Welcome to the ACMG Genomics Case Conference. This series has been planned by the ACMG Education Committee. Each month, the conference will rotate among laboratories, 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 their guardian. I'm Ian Campbell, the Chief Genetics Resident at Children's Hospital of Philadelphia, and I'm joined today by Donna McDonnell-McGinn, the Director of the 22Q&U Center at the Children's Hospital of Philadelphia. I'm Beata Nowakowska, and I'm the Head of the Cytogenetic Laboratory Warsaw in Poland Institute of Mother and Child. And this activity is intended solely for educational purposes. And here, again, is our names and our institutions. We do not have any financial conflicts of interest to disclose. Here are the planning committee members and their relevant disclosures. This session is approved for one unit of continuing medical education. Please complete the evaluation form at the end of the session if you would like to obtain credit. Here are the overall learning objectives for the genomic case conferences. And then these are the session learning objectives that we've made for this case series. At the end of the presentation, we will be available for a Q&A session. Please type your questions into the Q&A box in the top right corner of your screen. We will answer those questions verbally at the end of the presentation. So I would just like to thank the organizers for allowing us to present this session with you today. And once again, I'd like to disclose that, sadly, we have no financial conflicts. But we do come from beautiful Warsaw and Philadelphia. So the chromosome 22q and 1.2 deletion is the most common micro deletion syndrome with an estimated prevalence of 1 in 2,000 to 6,000 live births. It's also been identified in 1 in 992 unselected fetuses, so those without evidence of congenital heart disease or cleft palate. 22q and 1.2 deletion syndrome is the most common cause of syndromic palatal anomalies, the most common cause of schizophrenia, and the second most common cause of major congenital heart disease and developmental delay after Down syndrome. In fact, 22q and 1.2 deletion syndrome has been identified in 52% of patients with interrupted aortic arch type B, 34% with truncus arteriosus, and 16% with tetralogy of Fallot. So this is a more common cause of tetralogy of Fallot than Down syndrome. 22q and 1.2 deletion syndrome affects neural crest cell migration, causing structural abnormalities of the outflow track of the heart, leading to conotruncal cardiac anomalies, thymic hypoplasia leading to immunodeficiency, and hypoplasia of the parathyroid gland leading to hypocalcemia. The first description in the English language of findings now known to be caused primarily by a 22q and 1.2 deletion was in 1965 by Dr. Angelo DeGeorge in children presenting with the clinical triad of hypoparathyroidism and immunodeficiency, and a bit later congenital heart disease was added. 22q and 1.2 deletion syndrome is now known to have a heterogeneous presentation, including multiple other syndromic diagnoses, including velocardiofacial syndrome, which added the palatal abnormalities and some craniofacial dysmorphisms, conotruncal anomaly face syndrome as described by the Japanese, a subset of patients with autosomal-dominant OPGBB syndrome with hypertelarism, cleft lip and palate, hypostadius and laryngotracheal esophageal abnormalities, and Kähler cardiofacial syndrome as described by the Italians, which included asymmetric crying faces. Now most of these were described prior to the association with 22q and 1.2 deletion syndrome by clinicians concentrating on specific areas of expertise, so Dr. DeGeorge was an endocrinologist, Dr. Takao, a cardiologist, and Dr. Sprintzen, a speech pathologist. 22q and 1.2 deletion syndrome is now the preferred term as the single common denominator uniting all patients and their varied phenotypic presentations. 22q and 1.2 deletion syndrome is now also known to cause additional congenital anomalies, later onset conditions, cognitive delays, behavioral phenotypes, and psychiatric illness, all far extending the original description of DeGeorge syndrome. Now when we think about common significant associated features, those include the abnormalities that we've already talked about, including immunodeficiency, congenital heart disease, and palatal anomalies, but it has also been expanded to include gastrointestinal problems, endocrine abnormalities besides hypocalcemia, such as thyroid disease and growth hormone deficiency, skeletal abnormalities, in particular cervical spine anomalies and scoliosis, and renal differences, most often single or dysplastic kidneys and GU reflux. Most common but significant features include additional skeletal abnormalities, such as craniosynostosis and club foot, CNS differences, including idiopathic seizures, neural tube defects, polymicrogyria, and dystonia, autoimmune disease thought to be immune mediated, such as JRA and ITP, GU abnormalities that I mentioned already, ENT differences, such as laryngeal web, choanal atresia, and tracheal atresia, eye differences, such as coloboma and sclerocornea, which is considered an eye emergency resulting in corneal transplant, and malignancies in just under 6% of our population, including 43% in monozygotic twins and about 5% in dizygotic twins. And again, here we believe this is immune mediated. Now although common, lack of recognition of the condition and or lack of familiarity with genetic testing methods, as well as broad variability, delays diagnosis, which could improve outcomes, including in affected adults most often identified