Genetic Determinants of Facial Clefting: Analysis of 357 Candidate Genes Using Two National Cleft Studies from Scandinavia

Background Facial clefts are common birth defects with a strong genetic component. To identify fetal genetic risk factors for clefting, 1536 SNPs in 357 candidate genes were genotyped in two population-based samples from Scandinavia (Norway: 562 case-parent and 592 control-parent triads; Denmark: 235 case-parent triads). Methodology/Principal Findings We used two complementary statistical methods, TRIMM and HAPLIN, to look for associations across these two national samples. TRIMM tests for association in each gene by using multi-SNP genotypes from case-parent triads directly without the need to infer haplotypes. HAPLIN on the other hand estimates the full haplotype distribution over a set of SNPs and estimates relative risks associated with each haplotype. For isolated cleft lip with or without cleft palate (I-CL/P), TRIMM and HAPLIN both identified significant associations with IRF6 and ADH1C in both populations, but only HAPLIN found an association with FGF12. For isolated cleft palate (I-CP), TRIMM found associations with ALX3, MKX, and PDGFC in both populations, but only the association with PDGFC was identified by HAPLIN. In addition, HAPLIN identified an association with ETV5 that was not detected by TRIMM. Conclusion/Significance Strong associations with seven genes were replicated in the Scandinavian samples and our approach effectively replicated the strongest previously known association in clefting—with IRF6. Based on two national cleft cohorts of similar ancestry, two robust statistical methods and a large panel of SNPs in the most promising cleft candidate genes to date, this study identified a previously unknown association with clefting for ADH1C and provides additional candidates and analytic approaches to advance the field.

[1]  R. T. Lie,et al.  A population-based study of the risk of recurrence of birth defects. , 1994, The New England journal of medicine.

[2]  Y. Lan,et al.  Identification and developmental expression analysis of a novel homeobox gene closely linked to the mouse Twirler mutation. , 2006, Gene expression patterns : GEP.

[3]  I. Welsh,et al.  A dosage-dependent role for Spry2 in growth and patterning during palate development , 2007, Mechanisms of Development.

[4]  D. Darling,et al.  Analysis of Zfhx1a mutant mice reveals palatal shelf contact-independent medial edge epithelial differentiation during palate fusion , 2008, Cell and Tissue Research.

[5]  C. Weinberg,et al.  Identification of risk-related haplotypes with the use of multiple SNPs from nuclear families. , 2007, American journal of human genetics.

[6]  J. Shendure,et al.  Characterization of apparently balanced chromosomal rearrangements from the developmental genome anatomy project. , 2008, American journal of human genetics.

[7]  P. Sulima,et al.  The PDGF-C regulatory region SNP rs28999109 decreases promoter transcriptional activity and is associated with CL/P , 2009, European Journal of Human Genetics.

[8]  Tx Station.  Stata Statistical Software: Release 7. , 2001 .

[9]  K. Christensen,et al.  Changing Lifestyles and Oral Clefts Occurrence in Denmark , 2005, The Cleft palate-craniofacial journal : official publication of the American Cleft Palate-Craniofacial Association.

[10]  J. Murray,et al.  Sequence evaluation of FGF and FGFR gene conserved non‐coding elements in non‐syndromic cleft lip and palate cases , 2007, American journal of medical genetics. Part A.

[11]  M. Marazita,et al.  Medical Sequencing of Candidate Genes for Nonsyndromic Cleft Lip and Palate , 2005, PLoS genetics.

[12]  A. Beverdam,et al.  Vertebrate aristaless-related genes. , 1999, The International journal of developmental biology.

[13]  S Holloway,et al.  A chromosomal duplication map of malformations: regions of suspected haplo- and triplolethality--and tolerance of segmental aneuploidy--in humans. , 1999, American journal of human genetics.

[14]  D. Huylebroeck,et al.  Complementary expression pattern of Zfhx1 genes Sip1 and δEF1 in the mouse embryo and their genetic interaction revealed by compound mutants , 2006, Developmental dynamics : an official publication of the American Association of Anatomists.

[15]  T. Cox,et al.  Expression profiles of cIRF6, cLHX6 and cLHX7 in the facial primordia suggest specific roles during primary palatogenesis , 2006, BMC Developmental Biology.

[16]  R. T. Lie,et al.  Folic acid supplements and risk of facial clefts: national population based case-control study , 2007, BDJ.

[17]  V. Diewert.  Development of human craniofacial morphology during the late embryonic and early fetal periods. , 1985, American journal of orthodontics.

[18]  R. T. Lie,et al.  Familial risk of oral clefts by morphological type and severity: population based cohort study of first degree relatives , 2008, BDJ.

[19]  E. Pugh,et al.  Genome Scan, Fine-Mapping, and Candidate Gene Analysis of Non-Syndromic Cleft Lip with or without Cleft Palate Reveals Phenotype-Specific Differences in Linkage and Association Results , 2009, Human Heredity.

