© 1992 Oxford University Press
Human Molecular Genetics, Vol. 1, No. 9 765—766
A new PKU mutation associated with haplotype 12 Lourdes R.Desviat, Bel6n P6rez and Magdalena Ugarte* Centro de Biologfa Molecular 'Severe- Ochoa', Universidad Aut6noma de Madrid, Cantoblanco, 28049 Madrid, Spain Received October 16, 1992; Accepted October 20, 1992
* To whom correspondence should be addressed
MUTANT
NORMAL
G A T C
G A T C
fCG [.eu HTA-
GC | -GC P r o 2 4 4 ATj
UA
EXON 7 PAH GENE Figure 1. DNA sequence analysis of the missense mutation detected in exon 7 of the PAH gene of a Spanish PKU patient. The exon 7-containing region was amplified by PCR (4), and cloned into PUC8. A minimum of five independent clones were sequenced by the dideoxy method. The Figure shows the antisense sequence. The AGG (CCT triplet coding for Pro244) present in the normal sequence is changed in the mutant sequence for AAG (CTT triplet coding for Leu*").
1 2
3 4 NORMAL PROBE
12
3
4 MUTANT PROBE
Figure 2. Kindred analysis of the P244L mutation in a Spanish PKU family. 1, father; 2, mother; 3, sister; 4, PKU proband. Exon 7 of the PAH gene was amplified by PCR from the genomic DNA of each family member, dot-blotted onto a nylon membrane, and hybridized with ASO probes. The probes used to detect the P244L mutation were as follows: the normal probe (5'-CGCCTCCGACCTGTGGCTCG-3'), and the mutant probe (5'-CGCCTCCGACTTGGCTCG-3r). The underlined nucleotide is the mutated base.
appears to harbor different mutations, as occurs with haplotypes 1 and 4 (7). P244L is a new missense mutation occurring in exon 7 of the PAH gene. Many PKU mutations have been found in this exon.
Downloaded from http://hmg.oxfordjournals.org/ at James Cook University on March 15, 2015
Phenylketonuria (PKU) is an autosomal recessive disease that results from a reduction in phenylalanine hydroxylase (PAH) activity. The PAH gene has been cloned and mapped to chromosome 12, position 12q22—24.1 and it spans approximately 90 kb. The cDNA is 2.5 kb long and contains 13 exons that vary in length from 150 to 350 bp. The isolation of a PAH cDNA probe has permitted the characterization of restriction fragment length polymorphism (RFLP) haplotypes (1), studied in different populations. At the present time, more than 50 mutations in the human PAH gene have been reported. Most of them are associated with a specific haplotype and show a population-specific distribution. In Spain, 20% of the total of PKU mutations have been identified (2). The mutations found are prevalent in other Mediterranean countries. In order to search for new mutations that could account for the remaining Spanish PKU alleles we performed sequence analysis on one patient (NL). This patient had mutant haplotypes 12/9 and carried the previously described mutation I65T (3) on the haplotype 9 allele. Sequence analysis of exon 7 of patient NL revealed a new single base substitution of a C to a T at the second base of codon 244, causing a substitution of a Pro (CCT) for a Leu (CTT) (Figure 1). PCR amplification of exon 7 was performed using the primers and conditions described by DiLella et al. (4). The amplified product was purified by Gene Clean (Bio 101) and cloned into pUC8. Five individual recombinant colonies were selected for sequence analysis and the substitution was present in two of the clones. To ensure that the substitution was indeed a mutation and not a PCR artifact we amplified again exon 7 and repeated the analysis. The mutation was detected in 3 out of six independent clones. These results indicate that the patient is heterozygous for the mutant allele. To confirm the sequencing data and to determine the haplotype association, ASO analysis was performed on the whole family (Figure 2). The oligonucleotides probes used were: (5'-CGCCTCCGACCTGTGGCTCG-3') (Normal) and (5'-CGCCTCCGACTTGTGGCTCG-3') (Mutant) and the hybridization and washing temperature was 65 °C. The normal probe hybridized with each family member indicating successful amplification of all the DNA samples. The mutant