REVIEW ARTICLE
Methods in molecular cardiloy DHPLC
mutation detection analysis
R.J.E. Jongbloed, H. Smeets, P.A. Doevendans, A. van den Wijngaard
An increasing number of mutations have been identified in genes involved in cardiac disorders which has led to novel insights in the pathophysiology ofinherited cardiac diseases. As a result of these findings, techniques specialised in automated high-throughput analysis are implemented to handle the increasing number of diagnostic genetic requests. Denaturing high-performance liquid chromatography (DHPLC) is one such novel technique that fulfils the criteria of speed, sensitivity and accuracy. This issue focuses on the basic principle of the technique and illustrates how genetic alterations can be identified. (Neth Heart J2005;13:11-7.) Key words: molecular cardiology, mutation detection analysis
cardiac research an increasing number of genes have been reported that are involved in familial cardiovascular diseases. This ongoing progress forces a clinical diagnostic centre to improve the number and speed of DNA mutation analysis of genes that might be the underlying cause of the disease. In modern medicine, there is a great need for genetic evidence of a disease-causing nucleotide variant to support clinical diagnosis in affected family members. The identification of a genetic defect might also have a major n
RJ.E. Jongbloed University of Maastricht, Maastricht H. Smeets A. van den WlJngaard Departnent of Clinical Genetics, Maastricht University Hospital, Maastricht P.A. Doevendans Heart Lung Institute University Centre, University of Utrecht, Utrecht
Correspondence: R.J.E. Jongbloed Department of Genetics, University of Maastricht, Universiteitssingel 50, P0 Box 616, 6200 MD Maastricht E-mail: [email protected]
Netherlands Heart Journal, Volume 13, Number 1, January 2005
impact on strategies for clinical treatment and genespecific drug administration or other life-saving medical interventions. Until now, in routine DNA diagnostic analysis, several gel-based methods have been used for mutation detection of genes linked to heritable (mono)genetic diseases. Although sequencing analysis remains the 'golden standard' in mutation detection systems, novel prescreening techniques have been introduced to enhance reliable mutation detection analysis. Yet, improvement in speed and sensitivity remains an important issue in mutation detection analysis. If genes have to be fully analysed for a large number of samples, automation and development of high-throughput procedures need to be implemented. Denaturing high-performance liquid chromatography (DHPLC) is an example of such a rapid, automated and high-throughput mutation detection system. The method is based on DNA heteroduplex analysis similar to the widely used gel-based methods such as singlestrand conformation polymorphism analysis (SSCP), conformation-sensitive gel electrophoresis (CSGE) or denaturing gradient gel electrophoresis (DGGE). DHPLC implementation has significantly increased the number of samples that can be analysed successively. The improvement in time and sensitivity is an advantage as fast identification of the disease-causing genetic defect is warranted in clinical diagnosis. In the Netherlands, some laboratories have implemented the DHPLC technique as a diagnostic prescreening tool. In this article in the series on Methods in Cardiology, basic information about this method is presented as well as its application in a diagnostic setting.
Main principle of DHPLC-based mutation detection Denaturing high-performance liquid chromatography (DHPLC) is an easy and nondestructive, columnbased separation method. It is essential that a mixture of both wild-type and mutated DNA fragment is denatured (by heating up to 95 °C) to create singlestrand DNA fragments which can subsequently reanneal (by cooling down) to form homoduplexes and heteroduplexes of double-stranded DNA fragments (figure lA, 1B). Separation of homoduplexes and heteroduplexes is based on small 11
Methods in molecular cardiology: DHPLC mutation detection analysis
Singlestranded DNA
Double-stranded DNA Wld-type Mutant
11 11
Wild-ype
Heat 9500
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T a l
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Figure 1A. By beating the DNA up to 95 OCbothstrands are separated into individual DNA strands (denaturingstep). By cooling down to room temperature the singl-stranded (denatured) sense (+) DNA strands can form homodupexes by bybridising to the exact copy of the antiwnse (9 DNA strands, thesingek-stranded DNA (bybridisation). In the presence ofa DNA variant (mutant) a mismatcb bybridisation will occur of both wild-type and mutated strands which will lead to the formation of heteroduplex DNA mokcues.
differences in melting temperatures between normal wild-type and mutated DNA fragments and is generally successful in separating DNA duplexes that differ in the identity of one or more base pairs.' Basically, unpurified negatively charged double-strand DNA fragments are attached to positively charged ions of tri-ethyl ammonium acetate (TEAA) that are present in the liquid phase. The DNA-TEAA complexes bind to the neutral particles that are packed within the column cartridge. DNA can be eluted from the column by using a hydrophobic solution consisting of a linear acetonitril gradient. Analysis has to be performed at a
5
Heteroduplexes
Homoduplexes
Mv
0
-
Figure
B. The
Retention time (min)
intrtion of the DNA heterodupkxe with thesolid
phase in the DHPLC column is weaker than the interaction of the DNA homodupklxes. At a 'fragment specific' temperature and optmised acetonitril concentration, t heeroduplexs are eluted prior to the homodupkxes. By kaving the machine the eluted
fragments are detected by 260 nm LW absorption (mV) and visualised aspeaks. Tbefrontpeak contains unincorpratedprimers and deaoxibonucleoside triphosphates (dN7TPs).
12
temperature sufficient to partially denature DNA duplexes. For each PCR-generated fragment an analysing temperature just below melting temperature (Tm) is recommended and largely depends on the GC contents ofeach fragment. The concentration at which the fragments will be eluted from the solid phase depends on the lengths ofeach DNA fragment. Large DNA fragments are able to bind more TEAA ions than small fragments. Therefore, an increasing amount of acetonitril is needed to elute these DNA fragments from the solid phase. DNA heteroduplexes demonstrate a lower affinity for the solid phase than the homoduplexes. Consequently, the less stable heteroduplexes are eluted prior to the homoduplexes.2" Summarising, the analysing concentration of acetonitril is determined by the length of each DNA fragment while the analysing temperature is largely determined by its nucleotide sequence (AT-GC content). Quick and automated analysis is one ofthe major characteristics of the DHPLC system as injection and separation of samples occurs within minutes.4 Further, a remarkably high detection rate ofalmost 100% in blind trials has been reported.5 Since than, many examples of successful mutation detection analysis have been reported. Alist ofgenes that have been entirely or partially analysed by DHPLC is presented on the Internet site
http://insertion.stanford.edu/dhplc.genesl.html.6 Before getting staited Sensitivity of DHPLC analysis largely depends upon the size and sequence of the fragments. Mutation detection is optimal for relatively small DNA fragments (150 to 450 base pairs) and the success rate is influenced by the quality and the amount of the amplified product. Therefore, generating a unique, qualified amplicon is an important and initial goal. Amplicons should be verified by sequencing analysis to Netherlands Heart Journal, Volume 13, Number 1, January 2005
Methods in molecular cardiology: DHPLC mutation detection analysis
exclude inaccurate amplification products. The polymerase chain reaction (PCR) method was outlined in one ofthe previous artides in this series.7 As mentioned before, formation of DNA heteroduplexes is essential for reliable mutation detection and it should noted that both DNA duplexes (homo and hetero) are generated automatically during the repeated melting and reannealing steps embedded within the PCR amplification method. Primer design Gene-specific primer pair design is a critical element. For diagnostic purposes the design is mainly based upon reviewed reference sequences including exonic (coding) regions and intronic (noncoding) sequences of the gene of interest. Today, computer-based programmes enhance and facilitate unique primer set design.8 If primers have to be designed for DHPLC implementation, theoretically amplified products should be tested for the presence ofprovisional melting domains. To enhance time-consuming primer design DHPLC users profit from access to the website built and maintained by one ofthe DHPLC manufacturers.9 As DHPLC analysis is limited to 150 to 450 base pairs (bp), it is sometimes difficult to find a suitable combination of primers. For these particular cases, extended primers can be used to enlarge small fragments. On the other hand, sequences exceeding 450 base pairs can be minimised by using smaller overlapping fragments.
