1108

NEW VARIANT OF PHENYLKETONURIA WITH PROGRESSIVE NEUROLOGICAL ILLNESS UNRESPONSIVE TO PHENYLALANINE RESTRICTION B. E. CLAYTON O. H. WOLFF Departments of Child Health and Chemical Pathology, Institute of Child Health and the Hospital for Sick Children, London WC1 I. SMITH

Three children, two of them siblings, with an unusual type of phenylketonuria are described. The three patients, two of them observed from the neonatal period, had a progressive neurological illness which was unlike that of classical phenylketonuria, and which did not respond to a lowphenylalanine diet. The biochemical features suggested that the block in the conversion of phenylalanine to tyrosine was less severe than in the classical disease, and phenylalanine p-hydroxylase activity, measured in one patient, was normal. It is suggested that the patients have a disorder of biopterin metabolism possibly due to a defect of the enzyme dihydropteridine

Summary

reductase. Introduction AMONG three hundred patients with phenylketonuria seen at the Hospital for Sick Children between 1949 and 1974, there were three who had a severe, progressive neurological illness unlike that in untreated patients with classical phenylketonuria.1 A lowphenylalanine diet, which achieved good control of blood-phenylalanine levels, failed to modify the course of the clinical illness, and all three patients died of a

4. Kohner, E. M. J. R. Coll. Phys. 1972, 6, 259. 5. Kohner, E. M., Panisset, A., Cheng, H., Fraser, T. R. Diabetes, 6.

7. 8. 9.

10.

1971, 20, 816. Oakley, W. N., Pyke, D. A., Tattersall, R. B., Watkins, P. J. Q. Jl Med. 1974, 43, 145. Keen, H., Jarrett, R. J. in Atherosclerosis (edited by R. Jones); p. 435. Berlin, 1970. Albrink, M. J., Lavietes, P. H., Man, E. B. Ann. intern. Med. 1963, 39, 305. Elkeles, R. S., Lowy, C., Wyllie, A. D., Young, J. L., Fraser, T. R. Lancet, 1971, i, 880. Hunt, P. Presented at the Scientific Section of the British Diabetic

to their neurological features These clinical disease. suggested an unusual variant of phenylketonuria; the finding of normal phenylalanine p-hydroxylase activity in one patient confirms this possibility. A paper describing these cases has appeared in abstract form.2

bronchopneumonia secondary

Case-reports FIRST CASE

This girl, the fourth child of healthy parents, was born after a normal pregnancy and delivery, weighing 3 kg. Three older siblings, two girls and a boy, were normal. She was breast-fed for 12 days, when bottle feeding was introduced because of slow weight gain. After this she

gained weight normally, but feeding remained difficult She smiled at 4-5 weeks. At 7 weeks she had a choking attack during a feed and thereafter many similar attacks occurred. At 4 months she had a severe febrile illness with a prolonged convulsion and was admitted to hospital. Meningitis was ruled out; the only abnormal laboratory findings were a plasma-phenylalanine level of 45 mg. per 100 ml. and phenylpyruvic acid in the urine. The fever and signs of acute illness subsided after a few days, when it became clear that she

was

retarded and had abnormal

neurological signs (described below). Phenylketonuria diagnosed and a low-phenylalanine diet was begun, usingMinafen’ (Cow & Gate), with milk and vitamin supplements. Phenylalanine levels fell quickly (1 mg. per 100 ml. plasma after 2 days’ treatment), and an unusually high intake of cow’s milk (75-100 mg. per kg. per day) was required to maintain levels above 1 mg. per 100 ml. On this regimen the patient gained weight normally but made no further developmental progress, and suffered frequent convulsions and progressive neurological deterioration. Dietary treatment was therefore stopped at 12 months of age. A single phenylalanine level after was

reintroduction of a normal diet was 12 mg. per 100 ml. plasma. The patient died at the age of 6’5 years of

bronchopneumonia following repeated respiratory infections due to aspiration of feeds, having made no further developmental progress. SECOND CASE

