Unlocking answers with WES Trio

OK, hello everyone and welcome to today's webinar titled Unlocking Answers with Best Rio. This webinar is brought to you by Appropriate Genetic, A genetic knowledge company committed to providing an innovative approach to genetic testing and to ensure accurate and confident in your clinical practice. My name is Taina Vuopio and I have the privilege of hosting today's webinar. Please submit any questions you may have in the Qi box. You can submit them throughout the webinar and we will answer as many as possible at the end. We are excited to have two speakers today. Saija Ahonen and Kirsty Wells are senior geneticists at Blueprints Genetics, specializing in interpretation and reporting whole exome sequence data. Before joining Blueprint in 2019, Saija Avonen completed her PHPHD at the University of Helsinki in canine inherited disease, followed by postdoctoral research studying pediatric neurodegenerative disease at the Hospital for Sick Children in Canada. Kirsty Wells has a background in both research and diagnostics. Before joining Blueprint in 2018, Kirsty completed PhD and post doctoral research fellowships and undertook in depth training in genetic diagnostics in the UK's National Health Service. Kirsty is AUK certified clinical scientists. Hey, thank you for being here today. Please. Thank you, Taina. Good evening or good morning everyone. Thank you for joining us today for this webinar. Really great to see, see many online this evening. So as Taina said, my name is Kirsty Wells and and I'm joined also today by my colleague Saija Ahonen. And we're both really excited to be here today to talk to you specifically today about whole exome sequencing trios and how using a W trio can really help you to unlock answers for patients and families. So before we start talking about West Trios, I'll begin with a quick introduction to Blueprint Genetics. So at Blueprint Genetics, we deliver high quality genetic testing to the global clinical community. We have over 250 employees and we serve over 4000 clinicians in 70 countries. We have core operations in Helsinki in Finland, where we are speaking to you from today. And we offer a range of NGS based test types depending on your needs. So first of all gene panels, also whole exome sequencing, single gene testing, variant specific testing and also screening. But today in this webinar, we'll focus on whole exome sequencing. So let's start first of all with some background to whole exome sequencing in general to get us all on the same page. So whole exome sequencing, always for short, aims to sequence the protein coding regions of all known genes. So around 20,000 of them in a Wes then this means that between 1 and 2% of our DNA is covered because of course the majority of our genome is non coding. But nevertheless, Wes can be a very powerful diagnostic tool because the vast majority of known disease causing variants currently are in these protein coding regions that Wes covers. And also with Wes there's no restriction on what genes you can you, you choose to sequence as as you as you have with a panel. So Wes for this reason can be particularly useful for patients who have a very complex phenotype, for instance, or they may have an unclear clinical diagnosis and a patient like this may have had previously inconclusive genetic testing because it may have been difficult to choose what genes to sequence for that patient. So with Wes, you don't have to make that choice. It's a very comprehensive test. But it's important to realise that not all Wes tests are the same in terms of quality and performance. And the quality and performance of the West that you choose can really mean the difference between a patient receiving a molecular diagnosis from the testing or not. So at Blueprint Genetics, we really aim to optimize our W to give the best chance of identifying A molecular diagnosis for the patient. And this optimization encompasses a lot of different elements. So on the technical side, our wares includes high and uniform coverage. It also includes a large number of clinically non coding variants that are specifically targeted. It also includes optimized CMV detection and also the mitochondrial genome. So all of this is aiming to give you the most comprehensive performance possible. And then on the interpretation side, we know that having up to date gene disease associations in the system is really vital and also skilled interpretation of the variants that are detected by the test is really important. So all of these things work together to give us what we believe is a really best in class whole XM sequencing assay. So today though, we're going to focus on a really specific area of optimization, which is the value of running the your wares as a trio where possible for really optimized performance. So at Blueprint Genetics, we recommend choosing a trio of both parents and an affected child where possible. But using our Wes family product, it's possible to to sequence different family combinations. So first of all, with a duo you can sequence, for example, two siblings together or a parent and a child. With a trio, you the typical family arrangement is sequencing an effective affected child with two often unaffected parents. But it's also possible to do a parent and two children. Or it's also possible to do a quad where you sequence four family members together, so both parents and two children for instance, or one parent and three children. So a number of different family family combinations are possible, basically whichever, whatever works for your particular family situation. But what's really important is that whatever you choose