Presence of melting domains The next step shows how a theoretically amplified product behaves at various temperatures.10 The DHPLC method is believed to be most efficient near the melting temperature (Tm) of the duplex DNA at the specific site of the mutation. Simply by raising the temperature, the double-stranded DNA fragment will finally end up as separated stands or single-stranded DNA. The Tm is a guideline at which temperature a particular site has a 50% chance of being Watson-Crick bonded rather than open. According to the WatsonCrick model the DNA helix shows a triple hydrogen bond in GC coupling while AT couplings show double-hydrogen bonds. This explains why DNA fragments containing AT-rich domains are melted (separated) at lower temperatures than GC-rich domains. Some DNA fragments demonstrate only one melting domain while others may present two or even more (figure 2). In the case of multiple melting domains analysis should be performed at distinct temperatures corresponding to the various Tm's. At present, melting profiles of the entire sequence (including clamps if used) of DNA fragments is investigated using various algorithms to estimate the most accurate melting temperature and to ensure detection of all polymorphisms, such as: - Wavemaker utility control software provided by Transgenomics (Transgenomics Inc. San Jose);
Nethrlands Heart Journal, Volume 13, Number 1, January 2005
Heldmcod Frawton vs.Tmsrt6 l1±00
I
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45
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70 7e :) Temperature (0C'
llobn
PNJ
%
I
I 0
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i00
iA0
58 0C 63°C 65 °C
200 250 Base Position
Figure 2. Part ofthe WA VEMAKER software pngrammefor exon
10 (TNN72 gene) is illustrated in thisfigure. The upper window dispiaysthe helicalfraction vs. temperature. The arrows indicate the two distinctmeltingdomains (*57°Cand±62 °C) thatarepresent within the DNA fragment. The lower window displays predicted helicalfraction vs. basepairsatthreedistincttemperaturesallowing mutation detection analysis in a minimal temperature range. As demonstrated the initialpart ofthe sequences (up to 120base pairs) refcrsto the higher2nd melting domain, which isstillpresentin the double-belix form (100% helix) at all three temperatures (57-5859 °C.) The DHPLC Melt Programme recommends analysing temperatures at 58 °C and 63 OC
The DHPLC melting programme which is freely available on the internet site;" - By empirical data: the fragment specific melting curve can be constructed based on the retention time at nondenaturing condition (50 °C) in combination with the appropriate gradient profile and temperatures ranging from 56 to 80 'C. -
Heteroduplex analysis Successful DHPLC analysis depends on the presence of DNA heteroduplexes. Small insertions and deletions (21bp) will be easily detected as DNA fragments differ in length. Note that if large insertion/deletions are present they might also be recognised after electrophoresis when amplified products are verified for correct amplification. However, with a single nucleotide substitution it is difficult to predict whether a specific DNA variant will be detected at a specific temperature or not. Preferentially, positive controls of verified DNA variants are used to refine analysing conditions.
DHPLC Instrument The setup of the DHPLC instrument (Wave system 3500, 3500HT) consists of several individual elements. A schematic view ofthe initial elements is presented in 13
Methods in molecular cardiology: DHPLC mutation detection analysis
Figure 3. The schematic overpiew of the DHPLC technique illustrates the order of analysis. A small volume ofa DNA sample is injected into the liquid system and subsequently DNA-TEAA mokcules are attached onto the solid particles in the column. A permanentflow of a mixture ofBufferA and B is run through the system. Elution of thefragments occurs by raising the temperature and the concentration of BufferB. 77 released DNAfragments kave the column and are detcted by UV260absorption and ilustated aspeaks. Ifnecesary, afragment colkctor can be attached to colct separated DNA fragmentsforfurther analysis.
figure 3. Elements are put on top ofeach other to limit working space. Additionally, the machine is placed in the neighbourhood of an extractor fan to minimise inhalation of poisonous acetonitril fumes. Solid phase The solid phase consists of alkylated nonporous polystyrene-divinylbenzene partides (DNASep, DNASep'T, Transgenomics, San Jose, US) packed onto the column cartridge.'2 The hydrophobic partides can interact with the DNA triethylammonium acetate (DNA-TEAA) complexes. These DNA-TEAA complexes are formed by interaction of the negatively charged doublestranded DNA molecules with the positively charged TEAA molecules (figure 4). The DNA-TEAA complexes are eluted from the column by using an
DNA
increasing concentration of acetonitril as liquid phase. Large DNA particles can bind more TEAA molecules than small DNA fragments, and as a result they are more stably attached to the solid phase than small particles. This explains why large fragments will be eluted at a higher acetonitril concentration (longer elution time) than small DNA fragments. The lifetime of the DNASep column is limited, but can be regenerated after quite some time. An expected number of analyses of 8 to 10,000 runs/column is guaranteed by the manufacturer. Liquid phase The liquid phase consists of two solutions: buffer A and buffer B. BufferAis a mixture of 0.1M TEAA and a low concentration ofactonitril (0.25% ACN, Biosolve LTD. HPLC grade) while buffer B consists of 0.1 M TEAA and a high concentration of acetonitril (25% v/v ACN). Only HPLC grade TEAA (Transgenomics Inc.) is used to make up buffers A and B. Buffers are prepared with low-conductance miliQwater (18.2MW) and made up on a weekly basis or more frequently if required. The column washing solution (buffer C) contains a high concentration of acetonitril (75% v/v ACN) while the syringe washing solution (buffer D) contains 7.5% v/v acetonitril. Buffer C and buffer D are additional solutions and do not contain TEAA ions.
Oven, column pressure and needle injection port
syringe
Figure 4. The negatively charged DNA mokcules interact with the positively charged T7AA molecules. The bydropbobic compex allows the attachment to the hydrophobic particles that are packed onto the DHPLC column.
14
The temperature of the high-precision Peltier oven (Transgenomics Inc, type L7300 or L7300+) and column pressure is monitored on a regular basis. If pressure increases at a low flow rate (0.05 ml/min), the column is cleaned by using 100% buffer C (75% v/v acetonitril) at a flow rate of 0.9 ml/min at a temperature of 80 °C for 15 to 30 minutes. Prior to running samples through the system, the needle and injection port are washed (10 to 20 times) and the
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Methods in molecular cardiology: DHPLC mutation detection analysis
Table 1. DHPLC running conditions at a specific temperature.
