Welcome to the ACMG Genomics Case Conference. This series has been planned by the ACMG Education Committee. Today's webinar is titled Spectrum of Positive Findings Encountered During Expanded Carrier Screening. This webinar is hosted by Semaphore, which is a health information company that recently spun out from the Mount Sinai Health System in New York. This activity is supported by an unrestricted educational grant from QIAGEN Bioinformatics and the Ingenuity Clinical Decision Support Platform. My name is Ashley Birch. I'm an associate director in the Molecular Reproductive Genetics Division at Semaphore and an assistant professor in the Department of Genetics and Genomic Sciences at the ICAM School of Medicine at Mount Sinai. And my name is Bryn Webb. I'm a clinical and molecular geneticist and an assistant professor in the Department of Genetics and Genomic Sciences at Mount Sinai and a medical officer at Semaphore. So for our disclosures, we are an employee and a consultant respectively at Semaphore, which is a health information company that performs expanded carrier screening. And the planning committee has nothing to disclose. This session is approved for one CME credit. And if you purchase CME credits at the time of registration, you'll receive an email within 24 hours with a link to complete the CME certificate. Once complete, you'll receive a second email containing the certificate. And everyone is asked to please complete an evaluation at the end of the seminar. So the overall learning objectives for the ACMG Genomics Case Conference are listed below. The learning objectives from today's session are to discuss the history of carrier screening, including the development of expanded carrier screening panels, to critically assess the advantages and disadvantages of targeted genotyping versus full gene sequencing in carrier screening, to review advantages of performing ECF in all populations, regardless of ethnicity, to recognize the importance of the use of genetic testing in all populations, regardless of ethnicity, to recognize that accurate risk assessment and genetic counseling is dependent on assessing the detection rate and residual risk for the specific ECF panel used, as well as knowledge of the patient's personal and family history. So if you'd like to send us a question, please type it into the Q&A box in the lower left-hand corner of your control panel. And we'll respond to questions at the end of the presentation. So carrier screening is the practice of identifying individuals and couples who are at risk of conceiving children that are affected by autosomal recessive and X-linked recessive Mendelian diseases. It is expected that all individuals are carriers of approximately one in five children with Mendelian disease. 6 to 8 recessive diseases, and that most of these individuals do not have a known family history of the diseases that they carry. However, a woman's offspring are only at risk of inheriting an autosomal recessive disease if her partner, her reproductive partner, is also a carrier of the same disease. If so, they have a 1 in 4 chance in each pregnancy of having an affected child. If a female is a carrier of an X-linked recessive disease, she has a 1 in 4 chance of each pregnancy having an affected son, regardless of her reproductive partner's genotype. Carrier screening empowers couples who are at risk of having offspring with Mendelian disease to make choices regarding their family planning options. If a couple is identified to be at risk prior to conception, their options include in vitro fertilization followed by pre-implantation genetic diagnosis to minimize the risk of an affected fetus. They may also decide to use a GAMI donor or to adopt. If the couple is already pregnant, they can perform prenatal diagnosis by either chorionic villi sampling or amniocentesis, or they may decide not to perform testing, but instead to prepare for the possibility of a birth of a child with special needs. So there are many options, and with genetic counseling, a couple can decide what is the proper course of action for them. To give you some context into the key features of what carrier screening is and how it developed over time, we can begin by discussing carrier screening for Tay-Sachs disease. So this began in the 1970s. Tay-Sachs disease is considered to be an important health problem in the Ashkenazi Jewish community with about 1 in 3,000 infants being diagnosed with this disease that had no treatment and resulted in death in early childhood. Carrier screening was associated with educational programs and was widely supported by the Ashkenazi Jewish community. Although the genetic basis of Tay-Sachs disease had not been discerned at the time, biochemical evaluation of the hexosaminidase A enzyme could be used to accurately identify carriers of a deleterious allele with over 98% sensitivity. In the late 1980s, molecular analysis determined that three different pathogenic variants accounted for 92 to 98% of all Tay-Sachs carriers in the Ashkenazi Jewish population. This finding exemplifies the concept of founder mutation, when individuals of a defined ethnic background have a high frequency of specific deleterious variants due to the presence of a historical genetic bottleneck and subsequent population expansion. Founder mutations are at the heart of carrier screening because when a variant is located at a high allele frequency in a defined population, the disease will be a significant concern to the community and the molecular analysis of a small number of variants, traditionally by genotyping, was economical and had a high sensitivity. The carrier screening for Tay-Sachs disease in the Ashkenazi Jewish community has all but eliminated this disease in this population. So in 2001, both the American College of Obstetrics and Gynecology and the American College of Medical Genetics recommended that cystic fibrosis carrier screening be offered to pre-conceptual and prenatal patients in high-risk ethnic groups, such as people of Northern