Cristina Aguado

Analysis of the effect of tetrahydrobiopterin on PAH gene expression in hepatoma cells

Tetrahydrobiopterin (BH4)‐responsive phenylalanine hydroxylase (PAH) deficiency is a recently recognized variant of phenylketonuria, with a probable multifactorial molecular basis. In this study we have investigated the effect of BH4 on PAH gene expression in human hepatoma. Our results show that increased BH4 levels result in an enhancement of PAH activity and PAH protein, due to longer turnover rates, while PAH mRNA levels remain unchanged. This was confirmed for mutant PAH proteins (A309V, V388M and Y414C) associated to in vivo BH4 responsiveness, validating previous studies. We can conclude that there is no effect of the cofactor on PAH gene transcription, probably being the chemical chaperone effect of BH4 stabilizing mutant PAH proteins the major underlying mechanism of the response.

Functional and Structural Analysis of Five Mutations Identified in Methylmalonic Aciduria cbIB Type

ATP:cob(I)alamin adenosyltransferase (ATR, E.C.2.5.1.17) converts reduced cob(I)alamin to the adenosylcobalamin cofactor. Mutations in the MMAB gene encoding ATR are responsible for the cblB type methylmalonic aciduria. Here we report the functional analysis of five cblB mutations to determine the underlying molecular basis of the dysfunction. The transcriptional profile along with minigenes analysis revealed that c.584G>A, c.349‐1G>C, and c.290G>A affect the splicing process. Wild‐type ATR and the p.I96T (c.287T>C) and p.R191W (c.571C>T) mutant proteins were expressed in a prokaryote and a eukaryotic expression systems. The p.I96T protein was enzymatically active with a KM for ATP and KD for cob(I)alamin similar to wild‐type enzyme, but exhibited a 40% reduction in specific activity. Both p.I96T and p.R191W mutant proteins are less stable than the wild‐type protein, with increased stability when expressed under permissive folding conditions. Analysis of the oligomeric state of both mutants showed a structural defect for p.I96T and also a significant impact on the amount of recovered mutant protein that was more pronounced for p.R191W that, along with the structural analysis, suggest they might be misfolded. These results could serve as a basis for the implementation of pharmacological therapies aimed at increasing the residual activity of this type of mutations. Hum Mutat 31:1033–1042, 2010.

[An evaluation of glycosylated hemoglobin requesting patterns in a primary care setting: a pilot experience in the Valencian Community (Spain)].

Abstract Objective To assess the pattern of glycosylated hemoglobin (HbA1c) requests by clinicians from eight health care departments by calculating indicators of demand appropriateness. Methods A cross-sectional study of the number of HbA1c requests by primary care clinics in 2008 and 2009. The indicator of demand appropriateness was the proportion of HbA1c values lower than 6.5%. Variables were collected and indicators were automatically calculated. The number of HbA1c measurements that should theoretically have been requested according to known diabetes prevalence data was also calculated. Results A progressive increase was seen in the demand for HbA1c measurements. Approximately 54% of HbA1c values obtained in seven of the eight departments studied were lower than 6.5%. The number of theoretical HbA1c requests that would have been expected based on the known prevalence of diabetes was higher than the number of HbA1c requests in all departments. Conclusion The results appear to suggest that HbA1c requests in the health care departments studied were not always appropriate. HbA1c measurements were probably overused in patients without diabetes and underused in patients with diabetes.