actual cases and clinical and laboratory evaluations through the application of genome-scale testing for the patient. This is an activity of the Education Committee of the American College of Medical Genetics and Genomics. Each month, the conference will rotate among laboratories that perform genome-scale testing and the clinicians involved in the case. Use of all case materials that will be presented have been formally consented for use with the patient or guardian. Today we'll be talking about the clinical utility of whole exome sequencing in a patient with recurrent infections. This activity is supported by an unrestricted educational grant from QIAGEN Bioinformatics and the Ingenuity Clinical Decision Support Platform. The ACMG Genomics Case Conferences are intended strictly as an educational resource. ACMG does not warrant the accuracy, completeness, or timeliness of the information presented. In determining the propriety of any specific procedures, clinicians should apply their own professional judgment to the clinical circumstances of each patient. My name is Christina Ostensee. I'm a clinical genetics specialist involved in exome analysis at the Laboratory for Molecular Medicine. Joining me today are Sami Amr, who is a molecular geneticist and assistant director at the LMM, and Dr. Lael Yonker, co-director of the Pediatric Pulmonary Genetics Clinic at the Massachusetts General Hospital for Children. Both Sami and I are employees of the Lab for Molecular Medicine, which offers free fee-for-service molecular diagnostic testing. Here are the disclosures for the planning committee and staff of this series. The ACMG is accredited by the ACCME to provide continuing medical education for physicians. The ACMG designates this enduring material for a maximum of one credit. Physicians should claim only the credit commensurate with the extent of their participation in the activity. If you requested educational credit at the time of registration, you'll be sent an email within 24 hours of the live webinar containing a link. Click on the link within the email, fill in your information, and you'll get a certificate. To ask us a question, type your question into the Q&A box in the lower left corner of your control panel. We will answer the questions verbally at the end of the presentation. The overall learning objectives of the Genomic Case Conference are to describe the clinical utility of exome and genome sequencing, to identify clinical indications for exome and genome sequencing, to list determinants used to assess the probability of a variant's pathogenicity, and to elaborate on the importance of pretest counseling and consent. For this session, our objectives are to discuss the utility of exome sequencing in informing patient management in the pulmonary clinic, to compare differing exome filtration strategies, to explore the spectrum of genetic ideologies associated with common pulmonary phenotypes, and to explain how clinical labs will utilize phenotypic information from providers in exome interpretation. I will begin this session with an overview of the whole exome and whole genome sequencing process at the LMM. Then I'm going to hand over the webcam to Sammy Almer, who is going to go through a very interesting case that we want to discuss today. Sammy is going to review the clinical presentation of this case, as well as our analysis strategy and then the exome results. And we're also fortunate to have Dr. Lael Yonker with us today, who's going to go over the clinical perspectives from those molecular results. And then we'll leave time at the end for questions. So diving right into our overview of whole exome sequencing at the LMM, we offer both whole exome and whole genome sequencing. And the patients receiving these tests have a wide variety of clinical indications. However, they can be grouped into several major categories. The first category are those patients that have very complex disease presentations. And those may be patients for which there really isn't a clear panel test to order. The second group of patients are patients with genetic etiologies that have, maybe do apply to a panel test. There is a panel test available to them. However, they have exhausted all of those options. So they've received either negative or inconclusive results. And then they turn to exome sequencing in hopes of finding an answer. The last group of patients is an interesting one to us. These are patients that are starting to resort to exome sequencing as a first-line testing strategy. So maybe they haven't had any panel tests prior to running an exome. One disease area where we've seen this happening more frequently is pulmonary disease. And to date, we've actually sequenced eight pulmonary cases in whole exome sequencing. About half of these have been positive. So that means that we have found variants that are consistent with the patient's phenotype. Of the positive reports, actually all of them have had a suspected diagnosis of primary ciliary dyskinesia. So within those positive reports, actually two patients had variants in genes that are very well established for primary ciliary dyskinesia and may have been found on a panel test. Interestingly, one of the patients ended up with a variant in a gene that is more newly discovered as a