24 results on '"Ishorst N"'
Search Results
2. Identification of de novo variants in nonsyndromic cleft lip with/without cleft palate patients with low polygenic risk scores.
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Ishorst, N., Henschel, L., Thieme, F., Drichel, D., Sivalingam, S., Mehrem, S.L., Fechtner, A.C., Fazaal, J., Welzenbach, J., Heimbach, A., Maj, C., Borisov, O., Hausen, J., Raff, R., Hoischen, A., Dixon, M., Rada-Iglesias, A., Bartusel, M., Rojas-Martinez, A., Aldhorae, K., Braumann, B., Kruse, T., Kirschneck, C., Spanier, G., Reutter, H., Nowak, S., Gölz, L., Knapp, M., Buness, A., Krawitz, P., Nöthen, M.M., Nothnagel, M., Becker, T., Ludwig, K.U., Mangold, E., Ishorst, N., Henschel, L., Thieme, F., Drichel, D., Sivalingam, S., Mehrem, S.L., Fechtner, A.C., Fazaal, J., Welzenbach, J., Heimbach, A., Maj, C., Borisov, O., Hausen, J., Raff, R., Hoischen, A., Dixon, M., Rada-Iglesias, A., Bartusel, M., Rojas-Martinez, A., Aldhorae, K., Braumann, B., Kruse, T., Kirschneck, C., Spanier, G., Reutter, H., Nowak, S., Gölz, L., Knapp, M., Buness, A., Krawitz, P., Nöthen, M.M., Nothnagel, M., Becker, T., Ludwig, K.U., and Mangold, E.
- Abstract
Item does not contain fulltext, BACKGROUND: Nonsyndromic cleft lip with/without cleft palate (nsCL/P) is a congenital malformation of multifactorial etiology. Research has identified >40 genome-wide significant risk loci, which explain less than 40% of nsCL/P heritability. Studies show that some of the hidden heritability is explained by rare penetrant variants. METHODS: To identify new candidate genes, we searched for highly penetrant de novo variants (DNVs) in 50 nsCL/P patient/parent-trios with a low polygenic risk for the phenotype (discovery). We prioritized DNV-carrying candidate genes from the discovery for resequencing in independent cohorts of 1010 nsCL/P patients of diverse ethnicities and 1574 population-matched controls (replication). Segregation analyses and rare variant association in the replication cohort, in combination with additional data (genome-wide association data, expression, protein-protein-interactions), were used for final prioritization. CONCLUSION: In the discovery step, 60 DNVs were identified in 60 genes, including a variant in the established nsCL/P risk gene CDH1. Re-sequencing of 32 prioritized genes led to the identification of 373 rare, likely pathogenic variants. Finally, MDN1 and PAXIP1 were prioritized as top candidates. Our findings demonstrate that DNV detection, including polygenic risk score analysis, is a powerful tool for identifying nsCL/P candidate genes, which can also be applied to other multifactorial congenital malformations.
- Published
- 2023
3. MiRNA-149 as a Candidate for Facial Clefting and Neural Crest Cell Migration
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Stuessel, L. G., Hollstein, R., Laugsch, M., Hochfeld, L. M., Welzenbach, J., Schroeder, J., Thieme, F., Ishorst, N., Romero, R. Olmos, Weinhold, L., Hess, T., Gehlen, J., Mostowska, A., Heilmann-Heimbach, S., Mangold, E., Rada-Iglesias, A., Knapp, M., Schaaf, C. P., Ludwig, K. U., Stuessel, L. G., Hollstein, R., Laugsch, M., Hochfeld, L. M., Welzenbach, J., Schroeder, J., Thieme, F., Ishorst, N., Romero, R. Olmos, Weinhold, L., Hess, T., Gehlen, J., Mostowska, A., Heilmann-Heimbach, S., Mangold, E., Rada-Iglesias, A., Knapp, M., Schaaf, C. P., and Ludwig, K. U.
- Abstract
Nonsyndromic cleft lip with or without palate (nsCL/P) ranks among the most common human birth defects and has a multifactorial etiology. Human neural crest cells (hNCC) make a substantial contribution to the formation of facial bone and cartilage and are a key cell type in terms of nsCL/P etiology. Based on increasing evidence for the role of noncoding regulatory mechanisms in nsCL/P, we investigated the role of hNCC-expressed microRNAs (miRNA) in cleft development. First, we conducted a systematic analysis of miRNAs expressed in human-induced pluripotent stem cell-derived hNCC using Affymetrix microarrays on cell lines established from 4 unaffected donors. These analyses identified 152 candidate miRNAs. Based on the hypothesis that candidate miRNA loci harbor genetic variation associated with nsCL/P risk, the genomic locations of these candidates were cross-referenced with data from a previous genome-wide association study of nsCL/P. Associated variants were reanalyzed in independent nsCL/P study populations. Jointly, the results suggest that miR-149 is implicated in nsCL/P etiology. Second, functional follow-up included in vitro overexpression and inhibition of miR-149 in hNCC and subsequent analyses at the molecular and phenotypic level. Using 3 ' RNA-Seq, we identified 604 differentially expressed (DE) genes in hNCC overexpressing miR-149 compared with untreated cells. These included TLR4 and JUNB, which are established targets of miR-149, and NOG, BMP4, and PAX6, which are reported nsCL/P candidate genes. Pathway analyses revealed that DE genes were enriched in pathways including regulation of cartilage development and NCC differentiation. At the cellular level, distinct hNCC migration patterns were observed in response to miR-149 overexpression. Our data suggest that miR-149 is involved in the etiology of nsCL/P via its role in hNCC migration.
