Results of the German Q&A with Dr. Bohn held on July 3 2016


Q&A with Dr. Markus Bohn – Topic: Genetics of Connective Tissue Disorders (main interest in Tenascin X)

Part 1 – Results from the German Q&A held on July 3 2016

Translated by Karina Sturm

(No liability for correctness or completeness; no medical advice!)

1

Question

Which cell type or fiber type is mutated in hEDS?

Answer

What we know at the moment is that there is no special cell type affected but more a protein which is secreted by cell populations of connective tissue. Tenascin X is involved in the cross-linking of collagen fibers but the exact roll is unclear.

In this case the TNXB gene is mutated which is present in every cell of your body. It is believed that around 10 percent of all hypermobile EDS patients harbor a TNXB mutation. For the remaining 90 percent the causative gene still needs to be found.

2

Question

Is the mutation inheritance autosomal dominant or spontaneous?

Answer

The mutations we looked into in our study were all in the context of CAH-X, therefore all patients only had one normal functional TNXB allel. 2 of 3 patients had more than one mutation, all 3 were homozygous for at least 1. All were inherited by one or both parents.

3

Question

Is there a correlation with Marfan Syndrome?

Answer

At the moment it looks like TNXB mutations are occurring specifically in EDS.

4

Question

Is the mutation already included in NGS-CTD Panels?

Answer

Since the mutations we characterized have only been published 2 weeks ago and were not described before, it will probably take some time until they are going to be included in panels. But I do not know how long this process will be and how it works. I might ask someone in a diagnostic lab about it. In our patients we found those mutations all to be in the same terminal domain of TNX. It does look like this might be a hotspot which could be interesting for diagnostics.

5

Question

How high is the possibility of inheritance for spontaneous mutations?

Answer

We assume that mutations that are associated with EDS are systemically present in all cells of the body and develop through processes before birth (compared to for example drug resistance in breast cancer where the opposite happens). Because of this fact those mutations can be inherited. If both allels of the gene carry the mutation the probability is around 50 percent, if only one allel carries it, it is half. If this again is enough to lead to an EDS phenotype in the next generation depends on the EDS type.

6

Question

Since there are only a few HT patients where TNXB mutations can be detected, could the EDS HT type that is caused by TNXB maybe be a complete new and unique EDS type?

Additional question

Are there specific symptoms that only occur in EDS patients with TNXB mutations?

Answer

Not right now. It looks like even people with different TNXB mutations have a different phenotype but it all falls into the category of EDS HT at the moment.

Additionally I want to say that when I said TNXB mutations I was referring to the mutations we described in our recent study. There are patients that do have no detectable TNX Levels in blood and they do look like classical type EDS.

7

Question

In my case pseudogenes on TNXB have been found and were the reason why testing of the affected Exons wasn’t possible. What does this mean exactly? Could the underlying cause be right there?

Answer

Hmmm that is interesting. Does this mean the pseudogene is inserted in TNXB? Pseudogenes are non-functional regions that have characteristics of and were developed by protein-encoding genes, but which are not actively transcribed or translated. Some gene elements can actually „jump“ and for example deactivate a functional gene by for example inserting itself into Intron/Exon Splicing sites (in that case Exome Sequencing fails). It could actually be disease causing and even lead to haploinsufficiency. But this can only be certain after sequencing the whole gene.

Question

The exact wording was: Because of the existence of pseudogenes the Exons 32-44 of TNXB cannot be evaluated. Can 2 mutations on 2 different genes interfere with each other?

Answer

This sounds like it could be a methodological problem. If pseudogenes (that can occur everywhere) that have a high sequence-similarity with some TNXB exons are present during sequencing it could be impossible to differentiate pseudogenes from the actual TNXB exons. But it is really sad that this happened in the terminal exons. In our study exon 44 was the locus of causative mutations. And yes, if for example two proteins are part of the same process then mutations in both can interfere with the same process. Then mutations on different genes could even cancel each other out phenotypical. But that is very rare.

8

Question

The TNXB gene does not encode for collagen-modifying enzymes but maybe controls collagen-synthesis. How is that meant? Does it basically synthesis collagen but without a defect on those?

