So, welcome everybody. I'm Peter Bauer, Chief Genomic Officer at CentroGene and I welcome you to the talk on revolutionizing time-sensitive diagnostics for young patients, the framework of the 2021 ACMG Annual Meeting, which is really a topic that needs attention because the burden and the need for a diagnosis is so high in this cohort of patients. Let me guide you through the webinar today, which consists of a couple of steps where we look into the diagnostic challenges, the methods that can be applied to diagnose patients, and then some examples from our end where we can demonstrate a really high yield in diagnosis through exomogenome sequencing, and of course, the share for getting to new insights in science and innovation. In the end, the benefit for all those patients suffering from their disease and their families. Now, if we look into the diagnostic challenges, we are facing a job that is really not easy because genetic disorders in newborns are known to be a relevant fraction of these individuals. So, 3 to 4% of the newborns will have a genetic disease or a major birth defect, and among those, roughly 1% has a chromosomal abnormality. This is long, this is evidence known for a long time. However, since these genetic causes are not uncovered without a specific genetic test, still many babies and newborns are underdiagnosed and will not get the most appropriate attention and therapeutic options without a diagnosis. Furthermore, one-fifth of all infants with a birth defect or a genetic condition will die within the first month and years, and so there's a tremendous burden on these families to know about the diagnosis and take actions to help their children and as well avoid a repeat of these severe traumatic conditions. And from the angle of healthcare systems, more than 10% of the admissions in pediatric hospitals are due to children with genetic disorders or children with congenital abnormalities. And so, we have to look into this cohort to do our best very early and fast to help the families and our societies with a better outcome. Now, in this respect, we can be sure that every second counts, or at least every day counts, because we have very complex phenotypic presentations that are not easy to handle in the hospitals, and children are usually affected very severe with a risk that the situation escalates without proper treatment and proper measures. And there as well, the prognosis of diseases is usually defined by the genetic cause and therefore the information on the genetic underlying etiologies is so important. So, this is immediate, relevant, and very often leads as well to a change in treatment. I would like to share with you the two big technologies that are offered at the moment in the space of rare genetic diseases, which is exome and genome sequencing. And due to the very high pressure of finding the diagnosis and the unspecificity of symptoms, usually a single gene testing approach or as well copy number analysis through RA-CGH will not give you a high proportion of positive answers about the etiologies, and therefore we are working together in a field where we have rather the genome-wide complete assays, which is exome sequencing or genome sequencing, to help us identify the conditions underlying the symptoms in the patients. This is especially true for major birth defects, children with severe developmental delay, seizures, or children with severe muscular disease. If we look in more detail into the advantages and differences between exome and genome sequencing, we can state that exome sequencing with current technology is highly accurate because uniform coverage is meanwhile achieved through the most recent enrichment technologies. Exome sequencing covers all known protein coding regions, inter-exome boundaries, well, serious flies defects, and in principle, this technology is superior to any smaller panel or single exome approach since you save time and have a very comprehensive data set for the analysis and the correlation of phenotype-genotype observations. Furthermore, this test is shown to be very cost-efficient and can as well be enriched with add-ons that are or have not been included in exome sequencing in the past. This is especially that we enrich for the clinical and relevant genes where any coverage problem is improved through boosting the enrichment technology, adding additional baits for regions that are usually poorly covered on top. As well, all known non-co-NPC-causing variants can be included in the design of such an exome sequencing, customized exome sequencing approach, and as well, mitochondrial DNA fractions can be enriched by specific targets for the small genome of the small but important genome of the mitochondria. So, this boosted exome sequencing approach gives you the highest yield in established diagnosis and on top even, of course, adds genotypes on genes that have not been discovered yet, which I will address in the third part of this talk. On the other end, looking into genome sequencing, we have an even more uniform data set, which is completely unbiased things. The genes and the non-coding regions are captured without any enrichment technology in between. By that, we have very good and even coverage of coding and non-coding sequences and, of course, the best data set to look into copy number variation. This is as well copy