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Regulatory variant in FZD6 gene contributes to nonsyndromic cleft lip and palate in an African-American family.

Cvjetkovic N, Maili L, Weymouth KS, Hashmi SS, Mulliken JB, Topczewski J, Letra A, Yuan Q, Blanton SH, Swindell EC, Hecht JT - Mol Genet Genomic Med (2015)

Bottom Line: The variant C allele segregated with NSCLP in this family, through affected and unaffected individuals, and was found in one other NSCLP African-American family.Functional assays showed that this allele creates an allele-specific protein-binding site and decreases promoter activity.We hypothesize, therefore, that alteration in FZD6 expression contributes to NSCLP in this family by perturbing the WNT signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Texas Medical School at Houston Houston, Texas ; Graduate School of Biomedical Sciences, University of Texas Health Science Center Houston, Texas.

ABSTRACT
Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect affecting 135,000 newborns worldwide each year. While a multifactorial etiology has been suggested as the cause, despite decades of research, the genetic underpinnings of NSCLP remain largely unexplained. In our previous genome-wide linkage study of a large NSCLP African-American family, we identified a candidate locus at 8q21.3-24.12 (LOD = 2.98). This region contained four genes, Frizzled-6 (FZD6), Matrilin-2 (MATN2), Odd-skipped related 2 (OSR2) and Solute Carrier Family 25, Member 32 (SLC25A32). FZD6 was located under the maximum linkage peak. In this study, we sequenced the coding and noncoding regions of these genes in two affected family members, and identified a rare variant in intron 1 of FZD6 (rs138557689; c.-153 + 432A>C). The variant C allele segregated with NSCLP in this family, through affected and unaffected individuals, and was found in one other NSCLP African-American family. Functional assays showed that this allele creates an allele-specific protein-binding site and decreases promoter activity. We also observed that loss and gain of fzd6 in zebrafish contributes to craniofacial anomalies. FZD6 regulates the WNT signaling pathway, which is involved in craniofacial development, including midfacial formation and upper labial fusion. We hypothesize, therefore, that alteration in FZD6 expression contributes to NSCLP in this family by perturbing the WNT signaling pathway.

No MeSH data available.


Related in: MedlinePlus

FZD6 rs138557689/C creates an allele-specific protein-binding complex and decreases FZD6 promoter activity. (A) Electrophoretic mobility shift assays (EMSA) were performed using nuclear extract from Cos7 cells. Samples were incubated with P32-labeled oligonucleotides containing either the ancestral A or alternate C alleles, or with unlabeled ancestral A or alternate C serving as specific competitors. Poly-dCdG was used as a nonspecific competitor. Negative controls were run using labeled probes without the nuclear extract. Bands were observed with the C allele only and the alternate band was competed out with C competitor only. (B) Hek293T cells (100,000 cells/well) were seeded for 24 h and cotransfected with ancestral or alternate promoter construct and Renilla reporter construct. Luciferase activities were normalized to the internal Renilla control. Data represent mean values ± SD from three independent experiments. Alternate C allele showed significant decrease in activity (*P < 0.001, unpaired t-test).
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fig02: FZD6 rs138557689/C creates an allele-specific protein-binding complex and decreases FZD6 promoter activity. (A) Electrophoretic mobility shift assays (EMSA) were performed using nuclear extract from Cos7 cells. Samples were incubated with P32-labeled oligonucleotides containing either the ancestral A or alternate C alleles, or with unlabeled ancestral A or alternate C serving as specific competitors. Poly-dCdG was used as a nonspecific competitor. Negative controls were run using labeled probes without the nuclear extract. Bands were observed with the C allele only and the alternate band was competed out with C competitor only. (B) Hek293T cells (100,000 cells/well) were seeded for 24 h and cotransfected with ancestral or alternate promoter construct and Renilla reporter construct. Luciferase activities were normalized to the internal Renilla control. Data represent mean values ± SD from three independent experiments. Alternate C allele showed significant decrease in activity (*P < 0.001, unpaired t-test).

Mentions: EMSA was used to determine whether a protein-binding site was created by either allele. As shown in Figure2, there was one band present for the probe containing the rs138557689/C allele that was not present for rs138557689/A probe. This suggested that the rs138557689/C allele produces an allele-specific protein-binding site that cannot be competed out by the ancestral (A) allele (Fig.2A). As shown in Figure2B, luciferase reporter activity of FZD6 was reduced by approximately 80% (p < 0.001) with the rs138557689/C (alternate allele) promoter construct. Similar results were obtained for Cos7 cells (data not shown).


