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Clinical Chemistry 47: 1758-1762, 2001;
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(Clinical Chemistry. 2001;47:1758-1762.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

Fluorescence-based, Nonradioactive Method for Efficient Detection of the Pentanucleotide Repeat (TTTTA)n Polymorphism in the Apolipoprotein(a) Gene

Jill Rubin1, Thomas A. Pearson2, Roberta G. Reed3 and Lars Berglund1a

1 Department of Medicine, Columbia University, New York, NY 10032.

2 Department of Community and Family Medicine, University of Rochester, Rochester, NY 14642.

3 Bassett Research Institute, Cooperstown, NY 13326.

aAddress correspondence to this author at: Department of Medicine, Room PH 10-305, Columbia University, 630 West 168th St., New York, NY 10032. Fax 212-305-3213; e-mail lfb9{at}columbia.edu.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Background: The apolipoprotein(a) [apo(a)] gene is a major predictor of plasma lipoprotein(a) concentrations, an independent risk factor for cardiovascular disease. The apo(a) gene contains a pentanucleotide repeat (PNR) polymorphism, 1.4 kb upstream from the apo(a) gene reading frame. This polymorphism has been suggested to be important in control of apo(a) gene expression.

Methods: We developed a fluorescence-based, nonradioactive procedure to detect the PNR polymorphism. After amplification of the polymorphism by PCR, the respective PCR products were separated by denaturing polyacrylamide gel electrophoresis and detected using a 3'-end fluorescently labeled oligonucleotide as a probe. We used the method to characterize the PNR polymorphism pattern in 313 individuals, 195 Caucasians and 118 African Americans. The new method efficiently separated DNAs corresponding to the different PNR repeats.

Results: Among both ethnic groups, alleles containing eight PNRs were most common. Smaller PNRs were more common among African Americans, and larger PNRs were more common among Caucasians.

Conclusions: We developed a nonradioactive technique that separates the PNR polymorphism in the apo(a) gene and can be used in other studies involving closely sized polymorphisms.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Increased lipoprotein(a) [Lp(a)] 1 is an independent risk factor for cardiovascular disease (1)(2)(3)(4)(5)(6)(7)(8)(9)(11). Lp(a) is an LDL-like particle in which apolipoprotein B-100 is bound to another protein, apolipoprotein(a) [apo(a)], via a disulfide bond. apo(a) is present as several size isoforms produced by a variable number of kringle repeats (K4 repeats), so-called because of their resemblance to the kringles found in plasminogen (12). The size variation of the apo(a) protein, i.e., the number of K4 repeats, corresponds to the same size variation in the apo(a) gene. Plasma Lp(a) concentrations are genetically determined and partially regulated by apo(a) size; Lp(a) concentrations and apo(a) size are inversely related (13)(14). Although the apo(a) size polymorphism is an important predictor of Lp(a) concentrations, there is still a pronounced variation in Lp(a) concentrations among individuals carrying apo(a) isoforms of the same size. It is likely that a major part of this variation is attributable to additional genetic factors (14), but these factors have not been completely characterized in detail. Interestingly, although mean Lp(a) concentrations are twice as high in African Americans compared with Caucasians, several studies have failed to establish a significant association between increased Lp(a) and cardiovascular disease among African Americans (15)(16)(17). Recently, we demonstrated that the absence of this association in previous studies was attributable to differences in the distribution of Lp(a) across apo(a) sizes in Caucasians and African Americans (18). Thus, the combination of high Lp(a) concentrations and small-sized apo(a) was a risk factor in both ethnic groups.

In addition to the size variation, the apo(a) gene contains other polymorphisms, including a pentanucleotide repeat (PNR) polymorphism, (TTTTA)n, in the 5'-flanking region of the gene (19). Eight different alleles have been detected to date, with the number of TTTTA repeats ranging from 5 to 12 (20). Several studies have demonstrated an inverse relationship between PNR number and Lp(a) concentrations among Caucasians; alleles containing more repeats were generally associated with lower plasma Lp(a) concentrations (20)(21)(22). Studies on Blacks have been carried out mostly in South Africans and Khoi San, and data on African Americans are limited (23).

