J. Inher. Metab. Dis. t3 (1990) 739 750 © SSIEM and Kluwer AcademicPublishers. Printed in the Netherlands

Heterogeneity of Phenylketonuria at the Clinical, Protein and DNA Levels R. G. H. COTTON Olive Miller Protein Laboratory, The Murdoch Institute, Royal Children's Hospital, Melbourne, Victoria 3052, Australia

Summary: The cloning of the phenylalanine hydroxylase gene and cDNA has potentially allowed the complete characterization of patients with phenylketonuria and already many mutations have been defined. Parents of patients now have the option of prenatal diagnosis. The 18 mutations defined so far indicate enormous heterogeneity not only within particular populations but also between populations. These mutations give little indication as to the locations of the amino acid residues important in enzyme function but one-third of the mutations are in exon 7 which may be indicating the importance of the region coded by this exon in the protein.

The phenotype of phenylalanine hydroxylase (PAH, EC 1.4.16.i) deficiency has been known to be variable since the advent of the Guthrie and other tests allowed the study of large numbers of individuals with elevated serum and urinary phenylalanine and metabolites (McKusick 26160). This heterogeneity was detected first at the clinical level where some patients developed normally despite serum phenylalanine levels of greater than 20 #g//d (Hsia et al., 1968) and some individuals had intermediate phenylalanine levels (O'Flynn et al., 1967) now referred to as hyperphenylalaninaemia (HPA). During the 1970s liver biopsies were assayed for PAH enzyme activity and antibody reactive enzyme. Most recently since the isolation of a putative PAH cDNA (Robson et at., 1982), its authentication (Robson et al., 1984) and cloning of the human cDNA (Kwok et aI., 1985; Speeret at., 1986a, 1986b) and gene (DiLella et al., t986a), increasing numbers of mutations are being defined. All these studies indicate that there must be numerous mutations causing phenylketonuria (PKU) and HPA. The purpose of this work is to review the data of the last 10-15 years of molecular studies with particular emphasis on the DNA studies which allow exact and ultimate definition of the basic molecular defect. Detailed review of work before this time is contained in the following reviews and other reviews referred to therein: Cotton (1986), Scriver and Clow (1980) and Stanbury et al. (1983). In a heterogeneous disease like phenylalanine hydroxylase deficiency it would be a desirable end point of the current studies if the mutation(s) harboured by an MS received I1.5.89

Accepted 16.2.90

739

740

Cotton

individual could be used to predict outcome, i.e. in practical terms whether the diet was needed. A body of experience correlating clinical, metabotite, protein and DNA studies is necessary to fulfil this objective. The group having the most complete study in this regard is that initiated by Bickel in Heidelberg (reviewed in Cotton, 1986) and they are moving towards a near complete data set via their recent definition of mutations at the DNA level.

E N Z Y M E ACTIVITY STUDIES The most extensive studies correlating clinical phenotype with enzyme studies (a) in vitro, (b) in vivo and (c) with load tests have been from the Heidelberg group; these data have not been superseded in recent years and were reveiwed in detail earlier (Cotton, 1986). In this study of 71 patients with raised serum phenylalanine, all cases were classified as PKU or HPA on the basis of a standard load test. Two out of 37 of the cases of PKU had 1-5% of residual in vitro PAH activity, whereas the majority had values < 1% as expected. Of 27 cases of HPA, two were found who had < 1% activity but the majority had > 1 % , almost half having > 5 % residual activity. Thus it can be seen that the load test (used to define PKU or HPA) and in vitro activity do not always correlate. In vivo activity of 9 cases of PKU in the same group of patients using a deuterated phenylalanine load test showed good correlation with in vitro activity, i.e. < 1% for all in both assays. There were exceptions to the correlation in the 14 HPA cases studied. For example, one case had an unexpected 0.9% in vivo activity. These data indicate biochemical heterogeneity in the group.

