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Comparative semi-automated analysis of (CAG) repeats in the Huntington disease gene: use of internal standards
Molecular and Cellular Probes (1999) 13, 283–289 Article No. mcpr.1999.0248, available online at http://www.idealibrary.com on
Comparative semi-automated analysis of (CAG) repeats in the Huntington disease gene: use of internal standards L. C. Williams,1 M. R. Hegde,2 G. Herrera,3 P. M. Stapleton3 and D. R. Love1∗ 1
School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand, 2Molecular Genetics Laboratory, Auckland Hospital, Park Road, Grafton, Auckland, New Zealand and 3DNA Diagnostics Limited, 9 Mount Street Auckland, New Zealand (Received 22 September 1998, Accepted 10 May 1999) Huntington disease (HD) belongs to the group of neurodegenerative disorders characterized by unstable expanded trinucleotide repeats. In the case of HD, the expansion of a CAG repeat occurs in the IT15 gene. The detection of the expanded CAG repeats has usually involved the electrophoretic separation of polymerase chain reaction (PCR) amplification products using conventional agarose and acrylamide gel electrophoresis. We have undertaken the comparative analysis of sizing CAG repeats of the IT15 gene using radioactive and fluorescent PCR amplification, and the subsequent separation of these products by slab gel and capillary electrophoresis. The assays have been performed on both cloned and sequenced CAG repeats, as well as genomic DNA from HD patients with a wide range of repeat lengths. The mobility of the CAG repeat amplification products of the IT15 gene is greater using capillary electrophoresis compared to slab gel electrophoresis. The analysis of 40 DNA samples from HD patients indicates that the mobility difference increases with the length of the repeat. However, we have devised an allele ladder for sizing the CAG repeats. This ladder provides a mandatory internal calibration system for diagnostic purposes and enables the confident use of either capillary or slab gel electrophoresis for sizing HD alleles. 1999 Academic Press
KEYWORDS: trinucleotide repeat, HD gene, fluorescent PCR, capillary electrophoresis.
INTRODUCTION The accurate determination of the number of CAG repeats is pivotal in providing DNA-based predictive testing for at-risk individuals. To date, length estimates have been based on polymerase chain reaction (PCR) amplification of genomic DNA using primers flanking the CAG repeat region in the IT15 gene, and subsequent electrophoretic separation of the products in denaturing polyacrylamide gels. Unaffected individuals have repeat numbers of less than 30, while those at high-risk of developing Huntington disease (HD) have repeats in excess of 39. A range of 36–39
repeats has been designated as a zone of reduced penetrance,1 while individuals with 30–35 repeats have a risk of passing on repeats in the affected size range to their offspring.2–4 Polymerase chain reaction amplifications of the CAG repeat region have primarily been performed using 32P end-labelled primers, or by the incorporation of a32P-dNTPs.5,6 In some cases a fluorescently end-labelled primer has been used with subsequent sizing of amplification products in an Applied Biosystems (ABI) model 373A or PRISMTM 377 DNA sequencer,7,8 or an ABI PRISMTM 310 Genetic Analyser.9,10 The method of separation of
∗ Author to whom all correspondence should be addressed at: School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand. Tel: +649 373 7599; Fax: +649 373 7416; E-mail: [email protected]
0890–8508/99/040283+07 $30.00/0
1999 Academic Press
284
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Fig. 1. Location of IT15 gene primers. The sequence of the human IT15 gene is shown (Accession number L12392), together with the location and designation of the primers used in our study. The precise position of the 5′ end of each oligonucleotide is indicated. The CAG repeats are boxed.
amplification products for the 310 Genetic Analyser involves capillary electrophoresis, while the separation matrix for models 373A and 377 is denaturing polyacrylamide gels. Recently, Le et al.9 compared denaturing polyacrylamide gel and capillary electrophoresis in mutation testing for HD and three other inherited disorders. This study found CAG repeat numbers were consistently 1–2 repeats smaller using capillary electrophoresis compared to denaturing polyacrylamide gel electrophoresis over the range of 15–54 repeats. This sizing difference was thought to be partly explained by the different sieving properties of cross-linked polyacrylamide compared to the linear polymer used in capillary electrophoresis. We have undertaken the analysis of CAG repeat length by PCR amplification and subsequent electrophoresis using different labelling and separation methods. The sizing of cloned CAG repeats of the IT15 gene was analysed, and 40 HD patients with 14–70 CAG repeats were screened.
MATERIALS AND METHODS DNA isolation Genomic DNA was prepared from 3-ml samples of peripheral blood using PuregeneTM (Gentra Systems Inc, Minneapolis, USA) according to the manufacturers instructions.
Cloning of the region encompassing the CAG repeats of the IT15 gene Genomic DNAs of five HD patients with expanded CAG repeats were amplified using primers Hunt2A and HD514 (Fig. 1). The amplified DNA fragments in each 10 ll reaction were subsequently incubated with 1 U Klenow DNA polymerase (Life Technologies), 1 ll 10 m dNTPs, 1 ll 10 U/ll T4 polynucleotide kinase (Life Technologies) and 2 ll 1 m ATP at 37°C for 30 min. The fragments were separated by electrophoresis in an 8% polyacrylamide gel and
the lower and higher molecular weight fragments were excised from the gel and eluted overnight in elution buffer (0·5 Ammonium acetate, 1 m EDTA pH 8). The eluted fragments were ligated with Sma1 cut and dephosphorylated pUC18 (Pharmacia) and used to transform competent Escherichia coli DH5a cells. Plasmid DNA was isolated from ampicillinresistant transformant colonies and sequenced using fluorescently-labelled M13 forward primer and electrophoretic separation in an ABI373A DNA sequencer.
