Diagnostic kit for UGT1A1 gene variation

Abstract

The invention provides a diagnostic kit for UGT1A1 (UDP glucuronyl transferase 1 family polypeptide A1) gene variation, the diagnostic kit comprises a primer of an autonomously designed gene, a promoter TATA frame of the UGT1A1 gene, an exon and an intron region of 50-150bp near the upstream and downstream of the exon, and by observing all variation conditions of the coverage region of the UGT1A1 gene, the UDP glucuronyl transferase 1 family polypeptide A1 can be identified, and the UDP glucuronyl transferase 1 family polypeptide A1 can be identified. The prognosis condition after hepatitis B virus (HBV) infection is predicted. And the use of unstable bilirubin detection values is avoided.

Description

Technical Field

[0001] The invention relates to the field of genetic metabolism molecular biology, and in particular to a UGT1A1 (UDPglucuronosyltransferase family 1member A1, UDP glucuronosyltransferase 1 family polypeptide A1) gene variation diagnosis kit. Background Art

[0002] The outcomes of hepatitis B virus (HBV) infection include: negative conversion of surface antigen, virus carriage, chronic hepatitis, cirrhosis, and even hepatocellular carcinoma. The current treatment methods are: after the treatment indication is determined, specific treatment (multiple antiviral drugs) and liver protection treatment can be used, but neither can be a cure. A small number of patients can clear the virus, but the related factors are still unclear.

[0003] No study in this field has ever mentioned the protective effect of host bilirubin on the entire history of infection. Bilirubin is a decomposition metabolite of heme. The reasons for increased serum bilirubin concentrations may be excessive production, impaired uptake, binding or excretion, or reflux into the blood from damaged stem cells or bile ducts. Therefore, it is generally believed that increased serum bilirubin concentrations are more beneficial than harmful to the body, especially the liver. The difficulty in confirming the correlation between bilirubin and other diseases lies in the fact that this biochemical indicator is easily interfered with. Therefore, in addition to the inherent understanding of bilirubin, the volatility of bilirubin itself also makes its analysis a natural technical bottleneck.

[0004] By comparing people who are congenital carriers of Gilbert syndrome-related UGT1A1 gene mutations with wild-type people, it was found that people with gene mutations had better clinical prognosis after being infected with HBV virus, including: older age of disease diagnosis; significantly reduced end-stage liver disease events; and significantly more HBV virus clearance events. Further analysis in the gene mutation population revealed that as mutations accumulate or the frequency of mutations is ranked from common to rare, it is positively correlated with the age of diagnosis of end-stage liver disease. That is, the greater the impact of the patient's UGT1A1 gene mutation on the function of the encoded protein (the more mutations accumulate, or the rarer the mutation), the later the poor outcome after HBV infection occurs, and the better the infection outcome.

[0005] Therefore, the present invention replaces the judgment of abnormal bilirubin level of the body by observing the variation of UGT1A1 gene, and predicts the prognosis after hepatitis B virus infection according to the variation of UGT1A1 gene, and avoids using unstable bilirubin detection value. Summary of the invention

[0006] In order to overcome the above technical defects, the object of the present invention is to provide a diagnostic kit for UGT1A1 (UDPglucuronosyltransferase family 1 member A1, UDP glucuronosyltransferase 1 family polypeptide A1) gene mutation. The diagnostic kit for UGT1A1 gene mutation includes primers for amplifying the following genes: the promoter TATA box, exons, and the intron region 50-150 bp proximal to the upstream and downstream sides of the exons of the UGT1A1 gene. The primer sequences are shown in the following table:

[0007]

[0008] Compared with the prior art, the advantages of the present invention are:

[0009] The diagnostic kit for UGT1A1 gene mutation of the present invention contains independently designed primers to obtain the promoter TATA box, exons and introns of about 50-150 bp upstream and downstream of the UGT1A1 gene, and interprets these sequences to directly observe the mutation of the UGT1A1 gene, and predict the prognosis after hepatitis B virus infection based on the mutation. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 for Figure 1 The original peak diagram of sequencing using the primer pair of SEQ ID NO.1 to detect the heterozygous single-site variation of UGT1A1*6 (c.211G>A, rs4148323);

