Ctnnb1 mutation detection kit, method, and use in hcc detection and management

Abstract

Provided herein are a kit and method for detecting mutations in CTNNB1, and their use in detection and management of hepatocellular carcinoma (HCC). The kit comprises a first pair of primers, configured to specifically bind sequences flanking the genomic region for amplifying the genomic region in a first PCR reaction; and at least one clamp, each configured to bind to one first allele but not any second allele at an annealing temperature in the first PCR reaction to thereby selectively suppress amplification of the one first allele but still allow amplification of other second allele(s). A method for detecting or monitoring recurrence of HCC is further disclosed, which comprises determining levels of five DNA markers, including CTNNB1 mutations, hTERT mutations, TP53 mutations, RASSF1A methylation, and GSTP1 methylation.

1 . A kit for characterizing, in a biological sample containing at least one first allele of CTNNB1, at least one second allele in a genomic region of CTNNB1, the kit comprising:
 a first pair of primers, configured to specifically bind sequences flanking the genomic region to thereby allow amplification of at least one polynucleotide harboring the genomic region in a first PCR reaction; and   at least one clamp, each configured to bind to one of the at least one first allele but not any of the at least one second allele at an annealing temperature in the first PCR reaction to thereby selectively suppress amplification of the one of the at least one first allele but still allow amplification of the at least one second allele;   wherein:
 at least one of the first pair of primers comprises an oligonucleotide of an artificial sequence at a 5′-end thereof, configured to interrupt a secondary structure of DNA molecules of CTNNB1 or to increase a Tm of the at least one of the first pair of primers in the first PCR reaction using amplified products as templates to thereby increase an efficiency of the amplification of the at least one polynucleotide in the first PCR reaction. 
   
     
     
         2 . The kit of  claim 1 , wherein:
 the genomic region is hotspot region 1 encoding codons 32-37 of CTNNB1;   the at least one first allele comprises a wildtype allele of CTNNB1 in hotspot region 1; and   the at least one second allele comprises one or more mutant alleles of CTNNB1 in hotspot region 1.   
     
     
         3 . The kit of  claim 2 , wherein the first pair of primers respectively have a nucleotide sequence as set forth in SEQ ID NO: 1 and SEQ ID NO: 2. 
     
     
         4 . The kit of  claim 1 , wherein the at least one clamp comprises at least one of a bridged nucleic acid (BNA) clamp or a locked nucleic acid (LNA) clamp, configured to specifically target the wildtype allele of CTNNB1. 
     
     
         5 . The kit of  claim 4 , wherein the BNA clamp has a nucleotide sequence as set forth in SEQ ID NO: 3. 
     
     
         6 . The kit of  claim 2 , further comprising a second pair of primers and at least one probe, configured to allow characterization of one or more of the at least one second allele in a second PCR reaction over the at least one polynucleotide. 
     
     
         7 . The kit of  claim 6 , wherein the at least one probe comprises a hydrolysis probe having a nucleotide sequence as set forth in SEQ ID NO: 4, configured to allow detection or quantification of the one or more mutant alleles of CTNNB1 in a second PCR reaction over the at least one polynucleotide obtained from the first PCR reaction. 
     
     
         8 . The kit of  claim 7 , wherein the second pair of primers respectively have a nucleotide sequence comprising SEQ ID NO: 1 and SEQ ID NO: 2. 
     
     
         9 . The kit of  claim 8 , wherein the first pair of primers respectively have a nucleotide sequence as set forth in SEQ ID NO: 1 and SEQ ID NO: 2, and the at least one clamp comprises a bridged nucleic acid (BNA) clamp having a nucleotide sequence as set forth in SEQ ID NO: 3. 
     
