Multiple Copy Number Variations in a Patient with Developmental Delay ASCLS- March 31, 2016

Multiple Copy Number Variations in a Patient with Developmental Delay ASCLS- March 31, 2016 Marwan Tayeh, PhD, FACMG Director, MMGL Molecular Genetics Assistant Professor of Pediatrics Department of Pediatrics and Communicable Diseases Division of Pediatric Genetics, Metabolism and Genomics

Objectives Chromosomal microarray (CMA) types CMA applications and limitations Interpretation of Copy Number Variations (CNV) Genomic rearrangements and how they contribute to genetic diseases

Chromosomal Microarray (CMA) The American College of Medical Genetics and Genomics (ACMG) recommends CMA as the first-line genetic test for all children with autism spectrum disorders, developmental, intellectual disabilities, multiple congenital anomalies or birth defects. Miller et al. Am J Hum Genet 86:749 764, 2010

Chromosomal Microarray (CMA)

CMA Types Array CGH (acgh) SNP array

Array-CGH Protocol http://www.nature.com/scitable/content/41020/arraycgh_theisen_large_2.jpg

Log R 20.1 Mb Loss (NCBI36/hg18): arr 1q24.3q31.2(169165485-189301162)x1

Log R 537 Kb Gain (NCBI36/hg18): arr 15q13.3 (29,759,938-30,297,218)x3

SNP Array Protocols Alsolami et al. Per Med (2013) 10(4), 361 376.

6.5 Mb Loss (GRCh37/hg19): arr 11p14.1p13(29884050-36367117)x1 Log R B allele

1.6 Mb Gain at 3q29 (Chr3:195,738,409-197,346,566) Log R B allele

10.2 Mb Two-copy Gain at 15q11.1q13.2 & 2.0 Mb One-copy Gain at 15q13.2q13.3 Log R B allele

Objectives Chromosomal microarray (CMA) types CMA applications and limitations Interpretation of Copy Number Variations (CNV) Genomic rearrangements and how they contribute to genetic diseases

CMA Applications Structural variations (loss or gain) CMA has a higher sensitivity to detect structural variations than Karyotype or FISH analyses SNP- genotyping (SNP array only) GWAS, Linkage studies, and Homozygosity mapping Parent of origin Regions of SNP Homozygosity (ROH; also known as LOH) Incidental Findings

ROH McQuillan et al. Am J Hum Genet 83(3):359-372, 2008

6.15% SNP Homozygosity

ROH: 22.1 Mb (NCBI37/hg19): arr 3p24.3-p22.1 (18902798-41010253)x2 Log R B allele

Prader-Willi Syndrome: Maternal Uniparental Disomy 2.3% SNP Homozygosity

ROH: 40.6 Mb (NCBI37/hg19): arr 15q13.1-q23 (29424755-69985510)x2 Log R B allele

CMA Applications Structural variations (loss or gain) CMA has a higher sensitivity to detect structural variations than Karyotype or FISH analyses SNP- genotyping (SNP array only) GWAS, Linkage studies, and Homozygosity mapping Parent of origin Regions of SNP Homozygosity (ROH; also known as LOH) Incidental Findings

Incidental Findings Findings that are unrelated to the indication for ordering the test but of medical value for patient care http://www.acmg.net/

1.1 Mb Loss (GRCh37/hg19): arr 19p13.3(1214408-2285196)x1 Medical history: Hypoglycemia, Minor Dysmorphic Features, and Ventricular Septal Defect Log R B allele

Peutz-Jeghers Syndrome Nonsense mutations and deletions affecting STK11 gene have been associated with Peutz- Jeghers syndrome Peutz-Jeghers syndrome is an autosomal dominant disorder characterized by Melanocytic macules of the lips, buccal mucosa, and digits Multiple gastrointestinal hamartomatous polyps An increased risk of various neoplasms Hemminki et al. Nature Genet 15:87-90, 1997; Jenne et al. Nature Genet 18:38-43, 1998

CMA Limitations CMA cannot detect: Balanced rearrangement (translocation) Inversion Point mutations Exact location of insertion of copy number gains within the genome In tandem Dispersed

0.519 Mb and 6.1 Mb Gains (NCBI36/hg18): arr 15q11.2(20249886-20768955)x5, 15q11.2q13.1(20811896-26884937)x4 Medical History: Developmental Delay, Hypotonia, and Autism Spectrum Disorder Log R FISH probe

Metaphase FISH A courtesy of Diane Roulston, PhD. Director of the Cytogenetics laboratory at University of Michigan

Supernumeray Marker Chromosome (SMC) SMC(15) is the most common SMC in human SMC(15)s are almost always de novo in origin and maternally derived SMC(15)s syndrome are associated with: Autism Intellectual disability Ataxia Seizures Roberts et al. Am J Hum Genet 73(5):1061 1072, 2003

Objectives Chromosomal microarray (CMA) types CMA applications and limitations Interpretation of Copy Number Variations (CNV) Genomic rearrangements and how they contribute to genetic diseases

CMA Analysis Categories of clinical significance of Copy Number Variations (CNV): Pathogenic Documented as clinically significant Benign CNV reported in multiple publications or curated databases as a benign variant Uncertain clinical significance Likely pathogenic Likely Benign Uncertain clinical significance http://www.acmg.net/staticcontent/sgs/cnv.pdf

23 Kb Loss at 14q31.3 (Chr14:88,399488-88,422,442) Medical History: Hypotonia And Spasticity of Extremities, Developmental Delays, and Cerebral Palsy-like Neurologic Abnormalities Log R B allele

Krabbe Disease 23 Kb one copy loss that affects exons 11-17 of the GALC gene GALC pathogenic variants cause a deficiency of the galactocerebrosidase enzyme Deletion of Exons 11-17 is the most common pathogenic variant (70%) Homozygous or compound heterozygous pathogenic variants in GALC cause Krabbe disease

