Identification of functional variants for cleft lip with or without cleft palate in or near PAX7, FGFR2, and NOG by targeted sequencing of GWAS loci

Am J Hum Genet. 2015 Mar 5;96(3):397-411. doi: 10.1016/j.ajhg.2015.01.004. Epub 2015 Feb 19.

Abstract

Although genome-wide association studies (GWASs) for nonsyndromic orofacial clefts have identified multiple strongly associated regions, the causal variants are unknown. To address this, we selected 13 regions from GWASs and other studies, performed targeted sequencing in 1,409 Asian and European trios, and carried out a series of statistical and functional analyses. Within a cluster of strongly associated common variants near NOG, we found that one, rs227727, disrupts enhancer activity. We furthermore identified significant clusters of non-coding rare variants near NTN1 and NOG and found several rare coding variants likely to affect protein function, including four nonsense variants in ARHGAP29. We confirmed 48 de novo mutations and, based on best biological evidence available, chose two of these for functional assays. One mutation in PAX7 disrupted the DNA binding of the encoded transcription factor in an in vitro assay. The second, a non-coding mutation, disrupted the activity of a neural crest enhancer downstream of FGFR2 both in vitro and in vivo. This targeted sequencing study provides strong functional evidence implicating several specific variants as primary contributory risk alleles for nonsyndromic clefting in humans.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alleles
  • Amino Acid Sequence
  • Animals
  • Asian Continental Ancestry Group / genetics
  • Brain / abnormalities*
  • Carrier Proteins / genetics*
  • Carrier Proteins / metabolism
  • Cell Line
  • Cleft Lip / genetics*
  • Cleft Palate / genetics*
  • Epithelial Cells / metabolism
  • European Continental Ancestry Group / genetics
  • Gene Expression Regulation
  • Genetic Predisposition to Disease
  • Genome-Wide Association Study
  • Humans
  • Mice
  • Mice, Inbred C57BL
  • Molecular Sequence Data
  • Mutation, Missense
  • PAX7 Transcription Factor / genetics*
  • PAX7 Transcription Factor / metabolism
  • Polymorphism, Single Nucleotide*
  • Receptor, Fibroblast Growth Factor, Type 2 / genetics*
  • Receptor, Fibroblast Growth Factor, Type 2 / metabolism
  • Sequence Analysis, DNA
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Zebrafish / genetics

Substances

  • Carrier Proteins
  • PAX7 Transcription Factor
  • PAX7 protein, human
  • Transcription Factors
  • noggin protein
  • FGFR2 protein, human
  • Receptor, Fibroblast Growth Factor, Type 2

Supplementary concepts

  • Orofacial Cleft 1

Associated data

  • dbGaP/PHS000625.V1.P1