P R O T E I N STUDIES As with other diseases, two major mutant phenotypes are expected, i.e. patients with detectable levels of immunologically cross-reacting material (CRM +) and those with none ( C R M - ) . The former molecules :represent inactive PAH. The largest series is that of Bartholom6 from the Heidelberg group which indicated in 10 PKU patients the expected variety of protein phenotypes. However, he used an antibody which has not been fully authenticated, for example by preadsorption with pure enzyme (Cotton, 1986). Nine of the PKU patients were found to have protein with the same charge as wild type whilst the tenth patient had a more negatively charged protein than wild type. A later study from this laboratory indicated that two P K U patients produced PAH the same size as wild type but one had a different charge (Heimlich et al., 1985). All studies from other laboratories with antisera to rat and human found no mutant protein in PKU. These disparate data, which might be due to the patients studied, could also be due to different characteristics of the antisera. For example, the antiserum of Bartholom6 may in fact sample more epitopes on the native PAH molecule. Two different laboratories have detected PAH protein in HPA. One used antibodies to rat PAH to detect this protein in three cases. Bartholom6 found CRM in two d. lnher. Mefab. Dis. 13 (1990)

Heterogeneity in P K U

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cases, showing that one had the same charge as wild type and the other was more negative than wild type. In three cases attempts at expression of mutant cDNA in tissue culture have been made (DiLella et aI., 1986b, 1987; Lichter-Konecki et al., 1988a). In all three PKU mutations, expression of in vitro mutagenized PAH in cell culture resulted in no activity or protein detectable by antibody to rat PAH. This data cannot be regarded as definitive at the protein level at least because it could be that mutant PAH is not detectable by the particular antibody used. Stability of mutant protein in the cells used for expression of the mutant protein may also not be the same as its stability in human liver cells. Thus it is not certain whether the lack of activity in the patient's liver is due to normal quantities of mutant but inactive protein or low or zero levels of active or inactive protein.

mRNA STUDIES Recently it has been possible to define the messenger RNA phenotype of cases of PKU. In three cases studied by one laboratory, two patients produced phenylalanine hydroxylase mRNA but it was absent in the third patient. Both mRNA positive cases had the same quantity and quality of mRNA as wild type (DiLella et al., 1985; Ledley et al., 1988). Preliminary data from another laboratory showed the mRNA to be of the same size and amount as wild type (Cotton et al., 1986) in a case of PKU.

DNA STUDIES Haplotyping the PAH gene The initial studies to find polymorphic sites in the region of the PAH gene defined ten alleles with eight enzymes (Lidsky et at., 1985a) but eight sites using seven enzymes (Chakraborty et al., 1987) are now used extensively. The pattern of alleles at these eight sites has been taken as the haplotype (Chakraborty et al., 1987). Even though the PAH gene is 100kb long, there appears to be little evidence for recombination (Lidsky et al., 1985a; Daiger et al., 1986; Chakraborty et al., 1987; Reiss et al., 1987) but one case has recently been described (Reiss et al., 1989). Haplotype analysis in relation to race, and other allele studies Definition of particular haplotypes was first carried out in the Danish population (Chakraborty et aL, 1987) but has now been extended to the German (Aulehla-Scholz et al., 1988; Lichter-Konecki et al., 1988b; Reiss et al., 1988), Swiss (Sullivan et al., 1989), Scottish (Sullivan et al., t989), French (Rey et al., 1988), USA (Moore et at., 1988), Italian (Dianzani et al., 1989a, 1989b), Chinese (Chen et al., 1989), Turkish (Lichter-Konecki et aL, 1989), Belgian (Verelst et aL, 1988), French Canadian (John et aL, 1988) and Polynesian (Hertzberg et al., 1989) populations (summarized in Table 1). Haplotypes in black (Hofman et al., 1987) and Jewish (Avigad et al., 1988) populations have not yet been published in detail. So far it appears that at least 43 J. lnher. Metab. Dis. 13 (1990)

G G A -* TGA(B) G A A ~ AAA

CTT -* C C T TTT ~ T G T CCG ~ CTG(M)