     
         10 . A method for characterizing, in a biological sample containing at least one first allele of CTNNB1, at least one second allele in a genomic region of CTNNB1, by means of the kit according to  claim 1 , the method comprising a step of:
 (a) performing the first PCR reaction over a mixture of the biological sample, the first pair of primers, and the at least one clamp to thereby obtain a first PCR product, comprising a sub-step of:
 annealing the mixture at an annealing temperature for a time period such that each of the at least one clamp binds to one of the at least one first allele but not any of the at least one second allele to thereby selectively suppress amplification of the one of the at least one first allele but still allow amplification of the at least one second allele. 
   
     
     
         11 . The method according to  claim 10 , further comprising a step of:
 (b) performing a second PCR reaction over the first PCR product by means of a second pair of primers and at least one probe to allow characterization of one or more of the at least one second allele.   
     
     
         12 . The method according to  claim 11 , wherein the second PCR reaction in step (b) is configured to quantify one or more of the at least one second allele, wherein:
 a first cycle number for the first PCR reaction and a second cycle number for the second PCR reaction are configured to avoid non-specific amplification and to ensure amplification is within a range of linearity.   
     
     
         13 . The method according to  claim 10 , wherein:
 the genomic region is hotspot region 1 encoding codons 32-37 of CTNNB1;   the at least one first allele comprises a wildtype allele of CTNNB1 in hotspot region 1;   the at least one second allele comprises one or more mutant alleles of CTNNB1 in hotspot region 1;   the first pair of primers respectively have a nucleotide sequences as set forth in SEQ ID NO: 1 and SEQ ID NO: 2; and   the at least one clamp comprises a bridged nucleic acid (BNA) clamp having a nucleotide sequence as set forth in SEQ ID NO: 3.   
     
     
         14 . The method according to  claim 13 , wherein in the sub-step of annealing the mixture at an annealing temperature for a time period, the annealing temperature has a range of 52-56° C. 
     
     
         15 . The method according to  claim 13 , further comprising a step of:
 (b) performing a second PCR reaction over the first PCR product by means of a second pair of primers and a probe, respectively having nucleotide sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 4.   
     
     
         16 . A method of detecting or monitoring a recurrence of hepatocellular carcinoma (HCC), in a DNA sample obtained from a biological sample of a subject in need thereof, comprising the step of:
 (i) determining a level of mutation or methylation of one or more genes from a group consisting of TP53, CTNNB1, hTERT, RASSF1A, and GSTP1 in the biological sample; and   (ii) detecting a presence or an absence of HCC based on the level of mutation or methylation of the one or more genes.   
     
     
         17 . The method of  claim 16 , wherein step (i) comprises:
 determining a level of mutation of CTNNB1 in the biological sample, comprising the sub-steps of:
 performing a first PCR reaction over a mixture of the biological sample, a first pair of primers, and a clamp to thereby obtain a first PCR product, wherein the first pair of primers and the clamp respectively have a nucleotide sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and 
 performing a second PCR reaction over the first PCR product by means of a second pair of primers and a probe to determine the level of mutation of CTNNB1, wherein the second pair of primers and the probe respectively have a nucleotide sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 4. 
   
     
     
         18 . The method of  claim 17 , wherein the biological sample is a urine sample, and step (ii) comprises:
 detecting a presence of HCC, if the level of mutation of CTNNB1 in the urine sample is more than or equal to 10 copies of mutated CTNNB1 per 1,000 copies of CTNNB1 gene.   
     
     
         19 . The method of  claim 16 , wherein:
 step (i) comprises: determining a level of mutation or methylation of each of TP53, CTNNB1, hTERT, RASSF1A, and GSTP1 in the biological sample; and   step (ii) comprises: detecting a presence or an absence of HCC based on the level of mutation or methylation of each of TP53, CTNNB1, hTERT, RASSF1A, and GSTP1 in the biological sample.   
     
     
         20 . The method of  claim 19 , wherein:
 step (i) further comprises: determining a level of alpha-fetoprotein (AFP) in the biological sample; and   step (ii) comprises: detecting a presence or an absence of HCC based on the level of mutation or methylation of each of TP53, CTNNB1, hTERT, RASSF1A, and GSTP1, and the level of AFP, in the biological sample.