAGAAAGACTCC (SEQ ID NO: 105) 132305749-132305730 803 bp TGGGTCTACATGTCTATTGCTTTG (SEQ ID NO: 132304957-132304970 106) GGGAATGCCCATATTCATTG (SEQ ID NO: 107) 132305185-132305166 814 bp TGGAAACCCAAGTTCTTCTGA (SEQ ID NO: 108) 132304372-132304392 CCCGCAAAGTTGTTCTTTGT (SEQ ID NO: 109) 132304558-132304539 775 bp CCATGACACAGCCCTCAAG (SEQ ID NO: 110) 132303384-132303402 GCCTCTATCCCACGTTTTGA (SEQ ID NO: 111) 132304022-132304003 843 bp TGTCCTTTGGAGGGACATAGA (SEQ ID NO: 112) 132303180-132303200 TATACACGTGCCATGGTGGT (SEQ ID NO: 113) 132303452-132303433 792 bp TGACTTAATGAATAAGCCTGCTG (SEQ ID NO: 132302661-132302683 114) GCACCATATAAATGTGAGATTGGA (SEQ ID NO: 132302898-132302875 823 bp 115) 132302086-132302101 AAAAGAAGCTGAATTTGCTTTAAAAT (SEQ ID NO: 116) AGCAGGATACTGACAGGCAAA (SEQ ID NO: 117) 132302324-132302304 839 bp TCATTTAAAAATAAATCCCTCTGGA (SEQ ID NO: 132301486-132301510 118) TGTTTTCCATTTTTCAATCCAA (SEQ ID NO: 119) 132301639-132301618 772 bp CCAGGGTCCCATTCCTAGTT (SEQ ID NO: 120) 132300868-132300887 GAGGCTTGTGGAGCATTAGC (SEQ ID NO: 121) 132301074-132301055 829 bp AGCATGGGTTTCCATAGCAG (SEQ ID NO: 122) 132300246-132300265 TGTTTGGGATTGAGGTCCTT (SEQ ID NO: 123) 132300436-132300417 812 bp TGTGCAGTTCAAACCCATGT (SEQ ID NO: 124) 132299625-132299644 TGTGGTATTGGGTTGCCATT (SEQ ID NO: 125) 132299746-132299727 843 bp TTCTCCCTACAGGTCCCAGA (SEQ ID NO: 126) 132298904-132298923
TABLE-US-00004 TABLE 3 Primers for genotyping bp restriction enzyme analysis. Position according PCR Restric- Primer sequences to the product tion Polymorphism (forward and reverse) coordinate system. size Enzyme Expected profits rs6926879 ATGCACTACCACACTAGGCTGA 132304175-132304154 314 bp BseYI C/C genotype: (SEQ ID NO: 127) 164 + 122 + 28 bp AGCAGCATGAGACATCAATCAC 132303883-132303862 G/G genotype: (SEQ ID NO: 128) 192 + 122 bp rs34118837 ATGCACTACCACACTAGGCTGA 132304175-132304154 314 bp BseYI C/C genotype: (SEQ ID NO: 129) 164 + 122 + 28 bp AGCAGCATGAGACATCAATCAC 132303883-132303862 G/G genotype: (SEQ ID NO: 130) 192 + 122 bp rs1931003 TGATTCTTGAAATCAAACCTTGAA 132307324-132307301 273 bp NlaIII G/G genotype: (SEQ ID NO: 131) 273 bp GCTAGTAGGTTCCTCAGCAAGG 132307073-132307052 A/A genotype: (SEQ ID NO: 132) 170 + 103 bp rs12191459 ATGGCAATGCACACTTTCAC 132309590-132309571 243 bp BveI C/C genotype: (SEQ ID NO: 133) 150 + 93 bp GCTTTGTTGGAGTGCTAGGC 132309367-132309348 T/T genotype: (SEQ ID NO: 134) 243 bp rs12206863 CTTGCAGGCATACACACCAC 132310798-132310817 162 bp BsmAI A/A genotype: (SEQ ID NO: 135) 107 + 55 bp AACGGCCAGAGAGGTACAAA 132310656-132310637 C/C genotype: (SEQ ID NO: 136) 88 + 55 + 19 bp rs7768619 TGAGAGCCACTGAAGAATGG 132319120-132319101 224 bp KspAI T/T genotype: (SEQ ID NO: 137) 224 bp TAGGTGGAGCCTAGGGGACT 132318916-132318897 C/C genotype: (SEQ ID NO: 138) 127 + 97 bp rs10872386 GGGGACATTTTCCAGACACA 132323619-132323600 229 bp BanI C/C genotype: (SEQ ID NO: 139) 102 + 74 + 63 bp TGTCATCAAATTGCCACAGG 132323410-132323391 T/T genotype: (SEQ ID NO: 140) 165 + 74 bp rs2327184 AGCGAGACTCCGTCTCAAAA 132324882-132324863 225 bp BsuRI A/A genotype: (SEQ ID NO: 141) 225 bp CTTTGCTTTCCGCTGTGATT 132324677-132324658 G/G genotype: (SEQ ID NO: 142) 164 + 61 bp
EXAMPLES
Example 1
Association Between SNPs in the CTGF Gene Locus with Severe Hepatic Fibrosis (HF) in Two Chinese Populations' Samples
[0095] Data are provided for two independent Chinese samples (fishermen and farmers living in region endemic for S. japonicum). Disease phenotype included advanced hepatic fibrosis (fishermen) as described in Materials and Methods, or ascites and previous bleedings (farmers).
[0096] Association between genotypes and hepatic fibrosis (HF) phenotypes (described in Material and Methods) have been tested first using univariate analysis (upper part of the Table 4) and second by multivariate analysis (lower part of the Table 4) including SNPs or alterations rs12526196, rs1931002, rs3037970 and rs9402373 that showed the strongest associations when tested again SNPs from the same bin. Bins are correlation (r2>0.5) groups, genotype is the aggravating genotype, OR=Odd ratios, CI=Confidence interval of OR.
[0097] Fisherman sample: n=300, 99 cases and 201 controls; covariates: number of years fishing, being born on boat, gender, number of Praziquantel treatments. Chinese farmer sample: n=294, 113 controls and 181 cases; covariates place of birth is endemic or not endemic, cured or active HBV infection (p=0.05, OR=2.38).
[0098] Analysis of the various factors that influence the development of fibrosis in the fishermen population has shown that gender, exposure to infected waters and anti-schistosome treatments with Praziquantel were significantly associated with the risk of fibrosis. The testing of several covariates has shown that "the number of years fishing" and "being born on a fishing boat" were the best covariates to measure exposure. The "treatment" covariate is the number of Praziquantel treatments over the last twenty years.
Study Samples
[0099] Chinese fishermen were recruited (fisherman sample) on boats during a large field study, whereas farmers (farmer sample) were recruited from hospital records (cases) directly from their farms or villages (controls). Fishermen live on boat and or on small islands and fish since many years. They were much exposed to infection whereas most farmers have been infected a long time ago since parasite transmission in fields has been interrupted 15 to 20 years ago. Fishermen and farmers originate from different geographical regions. Fishermen are mostly from Jiangshu province and a few of them from Hubei and Jiangxi. Farmers have been living in Hunan province for several generations and for some of them were coming from the mountain area.
[0100] Hepatic fibrosis (HF) was evaluated accordingly to the WHO guidelines modified as indicated in (Arnaud et al, 2008). The fisherman sample comprised 300 subjects (201 controls, 99 cases). Cases exhibited either severe CentF (central fibrosis: CLH, D, E or F) or severe ParF (parenchymal fibrosis: GNH or GW) or both CLM and GNM. Controls had milder disease: CentFib.ltoreq.CLL and ParF.ltoreq.GNL. Covariates that could affect hepatic fibrosis in fishermen were exposure and the number of Praziquantel treatments. Exposure included two covariates: the number of years fishing and being born on a boat. The Chinese farmer sample included 294 subjects (113 controls and 181 cases). Cases had severe HF (as defined for the fishermen cases) and ascites and/or varicose veins. HBV and HCV infections could be evaluated in this sample; 3 subjects were infected with HCV and were not included, whereas >60% had an active or cured HBV infection. HBV infection was a significant covariate in the analysis (p<0.03), exposure and the number of treatments could not be evaluated accurately in Chinese farmers.
[0101] The inventors selected several SNPs for genotyping, two SNPs per bin and any SNPs in or outside the bins that could have a functional effect as determined by in silico analysis (see FIG. 1). These SNPs were then genotyped in a larger cohort of 450 individuals of the Chinese Fisherman cohort.
[0102] SNPs showing either positive or suggestive association with fibrosis are presented in Table 4. The correction factor to be applied to these data is <20 (see Materials and Methods). Since treatments could not be recorded accurately for all subjects, we show separately the data obtained with the 450 subjects for whom we knew exposure and with the 380 subjects for whom both exposure and treatments were known.
[0103] The inventors have first examined HF in fishermen of the Dong Ting Lake in central China that is highly endemic for S. japonicum. Covariates that influenced the development of HF were gender, exposure and anti-schistosome treatments. Using these covariates, we tested on 201 controls and 99 cases whether SNPs (3 SNPs per gene) in CCN2 and IFNGR1 were associated with HF. SNP rs9399005 close to CCN2 showed an association with HF (p=0.02). Sequencing CCN2 (3.2 kb) and 15.3 Kb upstream the starting codon and 14.1 Kb in the 3'UTR region revealed 61 SNPs (FIG. 1) among which 50 had been described before. The 53 SNPs with a MAF>20% were grouped in seven correlation (r2=0.5) bins (I to VII) (FIG. 1). We selected 22 SNPs for further genotyping, at least two SNPs per bin and SNPs (n=4) outside the bins that could have a functional effect as assessed by in silico analysis. SNPs associated with HF (Table 4) were SNP rs12527705 (p=0.02, OR=2.3) and SNP rs12526196 (p=0.02, or=2.2) in bin II, SNP rs9399005 (p=0.02, OR=2.2), SNP rs6918698 (p=0.02, OR=2) and D/I rs3037970 (p=0.003, OR=2.6) in bin III, SNP rs1931002 (p=0.004, OR=2.3) and rs21551532 (p=0.02, OR=1.98) in bin IV and SNP rs9402373 (p=0.015, OR=2) in bin VI. The analysis was adjusted on gender (p<0.001) exposure (fishing years: p<0.001, born on boat: p<0.01) and number of treatments (p<0.01). Deletion/insertion (D/I) rs3037970 excluded rs6918698 in a multivariate analysis testing simultaneously both SNPs. Likewise SNP rs1931002 excluded SNP rs21551532; SNP rs1256196 excluded SNP rs12527705 and SNP rs9399005. This indicated that SNPs rs12526196, rs30337970, rs1931002, rs9402373 had the strongest association with HF. When all four SNPs were tested in the same regression model (lower part Table 4), SNPs rs12526196 (p=0.007, OR=3), rs9402373 (p=0.002, OR=2.8) and rs1931002 (p=0.002, OR=2.8) showed independent associated with HF. An haplotype 1002C, 6196T, present in 53.9% controls and 67.5 cases was associated with HF (p<0.005). The inventors also tested a phenotype that associated advanced HF with evidence of portal hypertension. They found (154 controls and 151 cases) SNP rs9402373 (p=0.005, OR=2.6) and D/I rs3037970 (p=0.05, OR=2) were associated with that phenotype, SNP rs1256196 showed a trend for an association with this more severe disease phenotype (p=0.12). Gender (p=0.001) entered the model as covariate. This suggested that both rs9402373 and D/I rs3037970 could have a more important contribution to severe fibrosis.
