Welcome to the ACMG Genomics Case Conference. The title of today's session is Beyond Exome, Going the Extra Mile, and it's hosted by Greenwood Genetics Center. This activity is supported by an unrestricted educational grant from QIAGEN Bioinformatics and the Ingenuity Clinical Decision Support Platform. This conference is intended for educational purposes only. I'm Julie Jones. I'm the director of the Clinical Genomics Sequencing Program at Greenwood Genetics Center. And today's other presenters include Dr. Katie Spellesey and Dr. Kaja Ellsworth, who are two of our clinical molecular genetics fellows, and Dr. Jenny Lee, who is the lead director of our molecular diagnostic laboratory. The only disclosure we have is that GGC does receive revenue from diagnostic testing performed in our diagnostic laboratories. The planning committee and staff have no disclosures. This session is approved for one CME credit. And if those who purchased CME at the time of registration will receive an email with questions and a certificate within 24 hours. Everyone is asked to complete an evaluation. And at the end of the webinar, there will be a link to access the evaluation. And this link will also be emailed to you. These are the overall learning objectives for the Genomics Case Conference Series. And the learning objectives for today's session include demonstrate the importance of considering prior test results to help guide exome sequencing analysis, explain how exome sequencing results might facilitate additional testing that may ultimately help make the diagnosis, and in some cases, expand the phenotype of a disorder, and discuss the importance of close collaboration between the clinical and laboratory groups during exome analysis. To ask a panelist a question, type your question to the Q&A box in the lower left corner of your control panel. Questions will be answered verbally by the panelist at the end of the presentation. So during today's session, I will begin with an overview of GGC's experience with exome sequencing. We will then discuss four exome cases that were of particular interest to us. And although the exome results provided important information to help guide us in the right direction for these patients, these cases required us to go beyond exome sequencing to actually come to a diagnosis for these patients. The Greenwood Genetic Center is a private, nonprofit genetic institute that was established 43 years ago in Greenwood, South Carolina. The center provides clinical genetic services and has clinical offices located around the state, including offices in Florence, Charleston, Columbia, Greenville, and Greenwood. Diagnostic testing is performed in our laboratories based on our Greenwood campus. And the center is also heavily focused on both education and research. GGC began performing exome sequencing on a diagnostic basis on October 1 of 2014. All patients have genetic counseling both before and after exome sequencing. And during the pretest counseling session, the patient and or their parent decide whether or not they wish to receive secondary findings results. Also, as part of the exome request, the clinicians complete a clinical information form, and the exome analysis is performed based on the patient's phenotype. When we began exome sequencing at GGC, the Agilent SureSelect Human All Exome Version 5 Kit was used for exome enrichment. But in April of last year, we switched to the Agilent SureSelect Clinical Research Exome Kit, which provides deeper coverage for those genes that are known to be associated with human diseases. Paired end sequencing is performed on the Illumina NEC-SEQ 500 in the high-output flow cell configuration. Nine samples are included in each run. And with the CRE enrichment, the average full coverage is 176x, with 97% of the targeted regions covered at 10x or greater. This slide shows an overview of our bioinformatics pipeline. So once sequencing is complete, BCL files are initially sent to a Linux system for demultiplexing and generation of FASTQ files. The FASTQ files are then sent to the alignment cluster, and the NextGen software is used to merge the FASTQ files, delete duplicate reads, align the converted reads with the human reference sequence, and ultimately create BCL files. The multi-column BCL files are uploaded to Cartagena NGS Bench, and the Cartagena software is used to filter and classify the variants. Our bioinformaticists also use an in-house algorithm to detect copy number variants. And this information is provided to the analysts and the lab directors in a run summary report that also includes various other run metrics. During variant analysis, the analysts and lab directors work closely with the patient's clinical geneticist and genetic counselor to determine which variants should be confirmed and reported for the patient. Thus far, we have completed exome sequencing on 1,153 samples, of which 377 were ProVan samples. Analysis has been completed on 365 of these ProVans whose ages range at the time of testing from a few weeks to over 63 years. We have also completed analysis on three fetal PATH cases. The average age at the time of testing The average age at the time of testing was just over 10 years, with the median age just over eight years. A little over half of the ProVans were males, and most of the patients were Caucasians. The vast majority of the ProVans were run with family members as trios, but we have run them as singletons and also as part of duos, quads, and even quints. 