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CpG dinucleotides are mutation hot spots in phenylketonuria
GENOMICS
5,936-939
(1989)
SHORT COMMUNICATION CpG Dinucleotides
Are Mutation
Hot Spots in Phenylketonuria
V~RONIQUE ABADIE, STANISLAS LYONNET, NICOLE MAURIN,* MONIQUE BERTHELON, CATHERINE CAILLAUD, FRANCIS GIRAUD,* JEAN-FRANCOIS MATTEI,* JEAN REY, FRAN~OISE REY, AND ARNOLD MUNNICH Unit& de Recherches de G&&ique Me’dicale, INSERM U-12, Hdpital des Enfants-Malades, 149 rue de S&res, 75743 Paris Cedex 15, France; and *Unite’ de Recherches de Physiopathologie Chromosomique, INSERM U-242, and Centre de Ge’ne’tique Me’dicale, Ho”pital d’Enfants de la Timone, 13385 Marseille Cedex 5, France Received
March
31,
1989,
Inc.
Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by a deficiency in a liverspecific enzyme, phenylalanine hydroxylase (PAH, EC 1.14.16.1). Three-point mutations are known to be linked to particular mutant RFLP haplotypes at the PAH locus (DiLella et al., 1986, 1987; Lyonnet et al., 1989). Considering that two of these alter CpG dinucleotides, we hypothesized that CpG doublets might represent hot spots for mutations in PKU, as has been previously shown in other genetic diseases (reviewed in Cooper and Youssoufian, 1988). For this reason, we screened the most CpG-rich exon of the PAH gene (exon 7) in 20 PKU kindreds of Mediterranean ancestry. This resulted in the detection of o&3%7543/89 $3.00 Copyright 0 1989 by Academic Press, All rights of reproduction in any form
936 Inc. reserved.
June
16,
1989
two new missense mutations altering CpG dinucleotides, including one mutation linked to mutant haplotype 1, the most frequent mutant haplotype in all series reported thus far (Woo, 1988). This supports the view that CpG doublets might contribute to the emergence of mutations in PKU. All 40 children studied had severe PAH deficiency, with plasma phenylalanine levels over 20 mg/lOO ml, on both neonatal screening and reevaluation under normal diet at one month of age (protein intake, 2 g/ kg/day). Determination of the RFLP haplotypes at the PAH locus (Kwok et al., 1985), polymerase chain reaction (PCR) amplification (Saiki et al., 1988), and detection of point mutations by the allele-specific oligonucleotide (ASO) technique were performed as previously described (Saiki et al., 1986). Amplified exons were recovered from 1% low-melting agarose gel, phosphorylated at the 5’-OH termini, ligated into Ml3 mp18 phages, and sequenced using the T7 DNA polymerase (Sequenase, United States Biochemical Corp.). In order to rule out an erroneous replication by the enzyme Taq polymerase, a new set of experiments was carried out when a nucleotide change was detected. The experiment was repeated twice, starting from PCR amplification of the genomic DNA from the patients. The seventh exon of the PAH gene normally contains 5 of the 22 CpG dinucleotides included in the coding region of the PAH mRNA (Kwok et al., 1985). Four doublets belong to an arginine codon (CGX) and one overlaps two codons (Fig. 1). Our systematic procedure allowed direct exploration of three doublets and detected two new mutations in 4/20 PKU patients. The first mutation was a G to A transition at the second nucleotide of codon 261 (CGA, primers used for amplification: A-B), resulting in the replacement of an
The coding region of the phenylalanine hydroxylase (PAH) gene contains 22 CpG dinucleotides, including five doublets in the seventh exon of the gene. We hypothesized that CpG doublets could represent mutation hot spots in PAH deficiencies and we carried out the systematic sequence analysis of exon 7 in 20 unrelated PAH-deficient kindreds of Mediterranean ancestry. This procedure resulted in the detection of two novel missense mutations whose location and nature (CG to CA and CG to TG) were consistent with the accidental deamination of a 5-methylcytosine in a CpG doublet (codon 26 1 arg-g’n and codon 252arg+trp). Moreover, the codon 261 mutation was found to be associated with mutant restriction fragment length polymorphism (RFLP) haplotype 1, the most frequent mutant RFLP haplotype at the PAH locus in the studies reported thus far. However, since the mutation was detected in only 36% of haplotype 1 mutant alleles, it appears that this haplotype at the PAH locus is genotypically heterogeneous in Mediterranean countries. c