is really vital for the interpretation to know who out of those being sequenced is affected and who is unaffected. This is really important for us to be able to curate the variance effectively. So of all of these family combinations, as said, a trio of affected child and two parents is really we believe the most powerful. And the reasons for that are first of all that having the parental data really facilitates the interpretation. And I will show you a little bit more shortly as to how that works in practice. It facilitates, it allows de Novo variant detection. And this is really important because based on our internal data, approximately half of molecular diagnosis in West cases are de Novo variants. It enables us to see the phase of two heterozygous variants in the same gene. And with a W trio you get really valuable recurrence risk information, a more accurate variant classification straight away, and potentially all testing completed in one step. So let's take a look at some cases now to illustrate these different advantages of using a W trio and how these actually work in practice. So Sire and I will now show you 4 cases, which each illustrate these different advantages of using a Westrio. And all of these cases that we show you today, by the way, are based on real cases from the Blueprint Genetics Archive, but with some details changed or removed to protect patient confidentiality. OK, so let's start with the first case, which involved a small child with psychomotor delay, hypotonia and mild dysmorphic features, and there were no affected family members. So first of all, why is doing a Wes trio useful in this particular situation? Well first of all we have a patient with a very non specific phenotype of psychomotor delay, hypertonia and mild dysmorphic features. So this means that the differential diagnosis is very wise and this means that whole exome sequencing in general is an ideal test to choose as it is a very comprehensive 1. However, this non specific phenotype can make variant curation quite challenging as it can mean that the phenotype potentially overlaps at least partially with many different genetic diseases. So it becomes quite challenging, challenging to rule variants in or out based on the phenotype. But having the parental data really facilitates variant inclusion and exclusion. And I will demonstrate a bit more in a second about how exactly that works. And then also genetic diseases with with these kinds of clinical features can be caused by diseases with very many different inheritance patterns. Though syndromic neurodevelopmental disorders can be inherited in an autosomal dominant manner caused by a de Novo variant or can exhibit incomplete penetrance, variable presentation and even parent of origin effects. They can also be inherited in an autosomal recessive manner in an X linked manner or even in my through mitochondrial inheritance. So it's really important to have the parental data to be able to to see the inheritance pattern and to get recurrence risk evaluation. And then finally having the parental data can potentially impact the classification of the variance that you report. And again, we'll show that in in more detail exactly how that works. OK. So let's have a look now at how all this works in practice. So here on the slide, you can see some of the variants that were detected by whole exome sequencing in this patient. So this patient was found to have several extremely rare heterozygous missense variants in genes associated with autosomal dominant neurodevelopmental disorders and all of them had phenotypes that could potentially overlap with the phenotype of our current patient. And this situation is not unusual in a Wes analysis and it illustrates the difficulty potentially of a pro band only analysis. Without seeing the inheritance of these variants, it does make it a lot more challenging to decide whether to rule the variants in or to rule them out. And it's especially difficult if the patient has a non specific, maybe not very detailed phenotype like this current patient. So in this situation, you may end up having to report multiple variants as variants of uncertain significance and then potentially have more follow up family member testing to do to try to clarify these VUSS. But I said this, this analysis was run as a Wes trio, which included both unaffected parents. And because we had the parental data, sorry, I've jumped forward a little bit there, jumped back because we had the parental data, we could see straight away that this KMT 2A variant was not detected in either parents. So that's consistent with de Novo occurrence. So and then the other heterozygous mis sense variants were inherited from unaffected parents. So that meant that we could, we could rule them out. So this example is showing us how much having the parental data can help with inclusion or exclusion of variants in genes associated with autosomal dominant disorders. So taking a closer look at this gene, KMT 2A, pathogenic variants in this gene, KMT 2A cause Wiedemann Steiner syndrome, which is inherited in an autosomal dominant manner and is typically caused by de Novo variants. The typical clinical features include developmental delay, intellectual disability and hypertonia. So there is an overlap here with the reported phenotype of our current patient. Also short stature has been reported in about half of the patients and they can also be some additional congenital anomalies, but not in all patients. There have been some distinctive facial features that have been described, but again, not in all patients. And also hypertrochosis of the elbows was previously thought to be very