# Simple Detection of Large InDeLS by DHPLC: The ACE Gene as a Model

Authors: Renata Guedes Koyama; Rosa M. R. P. S. Castro; Marco Túlio De Mello; Sergio Tufik; Mario Pedrazzoli
Journal: Journal of Biomedicine and Biotechnology
 (2008)

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## Abstract

Insertion-deletion polymorphism (InDeL) is the second most frequent type of genetic variation in the human genome. For the detection of large InDeLs, researchers usually resort to either PCR gel analysis or RFLP, but these are time consuming and dependent on human interpretation. Therefore, a more efficient method for genotyping this kind of genetic variation is needed. In this report, we describe a method that can detect large InDeLs by DHPLC (denaturating high-performance liquid chromatography) using the angiotensin-converting enzyme (ACE) gene I/D polymorphism as a model. The InDeL targeted in this study is characterized by a 288 bp Alu element insertion (I). We used DHPLC at nondenaturating conditions to analyze the PCR product with a flow through the chromatographic column under two different gradients based on the differences between D and I sequences. The analysis described is quick and easy, making this technique a suitable and efficient means for DHPLC users to screen InDeLs in genetic epidemiological studies.


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## Body

## 1. Introduction

Insertion-deletion (InDel) polymorphisms are an important and abundant form of human genome variation; they are the second most frequent type of polymorphisms in the human genome and may be observed
at the level of single-base pairs, multibase pair expansions of repeat units,
random DNA sequence insertions and deletions, and as transposon insertions.
This kind of DNA polymorphism can be used for the purpose of genetic mapping
and diagnostics [1].It is well known that
translocation insertions and gross deletions (>100 pb) are important
causes of both cancer and inherited diseases. Microdeletions and
microinsertions (≤20 bp) account for 17% of all inherited diseases, as
reported in the May 2007 release of the human gene mutation database (www.hgmd.org).An example of such polymorphism is the insertion/deletion (I/D) in the angiotensin-converting
enzyme (ACE) gene that has been
associated with many diseases, particularly of the cardiovascular system.
Reports of such associations include risk of myocardial infarction and
cardiovascular disease, ischemic stroke, effects on response of human muscle to
strength training [2–6], and hypertension in subjects with mild to moderate degrees
of sleep apnea (5≤ apnea-hypopnea index ≤30) [7].In the renin-angiotensin system, ACE is a well-known zinc metallopeptidase
that is widely distributed on the surface of endothelial and epithelial cells.
ACE plays a role in the conversion of the inactive decapeptide, angiotensin I
(Ang I or Ang 1–10), into the active octapepptide and potent vasoconstrictor
angiotensin II (Ang II or Ang 1–8) as well as in the inactivation of the
vasodilator bradykinin [8, 9].The ACE InDel polymorphism is
characterized by the presence (insertion, I) or absence (deletion, D) of a 288 bp DNA sequence in intron 16 of the gene. This insertion sequence is an Alu
element (three Alu-repeat).Although several genetic studies of the ACE D allele have associated
polymorphism with cardiovascular disease, these data are questionable due to
probable mistyping of the ACE I
allele [10]. This problem occurs because of the different sizes of I and D
allele PCR products, which are 490 bp and 190 bp, respectively. But it happens
that the D (shorter) allele possess the property of being preferentially
amplified compared to the I allele, and this differential response to
amplification may lead to mistyping of the ID sample.Conflicting reports of ACE I/D frequencies have also been described,
given the difficult nature of this genotyping. The diversity of findings has
been attributed to methodological and technical variations in detection of the
polymorphisms [11].The first description of ACE I/D polymorphism genotyping proposed by
Rigat et al. [12] was a PCR method using a set of primers flanking the
insertion. However, as mentioned earlier, the D allele is preferentially
amplified, so the ID heterozygote can be mistyped as DD, thus causing a higher
frequency for this genotype. The probability of this mistyping has been
estimated to be 5–10% [13, 14].
Therefore, careful control is required and repeated testing is also often
necessary, especially when verifying the ID heterozygote.This method has been modified several times, and a confirmatory PCR has
