Randy C. Eisensmith

Molecular basis of phenotypic heterogeneity in phenylketonuria.

BACKGROUNDnPhenylketonuria is a metabolic disorder that results from a deficiency of the hepatic enzyme phenylalanine hydroxylase. Its clinical phenotype varies widely, and to date more than 10 mutations in the phenylalanine hydroxylase gene have been identified in persons with the disorder. We attempted to relate the clinical phenotype of patients to their genotype.nnnMETHODSnWe studied 258 patients with phenylketonuria from Denmark and Germany for the presence of eight mutations previously found in patients from these countries. The in vitro activity of the enzymes associated with these mutations was determined by expression analysis in heterologous mammalian cells. The level of activity was then used to predict the in vivo level of phenylalanine hydroxylase activity in patients with various combinations of mutant phenylalanine hydroxylase alleles.nnnRESULTSnThe eight mutations involved 64 percent of all mutant phenylalanine hydroxylase alleles in the patients. Expression analysis showed that these mutant enzymes produced from 0 to 50 percent of normal enzyme activity. The predicted level of phenylalanine hydroxylase activity correlated strongly with the pretreatment serum level of phenylalanine (r = 0.91, P less than 0.001 in the Danish patients and r = 0.74, P less than 0.001 in the German patients), phenylalanine tolerance in the Danish patients (r = 0.84, P less than 0.001), and the serum phenylalanine level measured after standardized oral protein loading in the German patients (r = 0.84, P less than 0.001).nnnCONCLUSIONSnOur results strongly support the hypothesis that there is a molecular basis for phenotypic heterogeneity in phenylketonuria. The establishment of genotype will therefore aid in the prediction of biochemical and clinical phenotypes in patients with this disease.

6 Molecular Genetics of Phenylketonuria: From Molecular Anthropology to Gene Therapy

Publisher Summary Classical phenylketonuria (PKU) was first recognized as a distinct metabolic disorder causing severe, irreversible mental retardation by the Norwegian biochemist and physician Asbjorn Folling. Folling observed increased levels of phenylpyruvate in the urine. Because of the structural similarity between phenylpyruvate and the essential amino acid phenylalanine, Folling hypothesized that phenylalanine was the most likely source of the urinary phenylpyruvate. Follings observations clearly implicated some defect of phenylalanine metabolism in phenylketonuric individuals. Subsequent studies reveal that phenylalanine hydroxylation (PAH) in humans is a complex reaction involving at least six different enzymes and their requisite cofactors. The cloning of the PAH cDNA provides the single most important reagent for the understanding of molecular genetics of PKU. The expression of this cDNA conclusively demonstrated that the human PAH protein is the product of a single gene. The knowledge of the molecular basis of PKU and the fact that it is primarily a single gene disorder has made it an attractive model for the development of new treatment modalities based on somatic gene transfer, which should also be applicable to a variety of monogenic diseases.

IFN-γ sensitization of prostate cancer cells to fas-mediated death: a gene therapy approach

Abstract While human prostate cancers and cell lines express Fas, most of these cell lines are resistant to Fas-mediated death. In the present studies we addressed the ability of IFN-γ to influence Fas-mediated cell death in prostate cancer cells. In vitro exposure of the human cell lines LNCaP and PC3 and the mouse cell line RM-1 to agonist anti-Fas antibody and/or soluble Fas ligand resulted in killing of only PC3 cells. However, preincubation with IFN-γ resulted in synergistic killing in all three cell lines. In vitro treatment of RM-1 with a replication-incompetent adenovirus expressing mouse FasL (Ad.FasL) resulted in maximal cell kill near 40%, which correlated with baseline Fas expression. The addition of IFN-γ enhanced cell kill to a degree consistent with the resulting higher levels of Fas and maintained synergistic killing at very low doses of vector. Co-inoculation of orthotopic RM-1 primary tumors with Ad.mFasL and an adenovirus expressing mouse IL-12 (Ad.mIL-12) to drive host production of IFN-γ negated the survival advantage of Ad.mIL-12 alone. However, the staggered injection of Ad.mIL-12 and Ad.FasL achieved almost threefold higher levels of apoptosis in primary tumor tissue and doubled median survival. Therefore, IFN-γ is capable of bestowing increased sensitivity to Fas-mediated cell death in prostate cancer cells and, in a gene therapy approach, may define a powerful tool to treat prostate cancers.

Molecular correlations in phenylketonuria : Mutation patterns and corresponding biochemical and clinical phenotypes in a heterogeneous California population

We studied 133 California phenylketonuria (PKU) patients and one obligate heterozygote to delineate the molecular basis of PKU in a population with greater ethnic diversity than in previous studies, and to determine whether a correlation exists between genotype and clinical phenotype, with the latter defined by both the diagnostic pretreatment blood phenylalanine (PHE) level and cognitive (IQ) test scores. To determine PAH genotypes, we used PCR-mediated amplification, denaturing gradient gel electrophoresis, and direct sequencing on dried whole blood samples. Where possible, mutation severity was defined according to predicted in vitro PAH enzyme activity estimated by using Cos cell expression analysis for a given mutation. We then asked whether mutation severity, as defined by such expression analysis, correlated with pretreatment PHE levels or with IQ test results. A mutation was identified in 236 (88%) of 267 mutant alleles. Seventeen new mutant alleles were found; A47E, T81P, I102T, E182G, T328D, Y343P, K371R, Y387H, A389E, E422K, IVS9nt5, IVS11nt20, delS70, del364–368/del198–220, delF299, delT323, and −1C/T. In striking contrast to a number of studies in other populations, in this study, based on predicted PAH activity, we observed no correlation between mutation severity and pretreatment PHE levels. There was also no correlation between genotype and IQ. We conclude that in samples collected from an ethnically heterogeneous population, there is no correlation of mutation severity with either pretreatment PHE levels or IQ measurement in treated patients. We caution that genetic counseling in PKU should incorporate the notion that prognosis may not be predicted with precision based on mutation analysis in a given patient.