- Published
- 2022
4. Extending the allelic spectrum at noncoding risk loci of orofacial clefting
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Thieme, F., Henschel, L., Hammond, N.L., Ishorst, N., Hausen, J., Adamson, A.D., Biedermann, A., Bowes, J., Zieger, H.K., Maj, C., Kruse, T., Buness, A., Hoischen, A., Gilissen, C., Kreusch, T., Jäger, A., Gölz, L., Braumann, B., Aldhorae, K., Rojas-Martinez, A., Krawitz, P.M., Mangold, E., Dixon, M.J., Ludwig, K.U., Thieme, F., Henschel, L., Hammond, N.L., Ishorst, N., Hausen, J., Adamson, A.D., Biedermann, A., Bowes, J., Zieger, H.K., Maj, C., Kruse, T., Buness, A., Hoischen, A., Gilissen, C., Kreusch, T., Jäger, A., Gölz, L., Braumann, B., Aldhorae, K., Rojas-Martinez, A., Krawitz, P.M., Mangold, E., Dixon, M.J., and Ludwig, K.U.
- Abstract
Contains fulltext : 237830.pdf (Publisher’s version ) (Open Access), Genome-wide association studies (GWAS) have generated unprecedented insights into the genetic etiology of orofacial clefting (OFC). The moderate effect sizes of associated noncoding risk variants and limited access to disease-relevant tissue represent considerable challenges for biological interpretation of genetic findings. As rare variants with stronger effect sizes are likely to also contribute to OFC, an alternative approach to delineate pathogenic mechanisms is to identify private mutations and/or an increased burden of rare variants in associated regions. This report describes a framework for targeted resequencing at selected noncoding risk loci contributing to nonsyndromic cleft lip with/without cleft palate (nsCL/P), the most frequent OFC subtype. Based on GWAS data, we selected three risk loci and identified candidate regulatory regions (CRRs) through the integration of credible SNP information, epigenetic data from relevant cells/tissues, and conservation scores. The CRRs (total 57 kb) were resequenced in a multiethnic study population (1061 patients; 1591 controls), using single-molecule molecular inversion probe technology. Combining evidence from in silico variant annotation, pedigree- and burden analyses, we identified 16 likely deleterious rare variants that represent new candidates for functional studies in nsCL/P. Our framework is scalable and represents a promising approach to the investigation of additional congenital malformations with multifactorial etiology.
- Published
- 2021
5. Nonsyndromic orofacial clefts - Identifying putative causative genes by CNV analysis of whole exome sequencing data
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Hoelzel, S., Ishorst, N., Greve, C., Degenhardt, F., Drichel, D., Maj, C., Nothnagel, M., Hehir-Kwa, J. Y., Veltman, J. A., Kruse, T., Reutter, H., Nowak, S., Carels, C., van Rooij, I., Ludwig, K. U., Mangold, E., Hoelzel, S., Ishorst, N., Greve, C., Degenhardt, F., Drichel, D., Maj, C., Nothnagel, M., Hehir-Kwa, J. Y., Veltman, J. A., Kruse, T., Reutter, H., Nowak, S., Carels, C., van Rooij, I., Ludwig, K. U., and Mangold, E.
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- 2020
6. Deletions and loss-of-function variants in TP63 associated with orofacial clefting
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Khandelwal, K., Boogaard, M. van den, Mehrem, S.L., Gebel, J., Fagerberg, C., Beusekom, E. van, Binsbergen, E. van, Topaloglu, O., Steehouwer, M., Gilissen, C., Ishorst, N., Rooij, I.A.L.M. van, Roeleveld, N., Christensen, K., Schoenaers, J., Berge, S.J., Murray, J.C., Hens, G., Devriendt, K., Ludwig, K.U., Mangold, E., Hoischen, A., Zhou, H., Dotsch, V., Carels, C.E.L., Bokhoven, H. van, Khandelwal, K., Boogaard, M. van den, Mehrem, S.L., Gebel, J., Fagerberg, C., Beusekom, E. van, Binsbergen, E. van, Topaloglu, O., Steehouwer, M., Gilissen, C., Ishorst, N., Rooij, I.A.L.M. van, Roeleveld, N., Christensen, K., Schoenaers, J., Berge, S.J., Murray, J.C., Hens, G., Devriendt, K., Ludwig, K.U., Mangold, E., Hoischen, A., Zhou, H., Dotsch, V., Carels, C.E.L., and Bokhoven, H. van
- Abstract
Contains fulltext : 204872.pdf (publisher's version ) (Closed access), We aimed to identify novel deletions and variants of TP63 associated with orofacial clefting (OFC). Copy number variants were assessed in three OFC families using microarray analysis. Subsequently, we analyzed TP63 in a cohort of 1072 individuals affected with OFC and 706 population-based controls using molecular inversion probes (MIPs). We identified partial deletions of TP63 in individuals from three families affected with OFC. In the OFC cohort, we identified several TP63 variants predicting to cause loss-of-function alleles, including a frameshift variant c.569_576del (p.(Ala190Aspfs*5)) and a nonsense variant c.997C>T (p.(Gln333*)) that introduces a premature stop codon in the DNA-binding domain. In addition, we identified the first missense variants in the oligomerization domain c.1213G>A (p.(Val405Met)), which occurred in individuals with OFC. This variant was shown to abrogate oligomerization of mutant p63 protein into oligomeric complexes, and therefore likely represents a loss-of-function allele rather than a dominant-negative. All of these variants were inherited from an unaffected parent, suggesting reduced penetrance of such loss-of-function alleles. Our data indicate that loss-of-function alleles in TP63 can also give rise to OFC as the main phenotype. We have uncovered the dosage-dependent functions of p63, which were previously rejected.
- Published
- 2019
7. Non-Syndromic Cleft Lip with or without Cleft Palate: Genome-Wide Association Study in Europeans Identifies a Suggestive Risk Locus at 16p12.1 and Supports SH3PXD2A as a Clefting Susceptibility Gene
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Rooij, I.A.L.M. van, Ludwig, K.U., Welzenbach, J., Ishorst, N., Thonissen, M., Galesloot, T.E., Ongkosuwito, E.M., Berge, S.J., Aldhorae, K., Rojas-Martinez, A., Kiemeney, L.A., Vermeesch, J.R., Brunner, H.G., Roeleveld, N., Devriendt, K., Dormaar, T., Hens, G., Knapp, M., Carels, C.E., Mangold, E., Rooij, I.A.L.M. van, Ludwig, K.U., Welzenbach, J., Ishorst, N., Thonissen, M., Galesloot, T.E., Ongkosuwito, E.M., Berge, S.J., Aldhorae, K., Rojas-Martinez, A., Kiemeney, L.A., Vermeesch, J.R., Brunner, H.G., Roeleveld, N., Devriendt, K., Dormaar, T., Hens, G., Knapp, M., Carels, C.E., and Mangold, E.