number disease and structural variants, which are not captured by enrichment technologies like exome sequencing. This high-end product is especially useful in heterogeneous genetic disorders, like I mentioned, intellectual disability, developmental delay, and epilepsy and other early onset neurological disorders. Furthermore, since we don't have to enrich, we save the time in the laboratory for these steps and are usually faster and more comprehensive with the genome sequencing data set, which is an ideal product for critically ill children and so helps to have a timely diagnosis and as early as possible treatment. Just to show that this is something we have successfully shown in practice, I would like to share with you this open access publication in 2020 seen in the European Journal of Human Genetics. We have shown that a series of more than 1,000 index patients can be managed and diagnosed through genome sequencing with a superior diagnostic yield and great insights into the practicability of this approach. We are proud that this paper has been selected by the editors and by the readers of the journal, one of the highlighted publications in 2020. Now, it's not only the type of analysis, but it's as well the setup you choose to go into the family you want to diagnose and there you can see in this chart that the trio analysis sometimes seems a little bit expensive because you have to run three tests in order to diagnose one patient, but the discrimination on the data sets is so powerful that we reduce ambiguity in results and come back with therefore with more robust interpretation of genotype-phenotype correlations and this is true for recessive diseases where we can see phasing in compound heterozygous index patients. We can see the recessive carriers shift of recessive mutation in parents in a homozygous trait and we can both see the de novo variants and the shared dominant variants in autosomal dominant monogenic diseases. So, the testing strategy we have established and as well described in this landmark paper of 2020 is that it's still a good start if time is not critical to choose exome sequencing in young patients with heterogeneous phenotypes and an unsuccessful testing history and then depending on the outcome you either have a genetic diagnosis or in a negative case you will consider genome sequencing as the most comprehensive choice for a diagnostic platform. For critically ill children in NICUs and PICUs, we would always recommend to choose the genome sequencing first just to not overlook as well genomic diseases that are not captured in exome sequencing and so our return data is already pointed out as fast as possible. Now let's look at an example that we could publish as well last year where the yield of the analysis was especially high and now again from our perspective yield is not a parameter of the laboratory of course all technical aspects have to be optimized but diagnostic yield is a function of a successful interaction of the referring physician and the laboratory and the medical experts at the laboratory. So, in principle we have five layers that need to come together. One is the patient selection that the referring physician clinicians is already suspecting genetic disease and does not want to diagnose an acquired disease with an expensive genetic test. Thereafter the decision whether a solo or trio design is supplied is important as I could show you. Sometimes parents are not available then of course there's no choice but if parents are available the power of the trio analysis is just striking. Furthermore, I shared with you some technical considerations which are in the responsibility of the laboratory and our experts in Wroclaw in Germany which always also includes the analysis of the data and accurate and curated variant repositories like CENTR-MD where we have curated all the insights and decisions we've taken in the past and in the end it's of course the decision and the expertise of the medical genetic people that approve the genotype-phenotype correlations in one picture with the data quality will trigger some autogeneral validation steps where necessary and we'll discuss with you outcomes and results in detail. Now, one of these diagnostic success stories is as well published in 2020 in Genomic Medicine where we showed that the analysis of almost 350 families used a very high diagnostic rate in Pakistan where children have not been touched with other genetic technologies before and therefore it gives us a very pure picture of the power of exome and genome sequencing. In this cohort, 1500 genetic tests have been done in 350 index patients and what we've seen is a very high diagnostic yield of more than 16 percent clear genetic diagnosis with pathogenic and like pathogenic variants. Only one-fifth, 18.9 percent were negative in this analysis pointing to rather non-genetic diseases and another fraction of one-fifth was a variant of uncertain significance still under observation or required further testing in the families and other individuals. What is really compelling is the high number of children that received and adjusted on new treatment after the genetic test which was more than 50 percent of this diagnosed cohorts. This also included offering prenatal or predictive testing in the families and in many, many cases allowed the referral to a