The primary molecular defects in phenylketonuria and its variants

REVIEW THE PRIMARY

MOLECULAR DEFECTS IN PHENYLKETONURIA AND ITS VARIANTS R. G. H. COTTON

Genetics

Research

Unit. Royal

Children’s Hospital Research Foundation, Victoria 3052. Australia (Receicrd

22 Norrtnhrr

INTRODUCTION

Ever since the first demonstration by Fiilling (1934) of the association of mental retardation and other characteristic symptoms with the excretion of phenylpyruvic acid there has been constant interest in the elucidation of the molecular defect. Because of the many difficulties in studying not only mutant human phenylalanine hydroxylase (PH) but also the native enzyme, progress has been slow, and thus very little is known of the molecular defect. The purpose of this review is to attempt to collect all the information which has contributed to the knowledge of the primary molecular defect in phenylketonuria (PKU) and its variants. This field has only been reviewed to a minor extent in the past (Kaufman, 1975, 1976; Knox, 1972). The large number of loading tests and the large body of clinical information will be omitted except where necessary but these have been extensively rcvirwed from time to time (for example see Knox. 1972; Hsia, 1970; Bickel rt ul., 1971). The important practical aspect of the distinction of PKU from other variants is not relevant to this review and has been frequently dealt with. The presumed secondary molecular defects such as deficient myelin formation will be excluded as it has been reviewed (Knox, 1972; Malmud, 1966).

Flemington

Road.

Parkville,

1976)

present in the neonatal period. In practical terms they are usually distinguished by the fact that the hyperphenylalaninemic tolerates a higher dietary phenylalanine during treatment which aims to keep the serum phenylalanine in the normal range. The implication is that the block in the phenylalanine elimination in HPA is not as severe as in PKU. Other categories of transient elevations of serum phenylalanine have also been described (see for example Aurebach et al., 1967) but will not be discussed, as information about the molecular defect is scant. More recently. a distinct variant has been identified in which the patient develops neurological symptoms despite good dietary control and eventually the condition may deteriorate so that death ensues well hcforc the first decade at ages ranging from 2 to 7 years. For brevity we will refer to this variant as lethal hypcrphenylalanincmia (LHPA). Phenylalanine hydroxylase will be distinguished from the “phenylalanine hydroxylating system”. The latter will refer to the sum of all the molecular components needed for maximum hydroxylation of phenylalanine, the former will be the smallest unit which shows activity when presented with its substrates.

GENETICS

NOMENCLATURE

From the observation of two cases in one family Penrose (1935) suggested PKU was recessively inherited and Jervis (1937) established an autosomal recessive inheritance. Hyperphenylalaninemia also appears to be an autosomal recessive (Kang, 1975) but the study was not extensive. Examination of the cases showing clinical evidence of lethal hyperphenylalaninemia indicates that it also is likely to be an autosomal recessive as in one family malt and female siblings were atTccted (Smith CI t/l.. 1975) and the fact that consanguinity is involved in 3 families (Kaufman et al., 1975a; Danks rt al., 1976). Some variability has been found in the abilities of both PKU and HPA individuals to metabolize phenylalanine (Woolf rt al., 1967, 1968; Rosenblatt & Striver. 1968). This might be expected due to the many amino acid substitutions that can ail’cct the activity of the components of the phenylalanine hydroxylating system.

The word phenylketonuria

is derived.from the early finding that this disease is characterized by high levels of the phenylketone. phenylpyruvic acid in the urine. High levels of serum and urinary phenylalanine are also characteristic. Before the dietary treatment was u\cd. ~;IIIC~IS with this disease progrcs~d 10 the characteristic symptoms. the most notable being mental retardation. ,Now that dietary treatment is used, the characteristic marker of mental retardation is not seen. Also Guthrie screening for elevated blood phenylalanine has revealed people with increased serum phenylalanine levels who, despite no dietary treatment. develop normally. This latter category (where phenylalanine elevation is not as severe as in PKU). will be referred to as hyperphenylalaninemia (HPA) and this arca has hcen thoroughly rcvicwed (Hsia et ccl.,1968). Thus the clinician may have difticulty distinguishing between the two when they arc 333