European background and Ashkenazi Jewish ancestry, where the carrier frequency is about 1 in 25 to about 1 in 30, and also be considered in other populations. Cystic fibrosis is unlike Tay-Sachs disease in that it is widespread in many populations and is caused by a wide variety of pathogenic variants that differ between ethnicities. So over 2,000 reportedly causative variants have been reported to date. While several variants, like the deletion of phenylalanine at codon 508, do account for the majority of pathogenic variants, if you want to achieve the same sensitivity that was achieved in Tay-Sachs carrier screening in the Ashkenazi Jewish population, you would need to screen many more variants. So the ACMG recommended a 23 mutation panel, which achieved a high sensitivity in the Caucasian and Jewish populations, but was significantly lower in the African, Asian, and other populations. The technology available in 2001 could not allow for timely and cost-effective detection of a high number of variants. So this example highlights the concept of residual risk following carrier screening. Residual risk is the chance that an individual is still a carrier after a negative carrier screening result. The residual risk depends on the carrier frequency of the disease in a population, as well as the detection rate that is provided by a genetic test. For example, an Ashkenazi Jewish individual may have an a priori risk of 1 in 28 for being a carrier of Tay-Sachs disease, but following carrier screening by biochemical and molecular testing that has a detection rate of 98%, the residual risk may only be 1 in 1300. However, an Asian individual may have a much lower a priori risk of 1 in 94 of being a carrier of cystic fibrosis, but with a much lower detection rate of 65%, the residual risk remains fairly high at 1 in 267. So as described in the previous slide, Tay-Sachs disease and cystic fibrosis are two examples that highlight the main features of classical carrier screening. So due to technological limitations, mutation testing focused on the most high-impact variants, those that were found at a high enough frequency to justify the cost of testing. These high-frequency variants tended to be founder mutations and therefore tended to be ethnic-specific. Carrier screening for these diseases in other populations would not justify the expense. As carrier sequencing in the 1990s and early 2000s was low throughput and not cost-effective, genotyping was the preferred technology to detect high-frequency pathogenic variants. That also meant that little variant curation was required, as only the desired variants were identified to be either present or absent. Lastly, few disorders were frequent enough in a population and were sufficiently clinically relevant to be considered for carrier screening programs. So once they were considered, they had to be amenable to testing, including having a distribution of pathogenic variants that resulted in a high sensitivity if tested. In the last 10 years, there have been many advances in carrier screening. In 2007, ACOG recommended that all individuals of African, Southeast Asian, and Mediterranean descent should be offered carrier screening for hemoglobinopathy. In 2008, ACMG recommended spinal muscular atrophy carrier screening for all couples, regardless of race or ethnicity. In 2008 and 2009, ACMG and ACOG, respectively, recommended carrier screening for up to nine diseases prevalent in the Ashkenazi Jewish population. By the early 2010s, next-generation sequencing was becoming more widely accepted and integrated into clinical practice. So this led to the first policy statement on expanded carrier screening being issued by ACMG in 2013. But before we get into this policy statement, we need to define what is expanded carrier screening. While there is no universally accepted definition for expanded carrier screening, it can generally be described as carrier screening for many disorders at a time, without regard to race or ethnicity. There is no set number of genes or mutations that need to be included for the test to be considered expanded. There are different ways that expanded carrier screening can be performed. It can use high-throughput genotyping, usually on an array, to test for a large number of population-based founder mutations, or pan-ethnic pathogenic variants. This method identifies known pathogenic variants only, and it may have a high detection rate in some populations but low in others. It can use next-generation sequencing, or NGS, but be analyzed bioinformatically for only a set of predetermined pathogenic variants. For this method, the technology is different, but the outcome is the same as the genotyping example described above. Or it can use next-generation sequencing, but all variants in the exons and intron-exon splice junctions are analyzed, and those determined to be pathogenic or likely pathogenic are reported, including novel variants. So this third type of expanded carrier screening is what we'll focus on for the rest of the talk. A radical shift in the technologies used in the clinic meant that laboratories were no longer limited to genotyping analysis for quick and cost-effective genetic testing. Using NGS, whole gene sequencing can be performed on many genes at the same time. Now it's no longer necessary to focus only on the most frequent mutations. Extremely rare variants, including variants that have never been recorded in the literature, can be detected for the same cost. It's also no longer necessary to focus only on diseases that were frequent in the target population, even genes causing relatively rare diseases can be included for only incremental increases in cost. And importantly, individuals of any ethnic background are eligible for expanded carrier screening, not just those with founder mutations. This is a very important point for the reasons that many individuals have extensive genetic admixture and or cannot accurately identify their ethnic background, and many diseases are understudied and