272 280

281 299 311

Del leu Del exon 12(U) arg ~ trp(U) arg --* pro

pro ~ leu phe --* cys leu -~ pro(U)

gly ~ S T O P glu -~ lys(L)

arg ~ gln

tyr --, cys arg --, S T O P arg ~ trp

met --* val phe -~ teu arg ---, S T O P Deletion arg---r gln

Amino acid change b

5 3 2 4

1,10

7 38,4

1

4

2 1 4 ? 4

Haplotype

?C C C C

C

C

C M

C

C C C

C C C C C

Phenotype ~ t 2 3 3 5

exon 11 intron 12 exon 12 exon 12

exon 7 exon 8 exon 9

exon 7 exon 7

exon 7

exon 6 exon 7 exon 7

exon exon exon exon exon

Localization J o h n et at., 1989 Forrest et al., 1989 W a n g et al., 1989a, 1989b Avigad et al., 1989 O k a n o et al., 1989a H o r s t et al., 1989 W a n g et al., 1989a O k a n o et al., 1989b O k a n o et al., 1989b Abadie et al., 1989 O k a n o et al., 1989b Abadie et al., 1989 Svensson et al., 1989 L y o n n e t et al., 1989 Abadie et al., 1989 O k a n o et al., 1989b O k a n o et al., 1989b O k a n o et al., 1989b Lichter-Konecki et al., 1988a Reiss et al., 1988 Verelst et af., 1988 H o f m a n et al., 1987 Svensson et al., 1989 DiLeUa et al., 1986b DiLella et al., 1987 W a n g et al., 1989a

Reference

"Where a restriction site change is affected it is designated (M = Mspl, B = B a m HI, H = Hind III) bWhere the effect on the enzyme is knoWn it is designated (U = unstable PAH, L = less active PAH) CPhenotype: where known it is designated (C = classical PKU, M = hyperphenylalaninemia). However, it does not necessarily mean it is proven that this mutation causes PAH deficiency by in vitro mutagenesis

CTC ~ del(H) G T --, AT CGG ~ TGG CGC ~ CCC

C G A ~ CAA

261

364 Splice 12 408 413

TAT --, T G T CGA ~ TGA C G G --, T G G

ATG ~ G T G T T C -~ T T G C G A ---,T G A ? CGG ~ CAG

1 39 111 Del exon 3 158

204 243 252

Base change a

Designation

Table 2 Mutations of phenylalanine hydroxylase

Heterogeneity in P K U

743

of the 512 possible variations have been defined (Aulehla-Scholz et al., 1988; Woo, 1988). However, many workers have reported haplotypes which are not in the original 12 and may be extra to the 43 so far collated by Woo (1988). Most mutant and normal alleles are contained in a small number of haplotypes as a rule in Caucasians; outside of these only one or a few alleles occur per haplotype. For example, in the largest series in Europe from the Heidelberg group (LichterKonecki et aI., 1988b) (100 normal and 100 mutant alleles) 87% of mutant and 59% of normal alleles are in haplotypes 1-4. For mutant alleles 25% are in haplotype 1, 24% in haplotype 2, 13% in haplotype 3 and 25% in haplotype 4. For normal alleles 25% are in haplotype 1, 10% in haptotype 2, 5% in haplotype 3 and t9% in haplotype 4. Further mutant and normal alleles are in I0 and t4 other haplotypes respectively. These distributions change little in the other two German studies and the French, Danish, Scottish, Belgian and Swiss populations. It is notable that 81% of French mutant alleles are in haplotypes 1, 2, 3,4 = 38 (in order of decreasing importance). However, this does not give a true picture of the French population as haplotype 38 mutations are carried by Algerians. The numbers are small in the Italian study but it can be said that the majority of mutant genes are in haplotypes nos. 1 and 6 (4 in each out of a total of 14). The increase in importance of haplotype 6 in Italy is consistent with the result in Turkey which is geographically close, where 39%' of the mutant alleles are in haplotype 6. In the Chinese study 10 out of 14 PKU genes are contained in haplotype 4 and it appears that over 50% of normal genes are contained in this haplotype. It is notable that there is a changed Xmn I fragment in this race. No mutant genes have been studied in Polynesia but it appears that 55% of normal genes are also in haplotype 4 (with 22% in 1 and 16% in 7) and this may reflect the Chinese ancestry of the Polynesians (Hertzberg et al., 1989). The details of numbers in particular haplotypes in the black population has not been published but it appears unremarkable in distribution. A particular mutation in Yemenite Jews appears in a single as yet undisclosed haplotype (Avigad et al., 1988). In summary, the predominant normal haplotypes appear to be 1, 2, 4, 5 and 7 in all races with 1, 2, 3 and 4 being prevalent for mutant alleles in Caucasians, and 6 being prevalent in Turks and Italians. Haplotype 4 is the prevalent mutant allele in Chinese and may turn out also to be so in Polynesians. The Yemenite Jews and Algerians appear to have particular mutant alleles of their own.