through a child born with congenital heart disease. We now believe the 22q and 1.2 deletion syndrome is significantly under-recognized. In fact, based on a prevalence of 1 in 2,000, there should be about 150,000 affected Americans, including 35,000 children and adolescents, which begs the question, where are they? And we believe that perhaps they're waiting to be diagnosed based on a variety of factors which will be discussed today. Now associated medical problems vary by age at ascertainment, and there are still some significant gaps requiring additional data. And the presence and severity of features varies by age and the focus changes. So in infancy and early childhood, everybody's worried about birth defects, associated medical issues, motor milestones, and speech and language. By school age, the focus shifts to academics and peer relations. And then in adolescence and adulthood, everybody is concerned about independence, the behavioral differences, and of course that all-important 50% recurrence risk. In the absence of typical features, the diagnosis may be missed, and this is especially true in adolescents, adults, and non-Caucasians, even in university-based medical centers. Which moves us on to case number one. At the time of evaluation, she was 14 months old. She had hypotonia and feeding difficulties that were noted at birth, and she stayed in the neonatal intensive care unit for about four weeks. She was placed into foster care from four months when she was adopted by her, eventually by her paternal cousin. Developmental delay was noted. She started rolling at seven months and was sitting around eight months, and was not yet walking independently during the evaluation at 14 months. Previous cardiac evaluation had shown a hemodynamically insignificant right aortic arch, which can be seen in this cardiac MRI, as well as an abnormal left pulmonary artery, which at that time had normal flow. Family history was provided by her foster mother, which revealed that the biological mother had suspected intellectual disability as well as mental health issues. A previous karyotype was 46XX, and subtilomeric fish evaluation was normal. On physical exam at age 14 months, her weight was below the first percentile, height below the first percentile, and head circumference at the fifth percentile. She had a high arch palate, left preauricular pit, had bilateral overfolded pinnae, as can be noticed in the pictures here. She also had an umbilical hernia, but by that time her tone had improved. At that time we recommended 22Q11.2 fish, which revealed that the child had a deletion of that locus. Subsequent research MLPA was performed, which revealed that the deletion was the typical A to D deletion. Additional medical problems were subsequently identified, including T cell lymphopenia, hypogamaglobulinemia, humoral deficits, and asthma. She also had juvenile scoliosis, myopia, astigmatism, and tortuous retinal vessels. She also had dysphagia, had crossbite, had belopharyngeal incompetence, and ultimately required a repair. She also had ADHD, receptive and expressive language issues, and her full-scale IQ was 72. At the time of her most recent evaluation, she is in the ninth grade in a special education classroom with a 1-to-1 aid, and is having some difficulty having mainstream education. The psychosocial history of the patient was complicated by several emergency room visits as a result of domestic disputes during the pregnancy, as well as IUGR and maternal hypothyroidism. Her biological mother was diagnosed with the 22Q11.2 deletion following her daughter's diagnosis. Mom's personal history was that of an umbilical hernia, status post-repair, and the hypothyroidism that I mentioned. She required special education classes in high school. She had difficulty obtaining employment, and she recently completed education towards becoming a pastry chef. The PROBAN has two maternal half-brothers, one without the 22Q11.2 deletion, who is reported to have some behavioral and learning deficits, and one with the deletion who has a history of a VSD, a PFO, and an imperforated anus status post-repair, which significantly complicated his care. As a result of that, he has chronic constipation and feeding problems, obstructive sleep apnea, asthma, anemia, speech, and developmental delay. Now, unlike the PROBAN, who has had the benefit of private instruction and opportunities to participate in a vast array of extracurricular activities, including intensive speech and language therapy, dance, music, art, singing, and sleep-away camp, et cetera, the affected maternal half-brothers' opportunities for such activities has been limited. Now, we previously reported the need for a social safety net in familial cases, in particular when there was an affected parent who was raising the affected child, and we suggested that this should best take the form of a social worker or committed relative, as observed in our PROBAN's family, again, as compared with her affected maternal half-brother. So, within the region, we have around 15 genes, which can be deleted if we have the full region deleted, and this deletion leads to a multi-system disorder. Most deletions are de novo, which is approximately 87%, but we have to think that if one of the parents is the carrier of the deletion, the recurrent risk for the pregnancy is 15%. So, the region is flanked by the low-copy repeats, which is called LCRs, and we have two LCRs on the two ends, LCR-A and LCR-B, which are the longest LCRs, and they are the most similar to each other, but between them, we have also LCR-B and LCR-C, and the similarity between the LCRs, they predispose