Hello, everyone. Welcome to the ACMG Genomics Case Conference. This session is featuring a session from the 2020 Annual Meeting Digital Edition, the Cardinal Signs of Common and Uncommon Disorders, featuring PLOD-3-related disorders, CHOP syndrome, and Primrose syndrome. This activity is intended for educational purposes only. My name is Dr. John Burnett from the University of Iowa. I'm a member of the planning committee for this case conference series. Our presenters in the broadcast today are Dr. Paul Hillman from the University of Texas, Dr. Kosuke Izumi from the Children's Hospital of Philadelphia, and Dr. Donita Velasco from the University of Nebraska Medical Center. Following the broadcast, I'll be joined for a live question and answer session by Dr. Hillman, Dr. Velasco, and Dr. Ian Krantz from the Children's Hospital of Philadelphia. All the presenters have nothing to disclose. These are the planning committee member financial disclosures. And this session is approved for one CME. We ask everyone to please complete the evaluation form at the end of this session by visiting the ACMG Genetics Academy. And these are the learning objectives for this session. As I mentioned, we'll have a live question and answer session following the broadcast. If you'd like to submit a question, please use the Q&A button in the Zoom chat. Platform. There is also a chat feature, but that's only to be used for indicating any technical issues. You can reach the ACMG staff to help out with technical issues with the chat function, but please use Q&A to ask any questions. There's also a feature to help prioritize a question by upvoting it by giving it a thumbs up. And with this, we'll start the broadcast. Hello, my name is Paul Hillman and I'm an assistant professor at McGovern Medical School at the University of Texas Health Science Center in Houston. I'd like to thank the ACMG for allowing me to talk to you today about my presentation Plot 3 Disease, Refining the Phenotype of a Clinically Recognizable Emerging Autosomal Recessive Collagenopathy. I have nothing to disclose. The objectives for today's talk is to review collagen function and its post-translation modification, review known disorders of collagen modification, define and identify Plot 3 Disease, and report on recommendations for management and testing availability. As many of you likely know, collagen is considered the glue that holds our bodies together. It has an important role in multiple different structures in humans and has also been associated with a number of different disorders. It is most prominently featured in the joints, cartilage, bone, muscle, eye, and skin. Lysylhydroxylases are a family of enzymes responsible for the post-translation modification of collagen, which is essential for the appropriate organization and extracellular cross-linking of collagen species. There are three known isoforms in humans, Lysylhydroxylase 1, 2, and 3. They have a great deal of amino acid homology and show significant evolutionary conservation. They also all share the similar Lysylhydroxylase activity. However, they do demonstrate tissue-specific expression profiles, although it has been proposed that there is some degree of functional redundancy between the three isoforms. LH3 is unique in possessing an additional functional domain capable of glycosylation. So not only can LH3 do the hydroxyl-lysyl-galactosyl transferase activity, but it can also do galactosyl-hydroxyl-lysyl-glucosyl transferase activity, making two modifications at the same position on a given collagen structure. All three of the Lysylhydroxylases have been previously associated with human disease. LH1, encoded by PLoD1 on chromosome 1, causes kyphoscoliotic Ehlers-Danlos syndrome, or EDS type 6. LH2, encoded by PLoD2 on chromosome 3, has been associated with a number of different skeletal disorders, including Brock syndrome, an autosomal recessive form of OI, kyphomelic dysplasia, a broad category of Brent bone disease, and mesomelic dysplasia, Kozlowski-Riordan type. LH3, encoded by PLoD3 on chromosome 7, has numerous reports associating its overexpression with cancer progression and metastasis, however, had not characteristically been associated with a hereditary disorder. This changed in 2008, when Salo et al. provided a case report of a female individual with biallelic pathogenic variants in PLoD3, leading to a connective tissue disorder phenotype. Her appearance was characterized by dysmorphic facial features, as well as numerous contractures of the large joints and fingers. Subsequently, this same family of patients was identified as having the miscarriage of a male fetus, which showed many of the same skeletal features upon autopsy. It would be 11 years before additional reports of disorders associated with PLoD3 mutations would be published. First, Madhuri Voola et al. used whole genome sequencing to confirm candidate genes in association with disease from autozygome. They reported on one sibship with homozygous mutations in the PLoD3 gene. They provide the phenotype of a single male that overlaps highly with the PLoD3 phenotype we'll be presenting later. Then, Fahed Nazad et al. reported on a single individual with a predominantly epidermolysis spilosa-like blistering phenotype, and showed abnormalities in type VII collagen function. However, this individual also had some dysmorphic features and skeletal findings consistent with those seen in the patients we will be presenting. Lastly, Ewens et al. provides a sibship with a distinct stickler-like phenotype arising from homozygous mutations in the PLoD3 gene. Now, 12 years later, with these