GENOMICS 14, 1-5 (1992)
Molecular Basis for Nonphenylketonuria Hyperphenylalaninemia EFFROSINIECONOMOU-PETERSEN,KAREN FRIISHENRIKSEN, PER GULDBERG,AND FLEMMING GOTTLER The John F. Kennedy Institute, DK-2600 Glostrup, Denmark Received March 5, 1992; revised May 2, 1992
Nonphenylketonuria hyperphenylalaninemia (nonP K U H P A ) is d e f i n e d as p h e n y l a l a n i n e h y d r o x y l a s e (PAH) deficiency with blood phenylalanine levels bel o w 6 0 0 # m o l / l i t e r (i.e., w i t h i n t h e t h e r a p e u t i c r a n g e ) o n a n o r m a l d i e t a r y i n t a k e . H a p l o t y p e a n a l y s i s at t h e PAH locus was performed in 17 Danish families with non-PKU HPA, revealing compound heterozygosity in all individuals. By allele-specific oligonucleotide (ASO) p r o b i n g for c o m m o n P K U m u t a t i o n s w e f o u n d 1 2 o f 1 7 non-PKU HPA children with a PKU allele on one chrom o s o m e . T o i d e n t i f y m o l e c u l a r l e s i o n s i n t h e s e c o n d allele, individual exons were amplified by polymerase chain reaction and screened for mutations by singlestrand conformation polymorphism. Two new missense mutations were identified. Three children had inh e r i t e d a G - t o - A t r a n s i t i o n at c o d o n 4 1 5 i n e x o n 1 2 o f the PAH gene, resulting in the substitution of asparagine for aspartate, whereas one child possessed an Ato-G t r a n s i t i o n at c o d o n 3 0 6 i n e x o n 9, c a u s i n g t h e r e p l a c e m e n t o f a n i s o l e u c i n e b y a v a l i n e i n t h e e n z y m e . It is f u r t h e r d e m o n s t r a t e d t h a t t h e i d e n t i f i e d m u t a t i o n s h a v e l e s s i m p a c t o n t h e h e t e r o z y g o t e ' s a b i l i t y to h y d r o x y l a t e p h e n y l a l a n i n e to t y r o s i n e c o m p a r e d to t h e parents carrying a PKU mutation. The combined effect on PAH activity explains the non-PKU HPA phenotype of the child. The present observations that PKU mutations in combination with other mutations result in the non-PKU HPA phenotype and that particular mutation-restriction fragment length polymorphism haplotype combinations are associated with this phenotype offer the possibility of distinguishing PKU patients from non-PKU individuals by means of molecular analysis of the hyperphenylalaninemic neonate and, conseq u e n t l y , o f d e t e r m i n i n g w h e t h e r a n e w b o r n c h i l d req u i r e s d i e t a r y t r e a t m e n t . © 1992 AcademicPress. Inc.
INTRODUCTION Phenylketonuria (PKU) is an autosomal recessive inherited deficiency of hepatic phenylalanine hydroxylase, which untreated causes severe, irreversible mental retardation (Scriver et al., 1989). Neonatal screening programs based on determination of phenylalanine in the blood (Guthrie and Susi, 1963) have been implemented throughout the world, except for some countries in the
Third World, and form the basis for dietary therapy. Within the first decade of screening for neonatal hyperphenylalaninemia (HPA) it became clear that H P A due to phenylalanine hydroxylase deficiency is phenotypically and genetically heterogeneous (Woolf, 1971). The question of whether dietary management should be initiated in all hyperphenylalaninemic neonates arose when it was recognized that children with serum phenylalanine concentrations below 600 #mol/liter (non-PKU HPA) appear to show normal intellectual and behavioral development without treatment (Woolf et al., 1961; Levy et al., 1971). Some neonates with nonP K U H P A present with serum phenylalanine values above 600 #mol/liter (O'Flynn et al., 1967; Castells et al., 1968; Gfittler and Wamberg, 1972; Gfittler, 1980). A 24-h phenylalanine load test at 6 months of age will eventually classify the children as having n o n - P K U H P A not needing therapy (Grittier, 1971). Initiation of dietary therapy soon after birth has important psychological and social implications (Mikkelsen et al., 1974). Therefore, it is imperative as early as possible to determine whether a newborn child requires a phenylalanine-restricted diet (PKU) or not (non-PKU HPA). Today much is known about the molecular basis for the phenotypic diversity of P K U (Giittler et al., 1987; Okano et al., 1991). Restriction fragment length polymorphism (RFLP) haplotypes associated with nonP K U H P A alleles have been described in 13 n o n - P K U H P A families (DiSilvestre et al., 1990), and more recent R F L P haplotype studies suggest that n o n - P K U H P A may result from compound heterozygosity for a P K U allele and an allele supposed to harbor a mutation with a milder effect (Avigad et al., 1991). The present study demonstrates that in 17 families with n o n - P K U HPA, 12 children had in fact inherited one of the common P K U mutations. Screening for mutations in individual exons of the phenylalanine hydroxylase (PAH) gene by polymerase chain reaction (PCR) in combination with single-strand conformation polymorphism (SSCP) (Orita et al., 1989) revealed two new mutations in four of these children. The tyrosine response following a phenylalanine loading suggests than n o n - P K U H P A results from mutant heterozygosity for a codominantly expressed P K U allele and a mutation that has less effect on phenylalanine hydroxylase activity. 0888-7543/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ECONOMOU-PETERSEN ET AL. TABLE 1