[0104] The inventors sought replication of these results in an additional independent sample of Chinese farmers who were infected by S. japonicum while working in fields and were recruited from the records of local hospitals. The SNPs that had showed some evidence of an association with HF in the fisherman sample were genotyped in the farmer sample (113 controls, 181 cases). An univariate analysis showed associations between severe HF and SNP rs9402373 (p=0.003, OR=2.23) and SNP rs1256196 (p=0.02, OR=1.85). Other SNPs were not associated (p>0.1). Multivariate analysis indicated that both SNPs rs9402373 (p=0.03, OR=2.26) and rs1256196 (p=0.02, OR=1.88) were independently associated with severe fibrosis. Covariates were birth place (endemic or not endemic, p=0.03) and infection with HVB (p=0.05).
TABLE-US-00005 TABLE 4 Chinese samples SNP or Controls % Cases % OR 95% CI p Controls % Cases % OR 95% CI p alteration Position Bins Genotype Chinese Fishermen Chinese Farmers 12527705 132304944 II AA 69.1 83.7 2.26 1.13-4.5 0.02 65.1 72 1.67 0.98-2.84 0.06 12526196 132305169 II TT 67.9 82.5 2.21 1.13-4.4 0.02 61.6 71.8 1.85 1.1-3.1 0.02 9399005 132310657 III CC 17.2 26.3 2.2 1.1-4.29 0.02 NS 6918698 132314950 III CC 26.6 35.1 2.0 1.1-3.7 0.02 NS 3037970 132316891 III -/- 27.3 38.8 2.59 1.4-4.8 0.003 NS 1931002 132320175 IV CC 52.5 66.7 2.32 1.3-4.13 0.004 NS 21551532 132316423 IV AA 55.4 65 1.98 1.12-3.5 0.02 NS 9402373 132319124 VI CC 50.3 60.6 2.04 1.15-3.54 0.015 25 43.1 2.23 1.32-3.76 0.003 12526196 II TT 3.0 1.4-6.6 0.007 1.88 1.13-3.19 0.018 9402373 VI CC 2.8 1.5-5.4 0.002 2.26 1.33-3.83 0.03 1931002 IV CC 2.81 1.47-5.37 0.002
Example 2
Association Between SNPs in the CTGF Gene Locus with Severe Hepatic Fibrosis (HF) in Sudaneses and Brazilians Infected with Schistosoma Mansoni
[0105] The two principal schistosome strains that cause HF are S. japonicum in Asia, and S. mansoni in Africa and in South America. We have investigated whether HF in S. mansoni endemic region of Sudan and Brazil was also affected by CTGF allelic variants. CTGF polymorphisms that were associated with HF in Chinese fishermen (Table 4) were genotyped in both samples. The phenotypes were either severe HF associated with portal hypertension in Sudanese or significant secondary portal branch thickening in Brazilians who were much less affected than Sudanese since transmission was lower in the Brazilians than in the Sudanese sample.
[0106] Associations between genotypes and HF phenotypes (described in Materials and Methods) have been tested first using univariate analysis (upper part of Table 5) and second by multivariate analysis (lower part of Table 5) including SNPs rs12526196, rs 12527705 and rs9402373. Genotype is the aggravating genotype, OR=Odd ratios, CI=Confidence interval of OR.
[0107] Sudanese farmers: 314: 219 controls and 95 cases. Brazilians 133: 60 cases and 73 controls.
Study Samples
[0108] The Sudanese sample (314 subjects, 95 cases and 219 controls) was recruited among farmers living in villages of the Wad Medani region of Sudan (Dessein et al., 1999). Severe phenotypes associated HF (grade.gtoreq.C) with evidence of portal hypertension as in (Dessein et al., 1999). All subjects were much exposed to infection with S. mansoni and had received no or few Praziquantel treatments. However, exposure and the number of treatments could not be accurately evaluated on this sample.
[0109] The Brazilian sample (133 subjects, 73 controls and 60 cases) was recruited in a village of North East Brazil among adult subjects with frequent contacts with a river populated with S. mansoni infested snails. Infection was much less, as assessed by schistosome egg excretion in teenagers, in Brazilians than in the Chinese and Sudanese. Severe HF with portal hypertension was observed in less than 2% of the population. All subjects showing clear evidence of periportal thickening of the secondary portal branches were cases, whereas the other subjects with similar contacts with the river were controls.
[0110] The inventors have investigated whether hepatic fibrosis in Brazilians infected with S. mansoni is also affected by CTGF allelic variants. The Brazilian cohort has been recruited for this study and has never been genotyped before. We have genotyped CTGF polymorphisms that yield a clear or a suggestive association with fibrosis in Chinese fishermen. In this association study cases were subjects with evidence of thickening of the wall of the secondary branches of the portal vein. The only confounding variable was age of the patients. All subjects with evidence of exposure to infection for many years were selected.
[0111] The inventors have investigated next whether HF caused by S. mansoni in endemic subjects in Sudan and Brazil was also affected by CTGF allelic variants. CTGF polymorphisms associated with HF in Chinese fishermen were genotyped in both samples. The phenotypes were either severe HF associated with portal hypertension in Sudanese or significant secondary portal branch thickening in Brazilians who were much less affected than Sudanese.
Conclusions:
[0112] Univariate analysis (Table 5 left) in Sudanese (219 controls, 95 cases) showed that SNPs rs9402373 (p=0.02, OR=2.7), rs1256196 (p=0.044, OR=3.2) and rs12527705 (p=0.05, OR=3) were associated with severe HF. The aggravating genotypes were identical in Sudanese and Chinese (Tables 4 and 5). Multivariate analysis indicated SNP rs9402373 (p=0.03, OR=2.7) and SNP rs1256196 (p=0.03, OR=3.5) were independently associated with the HF without additional covariates.
[0113] Univariate analysis of the genotyping data of the Brazilians (73 controls, 60 cases) showed (Table 5 right) associations between SNP rs9402373 (p=0.02, OR=2.57) and SNP rs6918698 (p=0.008, OR=3) with HF without additional covariates. The genotypes associated with disease for both SNPs were the same as in Chinese and Sudanese (Tables 4 and 5). Multivariate analysis indicated SNPs rs9402373 and rs6918698 were independently (p<0.001, OR>4) associated with HF in Brazilians.
[0114] In silico analysis had suggested that the allelic variants SNPs rs9402373 and rs12526196 might bind differently nuclear factors. We have demonstrated this with EMSA: the rs9402373C allele bound nuclear factors that were not bound by the G allele (FIG. 2A). This binding was competed with specific unbiotinylated probe (FIG. 2B). EMSA also showed a greater binding affinity of nuclear factors to the rs12526196T allele (FIG. 2A). EMSA did not reveal allele specific binding for rs12527705 and rs1931002 polymorphisms (FIG. 2A).
[0115] Then SNP rs9402373 was associated with HF in all four samples tested. This association was obtained in Chinese and Sudanese, using a strict fibrosis phenotype or a more severe phenotype (HF+evidence of portal blood hypertension). SNP rs1256196 was also independently associated with HF in Chinese and Sudanese. Our EMSA data indicate that allelic variants of SNP rs1256196 and SNP rs9402373 bind differently nuclear factor suggesting that they could affect the regulation of gene transcription or the stability of transcripts.
[0116] HBV infection was a significant covariate in the analysis of Chinese farmers indicating that HBV likely contributed significantly to severe disease. This also suggests that SNP rs1256196 and SNP rs9402373 might also modulate HF caused by HBV.
[0117] In conclusion the present study shows the association of polymorphisms with fibrosis, and identifies most critical steps in disease development indicating new therapeutical targets. It also provides valuable markers of HF progression.
TABLE-US-00006 TABLE 5 Sudanese sample Brazilian sample SNP or Controls Cases Controls Cases alteration Genotype % % OR 95% CI P SNP Genotype % % OR 95% CI P 12527705 AA 82.1 90.5 3 1-9.1 0.05 12527705 NS 12526196 TT 80.8 90.5 3.2 1-9.7 0.44 12526196 NS 3037970 -/- NS 3037970 -/- 80.5 90.2 0.12 6918698 CC NS 6918698 CC + CG 63 83.9 3.05 1.33-6.98 0.008 9402373 CC 80 89.8 2.75 1.17-6.5 0.02 9402373 CC 60.3 80.0 2.57 1.18-5.64 0.018 12526196 TT 3.5 1.1-10.9 0.03 6918698 CC 4.92 2.0-12 5 .times. 10-4 9402373 CC 2.76 1.2-6.7 0.026 9402373 CC 4.25 1.79-10.08 9 .times. 10-4
Example 3
Association Between SNPs in the CTGF Gene Locus with Hepatic Fibrosis (HF) in HCV Infected Subjects
[0118] Analysis of CTGF Genetic Variants in French Subjects Infected with HCV.
[0119] Example 1 describes SNP correlation bins (r2>0.8) in the CTGF encoding gene in a sample of Chinese population. These were defined over the entire gene and 10 Kb of the 3' and 5' flanking region. Only SNPs with a minor allele frequency>20% had been analyzed. There were seven bins of which four (groups II, III, IV and VI) showed some association with hepatic fibrosis caused by schistosome eggs in Chinese fishermen infected by Schistosoma japonicum. In Example 3, the inventors genotyped two SNPs in each of these four bins and an additional SNP rs9399005 (that showed suggestive associations with schistosomal fibrosis) in a French sample infected with HCV and determined whether any of them were associated with advanced hepatic fibrosis. They found evidence for association in bins III and VI but not in bins II and IV. SNP rs6918698 (p=0.03) and SNP rs3037970 (p=0.04) in bin IV, were associated with hepatic fibrosis. The CG and GG genotypes of SNP rs6918698 were associated with disease aggravation (OR=2.1, CI=1.1-4.0).
