comprehensive genetic approaches to cleft lip/palate jeff murray [email protected]
TRANSCRIPT
Disclosure Statement
• Our laboratory is funded in part by support from the NIDCR
• There will be no discussion or endorsement of any products in this presentation
Common, Complex Disorders of the Newborn
• Cleft lip and palate (1 in 700)• Congenital heart disease (1 in 200)• Neural tube defects (1 in 1000)• Preterm birth (1 in 8)
Non-syndromic 70% CLO and CL/P 1/1000 CPO 1/2500
Classic complex trait with genetic and environmental factors
Cleft Lip Cleft Palate
Varies by
Geography/SES
Gender
Sidedness (L > R)
Recurrences ~ 5% but vary by severity
Cleft lip and palatea model for interdisciplinary care
Surgery
Pediatrics
Dentistry
Speech
Psychology
Nutrition
Other specialties
Cardiology etc
Approaches to gene finding for complex diseases
• Epidemiology/Phenotypes/Biorepositories
• Candidate genes (Sequencing/Association)
• Location (Chromosomal/Linkage)
• Single gene models
• Genome Wide Association
• Genome Wide Sequencing
• Single case to cohorts of 100,000
Linkage in Families Linkage
Sib pair analysis
Identity by descent Identity by state
A/B A/B
*A/B A/C
A/C *A/C
A/C A/A
Transmission disequilibrium test
A1/A4 A2/A3
A1/A3
Transmitted allele
Non- transmitted allele
A1
A2
A3
A4
50
50
65
50
50
50
35
50
N families
Association Study
Healthy Control
Disease affected
Optimal Pediatric Study DesignEither infant or mother may be risk case
Allows for case/control, association, linkage, assessment of maternal effects
Collect bio-samples and phenotypes
Clinical Epidemiology and Biorepository:
Denmark/Christensen
Operation Smile
Iowa
>10,000 cases
~1200 familes (3 affecteds)
Sequence Evaluation of CL/P(Vieira, Riley, da Silva, Mansilla)
~200 cases for sequencing (70 genes to date)
Van den Boogaard, 2000; Jezewski, 2003 MSX1 (2%)Vieira, 2005 MSX2, FOXE1, GLI2, JAG2Mansilla, 2005 PTCH1Teti, 2006 FOXE1Alkuraya, 2006 SUMO1Yoshiura, 2007 RYKRiley, 2007 FGF8 and FGFR1Oseogawa, 2008 FGFR2Suzuki, 2009 BMP4
HumanCowMouseRatChickenFrog
A23V A34G E78V G91DMSX1
The arrow-head in the figure points to the discontinuity in the OOM of the proband
Child (L)has left microform cleft lip and cleft palate. Father (R) has subtle right microform cleft lip.
Microforms of CL/P and BMP4 mutations
(Phenotypic subtleties are important) (Marazita/Suzuki)
Mouse with “healed” cleft lip in tissue specific KO
8/1100 microforms/CLP
0/530 Controls p = 0.01
Lip prints, dental, cognitive
FGF8 - D73H (Riley) De novo mutations are highly suspect First example of a mutation in FGF8 in human disease Loss-of-function
Eliminates hydrogen bonding with FGFRs and destabilizes conformation of N-terminus
FGFRFGF8
FGFR1 - Syndromes can overlap with “non” syndromes
Nonsense mutation disrupts TK domain 1 Haploinsufficiency Loss-of-function
Proband = Kallmann Syndrome + CLP Father = CLP
FGFR2, Clefts and Breast Ca(Riley, Oseogawa, Bille)
FGFR2 point mutations in coding Deletion of FGFR2 in two generation CLP family Bille et al clefts associated with Breast and Lung Ca
FGFR2, Clefts and Breast Ca(Dietz, Grosen, Christensen, Erickson)
Association of FGFR2 with Breast Ca in GWAS Breast Ca and CLP association for same SNPs -
Beth Ericson p ~ 0.0001 Danish data from Statistics Denmark 1800 cleft cases, 5600 mothers, 2300 sisters 60,000-140,000 controls
Genome Wide Linkage – FOXE1 (Mansilla/Moreno/Marazita/Lidral/CIDR)
• Multiple Populations 600 families, 4000 individuals
Candidate Genes 9q22-q33 Region
Chromosome 9
http://www.ncbi.nih.gov
• 50 Mb region - 104 genes, predicted genes and ORF
• 8 strong candidates
ROR2 - Robinow syndrome, BDB1
PTCH - Gorlin Syndrome
FOXE1 - transcription factor, Bamforth-Lazarus Syndrome
TGFBR1 - growth factor receptor
9q Fine Mapping by Association
FOXE1 LD and Expression (~2000 trios p < 10-12
Marker OR (95%CI)
rs3758249 (13% O.T.) 1.41 (1.26-1.56)
rs4460498 (16%O.T.) 1.34 (1.21-1.49)
Expression in nasal prominences,
Missense mutations in rare families,
One LD block of 300Kb to search for the common variant
All cleft phenotypes affected
Van der Woude SyndromeMendel to Common Complex Trait
Schutte/Rahimov/Marazita/Leslie• Autosomal dominant• Phenotype
– Lip pits (85%)– Cleft lip – Cleft palate
• Deletion/linkage/microdeletion• MZ Twins in mutation detection• IRF6 (Interferon Regulatory Factor
6) gene • Mouse KO with clefts
(Dixon/Schutte)
SMIR/IAD