- Abstract
Contains fulltext : 215284.pdf (publisher's version ) (Open Access), Non-syndromic cleft lip with or without cleft palate (nsCL/P) ranks among the most common human congenital malformations, and has a multifactorial background in which both exogenous and genetic risk factors act in concert. The present report describes a genome-wide association study (GWAS) involving a total of 285 nsCL/P patients and 1212 controls from the Netherlands and Belgium. Twenty of the 40 previously reported nsC/LP susceptibility loci were replicated, which underlined the validity of this sample. SNV-based analysis of the data identified an as yet unreported suggestive locus at chromosome 16p12.1 (p-value of the lead SNV: 4.17 x 10(-7)). This association was replicated in two of three patient/control replication series (Central European and Yemeni). Gene analysis of the GWAS data prioritized SH3PXD2A at chromosome 10q24.33 as a candidate gene for nsCL/P. To date, support for this gene as a cleft gene has been restricted to data from zebrafish and a knockout mouse model. The present GWAS was the first to implicate SH3PXD2A in non-syndromic cleft formation in humans. In summary, although performed in a relatively small sample, the present GWAS generated novel insights into nsCL/P etiology.
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- 2019
8. Comprehensive analyses of genome-wide data reveal novel insights into distinct etiologies of cleft lip with/without celft palate, and cleft palate only
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Ludwig, K. U., Boehmer, A. C., Bowes, J., Nikolic, M., Ishorst, N., Wyatt, N., Hammond, N., Goelz, L., Thieme, F., Spielmann, M., Aldhorae, K., Rojas-Martinez, A., Noethen, M. M., Rada-Iglesias, A., Dixon, M. J., Knapp, M., Mangold, E., Ludwig, K. U., Boehmer, A. C., Bowes, J., Nikolic, M., Ishorst, N., Wyatt, N., Hammond, N., Goelz, L., Thieme, F., Spielmann, M., Aldhorae, K., Rojas-Martinez, A., Noethen, M. M., Rada-Iglesias, A., Dixon, M. J., Knapp, M., and Mangold, E.
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- 2018
9. Novel irf6 mutations detected in orofacial cleft patients by targeted massively parallel sequencing
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Khandelwal, K., Ishorst, N., Zhou, H., Ludwig, K.U., Venselaar, H., Gilissen, C.F., Thonissen, M., Rooij, I.A.L.M. van, Dreesen, K., Steehouwer, M., Vorst, J.M. van de, Bloemen, M., Beusekom, E. van, Roosenboom, J., Borstlap, W.A., Admiraal, R.J., Dormaar, T., Schoenaers, J., Poorten, V. Van der, Hens, G., Verdonck, A., Berge, S.J., Roeleveld, N., Vriend, G., Devriendt, K., Brunner, H.G., Mangold, E., Hoischen, A., Bokhoven, H. van, Carels, C.E.L., Khandelwal, K., Ishorst, N., Zhou, H., Ludwig, K.U., Venselaar, H., Gilissen, C.F., Thonissen, M., Rooij, I.A.L.M. van, Dreesen, K., Steehouwer, M., Vorst, J.M. van de, Bloemen, M., Beusekom, E. van, Roosenboom, J., Borstlap, W.A., Admiraal, R.J., Dormaar, T., Schoenaers, J., Poorten, V. Van der, Hens, G., Verdonck, A., Berge, S.J., Roeleveld, N., Vriend, G., Devriendt, K., Brunner, H.G., Mangold, E., Hoischen, A., Bokhoven, H. van, and Carels, C.E.L.
- Abstract
Contains fulltext : 191986.pdf (publisher's version ) (Closed access)
- Published
- 2017
10. Candidate Genes for Nonsyndromic Cleft Palate Detected by Exome Sequencing
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Hoebel, A.K., Drichel, D., Vorst, M. van de, Bohmer, A.C., Sivalingam, S., Ishorst, N., Klamt, J., Golz, L., Alblas, M., Maaser, A., Keppler, K., Zink, A.M., Dixon, M.J., Dixon, J., Hemprich, A., Kruse, T., Graf, I., Dunsche, A., Schmidt, G, Daratsianos, N., Nowak, S., Aldhorae, K.A., Nothen, M.M., Knapp, M., Thiele, H., Gilissen, C., Reutter, H., Hoischen, A., Mangold, E., Ludwig, K.U., Hoebel, A.K., Drichel, D., Vorst, M. van de, Bohmer, A.C., Sivalingam, S., Ishorst, N., Klamt, J., Golz, L., Alblas, M., Maaser, A., Keppler, K., Zink, A.M., Dixon, M.J., Dixon, J., Hemprich, A., Kruse, T., Graf, I., Dunsche, A., Schmidt, G, Daratsianos, N., Nowak, S., Aldhorae, K.A., Nothen, M.M., Knapp, M., Thiele, H., Gilissen, C., Reutter, H., Hoischen, A., Mangold, E., and Ludwig, K.U.