revised
SHORT
937
COMMUNICATION
Mutation codon 252
Mutation codon 261
TCGA
TCGA
I
I AS0
2
I q
FIG. 1. Top: Sequence analysis of the mutations Arg +Gln at codon 261 and Arg+Trp at codon 252. N, normal sequence. M, mutant sequence. The mutant nucleotide is indicated by an asterisk. Bottom: Nucleotide sequence of exon 7 of the PAH gene. Exon sequences are in capital letters and intron sequences in lowercase letters. The sequences of the primers (A, B, and C) used for PCR amplifications and the sequences of the allele-specific oligonucleotide (ASO) probes are underlined. CpG dinucleotides are indicated by boldface letters and by the codon number with respect to the normal cDNA sequence (7)
arginine by a glutamine (261arg’“‘“) in three unrelated children (Fig. 1 and Table 1, pedigrees l-3). The other original mutation in exon 7 was a C to T transition at the first nucleotide of codon 252, resulting in the replacement of an arginine by a tryptophan (252 ‘Igetrp, primers A-B, Fig. 1). One patient was found to be homozygous for this particular mutation. Finally, two patients homozygous for haplotype 4 and born to first-cousin Algerian parents were found to be homozygous for the 280p’“+1ys mutation (primers A-C, ASOl, Fig. 1) (Lyonnet et al., 1988). This observation invalidates the hitherto exclusive association of haplotype 38 with this point mutation. The three transitions reported here are therefore consistent with the deamination of a methylcytosine in a CpG doublet on either the coding or the noncoding
strand of the gene. No mutation altering the CpG doublet of codon 243 was found in our series and no other deleterious mutation in exon 7 was detected. The RFLP haplotypes of mutant PAH genes were determined in pedigrees l-3 (Table l), first reported to carry the mutation at codon 261. These patients were found to be either homozygous or heterozygous for RFLP haplotype 1 (Woo, 1988). In order to investigate the prevalence of the codon 261 mutat.ion in PKU, a total of 33 mutant and 16 normal haplotype 1 alleles were tested for this genotype by the AS0 technique (AS02, Fig. 1) in various pedigrees of Mediterranean ancestry. This mutation accounted for 36% (12/ 33) of all mutant RFLP haplotype 1 genes in our study and was never found in 16 haplotype 1 normal genes (P < 0.02). The mutation at codon 261 was also never
938
SHORT
TABLE
COMMUNICATION
1
Association of the Mutation Arg 261 with Mutant RFLP Haplotype Locus
+
Gln at Codon 1 at the PAH
Haplotype Pedigree 1 2 3 4 5 6 7 8 9 Note. The an asterisk.
Father
Mother
Proband
*1/5 417 *1/7 +1/7
l/12 *1/1 20/4 117 *l/l 28/41 *l/16 +I/4 *1/5
*1/1 4/1* *1/m $111 2/l* ‘l/28 16/l* 1/1* *l/l*
211 *1/9 1617 119 *l/l mutant
genotype
at codon
261 (CCA)
is indicated
by
found in 140 mutant genes of different haplotypes. Moreover, an association between the mutation at codon 261 and mutant RFLP haplotype 1 was noted, since the parent who transmitted this mutant genotype also transmitted the mutant haplotype 1 gene (Table 1, pedigrees l-9). Thus, there is a complete concordance between this mutation and mutant haplotype 1. In the past few years, it has become increasingly clear that CpG dinucleotides play a prominent role in the emergence of mutations in man (reviewed in Cooper and Youssoufian, 1988). In higher vertebrates, about half the cytosines of the genome are 5-methylated, mainly when they belong to a 5’ CpG 3’ doublet. The accidental deamination of a 5-methylcytosine leads to a transition to thymine, which may not be excised by the DNA repair system, thus resulting in an irreversible point mutation (Coulondre