1396–1400 Nucleic Acids Research, 1998, Vol. 26, No. 6
`
` 1998 Oxford University Press
`
`Denaturing high performance liquid chromatography
`(DHPLC) used in the detection of germline and
`somatic mutations
`Wanguo Liu, David I. Smith, Keri J. Rechtzigel, Stephen N. Thibodeau and C. David James*
`
`Department of Laboratory Medicine and Pathology, Division of Experimental Pathology, Mayo Clinic and Foundation,
`Rochester, MN 55905, USA
`
`Downloaded from
`
`http://nar.oxfordjournals.org/
`
` by guest on July 31, 2014
`
`this need, and these techniques have been reviewed on multiple
`occasions (1–4).
`A relatively new addition to DNA scanning methods uses
`denaturing high performance liquid chromatography (DHPLC;
`5–9). In its early stage of application to the analysis of nucleic
`acids, HPLC was shown to provide an effective means for
`separating oligonucleotides (10), PCR fragments (11) and for
`analyzing the products formed in competitive RT–PCR reactions
`to determine relative levels of gene expression (12).
`involves
`Mutation/polymorphism
`scanning by DHPLC
`subjecting PCR products to ion-pair reverse-phase liquid chroma-
`tography in a column containing alkylated non-porous particles.
`Under conditions of partial heat denaturation within a linear
`acetonitrile gradient, heteroduplexes that form in PCR samples
`having internal sequence variation display reduced column retention
`time relative to their homoduplex counterparts. In the majority of
`cases the elution profiles for such samples are distinct from those
`having homozygous sequence, making the identification of samples
`harboring polymorphisms or mutations a straightforward procedure.
`The major advantages of this method include the use of automated
`instrumentation, speed of analysis (~ 5 min per sample) and the size
`of the DNA fragment that can be analyzed (up to 1.5 kb).
`No previous report has addressed the accuracy of mutation/
`polymorphism detection by DHPLC analysis. One of the
`objectives of the investigation reported here was to determine the
`reliability of DHPLC for detecting inherited gene sequence
`variation. To accomplish this we used DHPLC to examine PCR
`fragments produced from several DNAs, having previously
`identified germline mutations or polymorphisms in the rearranged
`transforming proto-oncogene (RET) or the cystic fibrosis trans-
`membrane conductance regulator gene (CFTR). Our other major
`interest was to assess the usefulness of DHPLC for screening
`tumor DNAs for mutations of tumor suppressor genes (TSGs), a
`potentially powerful application of this technology that had not
`previously been examined. However, as the method requires
`heteroduplex DNA for detection of intra-sample sequence
`variation, it is reasonable to question whether mutations would
`escape detection in instances where loss of a wild-type TSG
`occurs in combination with mutation of the remaining allele since
`the predominant double-stranded DNA formed would be mutant
`homoduplex. To address this question, a large panel of malignant
`glioma DNAs were examined for phosphatase and tensin
`homologue deleted on chromosome ten gene (PTEN) mutations.
`
`Received December 17, 1997; Revised and Accepted January 27, 1998
`
`ABSTRACT
`
`Denaturing high performance liquid chromatography
`(DHPLC) has been described recently as a method for
`screening DNA samples for single nucleotide poly-
`morphisms and
`inherited mutations. Thirty-eight
`DNAs, 22 of which were heterozygous for previously
`characterized rearranged transforming gene (RET) or
`cystic fibrosis transmembrane conductance regulator
`gene (CFTR) mutations or polymorphisms, were
`examined using DHPLC analysis to assess the accuracy
`of this scanning method. Ninety-one per cent (20/22) of
`the PCR amplicons from specimens with heterozygous
`RET or CFTR sequence showed elution profiles distinct
`from corresponding homozygous normal patterns;
`whether the profiles for two amplicons containing
`heterozygous RET sequence were distinct from
`homozygous cases was equivocal. To investigate the
`usefulness of this method for detecting mutations in
`tumor DNAs, each of the phosphatase and tensin
`homologue deleted on chromosome ten gene (PTEN)
`exons were examined for mutations in 63 malignant
`gliomas. Seventeen PTEN PCR products from this
`series of brain tumors showed elution profiles
`indicating sample heterozygosity and in each instance
`conventional sequencing confirmed the presence of a
`mutation. PTEN amplicons containing exons 1, 3 and
`5 were sequenced for each of the 63 tumor DNAs to
`determine whether any mutations may have escaped
`DHPLC detection, and this analysis identified one such
`alteration in addition to the eight mutations that
`DHPLC had revealed. In total, DHPLC identified 37 of 40
`(92.5%) PCR products containing defined sequence
`variation and no alterations were indicated among 196
`amplicons containing homozygous normal sequence.
