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Identification of Disease Causing Mutations in Phenylketonuria by Denaturing Gradient Gel Electrophoresis Using the DCode System

Luc Michiels1, Baudouin Franois1, 2, and Jef Raus1, 2, 1 Dr. L. Willems-Instituut and 2 Limburgs Universitair Centrum, Universitaire Campus, B-3590 Diepenbeek, Belgium


Phenylketonuria (PKU) is a hereditary disease that gives rise to elevated blood levels of phenylalanine. The disease most frequently is caused by mutations in the phenylalanine hydroxylase gene. This gene is situated at the human chromosomal locus 12q22-q24.1 and encodes a hepatic enzyme that hydroxylates phenylalanine (PAH; phenylalanine 4-monooxygenase, EC The large variety of mutations described in this gene results in a broad range of plasma phenylalanine concentrations associated with phenotypic differences ranging from the severest form of PKU to the mildest hyperphenylalaninaemia. PKU is autosomal recessively inherited, carriers therefore are phenotypically normal, with no pronounced elevated phenylalanine or tyrosine blood levels. The need for a reliable method to identify persons carrying PAH mutations is evident. To identify mutations in each of the 13 exons of the PAH gene, Guldberg and coworkers (1993) reported a method based on DGGE.1 For the rapid screening of PKU patients and their relatives for mutations in the PAH exons and intron/exon boundaries we further refined this method by using multiplex PCR* amplifications combined with Multiplex Denaturing Gradient Gel electrophoresis analysis (Michiels et al., 1996).3



Genomic DNA of PKU patients was prepared from dried blood spots on Guthrie cards using the Chelex 100 extraction procedure (Walsh et al., 1991).5 Alternatively 0.5 ml fresh EDTA-treated blood sample was treated with 1 ml haemolysis buffer (HB) at 4 C for 10 minutes. White blood cells were pelleted (5 minutes at 2,500 rpm in a microfuge) and washed with HB until no red cell debris was left. After the removal of the supernatant, 500 l of 5% Chelex 100 (Bio-Rad, California) suspension in water was added and mixed thoroughly.

This step was followed by incubations at 56 C for 30 minutes and subsequently at 100 C for 5 minutes. After centrifuging (3 minutes at 2,500 rpm), the supernatant was stored at -20 C. Five l of these DNA preparations was used in a 20 l multiplex amplification reaction of the PAH exons and their intron-exon boundaries. GC-clamped primers for DGGE analysis of the PAH gene have been described by Guldberg et al., 1993.1 To achieve comparable PCR amplification efficiencies and non-overlapping, but high mutation resolution DGGE patterns, combinations of these primer sets were evaluated (Michiels et al. 1996).3 Amplifications were carried out in 20 l reaction mixtures, containing Perkin Elmer PCR reaction buffer with 1.5 mM MgCl2, 200 nM each dNTP, 800 nM each primer (MP 1: exon 1 + exon 4 + exon 13, MP2: exon 2 + exon 6+ exon 8, MP3 : exon 3 + exon 5 + exon 9, MP7 : exon 7 + exon 11, MP10: exon 10 + exon 12.) and 1.5 U of Taq DNA polymerase (Perkin Elmer). The Perkin Elmer 9600 thermal cycler was set at 5 minutes denaturation at 95 C followed by 40 cycles of 10 seconds denaturation at 95 C, 20 seconds annealing at 56 C and 40 seconds elongation at 72 C. After cycling, final elongation was done at 72 C for 5 minutes. Finall y, to generate heteroduplexes between heterozygous PAH gene fragments, the PCR fragments were incubated for 5 minutes at 95 C, 60 minutes at 65 C and 60 minutes at 37 C. Complete amplification mixes were used for DGGE analysis.