PCV causative gene and probably would not have been found on a panel test. And then the last positive case we've seen is the one we're going to discuss today. And although that patient had phenotypes very consistent with primary ciliary dyskinesia, the molecular results actually suggested a different diagnosis that we wouldn't have detected if we had just run a PCV gene panel. So this is the general exome workflow at the LMM. And I'm not going to belabor all of these points, but I do want to touch upon a few important concepts, both because they're important for exome sequencing as a whole, but also because they're important in the analysis of this particular case. So to begin with phenotype capture, accurate phenotype information is really essential to exome analysis, both in the formulation of an analysis strategy and also in the interpretation of the results. So we asked for quite a bit of phenotypic detail on our requisition form, and you're seeing a zoomed-in version of our form here. You can see that we're actually asking for essentially a full body scan in hopes that this prompts the physician to give us as much detail as they have on the patient. So this phenotype information can be very important, as I mentioned, in variant filtration. So what I mean by filtration, it's our mechanism of taking all of the variants found in that individual and narrowing them down to the ones that are most likely to be responsible for disease. So different labs can take a lot of different filtration strategies for exome, and I want to go through a few options of what we used today. The first is what I consider to be one of the most intuitive strategies, which is to build a patient-specific gene list. This gene list is built starting with all of those phenotypes that we got from the physicians and essentially searching various databases and literature to find genes that are associated with those phenotypes. So we can use tools like OMIM, HDMD, there are even web-based tools like Phenomizer, which will allow you to enter a number of different phenotypes, and it returns the genes that are most likely to be associated with a combination of those phenotypes. And so we use all these various resources to combine into a very large, or rather hopefully not too large, gene list for the patient. Once we have that gene list assembled, we can start our filtration. So if we start at the left with all of the variants found in the patient, it's quite a large number. What we're going to do with our gene list is use that to select only variants that are found within those genes that we think to be particularly important to that patient. So that's what these columns represent in the middle. This will reduce the number of variants quite significantly already. You can see it gets down to about 300 to 800 variants. However, that's still way too many for us to analyze. So we apply additional filtration methods, such as a minor allele frequency cutoff. Essentially, if there are any variants that are at a frequency that is higher than the prevalence of the disease in the general population, that's most likely to be benign variation, so we rule those out. We'll also apply other exclusions, such as variants that we have previously seen and classified as benign, to get down ultimately in the range of 30 to 50 variants that need analysis. So this is a way. It definitely reduces the number of variants for analysis quite significantly. However, it's still not terribly efficient. That's still a lot of variants to analyze. We find that most of the variants that come out of this filtration strategy are just very rare missense variants that have no information in the literature, and therefore we really can't classify them above the U.S. for the most part. So we also apply a different filtration strategy to all of our cases, which we call an expanded medical exome analysis. This filtration is really designed to return the variants that we are most likely to be able to reliably associate with a patient's disease or classify as pathogenic. So again, we're starting with all of the variants found in the patient. And on this top row of filtration, we're selecting for variants that have been previously published as disease-causing in either HDMD or ClinVar below a certain frequency cutoff, here 5%. We also have another group of variants that we are likely to be able to classify as pathogenic even if they haven't been previously published, and that's loss-of-function variants. So we will also pull out in this filtration novel loss-of-function variants. However, because everyone has quite a few loss-of-function variants, we restrict, at least in our first pass, we restrict our loss-of-function analysis to only variants within what we call the medical exome gene list. So what is that? Our medical – oops, sorry about that. Our medical exome gene list is actually designed to capture all genes that are associated with human disease. So we build this gene list by drawing essentially all of the genes found within multiple resources, including HDMD, ClinVar, OMIM, and Uniprot. And this list started off in the range of 4,000 to 5,000 genes and is currently at about 7,500 genes. And we do update it quarterly so that we're not missing any newly discovered disease genes. So back to our