36.4% of the ProVans had an abnormal result, and of those, a pathogenic or likely pathogenic variant was able to explain the patient's full phenotype in approximately 24% of the cases. And an additional 6%, the pathogenic variant that was detected could explain at least part of the phenotype. And in a little over 5%, a single heterozygous variant, a pathogenic variant was detected in a gene associated with an autosomal recessive disorder that had at least some overlap with the patient's clinical features. Approximately 21% of the ProVans had one or more variants of uncertain significance reported, and close to 40% had no variants reported. Two of the ProVans had two distinct disorders, and the pathogenic variants were identified for both disorders. And the vast majority of ProVans did consent to receive results for pathogenic variants in secondary findings genes, and about 3% of ProVans did have an abnormal secondary finding result. And the secondary findings gene list that was analyzed included the 56 genes recommended by the ACMG in 2013, as well as the PALV2 gene, and we are currently transitioning to version two. The first exome patient we will discuss today is a 19-year-old male with intellectual disability. He's microcephalic and has aortic stenosis by a custodial aortic valve, generalized muscle wasting, truncal hypotonia, as well as lower limb hypertonicity. He was appreciated to have developmental delay when he did not sit alone until 20 months and did not walk until two and a half years of age. He was referred to the Department of Health He was referred for genetic evaluation at 18 years of age because of a suspected muscular dystrophy that was suggested by thin habitus and an increased serum creatinine kinase level. At the time of his evaluation, his height was at the 10th centile, but his weight and head circumference were less than the third centile. Notice more facial features were observed. He was noted to have tight heel core, but significant truncal hypotonia, which led to a very broad date. Brain MRI scans showed abnormal T2 signals. And laboratory studies, which included whole gene sequencing and whole genome microarray analysis were normal. So here's a photo of our patient. And you can appreciate the thin habitus and the decreased muscle mass. Full exome sequencing was performed on the patient and his parents. An analysis in Cartagenia using a receptive classification tree identified two variants in the DPM2 gene in trans. One of the alterations was a nonsense change that was inherited from the father. The other was a missense change that was inherited from the patient's mother. And both of the alterations were confirmed by Sanger sequencing in the patient as well as the parents. Using the ACMG guidelines for variant interpretation, the nonsense alteration was classified as likely pathogenic since it is a null variant and it is absent from the public SNP databases, including exact. A missense change is also absent from the SNP databases. And it is predicted to be deleterious by several different in silico analysis algorithms, including mutation taster, LRT, polyphen-2, and SIFT. But based on the available evidence, this missense change was classified as a variant of uncertain significance. The DPM2 gene encodes the dolphyl phosphate manifold transferase subunit two, which is part of the DPMS complex. And the DPMS complex is composed of DPM1, DPM2, and DPM3. And the genes which encode these three proteins have all been associated with congenital disorders of glycosylation. The DPMS complex is a key enzyme in both N and O-linked glycosylation, as well as GPI anchor synthesis. Today, there's only been one published report describing patients with pathogenic alterations in the DPM2 gene. All three patients included in the report had a severe clinical presentation that's characterized by hypotonia, progressive muscle weakness and wasting, absent psychomotor development, seizures, and early death that occurred between seven months and three years of age. The patient in one family was a compound heterozygote and had a missense change, and on the other allele, had a splice variant. And the two siblings in the second family were homozygous for the same missense change that was identified in the first family. Although our patient's clinical presentation was much less severe than that described in these three patients, we felt there was enough overlap to pursue the exome findings further. So we contacted Dr. Eva Morava, one of the authors of this paper, to see if she would be willing to collaborate on additional studies for our patient. And she did graciously agree to help, so we therefore sent her some samples from our patient. So the first test that her lab performed was the transparent isoelectric focusing. Lane one and lane three through seven contained serum from healthy normal control. Lane two contained serum from a PGM1 deficient control. And our patient's serum is in lane eight. And the results for our patient are mildly abnormal with decreased tetracylotransparent and elevated