MA T E R I A L S A N D M E T H O D S Patients. Non-PKU HPA newborns were detected among approximately one million neonates screened for blood phenylalanine at Days 5-7 of life, born in Denmark from 1975 through 1990. The cutoff value for follow-up testing is 150 ttmol/liter. The children were diagnosed to have non-PKU HPA when blood phenylalanine levels persistently remained below 600 #mol/liter either from the neonatal period or within the first 12-18 months of life on a normal dietary intake of protein, and when no other reasons for hyperphenylalaninemia could be demonstrated (see Gfittler, 1980; Gfittler et al., 1992). In addition, a phenylalanine load test dose of 0.1 g phenylalanine/kg body wt must be eliminated within 24 h of the loading, with a tyrosine response showing either no or a slight increase in blood tyrosine within the first hour of the load (Giittler and Wamberg, 1977; Giittler, 1980). Clinical chemical analysis. Neonatal screening for blood phenylalanine was performed with the Guthrie microbiological assay at the State Serum Institute, Copenhagen, Denmark. Serum phenylalanine as well as serum tyrosine was confirmed fluorometrically (Grittier, 1980). Blood amino acids were analyzed by ion-exchange chromatography (Gfittler et al., 1978). Pterines in the urine were determined by HPLC (Niederwieser et al., 1980), and dihydropteridine reductase activity in cultured fibroblasts as previously described (Giittler et al., 1977). A phenylalanine load was given to the fasting individual as a pure solution (fully dissolved by sonication in 0.01 mol/liter HC1 to give a final concentration of 173 mmol/liter and pH 3.8), and blood specimens for analysis of phenylalanine and tyrosine were obtained 0, 15, 30, 45, and 60 min and 2, 3, 4, and 24 h after intake of 0.1 g phenylalanine/kg body wt. The tyrosine response to an oral phenylalanine load is not affected by gender, and females were loaded when not using oral contraceptives. The intraindividual variation was approximately 8% (Giittler, 1980). Genomic D N A analysis. DNA was isolated from peripheral blood leukocytes, and RFLP haplotyping was performed as previously described (Grittier and Woo, 1985; Chakraborty et al., 1987). Allele-specific oligonucleotide probing. Individual exons and flanking intronic sequences of the PAH gene were enzymatically amplified by PCR (Saiki et al., 1985). Primers for amplification of exons 5, 7, 9, and 12 have been described elsewhere (Okano et al., 1990; Hofman et al., 1989; DiLella et al., 1988). One-half microgram genomic DNA was included in a 25-td PCR mixture containing deoxynucleoside triphosphates (50 ttM each), 10 pmol each of the appropriate primers, 10 mM Tris-HC1 (pH 8.3), 50 mM KC1, 1.5 mM MgClz, 0.01% gelatin, and 0.8 U of AmpliTaq (Perkin-Elmer/Cetus). Amplification consisted of 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s using a PerkinElmer/Cetus Thermal Cycler. An aliquot (1 #l) of the reaction mixture was applied to a Zeta-probe membrane (Bio-Rad) in a dot-blot apparatus. Allele-specific oligonucleotide (ASO) probes specific for common mutant alleles were end-labeled with [~,-z2p]dATP, and procedures for hybridization and washing were as described (DiLella et al., 1988). P C R - S S C P analysis. For SSCP analysis, genomic DNA was amplified as described above, apart from the inclusion of 12.5 #Ci each of [a-zsS]dATP and [a-zSS]dCTP (NEN) in the reaction. Ten microliters of the amplification product was mixed with 40 ttl of 0.1% sodium dodecyl sulfate and 10 mM EDTA, and an aliquot (3 ttl) was supplemented with an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol. The samples were denatured at 80°C for 2 min, quenched on ice, and applied to a 6% polyacrylamide gel containing 10% glycerol in 1x TBE (90 mM Tris-borate, pH 8.3, 4 mM EDTA). Electrophoresis was performed at 35 W for 5-6 h using a fan to provide cooling. The gel was dried and exposed directly to Kodak XAR-5 film for 18-72 h at room temperature. Direct D N A sequencing. For identification of mutations causing conformation polymorphisms, genomic DNA was amplified as described above, except that the concentration of nucleotides was reduced to 8 ttM. The amplification product was sequenced directly without purification using end-labeled amplification primers as sequencing primers (Wong et al., 1987).
Distribution of Haplotypes and Identified Mutations in 17 Patients with Non-PKU HPA a Haplotype
nb
Mutation
nb
1
7
R261Q R408W D415N ND c
1 1 3 2
2
4
R408W ND
2 2
3
5
IVS-12 ND
3 2
4
10
R158Q Y414C I306V ND
1 4 1 4
a Eight alleles associated with haplotypes 5, 6, 12, 19, 25, and 31. b Number of alleles. c Not determined.