et al., 1978; Mandel and Chambon, 1979). This study reports on two new missensemutations, both of which altered a CpG doublet (252 arg+trpand 261”g’g’“), and one mutation in the CpG doublet of codon 280 (280g’““ys), originally reported by Lyonnet et al. (1988). Both the location of the three mutations within CpG doublets and the type of the nucleotide transition observed are consistent with an accidental deamination event on either a coding or a noncoding 5-methylcytosine. If confirmed, this mechanism might contribute to the variety of mutant genotypes in PKU. In keeping with this, the remarkable heterogeneity of clinical phenotypes in hyperphenylalaninemias might well be accounted for (at least in part) by the existence of numerous mutations, differing by the residual activity of the mutant gene product and by compound heterozygosity for these mutant genotypes at the PAH locus. In addition, the existence of mutation hot spots in PKU also helps in understanding why some mutant
genotypes are linked to specific RFLP haplotypes and why some are not. Along these lines, the present study clearly shows that, although the 280g’“+‘ysmutation is linked to an old RFLP haplotype of putative Saracen origin (haplotype 38, Lyonnet et al., 1989), the same mutation may well occur on a different RFLP haplotype, especially as a CpG hot spot is involved. Conversely, a given mutant RFLP haplotype at the PAH locus may possibly carry different mutations in the Mediterranean countries. Indeed, the present study suggests that mutant haplotype 1, the most frequent mutant RFLP haplotype at the PAH locus whatever the population studied (Chakraborty et al., 1987; Rey et al.,1988; Aulehla-Scholz et al., 1988; Lichter-Konecki et al., 1988), bears at least two mutations (and probably many more) in Mediterranean countries, including one prevalent mutation in a CpG doublet, which accounts for 36% of all mutant haplotype 1 genes in this series. The observation that one haplotype may carry several genotypes and, conversely, that one genotype may be carried by several haplotypes at the PAH locus greatly reinforces, in our opinion, the idea that more or less recent mutations (particularly mutat.ions in CpG doublets) might have contributed to the genetic diversity of PAH deficiencies. ACKNOWLEDGMENTS We are thankful to Monique Poussikre, Catherine Bona’iti, Jean Frezal, and Alan Strickland for their help in preparing this manuscript. V.A. is a recipient of a Guigoz Foundation grant. This study was supported by a grant of the Minis&e de la Recherche et de la Technologie (no 88X0179).
REFERENCES 1.
AULEHLA~CHOLZ, C., VORGERD, M., SAUTTEK, E., LEUPOLD, D., MAHLMANN, R., ULLRICH, K., OLEK, K., AND HORST, J. (1988). Phenylketonuria: Distribution of DNA diagnostic patterns in German families. Hum. Genet. 78: 353-355.
2.
CHAKRABORTY, R., LIDSKY, A. S., DAIGER, F., GUTTLER, F., SULLIVAN, S., DILELLA, A. G., AND Woo, S. L. C. (1987). Polymorphic DNA haplotypes at the human phenylalanine hydroxylase locus and their relationship with phenylketonuria. Hum.
3.
4.
5.
40-46.
322:
799-803.
DILELLA, A. G., MARVIT, J., BRAYTON, K., AND Woo, S. L. C. (1987). An amino-acid substitution involved in phenylketonuria is in linkage disequilibrium with DNA haplotype 2. Nature (London)
7.
76:
DILELLA, A. G., MARVIT, J., LIDSKY, A. S., GUTTLER, F., AND WOO, S. L. C. (1986). Tight linkage between a splicing mutation and a specific DNA haplotype in phenylketonuria. Nature (London)
6.
Genet.
COOPER, D. N., AND YOUSSOUFIAN, H. (1988). The CpG dinucleotide and human genetic disease. Hum. Genet. 78: 151155. COULONDRE, C.. MILLER, J. H., FARABANGH, P. J., AND GILBERT, W. (1978). Molecular basis of base substitution hot spots in Escherichia coli. Nature (London) 274: 775-780.
327:
333-336.
KWOK, S. C. M., LEDLEY, F. D., DILELLA, A. G., ROBSON, K. J. H., AND WOO, S. L. C. (1985). Nucleotide sequence of a full-length complementary DNA clone and amino acid sequence
SHORT
8.
9.
10.
11.