to the non-homologous recombination between the LCR, usually A and D, and this can cause the duplication of the region or the deletion, as you can see in the right bottom. So, as I said, we can have LCR-B and LCR-C, which is in the middle, and we also can obtain the small deletions between the LCR-A and B, A and C, and B and C, and C and D. So, between the region LCR-A and B, we have TBX1 gene, which was the key development gene and is thought to be a major cause of CHD in patients with the 22q deletion, and therefore the probes, which we, on the beginning, used for the fish, which detected the deletion, they are designed to cover the region between only LCR-A and B. So, as you can see, the common deletion between the LCR-A and D is the most frequent deletion and is observed in 84% of patients with the 22q11 deletion. Less common is the deletion between A and B, A and C, B and D, and C and D. However, when you look at the fish probes, which are located only between A and B, you can see that if we use this technique to detect the deletion, we will miss the cases where the deletion is smaller and doesn't cover the A-B region. So, we have to, we have to, remember that, which can happen sometimes, that if we want to confirm the deletion of C and D in the parental samples or in the relatives in the family, we cannot use the fish technology for the families to look for the deletion. So, the C and D, B and D deletion, is more commonly inherited, and this is also very important for the, for the consultation for the families. Only 13% of the A-D deletion is inherited from of the A-D deletion is inherited from the parents. This brings us to case number two. The patient was noted prenatally to have tetralogy of flow. At postnatal exam, he was noted to have normal growth parameters. He had slightly long fingers and squared off ears with a mild point on the left side. He also had a short forehead. Review of the chromosomal microarray that was performed prenatally via amniocentesis resulted in a report which, upon careful examination, showed that the patient had a 359 kilobase deletion of the 22q11.2 locus. And the report actually provided coordinates, as well as a number of genes that were deleted in the region. In order to further clarify what was going on, we pasted the coordinates of the deletion into the UCSC genome browser with the appropriate genome assembly, in this case, HG19. And then we reviewed which genes were in the deletion together with our previous knowledge, which showed this to be a typical C to D deletion. Further evaluation of the family revealed that the father also carried the C to D deletion. He had a master's degree in education, although he did have some learning deficits in mathematics. We also evaluated his older brother, who was also found to harbor the same C to D deletion. And an ultrasound of his kidney revealed that he had a right pelvic kidney. So overall, in this family, we had a father who had really no medical issues and a master's degree who passed on the C to D deletion to both of his children, one of whom had Tetralogy of Flow and the other with a right pelvic kidney. One of the important genes in the C to D deletion is CRKL or crackle. Recent evaluation in patients with kidney deficits in a New England Journal article showed that this was one of the key genes involved in kidney defects that are seen in 22q deletion syndrome. In addition to finding some patients with 22q deletions, they also found some patients with monoallelic pathogenic variants of the CRKL gene, which they felt were contributory to those patients' kidney malformations. Additionally, CRKL has been implicated in the etiology of congenital heart disease, as Rossetto and colleagues found that the CRKL deletion mice also had congenital heart defects. So we're constantly asking ourselves, why are these patients so complicated? And how can we explain this? And one of the areas of research that everyone has been working on has been modifier genes. But you can see from this slide that we even see broad variability within identical twins. One, for instance, with congenital heart disease and one without. And so, this just echoes the broad variability that we see both inter- and intrafamilial across the diagnosis. Now, this is complicated by dual diagnoses unrelated to 22q. So, we recently reported patients with a 22q11.2 deletion and a concomitant diagnosis completely unrelated to chromosome 22q, including a child with 22q in SCID, a child with 22q in cystic fibrosis, et cetera, et cetera. So, we always have to be thinking about the patient very broadly to be sure there isn't something else going on in the family, including something that would be inherited from a parent to a child or something that may be new in the child. Additionally, the patients that we follow are complicated by dual diagnoses related to the 22q11.2 deletion. So, for example, they have a deletion on one chromosome, and then they have a mutation or a copy number variant on the intact chromosome 22 allele that unmasks an autosomal recessive condition. And there have been a number of these conditions described most recently. Okay, so I'll present a few genes which were described recently, which mutation can cause additional phenotypic features in patients with 22q11. So, one of the gene is GP1BB, which caused the Bernard-Sylvier syndrome, and it's mostly associated with thrombocytopenia and megakaryocytes. Second gene is SCARF2, which was described in the patients in the Vanden-Gupta syndrome. And this syndrome is associated with camptodactyly, arachnodactyly, and some craniofacial problems. Another gene is