RESULTS Table I d e m o n s t r a t e s our results for R F L P haplotypi n g a n d A S O p r o b i n g o f P C R - a m p l i f i e d D N A f r o m 17 c h i l d r e n (7 girls a n d 10 b o y s ) w i t h n o n - P K U H P A . E i g h t y p e r c e n t of the m u t a n t alleles were associated w i t h h a p l o t y p e s 1 t h r o u g h 4. T w e n t y p e r c e n t w e r e a s s ociated with more rare haplotypes. I n 12 p a t i e n t s o n e o f t h e c o m m o n P K U m u t a t i o n s w a s d e t e c t e d by A S O probing. Six p a t i e n t s had a m u t a t i o n c o m m o n l y found in p a t i e n t s with severe classic P K U ( R 4 0 8 W a n d I V S - 1 2 ) ( O k a n o e t al., 1991). E x o n s 5, 7, 9, a n d 12 o f t h e P A H g e n e in t h e s e p a t i e n t s w e r e s c r e e n e d for m u t a t i o n s by S S C P a n d two new m u t a t i o n s associated with the second allele were discovered. T h r e e of the children with non-PKU HPA had inherited a D 4 1 5 N m u t a t i o n in e x o n 12, a n d o n e child, a I 3 0 6 V m u t a t i o n in e x o n 9. T h u s , 12 k n o w n P K U m u t a t i o n s w e r e f o u n d o n o n e c h r o m o s o m e i n 17 p r o b a n d s w i t h n o n P K U H P A and two new m u t a t i o n s a c c o u n t e d for 4 additional m u t a t i o n s on the second c h r o m o s o m e . T h e r e f o r e 20 o f 34 o r 59% o f t h e m u t a n t c h r o m o s o m e s in n o n - P K U H P A i n d i v i d u a l s ar e a c c o u n t e d f o r in t h i s s t u d y . T h e carrier status of the p a r e n t s was d e t e r m i n e d by direct s e q u e n c i n g o f e x o n s 9 a n d 12, a n d t h e s e g r e g a t i o n o f t h e m u t a n t a l l e l e s is s h o w n in Fig. 1. T h e A --~ G t r a n s i t i o n a t t h e f i r st p o s i t i o n o f c o d o n 306 i n e x o n 9 is i l l u s t r a t e d i n Fig. 2a. T h i s m u t a t i o n results in an isoleucine-to-valine substitution. T h e D 4 1 5 N m u t a t i o n i n e x o n 12 is d e p i c t e d in Fig. 2b. T h e G --~ A t r a n s i t i o n r e s u l t s i n t h e s u b s t i t u t i o n o f a s p a r t a t e b y a s p a r a g i n e . As t h e m u t a t i o n s r e s u l t in a m i n o a c i d substitutions, they most likely cause some degree of P A H d e f i c i e n c y . I n v i t r o e x p r e s s i o n s t u d i e s w o u l d suggest the degree of P A H depression. T h e i n v i v o c o n s e q u e n c e o f b e i n g h e t e r o z y g o u s f or eit h e r a m u t a t i o n a s s o c i a t e d w i t h c l a s s i c P K U or o n e o f
3
NON-PKU HYPERPHENYLALANINEMIA MUTATIONS Serum-tyrosine i~mol/I
160
Mother 1306Vcarrier
15014013012011010090
807060-
~
daughter 1306V/IVS-12
50-
F I G . 1. Segregation of classic P K U alleles (R408W, IVS-12) and alleles harboring m u t a t i o n s with a milder effect on phenylalanine hydroxylase activity (D415N, I306V) in three families with the nonP K U H P A phenotype.
the mutations described in the present paper is illustrated in Fig. 3. The parent heterozygous for the nonP K U HPA mutation (I306V) formed more tyrosine following a phenylalanine load of 0.1 g/kg body wt than the parent heterozygous for the IVS-12 splicing mutation causing classic P K U (Fig. 3). Their daughter, who had inherited the I306V and IVS-12 mutations, responded with no measurable increase in tyrosine (Fig. 3). However, she eliminated the phenylalanine load test dose within 24 h (data not shown). DISCUSSION The present data on haplotype distribution in patients with non-PKU HPA are consistent with recently published observations by DiSilvestre et al. (1990), who found that a majority of the alleles segregating with nonP K U HPA were associated with haplotype 1 or 4. The present haplotype data are also consistent with the data recently published by Avigad et al. (1991), who demona
b MUTANT
A
C
G
NORMAL
T
A
EXON
9
C
G
MUTANT
T
AC
G
NORMAL
T
A
C
G
T
Normal
5 '- T A C G A C C C A - 3
'
Mutant
5' . . . . A A C . . . . 3' Asn415
A~G T T
EXON
i l e 306
12 A s p 415
Normal
5 '- G A A A T T G G C - 3
'
Mutant
5 ' . . . . G T T . . . . 3' Va1306
F I G . 2. Sequence analysis of missense m u t a t i o n s in exons 9 and 12 of the h u m a n P A H gene. (a) T h e A-to-G transition in exon 9 causes the substitution of Ile 3 0 6 by Val 3 0 6 . (b) T h e G-to-A transition in exon 12 results in the substitution of Asp 415 by Asn ~15.
40
~ 1
6
i
2
I
3
4
Hours after phenylalanineloading F I G . 3. Tyrosine response following a load of 0.1 g phenylalan i n e / k g body wt in p a r e n t s heterozygous for one of the P A H m u t a tions described in this paper (I306V) or a m u t a t i o n causing classic P K U (IVS-12) and in their n o n - P K U H P A child carrying both mutations.
strated that in 6 of 27 families with non-PKU HPA, the affected child was a compound heterozygote for a haplotype associated with a classic P K U allele and a haplotype associated with an allele with an unknown mutation. Avigad et al. (1991) further demonstrated that in 2 of the 27 families a non-PKU HPA child had inherited a mutation causing moderate or classic P K U (R261Q and R408W, respectively). The present study of 17 non-PKU HPA families revealed that 12 children had a P K U mutation on one chromosome. In addition, we have identified two new mutations in 4 of 6 patients who had inherited a classic P K U mutation (IVS-12 or R408W). It is further demonstrated that the identified mutations (D415N in exon 12 and I306V in exon 9 of the PAH gene) have less impact on the carriers' ability to hydroxylate phenylalanine to tyrosine compared to the parents carrying a classic P K U mutation. This may explain why the combined effect causes non-PKU HPA. Phenylalanine hydroxylase activity in liver biopsies of patients with classic P K U is barely detectable, whereas the activity in patients with non-PKU HPA is 5-10% of normal (Friedman et al., 1973; Kaufman and Max, 1971; Bartholom~ et al., 1975). In accordance with the in vitro determinations of phenylalanine hydroxylase activity in liver biopsies, we observed that serum tyrosine levels either were unaffected (Fig. 3) or showed a slight increase within the first hour of a phenylalanine load in 12 children with non-PKU HPA, whereas serum tyrosine decreased in 40 patients with classic P K U following phenylalanine loads (Gfittler and Hansen, 1977). The decrease in serum tyrosine following a phenylalanine load in P K U patients may be explained by a sharp rise in plasma insulin, and glucagon, which is detectable within 20 min of ingestion of the phenylalanine test dose (Gfittler et al., 1978).
4
ECONOMOU-PETERSEN ET AL.