[0011] Figure 2 The original peak graph of sequencing of UGT1A1*28 (rs34983651) insertion mutation detected using the primer pair of SEQ ID NO.1;

[0012] Figure 3 The original peak graph of sequencing of the insertion variation of UGT1A1*63 (c.1091C>T, rs34946978) detected using the primer pair of SEQ ID NO.4;

[0013] Figure 4 This is a schematic diagram of the statistical results of clinical data in one embodiment of the present invention;

[0014] Figure 5 This is a schematic diagram of statistical results of clinical data in another embodiment of the present invention;

[0015] Figure 6 for Figure 5 A schematic diagram of the statistical results of clinical data in the illustrated embodiment. DETAILED DESCRIPTION

[0016] The advantages of the present invention are further described below in conjunction with the accompanying drawings and specific embodiments.

[0017] The present invention adopts polymerase chain reaction (PCR) and uses independently developed primers to amplify whole blood samples to obtain the promoter TATA box, exons and introns of about 50-150bp upstream and downstream of the UGT1A1 gene, and interprets these sequences to determine whether the sample has pathogenic related mutations.

[0018] The reagents and instruments used in the specific embodiments of the present invention are as follows:

[0019] 1. Reagents and their storage conditions are as follows: QIAamp Blood Mini Kit from QIAGEN, Germany, catalog number: 51106, 250 servings / box, stored at room temperature; Proteinase K, 1 bottle, stored at 4°C; Buffer; AllPrep DNA / RNA Mini Kit from QIAGEN, catalog number: 80204, 50 servings / box, stored at room temperature; Promega DNA polymerase, 100U / tube, stored at -20℃; dNTP Mixture from TAKARA, 2.5mM each, 1.28ml / tube, stored at -20℃; PrimeScript from TAKARA TM II 1st Strand cDNA synthesis kit, stored at -20℃; Shanghai Bioengineering agarose, 100 g / bottle, stored at room temperature; TAKARA's DL2000 DNA Marker kit, stored at -20℃.

[0020] Self-designed primer sequences (see Table 1):

[0021] Table 1. PCR primers

[0022]

[0023] 2. The instruments used include: Eppendorf 5415D centrifuge from Eppendorf; Nanodrop 2000 spectrophotometer from Thermoscientific; ProFlex TM 3X 32-well PCR system thermal cycler from ABI; Bio-rad electrophoresis instrument from Bio-Rad; Tanon 3500 gel imaging system from Tanon.

[0024] 3. Test conditions and steps:

[0025] DNA extraction: Take 200μl of the EDTA anticoagulated whole blood sample to be tested, 20μl of proteinase K, and 200μl of Buffer AL, mix and oscillate for 15 seconds, and place in a 56°C water bath for 30 minutes after sufficient mixing; add 200μl of anhydrous ethanol (96-100%), oscillate for 15 seconds, and centrifuge at 10,000 rpm for 1 minute in a centrifuge; transfer the above mixture to a QIAamp Spin Column and centrifuge at 10,000 rpm for 1 minute; replace the collection tube; add 500μl of Buffer AW1 and centrifuge at 10,000 rpm for 1 minute; replace the collection tube; add 500μl of Buffer AW2 and centrifuge at 12,000 rpm for 3 minutes, and replace with a 1.5ml centrifuge tube; add Buffer AE 200 μl was placed at room temperature for 5 minutes, centrifuged at 8000 rpm for 3 minutes to obtain genomic DNA, and stored at 4°C for later use; the concentration of the extracted genomic DNA was quantified using a spectrophotometer.

[0026] PCR reaction system :

[0027] Table 2: PCR reaction system

[0028] DNA template or cDNA template 3μl 5×Buffer 5μl dNTP Mixture 1μl GoTaq DNA polymerase 0.25μl Upstream primer 0.5μl Downstream primer 0.5μl Double distilled water supplement Total volume up to 25 μl

[0029] PCR reaction conditions:

[0030] 95℃2min

[0031] 95℃30s (35 cycles)

[0032] 55℃30s (35 cycles)

[0033] 72℃35s

[0034] 72℃6min

[0035] Quality Control

[0036] According to the quantitative requirements, the A260 / A280 ratio of DNA samples is controlled between 1.6-2.0; the laboratory room for PCR reaction system preparation and PCR reaction product identification is strictly separated from the PCR instrument working room; a negative control without DNA template is performed under the same conditions for each pair of primers in each batch of tests. All test systems are controlled by the ISO 15189 quality management system.

