Category Archives: Assignments

Group members’ names: Susan, Alanna, Rachel, Jane, Sam

Assigned paper:

Brison, N., Tylzanowski, P., Debeer, P. (2012) What the HOX is going on?

Questions:

1. Look at the diagram and table in Figure 2. Then, look at the variety of phenotypes associated with different mutations in HOXD13. Do you notice any patterns? Would you consider the wide spectrum of phenotypes to be an advantage or a disadvantage of using human mutant phenotypes to investigate the functions and mechanisms of action of HOXD13?

• seems that there are mainly missense mutations in Exon 2
• expansion/contraction in polyalanine region in Exon 1à webbing between 3-4^th finger (classical)
  • # of repeats correlates w/ severity of phenotype
• Frameshift/ nonsense mutations in homeodomain (Exon 2)
• Exon 1 mutations are dominant negative, homeodomain mutation= functional haploinsufficient

Disadvantage: more difficult to find a consistent phenotype and identify the actual gene’s function à hard to control for

– different combinations of phenotypes could be confusing

Advantage: wide range of phenotypes gives you a wider range of potential gene functions/ what it could control for (more possibilities)

2. Notice how there are only 3 reported mutations in Exon 1 outside the polyAla region, but 6 in the homeobox, even though the homeobox is much shorter than Exon 1. Propose two distinct hypotheses that would explain why this might be.

• people studying homeobox much more (more frequently) so may just happen to identify more
• phenotypes of mutations in Exon 1 may not attract attention (even though present)
• Exon 1 mutations may be lethal so no patient with mutation to report

3. In humans, Hoxd13 looks very much like it is haploinsufficient, and the mutations reported in the paper are typically dominant (or semi-dominant/incompletely dominant/co-dominant, depending on how we look at them) to wild-type Hoxd13. Before you attempt this question, please ensure that everyone in your group knows about the two ‘ways’ in which mutant alleles can be dominant to their wild-type counter-parts (i.e. dominant negatives and dominant due to simple haploinsufficiency) and how this relates to GOF and LOF.

a) At the molecular level, how do you think the Hoxd13-expanded polyAla tract mutant exerts its ‘super-dominant negative’?

• deformed cytoplasmic aggregates that prevents WT Hoxd13 from entering the nucleus in heterozygote
  • Negative since loses function of what Hoxd13 usually does
• “Super dominant” à also affects Hoxd11 and Hoxd12
  • polyalanine region acts as a binding domain for the cofactors of Hoxd11 and Hoxd12 à loss of sequence similarity/ structure similarity
    • Prevents Hoxd11 and Hoxd12 from doing what they usually do

b) The reported mutations in the homeodomain (~ the homeobox) of Hoxd13 typically result in LOFs and in specific phenotypes depending on the particular mutation in question. What does this suggest? Make sure to connect the mechanistic information in the paper with your view of autopod development.

• impairs the ability of Hoxd13 from recognizing consensus binding sequences
  • loses biding affinity
• critical for homeodomain to maintain its amino acid sequence for specific binding to DNA consensus sequences (Eg. Stability, affinity)
• if affect some very conserved regions in homeobox.. can prevent it from interacting w/ binding and TFs
  • depending on which amino acid you change.. could affect binding to different domains
• relative stability/ affinity of binding to Hoxd13 protein to target could create different levels of expression of it’s target, resulting in different phenotypes (dose dependent)
• different mutations result in different phenotypes à upstream of many regulatory pathways controlling autopod development

4. A great deal of what we know about the function(s) and role(s) of HOXD13, but also of several other HOX genes, is thanks to the study of families with limb (or other) malformations much through a ‘look’ (including, ‘look for correlations’) approach.

a) In addition, and complementing this source of information, we have model systems such as the mouse, where we can selectively mutate our genes of interest in any way we want, and the chicken, which can be locally infected with vectors carrying any gene of interest. We also have the power of bioinformatics.

What are the advantages and disadvantages of each system?