T h e tyrosine response following a p h e n y l a l a n i n e load i n c a r r i e r s of a m u t a t i o n c a u s i n g classic P K U v e r s u s c a r r i e r s of a m u t a t i o n c a u s i n g n o n - P K U H P A o b s e r v e d i n t h e p r e s e n t s t u d y is i n a g r e e m e n t w i t h p r e v i o u s o b s e r vations on heterozygous heterogeneity in the phenylalan i n e h y d r o x y l a s e d e f i c i e n c y t r a i t ( W a n g e t al., 1961; R o s e n b l a t t a n d Scriver, 1968; Gfittler, 1980; S v e n s s o n e t al., 1990). T h e p r e s e n t d a t a i n c o m b i n a t i o n w i t h r e c e n t k n o w l e d g e o n t h e m o l e c u l a r b a s i s for P K U ( O k a n o e t al., 1991) m a y e x p l a i n p r e v i o u s o b s e r v a t i o n s of a c o r r e l a t i o n b e t w e e n p a r e n t a l p h e n o t y p e s , as d e t e r m i n e d b y t h e t y r o s i n e r e s p o n s e to a p h e n y l a l a n i n e load, a n d p h e n o t y p e of t h e affected o f f s p r i n g ( J a c k s o n e t al., 1971; G i i t t l e r , 1980; T r e f z e t al., 1990). I n s u m m a r y , t h e p r e s e n t u n d e r s t a n d i n g of t h e g e n e t i c b a s i s for n o n - P K U H P A e n a b l e s u s to d i s t i n g u i s h between PKU and non-PKU HPA neonates and thereby to i d e n t i f y t h o s e w h o n e e d t r e a t m e n t .
ACKNOWLEDGMENTS P.G. is supported by Fellowship 99-9903 from the Danish Research Academy. The present study was supported by grants from the Danish Medical Research Council (12-0058, 12-7414, 5.17.4.2.53, and 129292), the Danish Biotechnological Research and Developmental Programme 1991-1995 (Grant 5.18.03), the Danish Health Insurance Foundation (H 11/210-89, H 11/282-90, and H 11/257-91), the Foundation of 1870, the Novo Foundation, Frantz Hoffmann's Memorial Fund, Frode V. Nyegaard's Fund, and Stenild and Else Hjorth's Fund.
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tion of the various types of phenylalanine hydroxylase deficiency in childhood. Acta Paediatr. Scand. Suppl. 280: 1-80. Gfittler, F., Guldberg, P., Henriksen, K. F., Holck, B., Lou, H., Mikkelsen, I., Rasmussen, V., and Toft, P. (1992). Follow-up testing and diagnosis of the newborn with hyperphenylalaninemia.I n "Neonatal Screening in the Nineties" (B. Wilcken and D. Webster, Eds.), Byrton Lawrence Printers, Sydney. Grittier, F., and Hansen, G. (1977). Serum tyrosine within the first hour after an oral load of phenylalanine. Scand. J. Clin. Lab. Invest. 37: 717-722. Gfittler, F., Kaufman, S., and Milstein, S. (1977). Phenylalanine has no effect on dihydropteridine reductase activity in phenylketonuria fibroblasts. Lancet 2: 1139-1140. Gfittler, F., Kfihl, C., Pedersen, L., and P~by, P. (1978). Effect of oral phenylalanine load on plasma glucagon, insulin, amino acid and glucose concentrations in man. Scand. J. Clin. Lab. Invest. 38: 255260. Gfittler, F., Ledley, F. D., Lidskey, A. S., DiLella, A. G., Sullivan, S. E., and Woo, S. L. C. (1987). Correlation between polymorphic DNA haplotypes at phenylalanine hydroxylase locus and clinical phenotypes of phenylketonuria. J. Pediatr. 110: 68-71. Gfittler, F., and Wamberg, E. (1972). Persistent hyperphenylalaninemia. Acta Paediatr. Scand. 61: 321-328. Gfittler, F., and Wamberg, E. (1977). On indications for treatment of the hyperphenylalaninemic neonate. Acta Paediatr. Scand. 66: 3239-3244. Gfittler, F., and Woo, S. L. C. (1985). Molecular genetics of PKU: Prenatal diagnosis and carrier detection by gene analysis. In "Recent Progress in the Understanding, Recognition and Management of Inherited Disease of Amino Acid Metabolism" (H. Bickel and U. Wachtel, Eds.), pp. 18-36, Thieme, Stuttgart. Hofman, K. J., Antonarakis, S. E., Missiou-Tsangaraki, S., Boehm, C. D., and Valle, D. (1989). Phenylketonuria in the Greek population: Haplotype analysis of the phenylalaninehydroxylase gene and identification of a PKU mutation. Mol. Biol. Med. 6: 245-250. Jackson, S. H., Hanley, W. B., Gero, T., and Gosse, G. D. (1971). Detection of phenylketonuric heterozygotes. Clin. Chem. 17: 538543. Kaufman, S., and Max, E. (1971). Studies on the phenylalanine hydroxylase system in human liver and their relationship to pathogenesis of PKU and hyperphenylalaninemia.In "Phenylketonuria, and Some Other Inborn Errors of Amine Acid Metabolism" (H. Bickel, F. P. Hudson, and L. I. Woolf, Eds.), pp. 13-19, Thieme, Stuttgart. Levy, H. L., Shih, V. E., Karolkewicz, V., French, W. A., Carr, J. R., Cass, V., Kennedy, J. L., and MacCready, R. A. (1971). Persistent mild hyperphenylalaninemia in the untreated state. A prospective study. N. Engl. J. Med. 285: 424-429. Mikkelsen, I., Scharling, E., Svendsen, F. U., and Wamberg, E. (1974). The influence of dietary treatment on the psycho-social conditions in families with phenylketonuric children. N~iringsforskning 18: 78-86. Niederwieser, A., Curtius, H.-Ch., Gitzelman, R., Otten, A., Baerlocher, K., Blehova, B., Berlow, S., GrSbe, H., Rey, F., Schaub, J., Scheibenreiter, S., Schmidt, H., and Viscontini, M. (1980). Excretion of pterins in phenylketonuria and phenylketonuria variance. Helv. Paediatr. Acta 35: 335-342. O'Flynn, M. E., Tillman, P., and Hsia, D. Y.-Y. (1967). Hyperphenylalaninemia without phenylketonuria. Am. J. Dis. Child. 113: 2230. Okano, Y., Eisensmith, R. C., Giittler, F., Lichter-Konecki, U., Konecki, D. S., Trefz, F. K., Dasovich, M., Wang, T., Henriksen, K., Lou, H., and Woo, S. L. C. (1991). Molecular basis of phenotypic heterogeneity in phenylketonuria. N. EngL J. Med. 324: 12321238. Okano, Y., Wang, T., Eisensmith, R. C., Steinmann, B., Gitzelmann, R., and Woo, S. L. C. (1990). Missense mutation associated with