CMA Analysis Categories of clinical significance of Copy Number Variations (CNV): Pathogenic Documented as clinically significant Benign CNV reported in multiple publications or curated databases as a benign variant Uncertain clinical significance Likely pathogenic Likely Benign Uncertain clinical significance http://www.acmg.net/staticcontent/sgs/cnv.pdf

Log R 537 Kb Gain (NCBI36/hg18): arr 15q13.3 (29,759,938-30,297,218)x3

Benign CNV chr10:135,254,554-135,371,866 http://genome.ucsc.edu/

Benign CNV chr10:135,254,554-135,371,866 http://genome.ucsc.edu/

CMA Analysis Categories of clinical significance of Copy Number Variations (CNV): Pathogenic Documented as clinically significant Benign CNV reported in multiple publications or curated databases as a benign variant Uncertain clinical significance Likely pathogenic Likely Benign Uncertain clinical significance http://www.acmg.net/staticcontent/sgs/cnv.pdf

CNVs of Uncertain Clinical Significance Likely pathogenic Described in a single case report Gene function relevant to reason for patient referral Likely Benign No genes in interval (usually reported because of a size criterion established by the laboratory) Described in the general population but does not represent a common variant Uncertain clinical significance no sub-classification Contains genes (irrelevant or not dosage sensitive) Described in multiple contradictory publications and/or databases http://www.acmg.net/staticcontent/sgs/cnv.pdf

VUS: 2.2 Mb Gain (NCBI37/hg19): arr 1p36.32-p36.31 (3386506-5620256)x3 Log R B allele

VUS Chr1:3386506-5620256 http://genome.ucsc.edu/

VUS Chr1:3386506-5620256 http://genome.ucsc.edu/

Objectives Chromosomal microarray (CMA) types CMA applications and limitations Interpretation of Copy Number Variations (CNV) Genomic rearrangements and how they contribute to genetic diseases

Complex Genomic Rearrangements (CGR) Complex Genomic (Chromosomal) rearrangement: At least three breakpoints involving more than a single chromosome that are associated with a variety of clinical phenotypes: Intellectual disability Developmental delay Complex congenital abnormalities (Kloosterman et al. Hum Mol Genet 20:1916-1924, 2011) Two or more breakpoints affecting a single or multiple chromosomes that are associated with genomic disorders (Liu et all. Cell 146:889-903, 2011)

Complex Genomic Rearrangements (CGR) 17 patients with Developmental Delay or Multiple Congenital Anomalies CMA testing detected multiple CGR in each patient All observed 17 CGR were affecting only one chromosome in each patient Liu et all. Cell 146:889-903, 2011

Medical History: Patient 1 Developmental delay Expressive speech disorder Scoliosis De novo 7q33 dup (46,XX,add(7)(q33)) detected previously by Karyotype analysis SNP array (CMA) was performed

Patient 1

Patient 1 A female chromosome profile with 10 aberrant regions of potential clinical significance: Xp11.4p11.3(40618904-45179762)x3 3p24.1p23(26797958-31529089)x3 4p11(48735747 49053522) x3 7q33q34(136624320-141093612)x3 8p21.2p12(25159885-29932284)x3 9p24.1p23(6027931-10603845)x3 9p21.3(20014872-24527357)x3 10q24.33q25.1(105666745-110387632)x3 15q11.2(22754322-23300172)x1 19q13.2q13.31(39379906-43465171)x3 Tayeh et al. Clin Case Rep 3(6):396-401, 2015

4.6 Mb Gain, 9p24.1p23(6027931-10603845)x3 4.5 Mb Gain, 9p21.3(20014872-24527357)x3 Log R B allele

546 Kb Loss, 15q11.2(22754322-23300172)x1 Log R B allele

Patient 1 Metaphase FISH data indicate that all aberrations are in tandem at the same site of genomic origin Tayeh et al. Clin Case Rep 3(6):396-401, 2015

Patient 1 Mechanism underlying such multiple genomic rearrangements De novo vs. inherited Random affecting both parental alleles Only affecting one of the parental alleles

Mother of Patient 1

Father of Patient 1

Father of Patient 1 546 Kb loss, 15q11.2(22754322-23300172)x1 Log R B allele

SNP Genotyping SNP haplotype analysis of the patient and her parents within genomic regions associated with copy number variations: Duplications arose on both maternal and paternal chromosomes B allele A,A B,B A,B A,A A,A A,A A,A A,B B,B A,A Patient 1: Paternal gain on 9p24.1p23 A,A,A B,B,A A,A,B A,A,B A,A,A

Patient 1 Tayeh et al. Clin Case Rep 3(6):396-401, 2015

Patient 1 Nine de novo duplications and an inherited 15q11.2 deletion Duplications arose on both maternal and paternal chromosomes A zygotic or post-zygotic origin The existence of a vulnerable period of chromosome instability during early development that may be influenced by genetic and non-genetic factors

Summary Chromosomal microarray (CMA) is a powerful tool to detect genomic copy number variations CMA cannot detect balanced translocations, inversions, point mutations and may not detect intragenic small deletions and duplications CMA cannot determine the location or orientation of where copy number gains are inserted in the genome Complex genomic rearrangements are associated with Intellectual disability, developmental delay, and complex congenital abnormalities

Acknowledgements Dr. Jeff Innis Dr. Diane Roulston Dr. Tom Glover Leslie Ernst MMGL Lab Todd Ackley Kristin Evon Tracy Rocco Jennifer Phenicie Alex Johnson Anna Sharkey Janean DeVaul Jessie Sluka Linda Haglund-Hazy Brittany Hannigan Rita Sanders Amit Rupani