Good afternoon, everybody, and thank you for joining us for the ACMG Genomics Case Conferences. As a reminder, please register. Don't forget to register for the 2017 conference that will be in Phoenix. So, today, we're here at the Children's Mercy Hospital hosting this, and our title is Surprises from the NGS Lab, Reconciling Genotype with Phenotype in the Era of Genomic Medicine. Just a few housekeeping things. This activity is supported by QIAGEN, so we want to thank them for their support. As a disclaimer, this is intended only for educational use. This is the list of presenters today. I'm Carol Saunders, the Clinical Director of the Center for Pediatric Genomic Medicine. I have with me four colleagues, Lauren Grody, Isabel Tiefoldt, Emily Farrow, and Jennifer Cannon. This is the planning committee and the staff, with no disclosures to make. We as presenters have nothing to disclose. This is available for one CME credit, and if you did sign up for CME, you'll get an e-mail and a survey to fill out. These are the overall learning objectives of these genomic case conferences, and these are the objectives that we have for this session. So, we have for you three cases today that we hope will illustrate the additional testing and clinical correlation that's needed to achieve accurate phenotype and genotype assignments. We'll be discussing the counseling challenges associated with expanding phenotypes and variable expression of rare genetic diseases, and we will describe the clinical utility of symptom-driven analysis in the context of whole-exome and whole-genome sequencing tests. If you have a question, you can type it in your Q&A box in the lower left corner, and they'll be addressed at the end of the session. So, just a bit about who we are. We established the Center for Pediatric Genomic Medicine in 2011, so working here with us, we have board-certified laboratory directors, analysts, genetic counselors, bioinformaticians, physician scientists, software engineers, lab personnel, and research coordinators. So, we're fairly self-contained. We do both research and clinical testing. They will be talking about a mix of those things. We have research coordinators who can enroll those patients. We have – this is our list of equipment. We have – we're largely focused on Illumina equipment. We have five HiSeqs, so we run all of our own sequencing. We can do a genome in about 24 – 25 hours, or 16 genomes in three days, depending on the run mode. Have I lost? Sorry, I seem to have lost the video. Okay. Or, depending on the run mode, we can run 16 genomes in three days. We also have two MiSeqs, and we just acquired a fluid IMC1 single-cell instrument that we're anxious to use for research purposes. So, not only do we run our own sequence, we also perform our own analysis bioinformatically. So, we have a lot of compute infrastructure in place to do that. So, this is a long list of what we use for that. So, as far as the clinical testing that we have available here, we've been doing – we've been doing a targeted test called – that we call TagScan, which is a targeted panel that's now retired, but we ran that for about five years. This has 571 genes, including the mitochondrial genome, and I mention it because one of the cases involves the use of that test. We also do exome sequencing, including the mitochondrial genome. We've been running that since March, where we can do virtual panels, whether they be symptom-driven or physician-selected. So, we don't really call it whole exome sequencing, since we're carving out panels bioinformatically. We do do whole genome sequencing. We've been doing this since July of 2015. This includes the mitochondrial genome, and for this, we do provide incidental findings if requested by the patient. So, to customize the data analysis, this is enabled by three different software programs that we designed here. They're called SAGA, RUNES, and Viking. So, SAGA stands for Sign and Symptom Assisted Genome Analysis. So, what this does is it maps the clinical symptoms to genes and diseases using SNOMED terms, standardized nomenclature, and this allows us to filter by symptoms. RUNES stands for Rapid Understanding of Nucleotide Variant Effects Software. So, this is our analysis pipeline. So, each variant is assessed for whether it's been seen before, either in population databases or disease databases like HGMD and ClinVar, in which case they would be flagged as having been reported previously as disease-causing mutation. We run it against EXAC and dbSNP and EBS, all the different population databases. It's assessed for splice prediction, translation impact prediction. All of this is consolidated into what we call a variant score, which is assigned based on the type of variant and all these other factors. And this data is loaded into our database, which we call the Variant Warehouse. So, Viking is the last piece of software, and this is our analysis tool, which allows us to integrate RUNES, the patient's variant file, and SAGA, the patient's phenotype. So, this allows us to prioritize the variants based on the clinical features and the variant score. And it also allows us to filter by frequency, inheritance filtering, and also to do trio analysis. So, the first case that we'll present today is a little girl who was born in Pakistan to a 28-year-old mother and 31-year-old