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I chose this assignment because I enjoyed discussing the aspects of this paper after studying for the quiz. It showed how much I actually know about the paper and also this assignment helped fill the holes in my understanding of the paper. This was a very difficult paper for me because of the clinical side of things.

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

  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?

Comparisons

  1. Both congenital disease, growth disorders
  1. Contrast
    1. SRS:
      1. Associated with growth restriction
      2. Facial characteristics altered
        • Shape, etc.
      3. BWS:
        1. Associated with overgrowth
        2. Minor details
          1. Lip size, nasal shape, ear crease
  • Internal defects

 

  1. 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.
    1. Looked up using the accession number: 130650
    2. States: Beckwith-Wiedemann Syndrome Chromosome Region, Included; BWCR included

 

 

  1. Look at the pedigree in Figure 1.
  2. What can immediately be concluded about BWS (even without knowing who inherits the mutant allele from whom)?
    1. Not sex linked; autosomal
    2. Roughly 50% of offspring inherit
      1. Makes sense because only on one of the inherited chromosomes

 

  1. 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.
    1. It depends on which maternally imprinted chromosome the offspring inherits
      1. For individual I-4, only one of her chromosomes has the microduplication. Both of the chromosomes are maternally imprinted.  This means that offspring have a 50% change of inheriting the microduplicated maternally imprinted chromosome.  If the offspring happen to inherit the microduplicated chromosome, they will have the BWS phenotype.

 

  1. 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.

  1. BWS:
    1. 160 kb inverted microduplication (in cis) consisting of ICR2 sequence and exons 12-15 of the KCNQ1 and 5’ 20kbp of the long ncRNA (KCNQ1OT1) in locus 11p15.5
    2. Methylation of duplicated ICR2: hypomethylated, which is inconsistent with the usual methylated status.
    3. This mutation results in the expression of a truncated long ncRNA and a distinct down regulation of the protein coding genes within the adjacent cluster (CDKN1C)
  2. SRS
    1. 2 Mbp inverted duplication (in cis) within the locus 11p15.5
    2. Methylation: is (mainly) consistent with the control group
      1. However, there is a double dosage of the protein coding genes from the maternal chromosome
      2. This results in twice the expression of these genes.

 

  1. Explain what the data in Figure 7 show, and how you interpret them.
  2. Figure 7: Looking at the interaction of the long ncRNA with the chromatin using qPCR analysis of ChRIP-purified RNA
    1. From the co-precipitated RNA, they differentiate between maternal and paternal alleles using specific SNPs.
    2. Figure 7a: Overall, compared to the controls, there is a significantly enriched interaction between the long ncRNA and the chromatin.
    3. Figure 7b: The paternal allele (full length RNA) is not significantly different from the control but the maternal copy is significantly more enriched for the patient showing the BWS phenotype. Most interestingly, this is consistent for a patient with no duplication but hypomethylation of ICR2 (BWS P4).  Majority of this difference is coming from the maternal allele as.  This hints at a common mechanism of action between the duplicated patients (P2 and P1) and patient P4.
    4. Figure 7c: They conduct sequence analysis at SNPs of cDNA obtained from ChRIP. Results are consistent with that seen in Figure 7a and 7b.  In the control, only see 1 full length copy, but in all BWS patients, you see the truncated as well as the full length copy of the long ncRNA.
  3. How to interpret:
    1. Need to compare the histograms (control vs. two different patient groups)
    2. Figure A and B: Is measuring fold enrichment of KCNQ1OT1 over IgG- quantitative
    3. Figure C: Is using sequencing to analysis expression- qualitative

 

  1. What valuable fundamental information was gained about imprinting control regions through the study of these patients?
    1. Imprinting is not only regulatory sequence dependent (eg. ICR2) but also requires information from the adjacent sequences.
    2. Imprinting mechanism in sperm and oocyte are distinctly different.

 

 

  1. 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.
    1. Although the authors concluded that the ICR2 sequence in the microduplication is insufficient for proper imprinting, what other factors are required?
    2. Pertaining to Figure 7: with the histograms. If Patient 4 had similar expressions of ICR2 since ICR2 is hypomethylated in both and with the microduplication, one copy of ICR2 is normally methylated, so the genetics seems like it should be of the same results (since they sort of equal to each other); why is there such a huge difference in chromatin enrichment in P4?
    3. How is imprinting erased and re-established during gametogenesis?

 

  1. Bonus Question
  • No: Because this is one specific case of this duplication. Other patients with BWS can inherit the disorder differently or have different epigenetic and genetic mutations in both coding and non-coding that cause this phenotype.  For example, Patient 4 in the paper has no duplication and instead just hypomethylation that causes the same sort of symptoms.

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