Mutations defined All mutations have so far been defined via recombinant DNA techniques and are summarized in Table 2. Eighteen defects in PKU genes have so far been described and it is known that three of these are most likely to give rise to unstable protein consistent with their causation of classical PKU; they have recently been reviewed in detail (Woo, 1989). Preliminary reports describe other mutations and all except the codon 280 change appear to be associated with classical PKU. Three are associated with restriction enzyme changes allowing easy detection.

J. Inher. Metab. Dis. 13 (1990)

e~

(10) 4

- (16)

1,2(14) 4,7= 1,5,2(64) 1,16 = 40(20) 4,1,7,6(622) 4,1,7,unclass(76)

1,4(14) 1,4,5 = 7(36)

1,4,5 = 7 = 8(3 l) 4 = 7,1,12(26) 1,4,7,2 = 5(100) 1,4,5,2(88) 4,1,3,2 = 5(38) 1,4,7,5(68)

~Haplotypes (Woo, 1988) given in descending order of prevalence bNumbers in brackets are the numbers of alleles studied ~Haplotypes 8 and 2 contained only two chromosomes aUnclassified indicates haplotype number not assigned

Blacks Welsh Gypsies

4,11 =unctassd(14)

1 =6,8(14) 6,1,4(64) 6,1,36(27) 38

Italian c Turkish

Algerian Yemenite Polynesians Chinese

1 =2,3,4(14) 1,4,2(38)

1,3,2(33) 2,3,1,4 = 8(25) 1 = 4,2,3(100) 2,1,4,3(88) 1,4,2,3(36) 1,2,3,4=38(74)

French Canadians Swiss

Scottish German 1 German 2 German 3 Belgian French

3,2,1,4(66) b

Danish

1,4,5=7(66)

Prominent haplotype a P K U-H PA Normal

Race and phenyiketonuria

Population

Table 1

311

280 Del exon 3 11t,204,413

l 408, splice 12 158,201 -

408, splice 12 31 l 408,311 408 311 252,261,280

408, splice 12

Mutations found

Dianzani et al, 1989a, 1989b Lichter-Konecki et al., 1989 Stuhrmann et al., 1989 Lyonnet et al., 1989 Avigad et al,, 1988 Hertzberg et al., 1989 Chen et aL, 1989 Hertzberg et aL, 1989 Wang et al., 1989a Hofman et al., 1987 Tyfield et al,, 1989

Chakraborty et al., 1987 Gfittler et aL, 1987 Sullivan et al., 1989 Reiss et al., 1988 Lichter-Konecki et aI., 1988b Aulehla-Scholz et al., 1988 Verelst et aL, 1988 Rey et al., 1988, Abadie et al., 1989 J o h n et al., t988, 1989 Sullivan et al., 1989