Time 0.0 0.1 5.0 5.1 5.7 5.8 8.0
% Buffer A % Buffer B 55 45 50 50 40 60 0 100 100 0 55 45 55 45
Equilibration column Start gradient Elution of fragment Column wash Column wash Equilibration column Equilibration column
Example of a DHPLC method. Running conditions are setup to achieve a linear acetonitrl gradient of 10% within five minutes (2% acetonitril/minute) and 0.9 ml/min flow rate. The gradient is built up by mixing buffer A and buffer B.
syringe is inspected regularly to check for signs of leakage and damage to the plunger seal. Intemal controls An appropriate DNA mutation standard can be used to verify the system (in particular the temperature of the oven) and the condition ofthe column. Depending on the application and method, various standard markers are commercially available. Fragment size standard pUC18/Hae 111 and mutation standard DYS271 are used as instrument controls (Transgenomics Inc, San Jose, US). These internal controls have to be run before and after the series of analysis and whenever maintenance occurs or when buffers, column, in-line filter etc. are changed, otherwise an instrument problem or a loss of heteroduplex resolution caused by an improper cartridge may remain unnoticed. Running negative controls is not necessary.
DHPLC analysis Heteroduplex formation Prior to DHPLC analysis, a reannealing step (TMHA: temperature mediated heteroduplex analysis) is introduced and although this step is not necessary, it may influence the reproducibility of the elution pattern. The following temperature parameters are recommended for efficient heteroduplex formation: 95 °C for five minutes, slowly cooling down (1 °C/min) to room temperature. Subsequently, PCRproducts are run on an appropriate agarose gel to check for efficient amplification, product length and intensities of the fragment bands. It is particularly important that the amount of PCR product is relatively uniform to be able to compare elution profiles within a run.
Sampling and running
Computer spreadsheets allow sample identification and handling to be linked to the appropriate analysing method. Samples are loaded on 96-well plates (Costar) and placed into the Wave apparatus. An aliquot of the
Netherlands Heart Journal, Vohune 13, Number 1, January 2005
unpurified PCR product (5-10 i) is injected on the preheated column. Fragments are eluted from the stationary phase using a fragment specific gradient. The gradient increases by 2% acetonitril per minute at a flow rate of 0.9 ml/min (1.5 ml/min for HT instruments). A final washing step (100% buffer B) finishes the analysing process, and remaining DNA molecules are removed from the column (table 1). The interposition of an accelerator significantly speeds up the final washing step. The gradient ensures that DNA fragments are eluted from the stationary phase at approximately three to four minutes. A gradient profile is usually set up for five minutes, which means a total increase of 10% buffer B (2%/min). For example if a fragment is eluted from the column at 66% of buffer B, the method is said to start at 56% and to end at 66%. Temperature setting As a guideline, the preferred analysing temperature is when 25% of the fragment is denatured, preferably 1 to 2 °C below the empirical calculated melting temperature. If available, positive controls such as known mutations or nucleotide variants are run under identical conditions.
Positive results Abnormal elution profiles or presence ofadditional or shoulder peaks are indicated as 'suspicious'. In practice, a reduced peak signal or peak broadening is suspicious but only if equal amounts of samples have been loaded to the stationary phase. AU abnormal elution profiles have to be verified by sequencing analysis to confirm the presence of DNA variants. To avoid crosscontamination and to eliminate the possibility of detecting PCR artifacts that give rise to the abnormal chromatograms, a second sequence reaction is prepared from a freshly prepared PCR product ofthe patient DNA sample. Alternatively, in case of a common polymorphism, the tested DNA sample can be equally mixed with a sample which is heterozygous for the variant. After heteroduplex formation the sample is reanalysed on the DHPLC. If no apparent change in the profile appears after the addition of the known polymorphism, it is suggested that no other mutation than the known variant is present. Another approach is to compare the tested sample with the profile of a known polymorphism at one or two injection temperatures. It is unlikely that these profiles remain identical at different injection temperatures if they are generated by a different mutation or polymorphism. False-positive results A multiple peak pattern can be produced by the combination of a low fidelity or non-proofreading polymerase or too many cycles during amplification. To circumvent inaccurate amplification and the formation of multiple peak patterns, a proofreading Taq polymerase (e.g. Optimase, Transgenomics Inc.) 15
Methods in molecular cardiology: DHPLC mutation detection analysis
B
A
C
Wild type llelO61le C>T Arg94Leu
D
Arg92Trp
Figure 5. Thisfigure illustrates distinct elution profiles that have been identified within exon 9 ofthe TNNT72gene, which is involved infamilial berrtrpphiccardiomyopathy (FHC). With respect to the elution pattern of the wild-type DNA fragment (A), abnormal elution profiles were observed in B to D. Arrows indicate aberrant and reduced UV,absoption signalsvisualisedaspeaksorshoulder peaks. Subsequently, sequencing analysis of the DNA showingfigure 5B identified a C>Ttransition in codon 106. The aberrant elution profiles in 5D and 5E are the result of two distinct pathogenic missense mutations Arg94Leu (CGC>CTC) and Arg92Trp (CGG>TGG). DNA fragments were eluted from the DNAsep column at 62 °C within 3 to 3.5 minutes using a lineargradient 54 to 64%.
Wild type
delE160
Figure 6. DHPLCprofik ofwild-te (kft) and mutatedfragment (right) illustrates a deletion of three nucleotides (578580delGAG>delE160) in exon 11 of the TNNT2 gene. Running conditions were performed at 57 0CQgradient 56 to 66% buffer B.
Application In cardiology In the Netherlands, DHPLC analysis has been successfully used for a number of genes involved in inherited cardiac disorders, with respect to genes involved in long-QT syndrome, hypertrophic cardiomyopathy and mitochondrial myopathies."'1-7 To
is recommended. This thermostable proofreading DNA polymerase has an efficient 3'->5' exonuclease activity and is advised for single nucleotide polymorphism (SNP) and mutation detection studies using DHPLC. An Optimase ProtocolWriter tool can be consulted for amplification conditions by simply entering the appropriate primers, the expected PCR product length, and selecting an appropriate PCR
protocol type.'3 False-negative results Make sure the mutation standards were analysed correctly, failure to detect mutations may arise for a number of reasons: Poor quality of the amplified product exhibits low signals and the ability to identify mutations is lost. Moreover very high yields can include a large number of mis-incorporations and hence increase difficulty in calling mutations. Incorporation of false nucleotides can occur if non-proofreading DNA poly-
used; Nucleotide changes that occur in a high melt GC-rich domain within the fragment: this inconvenience might be solved by redesigning amplification of fragments, preferably with the GC-rich regions towards the ends of the fragments or by using GC merases are
-
clamps;'4 -
Fragments too small: due to reduced sensitivity in fragments smaller than 150 base pairs sequence variations may not be detected. Redesigning the amplicon is recommended.