Answer

TNXB encodes for Tenascin X, an organizer of collagen. Collagen fibers have to have a defined degree of cross-linking and those links have to be in defined places to ensure the for us ideal elasticity and strength. TNX fulfills a function in this process but it is still unclear how. We do not have the methods to research it yet (and this irritates me a lot). Matrix biology is very difficult because there are so many factors that interact and we can hardly isolate all the single players to study all physiological relevant processes.

9

Question

TNXB mutations are related to EDS and CAH but how does the mutation lead to CAH and EDS? What EDS type is this then? Hypermobile, vascular, classical? Is the risk to develop POTS or another form of Dysautonomia increased with this type of EDS since the adrenal glands produce hormones like cortisol and aldosterone which regulate blood volume and blood pressure?

Answer

The affected gene in CAH lies next to TNXB. 9 percent of all CAH patients harbor deletions that bring together CYP21A2 with the neighboring TNXB and lead to a chimeric allel which does not function as CYP21A2 nor as TNXB. This is called CAH-X and is seen as a different EDS subtype.

There is only a small group of CAH patients who was characterized and in this group there was not seen an increased amount of POTS. We do not know of a direct correlation between TNXB mutations and Dysautonomia at the moment. But since 95 percent of all genes are regulated in any form at any point in our development through steroids it is very hard to find correlations for conditions like CAH. Cortisol simply influences too many things.

10

Question

A publication from 2015 states that TNX could be a possible marker for ovarian carcinoma. My mother was hypermobile with hematomas and died because of ovarian cancer in 2013. Is there a possible connection?

Additional question

Are there comorbid conditions like cancer in hEDS?

Answer

Not that it would be known by now. TNX was described in connection to neurofibromatosis and malign mesothelioma. But in those cases it did not come to TNX deficiency but more to an overproduction of TNX. This still does not mean that there is any connection. But this illustrates pretty good how critical dose effects are and that we cannot simply compensate the lack of TNX by replacing it with more TNX that then leads to another, different disease.

11

Question

Many EDSers have intolerances or high rates of side effects for medication. Often there are defects/mutations in Cytochrome P450 genes. I have 3 enzymes that metabolize intermediary because of mutations. Do EDS patients have problems with proteins overall?

Answer

There are no indications that EDS patients have a general problem with proteins or high mutation load. But there are areas in our genome that are less stable than others and were mutations more commonly occur and there are processes that can lead to similar mutations in different genes. Insofar different mutations can occur together.

12

Question

My skin biopsy is positive for EDS but the genetest is not. I am supposed to be tested again together with my daughters. Does it make sense to test again?

Answer

I am not a doctor which is why I unfortunately cannot say if diagnostic makes sense or not. If the skin biopsy was positive the cause could be on another gene than TNXB. Over 90 percent of all hypermobile type EDSers do not have a mutation on TNXB.

13

Question

I am suffering from vEDS and cEDS, both genetically proven but mostly I am having vEDS symptoms. Would an additional skin biopsy make sense?

Answer

Oh that sounds bad 🙁 I am not a MD and cannot say what counts as useful diagnostic test or not. But there is some kind of stepwise approach: clinical signs > tissue test > genetic testing. It is for sure good if all of those match but if you have clinical signs (especially in vEDS) and a known genetic mutation I do not see what additional diagnostic insight a biopsy would give.

14

Question

They are suspecting EDS HT in my case but still some doctors question this diagnosis because my elbows and knees are not hyperextensible and I do not have any skin involvement other than thick scarring. My fingers and hips are very hypermobile, my joint capsules are lax and I suffer from heart valve regurgitation. Are there other connective tissue disorders that should be taken into account?

Answer

There are many connective tissue disorders which all overlap at some point (which is why finding genetic markers is so important). Maybe, if there are new diagnostic criteria in the end of the year other joints might be included. If the orthopedic surgeon feels uncomfortable to operate on EDS patients I am not sure if he would feel better with another connective tissue disorder…

Question

Thank you for your answer. Are there databases or documents in which all connective tissue disorders and their symptoms are included?