Amplification and separation of radioactivelylabelled CAG repeats of the IT15 gene Primers HD1 and HD3 (see Fig. 1) were used with cycling conditions: 94°C for 2 min followed by 30 cycles of 94°C for 30 s, 60°C for 1 min and 72°C for 1 min. A final extension was performed at 72°C for 10 min. These amplifications were undertaken in a 15 ll reaction mix containing 1·5 m MgCl2, 0·2 m of each dNTP, 0·8 l of forward and reverse primers, 10% glycerol, 10% DMSO and 0·25 lCi a-P32dCTP. Polymerase chain reaction amplifications were performed using a Perkin Elmer 9600 or 2400 machine. One-microlitre samples of each amplification reaction mix were added to 9 ll of 9·5% formamide, 0·5% bromophenol blue and 0·5% xylene cyanol, heated at 95°C for 3 min, placed on ice and loaded onto denaturing 6% polyacrylamide sequencing gels; an M13 sequencing ladder was used as a size standard. In the calculation of CAG repeat length from the PCR product size, the CAA and CAG codons immediately 3′ to the CAG tract were considered to be constant sequence and not part of the CAG repeat.
Amplification and separation of fluorescentlylabelled CAG repeats of the IT15 gene Polymerase chain reaction amplifications in 10 ll reactions were undertaken using primers HD1 and HD3; the former labelled at its 5′ end with the fluorochrome 6-FAM. The buffer conditions were the
Repeats in the Huntington disease gene
same as those described by Whitefield et al.7 The cycling conditions involved an initial denaturation at 94°C for 4 min followed by 35 cycles of 94°C for 30 s, 65°C for 30 s and 72°C for 45 s, and a final extension at 72°C for 10 min. In the case of separation using ABI 373A and PRISMTM 377 DNA sequencers, a 1 ll sample of each reaction was mixed with 0·5 ll size standard (Tamra-350 labelled markers; Perkin Elmer, Applied Biosystems Division, Scoresby, Victoria, Australia), 0·5 ll loading buffer and 2 ll formamide. After denaturation at 90°C for 2 min, the samples were electrophoresed at 23 Watts in a denaturing 6% polyacrylamide gel (7 urea) using 24 cm well-to-read plates. GenescanTM and GenotyperTM software (Perkin Elmer, Applied Biosystems Division, Scoresby, Victoria, Australia) were used to determine CAG repeat lengths. In the case of separation using capillary electrophoresis in an ABI PRISMTM 310 Genetic Analyser, 1 ll of each PCR was mixed with 12 ll of deionized formamide and 0·5 ll Genescan Rox 500 labelled markers (Perkin Elmer, Applied Biosystems Division, Scoresby, Victoria, Australia). The mixture was denatured at 95°C for 2 min and cooled rapidly on ice. Polymerase chain reaction products were electrophoresed for 22 min at 60°C and 15 kV using a 47 cm×50 lm capillary containing Performance Optimized Polymer-4 (POP-4) (Perkin Elmer, Applied Biosystems Division, Scoresby, Victoria, Australia). The injection time was 5 s at 15·0 kV. The stuttering observed in the electropherograms was greater the larger the repeat. While this led to a broadening of the profile, the dominant peak determined either by peak height or peak area could be clearly assigned.
RESULTS Our initial comparative analysis of CAG repeat lengths in the IT15 gene was undertaken using cloned repeats and PCR amplification with primers HD1 and HD3 (Fig. 1). The amplification reactions were performed either in the presence of a-32P dCTP or with fluorescently end-labelled primer HD1. The former products were electrophoretically separated in a manual sequencing gel, while the latter were separated in Applied Biosystems (ABI) 373A and PRISMTM 377 DNA sequencers, and the ABI PRISMTM 310 Genetic Analyser. The ABI PRISMTM 310 Genetic Analyser uses a linear polymer separation matrix in contrast to a cross-linked polyacrylamide gel. This comparative analysis indicated that the lengths of the products, which comprised essentially a CAG repeat motif, were consistently shorter using the ABI PRISMTM 310
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Genetic Analyser (Fig. 2); the sizing in both matrices was found to be reproducible. Importantly, the lengths of the CAG repeats that were determined by denaturing polyacrylamide gel electrophoresis were within three base pairs of the length determined by sequencing, irrespective of the labelling method. The substitution of 7-deaza dGTP for dGTP in the PCR amplification of the CAG repeats of the IT15 gene had a negligible effect on mobility (data not shown). The above study was extended to the analysis of the CAG repeats of the IT15 gene from genomic DNA of 40 HD patients, covering a range of 14–70 CAG repeats. Fluorescently-labelled amplification products were separated in an ABI 373A DNA sequencer and PRISMTM 310 Genetic Analyser. Figure 3 shows the differing lengths obtained by the two separation methods. The