dicylotransparent isoform. DPM1 required DPM2 for its stable expression in the endoplasmic reticulum. Therefore, DPM1 western blot analysis was performed using fibroblast from either a healthy male control or fibroblast from our patient. And as you can see, this western blot detected no quantifiable DPM1 protein expression in the fibroblast from our patient. ICAM1 is a known hypoglycosylation biomarker in cells from patients with congenital disorders of glycosylation. And western blotting for ICAM1 revealed a significant decrease in ICAM1 protein expression in our patient's fibroblast. This demonstrates a decrease in linked glycosylation. Fibroblast from our patient also showed a significant decrease in ICAM1 protein expression by immunofluorescence staining, indicating a decrease in cell surface in linked glycosylation. So in summary, we've identified two alterations in the DPM2 gene in trans, and these alterations have not been previously reported. And these were identified in a patient that has microcephaly intellectual disability as well as clinical features that are consistent with the muscular dystrophy. Various studies showed that our patient does have a glycosylation defect. And based on these collective studies, we feel that the DPM2 variants that were identified by exome sequencing are responsible for the patient's clinical features. And since his features are much less severe than the clinical presentation for those patients previously reported, his diagnosis would lead to an expanded phenotype for DPM2-associated CDG. And now Dr. Katie Spellese will present our second case. Good afternoon. The proband in this second case was first seen at the Greenwood Genetics Center at the age of seven months old. He was born to a 34-year-old G4, P3 mother. And the reason for referral was to follow up on an abnormal newborn screen that showed low or borderline thyroxine, or T4, and low thyroid-stimulating hormone, or TSH. This biochemical finding is consistent with central hypothyroidism. For those who may not be familiar with central hypothyroidism, I included this schematic showing the feedback loop between the hypothalamus, the pituitary gland, and the thyroid gland. The type of hypothyroidism that most people are probably most familiar with is referred to as primary hypothyroidism, and it originates with abnormalities in the thyroid gland. And biochemical testing in this type of hypothyroidism typically shows low T4 and very high TSH. In central hypothyroidism, however, the abnormality can originate in two different places, either at the pituitary gland, which is referred to as secondary hypothyroidism, or in the hypothalamus, which is referred to as tertiary hypothyroidism. And in both of these situations, the biochemical testing typically shows low T4 and low or normal TSH, sometimes referred to as paradoxically normal TSH. And this is exactly what we observed in our patient, indicating he likely has an abnormality in either the pituitary gland or the hypothalamus. We followed up with this family a couple times over the next year or so. Our clinician noted that this proband had normal growth. Cranial MRI was also performed in order to check the pituitary gland for normal growth, as well as for abnormalities, and they didn't find anything abnormal. So this was a normal cranial MRI. They also performed ATTH testing, which was also normal. However, over the course of this year, our proband started to have these bony growths, both on its fingers and one on its patella. One of them occurred in the joint of his right index finger, causing this angulation of the finger. And I'll show a picture of that in just a few slides. This is the pedigree of our family. You can see our proband here, indicated by the arrow. He has two older brothers that are also affected with central hypothyroidism and who also have these unusual bony nodules. Of note in this pedigree is that the mother had a normal thyroid canal, but the maternal grandmother was diagnosed with hypothyroidism late in life at age 58 years old. The father was apparently unaffected with either phenotype, and as well as the maternal half-brother was also unaffected. Here's some pictures of these bony nodules in these brothers. On the far right here is our proband. You can see the bony nodules here on the side of the fifth finger, as well as this angulation due to the nodule in the joint on the index finger. The middle brother has this growth on his ankle, and the oldest brother has a couple of these growths on his digits. These are x-rays of the bony nodules, which reveal that they're actually end chondromas, which are growth originating in the cartilage tissue. Genetic testing was pursued in order to find the origin of the phenotypes in this family. The first test that was performed was a ray of PGH, and this revealed two duplications, one on 8q and one on 17q. Both of these were paternal in origin and were classified as variants of uncertain clinical significance. Neither of these duplications were present in the older affected brothers, so we concluded that these were likely not contributing to the phenotype of interest. Therefore, OLEPSOM sequencing was performed, and the sequencing was performed on the proband, his parents, and in the unaffected maternal