[0120] Bin VI only contains SNP rs9402373 that was also (p=0.007) associated with hepatic fibrosis in the French sample. Disease was aggravated by the CC genotype (OR=2.54; 1.3-4.96). Interestingly, the subjects homozygous C/C showed an average four-fold increase of the risk of severe hepatic fibrosis as compared to homozygous GG (OR=4.1 CI=1.1-14). Multivariate logistic regression analysis that included SNP rs6918698 and SNP rs9402373 indicated that the latter was the most strongly associated with hepatic fibrosis. Nevertheless in the presence of SNP rs9402373, SNP rs6918698 still showed a trend for an association with hepatic disease suggesting that SNP rs9402373 could not account for the full association of SNP rs6918698 with disease. Thus the inventors concluded both SNPs are independently associated with fibrosis.
CTGF Genetic Variants are Also Associated with Hepatic Fibrosis in Brazilians Infected with HCV.
[0121] The inventors then sought the confirmation of the above described findings by testing SNPs from the same four bins in Brazilians infected with HCV. They found that SNP rs6918698 (p=0.0004) was clearly associated with hepatic fibrosis. SNP rs3037970 showed a trend (p=0.07) for an association and SNP rs6918698 accounted for SNP rs3037970 association. The SNP rs6918698 aggravating genotype was G/G (OR 2.94; 1.5-7.7). As in the French sample, the inventors observed that SNP rs9402373 in bin VI, was strongly (p=0.003) associated with hepatic fibrosis (OR=2.94, 1.45-5.97) in the Brazilian sample. Of note, in this sample as in the French sample, C/C aggravated disease. Since subjects carrying the G/G genotype were few, the inventors could not compare the risk of severe fibrosis between homozygous subjects. Finally the inventors also showed a trend (p=0.06) for an association of SNPrs9399005 with hepatic fibrosis.
[0122] Logistic regression analysis showed that both SNP rs9402373 (p=0.029) and SNP rs6918698 (p=0.03) contributed independently to hepatic fibrosis with comparable relative risks (OR=2.4-2.5) and that the association of SNP rs9399005 with fibrosis was lost in the presence of the two other SNPs suggesting that the association of this SNP with hepatic fibrosis likely is entirely accounted for by its correlation with SNP rs9402373 and SNP rs6918698. It can not totally be excluded, however, that SNP rs9399005 could also be independently associated with hepatic fibrosis.
TABLE-US-00007 TABLE 6 SNPs rs 6918698 and rs9402373 are associated with hepatic fibrosis in HCV infected subjects French Sample GENOTYPE 95% Analysis SNP Position Bins (aggravating).sup.2 CONTROLS % CASES % OR CI P Univariate 1257705 132304944 II NS >1.4 12526196 132305169 II CT* NS >1.3 9399005 132310657 CC NS >0.3 3037970 132316891 III Del, del (French) 67.8 (78) 79.5 (89) 1.9 1.03-3.6 0.04 Del, del, del/wt (Brazilians) 6918698 132314950 III GG + CG 68.4 (78) 81.7 (89)) 2.1 1.1-4 0.025 (French) GG (Brazilians) 1931002 132320175 IV NS >0.5 2151532 132316423 IV NS >0.5 9402373 132319124 VI CC 51.3 (61) 70.8 (46) 2.54 1.3-4.96 0.007 12527379 AG + AA 57.1 (64) 67 (69) 1.7 0.94-2.97 0.08 Multivariate 6918698 C/G + G/G 1.54 0.77-3.1 0.22 (french) 9402373 G/G (Brazilians) 2.1 1.15-4 0.017 C/C Brazilian Sample GENOTYPE 95% Analysis SNP Position Bins (aggravating).sup.2 CONTROLS % CASES % OR CI P Univariate 1257705 132304944 II NS >1.4 12526196 132305169 II CT* NS >1.3 9399005 132310657 CC 57.4 (39) 72.4 (92) 1.9 0.97-3.7 0.06 3037970 132316891 III Del, del (French) 61 (41) 75 (93) 1.9 0.95-3.9 0.07 Del, del, del/wt (Brazilians) 6918698 132314950 III GG + CG 16.4 (11) 34.4 (44) 3.3 1.5-7.7 0.004 (French) GG (Brazilians) 1931002 132320175 IV NS >0.2 2151532 132316423 IV NS >0.2 9402373 132319124 VI CC 58.8 (40) 79.7 (102) 2.5 1.1-5 0.003 12527379 AG + AA 38.2 (26) 50.0 (64) 1.8 0.94-3.5 .08 Multivariate 6918698 C/G + G/G 2.5 1.1-5.9 0.03 (french) 9402373 G/G (Brazilians) 2.4 1.1-5 0.029 C/C French (n = 222) and Brazilians (n = 196) cohorts, infected with HCV: Covariates Sexe p < 10.sup.-3
[0123] The inventors then performed a Meta analysis of the data obtained with SNP rs9402373 in French and Brazilians infected with HCV.
[0124] Such an analysis was not performed with SNP rs6918698 because the fibrosis associated genotypes were different in French (G/G+C/G) and in Brazilians (G/G). The inventors found that SNP rs9402373 CC genotype was associated (p=0.00005, OR=2.72; 1.67-4.43) with hepatic fibrosis caused by HCV.
[0125] Since the association of this same CC genotype of SNP rs9402373 was shown with hepatic fibrosis in the above examples, the inventors also performed a Meta analysis of the studies testing the association of this SNP with HCV induced HF (two studies) and schistosomal induced HF (4 studies). The results of such a Meta analysis are presented in FIG. 3 and show that CC of the SNP rs9402373 is strongly associated (p<10-9) with severe hepatic fibrosis (OR=2.47; 1.8-3.25) caused by different pathogens HCV, S. mansoni and S. japonicum) in populations (Chinese, Sudanese, Brazilian and French) with markedly different genetic background.
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Sequence CWU
1
1
1421701DNAHomo sapiensmisc_feature(501)..(501)w=A/T 1cagatctcag ttcatctctt
tacgattggg agagtctgga agaaaaagat ctatctatat 60taatagagat tctttagagg
tgcaaatttt ccctcacaaa gaacaacttt gcggggccat 120ttcaaaatat ggcaaagaaa
catgttttgg ggaaatgcac tttttaattg atgtatattg 180ttccattata taaatattct
atactctatc aatttttctg tttacagaat tcagattttt 240gctttcagtt tttggatgga
tgaaactgct ttgagaattt tgtatgtctt ttgctcactt 300ctcttgaata tataccaaag
actaaaagtt attgtagtta tctaaattat acatgttaac 360ttgctgacaa aaattgtgtt
agaatttgtt gacctaatgt aaaagtttta tattcttgcg 420atgcaaattc taattaatca
atggtgctcc tcatttctta ctaactcttc tttgactctc 480ttttaaacat gtaatataga
wgatgggtct acatgtctat tgctttgaat cctatattta 540ggtttggaat ctgacagtat
tttaagaata tattttactt tgtttatttt gttcttagtc 600ttagaaacag tctatatata
attagtatga ttcatgaggt aaggaatagt tatacagagg 660aaaatcgttc accattttat
ttatggtata gagaaataga g 7012501DNAHomo
sapiensmisc_feature(251)..(251)Y=C/T 2ctctctttta aacatgtaat atagaagatg
ggtctacatg tctattgctt tgaatcctat 60atttaggttt ggaatctgac agtattttaa
gaatatattt tactttgttt attttgttct 120tagtcttaga aacagtctat atataattag
tatgattcat gaggtaagga atagttatac 180agaggaaaat cgttcaccat tttatttatg
gtatagagaa atagagtaaa agacaacact 240gaatatacaa ygaatatggg cattccctag
agattcgatg tgtgttgcat gtactcctag 300gcacgtggct aattaagata tagtggtttg
aatagctaag ttatttactt atttatgtat 360tgtgtattgt ttcatctaaa tgaaaatcat
aaaatacttg aacacttttt gacatattcc 420ttgctgtttt tttaatgaga tggaaatttg
ttttttaaaa aatctctttc ttataatgta 480tgttcatttc aaaaaatttg a
5013401DNAHomo
sapiensmisc_feature(201)..(201)Y=C/T 3aacggaatta tacaaaacaa gacttatgaa
atagctccat ttaaatgagg atttcatttc 60ataattttca gatttaacag taaggtaatg
aagttcagaa acagacctag agcataaaat 120ttttaaaaaa tgtaaacatt ttccacgtgc
aaggcacata gactagtttg tgatgtgaag 180ggttggaaac taacctataa yggccagaga
ggtacaaatg ccaaaaggac caagtgtaat 240acaacagggt taatggagac tgaagtaatc
tagagagaat tgaatttaaa gtgtctataa 300agttctgtga agataacaaa aaaaatggct
gtatagtcaa tgtggtgtgt atgcctgcaa 360gtctgtgata cttgattcag tgtgaagtat
gtttacataa a 4014601DNAHomo
sapiensmisc_feature(301)..(301)S=C/G 4ccccccaacc cttagcaatg atttgcgttt
tagaggctat aggctcttga aactctccaa 60agagtagaaa aggagtgagt tgcttttaac
catttgaaat cctcaagatg cctacctgta 120aaaccctgaa ttctatattt gagatttgat
