1. Genetics of Congenital Heart Disease 张咸宁 zhangxianning@zju.edu.cn Tel ： 13105819271; 88208367 Office: A705, Research Building 2012/02

2. Required Reading Thompson &Thompson Genetics in Medicine, 7 th Ed （双语版， 2009 ） ● Pages 91-92 、 168-169 、 356 Recommended: Weissman CG and Gelb BD. The genetics of congenital heart disease: a review of recent developments. Curr Opinion Cardiol 2007; 22:200-206. Kaltman JR, et al. Circulation 2010;121:2766-72. Kaltman JR, et al. Journal of the American College of Cardiology 2010;56:1262–5.

3. Learning Objectives To recognize familial patterns of CHD To understand developmental mechanisms of CHD To see CHDs as examples of the larger group of common disorders with common complex inheritance involving –Single genes –Multiple genes –Environmental influences

4. Overview Introduction to Congenital Heart Disease (CHD) Developmental Mechanisms –Flow Lesions –Problems in Cell Migration –Problems in Cell Death –Abnormalities in Extracellular Matrix –Abnormalities in Targeted Growth Summary

5. Introduction to CHD Relatively common birth defect –Liveborn infants 4-8/1,000 –Stillborns 10× higher or 8% –Miscarriages 15% in abortuses <24 weeks gestation

6. Introduction to CHD Variety of causes –Single gene –Chromosomal –Teratogen exposures Maternal rubella infection Gestational diabetes mellitus

7. Maternal Infections –Rubella: 35% affected Maternal Diseases –Diabetes Mellitus: 3-5% –Maternal PKU: 10% Teratogenic Substances –Alcohol: 25-35% –Dilantin （苯妥英） : 2-3% Congenital Heart Defects Environmental Component

8. Gross Chromosomal Defects –5-8% of Defects –Examples Trisomy 21: 35-50% Trisomy 18: 99% Turner syndrome: 20% Single-Gene Defects –3% of Defects Congenital Heart Defects Genetic Component

9. Familial Patterns of Recurrence CHD recurrence in a family –Affected individuals may not have identical anatomical heart abnormality –Will have lesions representing similarity in the developmental mechanism Should look for abnormalities outside of the cardiovascular system –May indicate a syndromic association with CHD

10. Developmental Mechanisms Flow Lesions Problems in Cell Migration Problems in Cell Death Abnormalities in Extracellular Matrix Abnormalities in Targeted Growth

12. Is Isolated CHD a Multifactorial Trait? Table 8-12: Population Incidence and Recurrence Risks for Various Flow Lesions VSD = Ventricular Septal Defect PDA = Patent Ductus Arteriosus ASD = Atrial Septal Defect AS = Aortic Stenosis DefectPop Incid (%) Freq in Sibs (%) λ sib VSD0.174.325 PDA0.0833.238 ASD0.0663.248 AS0.0442.659

13. Is Isolated CHD a Multifactorial Trait? For these flow lesions –Sib relative risk ratio (λ sib ) Support familial aggregation –Where genetic mutation not known Use empiric risk factors to counsel first degree relatives Rapid decrease in risk for second and third degree relatives to not much higher than population risks For families with CHD other than flow lesions –Reassure that recurrence risk is no greater than population risk Prenatal ultrasound can be used as part of counseling and often reassurance before birth

14. Flow Lesions Fig 8-9

15. Flow Lesions Large category of CHDs –Approximately 50% of all CHDs Up to 25% of flow lesion CHDs, particularly tetralogy of Fallot, have del22q11.2 –DiGeorge syndrome –Velocardiofacial syndrome –Conotruncal anomaly face syndrome

16. del22q11.2 Syndromes Autosomal dominant Variable expressivity Deletion of approximately 3Mb –Caused by homologous recombination of low copy repeat sequences One of the most common cytogenetic deletions with a significant phenotype –1 per 2,000-4,000 live births

17. Unequal Crossing Over Due to Homologous Recombination Fig 6-8

18. 22q11.2 Rearrangements Fig 6-9

19. del22q11.2 Syndromes Phenotypes may include –CHD –Craniofacial abnormalities –Mental retardation/developmental delay –Reduced circulating lymphocytes –Hypocalcemia –Schizophrenia