`
`INTRODUCTION
`
`It is of fundamental importance to both basic and clinical research
`to efficiently and accurately detect gene sequence variation
`within DNA samples. Several methods have been developed to
`scan DNAs for polymorphisms and mutations to accommodate
`
`*To whom correspondence should be addressed at: Mayo Clinic and Foundation, 200 First Street S.W., Hilton Building Room 820-D, Rochester, MN 55905, USA.
`Tel: +1 507 284 8989; Fax: +1 507 266 5193; Email: james.charles@mayo.edu
`
`GDX 1016
`
`

`

`1397
`
`Nucleic Acids Research, 1998, Vol. 26, No. 6
`Nucleic Acids Research, 1994, Vol. 22, No. 1
`
`1397
`
`Table 1. Comparison of mutation detection by DHPLC and by sequencing
`
`Downloaded from
`
`http://nar.oxfordjournals.org/
`
` by guest on July 31, 2014
`
`1Mobile phase temperature.
`2cDNA sequence location of alteration in parentheses.
`3HT, heterozygous; HM, homozygous.
`Italicized type indicates cases in which the results of DHPLC and conventional sequencing were discrepant. Altering
`the mobile phase temperature in these instances, however, resolved sample homoduplex and heteroduplex fractions
`(see Results and Discussion).
`
`The results of these analyses indicate that DHPLC offers a reliable
`approach for the detection of germline and somatic mutations.
`
`MATERIALS AND METHODS
`
`Amplicon synthesis
`
`DNAs from peripheral blood leukocytes and tumor tissue snap
`frozen by immersion in liquid nitrogen were isolated and purified
`as described (13). Samples used for mutation screening and
`sequencing were generated in 50 m
`l reaction volumes containing
`10–100 ng of genomic DNA, 20 pmol of forward and reverse
`primers for either PTEN exons 1–9 (14), CFTR exon 7 (15) or
`RET exon 10 (16), 200 m M dNTPs (Perkin-Elmer, Foster City,
`CA), 1.25 U of Taq polymerase (AmpliTaq Gold: Perkin-Elmer)
`and 1× buffer supplied by the manufacturer. PCR amplifications
`were for 35 cycles: 95_C for 30 s, 60_C for 30 s and 72_C for 1 min
`(final extension at 72_C for 10 min) following sample denaturation
`at 95_C for 9 min. Synthesis of appropriately sized PCR reaction
`products was confirmed by agarose gel electrophoresis.
`
`Denaturing HPLC analysis
`
`DHPLC analysis was carried out using automated instrumentation
`identical to that described by Underhill et al. (9). Four to seven m l
`of each PCR product, containing ~ 50–100 ng DNA, was
`denatured for 3 min at 95_C and then gradually reannealed by
`decreasing sample temperature from 95 to 65_C over a period of
`30 min. PCR products were then separated (flow rate of 0.9 ml/min)
`over a period of time and through a linear acetonitrile gradient, the
`values for which were determined by the size and G–C content of
`the amplicon (Table 1).