Regulatory variant in FZD6 gene contributes to nonsyndromic cleft lip and palate in an African-American family.

Cvjetkovic N, Maili L, Weymouth KS, Hashmi SS, Mulliken JB, Topczewski J, Letra A, Yuan Q, Blanton SH, Swindell EC, Hecht JT - Mol Genet Genomic Med (2015)

FZD6 rs138557689/C creates an allele-specific protein-binding complex and decreases FZD6 promoter activity. (A) Electrophoretic mobility shift assays (EMSA) were performed using nuclear extract from Cos7 cells. Samples were incubated with P32-labeled oligonucleotides containing either the ancestral A or alternate C alleles, or with unlabeled ancestral A or alternate C serving as specific competitors. Poly-dCdG was used as a nonspecific competitor. Negative controls were run using labeled probes without the nuclear extract. Bands were observed with the C allele only and the alternate band was competed out with C competitor only. (B) Hek293T cells (100,000 cells/well) were seeded for 24 h and cotransfected with ancestral or alternate promoter construct and Renilla reporter construct. Luciferase activities were normalized to the internal Renilla control. Data represent mean values ± SD from three independent experiments. Alternate C allele showed significant decrease in activity (*P < 0.001, unpaired t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4585452&req=5

fig02: FZD6 rs138557689/C creates an allele-specific protein-binding complex and decreases FZD6 promoter activity. (A) Electrophoretic mobility shift assays (EMSA) were performed using nuclear extract from Cos7 cells. Samples were incubated with P32-labeled oligonucleotides containing either the ancestral A or alternate C alleles, or with unlabeled ancestral A or alternate C serving as specific competitors. Poly-dCdG was used as a nonspecific competitor. Negative controls were run using labeled probes without the nuclear extract. Bands were observed with the C allele only and the alternate band was competed out with C competitor only. (B) Hek293T cells (100,000 cells/well) were seeded for 24 h and cotransfected with ancestral or alternate promoter construct and Renilla reporter construct. Luciferase activities were normalized to the internal Renilla control. Data represent mean values ± SD from three independent experiments. Alternate C allele showed significant decrease in activity (*P < 0.001, unpaired t-test).
Mentions: EMSA was used to determine whether a protein-binding site was created by either allele. As shown in Figure2, there was one band present for the probe containing the rs138557689/C allele that was not present for rs138557689/A probe. This suggested that the rs138557689/C allele produces an allele-specific protein-binding site that cannot be competed out by the ancestral (A) allele (Fig.2A). As shown in Figure2B, luciferase reporter activity of FZD6 was reduced by approximately 80% (p < 0.001) with the rs138557689/C (alternate allele) promoter construct. Similar results were obtained for Cos7 cells (data not shown).

Bottom Line: The variant C allele segregated with NSCLP in this family, through affected and unaffected individuals, and was found in one other NSCLP African-American family.Functional assays showed that this allele creates an allele-specific protein-binding site and decreases promoter activity.We hypothesize, therefore, that alteration in FZD6 expression contributes to NSCLP in this family by perturbing the WNT signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Texas Medical School at Houston Houston, Texas ; Graduate School of Biomedical Sciences, University of Texas Health Science Center Houston, Texas.

ABSTRACT
Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect affecting 135,000 newborns worldwide each year. While a multifactorial etiology has been suggested as the cause, despite decades of research, the genetic underpinnings of NSCLP remain largely unexplained. In our previous genome-wide linkage study of a large NSCLP African-American family, we identified a candidate locus at 8q21.3-24.12 (LOD = 2.98). This region contained four genes, Frizzled-6 (FZD6), Matrilin-2 (MATN2), Odd-skipped related 2 (OSR2) and Solute Carrier Family 25, Member 32 (SLC25A32). FZD6 was located under the maximum linkage peak. In this study, we sequenced the coding and noncoding regions of these genes in two affected family members, and identified a rare variant in intron 1 of FZD6 (rs138557689; c.-153 + 432A>C). The variant C allele segregated with NSCLP in this family, through affected and unaffected individuals, and was found in one other NSCLP African-American family. Functional assays showed that this allele creates an allele-specific protein-binding site and decreases promoter activity. We also observed that loss and gain of fzd6 in zebrafish contributes to craniofacial anomalies. FZD6 regulates the WNT signaling pathway, which is involved in craniofacial development, including midfacial formation and upper labial fusion. We hypothesize, therefore, that alteration in FZD6 expression contributes to NSCLP in this family by perturbing the WNT signaling pathway.

No MeSH data available.


Related in: MedlinePlus