NON-PKU HYPERPHENYLALANINEMIA MUTATIONS RFLP haplotypes 1 and 4 of the human phenylalanine hydroxylase gene. Am. J. Hum. Genet. 46: 18-25. Orita, M., Suzuki, Y., Sekiya, T., and Hayashi, K. (1989). Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5: 874-879. Rosenblatt, D., and Scriver, C. R. (1968). Heterogeneity in genetic control of phenylalanine metabolism in man. Nature 218: 677-678. Saiki, R. K., Sharf, S. J., Faloona, F. A., Mullis, K. B., Horn, G. T., Erlich, H. A., and Arnheim, N. (1985). Enzymatic amplification of fi-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350-1354. Scriver, C. R., Kaufman, S., and Woo, S. L. C. (1989). The hyperphenylalaninemias. In "The Metabolic Basis of Inherited Disease" (C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, Eds.), 6th ed., pp. 495-546, McGraw-Hill, New York. Svensson, E., von DSbeln, U., Eisensmith, R. C., Hagenfeldt, L., and Woo, S. L. C. (1990). Genotype versus phenotype in Swedish PKU and HPA patients. In "Abstracts of the International PKU Workshop, H6pital des Enfants Malades, Paris, November 16-17, 1990." H6pital des Enfants Malades, Paris.
5
Trefz, F. K., Lichter-Konecki, U., Batzler, U., Wendel, U., Endres, W., and Konecki, D. S. (1990). Genotype/phenotype correlation in phenylalanine hydroxylase deficiency: A preliminary report of the German PKU Collaborative Study. In "Abstracts of the International PKU Workshop, HSpital des Enfants Malades, Paris, November 16-17, 1990." H6pital des Enfants Malades, Paris. Wang, H. L., Morton, N. E., and Waisman, H. A. (1961). Increased reliability for the determination of the carrier state in phenylketonuria. Am. J. Hum. Genet. 13: 255-261. Wong, C., Dowling, C. E., Saiki, R. K., Higuchi, R. G., Erlich, H. A., and Kazazian, H. H., Jr. (1987). Characterization of fi-thalassemia mutations using direct genomic sequencing of amplified single copy DNA. Nature 330: 384-386. Woolf, L. I. (1971). Genetic of phenylalaninemia. In "Phenylketonuria, and Some Other Inborn Errors of Amino Acid Metabolism" (H. Bickel, F. P. Hudson, and L. I. Woelf, Eds.), pp. 103-108, Georg Thieme Verlag, Stuttgart. Woolf, L. I., Ounsted, C., Lee, D., Humphrey, M., Chesire, N. M., and Steed, G. R. (1961). Atypical phenylketonuria in sisters with normal offspring. Lancet 2: 464-465.
Molecular Basis for Nonphenylketonuria Hyperphenylalaninemia EFFROSINIECONOMOU-PETERSEN,KAREN FRIISHENRIKSEN, PER GULDBERG,AND FLEMMING GOTTLER The John F. Kennedy Institute, DK-2600 Glostrup, Denmark Received March 5, 1992; revised May 2, 1992
Nonphenylketonuria hyperphenylalaninemia (nonP K U H P A ) is d e f i n e d as p h e n y l a l a n i n e h y d r o x y l a s e (PAH) deficiency with blood phenylalanine levels bel o w 6 0 0 # m o l / l i t e r (i.e., w i t h i n t h e t h e r a p e u t i c r a n g e ) o n a n o r m a l d i e t a r y i n t a k e . H a p l o t y p e a n a l y s i s at t h e PAH locus was performed in 17 Danish families with non-PKU HPA, revealing compound heterozygosity in all individuals. By allele-specific oligonucleotide (ASO) p r o b i n g for c o m m o n P K U m u t a t i o n s w e f o u n d 1 2 o f 1 7 non-PKU HPA children with a PKU allele on one chrom o s o m e . T o i d e n t i f y m o l e c u l a r l e s i o n s i n t h e s e c o n d allele, individual exons were amplified by polymerase chain reaction and screened for mutations by singlestrand conformation polymorphism. Two new missense mutations were identified. Three children had inh e r i t e d a G - t o - A t r a n s i t i o n at c o d o n 4 1 5 i n e x o n 1 2 o f the PAH gene, resulting in the substitution of asparagine for aspartate, whereas one child possessed an Ato-G t r a n s i t i o n at c o d o n 3 0 6 i n e x o n 9, c a u s i n g t h e r e p l a c e m e n t o f a n i s o l e u c i n e b y a v a l i n e i n t h e e n z y m e . It is f u r t h e r d e m o n s t r a t e d t h a t t h e i d e n t i f i e d m u t a t i o n s h a v e l e s s i m p a c t o n t h e h e t e r o z y g o t e ' s a b i l i t y to h y d r o x y l a t e p h e n y l a l a n i n e to t y r o s i n e c o m p a r e d to t h e parents carrying a PKU mutation. The combined effect on PAH activity explains the non-PKU HPA phenotype of the child. The present observations that PKU mutations in combination with other mutations result in the non-PKU HPA phenotype and that particular mutation-restriction fragment length polymorphism haplotype combinations are associated with this phenotype offer the possibility of distinguishing PKU patients from non-PKU individuals by means of molecular analysis of the hyperphenylalaninemic neonate and, conseq u e n t l y , o f d e t e r m i n i n g w h e t h e r a n e w b o r n c h i l d req u i r e s d i e t a r y t r e a t m e n t . © 1992 AcademicPress. Inc.