SNAP29, which caused the Sednik syndrome. The mutations in SNAP29 cause polymicrogeria and keratoderma. And it's also seen in the patients with 22q11 deletion. Another syndrome and another gene is LZTR1, which is associated to autosomal recessive Lunan syndrome and cause a typical Lunan phenotype, CHD, short stutter, ptosis, and webbed neck. Another gene recently described by Donna and colleagues is CDC45, and this gene is associated with craniosynostosis, lymph abnormalities, mycrotia, and short stutter. Another gene is TANGO2, and mutation and or deletions of TANGO2 gene affect glycogen and long-chain fatty acid metabolism and mitochondrial ATP production. And from the literature, we know that this gene, this recessive syndrome can cause metabolic crisis, sudden death, seizures, hypoglycemia, and droid disease in the patients. This brings us to patient number three. He was evaluated at 10 months at another facility due to microcephaly, developmental delay, hypotonia, episodic ataxia, concern for polymicrogeria, an incomplete vascular ring, a submucosal cleft palate, mixed bilateral hearing loss, hypothyroidism, and constipation. The pregnancy was complicated by placenta previa, and the perinatal course was complicated by hyperbilirubinemia. His development was notably delayed. He walked at 18 months. He spoke his first words after the age of two, and at the time of our evaluation had very limited speech. On physical exam, he was on the shorter end of normal. He had low weight, but within the normal range. His head circumference was microcephalic. He did have mild down splints to the palpebral fissures, as you can see in these photographs, as well as mild hypertelorism. The nasal tip was broad, and he had hypoplastic alias nasi, and the collumella extends below the nares. He also had diastasis recti and slender fingers. MLPA revealed that he had a typical A to D deletion of 22q11.2. Parental studies were performed and were normal. As part of the evaluation, an MRI of the brain was obtained at Children's Hospital of Philadelphia, which was determined by our neuroradiologist to be structurally normal. However, MR spectroscopy, which was performed at the same time, showed a lactate peak on MRS that was obtained, that was seen in almost all the voxels on the MRI. As part of further evaluation, an organic analysis of urine organic acids showed that there were some carboxylic acids in the urine without ketosis, which the lab director noted could be seen in mitochondrial fatty acid beta oxidation defects, but that it was not consistent with MCAT. Unfortunately, at age five, the patient developed a viral illness. His mother found him the next morning choking and with decreased responsiveness. He was taken by ambulance to a local emergency room where he was noted to have extreme acidosis and was unresponsive with agonal breathing. Later, a head CT was performed, which demonstrated severe cerebral edema and uncle herniation with resultant posterior circulation strokes. Determination of brain death occurred three days after the event, unfortunately. We can see here that the lactic acid during the patient's hospitalization at the outside facility was quite notable. Whether that was due to an underlying illness or part of the aftermath of that event is not known. So, because of the clinical indication that the TANGO2 gene is involved in this case, we performed a whole exome sequencing for this patient, but we didn't find any mutation in the TANGO2, but we look further because the clinical picture was very clear. We look further and we look at the IGV and we saw that we don't have any rates for the exome from 3 to 9 for the TANGO2 gene. We confirmed this by other techniques and we looked also for different 77 patients where we performed the exome sequencing and also 127 patients where we performed the region-only sequenced and we identified two patients which had exactly the same deletion between exome 3 and 9 in the TANGO2 gene. So, this small deletion which encompass only a part of the TANGO2 is quite common in the general population and we have to think about it when we diagnose our patients. So, we also performed a qPCR to confirm the presence of the small deletion in the parental samples and we showed that one of the small deletion was inherited from the mother in the patient too. We also look for the mutations and the variants in different patients and we already identified seven patients with or deletion or mutation in TANGO2 which can cause the phenotype which was described before. So another gene which can also be deleted by additional small deletion in the cohort of patients with the 22q11 deletion is a small deletion encompassing two genes, PRODH and DGCR6. So, this is also a common deletion in the general population and even one of the paper mentioned that this deletion can occur with the frequency of the one in 250 normal people. So, when we have a full deletion of the PRODH gene which is shown on the right bottom corner, we perform RNA-seq to check the expression of this gene and we didn't notice expression of this gene in this patient. You can compare it to the three controls. So in this patient, we measured the proline level and it was approximately 600 micromole per liter and we know from the literature already in 2002, there was a first paper mentioned that the patients with the hyperprolinemia can be at very high risk of schizophrenia. So this was the 13-year-old male patient and we don't know if he's going to develop schizophrenia but he had a delayed speech but normal IQ which is very unusual for the mutations for the PRODH. So, looking at the current detection method