data indicate that the greater the number of (CAG) repeats then the greater the mobility of the products in an ABI PRISMTM 310 Genetic Analyser, compared to electrophoresis in a denaturing polyacrylamide gel. It is noteworthy that the size difference of the CAG repeats determined by separation of the amplification products through the two different matrices was not constant over the repeat range that was studied. Separate studies by us examining the mobility of amplification products encompassing diand tetra-nucleotide repeats have shown that the products migrate reproducibly faster using the ABI PRISMTM 310 Genetic Analyser compared to the ABI 373A DNA sequencer (data not shown). The analysis described above indicated that the amplification products of the cloned CAG alleles could provide sizing standards. Therefore, the cloned expanded alleles of patients DN0991 (35 CAG repeats) and DN0383 (41 CAG repeats) were amplified with HD3, and HD1 that was 5′ end-labelled with the fluorochrome TET. The number of CAG repeats corresponding to the expanded allele of patient DN0847 was determined relative to the TET-labelled allele ladder (Fig. 4). The number of CAG repeats was found to be identical following electrophoresis in an ABI PRISMTM 310 Genetic Analyser and an ABI PRISMTM 373A DNA sequencer, despite the mobility differences. Sizing of the CAG repeat amplification products was reproducible using the ABI PRISMTM 310 Genetic Analyser. Quadruplicate amplifications of genomic DNA from each of four HD patients yielded the same electropherograms (data not shown). Furthermore, the sizing of the amplification products was cycleindependent. Figure 5a shows the sizes of the CAG repeat amplification products of the IT15 gene using genomic DNA from nine HD patients; the DNAs were subjected to 20, 25, 30, 35, 40 and 45 rounds of amplification. The electropherograms of the higher
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Fig. 2. Sizing of cloned CAG repeats from Huntington disease (HD) patients. The expanded IT15 gene CAG repeats of four HD patients were introduced into pUC18 and subsequently polymerase chain reaction (PCR) amplified using primers HD1 and HD3. 32P-dCTP labelled amplification products were separated using manual sequencing gels. Fluorescently-labelled amplification products were separated in ABI 373A and PRISMTM 377 DNA sequencers, and an ABI PRISMTM 310 Genetic Analyser. The products were also sized by analysing the sequence of the amplification products. The sizes determined by separation in an ABI PRISMTM 310 Genetic Analyser, together with those determined by sequencing, are shown above the relevant bar graph. The cloned repeats correspond to CAG repeat lengths of 35 (DN0805 and DN0991), 37 (DN0847), 38 (DN0820) and 41 (DN0383) as determined by separation in an ABI 373A DNA sequencer.
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Fig. 3. Comparative sizing of CAG repeats of the IT15 gene of Huntington disease (HD) patients using an ABI 373A DNA sequencer and ABI PRISMTM 310 Genetic Analyser. Fluorescently-labelled polymerase chain reaction (PCR) products encompassing the IT15 gene CAG repeats were amplified using primers HD1 and HD3. The products were sized by separation in an ABI 373A DNA sequencer and ABI PRISMTM 310 Genetic Analyser. The difference in sizing is plotted against the allele sizes determined using the ABI 373A DNA sequencer. The vertical line represents the product size that corresponds to 36 CAG repeats.
molecular weight amplification product of patient DN0198 showed consistent relative peak heights over the course of 20 to 40 cycles of amplification (Fig. 5b).
DISCUSSION Our studies indicate that denaturing polyacrylamide gel electrophoresis is a reliable method for estimating
Repeats in the Huntington disease gene
DN0847 37 repeats 150.7 bp
DN0805 35 repeats 143.6 bp
DN0383 41 repeats 162.5 bp
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DN0847 37 repeats 157.8 bp DN0805 35 repeats 152.9 bp
DN0383 41 repeats 170 bp
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Fig. 4. Sizing of CAG repeats of the IT15 gene using an allele ladder. DNA of Huntington disease (HD) patient DN0847 was polymerase chain reaction (PCR) amplified using primers HD1 and HD3. These products were electrophoresed in an ABI PRISMTM 310 Genetic Analyser and an ABI model 373A DNA sequencer, together with the amplification products of the cloned expanded CAG repeats of patients DN0991 and DN0383. The former products were labelled with the fluorochrome 6-FAM, while the latter were labelled with TET. The arrows on the electropherograms indicate the CAG repeat values of the cloned repeats, which serve as the allele ladder, and of the test sample (patient DN0847).