boy,

This

the younger

sibling of

case

1,

was

born

Association, 1974. 11. Kemsley, W. F. E. Ann. Eugen. 1951, 16, 316. 12. Welborn, T. A., Fraser, T. R. Diabetologia, 1965, 1, 211. 13. Cramp, D. C., Robertson, G. Analyt. Biochem. 1968, 25, 246. 14. Levin, J., Zak, B. Clinica chim. Acta, 1964, 10, 381. 15. Kaufman, B. A., Harsoulis, P., Kuku, S. F., Tunbridge, W. M. G., Lowy, C., Fraser, T. R. Unpublished. 16. Walton, H. Clin. Chem. 1965, 11, 624. 17. Lewis, B., Chait, A., Wootton, I. D., Oakley, C. M., Krikler, D. M., Sigurdsson, G., February, A., Maurer, B. Lancet, 1974, i, 141. 18. Khachadurian, A. K., Uthman, S. M. J. Med. Leban, 1971, 24, 105. 19. Hayes, T. M. Clin. Endocr. 1972, 1, 247. 20. Avogoro, P., Capri, C., Gazzolato, R., Pais, M. Acta diabet. lat. 1972, 9, 540. 21. Lewis, B., Mancini, M., Mattock, M., Chait, A., Fraser, T. R. Eur. J. clin. Invest. 1972, 2, 445. 22. Kissebah, A. H., Adams, P. W., Wynn, V. Diabetologia, 1974, 10, 119. 23. Wing, D. R., Robinson, D. S. Biochem. J. 1968, 109, 841. 24. Nikkila, E. A., Pykalisto, O. Biochem. biophys. Acta, 1968, 152, 421. 25. Barso, L. V., Havel, R. J. J. clin. Invest. 1970, 49, 537. 26. Wieland, O., Weiss, L. Biochem. Biophys. Res. Commun. 1963, 10, 333. 27. Tubbs, P. K., Garland, P. B. Biochem. J. 1964, 93, 550. 28. Lowy, C., Kohner, E. M., Florey, C. du V., Young, J. L., HopeGill, H. F., Fraser, T. R. Excerpta med. 1973, no. 280, p. 184.

Fig. 1--Case

2:

plasma-phenylalanine

intake. P.A. =

phenylalanine.

levels and phenylalanine

1109 after a normal pregnancy and delivery, weighing 3 kg., A plasmaand was breast-fed until 5 months of age. phenylalanine level, estimated on the 7th day because of the family history, was 7’5 mg. per 100 ml., and repeated determinations over the next 4 months were in the range of 4 to 13’5 mg. (fig. 1). iPhenylpyruvic acid was absent from the urine. A low-phenylalanine diet was not given because the biochemical abnormality was in a range generally regarded as safe from the point of view When the patient was 2 months of brain development. old his mother noted choking episodes similar to those of his sister; at this stage physical and mental developAt 5 months he had an acute ment progressed normally. illness during which his mother noticed a sudden brief stiffening of his arms and unusual facial movements; a phenylalanine level on that day was found to be 27 mg., and he was admitted to hospital for introduction of a lowphenylalanine diet. As with his sister, a phenylalanine intake of 75 to 100 mg. per kg. body-weight was required to maintain plasma levels above 1 mg. (see fig. 1). During his stay in hospital neurological abnormalities similar to those shown by his sister were noted, and it became clear that developmental progress was no longer normal. Apart from the raised phenylalanine level, investigations were normal (haemoglobin, blood-film, urea, electrolytes, calcium, blood-sugar, liver-function tests, plasma-proteins, rubella and cytomegalovirus antibodies, blood and urine aminoacids, Wassermann reaction, and chromosomes). He made no further developmental progress and the diet was At the age of 5.5 years, he stopped at 13 months. recognised his parents and sometimes smiled but was unable to sit up or to make purposeful movements. He was difficult to feed, and had occasional grand-mal fits and recurrent respiratory infections. Phenylalanine levels, on the normal diet, were in the range 8 to 12’mg.; orthohydroxyphenylacetic acid was present in the urine but The patient died at 7 years of not phenylpyruvic acid. bronchopneumonia and gastroenteritis. THIRD CASE

This boy, unrelated to cases 1 and 2 though from the region of South-West England, was the second child of healthy parents. His sister was healthy. He was born after a normal pregnancy and delivery weighing 3-8 kg., and was bottle-fed with a dried cow’s milk formula (Full Cream Cow & Gate). The routine Guthrie test on the 7th day was positive (phenylalanine level 27 mg. per 100 ml.), and orthohydroxyphenylacetic acid He and phenylpyruvic acid were present in the urine. was treated with a low-phenylalanine diet using minafen with supplements of milk and vitamins. A phenylalanine intake of 75 to 150 mg. per kg. per day was required to maintain blood-levels above 1 mg. For the first 3-4 months his mother regarded his progress as normal, though he was difficult to feed and tended to choke during feeds. At 5 months he was admitted to hospital with an acute febrile illness lasting 2 days, during which the blood-phenylalanine level rose to 35 mg. During his stay in hospital it-became clear that he had not made normal developmental progress; though he smiled and responded to social advances, head control was poor and he made only few voluntary movements. From this time onwards, he made no further progress and became increasingly difficult to feed. At the age of 6 months extensive investigations (including those performed in case 2, and with the addition of urine analysis for metachromasia and mucopolysaccharides, blood analysis for acid phosphatase, measles and toxoplasma antibodies, creatinine kinase, hexosaminidase, /3-galactosidase, lactate and pyruvate, and electromyography) failed to identify The any abnormality other than the phenylketonuria. clinical picture closely resembled that of cases 1 and 2, same