been proposed by Shanmugan et al. [15] to minimize mistyping of the I allele as
a D allele. In this method, a new sense
primer is used inside the Alu sequence, resulting in amplification of a 408 bp
fragment of allele I. The D allele shows no amplification under this condition
due to lack of an annealing site for the new sense primer, so it works as an
additional amplification after the conventional method. The authors reported
100% accuracy with this methodology.
However, use of a primer annealing inside an Alu sequence may cause
nonspecific annealing due to structural differences in the I and D allele in
which there are three Alu sequences. Therefore, problems with preferential
amplification by PCR are not entirely excluded with this method [16]. Eight human genes specific to Alu insertion
polymorphisms have been described (ACE, TPA25, PV92, APO, FXIIIB, D1, A25, and B65) [17], and theoretically, any of
them can be amplified by PCR using a primer that anneals in the Alu sequence.
Moreover, this methodology generally involves various steps and is
time consuming.Other modifications have included a step-down PCR, as described by
Chiang et al. [16], which aims to increase the product and the detection rate
of the I allele. This method involves initial PCR annealing temperatures higher
than the melting point of the primers, followed by annealing temperatures
reduced stepwise to the melting point. In this technique with high
amplification failure, the results should be interpreted by two different
observers, and consensus opinions have to be obtained from a third observer
blinded to the results from the other two.The
low resolution of these methods can lead to difficulties in data
interpretation, and also requires various time-consuming steps. Therefore, a more efficient means of ACE genotyping is particularly desirable
for clinical and epidemiologic investigations.
Other techniques have been purposed, such as real-time PCR [18], to detect ACE ID polymorphism, but this has not
been used in genetic association studies, likely because of the need for
specific and more expensive reagents. The multiplex approach, proposed by Evans et al. [19], improves the
accuracy of ACE genotyping, but
presents difficulties in the post-PCR handling, such as agarose diagonal gel
electrophoresis. Moreover, to
our knowledge, the multiplex approach is specific to ACE InDel genotyping and cannot be extended to other systems.Here, we report a protocol using DHPLC (denaturating high-performance
liquid chromatography) to genotype large InDels employing the ACE gene I/D polymorphism as a model.
The major advantage of this method is the ease it provides in screening a large
number of samples while avoiding electrophoresis and gel analysis.The technique is based on automated detection of DNA segments by
ion-pair reverse-phase high performance liquid chromatography [20]. This approach normally compares two alleles
after denaturing and reannealing PCR amplicons. In this methodology, a
preliminary quality analysis of the amplicon is always done in nondenaturing
conditions at 50°C in order to quantify and verify the quality of the PCR
product. Under such nondenaturing conditions, the presence of InDels in
single-base pairs or small repeat units can be detected [21–24]. This method does not work well, however, in
detecting large InDels (>50 pb) because the gradient flow is designed for
small (deletion) sequences [25].In the present study, we
chose to analyze the amplicon using the DHPLC instrument at a nondenaturating
temperature, using the same PCR product to flow through the chromatography
column under two different gradients, which were based on the predicted D and I
allele sequences. We compared this DHPLC method with the conventional and
confirmatory methods for ACE I/D polymorphism
genotyping, and we propose this methodology for genotyping InDels throughout
the genome.


## 2. Materials and Methods

### 2.1. Samples

Genomic DNA was directly extracted from 3 mL of whole blood [26] from
335 volunteers with mixed ethnic backgrounds, after obtaining their written informed
consent.


### 2.2. Conventional PCR

Genotyping of the ACE gene was
performed in all 335 samples, as described by Rigat et al. [12]. The primers
anneal outside the insertion/deletion region in intron 16 of the ACE gene and yield a PCR product of 490
base pairs (bp) in the case of the insertion allele or 190 bp in the case of
deletion allele. Depending on the presence or absence of the insertion allele,
the genotype of the subjects was classified as II (homozygote for the insertion
allele), DD (homozygote for the deletion allele), or ID (heterozygote).