Answer

Unfortunately „connective tissue disorder“ is a large field and if we include the „non-hereditary“ as well even conditions like Lupus and other autoimmune disorders would count as CTD. Here is something that might help:

Murphy-Ryan M, Psychogios A, Lindor NM. Hereditary disorders of connective tissue: a guide to the emerging differential diagnosis. Genetics in Medicine. 2010 Jun 1;12(6):344-54.

or maybe this one is better: a comparison between EDS and other CTDs in Colombi M, Dordoni C, Chiarelli N, Ritelli M. Differential diagnosis and diagnostic flow chart of joint hypermobility syndrome/ehlers‐danlos syndrome hypermobility type compared to other heritable connective tissue disorders. InAmerican Journal of Medical Genetics Part C: Seminars in Medical Genetics 2015 Mar 1 (Vol. 169, No. 1, pp. 6-22).

15

Question

A skin biopsy showed positive results for EDS in my case but unfortunately the type is so far unclear. I might have a little bit of all main types of EDS. Clinically I am more of hypermobile/classical. Now a genetest should clarify those things. In the meantime I found out that there are many EDSer who are suffering from more than one type or rather, there are different types of EDS within the same family. Doctors keep telling me that this is very unlikely since there is usually the same gene mutation in the family which is then inherited. But to me it seems like there are many exceptions. Is there scientific insight that different types can be inherited in the same family or is this only coincidence?

Answer

Since the different EDS types are caused by different gene defects I wouldn’t know why they should not occur together. For sure one can have one mutation in CYP21A2 and one in TNXB. You can also inherit one from your mother and the other one from your father and through a crossing over you have both on one allele which again you can inherit (or the opposite happens and you pass on a completely healthy allel to your child and both mutations get lost). Also, every mutations has to be spontaneous at some point in family history before it can be inherited. This means there has to be one person in every family that is the first one with a new mutation that has not been seen in their parents.

16

Question

Can we see on a gene whether the mutation is spontaneous or do we always have to have the family history?

Answer

Only the family history shows whether the mutation is spontaneous or not. If your mutation is not found in your parents it is a spontaneous new mutation (or a failure during sequencing…)

17

Question

What can a patient do with a so far unpublished mutation?

Answer

The patient needs to then find doctors or scientists that are researching this mutation and hope to be included in a research study (even if this does not happen you can still learn something). Do not let yourself be limited by geographic because this kind of research is done all over the world.

18

Question

Can we for some mutations (not specifically TNXB) circumvent a metabolic blockade for example by using micro nutrients or a special diet? Can you tell in which cases this would work in which not?

Answer

In the case of TNX a special diet or nutrients would not work because TNX is a structural component but not involved in the collagen metabolism.

If the defect is integrated in an anabolic or catabolic pathway then we might be able to take some influence by changing the diet or taking nutrients (for example Vitamin C to help with collagen synthesis). It is maybe possible to improve your condition but it is no causative treatment. For example there was described that TNX Y production in chicken (chickens do not have TNX) reacts to mechanical stimulation. This gives an indication that sport can influence connective tissue forming. But I wouldn’t start kickboxing with EDS 🙂

Another example is CAH in which a mutation leads to deficiency of a hormone which can be replaced.

19

Question

What new developments are there in gene research (diagnostics, therapeutics)?

Answer

Diagnostics: All the new sequencing methods and expression statistics are getting cheaper and are going to be standard diagnostic methods at some point. Whole Genome methods are going to be standard.

Therapeutics: We are going to go into the direction of individualized medicine which means everything is based on the needs of the patient (for example drug metabolism testing).

Interesting: CRISPR/Cas9 Genome Editing (direct Genome editing system) is the scariest and most promising thing at the same time that molecular genetic has to offer.

20

Question

Which lab in Germany tests TNXB and are there biochemical tests for mutation analysis like protein analysis?

Answer

As scientist in the US I cannot say which lab offers those tests but theoretically every genetic lab should be able to perform standard genetests. Genetesting is genetesting. There is nothing special about it.

There is no biochemical test for mutation analysis. Biochemical tests detect protein function or presence. In case of TNX there is an ELISA (antibody based test) but it is hardly available commercially and mainly used in research. But since not every TNXB mutation leads to TNX deficiency the TNX ELISA cannot rule out TNXB mutations as cause of the symptoms.

