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Clinical Impact of Genomic Duplications: A Discussion of Reporting Practices
230 compared to the low risk group 3.2% (n = 62) p = 0.02. The incidence of VOUS was 0.5%. Average Turn around time was 11 days. Conclusion: Prenatal CMA detected clinically significant abnormalities in the high-risk group. Abnormal results were detected in 3.2% of the low risk group, which is higher than previously reported. A larger sample size is needed to confirm our findings and to establish the CMA as a primary tool for prenatal diagnosis.
3 Complex Cases: Putting It All Together Using the Tools in Your Toolbox (Microarray, FISH and Chromosome Analysis) Teresa A. Smolarek, Stephanie Balow, Xia Li, Bianca Russell, Leandra Tolusso, Lisa M. Dyer, Howard M. Saal
The American College of Medical Genetics and Genomics recommends use of microarray analysis as a first line test for suspected aneuploidy. However, many cases exist in which standard chromosome and fluorescence in situ hybridization (FISH) analyses also are essential for understanding chromosomal changes in which microarray may not fully explain the underlying genomic changes encountered and why microarray is needed to explain chromosome abnormalities. We present six cases which demonstrate the need for multiple technologies to clarify the genomic picture, and allow for better patient management and counseling. The first case was not analyzed using the array but standard chromosome analysis alone was not sufficient to illustrate the extreme complexity of the case. The three clones were identified with 45,X, a second with an isodicentric Y chromosome and an unbalanced t(Y;21) and a third clone that also had a ring Y chromosome. Analysis using the array would have been challenging due to the involvement of the sex chromosomes, and the complex mosaicism present. Case 2 was a patient with a known unbalanced translocation between chromosomes X and 22 with atypical Klinefelter syndrome and deletion 22q11.2 with the 22q11.2 deletion syndrome. Case 3 had a complex isoderivative chromosome Y composed of both X and Y chromosome material. Case 4 had a recombinant X chromosome with duplication of Xq, deletion of Xp and an additional interstitial duplication of Xq. A fifth case initially was interpreted as a balanced translocation, t(7;16)(p15.1;p11.2), but a clinically significant microdeletion of the Williams-Beuren syndrome critical region on 7q11.23 was identified on the derivative chromosome 7. The remaining case involved an unbalanced translocation between chromosomes 13 and 15 or der(13)t(13;15)(p13;q13.3), with no loss or gain involving the derivative chromosome13. The microarray identified only the deletion on chromosome 15.These cases demonstrate the challenges of interpreting chromosome by using only one analytic technology. There may be many cases mosaicism or complex chromosomal abnormalities which are not adequately interpreted because the full armament of cytogenetic technologies is not utilized.
4 Assessing Copy Number Variants Involving ACMGG Secondary Finding Genes Identified by Routine Chromosomal SNP Array in a Clinical Pediatric Population Jinbo Fan *, Surabhi Mulchandani, Matthew Dulik, Jinyun Chen, Adam Gleason, Pushkala Jayaraman, Mahdi Sarmady, Elaine Zackai, Minjie Luo, Nancy Spinner, Laura Conlin * Corresponding author.