R. G. H. COTTON

334 THE PHENYLALANINE

HYDROXYLATING

SYSTEM

This will not be reviewed exhaustively as this is not the purpose of this paper, but it has been reviewed extensively at regular intervals in the past (Kaufman, 1962; Kaufman, 1971; Kaufman & Fisher, 1974: Kaufman, 1976; Massey & Hemmerich, 1975). The current knowledge will be described and much of this stems from the careful work of Kaufman and his group. Only the information with the human enzyme using the natural cofactor will be detailed but where necessary this will be supplemented with information from other species where it will be indicated. Many properties of PH have been found to be markedly different with the synthetic cofactors as substrates (Friedman & Kaufman, 1973). While this does not make studies in the synthetic cofactors unimportant, any findings should ultimately be compared with those for the natural cofactor. The similarities of the properties and function of tyrosine and tryptophan hydroxylases to PH (Kaufman & Fisher, 1974) should also be emphasized at this stage. (a) Polyprptidr

componef7ts

Phenylalanine hydroxylase has a mol. wt of 108,000 but under certain conditions can exist as a 250,000 species (Woo et al., 1974). Work in our laboratory (Yap & Cotton, 1976) has indicated that the in vivo mol. wt of human, monkey and rat PH may be about 250,000. On SDS-electrophoresis a single mol. wt species has been found on 54,000 mol wt. Further work will be required to eliminate the possibility that two species of the same mol. wt are present. Two different peptides have been demonstrated on SDS in foetal human enzyme and monkey enzyme (Cotton & Danks, 1976) but the smaller may result from the proteolysis of the larger species. Two species have also been demonstrated in the case of the rat (Kaufman & Fisher, 1974) and again this could be due to proteolysis. Amino acid analysis has been performed on the purified rat enzyme (Kaufman & Fisher, 1974). Multiple forms of the enzyme have been reported by Barranger et al. (1972) after separation on calcium phosphate gel. Foetal enzyme showed two species and the adult three. In the rat these species have been

PHS

p*R

0% -

2

BH,Q I 0H,70

Fig. I. The phenylalanine hydroxylating system. The system comprises (I) Phenylalanine hydroxylase (phosphorylated form) (PHI. This is stimulated hy phenylalanine hydroxylase stimulating protein (PHS) in citro. (2) Dihydropteridine reductase (DHPR) keeps biopterin in the tetrahydro form (BH,). The recovery of 7.X-dihydrobiopterin BH, 7,8 (formed chemically from the quinonoid form [BH,Q] does not seem to be physiologically important. The point of entry of dietary and freshly synthesized biopterin is not clear.

shown to be of the same mol. wt (200,000). However chemical studies are needed to decide which of these are coded by separate genes or are post-translational modifications such as proteolyic modifications. Phenylalanine hydroxylase stimulating protein (PHS) from the rat has been shown to stimulate rat and human PH and this protein has been demonstrated as being present in normal and PKU human liver (Huang et al., 1973). The purification of rat PHS has revealed a mol. wt of 51,500 consisting of 4 subunits of 12,500. The function appears to be to catalyse the conversion of an intermediate to products (Huang & Kaufman. 1973) but the in vivo significance is not clear. Dihydropteridine reductase (DHPR) is an essential component which is needed to return the cofactor tetrahydrobiopterin. to the reduced form after conversion to the dihydro form during the reaction (see Fig. I). The enzyme from sheep is NADH-dependent and has been shown to have a mol. wt of 4142.800 with subunits of 21.300. the amino acid composition has been determined (Craine rot al., 1972). Dihydrofolate reductase (DHFR) has been suggested as being essential to normal phenylalanine metabolism because this NADPH dependent enzyme is thought to be able to scavenge any 7.8-dihydrobiopterin. see Fig. 1 (see below), which forms chemically from the quinonoid form or is produced by the organism or is derived from the diet (Kaufman it al., 1975~; Kaufman, 1976). It has also been suggested that a complete deficiency of this enzyme would be lethal (Kaufman et al., 1975a). However both these suggestions now seem incorrect with the identification of two cases of DHFR deficiency with 0% and approx 257: residual activity, respectively (Tauro et (I/.. 1976). These patients showed normal Guthrie tests (unpublished) and HVE analysis of the urine (Tauro rt ul.. 1976). Further evidence that DHFR is not important for normal phenylalanine metabolism comes from inhibition studies (Stone. 1976). As expected from earlier work using tyrosine hydroxylase (Lovenberg rt al.. 1975) PH has now been found to be a phosphoprotein and to be activated when phosphorylated (Milstein et al., 1976). The implication is that another essential component of the PH system might well be a “protein kinase”. This type of activation would have many analogies in other systems. Catalase has been shown to stimulate PH in citro (Bublitz, 1969; Nielsen, 1969) but the in vice significance is not clear. However as blood phenylalanine levels in two homozygote acatalasemic individuals were not different from normal levels (data kindly provided by H. Aebi) catalase appears unimportant. Another protein, Y protein, was isolated from monkey liver extract which stimulated activity (Cotton, 1971) and again the significance is not clear but on molecular weight considerations it appears distinct from catalase. (b) Non-protein