underreported in certain ethnicities. So with expanded carrier screening, all individuals get access to the same testing panel. So getting back to the ACMG 2013 policy statement, the authors were aware that expanded carrier screening tests were already being developed, but no guidelines had yet been published by a professional authority. They wanted to make sure that just because laboratories could offer carrier screening for any diseases that they chose to, doesn't necessarily mean that they should. So the ACMG outlined five criteria that should be met for a disorder to be included on an expanded carrier screening panel. These criteria include that most at-risk patients would consider having prenatal diagnosis to facilitate reproductive decision-making. For each condition, the causative genes, mutations, and mutation frequency should be known in the population being tested so that residual risk for those that test negative can be correctly assessed. There should be a validated clinical association between the mutations detected and the severity of the disorder. Patients must provide consent to screen for adult onset disorders, especially where there may be implications for the individuals or their family members. And the laboratories offering the tests should be in compliance with the ACMG standards and guidelines for clinical genetic laboratories. So in 2015, a more complete joint statement on expanded carrier screening was released by ACMG, ACOG, NSGC, Perinatal Quality Foundation, and the Society for Maternal-Fetal Medicine. The statement covered topics including consent, appropriate pre- and post-test counseling, using the most appropriate technologies for diseases that were not ideally tested for by NGS, and interpretation of molecular findings. So in the same year, the ACMG published their landmark guidelines for clinical variant interpretation. Using these guidelines, five different categories of variant classifications were described, pathogenic and likely pathogenic, benign and likely benign, and variants of uncertain significance. The 2015 joint statement on expanded carrier screening noted that variants of uncertain significance should not be reported back to the patient, but all pathogenic and likely pathogenic variants should be. So here we would like to describe the general workflow of expanded carrier screening. This test is typically ordered by a physician, often an OBGYN or sometimes a geneticist, and is completed prior to conception or early during pregnancy. Usually, testing begins with a screening of one partner, often the female partner. However, sometimes both partners are tested at the same time. Turnaround time for ECS panels vary, but it's most often around two weeks. Results are communicated to the patient by the physician or genetic counselor, and if recommended, and if necessary, screening of the second partner is ordered by the physician. This screening may include the full testing panel, or it may just include the disorders for which the female partner was carrier. This screening is typically completed prior to conception or early during pregnancy. Usually, testing begins with a screening of one partner, often the female partner. However, it may just include the disorders for which the female partner was carrier. If a couple is identified to be a carrier couple, options may include in vitro fertilization with PGD, if not yet pregnant, or prenatal testing via CVS or amniocentesis. So as of today, there are a number of expanded carrier screening tests on the market. So this slide describes some of the tests of which we are aware. These tests range in size from 128 to 327 genes. And we just want to give you a bit of background on the STEMI-4 expanded carrier screening test that was used to identify the cases that we're going to talk about today. Our test includes 281 genes, including 277 that are tested for by NGS. We perform copy number analysis using MLPA for Duchenne muscular dystrophy, alpha thalassemia, GND also includes NGS of the coding region. Fragile X testing is performed by a Shurgen PCR sizing of the TGG repeat, an NGS of the coding sequence, and southern blotting for pre-mutation and full-mutation alleles. We also perform genotyping on several genes, including Gaucher disease, which is not amenable to short resequencing. Several large insertions and deletions are also tested for using genotyping analysis. We also perform biochemical analysis for Tay-Sachs disease. So now we'll begin talking about the cases that we'd like to present today. So for case one, the patient was a 28-year-old Ashkenazi Jewish female, currently seven weeks pregnant via IVF, who presented for prenatal genetic counseling. The expanded carrier screening 281 gene panel was ordered. Her expanded carrier screening results were notable for a heterozygous likely pathogenic variant in DMD, a heterozygous pathogenic variant in IKZ-CAP, 502204 plus 6 TDC change, and a heterozygous pathogenic variant in phenylalanine hydroxylase. A laboratory GC informed the patient of her results and the patient stated that she was previously aware of her carrier status for Duchenne muscular dystrophy and familial dysautonomia, and her husband, who had Sephardi heritage, had previously tested negative for via carrier screening via genotyping for familial dysautonomia. So full gene sequencing for familial dysautonomia and phenylalanine hydroxylase deficiency was recommended for the partner and ordered by the referring physician. And the partner's results were notable for a heterozygous novel likely pathogenic variant in IKB-CAP, C1461 minus 2 A to G. This variant is not in NOMAD, and it's a variant at the splice acceptor site of exon 14 of 37. His testing results were also negative for phenylalanine hydroxylase deficiency. So the couple was therefore at a 25% risk for the pregnancy to be affected with familial dysautonomia. So familial dysautonomia is a neurological disorder due to defects in the development and survival of sensory, sympathetic, and parasympathetic