catttgtcac ttgaggtaac ggttttggga 180caagaaagag aacaaagacg cgtgtgactc
aggatgcagt ctcctggggc agatttccaa 240aactcttgct tagttttctc ttaatatatg
taggagtttt atataggcaa ggacaaggga 300sgagtggcca tcaatgtttc cagaaaaagg
gatccattgt tctatcagag caaatgattc 360tgtgttgggt aggtaggggc tctgggtgtc
agggtgggaa cactgggatg caaagggggt 420tcacctagga gcatttaaac acagcttcac
tagggtcttt gagaaatgag tgcattttct 480tatattaaaa cctataatct tcaaatgtgg
attggatttg gtttgtcttg ttaaaataag 540aagtctgatt ccattacaag agcagtgtac
cctgttgtat ctggatataa actaagattg 600a
6015984DNAHomo
sapiensmisc_recomb/insertion_seq(489)..(490)with or without insertion of
TAAAA between position 489 and 490 5gtactgggtt atagttagat ggcctaggtt
gtattcttgg aactgccttt cgcagatctc 60agggaagtca cacgtaggag ccatattccc
atttctgttt aaaaaatgaa agtaatcata 120atttttttaa tttaataggg ttcagtgagg
gctgagtaga tgaaaattcc agggaattgg 180tgctttattt ttttcagatt ttcttgattt
tttttcaatc tctgtgtata ataagtatat 240taccattctg ctctgttgtt aaaaatgtca
aaagaaagaa tttggtttta ctataaaatt 300ctcatagtcc ctcgaagtct caaaaaaatc
tatagtcagt tccaaataga tgattttaat 360gttgcttaag aggaaataag catattgaaa
tatatagaac atatgaaatc tcaggaaaga 420aaaagtttct gtgcattatc aggttagatt
gaggctttat gatcaaaact ctttttgaag 480gagttaaaag actattatta agagagtgac
acagagacag acagacacat acatgcacac 540acacccagag agagagagaa tgagagaaga
cacccaagac attcacagac tcagaagtat 600gtttttattg tgatatggac catcaaatgc
caggaaactc aaagggaaag gctgatactc 660catccataat tcatgccatg ggatctgaat
tcttgcacta ttatcaatga ctcagcaaat 720gtttgtgtgt gcctgggcgg taaagtggga
aatagtggct tcttataact tcatccaact 780ttgacttacc ccagcccctt tgattgacag
caggtcagag ttgggagaaa aagatctttg 840aatttgtgtg caaaatggtt aagatgcact
aattttgact tgtaagcaag tatttatcta 900gcaaatgctt tttagagagc attttttcct
cgtggtaaaa tgaggaatta actaagctgg 960ccactgagtc ctgtttacca agct
9846586DNAHomo
sapiensmisc_feature(228)..(228)R=A/G 6aaattaaaca atgcattagt tgacttttac
ttcaaatctg atgatgtcag gattttaaat 60agaatgtggt ttttcttcat atgtggtgaa
agttctgact tagaagataa gataccttct 120caaaaagcct tatccaggtg ctcagatttt
aatatatgat tcccataggc atggttattt 180aaagatttac acacaggaaa atagtttaga
aactctttgg atgattcraa caacttggtt 240tttagcattc tagaacatac tggctaccaa
gtttgctttt aatttgaaaa cttgtcacca 300tcatggtagc acagttcaca ttacaaaatg
aaatgaaaga aaactgtcaa cccaaatata 360tgtaaagtgt taaagtctgt agcatcacat
taagtaaaat accaccttgg tttccaagaa 420atacaaatct aaagaaaatg tgatggacac
aagcaatcat aattttttgg ttgtttcttt 480atcatttgca taccatattc ttattagtga
taagttacac atttttcact gaagaaagga 540agactgttag gaactcccta actctcccca
tcctccacaa atcaat 5867501DNAHomo
sapiensmisc_feature(251)..(251)Y=C/T 7tttgacattt ttaacaacag agcagaatgg
taatatactt attatacaca gagattgaaa 60aaaaatcaag aaaatctgaa aaaaataaag
caccaattcc ctggaatttt catctactca 120gccctcactg aaccctatta aattaaaaaa
attatgatta ctttcatttt ttaaacagaa 180atgggaatat ggctcctacg tgtgacttcc
ctgagatctg cgaaaggcag ttccaagaat 240acaacctagg ycatctaact ataacccagt
acaacaattt atcccctcca tgaatgagtc 300tccctgctgc aggcatgaag atggtggtag
ctttcagact ctcctgagga ttcagaaatt 360ccccaaccct tctcccacct acctcactct
gcaagctagg agagctcacc accaagccca 420catcccacct taggtgagac tcgcactgca
catccctacc cttccccagc caccacacct 480ttccaaccat tagaggtaga t
5018817DNAHomo
sapiensmisc_feature(317)..(317)S=C/G 8cccatcattc atccctggaa atgtgctccc
acctctagcc aagaggcctg aggtcccttc 60tcacaacttc aaactccctg gaataccatt
aggtggagcc taggggactg gctaggaggt 120gaaggtgtga gctccatacc tgccatttgc
ttgccagtga cctcagggaa gacacctact 180ctctctgagc ctgaaatttc ccacctgttc
acagttaatg tgcagctcaa ggagacaata 240tttatgaaag cactttcata aaggtattag
agttttaaaa ttgcccttct cttccattct 300tcagtggctc tcaaaastaa gcccaactca
gattgactgt aaaagcctgt cacagtttga 360ttttataaca gtgtccagct ctgcatgggt
tttagagtgt taaaactagg tgagagctca 420gagaaaatca aatcctacct cttatttatt
tatttatttt attagattag atattcagga 480ttgatagaga catttaacaa atgaaaactt
tttttctttt tgaaataggg tctggctctg 540tctcccaggg tggaatgtag tgccatgatc
ttggctcact gcaacctctg cctcctgggc 600tcaagtgatc ttcctgcctc agcctcccaa
gtagctggga tagctgggac tacaggtgtg 660caccaccata tccagctaat taaaagaatt
tttttttttt tttttgtaga gatggggttt 720tgccatgttg cccaggctgg tctcgaactc
ctgagctcaa gagatcagcc catctcagcc 780tcccaaagtg ctgggattac aggcgtaagc
ccctgcg 8179601DNAHomo
sapiensmisc_feature(301)..(301)R=A/G 9ataattgtat cattcactga taagctaccc
ttagggataa atatcatgac agagtctggg 60acaaaccatc agcactgaag caatgtctac
tatcaacata ggtttttcac gagtatcccc 120taataaacat gtcctgccat gttattgata
tttattttat gaacttgcct taactgaaaa 180tgcactttcc tcaaactgaa tatgctttgc
ctttcaggtt tacagctttc tccctctctc 240ctttaaccat ggaaggattt gatttgaatc
ataactagtt tcatgttgtt aatggaatgc 300ratttgtcat aactacacat ggcagagaga
gagggttgtc taggaatggt ggggttatga 360tgagggaatt ttcttattga cctgcacatg
tgtgtgaaag ttaacaacta caggattaca 420tttcatataa ccattgactt gctcatgtct
ggaaataata tcactattaa gctatttctc 480atatacagat gataaaaact tattttgtcc
aattgaatct tttgtggcat tttcatcatt 540tctaacgtct ctatgttggt tcttcttgat
tgtggttttc cctgctgttt ccgtatttga 600c
601101001DNAHomo
sapiensmisc_feature(501)..(501)R=A/G 10tgagaggcta tgagtggggt cagctttacc
caaagtacat gggctgaaac ttgggggaat 60aatccaccag agcaaagatc agcaattttt
tttcttgaaa agggccccat agtaaatatt 120tttagccttt gtgggctata cagtctgtca
ctcaattctg ccactgtagc tcaaaagcag 180ccatggatac tacatttttt taaaaaaggg
catgaccgtg taccaagaaa actttgttta 240caaaatcaga gagcaggcca gattcagatg
acaggccaga gtttgccaac ccctgcacta 300gagcgagatt aagtacagtt atcagactgg
aagtcaggct ataaaaataa gcaacagatg 360tccaccacat gcatagaata atatagatat
ccctgcttct ctgggtcttt agcttttctc 420tctctttgta ataagagtca taacagatga
ctttaggagg ctctttttct tccaaagttc 480tcctgacctc aaattaaaat rgtctactga
gagtccaggt ggtggctttt gcaaatgctt 540gggatccagg aggaaaggtg acagccttca
tggtagcaag cctatagaca gtagcagccc 600cagaatcaaa taatttgctt ctagatataa
cactttcaca tgctaaaata taaaacctct 660ctcctcagaa aaactaatct acttcttccc
tagtctttcc aatgccagat aacagcacca 720cagtttgtcc attcatttag gcaaaaagct
ggggtgatat actcattttc ctgttaatat 780ccctcagcca acctatcagc aagtcccatg
tgcagaatat gcactgaatc tgaccactcc 840atctacgtcc accaacacag ctcccacccg
tgtcaccatg cgcctctcct ggacttctcc 900agtagcctcc ccagtctccc tcctccctct
tgctcctcta ctgtcctttc tcaattcagc 960agccagaatg atctttctaa catctaaatc
ctcccgtgac c 100111861DNAHomo
sapiensmisc_feature(361)..(361)W=A/T 11agttctcctg acctcaaatt aaaatggtct
actgagagtc caggtggtgg cttttgcaaa 60tgcttgggat ccaggaggaa aggtgacagc
cttcatggta gcaagcctat agacagtagc 120agccccagaa tcaaataatt tgcttctaga
tataacactt tcacatgcta aaatataaaa 180cctctctcct cagaaaaact aatctacttc
ttccctagtc tttccaatgc cagataacag 240caccacagtt tgtccattca tttaggcaaa
aagctggggt gatatactca ttttcctgtt 300aatatccctc agccaaccta tcagcaagtc
ccatgtgcag aatatgcact gaatctgacc 360wctccatcta cgtccaccaa cacagctccc
acccgtgtca ccatgcgcct ctcctggact 420tctccagtag cctccccagt ctccctcctc
cctcttgctc ctctactgtc ctttctcaat 480tcagcagcca gaatgatctt tctaacatct
aaatcctccc gtgacctctc attatgctat 540ttatttattt attgagatgg agtcttcctc
tgtcacccag actggagtgc agtggcatga 600tttcggctca ctgcaacctc tgcctcccag
gttcaagcgg ttcttctacc tcagcctccc 660tagtagctgg gactacaggc atgcatctcc
acacctggct aatttttgta tttttagtag 720agagggggtt ttaccatgtc agccaggctg
gtctcaaact cctgagctca ggtgatctgc 780cctccttggc ctcccaaagt gctgggatta
caggcgtgag ccaccacgca cagcttcctt 840atgtttaaaa ctaaacctca g
861121249DNAHomo
sapiensmisc_feature(973)..(973)R=A/G 12aaaacctctc tcctcagaaa aactaatcta
cttcttccct agtctttcca atgccagata 60acagcaccac agtttgtcca ttcatttagg
caaaaagctg gggtgatata ctcattttcc 120tgttaatatc cctcagccaa cctatcagca
agtcccatgt gcagaatatg cactgaatct 180gaccactcca tctacgtcca ccaacacagc
