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A fluorescent multiplex ARMS method for rapid mutation analysis
Molecular and Cellular Probes (1995) 9, 447-451
A fluorescent multiplex ARMS method for rapid mutation analysis Johannes Zschocke* and Colin A. Graham
Department of Medical Genetics, Belfast City Hospital, Lisburn Rd., Belfast BT9 7AB (Received 20 June 1995, Accepted 7 July 1995) The amplification refractory mutation system (ARMS) is a powerful technique for the identification of mutations. We present a modification of this technique involving coamplification of normal and mutant alleles with primers that differ in length by three to five bases. Fluorescent labelling allows exact sizing of the amplification products on an automated DNA fragment analyser. We have used the fluorescent multiplex ARMS method for the identification of common phenylketonuria mutations in Northern Ireland (R408W, 165T and F39L) together with the analysis of a polymorphic short tandem repeat site at the human phenylalanine hydroxylase locus. This provides an efficient and inexpensive first step for diagnostic mutation analysis in our population. © 1995 Academic Press Limited
KEYWORDS:phenylketonuria, mutation, ARMS. INTRODUCTION Routine mutation analysis is now increasingly requested for many inherited disorders. It requires fast and inexpensive methods for mutation detection in patients and relatives. High sensitivity and specificity are of particular importance for a diagnostic test, and the analysis system must be able to handle small numbers of samples frequently. A large range of techniques for mutation detection is now available, 1"2 and the method of choice depends on many factors such as gene size and the nature and heterogeneity of mutations in a population. One aim of the Northern Ireland Phenylketonuria (PKU) Study was to design a system for comprehensive mutation analysis as a routine service in Northern Ireland. PKU is the commonest inborn error of amino acid metabolism in Europe and is particularly frequent in Ireland where 1 in 4500 children is affected. It is caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH), that leads to hyperphenylalaninaemia and severe mental retardation if
dietary treatment is not commenced in the neonate2 Routine mutation analysis in the newborn can assist in the management of families with PKU in that it confirms the diagnosis, gives information on the severity of the disease and enables carrier detection in the extended family. In a previous study we found that three mutations, R408W, 165T and F39L account for two thirds of PKU alleles in Northern Ireland, whilst a large number of rare mutations account for the rest.4 The analysis of a polymorphic short tandem repeat (STR) system in the PAH gene was found to be helpful for the identification of the rare mutations, and we have reported a method for the fluorescent analysis of this microsatellite marker, s Described here is a fluorescent multiplex ARMS method which allows the combination of STR analysis with screening for the three common mutations in our population. This provides a rapid, inexpensive and very efficient first step for PKU mutation analysis in the Northern Irish population.
*Author to whom correspondence should be addressed at: Universit~its-Kinderklinik, Deutschhausstr. 12, D-35033 Marburg,
Germany.