Abstracts Purpose: Unlike whole exome/genome sequencing, chromosomal microarray (CMA) testing is complicated by the fact that a copy number variant (CNV) may harbor genes associated with the patient’s phenotype as well as genes associated with a secondary finding. CMA is now the “first-tier” diagnostic test for individuals with developmental disabilities or congenital anomalies; however, the prevalence and significance of CNVs that encompass or disrupt one of the 56 American College of Medical Genetics and Genomics (ACMGG) secondary finding genes has not been systematically ascertained in a clinical pediatric population referred for routine diagnostic chromosomal SNP array analysis. Methods: We retrospectively compiled and reviewed 11,389 chromosomal SNP array cases from the Children’s Hospital of Philadelphia Cytogenetics Laboratory. Only 39 of these 56 ACMGG genes have been associated with a loss of function (LOF) mechanism, therefore eligible CNVs involving at least one of this subset of 39 genes were identified and analyzed to determine if the CNV explained the patient’s reason for study. We also compiled a list of historic findings reported from the clinical lab as incidental findings, including other medically actionable genes (e.g. DMD), excessive homozygosity suggesting incest, and carrier status (e.g. thalassemia trait). Results: Out of 11,389 patients examined by chromosomal SNP array, only 31 patients (0.27%) had CNVs that were predicted to result in a loss of function mechanism involving a deletion or intragenic duplication of an ACMGG gene. These predicted LOF CNVs included 25 whole gene deletions, 4 partial gene deletions, and 2 intragenic gene duplications. Among these CNVs, ten patients’ clinical indications can also be explained by the ACMGG genes. In total, only 21 of 11,389 (0.18%) patients had a reportable secondary finding that was not associated with their reason for study. In comparison, historic findings showed 1–2% of patients with an incidental finding. Conclusion: This study demonstrates that CNVs involving the 56 ACMGG secondary findings genes can be identified in routine chromosomal SNP array tests; however, the frequency of truly secondary findings (0.18%) is much lower than that reported for whole exome sequencing (~1%). When using the same criteria of incidental finding (but not applying a gene list), the percentage of incidental findings increased to a comparable percent. Thus, secondary findings tailored to chromosomal microarrays are warranted.
5 Clinical Impact of Genomic Duplications: A Discussion of Reporting Practices Cherisse A. Marcou *, Beth Pitel, Binu Porath, Umut Aypar, Nicole L. Hoppman, Erik C. Thorland, Hutton M. Kearney
The Mayo Clinic Cytogenetics Laboratory has performed microarray-based detection of copy number variation (CNVs) since 2006. Currently, postnatal CNV reporting criteria include deletions greater than 200 kb and duplications greater than 500 kb, as well as any known pathogenic CNV regardless of size. These thresholds are in place in an attempt to maximize diagnostic findings while minimizing the return of uncertain variants. However, our experience with small duplications (less than 2 Mb) reveals that the vast majority are inherited, challenging the clinical utility * Corresponding author.
Abstracts of reporting such variants. We performed a retrospective review of >4000 reported duplications in an effort to refine our reporting criteria for duplications. Our results suggest limited diagnostic utility for reporting duplications less than 1Mb that do not involve known triplosensitive genes. In addition, duplications are also evaluated for involvement of known haploinsufficient genes at the breakpoints, with concern for potential gene disruption. At least one recent study suggests that the majority of these duplication breakpoints represent nondisruptive/benign events. To further support this observation, we characterized four recurrent partial-gene duplications using wholegenome mate pair sequencing to refine the breakpoints and assess the orientation of the duplicated genomic material in order to assist in the assignment of pathogenicity. We found that our data support the conclusion made by others that the majority of partial gene duplication breakpoints are non-disruptive in nature. Considerations for adoption of revised postnatal reporting criteria for duplications and overall interpretive strategies for duplication CNVs will be discussed.
6 Re-Examination of Danish Carriers of Balanced Chromosomal Inversions Malene B. Rasmussen, Iben Bache, Mana M. Mehrjouy, Asli Silahtaroglu, Susanne Kjærgaard, Karen Brøndum-Nielsen, Peter K.A. Jensen, Ida Vogel, Jens Michael Hertz, Christina Fagerberg, Anders Bojesen, Michael B. Petersen, Jan Hansen, Christina Halgren, Mads Bak, Niels Tommerup
Molecular mapping of balanced chromosomal rearrangements (BCRs) such as translocations and inversions is a wellknown strategy for identifying new disease-associated genes, especially genes involved in early-onset and monogenic disorders. This strategy is especially applicable in Denmark, where we can link genomic and epidemiological data from nationwide medical registries. In a previous clinical and molecular reexamination of Danish translocation carriers and carriers of de novo BCRs using next-generation mate-pair sequencing, we have been able to identify new candidate disease-genes for both early and later onset disorders, as well as for monogenic and complex disorders. By linking medical registry and questionnaire data from >500 consented carriers of rare inversions, with mate-pair sequencing of 142 selected carriers, we have identified 60 unique rearrangements (corresponding to 121 inversion breakpoints). Consistent with previous findings, the breakpoints truncate all compartments of the genome, including protein-coding genes, noncoding genes, and known and candidate long-range acting regulatory domains. Some of the inversions segregate in surprisingly large families, and we have identified 10 founder inversions in the Danish population. In several of the large inversion families, we observe an apparent excess of neuropsychiatric disorders, where the molecular data link the phenotype to either a specific candidate gene, or to a candidate domain for long-range position effects. Thus, a paracentric founder inversion on chromosome 12p verified in 120 individuals from 36 families (including one homozygous individual), truncate a gene known to be associated with cardiac, skeletal and potentially cognitive problems. This unique cohort will allow a unique, unbiased register-based epidemiological assessment of the clinical significance of the 12pfounder inversion. Furthermore, the family histories indicate that this 12p-founder inversion could be present in North-America.
