High resolution melting analysis for gene scanning of adenomatous polyposis coli (APC) gene with oral squamous cell carcinoma samples

AUTHOR QUERY FORM

LIPPINCOTT

WILLIAMS AND WILKINS

JOURNAL NAME: PAI

ARTICLE NO: aimm_141590

QUERIES AND / OR REMARKS

CE: Sandhya

ED: Ma njunath

sr

R

A

High-resolution Melting Analysis for Gene Scanning of

Adenomatous Polyposis Coli (APC) Gene With Oral

Squamous Cell Carcinoma Samples

Ya-Sian Chang, PhD,*

w

Chien-Yu Lin, MS,

w

Shu-Fen Yang, BS,

w

Cheng Mao Ho, MD, PhD,

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and Jan-Gowth Chang, MD*

wz

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Background: There have been many different mutations reported

17 for the large adenomatous polyposis coli (APC) tumor suppressor gene. APC mutations result in inactivation of APC tumor

sup-19 pressor action, allowing the progression of tumorigenesis. The present study utilized a highly efficient method to identify APC

21 mutations and investigated the association between the APC genetic variants Y486Y, A545A, T1493T, and D1822V and

23 susceptibility to oral squamous cell carcinoma (OSCC). Methods: High-resolution melting (HRM) analysis was used to

25 _{characterize APC mutations. Genomic DNA was extracted}

from 83 patient specimens of OSCC and 50 blood samples from

27 _{healthy control subjects. The 14 exons and mutation cluster}

region of exon 15 were screened by HRM analysis. All

muta-29 _{tions were confirmed by direct DNA sequencing.}

31 Results: Three mutations and 4 single nucleotide polymo rphisms

(SNPs) were found in this study. The mutations were c.57 3T > C

33 (Y191Y) in exon 5, c.1005A > G (L335L) in exon 9, and

c.1488A > T (T496T) in exon 11. Two SNPs, c.4479 G > A

O

QUERY NO. Details Required Author’s Response

Q1 A running head short title was not supplied; please check if this one

is suitable and, if not, please supply a short title of up to 50

Q2 As per style gene names should be in italics. Please italiciz e (eg,

35 (T1493T) and c.5465A > T (D1822V), were located in exon 15, whereas c.1458T > C (Y486Y) and c.1635G > A (A545A) were

37 located in exon 11 and 13, respectively. There was no observe d

association between OSCC risk and genotype for any of the 4 APC

39 SNPs.

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Received for publication August 27, 2014; accepted October 8, 2014.

45 From the *Epigenome Research Center; wDepartment of Laboratory Medicine, China Medical University Hospital; and zSchool of

Conclusions: The mutation of APC is rare in Taiwanese patients with OSCC. HRM analysis is a reliable, accurate, and fast screening method for APC mutations.

Key Words: HRM, APC, OSCC, direct DNA sequencing (Appl Immunohistochem Mol Morphol 2014;00:000–000)

ral squamous cell carcinoma (OSCC) is one of the most frequently diagnosed malignancies worldwide, ranking sixth among all human cancers.1 _{Oral cancer is} also the fourth most common malignancy of Taiwanese males. Although diagnostic and therapeutic (surgery, radiation, and chemotherapy) technologies are well es-tablished for OSCC, patient survival has not significantly improved in decades.2,3 _{An understanding of the} molec-ular profile of OSCC is required for the development of molecular-targeted therapies and personalized medicine. In this regard, mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are of particular interest.

As with most cancers, the development of OSCC is the result of accumulated mutations in oncogenes and tu-mor suppressor genes, particularly those controlling cell proliferation, growth, differentiation, survival, apoptosis, and DNA repair.4,5 _{Next-generation sequencing has been} used to understand the molecular pathogenesis of OSCC.6,7 The molecular pathogenesis of OSCC includes at least 4 key functional pathways: cellular proliferation, squamous epithelial differentiation, cell survival, and invasion/meta-stasis.8 _{However, these pathways are influenced by many} genes. The cellular proliferation pathway includes p53, RB,

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Medicine, China Medical University, Taichung, Taiwan.