• Mouse: a lot more similar to humans (vs chicken)
• Chicken: less labor intensive than the mouse and faster!
  • Mutations in mouse and chicken that were the same in humans result in different phenotypes compared to humans (Hoxd13 function could be diff. from species to species based on the genes its involved in regulating)
• Human Studies: can only do studies on the specific mutations that exist
  • Results in phenotypes seen can be applied to the general population
• Bioinformatics: can look at conserved sequences to predict phenotypes

– but have to predict what the phenotype will be

b) In order for the human data to be used by researchers, the families involved have to provide informed consent. Why do you think families provide/do not provide consent? If they provide consent, what is the benefit for them? What is the benefit/value for the researchers?

• They provide consent because…
  • hopefully find treatment down the road since their children could be affected by the same disease
  • gain insight and provide opportunities for additional research and potential future treatments
• They don’t provide consent because…
  • Just don’t want to be part of a test –> want to keep their personal information private (cause they could be cloned..)
  • They’re just generally afraid.. scientists might use for other purposes without consent?

5. What did you find most surprising in this review article?

– the array of phenotypes with mutations in a single gene!

Reflection:

 I remember having some difficulties with this assignment as this paper was more complex than the first. But I felt like we were able to work out any questions or confusions we may have had as a group since the questions guided our thinking and we were able to build upon our own original ideas and thoughts. The part at the end regarding consent when conducting human research was different and we came up with  some interesting points as group.

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

Questions:

1. The authors report using OMIM to obtain some information for their research. Take a few minutes to look up Kcnq1ot1 on OMIM (http://www.omim.org) and see what information you get.

• associated with Beckwith- Wiedemann syndrome (BWS) and a variety of human cancers
• imprinted antisense transcript encoded by a 60-kb region
• expressed preferentially from the paternal allele
• produced in moth human tissues
• CpG island within intron 10 specifically methylated on the silent paternal allele
• When inherited maternally, deletion of LIT1 gene caused BWS with silencing p57(KIP2)

2. Look at the pedigree in Figure 1.

a) What are a few things that can immediately be concluded about BWS (even without knowing who inherits the mutant allele from whom)?

• Autosomal
• Affects both males and females
• Not lethal.. but there are also people who die

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.

• BWS is only on one allele.. since the child receives only one allele from each parent … the child only has 50% of inheritance of BWS

3. Both the BWS and the SRS patients have duplications of parts of the locus being studied, and so does Individual I-4, who has no syndrome. Using the information in Figure 5 and Figure 8, propose a hypothesis that explains the differences in phenotype (both macroscopic phenotype and the DNA methylation pattern phenotype) among the patients with BWS, SRS, and Individual I-4.

• figure 5 shows that I-4 duplication has minimal methylation whereas the SRS patient has more methylation of one of the duplicated alleles

–> duplicated on the paternal allele

• methylation on one of the duplicated alleles could contribute to the disease phenotype (macroscopic)
• Figure 8 shows that BWS and SRS patients both have duplications on the maternal allele whereas I-4 has duplication on the paternal allele
  • BWS: duplication of paternal allele on the maternal allele
  • SRS: duplication of the maternal allele on the maternal allele
  • I-4: duplication of the paternal allele on the paternal allele

–> any duplication occurring on maternal allele will cause a disease phenotype

(regardless of whether it is a maternal or paternal duplication)

4. Explain what the data in Figure 7 show, and how you interpret them in terms of the role of Kcnq1ot1 in the regulation of the imprinted cluster being studied.

• Kcnq1ot1 is involved in folding enrichment in BWS patients in the maternal allele
• Shows that Kcnq1ot1 RNA binds to H3
• B—significant difference in the amount binding between the BWS patients and the control (increased binding of BWS patients on the maternal allele)
  • Normally no expression on the maternal chromosome… so maybe production from the maternal allele is causing the disease phenotype

– not as much of a difference seen on the paternal allele… also see no effect phenotypically when inherit allele paternally

5. Optional/Time permitting: Take a look at the phenotypes of the SRS and BWS patients. 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? Knowing the effects at the molecular (DNA methylation/gene expression) of the various mutations, how could these macroscopic phenotypes be explained?

