Download Syndrome. Reviewing the diagnostic utility of genetic testing for EDS. My name is Dino Wapia, I’m working for Probrin Genetics, and I have the privilege of hosting the webinar. We are excited to have Professor Claire Franco Mano as a speaker today. Professor Franco Mano has been involved in the care of individuals with EDS throughout her career. During her years at the University Institute of Health, she managed along it to denial study on the Natural History of EDS that ran for over 20 years. Professor Franco Mano joined Indiana University in 2019 as a professor of medicine and molecular genetics in School of Medicine and director of the Residence Training Program in genetics at Indiana University. Thank you for being here today, Claire. Please, it’s a great pleasure. Thank you so much for having me and welcome everyone. Today we’re going to be discussing genetic testing for the Yelers Danlos syndromes and we’ll talk about the indications for testing, some of the methods and implications of genetic testing. Just a quick disclaimer, this presentation is for educational purposes only and the opinions or points of view expressed in this presentation represent my views and not necessarily those of Blueprint Genetics. So our presentation objectives today are to review the Ellers Danlo syndrome’s classification and inheritance, to discuss genetic testing options, to explore the limitations and uncertainties of genetic testing, and to emphasize the importance of genetic counseling. So we’ll start off with what are the Yeller’s Danlos syndromes. These are a group of heritable disorders of connective tissue, and today we recognize 13 different types that affect the collagen and the extracellular matrix, with the key features of joint hypermobility, skin hyperextensibility, and tissue fragility. The 13 different types of Ehlers Down Low syndrome have in common these things. They are all involve. They all involve the connective tissue and joint laxity is a feature in all of them. The skin is involved in most types with skin fragility, skin hyperextensibility, stretch Marks, and easy bruising. Predominant features and many other organ systems are involved to varying degrees in the different types of EDS. So why is it important to make an accurate diagnosis? Well, an accurate diagnosis informs risk management and medical management. It prevents misdiagnosis and delayed care. An accurate diagnosis is absolutely critical for surgical planning and emergency risk, and it enables reproductive counseling and family planning. Now genetic testing enables us to confirm the diagnosis in most types of vellers Download syndrome. It will identify the causative genetic variants in again in most types and clarify inheritance. And genetic testing is essential for family cascade testing, which means that if we have a patient who has multiple family members and there are questions about whether or not they might be affected, if we identify a pathogenic variant in our patient, we can use that information to test other members of the family. So the diagnostic classification for the different types of Ellers download syndrome was established in 2017, and that 2017 international classification recognized 13 different types of EDS. And the diagnosis for each of them is based on clinical features, inheritance, and the genetics. And this 2017 international classification replaced the 1997 nozology, which was called the Villefrage nozology. The 2017 International Classification is available at the Ehlers Danlos Society website, which is at the URL at the bottom of this slide. It’s ellers- danlos.com\ 2017- EDS- International- Classification. These are the 13 different types and the mode of inheritance for each of them. And I’m going to be talking a little bit more about each of these in turn. And this flower representation is from Joanna Amberger and her colleagues at Johns Hopkins, displaying the different types of Ehlers Danlos syndrome and the genes that are known to cause each one of those 13 different types. And you will notice that for the hypermobile type, the gene is listed as unknown here. And that unfortunately is still the case that the gene or genes underlying the hypermobile type of Ehlers Danlos Syndrome is or are still unknown. So the classical, vascular, hypermobile and arthrocholasia, myopathic and periodontal types are all inherited as autosomal dominant conditions. The remaining types are inherited as autosomal recessive disorders. And it’s important to remember that we can see and do see de Novo mutations and the vascular type of Ellers Danlos syndrome. So the fact that a child or a patient it’s not have an affected parent does not necessarily preclude the diagnosis of vascular Ellers Danlos. Now when the 2017 criteria were published, it was recognized that there were people with joint hypermobility who would not fulfill the diagnostic criteria for any of the different types of Ellers Danlos syndrome. And so there was a paper published looking at the classification of joint hypermobility. And I think it’s important to remember that there are going to be people out there with joint hypermobility who do not meet the diagnostic criteria for the Ehlers download syndromes. So when we think about the types of joint hypermobility, it the hypermobility may be localized, meaning that it involves only a limited number of joints. It may be peripheral, involving primarily the small joints of the hands and feet, with absence of large and axial joint involvement. You may see generalized joint hypermobility, which means joint hypermobility at more than five sites including the large and axial joints. And we generally use a scale called the bite and scale to assess for the presence of joint of generalized joint hypermobility. And finally, a person may have a history of joint