results, we can conclude that Madhuri Voola et al. have identified nine affected families representing 15 cases. Recently, we have also received additional pedigrees suggesting the possibility of a founder mutation in the PLoD3 gene. This is the case of a single male with a predominantly epidermolysis spilosa-like blistering phenotype, and showed abnormalities in type VII What is PLoD3 disease? First, it is characterized by distinctive dysmorphic facial features, which have been found in 100% of our patients. These include prominent eyes, down-sensing palpebral fissures, malar or mid-face hypoplasia, short, upturned nose, and large, protruding eyes. This is the case of a single male with a predominantly malar or mid-face hypoplasia, short, upturned nose, and low set ears. Visual defects are also found in approximately 100% of individuals with PLoD3 disease. These visual defects are predominated by the finding of myopia in 10 out of 10 individuals. However, other eye findings also include early cataracts, flat retina, optically empty vitreous, this was only reported at one kinship, as well as case reports of retinal detachment. Musculoskeletal abnormalities are also seen in 100% of individuals and are predominated by finger contractures, prominent knees, and scoliosis. However, hypermobility of the elbows, large joint contractures, and low bone mineral density are also common. Ectodermal abnormalities are also common with findings of reduced palmar creases, blistering of the skin, and rapidly growing nails. 90% of individuals were reported to have developmental delay, and 8 out of 12 individuals had below average stature. Another hallmark feature of the disease is the finding of sensory or hearing loss in most affected people. Other complications contribute significantly to the morbidity and mortality of the patient. Complications contribute significantly to the morbidity and mortality of the disease, with at least three individuals having been reported to have suffered aneurysms during the course of their life, and at least four individuals dying prematurely likely from complications of PLOD3 disease or these aneurysms. PLOD3 disease arises from biallelic mutations in the PLOD3 gene. Of the nine families identified, five arose from homozygous biallelic variants in known consanguineous relationships. These families constitute only four different variants, as two of the families were from Iran, and there is a proposed founder effect in the Iranian population. Four of the nine families were found to be compound heterozygotes, accounting for seven different variants, as two unrelated individuals were found to have the same genetic change. In total, we found 11 different variants. Five of those 11 variants were in the lysylhydroxylase domain. Five of the 11 variants were in the glucosal transferase domain, and one of the 11 variants was in the AC or linker domain, which lies between the two enzymatically active areas of the protein. All patients showed a reduction in the activity of at least one of the two functional domains of the protein. Colleagues at the University of Oulu in Finland transfected fall armyworm cells with our identified human variants in the PLOD3 gene, and then measured the activity of the lysylhydroxylase domain as well as the glucosal transferase domain for each mutation. You can see here that for all mutations, at least one domain shows significantly reduced activity. The LU627 proline mutation was unique in showing significant reduction in both enzymatic activities. Conversely, the premature stock codon at arginine 452, which truncates the protein prior to the lysylhydroxylase domain and presumably completely eradicates its function, could not be measured as this variant was not excreted from the insect cells being studied, and therefore the glucosal transferase activity could not be studied. Lastly, the PRO270LU variant, as reported by Ewins et al. previously, showed approximately a 50% reduction in the glucosal transferase activity, but this did not reach statistical significance. It was pointed out by Ewins that this due to the distance site of this with respect to the enzyme activities, this may be expected in close proximity to the enzyme activity, and this may not be the case for the proline variant. Greater numbers may be need to reach significance in this situation. The differential diagnosis for PLOT3 disease is related to the known disorders associated with other lysylhydroxylases, as well as those proposed by previous case studies. Overlapping features within the differential diagnosis are highlighted in red. As Ewins et al. pointed out, Stickler syndrome has a number of overlapping features with PLOT3 disease. These include in characteristic faces, sensorineural hearing loss, and scoliosis. However, the findings of cleft palate and vitreol retinal abnormalities were not commonly seen in PLOT3 patients. Furthermore, the contractures, vascular dissections, and eye features of cataracts and myopia common in PLOT3 are not typically associated with Stickler syndrome. Similarly, in EDS, vascular dissection and aneurysm, easy bruising, and scoliosis are all shared features. However, EDS is not typically associated with dysmorphic faces, hearing loss, or eye abnormalities. Furthermore, EDS is also not associated with joint contractures, more hypermobility. Lastly, osteogenesis impervecta was suggested in the initial case study as a possible differential due to the presence of bone fractures in that patient. However, bone fractures has not been seen as a major feature in other PLOT3 individuals. With review of the clinical information