neurons. Symptoms include autonomic crises, gastrointestinal dysfunction, recurrent pneumonia, altered sensitivity to pain, and cardiovascular instability. So the pathogenic variant in IKB-CAP C2204 plus 6 T to C that was identified in the female partner is responsible for virtually all cases of familial dysautonomia among the Ashkenazim. Additionally, only a total of four disease-causing variants for IKB-CAP are listed in HGMD. So here, molecular prenatal diagnostic testing for IKB-CAP was performed on DNA from the CVS sample, and the results were that the fetus was negative for both of the IKB-CAP variants. And so the couple had a healthy baby, and they're aware that testing for familial dysautonomia is indicated for future pregnancies. This case highlights the advantage of full gene pooling for IKB-CAP C2204 plus 6 T to C. And sequencing over genotyping. Rare pathogenic or likely pathogenic variants that are not known to be founder mutations will generally be missed when performing genotyping assays. So case two. So the patient's a 35-year-old female pregnant via IVF who presented to her OB for routine prenatal care. The expanded care screening 281 gene panel was ordered. And of note, previously the patient had received a smaller care screening panel. So her expanded care screening results were notable for a heterozygous known pathogenic variant in CEP290. This variant was C1219 to C1220 in dilution of AT. And a laboratory GC informed the patient of her results, and the referring physician ordered care screening for the partner for this condition. And the partner's results were notable for a heterozygous likely pathogenic variant in CEP290, a nonsense change at E238. And so the pregnancy was therefore at a 25% risk for labor congenital amaurosis X or other CEP290-related celiopathies. So the CEP290-related celiopathies include four overlapping autosomal recessive disorders, labor congenital amaurosis, Bardet-Beetle syndrome, Joubert syndrome, and Meckel syndrome. So labor congenital amaurosis manifests with vision loss at birth or early infancy. Bardet-Beetle syndrome is characterized by obesity, intellectual disability, kidney disease, loss of vision beginning with loss of night vision and progression to tunnel vision and blindness. And Joubert syndrome has features of intellectual disability, brain malformations, ocular problems including uncontrollable eye movements and loss of vision, and kidney cysts leading to end-stage renal disease. Meckel syndrome manifests with occipital encephalopathy, brain malformations, facial dysmorphism, renal genesis, and extra digits. And it's not currently possible to predict which phenotype a patient may have based on the variants inherited. So CVS was scheduled and a transabdominal ultrasound was completed at the time of the CVS. And notably on the ultrasound, they found a probable Dandy-Walker malformation. So the CEP290 prenatal diagnostic sequencing on the CVS sample revealed that both variants were inherited, both the heterozygous pathogenic variant C1219 to 1220 deletion AT and the E238 nonsense change. And the molecular and ultrasound findings here suggest a diagnosis of Joubert-Meckel syndrome. And so this pregnancy was terminated. This case highlights the utility of care screening with larger panels. CEP290 sequencing is not available on all ECS panels. And in this case, ECS enabled early diagnosis of the fetus and allowed time for the couple for reproductive decision-making. Additionally, the couple is informed of the 25% recurrence risk for CEP290-related celiopathies with each subsequent pregnancy. So case three. A Hispanic couple, each with hearing loss, presented to a reproductive endocrinologist for evaluation. And neither member of the couple had prior diagnostic molecular testing for hearing loss. And the couple was counseled that the care screening in this case may indeed reveal molecular diagnosis for their hearing loss. On the expanded care screening, 281 gene panel was ordered. The female partner's results were notable for two heterozygous pathogenic variants in GJB2 in trans, the C35 DILG variant and C94 C to T, as well as a heterozygous pathogenic variant in sarcoglycan. The male partner's results were notable for a homozygous pathogenic variant in GJB2, the C35 DILG, and a heterozygous pathogenic variant in sarcoglycan. So in the case of hearing loss, approximately 80% of prelingual hearing loss is genetic. And the most common cause of, molecular cause of hearing loss is GJB2. And so here, ECS did confirm that each partner had GJB2-related hearing loss. Also, expanded care screening here confirmed that this couple, the carrier couple for limb girdle muscular dystrophy type 2C. So limb girdle muscular dystrophy type 2C is an autosomal recessive condition which is more prevalent in individuals from Morocco or individuals of Roma descent. And this form of muscular dystrophy presents with weakness of the pelvic region and shoulder girdle in infancy or childhood with symptoms usually present by age 12 years. And progression is rapid and patients are usually wheelchair bound by adolescence. So this case, again, highlights the utility of larger panels. The ethnicity or family history of the couple did not suggest that they're at increased risk for child with limb girdle muscular dystrophy. And this couple was appreciative to learn that they're at this 25% risk for this condition, that PGD and or prenatal testing was available. Additionally, the expanded care screening results enabled acrogenic counseling regarding the risk of hearing loss in their offspring. As both individuals' hearing loss is due to GJP2 mutations, all children would be expected to have hearing loss. So case four, a 32-year-old Ashkenazi Jewish female who is currently pregnant and a known case of sex disease carrier presented to the OB for prenatal care. Molecular and enzyme testing was ordered for each partner. And the female's partner's results were notable for a heterozygous pathogenic variant in HEX-A, the C1274 to C1277 