INTRODUCTION Phenylketonuria (PKU) is an autosomal recessive inherited deficiency of hepatic phenylalanine hydroxylase, which untreated causes severe, irreversible mental retardation (Scriver et al., 1989). Neonatal screening programs based on determination of phenylalanine in the blood (Guthrie and Susi, 1963) have been implemented throughout the world, except for some countries in the
Third World, and form the basis for dietary therapy. Within the first decade of screening for neonatal hyperphenylalaninemia (HPA) it became clear that H P A due to phenylalanine hydroxylase deficiency is phenotypically and genetically heterogeneous (Woolf, 1971). The question of whether dietary management should be initiated in all hyperphenylalaninemic neonates arose when it was recognized that children with serum phenylalanine concentrations below 600 #mol/liter (non-PKU HPA) appear to show normal intellectual and behavioral development without treatment (Woolf et al., 1961; Levy et al., 1971). Some neonates with nonP K U H P A present with serum phenylalanine values above 600 #mol/liter (O'Flynn et al., 1967; Castells et al., 1968; Gfittler and Wamberg, 1972; Gfittler, 1980). A 24-h phenylalanine load test at 6 months of age will eventually classify the children as having n o n - P K U H P A not needing therapy (Grittier, 1971). Initiation of dietary therapy soon after birth has important psychological and social implications (Mikkelsen et al., 1974). Therefore, it is imperative as early as possible to determine whether a newborn child requires a phenylalanine-restricted diet (PKU) or not (non-PKU HPA). Today much is known about the molecular basis for the phenotypic diversity of P K U (Giittler et al., 1987; Okano et al., 1991). Restriction fragment length polymorphism (RFLP) haplotypes associated with nonP K U H P A alleles have been described in 13 n o n - P K U H P A families (DiSilvestre et al., 1990), and more recent R F L P haplotype studies suggest that n o n - P K U H P A may result from compound heterozygosity for a P K U allele and an allele supposed to harbor a mutation with a milder effect (Avigad et al., 1991). The present study demonstrates that in 17 families with n o n - P K U HPA, 12 children had in fact inherited one of the common P K U mutations. Screening for mutations in individual exons of the phenylalanine hydroxylase (PAH) gene by polymerase chain reaction (PCR) in combination with single-strand conformation polymorphism (SSCP) (Orita et al., 1989) revealed two new mutations in four of these children. The tyrosine response following a phenylalanine loading suggests than n o n - P K U H P A results from mutant heterozygosity for a codominantly expressed P K U allele and a mutation that has less effect on phenylalanine hydroxylase activity. 0888-7543/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
2
ECONOMOU-PETERSEN ET AL. TABLE 1
MA T E R I A L S A N D M E T H O D S Patients. Non-PKU HPA newborns were detected among approximately one million neonates screened for blood phenylalanine at Days 5-7 of life, born in Denmark from 1975 through 1990. The cutoff value for follow-up testing is 150 ttmol/liter. The children were diagnosed to have non-PKU HPA when blood phenylalanine levels persistently remained below 600 #mol/liter either from the neonatal period or within the first 12-18 months of life on a normal dietary intake of protein, and when no other reasons for hyperphenylalaninemia could be demonstrated (see Gfittler, 1980; Gfittler et al., 1992). In addition, a phenylalanine load test dose of 0.1 g phenylalanine/kg body wt must be eliminated within 24 h of the loading, with a tyrosine response showing either no or a slight increase in blood tyrosine within the first hour of the load (Giittler and Wamberg, 1977; Giittler, 1980). Clinical chemical analysis. Neonatal screening for blood phenylalanine was performed with the Guthrie microbiological assay at the State Serum Institute, Copenhagen, Denmark. Serum phenylalanine as well as serum tyrosine was confirmed fluorometrically (Grittier, 1980). Blood amino acids were analyzed by ion-exchange chromatography (Gfittler et al., 1978). Pterines in the urine were determined by HPLC (Niederwieser et al., 1980), and dihydropteridine reductase activity in cultured fibroblasts as previously described (Giittler et al., 1977). A phenylalanine load was given to the fasting individual as a pure solution (fully dissolved by sonication in 0.01 mol/liter HC1 to give a final concentration of 173 mmol/liter and pH 3.8), and blood specimens for analysis of phenylalanine and tyrosine were obtained 0, 15, 30, 45, and 60 min and 2, 3, 4, and 24 h after intake of 0.1 g phenylalanine/kg body wt. The tyrosine response to an oral phenylalanine load is not affected by gender, and females were loaded when not using oral contraceptives. The intraindividual variation was approximately 8% (Giittler, 1980). Genomic D N A analysis. DNA was isolated from peripheral blood leukocytes, and RFLP haplotyping was performed as previously described (Grittier and Woo, 1985; Chakraborty et al., 1987). Allele-specific oligonucleotide probing. Individual exons and flanking intronic sequences of the PAH gene were enzymatically amplified by PCR (Saiki et al., 1985). Primers for amplification of exons 5, 7, 9, and 12 have been described elsewhere (Okano et al., 1990; Hofman et al., 1989; DiLella et al., 1988). One-half microgram genomic DNA was included in a 25-td PCR mixture containing deoxynucleoside triphosphates (50 ttM each), 10 pmol each of the appropriate primers, 10 mM Tris-HC1 (pH 8.3), 50 mM KC1, 1.5 mM MgClz, 0.01% gelatin, and 0.8 U of AmpliTaq (Perkin-Elmer/Cetus). Amplification consisted of 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s using a PerkinElmer/Cetus Thermal Cycler. An aliquot (1 #l) of the reaction mixture was applied to a Zeta-probe membrane (Bio-Rad) in a dot-blot apparatus. Allele-specific oligonucleotide (ASO) probes specific for common mutant alleles were end-labeled with [~,-z2p]dATP, and procedures for hybridization and washing were as described (DiLella et al., 1988). P C R - S S C P analysis. For SSCP analysis, genomic DNA was amplified as described above, apart from the inclusion of 12.5 #Ci each of [a-zsS]dATP and [a-zSS]dCTP (NEN) in the reaction. Ten microliters of the amplification product was mixed with 40 ttl of 0.1% sodium dodecyl sulfate and 10 mM EDTA, and an aliquot (3 ttl) was supplemented with an equal volume of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol. The samples were denatured at 80°C for 2 min, quenched on ice, and applied to a 6% polyacrylamide gel containing 10% glycerol in 1x TBE (90 mM Tris-borate, pH 8.3, 4 mM EDTA). Electrophoresis was performed at 35 W for 5-6 h using a fan to provide cooling. The gel was dried and exposed directly to Kodak XAR-5 film for 18-72 h at room temperature. Direct D N A sequencing. For identification of mutations causing conformation polymorphisms, genomic DNA was amplified as described above, except that the concentration of nucleotides was reduced to 8 ttM. The amplification product was sequenced directly without purification using end-labeled amplification primers as sequencing primers (Wong et al., 1987).