which we can use in the laboratory to detect 22q11 deletion, we have to remember that we have different LCRs and also different methods which can detect only a part or the full region of the 22q11. So the best method which we are commonly using is the microarray which can detect all kind of length of the deletion because it has the probes between LCRA and to LCRD. We can also use the MLPA which has only three probes in the very basic kit. So this method can detect deletions between A and B and between C and D because there is also one probe there. Another method which is commonly used in many laboratories still is a FISH method which can detect only deletion between A and B which is also very important to remember when we verify the family and the parents. So another method which we recommend and I hope it will replace this methodology in the future, this is a whole region sequence. So just to remember that we still need to consider the full diagnosis in the patient and we still have to think about the genome-wide study to detect additional CNBs and for this we have to use a microarray just to be sure we don't miss any deletions or duplication on the chromosomes other than 22q11. We have to take careful attention to the phenotype of the family and the history to detect unrelated conditions like for example mutation in NF1 gene. And also we have to take a careful consideration of the mutation or CNB on the other allele and currently this methodology is available clinically only in Warsaw in our institute. So we have to ask ourselves how can clinicians and in fact families manage all of these challenges and we would suggest by first following the international healthcare guidelines which place an emphasis on ascertainment and comprehensive care. And considering additional laboratory testing in all patients with 22q11.2 deletion syndrome to ensure this appropriate care especially as these findings are medically actionable potentially altering long-term outcome as we've described with Tango 2. So in summary 22q11.2 deletion syndrome leads to significant morbidity with multi-organ system involvement. 22q is common affecting about one in a thousand pregnancies and providing an important window into understanding rare and frequent conditions in the general population including congenital heart disease, immunodeficiency, schizophrenia, autism, scoliosis, the list goes on and on. Currently touching healthcare providers in multiple settings including pediatrics, adult medicine, surgery, oncology, prenatal diagnosis and we hope in the near future newborn screening. And again the presence and severity of features varies by age and the focus changes and we always need to be thinking about this when we're speaking with families. And this wide variability hampers early diagnosis delaying interventions and leading to poorer prognosis sending families and healthcare providers on a diagnostic odyssey. Conversely early and comprehensive diagnosis offers medical and emotional preparedness reducing cost medically, emotionally and fiscally. So we'd like to thank our collaborators at our own institutions and around the globe because it really does take a village to care for a patient with 22q. We'd like to thank the patients and families for everything that they've taught us because it certainly always begins with a patient and we'd like to thank Rick Guidotti of Positive Exposure for all of the beautiful faces of sunshine that you've seen here today. And we'd like to ask you to consider joining your families on May 17th, Twin Awareness Day. It's definitely coming to a zoo near you. And if you love 22q consider attending the 12th Biennial International 22q Conference celebrating 22 years of education and collaboration in Split, Croatia this June. And thank you for your kind attention and if you have questions now you can type them into the Q&A box in the top right corner of your screen and the questions will be answered verbally by Beata, Ian and myself. Thank you again. All right. So I'd like to first start off by apologizing for a small editing error that occurred there. We previously recorded because of the different locations that the presenters were in and we'd now like to start answering your questions. The first one is from Albert Gold who asks, how do you explain the half-brother without the deletion but ostensibly with some of the difficulties? So Dr. Gold, thank you for the question. I believe if we think about some of the unaffected siblings in the families that we see, we do sometimes see differences in developmental outcome. And we believe this is related to the heritability of IQ that we would see in the general population. And so there are contributions of IQ genes from the parent with the deletion outside of the deletion region as well as her partner. So in essence, this is the result of assortative mating and we did not find any other abnormalities on microarray in that child. And so the second question comes from Hal Bass. He asks, what is the risk of childhood or adult cancer in 22q syndrome? As part of an analysis that was published by our group in 2018 on a subset of our patients, we found that 5.7% of patients had malignancies. And the types of malignancies that we found were hepatoblastoma, Wilms tumor, renal cell carcinoma, thyroid carcinoma, melanoma, and leukemia. Because we are pediatricians here at CHOP, we are biased towards childhood onset cancers, but some of those cancers were seen in adolescents and adults. So our next question comes from Albert Gold again. A small deletion like C to D is unlikely to be picked up by NIPT. Does this influence your view of