tcccacccgt gtcaccatgc gcctctcctg 240gacttctcca gtagcctccc cagtctccct
cctccctctt gctcctctac tgtcctttct 300caattcagca gccagaatga tctttctaac
atctaaatcc tcccgtgacc tctcattatg 360ctatttattt atttattgag atggagtctt
cctctgtcac ccagactgga gtgcagtggc 420atgatttcgg ctcactgcaa cctctgcctc
ccaggttcaa gcggttcttc tacctcagcc 480tccctagtag ctgggactac aggcatgcat
ctccacacct ggctaatttt tgtattttta 540gtagagaggg ggttttacca tgtcagccag
gctggtctca aactcctgag ctcaggtgat 600ctgccctcct tggcctccca aagtgctggg
attacaggcg tgagccacca cgcacagctt 660ccttatgttt aaaactaaac ctcagcagag
tttacaaggt ccctatgacc tagacttcct 720caaccctccc agcctcatct ctgaacactg
tctccttttt aactggagct gaaaggagga 780ggaataaagt aacttcaata aacaaccaga
acagtgcttt caaatgtcac ttttggtgat 840gatggaaatg ttctatatct gtgctgtcta
gtatggaagc cactagccgc atgtcactac 900tgagtgtttg aaatattgct actgtgacag
agaggctgac ttttacactg tacttaattt 960taattcattt aartgtaata gccacatttg
tctatcagct actatggaca gcaaaaaact 1020agaatatggg caaaactgat ataaatgata
aaataaagtt atgaacagta tttgttaggg 1080catctgaaag gaaagagggc tgctaggagg
ttatgggatt cagggtagta ttaaaaaatc 1140attcaaaaag tgacttctgg gaagggcttt
aaatgatgag tgaagttgac ttagatataa 1200atatatatgt gcttatatat atacatatac
acacatgcat tatgagaag 1249132706DNAHomo
sapiensmisc_feature(1997)..(1997)K=G/T 13ctatatttta gagcagttat gtacgtttat
gtatatctct gtgtgtatac atataaatat 60atatatatat atgcatgctt atgtataagt
gaaatgaatg acaacaatga tacaagggac 120aggagggagg aatttggatt acttgttatt
gcaaagtact tgtactatct atgaagctgt 180acagtgttat atgaaagtga acctggctgg
gccaggtgca gtggctcaag cctgtaatcc 240cagcactttg ggaggctgaa gtgggtgcat
cacaaggtca ggagctcgag accagcctgg 300ccaatatggt gaaaccctgt ctctactaaa
aatacaaaaa tgagcagggt gtggtggcag 360gtgcctgtag tcccagctac tcaggaggct
gaggcaggag aattgcttga acgcgggagg 420cagaggttgc agtgagccga gaatgcacca
ctgcactcca gcctgggtga cagagcgaga 480ttctgtctca agaaaaaaaa aaaaaagaaa
gaaagtgaac ttgggtttgt tgtaaatgtg 540tattgcaaat tctagagcaa ccgttaaaaa
agtttttaaa aaagtagaat tgatatgcta 600agaaaggaga gaaaatggaa tcatgtaaaa
tgctcagtta atgccacaaa agacaaaagc 660cctgtggaac acaagaagaa acaaagaaca
agagcaacac atagaaaaca gtaacaaata 720tggtagatgt taagccaact atatcaataa
tcactgtaga tgtcagtggt ctaaatatgc 780caattaaaag acagagcttg tcagagtata
ttaaaaacac accagccgtg gtggtgcact 840tgtgtaaccc cagctacctg ggatactgtg
tgcggaggat ataaagccta ggagttcatg 900accagcctgt acaacatagg gagaacctgt
ctcaaagaaa aacaaacaaa caaaaaatca 960agacccaact aattattgtc tgtaagaaac
ccatgttaaa cagattagac tctgacaaaa 1020cacgcatagg ttaaactcta gtaaaatact
ttctcccgag ggtaggccct gctaagaaga 1080gtacccagat gtatttcaaa atggttactt
tcctcctccc actgctggaa gagcaaggaa 1140atttttctcc aatattttgg gagataaaac
tccccaaaac atgtgtgggt ggagggacag 1200ggggcccatg actgtatctg ggaccacttt
ttatataaac aaatctctaa attgaagttc 1260aaatatttga gtgttggtga aaaagaaatg
gagttattct gatacttgtg actcattact 1320attatatata ttttttgctt aaattgtctc
aggtttaatt attgcgagct ccttcaagtt 1380gtcttttcag tatgaaccta acattttttt
cagcaatttc ttacttgctg gtgccacaag 1440tttttgcagg tttatcttgc attttctctg
cctgagccat gaaatcagcc atttttccaa 1500aaagccctag ttccttttat tggacattat
taaaatttgg gttctacata tgttaattgt 1560tctggggtgt cacagcacct aggatgatga
cagctcagtt gacagaaaat acacatacag 1620ctaagtgcat atataaaccc attctatatt
tatgtctgtg tatatctata catgcattaa 1680aaccatgagc tgtgctatgc ccaagccctc
acaattactc attacttacc cattgacttg 1740gcttgatcaa atgctagtca ggcttctccc
tacaggtccc agaactttat cttattccca 1800agcttttaag caagtgctaa gttacagaat
atcttctcaa tgacttactc caagaatcca 1860gtgaccaaag agaataaaaa tttactgtca
aagccactct catgccatct gcctacctcc 1920attcgcttgc cccactcttt cacaaacttc
ctgctagccc tatttacttc tccttataaa 1980agaaaggtct ttttctkttc gacctttaga
ctttgccact tctgcaatag agtattctcc 2040ctatttcaaa gttctcttcc ccatattgca
atgcagttgg tccttgaaaa acacaggggt 2100taggggtgct gaccccttgt gcagttaaaa
attcacgtaa aatttttgac tcaccataaa 2160cttaactact actagccgac tgttgactgg
aagctttacc aataacacaa acaatagatt 2220aacacatatt tgtgtgttat atatatttga
cactgaattc ttatagtaaa gtaagagaaa 2280ataaaatgaa aaaatgccat taagaacatt
atatggaaga gaaaatatat ttattactta 2340ttaaatggga atagatcatc agaaaggtct
ttatcctcat tgtcttcatg atgggcaggc 2400tgaggagaag agaaaagagt aggagttggt
cttgctgtcc cttggcgtaa ctattattga 2460aaaaaattca tgtataagtg gatttgtgca
gttcaaaccc atgttcttca agggtcaact 2520gtggcctttt tgaataaaat ctctatttta
atctggcttt taaaaaattt gactactggt 2580actactaatg gcaacccaat accacaggtg
tttgaattaa tcagttaatt gcctttattg 2640aaagaatgtc tatggtcttt tataatttat
attttcctca agatagattt gtactgaaat 2700aaaatc
270614701DNAHomo
sapiensmisc_feature(501)..(501)Y=C/T 14ttggtccttg aaaaacacag gggttagggg
tgctgacccc ttgtgcagtt aaaaattcac 60gtaaaatttt tgactcacca taaacttaac
tactactagc cgactgttga ctggaagctt 120taccaataac acaaacaata gattaacaca
tatttgtgtg ttatatatat ttgacactga 180attcttatag taaagtaaga gaaaataaaa
tgaaaaaatg ccattaagaa cattatatgg 240aagagaaaat atatttatta cttattaaat
gggaatagat catcagaaag gtctttatcc 300tcattgtctt catgatgggc aggctgagga
gaagagaaaa gagtaggagt tggtcttgct 360gtcccttggc gtaactatta ttgaaaaaaa
ttcatgtata agtggatttg tgcagttcaa 420acccatgttc ttcaagggtc aactgtggcc
tttttgaata aaatctctat tttaatctgg 480cttttaaaaa atttgactac yggtactact
aatggcaacc caataccaca ggtgtttgaa 540ttaatcagtt aattgccttt attgaaagaa
tgtctatggt cttttataat ttatattttc 600ctcaagatag atttgtactg aaataaaatc
taacttttat taaaactgaa aaagggaaat 660ggaggattca ttgacaatgg ggtgtttcta
tctgggtaac a 70115401DNAHomo
sapiensmisc_feature(201)..(201)R=A/G 15ggtttccata gcagcagctt ctagcagcca
ctcctattgg cctgggtcct taccacaagc 60agcacctccc tgcagatctt cctgctggtg
aatgctgctc caagtgatta atgacttcca 120ttcccctaga atctcattca gtgaatccta
aaatgttcaa ctttaaagga cctcaatccc 180aaacatcttc tctacaaaac rtctggattt
ctccaatagc tagagggagc atttgatttc 240tagcagcacc cccaagccag gtctcaatag
gctccttacg aagccctggg gttcagttag 300aatgccacac tgttttttca aagtccttta
ttatctccat cggctactat tcatacctcc 360ggtagaagag tcccattgac aggtgttctg
gacttcaggc t 40116601DNAHomo
sapiensmisc_feature(301)..(301)R=A/G 16atgtaaacag agtcctggca aatagcccag
acctcccagg gctccttaac ataagtacgc 60aagcttgcct aataatcctg agtttaaaat
tatgagtgac ataagataat cttcacactg 120ggagtcatcg gcccaaatta cggttaaagg
ttaaaaacaa acaaataaaa tgcaaaaaaa 180aatcacatac aaaccaggct tatgttgtag
aaaatatttc ctaagccctt ggatttaatg 240tcatttttag agtttgatga tacctcccta
gagagggtat aacaaatact gaacaaggat 300racacagatc agaaagaatt tagctaggga
ggaattcaga ttggggagaa aaataaggag 360ttgggcctat tctcatttaa aaataaatcc
ctctggaaag acataaaaga acatttttaa 420aaagactaaa agaatggcag tttatctttt
catttttaat gctaagtgtt tctattttaa 480agggcttcca tcccaagtat atacttggat
tgaaaaatgg aaaacagtta aaaatgaaga 540tttttaagtt caaaatgttg atcaaaattt
cctgattcaa attgcttttt tgactcttgg 600a
601171001DNAHomo
sapiensmisc_feature(501)..(501)R=A/G 17tatgcaaata aatatatatg ccaataaata
tatgcaaata aacaggcaga aatgttctac 60tttttacact gtatacagta ctttctcctt
tttaaccttc attcagtctt agttctaaag 120gtgagctata ttcaattctc actacagtcc
ttaggttagt atctctcaga catttgtgtt 180gtttgagctt gtttgctagt aggttcctca
gcaagggctc acaggaaaaa tatgccctga 240gttcttgtgt gtttttaaca gtttgtctgt
gtccttcctg ttcattgcag cactatccac 300gatagctaag ttatggaatc aacctaagtg
tccatcaaca gatggatggg taaagaagat 360gaggtatata tacacaatgg agtactattc
attaaaaaag aagggaatcc tgtcatttgt 420gacaacaggg atgaacctag tcattcaagg
tttgatttca agaatcaagt cattcttgaa 480aatcagtttt agaggataaa rgtttcttga
ctctcatttt ctttccttaa gtatcttaaa 540taagttattc catttttttc tgtcataaag
tattgctgta aaaaaaatct aacgatgagc 600taattttctt tttatttata aggctcatgt
tctttttgtc ttcatgttca aatagctttt 660cccttttctt taaactccag cagttttact
agaatgtgtt ttgctgttag ttgatttgtt 720taggcacatg gcacaccctt tcaacttgtg