`The column mobile phase consisted of a mixture of 0.1 M
`triethylamine acetate (pH 7.0) with (buffer A) or without (buffer
`B) 25% acetonitrile. The mobile phase temperatures required for
`optimal resolution of homoduplex and heteroduplex DNAs were
`determined empirically by injecting one PCR product for each
`exon at increasing temperatures until a significant decrease in
`sample retention time was observed. Specific values for the
`gradient ranges (buffer A component indicated), separation times
`and mobile phase temperatures used to analyze the amplicons
`described above are as follows: 57.0–64.2%, 4 min and 58_C for
`
`GDX 1016
`
`

`

`Downloaded from
`
`http://nar.oxfordjournals.org/
`
` by guest on July 31, 2014
`
`To determine whether DHPLC detection of sample hetero-
`zygosity is influenced by nucleotide identity at a specific site of
`sequence variation, several patient DNAs with heterozygous
`mutations effecting RET cysteine codons 609, 611, 618 and 620
`were examined. For nucleotide substitutions at position 1852 of the
`coding sequence, split-peak elution profiles distinct from the profiles
`associated with normal homoduplex DNAs were evident for T→A
`and T→G alterations (Fig. 1B). However, a T→C substitution at this
`position failed to produce a profile with multiple peaks; this was also
`the case for an amplicon containing a G→A alteration at base 1859
`(Table 1). The peaks for these two cases, however, were noticeably
`wider than control peaks, and thereby suggested the presence of
`homoduplexes and heteroduplexes in the corresponding eluates. An
`alternative DHPLC protocol (mobile phase temperature of 59_C)
`resulted in a slight resolution of homoduplex and heteroduplex
`fractions in each sample (see inset for the T→C substitution at base
`1852, Fig. 1B). Nine additional heterozygous samples with
`mutations effecting the cysteine codons showed unique profiles
`using the initial separation protocol, including two associated with
`different nucleotide substitutions at position 1853 (Fig. 1B).
`Significantly, there were no false positives associated with the
`analysis of either CF or RET sequence alterations.
`Mutation screening by DHPLC has not been applied to the
`analysis of DNA samples extracted from neoplastic tissue, and to
`assess the potential of this technology for analyzing tumor
`specimens, we examined DNAs from 63 malignant gliomas for
`mutation of the PTEN gene. Elution profiles indicated sequence
`variation within 17 of the 567 PCR products examined, and in
`each of these cases the presence of a mutation was determined by
`conventional sequencing (elution profiles for DNAs with exon
`5 mutations are shown in Fig. 2A). As opposed to the RET and
`CFTR germline mutations which involved single nucleotide sub-
`stitutions in all instances, the PTEN mutations included deletions and
`insertions, as well as several nucleotide substitutions (Table 1). The
`overall incidence of DHPLC-detected PTEN mutations among
`this series of tumors (17 of 63; 27%) compares favorably with
`those reported previously (14,17) and, as was the case for the
`analysis of DNAs for germline CFTR or RET mutations, there
`were no false positives associated with the PTEN analysis.
`To determine whether some alterations had escaped DHPLC
`detection, PTEN PCR fragments for exons 1, 3 and 5 were
`conventionally sequenced for all 63 cases. This analysis revealed
`a single exon 3 mutation that DHPLC had failed to identify.
`Interestingly, the exon 3 PCR product had the lowest G–C
`composition (27%) of any of the amplicons examined in this
`study. Comparison of mobile phase temperatures (MPTs) against
`corresponding amplicon G–C contents in Table 1 suggests that the
`temperature used to analyze exon 3 amplicons may have been too
`high and prevented mutation detection by ‘melting open’ sample
`duplexes. Consequently, we compared our empirically derived
`MPTs (Materials and Methods) against MPTs recommended by a
`recently installed internet program (http://lotka.stanford.edu/
`dhplc/melt.html ). This analysis revealed a close correspondence
`between experimental and recommended MPTs for all amplicons
`other than exon 3, for which the internet program recommended
`a temperature of 53_C. DHPLC analysis at this temperature
`clearly revealed homoduplex and heteroduplex fractions in the
`tumor specimen containing the exon 3 mutation (data not shown).