father. And there were no pregnancy complications. She was born at term, weighing 6 pounds. Seizures were first noted around eight to nine months of life, but went undiagnosed until the age of two. So, she was started on medication in Pakistan before moving here to the U.S., and she first presented to our neurology section around the age of three with intractable epilepsy, which presented in the form of tonic-clonic and complex partial seizures. She also had significant developmental delay. So, her medication regimen was altered when she arrived here, providing some relief and a decrease of seizures. An early brain RMI was performed that showed possible frontal cortical dysplasia, but was essentially read out as normal. She had a first vagal nerve stimulator placed in 2002, and this was turned off because it didn't really help. And a second was placed in 2015 and did provide some relief as far as decreasing her seizures. But despite numerous medications, she had very difficult-to-control epilepsy and had a very poor quality of life, according to the neurologist. So, her early language development was delayed, and currently she has very limited expressive language. She uses only a single word or short phrase on occasion. She can follow simple commands. She was diagnosed with autism, and she functions around the age of about a three- to four-year-old child. Her previous workup included an essentially normal karyotype, female karyotype. She had a few single-gene tests that were normal, and Fragile X was normal. This is the family history as we knew it at the time that she was referred to the Genome Center in 2013. So, she had intractable epilepsy. This is the patient here. She had intractable epilepsy, intellectual disability, and she's nonverbal, so a very severely affected girl. Interestingly, she had a younger brother who had focal epilepsy that's very easily well-controlled. He's an honor student. He was planning to go to medical school. We knew about two other siblings who were reportedly alive and well, as were her parents. So, they were enrolled in our undiagnosed – I'm sorry, she was enrolled in our undiagnosed disease program in 2013. So, we ran her sample alone, identifying a TSC1 variant in exon 4. So, this is a glutamine at codon 66 that was a nonsense mutation that we interpreted as likely pathogenic based on the fact that it's predicted to be truncating and was not present in any public databases. Despite this fairly convincing genotype, we were unsure what to call this diagnosis, given the fact that she had an essentially negative MRI and a lack of any skin findings, according to the neurologist, who said he did a skin exam and found no signs of anything related to TSC1. So, following these results, we requested samples from the parents and other affected family members. The patient had a renal ultrasound, which showed an enlarged and diffusely echogenic kidney. Subsequent to that, in 2014, the mother was diagnosed with a renal malignancy. And in 2016, we finally received samples from the other family members. I'll turn this over to Lauren Grody to discuss the rest of the case. So, you guys are all probably familiar with tuberous sclerosis, but I wanted to review it real briefly. To establish a clinical diagnosis, there are specific criteria in place. To have a definite diagnosis, you need two major features, or one major feature with two or more minor features, or a pathogenic variant in TSC1 or 2. And to get a suspected clinical diagnosis, you need one major feature or two or more minor features. Briefly, there are lots of different major features available to use for these criteria. Lots of them are skin findings, angiofibromas, ungle fibromas, sugaring patches. There are also cardiac findings with cardiac rhabdomyomas, and brain findings, cortical dysplasias, seventh-dimal nodules, SEGA, as well as eye findings and hamartomas as well. There are also a full list of minor features, confetti skin lesions, dental enamel pits, intraoral fibromas, renal cysts, et cetera. There are additional features that are included in the clinical criteria, which are seizures, which happen in over 80 percent of patients, the TSC-associated neuropsychiatric disorder, which includes things like autism, ADHD, learning and cognitive impairment, and disruptive behaviors and emotional issues, as well as renal cell carcinoma, which does occur in around 2 to 5 percent. This condition is autosomal dominant, and the penetrance is thought to be around 100 percent. Once you have a diagnosis, we do recommend many evaluations for each individual, and this is a list of them here, all screening for those major and minor features that were mentioned earlier. Back to our case, we went ahead and were able to sequence the siblings as well as both parents, and we found that the proband and all three sisters and her mother all tested positive for the TSC1 variant. The father tested negative. At that point, they were sent to clinical genetics, and we met with them to take a family history. There were several challenges getting the family to the clinic evaluation because they weren't keeping appointments, they were difficult to get in touch with, there was a language barrier, and the family was truly having some difficulty reconciling that their one daughter, who is severely affected, has the same thing as