filtration strategy. Again, we have selected for variants that are classified as pathogenic in either HDMD or ClinVar. We have also selected these novel loss-of-function variants within the medical exome. And this gets us down to a reasonable number of variants, but it's still quite a lot to analyze again. So again, we apply additional filters, and these include removing any variants that have been seen in more than 10% of the whole genome sequencing cases that we have seen, because these are most likely to be technical false positives. We'll also exclude variants that are in genes that are unrelated to the patient disease during our analysis, or any genes that really don't have any strong evidence for association with human disease. And finally, we'll perform some extra variant exclusions based again on variants that we have seen before and classified as non-pathogenic. So this method typically gets us down into the range of 5 to 10 variants that really need full review. And so this has been quite efficient for us, and this is actually the strategy that has returned all of the variants that we have reported so far. I will add as a caveat that if this method does not return a variant that we feel explains the patient disease, we will also go back and look at lots of function variants that are outside of our medical exome gene list. So I've mentioned that that method is the way that we have found all of the variants that we've reported so far. So just to touch on reporting quickly, we've decided as a lab to primarily report only pathogenic and likely pathogenic variants on our whole exome and whole genome sequencing reports. There are some exceptions to this rule. If we find a variant of uncertain significance that we find very suspicious or in a gene that is very consistent with the patient disease, we may report that. However, it doesn't come up very often. We will also report any pathogenic or likely pathogenic variants that are on the 56 gene list recommended by the ACMG for the reporting of incidental findings, and that's only if the patient selects to receive that information. Finally, if we do perform a gene-specific, a patient-specific gene list analysis, we will also report the coverage for all of those genes so that the physician is able to assess whether variants could have been adequately called in those genes. In terms of overall results, we call a case positive if we have found pathogenic or likely pathogenic variants that explain the patient disease, or negative if no such variants were found. So, at this point, I'm going to hand over the talk to Sami Amar, who's going to take us through this very interesting case. Thank you, Chrissy. So, for this case, this case was referred to us from the pulmonology clinic at MGH for exome sequencing, and this is the information that was provided to us at the time of testing. The probe man was a 10-year-old female. She presented with congenital unilateral hearing loss and acquired hearing loss developed in the opposite ear. There was bilateral scar tympanic membranes that were noted, and there was a history of recurrent infections, namely sinusitis, otitis media, and pneumonias. And the family history was – there was nothing significant in the family history. It was negative for cystic fibrosis and dysmotile cilia. The father did have asthma, allergies, and arthritis, and this patient did not have any siblings. So, the possible diagnoses that the clinicians suspected were one of two things, either primary ciliary dyskinesia based on the presence of recurrent respiratory tract and ear infections due to dysmotile cilia. However, the ciliary biopsy was inconclusive for this patient. And the second possible diagnosis was immunodeficiency. There's, again, the recurrent infections would point to that, to some defect in immune function. However, the patient had a normal immune workup based on a CBC. We ran the DNA for this patient through our standard whole exome sequencing workflow. And for the filtration approach, we applied both filters that Christy had talked about for this patient. So, we came up with a gene list for this patient based on the indications that were presented, and this included 530 genes that were associated with or involved with primary ciliary dyskinesia, immunodeficiency, as well as interstitial pulmonary fibrosis genes. And we looked at all the variants that had an allele frequency of less than 5% in the population databases. We also used the expanded medical exome analysis filtration method to look at the 7,000 or 7,000, more than 7,000 genes that have been implicated in human disease. And for these, we looked at all previously reported variants, as well as novel loss of function variants with, again, a minor allele frequency of less than 5% in the population databases. So, both of these filtration methods came up with two variants for this patient. One was a heterozygous CCDC151 missense variant, and the other was a splice variant in the – a heterozygous splice variant in the LANG. So now we'll look at these a little bit more closely. For the missense variant in CCDC151, this missense variant – this is a screenshot of the IGV sequencing read, so it's a heterozygous variant affecting this missense – affecting this amino acid. And for those of you who use