PRENATAL DIAGNOSIS, VOL. 9,421-425 (1989)

SHORT COMMUNICATION

PRENATAL DIAGNOSIS OF PHENYLKETONURIA BY HAPLOTYPE

ANALYSIS

K. WULFF, M. WEHNERT, M. SCHUTZ*, G. SEIDLITZ AND F. H. HERRMANN Institute of Medical Genetics, Ernst-Moritz-Arndt- University, Fleischmannstr. 42-44. Greif wald, 2200 G.D.R.; *University Clinic for Obsteirics and Gynaecology. Ernst-Moritz-Arndt- University. Greifwald,

2200, G.D.R.

SUMMARY Prenatal diagnosis of classic phenylketonuria (PKU) was performed after chorionic villus sampling by means of linked restriction fragment length polymorphisms (RFLPs) using the cDNA probe ph PAH 247 (Kwok et al. (1985) Biochemistry, 24,556-561). We report in this paper a PKU family who were only informative for RFLP analysis by a combination of two RFLPs on the basis of haplotype determination of the normal and mutant phenylalanine hydroxylase (PAH) alleles. The DNA analysis detected a PKU fetus homozygous for mutant PAH alleles and the mother opted for termination in the 12th week of gestation.

KEY WORDS Phenylketonuria Restriction fragment length polymorphism Prenatal diagnosis Haplotype analysis

INTRODUCTION

Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism due to a deficiency of hepatic phenylalanine-4-hydroxylase (PAH). Although PKU can be treated by dietary restriction of phenylalanine, 44 per cent of early treated classical PKU patients repeated one or more school years and 10 per cent were placed in special schools (Saudubray et al., 1987). Since the isolation of the cDNA clones of human PAH, prenatal diagnosis by linked restriction fragment length polymorphisms (RFLPs) has become possible for families at risk (Woo, 1984; Lidsky et al., 1985a,b; Speer et al., 1986; Guttler and Woo, 1986). We report a prenatal diagnosis in a PKU family in which full genomic predictability was reached by the combination of two RFLPs.

MATERIALS AND METHODS

In preparation for prenatal diagnosis by RFLP analysis, 13 families at risk from the northern part of the G.D.R. were analysed using eight restriction enzymes (Herrmann et al., 1988). Total genomic DNA was isolated from nucleated white blood cells (Davies et al., 1985). Twenty mg of chorionic villus sample was obtained

Addressee for correspondence: Dr K. Wulff, Institute of Medical Genetics, Ernst-Moritz-Arndt- University, Fleischmannstr. 4244, Greifswald, DDR-2200.

0197-385 1/89/06042145$05.00 0 1989 by John Wiley & Sons, Ltd.

Received 6 June 1988 Revised 3 October 1988

Accepted 26 November 1988

422 K. W L F F ETAL.

Table 1. RFLP haplotype analysis of PKU family Z. and prenatal diagnosis ~~

Haplotype (Giittler

Bgl Pvu Pvu Xmn Eco Msp Eco Hind andwoo, I1 IIa IIb I RI I RV I11 1986)

M Normal 1.7 19 11.5 6.5 11 23 30 4.2 7 Mutant M 3.6 6 11-5 6.5 11 23 30 4.2 3

P Mutant M 3.6 6 11.5 6.5 11 23 30 4.2 3 MutantF 1.7 19 9.1 9.4 11 1912.2 30 4.2 6

F MutantF 1.7 19 9.1 9.4 11 1912.2 30 4.2 6 + 1.8

+ 1.8 Normal 3.6 6 11.5 9.4 17 1914 30 4.2 1

P.d. Mutant M 3.6 6 11.5 23 3 Mutant F 1.7 19 9.1 1912.2 6

+ 1.8

Possible PAH allele combinations for the fetus Bgl Pvu Pvu RFLP pattern I1 IIa IIb Haplotype BglII PvuII

1. Heterozygous, Normal M 1.7 19 1 1.5 7 1.7kb 19kb carrier Mutant F 1.7 19 9.1 6 11.5 kb

9.1 kb 2. Heterozygous, Mutant M 3.6 6 11.5 3 3.6kb 11.5 kb

carrier Normal F 3.6 6 11.5 1 6kb 3. Homozygous, NormalM 1.7 19 11.5 7 3.6kb 19kb

healthy Normal F 3.6 6 11.5 1 1.7 kb 11.5kb 6kb

4. Homozygous, Mutant M 3.6 6 11.5 3 3.6kb 19kb PKU Mutant F 1.7 19 9.1 6 1.7 kb 11.5 kb

9.1 kb 6kb

M =mother; F = father; P =PKU patient; p.d. =result of the prenatal diagnosis.

at the 10th week of gestation for the prenatal diagnosis of family Z. DNA of trophoblast tissue was isolated as previously described (Wehnert et af., 1988).