16
illustrate what elution profiles may look like, examples of a single nucleotide substitution, a single nucleotide insertion and a deletion of three base pairs are demonstrated in figures 5, 6 and 7. Elution profiles generated by DNA variations in exon 9 ofthe troponin T (TNNT2) gene, involved in hypertrophic cardiomyopathy (CMH2), are shown in figures 5B to D. The single nucleotide substitution (ATC>ATT) representing the elution pattern in figure 5B does not affect the genetic information as both triplets encode for isoleucine (silent mutation) and are designated as coding single nucleotide polymorphism which is present within the normal population (refSNP ID:rs3729547). The elution profiles of two distinct pathogenic missense mutations, which have been identified within two unrelated Dutch families, are shown in figures 5C and D. Both pathogenic mutations had been described previously.18"19 Additionally a deletion ofthree nucleotides, encoding a glutamic acid (delE160), was identified in exon 11 (figure 6). The deletion had occurred within two apparently unrelated families with HCM and sudden death at young age (15 year).20 The MYBPC3 gene encodes the myosin-binding protein C which is involved in familial hypertrophic cardiomyopathy (CMH4). Elution profiles of the reference wild-type DNA and an insertion of a single nucleotide G in exon 25 of the MYBPC3 gene are shown in figure 7A. In the Netherlands the 2373insG has been identified as founder mutation which is present in almost 20% of the Dutch HCM popu-
Netherlands Heart Journal, Volume 13, Number 1, January 2005
Methods in molecular cardiology: DHPLC mutation detection analysis
lation.'6 Finally, using DHPLC analysis ofthe coding region ofthe nonsarcomeric PRKAG2 gene, a missense mutation (Arg302Gln) was identified in a family with clinical features ofWolff-Parkinson-White syndrome and hypertrophic cardiomyopathy (figure 7B).2' Conclusions The rapidly increasing number ofgenes that have been identified in inherited monogenetic cardiac diseases has forced the diagnostic genetic laboratories to implement fast and highly sensitive mutation detection systems. As mutations can now be easily detected by using advanced and high-throughput analysis systems, such as DHPLC, the challenge finding them becomes less teasing. Yet defining whether a variation indeed causes disease becomes more difficult. At the moment the number of naturally occurring validated DNA variants in the human genome is e 4.3 million (National Centre for Biotechnology Information (NCBI) dbSNP Build 120) indicating an occurrence ofapproximately 1 in 500 base pairs. This means that they are regularly found in routine screening causing an additional workload in the diagnostic centre as all aberrant DHPLC profiles have to be reamplified and checked by sequencing analysis. Summarising, the DHPLC technique turns out to be a very rapid and easy method but it should be realised that optimisation of an entire gene takes a lot of time as each fragment has to be optimised and verified by internal controls. A limitation of using highthroughput techniques is the huge amount of data that can be generated within a short period of time. To overcome misinterpretation, analysing software can be used for peak normalisation and automated detection of aberrant DHPLC profiles making mutation detection simpler and reliable which is essential in diagnostic genetic screening. U References Oefner PJ, Underhill P. DNA mutation detection using denaturing high-performance liquid chromatography (DHPLC). In: Dracopoli NC, Haines NC, Korf BR, Morton C, Seidman CE, Seidman JG, et al. (editors). Current Protocols in Human Genetics. New York: Wiley-Interscience; 1998:7(Suppl);10:1-12. 2 Underhill PA, Jin L, Zemans R, Oefier PJ, Cavalli-Sporza LL. A pre-Columbian Y chromosome-specific transition and its implications for human evolutionary history. Pros Natl Acad Sci USA 1993;93:196-200. 3 http://transgenomic.com/flash/DHPLCFlash2.asp. 4 Xiao W, Oefner P. Denaturing High-Performan ce Liquid Chromatography: A review. Hum Mut2001;17:439-74. 5 O' Donovan MC, Oefner PJ, Roberts SC, Austin J, Hoogendoom B, Guy C, et al. Blind Analysis of Denaturating High-Performance Liquid Chromatography as a Tool for Mutation Detection. Genomics 1998;52:44-9. 6 http://insertion.stanford.edu/dhplc_genesl.html. 7 Sonnemans DGP, de Wmdt L, de Muinck EM, Doevendans PA. Methods in molecular cardiology: the polymerase chain reaction. Neth HeartJ2002;10:412-8. 8 http://www.broad.mit.edu/cgi-bin/primer/primer3_www.cgi. 9 http://www.mutationdiscovery.com/. 1
Netherlands Heart Journal, Volume 13, Number 1, January 2005
B
A
Wild type
2373insG
Wild type
Arg3O2Gin
Figure 7. A. Elution patterns ofwild-type (left) and mutated DNA (right) of exon 25 of the MTBPC3 gene. The aberrant profile represents a single nucleotide insertion (2373insG). Note that the longer mutated DNA strand is eluted after the wild-type peak. Running conditions: at 63 OC; gradient 54 to 64% Buffer B. B. Missense mutation CGA>CAA (right profile) in exon 7 of the PRKAG2 gene identified by DHPLC analysis. Running conditions were performed at 59 0C; gradient 52 to 62% Buffer B. 10 Jones AC, Austin J, Hansen N, Hoogendoorn B, Oefner PJ, Cheadle JP, et al. Optimal temperature selection for mutation detection by denaturing high performance liquid chromatography and comparison to SSCP and heteroduplex analysis. Clin Chem 1999;45:1133-40. 11 http://insertion.stanford.edu/melt.html. 12 Bonn G, Hoefner C, Oefner P. Nucleic Acid Separation on Alkylated Nonporous Polymer Beads. US Patent 1996;5:236. 13 http://mutationdiscovery.com/screens/optimase/OptimaseInput.html. 14 Narayanawami G, Taylor P. Improved efficiency of mutation detection by denaturing high-performance liquid chromatography using modified primers and hybridization procedure. Genetic Testing 2001;5:9-16. 15 Jongbloed R, Marcelis C, Velter C, Doevendans P, Geraedts J, Smeets H. DHPLC analysis ofpotassium ion channel genes in congenital long QT syndrome. Hum Mut2002;20:382-91. 16 Alders M, Jongbloed R, Deelen W, Asidah F, van den Wijngaard A, Regitz-Zagrosek V, et al. The 2373insG mutation in the MYBPC3 gene is a founder mutation which accounts for nearly one-fourth of all HCM cases in the Netherlands. Eur Heart J 2003;24:1848-53. 17 Van den Bosch BJ, de Coo R, Scholte HR, Nijland JG, van den Bogaard R, de Visser M, et al. Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatograph. Nucleic Acids Res2000;28:E89. 18 Varnava A, Baboonian F, Davison F, De Cruz L, Elliot PM, Davies MJ, et al. A new mutation of the cardiac troponin T gene causing familial hypertrophic cardiomyopathy without left ventricular hypertrophy. Heart 1999;82:621-4. 19 Moolman JC, Corvield V, Posen B, Ngumbela K, Seidman C, Brink PA, et al. Sudden death due to Troponin T mutations. JAm Coll Cardiol 1997;29:549-55. 20 Watkins H, McKenna W, Thierfelder L, Suk HJ, Anan R, O'Donoghue A, et al. Mutations in the genes for cardiac troponin T and alpha-tropomyosin in hypertrophic cardiomyopathy. NEngl
JMed 1995;332:1058-64.
21 Gollob MH, Green S, Tang AS, Gollob T, Karibe A, Hassan AS, et al. Identification ofa gene resposable for familial Wolff-ParkinsonWhite syndrome. NEnglJMed 2001;344:1823-31.