the number of CAG repeats in the IT15 gene using PCR products that are either radioactively or fluorescently labelled. This conclusion is based on the analysis of five cloned CAG repeats from HD patients. The IT15 gene from which these repeats were amplified has been further characterized by us to carry two mutation events in the region bounded by the CAG and CCG repeats. These mutations result in the juxtaposition of pure CAG and CCG repeats (unpubl. data). However, the annealing of the HD3 primer appears to be unaffected by these mutation events based on data we obtained. Therefore, while patients DN0805 and DN0991 have 35 CAG repeats according to our standard amplification formula, they have in fact 37 contiguous CAG repeats. The presence of the two mutations does not militate against the cloned repeats serving as allele ladders. Altering the separation matrix from cross-linked
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polyacrylamide to a linear polymer results in CAG repeat-containing amplification products that migrate faster. The differential migration of DNA carrying repetitive sequences has also been observed for tetranucleotide repeats that are used in forensic and paternity studies.11 In the case of the CAG repeats of the IT15 gene, this differing mobility increases with increasing repeat length. This conclusion contrasts with the observations of Le et al.9 in which a constant size difference of 1–2 repeats was detected based on the analysis of 78 HD patients with CAG repeats of 15–54. In view of the faster migration of CAG-containing products in the ABI PRISMTM 310 Genetic Analyser, the cloned repeats that we analysed in our study offer a relevant allele ladder. We chose to amplify the cloned CAG repeats of patients DN0991 and DN0383, which provided peaks in the electropherogram corresponding to the range of 32–45 CAG repeats. This range incorporates the critical values of 36 and 39 CAG repeats that are the allele boundaries of the zone of reduced penetrance of HD. Our study involved the co-electrophoresis of the 6-FAM labelled test sample and the TET-labelled allele ladder using the ABI PRISMTM 310 Genetic Analyser and the ABI PRISMTM 373A DNA sequencer. However, the reference (TET-labelled allele ladder) and test sample could be electrophoresed separately and subsequently superimposed, using GenescanTM software, for sizing purposes. The use of this internal calibration system provides valid size standards that should be considered mandatory for HD-based diagnostics. The composition of the linear polymer used in our study is confidential to the manufacturer. It has been suggested that the different sieving effects of the linear polymer compared to cross-linked polyacrylamide, as well as the electro-osmotic effects generated in the former matrix, may account for discrepancies in band sizing.9 However, other factors such as the extent of denaturation of amplification products in the linear polymer used in capillary electrophoresis could also influence the rate of migration.12,13 In this regard, the manufacturer acknowledges that DNA migration in POP-4 appears to be influenced by the sequence. Particularly stable GC-rich sequences such as the CAG repeats of the IT15 gene would be more likely to be affected and that this effect would increase with increasing repeat length. The ABI PRISMTM 310 Genetic Analyser offers a rapid and simple semi-automated means of sizing CAG repeat lengths. These twin advantages contrast with the more conventional radioactive assay methods that rely on slab gel electrophoresis. The electrophoretic profiles achieved in an ABI PRISMTM
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Fig. 5. Polymerase chain reaction (PCR) cycle titration of the CAG repeats of the IT15 gene. DNA of nine Huntington disease (HD) patients was subjected to 20, 25, 30, 35, 40 and 45 cycles of PCR amplification using primers HD1 and HD3. The amplification products were separated in an ABI PRISMTM 310 Genetic Analyser and the sizes of the CAG repeats were scored according to the highest peaks of amplification in the electropherograms (a). The electropherograms of the upper CAG allele amplified from patient DN0198 are shown in (b). The peak of 242 bp was scored as the upper allele for each of the five PCRs.
310 Genetic Analyser are reproducible, and automated sizing reduces errors in scoring. Finally, the ABI PRISMTM 310 Genetic Analyser offers the capability of multiplexing such that different coloured fluorochromes can be detected simultaneously. In this respect, the analysis of CAG repeat numbers in several genes implicated in triplet repeat disorders, such as
the spinocerebellar ataxias, could be undertaken in one reaction by using differentially labelled primers.
ACKNOWLEDGEMENTS We thank Dr Karen Snow (Mayo Clinic) for critically reading this manuscript. This work was supported fin-
Repeats in the Huntington disease gene ancially by grants from the University of Auckland Research Committee, the Health Research Council of New Zealand, the Lottery Grants Board of New Zealand and the Oakley Mental Health Research Foundation. We gratefully acknowledge the assistance of the many clinicians and medical geneticists who have assisted in the identification of the HD patients used in the study.
REFERENCES 1. Andrew, S. E., Goldberg, Y. P. & Hayden, M. R. (1997). Rethinking genotype and phenotype correlations in polyglutamine expansion disorders. Human Molecular Genetics 6, 2005–10. 2. Duyao, M., Ambrose, C., Myers, R. et al. (1993). Trinucleotide repeat length instability and age of onset in Huntingtons disease. Nature Genetics 4, 387–92. 3. Snell, R. G., MacMillan, J. C., Cheadle, J. P. et al. (1993). Relationship between trinucleotide repeat expansion and phenotypic variation in Huntington’s disease. Nature Genetics 4, 393–7. 4. Andrew, S. E., Goldberg, Y. P., Kremer, B. et al. (1993). The relationship between trinucleotide (CAG) repeat length and clinical features of Huntington’s disease. Nature Genetics 4, 398–403. 5. Warner, J. P., Barron, L. H. & Brock, D. J. (1993). A new polymerase chain reaction (PCR) assay for the trinucleotide repeat that is unstable and expanded on Huntington’s disease chromosomes. Molecular and Cellular Probes 7, 235–9.
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6. MacDonald, M. E., Ambrose, C. M., Duyao, M. P. et al. (1993). A novel gene containing a trinucleotide repeat that is expanded in Huntington’s disease chromosomes. Cell 72, 971–83. 7. Whitefield, J. E., Williams, L., Snow, K. et al. (1996). Molecular analysis of the Huntington’s disease gene in New Zealand. New Zealand Medical Journal 109, 27–30. 8. Mangiarini, L., Sathasivam, K., Mahal, A., Mott, R., Seller, M. & Bates, G. P. (1997). Instability of highly expanded CAG repeats in mice transgenic for the Huntington’s disease mutation. Nature Genetics 15, 197–200. 9. Le, H., Fung, D. & Trent, R. J. (1997). Applications of capillary electrophoresis in DNA mutation analysis of genetic disorders. Journal of Clinical PathologyClinical Molecular Pathology Edition 50, 261–5. 10. Toth, T., Findlay, I., Nagy, B. & Papp, Z. (1997). Accurate sizing of (CAG)n repeats causing HuntingtonDisease by fluorescent PCR. Clinical Chemistry 43, 2422–3. 11. Sozer, A. C., Kelly, C. M. & Demers, D. B. (1997). Molecular Analysis of Paternity. In Current Protocols in Human Genetics (Dracopoli, N.C., Haines, J.L., Korf, B.R., et al., eds). NY: John Wiley and Sons, Inc. 12. Wenz, H., Robertson, J. M., Menchen, S. et al. (1998). High-precision genotyping by denaturing capillary electrophoresis. Genome Research 8, 69–80. 13. Rosenblum, B. B., Oaks, E., Menchen, S. & Johnson, B. (1997). Improved single-strand DNA sizing accuracy in capillary electrophoresis. Nucleic Acids Research 25, 3925–9.