their other children. When we did get them in, I was able to take the family history, and we found out that there is a third sister who also has seizures, which was unreported before. Sorry, a second sister with seizures, third sibling, and there was an additional sister who was still seizure-free at this point. We learned about mom's kidney cancer. She actually had bilateral renal cell carcinoma at age 45, and we took an extensive family history on mom's side to find out that there were no other people at least reported with seizures or developmental differences in mom's family. All of this history was complicated somewhat by the fact that this was a traditional Pakistani family, and so the father was the sole communicator during the case. He was answering the questions for all of the children and the mother, and was very quick to say everything was normal, even for the kiddos who had seizures, was not interested in giving a lot of information about what the seizure history was or if they had any other health issues. So, that certainly made it a little bit challenging. Once we met with them, we referred all of the children to the various evaluations that are typically recommended. So, for our proband here, this is her photograph, she had previously had a lot of the things she needed, dental exam, eye exam, EKG, and echocardiogram for various reasons, and of course, she had had numerous EEGs, which were abnormal based on her seizure history. We repeated her brain MRI, because it had been done many years prior, and this time they actually did notice some signal abnormalities compatible with either subcortical tubers or cortical dysplasias. She did not have any sub-ependymal nodules noted. We also referred her to dermatology. Because she was over 18, she was not able to be seen at our institution, so I unfortunately don't have any records from what her findings were. She had a renal imaging here, again, which noticed echogenic kidneys, and we had not done a urinalysis and LAME screening at this point, primarily due to ability to cooperate with these tests. The brother had previously had a dental exam and eye exam. We repeated his MRI, because that had also been done several years ago for seizure diagnosis. It was normal again. His EKG continues to be abnormal. He had never been seen by dermatology, so we were able to have him evaluated. He did have two hypomelanotic patches, a connective tissue nevus, and one periungual fibroma. His echocardiogram and EKG were all normal, and he was noted to have some renal cysts bilaterally on his abdominal MRI. The third child had previously had a normal dermatology evaluation, normal dental exam, and eye exam. Interestingly, she had some vocal cord dysfunction and some respiratory issues for which she was being worked up by pulmonology, so she's had some of the pulmonary function tests that we typically use the screen for LAM. She had a brain MRI after we asked for one, though she had never had any seizures before. They did see hyper-intense regions, which likely indicates subcortical tubers. Her EKG was not completed, because she wasn't actively having seizures, and neurology did not seem to think that it was necessary at this point, and she's doing well. She had a normal echo and EKG. We also did an abdominal MRI, which found a solitary renal mass. They actually followed that up with an ultrasound, and we were able to see that there was no They actually followed that up with an ultrasound to get another look, and while it seems atypical for an angiomyolipoma, they weren't able to exclude that, so she's being followed closely with nephrology currently over the next few months to see if anything's changing. Our last kiddo is the other individual who was having seizures. Her EEGs have been abnormal in the past. Her brain MRI was normal, and that was just done last year, so we did not repeat it this summer. Her dental exam and her dilated eye exam were also normal. We did an abdominal MRI for her, which showed two cysts in the left kidney, and her dermatology evaluation was also abnormal with several hypopigmented patches and one connective tissue nevus. This case was certainly challenging from a counseling perspective as far as trying to elicit all the information I needed from the family. Typically, when you're evaluating children and you ask if they have any skin differences, any spots, bumps, or bumps, kiddos are usually the first people to stick their arm out and show you what they have, and in this situation, I wasn't able to interact with the family in that way, given that dad was the sole communicator, and whether or not they would have noticed these changes that dermatology picked up, we're not sure. This diagnostic finding in the proland actually is more of an incidental finding for the rest of the family members. They did not have enough clinical features without doing all of these investigations to know that this is all related to each other, but we certainly changed management with this result. We found the explanation for mom's kidney cancer, and we're going to be screening all of these individuals in the family as well for additional cancer risk in the future. I'm going to pass this off to the next presenter. Hello. I'm going to present to you Case 2, which is a case of Wiedemann-Rautenstrauss Syndrome. The prenatal history for Case 2 