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Perspective interview - Niels Gregersen

Perspective interview - Niels Gregersen

PKU perspective - Professor Niels Gregersen

Niels Gregersen, Professor of Molecular Medicine at University of Aarhus in Denmark, believes there is a great potential for molecular biochemistry to lead to a fuller understanding of IEMs and their treatment.

Principally concerned with mitochondrial dysfunction disorders, such as beta-oxidation deficiencies, he spoke to the delegates at the 5th EPG Symposium about the lessons molecular research in PKU can learn from other diseases.

His comments were made against a background of concern that early hopes that a better characterization of genotype would lead to full understanding of the observed phenotype and thence to a plethora of new treatments might have been premature.

Riding the research roller-coaster

Prof Gregersen is an experienced rider of the roller-coaster of discovery, anticipation and, occasionally, disappointment that accompanies the introduction of new techniques that allow new avenues for investigation and the prospect of new treatments.

He told us ‘I first became involved with the study of IEMs in the middle of the 70s. It was an exciting time. I was employed as a chemist to take care of a big mass spectrometer which was used as a tool for diagnostics of organic acidurias. It was exciting because we participated in finding a number of mitochondrial beta-oxidation deficiencies which have characteristic urinary profiles.

‘Before the “genetic era”, the late ’80s, the beginning of the ’90s, the diagnostic tests involved looking in the urine, looking in the blood and measuring enzymes or enzyme pathways’ the professor told us.

The gene genie

‘But that all changed when the genes for MCAD and the other acyl-coenzymeA dehydrogenases in mitochondrial beta-oxidation were discovered in the middle of the ’90s. Then, these could be used for diagnoses. At that time, we thought that when we knew the gene defect then we would also know the disease: we would know the prognosis straight away, how serious it is and how it should be treated. But it’s turned out to be wrong. I think that the fatty-acid oxidation deficiencies were perhaps model diseases for this, in that the span in the prognosis, from seriousness to mildness for the same defects is very broad. It was first seen for the MCAD defect where there is a prevalent mutation (80% of the patients are homozygous for it), but despite having the defect, the patients could be asymptomatic or they could die in an attack.

‘So we realized that it was necessary to look in the cells; to look how the proteins were produced from the gene defect.’

This then, illustrates a downward swoop of the research roller-coaster – when a new discovery fails to herald a universal treatment. Was this demoralizing, we wondered.

Prof Gregersen replied ‘I have been so long in the field and in molecular medicine, so I would say people have been overly optimistic many times and, yes, there have been disappointments. You know the case of gene therapy in the beginning of the 90s. The view was “Now we can treat many diseases, also the genetic diseases, by gene therapy”. But how many diseases are treated by gene therapy today? I don’t think there are any.’

No quick fix

With the relationship between genotype and phenotype clearly not suitable as a simple diagnostic tool, is there, we wondered, any value in better understanding the relationship.

The professor told us ‘It’s important to investigate the relationship between genotype and phenotype, because it may yet lead to new treatments, particularly when we are looking at missense mutations, which can give rise to the protein, either with a distorted conformation, often leading to “mild” forms of the condition, or to folding intermediates which are very rapidly degraded and can effectively be a null mutation.

‘There is a continuum, where we have various forms of the protein which may have some residual activity. However, these proteins may also have a little distorted conformation. This can give rise to disturbances, toxicity in the cell. One of those disturbances is oxidative stress.’

Defeated by complexity?

There is a huge number of mutations capable of causing some form of phenylketonuria. Is it worthwhile, we asked, to search for genotypic markers that would allow us to predict a patient’s suitability for cofactor therapy.

Prof Gregersen: ‘With PKU you can read the responsiveness for the cofactor and for those who respond well to cofactor treatment. I think here you can say something about the prognosis. There is at least one case in the fatty acid beta oxidation: riboflavin responsive multiple acyl-CoA dehydrogenation deficiency, (glutaric acidemia, type 2) GA-2. In this disease we have found that some patients are riboflavin responsive and, if they are, then we can predict what the mutation is doing. If we have the knowledge in the database that a given mutation is present in a patient who is responsive, then we can apply that knowledge when looking at the next patient with the mutation.

This raises the question of where the priority should be for further molecular biochemistry research.

‘We need to look carefully at the effects of enhancing the misfolded proteins, for example with chemical chaperones’ said the professor. ‘In PKU, the cofactor works as a chemical chaperone and it can enhance the activity of the enzyme but there are other chaperones that could potentially do this job, possibly better. People are working with these chemicals but we have to be cautious because we have seen in a number of cases in other diseases that the chaperones can increase oxidative stress and the situation can arise where a “mild” case of a disease can give rise to greater oxidative stress than the more serious form.

‘We need to build a better picture of how this happens but perhaps, due to the distorted conformation of the protein, oxygen can go into the structure, leading to the production of super-oxides which of course are very toxic.

‘If it turns out (and we really don’t know) that chemical chaperones create molecules which are distorted, then we gain something but we lose something. We really have to find out what happens in the cells when you upregulate the amount of misfolded proteins.’

How relevant are concerns over oxidative stress to PKU, we wondered. ‘I think that it has turned out that in PKU there is oxidative stress in the cells. This stress has a lot of effects but these are affected by what exactly the species are and where they are produced’ Prof Gregersen said.

‘In the mitochondrial beta-oxidation deficiencies we are trying to find out where it comes from and we are looking for ways to treat it with anti-oxidant systems, with chemicals which enhance the endogenous defense systems. This is work that could be directly relevant to PKU.’

Early treatments… new treatments?

‘But I think the real challenge is to find out what is going on in the cells. For example these cells are creating oxidative stress and this can cause problems early in life, even during development of the fetus, leading to neurological deficiency. We see this in the beta-oxidation deficiencies. If it were possible to treat this very early, by somehow elevating the endogenous defence system, then this damage could be avoided. This is a whole new area of treatment.’

Once again, we see that the simple solution gives way to an appreciation that there is additional complexity involved. Is there anything we are sure of? ‘We have experienced in recent years that the better we get to look into the cells and into the genetics with new, large scale technologies, like next generation sequencing and with proteomics, protein profiling, we can certainly see that we create more questions than we made answers.’

The professor acknowledges ‘We have the knowledge that the misfolded proteins from missense mutations are doing something but we don’t really know what is starting the toxicity. What is going on within the cell, which pathways are affected? Although we know very little about this, if we can come a little further we might well be able to suggest new treatment compounds or regimes.’

Where next?

So, we asked ‘Where next? What will be the next upward move of the roller-coaster and what should our priorities be for further research?’

 ‘We need to continue, in our case, with protein profiling and looking at the different survival and death mechanisms in the cells. We have to study how they are distorted in the patients’ cells and see how we can manipulate that balance in order to try to suggest treatment, for example, with chaperones or anti-oxidant systems.’