- Abstract
Contains fulltext : 177554.pdf (publisher's version ) (Closed access), Nonsyndromic cleft palate only (nsCPO) is a facial malformation that has a livebirth prevalence of 1 in 2,500. Research suggests that the etiology of nsCPO is multifactorial, with a clear genetic component. To date, genome-wide association studies have identified only 1 conclusive common variant for nsCPO, that is, a missense variant in the gene grainyhead-like-3 ( GRHL3). Thus, the underlying genetic causes of nsCPO remain largely unknown. The present study aimed at identifying rare variants that might contribute to nsCPO risk, via whole-exome sequencing (WES), in multiply affected Central European nsCPO pedigrees. WES was performed in 2 affected first-degree relatives from each family. Variants shared between both individuals were analyzed for their potential deleterious nature and a low frequency in the general population. Genes carrying promising variants were annotated for 1) reported associations with facial development, 2) multiple occurrence of variants, and 3) expression in mouse embryonic palatal shelves. This strategy resulted in the identification of a set of 26 candidate genes that were resequenced in 132 independent nsCPO cases and 623 independent controls of 2 different ethnicities, using molecular inversion probes. No rare loss-of-function mutation was identified in either WES or resequencing step. However, we identified 2 or more missense variants predicted to be deleterious in each of 3 genes ( ACACB, PTPRS, MIB1) in individuals from independent families. In addition, the analyses identified a novel variant in GRHL3 in 1 patient and a variant in CREBBP in 2 siblings. Both genes underlie different syndromic forms of CPO. A plausible hypothesis is that the apparently nonsyndromic clefts in these 3 patients might represent hypomorphic forms of the respective syndromes. In summary, the present study identified rare variants that might contribute to nsCPO risk and suggests candidate genes for further investigation.
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- 2017
11. Candidate Genes for Nonsyndromic Cleft Palate Detected by Exome Sequencing
- Author
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Hoebel, A. K., Drichel, D., van de Vorst, M., Boehmer, A. C., Sivalingam, S., Ishorst, N., Klamt, J., Goelz, L., Alblas, M., Maaser, A., Keppler, K., Zink, A. M., Dixon, M. J., Dixon, J., Hemprich, A., Kruse, T., Graf, I., Dunsche, A., Schmidt, G., Daratsianos, N., Nowak, S., Aldhorae, K. A., Noethen, M. M., Knapp, M., Thiele, H., Gilissen, C., Reutter, H., Hoischen, A., Mangold, E., Ludwig, K. U., Hoebel, A. K., Drichel, D., van de Vorst, M., Boehmer, A. C., Sivalingam, S., Ishorst, N., Klamt, J., Goelz, L., Alblas, M., Maaser, A., Keppler, K., Zink, A. M., Dixon, M. J., Dixon, J., Hemprich, A., Kruse, T., Graf, I., Dunsche, A., Schmidt, G., Daratsianos, N., Nowak, S., Aldhorae, K. A., Noethen, M. M., Knapp, M., Thiele, H., Gilissen, C., Reutter, H., Hoischen, A., Mangold, E., and Ludwig, K. U.
- Abstract
Nonsyndromic cleft palate only (nsCPO) is a facial malformation that has a livebirth prevalence of 1 in 2,500. Research suggests that the etiology of nsCPO is multifactorial, with a clear genetic component. To date, genome-wide association studies have identified only 1 conclusive common variant for nsCPO, that is, a missense variant in the gene grainyhead-like-3 (GRHL3). Thus, the underlying genetic causes of nsCPO remain largely unknown. The present study aimed at identifying rare variants that might contribute to nsCPO risk, via whole-exome sequencing (WES), in multiply affected Central European nsCPO pedigrees. WES was performed in 2 affected first-degree relatives from each family. Variants shared between both individuals were analyzed for their potential deleterious nature and a low frequency in the general population. Genes carrying promising variants were annotated for 1) reported associations with facial development, 2) multiple occurrence of variants, and 3) expression in mouse embryonic palatal shelves. This strategy resulted in the identification of a set of 26 candidate genes that were resequenced in 132 independent nsCPO cases and 623 independent controls of 2 different ethnicities, using molecular inversion probes. No rare loss-of-function mutation was identified in either WES or resequencing step. However, we identified 2 or more missense variants predicted to be deleterious in each of 3 genes (ACACB, PTPRS, MIB1) in individuals from independent families. In addition, the analyses identified a novel variant in GRHL3 in 1 patient and a variant in CREBBP in 2 siblings. Both genes underlie different syndromic forms of CPO. A plausible hypothesis is that the apparently nonsyndromic clefts in these 3 patients might represent hypomorphic forms of the respective syndromes. In summary, the present study identified rare variants that might contribute to nsCPO risk and suggests candidate genes for further investigation.
- Published
- 2017
12. Novel mutations in LRP6 highlight the role of WNT signaling in tooth agenesis
- Author
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Ockeloen, C.W., Khandelwal, K.D., Dreesen, K., Ludwig, K.U., Sullivan, R., Rooij, I.A van, Thonissen, M., Swinnen, S., Phan, M., Conte, F, Ishorst, N., Gilissen, C., Roa Fuentes, L., van de Vorst, M., Henkes, A., Steehouwer, M., van Beusekom, E., Bloemen, M., Vankeirsbilck, B., Berge, S.J., Hens, G., Schoenaers, J., Vander Poorten, V., Roosenboom, J., Verdonck, A., Devriendt, K., Roeleveldt, N., Jhangiani, S.N., Vissers, L.E., Lupski, J.R., de Ligt, J., Von den Hoff, J.W., Pfundt, R., Brunner, H.G., Zhou, H., Dixon, J., Mangold, E., van Bokhoven, H., Dixon, M.J., Kleefstra, T., Hoischen, A., Carels, C.E., Ockeloen, C.W., Khandelwal, K.D., Dreesen, K., Ludwig, K.U., Sullivan, R., Rooij, I.A van, Thonissen, M., Swinnen, S., Phan, M., Conte, F, Ishorst, N., Gilissen, C., Roa Fuentes, L., van de Vorst, M., Henkes, A., Steehouwer, M., van Beusekom, E., Bloemen, M., Vankeirsbilck, B., Berge, S.J., Hens, G., Schoenaers, J., Vander Poorten, V., Roosenboom, J., Verdonck, A., Devriendt, K., Roeleveldt, N., Jhangiani, S.N., Vissers, L.E., Lupski, J.R., de Ligt, J., Von den Hoff, J.W., Pfundt, R., Brunner, H.G., Zhou, H., Dixon, J., Mangold, E., van Bokhoven, H., Dixon, M.J., Kleefstra, T., Hoischen, A., and Carels, C.E.