duplication of TATC. And the enzyme testing was consistent with the patient being a Tay-Sachs disease carrier. So the white blood cell HEX system indicates A person activity was 41.1 and the non-carrier range is 55 to 72. The male's partner's results were negative for the molecular testing, no likely pathogenic or pathogenic variants in HEX-A identified. But enzyme testing was notable for our patient being a carrier. The white blood cell HEX-A person injectivity was in the inconclusive range, 54.7%. But the plasma HEX-A person injectivity was within the carrier range, 47.5, when the reference range is 58 to 72. So the ordering provider requested unmasking of the ZEUS results for HEX-A for the male partner and a heterozygous variant of uncertain significance in HEX-A was reported. This variant was seen in NOMAD, but it had very low frequency, like 0.0051% overall, but 0.12% frequency in Ashkenazi Jewish individuals. So the couple received genetic counseling and given the male's results, he was treated as a carrier for Tay-Sachs disease. Tay-Sachs disease is a progressive neurodegenerative disorder, which is most often characterized by onset in infancy with developmental delay, followed by paralysis, dementia, and blindness, with death in the second or third year of life. A CVS was performed when molecular testing performed at an outside laboratory and enzymatic testing performed at semaphore. And the fetus was positive via molecular testing for at least the pathogenic variant. Because the couple has this pathogenic variant of ZEUS, enzymatic testing was necessary to determine affected status. So the prenatal enzyme test results are listed here. The fetus sample from CVS and normal control and affected control were tested. And the HEX-A specific activity for the fetus was 145.6, which is in the range for not affected. And the percent HEX-A percentage was 18.9, which was again in the range for not affected. So here, enzymatic testing on the cultured CVS sample revealed that the fetus was not expected to be affected. And our lab does not report Tay-Sachs disease carrier status on prenatal enzyme diagnostic testing. So this case highlights the advantages of using multiple cooperative technologies and expanded care screening to improve determination of carrier status. Tay-Sachs enzyme and molecular testing are complementary approaches. Additionally, prenatal enzyme testing for Tay-Sachs is diagnostic and most accurately identifies affected fetuses. So case five. An Ashkenazi Jewish couple presented for genetic evaluation after their two-month-old daughter was diagnosed with spinal muscular atrophy. So by molecular testing, she had zero copies of SMN1 detected. So the expanded care screening 281 gene panel was ordered for each partner. So estimated the progressive neurological disorder with symptoms of muscle weakness and atrophy resulting from degeneration, loss of anterior horn cells and spinal cord and brainstem nuclei. SMA has five subtypes numbered zero through four with variable age of onset and lifespan. So SMA0 has prenatal onset and lifespan less than six months. SMA1 and 2 are more common. SMA1 has onset less than six months of age and lifespan most often less than two years. SMA2 has onset between six to 18 months and 70% are alive at age 25. SMA3 is onset after 18 months in normal lifespan. And SMA4, the least severe, has onset in adulthood in normal lifespan. So her expanded care screening results were notable for SMN1 copy number of one in a hemizygous likely pathogenic variant SMN1. This variant was located in exon 7 of 8 and it was not seen in NOMAD. Additionally, she had a heterozygous pathogenic variant FAH and one copy of the HBA alpha 3.7 deletion detected. This expanded care screening results were notable for an increased risk of being an SMN1 2.0 silent carrier. The G27134 T to G SNP was identified and two copies of SMN1 were present. She also had a heterozygous pathogenic variant LOXHD1. So here's a cartoon illustrating the SM1 and SMN2 loci. So typically on each allele you have one copy of SMN1 and one copy of SMN2 in the wild type situation. And the situation of a silent carrier occurs when you have no copies of SMN1 on one allele but two copies of SMN1 on the other allele. And sometimes when there's two copies of SMN1 on this allele, this G27134 T to G SNP is present. And the HG19 coordinates for this SNP are listed here on chromosome 5, it's 70247901. And so an osteoporosis Jewish individual with two copies of SMN1, if this SNP is identified, this individual is likely an SMA carrier. So this carrier screening confirmed the couple was a carrier couple for SMA. Additionally, the 21-year-old female partner reports no symptoms of SMA, but given the results of the hemizygous-like pathogenic variant in SMN1, she may develop symptoms later in life and may be affected. So this case illustrates that the use of complementary methods, such as those that identify single nucleotide variants, as well as copy number variants, may improve carrier status determination. Additionally, the identification of at-risk haplotypes can improve detection rates. And testing may also identify genotypes with adult-onset disease. So case 6, the patient's a 32-year-old osteoporosis Jewish female who presented to her OB for preconception carrier screening, and the expanded carrier screening 281 gene panel was ordered, and her results were notable for a heterozygous pathogenic variant in MESV, this V726A variant, a heterozygous pathogenic variant in ATP7B, a heterozygous pathogenic variant in biotinidase, and a heterozygous likely pathogenic variant in COF483. A laboratory GC informed the patient of her results, and the referring provider ordered the full 281 gene panel on the partner. These results were notable for a heterozygous pathogenic variant in MESV, the same variant, the V726A change, as well as a heterozygous pathogenic variant in phenylalanine hydroxylase. So a laboratory GC informed the couple that they were at an increased risk, 25% chance with each pregnancy