In this post I’ll be looking at some of the different tools for diagnosing ASPA, or more specifically Canavan disease. The disease state cause by an ASPA variant. I’ll look at the different variants, different test available for Canavan disease, and possible restriction sites that could be used to check for ASPA variants.
Currently for ASPA there are twenty variants listed on ClinVar. Of the twenty total seventeen are listed as likely disease causing and three of them have uncertain significance. There definitely appears to be a large gap in clinical research into the different variants of ASPA. Of the twenty on ClinVar not a single variant is recognized by a professional society or an expert panel. All twenty are from a single submitter. This is semi surprising to me because of the nature of Canavan disease. Tragically once you are diagnosed with Canavan disease at birth there is no current treatment and life expectancy is very short, but parents can scan for the variant in their genome to know the risk of their child receiving Canavan disease. This is a very rare disease that only affects ~1/10,000 Ashenkazi Jews and is unknown in the general population which could explain the lack of research.
Currently there are quite a few tests to diagnose Canavan disease. According to NCBI Gene Testing reference there are fifty-four different labs around the world that will do testing for Canavan disease. Even though there are quite a few different laboratories that can perform Canavan disease screening most follow the same type of testing. All current testing involves checking for the disease causing variants or a deletion/duplication analysis. Some of the techniques are Multiplex Ligation-dependent Probe Amplification, Sanger, next gen sequencing, SNP detection, and microarray.
Another methods for diagnosing some genetic diseases is using restriction enzymes and running an agarose gel. This relies on there being a change to an enzyme cutting site caused from the variant in question. I took a look at the A854C variant in ASPA. This variant actually turns out to be a very good candidate for enzyme restriction analysis. There are three six cutter enzyme sites (NotI, EagI, and BsmI) that were created by the variant as you can see in the picture below. All of them are also unique so in my opinion any of these enzyme could be used for diagnosis.
I did run into a strange issue with GeneQuest when I was attempting to run an agarose gel simulations. When I added the enzymes mentioned above to GeneQuest the program recognized the new enzymes in the variant but when I ran the agarose simulation the gel was showing the same as the wild type. I tried the simulation with all three enzymes but the results were the same for all three. There was an additional enzyme site created (AciI) and this simulation can be seen below. In the laboratory this wouldn’t be particularly useful because of how small the last bands is. Further laboratory investigation would be needed to find out why none of the other sites were cutting the sequence in the simulation. The band sizes created by AciI are ~4, ~45, and ~1396.
michelconn 