- Abstract
Item does not contain fulltext
- Published
- 2016
13. Functional analysis of splice site mutations in the human hairless (HR) gene using a minigene assay
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Refke, M., Pasternack, S.M., Fiebig, B., Wenzel, S., Ishorst, N., Ludwig, M., Nothen, Markus, Seijger, M.M.B., Hamel, B.C.J., Betz, R.C., Refke, M., Pasternack, S.M., Fiebig, B., Wenzel, S., Ishorst, N., Ludwig, M., Nothen, Markus, Seijger, M.M.B., Hamel, B.C.J., and Betz, R.C.
- Abstract
Item does not contain fulltext, Background Congenital atrichia is a rare autosomal recessive form of isolated alopecia which is caused by mutations in the human hairless (HR) gene. Patients are born with normal hair that is shed almost completely and irreversibly during the first weeks of life. Objectives To investigate the molecular genetic basis of congenital atrichia in two patients, and to analyse the functional consequences of one newly identified and all seven previously identified HR splice site mutations using a minigene assay. Methods Molecular analysis of the HR gene was performed by direct DNA sequencing. To analyse the functional consequences of the splice site mutations, the respective sequences were cloned into a vector which allows directed splicing. After transfection of COS7 cells, isolation of RNA and cDNA synthesis, sequencing was performed to analyse the products. Results Two novel mutations were identified: an insertion in exon 2 (c.485insT; p.C162LfsX17), and a splice site mutation (c.2847-1G>A). In vitro analysis revealed aberrant splicing for all eight of the investigated HR splice site mutations. Comparison with the results of two biocomputational programs (neural network splice server and CRYP-SKIP) and calculation of consensus values revealed that the predictions of these two programs were consistent in only five and two of the eight mutations, respectively. Conclusions This is the first report to analyse the consequences of HR splice site mutations using a cell-based in vitro assay. The results highlight the importance of performing splicing experiments to clarify the consequences of putative splice site mutations.
- Published
- 2011
14. Bi-allelic variants in CELSR3 are implicated in central nervous system and urinary tract anomalies.
- Author
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Stegmann JD, Kalanithy JC, Dworschak GC, Ishorst N, Mingardo E, Lopes FM, Ho YM, Grote P, Lindenberg TT, Yilmaz Ö, Channab K, Seltzsam S, Shril S, Hildebrandt F, Boschann F, Heinen A, Jolly A, Myers K, McBride K, Bekheirnia MR, Bekheirnia N, Scala M, Morleo M, Nigro V, Torella A, Pinelli M, Capra V, Accogli A, Maitz S, Spano A, Olson RJ, Klee EW, Lanpher BC, Jang SS, Chae JH, Steinbauer P, Rieder D, Janecke AR, Vodopiutz J, Vogel I, Blechingberg J, Cohen JL, Riley K, Klee V, Walsh LE, Begemann M, Elbracht M, Eggermann T, Stoppe A, Stuurman K, van Slegtenhorst M, Barakat TS, Mulhern MS, Sands TT, Cytrynbaum C, Weksberg R, Isidori F, Pippucci T, Severi G, Montanari F, Kruer MC, Bakhtiari S, Darvish H, Reutter H, Hagelueken G, Geyer M, Woolf AS, Posey JE, Lupski JR, Odermatt B, and Hilger AC
- Abstract
CELSR3 codes for a planar cell polarity protein. We describe twelve affected individuals from eleven independent families with bi-allelic variants in CELSR3. Affected individuals presented with an overlapping phenotypic spectrum comprising central nervous system (CNS) anomalies (7/12), combined CNS anomalies and congenital anomalies of the kidneys and urinary tract (CAKUT) (3/12) and CAKUT only (2/12). Computational simulation of the 3D protein structure suggests the position of the identified variants to be implicated in penetrance and phenotype expression. CELSR3 immunolocalization in human embryonic urinary tract and transient suppression and rescue experiments of Celsr3 in fluorescent zebrafish reporter lines further support an embryonic role of CELSR3 in CNS and urinary tract formation., (© 2024. The Author(s).)
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- 2024
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15. Identification of de novo variants in nonsyndromic cleft lip with/without cleft palate patients with low polygenic risk scores.
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Ishorst N, Henschel L, Thieme F, Drichel D, Sivalingam S, Mehrem SL, Fechtner AC, Fazaal J, Welzenbach J, Heimbach A, Maj C, Borisov O, Hausen J, Raff R, Hoischen A, Dixon M, Rada-Iglesias A, Bartusel M, Rojas-Martinez A, Aldhorae K, Braumann B, Kruse T, Kirschneck C, Spanier G, Reutter H, Nowak S, Gölz L, Knapp M, Buness A, Krawitz P, Nöthen MM, Nothnagel M, Becker T, Ludwig KU, and Mangold E
- Subjects
- Humans, Genome-Wide Association Study, DNA-Binding Proteins genetics, Risk Factors, Cleft Palate genetics, Cleft Lip genetics
- Abstract
Background: Nonsyndromic cleft lip with/without cleft palate (nsCL/P) is a congenital malformation of multifactorial etiology. Research has identified >40 genome-wide significant risk loci, which explain less than 40% of nsCL/P heritability. Studies show that some of the hidden heritability is explained by rare penetrant variants., Methods: To identify new candidate genes, we searched for highly penetrant de novo variants (DNVs) in 50 nsCL/P patient/parent-trios with a low polygenic risk for the phenotype (discovery). We prioritized DNV-carrying candidate genes from the discovery for resequencing in independent cohorts of 1010 nsCL/P patients of diverse ethnicities and 1574 population-matched controls (replication). Segregation analyses and rare variant association in the replication cohort, in combination with additional data (genome-wide association data, expression, protein-protein-interactions), were used for final prioritization., Conclusion: In the discovery step, 60 DNVs were identified in 60 genes, including a variant in the established nsCL/P risk gene CDH1. Re-sequencing of 32 prioritized genes led to the identification of 373 rare, likely pathogenic variants. Finally, MDN1 and PAXIP1 were prioritized as top candidates. Our findings demonstrate that DNV detection, including polygenic risk score analysis, is a powerful tool for identifying nsCL/P candidate genes, which can also be applied to other multifactorial congenital malformations., (© 2022 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)
- Published
- 2023
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16. Prioritization of non-coding elements involved in non-syndromic cleft lip with/without cleft palate through genome-wide analysis of de novo mutations.