SUMMARY OF THE NEUROLOGICAL ILLNESS

At the age of 9 months low-phenylalanine stopped because it aggravated the feeding difficulties and had not prevented severe retardation. Subsequent phenylalanine levels ranged from 8 to 15 mg. per 100 ml. with orthohydroxyphenylacetic acid present in urine. During infections, levels exceeded 20 mg. per 100 ml. and phenylpyruvic At the age of 9 months acid appeared in the urine. liver biopsy was undertaken to determine phenylalanine p-hydroxylase activity (see biochemical findings), and a lumbar air encephalogram showed widening of the sulci. Brain biopsy at 14 months showed non-specific diffuse, patchy demyelination and active cellular degeneration. The patient died at 22 months of bronchopneumonia secondary to recurrent aspirations of feeds.

as

did the subsequent progress.

the

diet

was

Neurological Features The three cases presented similar features (see accompanying table): 1. Swallowing difficulty was the first and most persistent symptom and caused feeding difficulties, choking attacks, aspiration of feeds, and pulmonary infections.

recurrent

2. Developmental delay became obvious at 5 months of age and no developmental progress occurred after this time. Social responses, which were preserved initially, deteriorated at 7-8 months, and in cases 1 and 3 were lost between 9 and 14 months. 3. Convulsions began at 5 months in cases 1 and 2, at 7 months in case 3. Grand-mal attacks occurred occasionally throughout the clinical course, particularly Myoclonic attacks during intercurrent infections. occurred in bouts, sometimes several times a day and lasting several minutes, and consisted of sudden brief jerks of one or several limbs accompanied by facial grimacing or a sharp cry followed by a short period of vacancy. At 11-13 months they became less frequent and then stopped.

and

4. Neurological signs were first noted at 5 months and consisted of hypotonia of the trunk and lead-pipe rigidity Tendon jerks of the limbs; head control was poor. remained normal until the final stages when they became sluggish. Between 5 and 8 months the hypotonia of the trunk increased and the limb hypertonia lessened, particularly in the legs. At 8-9 months isolated choreiform movements of the arms, and occasionally the legs, were noted. At the same age episodes of oculomotor spasms occurred with lateral deviation and elevation of the eyes lasting 10-15 minutes. Up to that age the infants were restless and miserable; from then onwards they became

1110

passive and relaxed, with a blank facial expression, animated only occasionally by a smile elicited by tickling or the presence of the mother. Voluntary movements of face and limbs were few. At 8-10 months the patients adopted a characteristic posture when placed in the supine position. The arms lay loosely extended and internally rotated with the hands flexed at the wrist, thumbs held The legs remained loosely extended across the palm. The child or flexed at the hip in a "frog" position. lay still and withdrawn, except for occasional choreiform movements, but responded to painful stimuli by withdrawal. In the second year of life, the choreiform movements ceased and the clinical picture was of gross, generalised hypotonia and passivity. more

The neurological illness described above suggests a diffuse cerebral degeneration with early and severe involvement of the basal ganglia.

Electroencephalography The E.E.G., except for

paroxysmal changes during the

major convulsions, remained normal until the 2nd year of life when it became grossly and diffusely abnormal with persistent paroxysmal changes. The myoclonic convulsions and choreiform episodes were not

accompanied by

E.E.G.

changes.

febrile illnesses, and in case 1 when the protein intake exceeded 5 g. per kg., occur in atypical phenylketonuria when protein catabolism occurs during infections, or starvation, or when protein intake is high. 2. Response to oral phenylalanine-tolerance test.Cases 2 and 3, at 1 year and 9 months respectively, were given 100 mg. per kg. of pure L-phenylalanine by mouth (fig. 2). At 6 hours there was a sustained rise of phenylalanine with no rise of tyrosine, and phenylpyruvic acid and orthohydroxyphenylacetic acid appeared in the urine. This response was similar to that in classical phenylketonuria. After 24 hours, however, phenylalanine levels had returned to the pre-load values, whereas a more prolonged rise occurs in the classical disorder.

