Denaturing high-performance liquid chromatography (DHPLC) is a highly sensitive, semi-automated method for identifying mutations in the TSC1 gene

Denaturing high-performance liquid chromatography (DHPLC) is a highly sensitive, semi-automated method for identifying mutations in the TSC1 gene

J. Biochem. Biophys. Methods 47 (2001) 33–37 / locate / jbbm Denaturing high-performance liquid chromatography (DHPLC) is a highly s...

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J. Biochem. Biophys. Methods 47 (2001) 33–37 / locate / jbbm

Denaturing high-performance liquid chromatography (DHPLC) is a highly sensitive, semi-automated method for identifying mutations in the TSC1 gene a b a, a P.S. Roberts , S. Jozwiak , D.J. Kwiatkowski *, S.L. Dabora a

Division of Hematology, Brigham and Women’ s Hospital, 221 Longwood Ave., LMRC 301, Boston, MA 02115, USA b Department of Child Neurology, Children’ s Memorial Hospital, Warsaw, Poland Received 3 June 2000; accepted 9 June 2000

Abstract Sensitive and automated methods for the detection of DNA sequence variation are required for a wide variety of genetic studies. Diagnostic testing in human genetic disorders is one application of such methods. Tuberous sclerosis complex (TSC) is an autosomal dominant familial tumor syndrome characterized by the development of benign tumors (hamartomas) in multiple organs (OMIM [ 19110, [191092). There is a high frequency of sporadic cases and significant demand from patients and families for genetic testing information. Two TSC genes have been identified (TSC1 and TSC2 ) and together account for all cases [1,2]. Here we report our methods for DHPLC analysis of the TSC1 gene and demonstrate the high sensitivity of this method in a blinded analysis of 21 TSC patients with known TSC1 mutations. In this series, DHPLC detected 27 / 28 (96%) known TSC1 sequence variations. The only sequence variation not identified by DHPLC in this study is a mosaic case.  2001 Elsevier Science B.V. All rights reserved. Keywords: DHPLC; Tuberous sclerosis; Heterozygote mutation detection

Denaturing high-performance liquid chromatography (DHPLC) is a relatively new method for detection of heterozygote DNA sequence variation [3–5]. It is based on the separation of heteroduplexed DNA amplicons using reversed-phase ion chromatography *Corresponding author. Tel.: 11-617-278-0384; fax: 11-617-734-2248. E-mail address: [email protected] (D.J. Kwiatkowski). 0165-022X / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0165-022X( 00 )00149-4

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[6–8]. We are using DHPLC to detect small mutations (point mutations, insertions and deletions) in the coding exons of the genes which cause TSC. We have previously reported on the utility of DHPLC for detecting mutations in the TSC2 gene [9]. We have now optimized DHPLC run conditions for all coding exons of TSC1 and report here a blinded analysis of a series of samples with known TSC1 mutations identified previously using gel-based methods including single-stranded conformation analysis (SSCP), heteroduplex analysis (HD) and denaturing gradient gel electrophoresis (DGGE) [10,11]. DNA was obtained from TSC patients who provided informed consent and met diagnostic criteria [12]. Methods were similar to those used previously [9]. Briefly, primers were designed using the GCG v8 Primer program or the WI primer 3 program (http: / / / cgi-bin / primer / primer3]www.cgi) and are available at http: / / / projects / tsc /. Amplified fragments were 185–449 base pairs in length. The melting profile of each fragment was determined using a program written by N. F. Hansen and P. Oefner (http: / / / melt.html). Fragments whose melting temperature varied by more than 48 were modified by the addition of GC clamps (4–10 bp) to flatten the melting profile to less than 58 variation. PCR was done using AmplitaqR gold polymerase (Perkin-Elmer) on MJ Research PTC-100 thermal cyclers. Heteroduplexed PCR products were analyzed using the DHPLC machine WAVEE DNA Fragment Analysis System (Transgenomic, San Jose, CA). The buffers used for DHPLC consist of: buffer A, 0.1 M triethylammonium acetate (TEAA); and buffer B, 0.1 M TEAA with 25% acetonitrile. We used the 7.8-min run profile listed below. Five to 10 ml of each amplicon were injected and a flow rate of 0.9 ml / min was used. Time (min)


0 0.1 4.1 4.2 4.7 4.8 7.8

Y-10 Y-6 Y 100 100 Y-10 Y-10

The parameter Y was calculated according to the formula Y 5 19.24 1 [(53.6X) / 78.5 1 X] where X5number of base pairs of the amplicon. Each amplicon was run using this profile and a column temperature of 558C. For some amplicons, Y was empirically adjusted up or down by 1–2% to cause DNA elution at about 5.5 min. The elution profile of each amplicon was examined at temperatures of 558C, and a range of 58 surrounding the temperature recommended by the program written by Hansen and Oefner (http: / / / melt.html). An optimal temperature was chosen as