available for the patients to date, it is recommended that people affected with PLOT3 disease have regular evaluations by physical, occupational, and speech therapy, have evaluations with audiology, orthopedics, and ophthalmology. We would also recommend regular imaging evaluation of vasculature secondary to the risk of aneurysm and dissection, which lead to significant morbidity in those patients, as aneurysms have been found to occur in the carotids, aorta, coronaries, popliteal, and cerebellar arteries. Fortunately, PLOT3 is now included on panel testing by numerous different companies and included on panels testing for such disorders as Marfan syndrome, Louise Dietz, EDS, and aortopathy panels. In summary, PLOT3 disease is a clinically recognizable syndrome with key features, including characteristic facial features, sensorineural hearing loss, myopia, prominent knees, finger contractures, and skeletal abnormalities. Appropriate diagnosis is essential for morbidity associated with vascular risks of spontaneous rupture and dissection. The findings of developmental delay reported here may be attributed to hearing loss and or evidence of a history of brain bleeds in many patients. It is yet to be determined if developmental delay is an inherent abnormality of PLOT3 disease or subsequent of its other complications. At this time, there is insufficient patient numbers to extrapolate genotype-phenotype correlation, but there is evidence of both intra- and inter-familial variation. I would like to acknowledge all of the patients and family affected by PLOT3 disease, particularly those who agreed to provide patient samples and clinical reports for our ongoing study. Special recognition is given to the individual in the top two left-hand corner pictures. This is the index case from 2008 who agreed to be re-evaluated at the age of 26 in order to better expand our phenotypic description of PLOT3 disease. I would also like to recognize my collaborators from across the globe who provided important clinical information about patients that they have identified with PLOT3 disease. Special recognition goes to the University of Lulu at Finland group for their contribution of the biochemical assays looking at the functional impact of the patient variants that were identified. And these are my references. Thank you. Hello, my name is Kosuke Izumi from Children's Hospital of Philadelphia. I'm going to share our work on CHOP syndrome today. I have nothing to disclose. Our work on CHOP syndrome started when we met these three patients. These patients were referred to our hospital because they were suspected of having Cornelia de Lange syndrome. Although we did understand why they were thought to have Cornelia de Lange syndrome, we thought they had a different diagnosis. Based on their common features, we named this diagnosis as CHOP syndrome. We recently published an article describing clinical characteristics of CHOP syndrome, so I am going to share our findings of CHOP syndrome based on this article. CHOP syndrome is characterized by facial dysmorphism. I put our patient pictures together here. Although some differences exist, round faces, synophilus, and cross-facial features are commonly seen in individuals with CHOP syndrome. Developmental delay is universally seen in individuals with CHOP syndrome. Average age they started rolling over is at 12 months, started sitting at 18 months, and they started walking independently at 4.8 years. Congenital heart disease and cardiac problems are common in CHOP syndrome. PDA and VSD are the common type of congenital heart defect seen in CHOP syndrome. On the other hand, complex congenital heart disease have not been identified. One of the unique features of CHOP syndrome is its pulmonary involvement. Chronic Lange disease of unknown etiology was seen in 60% of children with CHOP syndrome. Subglottic stenosis, laryngomalacia, and sleep apnea were also seen. Growth profile of CHOP syndrome is also very unique. This is a growth chart of one of our CHOP syndrome patients. This girl has been ratioed, but her weight measurement has been following around fifth percentile, so her BMI numbers have been very high. This box plot demonstrates the distributions of their growth parameters. Y-axis is standard deviation from the mean. At the time of the birth, they are proportionally small in length, weight, and head circumference. However, as they got older, they maintained weight-height measurements as alarmingly low. Skeletal abnormalities are also common, but mainly mild skeletal differences such as brachydactyly. However, vertebral bone sheath abnormalities are also common. This is a graph of the skeletal abnormalities of CHOP syndrome patients. brachydactyly. However, vertebral bone sheath difference and hypoplasia of radius and fibula have been reported in children with CHOP syndrome. Our study reveals that cardinal features of CHOP syndrome are very common in children with CHOP syndrome. Cognitive impairment was seen in 100%, heart defect was 80%, obesity was 100%, pulmonary involvement was 80%, and short stature was seen in 100% of children with CHOP syndrome. CHOP syndrome is due to heterozygous missense mutations in AFF4 gene. Their missense mutations are clustered in a small region of AFF4 shown here. Here is a picture of AFF4 protein, and all the CHOP syndrome mutations are in the ALF homology domain of AFF4. AFF4 is a transcriptional regulatory protein and is a part of the super elongation complex SEC. This R258W mutation is a recurrent mutation and has been seen in 50% of children with CHOP syndrome. All the mutations identified are de