Supported by the Ministry of Science and Technology, Taiwan, R.O.C. (MOST 103-2314-B-039-001), China Medical University Hospital (DMR-103-091), Taiwan Ministry of Health and Welfare Cancer Research Center for Excellence (MOHW103-TD-B-111-03), and Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW103-TDU-B-212-113002).

The authors declare no conflict of interest.

Reprints: Jan-Gowth Chang, MD, Epigenome Research Center, China Medical University Hospital, 2 Yuh-Der Road, Taichung 404, Taiwan (e-mail: [email protected]).

Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Website, www. appliedimmunohist.com.

Copyright r 2014 by Lippincott Williams & Wilkins

CDKN2A, and CCND1. Squamous epithelial differ-entiation includes genes from the Notch/p63 axis. Cell survival includes EGFR, RAS, PIK3CA, PTEN, and CASP8. Invasion/metastasis includes TGF-b, SMAD, and FAT1.

APC is an important tumor suppressor located at chromosome 5q21-22. APC encodes a large multidomain protein with multiple cellular functions and interactions, including roles in the wingless/wnt signaling pathway, mediation of intercellular adhesion, and regulation of the cell cycle and apoptosis.9 _{The wingless/wnt family} con-tains glycogen synthase kinase 3b (GSK-3b), b-catenins and g-catenins, APC, axin, GSK-3b binding protein

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(GBP), and members of the T-cell factor/lymphoid with a LightCycler 480 Real-Time PCR System (Roche

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enhancer–binding factor-1 (TCF/LEF-1) family of tran-scription factors.10 _{In the presence of the wingless/wnt} signal, b-catenin is unable to bind APC, preventing the GSK-3b-dependent phosphorylation of b-catenin. Stabi-lized b-catenin can then translocate into the nucleus where it binds LEF-1/TCF to promote the transcription of wnt target genes such as cyclin D1, c-myc, and c-jun. In the absence of the wingless/wnt signal, phosphorylated b-catenin is degraded by the proteasome, resulting in a reduction in the b-catenin level. APC mutations may cause constitutive stimulation of the wingless/wnt sig-naling pathway, resulting in b-catenin accumulation. Therefore, the tumor suppressor APC is a negative reg-ulator of the wingless/wnt signaling pathway.

Most cases of familial adenomatous polyposis syn-drome result from germline mutations in the APC gene.11 APC mutations have also been described in patients with colorectal,12 _pancreatic,13 _{and lung cancers.}14 _{To date,} >1000 different germline and somatic mutations have been documented for APC.15 _{APC mutations appear to} concentrate between codons 1286 and 1513, which is named the mutation cluster region (MCR). However, a mutational analysis of the MCR and its vicinity in OSCC cases yielded a low frequency (0.0% to 12.5%) of the total APC mutations reported.16,17 _{The APC gene consists of} 15 exons panning 8538 bp, with exon 15 (6572 bp) ac-counting for >75% of the coding sequence of APC. We used high-resolution melting (HRM) analysis to screen exons 1 to 14 and part of exon 15, including the MCR region. The objective of this study was to assess the mu-tational spectrum of the APC gene in a Taiwanese pop-ulation with OSCC.

MATERIALS AND METHODS Sample Preparation and DNA Isolation

This study was approved by the Institutional Review Board of China Medical University Hospital (Taichung, Taiwan). Eighty-three patients diagnosed recently with OSCC were selected for participation in this study. Imme-diately after resection, tissue specimens were stored in liquid nitrogen before DNA extraction. Fifty peripheral blood samples were obtained from healthy control subjects. DNA extraction was performed as described previously.18 _The quality of the isolated genomic DNA was assessed using agarose gel electrophoresis, and the concentration was d

e-termined using a Nano-Drop-1000 instrument (Nano-Drop Technologies Inc., Wilmington, DE). The samples were then stored at À 201C until required for analysis.