Alamed, this is the Alamed output for this missense variant. And essentially, there's a couple of things to note here. First of all, that this variant has been observed previously in XAC, and the conservational – computational analysis prediction tools do not – do not suggest that this variant is causative. Additional information about this variant, the gene itself, CCDC151, has been associated with primary cellular dyskinesia and is expected to be inherited in an autosomal recessive pattern. However, this is a fairly new gene, and only one or two cases have been published to date. For this particular patient, there wasn't a second variant in that gene that was identified through our exome sequencing, and the variant spectrum does not really match up with a missense variant. And for those few cases that have been reported to date, it was primarily a loss of function – a loss of function variants that were described. So either a frameshift variant or a nonsense variant have been identified in individuals with PCD. The variant, as I mentioned before, has been seen in population databases, although it's not at a high enough frequency to rule out pathogenicity or to – to be confident that it's actually a benign variant. And conservation computational tools do not suggest pathogenicity. And this variant is – has also not been reported in any of the – in any literature or in affected individuals. So based on using this information and applying those to our ACMG standards and guidelines, you can see that the majority of data that's available for this variant, there's nothing to support a pathogenic role for this variant. And the only piece of evidence would actually suggest towards a benign role. However, it's not enough to call it likely benign or benign, so we classify this variant as a variant of unknown significance. The second variant that was identified was a splice variant in the ELAINE gene. The ELAINE gene has five exons, and this splice variant is in the three-prime end of the – of intron 4, which would presumably affect the splicing of exon 5. This variant has not been reported in the literature as well and was not seen in any population databases. And the ELAINE gene is associated with either severe congenital neutropenia or cyclic neutropenia. And this disease is inherited in autosomal dominant fashions. The clinical presentation for this individual is actually consistent with cyclic neutropenia, which is described to have recurrent – to have a history of recurrent infections, and we'll talk about that in a minute. And the variant spectrum for this gene and for this disease is enriched for lots of function variants, including non-sense frameshift and splice variants. And actually, several splice variants have been seen not at the three-prime end of intron 4, but actually at the five-prime end of intron 4. And those splice variants have been seen to segregate in individuals with cyclic neutropenia. So it is consistent with the features that have been seen in those individuals. Again, the variant itself has not been seen in any general population and not reported in the literature. So based on the information that was available to us, we had a few key pieces of evidence suggesting that this patient – this specific variant is a pathogenic variant and is related to this individual's phenotype. There are two additional pieces of evidence that would be also helpful to determine whether this is a causative variant for this individual's phenotype, and those included de novo testing in parents. Presumably, this individual did not have a positive family history for recurrent infections, so de novo testing or clinical assessment of the parents to determine if there is a phenotype here would be helpful to determine whether one of these two scenarios is the correct scenario. A little bit more about Elaine-related neutropenia. The Elaine gene codes for a serine protease that is found in neutrophils and in monocyte granules and is responsible for the protease activities of these cells. The first gene-disease association between neutropenia and this gene was first described in 1999, and actually in a family that had cyclic neutropenia. The prevalence of this disease is rare, and their inheritance is autosomal dominant. However, a large number of individuals were reported to be either sporadic and de novo variants were identified in several individuals. For the familial studies that were available, most studies suggest that the penetrance is full. However, there is a suggestion that there is anticipation with later generations. This is not always seen, but the common theme across familial studies was that you don't see more severe cases in parents than in children, but it may not get worse in children or in later generations. Finally, the Elaine gene and variants in the Elaine gene can be identified in 90 to 100 percent of individuals with autosomal dominant neutropenia. So, Elaine...so there's...the available literature on this gene and the disease and the neutropenia that's caused by this gene strongly suggests that there's really two types of neutropenia that is associated with the mutations that are distinct from each other in several ways. And those two types are listed here. One is the severe congenital neutropenia, and the second