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| Radiographs of engineered mice in Marfan Study. |
In this week's discussion we will delve into mouse genetics to try to determine the molecular mechanism of Marfan Syndrome. In lecture this week, we learned about different types of dominant mutations. We ased the question, How can a mutated gene be dominant? We also tried to delineate differences between gain of function, dominant negative, and haploinsufficiency mutations. The article here tries to address a conundrum: whether Marfan Syndrome is caused by a dominant negative mutation, or by another mechanism. Click onto link below:
Judge et al., "Evidence for a critical contribution of haploinsufficiency in the complex pathogenesis of Marfan syndrome", Jo Clin Invest, 2004k
I would like for you to think about whether evidence provided in the paper supports the authors' conclusion that Marfan Sydrome is due to haploinsufficiency, and not by a dominant negative mutation. Please come to class organized with notes you have taken that you feel answer this question.
For the blog please answer this question with a post:
Do you feel this study was appropriate for publication in the Journal of Clinical Investigation, or not, and why? (For this, think about quality of data, novelty of work, and overall impact on the field) Try to limit your response to 1/2 a page.
If you have trouble with the above link (which directs you to the paper on BlackBoard) try this one instead:
Judge et al. article
The Journal of Clinical Investigation is a publication of American Society for Clinical Investigation, which is self-described as “dedicated to the advancement of research that extends our understanding and improves the treatment of human diseases,” among other things (http://www.the-asci.org/aboutjci.shtml). As such, it seems to me highly appropriate that this article be a part of the JCI. The researchers’ approach to Marfan Syndrome is an innovative one. It not only enriches understanding of the disorder’s mechanisms, but it also reevaluates how treatment methods can and should be based on what types of genetic cues cause the disorder.
ReplyDeleteAs regards our recent work in this class, the paper is particularly articulate about historical support for the idea of dominant negative mutations’ role in Marfan Syndrome. A haploinsufficient subject tends to produce about 50% of a healthy amount of extracellular microfibrils, far fewer than those in patients with dominant negative mutation (Judge DP, et. al, 172). This fact alone, mentioned early in the paper, handily conveys the authors’ consideration of both the variety of genetic mechanisms for disease and this particular illness’s range of symptoms.
The intellectual results of their work enrich our shared understanding of haploinsufficiency, dominant negative mutation, and other meaningful genetic cues for illness. At the same time, these results advance how to approach and treat a disorder that is known to have a “wide range of clinical presentations” (Judge DP, et. al, 178). As such, it seems to me an ideal piece of research for the Journal of Clinical Investigation, and a model for investigating other such wide-ranging illnesses in the future.
Absolutely! The researchers did a great job explaining the history of the study of the mutated gene (FBN1). They carefully explained the evidence as to why it has been thought of as a dominant negative mutation and what new discoveries related to increasing understanding of fibrillin-1’s role in tissue growth suggest that perhaps the mutation is actually causing a loss of function allele.
ReplyDeleteI found it novel that they tested the dominant-negative hypothesis with new methods, even going so far as to explain why their testing method using “mouse models of MFS syndrome provide an ideal system to test these hypotheses.” (Mouse and human fibrillin-1 interact and coassemble, how convenient!) They presented the evidence for haploinsufficiently clearly and concisely, stating that even where there is more or an equal amount of the mutant protein to the WT protein, if a normal complement of WT protein is “added” to the mice, microfibrillar assembly proceeds well. This is very exciting for future treatment plans for patients with MFS.
Finally, another reason why I think this was a very worthy article, is that the researchers recognized their own limitations (that they were only studying the missense mutation of the gene, which produced a cysteine-substituded fibrillin-1) and cautioned readers to avoid generalizing their findings to all types of mutations of the FBN1 gene. They raise questions that they aren’t able to answer completely and suggest future topics for research.
The only problem that I see with this article is perhaps that they make reference to some data that is not yet published. The data is quite interesting and supports their hypothesis. They state: “preliminary analysis has revealed that mice heterozygous for a null Fbn1 allele are clinically and histologically indistinguishable from C1039G heterozygotes.” Pretty cool! But is it appropriate to reference unpublished findings?
