1. Based on the article, what are the known causes of SRS and BWS? Which of these causes are genetic, and which are epigenetic?

The known causes of SRS and BWS all involve defects in the 11p15.5 imprinted gene cluster, which contains two independent domains that are each under control of a different imprinting control region (ICR):

The genetic causes:

• Mutation of the DNA sequence of the cell growth inhibitor CDKN1C (5% of BWS cases)
• Loss-of-function mutation in a trans-acting factor
• Uniparental disomy
• Chromosomal abnormalities – maternal deletions, paternal duplications, balanced maternal translocations (BWS), maternal duplications (SRS)

The epigenetic causes are:

• Loss of DNA methylation on the maternal allele of ICR2
• DNA methylation defects at ICR1, associated with changes in IGF2-H19 expression

2. What do the data in Figure 5B show, and what do they tell us about the methylation state of the ICR2 region in individuals I-4, II-4 and III-6?

Figure 5B shows the methylation status of the ICR2 region, which is located within the imprinted gene cluster at 11p15.5, in three individuals in the BWS family. Two of these individuals carry the BWS phenotype (II-4 and III-6), while I-4 is normal phenotype. Individuals II-4 and III-6 have the maternal 160 kb duplication that was identified in the paper. The data show that I-4, II-4, and III-6 have hypomethylated ICR2 alleles relative to an unrelated control. The overall methylation pattern is quite similar across the three cases, but I-4 has the maternal allele almost entirely methylated, while the duplicated maternal allele is only partially methylated in II-4 and III-6.

3. 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?

Individuals II-4 and III-6 have the BWS phenotype, while I-4 does not. This difference can be explained based on the origin of the 160 kb duplication in ICR2. I-4 has a duplicated paternal allele, while II-4 and III-6 have duplicated maternal alleles. In both of the BWS patients, the maternal allele is only partially methylated, which indicates disruption of the imprinted methylation process in these individuals. In I-4, however, the maternal allele is still almost entirely methylated and the paternal allele is unmethylated. This difference suggests that the 160 kb duplication alters the imprinted methylation of ICR2 only when maternally transmitted, and that this is associated with the presence of the BWS phenotype.

4. Explain what Figure 7B shows and how you interpret the data.

This figure shows the interaction between the chromatin and the KCNQ1OT1 RNA transcript in four different cases: an unaffected control, two individuals with BWS from the analyzed pedigree (II-4 and III-6), and an individual with BWS that has ICR2 hypomethylation but no 160 kb microduplication. The data comes from chromatin RNA immunoprecipitation (ChRIP), a technique that precipitates RNA that is bound to specific regions of the chromatin. By synthesizing cDNA from the precipitated RNA transcripts and subsequently performing PCR, the authors were able to determine whether KCN1OT1 transcripts interact directly with the chromatin or exert their silencing through some other mechanism.

The data shows that the KCNQ1OT1 transcript is significantly enriched in the BWS patients relative to control (Fig 7A). The individual alleles were then analysed in Figure 7B, and the data show that the BWS maternal alleles’ interaction with chromatin is significantly higher relative to the control. There does not appear to be a significant difference in chromatin interaction with the paternal allele. Finally, in panel C the authors analyzed the sequence of the cDNA obtained from the ChRIP experiments. In the BWS samples, both parental alleles (A/G) were present, while only G was present in the control.

I interpret these data as indicating that the KCNQ1OT1 transcript interacts with chromatin, and that the maternal duplication of this region observed in the BWS cases correlates with increased interaction. This suggests that the duplicated region of the KCNQ1OT1 transcript is at least partly responsible for the interaction with the chromatin. Taken together with the other data presented in the paper, it also suggests that this chromatin interaction with the duplicated region may be the mechanism of action through which the transcript silences CDKN1C.

5. 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 Airn, Igf2r, and slc22a3, which of the two hypotheses is most likely and why?

Two possible mechanisms of action:

The mechanism that I think is more likely

Airn is also a cis-acting silencer – it controls the imprinting of the genes in Igf2r locus, which contains Igf2r, slc22a3, and Airn.

6. After reading this paper, how do you think clinical papers describing just a few patients can contribute to our understanding of the regulation of developmentally relevant genes?

Studies like this one are very useful when studying genetic conditions that are fairly rare, such as BWS and SRS. Silver-Russell syndrome’s exact incidence is unknown, but is estimated to affect between 1 in 30,000 to 1 in 100,000 people worldwide [1]. Beckwith-Wiedemann syndrome affects approximately 1 in 13,700 newborn infants worldwide, although this estimate is likely conservative since some individuals with mild phenotypes are never formally diagnosed [2]. Both of these conditions are therefore quite rare, and it can be challenging to find a sufficiently large population to perform large clinical studies. Furthermore, both of these conditions are quite heterogeneous in their etiology – see the causes listed under question 1. The authors also note in the paper that microduplications are rarer occurrences in both of these conditions, which further complicates the issue.

Using a few patients as case studies for specific molecular defects can provide interesting information that may be overlooked or unavailable from larger studies. Additionally, the fact that this data is clinical provides “real-world” examples of the disease’s molecular pathogenesis, which can sometimes be a limitation of animal model studies and cell culture experiments. Nevertheless, there are still limitations to this approach. The findings may not be applicable to . Additionally, since specific family pedigrees are usually studied, it is possible that typical familial resemblances in the genetic sequence could be misinterpreted as being correlated with the disease phenotype. Therefore, it is critical to couple these case studies with data from other studies. The authors of this paper attempted to do this, bringing in findings from mouse models regarding the function of the Kcnq1ot1 transcript to help support their own data. This paper demonstrates that there are many different ways to approach a scientific problem, and that examining specific biological questions from a variety of perspectives can be key to obtaining novel and important insights.

Reflection

I chose to showcase this assignment in particular since I enjoyed working through this paper. I do not typically work with rare genetic conditions and case studies of this nature, so it was interesting to see a different way of approaching the study of a molecular biology question. This paper also taught me quite a bit about the ChRIP technique, which I was not previously familiar with. I needed to do a bit of extra research on the technique in order to understand the results that were presented in Figure 7, which was helpful for me. I also looked into the prevalence of the two syndromes discussed in the paper, since I was curious to know how common it was in the general population. Once I realized how rare these conditions are, I understood more clearly how valuable familial case studies like this can be.