components

The cofactor in uivo is probably tetrahydrobiopterin (BH,) (Kaufman, 1963) but the possibility that it may be another 6 substituted tetrahydropterin like tetrahydroneopterin cannot be excluded. During oxidation this cofactor is converted to an unstable di-

The molecular defects in phenylketonuria hydro-form which is thought to be the quinonoid tautomer. This acts as a substrate for DHPR which regenerates the tetrahydro-form (Kaufman, 1964). This cofactor is also needed for tyrosine and tryptophan hydroxylation (Kaufman & Fisher, 1974). Synthetic tetrahydropterins have been used but work with tetrahydrobiopterin is obviously closest to the in viva situation. Lysoleicthin has heen shown to stimulate the human protein (Fisher & Kaufman. 1973) and the in vivo significance is not clear. Iron and Cu has been established as being present in the purified rat enzyme (Gillam et al., 1974; Fisher et al., 1972). Iron and Cu have been implicated in the human enzyme (Woo et al., 1974). The rat enzyme has been shown to contain FAD (Gillam et al., 1974). Kinetic studies of the human enzyme with natural cofactor have not been frequent. The apparent K, using partially purified enzyme have been shown to bephenylalanine.4 x 10m4M;BH4, 3 x IO-(‘M; 02, 0.950/o (Friedman & Kaufman, 1973). It is clear from this discussion that the impairment of hydroxylation of phenylalanine in the whole organism can result from decreases in levels of one of the many essential components of the complete system. Thus deficiency of PH, DHPR, BH,, phenylalanine hydroxylase stimulating protein and possibly phosphorylating protein would be expected to decrease hydroxylation. Less likely candidates in vivo are the metals, NADH and FAD. Thus the diseases we are considering may concern any one of these components. THE DEFECT

Residual phenylalanine

IN PHENYLKETONURIA

hydroxylation

in PK U

Since the correlation of mental retardation with phenylpyruvic acid excretion by Foiling (1934) there have been many studies implicating or demonstrating the impairment of the reaction converting phenylalanine to tyrosine, carried out by the PH system. With hindsight some of the early experiments were carried out under difficulties which did not allow the measurement of the exact diminution of activity in vitro. This is because (a) the optimum conditions for assay (Kaufman, 1969) were not known (and may still not be known), and (b) the lability of the human enzyme. Also small numbers were involved in each experiment which alone were not convincing but together they are. Recently two groups (Grimm rt al., 1975; Bartholome et al., 1975) have assayed liver tissue from large numbers of patients to demonstrate convincingly the extent of diminution of activity. The in viva and in vitro studies are listed in Table 1. In vivo studies. The first group to suggest the currently accepted position of the metabolic block (the conversion of phenylalanine to tyrosine) was that of Dann ct a!. (1943) whose evidence is rather obscure. However this finding was put on a sound basis 4 years later by Jervis (1947) who found Millon-positive substances (tyrosine and its derivatives) did not increase in PKU’s after ingestion of phenylalanine as they did in normals. On studying his data, 8 of his patients obviously had close to zero in riuo activity but the other two had some activity. The patients studied by Udenfriend and Bessman (1953) showed