Comparative semi-automated analysis of (CAG) repeats in the Huntington disease gene: use of internal standards L. C. Williams,1 M. R. Hegde,2 G. Herrera,3 P. M. Stapleton3 and D. R. Love1∗ 1
School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand, 2Molecular Genetics Laboratory, Auckland Hospital, Park Road, Grafton, Auckland, New Zealand and 3DNA Diagnostics Limited, 9 Mount Street Auckland, New Zealand (Received 22 September 1998, Accepted 10 May 1999) Huntington disease (HD) belongs to the group of neurodegenerative disorders characterized by unstable expanded trinucleotide repeats. In the case of HD, the expansion of a CAG repeat occurs in the IT15 gene. The detection of the expanded CAG repeats has usually involved the electrophoretic separation of polymerase chain reaction (PCR) amplification products using conventional agarose and acrylamide gel electrophoresis. We have undertaken the comparative analysis of sizing CAG repeats of the IT15 gene using radioactive and fluorescent PCR amplification, and the subsequent separation of these products by slab gel and capillary electrophoresis. The assays have been performed on both cloned and sequenced CAG repeats, as well as genomic DNA from HD patients with a wide range of repeat lengths. The mobility of the CAG repeat amplification products of the IT15 gene is greater using capillary electrophoresis compared to slab gel electrophoresis. The analysis of 40 DNA samples from HD patients indicates that the mobility difference increases with the length of the repeat. However, we have devised an allele ladder for sizing the CAG repeats. This ladder provides a mandatory internal calibration system for diagnostic purposes and enables the confident use of either capillary or slab gel electrophoresis for sizing HD alleles. 1999 Academic Press
KEYWORDS: trinucleotide repeat, HD gene, fluorescent PCR, capillary electrophoresis.
INTRODUCTION The accurate determination of the number of CAG repeats is pivotal in providing DNA-based predictive testing for at-risk individuals. To date, length estimates have been based on polymerase chain reaction (PCR) amplification of genomic DNA using primers flanking the CAG repeat region in the IT15 gene, and subsequent electrophoretic separation of the products in denaturing polyacrylamide gels. Unaffected individuals have repeat numbers of less than 30, while those at high-risk of developing Huntington disease (HD) have repeats in excess of 39. A range of 36–39
repeats has been designated as a zone of reduced penetrance,1 while individuals with 30–35 repeats have a risk of passing on repeats in the affected size range to their offspring.2–4 Polymerase chain reaction amplifications of the CAG repeat region have primarily been performed using 32P end-labelled primers, or by the incorporation of a32P-dNTPs.5,6 In some cases a fluorescently end-labelled primer has been used with subsequent sizing of amplification products in an Applied Biosystems (ABI) model 373A or PRISMTM 377 DNA sequencer,7,8 or an ABI PRISMTM 310 Genetic Analyser.9,10 The method of separation of
∗ Author to whom all correspondence should be addressed at: School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand. Tel: +649 373 7599; Fax: +649 373 7416; E-mail: [email protected]
0890–8508/99/040283+07 $30.00/0
1999 Academic Press
284
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Fig. 1. Location of IT15 gene primers. The sequence of the human IT15 gene is shown (Accession number L12392), together with the location and designation of the primers used in our study. The precise position of the 5′ end of each oligonucleotide is indicated. The CAG repeats are boxed.
amplification products for the 310 Genetic Analyser involves capillary electrophoresis, while the separation matrix for models 373A and 377 is denaturing polyacrylamide gels. Recently, Le et al.9 compared denaturing polyacrylamide gel and capillary electrophoresis in mutation testing for HD and three other inherited disorders. This study found CAG repeat numbers were consistently 1–2 repeats smaller using capillary electrophoresis compared to denaturing polyacrylamide gel electrophoresis over the range of 15–54 repeats. This sizing difference was thought to be partly explained by the different sieving properties of cross-linked polyacrylamide compared to the linear polymer used in capillary electrophoresis. We have undertaken the analysis of CAG repeat length by PCR amplification and subsequent electrophoresis using different labelling and separation methods. The sizing of cloned CAG repeats of the IT15 gene was analysed, and 40 HD patients with 14–70 CAG repeats were screened.
MATERIALS AND METHODS DNA isolation Genomic DNA was prepared from 3-ml samples of peripheral blood using PuregeneTM (Gentra Systems Inc, Minneapolis, USA) according to the manufacturers instructions.