half-brother as a quad analysis. This analysis identified two pathogenic nonsense variants, one in the protein tyrosine phosphatase non-receptor type 11 gene, or PTPN11 gene, and the second in the immunoglobulin superfamily member one gene, or the IGSF1 gene. These are the OMIM entries for these two genes. Everyone is probably familiar with PTPN11 because it's associated with both Noonan syndrome and Leopard syndrome, and these syndromes are caused by gain-of-function pathogenic variation in this gene. However, in 2011 and 2013, some publications came out in the literature demonstrating that loss-of-function heterozygous variants in PTPN11 lead to autosomal-dominant metachondromatosis. And this phenotype is characterized mostly by skeletal findings that include multiple exostoses, especially on the digits, and multiple enchondromas, and this is consistent with what we observed in the brothers of our family. The second condition is due to the IGSF1 gene, and it's X-linked recessive central hypothyroidism, also consistent with what we observed in this family and in the inheritance pattern in the pedigree. As far as inheritance, obviously the IGSF1 variant was maternally inherited. You can see in our Cartogenes software here that mother is heterozygous, the father and unaffected half-brother are wild-type G allele, and our patient is henozygous for this C allele. The PTPN11 variant, on the other hand, appears to be de novo. You can see mom, dad, and unaffected half-brother have only the wild-type T allele, and our patient is heterozygous for this G allele. However, given the three affected brothers, we suspected some sort of mosaicism, so we went back to the next-gen sequencing data and looked at this position in both of the parents. And sure enough, we found that dad had a very low level of this variant G allele at about 7% variant frequency, indicating that he's likely a low-level mosaic at this position. Our exoanalysis pipeline requires a threshold of 12% variant in order to call variants as heterozygous, and that's why dad appeared to be wild-type in this analysis. Here on the right are the Sanger confirmations of this variant. On the top is the forward and reverse read in our proband, and he is, as you can see, obviously heterozygous at this position. The bottom two reads are the Sanger confirmation in the father, and you can see just barely a hint of that G peak here in the reverse read. So this is consistent with the dad in this family being a low-level mosaic for this variant. In summary, we were able to establish a dual diagnosis in this family by a whole exome sequencing. The first diagnosis was X-linked central hypothyroidism due to a nonsense variant in the IGSF1 gene, which was inherited from the mother. The second diagnosis was autosomal-dominant metachondromatosis due to a pathogenic nonsense variant in the PTPN11 gene, which was inherited from an apparently mosaic father. And with that, I will turn it over to Dr. Kaja Ellsworth, and she will present our third case today. Good afternoon. I will present the case number three. This is a case of a seven-year-old female that was evaluated at the genetics clinic for history of speech and language delay and vision problems. The patient's medical records revealed her history of accommodative esotropia, amblyopia, and hyperopia. She had a long-standing history of Eustachian tube dysfunction, and recurrent acute otitis media was potential conductive low-frequency hearing loss. She also had recurrent severe urinary tract infections, therefore, renal ultrasound and VCOG were performed, and both were described as normal. Developmentally, she began crawling at six months and walking at 16 months. Speech delay was noted between age of 12 and 18 months. Around age of four, brain MRI was done, which was also reported as normal. At the time of evaluation, the patient had approximately 20 words, and upon physical examination, her height was in the seventh percentile, her weight was in the 60th percentile, and her head circumference was in the 64th percentile. She was described as mildly dysmorphic, with upslanting palate paper fissures, synophrys, everted lower lip, and thickened nasal tip. Her skin was described as soft and dolly, and previous genetic testing, including microarray, which was reported as normal. Therefore, next, whole-exome sequencing was ordered. Whole-exome sequencing identified a heterozygous de novo alteration in the PHIP gene, which stands for Plexstrain Homology Domain Interacting Protein. This gene is located on chromosome 6q14.1, and the identified variant was not present in any databases. Based on the literature review, at the time, there was only one publication that identified this gene, PHIP, as a candidate gene for intellectual disability. And in that particular publication, a patient had a de novo nonsense variant in this gene that was also identified via whole-exome sequencing. The patient had overlapping clinical features with our patient. In addition to that, there were several reports describing patients with microdeletions that were encompassing the PHIP gene that also had clinical features overlapping with our patient. However, some of those individuals had also additional clinical findings, most likely due to the deletion of adjacent