Comprehensive genetic approaches to
cleft lip/palate
Jeff Murray
jeff-murray@uiowa.edu
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
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Cleft lip and palate (1 in 700)
Congenital heart disease (1 in 200)
Neural tube defects (1 in 1000)
Preterm birth (1 in 8)
Cleft Lip
Varies by
Geography/SES
Gender
Sidedness (L > R)
Recurrences ~ 5% but
vary by severity
Cleft Palate
Non-syndromic
CLO and CL/P
CPO
70%
1/1000
1/2500
Classic complex trait with genetic
and environmental factors
Cleft lip and palate
a model for interdisciplinary care
Surgery
Pediatrics
Dentistry
Speech
Psychology
Nutrition
Other specialties
Cardiology etc
Approaches to gene finding for
complex diseases
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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
Linkage in Families
Sib pair analysis
A/B
A/C
A/B
*A/C
A/C
A/A
*A/B
A/C
Identity by descent
Transmission disequilibrium test
A1/A4
Identity by state
Association Study
A2/A3
N families
A1/A3
Transmitted
allele
Non- transmitted
allele
A1
50
50
A2
50
50
A3
65
35
A4
50
50
Healthy Control
Disease affected
Optimal Pediatric Study Design
Either 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
Vieira, 2005 MSX2, FOXE1, GLI2, JAG2
Mansilla, 2005 PTCH1
Teti, 2006 FOXE1
Alkuraya, 2006 SUMO1
Yoshiura, 2007 RYK
Riley, 2007 FGF8 and FGFR1
Oseogawa, 2008 FGFR2
Suzuki, 2009 BMP4
A23V
Human
Cow
Mouse
Rat
Chicken
Frog
A34G
MSX1 (2%)
MSX1
E78V
G91D
Microforms of CL/P and BMP4 mutations
(Phenotypic subtleties are important) (Marazita/Suzuki)
The arrow-head in the figure points to the
discontinuity in the OOM of the proband
Mouse with “healed” cleft lip in
tissue specific KO
8/1100 microforms/CLP
0/530 Controls
p = 0.01
Lip prints, dental, cognitive
Child (L)has left microform cleft lip and cleft palate. Father (R) has subtle right
microform cleft lip.
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
FGFR
FGF8
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
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1800 cleft cases, 5600 mothers, 2300 sisters
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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
• 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
http://www.ncbi.nih.gov
9q Fine Mapping by Association
FOXE1 LD and Expression
(~2000 trios
Marker
rs3758249 (13% O.T.)
rs4460498 (16%O.T.)
p < 10-12
OR (95%CI)
1.41
1.34
Expression in nasal prominences,
Missense mutations in rare families,
One LD block of 300Kb to search for the common
variant
All cleft phenotypes affected
(1.26-1.56)
(1.21-1.49)
Van der Woude Syndrome
Mendel 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)
A
VWS14
(E92X)
VWS25
(FTSKLLD290L)
PPS6
(R84C)
G A A T T C
T
C
547
T G C G C
T
365
150
132
303
244
Cleft palate
202
163
Cleft Lip
Lip Pits
B
DNA Binding
SMIR/IAD
Truncation
3
4
w ww
5
6
7
8
9
10
ww
A2V
V18M/A
P39A
*W60G
A61G
*K66T
G70R
*Q82K
*R84C/H
*N88H
*K89E
S90G
D98H
Missense
D430N
2
R250Q
Q273R
V274I
FTSKLLD290L
V297I
K320E
V321M
G325E
L345P
C347F
F369S
C374W
K388E
1
•Mutations in 210/300 independent VWS families ( 70%)
80 Truncations
120 Missense mutations
• Allelic disorder Popliteal Pterygium R84C
5 Deletions
5 splicing mutations
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
Marker
h1
h2
h3
h4
Allele
V-G
V-A
I-G
I-A
afreq
0.61
0.27
0.11
0.00
fam#
286
458.9
220.8
3.3
P value
0.7
3 x 10-8
0.000031 (-Z)
Cases
513
Norway/Denmark
1039 Trios
Controls
1245
P - value
Cleft type (N)
Freq
OR
95% CI
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
rs642961 disrupts AP2-α binding site
Human recombinant AP2-α
incubated with oligo probes
G allele binds to TF AP2-α
A allele cannot bind to AP2-α
Mouse Enhancer Assay (E11.5)
Facial and branchial
arch region
Limb ectoderm
Embryo at E11.5
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 Association
Enabled by “HapMap”
Etiologic Variant
1 Site on Chromosome
GWAS by Birnbaum et al, 2009 on CLP
Grant 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
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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)
Acknowledgments
Christensen, Marazita, Schutte, Lidral, Beaty, Lie
Many Students
Nurses, Genetic Counselors
Patients and families
Ann Marie and Ryan, Chris, Katie