335

about So/, of normal tyrosine formation under their conditions and the study of Curtius et al. (1972) showed no conversion of deuterated phenylalanine to tyrosine. These cases are probably reflecting the biochemical heterogeneity which is clearly revealed in the later extensive in vitro studies. In vitro studies. There were four studies (up to and including Kaufman, 1958) before it was accepted that the enzyme in normals was best measured by addition of tetrahydropterin cofactor and by use of [‘4C]phenylalanine as the substrate so that the results obtained cannot be regarded as accurate. However three of these showed conclusively that phenylalanine hydroxylase was markedly diminished in PKU. The activity shown in the case of Kaufman’s study probably being due to the proteolytic production of tyrosine. The other confirmed the finding of Wallace er al. (1957) that the “heat-stable enzyme” (probably DHPR as Mitoma et ~1.(1957) had showed it to be present in brain and the other possible candidate, DHFR, has been shown to be absent from brain (Makulu et al., 1973)) is not absent in PKU. At about the time of the work of Justice et al. (1967) difficulties arose in the classification of patients when it was realised that some people could develop normally despite elevated phenylalanine levels in serum. Thus in the later studies there is a chance of mis-classification as will become evident. Both the patients Justice er ctl. studied had zero activity. Grimm rt cd. (1975) using a tyrosine assay (rather than the more accepted radioactive assay) found 3 patients with no activity and the other 11 ranging from 0.4 to 4:; of normal. Most surprisingly they found that the small amount of residual activity in PKU was inhibitable by exogenous cofactor whereas residual activity from HPA was stimulated as might have been expected. This is a very important finding which needs confirmation with the radioactive assay although the result found in the case studied by Friedman et al. (1973) contradicts this finding but different cofactors were used in the two studies. Kaufman’s group (Friedman rt al.. 1972, 1973) latterly investigated 3 cases. two had no activity but one had 0.27Oi, of normal activity and its properties were thoroughly investigated (see below). Bartholome et al. (1975) studied 17 patients and obtained figures for residual activities from 0 to 5:;). Parker et al. (1975) studied 4 cases and stated that activity was less than 5%. It should be emphasised that in vitro activity demonstrated in these studies may not necessarily mean that the same activity is functional in vivo. It may well be zero in vivo and thus the correct diagnostic measurement might be the in vivo hydroxylation. Thus the deuterated study of Curtius et al. (1972) and a more recent version by Milstein and Kaufman (1975a) may be more relevant clinically. However the dietary phenylalanine load test or the dietary phenylalanine levels required to keep serum phenylalanine normal might be the simplest in practice. Characterisation

of a possible mutant proteiu

The first study to suggest the presence of a mutant protein was by Kaufman’s group who prepared antiserum to purified rat PH, showed precipitin lines with normal human liver extract but not with PKU liver

336

R. G. H. Table

Number of patients

Reference Jcrvis

(I 947):

Jervis (1953)*t Udenfriend & Bessman (1953)$ Mitoma er al. (1957)*-F Wallace et ~1. (1957) Kaufman (1958)

Kaufman (1958)t Justice et al. (1957) Woolf et ul. (1967): Grimm et (II. (1972. 1975)t

Curtius

et LII. (1975)

hydroxylation Basis for classification

Total Total

& Danks homozygotes heterozygotes

Other

Serum tyrosine decreased with phe in one

Nine with little activity, 18

2 2 1

MR PK MR MR PK

0. 0 7. 9

PK MR. HPA N.S. HPA

13, 15 0. 0 50 (average) 0. 0, 0, 0.4, 0.5. 0.6. I.l,I,2.2.2,3,4 15

I 2

2 2 31 hets 14

2

1 17 4

(1976)*t

Activity

(“,>of normal)

MR

DHPR

I

present

Cofactor increased 24 times. DHPR present in both

Added cofactor reisdual activity

inhibits

Deuterated phe not found in tyr metabolites

HPA

N.S. HPA, PK CB, MR

0.27 Twelve no activity. 1.5. 2.3. 2.4, 3.5 <5

NS

Change in isoenryme pattern A sibling (may not be a het) 60”:, of normal

40. 78

HPA.

findings

0

2 hets

Cotton

in phenylketonuria

10

I het 1

et al. (1972):

Friedman et al. (1972) Friedman et al. (1973) Bartholome et ul. (1975) Parker

1. Phenylalanine

COTTON

MR

No activity during affinity chromatography Range 0- 5 (RA assay) Range 15578

62 34

All studies were performed on biopsy material except where marked (*) where it was on autopsy material. Activity was estimated by formation of [‘4C]tyrosine from [‘4C]phenylalanine except where marked (i). All in t:itro studies after Justice er al. (1967) inclusrve. used added tetrahydropterin. 6,7-Dimethyl tetrahydropterin was used m ail cases except Friedman t’t al. (1973) and Bartholome et ctl. (1975) who used tetrahydrobiopterin. Kaufman rt al. (1958) used 7,8-dihydrobiopterin which was presumably converted to tetrahydrobiopterin. This addition of tetrahydropterin was found to be the most important factor influencing the magnitude of the results (Kaufman, 1969). Studies marked (3) were in ciao and the values given are approximate. Abbreviations: HPA. relatively high serum phe; MR. mental retardation; PK. phenylketones: NS. not stated; CB, clinical and biochemical parameters; het, heterozygote; RA. radioactive.