genes in that chromosomal location. So, based on that collective information and evidence, we decided to classify this variant as a variant of uncertain clinical significance. Although this identified change is a synonymous change, not predicted to affect the protein sequence and its amino acid position, given its proximity of this alteration to exome 24 and intron 24 boundary, we suspected that splicing of exome 24 might be affected. Therefore, first, we utilized some in silico prediction algorithms, as listed or shown over here, which are summarized on this slide. You can see that the maximum entropy models used here were significantly lower for the variant as compared to the wild type. Also, creation of an exonic splice silencer was predicted with alteration of the wild type donor site. The mutation taster predicted this change to be disease-causing. Moreover, the region where this variant is located is conserved among various species. So, based on that, we decided to perform RNA analysis to further characterize this variant, and fast tubes were obtained from the province, as well as from the province's parents. Once RNA was extracted and our cDNA was obtained, we used several different primer pairs, including one shown on this slide, that was flunking the entire region to amplify this region of interest. We first ran Ki-Xel-Gel prior to sequencing of our applicants, and we noticed an extra band that was present in the province, but was not observed in the mom or death sample. That suggested that possibly there's another shorter-than-expected product. When we sequenced that, we saw that the patient had a heterozygous deletion of an entire exome 24 region, so that was about 120 amino acids. Given that the synonymous change in the gene was the novel in the province, neither the mom or the dad had the same pattern on electrophoreogram. PHIP produces at least three proteins through alternative splicing, PHIP, NDRP, and PHIP. Domains of these proteins include a beta propeller forming the WD40-RP domain, nuclear localization signal, plexstrain homology domain binding region, and bromo domain. When we were performing our functional studies, at the same time a case report was published with a couple more variants identified in this gene. All of those published changes are indicated by those arrows. All of those variants are located in various domains of PHIP protein. Interestingly, PHIP1 is the only protein product disrupted by all of those four variants. Our variant shown through RNA studies leads to skipping of exome 24. This exome encodes for a functional protein domain called plexstrain homology domain binding region, to which PARP interacts with insulin receptor substrate, or IRS1, to propagate insulin signaling. Below are immunoprecipitation assay results showing this particular interaction between those two proteins in vitro. Moreover, PHIP has a high haploinsufficiency score, as well as intolerance to both loss of function variants in EXAC-PLI scores 1 and missus variants in EXAC-Z scores 5.2. Given that variants identified in this gene can lead to frame shifts or disruption of functional domains, as well as the fact that PHIP was in the region of larger chromosomal deletions, it was proposed the mechanism for the disease is autosomal dominant loss of function. This is a summary of clinical features of all of the patients that had variants identified in PHIP. In red are highlighted features that our patients shared with those other patients, and that includes the fact that this variant is a de novo change that was identified in all of those individuals. Those patients had some dysmorphic features, ophthalmologic issues, as well as delayed speech. Our patient, given her young age, she might still present with more features later in her life, especially when she is formally assessed for such. Lastly, there are only a limited number of cases published with variants in this gene and available photos. Therefore, genotype-phenotype correlation is not obvious yet in those individuals. Here is side-by-side comparison of a female patient that had a message change identified in this gene, as well as our patient. As you can see, both of those patients have mild dysmorphic features that include straight eyebrows. The patient to your left has deep-set eyes, as well as flashy ears. And our patient, she has up-slanted piperal fillers, as well as very mildly over-followed ear helices. And with that, I would like to say thank you. And Dr. Jenny Lee will present case number four. Okay, so I will present our final case. This is currently an eight-month-old African-American female who originally presented in the neonatal period, has progressed rapidly, and continues to be frequently hospitalized. On newborn screening, it was found that she had elevated tyrosine, which was two to three times the normal limit. And this was confirmed by her plasma amino acid profile. In addition, the patient had congenital posterior cleft palate, as well as mild micrognathia. Most salient of her features, as well, is the severe hypotonia that she exhibited, as well as severe weakness, anorexia, failure to thrive, a complete feeding failure that requires her to be G-tube dependent, as well as ventilator dependent. She was small on exam at one