Cloning of the region encompassing the CAG repeats of the IT15 gene Genomic DNAs of five HD patients with expanded CAG repeats were amplified using primers Hunt2A and HD514 (Fig. 1). The amplified DNA fragments in each 10 ll reaction were subsequently incubated with 1 U Klenow DNA polymerase (Life Technologies), 1 ll 10 m dNTPs, 1 ll 10 U/ll T4 polynucleotide kinase (Life Technologies) and 2 ll 1 m ATP at 37°C for 30 min. The fragments were separated by electrophoresis in an 8% polyacrylamide gel and
the lower and higher molecular weight fragments were excised from the gel and eluted overnight in elution buffer (0·5 Ammonium acetate, 1 m EDTA pH 8). The eluted fragments were ligated with Sma1 cut and dephosphorylated pUC18 (Pharmacia) and used to transform competent Escherichia coli DH5a cells. Plasmid DNA was isolated from ampicillinresistant transformant colonies and sequenced using fluorescently-labelled M13 forward primer and electrophoretic separation in an ABI373A DNA sequencer.
Amplification and separation of radioactivelylabelled CAG repeats of the IT15 gene Primers HD1 and HD3 (see Fig. 1) were used with cycling conditions: 94°C for 2 min followed by 30 cycles of 94°C for 30 s, 60°C for 1 min and 72°C for 1 min. A final extension was performed at 72°C for 10 min. These amplifications were undertaken in a 15 ll reaction mix containing 1·5 m MgCl2, 0·2 m of each dNTP, 0·8 l of forward and reverse primers, 10% glycerol, 10% DMSO and 0·25 lCi a-P32dCTP. Polymerase chain reaction amplifications were performed using a Perkin Elmer 9600 or 2400 machine. One-microlitre samples of each amplification reaction mix were added to 9 ll of 9·5% formamide, 0·5% bromophenol blue and 0·5% xylene cyanol, heated at 95°C for 3 min, placed on ice and loaded onto denaturing 6% polyacrylamide sequencing gels; an M13 sequencing ladder was used as a size standard. In the calculation of CAG repeat length from the PCR product size, the CAA and CAG codons immediately 3′ to the CAG tract were considered to be constant sequence and not part of the CAG repeat.
Amplification and separation of fluorescentlylabelled CAG repeats of the IT15 gene Polymerase chain reaction amplifications in 10 ll reactions were undertaken using primers HD1 and HD3; the former labelled at its 5′ end with the fluorochrome 6-FAM. The buffer conditions were the
Repeats in the Huntington disease gene
same as those described by Whitefield et al.7 The cycling conditions involved an initial denaturation at 94°C for 4 min followed by 35 cycles of 94°C for 30 s, 65°C for 30 s and 72°C for 45 s, and a final extension at 72°C for 10 min. In the case of separation using ABI 373A and PRISMTM 377 DNA sequencers, a 1 ll sample of each reaction was mixed with 0·5 ll size standard (Tamra-350 labelled markers; Perkin Elmer, Applied Biosystems Division, Scoresby, Victoria, Australia), 0·5 ll loading buffer and 2 ll formamide. After denaturation at 90°C for 2 min, the samples were electrophoresed at 23 Watts in a denaturing 6% polyacrylamide gel (7 urea) using 24 cm well-to-read plates. GenescanTM and GenotyperTM software (Perkin Elmer, Applied Biosystems Division, Scoresby, Victoria, Australia) were used to determine CAG repeat lengths. In the case of separation using capillary electrophoresis in an ABI PRISMTM 310 Genetic Analyser, 1 ll of each PCR was mixed with 12 ll of deionized formamide and 0·5 ll Genescan Rox 500 labelled markers (Perkin Elmer, Applied Biosystems Division, Scoresby, Victoria, Australia). The mixture was denatured at 95°C for 2 min and cooled rapidly on ice. Polymerase chain reaction products were electrophoresed for 22 min at 60°C and 15 kV using a 47 cm×50 lm capillary containing Performance Optimized Polymer-4 (POP-4) (Perkin Elmer, Applied Biosystems Division, Scoresby, Victoria, Australia). The injection time was 5 s at 15·0 kV. The stuttering observed in the electropherograms was greater the larger the repeat. While this led to a broadening of the profile, the dominant peak determined either by peak height or peak area could be clearly assigned.