the clinical utility of NIPT for 22q microdels? So another good question, Dr. Gold. Unfortunately, most NIPT studies would miss a nested deletion. There are a few SNP-based NIPT platforms that may pick up those patients. We just had an example of a child last week with a C to D deletion that was missed by NIPT. And so I think your question is well taken. As we mentioned at the end, our hope is that eventually 22q and 1.2 deletion syndrome will be added to newborn screening so that we obviate that diagnostic odyssey that many families have to deal with. And the testing that's been developed by Allie Tamita Mitchell at the University of Wisconsin is a QPCR-based platform, which is multiplex, so it includes both TBX1 and Crackle, and so we believe we would pick up patients even with small nested deletions. Certainly we encourage families, whenever there are findings prenatally, to consider invasive testing, but as you and others know, the uptake on those suggestions has decreased quite significantly, and families are relying on NIPT, so it certainly is an important consideration and discussion. Our next question is from Mahmoud Arabi. They ask, could you please comment on the 22Q11.2 distal deletions involving LCRs E through H? Thank you very much for that question. We do have a subset of patients that we now follow who we have what we call distal deletions. Certainly those children who have a D to E deletion have very similar characteristics one to another. They have significant short stature, microcephaly, congenital heart disease, preauricular tags and pits, and sensory neural and conductive hearing loss. Those that extend beyond E, our concern is always whether or not it extends into the region that includes SMART B1, and depending on the laboratory, some suggest that that begins at LCR F. Others, it will be called out at LCR G to H. Certainly those patients have a high risk for rhabdos, and so they're followed very closely by oncology. Those patients also have other structural anomalies, including congenital heart disease and other medical comorbidities, and so we believe that all of them require careful follow-up, but given that they don't overlap with this group that we're discussing today, they are actually quite different conditions and almost, you could say, as different as a child who had a 22Q13.3 deletion or Phelan McDermid, which is just another diagnosis. So our next question comes from Joanna Ma. She asks, loss of function variants of LZTR1 have been associated with schwannomatosis, and she wonders if that's the case in 22Q11.2 deletion syndrome patients. I would say that we think that haploinsufficiency of this gene does not seem to be associated with schwannomatosis in our cohorts. We also have two patients which have LZTR1 variants on the other allele, and neither of them have any schwannomas, so that's about as much as we can say on that. Our next question is from Katherine Nowak. She asks, do you recommend every patient be assessed for Bernard-Cillier syndrome? How about every patient undergoing surgery? She notes that some of her patients have large mean platelet volumes, but none have had abnormal specialized platelet studies yet. So another good question. I think we are always cognizant of taking a careful bleeding history. We know that patients with 22Q11.2 deletion syndrome have lowish platelets to begin with because of the haploinsufficiency. But of our 1,500 patients, we only have one with true Bernard-Cillier with a mutation on the other allele, and that was a patient who had a significant bleeding history. And when hematology looked at the blood smear under the microscope, they saw megakaryocytes. So I think that without that kind of a history, it's unlikely to be the case. But I think if anybody's concerned, they certainly can engage their hematology colleagues in the investigation. Next up, from Joanna Ma again. Are there any genes in the region that are responsible for hearing loss or hypocalcemia? As far as hearing loss is concerned, we haven't been able to identify any genes that explain the hearing loss, although it is seen at higher rates than in the general population. As far as hypocalcemia, I think that the variable expressivity that we see with regards to that phenotype, we haven't been able to identify any modifier genes as of yet, but that remains an ongoing part of our investigations. Next, from Theodore Wilson, we have a question. Are we at the point now that whole exome sequencing is also indicated for newborns with 22q11.2 deletions, or should additional genotyping still be used for other features that are present? So that's an important question that we're scratching our own heads about. I think given the cost of whole exome sequencing, the better part of valor would be to consider microarray in all patients. We no longer use FISH because we would miss the nested deletions, so a good microarray will rule out a CNV elsewhere. And then we're hoping that BAIDA studies, which are available clinically in Warsaw, essentially a slice of the other allele, will become available to our patients so that we can look specifically at those genes that are associated with the phenotype. So right now, we're not advocating whole exome sequencing, but certainly if a patient has atypical findings and the other allele testing is not available, it's an option. Certainly there are patients who we've seen with things like craniosynostosis who did not have any of the typical genes associated, for instance, the FGFRs or TWIS, and we have sent single gene testing for CDC45. The same is true for SNAP29 and TANGO2, which are all available clinically. The