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Methods in molecular cardiloy DHPLC
mutation detection analysis
R.J.E. Jongbloed, H. Smeets, P.A. Doevendans, A. van den Wijngaard
An increasing number of mutations have been identified in genes involved in cardiac disorders which has led to novel insights in the pathophysiology ofinherited cardiac diseases. As a result of these findings, techniques specialised in automated high-throughput analysis are implemented to handle the increasing number of diagnostic genetic requests. Denaturing high-performance liquid chromatography (DHPLC) is one such novel technique that fulfils the criteria of speed, sensitivity and accuracy. This issue focuses on the basic principle of the technique and illustrates how genetic alterations can be identified. (Neth Heart J2005;13:11-7.) Key words: molecular cardiology, mutation detection analysis
cardiac research an increasing number of genes have been reported that are involved in familial cardiovascular diseases. This ongoing progress forces a clinical diagnostic centre to improve the number and speed of DNA mutation analysis of genes that might be the underlying cause of the disease. In modern medicine, there is a great need for genetic evidence of a disease-causing nucleotide variant to support clinical diagnosis in affected family members. The identification of a genetic defect might also have a major n
RJ.E. Jongbloed University of Maastricht, Maastricht H. Smeets A. van den WlJngaard Departnent of Clinical Genetics, Maastricht University Hospital, Maastricht P.A. Doevendans Heart Lung Institute University Centre, University of Utrecht, Utrecht
Correspondence: R.J.E. Jongbloed Department of Genetics, University of Maastricht, Universiteitssingel 50, P0 Box 616, 6200 MD Maastricht E-mail: [email protected]
Netherlands Heart Journal, Volume 13, Number 1, January 2005
impact on strategies for clinical treatment and genespecific drug administration or other life-saving medical interventions. Until now, in routine DNA diagnostic analysis, several gel-based methods have been used for mutation detection of genes linked to heritable (mono)genetic diseases. Although sequencing analysis remains the 'golden standard' in mutation detection systems, novel prescreening techniques have been introduced to enhance reliable mutation detection analysis. Yet, improvement in speed and sensitivity remains an important issue in mutation detection analysis. If genes have to be fully analysed for a large number of samples, automation and development of high-throughput procedures need to be implemented. Denaturing high-performance liquid chromatography (DHPLC) is an example of such a rapid, automated and high-throughput mutation detection system. The method is based on DNA heteroduplex analysis similar to the widely used gel-based methods such as singlestrand conformation polymorphism analysis (SSCP), conformation-sensitive gel electrophoresis (CSGE) or denaturing gradient gel electrophoresis (DGGE). DHPLC implementation has significantly increased the number of samples that can be analysed successively. The improvement in time and sensitivity is an advantage as fast identification of the disease-causing genetic defect is warranted in clinical diagnosis. In the Netherlands, some laboratories have implemented the DHPLC technique as a diagnostic prescreening tool. In this article in the series on Methods in Cardiology, basic information about this method is presented as well as its application in a diagnostic setting.
Main principle of DHPLC-based mutation detection Denaturing high-performance liquid chromatography (DHPLC) is an easy and nondestructive, columnbased separation method. It is essential that a mixture of both wild-type and mutated DNA fragment is denatured (by heating up to 95 °C) to create singlestrand DNA fragments which can subsequently reanneal (by cooling down) to form homoduplexes and heteroduplexes of double-stranded DNA fragments (figure lA, 1B). Separation of homoduplexes and heteroduplexes is based on small 11
Methods in molecular cardiology: DHPLC mutation detection analysis
Singlestranded DNA
Double-stranded DNA Wld-type Mutant
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I
T
AT
QC
I I II
Figure 1A. By beating the DNA up to 95 OCbothstrands are separated into individual DNA strands (denaturingstep). By cooling down to room temperature the singl-stranded (denatured) sense (+) DNA strands can form homodupexes by bybridising to the exact copy of the antiwnse (9 DNA strands, thesingek-stranded DNA (bybridisation). In the presence ofa DNA variant (mutant) a mismatcb bybridisation will occur of both wild-type and mutated strands which will lead to the formation of heteroduplex DNA mokcues.
differences in melting temperatures between normal wild-type and mutated DNA fragments and is generally successful in separating DNA duplexes that differ in the identity of one or more base pairs.' Basically, unpurified negatively charged double-strand DNA fragments are attached to positively charged ions of tri-ethyl ammonium acetate (TEAA) that are present in the liquid phase. The DNA-TEAA complexes bind to the neutral particles that are packed within the column cartridge. DNA can be eluted from the column by using a hydrophobic solution consisting of a linear acetonitril gradient. Analysis has to be performed at a
5
Heteroduplexes
Homoduplexes
Mv
0
-
Figure
B. The
Retention time (min)
intrtion of the DNA heterodupkxe with thesolid
phase in the DHPLC column is weaker than the interaction of the DNA homodupklxes. At a 'fragment specific' temperature and optmised acetonitril concentration, t heeroduplexs are eluted prior to the homodupkxes. By kaving the machine the eluted
fragments are detected by 260 nm LW absorption (mV) and visualised aspeaks. Tbefrontpeak contains unincorpratedprimers and deaoxibonucleoside triphosphates (dN7TPs).
12
temperature sufficient to partially denature DNA duplexes. For each PCR-generated fragment an analysing temperature just below melting temperature (Tm) is recommended and largely depends on the GC contents ofeach fragment. The concentration at which the fragments will be eluted from the solid phase depends on the lengths ofeach DNA fragment. Large DNA fragments are able to bind more TEAA ions than small fragments. Therefore, an increasing amount of acetonitril is needed to elute these DNA fragments from the solid phase. DNA heteroduplexes demonstrate a lower affinity for the solid phase than the homoduplexes. Consequently, the less stable heteroduplexes are eluted prior to the homoduplexes.2" Summarising, the analysing concentration of acetonitril is determined by the length of each DNA fragment while the analysing temperature is largely determined by its nucleotide sequence (AT-GC content). Quick and automated analysis is one ofthe major characteristics of the DHPLC system as injection and separation of samples occurs within minutes.4 Further, a remarkably high detection rate ofalmost 100% in blind trials has been reported.5 Since than, many examples of successful mutation detection analysis have been reported. Alist ofgenes that have been entirely or partially analysed by DHPLC is presented on the Internet site
http://insertion.stanford.edu/dhplc.genesl.html.6 Before getting staited Sensitivity of DHPLC analysis largely depends upon the size and sequence of the fragments. Mutation detection is optimal for relatively small DNA fragments (150 to 450 base pairs) and the success rate is influenced by the quality and the amount of the amplified product. Therefore, generating a unique, qualified amplicon is an important and initial goal. Amplicons should be verified by sequencing analysis to Netherlands Heart Journal, Volume 13, Number 1, January 2005
Methods in molecular cardiology: DHPLC mutation detection analysis
exclude inaccurate amplification products. The polymerase chain reaction (PCR) method was outlined in one ofthe previous artides in this series.7 As mentioned before, formation of DNA heteroduplexes is essential for reliable mutation detection and it should noted that both DNA duplexes (homo and hetero) are generated automatically during the repeated melting and reannealing steps embedded within the PCR amplification method. Primer design Gene-specific primer pair design is a critical element. For diagnostic purposes the design is mainly based upon reviewed reference sequences including exonic (coding) regions and intronic (noncoding) sequences of the gene of interest. Today, computer-based programmes enhance and facilitate unique primer set design.8 If primers have to be designed for DHPLC implementation, theoretically amplified products should be tested for the presence ofprovisional melting domains. To enhance time-consuming primer design DHPLC users profit from access to the website built and maintained by one ofthe DHPLC manufacturers.9 As DHPLC analysis is limited to 150 to 450 base pairs (bp), it is sometimes difficult to find a suitable combination of primers. For these particular cases, extended primers can be used to enlarge small fragments. On the other hand, sequences exceeding 450 base pairs can be minimised by using smaller overlapping fragments.