hypermobility which is no longer present on examination and we call that historical joint hypermobility. So when we’re classifying joint hypermobility, there we may be people may be asymptomatic. So we might have asymptomatic people and they may have either localized peripheral or generalized joint hypermobility, in which case that’s what we call it localized joint hypermobility, peripheral joint hypermobility or generalized joint hypermobility. A person may have a very well defined syndrome associated with joint hypermobility, such as Ellers Danlos syndrome, if they meet one of the diagnostic criteria for one of the types of Ellers Danlos. Or there may be people with symptomatic joint hypermobility who do not meet diagnostic criteria for any other known hereditary syndrome associated with joint hypermobility. And these are the people in whom we diagnosed the hypermobility spectrum disorders. So the hypermobility spectrum disorders involve joint hypermobility plus one or more secondary musculoskeletal manifestations of joint hypermobility. The overall clinical picture for people with HSD does not meet the diagnostic criteria for any of the syndromes associated with joint hypermobility. And these, This hypermobility spectrum disorder category was intended as an alternative label for individuals with symptomatic joint hypermobility who do not have any of the rare types of EDS and do not meet the diagnostic criteria for hypermobile EDS. And the intention when this category was developed was it to identify discrete subtypes filling the full gap between asymptomatic joint hypermobility and hypermobile Ellers Danlos syndrome. So what I’m going to focus on next is the most common types of Ellers Danlos syndrome. We’re going to discuss the classical type in which we see joint hypermobility and very stretchy, fragile translucent skin. The vascular type in which joint hypermobility may be limited to the small joints in the hands. We may not see generalized joint hypermobility in the vascular type. In the vascular type, we also see aneurysmal dilation and rupture of the medium sized arteries and rupture of the hollow organs including the bowel, bladder and uterus. And then the hypermobile type of Ellers Danlos syndrome, which is the most common type of EDS characterized by joint hypermobility, less severe skin involvement than we see in the classical type. And as I mentioned earlier, we still do not have the underlying genes identified. So these are some of the cardinal clinical signs for the classical type of Ellers Danlos syndrome. We see this very, very stretchy skin, unusually stretchy skin and atrophic scarring. So widening of the scars of they don’t heal in a nice thin line, this unusual bruising of the shins and these in panel O here at the bottom left. These are called molluscoid pseudo tumors that are they just look like little growths of skin on the on the knees and sometimes we see them on the elbows and in the lower right hand corner of this illustration you see the features typical of the bite and scale. So looking for generalized joint hypermobility. So to meet the diagnostic criteria for the classical type of Ehlers Danlos syndrome, we have to have major criteria 1 and major criteria 2. So the major criteria are skin hyperextensibility and atrophic scarring and then generalized joint hypermobility. Or we can make a clinical diagnosis if they have the skin. The extensive skin hyperextensibility and atrophic scarring typical of the classical type of EDS and three or more of the minor criteria, which are easy bruising, soft doughy skin, skin fragility, or traumatic splitting of the skin. The molluscoid pseudo tumors I showed you in the previous slide, subcutaneous spheroids. They’re difficult to take a photograph of, but you can feel them underneath the skin. It kind of feels like there’s gravel underneath the skin when a person has subcutaneous spheroids, hernia or a history of hernia, epicanthal folds, complications of joint hypermobility including things like sprains, dislocations and subluxations, pain and flexible Pez plainus or flat foot. And then a family history of a first degree relative who meets the clinical criteria is also considered one of the minor criteria. So if we see one the the major criteria is skin involvement and three or more of the minor criteria, they meet the clinical diagnostic criteria for classical EDS. And we would then want to do the testing to see whether we can find a pathogenic or likely pathogenic variant and call 5A1 or call 5A2, which are the two genes that predominantly cause the classical type of EDS. Now these are the clinical cardinal signs for vascular Ehlers. Download Syndrome. These are illustrations from a paper which was published in 2011, demonstrating kind of typical facial features with a kind of long, thin nose, thin lips and very prominent eyes. We don’t always see this facial appearance, but when we do see it, it can be very helpful. And then kind of aged appearance of the hands and a very, very unusual bruising, really predisposition to extensive bruising. So when the diagnostic criteria were being published in 2017, the committee that was looking at vascular EDS did not put forward actual clinical diagnostic criteria, but rather clinical indications for genetic testing. And what they said was if one or more of the major criteria were present or if there were several of the minor criteria and they did not specify, but if you saw more, more than two or three of these minor criteria, that would be a clinical indication for genetic testing for vascular Ehlers download syndrome. So the major criteria. And if you see one or more of these, you should be thinking about testing for vascular EDS family history of VVEDS with a documented causative variant in Colt 3A1. That’s for sure. Like if if you know there’s a