Phenylketonuria (PKU) and mild hyperphenylalaninemia (MHP) are allelic disorders caused by mutations in the gene encoding phenylalanine hydroxylase (PAH). Previous studies have suggested that the highly variable metabolic phenotypes of PAH deficiency correlate with PAH genotypes. We identified both causative mutations in 686 patients from seven European centers. On the basis of the phenotypic characteristics of 297 functionally hemizygous patients, 105 of the mutations were assigned to one of four arbitrary phenotype categories. We proposed and tested a simple model for correlation between genotype and phenotypic outcome. The observed phenotype matched the predicted phenotype in 79% of the cases, and in only 5 of 184 patients was the observed phenotype more than one category away from that expected. Among the seven contributing centers, the proportion of patients for whom the observed phenotype did not match the predicted phenotype was 4%-23% (P<.0001), suggesting that differences in methods used for mutation detection or phenotype classification may account for a considerable proportion of genotype-phenotype inconsistencies. Our data indicate that the PAH-mutation genotype is the main determinant of metabolic phenotype in most patients with PAH deficiency. In the present study, the classification of 105 PAH mutations may allow the prediction of the biochemical phenotype in >10,000 genotypes, which may be useful for the management of hyperphenylalaninemia in newborns.

B- and T-lymphocyte attenuator (BTLA) is a lymphoid receptor that inhibits lymphocyte activation on interaction with its ligand, herpesvirus entry mediator (HVEM). We developed monoclonal antibodies against BTLA and HVEM to study their expression using immunohistochemical and flow cytometric analyses in human tissues. In reactive lymph nodes, they were both expressed in interfollicular T cells and in B cells from mantle and marginal zones. Within germinal centers, B cells were negative, whereas T follicular helper (TFH) cells were BTLA+ and follicular dendritic cells were HVEM+. BTLA was strongly expressed in chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL, 19 of 19 positive) when compared with other small B-cell lymphomas, including follicular lymphoma (0 of 24 positive), mantle cell lymphoma (0 of 10 positive), and marginal zone lymphoma (0 of 5 positive). Our results suggest that down-regulation of the BTLA-HVEM pathway may be involved in germinal center B-cell activation. The specific high expression of BTLA in B-CLL/SLL represents a new potential diagnostic tool. The BTLA positivity of TFH cells may be a basis for designing future immunotherapies.

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Previous studies have demonstrated the absence of viral replication of Vif ؊ mutants in stimulated primary blood mononuclear cells (PBMC). Human immunodeficiency virus type 1 strain NDK Vif ؊ mutants were propagated on the semipermissive CEM cell line, and the viral stock obtained was compared with the wild-type virus during a single cycle in PBMC. The Vif ؊ virus was able to enter PBMC with the same efficiency as the wild type, as demonstrated by quantification of the strong-stop cDNA, and retrotranscription was observed for both viruses within 4 h postinfection. Using a PCR assay with an Alu-long terminal repeat pair of primers, we detected integration for both the wild-type and Vif ؊ viruses. We then used qualitative and quantitative reverse transcription-mediated PCR techniques to study the steady-state level of intracellular and extracellular viral RNAs. All mRNA species were detected in PBMC infected with the wild-type virus or with the Vif ؊ virus 36 h postinfection. Furthermore, quantification of viral RNA released from infected cells demonstrated similar levels of virus produced after a unique cycle of replication. However, the Vif ؊ virus obtained after one replication cycle in PBMC was unable to initiate retrotranscription in permissive target cells. These data strongly suggest that the failure to infect target cells is due to a defect in the formation of the viral particle in PBMC.

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Twenty-two French patients with early treated phenylketonuria (PKU) off diet (no reduced phenylalanine, Phe) since their 5th birthday, 23 German patients on diet (reduced Phe), and 21 healthy control subjects from childhood to adulthood matched for age, sex, and IQ were investigated for visuomotor reaction time, sustained attention, and visual stimulus scanning. Determinations were made whether 1) the three groups showed different developmental trends in their reaction times, 2) the threshold of a Phe blood level of 360 mumol/L formulated in recent recommendations can be regarded as safe, 3) test performances are related to the quality of dietary control in the same way for all age groups, and 4) long-term elevated Phe levels result in aggravating effects of increasing differences between patients on and off diet and healthy controls. Results revealed that developmental trends were similar in all treatment groups. Only children with a mean Phe level below 360 mumol/L performed as well as control subjects. Differences between treatment groups decreased in adulthood, and no aggravating effect could be observed. Mean performance of patients with mild PKU off diet was in the same range as performance of patients with classical or mild PKU on diet, calling into question the benefit of treating these patients. It is concluded that it is preferably safer to maintain Phe blood levels below 360 mumol/L at least during the first 10 y of life.

The role of the accessory gene product Vpr during human immunodeficiency virus type 1 infection remains unclear. We have used the yeast two-hybrid system to identify cellular proteins that interact with Vpr and could be involved in its function. A cDNA clone which encodes the human uracil DNA glycosylase (UNG), a DNA repair enzyme involved in removal of uracil in DNA, has been isolated. Interaction between Vpr and UNG has been demonstrated by in vitro protein-protein binding assays using translated, radiolabeled Vpr and UNG recombinant proteins expressed as a glutathione S-transferase fusion protein. Conversely, purified UNG has been demonstrated to interact with Vpr recombinant protein expressed as a glutathione S-transferase fusion protein. Coimmunoprecipitation experiments confirmed that Vpr and UNG are associated within cells expressing Vpr. By using a panel of C-and N-terminally deleted Vpr mutants, we have determined that the core protein of Vpr, spanning amino acids 15 to 77, is involved in the interaction with UNG. We also demonstrate by in vitro experiments that the enzymatic activity of UNG is retained upon interaction with Vpr.

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