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

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.

Keywords: Frizzled-6; WNT pathway; nonsyndromic cleft lip and palate; regulatory variant.

Figure 1

Multiplex NSCLP African-American family. Pedigree depicts eleven individuals with NSCLP spanning three generations. Filled symbols denote affected individuals and asterisks (*) denote individuals included in the genome scan reported by Chiquet et al. (2009). Laterality is indicated as B for bilateral and U for unilateral. All of the examined individuals had cleft lip with cleft palate. C allele of rs138557689 segregates with affected individuals and is transmitted by four unaffected individuals originating from individual I-3. C allele is also present in 5 additional unaffected individuals.

Figure 2

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).

Figure 3

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.

Figure 4

Schematic model of WNT signaling by the FZD receptor(s). (A) In absence of WNT, β-catenin is degraded by AXIN-APC-GSK3β complex. (B) Binding of WNT to FZD receptor (plus specific other coreceptors) results in transcription of WNT target genes by the canonical pathway, mediation of tissue polarity control by noncanonical DVL-dependent pathway, and inhibition of RNA transcription of WNT target genes by noncanonical Ca²⁺- dependent pathway. APC, adenomatous polyposis coli; AXIN, axis inhibition protein; CAMKII, Calcium/calmodulin-dependent protein kinase II; DVL, disheveled; GSK3β, glycogen synthase kinase 3 beta; JNK, Jun N-terminal kinase; LRP5/6, low-density lipoprotein receptor-related protein 5/6; NLK, Nemo-like kinase; PIP2, Phosphatidylinositol-4,5-bisphosphate; Rho/Rac, Small GTP-binding proteins; TCF/LEF, T-cell factor/Lymphoid enhancer-binding factor; WNT, Wingless-type MMTV integration site family.