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

Elizabeth J. Leslie, Margaret Anne Taub, Huan Liu, Karyn Meltz Steinberg, Daniel C. Koboldt, Qunyuan Zhang, Jenna C. Carlson, Jacqueline B. Hetmanski, Hang Wang, David E. Larson, Robert S. Fulton, Youssef A. Kousa, Walid D. Fakhouri, Ali Naji, Ingo Ruczinski, Ferdouse Begum, Margaret M. Parker, Tamara Busch, Jennifer Standley, Jennifer Rigdon & 14 others Jacqueline T. Hecht, Alan F Scott, George L. Wehby, Kaare Christensen, Andrew E. Czeizel, Frederic W B Deleyiannis, Brian C. Schutte, Richard K. Wilson, Robert A. Cornell, Andrew C. Lidral, George M. Weinstock, Terri L Beaty, Mary L. Marazita, Jeffrey C. Murray

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)397-411
Number of pages15
JournalAmerican Journal of Human Genetics
Volume96
Issue number3
DOIs
StatePublished - Mar 5 2015

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Cleft Lip
Genome-Wide Association Study
Cleft Palate
Mutation
Neural Crest
Transcription Factors
Alleles
DNA
Proteins
In Vitro Techniques

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)
  • Medicine(all)

Cite this

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. / Leslie, Elizabeth J.; Taub, Margaret Anne; Liu, Huan; Steinberg, Karyn Meltz; Koboldt, Daniel C.; Zhang, Qunyuan; Carlson, Jenna C.; Hetmanski, Jacqueline B.; Wang, Hang; Larson, David E.; Fulton, Robert S.; Kousa, Youssef A.; Fakhouri, Walid D.; Naji, Ali; Ruczinski, Ingo; Begum, Ferdouse; Parker, Margaret M.; Busch, Tamara; Standley, Jennifer; Rigdon, Jennifer; Hecht, Jacqueline T.; Scott, Alan F; Wehby, George L.; Christensen, Kaare; Czeizel, Andrew E.; Deleyiannis, Frederic W B; Schutte, Brian C.; Wilson, Richard K.; Cornell, Robert A.; Lidral, Andrew C.; Weinstock, George M.; Beaty, Terri L; Marazita, Mary L.; Murray, Jeffrey C.

In: American Journal of Human Genetics, Vol. 96, No. 3, 05.03.2015, p. 397-411.

Research output: Contribution to journalArticle

Leslie, EJ, Taub, MA, Liu, H, Steinberg, KM, Koboldt, DC, Zhang, Q, Carlson, JC, Hetmanski, JB, Wang, H, Larson, DE, Fulton, RS, Kousa, YA, Fakhouri, WD, Naji, A, Ruczinski, I, Begum, F, Parker, MM, Busch, T, Standley, J, Rigdon, J, Hecht, JT, Scott, AF, Wehby, GL, Christensen, K, Czeizel, AE, Deleyiannis, FWB, Schutte, BC, Wilson, RK, Cornell, RA, Lidral, AC, Weinstock, GM, Beaty, TL, Marazita, ML & Murray, JC 2015, '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', American Journal of Human Genetics, vol. 96, no. 3, pp. 397-411. https://doi.org/10.1016/j.ajhg.2015.01.004
Leslie, Elizabeth J. ; Taub, Margaret Anne ; Liu, Huan ; Steinberg, Karyn Meltz ; Koboldt, Daniel C. ; Zhang, Qunyuan ; Carlson, Jenna C. ; Hetmanski, Jacqueline B. ; Wang, Hang ; Larson, David E. ; Fulton, Robert S. ; Kousa, Youssef A. ; Fakhouri, Walid D. ; Naji, Ali ; Ruczinski, Ingo ; Begum, Ferdouse ; Parker, Margaret M. ; Busch, Tamara ; Standley, Jennifer ; Rigdon, Jennifer ; Hecht, Jacqueline T. ; Scott, Alan F ; Wehby, George L. ; Christensen, Kaare ; Czeizel, Andrew E. ; Deleyiannis, Frederic W B ; Schutte, Brian C. ; Wilson, Richard K. ; Cornell, Robert A. ; Lidral, Andrew C. ; Weinstock, George M. ; Beaty, Terri L ; Marazita, Mary L. ; Murray, Jeffrey C. / 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. In: American Journal of Human Genetics. 2015 ; Vol. 96, No. 3. pp. 397-411.
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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.",
author = "Leslie, {Elizabeth J.} and Taub, {Margaret Anne} and Huan Liu and Steinberg, {Karyn Meltz} and Koboldt, {Daniel C.} and Qunyuan Zhang and Carlson, {Jenna C.} and Hetmanski, {Jacqueline B.} and Hang Wang and Larson, {David E.} and Fulton, {Robert S.} and Kousa, {Youssef A.} and Fakhouri, {Walid D.} and Ali Naji and Ingo Ruczinski and Ferdouse Begum and Parker, {Margaret M.} and Tamara Busch and Jennifer Standley and Jennifer Rigdon and Hecht, {Jacqueline T.} and Scott, {Alan F} and Wehby, {George L.} and Kaare Christensen and Czeizel, {Andrew E.} and Deleyiannis, {Frederic W B} and Schutte, {Brian C.} and Wilson, {Richard K.} and Cornell, {Robert A.} and Lidral, {Andrew C.} and Weinstock, {George M.} and Beaty, {Terri L} and Marazita, {Mary L.} and Murray, {Jeffrey C.}",
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T1 - 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

AU - Leslie, Elizabeth J.

AU - Taub, Margaret Anne

AU - Liu, Huan

AU - Steinberg, Karyn Meltz

AU - Koboldt, Daniel C.

AU - Zhang, Qunyuan

AU - Carlson, Jenna C.

AU - Hetmanski, Jacqueline B.

AU - Wang, Hang

AU - Larson, David E.

AU - Fulton, Robert S.

AU - Kousa, Youssef A.

AU - Fakhouri, Walid D.

AU - Naji, Ali

AU - Ruczinski, Ingo

AU - Begum, Ferdouse

AU - Parker, Margaret M.

AU - Busch, Tamara

AU - Standley, Jennifer

AU - Rigdon, Jennifer

AU - Hecht, Jacqueline T.

AU - Scott, Alan F

AU - Wehby, George L.

AU - Christensen, Kaare

AU - Czeizel, Andrew E.

AU - Deleyiannis, Frederic W B

AU - Schutte, Brian C.

AU - Wilson, Richard K.

AU - Cornell, Robert A.

AU - Lidral, Andrew C.

AU - Weinstock, George M.

AU - Beaty, Terri L

AU - Marazita, Mary L.

AU - Murray, Jeffrey C.

PY - 2015/3/5

Y1 - 2015/3/5

N2 - 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.

AB - 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.

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