A phenylalanine hydroxylase amino acid polymorphism with implications for molecular diagnostics.

Mutations in the gene encoding phenylalanine hydroxylase (PAH, EC 1.14.16.1) are associated with various degrees of hyperphenylalaninemia, including classical phenylketonuria (PKU). We examined the PAH gene in a Brazilian PKU family of African origin and identified three missense variants, R252W (c.754C --> T), K274E (c.820A --> G), and I318T (c.953T --> C), the two latter of which were transmitted in cis. Expression analyses in two different in vitro systems showed that I318T is associated with profoundly decreased enzyme activity, whereas the enzyme activity of K274E is indistinguishable from that of the wild-type protein. Detailed kinetic analyses of PAH expressed in E. coli showed that the K274E mutant protein has kinetic properties similar to that of the wild-type protein. Population studies have suggested that the K274E variant occurs on approximately 4% of African-American PAH alleles, whereas the neonatal screening incidence of PKU among African Americans is only 1:100,000. This is to our knowledge the first demonstration of a PAH missense variant with no apparent association to PAH deficiency. Awareness of this common variant may be helpful to laboratories that perform molecular diagnosis of PAH deficiency in populations of African origin.

Missense mutations in the N-terminal domain of human phenylalanine hydroxylase interfere with binding of regulatory phenylalanine.

Hyperphenylalaninemia due to a deficiency of phenylalanine hydroxylase (PAH) is an autosomal recessive disorder caused by >400 mutations in the PAH gene. Recent work has suggested that the majority of PAH missense mutations impair enzyme activity by causing increased protein instability and aggregation. In this study, we describe an alternative mechanism by which some PAH mutations may render PAH defective. Database searches were used to identify regions in the N-terminal domain of PAH with homology to the regulatory domain of prephenate dehydratase (PDH), the rate-limiting enzyme in the bacterial phenylalanine biosynthesis pathway. Naturally occurring N-terminal PAH mutations are distributed in a nonrandom pattern and cluster within residues 46-48 (GAL) and 65-69 (IESRP), two motifs highly conserved in PDH. To examine whether N-terminal PAH mutations affect the ability of PAH to bind phenylalanine at the regulatory domain, wild-type and five mutant (G46S, A47V, T63P/H64N, I65T, and R68S) forms of the N-terminal domain (residues 2-120) of human PAH were expressed as fusion proteins in Escherichia coli. Binding studies showed that the wild-type form of this domain specifically binds phenylalanine, whereas all mutations abolished or significantly reduced this phenylalanine-binding capacity. Our data suggest that impairment of phenylalanine-mediated activation of PAH may be an important disease-causing mechanism of some N-terminal PAH mutations, which may explain some well-documented genotype-phenotype discrepancies in PAH deficiency.

In vitro expression of 34 naturally occurring mutant variants of phenylalanine hydroxylase: correlation with metabolic phenotypes and susceptibility toward protein aggregation.

Phenylalanine hydroxylase (PAH) is a homotetrameric enzyme that catalyzes the conversion of phenylalanine to tyrosine, the rate-limiting step of phenylalanine disposal in humans. Primary dysfunction of PAH caused by mutations in the PAH gene results in hyperphenylalaninemia, which may impair cognitive development unless corrected by dietary restriction of phenylalanine. The mechanism(s) by which PAH missense mutations cause enzyme impairment has been studied in detail only in a small number of cases, but existing evidence points to a major role of enhanced proteolytic degradation due to aberrant folding of mutant polypeptides. We have used two heterologous in vitro expression systems (a mammalian cell-free transcription-translation system and the pET system of Escherichia coli) to examine 34 mutations that have been associated with PAH deficiency in the Danish population. These mutations represent a broad range of amino acid substitutions, functional enzyme domains, and metabolic phenotypes. In both systems, residual in vitro activities correlated broadly with metabolic phenotypes, however, with significant discrepancies. Analysis of E. coli extracts by nondenaturing polyacrylamide gel electrophoresis and storage experiments showed that (i) in general, mutations in the N-terminal regulatory domain are associated with relatively stable proteins compared to most mutations in the central catalytic domain, and (ii) for mutations in the catalytic domain, high levels of protein aggregation do not always correspond with a severe phenotype. Our data support and extend previous evidence that PAH mutations exert their pathogenic effects by several distinct mechanisms that may operate individually or in concert.

The retinoblastoma protein modulates expression of genes coding for diverse classes of proteins including components of the extracellular matrix.

The product of the retinoblastoma susceptibility gene, pRb, is a negative regulator of cell growth. It functions by regulating the activity of transcription factors. Rb represses some genes by sequestering or inactivating the positive transcription factor E2F and seems to activate some others by interacting with factors like Sp1 or ATF-2. However, there are only a few examples of genes which are positively regulated by pRb. In order to find out if there are common mechanisms for promoter regulation by pRb, we were interested to identify more genes which are either stimulated or repressed by pRb. Using the method of differential display (DDRT-PCR) in combination with nuclear run-on analyses we were able to detect a number of genes which are upregulated by ectopic expression of the Rb gene in Rb-deficient mammary carcinoma cells. We could demonstrate not only stimulation of the endogenous mutant Rb gene but also positive regulation of genes coding for diverse classes of proteins, including the endothelial growth regulator endothelin-1 and the proteoglycans versican and PG40. As a second approach, we investigated gene expression in cell lines established from Rb deficient heterozygous and homozygous knockout mouse embryos and normal mice. We have identified several genes the expression of which correlates positively or negatively with the presence of Rb. These data provide further evidence for pRb being a master regulator of a complex network of gene activities defining the difference between dividing and resting or differentiated cells.

Regulated expression of the retinoblastoma susceptibility gene in mammary carcinoma cells restores cyclin D1 expression and G1-phase control.

The product of the retinoblastoma susceptibility tumour suppressor gene, pRb, is a negative regulator of cell proliferation. In order to investigate the interaction between pRb and the cell cycle machinery in more detail, a functional Rb gene was reintroduced into the Rb-deficient human mammary carcinoma cell line Bt549. Since constitutive high level expression of Rb turned out to be difficult to maintain, the tetracycline-dependent gene expression system was used. A number of clones was generated which all showed low level expression in the noninduced state. Considerable induction rates were obtained. The low level of noninduced Rb expression was sufficient to induce the expression of cyclin D1 the level of which was not further increased by upregulation of Rb expression. Concomittantly, an increase in cell doubling time was observed due to retardation of the cell cycle in the G1-phase. The data suggest that limiting amounts of cyclin D1 determine, at least partly, the extent of growth-repressing properties of pRb. The inducible system allows for maintenance of Rb-reconstituted cells at a low level of expression and for their use in the investigation of downstream functions of pRb.