### 2.3. Confirmatory PCR

Each sample found to be of the DD genotype using the conventional method
was subjected to a second independent PCR amplification with a set of primers
that recognize the Alu insertion-specific sequence, as described by Shanmugan
et al. [15]. A PCR product of 408 bp
indicated the insertion allele.


### 2.4. DHPLC Analysis

DHPLC analysis was performed in each sample found to have the DD
genotype using the conventional method [12].Genomic DNA samples were
subjected to PCR using 10 mM of forward
primer and 10 mM of reverse primer, as
described by Rigat et al. [12], in a solution containing 1.0 mM MgCl+2,
2.5 mM Tris-HCl (pH 9.0), 1.0 mM each of dNTP, and 1 U of Platinum Taq DNA polymerase
(Invitrogen, SP, Brazil).We entered the two predicted sequences, representing the ACE gene I and D alleles (NCBI ref
X62855, SNP: rs13447447-Figure1), into the system to create two different gradients of buffers and
acetonitrile. The system control software (Transgenomic Navigator Software
version 1.5.4, Transgenomic Inc., USA) gave the flow rate of the
reagents based on the predicted sequences. The reverse phase gradient was
performed under two different conditions, one for each allele. In condition 1,
for the D sequence, the gradient was initially set to 47.2% 0.1 M TEAA, pH 7.0 (buffer A) mixed with 52.8% 0.1 M TEAA,
pH 7.0, v/v 25% acetronitrile (buffer B). At the end point (5 minutes), the
gradient was 38.2% buffer A and 61.8% buffer B. The gradient used for condition
2, for the large sequence (I), started with 39.2% of buffer A mixed with 60.8%
of buffer B, and at the end point (5 minutes) the gradient was composed of
30.2% buffer A and 69.8% buffer B.Observed chromatograms patterns in the DHPLC system
at 50°C after submitting the same samples to the two conditions. (a) ACE I
sequence and chromatogram. (b) ACE D sequence and chromatogram. The lines in the upper part of both chromatograms show I/I genotype, the line in the middle is the D/D genotype, and the line below is the I/D genotype.
The homozygotes show a peak when their sequences are analyzed, and the
heterozygotes show a peak in both analyses.
(a)(b)EightμL of PCR
product was applied twice to a DNASep column (Transgenomic-Wave 3500A DHPLC
system, ref DNA 99 3510, 4.6 mm × 50 mm, Transgenomic Inc., USA).
Elution of DNA was detected by 260 nm UV absorbance, and the chromatograms were
analyzed by the presence or absence of amplicon at 50°C with the two gradients
and flows (Figure 1).


## 2.1. Samples

Genomic DNA was directly extracted from 3 mL of whole blood [26] from
335 volunteers with mixed ethnic backgrounds, after obtaining their written informed
consent.


## 2.2. Conventional PCR

Genotyping of the ACE gene was
performed in all 335 samples, as described by Rigat et al. [12]. The primers
anneal outside the insertion/deletion region in intron 16 of the ACE gene and yield a PCR product of 490
base pairs (bp) in the case of the insertion allele or 190 bp in the case of
deletion allele. Depending on the presence or absence of the insertion allele,
the genotype of the subjects was classified as II (homozygote for the insertion
allele), DD (homozygote for the deletion allele), or ID (heterozygote).


## 2.3. Confirmatory PCR

Each sample found to be of the DD genotype using the conventional method
was subjected to a second independent PCR amplification with a set of primers
that recognize the Alu insertion-specific sequence, as described by Shanmugan
et al. [15]. A PCR product of 408 bp
indicated the insertion allele.