1 2 5 6 10ww
DNA Binding
A2
VV
18
M/A
P3
9A
*W6
0G
A6
1G
*K6
6T
G7
0R
*Q8
2K
*R8
4C
/H*N
88
H*K
89
ES
90
GD
98
H
Missense
TruncationR
25
0Q
Q2
73
RV
27
4I
V2
97
IK
32
0E
V3
21
MG
32
5E
L3
45
PC
34
7F
F3
69
SC
37
4W
K3
88
E
D4
30
N
FT
SK
LL
D2
90
L
4 7 8 9
365
202163
PPS6(R84C)
547
303244
VWS14(E92X)
A A T T CGT
A
B
VWS25(FTSKLLD290L)
132150
Lip PitsCleft Lip Cleft palate
C T CT
G CG
w ww
3
•Mutations in 210/300 independent VWS families ( 70%)
80 Truncations 120 Missense mutations 5 Deletions 5 splicing mutations
• Allelic disorder Popliteal Pterygium R84C One SNP that changes amino acid - V274I
IRF6 - Isolated CL/P
Similarity of phenotype of VWS and isolated clefts
V274I variant in 8000 individuals (1900 families)
Significant overtransmission (p<10-14)
Replicated in eight studies
Initial 12% Attributable Risk
Causal mutation likely in 140Kb LD block
Multispecies Comparison and AP2 site
AP2-α
V274I/rs642961(A/G) Associations
Cases Controls
Norway/Denmark 513 1245Cleft type (N) Freq OR 95% CI P - value
CL/P and CPO (513) 0.27 1.29 1.09-1.52 0.0032
CL/P (368) 0.30 1.52 1.26-1.82 <0.0001
CLP (221) 0.26 1.24 0.98-1.56 0.0752
CL (147) 0.36 2.01 1.56-2.59 <0.0001
CPO (145) 0.18 0.79 0.57-1.07 0.1284
Control (1245) 0.22
Attributable Risk Cleft lip only 15.0
Marker Allele afreq fam# P valueh1 V-G 0.61 286 0.7
h2 V-A 0.27 458.9 3 x 10-8
h3 I-G 0.11 220.8 0.000031 (-Z)h4 I-A 0.00 3.3
1039 Trios
rs642961 disrupts AP2-α binding site
Human recombinant AP2-αincubated with oligo probes
G allele binds to TF AP2-α
A allele cannot bind to AP2-α
Mouse Enhancer Assay (E11.5)
Embryo at E11.5
Facial and branchial
arch region
Limb ectoderm
IRF6 as cause of common clefts
• Disrupts the central dogma of clefting that cleft lip only and cleft lip/palate one entity
• “A” allele is additive in effect with AG ~ 1.7x and AA 2.4x increased risks
• AP2 binding site mutation as etiologic and AP2 and IRF6 in same developmental path
• Suggests a second common variant in South Asian populations
Genome Wide AssociationEnabled by “HapMap”
Etiologic Variant
1 Site on Chromosome
GWAS by Birnbaum et al, 2009 on CLPGrant et al, 2009 replication
Modest sizes suggest large effects
(250 cases and 400 controls)
One highly significant new locus at 8q24
8q24 replicates in Europeans but not in Asians
+ in Iowa, Denmark, Norwary
- in Philippines, Japan, Mongolia
GWAS by Beaty et al (CIDR in progress)(Christensen, Doheny, Lie, Marazita, Munger, Murray)
GENEVA consortium (14 sites)
dbGaP
2200 families and >7000 samples
Case parent trios with environmental exposure data
Preliminary data supports 8q24 in Europeans
Incidental findings issues
Caveats on GWAS
GWAS ideal when a common allele exists and works in a “hypothesis free” environment BUT
Population heterogeneity can mask positives
In the presence of allelic heterogeneity family studies and/or “complete” sequencing are needed
Need to incorporate the environment both as study variable and readily modifiable risk factor
GWAS CL/P All Ancestries
GWAS CL/P Population Specific
8q24 in Europeans
IRF6/MAFB/ABCA4 in Asians
Environment and CL/P
• Smoking• Alcohol• Nutrition• Teratogens (e.g.
phenytoin)• Geography• Social class
G x E (16 detox genes and maternal smoking)Min Shi
Denmark/Iowa (1500 cases/6000 samples)
GSTT1 null in fetus + mother smokes > 15 cigs/day
OR = 17 (p<0.001)GSTT1 null ~ 25% population
Forays into Clinical Trials
South American Sites
10 countries/100 hospitals
ECLAMC
Wehby/Castilla/Moretti-Ferriera/Felix
1. One month mortality
2. 2 year outcomes
3. Folate recurrence prevention trial
Brazil recurrence prevention
Folic acid: 400ug vs 4mg
16,000 case years randomized in two arms
1000 cases/ 150 births to date
Secondary Outcomes
Cleft Summary
Modest candidate gene successes by sequencing (MSX1, BMP4)
Modest linkage successes (FOXE1)
Candidate association success (IRF6)
Hiints of gene/environment interactions (GSTT1)
Good GWAS success (8q24, MAFB, ABCA4)
Little clinical impact yet of genetics
Cleft Future
Connections to outcomes - Breast Cancer
Connections to outcomes - Mental Health
Prevention Trials – Folate
Denmark gene/environment/subphenotype/outcomes
Microdeletions at genome scale
Mouse/Fish models (Schutte, Dixon, Cornell)
AcknowledgmentsChristensen, Marazita, Schutte, Lidral, Beaty, Lie
Many Students
Nurses, Genetic Counselors
Patients and families
Ann Marie and Ryan, Chris, Katie