For Southern blot analysis of genomic DNA, the recombinant plasmid ph PAH 247 was used containing the full length cDNA of the human phenylalanine hydroxy- lase gene (Kwok et af., 1985). The RFLPs of the enzymes Bgl I1 and Pvu I1 were detected with a 0-5 kb Bgl I1 fragment from the 5’ region of cDNA probe ph PAH 247.

Prehybridization and hybridization were done as described by Wulff et al. (1988).

RESULTS

Table 1 shows the RFLP haplotype profiles of PKU family Z which were established for each family member in preparation for prenatal diagnosis. In this family at risk,

PHENYLKETONURIA

Pvu II

423

Bgl II

b 11.5/1,5 t 11.5/9.1

1

II a 19/6 o 1976 b 11.5/9.1 b 11.5/91

-6

3.6/1.7

3.6/1.7 L 3. /1.7

Figure 1. Prenatal diagnosis of PKU family Z. by means of the F'vu I1 and Bgl I1 polymorphisms. Lane I- 1 , mother; lane 1-2, father; lane 11-1, PKU patient; lane 11-2, DNA from the fetus

we did not detect completely informative RFLPs, which would allow a prenatal diagnosis by only a single polymorphism. The RFLPs Pvu IIb, Eco RI, and Msp I (Table 1) are informative only for the father. Full genetic predictability in family Z was possible by a combination of the RFLPs Pvu I1 and Bgl 11. Association of the different RFLPs with the normal or mutant PAH alleles was only possible by complete haplotype analysis and confirmation of haplotype 3 with the specific oligonucleotide probe (DiLella et al., 1986). On this basis, it is evident that the maternal mutant PAH allele is associated with the polymorphic fragments 3.6 kb for Bgl I1 and 6 kb for Pvu IIa, with haplotype 3, and in the father with the 1.7 kb Bgl I1 fragment and the 19 kb Pvu IIa fragment, with haplotype 6 for the paternal mutant PAH allele.

Table 1 demonstrates the possible RFLP combinations in the prenatal diagnosis for the fetus in family Z . Figure 1 shows the result of the DNA diagnosis on chori- onic villus sampling in the 10th week of gestation. The fetus 11-2 has the same

424 K. W L F F ET AL.

polymorphic bands as the affected child with PKU (Table 1). This means that the fetus has inherited the four polymorphic Pvu I1 fragments, 19, 11.5,9.1, and 6 kb, and the polymorphic Bgl I1 fragments, 3.6 and 1.7 kb. This RFLP combination is only possible in a child affected with PKU (Table 1). Because of this result family Z decided on an abortion in the 12th week of pregnancy.

DISCUSSION

Early diagnosis by newborn screening followed by dietary intervention can prevent postnatal brain damage and the mental retardation process associated with PKU. Despite the good results of a low phenylalanine diet, 20 per cent of all PKU children in the G.D.R. put on this diet during the first month after birth must attend special schools for the mentally handicaped; some of the PKU children attending normal schools have learning disabilities (Seidlitz et al., 1988).

Thirteen families at risk from the northern part of the G.D.R. with at least one living patient were interested in prenatal diagnosis, because they wished to have healthy offspring. In 92 per cent of these families, the parental haplotypes were sufficiently informative to distinguish all mutant PAH alleles. Full genetic predict- ability was obtained in eight PKU families by use of only one RFLP. In addition, five of these families were informative for three different DNA poiymorphisms. Recently in one of these families, the prenatal diagnosis was performed after chori- onic villus sampling in the 10th week of gestation and a homozygous normal fetus was detected (Herrmann et al., 1988; Wulff et al., 1988). In four PKU families, full genetic predictability could be obtained only by a combination of at least two RFLPs. In the case reported here, the prenatal diagnosis was possible only by a combination of different RFLPs using complete haplotype analysis and including confirmation of haplotype 3 by a specific oligonucleotide probe.