novo. Interestingly, in our cohort, two patients were clinically suspected of having CHOP syndrome and they underwent AFF4 sequencing, resulting in CHOP syndrome diagnosis. Therefore, we think CHOP syndrome is a clinically recognizable diagnosis. As a mechanism of CHOP syndrome, we previously demonstrated that ALF homology domain mutations disrupt proteosomal degradation. ALF homology domain where CHOP syndrome mutation resides is conserved among AFF4 family protein shown here, and this domain is a Thia1E3-Rictin ligase target region. We demonstrated that Thia1 mediated proteosomal degradation differences between mutant AFF4 and wild-type using HEK293 T-cell system. We created AFF4 and Thia1 overexpression system. When wild-type AFF4 is transfected, the AFF4 and Thia1 overexpression system. When wild-type AFF4 is transfected, addition of Thia1 caused the disappearance of AFF4 band, but this was not the case with AFF4 mutant vectors. Addition of MG132, which is a proteosomal degradation inhibitor, recovered AFF4 band even with Thia1, confirming that this degradation is due to proteosomal degradation. Differential diagnosis considered for children with CHOP syndrome are Cornelia de Lange syndrome, Smith-Magnus syndrome, and mucopolysaccharidosis. Shared features between Cornelia de Lange syndrome and CHOP syndrome are developmental delay, facial dysmorphism, and short stature. Shared features between Smith-Magnus syndrome and CHOP syndrome are developmental delay and obesity. In CHOP syndrome patients with vertebral bone differences due to cross-facial features, mucopolysaccharidosis was considered as differential diagnosis. Using last a few slides, I'd like to share our work on the Using last a few slides, I'd like to share our work on the molecular overlap between Cornelia de Lange syndrome is a multi-system developmental disorder characterized by facial dysmorphism, limb anomalies, and other malformations. This syndrome is caused by heterozygous mutation in cohesin complex and regulatory molecule. We asked why CDLS and CHOP syndrome show similar clinical manifestations. First, we compared the transcriptome profiles between CDLS and CHOP syndrome. We established skin fibroblast cell lines from CDLS patient and CHOP syndrome patients. Then, we performed transcriptome analysis using CDLS, CHOP syndrome, and control sample. Basically, these box plots show the correlation between CHOPs and CDLS gene expression patterns. Left figure shows the gene expression levels of 250 CDLS differentially expressed gene level of CHOP syndrome sample. Right figure is CHOP syndrome differentially expressed gene expression level in CDLS patient sample. So basically, up-regulated genes and down-regulated genes are similar in CDLS and CHOP syndrome. This finding indicates the presence of common downstream target genes of AFF4 and cohesin. Then, we identified molecular interaction between AFF4 mutated in CHOP syndrome and cohesin complex mutated in CDLS. We performed immunoprecipitation western blot with SA1-Stag1 and SA2-Stag2 antibody. SA1 and SA2 are the cohesin complex components. Interestingly, super elongation complex components including AFF4, ELL2, cyclin T1, and CDK9 were pulled down with SA1 but not with SA2 antibody. Addition of DLB, which is a transcriptional elongation inhibitor, CDK9 inhibitor, slightly increased the amount of SEC associated with SA1. In summary, CHOP syndrome is a recognizable multi-system developmental disorder characterized by cognitive impairment, heart defect, obesity, pulmonary involvement, and short stature. Their mutations are clustered in ALF homology domain of AFF4. This is my last presentation slide. As I presented, CHOP syndrome mutations are clustered in the ALF homology domain. Question is whether AFF4 mutations in any other regions are associated with human genetic disorder. In NOMAD database, PLI score is 1, indicating that AFF4 is haploinsufficiency intolerant gene. Indeed, we have collected information about seven individuals with AFF4 variants outside of ALF homology domain. Their clinical features are mainly neurological and different from CHOP syndrome. So we think AFF4 mutations are likely associated with non-CHOP syndrome clinical phenotype as well. I'd like to thank all of my mentors, collaborators, as well as my funding sources. Thank you very much for your attention. Hello, my name is Danita Velasco. I'm from the Monroe Myers Institute at the University of Nebraska Medical Center, and I'll be discussing cardinal signs of Primrose syndrome today. I have no financial disclosures. Primrose syndrome is a recently characterized intellectual disability syndrome that has a recognizable pediatric phenotype, but has distinctive features that emerge over the lifespan, leading to a more dramatic presentation as an individual ages. It's caused by sporadic missense variants in the ZBTB20 gene located at chromosome 3q13.31. The history of Primrose syndrome helps to reveal that variation in the lifespan. The first report was in 1982, when Dr. Primrose described a 33-year-old Caucasian male who was institutionalized and had intellectual disability accompanied by cystic lesions of his humerus and femurs, muscle contractures, and muscular atrophy of his distal extremities, cataract, hearing loss, torus palatinus, and distinctively large ossified pinna. The picture on this slide shows that individual as a 12-year-old sitting with his unaffected brother. Following this description, between 1986 and 2011, there were several additional reports about adults who had these similar findings, including intellectual disability of variable degree, as well as