extracts

(Friedman

er ~11.. 1972) but in a later

publica-

tion (Friedman et ul., 1973) they studied an extract from a PKU liver with a residual 0.279/, of normal activity. They were able to show dependence on tetrahydrobiopterin. stimulation by lysolecithin (but less than normal), that the inhibition by amino acid analogues was characteristic of PH rather than of the other two aromatic hydroxylases that there was no inhibition by 0.2 mM phenylalanine whereas with normal enzyme there was inhibition, and that antiserum slightly inhibited the residual activity. Parker et al. (1975) in a preliminary communication found “a change in phenylalanine hydroxylase isoenzyme pattern” but too little information is given to judge its relevance. Cotton and Danks (1976) were able to show by affinity chromatography (Cotton & Grattan, 1975) on an unconjugated pteridine adsorbent (Cotton, 1974) the purification of a molecule from PKU autopsy liver, a protein of the same mol. wt as the smallest of two proteins present in active monkey and human foetal phenylalanine hydroxylase. This protein was

also purified from normal livers obtained at autopsy. The circumstantial evidence that this protein is. or is derived from, PH is strong but confirmation must await the protein chemistry. Technical problems have so far prevented comparison of charge of the normal and the PKU protein. No enzyme activity was found in the fractions during the affinity chromatography of the PKU liver extract whereas normal autopsy livers showed readily detectable activity during these experiments (unpublished). Kcccntly Bartholome and Ertel (1976) claim to have demonstrated cross reacting material in PKI’ licct extracts with antibodies prepared against monkey PH. This is a very important confirmation of earlier findings. Levels

qf

phenylalanine

hydroxq’lase

in hetewzygotrs

Very few estimations of enzyme activity have been made in heterozygotes. Woolf et al. (1968) made calculations after intravenous phenylalanine, loads and concluded they had an average of 51’:;, of the normal activity in 31 heterozygotes. Grimm rt al. (1975) in

337

The molecular defects in phenylketonuria a single case found only 15:;, of normal values in vitro. Parker et (I/. (1975) in two heterozygotes found 40 and 68”,, of normal in rirro activity.

Kaufman having demonstrated the role of “cofactor” in the PH reaction measured the level of cofactor (presumably 7,8-dihydrobiopterin) in two PKU liver extracts (Kaufman, 1958). He found increased levels (approximately 3 times normal levels) in the liver so he concluded the defect could not be due to lack of availability of cofactor in PKU. Unfortunately tetrahydrobiopterin has not been measured in a PKU liver extract. Kaufman’s group has also shown that the low activity in PKU in the case he studied was not due ;utl&r $ Fl$,i)(Friedman et al., 1973) or PHS Dihydropteridine reductase as mentioned above was shown to be present in PKU (Wallace c)t (II.. 1957; Mitoma et al.. 1957) but it has not been strictly quantitated in PKU since that time. We have studied this enzyme in fibroblasts (and Epstein Barr virus transformed lymphocytes) from PKU and normal people and find the levels are similar (Schlesinger rr a/.. 1976). Surprisingly when phenylalanine is added to the reaction mixture at levels commonly found in serum in PKU. the activity from normal cells is stimulated but that from PKU fibroblasts is inhibited (Table 2). Further studies will be required to decide whether there is a mutation in DHPR or whether another factor is influencing the DHPR activity in different ways in PKI’ and normal tissues. WC have evidence (Watson et al.. 1976) that this inhibition by phenylalanine may be occurring in uico in PKU as when serum phenylalanine levels are high a compound, thought to be dihydroxanthopterin, is excreted in the urine. When serum phenylalanine is low in PKU, this compound is not excreted. The only other circumstance where this compound is excreted is in DHPR deficiency (see below) where this enzyme is reduced to 25”<; of normal levels. These findings pose two questions (a) is it really PH which contains the primary mutation in PKU? and (b) are the neurological s! mptoms in PKIJ due to BH, deficiency in the brain as its production is inhibited by phenylalanine in PKU’s? Further work will be required to answer these questions.