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Zieger HK, Weinhold L, Schmidt A, Holtgrewe M, Juranek SA, Siewert A, Scheer AB, Thieme F, Mangold E, Ishorst N, Brand FU, Welzenbach J, Beule D, Paeschke K, Krawitz PM, and Ludwig KU
- Subjects
- Humans, Genome-Wide Association Study, Alleles, Mutation genetics, Cleft Palate genetics, Cleft Lip genetics
- Abstract
Non-syndromic cleft lip with/without cleft palate (nsCL/P) is a highly heritable facial disorder. To date, systematic investigations of the contribution of rare variants in non-coding regions to nsCL/P etiology are sparse. Here, we re-analyzed available whole-genome sequence (WGS) data from 211 European case-parent trios with nsCL/P and identified 13,522 de novo mutations (DNMs) in nsCL/P cases, 13,055 of which mapped to non-coding regions. We integrated these data with DNMs from a reference cohort, with results of previous genome-wide association studies (GWASs), and functional and epigenetic datasets of relevance to embryonic facial development. A significant enrichment of nsCL/P DNMs was observed at two GWAS risk loci (4q28.1 (p = 8 × 10
-4 ) and 2p21 (p = 0.02)), suggesting a convergence of both common and rare variants at these loci. We also mapped the DNMs to 810 position weight matrices indicative of transcription factor (TF) binding, and quantified the effect of the allelic changes in silico . This revealed a nominally significant overrepresentation of DNMs (p = 0.037), and a stronger effect on binding strength, for DNMs located in the sequence of the core binding region of the TF Musculin (MSC). Notably, MSC is involved in facial muscle development, together with a set of nsCL/P genes located at GWAS loci. Supported by additional results from single-cell transcriptomic data and molecular binding assays, this suggests that variation in MSC binding sites contributes to nsCL/P etiology. Our study describes a set of approaches that can be applied to increase the added value of WGS data., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)- Published
- 2022
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17. Author Correction: LAMP-Seq enables sensitive, multiplexed COVID-19 diagnostics using molecular barcoding.
- Author
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Ludwig KU, Schmithausen RM, Li D, Jacobs ML, Hollstein R, Blumenstock K, Liebing J, Słabicki M, Ben-Shmuel A, Israeli O, Weiss S, Ebert TS, Paran N, Rüdiger W, Wilbring G, Feldman D, Lippke B, Ishorst N, Hochfeld LM, Beins EC, Kaltheuner IH, Schmitz M, Wöhler A, Döhla M, Sib E, Jentzsch M, Moench EC, Borrajo JD, Strecker J, Reinhardt J, Cleary B, Geyer M, Hölzel M, Macrae R, Nöthen MM, Hoffmann P, Exner M, Regev A, Zhang F, and Schmid-Burgk JL
- Published
- 2022
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18. LAMP-Seq enables sensitive, multiplexed COVID-19 diagnostics using molecular barcoding.
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Ludwig KU, Schmithausen RM, Li D, Jacobs ML, Hollstein R, Blumenstock K, Liebing J, Słabicki M, Ben-Shmuel A, Israeli O, Weiss S, Ebert TS, Paran N, Rüdiger W, Wilbring G, Feldman D, Lippke B, Ishorst N, Hochfeld LM, Beins EC, Kaltheuner IH, Schmitz M, Wöhler A, Döhla M, Sib E, Jentzsch M, Moench EC, Borrajo JD, Strecker J, Reinhardt J, Cleary B, Geyer M, Hölzel M, Macrae R, Nöthen MM, Hoffmann P, Exner M, Regev A, Zhang F, and Schmid-Burgk JL
- Subjects
- Humans, COVID-19 diagnosis, COVID-19 Testing methods, Molecular Diagnostic Techniques methods, Nucleic Acid Amplification Techniques methods
- Abstract
Frequent testing of large population groups combined with contact tracing and isolation measures will be crucial for containing Coronavirus Disease 2019 outbreaks. Here we present LAMP-Seq, a modified, highly scalable reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. Unpurified biosamples are barcoded and amplified in a single heat step, and pooled products are analyzed en masse by sequencing. Using commercial reagents, LAMP-Seq has a limit of detection of ~2.2 molecules per µl at 95% confidence and near-perfect specificity for severe acute respiratory syndrome coronavirus 2 given its sequence readout. Clinical validation of an open-source protocol with 676 swab samples, 98 of which were deemed positive by standard RT-qPCR, demonstrated 100% sensitivity in individuals with cycle threshold values of up to 33 and a specificity of 99.7%, at a very low material cost. With a time-to-result of fewer than 24 h, low cost and little new infrastructure requirement, LAMP-Seq can be readily deployed for frequent testing as part of an integrated public health surveillance program., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2021
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19. Integrative approaches generate insights into the architecture of non-syndromic cleft lip with or without cleft palate.
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Welzenbach J, Hammond NL, Nikolić M, Thieme F, Ishorst N, Leslie EJ, Weinberg SM, Beaty TH, Marazita ML, Mangold E, Knapp M, Cotney J, Rada-Iglesias A, Dixon MJ, and Ludwig KU
- Abstract
Non-syndromic cleft lip with or without cleft palate (nsCL/P) is a common congenital facial malformation with a multifactorial etiology. Genome-wide association studies (GWASs) have identified multiple genetic risk loci. However, functional interpretation of these loci is hampered by the underrepresentation in public resources of systematic functional maps representative of human embryonic facial development. To generate novel insights into the etiology of nsCL/P, we leveraged published GWAS data on nsCL/P as well as available chromatin modification and expression data on mid-facial development. Our analyses identified five novel risk loci, prioritized candidate target genes within associated regions, and highlighted distinct pathways. Furthermore, the results suggest the presence of distinct regulatory effects of nsCL/P risk variants throughout mid-facial development and shed light on its regulatory architecture. Our integrated data provide a platform to advance hypothesis-driven molecular investigations of nsCL/P and other human facial defects., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)
- Published
- 2021
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20. Non-Syndromic Cleft Lip with or without Cleft Palate: Genome-Wide Association Study in Europeans Identifies a Suggestive Risk Locus at 16p12.1 and Supports SH3PXD2A as a Clefting Susceptibility Gene.