`PTEN mutations in malignant gliomas are often accompanied by
`loss of the remaining wild-type allele (14,17,18). To assess whether
`this had occurred in any of the tumors examined here, microsatellite
`
`
`
`1398
`
`Nucleic Acids Research, 1998, Vol. 26, No. 6
`
`CFTR exon 7; 53.0–59.3%, 3.5 min and 61_C or 53.0–59.3%,
`3.5 min and 59_C for RET exon 10; 55.2–56.2%, 5 min and 59_C
`for PTEN exon 1; 52.0–57.4%, 3 min and 58_C for PTEN exon
`3 and 54.5–60.8%, 3.5 min and 57_C for PTEN exon 5. Between
`sample analyses the column was regenerated with a 19:1 mixture
`of buffers A and B (40 s) and a solution whose buffer A content
`was 5% less than the low end of the desired gradient range (40 s).
`
`Sequence analysis
`Solutions (10 m
`l) were prepared with 10–20 ng of product from
`previous PCR reactions, 0.05 U of Taq polymerase, 1× buffer,
`10% DMSO, 400 m M ddATP, 600 m M ddTTP, 60 m M ddGTP,
`200 m M ddCTP, 10 m M each of dATP, dTTP and dCTP, 20 m M
`7-deaza-dGTP (Boehringer Mannheim) and 0.05 m M 5′-32P-labeled
`sequencing primer. Sequencing reactions were carried out for
`30 cycles at 95_C for 20 s, 58_C for 30 s and 72_C for 1 min,
`using a 1 min ramp time between annealing and elongation
`phases. Following sample denaturation, reaction products were
`loaded onto a 6% sequencing gel. Electrophoresis was at 75 W
`and room temperature for 1–3 h, after which the gels were dried
`and exposed to Kodak XAR film.
`
`Microsatellite analysis
`
`PCR reactions for determination of tumor loss of heterozygosity
`contained ~ 10 ng of genomic DNA, 8–10 pM forward and reverse
`primers for either the D10S541 or D10S1765 locus (Research
`Genetics, Huntsville, AL), 0.8 m Ci [a -32P]dCTP and 0.2–0.35 U
`of Taq polymerase in 10–15 m
`l of 1× buffer containing 200 m M
`dGTP, dATP and dTTP, and 25–34 m M dCTP. Samples were
`placed in 96-well plates and amplified at 95_C denaturation (30 s),
`55_C annealing (30 s) and 72_C extension (1 min) for 43 cycles.
`At completion of PCR, an equal volume of denaturing buffer was
`added to each reaction. Samples were then heated to 95_C and
`quenched on ice. Two m
`l of each sample were applied to 4 or 6%
`acrylamide sequencing gels and electrophoresed for 1.5–3 h at 75 W.
`Gels were dried and exposed to X-ray film for 4–48 h.
`
`RESULTS AND DISCUSSION
`
`PCR fragments were synthesized from 22 peripheral blood
`leukocyte specimens heterozygous for previously identified exon
`10 RET or exon 7 CFTR mutations or polymorphisms (Table 1).
`Each PCR reaction product was subjected to DHPLC analysis and
`their corresponding elution profiles were compared with patterns
`associated with homozygous normal sequence controls, nine of
`which were included for the analysis of CFTR sequence
`alterations and seven for the analysis of RET alterations.
`The elution profiles for the control CFTR PCR products were
`all highly similar and showed a single peak of homoduplex DNA.
`In contrast, each of the nine PCR products with internal CFTR
`sequence variation produced a distinct profile with multiple peaks
`due to the reduced column retention time of heteroduplex DNA
`(examples shown in Fig. 1A). All samples with heterozygous
`CFTR sequence were identified using the same separation
`conditions (Materials and Methods). G–C content of the 60 bases
`surrounding each CFTR alteration varied between 35 and 54%,
`suggesting that the detection of sample heteroduplex within a
`specific amplicon is not greatly influenced by differences in the
`melting point of sequences flanking the site of base mismatch.