The study of this variant has not been as intense as the study of PKU. Details are seen in Table 3. The first study was by Justice et al. (1967) where two patients were found to have 11 and 44% of normal activity. Kaufman’s group (Kang et al., 1970; Kaufman & Max. 1971; Kaufman et al., 19756; Friedman et ul., 1972) found a range of 4-7”, in 4 cases and Grimm et al. (1975) 8 and 10”; in 2 cases. Bartholome et al. (1975) with eight cases found a range from 0 to 34.5”, of normal. Interestingly Curtius er al. (1972) irk ciao found no production of tyrosine metabolites in hyperphcnylalaninemia (a single case). Of the few heterozygotes that have been studied

(Kang et ul., 1970; Kaufman rt al.. 19756) the figures are very interesting in that three couples show values of 8 and 15: 6 and IO: IO and 31”,, of normal activity. Thcsc are not LC’I-vdilTcrent from the Ic~vcla in their homo/ypote offspt’ing i.c. 4. 5 and 6’,, of normal. rcspectively. Woolf c’t ~1.(196X) using the I/V load tests calculated values on two pairs of parents of go’:/;,and approx 36: ; ; 34’:; and approx 35’:” of normal levels. The low levels of enzyme in heterozygotes (i.e considerably less than the expected 50’:, in heterozygotes) are interesting. If this is not an artefact due to ir? vitro assay (in riuo Woolf ct al. has found 34-36’:” of normal activity in 3 heterozygotes) it appears that the mutant gene or gene product somehow influences the activity of the wild type gene or gene product. Kaufman has attributed this to “negative co-operativity” (Kaufman et al., 19756) based on two dissimilar subunits but as there is no chemical evidence for two dissimilar subunits in the human (SW above) this speculation is premature. The only molecular characterisation study of the residual enzyme has been by Kaufman’s group. They found no precipitation of an extract of HPA liver with antiserum to purified rat enzyme even though this gave a precipitin line with normal human liccr extract. However inhibition of the residual activity by the antiserum was found. The apparent K, for synthetic cofactor Me,PH, was found to be 0.005 mM for both normal and residual activity but for phenylalanine the apparent K, was 0.62mM for the residual activity i.e. exactly half normal (Friedman (11(I/.. 1972). Similar values were obtained in earlier studies (Kaufman & Max 1971). Also the residual activity was shown to be more heat labile and less stimulable by lysolecithin than normal (Friedman et trl.. 1972). Thus a PH is present which appears to hc mutant. Cornparisorl qf PK L’ und HPA

It appears that there is a tendency for HPA to have higher levels of enzyme activity relative to those in PKU. Because there is a tendency to think of HPA as a mild form of PKU it is important to look at the clinical picture in cases of PKU where high levels of phenylalanine hydroxylase have been found and in cases of HPA where low levels have been found. Thi\ is to confirm that there is a true owl-lap in Table 2. Effect of phenylalanine reductase activity from normal fibroblasts

on dihydropteridine and PKU derived

Specific activity (nmole/min per mg protein) No 2mM phenylalanine phenylalanine Control fibroblasts PKU tibroblasts

4.65

6.47

3.24

2.05

These results are typical of our recent findings (Schlesinger et a/.. 1976). All PKU lines were from classical PKU patients. A total of 4 normal fibroblast lines and 4 PKU hbroblast lines have shown the effect illustrated. Inhibition in the case of PKU lines has ranged from 22 to 100~0 Stimulation in the case of normal lines has ranged from 22 to looon;.

338

R. G. H. COTTON Table 3. Phenylalanine hydroxylation in hyperphenylalaninemia

Reference Justice et al. (1967) Woolf et al. (1968)f Kang et el. (1970) Kaufman & Max (1971) Kaufman et al. (1975b) Friedman et al. (1972) Grimm et al. (1972, 1975)t Curtius et al. (1972):

Number of Basis of patients classification

Activity (V,,of normal)

2 LPA 4 hets LPA ON. LPA 3 3 x 2 hets

11, 44 80, -36; 34, -35 4, 5, 6 8. 15; 6, 10; IO, 31

1 2 1

N.S. LPA LPA

5-7 8, 10 0 0, 2, 2. 2, 2, 2. 3. 6, 9. 15, 32. 35

Bartholomt rt al. (1975)8

12

CB, LMR

Total homozygotes Total heterozygotes

21 10

Range &44

Other findings

Parents respectively of homozygotes above Lack of deuteration of tyrosine metabolites