HRM Analysis

We designed the primer sets on the APC DNA and mRNA sequences (NCBI reference sequences: NG_008481 and NM_000038). HRM analysis was per-formed to identify mutations in APC using 20 pairs of primers designed with Primer3 software (Supplementary Table 1, Supplemental Digital Content, http://links. lww.com/AIMM/A62). HRM analysis was performed

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Diagnostics, Penzberg, Germany) and LightCycler 480 High Resolution Melting Master Kit (Roche Diagnostics) according to the manufacturer’s protocols. Thermal cy-cling consisted of an initial denaturation step (951C for 10 min) followed by a 45-cycle program (denaturation at 951C for 15 s, annealing at 601C for 15 s, and elongation at 721C for 15 s, with reading of the fluorescence; ac-quisition mode: single). The melting program included 3 steps: denaturation at 951C for 1 minute, annealing at 401C for 1 minute, and subsequent melting, which consisted of a continuous fluorescence reading from 60 to 901C at a rate of 25 acquisitions/1C.

Gene Scanning

The melting curve analysis was subsequently per-formed using LightCycler 480 Gene Scanning Software, version 1.5 (Roche Diagnostics). The gene scanning soft-ware utilized 3 steps. The first step was normalization of the melting curves by setting the initial and final fluorescence signals of all samples to uniform values. In the next step, the normalized curves were shifted along the temperature ax is

to equalize the point at which the dsDNA in each sample was completely denatured. The final step involved sub-tracting the shifted normalized curves from a referenc e

curve to display the difference in shape of the melting curve. In the resulting difference plots, the samples were clustered into groups of similar melting curve shape.

Direct DNA Sequencing

After HRM analysis, the samples were purified us-ing the PCR-M Clean-Up System (Viogen, Sunnyvale, CA) before direct sequencing with the same primers used for PCR. These PCR products were then subjected to direct sequencing using the same primers. The sequence reaction was performed in a final volume of 10 mL, in-cluding 1 mL of purified PCR product, 2.5 mmol/L of PCR primer, 2 mL of ABI PRISM Terminator Cycle Sequenc-ing Kit v3.1 (Applied Biosystems, Foster City, CA), and 2 mL of 5 Â sequence buffer. The sequencing program consisted of a denaturation step (961C for 1 min) followed by a 25-cycle PCR program (denaturation at 961C for 10 s, annealing at 501C for 5 s, and elongation at 601C for 4 min). Direct DNA sequencing was performed using an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) according to the manufacturer’s protocol.

Statistical Analysis

Data analysis was performed using SPSS version 17.0 software (SPSS Inc., Chicago, IL). The w2 _{test was} used to analyze APC genotype distributions between the case and control groups. P < 0.05 was considered to indicate a statistically significant difference.

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RESULTS _{(Fig. 3A). Homozygotes for 1458T and 1458C could not be}

3 Screening for APC Mutations in Exons 5, 9, 11, and 13

Specific primer pairs were designed for each exon region to identify mutations in exons 5, 9, 11, and 13, as shown in Supplementary Table 1 (Supplemental Digital Content, http://links.lww.com/AIMM/A62). Because of size (379 nucleotides), it was difficult to screen exon 9 using only 1 primer pair; thus, 2 sets of primers were designed for exon 9.

HRM analysis of APC exon 5 revealed a hetero-zygous mutation at position 573 (573 T > C; rs185154886) that could be identified by a high upcurved plot that differed from that of wild type (Fig. 1A). The results of the HRM analysis were confirmed by direct sequencing (Figs. 1B, C). This mutation was not predicted to result in an amino acid substitution (Y191Y).

Similarly, HRM analysis of APC exon 9 showed a heterozygous mutation at position 1005 (1005A > G; rs3797704) that could be identified by a high upcurved plot that differed from that of wild type (Fig. 2A). The results of the HRM analysis were confirmed by direct sequencing (Figs. 2B, C). This mutation was also silent (L335L).