Phenylalanine p-hydroxylase activity in liver.-This measured only in case 3, using the method of Justice et a1.3 as adapted by McClean et al.4 It was normal (31’2 micromoles tyrosine per hour per g. of protein, normal range 29±S.D. 15); in classical phenylketonuria activity is very low or absent.3 4. Maternal phenylalanine levels.-Both mothers had 3.

was

less than 1 mg. per 100 ml. blood. The in a fasting blood specimen from the mother of case 3, was compatible with heterozygous state for classical phenylketonuria.

levels which

were

phenylalanine/tyrosine ratio,

Discussion Biochemical

Findings

The three cases had certain biochemical features which suggest the diagnosis of atypical rather than classical phenylketonuria: 1. Relatively high tolerance of natural protein.-Case 2 (fig. 1) when breast-fed had plasma-phenylalanine levels between 7 and 13-5 mg. per 100 ml. until they rose to 27 mg. per 100 ml. during a febrile illness; all three cases on a low-phenylalanine diet required 75-100 mg. of phenylalanine per kg. per day throughout the 1st year of life In classical phenylto maintain levels above 1 mg. ketonuria the requirements during the second half of the 1st year are 25-50 mg. per kg. per day. When a normal diet was reintroduced, blood-levels ranged between 8 and 15 mg., whereas in the classical condition corresponding levels are above 20 and usually above Levels above 20 mg. which occurred during 30 mg.

The biochemical findings in the patients of persistently raised plasma or blood phenylalanine levels, phenylketones in the urine, and failure of plasmatyrosine to rise after a phenylalanine load, point to a persistent defect in the hydroxylation of phenylalanine to tyrosine. The relatively high tolerance of natural protein, and the rapid return to normal of the phenylalanine levels after a phenylalanine load, suggest an atypical rather than the classical form of the disease. Patients with classical phenylketonuria have very low or absent phenylalanine p-hydroxylase activity in the liver 3the presence of the normal activity in one patient confirms the atypical nature of the biochemical defect in this case. HOH

OH

CH-OH.CH.OH.CH3 HN CH.OH-CH Of H

H

3,4,7,8,Tetrahydrobiopterin (XH4)

’,8,Dihydrobiopterin (XH2)

Fig. 3-Biopterin in the dihydro-(XH,) and tetrahydro-(XH,) -

Atypical phenylketonuria is often associated with or only slightly impaired intelligence. Our and unusual patients’ progressive neurological illness, which failed to respond to a low-phenylalanine diet, is unlike that in the classical defect. Since our original paper,2 Bartholom6lhas reported brief details of a similar patient, and a search of published reports brought to light a case6 with a somewhat similar neurological picture. Such cases are likely to be rare; in our regional neonatal programme we have failed to find one amongst forty patients diagnosed since 1969. It is likely that these cases represent a new variant of phenylketonuria, probably due to a defect in the metabolism of biopterin (fig. 3), the natural co-factor normal

0

1

2

3

4

5

6

24

Hours aFter oral P.A.

Fig. 2-Oral phenylalanine load per kg.)

test

P.A.=phenylalanine. Tyr.=tyrosine.

(100

mg.

phenylalanine

forms.

’

1111 REFERENCES

Phenylketonuria (edited by F. Lyman); p. 52. Springfield, Ill., 1963. Smith, I. Archs Dis. Childh. 1974, 49, 245. Justice, P., O’Flynn, M. E., Hsia, D. Y. Lancet, 1967, i, 928. McClean, A., Marwick, M. J., Clayton, B. E. J. clin. Path. 1973,

Jervis,

1. 2. 3. 4.

G. A. in

26, 678. 5. Bartholomé, K. Lancet, 1974, ii, 1580. 6. Crome, L. J. Neurol. 1962, 25, 149. 7. Kaufman, S. Adv. Enzymol. 1971, 35, 245. 8. Blakeley, R. L. The Biochemistry of Folic Acid and Related Pteridines): vol. XIII in the series Frontiers of Biology (edited by A. Neuberger and E. L. Tatum). Amsterdam, 1969. 9. Jacobson, W. in Some Recent Advances in Inborn Errors of Metabolism: Symposium 4 of the Society for the Study of Inborn Errors of Metabolism (edited by K. S. Holt and V. P. Coffey);

p. 1. Edinburgh, 1968. 10. Jacobson, W. Personal communication.