231 7 Evaluation of a Cystic Placenta: Spectrum of Genomic Changes Including GRB10 Microdeletion Urvashi Surti, Svetlana Yatsenko, Jie Hu, Daniel Bellissimo, W. Tony Parks, Lori Hoffner
Placental abnormalities can affect fetal growth and development. Cystic villous placentas associated with complete mole (diploid androgenetic), partial mole (diandric triploidy), mutations in two maternal-effect genes NLRP7 and KHDC3L resulting in familial diploid biparental molar pregnancies, and placental mesenchymal dysplasia with or without a live-born (androgenetic/ biparental mosaicism). We examined several unpublished cases of cystic placentas and determined their unusual genetic etiologies, ranging from genome-wide changes to microdeletions. These include: 46,XY(androgenetic)/46,XX(biparental) placental mosaicism; 47,XX,+9(androgenetic)/46,XX(biparental) fetal-placental mosaicism; 46,XX(androgenetic)/69,XXX fetal-placental mosaicism; 46,XY arr(1-22)x2(XY)x1 /46,XY arr[hg19] 7p12.1(50,697,878– 50,757,661)x1 placental mosaicism. This last case involved partial deletion of GRB10, which was detected only in the enlarged cystic portion of the placenta. The 30-week placenta of a live-born male was >90th percentile with a focus of enlarged cystic villi involving 30% of the placenta. Cytogenetic analysis and FISH revealed 46,XY nuc ish(DXZ1x1,DYZ3x1,D18Z1x2) for both the “normal appearing” and “enlarged cystic” villi. Whole-genome CGH+SNP microarray analysis was normal for the “normal appearing” villi, however, a 60 Kb deletion in GRB10 (GROWTH FACTOR RECEPTORBOUND PROTEIN 10) was detected in the “enlarged cystic” villi. In mice, Grb10 is paternally expressed in brain and maternally expressed in all other tissues, and modulates placental size and efficiency. Disruption of the maternal Grb10 results in overgrowth of both the embryo and placenta, indicating that GRB10 is a potent growth inhibitor. Mice with a disrupted paternal Grb10 show normal growth. In humans, GRB10 shows biallelic expression in most tissues, isoform-specific paternal expression in brain, and maternal expression in placental trophoblast. We hypothesize that deletion of maternal GRB10, in this case, resulted in placental overgrowth. Ancillary techniques should be utilized to investigate cystic placentas with atypical etiologies, due to the possibility of androgenetic cells or deleted growth suppressor genes in the fetus that could present a childhood malignancy risk or overgrowth syndrome.