Range 680

All assays were ill 111tr0on biopsy specimens except the studies marked (z) which were bl uico. All studies except that marked (+) were obtained by measurement of [14C]tyrosine formed from [‘4C]phenylalanine. Only the study indicated (“) used tetrahydrobiopterin as cofactor, the others used 6,7-dimethyl tetrahydropterin as cofactor. Abbreviations: LMR, lack of mental retardation; LPA. relatively low serum serum phe; ON, ortho hydroxy phenyl pyruvic acid negative without loading; N.S.. not stated. amounts of residual activity (measured in vitro) in PKU and HPA. In the study of Bartholomk et al. (1975) three seriously retarded cases i.e. clinically PKU’s were found to have activities of 2.4, 3 and S”,,; of normal. In contrast in an HPA patient with no residual in citro activity’ a protein loading of 180mg phe/‘kg for 3 days did not raise the serum phenylalanine levels over 13 mg/lOO ml, a figure recognised as harmless. A healthy brother has a similar picture. Thus according to in vitro enzyme assay there is considerable overlap of the PKU and HPA groups. Perhaps in vitro enzyme assay is not the best way of distinguishing between these two groups, an important practical point. The in viuo assay has in fact been done for years in the form of the phenylalanine load test. It would be interesting to do load tests on the cases of Bartholomit or alternatively to find the dietary level of phenylalanine needed for maintenance of normal serum levels. It is interesting to note that Curtius et al. (1972) in vioo found no difference between the hydroxylation capacity of a PKU and an HPA. It should be emphasised that whatever measurements and subsequent predictions are made regarding classification of PKU and HPA it should be remembered that an originally described marker for PKU, mental retardation, will not be found because all neonates suspected of having PKU will be placed on the diet. In both PKU and HPA no precipitating material has been found with antisera to rat phenylalanine hydroxylase even though there is some evidence that mutant molecules may be present. This may be explained by the relevant antigenic determinant being removed by the mutation or being buried in the molecule as a result of changes in the secondary structure. This assertion is strengthened by the preliminary results of Bartholomt: and Ertel (1976). Lethal hyperphenylalanineria

This disease was first alluded to by Udenfriend and Bessman as early as 1953, i.e. “the amount of L-phenylalanine oxidase may be normal but certain cofactors might be missing to activate the enzyme”, further “a

cofactor or inhibitor of phenylalanine oxidase might also influence another metabolic system which could be essential for normal brain metabolism”. Recently Kaufman et al. (1975a) demonstrated a BH, deficiency in the liver of a patient. The examination of enzymes of the PH system however does not give us a simple picture. The DHPR was less than 1% of normal in liver and fibroblasts but also the liver DHFR was 25% of normal and the liver PH was 20% of normal. However the disease is said to be due to the DHPR deficiency even though there seem to be multiple alterations in enzyme levels. A defect in DHPR seems likely as antiserum to sheep liver DHPR failed to detect cross reacting material in the liver of this patient (Milstein & Kaufman, 1975b). Other patients with similar clinical picture, i.e. a neurological disease progressing to early death despite excellent control of phenylalanine levels, had appeared in the literature earlier. Smith and co-workers (Smith, 1974; Smith et al., 1975) described 3 patients who died at 2, 6+ and 7 years of progressive neurological disease despite excellent dietary control of the serum phenylalanine levels. In one of these a normal phenylalanine hydroxylase level was demonstrated (Smith, 1974). They postulated a defect in biopterin metabolism suggesting a deficiency of DHPR as a possible candidate (Smith et al., 1975). No further chemical studies were performed. Bartholomi: (1974) described a similar case in which PH was normal but did not suggest an explanation. Later this patient was shown to have normal levels of DHPR, PHS protein and BH4 in a liver biopsy (Kaufman et al., 1975~). Thus as yet the defect is unexplained. However the absolute level of Crithidia active pterins is low (Leeming et al., 1976) and the patient responded to therapy which included L-DOPA and hydroxytryptophan (Bartholomk & Byrd, 1975). These latter compounds are expected to be and have been demonstrated to be low in BH4 deficient patients. Thus the in vitro estimations may not be reflecting the true in ciao lesion as adequate