HRM analysis of APC exon 11 revealed 2 genotypic variations: a thymine to cytosine substitution at position 1458 (1458T > C; rs2229992), and an adenine to thymin e

substitution at position 1488 (1488A > T; rs9282599) 31

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distinguished by traditional HRM methods. However, these 2 homozygous genotypes could be clearly distinguished by generating artificial heterozygotes using a 1:1 mixture of the tested DNA and homozygous CC DNA (Fig. 3B). The results of the HRM analysis were confirmed by direct sequencing (Figs. 3C–E). The exon 11 polymorphism and mutation were not predicted to result in amino acid sub-stitutions (Y486Y and T496T, respectively).

HRM analysis of APC exon 13 showed a hetero-zygous polymorphism at position 1635 (1635G > A; rs351771) that could be identified by a high upcurved plot that differed from a homozygous genotype at this position (Fig. 4A). Homozygous 1635G and 1635A could not be distinguished by traditional HRM methods. However, these 2 homozygous genotypes could be clearly dis-tinguished by generating artificial heterozygotes using a 1:1 mixture of the tested DNA and homozygous AA DNA (Fig. 4B). The results of the HRM analysis were confirmed by direct sequencing (Figs. 4C, D). This poly-morphism was predicted to be silent (A545A).

Screening for APC Mutations in Exon 15

Three sets of primers (1281-1352, 1352-1441, and 1442-1522) were used to investigate the MCR region of APC. On the basis of the UMD-APC mutations database, the most common germline mutations occur at codons 1061 and 1309 of exon 15. One primer set (1034-1086) was designed to detect codon 1061 and another set (1802-1846) was designed to detect codon 1822.

HRM analysis of codons 1442-1522 revealed a het-erozygous polymorphism at position 4479 (4479G > A;

61 63 65 67 69 71 73 75 77 79 81 83 85 87 89 91 93 35 95 37 97 39 99 41 101 43 103 45 105 47 107 49 109 51 111 53 113 55 _{FIGURE 1. HRM assays for APC exon 5. A, The normalized and}

temperature-shifted difference plot showing 2 different

melt-57 ing profiles; the wild-type (WT) sample is blue, and mutations are other colors. Sequencing results confirm the (B) WT and

59 the presence of the APC exon 3 mutation: (C) c.573T > C.

FIGURE 2. HRM assays for APC exon 9. A, The normalized and temperature-shifted difference plot showing 2 different melting profiles; the wild-type (WT) sample is blue, and mutations are other colors. Sequencing results confirm the (B) WT and the presence of the APC exon 9 mutation: (C) c.1005A > G.

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FIGURE 3. HRM assays for APC exon 11. A, The normalized and temperature-shifted difference plot showing that HRM can distinguish the heterozygous genotype (T/C) from homozygous genotypes (T/T or C/C) at position 1458. Concomitant position 1458 and 1488 mutations were observed in 1 patient. B, Homozygous genotypes (T/T or C/C) were discriminated by generating heterozygotes with 1:1 mixture. Sequencing results confirm the (C) TT genotype, (D) TC genotype, (E) CC genotype, and (F) 2 distinct mutations at the position 1458 and 1488.

93 95 97 99 41 rs41115) that could be identified by a high upcurved plot tested DNA and homozygous AA DNA (Fig. 6B). The

that differed from a homozygous genotype in this position results of the HRM analysis were confirmed by direct se-101 43 (Fig. 5A). Homozygous 4479G and 4479A could not be quencing (Figs. 6C, D). This polymorphism was predicted

distinguished by traditional HRM methods. However, to result in an amino acid substitution (D1822V) in APC. 103 45 these 2 homozygous genotypes were clearly differentiated HRM analysis of codons 1034-1086, 1281-1352, and

by generating artificial heterozygotes using a 1:1 mixtur

e 1352-1441 revealed no nucleotide changes in these regions 105

47 of the tested DNA and homozygous AA DNA (Fig. 5B). (Supplementary Data, Supplemental Digital Content,

The results of the HRM analysis were confirmed by direct http://links.lww.com/AIMM/A62). 107 49

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sequencing (Figs. 5C, D). The 4479G > A polymorphism was not predicted to result in an amino acid substitution (T1493T).