COMMUNICATION
of human phenylalanine hydroxylase. Biochemistry 24: 446561. LIGHTER-KONECKI, U., KONECKI, D. S., DILELLA, A. G., BRAYTON, K., MARVIT, J., HAHN, T. M., TREFZ, F. K., AND Woo, S. L. C. (1988). Phenylalanine hydroxylase deficiency caused by a single base substitution in an exon of the human phenylalanine hydroxylase gene. Biochemistn, 27: 2881-2885. LYONNET, S., CAILLAUD, C., REY, F., BERTHELON, M., FREZAL, J., REY, J., AND MUNNICH, A. (1988). Guthrie cards for detection of point mutations in phenylketonuria. Lancet 2: 507. LYONNET. S., CAILLAUD, C., REY, F., BERTHELON, M., FREZAL, J., REY, J., AND MUNNICH, A. (1989). Molecular basis of phenylketonuria in Mediterrranean countries: A mutation associated with partial phenylalanine hydroxylase deficiency. Amer. J. Hum. Genet. 44: 511-517. MANDEL, J.-L., AND CHAMBON, P. (1979). DNA methylation: Organ specific variations in the methylation pattern within and
12.
13.
14.
15.
939
around ovalbumin and other chicken genes. Nucleic Acids Res. 7: 2081&2102. REY, F., BERTHELON, M., CAILLAUD, C.. LYONNET, S., AHAIXE, V., BLANDIN-SAVOJA, F.. FEINGOLD, J., SAUDUBRAY, J.-M., FREZAL, J., MUNNICH, A., AND REY, J. (1988). Clinical and molecular heterogeneity of phenylalanine hydroxylase deficiencies in France. Amer. J. Hum. Genet. 43: 914-921. SAIKI, R. K., BUGAWAN, T. L., HORN, G. T., MULIZ, K. B., AND ERLICH, H. A. (1986). Analysis of enzymatically amplified beta-globin and HLA-DQalpha DNA with allele specific oligonucleotide probes. Nature (London) 324: 163-166. SAIKI, R. K., GELFAND, D. H., STOFFEL, S., SCHARF,S. J., HIGUCHI,R., HORN, G. T., MULLIS, K. B., AND ERLICH, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491. WOO, S. L. C. (1988). Collation of RFLP haplotypes at the human phenylalanine hydroxylase (PAH) locus. Amer. J. Hum. Genet. 43: 781-783.
5,936-939
(1989)
SHORT COMMUNICATION CpG Dinucleotides
Are Mutation
Hot Spots in Phenylketonuria
V~RONIQUE ABADIE, STANISLAS LYONNET, NICOLE MAURIN,* MONIQUE BERTHELON, CATHERINE CAILLAUD, FRANCIS GIRAUD,* JEAN-FRANCOIS MATTEI,* JEAN REY, FRAN~OISE REY, AND ARNOLD MUNNICH Unit& de Recherches de G&&ique Me’dicale, INSERM U-12, Hdpital des Enfants-Malades, 149 rue de S&res, 75743 Paris Cedex 15, France; and *Unite’ de Recherches de Physiopathologie Chromosomique, INSERM U-242, and Centre de Ge’ne’tique Me’dicale, Ho”pital d’Enfants de la Timone, 13385 Marseille Cedex 5, France Received
March
31,
1989,
Inc.
Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by a deficiency in a liverspecific enzyme, phenylalanine hydroxylase (PAH, EC 1.14.16.1). Three-point mutations are known to be linked to particular mutant RFLP haplotypes at the PAH locus (DiLella et al., 1986, 1987; Lyonnet et al., 1989). Considering that two of these alter CpG dinucleotides, we hypothesized that CpG doublets might represent hot spots for mutations in PKU, as has been previously shown in other genetic diseases (reviewed in Cooper and Youssoufian, 1988). For this reason, we screened the most CpG-rich exon of the PAH gene (exon 7) in 20 PKU kindreds of Mediterranean ancestry. This resulted in the detection of o&3%7543/89 $3.00 Copyright 0 1989 by Academic Press, All rights of reproduction in any form