ACKNOWLEDGEMENTS

We are very much obliged to Professor Dr S. L. C. Woo (Howard Hughes Medical Institute and Institute of Molecular Genetics, Houston) for providing us with the ph PAH 247 cDNA clone isolated in this laboratory. We thank Mrs Dr U. Lichter- Konnecki and Priv. Doz. Dr F. K. Trefz (University Clinic of Pediatrics, Heidelberg) for the investigations with the specific oligonucleotide probe for the mutant haplotype 3 PAH alleles, and Mrs R. Diedrich and Mrs A. Steinmann for expert technical assistance.

REFERENCES

Davies, K.E., Speer, A., Herrmann, F.H., Spiegler, A.W.J., McGlade, S., Hofker, M.H., Briand, P., Hanke, R., Schwartz, M., Steinbicker, V., Szibor, R., Komer, H., Sommer, D., Pearson, P.L., Coutelle, Ch. (1985). Human X-chromosome markers and Duchenne muscular dystrophy, Nucle. Acids Res., 13,3419-3425.

DiLella, A.G., Marvit, L., Lidsky, A.S., Guttler, F., Woo, S.L.C. (1986). Tight linkage between a splicing mutation and a specific DNA haplotype in phenylketonuria, Nature,

Guttler, F., Woo, S.L.C. (1986). Molecular genetics of PKU, J . Znher. Metab. Dis., 9,5848. 322,799-803.

PHENYLKETONURIA 425

Herrmann, F.H., Wulff, K., Wehnert, M., Seidlitz, G., Guttler, F. (1988). Haplotype analysis of classical and mild phenotype of phenylketonuria in the German Democratic Republic, Clin. Genet., 34, 176-180.

Kwok, S.C.M., Ledley, F.D., DiLella, A.G., Robson, K.J.H., Woo, S.L.C. (1985). Nucleotide sequence of a full-length cDNA clone and amino acid sequence of human phenylalanine hydroxylase, Biochemistry, 24,556-561.

Lidsky, A.S., Guttler, F., Woo, S.L.C. (1 985b). Prenatal diagnosis of classic phenylketonuria by DNA analysis, Lancet, 1,549-551.

Lidsky, A.S., Ledley, F.D., DiLella, A.G., Kwok, S.C.M., Daiger, S.P., Robson, K.J.M., Woo, S.L.C. (1985a). Extensive restriction site polymorphism at the human phenylalanine hydroxylase locus and application in prenatal diagnosis of phenylketonuria, Am. J. Hum. Genet., 37,619434.

Saudubray, J.M., Rey, F., Ogier, H., Abadie, V., Farriaux, J.P., Ghisolti, J., Guidaud, P., Rey, J., Vidailhet, M. (1 987). Intellectual and school performances in early-treated classical PKU patients. The French collaborative study, Eur. J. Pediutr., 146, A2GA22.

Seidlitz, G., Machill, G., Grimm, U., Knapp, A., Ebener, U., Herrmann, F.H. (1988). Phenylketonurie aus heutiger Sicht, Z . Aerztl. Fortbild., 82, 1123-1 125.

Speer, A., Bollman, R., Michel, A., Neumann, R., Bommer, Ch., Hanke, R., Riess, O., Cobet, G., Coutelle, Ch. (1986). Prenatal diagnosis of classical phenylketonuria by linked restric- tion fragment length polymorphism analysis, Prenat. Diagn., 6,447450.

Wehnert, M., Kiessiing, U., Schutz, M., Machill, G., Wulff, K., Herrmann, F.H. (1988). DNA-Praparation aus kultivierten Fruchtwasserzellen und Choriongewebe, Z . Med. Labor. Diagn., 28, 126-127.

Woo, S.L.C. (1984). Prenatal diagnosis and carrier detection of classic phenylketonuria by gene analysis, Pediatrics, 74,412423.

Wulff, K., Herrmann, F.H., Wehnert, M., Seidlitz, G., Schutz, M. (1988). Genomische Diagnostik bei klassischer Phenylketonurie, 2. AerzfZ. Fortbild., 82, 1127-1 13 1.