distinctively large and calcified auriculocartilage. In 2014, ZBTB20 was identified as the causative gene for Primrose syndrome, and at that time, the first reports of Primrose syndrome in pediatric patients also emerged. The first report was in 2014, when Dr. Primrose described a 12-year-old male. Since then, with the advent of whole exome sequencing, there have been several more reports that have come out and are primarily of pediatric patients, although a few additional adults have also been reported. As of April of 2020, there are about 50 individuals reported in the literature, and about 20 of them have been reported only in the last two months. The majority of these individuals are in the pediatric age group, although the age range in general of those reported ranges from a 35-week gestation fetus to the oldest individual with molecularly confirmed Primrose syndrome, who was 49 years old at the time of report. The phenotype does change with time, and so the cardinal signs in each age group are slightly different. For the pediatric age group, which includes individuals less than 20 years old who have a molecularly confirmed missense variant in ZBTB20, cardinal features include global developmental delay or intellectual disability that does vary in its severity. Almost all individuals have a history of hypotonia early in life, and many of them are very delayed in gaining motor milestones. Some of them do not begin walking until the age of four or five or even older. Speech is also delayed with this. Macrocephaly is another feature that's almost universal. Although the majority of individuals have an absolute macrocephaly with head circumference greater than two standard deviations above the mean at the time of report, there are a few who, even without absolute macrocephaly, have a relative macrocephaly when comparing their head circumference to their other growth parameters. Hearing loss is another frequently seen feature of the pediatric age group. It is most often described as a sensorineural hearing loss, and there are individuals who have sensorineural hearing loss and primrose syndrome, but who pass their newborn hearing screen, indicating that this is not always a congenital hearing loss and that monitoring of hearing will be important for these individuals. Corpus callosum abnormalities have been seen in the majority of pediatric patients who have had brain MRI. Like other features, in this syndrome, this does vary, with the most mild presentation involving a nick at the posterior region of the corpus callosum and including, on the more severe end, complete agenesis of the corpus callosum. While the initial reports in adults universally involved ear abnormalities, in the pediatric age group, this is present in only about half of the patients. The ears are not calcified when children are young. They tend to be large and prominent ears, and calcification occurs later in life, although the exact time when this would be anticipated is still unclear. Other bony abnormalities are also commonly described and somewhat heterogeneous. Delayed bone age is seen frequently, as is decreased bone mineral density, even in pediatric patients. There have been a number of patients who are described with scoliosis and a few who have other epiphyseal changes that are variable and not consistent between individuals. When reports have included description of the distal extremities and the nails, a number of individuals have been noted to have thin or dysplastic nails. There are many reports that don't include any description of this, so this may be an emerging feature in Primrose syndrome. Hypothyroidism has been reported in a handful of pediatric patients as well. In two of those individuals, it was congenital hypothyroidism, but for the others, it's unclear whether it was congenital or emerged later in life. Finally, autism and behavior problems are described in many individuals with Primrose syndrome, and variable in how it's reported in the literature. The behavior problems included range from anxiety, increased sensitivity to stimuli, mild sleep problems, to more severe self-injurious behavior. And the final feature in the pediatric population includes distinctive craniofacial features. For adults with Primrose syndrome, there are 10 in the literature who are confirmed to have missense variants in CBTB20 and over the age of 20 years old. Intellectual disability is universal, although the severity, again, ranges from mild to severe. The majority of individuals do have a moderate to severe intellectual disability. Abnormalities of the external ear is also a universal finding. The majority do have calcification of the pinna as can be seen on this CT scan from an individual reported in 2002. However, some individuals are noticed to have a severe pinna. However, some individuals are noted just to have macrosia, and then a clinical description including firm, immobile pinna. Distal muscle wasting and joint contractures is another common feature in the adults with Primrose syndrome. It appears that onset is sometime after puberty, but again, varies from person to person, and there are some adults who have not been reported to have this at all. Hearing loss in the adult population, similar to the pediatric population, is quite common. Impaired glucose metabolism is another common feature and ranges from fasting, elevated fasting blood glucose, to type 2 diabetes requiring medical management. Brain calcifications have been seen in a number of individuals, most often in those who were reported previously and had a CT scan rather than MRI scan. The location of calcifications did