is the cyclic neutropenia or cyclic hematopoiesis. For the severe congenital form, the absolute...one thing that is noted is that the absolute neutrophil count is less than 500 neutrophils per microliter. For a normal individual, the normal number of...the normal AMC count is from 1,500 and above. And in severe congenital neutropenia, for most individuals, this is recognized at birth or shortly after due to recurrent fevers or severe bacterial infections. And the common first sign is on...ontholitis, which is an infection of the umbilical stump. In some cases, having severe congenital neutropenia predisposes individuals for leukemic events, which is exacerbated by the granulocyte colony-stimulating factor, which is one of the modes of treatment for severe congenital neutropenia. The second type is the cyclic neutropenia, and this is characterized by oscillating neutropenia the last three to five days in a 21-day period cycle. So this figure here shows how different AMC counts at different time points will show the cyclic events where you have three to five days of actual neutropenia, and for the rest of the time, the neutropenia levels are...are either slightly below normal or at normal levels. However, again, for these individuals, even with cyclic neutropenia, these are usually diagnosed soon after birth or within the first year of life, based on a pattern of recurrent fever and oral ulcerations with serial blood counts showing this cyclic neutropenia activity. And one thing that was noted in cyclic neutropenia was that the symptoms are more severe in children than they are in adults, and they seem to get better with age. One common theme that was shared between cyclic and...the cyclic form, as well as the severe congenital form, just based on the literature, was that individuals have an early onset or can be detected earlier in life. There is a history of fever and recurrent skin and oropharyngeal inflammation. Many studies said that either mouth ulcers or gingivitis were some of the key symptoms that helped identify or help class...help diagnose the disease in individuals. The variant spectrum for both cyclic and severe congenital neutropenia is shown here. The variants that are shown at the top here are for severe congenital neutropenia, and the ones that are shown at the bottom here are for cyclic neutropenia. The spectrum includes missense variants, loss-of-function variants, a very broad spectrum of variants for both cyclic and severe neutropenia. And for the most part, variants are either cause one or the other. About 70 to 80 percent of them are exclusive to either severe or...severe congenital neutropenia or the cyclic form. However, the ones that are shown in bold are actually shared between severe congenital neutropenia as well as cyclic neutropenia. And in a couple of studies, it was shown that even within the same family, individuals can have...carrying the same variant can have different types of neutropenia, which suggested that there may be other factors, environmental or other genetic background may influence the actual...the actual presentation of the disease in individuals. One thing to note is that the majority of missense variants occurring in the earlier exons of the gene are mostly seen in the congenital neutropenia, and that loss-of-functions or splice variants affecting NTRN4 are common to cyclic neutropenia. So based on this information, we've classified this variant as pathogenic. However, we wanted to see whether this would...this pathogenic variant is actually consistent with the...with the phenotype that's seen in the...in the patient, given the information that we had at the time of testing, and given the available information about neutropenia and the different types of neutropenia, the phenotypic spectrum that's been associated with ELAIN-related neutropenia, whether it...that information was consistent with the...with what we were seeing in the patient. So the patient did have recurrent infections, and this is consistent with...with what is seen with ELAIN-related neutropenia, but it...in the requisition form, it wasn't clear whether the common infections that were...that were present in the literature, that were highlighted in the literature, were present in this individual. So the two common infections that were mentioned over and over again were the recurrent skin and the...the mouth ulcers, the gingivitis, and the, in general, the...the oropharyngeal inflammation that was noted. In addition, the patient had an immune workup, and that was...that was normal at the time of testing. But as we know, with cyclic neutropenia, there are bouts of...of normal or subnormal levels of absolute neutrophil counts. And finally, the patient did not present with an autosomal dominant family history, but of course, it could be de novo. De novo cases have been seen in individuals with ELAIN-related neutropenia, and the variant is a splice variant affecting intron 4, and splice variants in...affecting intron 4 of the ELAIN gene have been seen in other individuals with...with neutropenia. And so, at this point, using this information, we have two ways we can report this based on the...the classification of the variant, but also whether we felt the...this variant is consistent with this individual's disease, and