0890-8508/951060447 + 05 $12.00/0
447
© 1995 AcademicPressLimited
448
J. Zschocke and C. A. Graham Table 1. Sequencesof primers used for the analysis of PKU
mutations R408W, 165T and F39L. '-N' primers amplify the normal allele and '-M' primers the mutant allele; altered and allele-specific bases are underlined. '-C' denotes the common primers, labelled at their 5' ends with the blue-coloured fluorescent dye 6-FAM ('*-9. Sequencesof the primers used for the amplification of the STR site are those recommended by Goltsov et al. ~8 with the addition of a fluorescent label to one of the primerss R408W-N: R408W-M: R408W-C: 165T-N: 165T-M: 165T-C: F39L-N: F39L-M: F39L-C:
5'GTCGTAGCGAACTGAGAAGGGGCG-3' 5'-TATGGGTCGTAGCGAACTGAGAAGGGGCA-3' 5'-*-CTCCAAATGGTGCCCTTCACTCAAGC-3' 5'-G GAGAATGATGTAAACCTGACCCAGAT-3' 5'-CTAGGAGAATGATGTAAACCTGACCCAGAC-3' 5'-*-GATCTTGATGATGTTTGTCAGAGCAGGC-3' 5'-TGCACCAACTTCTTCTTTGAGTTAG-3' 5'-GCCAATGCACCAACTTCTTCTTTGAGTTAC-3' 5'-*-GAAAGAGTTCATGCTTGCTTTGTCC-3'
The method relies on selective amplification of specific alleles during polymerase chain reaction (PCR), a technique described independently by several groups in 1989 and known as amplification refractory mutation system (ARMS), PCR amplification of specific alleles (PASA), allele-specific amplification (ASA) or allele-specific PCR (ASPCR).°-9 Selectivity is achieved by using PCR primers that end at a mutation site and contain a deliberate mismatch usually at the third last base. ARMS primers with the normal base at their 3" end amplify normal alleles but not mutant alleles, whilst primers corresponding to the mutant sequence do not anneal to normal alleles. Amplification products are generally visualized by ethidium bromide staining of agarose or acrylamide gels. The technique has been widely used and has been adapted to elucidate haplotypes from single individuals (double ARMS or double PASA)l°'u or to simultaneously screen multiple cystic fibrosis or betathalassaemia mutations (multiplex ARMS). 12'13 Competitive priming with two primers specific for two alleles of a single site has been described as PAMSA; TM amplification products in this approach were distinguished by adding 30 or more non-complementary bases to one of the primers. Labelling of primers with different fluorescent dyes has been used in the detection of a small deletion, the commonest cystic fibrosis mutation AFS08. ~s'~6In the present approach, we employ competitive priming with two ARMS primers that differ in length by three to five bases and use a common primer labelled with a fluorescent dye. This allows multiplexing and accurate fragment sizing on a fluorescent DNA analysis system.
MATERIALS A N D M E T H O D S
Eighty-two Northern Irish families with hyperphenylalaninaemia (patients and, where available, both parents) were investigated with the fluorescent multiplex ARMS method. The subjects had not been studied previously and included patients with both PKU and mild hyperphenylalaninaemia not requiring diet (MHP). One child with dihydrobiopterin reductase deficiency was excluded. Using standard procedures, genomic DNA was isolated from leukocytes. Sequences of primers used in the assay are given in Table 1. ARMS primers specific for mutant alleles were three to five bases longer than those for normal alleles, and the lengths of amplification products were as follows. R408W: 90 bp (normal alleles)/95 bp (mutant alleles); 165T: 124 bp/127 bp; F39L: 176 bp/ 181 bp. Amplifications were performed in a Perkin Elmer Cetus DNA thermal cycler. The amplification reaction mixture contained 1.5 mM MgCl2, 200 I~M dNTP (Pharmacia) and 0-1 U Taq polymerase (Gibco BRL) in 10 I.d standard PCR buffer (Gibco BRL). Primer Table 2
Frequenciesof the commonest mutations causing hyperphenylalaninaemia in Northern Ireland, and their association with different STR alleles. A total of 121 patients was investigated Mutation
No.
Frequency
STR
R408W 165T F39L Total
67 49 23 242
28% 20% 9.5%
242, 238, 230 246, 250, 242, 238 238, 242, 246
Fluorescent m u l t i p l e x A R M S m e t h o d R408W 71.
el.
" + 91 ,lo,z ,HI
165T
F39L
449 STR
-+ - + microsatellite ,121 ,1~1. ,1~1 ,151 ,l~z, ,17.z ,18} ,191 ,2o! ,21! ?2.z ?3z ?4z ,=sJ: 26z, ?Tz,
fragment length in bp
,
a)
mutations R408W and F39L (STR 242, 246)
b)
mutation F39L (STR 246)
c)
mutation R408W (STR 242)
Fig. 1. Identification of mutations R408W and F39L in a family with PKU. The mother is carrier for F39L-STR 246 (b), the father is carrier for R408W-STR 242 (c).
71.