month, approximately the first percentile, and questionably had seizures. So she had some prior testing performed. She was negative for Angelman and Prader-Willi syndromes, as well as myotonic dystrophy. Also normal were her echo, renal ultrasound, brain MRI, and CK level. Her urine ologosaccharides were normal, which ruled out Pompeii. And her succinyl acetone was also normal, which ruled out tyrosinemia type one. She also had a normal hepatic panel, which ruled out liver disease. She showed an abnormal flowing on the EEG, which is a nonspecific finding. However, it was not consistent with the presence of seizures. In addition, she had microarray analysis performed, which revealed approximately 20% autosomal absence of heterozygosity, as well as a gain on chromosome 13 that was determined to be a variant of uncertain significance. In the region of homozygosity included genes for both tyrosinemia type two and three on chromosome 12. This could explain the elevated tyrosine. However, this finding could not explain severe hypotonia or cleft palate. The presence of such homozygosity in this patient does suggest autosomal recessive inheritance. However, consanguinity was denied in this family. At this point in time, the physician had suspected that there was more than one autosomal recessive condition for the patient. Given the complex clinical picture, at this time, whole exome sequencing was ordered. The final cause were the elevated tyrosine, the severe hypotonia, and the cleft palate. At this point in time, suspected were still tyrosinemia types two and three, as well as transient tyrosinemia, because other causes were already ruled out. Whole exome sequencing analysis was performed on proband and mother, and father was not available. By this analysis, we did have one finding in the HPD gene, which includes the enzyme 4-hydroxyphenylpyruvate dioxygenase. This change was a homozygous ring shift alteration, causing the duplication of four nucleotides. This change was shown to be maternal and presumed to be paternal, but the father was not tested. It is located in a region of homozygosity on chromosome 12. By ACMG criteria, we could assign PBF1, because this is a null variant, as well as PM2, because it was absent from all online databases. Although there was prior enzyme testing, those results were not specific to the HPD gene, and therefore we could not use them as functional evidence. Taken together, we determined that this change is likely pathogenic. Pathogenic alterations in the HPD gene are responsible for tyrosinemia type 3. This is an autosomal recessive condition and the least frequent form of tyrosinemia. The mechanism is a deficient HPD enzyme, which can't break down tyrosine, causing toxicity, nerve cell damage, and nerve cell death. Tyrosinemia type 3 has a variable clinical picture, including intellectual disability, seizures, and intermittent ataxia. In our particular patient, seizures were not observed. However, the patient was still quite young to exhibit the other features. This patient is being treated with a medical formula that is restricted in both tyrosine and phenylalanine. We also had a second finding by whole exome sequencing in the SNPD1 gene. This encodes acid sphingomyelinase. The change is a homozygous missense alteration that was maternal and presumed to be paternal and is well located in a region of homozygosity on chromosome 11. By ACMG criteria, we were able to assign PM2 because the allele has very low allele frequency. It was found in dbSNP and EXAC, but absent from EDS and HTMD. We could also assign PP3 because in silico programs, it's predicted to be deleterious. Taken together, we were able to assign this change as a variant of uncertain clinical significance. Pathogenic alterations in the SNPD1 gene are associated with pneumatic disease type A and B. While pneumatic disease could explain the patient's hypotonia, there are other features associated with pneumatic disease that did not fit the clinical picture. Therefore, this variant was thought to be non-contributory. As previously mentioned, as part of the whole exome sequencing analysis, we also do parallel analysis for copy number variation. By this analysis, we normalize the mean depth of coverage for consecutive coding exomes for a given patient. We take that information and compare it to a normalized database average and look at the standard deviation of the mean. That data is shown here on the lower right. There's a region of the genome in this patient that appears to be deleted. Interestingly, within this region is the SMN1 gene. If we also look at the raw reads for the SMN1 gene, it appears to have low coverage. I should point out at this time that there's also the presence of a highly homologous SMN2 gene. It is possible that some of the reads that we see are from that gene instead. This region that is potentially deleted is located in a region of homozygosity and chromosome 5. At this point, we had decided to recommend comprehensive molecular testing for spinal muscular atrophy. Based on this recommendation, this further testing was performed by MLPA. It was found that there are zero copies of SMN1 showing a homozygous loss. There are no copy number of SMN2. This most closely fits with this clinical picture of SMA type 