vary and included the basal ganglia, the thogs, and other regions of the brain. Taurus palatinus is Taurus palatinus is another distinctive feature that's only been described primarily in adult patients, although there's one recent report of a pediatric patient with Taurus palatinus. And finally, adults carry the same distinctive craniofacial features. There are only a couple of adults who have been reported to have also developed cataracts. To describe the entire lifespan, I'd also like to point out the one report of a fetus who was born, still birth, 35 weeks gestation. This infant had normal head circumference, but on autopsy was noted to have megalencephaly and had subtotally genesis of the corpus callosum. Genetic testing for that baby did confirm a variant in ZBTV-20. The craniofacial features seen in Primrose syndrome are variable from person to person, but do seem to have some distinctive findings that are commonly present. In addition to having macrocephaly, patients are also described with brachycephaly and occasionally with some bi-temporal pinching. The forehead tends to be broad and eyes are commonly deep set with somewhat narrow and down slanting palpebral fissures. Nasal bridge is frequently but not universally depressed and the nasal tip is comparatively broad. As you can see in these pictures, many of the individuals here have rather large ears and prominent ears. In the majority of individuals who have had genetic testing and have been found to have Primrose syndrome, they are of Caucasian descent and so there are only a few reports of individuals who are Brazilian and then the patient in the bottom row of these photographs who's of Mexican-American descent. It's possible that in different ethnicities, the craniofacial features may present slightly differently and will be an area that deserves further attention as more cases of Primrose syndrome are reported. The growth pattern in Primrose syndrome also deserves additional attention. Early reports of Primrose syndrome described it as an overgrowth syndrome, but in looking at the growth parameters and reported patients at the time of last report and dividing them in their pediatric in the light blue versus adult in the dark blue subgroups, you can see that height actually follows a bell-shaped curve. There are individuals who are more than two standard deviations below the mean as well as individuals who are above two standard deviations greater than the mean. For adults, they have a similar distribution but tend to cluster more into the upper limit of normal height. The weight trend for the pediatric patients also shows this bell-shaped curve where there are a number of patients whose weight is more than two standard deviations below the mean, but then many who fall within the plus or minus two standard deviations and a few whose weight is higher. Adults, on the other hand, show in general a clustering towards having a weight more than two standard deviations above the mean, which does not correlate with their height, indicating there's a trend towards obesity in the adult population. Finally, if you examine head circumference, you can see that absolute macrocephaly is common within individuals with Primrose syndrome. There are a few who have a smaller head circumference, and in all but one of these individuals, when you compare head circumference to their height and weight, they do have a relative macrocephaly. This is true for the adult population as well. The gene involved in Primrose syndrome is CBTB20. This gene is a transcription repressor in the zinc finger bric-a-brac tram track family of transcription repressors and has been shown to play a role in brain development, somatic growth, and glucose metabolism. The gene is located on chromosome 3 on the long arm Q13.31. The diagram included here shows the variants that have been reported in the literature as of February of 2020. There are now many more additional variants that have been reported, but you can see that the trend prior to February and still ongoing is that the missense variants cluster in the zinc finger domains on this gene. The BTB domain at the N-terminus is involved in protein-protein interactions, while the zinc finger domains are involved in protein-DNA binding. It's been proposed that the effect from missense variants in the zinc finger domains leads to a dominant negative effect on the action of this transcription repressor. The majority of patients who have been found to have Primrose syndrome have been identified through whole exome sequencing, and there are no known transmissions of this from apparent to a child. Everyone in whom parental information is known has had a sporadic variant. The differential diagnosis for Primrose syndrome is broad, depending on which features you look at in most detail. Overlapping with large ears, developmental delay, and macrocephaly would be Phelan-McDermid syndrome and 3q13.31 deletion syndrome. Kudel syndrome, chondrocalcinosis, and paroxysemibiogenesis disorders all have ectopic calcification and may present with calcification of the pinna. There are a number of syndromes that are associated with developmental delay or autism and macrocephaly, and those are listed as well. But of these, the one that should be discussed a little is 3q13.31 microdeletion syndrome. This deletion syndrome was described prior to identification of ZBTB20 as the gene responsible for Primrose syndrome, and you can see that there's significant overlap in the description of 3q13.31 microdeletion syndrome and those craniofacial features seen in Primrose syndrome. Developmental delay is common in this deletion