R408W 165T F39L STR - + -+ - + microsatellite ,sl ,91. ,lol. ,111. ~2~ ,13z ,141 ,15z, 16z ,lv l ,18.z ,19,z 7o.1 71.1 721 ~z~ 3.~ ,=5~ ?6z, 771,
fragment length in bp
a)
homozygote for mutation 165T - STR 246
b)
heterozygote for 165T (STR 246)
c)
heterozygote for 165T (STR 246)
Fig. 2. Identification of homozygosity for 165T (STR 246) in a patient with PKU (a). Both parents are carriers for 165T (b and c). concentrations were 5 pM for the ARMS primers specific for normal alleles, and 2.5 pM for the other primers. Two separate PCR amplifications with different annealing temperatures (69°C for mutations R408W and 165T, 58°C for F39L and STR analysis) were required. Amplification conditions were: denaturation at 94°C for 5 min, followed by 30 cycles of 58°C (69°C) for 30 s, 72°C for 30 s and 94°C for 30 s, followed by final extension at 72°C for 5 min and rapid cooling to 20°C. Amplifications were thus completed within 2 h. The products of both amplification reactions were combined, and 1 I~1 of the mixture was added to
2.5 pl deionised formamide and 1.5 I~1 loading buffer containing Genescan-2500 ROX internal lane standard (4fmol of a Pstl digest of phage lambda DNA labelled with the red fluorescent dye 6-carboxyrhodamine, Applied Biosystems). The loading mix was denatured at 90°C for 2 rain, loaded onto a 6% polyacrylamide sequencing gel (Sequagel, National Diagnostics) and electrophoresed at 40 W for 4 h on an Applied Biosystems 373a fragment analyser which detects fluorescently labelled DNA by laser scanning. Allele sizes were determined automatically with Genescan 672 software (Applied Biosystems).
450
J. Zschocke and C. A. Graham
RESULTS A N D DISCUSSION
Mutation analysis in 82 Northern Irish families with hyperphenylalaninaemia using fluorescent multiplex ARMS method confirmed R408W, 165T and F39L as the commonest mutations in Northern Ireland. Including previously investigated families 4 they account for 28%, 20% and 9.5% of mutant alleles (Table 2). All three mutations are associated with several different STR alleles. STR analysis in all patients subsequently helped to target screening for other mutations, and we rapidly achieved a mutation detection rate of 99.6%? 7 Normal and mutant alleles are distinguished in the fluorescent multiplex ARMS technique by the length of the amplification products, visualized as two distinct peaks in the chromatogram profiles (Figs 1 and 2). The increased length of mutant ARMS primers can lead to preferential amplification of the mutant alleles in the heterozygote (mutation F39L, Fig. 1). However, this effect is consistent in all samples and homozygosity is easily recognized by complete absence of the normal allele peak in a patient and presence of the mutation in both parents (Fig. 2). Preferential amplification was reduced by changing the ratio of normal: mutant primer and may have been completely avoided if a non-complementary T-tail of 3-5 bases was used to increase the length of the mutant ARMS primer. The fluorescent multiplex ARMS method is very specific, not least because of coamplification of normal and mutant alleles. Intrinsic control is further increased by multiplex amplification of different sites. In a diagnostic setting, DNA samples of patients should always be analysed together with samples of both parents. This provides independent confirmation of the results and enables accurate assignment of STR alleles. The presence of a different mutation at one of the investigated sites can lead to non-amplification of both normal and mutant alleles, and this was observed with the mutation R408Q. Homozygosity for R408Q could be recognised by complete absence of amplification at the R408W site, normal amplification at the 165T site, and homozygosity of STR allele 234. Two patients were compound heterozygote for R408W and R408Q, and the absence of the normal allele peak together with presence of an R408W peak in these cases could have been misinterpreted as homozygosity for R408W. However, the R408W peak was relatively small, R408W was only identified in one of the parents and the subjects were heterozygous at the STR site. The finding of an STR 234 allele suggested the presence of R408Q which was then confirmed by restriction enzyme analysis (not shown).