causative variant in a family member, we go look for that causative variant. Arterial rupture at a young age. Spontaneous sigmoid:. Perforation in the absence of known diverticular disease or other bowel pathology. A uterine rupture during the third trimester in the absence of previous C-section or severe peripartum or perineal tears, and then finally the carotid cavernous sinus. Fistula formation in the absence of trauma. So any of those would be an indication for genetic testing. And then if we see a combination of these minor criteria, we would also think about genetic testing, and these include bruising unrelated to identified trauma, thin translucent skin with increased venous visibility, characteristic facial appearance, which I described to you earlier. Spontaneous pneumothorax or collapse of the lung. Akrigeria is the word for aged appearance, particularly in the hands. Talpesaquinovaris or clubfoot. Congenital hip dislocation, hypermobility of the small joints of the hands and feet. Tendon and muscle rupture. Keratoconus gingival recession and gingival fragility and the early onset of varicose veins meaning underage 30 and starting before pregnancy in women. Now thinking about the hypermobile type of Ehlers Danlos syndrome and the hypermobility spectrum disorders. These are clinical diagnosis because we do not have genetic testing to offer for these diagnosis as of this time. You can find the diagnostic checklist by going to ellers danlos dot dot org, Ellers- danlos.org then look for professionals, the heds diagnostic checklist. So just working your way through on the society web page. And this is the full URL that will take you to this heds diagnostic checklist. The first criterion is that of generalized joint hypermobility. And as I mentioned earlier, we’re using a score called the Biden score to assess for the presence of generalized joint hypermobility. The we look for hyperextensibility at the metacarpal phalangeal joint of the fifth finger. If this extends beyond 90°, we give one point for each side, right and left. If the person is able to touch their thumb to their forearm, they get one point for the right and one point for the left. A hyperextension of the elbows beyond 190°, so 10° more than 10° beyond the straight extension gets one point each, and then hyperextension of the knees beyond 190° also one point each. And finally, if the person is able to put their palms on the floor without bending their knees, they get one point. And we have age specific cut offs for defining generalized joint hypermobility in prepubertal children and adolescents. We need a score of 6 or more. For men and women up to the age of 50, we need a score of five or more. And for men and women over the age of 50, a score of four or more defines generalized Joint Hypermobility Criterion 2 for hypermobile alerts. Download Syndrome requires 2 out of 3 features. We have feature A, Feature B, and Feature C Feature A is a list of 12 criteria, 12 elements that we can either see on physical examination or inquire about from history, and these include unusually softer velvety skin, mild skin, hyperextensibility, unexplained stretch marks, bilateral piazogenic papules, and I’ll show you a picture of those in a minute. Recurrent multiple abdominal hernias, Atrophic scarring, prolapse of the pelvic floor, ****** and or uterus, dental crowding, and a high narrow palette. Arachnodactyly, which is defined as a positive wrist or positive thumb sign bilaterally. An arm stand to height ratio of more than or equal to one point O 5 and the presence of mitral valve prolapse or aortic root dilatation with AZ score of more than +2 on echocardiogram. So if there are 5 or more of those elements, we can say that feature A is met. Feature B is a positive family history with one or more first degree relatives independently meeting the criteria for hypermobile Ehlers download Syndrome. And Feature C is the presence of either musculoskeletal pain in two or more limbs recurring daily for at least three months, chronic widespread pain for more than three months, or recurrent joint dislocations or frank joint instability in the absence of trauma. And one or more of these will qualify for Feature C to be met. So we need 2 out of these three features to say that criteria 2 is met. And these are just some illustrations of the features that we’re looking for in feature. In feature a mildly stretchy skin, the piasogenic papules are these little bumps on the heels. Panel C illustrates the atrophic scarring that we can see with widening of the scars and the kind of wrinkly appearance to the skin in between the edges of the scars. And then Panel D illustrates the Street E or stretch Marks, and we’re looking for those in the absence of weight gain and in women prior to pregnancy. So criteria 3 says that we have to exclude other possible diagnosis. It just says that we have done our due diligence and really thought about all the other possible diagnosis that might explain our patient’s presentation. So we want to exclude the unusual skin fragility which should prompt consideration of other types of EDS. We want to exclude other heritable and acquired connective tissue disorders that might explain the person’s pain and exclude alternative diagnosis that such as diagnosis that might involve the muscle or hypotonia. So we we want to make sure that we’ve really, really thought about all the other possible diagnosis. So this brings us to this really pivotal question of when should we test? And I have to credit Dr. Allen Hakeem, who came up with this wonderful mnemonic telling us to take a focused history. And so the things that we want to really think about, is there a family history of a confirmed diagnosis of another type of EDS or other hereditary disorder of connective tissue? Are there organ or internal problems