ctttcaaata cttttttatt ctagtaaagt 780tttttttgac ttatagcttt taggattttt
ttttcttctt taagaatttc taccctccac 840atattggatt ttctttgcct gtccacaata
tttattactt tttattgcaa tatgtttatt 900tcttctttta tatttaaact tgttttctta
tctgtaagag tatattcact cttatgctcc 960ttgtagtttg atatagttcc tttatttcta
aatctctgtt g 100118601DNAHomo
sapiensmisc_feature(301)..(301)S=C/G 18tgtgtgcaga tgttgaatat ggccaataaa
cctaagcata tctccatttc ttaatatagt 60gctacttagc ttcactaaag acccaactaa
ttcactgtgc tttcatttgg aatctatgtt 120gtttagtggc ttttttgtta atacctccag
ttcccatttt atataaagag agtgattcta 180gggccatacc atttttgtat tctgtaatgc
cgcatcattt gacattggtg ttaccaatga 240gtagattcct ttaagttgtt gatgcttcag
agcatgggtt caagataagc atatctgttt 300stggtgcaaa gatcggcttt agaaacaatg
cattatagat ttatcatttg aaaacagcct 360gtttaaactg atcaaatttt taaagtttca
gtacagtctg ttgaaaaata gccaagaaga 420aatataacca ctccaacttc agcatagagc
atatcatctg tgcacgatta cctcgttaag 480ccagccacat gttatagagt gtagttgcat
gtcaatagtg tgaatggcca gcaaaggggt 540gggggtgccc tctgaagctc aggaagaatt
ttcgtcattg ttatttaatg tcctttccaa 600g
60119602DNAHomo
sapiensmisc_feature(333)..(333)M=A/C 19agctgaacat cttacaagac ttaaccaaac
gaccagtaca aatgatgccc tagtagacac 60acacacatac cttatatagc ttggtaaaca
ggactcagtg gccagcttag ttaattcctc 120attttaccac gaggaaaaaa tgctctctaa
aaagcatttg ctagataaat acttgcttac 180aagtcaaaat tagtgcatct taaccatttt
gcacacaaat tcaaagatct ttttctccca 240actctgacct gctgtcaatc aaaggggctg
gggtaagtca aagttggatg aagttataag 300aagccactat ttcccacttt accgcccagg
camacacaaa catttgctga gtcattgata 360atagtgcaag aattcagatc ccatggcatg
aattatggat ggagtatcag cctttccctt 420tgagtttcct ggcatttgat ggtccatatc
acaataaaaa catacttctg agtctgtgaa 480tgtcttgggt gtcttctctc attctctctc
tctctgggtg tgtgtgcatg tatgtgtctg 540tctgtctctg tgtcactctc ttaataatag
tcttttaact ccttcaaaaa gagttttgat 600ca
60220601DNAHomo
sapiensmisc_feature(301)..(301)Y=C/T 20tgtttgctta agatgaaggt taaggagagg
gcgtacttgc tgggagttca tggcggggaa 60gataaacgcg gatgtttact atctaaatta
cattcattac cttcatgcct tttcctcctt 120ataattagtt ctctgacttt cttaggatat
tttctaatgc aaaggtattt gtcagaaggc 180agtcaaggcc gttgcatgtg gtttgtgttt
tatgtttgcc tgttcctcgc ttgtccatag 240agttggaggc cagaggggtg agaaaggaag
ttttgttttg taagatgata tcccttcaac 300ytactgtttg tttcacttat tttgttaatt
tgactctttt tttttttaaa gtagtttact 360tagaatcctg tgttgcctct agtcagcttt
tgttcatcta agatagtgtc agatgaggat 420gctgctaggt aggaatgagc ctggtggtct
cttacactgt cctcgacagg ttagaatctc 480aaattcatca ccgtttagca accaggagta
tttacagccc cttgcttcat cagggctaaa 540tgtgaagcat ccccttggtg atacttaagg
tgatatttaa gtgagtggga atgaaaaaca 600g
60121401DNAHomo
sapiensmisc_feature(201)..(201)R=A/G 21aagatagtgt cagatgagga tgctgctagg
taggaatgag cctggtggtc tcttacactg 60tcctcgacag gttagaatct caaattcatc
accgtttagc aaccaggagt atttacagcc 120ccttgcttca tcagggctaa atgtgaagca
tccccttggt gatacttaag gtgatattta 180agtgagtggg aatgaaaaac rgaggaaata
agcatttctt aagatgtgtg atttggggac 240acccaatcaa taggtttagg aggaagagag
aattgggact agctatctta tctcaaataa 300tagaagtaag tgaaaggata aatattttga
aaggactagt gtgaaatata catgaatgta 360ttcctaggga ttctgctgcg gggtgggaaa
ggggtgcaga t 40122601DNAHomo
sapiensmisc_feature(301)..(301)R=A/G 22aggatgctgc taggtaggaa tgagcctggt
ggtctcttac actgtcctcg acaggttaga 60atctcaaatt catcaccgtt tagcaaccag
gagtatttac agccccttgc ttcatcaggg 120ctaaatgtga agcatcccct tggtgatact
taaggtgata tttaagtgag tgggaatgaa 180aaacagagga aataagcatt tcttaagatg
tgtgatttgg ggacacccaa tcaataggtt 240taggaggaag agagaattgg gactagctat
cttatctcaa ataatagaag taagtgaaag 300rataaatatt ttgaaaggac tagtgtgaaa
tatacatgaa tgtattccta gggattctgc 360tgcggggtgg gaaaggggtg cagattaagc
cagaccccat cattcatccc tggaaatgtg 420ctcccacctc tagccaagag gcctgaggtc
ccttctcaca acttcaaact ccctggaata 480ccattaggtg gagcctaggg gactggctag
gaggtgaagg tgtgagctcc atacctgcca 540tttgcttgcc agtgacctca gggaagacac
ctactctctc tgagcctgaa atttcccacc 600t
60123501DNAHomo
sapiensmisc_feature(251)..(251)R=A/G 23aaaaaaaaag aatgtcatga cagtttgtat
tgacagcctt tttctagcaa gaaacatacc 60gttattttga acttgtgaat gatttatgaa
ttgtgattct gtcttcagga tcctgttttt 120ttctgttttg tttttgtttt tgtcatcaaa
ttgccacagg ggggagtata agttcttgaa 180agcagcaaga aatacagttt aagtaatttg
gtaccaatta tcattattat tagaaacgtc 240catatctgaa rcatctaaac agattagatt
aatacctatg tgaaagtaga aatatttata 300gtaaaaaatt gggcaccgtt taatatagga
tttattaatt ttgtgatagt tgcaaaagct 360gtgtctggaa aatgtcccca tgggtgaatg
tgtgggcgtg cacatgcatg tgggtaatat 420taatgctgta ggagaaggga gaaaatgtaa
gtttctagtt tttgccatta aagtcatgac 480gtgaaagaga gagggagaga g
50124601DNAHomo
sapiensmisc_feature(301)..(301)R=A/G 24agacaaaggc tggaggtttt cacacctggt
agtctcatgt agaaaggaag gctgatcagc 60agggaggcag aggaagggtt gacagggtag
gaccttggag ggaagcagtt aggcttggaa 120gggacacaaa ggaaaggatg gcaaagaact
gaccagagca actaaaagca caacatagta 180gaaccgagga cacatctgag actagagacc
acaaatgtat ggcagacact ataggaacaa 240ctaaggcagg cactggcttg aagaaaggtc
atgaggccaa aggccagaag tgtaacaagg 300rgctgttggc ataaaacagt atgccatgga
gagagaggaa gcaaaaccgt gtgtccagga 360gaaaaggaag gggcaagaga ccaaagtcac
gatcaggtca aaacattgat gtgaagaata 420aatgagcaag agaggccggg ggcggtggct
cacgcctgta atcccagcac tttgggagac 480cgaggtgggc agatcatgag gtcaggagat
ggagaccatc ctggctaaca cggtgaaacc 540ccgtctctac taaaagtaca aaaaacttag
ccaggcgtgg tggcgggcgc ctgtagtccc 600a
601251982DNAHomo
sapiensmisc_feature(1102)..(1102)M=A/C 25caggaactca tcttaaatat ttaaaaataa
gttttttttt ctaattgcaa ggaataagta 60ctctttgtaa atgatatgga aagtatggaa
tataataaag atgaaaaaga taactcctag 120ataatcactt ttaatgtttt ggcaaaattc
tttttaaaaa gtgctaaaga aaatgtggca 180catatacatc atggaatact atgcatccat
aaaaaagaat gagttcatgt cctttggagg 240gacatagatg aagctgaaag ccatcattct
tagcaaacta acccaggaac agaaaaccaa 300acaccgcata ttctcactca taagtgggag
ttgaacaatg agaacacatg gacacaggga 360ggggaacatc acacatgggg gcctgttggg
gggtgggggc caaggaaagg gaaaacatta 420ggacaaatac ctaatgtatg cagagttaaa
atctagatga cgggttgata ggtgcagcaa 480accaccatgg cacgtgtata cctatgtaac
aaatctgcac attctgcaca tgtatcccag 540aacttaaagt aaaaaaaaaa aaagtgcttg
aatatattta atttttaaac tttttaattg 600aaatataata gagatttaac aatatgtgcc
aagttctctc atgcccccac ctccaaagta 660accactaccc tgcctttatc actatttagt
tttgcttcct cttcaatttc atatagatga 720cattatgcag tgtgtatgct tttgtgcctg
acttctttca ctcaccataa tagtgctttt 780cttttttata tataagagat ttattctgag
ccaaatatga gtgaccatgg cccatgacac 840agccctcaag aggtcctgag aacttgtgcc
caaggtggtc tgggggcagc ttggttttat 900acattttaga gcagcatgag acatcaatca
catccgttta agaaatacat tggtttggtc 960cagatgtgga gcgggtggtg ggggaagggg
aggcttccag gttataggtg aatttaaaca 1020ttttctggtt gacaattggt tgagtttgtc
tcaaaacgtg ggatagaggc tgggcgcagt 1080ggctcacgcc tataatccca gmactttggg
aggccgaggc gggcggatca cctgagctca 1140gaagttcaag accagcctgg gcaacatggt
gaaaccccgt ctctattaaa aatacaaaaa 1200atcagcctag tgtggtagtg catgcctgta
atcccagcta ctcgggaggc tgaggcagga 1260gaatcacttg aacccgggag gcagaggttg
cagtgagtcg agatcacgcc actgcactcc 1320ggcctgggcg acagagagag actttgtatc
aaaaaaaaaa aaaaaaaaaa aaggcctggg 1380atagatagaa agggaatgtt caggttaaga
taaacattgt ggaaacccaa gttcttctga 1440ggtcttatag tggctgcctg tagaggcaag
aagtgacaaa tgtttcctat tcagatctca 1500gttcatctct ttacgattgg gagagtctgg
aagaaaaaga tctatctata ttaatagaga 1560ttctttagag gtgcaaattt tccctcacaa
agaacaactt tgcggggcca tttcaaaata 1620tggcaaagaa acatgttttg gggaaatgca
ctttttaatt gatgtatatt gttccattat 1680ataaatattc tatactctat caatttttct
gtttacagaa ttcagatttt tgctttcagt 1740ttttggatgg atgaaactgc tttgagaatt
ttgtatgtct tttgctcact tctcttgaat 1800atataccaaa gactaaaagt tattgtagtt
