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IVE commentary

In Vitro Expression analysis: an update and brief commentary on the data
Paula J. Waters (IVE curator) - June 17th 2002.

New data

Data concerning in vitro expression analysis of naturally-occurring mutations in the human PAH gene have recently been extensively updated online, to include a large number of findings published over the last year. As of June 2002, the searchable table entitled "in vitro expression (human)" summarises analysis of 81 different PAH mutations, the vast majority of which are missense alleles. There are currently a total of 225 individual records of analysis, reflecting the fact that frequently a given mutation may have been studied in multiple expression systems and/or by different research groups.

Rationales for expression analysis

In vitro expression analysis has been driven by three linked rationales:

  1. To confirm that a "disease-associated" mutation is truly pathogenic (rather than a coincident benign change).
  2. To assess the severity of a mutation's impact (and aid the correlation of PAH genotype with hyperphenylalaninemia phenotype).
  3. To help understand how a mutation exerts its deleterious effects (elucidate the molecular mechanisms involved).

While all three continue to be valuable lines of investigation, over recent years the emphasis has shifted heavily towards the third of these goals. In the process, our understanding of the mechanisms has reshaped our thinking on how best to identify, and attempt to quantitate, a mutation's pathogenicity.

Expression systems

Various expression systems have been applied to study PAH mutations, by placing the mutant (and wild-type) cDNAs into plasmid vectors and introducing these into host cells. The systems include:

  1. Transiently transfected human or other mammalian host cells - being probably the closest available approximation to the in vivo situation.
  2. Transformed bacterial (E. coli) cells - where high protein production often permits enzyme purification and thus its detailed characterisation.
  3. in-vitro transcription-translation systems - allowing synthesis of radiolabelled PAH, the fate of which can be followed over time.
  4. Two-hybrid systems (in yeast or mammalian cells) - allowing the study of interactions between PAH monomers.

Often the study of a single mutation in multiple complementary expression systems allows the piecing together of a coherent picture of the mutation's mode of action and the severity of its impact on the function of the expressed protein.

(It must however be noted that these classic IVE expression systems only examine the effects of nucleotide changes in the context of cDNA, not of genomic DNA. Therefore any effects of apparent missense or silent substitutions upon RNA splicing in vivo will not be observed using these methods).

Parameters included in the IVE(human) table in PAHdb

Each record in the IVE (human) table has twelve listed parameters, as follows:

  1. Mut. name: The mutation name, using the trivial (amino acid) nomenclature.
  2. IVEhID: A unique identifier for the individual record of expression analysis.
  3. Plasmid: The plasmid vector used.
  4. Host: The host cell type used.
  5. PAH Enzyme: Refers to PAH enzyme activity measured in cell lysates (as a percentage of the wild-type value).
  6. PAH Immuno: Refers to the amount of (immunoreactive) PAH protein present in cell lysates (again, as a percentage of the wild-type value).  Frequently a decrease in cellular PAH enzyme activity is in fact accounted for simply by a decrease in the PAH protein level.
  7. PAH mRNA: Refers to PAH mRNA levels in cell lysates (as a percentage of the wildtype value).In practice this has not yet been found to be decreased for any mutation studied in these systems, thus is not the explanation for observed decreases in PAH protein levels. Note: Columns 5,6 and 7 (Pah Enzyme, PAH Immuno, PAH mRNA) were inadvertently omitted from the print versions of the IVE(human) tables included in recent Newsletters; they are however present and correct in the data viewable online.
  8. Spec. act.: Refers to enzyme specific activity, i.e. the ratio of PAH activity / PAH protein. Where this is less than 100% relative to wild-type, it is highlighted; this finding is especially pertinent in mammalian cell systems, since it may provide a pointer to mutations which affect PAH enzyme activity independently of an effect on cellular PAH protein levels.
  9. Note: Refers to methodological details which have a bearing on interpretation of the observed results.
  10. Addit. Observ.: Includes additional observations on the effects of a mutation, including insights into mechanisms.
  11. Date entered: The date of entry online.
  12. Reference: The full citation for the original research; usually a published paper, occasionally the record of a direct submission to the PAH Consortium.

As the sophistication and complexity of IVE data has tended to increase, the conversion of literature data to a simple format compatible with their concise summary in tabular form has become progressively more challenging! The interested visitor to the PAHdb IVE tables is therefore always referred back to the primary citation for further details.

Take-home messages from IVE (human) data

Accumulated data from various researchers together illustrates some important general themes:

  1. A common type of mechanism now appears to be implicated in the pathogenicity of numerous PAH missense mutations. Such mutations promote misfolding of the PAH monomer and oppose correct assembly of monomers into the native tetrameric enzyme. The resulting structural aberrations trigger cellular defences, provoking accelerated degradation of the abnormal protein by proteases. The intracellular steady-state levels of the mutant PAH protein are therefore reduced, leading to an overall decrease in phenylalanine hydroxylation within cells, and thus to hyperphenylalaninemia.
  2. The effects of such mutations may well be modulated in vivo by modification of the cellular handling (folding, assembly and degradation) of the mutant enzyme. This has major implications, first for our understanding of genotype-phenotype correlations (and their imperfections), secondly for possibilities of novel therapeutic approaches.
  3. Certain PAH mutations have more direct adverse effects on enzyme activity - for instance, by affecting binding of substrate or cofactor to the enzyme's catalytic or regulatory sites, or by affecting residues with other critical roles in the catalytic process.
  4. The pathogenicity of some PAH missense mutations appears to reflect a combination of different mechanisms acting in concert. Dissecting out their relative in vivo significance is an ongoing challenge.

Artificial mutations and rat PAH mutations

PAHdb also contains two smaller tables of data related to in vitro expression analysis. (These are pre-queried, rather than searchable by individual mutation). The "in vitro expression (artificial)" table documents artificially-created mutations in the human PAH gene (not corresponding to naturally-occurring mutations). The "in vitro expression (rat)" table documents artificially-created mutations in the rat PAH gene (a few of which do correspond to natural mutations in the homologous human gene). For both of these datasets, the rationale for the studies is slightly different than for studies on naturally-occurring mutations - where the emphasis is on understanding the direct relevance of those mutations to the associated disease phenotypes observed in patients. The artificial mutations (both human and rat) instead have been designed specifically to examine structure / function relationships in the wild-type enzyme, by targeting residues hypothesised to have key functional roles. Analysis accordingly has focussed on the properties of the isolated mutant protein (usually purified from expression in E. coli), rather than on the fate of the protein in its "in vivo" cellular context. Design of these two IVE tables therefore differs somewhat from that of the primary IVE (human) table.

The IVE (artificial) and IVE (rat) tables should also be updated online by the end of July 2002.

Copyright 2003 DeBelle Laboratory - Created [2002.10.17.208076] - Updated [2009.08.31]