Reference

-..q

Heterogeneity in P K U

745

Relationship of these mutations to haplotype From the thalassaemia precedent it is expected that particular mutations may occur only in a particular haplotype. This has been found in the Danish population for mutations Splice 12 and 408. No examples of these were found outside haplotypes 3 and 2 respectively in 66 families and all mutant genes of haplotypes 3 and 2 contained only mutations Splice 12 and 408 respectively in those families (DiLella et al., 1986b, 1987). However, in one German study of 10 PKU alleles of haplotype 3, the Splice 12 mutation could not be found using synthetic oligonucleotides in one of the alleles (Aulehla-Seholz et al., 1988) but 13 patients with haplotype 3 in the other German study all had the Splice 12 mutation (Lichter-Konecki et al., 1988a). Concordance of Splice 12 and 408 mutations with haplotypes 3 and 2 respectively has also been shown in the Swiss and Scottish populations (Sullivan et al., 1989). Preliminary data from the Italian population (Dianzani et al., 1989a, 1989b) shows that only 1 out of 8 haplotype 3 chromosomes have the Splice 12 mutation and 2 out of 4 haplotype 2 show mutation 408. Further study in the Chinese population (Wang et al., 1989a) has shown mutation 111 to be present in 2 out of 9 P K U alleles while in the Swiss population 2 out of 6 haplotype 4 PKU alleles show mutation 158 and 13 out of 18 haplotype 1 show mutation 201 (Okano et al., 1989a). The only other data in this regard is that the exon 3 deletion is contained in the same haplotype in five Yemenite Jewish families, We will have to await further studies to see if haplotype 6 of the Turkish and the Italian populations contains a single mutation. It can be seen that on the whole haplotypes 1-4 each contain more than one mutation. It is possible that there may still be a particular mutation which is inclusively and exclusively linked to a particular haplotype, but heterogeneity appears to be the rule.

Relationship of mutations, haplotypes and phenotypes Five studies have related the degree of severity of the effect of the mutation to the haplotype of the mutant genes in a number of patients (Guttler et al., 1987; John et al., 1988; Lichter-Konecki et al., 1988b; Rey et al., 1988; Trefz et al., 1988; Verelst et al., 1988). The large Heidelberg study (Lichter-Konecki et al., 1988b, Trefz et al., 1988), however, has not detailed the numbers in each haplotype combination. The results of four of these studies are summarized in Table 3. It is clear at this time that 3/3 or 2/3 may be exclusively associated with classical PKU but exceptions are sure to be found: more precisely, if the Splice 12 mutation is present in the homozygous state or in combination with 408 classical PKU results. Haplotype 3 has been described as having a mutation other than Splice 12 (AulehlaScholz, et al. 1988) but it still appears to be associated with classical PKU. From the data it would appear that haplotype 3 (mainly the Splice mutation) in combination with any other allele (23 out of 30 cases) commonly gives rise to classical PKU, the exceptions being in the Danish data where mild cases occur in 3/1 and 3/4 combinations. This could simply reflect the different mutations present in the 1 and 4 alleles between the populations. It appears that haplotypes 1 and 4 in combination with themselves or each other can give rise to classical or mild PKU, again reflecting the heterogeneity of the mutations in these haplotypes. The German data indicates J. lnher. Metab. Dis. 13 (1990)

746

Cotton

Table 3 Correlation of haplotype with phenotype 1

2

3

4

X

M c(5) M C M C,M C, M(3) C,M C(2) c(3) M(3) C

X

C, M(3) C(2) C

C(2) C(2) C(2) C

C(2) C, M(2) M(2)

C M

C, M(4) C(4)

C(3)a, M(2) C

C(3) C

C(5)

C(2), M(4) C C C(2)

C(2)", M C,M

C, M C(3), M(4)

The top line in each box represents the Belgian data (Verelst et al., 1988).The second line represents the data from 74 mutant alleles studied in the French population (Rey et al., 1988). The third line represents data from the Danish population (Grittier et aL, 1987). The fourth line represents the French Canadian data (John et aL, I988). The numbers in brackets are the number of cases in that category.Where no number is given it represents one case. In this data C = classical PKU, M = HPA based on load tests. aTwo cases in each classical category were compound heterozygotes of haplotype no. 27. X represents haplotypes other than 1-4 HPA in 4/2 and 4/4 combinations which is the main difference from that shown in Table 3. The absence of complete numbers in the German studies precludes estimation of the numbers of minimum possible mutations in the PAH genes indicated so far. Outside haplotypes 1-4 one can estimate that there are at least nine different mutations in the biggest German study (Lichter-Konecki et al., 1988) if each haplotype represents a different mutation. The French study (Rey et aL, 1988) indicates at least eight mutations outside haplotypes 1-4. In fact, it is highly likely that there will be 50-100 mutations found. It wilt be some time before the mutations and their combinations which cause P K U are elucidated.