The fluorescent multiplex ARMS approach is a useful first step in routine mutation analysis, particularly in a population where a few common mutations are found together with a large number of rare mutations. The method is simple, fast, inexpensive and is particularly well suited to the frequent analysis of few samples. Very little hands-on time is required and comprehensive results are obtained within one day. Very small PCR volumes are sufficient. The method employs an expensive automated fragment analyser but this equipment is increasingly becoming available in diagnostic laboratories. Only three primers are necessary for each mutation, only one of them has to be fluorescently labelled, and the same label can be used for all mutations. This reduces the initial costs in the set-up of the technique. It should be possible to further improve the technique by designing primers which use the same annealing temperatures and by adding other mutations and polymorphic sites to the assay. The method can then easily be modified for different populations by tailoring the multiplex combination to suit the respective mutation spectrum.
ACKNOWLEDGEMENTS
We are grateful to Dr John Old for advice on designing ARMS primers and to the staff at the UK--Human Genome Mapping Project Resource Centre in Harrow, England, for kindly synthesizing the primers. J. Z. was supported by research scholarships of the Deutsche Forschungsgemeinschaft (Zs 1711-1 and 1-2). The study was partly funded by the Medical Research Council.
REFERENCES
I. Grompe, M. (I 993). The rapid detection of unknown mutations in nucleic acids. Nature Genetics 5, 111-I 7. 2. Cotton, R. G. H. (1993). Current methods of mutation detection. Mutation Research 285, 125-44. 3. Scriver, C. R., Kaufman, S., Eisensmith, R. C. & Woo, S. L. C. (1995). The Hyperphenylalaninaemias. In The Metabolic and Molecular Basis of Inherited Disease.
(Scriver, C. R., Beaudet, A. L., Sly, W. S. and Valle, D., eds), Pp. 1015-1077. New York: McGraw-Hill. 4. Zschocke, J., Graham, C. A., Stewart, F. J., Carson, D. J. & Nevin, N. C. (1994). Automated sequencing detects all mutations in Northern Irish patients with phenylketonuria and mild hyperphenylalaninaemia. Acta Paediatrica Suppl 407, 37-38. 5. Zschocke, J., Graham, C. A., McKnight, J. J. & Nevin, N. C. (1994). The STR system in the human phenylalanine hydroxylase gene: true fragment length obtained with fluorescent labelled PCR primers. Acta Paediatrica Suppl 407, 41-42. 6. Newton, C. R., Graham,A., Heptinstall, L. E. et al. (1989). Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Research 17, 2503-16.
Fluorescent multiplex ARMS method
7. Sommer, S. S., Cassady, J. D., Sobell, J. L. & Bottema, C. D. K. (1989). A novel method for detecting point mutations or polymorphisms and its application to population screening for carriers of phenylketonuria. Mayo Clinic Proceedings 64, 1361-72. 8. Okayama, H., Curiel, D. T., Brantly, M. L., Holmes, M. D. & Crystal, R. G. (1989). Rapid, nonradioactive detection of mutations in the human genome by allelespecific amplification. Journal of Laboratory and Clinical Medicine 114, 105-13. 9. Wu, D. Y., Ugozzoli, L., Pal, B. K. & Wallace, R. B. (1989). Allele-specific enzymatic amplification of Bglobin genomic DNA for diagnosis of sickle cell anemia. Proceedings of the National Academy of Science of the U.S.A. 82, 2757-60. 10. li, S. & Sommer, S. S. (1993). The high frequency of TTR M30 in familial amyloidotic polyneuropathy is not due to a founder effect. Human Molecular Genetics 2, 1303-5. 11. Lo, Y. M. D., Patel, P., Newton, C. R., Markham, A. F., Fleming, K. A. & Wainscoat, J. S. (1992). Direct haplotype determination by double ARMS: specificity, sensitivity and genetic applications. Nucleic Acids Research 19, 3561-7. 12. Ferrie, R. M., Schwarz, M. J., Robertson, N. C. et al. (1992). Development, multiplexing and application of ARMS tests for common mutations in the CFTR gene. American Journal of Human Genetics 51, 251-62.