RESULTS Our initial comparative analysis of CAG repeat lengths in the IT15 gene was undertaken using cloned repeats and PCR amplification with primers HD1 and HD3 (Fig. 1). The amplification reactions were performed either in the presence of a-32P dCTP or with fluorescently end-labelled primer HD1. The former products were electrophoretically separated in a manual sequencing gel, while the latter were separated in Applied Biosystems (ABI) 373A and PRISMTM 377 DNA sequencers, and the ABI PRISMTM 310 Genetic Analyser. The ABI PRISMTM 310 Genetic Analyser uses a linear polymer separation matrix in contrast to a cross-linked polyacrylamide gel. This comparative analysis indicated that the lengths of the products, which comprised essentially a CAG repeat motif, were consistently shorter using the ABI PRISMTM 310
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Genetic Analyser (Fig. 2); the sizing in both matrices was found to be reproducible. Importantly, the lengths of the CAG repeats that were determined by denaturing polyacrylamide gel electrophoresis were within three base pairs of the length determined by sequencing, irrespective of the labelling method. The substitution of 7-deaza dGTP for dGTP in the PCR amplification of the CAG repeats of the IT15 gene had a negligible effect on mobility (data not shown). The above study was extended to the analysis of the CAG repeats of the IT15 gene from genomic DNA of 40 HD patients, covering a range of 14–70 CAG repeats. Fluorescently-labelled amplification products were separated in an ABI 373A DNA sequencer and PRISMTM 310 Genetic Analyser. Figure 3 shows the differing lengths obtained by the two separation methods. The data indicate that the greater the number of (CAG) repeats then the greater the mobility of the products in an ABI PRISMTM 310 Genetic Analyser, compared to electrophoresis in a denaturing polyacrylamide gel. It is noteworthy that the size difference of the CAG repeats determined by separation of the amplification products through the two different matrices was not constant over the repeat range that was studied. Separate studies by us examining the mobility of amplification products encompassing diand tetra-nucleotide repeats have shown that the products migrate reproducibly faster using the ABI PRISMTM 310 Genetic Analyser compared to the ABI 373A DNA sequencer (data not shown). The analysis described above indicated that the amplification products of the cloned CAG alleles could provide sizing standards. Therefore, the cloned expanded alleles of patients DN0991 (35 CAG repeats) and DN0383 (41 CAG repeats) were amplified with HD3, and HD1 that was 5′ end-labelled with the fluorochrome TET. The number of CAG repeats corresponding to the expanded allele of patient DN0847 was determined relative to the TET-labelled allele ladder (Fig. 4). The number of CAG repeats was found to be identical following electrophoresis in an ABI PRISMTM 310 Genetic Analyser and an ABI PRISMTM 373A DNA sequencer, despite the mobility differences. Sizing of the CAG repeat amplification products was reproducible using the ABI PRISMTM 310 Genetic Analyser. Quadruplicate amplifications of genomic DNA from each of four HD patients yielded the same electropherograms (data not shown). Furthermore, the sizing of the amplification products was cycleindependent. Figure 5a shows the sizes of the CAG repeat amplification products of the IT15 gene using genomic DNA from nine HD patients; the DNAs were subjected to 20, 25, 30, 35, 40 and 45 rounds of amplification. The electropherograms of the higher
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Fig. 2. Sizing of cloned CAG repeats from Huntington disease (HD) patients. The expanded IT15 gene CAG repeats of four HD patients were introduced into pUC18 and subsequently polymerase chain reaction (PCR) amplified using primers HD1 and HD3. 32P-dCTP labelled amplification products were separated using manual sequencing gels. Fluorescently-labelled amplification products were separated in ABI 373A and PRISMTM 377 DNA sequencers, and an ABI PRISMTM 310 Genetic Analyser. The products were also sized by analysing the sequence of the amplification products. The sizes determined by separation in an ABI PRISMTM 310 Genetic Analyser, together with those determined by sequencing, are shown above the relevant bar graph. The cloned repeats correspond to CAG repeat lengths of 35 (DN0805 and DN0991), 37 (DN0847), 38 (DN0820) and 41 (DN0383) as determined by separation in an ABI 373A DNA sequencer.
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Fig. 3. Comparative sizing of CAG repeats of the IT15 gene of Huntington disease (HD) patients using an ABI 373A DNA sequencer and ABI PRISMTM 310 Genetic Analyser. Fluorescently-labelled polymerase chain reaction (PCR) products encompassing the IT15 gene CAG repeats were amplified using primers HD1 and HD3. The products were sized by separation in an ABI 373A DNA sequencer and ABI PRISMTM 310 Genetic Analyser. The difference in sizing is plotted against the allele sizes determined using the ABI 373A DNA sequencer. The vertical line represents the product size that corresponds to 36 CAG repeats.
molecular weight amplification product of patient DN0198 showed consistent relative peak heights over the course of 20 to 40 cycles of amplification (Fig. 5b).
DISCUSSION Our studies indicate that denaturing polyacrylamide gel electrophoresis is a reliable method for estimating
Repeats in the Huntington disease gene
DN0847 37 repeats 150.7 bp
DN0805 35 repeats 143.6 bp
DN0383 41 repeats 162.5 bp
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DN0847 37 repeats 157.8 bp DN0805 35 repeats 152.9 bp
DN0383 41 repeats 170 bp
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Fig. 4. Sizing of CAG repeats of the IT15 gene using an allele ladder. DNA of Huntington disease (HD) patient DN0847 was polymerase chain reaction (PCR) amplified using primers HD1 and HD3. These products were electrophoresed in an ABI PRISMTM 310 Genetic Analyser and an ABI model 373A DNA sequencer, together with the amplification products of the cloned expanded CAG repeats of patients DN0991 and DN0383. The former products were labelled with the fluorochrome 6-FAM, while the latter were labelled with TET. The arrows on the electropherograms indicate the CAG repeat values of the cloned repeats, which serve as the allele ladder, and of the test sample (patient DN0847).