`
`GDX 1016
`
`

`

`1399
`
`Nucleic Acids Research, 1998, Vol. 26, No. 6
`Nucleic Acids Research, 1994, Vol. 22, No. 1
`
`1399
`
`Downloaded from
`
`http://nar.oxfordjournals.org/
`
` by guest on July 31, 2014
`
`Figure 1. DHPLC detection of CFTR and RET germline mutations. Elution profiles associated with the DHPLC analysis of PCR amplicons containing either CFTR
`exon 7 (A) or RET exon 10 (B). Retention times for homoduplex and heteroduplex fractions are indicated above the CFTR elution peaks. For the RET exon 10 results,
`only the portion of the profile containing the homoduplex and heteroduplex peaks are shown. cDNA sequence location of mutations identified previously in each
`sample are indicated. The inset profile shown for the T→C substitution at base 1852 was obtained using a mobile phase temperature of 59_C.
`
`analysis was performed on 14 samples with PTEN mutations for
`which there was corresponding normal DNA available. This
`analysis revealed loss of heterozygosity in 13 instances and,
`consequently, these results suggest that DHPLC can detect the
`formation of heteroduplex even when the ratio of normal:mutant
`DNA sequence in a tumor DNA is quite low. To formally test this
`hypothesis, we mixed varying amounts of DNA from normal tissue
`and cell line U251, homozygous for a dinucleotide insertion
`mutation in PTEN exon 7 (14), and analyzed resulting exon 7 PCR
`amplicons. Elution profiles from the DHPLC analysis of these
`samples indicate that substantial heteroduplex is formed even in
`instances where the tumor DNA is 4-fold more abundant than
`normal (Fig. 2B); similar results were obtained for the reverse
`situation where normal DNA represented the majority component of
`
`the mixture. Taken together, these results indicate that DHPLC
`requires between 10 and 20% of the minority DNA species for
`detecting heteroduplex DNA, and extend the use of this method to
`tumor mutational analysis. In addition, this experiment shows that
`DHPLC can be used to detect alterations in a homogenous mutant
`DNA (e.g. cell line) sample by adding an approximately equal
`amount of normal DNA to the clonal, mutant specimen.
`In summary, this survey of different exon sequences indicates that
`DHPLC offers a reliable and sensitive means for the detection of
`germline and somatic mutations. The few exceptions encountered
`may relate to the extreme G–C content of the associated
`amplicons (64 and 27% for RET exon 10 and PTEN exon 3,
`respectively). The differential sensitivity for detecting the
`transversion and transition mismatches at RET positions 1852 and
`
`GDX 1016
`
`

`

`Downloaded from
`
`http://nar.oxfordjournals.org/
`
` by guest on July 31, 2014
`
`ACKNOWLEDGEMENTS
`
`This work was supported by NCI grants CA-55728 (C.D.J.) and
`CA-48031 (D.I.S.).
`
`REFERENCES
`
`1 Grompe, M. (1993) Nature Genet., 5, 111–117.
`2 Forrest, S., Cotton, R., Landegren, U. and Southern, E. (1995)
`Nature Genet., 10, 375–376.
`3 Mashal, R.D. and Sklar, J. (1996) Curr. Opin. Genet. Dev., 6, 275–280.
`4 Cotton, R.G.H. (1997) Trends Genet., 13, 43–66.
`5 Oefner, P.J. and Underhill, P.A. (1995) Am. J. Hum. Genet., 57 (Suppl.),
`A266.
`6 Underhill, P.A., Jin, L., Zemans, R., Oefner, P.J. and Cavalli-Sforza, L.L.
`(1996) Proc. Natl. Acad. Sci. USA, 93, 196–200.