syndrome, as is a broad prominent forehead, downslanting palpebral fissures, and necrosia. Individuals with 3q13.31 are also generally described as being overgrown, and an additional feature has been kind of distinctive morphology of the mouth and lips. What distinguishes this deletion syndrome from Primrose syndrome is that there does not appear to be the same progressive nature to the deletion syndrome as there is in Primrose syndrome. Individuals with 3q13.31 microdeletion syndrome have not been reported to go on and develop similar problems with muscular atrophy, hearing loss, or other ectopic calcifications, including calcification of the pinna. For individuals with Primrose syndrome, some initial management suggestions include hearing and vision screens throughout the lifespan, given the propensity towards sensorineural hearing loss and the potential risk for developing cataracts as they age. Thyroid screening is also indicated within the pediatric population and beyond. Skeletal imaging may be helpful in identifying those who have scoliosis or have decreased bone mineral density to allow for better management in a timely fashion. And finally, given that propensity towards developing abnormalities in glucose metabolism as well as towards developing obesity, proactive weight management programs and monitoring should be initiated. At this time, there's no clear increased risk for malignancy associated with Primrose syndrome. There are two adult males who have developed testicular cancers, although the exact link between those testicular cancers to ZBTB20 is unclear. AFP has been followed in individuals with Primrose syndrome. It has been noted to be increased in the pediatric population, but the role in that towards the other features of this syndrome is also a feature that deserves ongoing investigation. I'd like to thank the patients and their families for allowing their inclusion in this presentation, and I'd also like to thank Dr. Lois Starr and his team. All right, so as we're assembling our virtual panel here, I just want to apologize for any of the differences in volume with the different speakers here. We'll work on remastering this for the home video version. We have a number of questions that have come in, and just a reminder that we can please submit your questions through the Q&A function on the Zoom here. And I think we'll just have our other panelists, if they can turn on their videos, to join us here. So, we have Dr. Paul Hillman from the University of Texas, who presented on PLoD 3-related disorders. We have Dr. Danita Valesko, who just had presented on her topic, which, I'm sorry, it just escaped me here. Primos. Primos. Thank you. And then, in place of Dr. Azumi, we have Dr. Ian Krantz from Children's Hospital of Pennsylvania, of Philadelphia, with CHOPS. So, we have a number of questions. Let's get started. We'll start with a few here on PLoD 3. So, Dr. Hillman, how severe is the myopia in PLoD 3? We don't have exact numbers on all of our patients, but we did have myopia numbers for four of them, and it ranged anywhere from minus 5 to minus 15 diopters, with some of them showing a progressive nature into adolescence. Okay. And then, going along with that, how often do you see consanguinity as a feature? Yeah. So, of our 14 cases, 10 of them originated in consanguineous families. So, five of the eight families had known consanguinity, leading to PLoD 3 disease. But we do have three patients, and then actually, courtesy of this presentation, was contacted yesterday for the addition of a fourth patient with compound heterozygote changes in non-consanguineous families, leading to PLoD 3 disease. All right. And then, one question here, did you observe prenatal anomalies, AMC, I'm guessing that's arthrogryposis multiplex congenita, in your families? So, not with respect to the arthrogryposis. There were, our prenatal information is probably the one that we have the least amount of information for, given the retrospective review that we've been trying to collect this information from. But a number, several of the patients were born prematurely with concerns of IUGR and oligohydramnios prenatally. All right. Now, here's a question that's really to all of the panelists here. Question is, for any of these conditions, have they, has mosaicism been detected? And if you have, how is the phenotype different? Maybe we'll start with Dr. Krantz. Sure. Well, we haven't detected mosaicism in CHOP syndrome, but as many of you may know, in chronic delying syndrome, which is closely related and also caused by de novo mutations, there's a relatively high rate of mosaicism, maybe as high as 15 to 20%, or it's been observed in almost all of the known genes that cause CDLS. So I would just recommend CHOP syndromes fairly rare, but if you saw a kid that really felt like they matched the phenotype and you didn't see something on blood, I would definitely check saliva or a buccal swab. Great. How about Dr. Velasca with Primrose? There have not been any reports of a syndrome mosaic state that I'm aware of. However, I will say from February of 2020 through October, there's been a number of additional reports that have come out following patients globally. So it's possible that there has been one that's not accounted for within my talk. I didn't see any mention of that specifically, though. Okay. And then Dr. Hillman. Yeah, and certainly with plethora being a recessive disorder, no cases of mosaicism reported in the cases that I've presented today are the sum total of what is available in the literature. And all of them have identifiable germline changes. All