atctaaatta tacatgttaa cttgctgaca 1860aaaattgtgt tagaatttgt tgacctaatg
taaaagtttt atattcttgc gatgcaaatt 1920ctaattaatc aatggtgctc ctcatttctt
actaactctt ctttgactct cttttaaaca 1980tg
198226978DNAHomo
sapiensmisc_feature(679)..(679)R=A/G 26aactatatca aactacaagg agcataagag
tgaatatact cttacagata agaaaacaag 60tttaaatata aaagaagaaa taaacatatt
gcaataaaaa gtaataaata ttgtggacag 120gcaaagaaaa tccaatatgt ggagggtaga
aattcttaaa gaagaaaaaa aaatcctaaa 180agctataagt caaaaaaaac tttactagaa
taaaaaagta tttgaaagca caagttgaaa 240gggtgtgcca tgtgcctaaa caaatcaact
aacagcaaaa cacattctag taaaactgct 300ggagtttaaa gaaaagggaa aagctatttg
aacatgaaga caaaaagaac atgagcctta 360taaataaaaa gaaaattagc tcatcgttag
atttttttta cagcaatact ttatgacaga 420aaaaaatgga ataacttatt taagatactt
aaggaaagaa aatgagagtc aagaaacttt 480tatcctctaa aactgatttt caagaatgac
ttgattcttg aaatcaaacc ttgaatgact 540aggttcatcc ctgttgtcac aaatgacagg
attcccttct tttttaatga atagtactcc 600attgtgtata tatacctcat cttctttacc
catccatctg ttgatggaca cttaggttga 660ttccataact tagctatcrt ggatagtgct
gcaatgaaca ggaaggacac agacaaactg 720ttaaaaacac acaagaactc agggcatatt
tttcctgtga gcccttgctg aggaacctac 780tagcaaacaa gctcaaacaa cacaaatgtc
tgagagatac taacctaagg actgtagtga 840gaattgaata tagctcacct ttagaactaa
gactgaatga aggttaaaaa ggagaaagta 900ctgtatacag tgtaaaaagt agaacatttc
tgcctgttta tttgcatata tttattggca 960tatatattta tttgcata
97827701DNAHomo
sapiensmisc_feature(501)..(501)Y=C/T 27tcttttcaac atactgactt ctttttcatt
gagtgtatac ccagtaatgg gattgctata 60tcatatagta attgtattct taatttttta
tggaactttc tgtttttcat aatggccgta 120ctaatttaca ttcctgccaa caatgcacag
ggtttttttt cctccacatc cttgctgaca 180gttttgttat cttttgtttt tttgatagta
gccattttaa caggtgtgaa gtgatagctc 240attgtggttt tgatttgcat ttccctgatg
gttagtaatg ttgaacattt tttcatatac 300ttgttggcca tttgtgtgtc ttcttttgag
aaacatctgt ctggtgacct gctcaattta 360gattccactt ccaggagtat gaggatcttc
tcctgggagg cagccctggc ttgtaagctt 420tgttggagtg ctaggccaaa cccctactag
attcaacagc tgctgtcagc ctggccttct 480atatatctag tgactacctg ytggctgttt
gagagctctc gtgttctcag aagcatcaga 540taccttgttg tttctctgct tttctcacac
atgtgctgac acggtgcaga tcttacggtg 600gttgatgatt tggcaccccc tacctccgat
atttgggggg tgaaagtgtg cattgccatc 660taatttttgt agaaacattg cctgtgggat
ttttgttttg c 70128601DNAHomo
sapiensmisc_feature(301)..(301)M=A/C 28attggttatc atggcaacca aaaacggaat
tatacaaaac aagacttatg aaatagctcc 60atttaaatga ggatttcatt tcataatttt
cagatttaac agtaaggtaa tgaagttcag 120aaacagacct agagcataaa atttttaaaa
aatgtaaaca ttttccacgt gcaaggcaca 180tagactagtt tgtgatgtga agggttggaa
actaacctat aacggccaga gaggtacaaa 240tgccaaaagg accaagtgta atacaacagg
gttaatggag actgaagtaa tctagagaga 300mttgaattta aagtgtctat aaagttctgt
gaagataaca aaaaaaatgg ctgtatagtc 360aatgtggtgt gtatgcctgc aagtctgtga
tacttgattc agtgtgaagt atgtttacat 420aaaaattcta tggaataatc aatttattaa
tgtatagaat aattaaagat atttagaaaa 480ttattggtga attatcttat gattaataag
aacttcctat ttatgctcaa gaaaactcag 540aaatgtttag aaggataaat gaataaccaa
gccctttata gaaaaaatat acactttatt 600t
60129406DNAHomo
sapiensmisc_feature(206)..(206)Y=C/T 29cctggaaatg tgctcccacc tctagccaag
aggcctgagg tcccttctca caacttcaaa 60ctccctggaa taccattagg tggagcctag
gggactggct aggaggtgaa ggtgtgagct 120ccatacctgc catttgcttg ccagtgacct
cagggaagac acctactctc tctgagcctg 180aaatttccca cctgttcaca gttaaygtgc
agctcaagga gacaatattt atgaaagcac 240tttcataaag gtattagagt tttaaaattg
cccttctctt ccattcttca gtggctctca 300aaactaagcc caactcagat tgactgtaaa
agcctgtcac agtttgattt tataacagtg 360tccagctctg catgggtttt agagtgttaa
aactaggtga gagctc 40630438DNAHomo
sapiensmisc_feature(201)..(201)Y=C/T 30ttttttctgt tttgtttttg tttttgtcat
caaattgcca caggggggag tataagttct 60tgaaagcagc aagaaataca gtttaagtaa
tttggtacca attatcatta ttattagaaa 120cgtccatatc tgaaacatct aaacagatta
gattaatacc tatgtgaaag tagaaatatt 180tatagtaaaa aattgggcac ygtttaatat
aggatttatt aattttgtga tagttgcaaa 240agctgtgtct ggaaaatgtc cccatgggtg
aatgtgtggg cgtgcacatg catgtgggta 300atattaatgc tgtaggagaa gggagaaaat
gtaagtttct agtttttgcc attaaagtca 360tgacgtgaaa gagagaggga gagagagaaa
gaaagtgaga aagtgctagt ctgtgtaaaa 420tgaccatgaa agggcttg
438311001DNAHomo
sapiensmisc_feature(501)..(501)R=A/G 31tgtgtatagt atctcatata tatatatata
tatatatttt ttcgggtttt cacgctcaaa 60gtcctgccac catcgtaagt taccccaata
tccacccaaa tcactatcca aactcacttc 120taagctcctc ttgcctgtct cttctaagct
agtcaggatt tgccatacac cctgccattt 180catagaactt ctgcaatccc cttgggtcct
tgatatggtt tggctctgtg tccccaccaa 240atctcatgta agattgtaat ccccagtgtt
ggaggtggag ctggtgggag gtgattggat 300cataggggtg gtttctgaag gtttagcacc
atcaccctgg tgctgtctca tgagagagtt 360ctcatgaaat ctgcttgttt aaaagtgtcc
ccctttgttc ttttcttcct gctccagcca 420tgtaggacgt gtctctttcc tctttgcttt
ccgctgtgat tgcaagtttc ccgagacctc 480cccagtcatg cttcctttac rgcctgcaga
accatgaacc aattaaacct ctcttcttta 540tagattatcc agtctcacgt agttctttat
agcaatgtga gtacagacta atacagccct 600acatttctgt cctctcctgc tctctcttgc
tcatttattc tttttttttt gagacggagt 660ctcgctctgt cgcccaggct ggagtgcagt
tgggcgatct cggctcactg caagctccac 720ctcctgggtt cacgccattc tcctgcctca
gcctcccgag cagctgggac tacaggcgcc 780cgccaccacg cctggctaag ttttttgtac
ttttagtaga gacggggttt caccgtgtta 840gccaggatgg tctccatctc ctgacctcat
gatctgccca cctcggtctc ccaaagtgct 900gggattacag gcgtgagcca ccgcccccgg
cctctcttgc tcatttattc ttcacatcaa 960tgttttgacc tgatcgtgac tttggtctct
tgccccttcc t 100132601DNAHomo
sapiensmisc_feature(301)..(301)S=C/G 32aatagtgctt ttctttttta tatataagag
atttattctg agccaaatat gagtgaccat 60ggcccatgac acagccctca agaggtcctg
agaacttgtg cccaaggtgg tctgggggca 120gcttggtttt atacatttta gagcagcatg
agacatcaat cacatccgtt taagaaatac 180attggtttgg tccagatgtg gagcgggtgg
tgggggaagg ggaggcttcc aggttatagg 240tgaatttaaa cattttctgg ttgacaattg
gttgagtttg tctcaagacg tgggatagag 300sctgggcgca gtggctcacg cctataatcc
cagcactttg ggaggccgag gcgggcggat 360cacctgagct cagaagttca agaccagcct
gggcaacatg gtgaaacccc gtctctatta 420aaaatacaaa aaatcagcct agtgtggtag
tgcatgcctg taatcccagc tactcgggag 480gctgaggcag gagaatcact tgaacccggg
aggcagaggt tgcagtgagt cgagatcacg 540ccactgcact ccggcctggg cgacagagag
agactttgta tcaaaaaaaa aaaaaaaaaa 600a
6013323DNAArtificial SequenceSynthetic
oligonucleotide 33gctctcaaaa ctaagcccaa ctc
233423DNAArtificial SequenceSynthetic oligonucleotide
34gagttgggct tagttttgag agc
233521DNAArtificial SequenceSynthetic oligonucleotide 35gtaatataga
agatgggtct a
213621DNAArtificial SequenceSynthetic oligonucleotide 36gtaatataga
tgatgggtct a
213721DNAArtificial SequenceSynthetic oligonucleotide 37gaatatacaa
cgaatatggg c
213821DNAArtificial SequenceSynthetic oligonucleotide 38gaatatacaa
tgaatatggg c
213921DNAArtificial SequenceSynthetic oligonucleotide 39tggatgattc
aaacaacttg g
214021DNAArtificial SequenceSynthetic oligonucleotide 40tggatgattc
gaacaacttg g
214120DNAArtificial SequenceSynthetic oligonucleotide 41aaggggcaag
agaccaaagt
204221DNAArtificial SequenceSynthetic oligonucleotide 42caccctgcca
tttcatagaa c
214320DNAArtificial SequenceSynthetic oligonucleotide 43accagggtga
tggtgctaaa
204420DNAArtificial SequenceSynthetic oligonucleotide 44aatgaccatg
aaagggcttg
204520DNAArtificial SequenceSynthetic oligonucleotide 45tgcccccata
tgtacagaaa
204620DNAArtificial SequenceSynthetic oligonucleotide 46gggacatttt
gcaagggtta
204721DNAArtificial SequenceSynthetic oligonucleotide 47ttgctagaaa
aaggctgtca a
214820DNAArtificial SequenceSynthetic oligonucleotide 48cagcccaata
ttccaccaag
204921DNAArtificial SequenceSynthetic oligonucleotide 49gccaagttta
ttttggcagg t
215023DNAArtificial SequenceSynthetic oligonucleotide 50ttggttcttc
ttgattgtgg ttt