was that the prorand was the first pregnancy to a 34-year-old G1P0 mother and a 40-year-old father as a couple. Prenatally, their ultrasounds had revealed inter-uterine growth restriction with all growth measuring consistently less than a third percentile, a strawberry-shaped skull, splaying of the cervical spine, micrognathia, rocker-bottom feet, and fish for trisomy 13, 18, and 21, as well as a snip array, were all normal. And in total, prenatal genetic consultation did not result in a diagnosis. Our patient was born at 32 weeks, 3 days due to complications from Maternal Health Syndrome. A physical examination at birth was remarkable for, as expected, small for gestational age with both weight, length, and OFC, again, less than a third percentile. Pseudohydrocephalus, she was noted to have decreased subcutaneous fat, a triangular face with frontal and parietal bossing, a wide fontanelle with prominent scalp veins, sparse hair, low-set dysplastic ears, a beak, nose, and, interestingly, two natal teeth. She also was found to have a persistent PDA, a restricted airway that required mechanical ventilation, and an anal structure that required dilation by the surgical team. She was G2-independent, and due to instability, an MRI was not performed. A brain ultrasound was able to be done at day one in six weeks of life, which identified no structural abnormalities. The patient, unfortunately, died at seven months of age from complications of her features. And this phenotype was consistent with a clinical diagnosis of Wiedemann-Rautenstrauss Syndrome, which is also known as neonatal progeria. These are two pictures of the patient, both at birth and then at four-and-a-half months before she passed away, where you can see her strawberry-shaped skull and the prominent scalp veins and many of the features that were noted. The maternal – the family history was really largely non-contributory. The mother reported difficulties with reading comprehension, as well as difficulty with The mother reported difficulties with reading comprehension, as well as one brother and one sister. The father had one brother born with an imperfect anus, but there was no known consanguinity, and certainly no one in the family with any features similar to our patient. So, Wiedemann-Rautenstrauss Syndrome, or WRS, was first described in 1977 and then further defined by Wiedemann in 1979. Again, it's called neonatal progeria syndrome, which is classically thought to – patients have diminished subcutaneous fat with neonatal teeth and pseudohydrocephalus. And this is a picture of a patient that has been reported in the literature. You can see the similarities between our patient and previously reported patients. And some at the time of – when this child was referred to us, approximately 40 cases had been reported, all with consistent phenotypes of WRS. Again, they had the characteristic physical findings, including neonatal progeria appearance, sparse scalp hair, prominent veins, lipoatrophy. In addition, neonatal teeth are often a distinctive finding. And the inheritance pattern of this disorder has been postulated to be autosomal recessive, although a specific gene at the time has not been identified. And because of this, we enrolled them into our undiagnosed disease research program. Now, I'll pass it off to my colleague, Isabel, to describe the molecular findings. So, using West 3.0 analysis and our software, Viking, we relaxed our filter and we're looking basically at first for the analysis of the novel gene. However, we uncovered a diagnostic genotype in a gene called Pol-R3a. So, the problem at a maternally inherited non-pathogenic mutation, it's a splicing mutation which is actually at 18 nucleotides from the exon. So, this particular mutation has been reported before and our functional studies show that actually this self-mutation creates a retention of 19 nucleotides in the entrant protein, which creates a premature stop codon at position 650. Also, the RNA studies show that both the normal and the aberrant splice product are produced, suggesting that some level of the normal protein may be expressed. This particular variant is absent from population databases and it's been previously reported in six individuals affected from a very large Syrian family and published in 2014 by Bernard. Also, this particular mutation, if you look at computational evidence, is predicted to be deleterious for splicing. The second variant that we identified, the paternally inherited, is also a truncating variant. If we use the CMG categorization, so this particular mutation creates a nonsense mutation. It's not been reported in affected individuals, but it's the type that is expected to be pathogenic. This particular variant has been reported in four psoriasis individuals in the exact databases, but it's absent in the other databases. So, when we found this particular variant, we were kind of surprised because the gene has been associated with a very specific phenotype, which is mainly leukodystrophy. It's been also reported in different individuals with a slightly different clinical feature, which can include cerebral atrophy, apoplexia of the corpus callosum, and oligodontia. This particular gene has been first described in French-Canadian patients, which were very much more homozygous and they were presenting pretty much the very