936 Inc. reserved.
June
16,
1989
two new missense mutations altering CpG dinucleotides, including one mutation linked to mutant haplotype 1, the most frequent mutant haplotype in all series reported thus far (Woo, 1988). This supports the view that CpG doublets might contribute to the emergence of mutations in PKU. All 40 children studied had severe PAH deficiency, with plasma phenylalanine levels over 20 mg/lOO ml, on both neonatal screening and reevaluation under normal diet at one month of age (protein intake, 2 g/ kg/day). Determination of the RFLP haplotypes at the PAH locus (Kwok et al., 1985), polymerase chain reaction (PCR) amplification (Saiki et al., 1988), and detection of point mutations by the allele-specific oligonucleotide (ASO) technique were performed as previously described (Saiki et al., 1986). Amplified exons were recovered from 1% low-melting agarose gel, phosphorylated at the 5’-OH termini, ligated into Ml3 mp18 phages, and sequenced using the T7 DNA polymerase (Sequenase, United States Biochemical Corp.). In order to rule out an erroneous replication by the enzyme Taq polymerase, a new set of experiments was carried out when a nucleotide change was detected. The experiment was repeated twice, starting from PCR amplification of the genomic DNA from the patients. The seventh exon of the PAH gene normally contains 5 of the 22 CpG dinucleotides included in the coding region of the PAH mRNA (Kwok et al., 1985). Four doublets belong to an arginine codon (CGX) and one overlaps two codons (Fig. 1). Our systematic procedure allowed direct exploration of three doublets and detected two new mutations in 4/20 PKU patients. The first mutation was a G to A transition at the second nucleotide of codon 261 (CGA, primers used for amplification: A-B), resulting in the replacement of an
The coding region of the phenylalanine hydroxylase (PAH) gene contains 22 CpG dinucleotides, including five doublets in the seventh exon of the gene. We hypothesized that CpG doublets could represent mutation hot spots in PAH deficiencies and we carried out the systematic sequence analysis of exon 7 in 20 unrelated PAH-deficient kindreds of Mediterranean ancestry. This procedure resulted in the detection of two novel missense mutations whose location and nature (CG to CA and CG to TG) were consistent with the accidental deamination of a 5-methylcytosine in a CpG doublet (codon 26 1 arg-g’n and codon 252arg+trp). Moreover, the codon 261 mutation was found to be associated with mutant restriction fragment length polymorphism (RFLP) haplotype 1, the most frequent mutant RFLP haplotype at the PAH locus in the studies reported thus far. However, since the mutation was detected in only 36% of haplotype 1 mutant alleles, it appears that this haplotype at the PAH locus is genotypically heterogeneous in Mediterranean countries. c
revised
SHORT
937
COMMUNICATION
Mutation codon 252
Mutation codon 261
TCGA
TCGA
I
I AS0
2
I q
FIG. 1. Top: Sequence analysis of the mutations Arg +Gln at codon 261 and Arg+Trp at codon 252. N, normal sequence. M, mutant sequence. The mutant nucleotide is indicated by an asterisk. Bottom: Nucleotide sequence of exon 7 of the PAH gene. Exon sequences are in capital letters and intron sequences in lowercase letters. The sequences of the primers (A, B, and C) used for PCR amplifications and the sequences of the allele-specific oligonucleotide (ASO) probes are underlined. CpG dinucleotides are indicated by boldface letters and by the codon number with respect to the normal cDNA sequence (7)
arginine by a glutamine (261arg’“‘“) in three unrelated children (Fig. 1 and Table 1, pedigrees l-3). The other original mutation in exon 7 was a C to T transition at the first nucleotide of codon 252, resulting in the replacement of an arginine by a tryptophan (252 ‘Igetrp, primers A-B, Fig. 1). One patient was found to be homozygous for this particular mutation. Finally, two patients homozygous for haplotype 4 and born to first-cousin Algerian parents were found to be homozygous for the 280p’“+1ys mutation (primers A-C, ASOl, Fig. 1) (Lyonnet et al., 1988). This observation invalidates the hitherto exclusive association of haplotype 38 with this point mutation. The three transitions reported here are therefore consistent with the deamination of a methylcytosine in a CpG doublet on either the coding or the noncoding
strand of the gene. No mutation altering the CpG doublet of codon 243 was found in our series and no other deleterious mutation in exon 7 was detected. The RFLP haplotypes of mutant PAH genes were determined in pedigrees l-3 (Table l), first reported to carry the mutation at codon 261. These patients were found to be either homozygous or heterozygous for RFLP haplotype 1 (Woo, 1988). In order to investigate the prevalence of the codon 261 mutat.ion in PKU, a total of 33 mutant and 16 normal haplotype 1 alleles were tested for this genotype by the AS0 technique (AS02, Fig. 1) in various pedigrees of Mediterranean ancestry. This mutation accounted for 36% (12/ 33) of all mutant RFLP haplotype 1 genes in our study and was never found in 16 haplotype 1 normal genes (P < 0.02). The mutation at codon 261 was also never
938
SHORT
TABLE
COMMUNICATION
1
Association of the Mutation Arg 261 with Mutant RFLP Haplotype Locus
+
Gln at Codon 1 at the PAH
Haplotype Pedigree 1 2 3 4 5 6 7 8 9 Note. The an asterisk.