Reflection: I chose to include this assignment since we struggled to make sense of the pedigree at first but overcame that as we worked through the assignment as a group. It was also interesting to read a paper on a specific disease as I feel like I am always more interested in reading things when they pertain to disease phenotypes.. Not sure why. But overall, I thought we worked well as a group to overcome any confusions we may have had in the beginning and ended up clarifying and questions we had while we worked though the assignment.

1. Names and contributions of group members:

Alanna: Research, Write-up, Powerpoint

Susan: Research, Write-up, Powerpoint

Rachel: Research, Write-up, Powerpoint

Jane: Research, Write-up, Powerpoint

Sam: Research, Write-up, Powerpoint

2. Technique chosen:

RT-PCR: Real time PCR or Quantitative PCR (qPCR)

3. What does this technique ‘do’?

Amplification and detection of DNA simultaneously in Real Time.

4. What applications is this technique employed for?

• Gene expression analysis or mRNA analysis
  • Hein et al. (2001) employed real time PCR to analyze murine gamma interferon-gamma mRNA (a cytokine mRNA) expression in splenocytes
• Detection of GMOs in food
  • Zeitler et al. (2002) identified real time PCR as a method for quantifying transgenic contaminants with herbicide resistance in conventional rape seed.
• Cancer or disease detection
  • Multiplex real-time reverse transcriptase PCR is an applicable method for the detection, identification, and quantification HBV, HCV and HIV-1
  • Bernard and Wittwer (2002) used real-time PCR for detection of multiple breast cancer molecular markers
• Genetic variation analysis
  • Real-time PCR is able to detect mutations in the sequence, including single nucleotide polymorphisms (SNPs) through its melting point analysis.

5.  What questions relating to gene regulation and/or development can be addressed using this technique? Provide two examples (peer-reviewed papers) that use this technique.

Cytokine gene expression and regulation during infection:

Example: Th1 and Th2 cytokine gene expression in primary infection and vaccination against Fasciola gigantica in buffaloes by real-time PCR.

Cytochrome expression and regulation in different life stages (unfertilized eggs, embryos/larvae and adult tissue) and its role on neurodevelopment and plasticity:

Example: Real-time PCR analysis of cytochrome P450 aromatase expression in zebrafish: Gene specific tissue distribution, sex differences, developmental programming, and estrogen regulation.

6. What critical reagents are required to use this technique?

• Taqman based real-time PCR
  • Primers, Taq polymerase, Mg2+, Nuclease free H2O, dNTPs, Reverse transcriptase (if starting material is RNA), Taqman probes, appropriate buffers and DNA template.
• SYBR Green based real-time PCR
  • Primers, thermophilic DNA polymerase, Mg2+, SYBR green I dye, Nuclease free H2O, dNTPs, Reverse transcriptase (if starting material is RNA), appropriate buffers and DNA template.

• Other materials include: Real-time PCR machine that is able to complete thermal cycling steps for PCR and collect fluorescence data simultaneously, a computer, and suitable computer software for data collection and analysis.

7. What critical information is required to be able to employ this technique?

• Sequence data on the target sequence to design proper probes and primers.
• Melting temperatures for PCR products for melting curve analysis to determine amplification specificity.

8. References:

Bernard PS, Wittwer CT. Real-time PCR technology for cancer diagnostics. Clin Chem 2002; 48:1178–1185.

Bustin SA, Mueller R. Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis. Clinical Science 2005; 109:365-379.

Hein J, Schellenberg U, Bein G, Hackstein H. Quantification of murine IFN-γ mRNA and protein expression: impact of real-time kinetic RT-PCR using SYBR Green I dye. Scand J Immunol 2001; 54:285–291

Kumar N, Raina OK, Nagar G, Prakash V, Jacob SS. Th1 and Th2 cytokine gene expression in primary infection and vaccination against Fasciola gigantica in buffaloes by real-time PCR. Parasitol Res 2013; 112:3561-3568.