451
13. Fortina, P., Dotti, G., Conant, R. etal. (I 992). Detection of the most common mutations causing beta-thalassemia in Mediterraneans using a multiplex amplification refractory mutation system (MARMS). PCR Methods and Applications 2, 163-6. 14. Dutton, C. & Sommer, S. S. (1991). Simultaneous detection of multiple single-base alleles at a polymorphic site. Bio Techniques 11,700-2. 15. Chehab, F. F., Wall, J. & Kan, Y. W. (1992). Amplification and detection of specific DNA sequences with fluorescent PCR primers: application to ~F508 mutation in cystic fibrosis. Methods in Enzymology 216. 16. Fortina, P., Conant, R., Parrella, T. et al. (1992). Fluorescence-based, multiplex allele-specific PCR (MASPCR) detection of the delta F508 deletion in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Molecular and Cellular Probes 6, 353-6. 17. Zschocke, J., Graham, C. A., Carson, D. J. & Nevin, N. C. (1995). Phenylketonuria mutation analysis in Northern Ireland: a rapid stepwise approach. American Journal of Human Genetics 57, in press. 18. Goltsov, A. A., Eisensmith, R. C., Naughton, E. R., Jin, L., Chakraborty, R. & Woo, S. L. C. (1993). A single polymorphic STR system in the human phenylalanine hydroxylase gene permits rapid prenatal diagnosis and carrier screening for phenylketonuria. Human Molecular Genetics 3, 577-81.
A fluorescent multiplex ARMS method for rapid mutation analysis Johannes Zschocke* and Colin A. Graham
Department of Medical Genetics, Belfast City Hospital, Lisburn Rd., Belfast BT9 7AB (Received 20 June 1995, Accepted 7 July 1995) The amplification refractory mutation system (ARMS) is a powerful technique for the identification of mutations. We present a modification of this technique involving coamplification of normal and mutant alleles with primers that differ in length by three to five bases. Fluorescent labelling allows exact sizing of the amplification products on an automated DNA fragment analyser. We have used the fluorescent multiplex ARMS method for the identification of common phenylketonuria mutations in Northern Ireland (R408W, 165T and F39L) together with the analysis of a polymorphic short tandem repeat site at the human phenylalanine hydroxylase locus. This provides an efficient and inexpensive first step for diagnostic mutation analysis in our population. © 1995 Academic Press Limited
KEYWORDS:phenylketonuria, mutation, ARMS. INTRODUCTION Routine mutation analysis is now increasingly requested for many inherited disorders. It requires fast and inexpensive methods for mutation detection in patients and relatives. High sensitivity and specificity are of particular importance for a diagnostic test, and the analysis system must be able to handle small numbers of samples frequently. A large range of techniques for mutation detection is now available, 1"2 and the method of choice depends on many factors such as gene size and the nature and heterogeneity of mutations in a population. One aim of the Northern Ireland Phenylketonuria (PKU) Study was to design a system for comprehensive mutation analysis as a routine service in Northern Ireland. PKU is the commonest inborn error of amino acid metabolism in Europe and is particularly frequent in Ireland where 1 in 4500 children is affected. It is caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH), that leads to hyperphenylalaninaemia and severe mental retardation if
dietary treatment is not commenced in the neonate2 Routine mutation analysis in the newborn can assist in the management of families with PKU in that it confirms the diagnosis, gives information on the severity of the disease and enables carrier detection in the extended family. In a previous study we found that three mutations, R408W, 165T and F39L account for two thirds of PKU alleles in Northern Ireland, whilst a large number of rare mutations account for the rest.4 The analysis of a polymorphic short tandem repeat (STR) system in the PAH gene was found to be helpful for the identification of the rare mutations, and we have reported a method for the fluorescent analysis of this microsatellite marker, s Described here is a fluorescent multiplex ARMS method which allows the combination of STR analysis with screening for the three common mutations in our population. This provides a rapid, inexpensive and very efficient first step for PKU mutation analysis in the Northern Irish population.