Relation to enzyme function Mutations in genes often indicate which amino acid residues are important for enzyme function. However, mutations could make the m R N A unstable, the protein unstable (proteolytically or structurally), or in fact make the mature mutant protein inactive. So far only one described mutation appears to have allowed expression of

J. lnher. Metab. Dis. t3 (1990)

Heterogeneity in P K U

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protein which is stable (Lyonnet et al., 1989). Three mutations (Splice 12, 408 and 311) probably express unstable protein in liver. The other mutations have not been expressed so it is not clear if large quantities of an inactive protein are produced in vivo. Strictly none of the mutations have yet been shown to cause PKU, only to be associated with it. Thus the mutations so far described provide little assistance towards the quest to identify the amino acids important in catalytic function and substrate binding. However, by reference to Table 2 it can seem that exon 7 contains 6 of the t8 described mutations. It is not yet clear if the position of the protein coded by this region is important in enzyme function.

Prenatal diagnosis As expected, the cloning of PAH quickly led to prenatal diagnosis of fetuses at risk of P K U (Lidsky et aL, 1985b; Speer et al., 1986b) as no other reliable method was available. With eight polymorphic loci there is a good chance of identifying an informative polymorphism. The Hind III (Lidsky ee al., 1985b; Speer et al., 1986b) and EcoRI potymorphisms (Lidsky et aL, 1985b) have been successfully used to predict the status of a fetus (Lidsky et al., 1985b; Speer et al., 1986b). It is predicted that prenatal diagnosis can be confidently applied to 90% of Caucasian families with previously affected children (Lidsky et at., 1985b). However, the situation appears different in the Chinese population as the locus is less heterogeneous for these alleles, and it is expected that 42% of Chinese PKU families are informative for prenatal diagnosis (Chert ee al., 1989). The Japanese population has also been found to be less polymorphic (Trefz et al., 1989). FUTURE DIRECTIONS Already oligonucleotides are widely used to detect known mutations in PKU and more recently the polymerase chain reaction technique has been applied to genomic DNA from white blood cells (DiLella et al., 1988; Lichter-Konecki et al., 1988b) and even to Guthrie blotters (Lyonnet et al., 1988). Thus known mutation detection will be easy. It is not yet clear whether in PKU it will be more efficient, when a couple presents for prenatal diagnosis, to screen for known mutations using oligonucleotides in the first affected child or to establish the identity (by RFLPs) of the affected parental genes in the first affected child for subsequent prenatal diagnosis. As with all genetic disease, somatic therapy with safety is probably at least 10-20 years away for PKU.

ACKNOWLEDGEMENTS Dr Susan Forrest and Dr Irma Dianzani are thanked for criticisms of the manuscript.

REFERENCES Abadie, V., Lyonnet, S., Maurin, N., Berthelon, M., Caillaud, C., Giraud, F., Mattei, J. F., Rey, J., Rey, F. and Munnich, A. CpG dinucleotides are mutation hot spots in phenylketonuria. Am. J. Hum. Genet. 45 (1989) Abstract 661 Aulehla-Scholz, C., Vorgerd, M., Sautter, E., Leupold, D., Mahlmann, R., Ullrich, K., Olek, J. lnher. Metab. Dis. 13 (1990)