Distribution of Haplotypes and Identified Mutations in 17 Patients with Non-PKU HPA a Haplotype
nb
Mutation
nb
1
7
R261Q R408W D415N ND c
1 1 3 2
2
4
R408W ND
2 2
3
5
IVS-12 ND
3 2
4
10
R158Q Y414C I306V ND
1 4 1 4
a Eight alleles associated with haplotypes 5, 6, 12, 19, 25, and 31. b Number of alleles. c Not determined.
RESULTS Table I d e m o n s t r a t e s our results for R F L P haplotypi n g a n d A S O p r o b i n g o f P C R - a m p l i f i e d D N A f r o m 17 c h i l d r e n (7 girls a n d 10 b o y s ) w i t h n o n - P K U H P A . E i g h t y p e r c e n t of the m u t a n t alleles were associated w i t h h a p l o t y p e s 1 t h r o u g h 4. T w e n t y p e r c e n t w e r e a s s ociated with more rare haplotypes. I n 12 p a t i e n t s o n e o f t h e c o m m o n P K U m u t a t i o n s w a s d e t e c t e d by A S O probing. Six p a t i e n t s had a m u t a t i o n c o m m o n l y found in p a t i e n t s with severe classic P K U ( R 4 0 8 W a n d I V S - 1 2 ) ( O k a n o e t al., 1991). E x o n s 5, 7, 9, a n d 12 o f t h e P A H g e n e in t h e s e p a t i e n t s w e r e s c r e e n e d for m u t a t i o n s by S S C P a n d two new m u t a t i o n s associated with the second allele were discovered. T h r e e of the children with non-PKU HPA had inherited a D 4 1 5 N m u t a t i o n in e x o n 12, a n d o n e child, a I 3 0 6 V m u t a t i o n in e x o n 9. T h u s , 12 k n o w n P K U m u t a t i o n s w e r e f o u n d o n o n e c h r o m o s o m e i n 17 p r o b a n d s w i t h n o n P K U H P A and two new m u t a t i o n s a c c o u n t e d for 4 additional m u t a t i o n s on the second c h r o m o s o m e . T h e r e f o r e 20 o f 34 o r 59% o f t h e m u t a n t c h r o m o s o m e s in n o n - P K U H P A i n d i v i d u a l s ar e a c c o u n t e d f o r in t h i s s t u d y . T h e carrier status of the p a r e n t s was d e t e r m i n e d by direct s e q u e n c i n g o f e x o n s 9 a n d 12, a n d t h e s e g r e g a t i o n o f t h e m u t a n t a l l e l e s is s h o w n in Fig. 1. T h e A --~ G t r a n s i t i o n a t t h e f i r st p o s i t i o n o f c o d o n 306 i n e x o n 9 is i l l u s t r a t e d i n Fig. 2a. T h i s m u t a t i o n results in an isoleucine-to-valine substitution. T h e D 4 1 5 N m u t a t i o n i n e x o n 12 is d e p i c t e d in Fig. 2b. T h e G --~ A t r a n s i t i o n r e s u l t s i n t h e s u b s t i t u t i o n o f a s p a r t a t e b y a s p a r a g i n e . As t h e m u t a t i o n s r e s u l t in a m i n o a c i d substitutions, they most likely cause some degree of P A H d e f i c i e n c y . I n v i t r o e x p r e s s i o n s t u d i e s w o u l d suggest the degree of P A H depression. T h e i n v i v o c o n s e q u e n c e o f b e i n g h e t e r o z y g o u s f or eit h e r a m u t a t i o n a s s o c i a t e d w i t h c l a s s i c P K U or o n e o f
3
NON-PKU HYPERPHENYLALANINEMIA MUTATIONS Serum-tyrosine i~mol/I
160
Mother 1306Vcarrier
15014013012011010090
807060-
~
daughter 1306V/IVS-12
50-
F I G . 1. Segregation of classic P K U alleles (R408W, IVS-12) and alleles harboring m u t a t i o n s with a milder effect on phenylalanine hydroxylase activity (D415N, I306V) in three families with the nonP K U H P A phenotype.