probe hybridized with the amplified DNA from the father, the proband and another sibling, indicating that the mutation was inherited from the father, who carries a mutant haplotype 12 allele. Therefore, the P244L mutation is associated with haplotype 12 in this family. To establish the frequency of this mutation in Spain, a total of 24 PKU patients was screened by ASO analysis. None of these other patients carried a mutant haplotype 12, and, accordingly, the mutation was not detected on any of the 48 mutant chromosomes. Recently, two mutations have been described present on haplotype 12 alleles (5,6). Haplotype 12, therefore,
766 Human Molecular Genetics, Vol. 1, No. 9 There are several factors that may account for this. Exon 7 lies in the most highly conserved region between human and other species PAH (8) and among related aromatic aminoacid hydroxylases. On the other hand, exon 7 is rich in CpG dinucleotides which sustain a high frequency of mutation, probably resulting from methylation and subsequent deamination of cytosine to thymidine. The P244L mutation causes a non-conservative amino acid substitution and secondary structure predictions show that the change may affect the conformation of die molecule in that region. The expression analysis of the mutant protein will allow us to determine the effect of this substitution on the activity and stability of the protein. ACKNOWLEDGEMENTS
REFERENCES 1. Woo.S.L.C. (1989) Biochemistry 28, 1-7. 2. PeYez.B., Desviat.L.R., Die,M. and Ugarte.M. (1992) Hum. Genet. 89, 341-342. 3. John.S.W.M., Scnver,C.R., Laframboise.R. and Rozen,R. (1992) Hum. Mut. 1, 147-153. 4. DiLella.A.G., Huang.W.M. and Woo.S.L.C. (1988) Lancet I, 497-498. 5. Eiken.H.G., Stangeland.K., Skjelvale.L., Knappskog.P.M., Boman,H. and ApokU. (1992) Hum. Genet. 88, 608-612. 6. Svensson.E., Eisensmith,R.C, Dworniczak.B., von D6beln,U., Hagenfeldt.L., Horst.J. and Woo.S.L.C. (1992) Hum. Mut. 1, 129-137. 7. Konecki.D.S. and Lichter-Konccki.U. (1991) Hum. Genet. 87, 377-388. 8. Morales.G., RequenaJ.M., Jimenez-Ruiz.A., Lopez,M.C., Ugarte.M. and Alonso,C. (1990) Gene 93, 213-219.
Downloaded from http://hmg.oxfordjournals.org/ at James Cook University on March 15, 2015
The financial support of the Fundacion Ram6n Areccs to the Centra de Biologfa Molecular is gratefully acknowledged. This work was supported by a grant from the Consejerfa de Educacidn de la Comunidad dc Madrid.
Human Molecular Genetics, Vol. 1, No. 9 765—766
A new PKU mutation associated with haplotype 12 Lourdes R.Desviat, Bel6n P6rez and Magdalena Ugarte* Centro de Biologfa Molecular 'Severe- Ochoa', Universidad Aut6noma de Madrid, Cantoblanco, 28049 Madrid, Spain Received October 16, 1992; Accepted October 20, 1992
* To whom correspondence should be addressed
MUTANT
NORMAL
G A T C
G A T C
fCG [.eu HTA-
GC | -GC P r o 2 4 4 ATj
UA
EXON 7 PAH GENE Figure 1. DNA sequence analysis of the missense mutation detected in exon 7 of the PAH gene of a Spanish PKU patient. The exon 7-containing region was amplified by PCR (4), and cloned into PUC8. A minimum of five independent clones were sequenced by the dideoxy method. The Figure shows the antisense sequence. The AGG (CCT triplet coding for Pro244) present in the normal sequence is changed in the mutant sequence for AAG (CTT triplet coding for Leu*").
1 2
3 4 NORMAL PROBE
12
3
4 MUTANT PROBE
Figure 2. Kindred analysis of the P244L mutation in a Spanish PKU family. 1, father; 2, mother; 3, sister; 4, PKU proband. Exon 7 of the PAH gene was amplified by PCR from the genomic DNA of each family member, dot-blotted onto a nylon membrane, and hybridized with ASO probes. The probes used to detect the P244L mutation were as follows: the normal probe (5'-CGCCTCCGACCTGTGGCTCG-3'), and the mutant probe (5'-CGCCTCCGACTTGGCTCG-3r). The underlined nucleotide is the mutated base.
appears to harbor different mutations, as occurs with haplotypes 1 and 4 (7). P244L is a new missense mutation occurring in exon 7 of the PAH gene. Many PKU mutations have been found in this exon.