to have a child with familial Mediterranean fever. So familial Mediterranean fever is a disorder characterized by recurrent attacks of fever and inflammation in the peritoneum, synovium, or pleura accompanied by pain. Amyloidosis with renal failure is a complication and may develop without overt crises. And so during the counseling session, the couple actually reported that they have a child with episodes of unexplained fever. So the couple was encouraged to have a clinical genetics evaluation scheduled for the child, and it was also noted that treatment with colchicine is available for FMF, and if the child was identified to have FMF, may be a helpful treatment. So the child with a history of unexplained fevers did later receive diagnostic testing in our lab, and was identified to be homozygous for the pathogenic variant, the V726A change, confirming the diagnosis of familial Mediterranean fever. So this case highlights that expanded care screening may not only lead to clinical diagnoses and assessment for current and future pregnancies, but may lead to clinical diagnosis and a family member not tested. So for case seven, so the next case involves a pregnant 31-year-old female who presented to her OB for routine prenatal care, and the full ECS 281 gene panel was ordered. So her results were notable for a heterozygous pathogenic non-sense variant in HBB causing beta thalassemia, and a heterozygous alpha 3.7 triplication of the HBA locus, resulting in five copies of HBA instead of the regular four. So HBH triplication alleles, which are represented like this, are generally considered to be benign variants, unless found in a patient that also carries an HBB allele causing beta thalassemia, which together can cause imbalance of the alpha and beta globin subunits, and result in clinical thalassemia intermedia. Therefore, reproductive partners of patients carrying HBB pathogenic variants are recommended to get not only HBB carrier screening, but also HBA testing in order to identify a triplication allele. Likewise, the spouses of patients who carry HBA triplications are recommended to get HBB sequencing. So a laboratory GC informed the patients of her results. She disclosed that she did have a clinical diagnosis of thalassemia intermedia and had chronic persistent anemia and was under the care of a hematologist. So this case highlights the necessity for clinical expertise in order to accurately interpret expanded carrier screening results. So in this particular case, while the patient who was undergoing ECS was the one who carried both the HBA and the HBB alleles that interact with each other, it does parallel the cases where we have one spouse who carries an HBB pathogenic allele and the other carries an HBA triplication allele. So case 8 was an Arabic-speaking couple who were both 28 years old who were seen in a reproductive clinic. The ECS 281 gene panel was ordered and their specific ethnicity was not provided. So the female partner was found to have a homozygous pathogenic start-loss variant in DHCR7, which causes Smith-Limley-Opitz syndrome. She also carried a heterozygous pathogenic missense variant in VSX2. The male partner also carried the same start-loss variant in DHCR7, but in the heterozygous form. So a laboratory GC informed the patient of her results and inquired whether the female patient exhibited any clinical symptoms of Smith-Limley-Opitz syndrome, and she denied symptoms. So the start-loss variant was classified as pathogenic due to it being rare in population databases with an allele frequency of 0.001% in NOMAD and has also been reported in at least six unrelated families, each compound heterozygous with severe variants. And most of these patients were reported to have milder types of disease. So homozygotes for this variant had not been reported in the literature or in population databases. So when we looked a bit further, we saw that functional studies showed that a methionine later in the transcript at position 59 was shown to be useful to produce a partially functional protein, and this can explain why this variant has a milder effect. So this case highlights the importance of collecting clinical information obtained from performing expanded carrier screening. So as noted in the 2015 joint statement on ECS, continual improvement of carrier testing can occur through the communication of findings obtained during testing. At the time we reported these cases, there was no information available on the phenotype of homozygotes for this variant, but now that we've got this information, we can include this in the description of the variant when reporting in future cases. So cases 9 to 12, I'll discuss as a group. So on average, 6.5% of individuals carry at least one novel variant that can be classified as likely pathogenic on our 281 gene panel. The likelihood of a couple being at risk of having a child with a rare disease increases dramatically when they're consanguineous. For example, if a couple is composed of two first cousins and one partner is found to be a carrier, the second partner now has a 12.5% chance, or 1 in 8, of carrying the same variant. A 1 in 8 chance is much higher than any of the population frequencies for the diseases on our panel. The below cases are examples of consanguineous couples that we've tested, which are generally first cousins, that carry rare or novel variants for diseases that are not known to be at high frequency in their ethnic background. So in these cases, two couples were South Asian, one was Middle Eastern, and the fourth was from South America. So these genes include TAPN3, which causes limb-girdle muscular dystrophy type 2A, CNGB3, which causes achromatopsia, TRMU, which causes acute infantile liver failure, and PHGDH, which causes 3-phosphoglycerate dehydrogenase deficiency. So these cases highlight the benefit of expanded carrier screening to consanguineous couples. These diseases may be very rare in certain parts of the world, but because of the significant rate of private or rare mutations in