## 2.4. DHPLC Analysis

DHPLC analysis was performed in each sample found to have the DD
genotype using the conventional method [12].Genomic DNA samples were
subjected to PCR using 10 mM of forward
primer and 10 mM of reverse primer, as
described by Rigat et al. [12], in a solution containing 1.0 mM MgCl+2,
2.5 mM Tris-HCl (pH 9.0), 1.0 mM each of dNTP, and 1 U of Platinum Taq DNA polymerase
(Invitrogen, SP, Brazil).We entered the two predicted sequences, representing the ACE gene I and D alleles (NCBI ref
X62855, SNP: rs13447447-Figure1), into the system to create two different gradients of buffers and
acetonitrile. The system control software (Transgenomic Navigator Software
version 1.5.4, Transgenomic Inc., USA) gave the flow rate of the
reagents based on the predicted sequences. The reverse phase gradient was
performed under two different conditions, one for each allele. In condition 1,
for the D sequence, the gradient was initially set to 47.2% 0.1 M TEAA, pH 7.0 (buffer A) mixed with 52.8% 0.1 M TEAA,
pH 7.0, v/v 25% acetronitrile (buffer B). At the end point (5 minutes), the
gradient was 38.2% buffer A and 61.8% buffer B. The gradient used for condition
2, for the large sequence (I), started with 39.2% of buffer A mixed with 60.8%
of buffer B, and at the end point (5 minutes) the gradient was composed of
30.2% buffer A and 69.8% buffer B.Observed chromatograms patterns in the DHPLC system
at 50°C after submitting the same samples to the two conditions. (a) ACE I
sequence and chromatogram. (b) ACE D sequence and chromatogram. The lines in the upper part of both chromatograms show I/I genotype, the line in the middle is the D/D genotype, and the line below is the I/D genotype.
The homozygotes show a peak when their sequences are analyzed, and the
heterozygotes show a peak in both analyses.
(a)(b)EightμL of PCR
product was applied twice to a DNASep column (Transgenomic-Wave 3500A DHPLC
system, ref DNA 99 3510, 4.6 mm × 50 mm, Transgenomic Inc., USA).
Elution of DNA was detected by 260 nm UV absorbance, and the chromatograms were
analyzed by the presence or absence of amplicon at 50°C with the two gradients
and flows (Figure 1).


## 3. Results

From the initial 335 samples,
95 were found to be of the DD genotype. These were reevaluated using the
conventional genotyping method, as well as the confirmatory and DHPLC methods
for comparison. We confirmed that 81.05% of the 95 samples were the DD
genotype. However, in using the DHPLC and confirmatory methods, 18.95% of the
samples proved to be the ID genotype. In two cases (2.1%) only the DHPLC
detected the ID genotype and in another two cases (2.1%) only confirmatory PCR
detected the ID genotype (Table1).Table 1
Frequencies of DD only genotype using DHPLC and confirmatory methods.
Genotype% (n)Confirmatory % (n)DHPLC % (n)DHPLC + Confirmatory % (n)ConcordancesDifferencesDD100 (95)81.05 (77)81.05 (77)78.95 (75)2.11 (2)ID018.95 (18)18.95 (18)16.84 (16)2.11 (2)An example of the pattern found using the DHPLC method is shown in
Figure1. The DD genotype was only detected by the specific DHPLC gradient and
flow designed for the shorter PCR product (see Mat&Meth DHPLC condition 1), and the II genotype
was detected only by the specific DHPLC gradient and flow designed for the
larger PCR product (see Mat&Meth
DHPLC condition 2). When we had heterozygosity (I/D genotype sample), we
observed peaks in the two different gradient and flow conditions.