I agree that it is appropriate for this article to be included in the JCI. The authors are testing an inference about the mechanism of MFS (dominant-negative) and proposing and testing an alternate hypothesis (one of haploinsufficiency). Their work suggests exciting implications for the clinical treatment of MFS (e.g. therapeutic strategies that address the primary cause of protein insufficiency, not just secondary events). The authors also make a claim for novelty of technique, but having little exposure to mouse research techniques, I’m not sure I can critique the accuracy of this claim. I think the authors do a good job of summarizing past data that suggested a dominant-negative mechanism for MFS and laying out their own evidence for a haploinsufficiency model. Most importantly, I think they address the limitations of their study in the discussion section.
ReplyDeleteI appreciate that they recognize that haploinsufficiency alone can not explain the variety in clinical presentations of MFS and that “additional genotype-modified events” likely play a role in the disease. One of the questions I had about their experiment to counter the dominant-negative hypothesis was that it didn’t seem to accurately capture what is happening in patients with MFS. The introduction of either a WT FBN1 or a mutant FBN1 onto a healthy background would leave the mouse with a total of three alleles, two of which are functioning normally. Even if the mutant allele is producing 100-200% the protein of the other two, can this experimental situation directly translate to what’s actually happening with MFS? The researchers conclude that absence of an MFS phenotype in these mice means the disorder is not dominant-negative, but go on to say in the discussion that the very activity of this transgene may create an environment that “never reaches the threshold loss of function needed for clinical expression in the lifetime of the animal”(178). I’m also skeptical that using different strains of mice is enough to effectively mimic the complexity of MFS, which the authors describe as a “dynamic interplay between primary tissue predisposition, physiologic stress, time, and both productive and deleterious compensatory events”(177).
In agreement with my classmates, I think this seems like an appropriate topic for the Journal of Clinical Investigation. The introduction and discussion both make a case for the relevance of investigating the genetic mechanism for MFS to clinical treatment. As described in the discussion, the dominant-negative model of MFS precluded many potentially effective therapies including boosting expression of WT fibrillin-1 because it was thought that the mutant protein would “neutralize even highly efficient strategies” to do so (p. 178). It’s very exciting that MFS may conform to a dose-dependent model and that there is potential to rescue the phenotype of an individual through gene therapy. The authors also effectively describe their own limitations and suggest the role of other genes in the progression of the disease, which is consistent with the fact that MFS has multiple clinical presentations. Overall, along with the NYT article about Leukemia, there are many reasons to be positive about the advancement of genetic techniques. With all of that said, I do think that the realities of publishing often include trying to publish in the journal with the widest reach, most prestige, and to select a journal which you believe will be likely to accept your work. This kind of genetic research is becoming increasingly relevant in a clinical setting and I would expect that journals such as JCI are trending more and more toward research on genetic mechanisms.
ReplyDeleteI also agree with the above posts and think that the article fits well into the Journal of Clinical Investigation. Not only does it broaden our perspective on dominant mutations and disease processes, but it contributes significantly to the literature on MFS. After looking at a couple of articles on MFS published in the Journal in 1992 and then another in 2004, much of this earlier work was well-summarized in the Judge et. al paper. The 1992 article cites the hypothesis that MFS results from gene mutations and the 2004 article extends this research by looking at a specific pathway for the manifestation of MFS (stemming off of the dominant-negative mutation theory). The Judge et. al paper adds to this pool of thought that haploinsufficiency is the real cause of MFS, not a dominant-negative mechanism. This experiment gave ample evidence to support the final conclusion especially by investigating alternate explanations for outcomes throughout the experiment process. For example, when the mutant human protein did not show any dominant-negative interference, researchers addressed the possibility that it could have resulted from the transgene not being expressed in the appropriate tissues at the appropriate time. This kind of detail and open-minded thinking is what makes this article significant in its contributions to the Journal as a whole.
ReplyDeleteThe finding that MFS could potentially be partially treated by the addition of a normal allele is exciting and intriguing. How would such a process work on a molecular level? Gene-transfer strategies suggest that insertion of the gene as is or through a virus are viable options. However, as is true with most (if not all) invasive treatments, there could be side effects. These side effects could manifest themselves as anything from over-expressing other genes to wiping out expression of certain phenotypes. Further investigation of the applications of a haploinsufficiency-related mechanism seems to be the next potential journal article in continuing research in the field.
