Download Monday March 10th in-class “assignment”: studying SRS and BWS

Monday March 10th in-class “assignment”:
studying SRS and BWS patients as a way to elucidate regulatory mechanisms of imprinted loci
Assigned paper:
Chiesa et al. (2012) The KCNQ1OT1 imprinting control region and non-coding RNA: new
properties derived from the study of Beckwith–Wiedemann syndrome and Silver–Russell
syndrome cases
You are welcome to work on this set of questions collaboratively or individually, and to request help as/if needed.
You will hand in your answers at the end of class (one set of answers per group if you work collaboratively).
1. Compare and contrast the phenotypes of the SRS vs. BWS patients (you will need to look at Table 1). Do
you notice any trends? Knowing that all the patients studied in this article have mutations in the same
imprinted cluster, what could explain the differences in phenotypes?
SRS – Growth limiting.
BWS – overgrowth.
-Trends: BWS and SRS facial characteristics show opposite phenotype.
Other explanations for the differences in phenotypes could be: environmental, or differences in other
areas of the genome.
2. The authors report using OMIM to obtain some information for their research. Take a few minutes to look
up Kcnq1ot1 on OMIM and see what information you get.
-OMIM tells us that this gene is imprinted, and imprinting dysfunction has been associated with both the BWS
and SRS conditions.
- Kcnq1ot1 overlaps with Kcnq1 in antisense orientation
-“ Kcnq1ot1 was associated with imprinting of nearby genes, including Cdkn1c, Kcnq1,
Slc22a18 (602631), and Phlda2 (602131), and also with more distant genes, including Cd81
(186845), Ascl2 (601886), Tspan32 (603853), and Tssc4 (603852)” –OMIM
3. Look at the pedigree in Figure 1.
a) What can immediately be concluded about BWS (even without knowing who inherits the mutant allele
from whom)?
-It is associated with miscarriage
-It is present in both genders
-Not necessarily lethal
-Individuals with the syndrome are fertile
b) II-2 and II-4 both have BWS, and both have one child with BWS and one child without BWS. Briefly
explain how this is possible.
-Affected individuals II-2 and II-4 are both female and both maternally imprint the genes in their gametes.
They may pass on the chromosome with the micro-duplication(causing BWS) or the chromosome without
the micro-duplication(not casuing BWS.) M
4. Briefly describe the mutation detected in the BWS patients and the mutation detected in the SRS patient,
and their respective effects at the molecular, cellular, and organismal levels (use figures 2, 3, and 8, as
well as your answer to Question 1).
NOTE: molecular level = DNA sequence, DNA methylation, gene expression; cellular = proteins present in
the cell, potential effects on the cell; organismal = effects on the entire organism.
-At the molecular level, BWS can be caused by a 160kb inverted duplication involving ICR2 and the 5` most 20kb
region of the Kcnq1ot1. As a result the maternal copy of the ICR2 is hypomethylated because
the duplicated ICR2 is not methylated. Kcnq1ot1 is transcribed usually only from the paternal
chromosome but in the BWS case Kcnq1ot1 is also ectopically transcribed from the maternal
chromosome, repressing CDKN1C in cis. As a result CDKN1C, a cell growth inhibitor, is biallelicly silenced and CDKN1C is present at extremely low levels causing over-growth of the
organism.
-At the molecular level SRS can be caused by a 1.2mb duplication of the entire 11p15.5 cluster including ICR1,
ICR2, and CDKN1C. The duplicated ICR2 when inherited maternally is also methylated causing Kcnq1ot1 is
not transcribed maternally. However since CDKN1C is doubled, and not repressed in cis by
Kcnq1ot1, more CDKN1C protein is present within the cell. CDKN1C repressed growth of the
cell, leading to the phenotype observed, growth restriction of the organism.
5. Explain what the data in Figure 7 show, and how you interpret them.
FIG A – Shows BWS patients have increased interaction Kcnq1ot1 RNA with the region 500 bp
downstream of the CDKN1C transcription start site
From that we can conclude that Kcnq1ot1 from the maternal allele as well as the paternal
allele interact with chromatin in the BWS patient.
FIG B – Shows that the paternal Kcnq1ot1 RNA interacts with chromatin in BWS cases and in
controls, but the maternal Kcnq1ot1 RNA interacts with chromatin much more in BWS cases
than in controls.
FIG C – Shows that in the control only Kcnq1ot1 RNA from one parent is present. But in BWS
cases Kcnq1ot1 RNA from both parents is present as indicated by a SNP that differs between
the two parents.
6. What valuable fundamental information was gained about imprinting control regions through the study of
these patients?
7. Please list any questions and points of confusions relative to imprinting and the regulation of Hox genes (if
nothing much comes to mind, please think about it and post on FB or on the Connect Discussion board).
We will take the time to clear up your questions before starting on X inactivation.
BONUS Q: Do you think the phenotypes of BWS patients 1-3 are representative of BWS? Justify?