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K. and Horst J. Phenylketonuria: distribution of DNA diagnostic patterns in German families. Hum. Genet. 78 (1988) 353-355 Avigad, S., Kleiman, S., Cohen, B. E., Orgad, S., Holtzer, L., Schwartz, G., Gazit, E. Lyonnet, S., Woo, S. L. C. and Shiloh, Y. Molecular population genetics of phenylketonuria in Israel. Am. J. Hum. Genet. 43 (1988) Abstract 838 Chakraborty, R., Lidsky, A. S., Daiger, S. P., Gfittler, F., Sullivan, S., DiLetla, A. G. and Woo, S. L. C. Polymorphic DNA haplotypes at the human phenylalanine hydroxylase locus and their relationship with phenylketonuria. Hum. Genet. 76 (1987) 40-46 Chen, S.-H., Hsiao, K.-J., Lin, L.-H., Liu, T.-T., Tang, R.-B. and Su, T.-S. Study of restriction fragment length polymorphisms at the human phenylalanine hydroxylase locus and evaluation of its potential application in prenatal diagnosis of phenylketonuria in Chinese. Hum. Genet. 81 (1989) 226-230 Cotton, R. G. H. Inborn errors of pterin metabolism. In: Blakley, R. L. and Whitehead, V. M. (eds), Pterins and Folates, Nutritional Pharmacologic and Physiological Aspects of Folates and Pterins, vol. 2, John Wiley, New York t986, pp. 359-412 Cotton, R. G. H., Dahl, H. H. M., Mercer, J. F. B., Jennings, I., Haan, E. A., Chow, C. W., Danks, D. M. and Morgan, F. J. Molecular biology of phenylalanine hydroxylase. J. Inher. Metab. Dis. 9 (t986) 206-208 Daiger, S. P., Chakraborty, R., Giittler, F., Lidsky, A. S., Koch, R. and Woo, S. L. C. Polymorphic DNA haplotypes at the phenylalanine hydroxylase locus in prenatal diagnosis of phenylketonuria. Lancet 1 (1986) 229-323 Dianzani, I., Farinasso, L., Fortina, P., Camaschella, C., Ponzone, R., Dahl, H. H. M., Cotton, R. G. H. and Ponzone, A. RFLP of the phenylalanine hydroxylase (PAH) gene in the Italian population. J. Inher. Metab. Dis. 12 (1989a) 162-165 Diazani, I., Camaschelta, C., Saglio, G., Ferrero, G. B., Romeo, G., Devoto, M., Romano, C., Cerone, R., Giovannini, M., Riva, E., Trefz, F. K., Lichter-Konecki, U. and Woo, S. L. C. Haplotype distribution and molecular defects of PKU in Italy, Abstract, SSIEM, London, 1989b Di Lella, A. G., Ledley, F. D., Rey, F., Munnich, A. and Woo, S. L. C. Detection of phenylalanine hydroxylase messenger RNA in liver biopsy samples from patients with phenylketonuria. Lancet 1 (1985) 160 161 DiLella, A. G., Kwok, S. C. M., Ledley, F. D., Marvit, J. and Woo, S. U C. Molecular structure and polymorphic map of the human phenylalanine hydroxylase gene. Biochemistry 25 (1986a) 743-749 DiLella, A. G., Marvit, J., Lidsky, A. S., Giittler, F. and Woo, S. L. C. Tight linkage between a splicing mutation and a specific DNA haplotype in phenylketonuria. Nature 322, (1986b) 799-803 DiLella, A. G., Marvit, J., Brayton, K. and Woo, S. L. C. An amino-acid substitution involved in phenylketonuria is in linkage disequilibrium with DNA haplotype 2. Nature 327 (1987) 333-336 DiLella, A. G., Huang, W.-M. and Woo, S. L. C. Screening for phenylketonuria mutations by DNA amplification with the polymerase chain reaction. Lancet 1 (1988) 497-499 Forrest, S. M., Howells, D. W., Dahl, H.-H. and Cotton, R. G. H. Use of the chemical cleavage method to detect mutations causing phenylketonuria and dihydropteridine reductase deficiency. Am. J. Hum. Genet. 45 (1989) Abstract 186 Grittier, F., Ledley, F. D., Lidsky, A. S., DiLella, A. G., Sullivan, S. E. and Woo, S. L. C. Correlation between polymorphic DNA haplotypes at phenylalanine hydroxylase locus and clinical phenotypes of phenylketonuria. J. Pediatr. 110 (1987) 68-71 Heimlich, J., Kopietz-Schulte, E., Olek, K. and Bartholom6. Mutant phenylalanine hydroxylase characterized by the immunoblotting technique. Eur. J. Pediatr. 143 (1985) 243 Hertzberg, M., Jahromi, K., Ferguson, V., Dahl, H. H. M., Mercer, J., Mickleson, K. N. P. and Trent, R. J. Phenylalanine hydroxylase gene haplotypes in Polynesians; evolutionary origins and absence of alleles associated with severe phenylketonuria. Am. J. Hum. Genet. 44 (1989) 382-387 J. Inher. Metab. Dis. 13 (1990)

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