individuals, consanguineous couples are at a high risk of carrying deleterious variants in the same gene. So in conclusion, expanded carrier screening is a valuable tool that helps couples understand what diseases their children may be at risk for. Full gene sequencing allows individuals of all ethnic backgrounds to take advantage of carrier screening, rather than those from populations with significant founder effects. While many diseases are individually rare, large ECF panels may collectively identify many more carrier couples when screening ethnically diverse populations, including those with a higher incidence of consanguinity. Varied technologies, in addition to NGS, may be required for comprehensive determination of carrier status. And expanded carrier screening may inform on the health of adult patients who are being tested, as well as their family members. So we have a few slides of references, and we'd like to acknowledge everyone who has a semaphore who's been involved in developing and producing this test. So thank you for your time and attention. Just to remind you that if you would like to ask us a question, please type it into the Q&A box in the lower left-hand corner of the screen. As well, if you'd like to email us after the webinar, our email addresses are here, and you should have them in your handout that you can download as well. So finally, thank you to QIAGEN for supporting this program and the Ingenuity Clinical Decision Support Platform. And again, just a reminder to join us at ACMG 2018. So we'll take a look at some of the questions now. So the first question is, are there agreed-upon public resources for the frequencies needed to calculate residual risk for most or many diseases? So unfortunately, there's no single database where we can go to to get the carrier frequencies that we need in order to calculate accurate residual risk. So we can go to the literature. We can go to databases like Orphanet, which have kind of general disease frequencies. For better-studied diseases, there's usually some information in gene reviews. But it does take a bit of searching, usually to find this information, and it can be in different areas. We are, as said before, we are able to use kind of our previous experience with carrier screening. We have now tested over 180,000 individuals. We can kind of start deriving our own carrier frequencies, which we'll publish. So as time goes on, these resources, I think, will become more widely available to everybody. Okay. So another question is, are there any laboratory performance tests available for ECS? So I think proficiency testing. So my understanding is that there's proficiency testing available on a gene-by-gene basis, not for every gene. But because every single carrier screening test is different, and there's some genes that are on our panel that are not on other labs' panels, et cetera, that I don't think there's one, you know, proficiency test for expanded carrier screening. But I think that there are multiple genes that are on expanded carrier screening panels that can be tested for with proficiency testing. Oh, okay. So how often would Boost be unmasked at the request of the physician when the other parent is a carrier? So if it comes at request of the physician, then we can unmask the Boosts. So if the question is, how often would they be unmasked if requested, I think the answer is all the time. If the question is, how often are we asked to unmask Boosts because the other spouse is a carrier, it's not terribly frequent. It's usually more when there's a family history of the disease. Maybe they already had an affected child or they have an affected niece or nephew and they want to, you know, try to cover all their bases. But usually the most amount of the time we're not asked to unmask Boosts in these cases. But we will if we're requested. You can do this similar one too. Do labs have uniform policies on reporting of Boosts? So most labs, you know, choose not to report Boosts. There are a few labs in certain instances will report Boosts on carrier screening. But in accordance with the 2015 joint statement on expanded carrier screening, it's advised not to report variants of uncertain significance. It's generally considered to be putting the patient in a position where they don't know how to act on information that we don't know how to interpret. So it's made clear in the report that only pathogenic and likely pathogenic variants are reported. And the fact that there are Boosts that are not reported are reflected in the fact that there's a residual risk where the patient may still be a carrier even after they're positive, even after they're negative. We can answer this one. Okay. So in the Tay-Sachs case, was molecular testing also performed for the fetus in addition to the enzymatic testing? And so the molecular testing on the prenatal sample was actually performed in an outside lab, but we were aware that at least the patient was... the fetus had at least the pathogenic change. Otherwise, we wouldn't have received the fetal sample for enzymatic testing. There's a question just above. Sorry. How do you calculate the residual risk with ECF? So in that case, what you need, you need a carrier frequency and you need a detection rate. And so how you calculate those can be... can probably vary between laboratories. I'm sure it does. We have used population databases, literature review, internal data to calculate our carrier frequencies and detection rates. And we also do take into account the fact that Boosts are not reported. And so for SOMA4, our detection rates are not, you know, 99%, even if we think that we can actually detect the variants in the gene. If we think we may not report them due to the fact that there's not enough clinical information, we will reflect that in a lower detection rate and a lower residual risk or higher residual risk. That seems like a question we can answer. Sure. So are discussions ongoing at SOMA4 Sinai related to incorporating autosomal dominant diseases, for