*Author to whom correspondence should be addressed at: Universit~its-Kinderklinik, Deutschhausstr. 12, D-35033 Marburg,
Germany.
0890-8508/951060447 + 05 $12.00/0
447
© 1995 AcademicPressLimited
448
J. Zschocke and C. A. Graham Table 1. Sequencesof primers used for the analysis of PKU
mutations R408W, 165T and F39L. '-N' primers amplify the normal allele and '-M' primers the mutant allele; altered and allele-specific bases are underlined. '-C' denotes the common primers, labelled at their 5' ends with the blue-coloured fluorescent dye 6-FAM ('*-9. Sequencesof the primers used for the amplification of the STR site are those recommended by Goltsov et al. ~8 with the addition of a fluorescent label to one of the primerss R408W-N: R408W-M: R408W-C: 165T-N: 165T-M: 165T-C: F39L-N: F39L-M: F39L-C:
5'GTCGTAGCGAACTGAGAAGGGGCG-3' 5'-TATGGGTCGTAGCGAACTGAGAAGGGGCA-3' 5'-*-CTCCAAATGGTGCCCTTCACTCAAGC-3' 5'-G GAGAATGATGTAAACCTGACCCAGAT-3' 5'-CTAGGAGAATGATGTAAACCTGACCCAGAC-3' 5'-*-GATCTTGATGATGTTTGTCAGAGCAGGC-3' 5'-TGCACCAACTTCTTCTTTGAGTTAG-3' 5'-GCCAATGCACCAACTTCTTCTTTGAGTTAC-3' 5'-*-GAAAGAGTTCATGCTTGCTTTGTCC-3'
The method relies on selective amplification of specific alleles during polymerase chain reaction (PCR), a technique described independently by several groups in 1989 and known as amplification refractory mutation system (ARMS), PCR amplification of specific alleles (PASA), allele-specific amplification (ASA) or allele-specific PCR (ASPCR).°-9 Selectivity is achieved by using PCR primers that end at a mutation site and contain a deliberate mismatch usually at the third last base. ARMS primers with the normal base at their 3" end amplify normal alleles but not mutant alleles, whilst primers corresponding to the mutant sequence do not anneal to normal alleles. Amplification products are generally visualized by ethidium bromide staining of agarose or acrylamide gels. The technique has been widely used and has been adapted to elucidate haplotypes from single individuals (double ARMS or double PASA)l°'u or to simultaneously screen multiple cystic fibrosis or betathalassaemia mutations (multiplex ARMS). 12'13 Competitive priming with two primers specific for two alleles of a single site has been described as PAMSA; TM amplification products in this approach were distinguished by adding 30 or more non-complementary bases to one of the primers. Labelling of primers with different fluorescent dyes has been used in the detection of a small deletion, the commonest cystic fibrosis mutation AFS08. ~s'~6In the present approach, we employ competitive priming with two ARMS primers that differ in length by three to five bases and use a common primer labelled with a fluorescent dye. This allows multiplexing and accurate fragment sizing on a fluorescent DNA analysis system.
MATERIALS A N D M E T H O D S
Eighty-two Northern Irish families with hyperphenylalaninaemia (patients and, where available, both parents) were investigated with the fluorescent multiplex ARMS method. The subjects had not been studied previously and included patients with both PKU and mild hyperphenylalaninaemia not requiring diet (MHP). One child with dihydrobiopterin reductase deficiency was excluded. Using standard procedures, genomic DNA was isolated from leukocytes. Sequences of primers used in the assay are given in Table 1. ARMS primers specific for mutant alleles were three to five bases longer than those for normal alleles, and the lengths of amplification products were as follows. R408W: 90 bp (normal alleles)/95 bp (mutant alleles); 165T: 124 bp/127 bp; F39L: 176 bp/ 181 bp. Amplifications were performed in a Perkin Elmer Cetus DNA thermal cycler. The amplification reaction mixture contained 1.5 mM MgCl2, 200 I~M dNTP (Pharmacia) and 0-1 U Taq polymerase (Gibco BRL) in 10 I.d standard PCR buffer (Gibco BRL). Primer Table 2
Frequenciesof the commonest mutations causing hyperphenylalaninaemia in Northern Ireland, and their association with different STR alleles. A total of 121 patients was investigated Mutation
No.
