Presenter: Dean Danner, PhD
Since 1973, Dr. Danner has been studying mutations that cause MSUD. He is a professor in the Dept. of Genetics and Molecular Medicine at Emory University, Atlanta, Georgia. He sincerely thanked the families for their help and cooperation which makes gene therapy possible. He answered technical questions in a relaxed and easy to understand manner.
1) What do we know about MSUD?
The function within the cell that is not working is the enzyme, branched-chain alpha-keto acid dehydrogenase (BCKD). It breaks down the amino acids in proteins. When it doesn't work, the branched-chain amino acids (BCAA) and their keto acids (BCKA) build to toxic levels in tissues and fluids.
There is no racial or ethnic preference. Dr. Danner has cell lines from China, Japan, Africa, Saudi Arabia and Turkey.
How often does it occur? The incidence is one in 176 in the Mennonite community, 1 in 100,000 in Georgia and 1 in 185,000 in the general population. There is a common mutation in the Mennonite community, but no other common mutations have been found. There was a different mutation in each of the other families he tested.
2) What have we learned in molecular genetics?
BCKD is found in the mitochondria. Every cell of the body has mitochondria except red blood cells. The amino acids have to get into the mitochondria and the BCKD must be available to break down the amino acids to produce energy. The chemistry is very complicated. The enzyme is in active form at only 40% in the kidney cells, 3% in muscle cells and maybe 30% in the brain cells.
3) How do we detect mutations within families?
Dr. Elsas used the breath test at the Symposium in Toronto. It tests the ability to oxidize leucine. Many of those analyses are still being done because it takes a long time.
From blood samples, Dr. Danner transforms white blood cells so they grow in culture. It takes 6 to 12 weeks until they are ready to study. After that he does an enzyme activity and a Western Blot to look for the presence of the E2, E1-alpha or E1-beta protein to see which is missing. (The mutation in MSUD can occur at any of these three subunits of the enzyme complex.) He also looks at the RNA which is produced from those genes and then checks the parent cells to see if they really got this change from Mom and Dad. Using illustrations Dr. Danner briefly explained the complicated tests he uses to find the defective gene in individuals.
The combined information from several laboratories has identified 20 mutations on the E1-alpha subunit, 7 on the E1-beta subunit, and 11 on the E2 subunit. One parent may have a different mutation from the other but in the same subunit.
4) What has this knowledge done for MSUD families?
Since MSUD is now treated with the protein restricted diet, the old clinical classifications are useless. We need to determine the best treatment for a specific type of mutation.
Prenatal monitoring is possible when we know the mutation within that family. Any other pregnancy in that family can be monitored by enzyme assay or DNA analysis. Also other relatives can be tested for the mutation but only at the DNA level.
Thiamine responsive MSUD is difficult to identify and is not well understood. It is not a deficiency of vitamin B1 but requires pharmaceutical doses of 100 to 1000 times the normal amount of thiamine (B1). Some clinics give it to all the MSUD patients because of the possibility it might help and it is not dangerous. Patients proven to be thiamine responsive have an E2 mutation.
Two other things we've learned are that newborn screening is very important in averting the consequences of MSUD, and that the diet is necessary for life.
5) What questions remain? Are there new questions?
We want to identify all the mutations. The technology is there but we need hands and dollars to do it. As a geneticist, I want to know why these changes cause a dysfunction so better management can be provided. We want to relate genotype to therapy and also identify which is the most important tissue for BCKD function in humans, so we know which tissue to target for gene replacement therapy.
The new problem is maternal MSUD. Being on diet, young women with MSUD are capable of reproduction. How do they nurture a fetus who needs a high protein diet to develop? It is necessary to identify when, in the embryonic development, the BCKD is turned on in the fetus. There is a chance the fetus can help the MSUD mom metabolize the BCAAs during pregnancy so she does not get sick. Only one pregnancy of a woman with MSUD has been reported in medical literature and she had a normal baby.
6) Future Goals:
Enzyme replacement is probably not possible, but gene replacement or organ transplant (like kidney) is a possibility. However, after identifying the best tissue, how do we deliver the corrected gene and how often will replacement be necessary? There is hope that gene replacement could be possible - it is possible now in the lab - but it won't happen tomorrow.
Dr. Danner is developing an MSUD mouse. An animal model is needed to study the neurological complications in gene replacement therapy and to detect during pregnancy when the fetus can begin to handle the leucine overload so the MSUD mother can increase dietary protein.
Enzyme activity is no gauge of phenotype or how well protein is tolerated. One carrier father has 2% activity level and is perfectly normal. Both carrier parents of two children severely affected have 100% enzyme activity. A child with severe clinical signs of MSUD can have activity level at 20 to 30%. Another child can be doing very well with less than 1% enzyme activity. We don't know that much about the enzyme activity. Other genes interact and persons respond differently.
Are persons with MSUD missing something by not getting energy from protein? Biochemically it takes more effort to make energy from protein than from carbohydrates. So most energy comes from carbohydrates, except when they are lacking, then it comes from protein. Muscles may fatigue more quickly because they cannot activate the enzyme to get rid of toxic products. Muscles have only 1 to 2% of the active form of the enzyme.
In comparison to PKU research, we are not as advanced. PKU involves one organ, the liver, and 1 gene. MSUD involves 3 genes, and we still do not know the organ to target.
Questions were asked of Dr. Danner about how his research was funded. All of his research funds now come from NIH and it is getting tougher to get funding. Companies will not fund research for such a small group of patients as MSUD. We can write to our Senators and Congressmen to continue and increase the funding for NIH. It is urgent that funding continue to ensure further research efforts. Dr. Danner would gladly provide anything needed to raise funds for his research.