example, long QT into standard ECFs, why do dominant diseases tend not to be included today? So we can just say, like, traditionally, carrier screening is, you know, thought to identify, like, recessive diseases when parents are... when the partners are thought to be unaffected. The main intent of carrier screening is not to be diagnostic of either one of the partners. So that's one of the main reasons not to include dominant disease. Yeah. And I would think that for diseases that may have reduced penetrance, where maybe the parent is a carrier and the child may have a different phenotype, those are very hard to counsel on because if you have a reduced penetrance allele and the parent carries it, it's very hard to know, I think, what to do in the pregnancy. But as Bryn said, the main reason is because it's not meant to be diagnostic and in cases of high-penetrant alleles, the family member or the parent will have already been diagnosed. This is a longer question. I'm just reading it. Um, we can... Okay, so let's... So the question is, let's say you have one parent with a confirmed pathogenic variant and another with a variant of endosignificance not previously reported in the literature that's predicted to be pathogenic by in silico analysis. Would you report this variant to the family? So at Semaphore, this would be considered to be a variant of endosignificance and to be a variant of endosignificance. If the only indication that it could possibly be pathogenic is in silico data, that's not enough using the ACMG criteria for variant curation to bump that up into the likely pathogenic range. So that would consider... That would be considered a voof and would not be reported back unless specifically requested. Yeah, so I guess we could add that on the typical report, because it's a voof, it wouldn't be reported. But once... If the physician asks for unmasking, we would give out another report saying the voof. And so once you have that clinical report, the question is, do you tell that to the family? I think once you have the report, you're kind of obligated to tell that to the... to share that information with the family. Right. So I'm thinking about it in terms of the lab. We would not send it in a report to the physician. But if it's asked. But if it was ordered by the physician to unmask vooses, then that would be reported. Someone said, could you go over the residual risk for an Asian couple who screened negative for CF? Okay. So I think... Yeah, so I mentioned something earlier. So the... I'm not sure. Yeah. So in... Okay, so you have... Yeah, we have the information. Yeah. Okay, thank you. Okay. So an Asian individual, let's say... It's an individual. They have an a priori risk, so a risk before being tested, of, let's say, 1 in 94. I think that's the number that we have in our... Yeah, 1 in 94. Of being a carrier. But using a standard 23 gene mutation panel, the detection rate may only be 65%. So using a Bayesian analysis, starting with your a priori of 1 in 94 and your detection rate of 65%, you end up with a residual risk of 1 in 267. So I think the point that we were kind of trying to make with this example was that you can have a relatively lower a priori. So the chance that someone who's of Asian background is a carrier for cystic fibrosis is not nearly as high as someone who's Caucasian or Ashkenazi Jewish being a carrier. But because their testing is... Detection rate. The detection rate is not as high, then you end up, even after testing, being more likely than the person who had a higher carrier frequency, but a better detection rate. I mean, it depends on the actual numbers, but sometimes it's... In order to have a lower residual risk, you... The main thing is to have a high detection rate. Okay. The question is, do you confirm every variant, including boosts, that are reported by a second method? So for variants that are reported in the report, so pathogenic and likely pathogenic variants, we confirm them by a second method if required by New York State guidelines. So when we first began our carrier screening, New York State guidelines said we had to confirm every variant with a second method, which we did. As time went on, we now no longer have to confirm certain classes of variants, as long as they meet very strict cutoffs for quality. We only say or confirm about a quarter of our variants that we report at the moment. All the other ones have met all of the criteria responsible not to be reported. And as far as boosts go, there are times when we do say or confirm boosts if it's a complex variant, and we just want to be sure that the variant is the variant that we think it is. So I think we covered the majority of the questions that were typed in. Some of them are a little bit redundant. So there was a question saying, do you quote greater than 95% or to 99% pan-ethnic detection rate on your complete gene sequencing for the rare genes on your panel? So we can say that we do not quote greater than 95%. So we calculate our detection rate generally based on the known mutations, but we also quote an analytical detection rate, which takes into account even the variants that if we were to report boosts, if we were to ask to report every variant back, what would that be? So our analytical detection rate is usually much higher. Well, not much higher. It's generally higher, but we always kind of cap our detection rates at greater than 95%, because even if we detect everything that's reported in the literature, we don't know what alleles haven't been reported, and so we don't want our residual risk to be too low. I think that's it on the question. Okay. So just a reminder to do the survey at the end. Do the survey, and if there are any questions that you still have that we didn't answer or that you think of afterwards, then please feel free to email us.

Spectrum of Positive Findings

Expanded Carrier Screening

ACMG Genomics Case Conference

Semaphore

Ashley Birch

Bryn Webb

Mount Sinai Health System

Genetic Counseling

Comprehensive Testing Panels