The reports on the effects of apo(a) polymorphisms on Lp(a) concentrations and heart disease have led to interest in rapid genotyping of the apo(a) gene for population studies. Although there is no universally accepted procedure to detect the PNR polymorphism, until recently, the primarily used method has been PCR followed by denaturing polyacrylamide gel electrophoresis (PAGE) and detection using a radiolabeled DNA probe. The caveats to using this technique include the radioactivity decay and potential health hazards. In addition, PCR followed by nondenaturing PAGE has been used to detect the PNR polymorphism. However, in the latter method, because of the small 5-bp size difference between adjacent PNR repeats, the separation of bands corresponding to the individual alleles is difficult and interpretations are challenging. To improve this, we have devised a method that does not require radioactivity and adequately separates alleles with a 5-bp size difference. To validate this method, we used our methodology to characterize the PNR frequency in a sample of Caucasians and African Americans.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
labeled oligonucleotide
The probe used for hybridization with the DNA consisted of 11 TTTTA repeats with CC bookends (5'-CCT TTT ATT TTA TTT TAT TTT ATT TTA TTT TAT TTT ATT TTA TTT TAT TTT ATT TTA CC-3'). The oligonucleotide was reconstituted in Tris-EDTA buffer at a final DNA concentration of 100 mmol/L and labeled using the Gene Images 3'-oligolabeling module (Amersham Pharmacia Biotech). 3'-End labeling is optimal for DNA 15–100 bp in size; our probe contained 59 bp. Oligonucleotide (200 pmol) was added to the labeling reaction mixture, and the total reaction (80 µL) was incubated at 37 °C for 90 min. Incorporation of the label was tested by dotting a 5-µL sample of the reaction mixture onto a strip of Hybond N+ (Amersham Pharmacia Biotech) and washing the strip with 2x standard saline citrate (SSC; 1x = 0.15 mol/L NaCl, 0.015 mol/L sodium citrate) for 20 min at 60 °C. Visualization of the Hybond N+ strip on an ultraviolet (UV) transilluminator (FOTO/PREP Fotodyne Inc.) allowed us to check for the presence and the intensity of the incorporated label. The fluorescently labeled probe was stored in the dark at -20 °C for a maximum of 6 months before use.

amplification
Leukocyte DNA was extracted and amplified by PCR in a DNA Thermal Cycler (GeneAmp PCR System 9600; Perkin-Elmer) with oligonucleotide primers -1442 to -1419 (5'-GAA TTC ATT TGC GGA AAG AT-3') and -1336 to -1355 (5'-CAC GTC AGT GCA CTT CAA CC-3'). In addition to the buffers and nucleotide components as recommended by the Taq polymerase distributors (Gibco BRL), each amplification reaction contained 1µg of DNA, 20 pmol of each primer, and 1.5 U of Taq polymerase in a final reaction volume of 50 µL. Each reaction mixture was heated to 94 °C for 7 min for initial denaturation, after which time the Taq polymerase was added and the final reaction volume was reached. This was followed by 2 min at 94 °C, 1.5 min at 50 °C, and 1 min at 72 °C for 35 cycles and a 10-min final extension at 72 °C.

southern transfer
Ten microliters each of the PCR product and the loading buffer (980 mL/L formamide, 0.1 g/L bromphenol blue, 0.1 g/L xylene cyanol, and 10 mmol/L EDTA) were mixed and boiled in water for 5 min. A 15-µL portion of each reaction mixture was loaded onto a prerun 6% denaturing polyacrylamide gel (42 g of urea, 43.5 mL of water, 10 mL of 10x Tris-borate-EDTA, 15 mL of 400 g/L acrylamide, 1 mL of 100 g/L ammonium persulfate, and 30 µL of TEMED) and electrophoresed for 2 h at 250 V on an SE600 series vertical Slab Gel unit (Hoefer Pharmacia Biotech). After electrophoresis, the gel was depurinated in 250 mmol/L HCl for 15 min, denatured in 0.5 mol/L NaCl–1.0 mol/L NaOH for 20 min, and neutralized in 0.5 mol/L Tris-HCl (pH 7.4)–1.5 mol/L NaCl. The DNA was transferred to Hybond N+ in 1x Tris-acetate-EDTA buffer for 2 h under constant current (1 A) at room temperature. Following transfer, the DNA was UV-fixed to the membrane by a UV cross-linker (Hoefer Pharmacia Biotech).