Father
Mother
Proband
*1/5 417 *1/7 +1/7
l/12 *1/1 20/4 117 *l/l 28/41 *l/16 +I/4 *1/5
*1/1 4/1* *1/m $111 2/l* ‘l/28 16/l* 1/1* *l/l*
211 *1/9 1617 119 *l/l mutant
genotype
at codon
261 (CCA)
is indicated
by
found in 140 mutant genes of different haplotypes. Moreover, an association between the mutation at codon 261 and mutant RFLP haplotype 1 was noted, since the parent who transmitted this mutant genotype also transmitted the mutant haplotype 1 gene (Table 1, pedigrees l-9). Thus, there is a complete concordance between this mutation and mutant haplotype 1. In the past few years, it has become increasingly clear that CpG dinucleotides play a prominent role in the emergence of mutations in man (reviewed in Cooper and Youssoufian, 1988). In higher vertebrates, about half the cytosines of the genome are 5-methylated, mainly when they belong to a 5’ CpG 3’ doublet. The accidental deamination of a 5-methylcytosine leads to a transition to thymine, which may not be excised by the DNA repair system, thus resulting in an irreversible point mutation (Coulondre et al., 1978; Mandel and Chambon, 1979). This study reports on two new missensemutations, both of which altered a CpG doublet (252 arg+trpand 261”g’g’“), and one mutation in the CpG doublet of codon 280 (280g’““ys), originally reported by Lyonnet et al. (1988). Both the location of the three mutations within CpG doublets and the type of the nucleotide transition observed are consistent with an accidental deamination event on either a coding or a noncoding 5-methylcytosine. If confirmed, this mechanism might contribute to the variety of mutant genotypes in PKU. In keeping with this, the remarkable heterogeneity of clinical phenotypes in hyperphenylalaninemias might well be accounted for (at least in part) by the existence of numerous mutations, differing by the residual activity of the mutant gene product and by compound heterozygosity for these mutant genotypes at the PAH locus. In addition, the existence of mutation hot spots in PKU also helps in understanding why some mutant
genotypes are linked to specific RFLP haplotypes and why some are not. Along these lines, the present study clearly shows that, although the 280g’“+‘ysmutation is linked to an old RFLP haplotype of putative Saracen origin (haplotype 38, Lyonnet et al., 1989), the same mutation may well occur on a different RFLP haplotype, especially as a CpG hot spot is involved. Conversely, a given mutant RFLP haplotype at the PAH locus may possibly carry different mutations in the Mediterranean countries. Indeed, the present study suggests that mutant haplotype 1, the most frequent mutant RFLP haplotype at the PAH locus whatever the population studied (Chakraborty et al., 1987; Rey et al.,1988; Aulehla-Scholz et al., 1988; Lichter-Konecki et al., 1988), bears at least two mutations (and probably many more) in Mediterranean countries, including one prevalent mutation in a CpG doublet, which accounts for 36% of all mutant haplotype 1 genes in this series. The observation that one haplotype may carry several genotypes and, conversely, that one genotype may be carried by several haplotypes at the PAH locus greatly reinforces, in our opinion, the idea that more or less recent mutations (particularly mutat.ions in CpG doublets) might have contributed to the genetic diversity of PAH deficiencies. ACKNOWLEDGMENTS We are thankful to Monique Poussikre, Catherine Bona’iti, Jean Frezal, and Alan Strickland for their help in preparing this manuscript. V.A. is a recipient of a Guigoz Foundation grant. This study was supported by a grant of the Minis&e de la Recherche et de la Technologie (no 88X0179).
REFERENCES 1.