Screening for APC Mutations in Exons 1, 2, 3, 4, 109 6, 7, 8, 10, 12, and 14

HRM analysis of codons 1802-1846 revealed a het- The results of HRM for exons 1, 2, 3, 4, 6, 7, 8, 10, 111 53 erozygous polymorphism at position 5465 (5465A > T; 12, and 14 revealed no nucleotide changes in these regions

rs459552) that could be identified by a high upcurved plot (Supplementary Data, Supplemental Digital Content, 113 55 that differed from homozygous genotypes in this position http://links.lww.com/AIMM/A62).

57 distinguished by traditional HRM methods. However, these detected in the APC gene in OSCC: 3 silent mutations and 2 homozygous genotypes were clearly differentiated b

y 4 single nucleotide polymorphisms (SNPs). The minor 117

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1 61 3 63 5 65 7 67 9 69 11 71 13 73 15 75 17 77 19 79 21 81 23 83 25 85 27 FIGURE 4. HRM assays for APC exon 13. A, The normalized and temperature-shifted difference plot showing that HRM can

distinguish the heterozygous genotype (G/A) from homozygous genotypes (G/G or A/A). B, Homozygous genotypes (G/G or

29 A/A) were discriminated by generating heterozygotes with 1:1 mixture. Sequencing results confirm the (C) GG genotype, (D) GA genotype, and (E) AA genotype.

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87 89 D1822V SNPs in Taiwanese patients with OSCC were

33 28.92%, 16.27%, 16.87%, and 10.84%, respectively. 35 APC Polymorphisms in Normal Individuals as

Shown by HRM Analysis

We recruited 50 healthy Taiwanese subjects from China Medical University Hospital. Four SNPs were iden-39 tified by HRM analysis and confirmed by direct DNA

sequencing: c.1458T > C, c.1635G > A, c.4479G > A, and 41 c.5465A > T. The minor allele frequencies of these

nucleo-tide changes were 33%, 15%, 15%, and 6%, respectively .

43 The frequencies of these 4 SNPs in the general population were similar to those of Taiwanese patients with OSCC. 45

47

Susceptibility to OSCC

We examined the association between each of the APC SNPs and OSCC risk (Supplementary Table 2, 51 Supplemental Digital Content, http://links.lww.com/

AIMM/A62). No association was observed between OSCC

53 risk and any of the 4 APC SNPs at each individual lo cus. Using the P CR si ngle-stran d con form ation poly morp hism 37

Association Between APC Y486Y, A545A, T1493T, and D1822V Polymorphisms and 49

(PCR-SSCP) method and direct sequencing, Uzawa et al19 reported that the prevalence of APC mutations was 12.5% in 24 OSCC patients. Sakata20 _{used the same technique to} analyze mutations at codons 1260-1673 of the APC gene in 23 OSCCs and no mutations were found. Kok et al16 used PCR-SSCP and direct sequencing to identify muta-tions at codons 279-1673 of the APC gene in 40 OSCC samples and found 6 mutations in 5 samples (12.5%). Tsuchiya et al17 _{found no mutations in and around the} APC MCR region in 23 OSCCs. Iwai et al21 _{found no} mutations in APC in 20 OSCCs. However, silent APC mutations were found in 3 of the 4 cell lines.21 _{We found 3} APC gene mutations in 4 of 83 OSCCs, but all were silent. Our study is the most comprehensive study in OSCC in comparison with the known studies.16,17,19–21

In this study, we analyzed the latest upper digestive tract tumors (squamous cell carcinoma) data in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. The results showed the 3 cases of APC muta-tion in the 333 cases of upper digestive tract tumors (squamous cell carcinoma), including OSCC. These

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55 DISCUSSION sults suggest that mutations of the APC gene may notplay an important role in OSCC development. 113 APC gene mutations have been described in

color-57 ectal tumors and other tumors, including OSCC. How-ever, the mutation of APC in OSCC has only been 59 reported in 4 studies from Japan and 1 from Taiwan.

