HUMAN MUTATION 5:188-190 (1995)

MUTATION IN BRIEF

E ~ r e s s i o n Analysis of Mutation P24$k, M i c h causes Mild Hyperphenylalaninemia Bekn Perez, Lourdes R. Desviat, and Magdalena Ugarte* Centro de Biologia Molecular Severo Ochoa CSIC-UAM, Unwersidud Aut6noma de Madrid, Cantoblanco, 28049 Madrid, Spain; Fax: 34-1-397-4870

Communicated by Charks R. Scriver

INTRODUCTION

Phenylketonuria (PKU) is characterized by a high level of biochemical and clinical heterogene- ity with an underlying genetic variability. Up to now, 130 mutations (mostly single base substitu- tions) have been described in the phenylalanine hydroxylase (PAH) gene, associated with specific haplotypes and populations (Eisensmith and Woo, 1992). Expression analysis confirm that these changes are responsible for the PKU phenotype and enable the establishment of a correlation be- tween the genotype and the clinical severity of the disease (Okano et al., 1991; Trefz et al., 1993).

In Spain, 46% of the mutations have been iden- tified (Desviat et al., 1993). The two major mu- tations, IVSlO and I65T, have frequencies of 20% and lo%, respectively, and seven other mutations are present in low frequencies. Mutation P244L was identified by sequence analysis in one family (Desviat et al., 1992). In this study we describe the expression analysis of this mutation found on a haplotype 12 background.

MATERIALS AND METHODS

The PKU proband with the P244L mutation has mutant haplotypes 12/9 and carries mutation I65T on the haplotype 9 allele. The patient exhibits a mild form of PKU, with a plasma Phe level of 720 kmol/L and a phenylalanine tolerance of 900 mg/ day. Two untreated siblings, born before routine mass screening, have the same genotype and are clinically normal (plasma Phe levels of 500 and 900 pmol/L, respectively). We have analyzed by allele-specific oligonucleotide (ASO) screening 67 patients for the presence of the P244L mutation. In 36 of them, the source of DNA was dried blood spots. Polymerase chain reaction (PCR) condi- tions and experimental procedures for AS0 anal-

0 1995 WILEY-LISS, INC.

ysis have been described (Pkrez et al., 1993; Des- viat et al., 1992).

Expression analysis was performed in COS cells using the human expression vector pRc/CMV (In- Vitrogen). The constructs with the polyaromatic hydrocarbon (PAH) cDNA and the P-galactosi- dase cDNA inserted in the pRc/CMV vector were kindly provided by Dr. S.L.C. Woo. The oligonu- cleotide-directed mutagenesis kit from Amersham was used to introduce the P244L mutation in the PAH sequence. The mutagenic oligonucleotide was 5’CGCCTCCGACTTTGGCTCG 3‘.

COS cells were transfected with the lipofectin reagent (B. R. L. ) following the manufacturer’s rec- ommendations. P-Galactosidase activity was de- termined as described by Nielsen et al. (1983). PAH enzymatic activity in transfected cells and in rat liver was assayed essentially as described by Okano et al. (1991). Total cellular RNA was iso- lated by the guanidium thiocyanate method of Chomczynski and Sacchi ( 1987). Western blots were prepared according to standard procedures (Harlow and Lane, 1988).

Anti-PAH antibodies were produced in rabbit by immunization with six synthetic peptides cor- responding to different regions in the PAH pro- tein, selected according to their hydrophylicity , using the program PEPTIDESTRUCTURE de- veloped by the University of Wisconsin Genetics Computer Group. The following peptides were used: RLKKDEYEFFTHLDK, WFPRTIQELDRF- ANQILS, IPQLEDVSQFLQTC, PEPDICHELLG- HVPLF, SFGELQYCLSEKP, and FSVRYDPYTQ- RIEVL.

Received March 11, 1994 accepted April 28, 1994 *To whom reprint requestsicorrespondence should be ad-

dressed.

MUTATION P244L AND HYPERPNEHYLALANINEMIA 189

FIGURE 1. Relative PAH activity was assayed measuring the conversion of 14C-Phe to 14C-Tyr. Lanes 1-4, PAH activity in liver extracts, COS cells transfected with normal cDNA, which mutant cDNA and with p-galactosidase cDNA, re- spectively.

RESULTS AND DISClUSSION

The P244L mutation was identified through se- quence analysis in a Spanish PKU patient. AS0 analysis of the parental sampkj showed that the mutation segregated with haplotype 12 (Desviat et al., 1992). This is a rare mutation, which we have found on only one of the 134 mutant alleles tested.

The functional significance c,f the P244L muta- tions was assayed by transient expression of the mutant cDNA in COS cells. PAH enzymatic ac- tivity was measured as conversion of l4C-pheny1a- lanine to 14C-tyrosine. The relative enzyme activ- ity of the mutant PAH was expressed as a percentage of wild-type PAH activity, and the mean value was determined from three indepen- dent transfection experiments. Extracts from cells expressing the P244L mutation had 70% control activity (Fig. 1). RNA and Western blot analysis revealed similar levels of PAH inRNA and immu- noreactive protein in cells transfected with the mutant and wild-type PAH cDNA (data not shown). Consequently, the mutation does not al- ter the stability of the protein, but it does cause a moderate reduction in the specific activity of the enzyme.

The sequence comparison berween human PAH and PAH from Drosophila rnelanogaster has shown a high homology (more than 880/) from amino acids 236-323 (Morales et al., 1990). The highest ho- mology with tyrosine hydroxylase and rat PAH also lies within this region, so tlhis part of the mol- ecule has been suggested to include the catalytic core (Ledley et al., 1985; Dahl and Mercer, 1986). Nevertheless, the Pro to Leu substitution only has

a moderate effect on the protein activity. It is im- portant to note that the mutation is close to the binding region of many ligands, such as the cofac- tor, BH,, and the nonheme iron (Jennings et al., 1991; Gibbs et al., 1993). Even though the sec- ondary structure predictions show that the Pro to Leu substitution may change the conformation of the molecule and thus affect the affinity for some ligand, this effect would not be detected due to the excess of the hydroxylation components used in the in vitro PAH assay.

The patient bearing the P244L mutation carries mutation I65T on the other chromosome. This mutation results in a 26% mean residual activity when compared to the wild-type enzyme (John et al. , 1992). The patient exhibits a mild phenotype, consistent with the findings of the high in vitro activity of the P244L mutation. In the same fam- ily, there are two other PKU siblings, never dietary treated, with the same genotype, similar biochem- ical phenotype and clinically normal. Okano and co-workers ( 1991) have related the predicted value of PAH activity to the biochemical phenotypes of patients. However, it has been repeatedly dis- cussed that, in some mutations, the in vitro activ- ities in COS cells are most likely higher than the corresponding in vivo values (Svensson et al., 1993; Okano et al., 1991). We believe this could be the case of the P244L mutation. To confirm this hypothesis, kinetic experiments with the purified protein evaluating the affinity of the mutant en- zyme for the cofactor or other ligands should be performed.

ACKNOWLEDGMENTS

We thank Dr. S.L.C. Woo and Dr. R.C. Eisen- smith for their collaboration, Dr. M.E. Patarroyo for providing the immunogenic peptides, and C. Hernfindez for her expert technical assistance. The financial support of the Fundaci6n Ram& Areces to the Centro de Biologia Molecular Severo Ochoa is gratefully acknowledged. This work was sup- ported by grant SAF93-0076 from the CICYT (Comisibn Interministerial de Ciencia y Tecnolo- gia).

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