Three polymorphisms but no disease-causing mutations in the proximal part of the promoter of the phenylalanine hydroxylase gene.

The proximal promoter region of the human phenylalanine hydroxylase (PAH) gene was analyzed for the presence of mutations in 122 European phenylketonuria (PKU) and hyperphenylalaninemia patients having altogether 187 uncharacterized mutant PAH alleles. This promoter fragment, which contained the most 5′ transcription start site and about 300 bp upstream, was sequenced directly from polymerase-chain-reaction-amplified genomic DNA. No disease-causing mutations but three neutral nucleotide substitutions were found. A −195 T-to-C transition was present on 1% of 441 normal and 0.3% of 653 mutant chromosomes. All chromosomes that carried this transition and to which a restriction fragment length polymorphism (RFLP) haplotype had been assigned were of haplotype 1. A −71 G-to-A change and a +7 C-to-T change were always observed together and were found on 1% of 425 normal and 4% of 681 mutant chromosomes. In addition, these two transitions were found in seven heterozygote samples where the phase could not be established due to incomplete family samples. In individuals where RFLP haplotyes were known and phase could be established, these linked substitutions were associated with RFLP haplotype 9. The relatively high frequency (10–20%) of these two polymorphisms on PKU chromosomes from Great Britain, Ireland and France may reflect a relative concentration of haplotype 9 alleles among PKU chromosomes from these countries compared to the rest of Europe. The absence of disease-causing mutations within a region of the PAH gene that possesses basal promoter activity suggests that transcriptional mutations are not likely causes of PKU in Caucasian populations.

Origins of hyperphenylalaninemia in Israel.

Mutations and polymorphisms at the phenylalanine hydroxylase (PAH) gene were used to study the genetic diversity of the Jewish and Palestinian Arab populations in Israel. PAH mutations are responsible for a large variety of hyperphenylalaninemias (HPAs), ranging from the autosomal recessive disease phenylketonuria to various degrees of nonclinical HPA. Seventy-two Jewish and 36 Palestinian Arab families with various HPAs, containing 115 affected genotypes, were studied by haplotype analysis, screening for previously known PAH lesions and a search for novel mutations. Forty-one PAH haplotypes were observed in this sample. Four mutations previously identified in Europe (IVS10nt546, R261Q, R408W and R158Q) were found, and were associated with the same haplotypes as in Europe, indicating possible gene flow from European populations into the Jewish and Palestinian gene pools. Of particular interest is a PAH allele with the IVS10nt546 mutation and haplotype 6, that might have originated in Italy more than 3,000 years ago and spread during the expansion of the Roman Empire. These results, together with previous identification of three PAH mutations unique to Palestinian Arabs [IVSnt2, Edel(197–205) and R270S], indicate that the relatively high genetic diversity of the Jewish and Palestinian populations reflects, in addition to genetic events unique to these communities, some gene flow from neighboring and conquering populations.

Phenylketonuria in Costa Rica: preliminary spectrum of PAH mutations and their associations with highly polymorphic haplotypes

A preliminary evaluation of the molecular basis of phenylketonuria (PKU) in Costa Rica was made by performing mutational analyses in the six PKU families identified to date. These studies revealed the presence of the previously reported European mutations IVS1nt5, L48S, E221G and IVS12ntl as well as the novel mutation IVS7nt3. The combined use of the STR, VNTR and XmnI polymorphic systems for the PAH gene resulted in a discriminant distribution of haplotypes among normal and mutant chromosomes and suggests its potential usefulness for future diagnostic applications in Costa Rican PKU kindreds. This is the first report of a genetic analysis in a Central American PKU population.

Complete spectrum of PAH mutations in Tataria: presence of Slavic, Turkic and Scandinavian mutations.

Phenylketonuria (PKU) is an autosomal recessive disorder associated with a deficiency of hepatic phenylalanine hydroxylase (PAH). Although the molecular lesions present in the PAH genes of PKU patients have previously been determined in several Slavic populations, little is known regarding the molecular basis of PKU in the non-Slavic populations of the former Soviet Union. Guthrie card samples from twenty-one classical PKU patients residing in the Tatarian Republic were examined by a combination of denaturing gradient gel electrophoresis and direct sequence analysis. Twelve patients were of Tatarian ancestry, five were of Russian ancestry, and four were of mixed Tatarian and Russian ancestry. Two of the Tatarian patients were related, sharing one mutant allele. The single major allele identified in this study was R408W/RFLP haplotype 2/VNTR 3, which was present on 11/14 or 78.6% of all mutant chromosomes of Slavic origin, but on only 10/27 or 37.0% of mutant chromosomes of Tatarian origin. This result suggests that this allele was introduced into central Asian populations during the eastward expansion of Slavs across the Eurasian landmass. A significant influence of Turkic peoples on Tatars can be inferred from the presence of R261Q, IVS10nt546g→a, L48S, IVS2nt5g→c: and P281L, all of which are relatively common among Turks or have been observed in Mediterranean populations. Together, these alleles are present on 11/27 or 40.7% of all mutant chromosomes in ethnic Tatars. Surprisingly, the common Scandinavian mutation IVS12nt 1g→a was also present in Tataria, as was the ΔagE221D222fs mutation found previously only in Denmark. Thus, some direct or indirect gene flow from Scandinavian into Tataria seems evident. Finally, the complete absence of PAH mutations previously observed in Oriental populations suggests that there was little gene flow into Tataria from Eastern Asia.