Frequency
STR
R408W 165T F39L Total
67 49 23 242
28% 20% 9.5%
242, 238, 230 246, 250, 242, 238 238, 242, 246
Fluorescent m u l t i p l e x A R M S m e t h o d R408W 71.
el.
" + 91 ,lo,z ,HI
165T
F39L
449 STR
-+ - + microsatellite ,121 ,1~1. ,1~1 ,151 ,l~z, ,17.z ,18} ,191 ,2o! ,21! ?2.z ?3z ?4z ,=sJ: 26z, ?Tz,
fragment length in bp
,
a)
mutations R408W and F39L (STR 242, 246)
b)
mutation F39L (STR 246)
c)
mutation R408W (STR 242)
Fig. 1. Identification of mutations R408W and F39L in a family with PKU. The mother is carrier for F39L-STR 246 (b), the father is carrier for R408W-STR 242 (c).
71.
R408W 165T F39L STR - + -+ - + microsatellite ,sl ,91. ,lol. ,111. ~2~ ,13z ,141 ,15z, 16z ,lv l ,18.z ,19,z 7o.1 71.1 721 ~z~ 3.~ ,=5~ ?6z, 771,
fragment length in bp
a)
homozygote for mutation 165T - STR 246
b)
heterozygote for 165T (STR 246)
c)
heterozygote for 165T (STR 246)
Fig. 2. Identification of homozygosity for 165T (STR 246) in a patient with PKU (a). Both parents are carriers for 165T (b and c). concentrations were 5 pM for the ARMS primers specific for normal alleles, and 2.5 pM for the other primers. Two separate PCR amplifications with different annealing temperatures (69°C for mutations R408W and 165T, 58°C for F39L and STR analysis) were required. Amplification conditions were: denaturation at 94°C for 5 min, followed by 30 cycles of 58°C (69°C) for 30 s, 72°C for 30 s and 94°C for 30 s, followed by final extension at 72°C for 5 min and rapid cooling to 20°C. Amplifications were thus completed within 2 h. The products of both amplification reactions were combined, and 1 I~1 of the mixture was added to
2.5 pl deionised formamide and 1.5 I~1 loading buffer containing Genescan-2500 ROX internal lane standard (4fmol of a Pstl digest of phage lambda DNA labelled with the red fluorescent dye 6-carboxyrhodamine, Applied Biosystems). The loading mix was denatured at 90°C for 2 rain, loaded onto a 6% polyacrylamide sequencing gel (Sequagel, National Diagnostics) and electrophoresed at 40 W for 4 h on an Applied Biosystems 373a fragment analyser which detects fluorescently labelled DNA by laser scanning. Allele sizes were determined automatically with Genescan 672 software (Applied Biosystems).