8 Is Intrachromosomal Amplification of Chromosome 21 (iAMP21) Always Intrachromosomal? Karen Tsuchiya, Billy Davis, Rebecca Gardner
Recurrent chromosomal abnormalities in childhood precursor B-cell acute lymphoblastic leukemia (pre B-ALL), such as t(12;21)(p13;q22) that results in the ETV6/RUNX1 fusion, provide prognostic information that is useful in determining treatment stratification. iAMP21 is a more recently described cytogenetic entity of pre B-ALL that is frequently associated with an older age at presentation, a low white count, and a high risk of relapse with standard risk treatment. iAMP21 was originally described as multiple copies of the RUNX1 gene on a structurally abnormal chromosome 21, and subsequent studies elucidated a common
3 Complex Cases: Putting It All Together Using the Tools in Your Toolbox (Microarray, FISH and Chromosome Analysis) Teresa A. Smolarek, Stephanie Balow, Xia Li, Bianca Russell, Leandra Tolusso, Lisa M. Dyer, Howard M. Saal
The American College of Medical Genetics and Genomics recommends use of microarray analysis as a first line test for suspected aneuploidy. However, many cases exist in which standard chromosome and fluorescence in situ hybridization (FISH) analyses also are essential for understanding chromosomal changes in which microarray may not fully explain the underlying genomic changes encountered and why microarray is needed to explain chromosome abnormalities. We present six cases which demonstrate the need for multiple technologies to clarify the genomic picture, and allow for better patient management and counseling. The first case was not analyzed using the array but standard chromosome analysis alone was not sufficient to illustrate the extreme complexity of the case. The three clones were identified with 45,X, a second with an isodicentric Y chromosome and an unbalanced t(Y;21) and a third clone that also had a ring Y chromosome. Analysis using the array would have been challenging due to the involvement of the sex chromosomes, and the complex mosaicism present. Case 2 was a patient with a known unbalanced translocation between chromosomes X and 22 with atypical Klinefelter syndrome and deletion 22q11.2 with the 22q11.2 deletion syndrome. Case 3 had a complex isoderivative chromosome Y composed of both X and Y chromosome material. Case 4 had a recombinant X chromosome with duplication of Xq, deletion of Xp and an additional interstitial duplication of Xq. A fifth case initially was interpreted as a balanced translocation, t(7;16)(p15.1;p11.2), but a clinically significant microdeletion of the Williams-Beuren syndrome critical region on 7q11.23 was identified on the derivative chromosome 7. The remaining case involved an unbalanced translocation between chromosomes 13 and 15 or der(13)t(13;15)(p13;q13.3), with no loss or gain involving the derivative chromosome13. The microarray identified only the deletion on chromosome 15.These cases demonstrate the challenges of interpreting chromosome by using only one analytic technology. There may be many cases mosaicism or complex chromosomal abnormalities which are not adequately interpreted because the full armament of cytogenetic technologies is not utilized.
4 Assessing Copy Number Variants Involving ACMGG Secondary Finding Genes Identified by Routine Chromosomal SNP Array in a Clinical Pediatric Population Jinbo Fan *, Surabhi Mulchandani, Matthew Dulik, Jinyun Chen, Adam Gleason, Pushkala Jayaraman, Mahdi Sarmady, Elaine Zackai, Minjie Luo, Nancy Spinner, Laura Conlin * Corresponding author.
Abstracts Purpose: Unlike whole exome/genome sequencing, chromosomal microarray (CMA) testing is complicated by the fact that a copy number variant (CNV) may harbor genes associated with the patient’s phenotype as well as genes associated with a secondary finding. CMA is now the “first-tier” diagnostic test for individuals with developmental disabilities or congenital anomalies; however, the prevalence and significance of CNVs that encompass or disrupt one of the 56 American College of Medical Genetics and Genomics (ACMGG) secondary finding genes has not been systematically ascertained in a clinical pediatric population referred for routine diagnostic chromosomal SNP array analysis. Methods: We retrospectively compiled and reviewed 11,389 chromosomal SNP array cases from the Children’s Hospital of Philadelphia Cytogenetics Laboratory. Only 39 of these 56 ACMGG genes have been associated with a loss of function (LOF) mechanism, therefore eligible CNVs involving at least one of this subset of 39 genes were identified and analyzed to determine if the CNV explained the patient’s reason for study. We also compiled a list of historic findings reported from the clinical lab as incidental findings, including other medically actionable genes (e.g. DMD), excessive homozygosity suggesting incest, and carrier status (e.g. thalassemia trait). Results: Out of 11,389 patients examined by chromosomal SNP array, only 31 patients (0.27%) had CNVs that were predicted to result in a loss of function mechanism involving a deletion or intragenic duplication of an ACMGG gene. These predicted LOF CNVs included 25 whole gene deletions, 4 partial gene deletions, and 2 intragenic gene duplications. Among these CNVs, ten patients’ clinical indications can also be explained by the ACMGG genes. In total, only 21 of 11,389 (0.18%) patients had a reportable secondary finding that was not associated with their reason for study. In comparison, historic findings showed 1–2% of patients with an incidental finding. Conclusion: This study demonstrates that CNVs involving the 56 ACMGG secondary findings genes can be identified in routine chromosomal SNP array tests; however, the frequency of truly secondary findings (0.18%) is much lower than that reported for whole exome sequencing (~1%). When using the same criteria of incidental finding (but not applying a gene list), the percentage of incidental findings increased to a comparable percent. Thus, secondary findings tailored to chromosomal microarrays are warranted.