Presence of melting domains The next step shows how a theoretically amplified product behaves at various temperatures.10 The DHPLC method is believed to be most efficient near the melting temperature (Tm) of the duplex DNA at the specific site of the mutation. Simply by raising the temperature, the double-stranded DNA fragment will finally end up as separated stands or single-stranded DNA. The Tm is a guideline at which temperature a particular site has a 50% chance of being Watson-Crick bonded rather than open. According to the WatsonCrick model the DNA helix shows a triple hydrogen bond in GC coupling while AT couplings show double-hydrogen bonds. This explains why DNA fragments containing AT-rich domains are melted (separated) at lower temperatures than GC-rich domains. Some DNA fragments demonstrate only one melting domain while others may present two or even more (figure 2). In the case of multiple melting domains analysis should be performed at distinct temperatures corresponding to the various Tm's. At present, melting profiles of the entire sequence (including clamps if used) of DNA fragments is investigated using various algorithms to estimate the most accurate melting temperature and to ensure detection of all polymorphisms, such as: - Wavemaker utility control software provided by Transgenomics (Transgenomics Inc. San Jose);
Nethrlands Heart Journal, Volume 13, Number 1, January 2005
Heldmcod Frawton vs.Tmsrt6 l1±00
I
At
50-
45
50
55
Hlical Fraction vs. las %ii 1
60
65
70 7e :) Temperature (0C'
llobn
PNJ
%
I
I 0
50
i00
iA0
58 0C 63°C 65 °C
200 250 Base Position
Figure 2. Part ofthe WA VEMAKER software pngrammefor exon
10 (TNN72 gene) is illustrated in thisfigure. The upper window dispiaysthe helicalfraction vs. temperature. The arrows indicate the two distinctmeltingdomains (*57°Cand±62 °C) thatarepresent within the DNA fragment. The lower window displays predicted helicalfraction vs. basepairsatthreedistincttemperaturesallowing mutation detection analysis in a minimal temperature range. As demonstrated the initialpart ofthe sequences (up to 120base pairs) refcrsto the higher2nd melting domain, which isstillpresentin the double-belix form (100% helix) at all three temperatures (57-5859 °C.) The DHPLC Melt Programme recommends analysing temperatures at 58 °C and 63 OC
The DHPLC melting programme which is freely available on the internet site;" - By empirical data: the fragment specific melting curve can be constructed based on the retention time at nondenaturing condition (50 °C) in combination with the appropriate gradient profile and temperatures ranging from 56 to 80 'C. -
Heteroduplex analysis Successful DHPLC analysis depends on the presence of DNA heteroduplexes. Small insertions and deletions (21bp) will be easily detected as DNA fragments differ in length. Note that if large insertion/deletions are present they might also be recognised after electrophoresis when amplified products are verified for correct amplification. However, with a single nucleotide substitution it is difficult to predict whether a specific DNA variant will be detected at a specific temperature or not. Preferentially, positive controls of verified DNA variants are used to refine analysing conditions.
DHPLC Instrument The setup of the DHPLC instrument (Wave system 3500, 3500HT) consists of several individual elements. A schematic view ofthe initial elements is presented in 13
Methods in molecular cardiology: DHPLC mutation detection analysis
Figure 3. The schematic overpiew of the DHPLC technique illustrates the order of analysis. A small volume ofa DNA sample is injected into the liquid system and subsequently DNA-TEAA mokcules are attached onto the solid particles in the column. A permanentflow of a mixture ofBufferA and B is run through the system. Elution of thefragments occurs by raising the temperature and the concentration of BufferB. 77 released DNAfragments kave the column and are detcted by UV260absorption and ilustated aspeaks. Ifnecesary, afragment colkctor can be attached to colct separated DNA fragmentsforfurther analysis.
figure 3. Elements are put on top ofeach other to limit working space. Additionally, the machine is placed in the neighbourhood of an extractor fan to minimise inhalation of poisonous acetonitril fumes. Solid phase The solid phase consists of alkylated nonporous polystyrene-divinylbenzene partides (DNASep, DNASep'T, Transgenomics, San Jose, US) packed onto the column cartridge.'2 The hydrophobic partides can interact with the DNA triethylammonium acetate (DNA-TEAA) complexes. These DNA-TEAA complexes are formed by interaction of the negatively charged doublestranded DNA molecules with the positively charged TEAA molecules (figure 4). The DNA-TEAA complexes are eluted from the column by using an
DNA
increasing concentration of acetonitril as liquid phase. Large DNA particles can bind more TEAA molecules than small DNA fragments, and as a result they are more stably attached to the solid phase than small particles. This explains why large fragments will be eluted at a higher acetonitril concentration (longer elution time) than small DNA fragments. The lifetime of the DNASep column is limited, but can be regenerated after quite some time. An expected number of analyses of 8 to 10,000 runs/column is guaranteed by the manufacturer. Liquid phase The liquid phase consists of two solutions: buffer A and buffer B. BufferAis a mixture of 0.1M TEAA and a low concentration ofactonitril (0.25% ACN, Biosolve LTD. HPLC grade) while buffer B consists of 0.1 M TEAA and a high concentration of acetonitril (25% v/v ACN). Only HPLC grade TEAA (Transgenomics Inc.) is used to make up buffers A and B. Buffers are prepared with low-conductance miliQwater (18.2MW) and made up on a weekly basis or more frequently if required. The column washing solution (buffer C) contains a high concentration of acetonitril (75% v/v ACN) while the syringe washing solution (buffer D) contains 7.5% v/v acetonitril. Buffer C and buffer D are additional solutions and do not contain TEAA ions.
Oven, column pressure and needle injection port
syringe
Figure 4. The negatively charged DNA mokcules interact with the positively charged T7AA molecules. The bydropbobic compex allows the attachment to the hydrophobic particles that are packed onto the DHPLC column.
14
The temperature of the high-precision Peltier oven (Transgenomics Inc, type L7300 or L7300+) and column pressure is monitored on a regular basis. If pressure increases at a low flow rate (0.05 ml/min), the column is cleaned by using 100% buffer C (75% v/v acetonitril) at a flow rate of 0.9 ml/min at a temperature of 80 °C for 15 to 30 minutes. Prior to running samples through the system, the needle and injection port are washed (10 to 20 times) and the
Netherlands Heart Journal, Volume 13, Number 1, January 2005
Methods in molecular cardiology: DHPLC mutation detection analysis
Table 1. DHPLC running conditions at a specific temperature.