it was causative for the phenotype that was present in...that was observed in the clinic. In our discussions with the...with the physicians and the clinicians, they had agreed that the...this variant and the available information, along with the...the clinical history of this individual, is consistent with ELAIN-related neutropenia, despite the fact that, you know, the...the fact of the normal immune workup, as well as the...the more...the absence of pathopneumonic infections, such as the recurrent skin and the mouth...the mouth...the mouth inflammation. And so, based on that, we reported this...this report out as...as positive, and we felt that this variant was causative for this individual's disease. And so, this experience led us to kind of...or point...highlighted some...some...some key factors that...that we experienced in the laboratory. So, one of those things is that the two different variant filtration approaches, either using a gene-based list or the medical exome list, gave us the same type of data. So, both of those filtration methods gave us the ELAIN variant that we found to be causative for this...for this individual's phenotype. So, it...so, it may...one consideration is that the time spent curating genes for these gene lists, which we sometimes...which are unclear or sometimes missing, do not always...is not always the best filtration approach to use. And the second piece was that the expanded analysis of the medical exome genes can also identify these causative variants. The second realization or conclusion from this case was that there is in the ELAIN-related neutropenia, as is the case with many genetic diseases. There are classic diagnostic criteria that bend certain features into either one type or the other. However, with molecular diagnostics, that's changing. And this is what we found that...this is the case with ELAIN-related neutropenia. And so, in this case, we felt that the mole...the molecular diagnosis of this case highlights that there is variability and overlap between these diseases, and there may not be truly two types of neutropenia, severe congenital and cyclic neutropenia, but they are part of the same spectrum. And the third piece was...this case highlights the importance of having communication with the clinicians to be able to essentially gather additional clinical information and family history, which is necessary to identify the primary cause of disease, but also allow for follow-up testing. In this case, we asked for additional serial absolute neutrophil counts, as well as de novo testing for parents. And now I will turn it over to Dr. Yonker, who will also provide the clinical perspective for this case. Thank you. So to give you a background and a picture of what the patient looks like, she's an 11-year-old girl, and she's really well-appearing, very pleasant, cooperative, and clinic, and no distress, no work of breathing. Her ears, though, are significant in the fact that they're severely scarred, so much that she has hearing aids in both ears. She was born with hearing loss, or hearing loss was picked up early in one ear, and then she eventually developed hearing loss in the other side, so it would likely be related to the recurrent ear infection. Her lungs were clear, no crackles or any, you know, abnormal breath sounds. And the other notable thing was that she did have some clubbing on her fingers, which is sort of a roundness to the fingertips, which can go along with, you see it a lot in cystic fibrosis or diseases where you have chronic infection and inflammation in the lungs. And here's a picture of her chest X-ray. And so we got this when she was well, and we were a little bit surprised at what we saw. It's hard to see on the, you know, on this projection, but it is hyperinflated, more hyperinflated than you would expect in a healthy child. And if you, let's see, if you look sort of near the central airways, there's some slight dilation, and also up here, which, dilation of the airways, which seem to be, at least in the central airways, larger than the corresponding vessel, which would be suggestive of bronchiectasis. And also there's some small cyst-like areas, which seem to be suggestive of bronchiectasis. Which, you know, would be, would be, which surprised us. Because of the hearing, you know, the hearing loss with the severe infections and the bronchiectasis suggestive of significant pneumonia, we decided to do an immune workup. And this was essentially normal. We looked at total white count, which was fine within normal differential. Notably there were some eosinophils, and she had a relatively elevated IgE with a positive allergy test to dust mites, so she does have some allergy and asthma, but that wouldn't correspond with this clinical picture. We also, we also looked at her B-cell and T-cell response by looking at immunoglobulin levels and response to various vaccine titers, and those were normal. Assuming this was primary ciliary dyskinesia, we did a ciliary biopsy in the nose, the nasal mucosa in the office, and that, unfortunately, was inconclusive. It's an unpleasant procedure for the patient, so it's really hard to have to do it multiple times, but unfortunately there were just too few cilia to comment on. We also did a sweat test to assess for cystic