[20]  David N. Messina,et al.  Gene expression in pharyngeal arch 1 during human embryonic development. , 2005, Human molecular genetics.

[21]  J. Ioannidis,et al.  Replication validity of genetic association studies , 2001, Nature Genetics.

[22]  D. Wyszynski.  Cleft lip and palate : from origin to treatment , 2002 .

[23]  B. Maher,et al.  Genome scan for loci involved in cleft lip with or without cleft palate, in Chinese multiplex families. , 2002, American journal of human genetics.

[24]  R. T. Lie,et al.  First-trimester maternal alcohol consumption and the risk of infant oral clefts in Norway: a population-based case-control study. , 2008, American journal of epidemiology.

[25]  Jeffrey C. Murray,et al.  Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes , 2002, Nature Genetics.

[26]  J R O'Connell,et al.  PedCheck: a program for identification of genotype incompatibilities in linkage analysis. , 1998, American journal of human genetics.

[27]  J. Guénet,et al.  Mouse Alx3: an aristaless-like homeobox gene expressed during embryogenesis in ectomesenchyme and lateral plate mesoderm. , 1998, Developmental biology.

[28]  E. Spjøtvoll,et al.  Plots of P-values to evaluate many tests simultaneously , 1982 .

[29]  G. Abecasis,et al.  Using haplotype blocks to map human complex trait loci. , 2003, Trends in genetics : TIG.

[30]  B. Maher,et al.  Impaired FGF signaling contributes to cleft lip and palate , 2007, Proceedings of the National Academy of Sciences.

[31]  M. Ehm,et al.  Detecting marker-disease association by testing for Hardy-Weinberg disequilibrium at a marker locus. , 1998, American journal of human genetics.

[32]  Jeffrey C Murray,et al.  Genetic approaches to identify disease genes for birth defects with cleft lip/palate as a model. , 2004, Birth defects research. Part A, Clinical and molecular teratology.

[33]  J. Mulliken,et al.  Association of chromosomal regions 3p21.2, 10p13, and 16p13.3 with nonsyndromic cleft lip and palate , 2004, American journal of medical genetics. Part A.

[34]  Jing Han,et al.  PDGF-C controls proliferation and is down-regulated by retinoic acid in mouse embryonic palatal mesenchymal cells. , 2006, Birth defects research. Part B, Developmental and reproductive toxicology.

[35]  L. Nelles,et al.  Mice lacking ZFHX1B, the gene that codes for Smad-interacting protein-1, reveal a role for multiple neural crest cell defects in the etiology of Hirschsprung disease-mental retardation syndrome. , 2003, American journal of human genetics.

[36]  T. Beaty,et al.  High throughput SNP and expression analyses of candidate genes for non-syndromic oral clefts , 2005, Journal of Medical Genetics.

[37]  Nicholas J Schork,et al.  Methods for handling multiple testing. , 2008, Advances in genetics.

[38]  H. Edenberg.  The Genetics of Alcohol Metabolism: Role of Alcohol Dehydrogenase and Aldehyde Dehydrogenase Variants , 2007, Alcohol research & health : the journal of the National Institute on Alcohol Abuse and Alcoholism.

[39]  Wen-Chung Lee.  Detecting population stratification using a panel of single nucleotide polymorphisms. , 2003, International journal of epidemiology.

[40]  Axel Visel,et al.  Disruption of an AP-2α binding site in an IRF6 enhancer is strongly associated with cleft lip , 2008, Nature Genetics.

[41]  A. Jugessur,et al.  Orofacial clefting: recent insights into a complex trait. , 2005, Current opinion in genetics & development.

[42]  Klaus Meyer,et al.  Oral facial clefts and gene polymorphisms in metabolism of folate/one‐carbon and vitamin A: a pathway‐wide association study , 2009, Genetic epidemiology.

[43]  A. S. Knight,et al.  Developmental expression analysis of the mouse and chick orthologues of IRF6: The gene mutated in Van der Woude syndrome , 2006, Developmental dynamics : an official publication of the American Association of Anatomists.

[44]  Rory A. Fisher,et al.  Statistical Methods for Research Workers. , 1956 .

[45]  Mark Daly,et al.  Haploview: analysis and visualization of LD and haplotype maps , 2005, Bioinform..

[46]  L. Garavelli,et al.  Mowat-Wilson syndrome , 2007, Orphanet journal of rare diseases.

[47]  H. Kondoh,et al.  DeltaEF1, a zinc finger and homeodomain transcription factor, is required for skeleton patterning in multiple lineages. , 1998, Development.

[48]  Philip Stanier,et al.  FGF signalling and SUMO modification: new players in the aetiology of cleft lip and/or palate. , 2007, Trends in genetics : TIG.

[49]  M. Melnick.  Cleft Lip and Palate: From Origin to Treatment. , 2003 .

[50]  A. Lidral,et al.  Progress toward discerning the genetics of cleft lip , 2005, Current opinion in pediatrics.

[51]  K. Henkel.  Cleft Lip and Palate from Origin to Treatment , 2004 .