characteristic of this condition, but has actually more recently been reported to be found in just over half of the patients. So the point here is that these days the phenotype associated with this gene is actually known to be quite variable and non specific. So this disease can perhaps be hard to diagnose clinically. But with a W trio the molecular diagnosis pops out straight away. So how about the classification of this de Novo misense variant in KMT 2A? In this patient we found a mistense variant that was absent in Nomad. So the reference population database that we that we use indicating that it is extremely rare in the general population. The phenotype is consistent in our patient with the the disease associated with this gene. And also the variant was predicted to be deleterious by the Insilico tool that we use, which is called Revel. And there were no other reports of the variant though in other patients in the literature or on databases to our knowledge. So based on this evidence and without any additional evidence, a variant like this would be classified as a variant of uncertain significance. But in the West trio, we could see that the variant had occurred de Novo in the setting of a novel disease in the family. And in our classification scheme which is based on the ACMG classification scheme, which is is very widely adopted in the field, de Novo occurrence is considered as very good evidence in support of pathogenicity. So de Novo occurrence gives us an extra point in the classification scheme and this allows us to classify this variant as likely pathogenic. And in general, being able to detect de Novo variants in genetic disorders is very useful because the literature supports that de Novo variants are a very frequent cause of genetic disease and particularly neurodevelopmental disorders. In this recent study, a very recent Wes trio study of 173 children with intellectual disability and developmental delay, half of the diagnostic variants in the cohort were de Novo, which also tracks with our internal data as well. So it's definitely advised to do a Wes Trio if the patient has a neurodevelopmental phenotype particularly. OK. So to summarize this case, Wes Trio identified A molecular diagnosis of KM2KMT2A related disease which was due to a de Novo variant which comes with a low recurrence risk and facilitates genetic counselling and management. And Wes Trio unlocked the answer here by facilitating the interpretation leading to a simpler report and immediate likely pathogenic classification and all testing completed in one step. OK, so I will hand over now to to Sia who is going to share an interesting a second interesting case with you demonstrating the advantage of ways Trio in autosomal recessive disorders. Hi everyone also on my behalf and welcome to the webinar.  So our next case example is also a small child who presented with a muscle weakness ataxia with some vision problems and also had a slurred speech motor delay and diagnosed with demyelinating sensory motor polyneuropathy. And no affected family members were reported in this family. So why in this case was trio is then useful? The differential diagnosis for this patient is broad and several different genes could be the cause of the broadband phenotypes. And including the parents also in this case facilitates the variant reporting and possibly also the variant classification. In addition, there is no clear inheritance pattern for this broadband, but the genetic cost if we do the different inheritance model that can be possibly be revealed using the West Trio. So in this case, based on the West Trio analysis, we identified 2 variants in SU R1 gene and based on the parental data, the patient was compound heterocycles for these variants. So the first variant identified was the truncating variant in the last X and of the gene which was inherited from the patient's mother. The variant was rare in Contra population that we use and also other disease causing truncating variants had been previously reported in the literature in patients with SURF 1 related phenotypes indicating that the last X and truncating variants are disease causing and thus according to our classification sheet for recessive disease. Now this variant was classified as like the pathogenic. The other variant identified in this patient was the missus variant that was inherited from the patient's father. This variant was also rare in Contra population and predicted deleterious by the insilical 2 rebel that we use. The variant had not been previously reported, but it was detected as compound heterocycles with another likely pathogenic variant, so it was also classified as likely pathogenic in this program. Pathogenic variant variants in SUR F1 gene have been associated with all the similar recessive demyelinating Chargot Marie 2 disease type 4K. And the clinical signs associated with this disease include for example, muscle weakness and muscle atrophy, cerebral ataxia, mitochondria complex for deficiency and the myelinating sensory motor peripheral neuropathy. And the onset onset of the disease is usually in the first decade of life. And these clinical signs matched well, very well with the reported phenotype of the propane and thus confirmed that the SURF one was the molecular diagnosis for this patient. So why were we then able to classify the identified distance variant as likely pathogenic based on our classification sheet, which is based on the ACMG guidelines for variant classification? And the ACMG guideline state that when 2 heterocycles variants are identified in a gene for a recessive disorder, if one of the variant is known to be likely pathogenic or pathogenic, then