Sawyer SJ, Gerstner KA, Callard GV. Real-time PCR analysis of cytochrome P450 aromatase expression in zebrafish: Gene specific tissue distribution, sex differences, developmental programming, and estrogen regulation. General and comparative Endocrinology 2006; 147:108-117.

Valasek MA, Repa, JJ. The power of real-time PCR. Adv Physiol 2005; 29: 151-159.

Zeitler R, Pietsch K, Waiblinger H. Validation of real-time PCR methods for the quantification of transgenic contaminations in rape seed. Eur Food Res Technol 2002; 214:346-351.

Our revised question: 

Which of the following statements are TRUE?

A) One advantage of real time PCR is that it can be performed at a single temperature without the need for specialized equipment.

B) If a DNA polymerase other than Taq polymerase was placed in the TaqMan qPCR reaction, the target sequence will be amplified but it will not be detected by the machine.

C) In an experiment, we want to find a mutation in our gene of interest by melt curve analysis, we can use SYBR Green method of qPCR.

D) Use of gel, low sensitivity and poor precision are all draw backs of traditional PCR

E) Contamination by DNA fragments with no sequence homology to the target sequence can show up as a false positive in TaqMan method but not in SYBR Green Method.

Link to powerpoint presentation:  https://docs.google.com/presentation/d/150EOQskwstkJAW_c45nkrYdH_an7zSkDElTfQb3a3Dg/edit#slide=id.gbd6576bcc_0_25

Reflection:

I chose our techniques project as one of my top assignments as I felt like it was different from all the others since it was the first assignment that really forced us to work on something as a collective group. Starting with our research for the qRT-PCR, I learned to search and extract relevant information literature effectively and most importantly simplify all the technical details of the method so that it could be better understood. As a group we were able to identify key characteristics and traits of the technique to include in our presentation and we tried to communicate that information clearly during the actual presentation by outlining key facts and points we found to be of particular importance. This project also let me assess my own knowledge of qRT-PCR which was introduced to me in BIOL 335 and I found that I had to relearn the procedures for the technique to fill in any gaps from what I could remember. Overall, this project was a great group effort and really taught us how to collaborate and work together as a group for the rest of the semester. We also established a facebook group as a way to communicate our ideas and collectively work on assignments if we couldn’t meet in person. I also really enjoyed the Techniques Café part of the project in that it allowed us to see what other groups had done and how they chose to present their projects.

1) How does a mother’s hormones/ diet affect the development of a fetus?

2) What are the epigenetic changes involved during development that lead to mental illnesses and other forms of abnormal human development?

I’ve always been particularly interested in this question as there has always been many articles that advise mothers to tweak their diet during pregnancy to ensure a healthy baby. I’m curious to find out if these tips actually have any impact on a developing fetus and what specific types of hormones/ foods are involved in such epigenetic changes that could alter normal development. A better understanding of this could possibly help to lower rates of postnatal complications and reduce the incidents of developmental diseases/ abnormal development. It will also allow future mothers to gain more awareness and control over their baby’s development as to avoid any unexpected developmental disabilities.

Reflection:

I’ve decided to include this particular assignment since it was the very assignment we did for the class and really set the tone for the rest of course. It really forced my to think about what I was curious about in the field of developmental genetics and actually formulating a question was more difficult than I thought. Since the question had to ideally be testable, it had to be specific and address a certain area in genetics or development that could possibly be beneficial and insightful to the general public. This assignment also forced me to think back to what I had learned in previous genetics and developmental biology courses to ultimately tie those topics together. After thinking about it for a while, I finally came up with a question that addressed both fields of biology and could be of interest to the scientific community and the general public. This assignment also eased me into the process of finding a question for my final project!