beauty of BAIDA's test is that she does them all at once so that you don't have to sort of cherry-pick or pay per gene. Conversely, whole exome may get us that, although our patient who succumbed at 5 years of age with the TANGO2 deletion was essentially missed for 2 years because we did send studies to look at the other allele, and they kept telling us there was no mutation, but in fact, there was a small deletion, which I'm not sure if the whole exome would be as reliable as somebody like BAIDA, who knows the region so well, looking carefully. And so the next question we have from Kamar Tezcan, which is also very similar to that, which was from Robin Clark. They wonder, what percentage of patients have a dual diagnosis due to mutations on the other allele? So we have only looked at a subset of patients who either had atypical features or they were part of a brain behavior study, were included in the International Brain Behavior Consortium, where we were doing whole genome sequencing to look for modifier genes of behavioral differences. So we looked at about 400 patients in the laboratory of Dr. Nowakowska, and we found a mutation of import on the other allele or a deletion in 4 percent of those patients. But again, it was enriched by some patients who had atypical features. So we really don't have a good handle on the whole cohort as yet. And so next we have a question from Dr. Subhadra Ramanathan. She asks, are there any plans for offering clinical whole region sequencing at CHOP or in the U.S., particularly since we were just commenting on how it's available in the European Union? So we definitely would like to offer this in the U.S. My preference would be to figure out a way to have BAIDA do it in her lab because she's such an expert in this area, and there are not so many around the world that know this region so intimately. But it's unclear if that's going to work out or not. I think one way or another, we have to come up with a solution because I do think all patients will need these studies. Next we have a question from Rana Sherik. Is there any evidence the 22q deletions are increased in spontaneous abortions? That's a good question. There is a paper by Melissa Meisenbacher who works for the NIPT company, Natera, and they looked at products of conception. But I have to say, I don't remember the data offhand. I believe it was minimally elevated, but I couldn't swear to it. But I would suggest if you search for Melissa Meisenbacher's paper, you'll find that answer. And there is some interesting data from the reproductive rates of females with 22q deletions, and they do appear to have slightly decreased fecundity. But whether that may be due to mental illness or socioeconomic status or heart defects, that remains to be fully elucidated. Next up, from Deborah Friedenberg, we have immunologists often refer to complete and incomplete to George. Can you comment on the designation? So that is 100% true that immunologists use those terms. I think, you know, we always have to be certain we're talking about patients with 22q11.2 mutation syndrome and not other causes of George, such as an infant of a diabetic mother or someone with a CHD7 mutation who has CHARGE syndrome. In general here, they don't use that language any longer because whether or not you see a thymus on ultrasound or a chest X-ray doesn't really determine the associated immunodeficiency or the level of immunodeficiency. We have really had very few patients who have had really significant immunodeficiency requiring IVIG or a thymic transplant. I think maybe one out of our 1,500 required a thymic transplant, as opposed to those patients who had CHARGE syndrome that were much more significantly affected from an immune perspective. So we don't use the term, but I know that it's something that immunologists are quite familiar with, and they use it interchangeably all the time. Next from Robin Clark, we have the question, is socioeconomic status a separate factor in outcomes? So, Robin, that's a really good question, too. We believe it's the case. We have not done a prospective study looking at that, but it's certainly something that we need to do. Again, if you think about heritability of IQ and relationship with socioeconomics, you could imagine that there would be a relationship, but we just haven't done the studies as yet. Next up, from Adrielle Kim, we have some patients with 22q deletion syndrome percent with ASD, which I will assume she means autism spectrum disorder. Is there a unique genetic profile, such as recurring location, size of the deletion, copy number variation within 22q11.2, et cetera, in these patient groups? I would say that in our review of our cohort most recently in 2018, we saw that 19 percent of patients had a diagnosis of autism spectrum disorder, and since the vast majority of our patients have the typical A to D deletion, that we didn't have enough statistical power to determine which regions within the 22q11.2 region are causative of autism spectrum disorder. However, that is a focus of ongoing investigation. I would also add to that that we have seen a difference in IQ in those patients with an A to B deletion compared to the A to D deletion, and that was a paper that came out of the International Brain Behavior Consortium, and we have seen that anecdotally in our own subset. So, it is important to size the deletion moving forward because we may be able to give that kind of detailed counseling to families as it becomes available. Next, from Nicole Safina, we have, how often should we be looking for additional genetic conditions in affected individuals in 22q11.2? So, that's a