21

Question

What causes early cartilage degeneration in EDS?

Answer

This is more of a clinical question (but I do not know anyone who is recently researching EDS and cartilage/ligaments) As far as I know degeneration of cartilage is a purely mechanical process which develops through friction between joints (which is why it occurs increasingly in older people). With hypermobility the degeneration is higher (because of the laxity of ligaments). But I would not be surprised if they found out that the genetic cause of EDS would also influence cartilage.

22

Question

Are there mixed/overlapping types of hEDS and vEDS at the same time?

Answer

In general every form of overlap is possible. For example one COL1A1 mutation was described that led to an overlap of classical and vascular EDS. Or a COL1A1/COL1A2 mutation has been found to cause an overlapping type of EDS and Osteogenesis imperfecta.

De Paepe A, Malfait F. The Ehlers-Danlos Syndrome, a disorder with many faces. Clin Genet 2012; 82; 1-11.

23

Question

I participated in a big POTS questionary study accomplished by Vanderbilt University. They asked about everything from my way to diagnosis, misdiagnosis, type of diagnostic, symptoms, medication, additional therapeutic measures, severity, comorbidities, limitations during everyday life, social and economic impact.

The benefit was that it was an online survey that everyone worldwide could participate in which made it possible to reach more people. Would a survey  that could be used to find new research topics for clinical studies based on similar occuring answers be useful for EDS as well? (They would for sure discover many similarities in different groups that nobody expected before)

Answer

Statistical questionaries around the topic misdiagnoses would be very interesting as well (maybe even with a map to see if they might occur in one area more frequently). Those surveys could even be created by individuals because they do not need a clinical infrastructure.

24

Question

How does research look like and what are the benefits of biomedical research?

Answer

We don’t just see association between a special genotype with a phenotype but we are in a position to find causative relationships and develop new options for diagnostics and treatments.

It is nice to know what you are suffering from but it is even better if there is something we can do about it and in EDS we do not have causative treatments at the moment.

25

Question

How does a genetic predisposition lead to a phenotype?

Answer

Example: TNX C>G in Exon 40

The Cysteine is replaced by a Tryptophan. The replacement of Cysteine with a Tryptophan leads to  breakage of a disulfide bond disrupting the folding of secondary structure. This again leads to failure in organizing fibrillin fibers in tissue and then to the EDS tissue phenotype.

26

Question

I have another question about pseudogenes. Is it possible that a different lab could sequence all exons? Or is it impossible to sequence TNXB due to the pseudogenes no matter what lab?

Answer

Most of the sequencing protocols are very similar because many labs use the same machines (Illumina is common) and probably also similar molecular probes. Without being ensured by someone that the same problem does not occur during resequencing I would at least not pay it out of pocket. Whole Genome Sequencing would be an alternative. My favorite mutation is 12174C>G which is discussed in our paper because it is very obvious.

27

Question

What is the recent state of research around TNXB? Which Universities do study it?

Answer

We never know enough. 🙂 We know that TNX is some sort of organizer but that’s about it. The exact connection of connective tissue building of the extracellular matrix are not known and we do not have the methods to study it.

What we need in research is a system that we want to understand, in this case TNX and the extracellular matrix, and techniques that enable us to study it. We cannot differentiate all connections at the moment.

Groups at the NIH do research TNXB at the moment but since this changes a lot and there is no uniform database it is very hard to give any information about that.

To receive recent studies and to see who currently works on it one can create an alert on Pubmed.

28

Question

What are the consequences of more than one copy of a gene on a chromosome? And how does this happen?

Answer

There are genes we want to have more copies of (for example antibody fragments) and there are others where more copies lead to dose effects that are not wanted (like trisomy).

Copies develop through unequal crossing over during meiosis and some ares of our genome are hotspots for gene-duplications (for example RCCX).

29

Question

Are there next generation sequencing panels that can test all possible genes at once? Costs?

Answer

There are panels that test a significant part of all known causative genes for many genetic connective tissue disorders. Of course they are based on already described markers and do not include new mutations.

I cannot say anything about costs because I do not work in a routine lab.