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the highest temperature at which homoduplex DNA eluted as a narrow peak with a significant shift from the non-denaturing temperature of 558C. Column temperature and %B for exons 3–23 of TSC1 can be found at http: / / / projects / tsc /. DNA fragment elution profiles were captured on line and visually displayed using the manufacturer’s software. In exons where an expected shift was identified, resequencing was not performed. Sequencing was performed in five exons where a DHPLC shift was observed in a sample not previously known to contain a variation in that exon using Big Dye chemistry on the ABI 377. Gap4 of the Staden package was used for sequence analysis as described previously [13]. Twenty-one DNA samples from TSC patients previously found to have mutations in TSC1 by HD, SSCP, and 2D DGGE were studied using DHPLC in a blinded manner [10,11]. The DHPLC runs and elution profile analysis were done by one author (PR) who was not involved with previous mutation detection analyses on this set of patients. The 28 known sequence variants present in these DNA samples are shown in Table 1 and consisted of 12 small deletions, one insertion and 15 point mutations in 11 different Table 1 TSC1 sequence variations identified using DHPLC in blinded analysis Patient


Sequence variation


Mutation or polymorphism

1 13 14 2 9 15 3 9 15 15 6 16 8 17 17 18 5 7 19 20 21 4 9 10 11 12 3 12

Exon 7 Exon 8 Exon 9 Exon 9 Exon 10 Exon 10 Exon 10 Exon 14 Exon 14 Exon 14 Exon 15 Exon 15 Exon 15 Exon 15 Exon 15 Exon 15 Exon 15 Exon 15 Exon 15 Exon 15 Exon 15 Exon 15 Exon 17 Exon 18 Exon 18 Exon 20 Exon 22 Exon 23

868–869delTT 903C.T, R228X 1112T.G, Y297X 1122–113411 del14 1186C.T, silent 1186C.T, silent 1209–1210delCT 1556A.G, silent 1654insGA 1556A.G 1746C.T, R509X 1746C.T, R509X 1929–1930delAG 1890C.T, silent 1950, E587X 2044–2045delTT 2105delAAAG 2105delAAAG 2105delAAAG 2126delAG 2176delTG 2181C.T, Q554X 2394delAAAG 2448C.T, Q743X 2577C.T, R786X 2729delAAAC 3050C.T, silent 3543G.A, G1108S

del point point del point point del point ins point point point del point point del del del del del del point del point point del point point

M M M M P P M P M P M M M P M M M M M M M M M M Ma M P P


Patient 11, exon 15 mutation is a mosaic case that was not identified using DHPLC.

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Table 2 TSC1 single base substitution polymorphisms identified using DHPLC Patient

Intron / exon

Sequence variation


Mutation or polymorphism

19 12 5 15

Exon 21 Exon 15 Exon 22 Intron 14

2920A.G, Q900R 1981A.G, K587R 3050C.T, silent 1660–37C.T

point point point point


exons of TSC1. The DHPLC analysis identified abnormal tracings in 27 / 28 (96%) of these known sequence variants. The one sequence variant that was missed had been detected by SSCP only, and was identified after isolation and reamplification of the variant band [10]. This patient was a mosaic for that mutation as noted previously (same as patient 2 in Dabora et al. [11]). In addition to the 27 known sequence variants that were detected, an additional seven DHPLC shifts in DNA samples / amplicons not known to contain a sequence variation were identified. Four of these have been sequenced and identified as single base substitution polymorphisms which are listed in Table 2. Comprehensive mutation analysis for genetic testing in TSC presents a significant challenge. There are two genes with a large number of coding exons (21 in TSC1 and 41 in TSC2 ). The mutation spectrum is varied and mutations have been reported in all coding exons of TSC1 and all but three exons of TSC2 (http: / / / projects / tsc / ). Although some mutations have been seen recurrently, none of these accounts for more than 5% of cases. Comprehensive mutation analysis therefore requires a sensitive method of scanning all coding exons for unknown mutations. We have previously used several gel-based mutation detection methods including SSCP, HD, and DGGE for scanning TSC1 exons for mutations [10,11]. The blinded analysis of TSC1 sequence variations reported here confirms the high sensitivity of DHPLC. The only mutation that was missed in this study was a mosaic case in which we estimate the level of mosaicism at less than 25%. Mosaicism is well documented in TSC and is a known potential cause for failure of genetic diagnosis [14–17]. Although the initial expense of a DHPLC instrument is a barrier, we favor its use over gel-based mutation scanning methods because of the high sensitivity, reproducibility, and automated format.