determining that the other variant is in trans, meaning compound heterocycles, can be considered as evidence for pathogenicity of the larger variant. So although the missus variant identified in this program had not been reported in the literature before we were, we were able to use this ACMG guideline as additional evidence towards pathogenicity as the missus variant was identified as compound heterocycles with the likely pathogenic variant and thus classify the missus variant also as likely pathogenic in three OS. Analysis of parental data is very informative when when we evaluate the phase of the variants such as in this case, as the two variants were inherited from different parents in three OS, we can use the parental data to exclude variants in orders and more recessive genes. When variants are identified, when the variants that we have identified are inherited from the same parent and that's not causative for recessive disease. And this then reduces the number of reported variants when we can, when we know the phase based on the parental data that is included in the West trio. So how in this case, the West unlocked the answer for for the patient, the molecular diagnosis was established as SUR 1 related disease and as the genetic course was identified. Also, the recurrence risk is now known for the future siblings and can be considered in family planning by offering the parents genetic counselling. If this case would have been a pro band only exome, the SURF 1 missense variance would have been remained as a variant of uncertain significance as the face of the variance could not have been determined in pro band only case and thus the molecular diagnosis would not have been established based on the pro band only W analysis. Now as the parents were included in this trio example, it enabled an immediate likely pathogenic classification for both of the identified variants in this one step test for the program and then no further parental testing is then required. And now Kirsty will present our next example case. Yes, thank you, Sia. Let's take a look then at a third case now. And this case involved a baby with microcephaly and the mother was also said to have microcephaly and with cleft palate. And then there was a mention that there was a maternal relative who had a small jaw. And again, this analysis was run as a Wes trio. So again, why is West trio useful in this particular situation? So here we have a slightly different situation in that one of the parents is potentially also affected as well as as the child. And and in this kind of situation, W trio can can also be really useful in this situation. With these clinical features, it's not completely clear if the mother and the child have the same disease. Both have microcephaly and additionally the mother has cleft palate. But microcephaly has potentially very many different genetic and perhaps non genetic causes. So it's not a given that the the the disease is the same in the mother and child. So testing with the trio in this situation can help us determine if they have the same genetic disease and what the inheritance pattern is.  And here it's useful to do a trio rather than testing just the mother and child in a duo, because doing a trio, it's possible to see if the baby's phenotype has a different cause caused by perhaps a de Novo ovarian, for instance. So by testing with a trio, we will be able to see that information. So what did we find in this family? The trio has identified A heterozygous likely pathogenic deletion of a single exon of a gene called EFTU D2. This deletion was approximately 490 base pairs in size and the deletion involved an out of frame exon, meaning that the deletion of this exon would be predicted to lead to a frame shift and a loss of protein function. And loss of function is an established disease mechanism in EFTU D2 related disease, meaning that we could classify this deletion immediately as likely pathogenic. And since we had the parental samples, we could see that this deletion was also identified as heterozygous in the baby's mother. So taking a look closer look at this gene EFTU D2 pathogenic heterozygous variants in this gene cause mandibulofacial dystosis with microcephaly. This is inherited in an autosomal dominant man and the typical clinical features include malar and mandibular hyperplasia, microcephaly, ear malformations and or hearing loss and also other associated craniofacial malformations. And there can also be intellectual disability with variable severity. So we can see straight away here that we have some overlap with the phenotype in in in this baby also the mother and potentially also the more distant maternal family member. So what's interesting about this disease though, is that even though it's highly penetrant, it's very variably expressive, which potentially explains the slightly different phenotype in the mother and child and also could explain the possible mandibular hyperplasia in the more distant maternal family member. So where's Trio here identified A molecular diagnosis of autosomal dominant EFTU. The two related disease and the parent and child were determined to have the same variants of the same genetic disease. So this facilitates testing of extended family members and leads to makes possible genetic counselling and management. So in a pro band only analysis, if we had only tested the baby here, it still would not be clear if this deletion was inherited from the potentially also affected mother or if it was de Novo. So doing a trio in this situation was was really helpful. So finally, I will hand back now to Saija to show you one last interesting case, which demonstrates