same clinical feature which is hypomelanation, apodontia, apogonotropic, apogonatism. So, at the time of testing, no patient has been reported with homozygous or compound heterozygous truncating variants. Most of the patients, if they had one truncating variant, the second variant was a misspence. So, we contacted Dr. Geneviève Bernard, which is the primary investigator of that gene, and she asked us at first if the patient has neonatal PEEP because it's actually also a feature of polarity-related disease. Interestingly, she also told us that the mouse model is actually embryonic-lethal if you have two truncating variants. The other interesting fact is that, at the time, that was the first patient that was representing an extension of the clinical spectrum and the severity, but additionally, there's two additional cases which presented with two truncating variants in that gene that confirmed that actually we had the right diagnosis. So, this case illustrates the extension of phenotype, but also the need for expert consultation and also the importance of relaxing filtering strategies when you're looking for a rare phenotype. I'm going to pass now for the case number three. Hi, I'm Jennifer Gannon. Case three is a 9-year-old boy with a 3-week history of fever, vomiting, abdominal pain, and generalized erythroderma who had a significant past medical history for generalized pustular psoriasis, and this rash first appeared when he was 4 months old and was diagnosed as this at 10 months of age. It was very refractory to treatment, and he had iatrogenic adrenal insufficiency, hypertension, and short stature from this appropriately aggressive treatment. Because he had skin integrity issues because of this rash, he had recurrent bacterial and fungal skin infections, and two prior hospitalizations for septic shock, lactic acidosis. Interestingly, he had no other symptoms other than the rash, really no other problems. He had normal intellect and development. Just briefly, the family history, we can see that he is the fourth of seven children and that he had an older sibling who died at 4 days of age because of complications of a febrile illness. He also has a younger sibling who also has a diagnosis of ichthyosiform dermatitis. There's no known consanguinity in this family. However, they're from the same small area in South Sudan. So this patient was ill appearing at presentation. He had anti-gap metabolic acidosis with a pH of 7.03 and lactate of 4.6. He had leukocytosis and thrombocytopenia and evidence of coagulopathy. There was concern that he had either a toxin-mediated process or septic shock. He was admitted to the pediatric ICU and had quite rapid decompensation and required ventilation and dialysis with inotropic support. We're just going to double check our video for a moment. Thank you. So just a few points about his work prior to hospitalization because the skin rash was thought to be the underlying problem that led to his bacterial infection which was presumed and shock. He was diagnosed, as I mentioned, at 10 months of age on skin biopsy. It was found to be most consistent with pustular psoriasis. At 18 months of age, he had SPNC5 molecular testing because of concern that maybe this was actually Netherton syndrome and that was normal. At 9 1⁄2 years of age, he had a microarray. That was normal. Notably, there are no large areas of loss of heterozygosity. He had a 39-gene panel for congenital ichthyosis that was also negative. So back to the current hospitalization. There was an extensive workup to find out what the underlying source of this sepsis was or apparent sepsis. Initially, all that was found was a topical candidal infection. But then he had a tissue biopsy of skin that appeared to be necrotic and this was found to grow fungus. So he was diagnosed with mucormycosis. The interesting thing about mucormycosis is we generally think of that as being associated with an underlying immunodeficiency, whether acquired or inherent to that individual. And he was also found to have reactivation of HHV-6 and CMV, which further supported the underlying immunodeficiency theory. He was found to be HIV-negative and he had a bone marrow biopsy with cytogenetic testing which showed no evidence of malignancy. And so he was also far enough from any kind of immunosuppressant therapy he had received for his rash that people didn't think that was the etiology. So he had a large immunodeficiency workup that was non-diagnostic and a 41-gene panel for periodic fever disorders that was normal. So further molecular testing was recommended at that time to evaluate for an autoinflammatory disorder or an immune dysregulation disorder, particularly those that have dermatitis associated with them. I'm going to pass off to Carol for the results. Thank you. Okay, so while the patient was in the hospital, the immunology team ordered our targeted panel, our targeted NGS panel from him. So this includes 571 genes, but as I explained earlier, the phenotype is going to drive what is analyzed. So for this particular patient, we were largely focused on the immunological picture. So we used nine terms that included fever, dermatitis, scaly skin, bacterial infectious disease, and other immunological concerns. These nine terms pulled 144 genes for analysis, and there were 29 variants that were a part of that. In the