- Author
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van Rooij IA, Ludwig KU, Welzenbach J, Ishorst N, Thonissen M, Galesloot TE, Ongkosuwito E, Bergé SJ, Aldhorae K, Rojas-Martinez A, Kiemeney LA, Vermeesch JR, Brunner H, Roeleveld N, Devriendt K, Dormaar T, Hens G, Knapp M, Carels C, and Mangold E
- Subjects
- Animals, Belgium, Chromosomes, Human, Pair 10 genetics, Female, Genome-Wide Association Study, Humans, Male, Mice, Mice, Knockout, Netherlands, Risk Factors, Zebrafish, Adaptor Proteins, Vesicular Transport genetics, Chromosomes, Human, Pair 16 genetics, Cleft Lip genetics, Cleft Palate genetics
- Abstract
Non-syndromic cleft lip with or without cleft palate (nsCL/P) ranks among the most common human congenital malformations, and has a multifactorial background in which both exogenous and genetic risk factors act in concert. The present report describes a genome-wide association study (GWAS) involving a total of 285 nsCL/P patients and 1212 controls from the Netherlands and Belgium. Twenty of the 40 previously reported nsC/LP susceptibility loci were replicated, which underlined the validity of this sample. SNV-based analysis of the data identified an as yet unreported suggestive locus at chromosome 16p12.1 ( p -value of the lead SNV: 4.17 × 10
-7 ). This association was replicated in two of three patient/control replication series (Central European and Yemeni). Gene analysis of the GWAS data prioritized SH3PXD2A at chromosome 10q24.33 as a candidate gene for nsCL/P. To date, support for this gene as a cleft gene has been restricted to data from zebrafish and a knockout mouse model. The present GWAS was the first to implicate SH3PXD2A in non-syndromic cleft formation in humans. In summary, although performed in a relatively small sample, the present GWAS generated novel insights into nsCL/P etiology.- Published
- 2019
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21. Deletions and loss-of-function variants in TP63 associated with orofacial clefting.
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Khandelwal KD, van den Boogaard MH, Mehrem SL, Gebel J, Fagerberg C, van Beusekom E, van Binsbergen E, Topaloglu O, Steehouwer M, Gilissen C, Ishorst N, van Rooij IALM, Roeleveld N, Christensen K, Schoenaers J, Bergé S, Murray JC, Hens G, Devriendt K, Ludwig KU, Mangold E, Hoischen A, Zhou H, Dötsch V, Carels CEL, and van Bokhoven H
- Subjects
- Adult, Amino Acid Substitution, Cohort Studies, Female, Humans, Male, Middle Aged, Mutation, Missense, Alleles, Base Sequence, Cleft Lip genetics, Cleft Palate genetics, Loss of Function Mutation, Sequence Deletion, Transcription Factors genetics, Tumor Suppressor Proteins genetics
- Abstract
We aimed to identify novel deletions and variants of TP63 associated with orofacial clefting (OFC). Copy number variants were assessed in three OFC families using microarray analysis. Subsequently, we analyzed TP63 in a cohort of 1072 individuals affected with OFC and 706 population-based controls using molecular inversion probes (MIPs). We identified partial deletions of TP63 in individuals from three families affected with OFC. In the OFC cohort, we identified several TP63 variants predicting to cause loss-of-function alleles, including a frameshift variant c.569_576del (p.(Ala190Aspfs*5)) and a nonsense variant c.997C>T (p.(Gln333*)) that introduces a premature stop codon in the DNA-binding domain. In addition, we identified the first missense variants in the oligomerization domain c.1213G>A (p.(Val405Met)), which occurred in individuals with OFC. This variant was shown to abrogate oligomerization of mutant p63 protein into oligomeric complexes, and therefore likely represents a loss-of-function allele rather than a dominant-negative. All of these variants were inherited from an unaffected parent, suggesting reduced penetrance of such loss-of-function alleles. Our data indicate that loss-of-function alleles in TP63 can also give rise to OFC as the main phenotype. We have uncovered the dosage-dependent functions of p63, which were previously rejected.
- Published
- 2019
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22. Imputation of orofacial clefting data identifies novel risk loci and sheds light on the genetic background of cleft lip ± cleft palate and cleft palate only.
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Ludwig KU, Böhmer AC, Bowes J, Nikolic M, Ishorst N, Wyatt N, Hammond NL, Gölz L, Thieme F, Barth S, Schuenke H, Klamt J, Spielmann M, Aldhorae K, Rojas-Martinez A, Nöthen MM, Rada-Iglesias A, Dixon MJ, Knapp M, and Mangold E
- Subjects
- Animals, Cleft Lip metabolism, Cleft Lip pathology, Cleft Palate metabolism, Cleft Palate pathology, Female, Humans, Male, Mice, Chromosomes, Human genetics, Cleft Lip genetics, Cleft Palate genetics, Databases, Genetic, Genetic Loci, Genome-Wide Association Study, Polymorphism, Single Nucleotide
- Abstract
Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is among the most common human birth defects with multifactorial etiology. Here, we present results from a genome-wide imputation study of nsCL/P in which, after adding replication cohort data, four novel risk loci for nsCL/P are identified (at chromosomal regions 2p21, 14q22, 15q24 and 19p13). On a systematic level, we show that the association signals within this high-density dataset are enriched in functionally-relevant genomic regions that are active in both human neural crest cells (hNCC) and mouse embryonic craniofacial tissue. This enrichment is also detectable in hNCC regions primed for later activity. Using GCTA analyses, we suggest that 30% of the estimated variance in risk for nsCL/P in the European population can be attributed to common variants, with 25.5% contributed to by the 24 risk loci known to date. For each of these, we identify credible SNPs using a Bayesian refinement approach, with two loci harbouring only one probable causal variant. Finally, we demonstrate that there is no polygenic component of nsCL/P detectable that is shared with nonsyndromic cleft palate only (nsCPO). Our data suggest that, while common variants are strongly contributing to risk for nsCL/P, they do not seem to be involved in nsCPO which might be more often caused by rare deleterious variants. Our study generates novel insights into both nsCL/P and nsCPO etiology and provides a systematic framework for research into craniofacial development and malformation., (© The Author 2017. Published by Oxford University Press.)