fibrosis, which was normal, and then we went back and reviewed her newborn screen, which was also normal for cystic fibrosis. So what was next? We asked a little bit more about her family history. I'm sorry. Well, what ended up happening next was we ended up sending, deciding to send the genetics, and we, what we ended up doing was sending this whole exome, and the reason we chose the whole exome was because it wasn't clear to us. We had high suspicion for primary ciliary dyskinesia or an immune defect, and the panels don't capture all of the PCV genes at this point, so we decided to go whole exome, and so we sent that, and then we got back this result. We had mostly interacted with dad for the family history before we got the result, and then when mom came back in, when both parents came back in to go over the results, mom gave slightly different history, and she did say that when she was younger, she used to get sick quite a bit, and there were also other family members who just got sick a lot. So take that for what it's worth. Mom was noted to have some clubbing in her fingertips as well, though. So because we also had this mutation, we decided to look at her neutropenia. She's not followed primarily in our hospital. She's followed at a private pediatrics center, so we had, we asked her to do some serial CBCs, so this child went, and every three days went and had a blood draw, and there was no, there, she never really got into the neutropenic area. Her ANC was greater than 2,000, and so really, to have neutropenia, you'd expect an ANC less than 500, but she wasn't sick at all during these blood draws, and she did it for about a 20-day period. So as Sammy mentioned, the periods between neutropenia can oscillate and sort of extend over time, so perhaps we just didn't capture her neutropenic period. And then there's a, he also brought up a good point that perhaps there's some, there's a spectrum of disease, and perhaps her mutation and the location of it may correspond with a milder presentation. So there is a registry for severe chronic neutropenia that was just published last year, and what they described was, granted, this is, there is some selection bias, because these are going to be the more sick patients along the spectrum who are picked up, but what they described was the majority of these patients had mouth ulcers, about 49% had pneumonia, 17% had sepsis, and 12% had cellulitis. So just thinking about our patient, she did have several episodes of pneumonia. She had sinusitis and ear infections. We didn't document any mouth ulcers, but, you know, she, you think to the, you know, a typical child who's in daycare, as she was, they frequently come home on a monthly, at least monthly, if not every other week, with something new, ulcers, pneumonia, ear infections, these sort of things. So it could have been overlooked and not picked up. So now that's sort of where we stand. I want to talk more about the disease as a whole, and then we'll get back to the patient. So for cyclic neutropenia, the treatment options are, you can, really, the recommended treatment is treating with GCSF, which helps boost neutrophil counts, and the dose is depending on what your starting level of neutropenia is and how resistant the neutropenia is to the medicine. And it can be, so anyways, and there are ways of augmenting the treatment as well. And if you really fail to respond to the GCSF, you can ultimately go on to bone marrow transplant. So the other option is no treatment. So though this is generally not what the patients in the registry, what they did, there were 66 patients in the registry who were not on treatment, and when they looked at how those patients did without treatment, the patients with cyclic neutropenia, there were eight deaths, three who died of sepsis, one who needed a hematopoietic transplant for a reason other than AML, which I'll talk about, which is a potential complication of cyclic neutropenia. And then others from typical sort of general population issues, cancer, heart disease, stroke, other causes. And the more severe cyclic neutropenia patients, the severe congenital neutropenia, had a higher number of deaths, and sorry, the number is wrong here, but the vast majority of them passed away from this myelodysplastic syndrome, or AML. And as I mentioned, these more severe patients often required transplants for failure to respond to GCSF, and a few of them died following that. So the outcomes, I mentioned that the neutrophil oscillations may persist, but the neutropenic periods are shortened, and of note, the milder disease tended to have a higher nader of ANC. Also, I briefly mentioned that there's a risk of developing myelodysplastic syndrome, or AML, and this really only occurred, really was specific to the severe congenital neutropenia group, or 16% of those patients developed this. There were no cases reported of AML or MDS in the cyclic neutropenia group. And then if you look at overall serious events, incidents such as death, transplant, or this AML over a 20-year cumulative time period, the outcomes were much worse in the severe congenital neutropenia, with 50% almost having a more significant event where it compared to 7% in cyclic neutropenia. And then when you look at the treatment of AML, I'm sorry, the