Time 0.0 0.1 5.0 5.1 5.7 5.8 8.0
% Buffer A % Buffer B 55 45 50 50 40 60 0 100 100 0 55 45 55 45
Equilibration column Start gradient Elution of fragment Column wash Column wash Equilibration column Equilibration column
Example of a DHPLC method. Running conditions are setup to achieve a linear acetonitrl gradient of 10% within five minutes (2% acetonitril/minute) and 0.9 ml/min flow rate. The gradient is built up by mixing buffer A and buffer B.
syringe is inspected regularly to check for signs of leakage and damage to the plunger seal. Intemal controls An appropriate DNA mutation standard can be used to verify the system (in particular the temperature of the oven) and the condition ofthe column. Depending on the application and method, various standard markers are commercially available. Fragment size standard pUC18/Hae 111 and mutation standard DYS271 are used as instrument controls (Transgenomics Inc, San Jose, US). These internal controls have to be run before and after the series of analysis and whenever maintenance occurs or when buffers, column, in-line filter etc. are changed, otherwise an instrument problem or a loss of heteroduplex resolution caused by an improper cartridge may remain unnoticed. Running negative controls is not necessary.
DHPLC analysis Heteroduplex formation Prior to DHPLC analysis, a reannealing step (TMHA: temperature mediated heteroduplex analysis) is introduced and although this step is not necessary, it may influence the reproducibility of the elution pattern. The following temperature parameters are recommended for efficient heteroduplex formation: 95 °C for five minutes, slowly cooling down (1 °C/min) to room temperature. Subsequently, PCRproducts are run on an appropriate agarose gel to check for efficient amplification, product length and intensities of the fragment bands. It is particularly important that the amount of PCR product is relatively uniform to be able to compare elution profiles within a run.
Sampling and running
Computer spreadsheets allow sample identification and handling to be linked to the appropriate analysing method. Samples are loaded on 96-well plates (Costar) and placed into the Wave apparatus. An aliquot of the
Netherlands Heart Journal, Vohune 13, Number 1, January 2005
unpurified PCR product (5-10 i) is injected on the preheated column. Fragments are eluted from the stationary phase using a fragment specific gradient. The gradient increases by 2% acetonitril per minute at a flow rate of 0.9 ml/min (1.5 ml/min for HT instruments). A final washing step (100% buffer B) finishes the analysing process, and remaining DNA molecules are removed from the column (table 1). The interposition of an accelerator significantly speeds up the final washing step. The gradient ensures that DNA fragments are eluted from the stationary phase at approximately three to four minutes. A gradient profile is usually set up for five minutes, which means a total increase of 10% buffer B (2%/min). For example if a fragment is eluted from the column at 66% of buffer B, the method is said to start at 56% and to end at 66%. Temperature setting As a guideline, the preferred analysing temperature is when 25% of the fragment is denatured, preferably 1 to 2 °C below the empirical calculated melting temperature. If available, positive controls such as known mutations or nucleotide variants are run under identical conditions.
Positive results Abnormal elution profiles or presence ofadditional or shoulder peaks are indicated as 'suspicious'. In practice, a reduced peak signal or peak broadening is suspicious but only if equal amounts of samples have been loaded to the stationary phase. AU abnormal elution profiles have to be verified by sequencing analysis to confirm the presence of DNA variants. To avoid crosscontamination and to eliminate the possibility of detecting PCR artifacts that give rise to the abnormal chromatograms, a second sequence reaction is prepared from a freshly prepared PCR product ofthe patient DNA sample. Alternatively, in case of a common polymorphism, the tested DNA sample can be equally mixed with a sample which is heterozygous for the variant. After heteroduplex formation the sample is reanalysed on the DHPLC. If no apparent change in the profile appears after the addition of the known polymorphism, it is suggested that no other mutation than the known variant is present. Another approach is to compare the tested sample with the profile of a known polymorphism at one or two injection temperatures. It is unlikely that these profiles remain identical at different injection temperatures if they are generated by a different mutation or polymorphism. False-positive results A multiple peak pattern can be produced by the combination of a low fidelity or non-proofreading polymerase or too many cycles during amplification. To circumvent inaccurate amplification and the formation of multiple peak patterns, a proofreading Taq polymerase (e.g. Optimase, Transgenomics Inc.) 15
Methods in molecular cardiology: DHPLC mutation detection analysis
B
A
C
Wild type llelO61le C>T Arg94Leu
D
Arg92Trp
Figure 5. Thisfigure illustrates distinct elution profiles that have been identified within exon 9 ofthe TNNT72gene, which is involved infamilial berrtrpphiccardiomyopathy (FHC). With respect to the elution pattern of the wild-type DNA fragment (A), abnormal elution profiles were observed in B to D. Arrows indicate aberrant and reduced UV,absoption signalsvisualisedaspeaksorshoulder peaks. Subsequently, sequencing analysis of the DNA showingfigure 5B identified a C>Ttransition in codon 106. The aberrant elution profiles in 5D and 5E are the result of two distinct pathogenic missense mutations Arg94Leu (CGC>CTC) and Arg92Trp (CGG>TGG). DNA fragments were eluted from the DNAsep column at 62 °C within 3 to 3.5 minutes using a lineargradient 54 to 64%.
Wild type
delE160
Figure 6. DHPLCprofik ofwild-te (kft) and mutatedfragment (right) illustrates a deletion of three nucleotides (578580delGAG>delE160) in exon 11 of the TNNT2 gene. Running conditions were performed at 57 0CQgradient 56 to 66% buffer B.
Application In cardiology In the Netherlands, DHPLC analysis has been successfully used for a number of genes involved in inherited cardiac disorders, with respect to genes involved in long-QT syndrome, hypertrophic cardiomyopathy and mitochondrial myopathies."'1-7 To
is recommended. This thermostable proofreading DNA polymerase has an efficient 3'->5' exonuclease activity and is advised for single nucleotide polymorphism (SNP) and mutation detection studies using DHPLC. An Optimase ProtocolWriter tool can be consulted for amplification conditions by simply entering the appropriate primers, the expected PCR product length, and selecting an appropriate PCR
protocol type.'3 False-negative results Make sure the mutation standards were analysed correctly, failure to detect mutations may arise for a number of reasons: Poor quality of the amplified product exhibits low signals and the ability to identify mutations is lost. Moreover very high yields can include a large number of mis-incorporations and hence increase difficulty in calling mutations. Incorporation of false nucleotides can occur if non-proofreading DNA poly-
used; Nucleotide changes that occur in a high melt GC-rich domain within the fragment: this inconvenience might be solved by redesigning amplification of fragments, preferably with the GC-rich regions towards the ends of the fragments or by using GC merases are
-
clamps;'4 -
Fragments too small: due to reduced sensitivity in fragments smaller than 150 base pairs sequence variations may not be detected. Redesigning the amplicon is recommended.