I believe this article does merrit being published in the Journal of Clinical Investigation on several accounts. The results that these scientists obtained with their transgenic mice illuminates the nature of fibrillin-1, the FBN1 gene, MFS, and how they all interact. Although I had never heard of Marfar syndrome before, the article says that "the pathogenesis of the disorder has been the subject of intense investigation and discovery," and I feel like the scientists here have made great strides forward in discovering not only the nature of the pathogenesis of the disorder, but also a possible treatment method that is novel and appears to be successful in their murine trials.
ReplyDeleteThe scientists clearly layed out the previous beliefs about MFS and fibrillin-1 and then showed how their studied challenged these old beliefs and presented hope for progress in finding a treatment. Because of old and incorrect conceptions about fibrillin-1, scientists believed that "individuals affectwed with MFS are born with obligate predisposition for postnatal failure." But beliefs such as these, which did not bode well for the treatment of MFS patients has been challenged by their studies on fibrillin-deficient mice.
Genetics medicine brings hope for many people afflicted with disorders that have never before been fully understood, and never before been treatable. The data from tthis study suggest that "haploinsufficiency for WT fibrillin-1, rather than the production of mutant fibrillin-1, may be the critical determinant of impaired microfibrillar deposition." This is huge, because this means that the transgene approach the scientists took - of introducing the WT gene into the affected mouse - could supply enough of the necessary protein and rescue the rate that would otherwise present Marfan syndrome. This difference between haploinsuffiency versus a dominant-negative mechanismharkens back to discussions we recentlty have had in class. It brings me a rush to see how a concept such as this is being applied in the lab, and how it makes a critical difference in understanding and treating disease!
I feel this paper was appropriate for the JCU. It was a specific study that looked at a hypothesis concerning the interaction of genetic mechanisms and the researchers clearly outline how their applied technique and application is different from previous studies. I think there is significant impact offered in terms of better understanding the processes involved in Marfan's. I'm uncertain as to whether or not this has had an impact on the field, however, there are 37 articles that site it and several that mention using heterozygous mice with the C1039G mutation. (Fibrillin-1 genetic deficiency leads to pathological ageing of arteries in mice B. et al) My partner is affected by Marfan's so this article was particularly interesting to me.
ReplyDeleteIt's interesting in that this paper is from 2004 and yet, the wikipedia page and a lot of the Marfan Support websites still say that the issue is due to dominant negative mutation and haploinsufficiency whereas this article makes the case that it is solely haploinsuffiency. There is also some new research going on that points to Marfan's being more of a spectrum disorder as opposed to a missense mutation that causes direct, specific phenotypes. It may undergo a re-categorization to allow for a broader definition of this disorder.
Though I won't pretend to be an avid reader of the Journal of Clinical Investigation, I am sure that this article is a fitting example of the high-quality analysis the journal no doubt demands. I'm still kind of baffled by how incredibly clever molecular geneticists can be, however, and so right now I'm fairly easy to impress.
ReplyDeleteWhile admittedly most of the article went straight over my head (despite having to stop every five seconds to Google a new word), the experiments themselves were strikingly simple. It seems rare in genetics for scientists to be able to construct such straightforward and reliable experiments with the potential to prove so beneficial to those afflicted by disease or disability. As Allison mentioned above, this article is comparable to the previous one concerning Dr Wartman's leukemia diagnosis; both hold much promise for the future, while leaving us with many more questions (what other gene modifiers may be affecting Fbn1 expression? what treatment options are now available for those afflicted? and as Mo wisely points out above, how can we begin to recategorize Marfan's armed with this new information?). Nevertheless, I'm optimistic.
I agree that it was appropriate for the Journal of Clinical Investigation to publish this study. The findings of the study helped to shed light on the genetic expression that results in MFS and have implications for potential improvement in treating the syndrome. Looking at some of the titles of other studies published by this periodical, the MFS study appears to be a good fit and would reach an interested, appropriate audience. I can't say whether the researcher's methods were novel in 2004. The researchers did seem to provide adequate evidence that they achieved sufficient gene expression from their transgenes to indicate that increasing the production of mutant fibrillin-1 did not adversely affect mice with normal endogenous genes, and that introducing a transgene with a normal allele to a heterozygous mouse could mitigate the phenotypic expression of the defective allele. I would think these findings would have a huge impact on the field of genetically inherited disorders. I am curious to know what developments have taken place in the seven years since this study was published.