right, great. So a few more here for Dr. Velasco. Someone mentions having a case with acrocephaly and mild autistic features and developmental delay that's improving. No particular dysmorphic features, normal brain MRI with no brain calcifications and then no calcified PINA. This patient had whole exome sequencing that had a variant of uncertain significance for Primrose and it appeared to be a de novo mutation. So do you think this might be enough to be suspicious for Primrose? I mean, certainly there are features that overlap there. I'd hesitate a little bit on how much significance to assign lack of calcification depending on the age as we know that that's something that emerges over time and is not generally present in the pediatric age group. You could consider checking an AFP level because it does seem to be elevated often in Primrose syndrome. It's certainly not something that is within the standard guidelines and may or may not be really directive for you. I would also look at where that variant lies as it really is those zinc fingers that seem to be involved in the cases that are clinically most overlapping with the Primrose syndrome phenotype. All right. And then also for Dr. Velasco, the macrocephaly seems to be less common in adults with Primrose compared with infants while overweight or obesity is more common. Could this be associated with worsening short stature? So the adults are often still within the normal range for their final adult height and they have normal head size to an elevated head size. So it's less that people fall off of the growth curve. It seems that there's just a slightly different growth pattern within Primrose syndrome if that makes sense in answering your question. Okay. And Dr. Velasco, how is the clinical presentation of those with the microduplication 3q13.31 compared to the microdeletion and to Primrose missense variants? I would have to look a little bit more at what that microduplication phenotype looks like comparatively. I don't know that that's reported as commonly as the microdeletion syndrome. A key feature with that being that it's mainly in the pediatric group again and so we don't have as much data about adults with that 3q13 deletion syndrome. Okay. So Dr. Hillman, going back to plot three, could you comment on epidermolysis bullosa like features? How commonly do you see this and what could the potential mechanism be? So in the personal cohort that we've assembled, there was increased blistering in the index case, primarily I believe the fingertips. The report that linked it to an epidermolysis lysis like phenotype was only that single patient and does not seem to be one of the more predominant features that we see in the disease. But we do see there is a good degree of phenotypic variability across the families that we've assembled. So I think it's something to be considered, but not necessarily a prominent feature. And as far as the mechanism goes, I believe it is, they've linked it to abnormal post-translational modification of collagen 7, I believe in the basement membrane, but I'd have to look back at the paper. Okay. Thank you. For Dr. Krantz, have you observed any limb anomalies similar to those seen in Cornelia de Lange patients, in patients with CHOPs, thinking that the pathways could be related? No, we haven't. I mean, they have brachydactyly, short fingers, other skeletal involvement, but not the typical sort of ulnar limb reduction differences that we see in CDLS. There is significant overlap and we've shown molecularly, I think Dr. Azumi showed a slide that showed sort of global transcriptional dysregulation with many genes that overlap, but there's definitely differences. So whatever the downstream targets of cohesin disruption that affects limb development likely isn't as major a manifestation in CHOP syndrome. Thank you. And then for Dr. Velasco, there is a clinician who has a pediatric patient with a likely diagnosis of Primrose syndrome due to characteristic features and a suspicious variant in ZBTV20. He has had severe constipation requiring sigmoid resection and also prominent abnormal breathing patterns, hyperventilation and breath holding. Do you know, have either of these features been reported in other Primrose patients? Some mild GI findings have been reported within those larger cohorts. And I think there's been one case report with an overlap in the breath holding spells. If I remember right, that was out of a Japanese case series with like two patients. So I think, again, I wish I don't have that pulled up right now to tell you exactly, but I think that would be worth reviewing potentially to see if there's that overlap in the breathing patterns. They're not common findings though. All right. Thank you. That's all the questions that I'm seeing. Maybe I'll hold for another 15 seconds here. Any last questions coming in? All right. So I just want to thank our live panelists for joining us here today. So I just want to take a second to let everyone know that the 2021 ACMG Annual Clinical Genetics Meeting Call for Abstracts is now open. We welcome all abstracts. Be submitted at www.acmgmeeting.net. The Undiagnosed Diseases Network, UDN Grand Rounds, next session will be on December 10th, hosted by the Baylor College of Medicine clinical site, and you can register for this and other Grand Rounds sessions at udnconnect.org slash Grand Rounds. Thank you very much for joining us today, and thank you to our panelists.

ACMG Genomics Case Conference

PLOD-3-related disorders

CHOP syndrome

Primrose syndrome

developmental delay

distinctive craniofacial features

ZBTB20 gene

macrocephaly

autism