Cliriical Genetics 1977: 11: 137-146

Heterozygote detection in phenylketonuria FLEMMING G+ER

AND

GERTHANSEN

John F. Kennedy Institute, Glostrup, Denmark Phenylalanine loading was carried out on 105 parents of children with phenylalanine hydroxylase deficiency and 33 apparently normal individuals with no family history of phenylketonuria. The best discriminant was found to be the logarithmic transformation of the slope of the rise in serum tyrosine multiplied by the maximum serum tyrosine concentration over the maximum serum phenylalanine concentration obtained after an oral load with a pure solution of L-phenylalanine. The overlap between heterozygotes for phenylketonuria and normal homozygotes was 2.4 %. The distribution of the discriminant values suggested three heterozygous phenotypes for phenylalanine hydroxylase deficiency, and the phenotypic combination of parents could be correlated to the phenotype of their affected offspring, i.e. classical phenylketonuria, mild phenylketonuria or hyperphenylalaninemia. The probability of heterozygosity for phenylketonuria was determined by means of the distribution of the discriminant values of the heterozygotes and that of normal homozygotes. The likelihood of being a heterozygote was corrected for the genetic background of the person requiring genetic counseling, and was finally expressed as the percentage probability of being a heterozygote for phenylketonuria. Received I 0 September, accepted for pirblicntiori 30 September I976

The enzyme deficient in phenylketonuria, phenylalanine hydroxylase, occurs in the liver (Kaufman 1969), but not in phytohemagglutinin-stimulated human lymphocytes (Horn & Giittler 1976). Heterozygous carriers for this deficiency are therefore preferably detected by indirect methods, e.g. by the administration of a loading dose of phenylalanine (Hsia et al. 1956. Rampini et al. 1969, Westwood 8: Raine 1973). In the last decade, a growing number of observations have shown heterogeneity in phenylketonuria. Instead of a single clinical and biochemical entity, a spectrum of disorders in the hydroxylation of phenylala-

nine to tyrosine has been recognized (Rampini et al. 1969, Menkes & Holtzman 1970, Blaskovics et al. 1974). T h e aim of the present investigation was t o develop a test of heterozygosity f o r phenylketonuria with high discriminatory ability, in order to examine whether the heterogeneity among affected homozygotes, mentioned above, is reflected by a similar heterogeneity of the heterozygous phenotypes.

Material and Methods

The criteria for distinguishing classical ver-

Part of this material was presented at The Fourth International Congress for the International Association for the Scientific Study of Mental Deficiency, Washington, D.C., August 1976.

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GUTTLER AND HANSEN

Fasling serum phenylalanine correlaled lo the eeuvalent ralio of phenylalanins to tyrosine 82 Helerozygoles lor P K U A 33 Controls

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sus mild phenylketonuria, and these forms versus persistent hyperphenylalaninemia, have been described in previous papers (Giittler & Wamberg 1972, Giittler & Hansen 1976). Briefly, to keep serum phenylalanine levels within 180-425 prnolil (3-7 mg/100 ml), children with classical phenylketonuria tolerate 9-18 % and children with the mild form of phenylketonuria 22-36 % of a normal daily intake of phenylalanine. This distinction cannot be made until the child is more than 2% years of age (c.f. Giittler & Hansen 1976). Children with persistent hyperphenylalaninemia show serum phenylalanine values within

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Fig. 1. Distribution of molar phenyla1anine:tyrosine ratios plotted against the equivalenl phenylalanine values ((,mol/l) in fasting serum of 82 heterozygotes for phenylketonuria (0) and 33 control subjects (A). Dotted lines define range of normal population.

the levels mentioned above on a normal dietary intake of phenylalanine. After they had fasted overnight, a sample of venous blood was drawn from 105 parents of these children and 33 individuals with no family history of phenylketonuria. A load of a pure solution of 0.6 mmol Lphenylalanine per kg body weight was then given orally, and venous bIood was sampled hourly for the next 4 hours. The serum was stored within 30 min at -20°C until analyzed. Serum phenylalanine and tyrosine were determined fluorimetrically, as described previously (Giittler & Wamberg 1972, Giittler & Hansen 1976).

HETEROZYGOTE DETECTION IN PHENYLKETONURIA

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---- Serum phenylalanine Median1 -Serum

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Flg. 2. Median serum phenylalanine (!rnol/l) - - - - - - - and median serum tyrosine (pmolll) __response to an oral load of L-phenylalanine in 82 heterozygotes for phenylketonuria (W), 23 heterozygotes for hyperphenylalaninemia (A) and 33 control subjects (0).

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33 Controls 23 Helerozygoles for H PA 82 Helerozygoles lor P K U

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Results

Fasting serum phenylalanine/tyrosine ratio. The present study showed a median serum phenylalanine/tyrosine ratio for 82 fasting heterozygotes for PKU of 2.39 (range 1.264.05) and for 33 fasting normal homozygotes of 1.83 (range 1.21-2.32), with an overlap of 39 %. When the distribution of the phenyla1anine:tyrosine ratios was plotted against the equivalent serum phenylalanine values in the fasting serum of the 115 subjects, 22 subjects or 20 % could not be accurately classified (Fig. 1).

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The response to an oral load of phenylalanine. The responses to a phenylalanine load of 82 heterozygotes for phenylketonuria, 23 heterozygotes for hyperphenylalaninemia and 33 controls are shown in Figure 2. The following discriminant based on phenylalanine loading was found to be the most powerful: the rate of tyrosine formation in pmol per 1 of serum per hour (btyr, Fig. 3) multiplied by the maximum value of serum tyrosine (tyr,, Fig. 3) in ymol per 1 over the maximum value of serum phenylalanine in pmol per 1 (phe,,

GOTTLER AND HANSEN

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Fig. 3. Serum phenylalanine (umoi;l and serum tyrosine ((tmol I) (0 response to an oral load of a pure solution of 0 6 mmol phenylalanine per kg of body weight. Rate of tyrosine formation, b , > r ;maximum value of serum phenylalanine, phe,,,; maximum value of serum tyrosine, tyr,,,.

Fig. 3), according to the function: btyr (tYr,,/phe,,) ' Four successive phenylalanine-loading tests of the same person showed discriminant values of 8.26, 8.82, 9.29 and 11.0. Four heterozygotes were loaded twice. The coefficient of variation was 5.0 %, 5.3 52, 5.7 % and 11.4 %, respectively. The median

value of the discriminant for 82 heterozygotes of phenylketonuria was 2.9 (range 0.1-1 1.8) and for 33 normal homozygotes 18.4 (range 9.3-60.8), P 0.001, with an overlap of 2.4 '2 (Fig. 4). The discriminatory power according to Penrose (1951) was 3.5.


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