5 Clinical Impact of Genomic Duplications: A Discussion of Reporting Practices Cherisse A. Marcou *, Beth Pitel, Binu Porath, Umut Aypar, Nicole L. Hoppman, Erik C. Thorland, Hutton M. Kearney
The Mayo Clinic Cytogenetics Laboratory has performed microarray-based detection of copy number variation (CNVs) since 2006. Currently, postnatal CNV reporting criteria include deletions greater than 200 kb and duplications greater than 500 kb, as well as any known pathogenic CNV regardless of size. These thresholds are in place in an attempt to maximize diagnostic findings while minimizing the return of uncertain variants. However, our experience with small duplications (less than 2 Mb) reveals that the vast majority are inherited, challenging the clinical utility * Corresponding author.
Abstracts of reporting such variants. We performed a retrospective review of >4000 reported duplications in an effort to refine our reporting criteria for duplications. Our results suggest limited diagnostic utility for reporting duplications less than 1Mb that do not involve known triplosensitive genes. In addition, duplications are also evaluated for involvement of known haploinsufficient genes at the breakpoints, with concern for potential gene disruption. At least one recent study suggests that the majority of these duplication breakpoints represent nondisruptive/benign events. To further support this observation, we characterized four recurrent partial-gene duplications using wholegenome mate pair sequencing to refine the breakpoints and assess the orientation of the duplicated genomic material in order to assist in the assignment of pathogenicity. We found that our data support the conclusion made by others that the majority of partial gene duplication breakpoints are non-disruptive in nature. Considerations for adoption of revised postnatal reporting criteria for duplications and overall interpretive strategies for duplication CNVs will be discussed.
6 Re-Examination of Danish Carriers of Balanced Chromosomal Inversions Malene B. Rasmussen, Iben Bache, Mana M. Mehrjouy, Asli Silahtaroglu, Susanne Kjærgaard, Karen Brøndum-Nielsen, Peter K.A. Jensen, Ida Vogel, Jens Michael Hertz, Christina Fagerberg, Anders Bojesen, Michael B. Petersen, Jan Hansen, Christina Halgren, Mads Bak, Niels Tommerup
Molecular mapping of balanced chromosomal rearrangements (BCRs) such as translocations and inversions is a wellknown strategy for identifying new disease-associated genes, especially genes involved in early-onset and monogenic disorders. This strategy is especially applicable in Denmark, where we can link genomic and epidemiological data from nationwide medical registries. In a previous clinical and molecular reexamination of Danish translocation carriers and carriers of de novo BCRs using next-generation mate-pair sequencing, we have been able to identify new candidate disease-genes for both early and later onset disorders, as well as for monogenic and complex disorders. By linking medical registry and questionnaire data from >500 consented carriers of rare inversions, with mate-pair sequencing of 142 selected carriers, we have identified 60 unique rearrangements (corresponding to 121 inversion breakpoints). Consistent with previous findings, the breakpoints truncate all compartments of the genome, including protein-coding genes, noncoding genes, and known and candidate long-range acting regulatory domains. Some of the inversions segregate in surprisingly large families, and we have identified 10 founder inversions in the Danish population. In several of the large inversion families, we observe an apparent excess of neuropsychiatric disorders, where the molecular data link the phenotype to either a specific candidate gene, or to a candidate domain for long-range position effects. Thus, a paracentric founder inversion on chromosome 12p verified in 120 individuals from 36 families (including one homozygous individual), truncate a gene known to be associated with cardiac, skeletal and potentially cognitive problems. This unique cohort will allow a unique, unbiased register-based epidemiological assessment of the clinical significance of the 12pfounder inversion. Furthermore, the family histories indicate that this 12p-founder inversion could be present in North-America.