20. Sidebar: del22q11.2 and Schizophrenia An estimated 25% of patients with del22q11.2 develop this –Even in the absence of many or most of the physical signs of DGS 1 per 2-4,000 live births with del22q11.2 –Therefore, 1 per 8-16,000 live births may develop schizophrenia due to this deletion Most common genetic mechanism for schizophrenia known at this time –Mechanism is unknown

21. del22q11.2 and CHD Responsible for between 5% and 12.5% of CHDs Particularly common in certain CHDs – >40% of patients with tetralogy of Fallot (TOF) and pulmonary atresia (PA) – >60% of patients with TOF and absent pulmonary valve

22. DGS TDR (Typically Deleted Region) 3Mb deletion –Loss of approximately 30 genes Smaller 1.5 Mb deletion –Seen in approximately 10% of patients TBX1 maps in DGS TDR –Encodes transcription factor involved in pharyngeal arch development –Haploinsufficiency implicated in DGS –Mutated in patients with similar phenotype who do not have del22q11.2

23. Apoptosis and CHD TBX1 may be involved in apoptosis, a mechanism known to be involved in normal cardiac and lymphocyte development –Foxp1 in mice Required for remodeling of endocardial cushions (portions of ventricular septum and cardiac outflow tract) To position aortic and pulmonary vessels normally by eliminating certain cells to shift the cushions’ positions –Apoptosis occurs during immune system development To eliminate lymphocytic lineages that react to self Required for protection against autoimmune disease

24. Apoptosis and CHD If TBX1 causes the conotruncal defects (e.g. TOF) associated with del22q11.2, and if the mechanism is apoptosis, then what does that do to our “developmental mechanisms” outlined at the beginning –del22q11.2 causes the largest proportion of flow lesions, but may be a problem in cell death See a shift in concepts of pathogenesis –From a physiological view (flow) –To a molecular view (defect in apoptosis)

25. 4-m.o. Female Infant –CHF from a Large VSD –Dysmorphic Appearance –Family History: Sib and Half-Sib with CHD –Mother with Multiple Psychiatric Admissions Case #1 Truncus Arteriosus TOFVSD

26. DiGeorge (not DiGeorge’s) Syndrome Features Include: –Cardiac: Conotruncal Defects –Immunologic: Thymic Aplasia or Hypoplasia –Hypocalcemia: Parathyroid Absence or Hypoplasia –Dysmorphism: Hypertelorism, Short Philtrum, Cupid’s Bow Mouth, Ear Anomalies DiGeorge Syndrome

27. Features Include: –Cardiac: VSD, Tetralogy of Fallot, Rt. Aortic Arch –Cleft Palate: Overt or Submucosal –Development Delay: Mild-to-Moderate, esp. Speech –Dysmorphisms: Prominent Nose, Abnormal Ears, Abundant Hair, Tapered Fingers VeloCardioFacial (VCF) Syndrome

28. VCF/DG SYNDROMES Clinical Overlap Cleft Palate Dev. Delay DGS Cleft Palate Dev. Delay VCF Facies CHD Dev. Delay VCF Facies

30. Deleted Chr 22 Normal Chr 22 FISH for del22q11.2 Detects 85-90% of patients

31. Molecular Cytogenetics Single nucleotide polymorphism microarrays (SNP chips) can determine copy number variations (CNVs) Billions of features to evaluate 2M SNPs on a chip

32. Constructing and Analyzing Microarrays Photolihography

33. DNA: Match vs. Mismatch Match Mismatch Hybridize Heat Double Stranded DNA Single Stranded DNA

34. Microarray: Match vs. Mismatch Match Mismatch Hybridize Heat Target DNA Captured Target DNA Lost Wash Labeled Target Attached Capture Probe

35. Microarray Analyses SNPs –AA, AB and BB Where A and B are A, T, G or C –Original use: genome-wide association studies (GWAS) –Software can determine if A/null or B/null Copy number variations (CNVs)

