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Comprehensive genetic approaches to cleft lip/palate

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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Comprehensive genetic approaches to cleft lip/palate

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  1. Comprehensive genetic approaches to cleft lip/palate Jeff Murray jeff-murray@uiowa.edu

  2. 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

  3. 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)

  4. Cleft Lip Cleft Palate Non-syndromic 70% CLO and CL/P 1/1000 CPO 1/2500 Classic complex trait with genetic and environmental factors Varies by Geography/SES Gender Sidedness (L > R) Recurrences ~ 5% but vary by severity

  5. Cleft lip and palatea model for interdisciplinary care Surgery Pediatrics Dentistry Speech Psychology Nutrition Other specialties Cardiology etc

  6. 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

  7. Linkage Sib pair analysis Linkage in Families A/B A/C A/B *A/C A/C A/A *A/B A/C Identity by descent Identity by state Transmission disequilibrium test Association Study A1/A4 A2/A3 N families Transmitted allele Non- transmitted allele A1 A2 A3 A4 50 50 65 50 50 50 35 50 A1/A3 Healthy Control Disease affected

  8. 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

  9. Clinical Epidemiology and Biorepository: Denmark/Christensen Operation Smile Iowa >10,000 cases ~1200 familes (3 affecteds)

  10. 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, 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 A34G MSX1 E78V G91D Human Cow Mouse Rat Chicken Frog

  11. 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 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.

  12. FGFR FGF8 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

  13. FGFR1 - Syndromes can overlap with “non” syndromes • Nonsense mutation disrupts TK domain 1 • Haploinsufficiency • Loss-of-function • Proband = Kallmann Syndrome + CLP • Father = CLP

  14. 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

  15. 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

  16. Genome Wide Linkage – FOXE1 (Mansilla/Moreno/Marazita/Lidral/CIDR) • Multiple Populations 600 families, 4000 individuals

  17. Chromosome 9 http://www.ncbi.nih.gov Candidate Genes 9q22-q33 Region • 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

  18. 9q Fine Mapping by Association

  19. FOXE1 LD and Expression(~2000 trios p < 10-12 Expression in nasal prominences, Missense mutations in rare families, One LD block of 300Kb to search for the common variant All cleft phenotypes affected

  20. Van der Woude SyndromeMendel to Common Complex TraitSchutte/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)

  21. A VWS14 (E92X) VWS25 (FTSKLLD290L) PPS6 (R84C) G T A A T T C C T G C T G C 547 365 150 132 202 303 163 Cleft palate Cleft Lip 244 Lip Pits B DNA Binding SMIR/IAD R250Q Q273R V274I V297I K320E V321M G325E L345P C347F F369S C374W K388E D430N Truncation A2V V18M/A P39A *W60G A61G *K66T G70R *Q82K *R84C/H *N88H *K89E S90G D98H 1 2 3 4 5 6 7 8 9 10 w w w w w Missense FTSKLLD290L • 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

  22. 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

  23. Multispecies Comparison and AP2 site AP2-α

  24. V274I/rs642961(A/G) Associations 1039 Trios CasesControls Norway/Denmark 513 1245 Attributable Risk Cleft lip only 15.0

  25. 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

  26. Mouse Enhancer Assay (E11.5) Facial and branchial arch region Limb ectoderm Embryo at E11.5

  27. 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

  28. Genome Wide AssociationEnabled by “HapMap” Etiologic Variant 1 Site on Chromosome

  29. 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

  30. 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

  31. 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

  32. GWAS CL/P All Ancestries

  33. GWAS CL/P Population Specific 8q24 in Europeans IRF6/MAFB/ABCA4 in Asians

  34. Environment and CL/P • Smoking • Alcohol • Nutrition • Teratogens (e.g. phenytoin) • Geography • Social class

  35. G x E (16 detox genes and maternal smoking)Min ShiDenmark/Iowa (1500 cases/6000 samples)GSTT1 null in fetus + mother smokes > 15 cigs/day OR = 17 (p<0.001)GSTT1 null ~ 25% population

  36. 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

  37. Brazil recurrence prevention Folic acid: 400ug vs 4mg 16,000 case years randomized in two arms 1000 cases/ 150 births to date Secondary Outcomes

  38. 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

  39. 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)

  40. Acknowledgments Christensen, Marazita, Schutte, Lidral, Beaty, Lie Many Students Nurses, Genetic Counselors Patients and families Ann Marie and Ryan, Chris, Katie

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