[52]  E. Jabs,et al.  Genomic, cDNA and embryonic expression analysis of zebrafish IRF6, the gene mutated in the human oral clefting disorders Van der Woude and popliteal pterygium syndromes. , 2005, Gene Expression Patterns.

[53]  D. Beier,et al.  Genetic Mapping and ENU Mutagenesis , 2004, Genetica.

[54]  Jennifer H Barrett,et al.  Association studies. , 2002, Methods in molecular biology.

[55]  S Holloway,et al.  A chromosomal deletion map of human malformations. , 1998, American journal of human genetics.

[56]  H. Jörnvall,et al.  The gamma 1 and gamma 2 subunits of human liver alcohol dehydrogenase. cDNA structures, two amino acid replacements, and compatibility with changes in the enzymatic properties. , 1986, European journal of biochemistry.

[57]  M. McCarthy,et al.  Genome-wide association studies for complex traits: consensus, uncertainty and challenges , 2008, Nature Reviews Genetics.

[58]  M. Pisano,et al.  Developmental gene expression profiling of mammalian, fetal orofacial tissue. , 2004, Birth defects research. Part A, Clinical and molecular teratology.

[59]  J. Wilson-Rawls,et al.  Mohawk is a novel homeobox gene expressed in the developing mouse embryo , 2006, Developmental dynamics : an official publication of the American Association of Anatomists.

[60]  R. T. Lie,et al.  Case‐parent Triads: Estimating Single‐ and Double‐dose Effects of Fetal and Maternal Disease Gene Haplotypes , 2006, Annals of human genetics.

[61]  C. Betsholtz,et al.  A specific requirement for PDGF-C in palate formation and PDGFR-alpha signaling. , 2004, Nature genetics.

[62]  T. Beaty,et al.  Meta-analysis of 13 genome scans reveals multiple cleft lip/palate genes with novel loci on 9q21 and 2q32-35. , 2004, American journal of human genetics.

[63]  Jacqueline K. Wittke-Thompson,et al.  Rational inferences about departures from Hardy-Weinberg equilibrium. , 2005, American journal of human genetics.

[64]  C. Perret,et al.  Interaction between the ADH1C polymorphism and maternal alcohol intake in the risk of nonsyndromic oral clefts: an evaluation of the contribution of child and maternal genotypes. , 2005, Birth defects research. Part A, Clinical and molecular teratology.

[65]  K. Christensen,et al.  Complete sequencing shows a role for MSX1 in non-syndromic cleft lip and palate , 2003, Journal of medical genetics.

[66]  N Risch,et al.  The Future of Genetic Studies of Complex Human Diseases , 1996, Science.

[67]  Paola Sebastiani,et al.  Minimal haplotype tagging , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[68]  J. Mansell,et al.  Microarray analysis of murine palatogenesis: Temporal expression of genes during normal palate development , 2003, Development, growth & differentiation.

[69]  M. Goossens,et al.  Mowat-Wilson syndrome , 2003, Journal of Medical Genetics.

[70]  R. T. Lie,et al.  Genetic variants in IRF6 and the risk of facial clefts: single‐marker and haplotype‐based analyses in a population‐based case‐control study of facial clefts in Norway , 2008, Genetic epidemiology.

[71]  G. Chenevix-Trench,et al.  Evidence for an association between nonsyndromic cleft lip with or without cleft palate and a gene located on the long arm of chromosome 4. , 1995, American journal of human genetics.

[72]  T. Gong,et al.  Identification of Markers of the Midface , 2005, Journal of dental research.

[73]  Krishanu Mukherjee,et al.  Comprehensive Analysis of Animal TALE Homeobox Genes: New Conserved Motifs and Cases of Accelerated Evolution , 2007, Journal of Molecular Evolution.

[74]  CRISPLD2: a novel NSCLP candidate gene. , 2007, Human molecular genetics.

[75]  M. Kendall.  Statistical Methods for Research Workers , 1937, Nature.

[76]  K H Buetow,et al.  Association of genetic variation of the transforming growth factor-alpha gene with cleft lip and palate. , 1989, American journal of human genetics.

[77]  M. Lovett,et al.  Abnormal skin, limb and craniofacial morphogenesis in mice deficient for interferon regulatory factor 6 (Irf6) , 2006, Nature Genetics.

[78]  T. Foroud,et al.  Possible localization of a major gene for cleft lip and palate to 4q , 1994, Clinical genetics.

[79]  X. Nie,et al.  FGF signalling in craniofacial development and developmental disorders. , 2006, Oral diseases.

[80]  J. Korving,et al.  Severe nasal clefting and abnormal embryonic apoptosis in Alx3/Alx4 double mutant mice. , 2001, Development.

[81]  S. Gabriel,et al.  Efficiency and power in genetic association studies , 2005, Nature Genetics.

[82]  Michael J Dixon,et al.  Irf6 is a key determinant of the keratinocyte proliferation-differentiation switch , 2006, Nature Genetics.