Seminar paper 2: Gaucher disease Flashcards
What is Gaucher disease?
Autosomal recessive lysosomal storage disorder characterized by an acid beta-glucosidase gene deficiency
What was the purpose of this paper?
Determine the underlying genetic cause of a 43-year-old male’s Gaucher disease
What was the main finding of this paper?
This patient was a compound heterozygote, with one previously uncharacterized deletion allele
What mutant allele was first identified in the patient?
G1226A/N370S - a common missense mutation in Gaucher patients
Why did the authors suspect there was another mutant allele that their PCR wasn’t detecting besides the N370S allele?
They weren’t getting any WT sequences, only the N370S. And this is a recessive condition, so would need two copies to show the disease phenotype. They were also getting abnormal restriction fragment sizes
Why did the authors suspect it was the 5’ end of the gene was deleted in the suspected deletion allele? How did they determine this?
No initial PCR amplification. When they used a primer that bound in intron 2, they found another novel base substitution that resulted in a silent mutation (P32P). Then when they tried to amplify using primers binding in the 5’ UTR, there was nothing amplified of the allele with P32P, but the N370S allele was amplified
Where any of the primers listed in that supplementary document used to generate the Southern blot probe?
Nope. All the possible PCR fragments with the right sequences are all too small or too large
What fragments were seen in the WT controls for the Southern blot after digestion with XbaI?
A large 17 kb fragment corresponding to the XbaI site in the nearby pseudogene, and the 4 kb fragment corresponding to XbaI sites within the GBA1 gene
What fragments were seen in the patient for the Southern blot after digestion with XbaI?
He also had the 17kb pseudogene fragment and the 4 kb gene fragment from the N370S allele, but had a third 12 kb fragment from the deletion allele. The deletion had removed one XbaI site and 4 kb of DNA
How would the deletion in this patient have been generated?
Unequal crossing over between the Alu sequences in the GBA1 gene to generate a deletion and a duplication
What is the maximum size of a PCR fragment that can be reliably generated?
About 3000 bp
What was the second way the authors showed the deletion allele was present in the patient?
PCR amplication of the gene from the 5’ UTR to the 4th intron. The WT fragment is too large for PCR to amplify, but the deletion can be. And this band was in the patient and not in the controls
Why did the authors think the deletion allele wouldn’t be expressed?
A lot of important sequences are deleted
Was the deletion allele expressed? How did the authors determine that?
Nope. The authors used RT-qPCR using primers from exons 3 to 9, that would bind with or without the deletion. They found the N370S allele, but didn’t find P32P, showing that the deletion isn’t transcribed