[0037] Mutation interpretation

[0038] The PCR amplification positive products were sent to the sequencing company for sequencing, and the primers related to the tested items were used as sequencing primers. The sequencing results were analyzed using CodonCode Aligner software, and the relevant test items were compared with their respective genomic DNA and reading frame DNA sequences as reference sequences. The disease mutation databases were referenced to the HGMD (http: / / www.hgmd.cf.ac.uk / ac / index.php), OMIM (http: / / www.ncbi.nlm.nih.gov / omim), dSNP and other databases.

[0039] HBV infection testing :

[0040] HBV serological indicators include HBsAg, anti-HBs, HBeAg, anti-HBe, and anti-HBc.

[0041] Diagnosis of different stages of the natural history of HBV infection :

[0042] Surface antigen clearance, HBV carrier, hepatitis B, cirrhosis, liver cancer.

[0043] The above basis - "Guidelines for the Prevention and Treatment of Chronic Hepatitis B (2022 Edition)" requires the combination of serology, biochemistry, and imaging for corresponding diagnosis.

[0044] The kit of the present invention can realize HBV exposure detection; UGT1A1 gene variation detection: promoter TATA box of UGT1A1 gene, all exons and intron regions 50-150bp proximal to the upstream and downstream of exons; and UGT1A1 gene variation analysis: classification of mutation sites, quantity and properties after detection; and judgment of HBV prognosis.

[0045] in, Figure 1 The original peak diagram of sequencing using the primer pair of SEQ ID NO.1 to detect the heterozygous single-site variation of UGT1A1*6 (c.211G>A, rs4148323); Figure 2 The original peak graph of sequencing of UGT1A1*28 (rs34983651) insertion mutation detected using the primer pair of SEQ ID NO.1; Figure 3 The original peak graph of sequencing of UGT1A1.63 (c.1091C>T, rs34946978) insertion variation detected using SEQ ID NO.4 primer pair.

[0046] Clinical Trials

[0047] Example 1: Patients with UGT1A1 mutations have a poorer HBV (hepatitis B virus) outcome at an older age (age without UGT1A1 mutation vs. age with mutation), and the specific sample data are as follows: Figure 4shown.

[0048] according to Figure 4 It can be seen that compared with patients without UGT1A1 mutations, patients with UGT1A1 mutations generally develop HBV liver cancer or cirrhosis at a later age.

[0049] Example 2: Patients with UGT1A1 mutations are less likely to have a worse outcome in HBV (hepatitis B virus) (UGT1A1 without mutations vs. with mutations). The clinical data are shown in Table 3. Compared with patients without UGT1A1 gene mutations, patients with UGT1A1 mutations have a lower chance of progressing to liver cancer or cirrhosis.

[0050] Table 3 Sample data of liver cancer or cirrhosis in patients without UGT1A1 gene mutation and patients with mutation

[0051]

[0052]

[0053] Example 3: Patients with UGT1A1 mutations had an ideal HBV outcome (UGT1A1 without mutation vs. with mutation). The clinical data are shown in Table 4. Compared with patients without UGT1A1 gene mutations, patients with UGT1A1 gene mutations had a significantly higher probability of viral clearance.

[0054] Table 4 Sample data of HBV clearance in patients without UGT1A1 gene mutation and patients with mutation

[0055]

[0056] Example 4: The more UGT1A1 mutations a patient accumulates or the rarer mutations a patient carries, the better the outcome of HBV infection.

[0057] It should be noted that the embodiments of the present invention have better practicability and do not impose any form of limitation on the present invention. Any technician familiar with the field may use the technical content disclosed above to change or modify it into an equivalent effective embodiment. However, any modification or equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A diagnostic kit for UGT1A1 gene mutation, characterized in that: The primers for amplifying the following genes are included: the promoter TATA box, exons and the intron region 50-150 bp proximal to the upstream and downstream sides of the exons of the UGT1A1 gene. The primer sequences are shown in the following table:

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