such as bowel rupture or bladder rupture? Have there been cardiovascular problems like extreme extreme valvular issues that might point to the cardiac valvular type of Ellers download syndrome or have there been aortic aneurysms or arterial aneurysms that might point to the vascular type of Ehlers Dallow syndrome or other aortopathies? Is there in family history of unexplained sudden death at a young relatively young age of less than 50 years? Are there skeletal abnormalities such as congenital hip dislocations or club feet that might point to other types of Ehlers Danlos syndrome or the kyphoscoliosis that would point towards the kyphoscoliotic type of EDS, for example, And then other skin abnormalities. So is the skin typical of the hypermobile type of Iller Stanlow syndrome or is it more stretchy than we would expect it to be or more fragile? Or are there those really unusual molluscular pseudo tumors or the subcutaneous spherules that are typical of the classical type for eye abnormalities? We think about the brittle cornea that we see in the brittle cornea type or if there are dislocated lenses that would point us to Marfan syndrome, the there are other blocking on the word. We can also see dental abnormalities that may point us to the periodontal type of Ellers Danlow syndrome or the gingival abnormalities that may be seen in the vascular type. And also if we see dentinogenesis imperfecta or very fragile teeth with the abnormal dentin, that may point us to osteogenesis imperfecta, another hereditary disorder order of connective tissue. So if we think about this mnemonic focused and you can remember family history, organs, cardiovascular, unexplained sudden deaths, skeletal abnormalities, skin abnormalities, eye and dental, if you see any of those in your patient or in their family history, this would point towards testing. So then we have to choose the right test and really our clinical phenotype is going to determine our testing strategy. And we have to think about do we want to do single gene testing versus panel testing versus sequencing of the either the exome or the whole genome. Now we might think about single gene testing if we have a very, very high suspicion for one particular type of Ellers Danlos syndrome. So if the person is really meeting like every criteria clinically for classical Ellers Danlos syndrome, we might choose to just do single gene testing for well 2 gene testing for Col. 5A1 and Col. 5A2. If we have an extremely high suspicion for the vascular type of Ellersdale syndrome, we might do single gene testing for call 3A1. And similarly for the very rare recessive types, we can do single gene testing for the specific genes for those types. But honestly in 2025, we usually are not doing single gene testing. Most commonly we will go to multi gene panels and these are gene gene panels that depending on the lab will include between 10 and 95 connective tissue disorder genes. And these are very efficient if we if it’s possible that the clinical presentation could be one of any number of different types of connective tissue disorders. So typically this, this is honestly the one that we’re using most often in my clinic these days is the multi gene panel for hereditary disorders of connective tissue. Now we do exome or genome sequencing in some situations where we really want more comprehensive coverage. Remember the exome is gonna give us all the information of the known genes, the coding regions of the known genes. So this represents between 22 and 25,000 genes and the coding region. So the exons of those genes and the genome is going to give us the 3 billion base pairs, the whole genome that’ll give us regulatory regions and the introns of the genes that we do not see in the exome. Both of these are much more comprehensive than the panel testing. They’re useful for novel or unclear cases or if we’ve done a panel test and we haven’t found anything. But we really think there’s something there that we’ve just not been able to find with the panel test. And the important thing about the exome and genome is that this may detect incidental findings. And it’s very important in your counseling to talk about those incidental findings with the family. We can also do copy number variant analysis and this this strategy is important for detecting large deletions. For example, in TNXB or a gene called TPS AB1, we may see large deletions or rearrangements that may be missed by sequencing alone. So that’s another type of testing that we may have to consider. Now when we’re talking to the patient and their family about the possible outcomes of genetic testing, we tell them that they they we may not find any variants and that would be what we call a completely negative test. Or there may be variants identified, and these may be benign or likely benign, pathogenic, or likely pathogenic, or variants of uncertain significance. And we use the standards and guidelines for the interpretation of sequence variance from this paper that was published by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. And this paper gives us considerations for classification of the variance. And the things that we think about are the frequency of the variant in large population databases. So if the variant is extremely rare or has never been seen before, it’s more likely to be pathogenic than if we see it in a third of the population. For example. We look at in silico predictions, the computer based predictions of whether the variant will be damaging to the protein. We look at available functional data, segregation data in families, information about whether the variant is de Novo or not, and information about whether or not it’s allelic. So is it in trans with a known pathogenic variant or CIS for example? And this table really gives us sort of the like the way people