16
illustrate what elution profiles may look like, examples of a single nucleotide substitution, a single nucleotide insertion and a deletion of three base pairs are demonstrated in figures 5, 6 and 7. Elution profiles generated by DNA variations in exon 9 ofthe troponin T (TNNT2) gene, involved in hypertrophic cardiomyopathy (CMH2), are shown in figures 5B to D. The single nucleotide substitution (ATC>ATT) representing the elution pattern in figure 5B does not affect the genetic information as both triplets encode for isoleucine (silent mutation) and are designated as coding single nucleotide polymorphism which is present within the normal population (refSNP ID:rs3729547). The elution profiles of two distinct pathogenic missense mutations, which have been identified within two unrelated Dutch families, are shown in figures 5C and D. Both pathogenic mutations had been described previously.18"19 Additionally a deletion ofthree nucleotides, encoding a glutamic acid (delE160), was identified in exon 11 (figure 6). The deletion had occurred within two apparently unrelated families with HCM and sudden death at young age (15 year).20 The MYBPC3 gene encodes the myosin-binding protein C which is involved in familial hypertrophic cardiomyopathy (CMH4). Elution profiles of the reference wild-type DNA and an insertion of a single nucleotide G in exon 25 of the MYBPC3 gene are shown in figure 7A. In the Netherlands the 2373insG has been identified as founder mutation which is present in almost 20% of the Dutch HCM popu-
Netherlands Heart Journal, Volume 13, Number 1, January 2005
Methods in molecular cardiology: DHPLC mutation detection analysis
lation.'6 Finally, using DHPLC analysis ofthe coding region ofthe nonsarcomeric PRKAG2 gene, a missense mutation (Arg302Gln) was identified in a family with clinical features ofWolff-Parkinson-White syndrome and hypertrophic cardiomyopathy (figure 7B).2' Conclusions The rapidly increasing number ofgenes that have been identified in inherited monogenetic cardiac diseases has forced the diagnostic genetic laboratories to implement fast and highly sensitive mutation detection systems. As mutations can now be easily detected by using advanced and high-throughput analysis systems, such as DHPLC, the challenge finding them becomes less teasing. Yet defining whether a variation indeed causes disease becomes more difficult. At the moment the number of naturally occurring validated DNA variants in the human genome is e 4.3 million (National Centre for Biotechnology Information (NCBI) dbSNP Build 120) indicating an occurrence ofapproximately 1 in 500 base pairs. This means that they are regularly found in routine screening causing an additional workload in the diagnostic centre as all aberrant DHPLC profiles have to be reamplified and checked by sequencing analysis. Summarising, the DHPLC technique turns out to be a very rapid and easy method but it should be realised that optimisation of an entire gene takes a lot of time as each fragment has to be optimised and verified by internal controls. A limitation of using highthroughput techniques is the huge amount of data that can be generated within a short period of time. To overcome misinterpretation, analysing software can be used for peak normalisation and automated detection of aberrant DHPLC profiles making mutation detection simpler and reliable which is essential in diagnostic genetic screening. U References Oefner PJ, Underhill P. DNA mutation detection using denaturing high-performance liquid chromatography (DHPLC). In: Dracopoli NC, Haines NC, Korf BR, Morton C, Seidman CE, Seidman JG, et al. (editors). Current Protocols in Human Genetics. New York: Wiley-Interscience; 1998:7(Suppl);10:1-12. 2 Underhill PA, Jin L, Zemans R, Oefier PJ, Cavalli-Sporza LL. A pre-Columbian Y chromosome-specific transition and its implications for human evolutionary history. Pros Natl Acad Sci USA 1993;93:196-200. 3 http://transgenomic.com/flash/DHPLCFlash2.asp. 4 Xiao W, Oefner P. Denaturing High-Performan ce Liquid Chromatography: A review. Hum Mut2001;17:439-74. 5 O' Donovan MC, Oefner PJ, Roberts SC, Austin J, Hoogendoom B, Guy C, et al. Blind Analysis of Denaturating High-Performance Liquid Chromatography as a Tool for Mutation Detection. Genomics 1998;52:44-9. 6 http://insertion.stanford.edu/dhplc_genesl.html. 7 Sonnemans DGP, de Wmdt L, de Muinck EM, Doevendans PA. Methods in molecular cardiology: the polymerase chain reaction. Neth HeartJ2002;10:412-8. 8 http://www.broad.mit.edu/cgi-bin/primer/primer3_www.cgi. 9 http://www.mutationdiscovery.com/. 1
Netherlands Heart Journal, Volume 13, Number 1, January 2005
B
A
Wild type
2373insG
Wild type
Arg3O2Gin
Figure 7. A. Elution patterns ofwild-type (left) and mutated DNA (right) of exon 25 of the MTBPC3 gene. The aberrant profile represents a single nucleotide insertion (2373insG). Note that the longer mutated DNA strand is eluted after the wild-type peak. Running conditions: at 63 OC; gradient 54 to 64% Buffer B. B. Missense mutation CGA>CAA (right profile) in exon 7 of the PRKAG2 gene identified by DHPLC analysis. Running conditions were performed at 59 0C; gradient 52 to 62% Buffer B. 10 Jones AC, Austin J, Hansen N, Hoogendoorn B, Oefner PJ, Cheadle JP, et al. Optimal temperature selection for mutation detection by denaturing high performance liquid chromatography and comparison to SSCP and heteroduplex analysis. Clin Chem 1999;45:1133-40. 11 http://insertion.stanford.edu/melt.html. 12 Bonn G, Hoefner C, Oefner P. Nucleic Acid Separation on Alkylated Nonporous Polymer Beads. US Patent 1996;5:236. 13 http://mutationdiscovery.com/screens/optimase/OptimaseInput.html. 14 Narayanawami G, Taylor P. Improved efficiency of mutation detection by denaturing high-performance liquid chromatography using modified primers and hybridization procedure. Genetic Testing 2001;5:9-16. 15 Jongbloed R, Marcelis C, Velter C, Doevendans P, Geraedts J, Smeets H. DHPLC analysis ofpotassium ion channel genes in congenital long QT syndrome. Hum Mut2002;20:382-91. 16 Alders M, Jongbloed R, Deelen W, Asidah F, van den Wijngaard A, Regitz-Zagrosek V, et al. The 2373insG mutation in the MYBPC3 gene is a founder mutation which accounts for nearly one-fourth of all HCM cases in the Netherlands. Eur Heart J 2003;24:1848-53. 17 Van den Bosch BJ, de Coo R, Scholte HR, Nijland JG, van den Bogaard R, de Visser M, et al. Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatograph. Nucleic Acids Res2000;28:E89. 18 Varnava A, Baboonian F, Davison F, De Cruz L, Elliot PM, Davies MJ, et al. A new mutation of the cardiac troponin T gene causing familial hypertrophic cardiomyopathy without left ventricular hypertrophy. Heart 1999;82:621-4. 19 Moolman JC, Corvield V, Posen B, Ngumbela K, Seidman C, Brink PA, et al. Sudden death due to Troponin T mutations. JAm Coll Cardiol 1997;29:549-55. 20 Watkins H, McKenna W, Thierfelder L, Suk HJ, Anan R, O'Donoghue A, et al. Mutations in the genes for cardiac troponin T and alpha-tropomyosin in hypertrophic cardiomyopathy. NEngl
JMed 1995;332:1058-64.
21 Gollob MH, Green S, Tang AS, Gollob T, Karibe A, Hassan AS, et al. Identification ofa gene resposable for familial Wolff-ParkinsonWhite syndrome. NEnglJMed 2001;344:1823-31.
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