1. Web sites http: / / / projects / tsc / http: / / / melt.html http: / / / cgi-bin / primer / primer3]www.cgi http: / / / omim /

Acknowledgements We wish to thank the TSC patients and families for contributing blood samples and / or

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financial support for this project. This work was supported by NIH grants CA71445 (SD) and NS 31535 (DK), and the National Tubersous Sclerosis Association. References [1] Consortium ECTS. Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell 1993;75:1305–15. [2] van Slegtenhorst M, de Hoogt R, Hermans C, Nellist M, Janssen B, Verhoef S, Lindhout D, van den Ouweland A, Halley D, Young J, Burley M, Jeremiah S, Woodward K, Nahmias J, Fox M, Ekong R, Osborne J, Wolfe J, Povey S, Snell RG, Cheadle JP, Jones AC, Tachataki M, Ravine D, Kwiatkowski DJ. Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science 1997;277:805–8. [3] McCallum CM, Comai L, Greene EA, Henikoff S. Targeted screening for induced mutations. Nat Biotechnol 2000;18:455–7. [4] Wagner TM, Hirtenlehner K, Shen P, Moeslinger R, Muhr D, Fleischmann E, Concin H, Doeller W, Haid A, Lang AH, Mayer P, Petru E, Ropp E, Langbauer G, Kubista E, Scheiner O, Underhill P, Mountain J, Stierer M, Zielinski C, Oefner P. Global sequence diversity of BRCA2: analysis of 71 breast cancer families and 95 control individuals of worldwide populations. Hum Mol Genet 1999;8:413–23, [published erratum appears in Hum Mol Genet 1999 Apr;8(4):717–9]. [5] Wagner TM, Moslinger RA, Muhr D, Langbauer G, Hirtenlehner K, Concin H, Doeller W, Haid A, Lang AH, Mayer P, Ropp E, Kubista E, Amirimani B, Helbich T, Becherer A, Scheiner O, Breiteneder H, Borg A, Devilee P, Oefner P, Zielinski C. BRCA1-related breast cancer in Austrian breast and ovarian cancer families: specific BRCA1 mutations and pathological characteristics. Int J Cancer 1998;77:354–60. [6] Underhill PA, Jin L, Lin AA, Mehdi SQ, Jenkins T, Vollrath D, Davis RW, Cavalli-Sforza LL, Oefner PJ. Detection of numerous Y chromosome biallelic polymorphisms by denaturing high-performance liquid chromatography. Genome Res 1997;7:996–1005. [7] Liu W, Smith DI, Rechtzigel KJ, Thibodeau SN, James CD. Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations. Nucleic Acids Res 1998;26:1396–400. [8] O’Donovan MC, Oefner PJ, Roberts SC, Austin J, Hoogendoorn B, Guy C, Speight G, Upadhyaya M, Sommer SS, McGuffin P. Blind analysis of denaturing high-performance liquid chromatography as a tool for mutation detection. Genomics 1998;52:44–9. [9] Choy Y, Dabora S, Hall F, Ramesh V, Niida Y, Franz D, Kasprzyk-Obara J, Reeve M, Kwiatkowski DJ. Superiority of denaturing high performance liquid chromatography over single-stranded conformation and conformation-sensitive gel electrophoresis for mutation detection in TSC2. Ann Hum Genet 1999;63:383–91. [10] Kwiatkowska J, Jozwiak S, Hall F, Henske EP, Haines JL, McNamara P, Braiser J, Wigowska-Sowinska J, Kasprzyk-Obara J, Short MP, Kwiatkowski DJ. Comprehensive mutational analysis of the TSC1 gene: observations on frequency of mutation, associated features, and nonpenetrance. Ann Hum Genet 1998;62:277–85. [11] Dabora SL, Sigalas I, Hall F, Eng C, Vijg J, Kwiatkowski DJ. Comprehensive mutation analysis of TSC1 using two-dimensional DNA electrophoresis with DGGE. Ann Hum Genet 1998;62:491–504. [12] Roach ES, Gomez MR, Northrup H. Tuberous sclerosis complex consensus conference: revised clinical diagnostic criteria. J Child Neurol 1998;13:624–8. [13] Dabora SL. Heterozygote detection using the ABI. Curr Prot Hu Genet 2000:Unit 7.12. [14] Verhoef S, Bakker L, Tempelaars AM, Hesseling-Janssen AL, Mazurczak T, Jozwiak S, Fois A, Bartalini G, Zonnenberg BA, van Essen AJ, Lindhout D, Halley DJ, van den Ouweland AM. High rate of mosaicism in tuberous sclerosis complex. Am J Hum Genet 1999;64:1632–7. [15] Verhoef S, Vrtel R, van Essen T, Bakker L, Sikkens E, Halley D, Lindhout D, van den Ouweland A. Somatic mosaicism and clinical variation in tuberous sclerosis complex. Lancet 1995;345:202. [16] Kwiatkowska J, Wigowska-Sowinska J, Napierala D, Slomski R, Kwiatkowski DJ. Mosaicism in tuberous sclerosis as a potential cause of the failure of molecular diagnosis. New Engl J Med 1999;340:703–7. [17] Yates JR, van Bakel I, Sepp T, Payne SJ, Webb DW, Nevin NC, Green AJ. Female germline mosaicism in tuberous sclerosis confirmed by molecular genetic analysis. Hum Mol Genet 1997;6:2265–9.