the value of Wes trio in a slightly different diagnostic situation. Thank you, Kirsty. So our last example also considers a small child who presented with developmental delay, dysmorphic morphic features, some behavioural problems, first degree heart block and the clinician suspected a Noonan syndrome is in this patient and there were no affected family members in this family. So why in this case West Trio is very useful? Again, there is no clear diagnosis, although a syndrome is suspected, but it's also not clear what the inheritance model would be in this case. And using a restrio and having the parental data included may sometimes reveal something unexpected for the clinicians. So based on the Restrio analysis, we identified A mitochondrial variant in this patient in a mitochondrial gene called MTTE and the patient had this had this diagnostic variant in this gene with the heteroplasty level of 20% in the tested DNA sample. The variant was also detected in the mother sample who was reportedly unaffected, but the heteroplasma level in the mother was very low. So the MTTE variant that we identified in the propane, it's a known disease causing variant and it has been reported in the literature in multiple patients. The associated phenotype is very variable and also the age of onset is varies a lot from birth to adulthood and even within families with the same variant. There is a very broad broad range of associated phenotypic features which include muscle phenotype, diabetes, hearing impairment, neurological phenotypes, some ophthalmological phenotype, cardio related phenotype, epilepsy and also developmental delay and behavioural issues have been reported in association with this variant and some of these features also match with our patient in question. In addition, this variant is already annotated as confirmed disease causing variant in the MiTo map database and it has been reported there in association with different mitochondrial phenotypes. So in this case, the molecular diagnosis was identified as an MTDE related mitochondrial disease. As the variant was also detected in the mother, the mother can now have a clinical evaluation to detect possible clinical signs of the disease. In addition, the recurrence risk is now known for this family and future siblings. As the variant is mitochondrial so it is maternally inherited and the mother passes the variant to her offsprings and genetic counselling and management can be now provided for this family. And now I let Kirsty to continue with what if you what can you do if you can't do a trio? Yeah, thank you, Saija. So yeah, we've we've shown you 4 cases today which which illustrate the value of doing your Wes analysis as a child and both parents trio. But the question remains, what if you can't actually do a trio? Because of course, we recognize that it's not always possible to test samples from both parents. So. So what are your options in that situation? Well, of course, it's always possible to do a pro band only analysis. So only analysing data from the affected individual and no family members. So with a pro band only case, nothing is missed. The relevant finding or findings will still be reported. But as shown with the first case that we showed today, you may also you may also get other VU s s on on the report and also the classifications of the reported variants are more likely to stay as VUS without the parental samples to show de Novo status or phase. Also duo is a a good option and having just one parent available does help with ruling variants in or out based on inheritance or not from the sequenced parent, but obviously not as much as if you had data from both parent. And of course with a duo it's generally not. It's not possible to see de Novo status of varying variants and it's not possible to know for sure the phase of two heterozygous variants in the same gene. Although having data from one parent can suggest phase, but we cannot normally confirm it with data from just one parent. So you may wonder if you have performed a pro band only where's or a duo where's and Avus has been reported. How can you get that VUS to likely pathogenic or pathogenic if there isn't any parental data available? Well, the additional evidence that you would need is typically additional reports of the variant in more patients in the literature or on databases or functional studies as well. But it's, it's important to be aware that this kind of data may not appear in the literature for a long time, especially with really rare diseases and it may never appear at all. So, so basically having data from both parents is the best way to get you to the molecular diagnosis in the fastest way. OK, so to summarize what we've shown you today, we've shown you that Wes Trio can unlock answers for patients and families by facilitating the interpretation of variants, by enabling the identification of de Novo variants, inherited variants, and also showing you the phase of two variants in the same gene. It also gives you valuable recurrence risk information and often a more accurate variant classification straight away enabling you to potentially complete all the testing that you need to do in one step without additional family member testing. OK. And then finally, we would like to acknowledge the clinical interpretation team at Blueprint Genetics. We are a large and happy team, as you can see, working everyday on patient cases like the ones that we've shown you today and all driven by a desire to unlock answers for patients and families. And thank you to you all for listening. And Sarah and I are now very happy to take any questions or comments on today's today's material.