36. Options in Whole Genome Analysis Current genome sequencing –Complete Genomics (Mountain View, CA) Announced February 5, 2010 Beginning June 2010, offering sequencing at $5,000/genome (http://www.bloomberg.com/apps/news?sid=aEUlnq6ltPpQ&pid=20601124#) –NHGRI: Revolutionary Sequencing Technologies – The $1,000 Genome (R01) SNP (Single Nucleotide Polymorphism) Microarrays –Affymetrix 6.0 array >906,600 SNPs >946,000 Copy Number Probes (CNPS) Human Genome Project, 2003 First genome sequenced in 3 yrs At a cost of $2.5B

37. Whole Genome Analysis CNPs –202,000 probes targeting 5,677 known CNV regions –744,000 evenly spaced probes across the genome Overall –Average approximately 1probe/1,500 bp –Median inter-marker spacing of 696 bp http://www.sanger.ac.uk/humgen/cnv/data /

38. Whole Genome Data Is Acquired Patient below without any known genetic disease All chromosomes but Y represented

39. Multiple Patients with del22q11.2 Syndrome Show Similar Deletions in DGCR Five patients with del22q11.2 show similar 3 Mb TDR Not seen in five patients without del22q11.2 syndrome

40. Whole Genome Analysis Microarrays –Proxy for genomic sequencing –Learning to live with Uncertainty –Error vs. benign change –Correlations with clinical findings Huge volumes of data –Storage –Processing

41. DNA Hybridization as Nanotechnology SNP Microarrays or “SNP Chips” –NanoPediatrics core uses the Affymetrix platform –SNP “feature” is a 20-mer that will identify a specific SNP –If SNP present in person’s DNA, then form a double helix in the chip –The double helix shown here is made up of hybridized 20-mers Each 20-mer in the double helix configuration is 6.8 nm long with a diameter of 2 nm

42. Whole Genome Microarrays: Clinical Diagnostics UCLA –Medical Genetics using this technology clinically since 2006 –September 1, 2009 announced Personalized Genetics Medicine Center Joint venture between Pathology and Pediatrics Integrates laboratory diagnostic and genetic counseling services Important to gain this experience as we approach the clinical use of whole genome sequencing

43. Problems in Cell Migration: Patent Ductus Arteriosus (PDA) 1 in 2,000 Fullterm Infants 10% of CHD 2:1 Female to Male Ratio Multifactorial Etiology: Genes and Environment

44. Familial PDA 2-y.o. Palestinian Boy –Patent Ductus Arteriosus –Positive Family History PDA

45. Char Syndrome Described in 1978 by Florence Char, Univ of Arkansas Features –Patent Ductus Arteriosus (PDA) –Facial Dysmorphism –5th Finger Abnormalities Autosomal Dominant Inheritance –Complete Penetrance –Variable Expression

46. CHAR SYNDROME

47. 213456 12 1112 9 456 8131516171918312 71410 2425234615161719212315789101418202213 2346157 ARKANSAS CHAR KINDRED III IV V VI II I

48. Char Syndrome Disease Gene Discovery Linked to Chromosome 6p12 Candidate Gene Approach –Transcription Factor AP-2  Relevant for Neural Crest Development Missense Mutations in Several Unrelated Patients Dominant Negative Mechanism

49. Neural Crest Cell Migration and Cardiac Development

50. Cardiac Genetics Population Perspective Developing Innovative Therapies –Postnatal Interventions Marfan Syndrome: Anti-TGF  –Prenatal Interventions Folate Improving Clinical Trials Research –Cardiology Emulating Heme/Onc –Primary Endpoints - Function, Not Survival –Better Statistical Power

52. Summary: CHD Relatively common birth defect –4-8/1,000 live births Familial CHD –May not have identical anatomic abnormality Variety of developmental mechanisms –Undergoing revision as we understand molecular pathogenesis

53. CHD Recurrence risk –If familial, identify inheritance pattern –If not familial, use empiric risk data del22q11.2 is a common cause of CHD –Up to 25% of flow lesions –Flow lesions represent 50% of all CHD –Therefore, 12.5% of all CHD

54. Acknowledge （ PPT 特别鸣谢！） UCLA David Geffen School of Medicine www.medsch.ucla.edu/ANGEL/ Prof. McCabe E (Mattel Children’s Hospital UCLA; Dept of Pediatrics, Human Genetics, and Bioengineering, UCLA), et al.Prof. McCabe E (Mattel Children’s Hospital UCLA; Dept of Pediatrics, Human Genetics, and Bioengineering, UCLA), et al.