think about how you interpret the evidence for population data, computational and protective data, functional data, segregation data, and so on. And I recommend this paper to you as an as a way of familiarizing yourself with the kinds of thinking that goes into establishing whether a variant is pathogenic, likely pathogenic, benign, or likely benign, or if we just don’t have enough information to say we call it a variant of uncertain significance. And there are a number of resources out there that can help assess whether or not a variant is benign or pathogenic. And these include Variant Matcher, the Franklin Variant Analyzer, and Verisom, which can help all of these. You can find them on the web and put your variant in there and it will give you information about whether how it’s classified and how it came to that conclusion. So I’ve got a couple of cases to discuss here. The first one is a case of suspected classical Ellers Danlos Syndrome and a 25 year old gentleman with a lifelong history of joint hypermobility, exceptionally stretchy skin, hemosiderotic scars on the shins and molluscoid pseudo tumors on the knees and a mother and brother with similar findings. So in this case, we did directed or directed genetic testing of CALL 5A1 and CALL 5A2 and found a path likely pathogenic variant in CALL 5A1 and cascade testing confirmed the presence of this variant and the affected mother and brother. So this is a very straightforward situation where the clinical presentation was very, very specific. We could do genetic testing of two very specific genes. We found a likely pathogenic variant and confirm the presence of the variant in two affected family members in the next case. This was suspected vascular type of Ehlers Danlos syndrome. A 42 year old woman who was well but presented with an acute coronary event due to spontaneous dissection of the right coronary artery, and on exam she had translucent skin and hypermobile joints in her hands. But she did not have generalized joint hypermobility because her Biden score was only three out of nine. So we had a suspicion for the vascular type of Ellers download syndrome, but in this case we selected a panel test. Because the differential diagnosis does include a large number of hereditary disorders of connective tissue causing a vasculopathy, So we opted for a hereditary disorder of connective tissue panel. And in this case, we did find a pathogenic variant in Call 3A1, which was not found in either of her parents. So this represents a de Novo variant in Call 3A1 causing the vascular type of Ehlers Danlos syndrome. Now genetic testing does have certain limitations particularly when we’re thinking about hypermobile Ehlers Danlos syndrome or hypermobility spectrum disorder. We don’t have genes to test for. And so if we do that focused history and there are no suspicions, nothing, no red flags that suspect that of for a suspected other hereditary disorders of connective tissue or other types of EDS. We may not test if we’re we’re confident that the clinical diagnosis of hypermobile Ehlers Stanlow syndrome or HSD is what we’re going with the variance of uncertain significance are they leave us with question Marks and that we just can’t really make a diagnosis or exclude A diagnosis when we find these variants of uncertain significance and there are technical gaps and missed variants. So we’re not going to find as I mentioned about the the copy number variants, our current sequencing technology is going to miss rearrangements and large deletions and so on. So we still are not finding everything. And it’s important to recognize that a negative test is not necessarily exclude A diagnosis. So when we’re thinking about the variance of uncertain significance, as I said, we can’t confirm or exclude diagnosis based on these variance of uncertain significance and they require follow up and future reanalysis. And I always tell my patients, please, please, please make sure you’re talking with your, your physician, you know, come back and let’s discuss this at follow up. And we will, we will do a reanalysis in the future to see whether additional information is available about that particular variant because the databases are changing all the time now. Our genetic counselors are absolutely crucial members of the care team. It’s very, very important to discuss the goals, limitations, and possible outcomes of genetic testing before the testing is done and to obtain informed consent for the testing. And then after the testing, we want to make sure the results are explained very clearly. We provide written summaries and address the family implications for whatever testing result is obtained. So our key takeaways are the genetic testing can clarify, but not always confirm a diagnosis. Clinical correlation is essential and genetic counseling will support the interpretation of the genetic test. There’s a lot of work ongoing in this area. Many many people trying to find genes underlying the hypermobile type of Ehlers Danlos syndrome and HSDS. Whole genome sequencing, RNA seq and long read technologies are methodologies that are likely to assume more and more importance and become more readily available and help us in making diagnosis. And the global registries and functional studies research efforts that are going on now are, are going to be very, very helpful in establishing which variants are pathogenic and likely pathogenic and, and to help us in the interpretation of the sequencing data. So there’s a lot, a lot of work that’s moving things forward. Thank you very much for your attention. It’s been a pleasure being with you here today. Thank you, Claire. Excellent presentation. Really really interesting. And I really, really loved all the the patient cases you showed. So really, really thank you. It’s a pleasure. Thanks for having me.