MP-DGGE was performed under two different electrophoresis conditions. A 6% polyacrylamide gel containing a 2070% (for MP2, MP3 and MP10) or a 3080% (for MP1 and MP7) gradient (Model 475 Gradient Delivery system, Bio-Rad) of urea and formamide (100% is 7 M urea and 40% formamide) was run at 60 C in TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0). Electrophoresis conditions were 130 V for 6 hours on the DCode apparatus (Bio-Rad). Gels were stained in 1 g/ml ethidium bromide and visualized by UV transillumination.

The point mutations were confirmed by sequencing the PCR amplified fragments with the non-GC clamped primer of each exon, using the Applied Biosystems Model 373A DNA Sequencing System and the PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems).


Denaturing gradient gel electrophoresis allows detection of more than 95% of the PKU causing mutations. In the Belgian population, we observed about 100 different mutations using DGGE technology. Among these, seven new mutations were identified (Michiels et al.,1998).4 The high mutation resolving power of DGGE is illustrated in Figure 1. This figure shows all the mutations or mutation combinations we located in exon 7 of Belgian PKU patients. The screening of PKU patients and their relatives is rather laborious when exon specific amplification and DGGE conditions are used. Therefore, to obta in compatible co-amplifications with comparable efficiencies, different exon PCR amplifications were combined. Moreover, to make sure that the resolving power to identify the exon specific mutations remains intact and that the DGGE patterns of the combined exons do not interfere with each other, the multiplex PCR amplifications have to be resolved subsequently on denaturing gradient gels. This resulted in a multiplex DGGE (MP-DGGE) of the PAH gene (Michiels et al. 1996).3 Examples of such an MP-DGGE are shown in Figure 2. The 13 different PAH exons are analyzed in 5 distinct multiplex PCR and DGGE patterns. Each exon shows distinct mutations, demonstrating that the resolving power of the DGGE gel remains intact. In addition, the DGGE patterns for the different exons do not overlap. Cycle sequencing of the DGGE DNA bands excised from the gel as described in the methods allows the identification of new mutations.

Several DNA preparation protocols have been tested: home made (see Methods) or commercially available kits such as Split Second from Boehringer Mannheim, Germany and DNA Easy-Prep from Lifecodes Corporation, USA, and can all be used in the MP-DGGE assay. All of them gave comparable results in the multiplex PCR/DGGE experiments.


In this report we demonstrate the use of powerful multiplex PCR amplification combined with DGGE analysis to rapidly identify mutations causing PKU. This MP-DGGE allows analysis of the complete PAH gene for three different individuals on one gel, whereas previously such analysis would take three gel runs. Moreover, the multiplex DGGE analysis is compatible with all the tested rapid DNA iso lation methods commercially available. With this procedure DNA extracted from dried blood spots on Guthrie cards can be successfully analyzed.


This work was carried out within the framework of Biomed 1 GENE-CT93-0081. Further support was obtained from the Fonds ter Bevordering van het Wetenschappelijk Onderzoek in het DWI (FWI).

1. Guldberg P., Henriksen K.F. and Gttler F. Molecular Analysis of Phenylketonuria in Denmark: 99 % of the Mutations Detected by Denaturing Gradient Gel Electrophoresis. Genomics, 17, 141146 (1993).

2. Gttler F., Hyperphenylalaninemia: Diagnosis and classification of the various types of phenylalanine hydroxylase deficiency in childhood. Acta Pediatr. Scand. 280 (Suppl): 180 (1989).

3. Michiels L., Franois B., Raus J. and Vandevyver C. Rapid identification of PKU-associated mutations by multiplex DGGE analysis of the PAH gene. J. of Inherited Metabolic Disease, 19, 735738, (1996).

4. Michiels L., Franois B., Raus J. and Vandevyver C. The identification of 7 new mutations in the phenylalanine hydroxylase gene associated with hyperphenylalanemia in the Belgian population. Human Mutation, Supplement 1, S123124, (1998).

5. Walsh P.S., Metzger D.A. and Higuchi R. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 10, 506513 (1991).

The Polymerase Chain Reaction (PCR) process is covered by patents owned by Hoffmann-LaRoche. Use of the PCR process requires a license.

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