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Friday, May 18, 2012

Likely Cause of CMT4 Identified: SH3TC2

Extremely Likely Cause for CMT4 Identified

I received an email from Brad Chapman who has an awesome blog that explains how to deal with next-generation sequencing data. He generously offered his assistance in tracking down the CMT4 causal variant(s) in Stacy. As opposed to manually inspecting each known causal variant, he proposed scanning all low frequency mutations within known causal genes. Thanks to his help, we now know that Stacy has two mutations in the known CMT4 gene SH3TC2  that are so rare, they never occur in the 1000 genomes dataset. These two mutations are...

  • Arg954X (Known CMT4C causal mutation)
  • Gly843Glu (Potential Novel CMT4C causal mutation)

These two mutations are on different haplotype blocks, making it very likely that they are compound heterozygotes.

Visualization of the Likely Compound Heterozygotes

***Note:  I "flipped the strand" on the reference genome since SH3TC2 is on the reverse strand. This means we have to read each codon in reverse and we must also reverse complement Stacy's reads. This means that the "A" you see below should be considered a "T". 

Here is Stacy's Arg954X mutation:

Here is Stacy's Gly843Glu mutation:

Gly843Glu is Likely a Novel CMT4C Causing Mutation

After some digging around, I found that Arg954X is a known recessive CMT4C causal mutation. Since Gly843Glu is on the other haplotype block, it is likely disrupting function on the second copy of the gene, leading to two loss of function mutations (one in each copy of the gene). However, Gly843Glu is not showing up in any database I have found nor in MedLine searches as a known CMT4C causal mutation. It is very likely that Gly843Glu is a novel recessive CMT4C causal mutation. I say "very likely" because although this appears to be (by far) the most probable explanation for Stacy's CMT4, I intend to find another individual with CMT4 who is missing all known causal variants, but is homozygous for Gly843Glu.


I am extremely grateful for Brad Chapman's assistance as well as for 23andMe's commitment to giving people access to their own genomes. We can now be almost 100% sure that Stacy's compound heterozygous mutations have been identified. I would like to point out that this process was a lot cheaper than the $3000 that Stacy was going to be charged out of pocket by doing this via a hospital and we learned of the causal mutation 11 days after getting her exome from 23andMe. We not only know more about her disease, but can also examine thousands of other genes as well. I will go through more details in future blog posts about how to replicate this entire process that has led to these findings. I intend to be thorough, so I will continue to search for anything suspicious in any of the known CMT causal genes as well finish inspecting the remaining known CMT4 causal mutations in Stacy's exome. I also intend to add citations and tidy up a lot of the work that has been done. My goal was to move quickly, but I can now add some polish now that I believe the causal mutations have been identified. I wilI also discuss the next steps in validating the potentially novel causal CMT4C mutation Gly843Glu as well as researching the pathophysiological effects of these mutations.


  1. Awesome news, and awesome post! I'm thrilled to hear of Brad's help -- he's one of my heroes in the grassroots bio movement, and yes, he does have an awesome blog! This whole discovery process has been so impressive.

    I'm particularly struck by Stacy's combination of incredibly bad AND good luck: bad because she managed two different mutations in the same gene, and good because of your exemplary concern (which many might show) combined with extraordinary intelligence applied to the requisite domain (genetics). In other words, it's not just that you take care of her (already a big deal), but you can also materially impact the outcome of her disease. Just amazing. Hats off!

  2. Thank you for the kind words. This whole project has been thrilling and empowering. Brad has been extremely helpful. I'm a fan of his blog and his awesome work to show everyone how to explore next-generation sequence data.

    The goal was was to identify the disfunction at the molecular level. The fact that a potentially novel CMT4C mutation has been identified is a bonus. Just recently, a study showed what goes wrong when both copies of SH3TC2 have loss-of-function mutations. Here is the abstract of the article:

    ABSTRACT: http://www.ncbi.nlm.nih.gov/pubmed/20028792
    Mutations in the functionally uncharacterized protein SH3TC2 are associated with the severe hereditary peripheral neuropathy, Charcot-Marie-Tooth disease type 4C (CMT4C). Similarly, to other proteins mutated in CMT, a role for SH3TC2 in endocytic membrane traffic has been previously proposed. However, recent descriptions of the intracellular localization of SH3TC2 are conflicting. Furthermore, no clear functional pathogenic mechanisms have so far been proposed to explain why both nonsense and missense mutations in SH3TC2 lead to similar clinical phenotypes. Here, we describe our intracellular localization studies, supported by biochemical and functional data, using wild-type and mutant SH3TC2. We show that wild-type SH3TC2 targets to the intracellular recycling endosome by associating with the small GTPase, Rab11, which is known to regulate the recycling of internalized membrane and receptors back to the plasma membrane. Furthermore, we demonstrate that SH3TC2 interacts preferentially with the GTP-bound form of Rab11, identifying SH3TC2 as a novel Rab11 effector. Of clinical pathological relevance, all SH3TC2 constructs harbouring disease-causing mutations are shown to be unable to associate with Rab11 with consequent loss of recycling endosome localization. Moreover, we show that wild-type SH3TC2, but not mutant SH3TC2, influences transferrin receptor dynamics, consistent with a functional role on the endocytic recycling pathway. Our data therefore implicate mistargeting of SH3TC2 away from the recycling endosome as the fundamental molecular defect that leads to CMT4C.