relevant specialist in the area. So, even in countries where you would suppose or usually suppose that it's hard to find then treatment and specialists, we have demonstrated together with our partners in Lahore that this is possible in such a scenario and that genetic analysis is opening these doors for the parents and their families. I just received this morning an email from a couple that had prenatal testing of the diagnosis of an infected child before and they were so happy now to have their daughter born without any concern about the disease they suffered with their first child. Now, this is the high quality and the services we are offering for you in a diagnostic environment but exome sequencing and genome sequencing of course helps us as well to go beyond diagnostic because insights into yet undiagnosed families helps us to understand new conditions of disease and there we see a high impact on science and innovation which we are driving in international collaboration. Just there to share with you our approach, we have usually the diagnostic evaluation which ends up with a positive or sometimes negative record. If the family and the individuals consented to use data and material for research, we go through a process where we apply filtering on variance and impact of variance as you can see in the panel of the sequence on the right side where all the high quality variants are then put together and then compared with phenotypic pictures that would hint us towards a gene that could cause a novel entity of phenotypes yet this is a new representing a new disease gene. Just three examples of the last year as well where we collaborated with the colleagues in the Netherlands on Osteets-like syndrome with loss of function in IPO8 where we could contribute those three families with a striking similarity in phenotype. We see the skeletal malformation and some facial dysmorphology and for all families in the paper published we have shown that they have a homozygous variant loss of function variant in IPO8 which is interestingly a factor interacting with SMAD1, SMAD3, SMAD4 which are central contributors of the pathophysiology of Osteets syndrome. So this is just in publication and so showed how we can swiftly and very rapidly contribute to research networks. Another example with even more than 30 families is just accepted for publication with a loss of function in ZNF699. Here you can see that in those individuals a typical facial appearance and once again some skeletal malformations as you can see in the thumb of the patient, index patient, number 27, dated to a joint etiology. This is a large collection where more than 30 individuals have contributed to the publication and I think that's the way forward to come back with a broad phenotype description and good evidence on the robustness of the solution. Still we don't know how this gene is exerting its functional effects and how the abrogation of protein leads to a malformation. This is currently under investigation. And finally just published these days in NSF Neurology we also contributed three families in a novel recessive factor leading to early onset generalized dystonia. Families B, C, and D have been found and identified through the database research at CenterMD and our internal resources and this study helped beyond the definition of a new disease gene as well in the elucidation of pathways that integrate our knowledge on the dystonia symptoms in patients. There is a disturbed integrated stress response like you can see on the chart on the right side that now this UIF2AK2 is unknown, an additional factor now with monogenetic contributions. So looking into the broad picture it's a continuum from serving the patients but as well serving science and the communities to identify and diagnose the patients to adjust treatment and refine as well treatment to be more efficient in the resources we offer to those families with rare diseases in the end as well to go beyond the state of the art and innovate our rare disease landscape to ultimately be strong in understanding genetic disease and going for targets and pathways that are worth addressing in therapeutic development. So all this knowledge of course in the end should help to define and develop the treatments for the children and their families. So the summary is that with this high quality broad genetic tests we have an answer to address the time sensitivity in predated collegial children to readjust the healthcare resources to make best of everything we have at hand and use it for those children that really need it, avoid spending and other diagnostic options that are not in the ends contributing to the diagnosis and a set to help build up biobanks and insights for targeted treatment developments. So I thank you for your interest today. Since this is offline you will be able to reach out with questions or any collaboration ideas through the customer support etc. as you can see in this email and so as of today I wish you all that you stay healthy that you manage the next weeks and months in the pandemic and then hopefully we return to a new normality that also includes meetings and on-site discussions in the very near future. Thank you and have a good day. Bye-bye.

time-sensitive diagnostics

young patients

genetic disorders

major birth defects

exome sequencing

genome sequencing