the number of CAG repeats in the IT15 gene using PCR products that are either radioactively or fluorescently labelled. This conclusion is based on the analysis of five cloned CAG repeats from HD patients. The IT15 gene from which these repeats were amplified has been further characterized by us to carry two mutation events in the region bounded by the CAG and CCG repeats. These mutations result in the juxtaposition of pure CAG and CCG repeats (unpubl. data). However, the annealing of the HD3 primer appears to be unaffected by these mutation events based on data we obtained. Therefore, while patients DN0805 and DN0991 have 35 CAG repeats according to our standard amplification formula, they have in fact 37 contiguous CAG repeats. The presence of the two mutations does not militate against the cloned repeats serving as allele ladders. Altering the separation matrix from cross-linked
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polyacrylamide to a linear polymer results in CAG repeat-containing amplification products that migrate faster. The differential migration of DNA carrying repetitive sequences has also been observed for tetranucleotide repeats that are used in forensic and paternity studies.11 In the case of the CAG repeats of the IT15 gene, this differing mobility increases with increasing repeat length. This conclusion contrasts with the observations of Le et al.9 in which a constant size difference of 1–2 repeats was detected based on the analysis of 78 HD patients with CAG repeats of 15–54. In view of the faster migration of CAG-containing products in the ABI PRISMTM 310 Genetic Analyser, the cloned repeats that we analysed in our study offer a relevant allele ladder. We chose to amplify the cloned CAG repeats of patients DN0991 and DN0383, which provided peaks in the electropherogram corresponding to the range of 32–45 CAG repeats. This range incorporates the critical values of 36 and 39 CAG repeats that are the allele boundaries of the zone of reduced penetrance of HD. Our study involved the co-electrophoresis of the 6-FAM labelled test sample and the TET-labelled allele ladder using the ABI PRISMTM 310 Genetic Analyser and the ABI PRISMTM 373A DNA sequencer. However, the reference (TET-labelled allele ladder) and test sample could be electrophoresed separately and subsequently superimposed, using GenescanTM software, for sizing purposes. The use of this internal calibration system provides valid size standards that should be considered mandatory for HD-based diagnostics. The composition of the linear polymer used in our study is confidential to the manufacturer. It has been suggested that the different sieving effects of the linear polymer compared to cross-linked polyacrylamide, as well as the electro-osmotic effects generated in the former matrix, may account for discrepancies in band sizing.9 However, other factors such as the extent of denaturation of amplification products in the linear polymer used in capillary electrophoresis could also influence the rate of migration.12,13 In this regard, the manufacturer acknowledges that DNA migration in POP-4 appears to be influenced by the sequence. Particularly stable GC-rich sequences such as the CAG repeats of the IT15 gene would be more likely to be affected and that this effect would increase with increasing repeat length. The ABI PRISMTM 310 Genetic Analyser offers a rapid and simple semi-automated means of sizing CAG repeat lengths. These twin advantages contrast with the more conventional radioactive assay methods that rely on slab gel electrophoresis. The electrophoretic profiles achieved in an ABI PRISMTM
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(b) 40 cycles 35 cycles 30 cycles 25 cycles 20 cycles
Fig. 5. Polymerase chain reaction (PCR) cycle titration of the CAG repeats of the IT15 gene. DNA of nine Huntington disease (HD) patients was subjected to 20, 25, 30, 35, 40 and 45 cycles of PCR amplification using primers HD1 and HD3. The amplification products were separated in an ABI PRISMTM 310 Genetic Analyser and the sizes of the CAG repeats were scored according to the highest peaks of amplification in the electropherograms (a). The electropherograms of the upper CAG allele amplified from patient DN0198 are shown in (b). The peak of 242 bp was scored as the upper allele for each of the five PCRs.
310 Genetic Analyser are reproducible, and automated sizing reduces errors in scoring. Finally, the ABI PRISMTM 310 Genetic Analyser offers the capability of multiplexing such that different coloured fluorochromes can be detected simultaneously. In this respect, the analysis of CAG repeat numbers in several genes implicated in triplet repeat disorders, such as
the spinocerebellar ataxias, could be undertaken in one reaction by using differentially labelled primers.
ACKNOWLEDGEMENTS We thank Dr Karen Snow (Mayo Clinic) for critically reading this manuscript. This work was supported fin-
Repeats in the Huntington disease gene ancially by grants from the University of Auckland Research Committee, the Health Research Council of New Zealand, the Lottery Grants Board of New Zealand and the Oakley Mental Health Research Foundation. We gratefully acknowledge the assistance of the many clinicians and medical geneticists who have assisted in the identification of the HD patients used in the study.
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6. MacDonald, M. E., Ambrose, C. M., Duyao, M. P. et al. (1993). A novel gene containing a trinucleotide repeat that is expanded in Huntington’s disease chromosomes. Cell 72, 971–83. 7. Whitefield, J. E., Williams, L., Snow, K. et al. (1996). Molecular analysis of the Huntington’s disease gene in New Zealand. New Zealand Medical Journal 109, 27–30. 8. Mangiarini, L., Sathasivam, K., Mahal, A., Mott, R., Seller, M. & Bates, G. P. (1997). Instability of highly expanded CAG repeats in mice transgenic for the Huntington’s disease mutation. Nature Genetics 15, 197–200. 9. Le, H., Fung, D. & Trent, R. J. (1997). Applications of capillary electrophoresis in DNA mutation analysis of genetic disorders. Journal of Clinical PathologyClinical Molecular Pathology Edition 50, 261–5. 10. Toth, T., Findlay, I., Nagy, B. & Papp, Z. (1997). Accurate sizing of (CAG)n repeats causing HuntingtonDisease by fluorescent PCR. Clinical Chemistry 43, 2422–3. 11. Sozer, A. C., Kelly, C. M. & Demers, D. B. (1997). Molecular Analysis of Paternity. In Current Protocols in Human Genetics (Dracopoli, N.C., Haines, J.L., Korf, B.R., et al., eds). NY: John Wiley and Sons, Inc. 12. Wenz, H., Robertson, J. M., Menchen, S. et al. (1998). High-precision genotyping by denaturing capillary electrophoresis. Genome Research 8, 69–80. 13. Rosenblum, B. B., Oaks, E., Menchen, S. & Johnson, B. (1997). Improved single-strand DNA sizing accuracy in capillary electrophoresis. Nucleic Acids Research 25, 3925–9.