## 4. Discussion

In this report, we established the use of DHPLC for rapid screening of
large InDels such as ACE I/D
polymorphisms. Compared to other genotyping techniques, DHPLC offers several
technical advantages. In a large-scale genotyping setting such as population
screening, the adaptability of DHPLC, along with its high throughput, should
significantly reduce overall processing time. Also, the autorun mode of DHPLC
significantly decreases handling time without the loss of assay specificity
[27–29],  and screening is relatively quick and easy (8 minutes for total run
per sample after PCR, including sample injection, column equilibration, and
cleaning).The method
presented here offers several specific advantages over the other methods used
to genotype ACE I/D polymorphisms, in
which the false-positive mistyping of the DD genotype is a concern. The
probability of mistyping has been cited by some authors to range from 5–10%
[13, 14], but in our samples we detected a discrepancy in 19% of cases. Certainly, this accuracy depends on
the observer’s expertise in performing PCR gel electrophoresis analyses. The
confirmatory PCR method, used to overcome this problem, is time consuming since
two PCRs must be performed, and some believe that the results (the PCR bands in
the gel) should be assessed by more than one observer to avoid
misinterpretation [15]. This method has been reported to have 100% accuracy in
genotyping the ID heterozygote. However, in our study, the ID heterozygote was
not detected in 2% of all samples. Chiang et al. [16] also showed decreased
detection of the I/D heterozygote using this method relative to the step-down
approach. DHPLC has the advantage of genotyping many samples without
supervision and the DHPLC can run overnight with automatically generated
results. If the PCR
conditions is carefully optimized
and the injected
amplicon is unique and pure, the exit pattern shows only one clear and
recognizable peak, thus avoiding the necessity of analysis by two independent
observers.DHPLC methodology makes genotyping of
slightly modified DNA, like SNPs and
small InDels (1 to 50 bp) feasible for numerous samples and has been used in
many studies [30–32]. Traditionally, DHPLC has not been recommended for large
InDels, because the gradient flow is designed for one sequence (deletion, e.g.), thus rendering the other (insertion) unrecognizable [24, 25]. We
observed this phenomenon when we analyzed the ACE amplicon in a single DHPLC
run, but in this study we demonstrated that it is possible to overcome this
problem with two separate runs using different gradient flows for each allele.
Therefore, DHPLC may be used to detect large InDels (up to ~200 bp) if one
analyzes the amplicon using two nondenaturating runs with two buffer gradients
and two flow adjustments.To the best of our knowledge, this is the first report of a DHPLC
selective approach to genotyping large InDels using two sequences input for two
gradients of buffers and two flow adjustments to analyze both sequences
separately. A multiplex-PCR coupled to HPLC analysis under nondenaturating
conditions for detection of large InDels [33] has already been proposed,
but small nonspecific peaks might hamper the analysis.We have shown in this study that this DHPLC method is highly efficient
and reproducible in the detection and genotyping of the ACE ID polymorphism, which has been a challenging goal to achieve. In our study, two
cases with the ID genotype were not detected by DHPLC but were detected by
confirmatory PCR. In another two cases, only DHPLC, but not confirmatory PCR,
detected the ID genotype. There is no clear explanation for this, but we
conclude that identification of large InDels, like the ACE ID, are very difficult to genotype using only one methodology. Moreover,
in confirmatory
PCR a primer inside the Alu sequence is used, and the two
samples not detected by
DHPLC could have amplified other genes to Alu insertion
polymorphisms.Comparing DHPLC and the confirmatory PCR methodology used for the same
purpose, DHPLC has the advantages of ease of handling and significantly fewer
problems with data interpretation. Besides its application in ACE I/D polymorphism, DHPLC is a
powerful tool for clinical and population analysis of InDels in general, which
may greatly increase laboratory throughput.


## 5. Conclusions

InDels may be observed as one- or multibase pair expansions of repeat
units, besides random DNA sequences as transposon insertions and deletions and
their detection is used for the purpose of genetic mapping and diagnostics. The
DHPLC method usually can detect small repeat units under nondenaturating
conditions. With the approach described in the present study, DHPLC can be used
to genotype large InDels, as demonstrated in the detection of ACE gene I/D polymorphisms. This method could be useful in clinical applications, and
carries the advantage of avoiding possible gel misinterpretation and, thus,
genotype misinterpretations. In
conclusion, the DHPLC design described in this study is an alternative approach
for large InDel detection and could be used in studies that evaluate this kind
of polymorphism.



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*Source: 562183-2008-04-20.xml*