231 7 Evaluation of a Cystic Placenta: Spectrum of Genomic Changes Including GRB10 Microdeletion Urvashi Surti, Svetlana Yatsenko, Jie Hu, Daniel Bellissimo, W. Tony Parks, Lori Hoffner
Placental abnormalities can affect fetal growth and development. Cystic villous placentas associated with complete mole (diploid androgenetic), partial mole (diandric triploidy), mutations in two maternal-effect genes NLRP7 and KHDC3L resulting in familial diploid biparental molar pregnancies, and placental mesenchymal dysplasia with or without a live-born (androgenetic/ biparental mosaicism). We examined several unpublished cases of cystic placentas and determined their unusual genetic etiologies, ranging from genome-wide changes to microdeletions. These include: 46,XY(androgenetic)/46,XX(biparental) placental mosaicism; 47,XX,+9(androgenetic)/46,XX(biparental) fetal-placental mosaicism; 46,XX(androgenetic)/69,XXX fetal-placental mosaicism; 46,XY arr(1-22)x2(XY)x1 /46,XY arr[hg19] 7p12.1(50,697,878– 50,757,661)x1 placental mosaicism. This last case involved partial deletion of GRB10, which was detected only in the enlarged cystic portion of the placenta. The 30-week placenta of a live-born male was >90th percentile with a focus of enlarged cystic villi involving 30% of the placenta. Cytogenetic analysis and FISH revealed 46,XY nuc ish(DXZ1x1,DYZ3x1,D18Z1x2) for both the “normal appearing” and “enlarged cystic” villi. Whole-genome CGH+SNP microarray analysis was normal for the “normal appearing” villi, however, a 60 Kb deletion in GRB10 (GROWTH FACTOR RECEPTORBOUND PROTEIN 10) was detected in the “enlarged cystic” villi. In mice, Grb10 is paternally expressed in brain and maternally expressed in all other tissues, and modulates placental size and efficiency. Disruption of the maternal Grb10 results in overgrowth of both the embryo and placenta, indicating that GRB10 is a potent growth inhibitor. Mice with a disrupted paternal Grb10 show normal growth. In humans, GRB10 shows biallelic expression in most tissues, isoform-specific paternal expression in brain, and maternal expression in placental trophoblast. We hypothesize that deletion of maternal GRB10, in this case, resulted in placental overgrowth. Ancillary techniques should be utilized to investigate cystic placentas with atypical etiologies, due to the possibility of androgenetic cells or deleted growth suppressor genes in the fetus that could present a childhood malignancy risk or overgrowth syndrome.
8 Is Intrachromosomal Amplification of Chromosome 21 (iAMP21) Always Intrachromosomal? Karen Tsuchiya, Billy Davis, Rebecca Gardner
Recurrent chromosomal abnormalities in childhood precursor B-cell acute lymphoblastic leukemia (pre B-ALL), such as t(12;21)(p13;q22) that results in the ETV6/RUNX1 fusion, provide prognostic information that is useful in determining treatment stratification. iAMP21 is a more recently described cytogenetic entity of pre B-ALL that is frequently associated with an older age at presentation, a low white count, and a high risk of relapse with standard risk treatment. iAMP21 was originally described as multiple copies of the RUNX1 gene on a structurally abnormal chromosome 21, and subsequent studies elucidated a common