450
J. Zschocke and C. A. Graham
RESULTS A N D DISCUSSION
Mutation analysis in 82 Northern Irish families with hyperphenylalaninaemia using fluorescent multiplex ARMS method confirmed R408W, 165T and F39L as the commonest mutations in Northern Ireland. Including previously investigated families 4 they account for 28%, 20% and 9.5% of mutant alleles (Table 2). All three mutations are associated with several different STR alleles. STR analysis in all patients subsequently helped to target screening for other mutations, and we rapidly achieved a mutation detection rate of 99.6%? 7 Normal and mutant alleles are distinguished in the fluorescent multiplex ARMS technique by the length of the amplification products, visualized as two distinct peaks in the chromatogram profiles (Figs 1 and 2). The increased length of mutant ARMS primers can lead to preferential amplification of the mutant alleles in the heterozygote (mutation F39L, Fig. 1). However, this effect is consistent in all samples and homozygosity is easily recognized by complete absence of the normal allele peak in a patient and presence of the mutation in both parents (Fig. 2). Preferential amplification was reduced by changing the ratio of normal: mutant primer and may have been completely avoided if a non-complementary T-tail of 3-5 bases was used to increase the length of the mutant ARMS primer. The fluorescent multiplex ARMS method is very specific, not least because of coamplification of normal and mutant alleles. Intrinsic control is further increased by multiplex amplification of different sites. In a diagnostic setting, DNA samples of patients should always be analysed together with samples of both parents. This provides independent confirmation of the results and enables accurate assignment of STR alleles. The presence of a different mutation at one of the investigated sites can lead to non-amplification of both normal and mutant alleles, and this was observed with the mutation R408Q. Homozygosity for R408Q could be recognised by complete absence of amplification at the R408W site, normal amplification at the 165T site, and homozygosity of STR allele 234. Two patients were compound heterozygote for R408W and R408Q, and the absence of the normal allele peak together with presence of an R408W peak in these cases could have been misinterpreted as homozygosity for R408W. However, the R408W peak was relatively small, R408W was only identified in one of the parents and the subjects were heterozygous at the STR site. The finding of an STR 234 allele suggested the presence of R408Q which was then confirmed by restriction enzyme analysis (not shown).
The fluorescent multiplex ARMS approach is a useful first step in routine mutation analysis, particularly in a population where a few common mutations are found together with a large number of rare mutations. The method is simple, fast, inexpensive and is particularly well suited to the frequent analysis of few samples. Very little hands-on time is required and comprehensive results are obtained within one day. Very small PCR volumes are sufficient. The method employs an expensive automated fragment analyser but this equipment is increasingly becoming available in diagnostic laboratories. Only three primers are necessary for each mutation, only one of them has to be fluorescently labelled, and the same label can be used for all mutations. This reduces the initial costs in the set-up of the technique. It should be possible to further improve the technique by designing primers which use the same annealing temperatures and by adding other mutations and polymorphic sites to the assay. The method can then easily be modified for different populations by tailoring the multiplex combination to suit the respective mutation spectrum.
ACKNOWLEDGEMENTS
We are grateful to Dr John Old for advice on designing ARMS primers and to the staff at the UK--Human Genome Mapping Project Resource Centre in Harrow, England, for kindly synthesizing the primers. J. Z. was supported by research scholarships of the Deutsche Forschungsgemeinschaft (Zs 1711-1 and 1-2). The study was partly funded by the Medical Research Council.
REFERENCES
I. Grompe, M. (I 993). The rapid detection of unknown mutations in nucleic acids. Nature Genetics 5, 111-I 7. 2. Cotton, R. G. H. (1993). Current methods of mutation detection. Mutation Research 285, 125-44. 3. Scriver, C. R., Kaufman, S., Eisensmith, R. C. & Woo, S. L. C. (1995). The Hyperphenylalaninaemias. In The Metabolic and Molecular Basis of Inherited Disease.
(Scriver, C. R., Beaudet, A. L., Sly, W. S. and Valle, D., eds), Pp. 1015-1077. New York: McGraw-Hill. 4. Zschocke, J., Graham, C. A., Stewart, F. J., Carson, D. J. & Nevin, N. C. (1994). Automated sequencing detects all mutations in Northern Irish patients with phenylketonuria and mild hyperphenylalaninaemia. Acta Paediatrica Suppl 407, 37-38. 5. Zschocke, J., Graham, C. A., McKnight, J. J. & Nevin, N. C. (1994). The STR system in the human phenylalanine hydroxylase gene: true fragment length obtained with fluorescent labelled PCR primers. Acta Paediatrica Suppl 407, 41-42. 6. Newton, C. R., Graham,A., Heptinstall, L. E. et al. (1989). Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Research 17, 2503-16.
Fluorescent multiplex ARMS method
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