hybridization and detection
The membranes were washed in 900 mL/L ethanol for 10 min, rinsed twice with 5x SSC, and added to hybridization tubes containing 0.25 mL/cm2 preheated (42 °C) hybridization buffer [5x SSC, 1 g/L sodium dodecyl sulfate (SDS), 5 g/L dextran sulfate, and a 20-fold dilution of liquid block (Gene Images CDP-star detection module, Amersham Pharmacia Biotech)]. The blot was prehybridized at 42 °C for 1 h, and 5 µg/L denatured probe was then added. After overnight hybridization, the blot was washed twice with 5x SSC containing 1 g/L SDS for 5 min at room temperature and twice with preheated 1x SSC containing 1 g/L SDS for 15 min at 48 °C. The membrane was transferred to a clean Pyrex dish and incubated for 1 h at room temperature with 1 mL/cm2 of a 1:10 dilution of liquid block in buffer A (100 mmol/L Tris-HCl, 300 mmol/L NaCl, pH 9.5). This was followed by a 1-h incubation with 0.3 mL/cm2 of a 5000-fold dilution of the alkaline phosphatase (AP) conjugate in buffer A, followed by three 10-min washes using 2 mL/cm2 of 3 mL/L Tween 20 in buffer A. Detection reagent (40 µL/cm2; Gene Images CDP-star detection module) was added for 5 min, at which time the membrane was exposed to Kodak X-omat film. The probe-bound AP conjugate catalyzes light production by enzymatic decomposition of a stabilized dioxetane substrate. The Gene Images CDP-star detection module is an improved version of the Fluorescein Gene Images dioxetane module (Amersham Pharmacia Biotech): more rapid light output produces stronger signals in less time. All hybridization and detection steps were carried out in ethanol-rinsed dishes and with autoclaved reagents to avoid contamination of the membranes by exogenous AP.

The PCR products were determined using calibrators (PNR 8/9 and 8/11) that consistently were included in all experiments. The size of the calibrators was determined using DNA molecular weight markers that span the entire range of the PNR allele sizes in 12% nondenaturing PAGE.

study population
DNA samples from 313 participants in the Harlem-Bassett Study were included in the present study. The design of the study and the recruitment procedure have been described in detail previously (18)(24). In all, 648 patients (232 African Americans, 344 Caucasians, and 72 others) were recruited in the Harlem-Bassett Study, and the present study is based on a random sample of 118 African Americans and 195 Caucasians. The study was approved by the Institutional Review Boards at Harlem Hospital, the Mary Imogene Bassett Hospital, and Columbia University College of Physicians and Surgeons. Proportions were compared between the two ethnic groups using {chi}2 analyses.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
A typical example of the efficient separation of adjacent PNR repeats obtained using the technique described here is illustrated in Fig. 1 . The genotype patterns from 10 different individuals, 6 heterozygotes and 4 homozygotes, are presented. The excellent separation of bands with a difference of only a few bases can be seen in lanes 3 and 4 of Fig. 1 , in which individuals with an 8- and a 9-PNR allele are represented, and in lane 10, in which an individual with a 9- and a 10-PNR allele is represented; there is a clearly distinct partition between the bands. This pattern was also easily distinguishable from the patterns in lanes 1 and 9, which represent individuals heterozygous for the 8- and 11-PNR alleles; and lane 7, which represents an individual heterozygous for the 8- and 10-PNR alleles. The presence of homozygosity, expressed as a single band, such as 8/8 in lanes 2, 6, and 8 or 10/10 in lane 5 of Fig. 1 , was also easy to distinguish from a heterozygous pattern with adjacent allele sizes. Furthermore, the two alleles from the 8/10 heterozygote in lane 7 (Fig. 1 ) correspond nicely to the 10/10 and 8/8 homozygotes in lanes 5 and 6, respectively.



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  		Figure 1. Genomic blot of PCR products after electrophoresis on a 6% denaturing gel.

The blot was hybridized with a 3'-end fluorescence-labeled PNR probe and detected using CDP-star detection module (as described in Materials and Methods). Presented are two different experiments; lanes 1–4 are from one gel, and lanes 5–10 are from another gel. The numbers above each lane represent the number of PNRs on each of the apo(a) alleles and were determined using 8/9 and 8/11 PNR size calibrators, previously determined using nondenaturing gel techniques as described in Materials and Methods.