ReplyDeleteI thought the article was interesting and informative, but unless I'm missing something, I'm not sure that it was as conclusive as the author claims it to be. The reason that I question his conclusion derives fromt he following statement:
ReplyDelete"The mutant YAC transgene was engineered to produce fibrillin-1 with a cysteine substitution in a cbEGF-like domain (C1663R). This particular mutation had been described in a patient with a classic form of the disease (23, 24)."
The use of one case study as a basis for creating the variable mutation seems like a pretty big limitation to me. The author is asserting that because this mutation, which was observed in this single patient, was correlated with Marfan syndrome. Manipulating fibrillin-1 to create a mutation makes sense, but what if a different mutation could have produced a dominant negative effect? How can he assert that, because that particular mutation did not produce such an effect, it must occurt by an alternate mechanism?
Whether or not the point about the mutation if valid (honestly I'm not sure I totally got that part of the article) I'm still not sure that the absence of one effect is proof of another mechanism.What would have made the article more conclusive, in my view, would have been a study design that included supplementation of the normal protein, in cells that possess the mutation, to see whether the mutation has an affect when higher levels of normal protein are present.
I'm in agreement that this study befits the Journal of Clinical Investigation. I don't know how paper publishing criteria varies from journal to journal, but I would imagine that any study representative of the advancement in understanding of unknown mechanisms in a given field would be permissible, first of all. Secondly, any paper that may drastically affect the studies conducted by other scientists operating in the same field should probably be published. Does this study posit its hypothesis using the quality of evidence needed in order for other scientists to shift their views regarding the mechanisms of expression of the FBN1 gene? Seems to me that it does. And finally, the last component a paper needs to get published is an extremely novel idea. I think by virtue of the fact that rudimentary ideas about the origin of this disease are being challenged, that ideas prematurely precluding other possibilities are being re-evaluated, that this study generated enough intellectual curiosity for this to be considered a novel concept. I look forward to hearing everyone's opinions Friday when we discuss this further in class, and hope to learn why this article might NOT have been an ideal candidate for publishment in this journal.
ReplyDeleteI am in agreement with my classmates that this is an appropriate article for the Journal of Clinical Investigation. The article clearly articulates new research findings on the genetic mechanism that results in MFS, as well as makes a clear statement of how these findings could impact the future treatment of MFS. Given the aim of the JCI to present articles with novel research findings that help us to further understand human disease with the aim to improve clinical treatment, I feel this paper is spot on and highly appropriate for the inclusion in the journal.
ReplyDeleteThe paper presented the historical research that pointed to a dominant-negative model for the genetic mechanism of MFS, clearly explaining the reasons this model was previously accepted. This information helped me better understand how researchers and clinicians were thinking about the mechanism for MFS at the time, as well as the available treatment options available to those with MFS based on the idea of a dominant-negative genetic model. The researchers offered an alternative hypothesis of a haploinsufficiency model for MFS, clearly presenting results that suggest haploinsufficiency to be, at least partially, the genetic mechanism responsible for the expression of MFS. They clearly stated the impact this different genetic model could have on viable treatment options. With this dosage-dependent model, they make a case that is is possible future treatment of MFS could involve primary prevention via transgene therapy, rather than secondary support. I felt overall that the paper presented information in an unbiased way with careful consideration and testing of the dominant-negative. Additionally, I feel that by discussing the issue of there being many different clinical presentations of MFS and the likelihood of other modifying genes responsible for this variation in expression, they made it very clear that there are limitations to their findings and that there is still much more research that needs to be done to fully understand the complexity of this disease.
Again, a very exciting glimpse at the future of medicine as we continue to understand more about the genetic basis of disease. I am looking forward to discussing in greater depth how transgene therapy can be implemented, as well as any potential risks that may arise with such a treatment option.
