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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

Knockdown and overexpression of FZD6 protein in zebrafish results in craniofacial defects. Both nonoverlapping morpholinos caused the same spectrum of craniofacial abnormalities. Only MO1-injected embryos are shown as an example. (A, D, G, J, and M) are uninjected control (UIC) embryos. (B, E, H, K, and N) show the phenotypes associated with knockdown of fzd6. (C, F, I, L, and O) show phenotypes associated with overexpression of fzd6. (A) UIC embryos at 24 hpf. (B) fzd6 MO-injected embryos at 24 hpf. (C) fzd6 mRNA-injected embryos at 24 hpf. (D) UIC embryos at 6 dpf. (E) fzd6 MO-injected embryos at 6 dpf. (F) fzd6 mRNA-injected embryos at 6 dpf. G-O show Alcian blue and alizarin red-stained embryos at 6 dpf. Jaw and palatal abnormalities due to fzd6 knockdown are shown (red arrows) in (K and N), respectively. Specifically in (K), arrows point to abnormal Meckel’s and ceratohyal cartilage in the lower jaw. In (N), arrows point to a reduced ethmoid palatal plate. Jaw and palatal abnormalities due to fzd6 overexpression are shown (red arrows) in (L and O), respectively. Specifically in (L), arrows point to abnormal Meckel’s and ceratohyal cartilage in the lower jaw. In (O), arrow points to a loss of the ethmoid plate in the palate.
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fig03: Knockdown and overexpression of FZD6 protein in zebrafish results in craniofacial defects. Both nonoverlapping morpholinos caused the same spectrum of craniofacial abnormalities. Only MO1-injected embryos are shown as an example. (A, D, G, J, and M) are uninjected control (UIC) embryos. (B, E, H, K, and N) show the phenotypes associated with knockdown of fzd6. (C, F, I, L, and O) show phenotypes associated with overexpression of fzd6. (A) UIC embryos at 24 hpf. (B) fzd6 MO-injected embryos at 24 hpf. (C) fzd6 mRNA-injected embryos at 24 hpf. (D) UIC embryos at 6 dpf. (E) fzd6 MO-injected embryos at 6 dpf. (F) fzd6 mRNA-injected embryos at 6 dpf. G-O show Alcian blue and alizarin red-stained embryos at 6 dpf. Jaw and palatal abnormalities due to fzd6 knockdown are shown (red arrows) in (K and N), respectively. Specifically in (K), arrows point to abnormal Meckel’s and ceratohyal cartilage in the lower jaw. In (N), arrows point to a reduced ethmoid palatal plate. Jaw and palatal abnormalities due to fzd6 overexpression are shown (red arrows) in (L and O), respectively. Specifically in (L), arrows point to abnormal Meckel’s and ceratohyal cartilage in the lower jaw. In (O), arrow points to a loss of the ethmoid plate in the palate.

Mentions: To examine the role of FZD6 during craniofacial development, two nonoverlapping antisense MOs directed against the translation start site of zebrafish fzd6 were injected into one-cell embryos to knock down production of Fzd6 protein. Wild-type embryos injected with the nonoverlapping MOs resulted in necrosis at 24 hours postfertilization (hpf) (arrows in Fig.3A and B). By 6 dpf, fzd6 MO-injected embryos exhibited abnormalities of craniofacial elements when compared to wild-type controls (Fig.3D and E). All fzd6 MO-injected embryos developed abnormal Meckel’s and ceratohyal cartilage in the lower jaw (arrows in Fig.3H and K), and a reduced ethmoid plate (arrow in Fig.3N). No abnormalities were found upon injection of the mismatched MO. Full-length zebrafish fzd6 mRNA was injected into one-cell embryos and allowed to develop to 6 dpf. Interestingly, overexpression of wild-type fzd6 in zebrafish also resulted in a severe craniofacial phenotype with cyclopia when compared to wild-type controls (Fig.3C, F, I, L, and O). Compared to wild-type controls, fzd6 mRNA-injected embryos develop severely abnormal Meckel’s and ceratohyal cartilage in the lower jaw (arrows in Fig.3I and L) and a complete loss of the ethmoid plate (arrow in Fig.3O).