This clarity in genotyping contrasted to results obtained with a more conventional nondenaturing gel electrophoresis technique as seen in Fig. 2 . The latter method could not easily distinguish heterozygotes with adjacent PNR sizes from homozygotes. For example, an individual heterozygous for the 8/9-PNR alleles could be inappropriately characterized as an 8/8 or 9/9 homozygote, and conversely, an 8/8 or 9/9 homozygote could be inappropriately characterized as an 8/9 heterozygote. In addition, separation of heterozygotes with close allele size differences, e.g., individuals heterozygous for the 8/9- and 8/10-PNR alleles, was also quite difficult in the nondenaturing gel electrophoresis technique, leading to uncertain interpretation. The results in Fig. 2 obtained with the conventional nondenaturing gel electrophoresis technique illustrate the problems of resolution of alleles that differ in size by only 5 bp. As seen in Fig. 2 , the boundaries of the DNA bands were not well defined and there was no clear space between the bottom of one DNA band and the top of an adjacent DNA band. Even for homozygotes, where the band thickness was commonly less than for heterozygotes, we could not with absolute confidence determine the pattern. It is clear from Figs. 1Up and 2 that there was a striking difference in the efficiency of separation that was observed. Accordingly, the results obtained with the methodology illustrated in Fig. 1Up can be presented much more confidently than those obtained with the technique in Fig. 2 . Efforts to improve the conventional approach by use of various polyacrylamide concentrations between 10% and 20% did not improve the separation (data not shown).



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  		Figure 2. Analysis of PCR products after nondenaturing gel electrophoresis on a 12% polyacrylamide gel.

PCR products were electrophoresed overnight, and the gel was stained with ethidium bromide and visualized on a UV illuminator. A DNA ladder, spanning the 93- to 128-bp range of the PNR, was used to determine the number of PNRs on each of the alleles.

To validate the present method and to extend previous studies to an African-American sample, we genotyped random samples from patients participating in the Harlem-Bassett Study (18)(24). In previous studies, the range of alleles for the PNR polymorphism spanned 5 to 12 TTTTA repeats, the 8-PNR allele being the most common. In the studied population, the PNR size range was between 5 and 11 PNR repeats. The relative distribution of the PNR polymorphism for the 195 Caucasians and 118 African Americans is shown in Fig. 3 . In agreement with previous studies (20)(21)(22)(23), we found that an allele with 8 repeats was the most common among Caucasians (58%). Previous studies in African populations have found similar dominance of the 8-PNR allele; in our African-American sample, the 8-PNR allele constituted an allele frequency of 62%. In addition, we found that smaller PNRs (5–7 repeats in length) were more common among African Americans than among Caucasians (15% vs 2.6%), and larger PNR (9–11 repeats in length) were more common among Caucasians than among African Americans (39.5% vs 22.5%: P <0.0001). Our results for Caucasians are in line with previously published studies, in support of the validity of the present technique. Although there are virtually no data reported for African Americans, our results were generally in concert with those reported for South Africans (23).



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  		Figure 3. Distribution of the PNR polymorphism among Caucasians and African Americans.

The most common allele in both populations was the 8-PNR allele (58% and 62% in Caucasians and African Americans, respectively). Smaller PNRs were observed more among the African-American population and larger PNRs among the Caucasian population.

Although it is known that genetic variation at the apo(a) gene locus predicts a major part of the variation in plasma Lp(a) concentrations, the exact mechanisms behind this regulation remain to be clarified (14). Interestingly, the genetic control seems to differ between Africans and Caucasians (25). In both groups, the apo(a) size variation is the quantitatively most important variation, but it corresponds to only approximately one-half of the genetic variation. Because of the possibility of linkage disequilibrium, it is likely that detailed analysis of apo(a) gene expression in future studies will require simultaneous determinations of several polymorphisms (22)(23)(26). For such studies, the present method offers a robust and easily interpretable procedure to assess the PNR polymorphism.

In conclusion, we believe the method described here allows for successful genotyping of the PNR polymorphism in the apo(a) gene. Furthermore, we believe our approach could also be applied to other genes containing polymorphisms with closely sized alleles for which common genotyping techniques do not provide efficient separation.


   Acknowledgments
 
The project was supported by Grants 49735 and 62705 from the National Heart, Lung and Blood Institute.


   Footnotes
 
1 Nonstandard abbreviations: Lp(a), lipoprotein(a); apo(a), apolipoprotein(a); PNR, pentanucleotide repeat; PAGE, polyacrylamide gel electrophoresis; SSC, standard saline citrate; UV, ultraviolet; SDS, sodium dodecyl sulfate; and AP, alkaline phosphatase.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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