235120DNAArtificial SequenceSynthetic oligonucleotide 51gcaaagcaac
attggttcaa
205220DNAArtificial SequenceSynthetic oligonucleotide 52ggtatgcttt
gggaggctta
205320DNAArtificial SequenceSynthetic oligonucleotide 53tctcagccga
aacaagactg
205420DNAArtificial SequenceSynthetic oligonucleotide 54tttagcggga
acgcttctta
205520DNAArtificial SequenceSynthetic oligonucleotide 55accgtgaaag
ggctttgtaa
205622DNAArtificial SequenceSynthetic oligonucleotide 56tgctcagctt
atttttgtca cc
225720DNAArtificial SequenceSynthetic oligonucleotide 57ccctaactct
ccccatcctc
205820DNAArtificial SequenceSynthetic oligonucleotide 58atgtgcagct
caaggagaca
205923DNAArtificial SequenceSynthetic oligonucleotide 59ttgattttct
ctgagctctc acc
236020DNAArtificial SequenceSynthetic oligonucleotide 60taggaatgag
cctggtggtc
206120DNAArtificial SequenceSynthetic oligonucleotide 61ggtgtcccca
aatcacacat
206220DNAArtificial SequenceSynthetic oligonucleotide 62tatgggcatc
tggacagtga
206320DNAArtificial SequenceSynthetic oligonucleotide 63acgccctctc
cttaaccttc
206419DNAArtificial SequenceSynthetic oligonucleotide 64agcccctttg
attgacagc
196523DNAArtificial SequenceSynthetic oligonucleotide 65gttaattcct
cattttacca cga
236621DNAArtificial SequenceSynthetic oligonucleotide 66agtccctcga
agtctcaaaa a
216723DNAArtificial SequenceSynthetic oligonucleotide 67gcacagaaac
tttttctttc ctg
236820DNAArtificial SequenceSynthetic oligonucleotide 68aagggtaggg
atgtgcagtg
206920DNAArtificial SequenceSynthetic oligonucleotide 69caggcatgaa
gatggtggta
207020DNAArtificial SequenceSynthetic oligonucleotide 70ggtgcaaaga
tcggctttag
207124DNAArtificial SequenceSynthetic oligonucleotide 71tcttcttggc
tatttttcaa caga
247220DNAArtificial SequenceSynthetic oligonucleotide 72ggggttcacc
taggagcatt
207322DNAArtificial SequenceSynthetic oligonucleotide 73caagacaaac
caaatccaat cc
227420DNAArtificial SequenceSynthetic oligonucleotide4 74tatcctcttc
gcaccactcc
207520DNAArtificial SequenceSynthetic oligonucleotide 75gtggacagaa
cagggcaaac
207618DNAArtificial SequenceSynthetic oligonucleotide 76gcttacccgg
ctgcagag
187718DNAArtificial SequenceSynthetic oligonucleotide 77acagccccga
gacgacag
187820DNAArtificial SequenceSynthetic oligonucleotide 78gcagcagctg
gagaaagaaa
207920DNAArtificial SequenceSynthetic oligonucleotide 79aaaaagaaac
cgctcggact
208020DNAArtificial SequenceSynthetic oligonucleotide 80ccaggcagtt
ggctctaatc
208120DNAArtificial SequenceSynthetic oligonucleotide 81tcagggtcgt
gattctctcc
208220DNAArtificial SequenceSynthetic oligonucleotide 82tggagatttt
gggagtacgg
208320DNAArtificial SequenceSynthetic oligonucleotide 83tcaaacttcc
tcccctcaaa
208420DNAArtificial SequenceSynthetic oligonucleotide 84tgctcctaaa
gccacacctt
208520DNAArtificial SequenceSynthetic oligonucleotide 85ggaaaagatt
cccacccaat
208620DNAArtificial SequenceSynthetic oligonucleotide 86ccatttcatg
ctttgaacga
208722DNAArtificial SequenceSynthetic oligonucleotide 87ttgtgtgtgt
gtgtgtgtgt gt
228820DNAArtificial SequenceSynthetic oligonucleotide 88aatgccaaaa
ggaccaagtg
208920DNAArtificial SequenceSynthetic oligonucleotide 89cagacttgca
ggcatacaca
209020DNAArtificial SequenceSynthetic oligonucleotide 90tggtttgtgg
tgtggacagt
209121DNAArtificial SequenceSynthetic oligonucleotide 91ccacatgagg
atagctgagg a
219220DNAArtificial SequenceSynthetic oligonucleotide 92gctttgttgg
agtgctaggc
209320DNAArtificial SequenceSynthetic oligonucleotide 93ccaaatcatc
aaccaccgta
209423DNAArtificial SequenceSynthetic oligonucleotide 94tttctgtaac
ctggcttatt tca
239520DNAArtificial SequenceSynthetic oligonucleotide 95tctgcacctc
catgttcatt
209620DNAArtificial SequenceSynthetic oligonucleotide 96tttgtaggga
tttggcttca
209720DNAArtificial SequenceSynthetic oligonucleotide 97attgagtggg
tcagggacaa
209822DNAArtificial SequenceSynthetic oligonucleotide 98cccttttctt
taaactccag ca
229920DNAArtificial SequenceSynthetic oligonucleotide 99tggacaggca
aagaaaatcc
2010022DNAArtificial SequenceSynthetic oligonucleotide 100tgcaaataaa
caggcagaaa tg
2210120DNAArtificial SequenceSynthetic oligonucleotide 101gcccttgctg
aggaacctac
2010220DNAArtificial SequenceSynthetic oligonucleotide 102acacatttct
tgccccagac
2010320DNAArtificial SequenceSynthetic oligonucleotide 103tccccctaca
tctgcctaca
2010420DNAArtificial SequenceSynthetic oligonucleotide 104agcctgtctt
ctgaggcact
2010520DNAArtificial SequenceSynthetic oligonucleotide 105gggggacaga
gaaagactcc
2010624DNAArtificial SequenceSynthetic oligonucleotide 106tgggtctaca
tgtctattgc tttg
2410720DNAArtificial SequenceSynthetic oligonucleotide 107gggaatgccc
atattcattg
2010821DNAArtificial SequenceSynthetic oligonucleotide 108tggaaaccca
agttcttctg a
2110920DNAArtificial SequenceSynthetic oligonucleotide 109cccgcaaagt
tgttctttgt
2011019DNAArtificial SequenceSynthetic oligonucleotide 110ccatgacaca
gccctcaag
1911120DNAArtificial SequenceSynthetic oligonucleotide 111gcctctatcc
cacgttttga
2011221DNAArtificial SequenceSynthetic oligonucleotide 112tgtcctttgg
agggacatag a
2111320DNAArtificial SequenceSynthetic oligonucleotide 113tatacacgtg
ccatggtggt
2011423DNAArtificial SequenceSynthetic oligonucleotide 114tgacttaatg
aataagcctg ctg
2311524DNAArtificial SequenceSynthetic oligonucleotide 115gcaccatata
aatgtgagat tgga
2411626DNAArtificial SequenceSynthetic oligonucleotide 116aaaagaagct
gaatttgctt taaaat
2611721DNAArtificial SequenceSynthetic oligonucleotide 117agcaggatac
tgacaggcaa a
2111825DNAArtificial SequenceSynthetic oligonucleotide 118tcatttaaaa
ataaatccct ctgga
2511922DNAArtificial SequenceSynthetic oligonucleotide 119tgttttccat
ttttcaatcc aa
2212020DNAArtificial SequenceSynthetic oligonucleotide 120ccagggtccc
attcctagtt
2012120DNAArtificial SequenceSynthetic oligonucleotide 121gaggcttgtg
gagcattagc
2012220DNAArtificial SequenceSynthetic oligonucleotide 122agcatgggtt
tccatagcag
2012320DNAArtificial SequenceSynthetic oligonucleotide 123tgtttgggat
tgaggtcctt
2012420DNAArtificial SequenceSynthetic oligonucleotide 124tgtgcagttc
aaacccatgt
2012520DNAArtificial SequenceSynthetic oligonucleotide 125tgtggtattg
ggttgccatt
2012620DNAArtificial SequenceSynthetic oligonucleotide 126ttctccctac
aggtcccaga
2012722DNAArtificial SequenceSynthetic oligonucleotide 127atgcactacc
acactaggct ga
2212822DNAArtificial SequenceSynthetic oligonucleotide 128agcagcatga
gacatcaatc ac
2212922DNAArtificial SequenceSynthetic oligonucleotide 129atgcactacc
acactaggct ga
2213022DNAArtificial SequenceSynthetic oligonucleotide 130agcagcatga
gacatcaatc ac
2213124DNAArtificial SequenceSynthetic oligonucleotide 131tgattcttga
aatcaaacct tgaa
2413222DNAArtificial SequenceSynthetic oligonucleotide 132gctagtaggt
tcctcagcaa gg
2213320DNAArtificial SequenceSynthetic oligonucleotide 133atggcaatgc
acactttcac
2013420DNAArtificial SequenceSynthetic oligonucleotide 134gctttgttgg
agtgctaggc
2013520DNAArtificial SequenceSynthetic oligonucleotide 135cttgcaggca
tacacaccac
2013620DNAArtificial SequenceSynthetic oligonucleotide 136aacggccaga
gaggtacaaa
2013720DNAArtificial SequenceSynthetic oligonucleotide 137tgagagccac
tgaagaatgg
2013820DNAArtificial SequenceSynthetic oligonucleotide 138taggtggagc
ctaggggact
2013920DNAArtificial SequenceSynthetic oligonucleotide 139ggggacattt
tccagacaca
2014020DNAArtificial SequenceSynthetic oligonucleotide 140tgtcatcaaa
ttgccacagg
2014120DNAArtificial SequenceSynthetic oligonucleotide 141agcgagactc
cgtctcaaaa
2014220DNAArtificial SequenceSynthetic oligonucleotide 142ctttgctttc
cgctgtgatt 20
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