end, we reported two variants of unknown significance. One of them was in the DOCK8 gene. It was a heterozygous variant, a heterozygous missense variant predicted to be tolerated benign by SIFT and polyphen, and DOCK8 is associated with hyper-IgE recurrent infection syndrome, which he didn't have. So the only other variant on this panel that was interesting was a homozygous variant in the HLCS gene, which is associated with autosomal recessive holocarboxylase synthetase deficiency. Now this is a missense variant. It's a histidine at 669 to a tyrosine. It was predicted to be deleterious and absent from any database that we could find. So if you look at the criteria for sequencing interpretation, the 2015 ACMG criteria, really there's not much to say about this variant other than, you know, you have one moderate and one supporting criteria for a variant of unknown significance, so that it's absent in population databases and multiple lines of computational evidence support a deleterious effect on the protein. So being a molecular lab and knowing only enough to be dangerous in the metabolic world, you know, we thought it was worth, you know, talking to the metabolic folks about their thoughts on this variant. So they didn't completely blow us off. They thought it could be interesting, although the skin rash was not at all what they would expect to see with this condition. He did have a skin rash, and he did have intermittent lactic and metabolic acidosis. So they recommended an acylcarnitine and urine organic acid profile, which was performed and showed massively elevated C3 and C5 OH on the plasma acylcarnitine profile. No urine organic acid was done on because the patient was on dialysis and was not producing any urine. So based on that functional evidence of the biochemical testing, we upgraded the variant to pathogenic, and some other findings that we'll discuss later, we upgraded the variant to pathogenic. So we were able to have a strong and a couple of moderate and a few supporting criteria to support that. And I'm going to pass it back to Dr. Gannon to discuss the rest of this case. Okay, so briefly about holocarboxylase synthetase deficiency. It's a rare autosomal disorder. HLCS activates four biotin-dependent enzymes by covalently bonding biotin to them, so it's a form of multiple carboxylase deficiency. The classic neonatal presentation is similar to probiotic acidemia or pyruvate carboxylase deficiency, as these two enzymes are deficient in this condition. And it usually presents with vomiting, lethargy, poor feeding, low tone, and seizures, usually with metabolic acidosis, and in some cases, hyperammonemia. These infants can get very sick, and they're often thought of as potentially dying in the neonatal period. The long-term symptoms in individuals who remain untreated include rash, alopecia, developmental delay, optic atrophy, hearing loss, and seizures. So left untreated, these individuals can also have periodic metabolic decompensation with intercurrent illness. So on the right, you see a photo of an infant with a typical rash, and this infant has decompensated, actually, and he had been treated, which is why he has hair there. As has already been stated by Carol, the lab diagnosis involves acyl carnitine profile analysis and urine organic acids. And therefore, you can actually screen for this on newborn screening by looking at C5OH as the primary analyte, and this condition is on the RASP as one of the core conditions. This patient was born prior to expanding newborn screening, so he wouldn't have been able to benefit from that. It's important to remember that not all patients have a classic presentation. Our patient was well in the neonatal period and didn't begin with rash until he was four months of age. He had no neurologic involvement at all and actually was quite a bright child who did well in school. There have been a few reports over the past 20-plus years of children who present with atypical rash, and there have been reports of children presenting as late as eight years of age with HLCS. I list there three of the presentations for skin rash that have been reported in the literature. And the treatment for this is relatively simple in the long term, which is treatment with oral biotin. So, unfortunately, this child died about two weeks after the diagnosis was made, and so we did have the opportunity to treat the younger brother. And when I first met him about a month after his brother died, he was two years old. He had a diagnosis of congenital ichthyosis that first began when he was about 12 months of age. And he had had no hospitalizations, no neurologic problems, had no concerns in terms of development, and so the rash was really the primary manifestation, which also was very refractory to treatment. Interestingly with him, he had an initial newborn screen that we were not aware of that was flagged for C5OH and was considered low risk by the state newborn screening lab. So they didn't refer the screen to a metabolic center, but rather asked the PCP for a repeat screen, which was then within normal limits. You can see that his rash is an ichthyosiform, a desquamating rash, and his acyl carnitine profile showed elevations of C3 and C5OH with no elevations of other acyl carnitine species. Urine organic acids were not done on this child because he had significant