Addendum Fig. 4-Hydroxylation of phenylalanine biopterin (after Kaufman’).

to

tyrosine and role of

XH,=dihydrobiopterin. XH4=tetrahydrobiopterin. This compound, in for phenylalanine hydroxylase. its active tetra-hydro form, donates hydrogen ions during the hydroxylation reactionand is converted to quinonoid-dihydropteridine. This is converted back to tetrahydrobiopterin by the enzyme dihydroThe same co-factor system pteridine reductase (fig. is used in neural tissue for the hydroxylation of tyrosine to dihydroxyphenylalanine (levodopa) in the synthesis of the amine transmitters, dopamine, noradrenaline, and adrenaline, and also of tryptophan to 5-hydroxytryptophan in the synthesis of serotonin. Thus a deficiency of biopterin or of the enzyme dihydropteridine reductase, would not only impair the hydroxylation of phenylalanine to tyrosine, but also interfere

4).

with the production of neurotransmitters in the brain. Such a defect might be expected to cause severe neurological disease. Phenylalanine restriction, which would correct the hyperphenylalaninaemia without affecting the block in transmitter production, would be unlikely to benefit the neurological symptoms. Levodopa and dopamine play an important part in the function of the basal ganglia, and clinically this part of the brain appeared to be involved early in the course of the illness. All the features of these cases can be explained on the basis of a defect in biopterin metabolism. Jacobsonhas pointed out that pterins may have a part to play in the treatment of phenylketonuria, and an even stronger argument exists for a trial of such compounds in patients with a suspected co-factor defect. We were unable to obtain biopterin in sufficient quantities for a clinical trial when our patients were still alive, but we have since learnt 10 that 6,7-dimethylpterin, an analogue of biopterin, can be synthesised with relative ease. This compound is active in vitro, though less so than biopterin, and it could be used in a therapeutic trial. We thank Dr Ann McLean for her assay of phenylalanine p-hydroxylase; Dr J. Wilson, Dr E. Brett, and Dr K. Holt for their comments on the patients’ neurological illness; Dr G. Pampiglione for the E.E.G. reports; Dr J. N. Montgomery and Dr T. H. Hughes-Davies for referring the patients to us; and the Department of Health and Social Security and the

Wellcome Trust for financial support. Requests for reprints should be addressed to 1. S., Department of Child Health, Institute of Child Health, 30 Guilford Street, London WC1N lEH.

In an abstract (Pediat. Res. 1975,9,348) 1. J. Butler, N. A. Holtzman, S. Kaufman, S. H. Koslow, A. Krumholz, and S. Milstien have lately reported dihydropteridine-reductase deficiency in a patient with phenylketonuria unresponsive to dietary treatment.

FACTOR VIII AND GLOMERULONEPHRITIS M. EKBERG

I. M. NILSSON

Renal Unit and Coagulation Laboratory, Department Internal Medicine, University of Lund, Allmänna Sjukhuset, Malmö, Sweden

of

To find out if determination of factor VIII, which most probably is synthetised in the intima of blood-vessels, is of value for predicting the severity of vessel damage in glomerulonephritis, factor-VIII activity, factor-VIII-related antigen, and glomerular filtration-rate were estimated in 85 patients with early glomerulonephritis on admission, and in 70 of these at follow-up for up to 4 years.

Summary

The levels of factor-VIII activity and factor-VIII-related antigen on admission were normal in those patients who recovered. Where renal function was impaired or became so during follow-up, factor high. Determination of factor VIII might thus be of prognostic value in early glomerulonephritis.

on

admission

VIII was

Introduction

Bloom et aLl and Hoyer et al.2.3 have identified factor-vni-related antigen in normal vessel walls and glomerular capillary endothelium by an indirect immunofluorescent technique. The antigenic protein in the intima is not due to adhesion of the factor from the blood; Holmberg et al found that infusion of large doses of factor-vm concentrate to four patients with von Willebrand’s disease, in which intimal factor vm is totally lacking, did not result in the appearance of any specific fluorescence in the intima. This

suggested synthesis of the protein by the endothelial cells of the vessel wall, and corroborated the finding of factor-vm-antigen production in cultures of fetal endothelial cells by Jaffe et al. In glomerulonephritis, the endothelium of the glomerular vessels is damaged by deposition of complement and the consequent inflammatory reaction,6 which is followed by aggre-