1. & Beckwith-Wiedemann Syndrome
p57
April 2nd 2013
& Beckwith-Wiedemann Syndrome
Introduce myself and my topic of discussion…need to figure out how to correctly pronounce the disease of interest (check online)…
Brandon Ballance

2. Beckwith-Wiedemann Syndrome
Beckwith-Wiedemann syndrome is an inheritable genetic overgrowth disorder in children. 1:~14,000 children (worldwide) develop the disease (rare). The most common features of BWS include 90th percentile body weight (so basically, very large babies!), macroglossia (large tongue), abdominal wall defects (umbilical hernia), and most importantly (or at least in terms of relevance to our course), an increased risk for childhood tumors (7.5%; these include: kidney, adrenal gland, liver, etc.). There is no cure for BWS as of today, however, if detected through embryonic screening, children can be closely monitored for cancer development. So, as I said, the disorder is thought to be primarily genetic, the question now is: what genes are involved?
Risk of developing cancer:
7.5%

3. Genes Associated with BWS
CDKN1C
The disorder is thought to be caused by the incorrect regulation of a set of imprinted genes found on Chromosome 11 at location p15.5 (which is on the short arm of the chromosome), specifically, it seems to be primarily caused by mutations resulting in a non-functioning CDKN1C (the gene encoding for the protein p57). Yet, before we go into the specifics of the protein itself, it is important to understand the genetic imprinting and how it relates to CDKN1C and BWS.

4. Only MATERNAL allele expressed!
Genetic Imprinting
1 in 13,700
Children
Only MATERNAL allele expressed!
Generally speaking, gene imprinting is when two alleles are not expressed at the same level in a given cell. This is accomplished by effectively knocking-out the function of one of the parental alleles through methylation (the addition of methyl groups to the gene itself or surrounding regulating genes). In the case of CDKN1C, the gene thought to be important in BWS, the allele from one’s father is methylated and hence, inactivated. Due to this, cells have only one working copy of the gene (the maternal copy). As I said in the previous slide, the loss of CDKN1C function appears to lead to BWS. So, since there is only one functioning allele (the mother’s), only that one allele needs to be knocked out in order for CDKN1C to no longer be expressed and for an individual to have BWS. It turns out that this is indeed the case and we can see it very clearly in family pedigrees of BWS patients in the next slide…
CH3
CH3

5. Familial Inheritance Pattern
Mutated maternal allele = BWS
Mutated paternal allele = CARRIER
UPD
As you can see in these pedigrees, only those individuals who inherit the mutated allele from their mother can actually develop BWS. If an individual inherits a mutation that affects the CDKN1C gene from their father (assuming they get a normal allele from their mother) they will only be carriers for BWS. Although this is the general pattern for inheritance, there are some instances where uniparental disomy (UPD) plays a role. This is a mistake in which some cells have two alleles from one parent instead of both. As you could assume, if both alleles come from the father, they both will be methylated, no CDKN1C will be transcribed, and the individual will likely develop BWS. You can see this problem in BWS Family C above. So now that you know about the gene CDKN1C, we can go back and discuss its encoded protein p57 (sometimes called KIP2).

6. What Does the Protein Do?
CDKN1C
p57 (KIP2)
G1 Cyclin-CDK Complexes
CDKN1C’s encoded protein (p57) is an inhibitory protein that acts on G1 Cyclin-CDK Complexes. As we learned Cyclin-CDK complexes are necessary to continue through the various checkpoints of the cell cycle. So, you can infer that if p57 inhibits these complexes, it must be a tumor suppressor candidate. This inhibitory property of p57 is shown in the western blot assay on the right. Here CDKNIC was made sensitive to doxycycline (an antibiotic). When the antibiotic was present, p57 was transcribed/translated at a higher rate…hence, the darker band (showing more protein) in the second column for p57. The other proteins become lighter because they are being inhibited by p57 when it is expressed at higher levels. Now you know what it does in cell cultures, what does it do in an actual model organism?
p57
= TUMOR SUPPRESSOR

7. Model Organism: Knockout Mice
As we know, if you want to know what a protein does in an organism all you have to do is make mutants! In this experiment, researchers made homozygous mutants. They did this because they wanted to make sure absolutely no p57 was expressed. You can see in the image that null p57 mice had many of the traits observed in children with CWS…for instance, they saw that null mice had higher average weight than WT mice. This feature can also be seen in the graph on the right were mice clearly are heavier if they express no p57. Although they observed many similarities, they saw absolutely no neoplastic (tumor) development in any of the null mice! I looked through a variety of other experiments and no other researchers were able to induce the generation of tumors by simply knocking out p57 expression. Researchers hypothesized that they were not seeing tumor development because mice were simply not living long enough. Hence, in order to have the mice live longer and hopefully develop cancer, researchers grafted organs from KO mice and put them into nude mice, low and behold, cancer developed in those organs within the nude mice. This can be seen on the following slide.
NO tumors developed

8. Grafted Prostates from Null Mice
Tumors!
Although null mice did not develop cancer on their own, this experiment shows that their grafted organs (in this case a prostate) would develop cancer in put into a nude mice (as you can see in the top image). These researchers thought this was the case because the null mice on their own simply did not live long enough (most only lived 2 wks.) to develop the disease. This experiment also shows another interesting feature of cancers caused by p57 KO. As was the case in the previous image (point to the western blot shown before). This experiment made the CDKN1C gene sensitive to doxycycline. As you can see, when doxycycline was given to the mice, p57 expression increased, and their prostate tumors became much less invasive. The researchers speculated that this may hold great value for future drug development for such disorders as BWS (since there are currently no drugs for treatment available). Thank you very much, do you all have any questions?

9. Works Cited:
"Beckwith-Wiedemann Syndrome." Humpath.com. N.p., 17 Sept Web. < Hatada, I., Mukai, T. Genomic imprinting of p57(KIP2), a cyclin-dependent kinase inhibitor, in mouse. Nature Genet. 11: , Jin RJ, et al. Down-regulation of p57(Kip2) induces prostate cancer in the mouse. Cancer Res. 2008;68: Lee, M.-H., Reynisdottir, I., Massague, J. Cloning of p57(KIP2), a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution. Genes Dev. 9: , Pateras IS, Apostolopoulou K, Niforou K, Kotsinas A, Gorgoulis VG. p57KIP2: “Kip”ing the cell under control. Mol. Cancer Res. 2009;7: Pilu, Gianluigi. "The Global Library of Women’s Medicine." Ultrasound Atlas. The International Society of Ultrasound in Obstetrics & Gynecology, n.d. Web. < Sparago A., Russo S., Cerrato F., Ferraiuolo S., Castorina P., Selicorni A., Schwienbacher C., Negrini M., Ferrero G.B., Silengo M.C., et al. Mechanisms causing imprinting defects in familial Beckwith–Wiedemann syndrome with Wilms’ tumour. Hum. Mol. Genet. 2007;16: Tunster SJ, Van De Pette M, John RM. Fetal overgrowth in the Cdkn1c mouse model of Beckwith-Wiedemann syndrome. DMM Disease Models and Mechanisms. 2011;4(6):814–821. "What Is Beckwith Wiedemann Syndrom?" Assisting Families and Individuals Affected with BWS. Beckwith Wiedemann Children's Assn of NZ (Inc), n.d. Web. <
Works cited page, don’t think I have to do anything special here…