the mutations described in the present paper is illustrated in Fig. 3. The parent heterozygous for the nonP K U HPA mutation (I306V) formed more tyrosine following a phenylalanine load of 0.1 g/kg body wt than the parent heterozygous for the IVS-12 splicing mutation causing classic P K U (Fig. 3). Their daughter, who had inherited the I306V and IVS-12 mutations, responded with no measurable increase in tyrosine (Fig. 3). However, she eliminated the phenylalanine load test dose within 24 h (data not shown). DISCUSSION The present data on haplotype distribution in patients with non-PKU HPA are consistent with recently published observations by DiSilvestre et al. (1990), who found that a majority of the alleles segregating with nonP K U HPA were associated with haplotype 1 or 4. The present haplotype data are also consistent with the data recently published by Avigad et al. (1991), who demona
b MUTANT
A
C
G
NORMAL
T
A
EXON
9
C
G
MUTANT
T
AC
G
NORMAL
T
A
C
G
T
Normal
5 '- T A C G A C C C A - 3
'
Mutant
5' . . . . A A C . . . . 3' Asn415
A~G T T
EXON
i l e 306
12 A s p 415
Normal
5 '- G A A A T T G G C - 3
'
Mutant
5 ' . . . . G T T . . . . 3' Va1306
F I G . 2. Sequence analysis of missense m u t a t i o n s in exons 9 and 12 of the h u m a n P A H gene. (a) T h e A-to-G transition in exon 9 causes the substitution of Ile 3 0 6 by Val 3 0 6 . (b) T h e G-to-A transition in exon 12 results in the substitution of Asp 415 by Asn ~15.
40
~ 1
6
i
2
I
3
4
Hours after phenylalanineloading F I G . 3. Tyrosine response following a load of 0.1 g phenylalan i n e / k g body wt in p a r e n t s heterozygous for one of the P A H m u t a tions described in this paper (I306V) or a m u t a t i o n causing classic P K U (IVS-12) and in their n o n - P K U H P A child carrying both mutations.
strated that in 6 of 27 families with non-PKU HPA, the affected child was a compound heterozygote for a haplotype associated with a classic P K U allele and a haplotype associated with an allele with an unknown mutation. Avigad et al. (1991) further demonstrated that in 2 of the 27 families a non-PKU HPA child had inherited a mutation causing moderate or classic P K U (R261Q and R408W, respectively). The present study of 17 non-PKU HPA families revealed that 12 children had a P K U mutation on one chromosome. In addition, we have identified two new mutations in 4 of 6 patients who had inherited a classic P K U mutation (IVS-12 or R408W). It is further demonstrated that the identified mutations (D415N in exon 12 and I306V in exon 9 of the PAH gene) have less impact on the carriers' ability to hydroxylate phenylalanine to tyrosine compared to the parents carrying a classic P K U mutation. This may explain why the combined effect causes non-PKU HPA. Phenylalanine hydroxylase activity in liver biopsies of patients with classic P K U is barely detectable, whereas the activity in patients with non-PKU HPA is 5-10% of normal (Friedman et al., 1973; Kaufman and Max, 1971; Bartholom~ et al., 1975). In accordance with the in vitro determinations of phenylalanine hydroxylase activity in liver biopsies, we observed that serum tyrosine levels either were unaffected (Fig. 3) or showed a slight increase within the first hour of a phenylalanine load in 12 children with non-PKU HPA, whereas serum tyrosine decreased in 40 patients with classic P K U following phenylalanine loads (Gfittler and Hansen, 1977). The decrease in serum tyrosine following a phenylalanine load in P K U patients may be explained by a sharp rise in plasma insulin, and glucagon, which is detectable within 20 min of ingestion of the phenylalanine test dose (Gfittler et al., 1978).
4
ECONOMOU-PETERSEN ET AL.
T h e tyrosine response following a p h e n y l a l a n i n e load i n c a r r i e r s of a m u t a t i o n c a u s i n g classic P K U v e r s u s c a r r i e r s of a m u t a t i o n c a u s i n g n o n - P K U H P A o b s e r v e d i n t h e p r e s e n t s t u d y is i n a g r e e m e n t w i t h p r e v i o u s o b s e r vations on heterozygous heterogeneity in the phenylalan i n e h y d r o x y l a s e d e f i c i e n c y t r a i t ( W a n g e t al., 1961; R o s e n b l a t t a n d Scriver, 1968; Gfittler, 1980; S v e n s s o n e t al., 1990). T h e p r e s e n t d a t a i n c o m b i n a t i o n w i t h r e c e n t k n o w l e d g e o n t h e m o l e c u l a r b a s i s for P K U ( O k a n o e t al., 1991) m a y e x p l a i n p r e v i o u s o b s e r v a t i o n s of a c o r r e l a t i o n b e t w e e n p a r e n t a l p h e n o t y p e s , as d e t e r m i n e d b y t h e t y r o s i n e r e s p o n s e to a p h e n y l a l a n i n e load, a n d p h e n o t y p e of t h e affected o f f s p r i n g ( J a c k s o n e t al., 1971; G i i t t l e r , 1980; T r e f z e t al., 1990). I n s u m m a r y , t h e p r e s e n t u n d e r s t a n d i n g of t h e g e n e t i c b a s i s for n o n - P K U H P A e n a b l e s u s to d i s t i n g u i s h between PKU and non-PKU HPA neonates and thereby to i d e n t i f y t h o s e w h o n e e d t r e a t m e n t .
ACKNOWLEDGMENTS P.G. is supported by Fellowship 99-9903 from the Danish Research Academy. The present study was supported by grants from the Danish Medical Research Council (12-0058, 12-7414, 5.17.4.2.53, and 129292), the Danish Biotechnological Research and Developmental Programme 1991-1995 (Grant 5.18.03), the Danish Health Insurance Foundation (H 11/210-89, H 11/282-90, and H 11/257-91), the Foundation of 1870, the Novo Foundation, Frantz Hoffmann's Memorial Fund, Frode V. Nyegaard's Fund, and Stenild and Else Hjorth's Fund.
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