AULEHLA~CHOLZ, C., VORGERD, M., SAUTTEK, E., LEUPOLD, D., MAHLMANN, R., ULLRICH, K., OLEK, K., AND HORST, J. (1988). Phenylketonuria: Distribution of DNA diagnostic patterns in German families. Hum. Genet. 78: 353-355.
2.
CHAKRABORTY, R., LIDSKY, A. S., DAIGER, F., GUTTLER, F., SULLIVAN, S., DILELLA, A. G., AND Woo, S. L. C. (1987). Polymorphic DNA haplotypes at the human phenylalanine hydroxylase locus and their relationship with phenylketonuria. Hum.
3.
4.
5.
40-46.
322:
799-803.
DILELLA, A. G., MARVIT, J., BRAYTON, K., AND Woo, S. L. C. (1987). An amino-acid substitution involved in phenylketonuria is in linkage disequilibrium with DNA haplotype 2. Nature (London)
7.
76:
DILELLA, A. G., MARVIT, J., LIDSKY, A. S., GUTTLER, F., AND WOO, S. L. C. (1986). Tight linkage between a splicing mutation and a specific DNA haplotype in phenylketonuria. Nature (London)
6.
Genet.
COOPER, D. N., AND YOUSSOUFIAN, H. (1988). The CpG dinucleotide and human genetic disease. Hum. Genet. 78: 151155. COULONDRE, C.. MILLER, J. H., FARABANGH, P. J., AND GILBERT, W. (1978). Molecular basis of base substitution hot spots in Escherichia coli. Nature (London) 274: 775-780.
327:
333-336.
KWOK, S. C. M., LEDLEY, F. D., DILELLA, A. G., ROBSON, K. J. H., AND WOO, S. L. C. (1985). Nucleotide sequence of a full-length complementary DNA clone and amino acid sequence
SHORT
8.
9.
10.
11.
COMMUNICATION
of human phenylalanine hydroxylase. Biochemistry 24: 446561. LIGHTER-KONECKI, U., KONECKI, D. S., DILELLA, A. G., BRAYTON, K., MARVIT, J., HAHN, T. M., TREFZ, F. K., AND Woo, S. L. C. (1988). Phenylalanine hydroxylase deficiency caused by a single base substitution in an exon of the human phenylalanine hydroxylase gene. Biochemistn, 27: 2881-2885. LYONNET, S., CAILLAUD, C., REY, F., BERTHELON, M., FREZAL, J., REY, J., AND MUNNICH, A. (1988). Guthrie cards for detection of point mutations in phenylketonuria. Lancet 2: 507. LYONNET. S., CAILLAUD, C., REY, F., BERTHELON, M., FREZAL, J., REY, J., AND MUNNICH, A. (1989). Molecular basis of phenylketonuria in Mediterrranean countries: A mutation associated with partial phenylalanine hydroxylase deficiency. Amer. J. Hum. Genet. 44: 511-517. MANDEL, J.-L., AND CHAMBON, P. (1979). DNA methylation: Organ specific variations in the methylation pattern within and
12.
13.
14.
15.
939
around ovalbumin and other chicken genes. Nucleic Acids Res. 7: 2081&2102. REY, F., BERTHELON, M., CAILLAUD, C.. LYONNET, S., AHAIXE, V., BLANDIN-SAVOJA, F.. FEINGOLD, J., SAUDUBRAY, J.-M., FREZAL, J., MUNNICH, A., AND REY, J. (1988). Clinical and molecular heterogeneity of phenylalanine hydroxylase deficiencies in France. Amer. J. Hum. Genet. 43: 914-921. SAIKI, R. K., BUGAWAN, T. L., HORN, G. T., MULIZ, K. B., AND ERLICH, H. A. (1986). Analysis of enzymatically amplified beta-globin and HLA-DQalpha DNA with allele specific oligonucleotide probes. Nature (London) 324: 163-166. SAIKI, R. K., GELFAND, D. H., STOFFEL, S., SCHARF,S. J., HIGUCHI,R., HORN, G. T., MULLIS, K. B., AND ERLICH, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491. WOO, S. L. C. (1988). Collation of RFLP haplotypes at the human phenylalanine hydroxylase (PAH) locus. Amer. J. Hum. Genet. 43: 781-783.