`7 Ophoff, R.A., Terwindt, G.M., Vergouwe, M.N., van Eijk, R., Oefner, P.J.,
`Hoffman, S.M., Lamerdin, J.E., Mohrenweiser, H.W., Bulman, D.E.,
`Ferrari, M., Haan, J., Lindhout, D., van Ommen, G.J., Hofker, M.H.,
`Ferrari, M.D. and Frants, R.R. (1996) Cell, 87, 543–552.
`8 Hayward-Lester, A., Chilton, B.S., Underhill, P.A., Oefner, P.J. and Doris, P.S.
`(1996) In Ferre, F. (ed.), Gene Quantification. Birkhauser Verlag, Basel
`Switzerland, pp. 44–77.
`9 Underhill, P.A., Jin, L., Lin, A.A., Mehdi, S.Q., Jenkins, T., Vollrath, D.,
`Davis, R.W., Cavalli-Sforza, L.L. and Oefner, P.J. (1997) Genome Res., 7,
`996–1005.
`10 Huber, C.G., Oefner, P.J. and Bonn, G.K. (1993) Anal. Biochem., 212,
`351–358.
`11 Huber, C.G., Oefner, P.J., Preuss, E. and Bonn, G.K. (1993)
`Nucleic Acids Res., 21, 1061–1066.
`12 Hayward-Lester, A., Oefner, P.J., Sabatini, S. and Doris, P.A. (1995)
`Genome Res., 5, 494–499.
`13 James, C.D., Carlbom, E., Dumanski, J., Hansen, M., Nordenskjold, M.,
`Collins, V.P. and Cavenee, W.K. (1988) Cancer Res., 48, 5546–5551.
`14 Steck, P.A., Pershouse, M.A., Jasser, S.A., Yung, W.K.A, Lin, H.,
`Lignon, A.H., Langford, L.A., Baumgard, M.L., Hattier, T., Davis, T.,
`Frye, C., Hu, R., Swedlund, B., Teng, D.H.F. and Tavtigian, S.V. (1997)
`Nature Genet., 15, 356–362.
`15 Zielenski, J., Rozmahel, R., Bozon, D., Kerem, B., Grzelczak, Z., Riordan,
`J.R., Rommens, J. and Tsui, L.C. (1991) Genomics, 10, 214–228.
`16 Tsai, M.S., Ledger, G.A., Khosla, S., Gharib, H. and Thibodeau, S.N.
`(1994) J. Clin. Endocrinol. Metab., 78, 1261–1264.
`17 Rasheed, B.K.A., Stenzel, T.T., Mclendon, R.E., Parsons, R., Friedman, A.H.,
`Friedman, H.S., Bigner, D.D. and Bigner, S.H. (1997) Cancer Res., 57,
`4187–4190.
`18 Li, J., Yen, C., Liaw, D., Podsypanina, K., Bose, S., Wang, S.I., Janusz, P.,
`Miliaresis, C., Rodgers, L., McCombie, R., Bigner, S.H., Giovanella, B.C.,
`Ittmann, M., Tycko, B., Hibshoosh, H., Wigler, M.H. and Parsons, R.
`(1997) Science, 275, 1943–1947.
`
`
`
`1400
`
`Nucleic Acids Research, 1998, Vol. 26, No. 6
`
`Figure 2. DHPLC detection of PTEN mutations in tumor DNAs. Portions of
`elution profiles are shown for homoduplex and heteroduplex peaks resulting
`from the analysis of normal or tumor PTEN exon 5 PCR products (A) and for
`PTEN exon 7 PCR products synthesized from a mixed sample containing
`homozygous normal DNA and DNA from a glioblastoma cell line with a PTEN
`exon 7 mutation (B). Locations of corresponding exon 5 mutations are shown
`with the profiles to the left (A) and corresponding proportions of cell
`line:normal DNA are indicated for the profiles shown to the right (B).
`
`1853 are surprising, but imply that DHPLC profiles may serve as a
`type of ‘fingerprint’ revealing the precise sequence alteration
`associated with sample heterogeneity. At a minimum, the accuracy
`of the method suggests a potential for increased use.
`
`GDX 1016
`
`