1. SMA, or spinal muscular atrophy, is a neuromuscular disorder characterized by degeneration of the anterior horn cells of the spinal cord leading to symmetrical muscle weakness and atrophy. It is an autosomal receptive condition caused by mutation, gene conversion, or in this case deletion of the SMN1 gene. We thought that in this particular patient, it could explain the severe hypotonia that she was displaying. We were able to confirm a diagnosis of SMA type 1. In summary, this is another example of a dual diagnosis. For this case, we felt that prior testing, which included microarray analysis as well as enzyme studies, had guided the exome sequencing analysis. We felt that this exome sequencing analysis was also able to guide further follow-up testing that was needed to make the diagnosis. By whole exome sequencing, we identified a homozygous spring shift alteration in the HPD gene, which would explain the elevated tyrosine seen for this patient. This alteration is likely pathogenic and confirms the diagnosis of tyrosinemia type 3. We also suspected by whole exome sequencing a dilution including the SMN1 gene. This dilution was later confirmed by MLPA studies, which confirmed the diagnosis of spinal muscular atrophy type 1. And lastly, by whole exome sequencing analysis, we identified a homozygous mismatch change in the SMPD1 gene, which was determined to be a variant of uncertain significance and thought to be non-contributory. This concludes the cases that we plan to present to you today. We would like to acknowledge the patients and their families, as well as the clinicians and counselors who have referred patients for clinical testing. Lastly, we recognize the contributions of our laboratory staff and the clinical team at GGC for their considerable efforts. At this time, I'd like to open up this platform for questions. To ask a panelist a question, please type your question into the Q&A box in the lower left-hand corner of your screen. So we have a question for your case. Okay. Dr. Lee, were the parents in Case 4 consanguineous? We assume that they were, although that was denied when that was questioned. I think at that level of homozygosity, I think there has to be some level of consanguinity. Okay. And a couple of other questions regarding Case 4. SMN1 is located in the segmentally duplicated region. Do you handle the normalization of such regions differently than unique regions? I'm told by our bioinformatics specialist that we don't handle those regions differently. And another question for Case 4. The reads that did map to SMN1, were these assumed to be coming from SMN2? Yes. We presume that the reads, because of the homozygous loss that was confirmed by MLPA, that any reads that were present would have been from SMN2. Okay. And we have a question for Dr. Spellacy's case. So the question is, were both of the older affected brothers of Case 2 confirmed to carry both variants? Yes. We performed family studies by finger sequencing, and both variants segregated with disease in this family. And so I don't see any other questions at this time. So we'd like to again thank QIAGEN Bioinformatics and the Injunuity Clinical Decision Support Platform for the support of this program. A recording of the seminar will be available in the ACMG Genetics Academy in a couple of weeks. There were a couple of more questions that were just submitted, so we'll go ahead, since we have time, and take those. So the next question is also about Case 4. How many nucleotide differences were there between SMN1 and SMN2 in this patient? And does mapping typically differentiate between these two very similar genes? Dr. Lee is going to come and answer that question. I think the first part of the question was how many nucleotide differences were there. Well, SMN1 had a homozygous deletion, and so there was really no sequence to compare SMN2 to. And what was the second part of the question again? Does mapping typically differentiate between these two very similar genes? Okay. I would say relatively. I mean, if there's a difference between the nucleotide level, then that's about it. The answer would be that they somewhat differ at the nucleotide level. Yeah. Reads that map to both are deleted out. And reads that map to both are deleted out. OK. There's another question that says, what percentage of your positive exome cases have more than one etiology? And that was, we had two cases out of the probands that we've completed analysis, and that is 0.6%. Another question, was obligate carrier status for SMN1 confirmed in both parents? I think the parents were not tested, to my knowledge, for SMN1 or 2. And it's just presumed that, because the SMN1 gene is located in the region of homozygosity in the patient, that both parents would be carriers. But that was not confirmed. I don't see any other questions at this time, so we thank everyone for joining us and just a reminder that a recording of this seminar will be available within a few weeks at the ACMG Genetics Academy and also please remember to complete the evaluation. Thank you very much. Thank you.

ACMG Genomics Case Conference

Beyond Exome, Going the Extra Mile

Greenwood Genetics Center

QIAGEN Bioinformatics

educational purposes

whole exome sequencing

genetic variants

Q&A session