Several methods, including the protein truncation t est

(PTT),22–24 _{DNA sequencing,}22–24 _PCR-SSCP,22 _quantitative multiplex PCR of short fluorescent fragments (QMPSF),25 conformation-sensitive gel electrophoresis (CSGE),24 _multip lex

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FIGURE 5. HRM assa ys for APC codons 14 42 to 152 2. A, The n ormalized and temp erature-shifted di fference p lot showin g that

HRM can distinguish the heterozygous genotype (G/A) from homozygous genotypes (G/G or A/A). B, Homozygous genotypes (G/G or A/A) were discriminated by generating heterozygotes with 1:1 mixture. Sequencing results confirm the (C) GG genotype, (D) GA genotype, and (E) AA genotype.

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ligation-dependent probe amplification (MLPA),24 _real-time quantitative multiplex (RQM)-PCR,26 _{and HRM,}27 _{have been} developed to detect APC mutations and deletions. The QMPSF, MLPA, and RQM-PCR methods are able to detect deletions in APC. MLPA is a widely used method; however, it required a large number of probes. QMPSF is a simple sem-iquantitative method, but it requires 3 PCRs. RQM-PCR has been successfully developed to detect whole-gene APC

dele-tions, but the detection device and consumables are expensi ve,

and setting up for the assay is labor intensive. The PTT, DNA sequencing, PCR-SSCP, CSGE, and HRM methods can be used to detect mutations in APC. PTT requires the use of radioactive labels for protein detection and DNA sequencing is then performed on samples with positive PTT results. PCR-SSCP is simple to perform but is time consuming and has less sensitivity for the detection of genetic variations. CSGE h as

high efficiency and relatively high sensitivity, but it is tim e

consuming. Thus, all of the available methods have adva

n-tages and disadvann-tages.

The HRM method is rapidly becoming the most im-portant mutation scanning methodology. It is a closed-tube

method, indicating that PCR amplification and subsequent analysis are sequentially performed in 1 well. This makes HRM more convenient than other scanning methodologies .

However, although the HRM method is a powerful screening tool it also has some limitations. For example, the presence o f

unexpected polymorphisms in the mutation of interest m ay

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interfere with genotyping. To mitigate this risk, we desig ned

amplicon lengths of 100 to 300 bp. As the amplicon lengt h

increased, the wild-type and heterozygote curves beca me

smaller and more difficult to distinguish. Obviously, the HRM method is also not able to detect mutations encompassing a n

entire exon or deletions of entire genes and exons.28 Four SNPs (rs2229992, rs351771, rs41115, and rs459552) were detected using the HRM primers designed in this study. The 4 APC SNPs (Y486Y, A545A, T1493T, and D1822V) had a minor allele frequency of 28.92%, 16.27%, 16.87%, and 10.84%, respectively. This is the first inves-tigation of the association between SNPs in APC and OSCC. Wong et al29 _{investigated the association between} APC SNPs and colorectal adenoma risk in non-Hispanic whites. In this study, the minor allele frequencies of 8 APC SNPs (rs2304793, rs2229992, rs548710, rs2909786, rs4111 5,

rs42427, rs459552, and rs2229995) ranged from 2.2% to 48.4% among the cases and controls. Of the 8 APC SNPs, rs2229992, rs41115, and rs459552 had minor allele fr

e-quencies of 40.2%, 37.1%, and 22.4%, respectively.29 In-triguingly, the minor allele frequencies in our population were lower than those of the population studied by Wong et al.29 _{Feng et al}30 _{reported that the T allele of the} APC D1822V polymorphism may be a protective factor for colorectal cancer. However, we acknowledged that our study population was small and larger studies were required to validate our findings.

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27 FIGURE 6. HRM assays for APC codons 1802 to 1846. A, The normalized and temperature-shifted difference plot showing that HRM can distinguish the heterozygous genotype (T/A) from homozygous genotypes (A/A or T/T). B, Homozygous genotypes (A/A or T/T) were discriminated by generating heterozygotes with 1:1 mixture. Sequencing results confirm the (C) AA genotype,

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In conclusion, the method presented in this report provides a reliable, accurate, and fast way to identify APC mutations for clinically diagnosing human cancer.

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