At the risk of sounding repetitive, I will also say that I believe this piece of research finds a good home in the Journal of Critical Investigation. Upon cursorily researching the JCI, I found that it is a peer-reviewed publication that seeks to provide free access of its research articles to a widest possible audience. I liked this designation, as I feel that articles and research such as this one aren't always available to the average web-surfer. It is clearly a reputable publication with an emphasis on accessibility, and thus seems to be an appropriate forum for research on Marfan's Syndrome.
ReplyDeleteAlthough most of this article was over my head content-wise, I appreciated the forward-thinking, forward-moving nature of this "investigatory research." I like that this article strives to counter previously held ideas that MFS results from a dominant-negative mutation and posit the idea that it results rather from haploinsufficiency. I am in agreement with several of my classmates who have iterated the exciting nature of research like this and its implications for the betterment of MFS treatment and possible cures.
On the other hand, I appreciated Jessica's comment about the lack on conclusiveness of this research. While it is clear that there is verifiable support for the claims these researchers are making, their ideas seems to encompass only one particular phenotypic expression of the kind of gene activity that they are discussing. Even as an elementary reader of research like this, I found myself wondering if a more general, and therefore more conclusive approach could have been explored before publication.
I appreciate the study conducted by the research team. Though I, too, had trouble understanding every other word in the article (which reminds me to study harder for biology and especially genetics), I am with agreement with my classmates that not only is this study appropriate for the JCL, but also the study promotes promising treatments for future Marfan Syndrome (MFS) patients.
ReplyDeleteThis study is appropriate because, in 1991, mutations in the FBN1 gene encoding fibrillin-1 were found to cause MFS, but this 2004 study indicates that overexpression of the mutant protein were not shown any abnormalities by mice. This suggests that several factors can cause MFS, which is why I appreciate this research team for carrying out this study. Like everyone else who blogged about this, I remain optimistic that these study will be one of the pioneers for the potential cure and prevention/management of MFS.
Though, I am curious to learn more about the methodology of the experiment. While I do not know what these YAC mutagenesis, YAC transgenesis, northern blot analysis, and coimmunoprecipitation entails, in order for me to truly appreciate genetics, I would like to carry out some experimentations in the lab (I am disappointed that this class does not include lab). Yes, I do remain optimistic about the NFS study, but my understanding of the study would truly be supported by getting a glimpse of what they did in the lab.
I think that this study was appropriate for the Journal of Clinical Investigation because it is addresses the operational methods behind MFS. With increasing knowledge and awareness surrounding gene sequencing, and the influence over our genetic material, it is important to have studies marked by pertinent research to investigate the proper/improper functions (or lack of) associated with genes. Understanding the pathways associated with proper function allows physicians and patients alike the ability to promote further understanding of why we can influence (or event turn on/off) the body’s ability to perform specific operations. Likewise, it should be noted that understanding the pathway for syndromes, such as Marfan syndrome, can be exploited through genetic variation and manipulation. Using the mouse as a model organism, this study was able to produce sufficient data to suggest that MFS can be combated by boosting fibrillin-1 expression. This fact alone, amongst the myriad of findings within the study, leads me to believe that this was a study worthy of a spot in the Journal of Clinical Investigation.
DeleteAs my classmates have so comprehensively determined, one would be hard pressed to find reasons to exclude this study from the JCI. The JCI is a free, high-impact journal for advances in biomedical research, and this article not only overturns previous notions of the genetic etiology of a not-uncommon debilitating medical syndrome, its findings indicate future targets for its direct treatment and potential prevention. This is especially significant in that the overturned pathological framework predicted "an obligate predisposition for postnatal tissue failure... and an inherent loss of biomechanical integrity", which "boded poorly for the development of productive treatment strategies" (Judge et al., 2004). That being said, the authors admit that microfibrillar assembly failure due to haploinsufficiency is not the only mechanism behind the MFS phenotype. If these results can be used to treat even a small subset of MFS patients, this article has more than earned its publication here.
ReplyDeleteAn interesting caveat is that (according to Wikipedia, admittedly), yeast artifical chromosome techniques, as used in this study to insert the gene to produce WT and mutant fibrillin-1, seem to have fallen out of favor since the time of this article due to a high incidence of artifacts, including the deletion, rearrangement, and duplication of genomic segments being studied. The authors do, however, appear to have taken the proper precautions to rule out these artifacts, as indicated in the Methods section, by analyzing the DNA sequences at each step.