Downloaded from http://hmg.oxfordjournals.org/ at James Cook University on March 15, 2015
Phenylketonuria (PKU) is an autosomal recessive disease that results from a reduction in phenylalanine hydroxylase (PAH) activity. The PAH gene has been cloned and mapped to chromosome 12, position 12q22—24.1 and it spans approximately 90 kb. The cDNA is 2.5 kb long and contains 13 exons that vary in length from 150 to 350 bp. The isolation of a PAH cDNA probe has permitted the characterization of restriction fragment length polymorphism (RFLP) haplotypes (1), studied in different populations. At the present time, more than 50 mutations in the human PAH gene have been reported. Most of them are associated with a specific haplotype and show a population-specific distribution. In Spain, 20% of the total of PKU mutations have been identified (2). The mutations found are prevalent in other Mediterranean countries. In order to search for new mutations that could account for the remaining Spanish PKU alleles we performed sequence analysis on one patient (NL). This patient had mutant haplotypes 12/9 and carried the previously described mutation I65T (3) on the haplotype 9 allele. Sequence analysis of exon 7 of patient NL revealed a new single base substitution of a C to a T at the second base of codon 244, causing a substitution of a Pro (CCT) for a Leu (CTT) (Figure 1). PCR amplification of exon 7 was performed using the primers and conditions described by DiLella et al. (4). The amplified product was purified by Gene Clean (Bio 101) and cloned into pUC8. Five individual recombinant colonies were selected for sequence analysis and the substitution was present in two of the clones. To ensure that the substitution was indeed a mutation and not a PCR artifact we amplified again exon 7 and repeated the analysis. The mutation was detected in 3 out of six independent clones. These results indicate that the patient is heterozygous for the mutant allele. To confirm the sequencing data and to determine the haplotype association, ASO analysis was performed on the whole family (Figure 2). The oligonucleotides probes used were: (5'-CGCCTCCGACCTGTGGCTCG-3') (Normal) and (5'-CGCCTCCGACTTGTGGCTCG-3') (Mutant) and the hybridization and washing temperature was 65 °C. The normal probe hybridized with each family member indicating successful amplification of all the DNA samples. The mutant probe hybridized with the amplified DNA from the father, the proband and another sibling, indicating that the mutation was inherited from the father, who carries a mutant haplotype 12 allele. Therefore, the P244L mutation is associated with haplotype 12 in this family. To establish the frequency of this mutation in Spain, a total of 24 PKU patients was screened by ASO analysis. None of these other patients carried a mutant haplotype 12, and, accordingly, the mutation was not detected on any of the 48 mutant chromosomes. Recently, two mutations have been described present on haplotype 12 alleles (5,6). Haplotype 12, therefore,
766 Human Molecular Genetics, Vol. 1, No. 9 There are several factors that may account for this. Exon 7 lies in the most highly conserved region between human and other species PAH (8) and among related aromatic aminoacid hydroxylases. On the other hand, exon 7 is rich in CpG dinucleotides which sustain a high frequency of mutation, probably resulting from methylation and subsequent deamination of cytosine to thymidine. The P244L mutation causes a non-conservative amino acid substitution and secondary structure predictions show that the change may affect the conformation of die molecule in that region. The expression analysis of the mutant protein will allow us to determine the effect of this substitution on the activity and stability of the protein. ACKNOWLEDGEMENTS
REFERENCES 1. Woo.S.L.C. (1989) Biochemistry 28, 1-7. 2. PeYez.B., Desviat.L.R., Die,M. and Ugarte.M. (1992) Hum. Genet. 89, 341-342. 3. John.S.W.M., Scnver,C.R., Laframboise.R. and Rozen,R. (1992) Hum. Mut. 1, 147-153. 4. DiLella.A.G., Huang.W.M. and Woo.S.L.C. (1988) Lancet I, 497-498. 5. Eiken.H.G., Stangeland.K., Skjelvale.L., Knappskog.P.M., Boman,H. and ApokU. (1992) Hum. Genet. 88, 608-612. 6. Svensson.E., Eisensmith,R.C, Dworniczak.B., von D6beln,U., Hagenfeldt.L., Horst.J. and Woo.S.L.C. (1992) Hum. Mut. 1, 129-137. 7. Konecki.D.S. and Lichter-Konccki.U. (1991) Hum. Genet. 87, 377-388. 8. Morales.G., RequenaJ.M., Jimenez-Ruiz.A., Lopez,M.C., Ugarte.M. and Alonso,C. (1990) Gene 93, 213-219.
Downloaded from http://hmg.oxfordjournals.org/ at James Cook University on March 15, 2015
The financial support of the Fundacion Ram6n Areccs to the Centra de Biologfa Molecular is gratefully acknowledged. This work was supported by a grant from the Consejerfa de Educacidn de la Comunidad dc Madrid.