good question. Certainly, we should be looking at it for any patient who has atypical features, but given that we have a number of patients who don't have atypical features but have additional CNVs, some of which are familial, we would advise undergoing microarray for all patients and considering additional genomic studies based on the phenotype. Next up, from Jia Wang, we have, how much is the percentage of asymptomatic, no congenital anomalies, typical cognitive abilities in individuals with C to D deletions? So, we have a very small cohort with B to D or C to D deletions. We have one patient with a B to D who has an IQ of 57. We have other patients who have very high IQs. I think the numbers are just too small. We are pooling our data with our international colleagues, and so we may have additional information to share in the future, but I think it's going to take a little time until we have large enough numbers to say definitively. We certainly see more patients with A to B deletions than we do with the B to D or C to D deletions. Next up, from Robin Clark, we have, are there any genetic modifiers such as gain-of-function variants that improve the outcome in 22q11.2 deletion syndrome? I think that that's a very fascinating question. Could there be positive genetic modifiers? Unfortunately, we've mostly been spending our efforts on trying to identify the negative genetic modifiers, but that is definitely a question which we could look to in the future. Next up, from Catherine Nowak, she has a follow-up question about platelets. Is there a different reason why patients with 22q deletions have elevated mean platelet volumes? Many of them don't have a bleeding history, and so she wonders if it's reasonable not to pursue platelet function testing in those people. I think that we don't know the exact reason why some patients have elevated platelet volumes, and I would refer back to our previous answer about speaking to your hematology colleagues about their recommendations. Next up, we have a question from Teresa Greb. Have you had any first cousins affected with 22q whose parents are not affected? Any cases where parents with balanced translocations? So I'll go first with the balanced translocation because the original report by Rick Kelly et al. in 1982 was a family where the child had an unbalanced translocation involving chromosome 10 and 22, and the father had a balanced translocation. And there have been a handful of those cases over the years, but not too many. In reference to cousins, we actually have a report from Shalagna Saida where we saw a child, and we were following her for several years, and the parental studies were normal. The mother called and said, I really think my nephew has the deletion, and in general, parents definitely know best. And so we suggested the child have deletion testing, and sure enough, he was deleted. And then in the laboratory of Beverly Emanuel's laboratory, Shalagna found that the deletion occurred in the unrelated sister-in-laws. So it was just by chance alone, and I think that reflects how common this deletion really is amongst the general population, and I think the patients we're seeing are really the tip of the iceberg. And to follow up on those points, it is important to remember that while there are these exceptions to the rule, because these deletions occur via non-allelic homologous recombination, which occur during meiosis, we should expect that those result in the deletions and wouldn't be creating balanced insertional translocations. Although there have been rare reports of seemingly mitotic, so during embryogenesis deletions, but the vast majority should occur during meiosis and not result in those types of balanced rearrangements. Next up, we have from Jun Shen, what is the prevalence of hearing loss in 22Q11.2 deletion patients? So it's a fabulous question. We just pulled audiograms on 1,000 patients, and we're looking at it now. Certainly conductive hearing loss is common because these are kids with palatal insufficiency, so they have lots of otitis media. A subset have sensorineural hearing loss, some have mixed, and certainly we have a small subset with cochlear abnormalities. So it's something that I think requires additional investigation. But certainly whenever we see a child with 22Q, we recommend an audiogram. And certainly when they have delay in emergence of language, even though we're used to seeing a mean age of language of about two and a half, we want to make sure that their language isn't delayed because they have a hearing deficit. And then she has a follow-up question. Do the patients typically pass their newborn hearing screen? And they typically do. And then a final question from Jun Shen, they ask, can genome sequencing reliably detect 22Q11.2 deletions? And I would say that the answer is yes. Any laboratory that is calling copy number variants off of their genome sequencing data should have no trouble identifying these deletions, both from a read depth perspective as well as the runs of homozygous variants that will occur on the other allele. And so with that, those are all the questions that we've received. And so thank you for being with us. And a recording of this seminar will be available in the ACMG Genetics Academy at the website that you see there on your screen. And if you have any questions, you can, of course, email any of the three of us. Our emails were on the screen earlier. Thank you and have a good day.

ACMG Genomics Case Conference

22q11.2 deletion syndrome

prevalence

associated features

genetic factors

early diagnosis

comprehensive care

additional testing

ongoing research