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)

Knockdown and overexpression of FZD6 protein in zebrafish results in craniofacial defects. Both nonoverlapping morpholinos caused the same spectrum of craniofacial abnormalities. Only MO1-injected embryos are shown as an example. (A, D, G, J, and M) are uninjected control (UIC) embryos. (B, E, H, K, and N) show the phenotypes associated with knockdown of fzd6. (C, F, I, L, and O) show phenotypes associated with overexpression of fzd6. (A) UIC embryos at 24 hpf. (B) fzd6 MO-injected embryos at 24 hpf. (C) fzd6 mRNA-injected embryos at 24 hpf. (D) UIC embryos at 6 dpf. (E) fzd6 MO-injected embryos at 6 dpf. (F) fzd6 mRNA-injected embryos at 6 dpf. G-O show Alcian blue and alizarin red-stained embryos at 6 dpf. Jaw and palatal abnormalities due to fzd6 knockdown are shown (red arrows) in (K and N), respectively. Specifically in (K), arrows point to abnormal Meckel’s and ceratohyal cartilage in the lower jaw. In (N), arrows point to a reduced ethmoid palatal plate. Jaw and palatal abnormalities due to fzd6 overexpression are shown (red arrows) in (L and O), respectively. Specifically in (L), arrows point to abnormal Meckel’s and ceratohyal cartilage in the lower jaw. In (O), arrow points to a loss of the ethmoid plate in the palate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: Knockdown and overexpression of FZD6 protein in zebrafish results in craniofacial defects. Both nonoverlapping morpholinos caused the same spectrum of craniofacial abnormalities. Only MO1-injected embryos are shown as an example. (A, D, G, J, and M) are uninjected control (UIC) embryos. (B, E, H, K, and N) show the phenotypes associated with knockdown of fzd6. (C, F, I, L, and O) show phenotypes associated with overexpression of fzd6. (A) UIC embryos at 24 hpf. (B) fzd6 MO-injected embryos at 24 hpf. (C) fzd6 mRNA-injected embryos at 24 hpf. (D) UIC embryos at 6 dpf. (E) fzd6 MO-injected embryos at 6 dpf. (F) fzd6 mRNA-injected embryos at 6 dpf. G-O show Alcian blue and alizarin red-stained embryos at 6 dpf. Jaw and palatal abnormalities due to fzd6 knockdown are shown (red arrows) in (K and N), respectively. Specifically in (K), arrows point to abnormal Meckel’s and ceratohyal cartilage in the lower jaw. In (N), arrows point to a reduced ethmoid palatal plate. Jaw and palatal abnormalities due to fzd6 overexpression are shown (red arrows) in (L and O), respectively. Specifically in (L), arrows point to abnormal Meckel’s and ceratohyal cartilage in the lower jaw. In (O), arrow points to a loss of the ethmoid plate in the palate.
Mentions: To examine the role of FZD6 during craniofacial development, two nonoverlapping antisense MOs directed against the translation start site of zebrafish fzd6 were injected into one-cell embryos to knock down production of Fzd6 protein. Wild-type embryos injected with the nonoverlapping MOs resulted in necrosis at 24 hours postfertilization (hpf) (arrows in Fig.3A and B). By 6 dpf, fzd6 MO-injected embryos exhibited abnormalities of craniofacial elements when compared to wild-type controls (Fig.3D and E). All fzd6 MO-injected embryos developed abnormal Meckel’s and ceratohyal cartilage in the lower jaw (arrows in Fig.3H and K), and a reduced ethmoid plate (arrow in Fig.3N). No abnormalities were found upon injection of the mismatched MO. Full-length zebrafish fzd6 mRNA was injected into one-cell embryos and allowed to develop to 6 dpf. Interestingly, overexpression of wild-type fzd6 in zebrafish also resulted in a severe craniofacial phenotype with cyclopia when compared to wild-type controls (Fig.3C, F, I, L, and O). Compared to wild-type controls, fzd6 mRNA-injected embryos develop severely abnormal Meckel’s and ceratohyal cartilage in the lower jaw (arrows in Fig.3I and L) and a complete loss of the ethmoid plate (arrow in Fig.3O).

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