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Technical Briefs |
1
I. Dept. of Obstet. and Gynaecol., Semmelweis Univ. Med. School, Budapest, Hungary;
2
Dept. of Molec. Pathol., Inst. of Pathol., Algernon Firth Bldg., Univ. of Leeds, Leeds, UK;
Huntington disease (HD) is an autosomal dominant progressive neurodegenerative disorder characterized by motor disturbance, cognitive loss, and psychiatric manifestations. Mapping of the putative HD gene to chromosome 4 in 1983 [1] facilitated presymptomatic testing of people at risk for HD by using linked polymorphic DNA markers. This method required DNA from related individuals to track the putative HD allele within a family. The situation changed after the gene responsible for HD was identified in 1993 [2], and a new method, based on PCR, became available for the detection of the disorder. This new method also enabled direct mutation analysis and genetic counseling for new mutation HD families. The mutation mechanism was found to be the expansion of a CAG repeat in the 5'-translated region of the HD gene. The mechanism by which the increased trinucleotide repeat length leads to the characteristic clinical symptoms and neuropathology of HD is, as yet, unknown. The CAG repeat of the HD gene is polymorphic in the population, varying between 8 and 36 repeats on normal chromosomes and is expanded with >37 repeats in chromosomes of HD-affected individuals [3].
Since the first description of a PCR method to detect the mutation in the HD gene was published, there have been difficulties reproducing these results (2). The difficulties in the PCR are caused by the composition of the DNA sequence 3' to the CAG repeat, which has a high GC content and is also highly repetitive. The other difficulty with the initial method was that it utilized primers flanking a region of a polymorphic CCG repeat as well as the CAG repeat at the 5' end of the gene. This leads to a PCR test being developed that is easy to use and that does not amplify the CCG region (4). This detection of the number of CAG repeats allows the direct presymptomatic diagnosis of the disease.
Although this modified method is more accurate and produces cleaner gel bands than can be obtained by the first primer set, the measurement of the length of the trinucleotide repeat remains very difficult. The repeat length detection requires radioactive analysis or silver staining of polyacrylamide gels, which produces several shadow bands around the main allele band due to the nature of the repeat amplifications. In addition, it is often very difficult to distinguish between the main and shadow bands when they have similar intensities. Although allele sizes can be determined by using appropriate size markers and controls with known numbers of CAG repeats, it is difficult to size the alleles for two reasons. First, if the product yield is low, it produces a very faint signal, and second, as the migration rate across the gel is different in different lanes, the calibrator size values may be rendered inaccurate.
Because the CAG repeat can expand over 60 units or even more, the intensity of larger HD alleles can be extremely weak and therefore cannot be detected by conventional methods. In these cases the amount of PCR products cannot be increased by increasing the number of PCR cycles, as this causes high background (5) and confuses the detection of the products on the gel. Therefore conventional PCR and the usual detection of products are generally not suitable for appropriate presymptomatic diagnosis of individuals at risk for HD. Particular difficulties occur when the number of CAG repeats are in the range of 35 to 38. This requires a very exact measurement for predicting the risk of the individual or when a weak PCR signal is obtained from an expanded allele.
We report a suitable method to solve these problems by amplifying alleles from the CAG region of the HD gene by fluorescent PCR.
The fluorescent PCR method (6) has particular advantages in the detection of HD alleles compared with the conventional method: It is more sensitive, it can be used for quantitative measurement of bands, and fewer PCR cycles are required for the same level of detection, resulting in cleaner band pictures and easier interpretation. Further advantages are that signals can be easily visualized even if very weak, allowing the detection of alleles with high numbers of CAG repeats, and that highly accurate molecular mass determination is possible because of the inclusion of internal size calibrators.
We report our fluorescent PCR-based method for accurate detection of CAG numbers from the 5' region of the HD gene. Our previously described PCR conditions (7)(8) for the detection of CAG repeats have been modified to take into account the requirements of fluorescent PCR and use of accurate sizing of alleles. The primers were 5'-5-carboxyfluorescein (FAM)-ATG AAG GCC TTC GAG TCC CTC AAG TCC TTC-3' and 5'-GGC GGT GGC GGC TGT TGC TGC TGC TGC TGC-3' (4). PCR amplification was performed in 40 µL volume, and the reaction mixture consisted of 300–500 ng of DNA, 0.625 mmol/L magnesium chloride, 50 mmol/L potassium chloride, 240 µmol/L of each dNTP, 120 g/L dimethyl sulfoxide, 20 pmol of each primer (one labeled with FAM), and 1.25 U of AmpliTaq (Perkin-Elmer). After initial denaturation at 95 °C for 5 min, denaturation, annealing, and extension were carried out at 95 °C (30 s), 64 °C (45 s), and 72 °C (45 s), respectively, for 28 cycles in a Perkin-Elmer thermocycler 2400 followed by a 10-min extension at 72 °C. Two microliters of PCR products were mixed with 24 µL of formamide and 1 µL of Genescan-500 TAMRA size calibrator. The mixture was denatured at 95 °C for 3 min and placed on ice until analysis. Electrophoretic analysis was performed with POP4 gel and a ABI 310 Genetic Analyzer (Applied Biosystems). The amplified products were analyzed by Genescan Analysis 2.1 software (Applied Biosystems). Accurate sizing of alleles was determined by using the internal size marker, allowing relative allele peak areas to be calculated.
Figure 1
demonstrates the analysis result of a patient (family member V)
at risk for HD. The patient's brother (family member IV) has 17/38 CAG
repeats and shows clinical signs of the disease. His brothers and
sisters asked for predictive testing, and because of the borderline
repeat number and the possibility of contraction of this repeat, it was
necessary that an accurate measurement be performed. This measurement
was carried out with the above fluorescent PCR method and a contracted
allele with 35 CAG repeat units was detected, thereby indicating that
the patient is at low risk for developing HD. CAG repeat numbers of
further family members are: family member I, 17/19 repeats; family
member II, 13/36 repeats; and family member III, 1336 repeats.
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Table 1
illustrates the peak heights of alleles corresponding to
different CAG repeat units. These results indicate the reliability of
this fluorescent PCR method for very accurate size detection of
Huntington alleles in the intermediate and also in the expanded range.
The method is simple, rapid, and, we believe, is more accurate than any
other techniques currently used for sizing the HD alleles. The method
allows analysis within a few hours and in addition does not require
isotopic manipulation and polyacrylamide gel electrophoresis, thereby
making the method much safer.
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Footnotes
a address for reprint requests and correspondence: I. Dept. of Obstet. and Gynaecol. Semmelweis Univ., Baross u. 27., Budapest, H-1088, Hungary 
References
The following articles in journals at HighWire Press have cited this article:
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  C. R. L. Teo, W. Wang, H. Yang Law, C. G. Lee, and S. S. Chong Single-Step Scalable-Throughput Molecular Screening for Huntington Disease Clin. Chem., June 1, 2008; 54(6): 964 - 972. [Abstract] [Full Text] [PDF]
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 A. Gambardella, M. Muglia, A. Labate, A. Magariello, A. L. Gabriele, R. Mazzei, D. Pirritano, F. L. Conforti, A. Patitucci, P. Valentino, et al. Juvenile Huntington's disease presenting as progressive myoclonic epilepsy Neurology, August 28, 2001; 57(4): 708 - 711. [Abstract] [Full Text] [PDF]
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 D. Blake, S. L. Tan, and A. Ao Assessment of multiplex fluorescent PCR for screening single cells for trisomy 21 and single gene defects Mol. Hum. Reprod., December 1, 1999; 5(12): 1166 - 1175. [Abstract] [Full Text] [PDF]
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