involvement of the diaper area with an active infection and completely desquamated skin. So that day, we recommended biotin of 40 milligrams per day, high dose, and his father started it that afternoon for him. And at four weeks, he came back, and this is a photograph of his back from when he came back. The rash was completely resolved over his entire body, and the areas that had been exposed that had been desquamated had completely healed. After 10 weeks, we saw him again in follow-up. We did an acyl carnitine profile and urine organic acids. The acyl carnitine profile had normalized, and the organic acids were normal. Eleven months later, he's had no recurrence to the rash, remains healthy, and has only had one visit to the ED for vomiting, which there was no acidosis associated with that. Molecular testing showed that he was homozygous for the familial mutation. So these are just photos before he was treated. That was the day we first evaluated him in clinic, and the bottom row is after four weeks of biotin therapy. So case three illustrates the following. It's an example of how additional data can change your interpretation of a VUS. Specifically in this case, the biochemical findings and the weight of the clinical features in this patient, particularly the rash and the lactic acidemia, were able to be weighed in the variant interpretation. And it's important to consider the specificity of the features and the degree of locus heterogeneity of the condition in question. I'm going to hand it back over to Dr. Sommers. Thank you. So we feel like all of these three cases illustrate the need for a close relationship between the – an open dialogue between the lab and the clinical team, and sometimes even outside researchers. Also, the importance of additional studies, which can sometimes be a little bit more complicated Also, the importance of additional studies, which can sometimes end up bleeding into the research world. It also shows us our limitations of our current knowledge of the clinical spectrum of genetic diseases, whether they're well-known diseases like tuberous sclerosis, whether they're ultra-rare, or whether they're brand new. So it's a really exciting time to be doing what we're doing, and we thank you for your attention. And we will look at the questions that we have here and respond to those. Let's see. So the first question is, can we comment about how we're able to obtain a consolidated variant score? I may have misspoke there. The only way to get a consolidated variant score in terms of combining things from the literature, whether the variant's been reported, looking at the bioinformatic tools, is from a human. And so that would be how we consolidate the score. And we do curate our variants in our database. So I hope that answers the question. The second question is, do we have any insights as to why the proband in case one is more severely affected than the siblings? And does she have any other de novo variants? She did not have any other de novo variants. There has been significant variability noted for tuberous sclerosis in the literature. And there's different theories out there as far as it could be modifying genes. You know, there can always be mosaicism, which is obviously not what's happening in this family. And there's also some thoughts about loss of heterozygosity. Let's see. Another question is, did we treat the older child with biotin? And I believe he was treated for two weeks with biotin and unfortunately passed away. He had overwhelming multi-organ failure and sepsis due to – he ended up developing fungemia. And it was – he was just so sick that he wasn't responding well to treatment. Oops, there's another one. Oh. Let's see. Another question. Obviously, hindsight is 20-20. But for cases number three, why was metabolic not involved sooner for lactic acidosis? Wouldn't biochemical testing early have led to the diagnosis? Yeah, I agree that hindsight is 20-20, particularly in that case. I don't know the answers to why we weren't involved sooner. We never were consulted on this child to the best of our knowledge in looking at the chart. He had – those episodes he was admitted for before, he had lactate as high as 15, 16. And from the chart, it appears it was chalked up to septic shock. We have folks that consult us for lactates of four. So, I think that people have learned from this case and from other cases in which they're much more sensitive now to lactic acidemia and having it worked up. And metabolic testing would have diagnosed him at that time. And other questions. Will we receive a copy of the presentation or get access to the reporting? And I – we switched this. The slide here is saying a recording of this seminar is available in the – on the web. So, yes. Yeah, I put it – and I just want to acknowledge – go back and acknowledge – obviously, this is a big team effort here, and I just want to acknowledge my co-presenters here and all of our colleagues at the Center for Pediatric Genomic Medicine. And clinical genetics and other sub-specialists here at the hospital. So, thank you for your attention. If you – if there's any other questions, we'll stick around for a couple minutes.

ACMG Genomics Case Conferences

genetic diagnoses

TSC1 gene

tuberous sclerosis complex

neonatal progeria syndrome

Pol-R3a gene

pustular psoriasis

HLCS gene