treatment of cyclic neutropenia with GCSF, the treatments, the sort of cumulative exposure to GCSF, and you compare it to the development of MDS or AML, there is an increase. So the longer you're on the GCSF, the more likely you are to develop AML, and whether or not that speaks to the GCSF or the underlying disease is not clear to me by these graphs, but certainly there's an increased risk, and the worse your disease, the more likely you are to need GCSF, and the more likely you are to develop this negative outcome. So for our patients, what are our treatment options? Should we do GCSF? Her ANCs are currently normal, and there is a risk that in cancer patients that there is some immune dysregulation over time on GCSF, so no, it's not without risk, and it's not really warranted at this point, so we are not treating her GCSF, but we will keep a close eye on her, knowing that she's at risk for developing neutropenia. And then also we'll have a higher threshold for being more aggressive if she does develop an infection, and we would want to check a CBC and check her neutrophil counts when she is sick and with fevers. We'll just monitor her closely. We're testing the parents. Those results haven't come back yet, but we're testing mom and dad, and mom seems to have had this course where she was sick a lot when she was younger, and if she does carry the mutation, she's had a relatively benign course, so hopefully that would bode well as well for the patient. And then we'd monitor her for this MDS AML, and I think with a more mild disease, it's much less likely, but I would, you know, at most do a yearly CBC and a slow cytometry, but probably that's not even warranted, probably just periodic screening. And I think the most important component of this, of the information that we gained from this case, is family planning. So this allows for preconception counseling if, you know, she goes on to want to have kids when she's older, and then also if she does have children with or without genetic testing, it raises our threshold for keeping a close eye on them as well and treating them aggressively with antibiotics if they develop illness. With that, we'll open it up to questions. So one question that we've received was, what was the workup for the congenital hearing loss? I think Dr. Younger would be most appropriate to answer that. So the, I don't, I have not seen any workup in her medical records for the hearing loss, and I think it was felt since it was unilateral, they, you know, I'm not sure if they assumed that it was related to some in utero and filter exposure. She did have hearing in one ear, but that progressed over time. So I don't know, you know, what further workup has been done, and if it was just related to the scarring. Our sense now is that it was related to this chronic scarring from her recurrent ear infection. Second question is, why was the VUS reported? So we did, we did report both the Elaine variant as well as the variant in the PCV gene, which was classified as a VUS, and a couple of the different things led us to report the VUS variant. As Christy mentioned, we rarely report VUS variants that we don't think are associated with disease, but the one, the first thing was that there was a strong indication in the, in the requisition form, or in the clinical information, that it was a PCV-related disease, and even though we felt that Elaine does explain, is consistent with the patient's phenotype, and is likely to be the primary cause, we didn't want to rule out, given that we don't have, there is no dominant family history, or at least in the beginning, we didn't get a sense that there was a family history, and the fact that we didn't know whether the Elaine variant was de novo or not, we didn't want to rule out that there might be a secondary cause of disease, given that this is a VUS, it's a, in a PCV gene that may or may not be related, so we just wanted to indicate to the physician that there was this other variant that is related to PCV that was also identified, but at the same time, we felt strongly that the primary cause of disease is because of the Elaine variant. The next question is about how many mutations, CF mutations were tested as a newborn, and where CF mutations looked at in the whole exome sequence, and I think that's a great question, and that was definitely top of our list as well. In Massachusetts, the newborn screen has been around for several years. It was an opt-in program for a while, and now it's standard, and currently, we look for the most common, the 36 most common mutations on the newborn screen. In the whole exome, they did look at the CF mutations, so Sam is going to talk more about that. So the cystic fibrosis gene is covered within our exome, and all coding regions as well as splice sites and known variants in the CF gene were, would be picked up by our testing method, but we didn't identify any cystic fibrosis variants in this individual. All right, there's no additional questions, so with that, I'd like to thank you all for attending and to thank the ACMG for the opportunity to present our case and share it with you. Thank you very much.

clinical case

recurrent infections

hearing loss

sinusitis

otitis media

pneumonias

primary ciliary dyskinesia

immunodeficiency

neutropenia