Chiesa et al. reading
What can you deduce about BWS from the pedigree shown in the article?
BWS is passed down by a maternal dominant mutation/allele that is not x-linked.
from the pedigree, female affected patients have decreased fitness from miscarriages.
1. Please list the names of all the group members who participated:
Rosalie Ho
Giulio Sucar Pregnolato
2. General question: Based on the article, what are the known causes of SRS and BWS? Which of these causes are genetic, and which are epigenetic?
Both are genetic defects due to duplication of the locus; these genes are also imprinted, so epigenetic factors determine phenotypes in each patient or carrier.
3. Consult Figure 8 to remind yourself of what/where ICR2 is. Then consider the data shown in Figure 5B.
A. What did the authors do?
The authors ran a methylation sequencing of the ICR2 region of multiple individuals, differentiating paternal and maternal alleles.
B. What were the results?
Control individuals had all paternal alleles unmethylated, and maternal alleles methylated (in general).
I-4 had a duplication of the region, but the normal methylation pattern of paternal unmethylated and maternal methylated genes.
II-4 and III-6 had duplications of maternal genes, where half of the genes were methylated and the other half was not.
C. What do the data tell us about the methylation state of the ICR2 region in individuals I-4, II-4 and III-6?
I-4 has the expected pattern of paternal imprinting, whereas II-4 and III-6 have an unexpected pattern of having one duplicate methylated and the other unmethylated.
4. Notice how I-4, II-4 and III-6 all have the same number and methylation pattern of ICR2 ‘loci’. How can their difference in terms of having vs. not having BWS be explained?
II-4 and III-6, patients of BWS, have an insertion of KCNQ1OT1 inside a region usually inactivated by maternal ICR2 methylation, which leads to a second expressed locus of KCNQ1OT1. In contrast, a BWS carrier I-4 has this duplication of KCNQ1OT1 inside the normal KCNQ1OT1 locus, in the hypomethylated paternal chromosome. Thus, even if the duplicate is expressed, it would block the expression of the regular copy, which creates a regular phenotype.
5. Consider Figures 7A and 7B.
A. What did the authors do?
The authors ran ChRIP seq to confirm the presence of maternal or paternal alleles, then ran a q-PCR to evaluate the expression of KCNQ10T1 in maternal and paternal alleles for each BWS patient considered.
B. What were the results?
BWS patients all demonstrate an overexpression of KCNQ10T1. Paternal alleles of KCNQ10T1 in BWS patients seem to be expressed at similar levels of healthy controls, evidenced by the statistically insignificant differences between the reported values. Furthermore, maternal alleles of KCNQ10T1 in BWS patients seem to be overexpressed, as evidenced by statistically significant differences in all patients tested.
C. What can we directly conclude?
We can conclude that maternal alleles of KCNQ10T1 are overtranscribed in BWS patients, indicating that their overexpression is a cause of the syndrome.
D. Provide an interpretation for the results.
Due to methylation patterns of this region, a paternal allele with the duplication is harmless, since it duplicates the KCNQ10T1 gene promoter inside its own normal locus, which prevents overexpression. However, the syndrome is expressed when the gene is inherited maternally because the duplicate is inside a region that is normally inactivated by methylation, so when the KCNQ10T1 duplicate is transcribed, it overexpresses the gene, thus leading to the observed phenotypes.
6. One of the authors’ hypothesis is that many of the physical phenotypes associates with the BSW patients are due to reduced expression of CDKN1C. Propose two possible mechanisms that would explain how the duplication of ICR2 in these patients causes a reduction in the expression of CDKN1C. Based on what you know about imprinted loci, which of the two mechanisms is most likely and why?
One possible mechanism is that while KCNQ10T1 is transcribed, it covers the coding region of CDKN1C, which is consequentially not expressed.
Another model is that CDKN1C is repressed by KCNQ10T1 RNA.
Since an overexpression of maternal KCNQ10T1 seems to cause a repression of maternal CDKN1C, but no alteration in paternal expression of this region, the first mechanism is more likely.
7. Time permitting: what do you think is the value of clinical studies? Who are they valuable for?
They are essential in the development of applications for all the biological tools and models being developed in research. Were it not for clinical studies, the return on investment of biological research would be too small to justify the effort and capital.