- Published
- 2017
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23. Novel mutations in LRP6 highlight the role of WNT signaling in tooth agenesis.
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Ockeloen CW, Khandelwal KD, Dreesen K, Ludwig KU, Sullivan R, van Rooij IALM, Thonissen M, Swinnen S, Phan M, Conte F, Ishorst N, Gilissen C, RoaFuentes L, van de Vorst M, Henkes A, Steehouwer M, van Beusekom E, Bloemen M, Vankeirsbilck B, Bergé S, Hens G, Schoenaers J, Poorten VV, Roosenboom J, Verdonck A, Devriendt K, Roeleveldt N, Jhangiani SN, Vissers LELM, Lupski JR, de Ligt J, Von den Hoff JW, Pfundt R, Brunner HG, Zhou H, Dixon J, Mangold E, van Bokhoven H, Dixon MJ, Kleefstra T, Hoischen A, and Carels CEL
- Subjects
- Adolescent, Anodontia pathology, Child, Female, Frameshift Mutation genetics, Humans, Male, Mutation, Missense genetics, Pedigree, Sequence Analysis, DNA, Wnt Signaling Pathway genetics, Anodontia genetics, Exome genetics, Genetic Predisposition to Disease, Low Density Lipoprotein Receptor-Related Protein-6 genetics
- Abstract
Purpose: We aimed to identify a novel genetic cause of tooth agenesis (TA) and/or orofacial clefting (OFC) by combining whole-exome sequencing (WES) and targeted resequencing in a large cohort of TA and OFC patients., Methods: WES was performed in two unrelated patients: one with severe TA and OFC and another with severe TA only. After deleterious mutations were identified in a gene encoding low-density lipoprotein receptor-related protein 6 (LRP6), all its exons were resequenced with molecular inversion probes in 67 patients with TA, 1,072 patients with OFC, and 706 controls., Results: We identified a frameshift (c.4594delG, p.Cys1532fs) and a canonical splice-site mutation (c.3398-2A>C, p.?) in LRP6, respectively, in the patient with TA and OFC and in the patient with severe TA only. The targeted resequencing showed significant enrichment of unique LRP6 variants in TA patients but not in nonsyndromic OFC patients. Of the five variants in patients with TA, two affected the canonical splice site and three were missense variants; all variants segregated with the dominant phenotype, and in one case the missense mutation occurred de novo., Conclusion: Mutations in LRP6 cause TA in humans.Genet Med 18 11, 1158-1162.
- Published
- 2016
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24. Sequencing the GRHL3 Coding Region Reveals Rare Truncating Mutations and a Common Susceptibility Variant for Nonsyndromic Cleft Palate.
- Author
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Mangold E, Böhmer AC, Ishorst N, Hoebel AK, Gültepe P, Schuenke H, Klamt J, Hofmann A, Gölz L, Raff R, Tessmann P, Nowak S, Reutter H, Hemprich A, Kreusch T, Kramer FJ, Braumann B, Reich R, Schmidt G, Jäger A, Reiter R, Brosch S, Stavusis J, Ishida M, Seselgyte R, Moore GE, Nöthen MM, Borck G, Aldhorae KA, Lace B, Stanier P, Knapp M, and Ludwig KU
- Subjects
- Abnormalities, Multiple diagnosis, Abnormalities, Multiple genetics, Alleles, Case-Control Studies, Cleft Lip diagnosis, Cleft Lip genetics, Cleft Palate diagnosis, Cysts diagnosis, Cysts genetics, Humans, Lip abnormalities, Mutation, Polymorphism, Single Nucleotide, Racial Groups genetics, Cleft Palate genetics, DNA-Binding Proteins genetics, Genetic Predisposition to Disease, Open Reading Frames, Transcription Factors genetics
- Abstract
Nonsyndromic cleft lip with/without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO) are the most frequent subphenotypes of orofacial clefts. A common syndromic form of orofacial clefting is Van der Woude syndrome (VWS) where individuals have CL/P or CPO, often but not always associated with lower lip pits. Recently, ∼5% of VWS-affected individuals were identified with mutations in the grainy head-like 3 gene (GRHL3). To investigate GRHL3 in nonsyndromic clefting, we sequenced its coding region in 576 Europeans with nsCL/P and 96 with nsCPO. Most strikingly, nsCPO-affected individuals had a higher minor allele frequency for rs41268753 (0.099) than control subjects (0.049; p = 1.24 × 10(-2)). This association was replicated in nsCPO/control cohorts from Latvia, Yemen, and the UK (pcombined = 2.63 × 10(-5); ORallelic = 2.46 [95% CI 1.6-3.7]) and reached genome-wide significance in combination with imputed data from a GWAS in nsCPO triads (p = 2.73 × 10(-9)). Notably, rs41268753 is not associated with nsCL/P (p = 0.45). rs41268753 encodes the highly conserved p.Thr454Met (c.1361C>T) (GERP = 5.3), which prediction programs denote as deleterious, has a CADD score of 29.6, and increases protein binding capacity in silico. Sequencing also revealed four novel truncating GRHL3 mutations including two that were de novo in four families, where all nine individuals harboring mutations had nsCPO. This is important for genetic counseling: given that VWS is rare compared to nsCPO, our data suggest that dominant GRHL3 mutations are more likely to cause nonsyndromic than syndromic CPO. Thus, with rare dominant mutations and a common risk variant in the coding region, we have identified an important contribution for GRHL3 in nsCPO., (Copyright © 2016 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
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