The molecular defects in phenylketonuria BH4 levels in liver with elevated serum phenylalanine is irreconcilable on current knowledge. Unfortunately, the most important measurement, brain BH, levels are unlikely to be measured in this patient. The case seen at our hospital (Danks et al., 1975, 1976a, h) has 2.5% of normal DHPR in fibroblasts. That it is deficient in BH, is demonstrated by a marked lowering of serum phenylalanine on I/v administration of BH,. Serum Crithidia factors are increased and a marked fluorescent spot is seen on HVE of urine which has properties of dihydroxanthopterin (Watson et al., 1976). This material is not detectable in normal urine. Thus it appears the pterin metabolism is disturbed probably as a result of the DHPR deficiency. Another case seen at our hospital has no DHPR activity in an autopsy liver specimen (Danks et al., 19766) under certain conditions. The most recent reported case has been investigated by Leeming rt al. (1976). The first chemical studies indicated that Crithidia active substances in the serum and urine were low. Also DHPR, PH, and PHS were normal (K. BartholomC, personal communication). Because B and BH, cochromatographed in their study it is impossible to say if there was B or BH, present in serum. As 7,8-dihydrobiopterin was down in urine maybe there is a 7,8-dihydrobiopterin deficiency from defective synthesis leading to a BH, deficiency. Clearly the chemical evidence is rather sketchy but it is evident that the cases seen by us and Kaufman are BH4 deficient probably in both cases because of a DHPR deficiency. In the cases seen by Leeming et al. (1976) and Bartholomk et al. (1974) the only clear data are that the urine and serum pterins are low and it is tempting to postulate that biopterin synthesis is below normal but this is contradicted by the finding of normal BH, levels in the liver of BarthoIomC’s patient. The brain symptoms in all cases could be explained by a BH, deficiency in the brain. There is much data yet to be gathered but of course there are likely to be multiple causes of a functional or absolute deficiency of BH, at the site of action of the enzymes involved. The frequency of this disease in the phenylketonurics seen by Smith et al. (1975) is 3 in 300, in Australia it is 7 in 258 (Danks et al., 1976~). Analoyues and in PK U. etc.

models for

the primary

genetic

defect

Numerous studies have been made by either feeding vast quantities of phenylalanine to animals or inhibiting PH by chlorophenylalanine but as these are aimed at studying the secondary brain lesion they are not relevant to this review. Two inherited diseases of the mouse, dilute lethal and wabbler lethal, have been looked at as possible ‘models’ for PKU. It is clear that the dilute lethal mouse is not a good analogue of PKU if only because of the lack of elevation of serum phenylalanine (Woolf et al., 1970) and in fact that the mutation is lethal. There has been much debate about the parameters of phenylalanine metabolism that might be altered. The wabbler lethal mouse also appears to have abnormal phenylalanine metabolism (Siegel & Rauch, 1969). We have approached the problem by isolating

339

mutants of rat hepatomas which are deficient in phenylalanine hydroxylating ability, i.e. they are tyrosine auxotrophs (Choo & Cotton, 1976). This will allow us to isolate mutations in all the genetically determined components which are essential for the PH system. Phenylalanine hydroxylase in these auxotrophs has ranged from 0 to about 70% of wild type activity. As we are also likely to isolate analogues of lethal HPA. these may be amongst the clones which have higher levels of phenylalanine hydroxylase activity. Thus they might be defective in the synthesis of BH,. It is premature to speculate that the mutations carried by these auxotrophs are analogous to inherited diseases of phenylalanine metabolism of man. CONCLUSION

In both PKU and HPA it is quite clear that PH activity is decreased sometimes to zero as measured in uiuo and in vitro. This effect is more marked in PKU. There is some evidence that mutant proteins with lowered or zero activity is present in both PKU and HPA but confirmation will have to await chemical studies of the polypeptide chain(s). What is quite unclear is why some individuals (HPA) with little or no in citro PH activity are able to (a) avoid mental retardation and (b) metabolize relatively more phenylalanine, whereas others (PKU) with similar PH levels (in vitro) are mentally retarded and not able to metabolize as much phenylalanine. This may be reflecting (a) inadequacy of the in citro assay, (b) operation of alternate pathways for the metabolism of phenylalanine in HPA, (c) primary amino acid substitution in different polypeptide chains or different amino acid substitutions in the same polypeptide chain, or (d) different secondary effects of the primary mutation. Recent findings of Schlesinger et a/. (1976) must question the accepted hypothesis (Kaufman, 1976) that a mutation in PH is the primary defect in PKU. In LHPA the primary defect appears to be a mutation in the DHPR gene in two cases but in others may be a block in biosynthesis of BH, at an earlier step than DHPR. Methods are now available which should resolve some of these problems in the near future. Acknowledyemrrlts---Professor D. M. Danks and Mr. K. H. Choo are thanked for criticism of the manuscript. Work on the present subject has been supported by g Queen Elizabeth II Scholarship. National Health and Medical Research Council, Royal Children’s Hospital Research Foundation, and the University of Melbourne. REFERENCES

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