Download Entry slip BL 610B Congenital Heart Disease paper names _ Smith

Entry slip BL 610B Congenital Heart Disease paper names __________________
Smith, K.A. et al. (2009) Dominant-negative ALK2 allele associates with congenital heart defects. Circulation
119:3062-3069.
Authors: where are these authors located?
Summary/ Background. What is an atrioventricular septum (not described)?
Problems in AVS development lead to heart disease, but what genes are involved?
Congenital heart defects (CHD) can arise from many causes in development of primitive heart, but what are
some signaling pathways that are implicated?
Therefore, the authors used a candidate gene approach: they sequenced coding regions of 32 candidate genes
that might be involved, comparing patients with CHD history and normal controls. What are cSNPs, and how
can these help in genetic disease association studies?
They found some novel cSNPs, in total ____ genetic lesions in _____ different genes predicted to affect protein
function.
They focused on the ALK2 gene. What does this gene do, and through what signaling pathway does it work?
** Presenters, please look up information (as from developmental biology) about how ALK2 and this signaling
pathway work; where is ALK2 protein normally located, for example
..
They tested the WT and altered ALK2 genes for function in new model organism,? _____________________
What is the scientific name of this organism? (not given)___________________________
What was their overall conclusion in the summary?
What is a dominant-negative allele?
Methods: how did they get the DNA and determine the sequences of 32 candidate genes?
How did they determine which patients had CHD, versus normal controls?
Results: How many patients with CHD were analyzed? ______
What did they find for the 32 genes analyzed?
Some of the cSNPs had been found in normal people and were not interesting, namely ________
Other cSNPs were unique to the patients, namely ________. Computer programs predicted _____ of these
cSNPs would affect protein function, of which _____ were patient-specific (the Table).
Table: How many different genes were identified by these mutations affecting function? _____
Figure 1. They focused on the ALK2 gene, and had found 3 SNPs. Why did they only focus on L343P for the
rest of the paper?
Supplemental Figure 1. What did the pedigree analysis tell them about the cSNPs in ALK2?
Fig. 2. They tested the activity of the WT ALK2 and various mutant proteins by constructing plasmids carrying
the different genes. They introduced these genes into bovine endothelial cells in tissue culture and tested the
activation of a Luciferase reporter with a BMP-responsive element in the promoter. They tested each ALK2
gene alone, or after inducing BMP6 protein. Controls were no ALK2 (vector), a known Dominant-negative
mutation and CA, constitutively active variant.
What did they find for the expression of the luciferase reporter in response to the different ALK2s +/- BMP?
What did they find for kinase activity of the different variants?
Fig. 3. Why did they introduce the different ALK2 constructs into the zebrafish embryos? How did they
introduce the mutant sequences, and what did they observe and measure?
What are the conclusions from Fig. 3?
Why did they also do a Western blot (Fig. 3e,f)?
Fig. 4. What did they find for the hearts of the zebrafish? And the effect of ALK2 L343P mutation?
Overall conclusions, questions?