Genetics of Congenital Heart Disease

张咸宁zhangxianning@zju.edu.cn

Tel ： 13105819271; 88208367 Office: A709, Research Building

2011/03

Required ReadingThompson &Thompson Genetics

in Medicine, 7th 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

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

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

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

Introduction to CHD

• Variety of causes– Single gene– Chromosomal– Teratogen exposures

• Maternal rubella infection

• Gestational diabetes mellitus

• 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 DefectsEnvironmental Component

• 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 DefectsGenetic Component

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

Developmental Mechanisms

Flow Lesions

Problems in Cell Migration

Problems in Cell Death

Abnormalities in Extracellular Matrix

Abnormalities in Targeted Growth

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

Defect Pop

Incid (%)

Freq in Sibs (%)

λsib

VSD 0.17 4.3 25

PDA 0.083 3.2 38

ASD 0.066 3.2 48

AS 0.044 2.6 59

Is Isolated CHD a Multifactorial Trait?

• For these flow lesions– Sib relative risk ratio (λsib)

• 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

Flow LesionsFig 8-9

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

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

Unequal Crossing Over Due to Homologous Recombination

Fig 6-8

22q11.2 RearrangementsFig 6-9

del22q11.2 Syndromes

• Phenotypes may include – CHD– Craniofacial abnormalities– Mental retardation/developmental delay– Reduced circulating lymphocytes– Hypocalcemia– Schizophrenia

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

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

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

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

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)

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

TruncusArteriosus

TOF VSD

• 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

• 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

VCF/DG SYNDROMESClinical Overlap

Cleft PalateDev. Delay

DGS

Cleft PalateDev. DelayVCF Facies

CHDDev. DelayVCF Facies

DeletedChr 22

NormalChr 22

FISH fordel22q11.2

Detects 85-90%of patients

Molecular Cytogenetics

• Single nucleotide polymorphism microarrays (SNP chips) can determine copy number variations (CNVs)

• Billions of features to evaluate 2M SNPs on a chip

Constructing and Analyzing Microarrays

Photolihography

DNA: Match vs. Mismatch

Match

Mismatch

Hybridize

Hybridize

Heat

Heat

Double Stranded DNA

Single Stranded DNA

Microarray: Match vs. Mismatch

Match

Mismatch

Hybridize

Hybridize

Heat

Heat

Target DNACaptured

Target DNALost

Wash

Wash

Labeled Target

Attached Capture

Probe

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)

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, 2003First genome sequenced in 3 yrsAt a cost of $2.5B

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/

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

Multiple Patients with del22q11.2 SyndromeShow Similar Deletions in DGCR

Five patients with del22q11.2 show similar 3 Mb TDR Not seen in five patients without del22q11.2 syndrome

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

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

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

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

Familial PDA

• 2-y.o. Palestinian Boy– Patent Ductus Arteriosus

– Positive Family History

PDAPDA

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

CHAR SYNDROME

21 3 4 5 6

12

11

1 2 94 5 6 8 13 15 16 17 19183 12 7 1410

24 252 3 4 6 15 16 17 19 21 231 5 7 8 9 10 14 18 20 2213

2 3 4 61 5 7

ARKANSAS CHAR KINDRED

III

IV

V

VI

II

I

Char SyndromeDisease 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

Neural Crest Cell Migration and Cardiac Development

Cardiac GeneticsPopulation 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

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

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

Acknowledge （ PPT特别鸣谢！）

• UCLA David Geffen School of Medicine

• www.medsch.ucla.edu/ANGEL/

• Prof. McCabe E (Mattel Children’s Prof. McCabe E (Mattel Children’s Hospital UCLA; Dept of Pediatrics, Hospital UCLA; Dept of Pediatrics, Human Genetics, and Bioengineering, Human Genetics, and Bioengineering, UCLA), et al.UCLA), et al.