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. 2011 Jun 10;88(6):852–860. doi: 10.1016/j.ajhg.2011.05.013

Mutations in Frizzled 6 Cause Isolated Autosomal-Recessive Nail Dysplasia

Anne-Sophie Fröjmark 1, Jens Schuster 1,6, Maria Sobol 1,2,6, Miriam Entesarian 1,5,6, Michaela BC Kilander 3, Dana Gabrikova 1,7, Sadia Nawaz 4, Shahid M Baig 4, Gunnar Schulte 3, Joakim Klar 1, Niklas Dahl 1,∗
PMCID: PMC3113248  PMID: 21665003

Abstract

Inherited and isolated nail malformations are rare and heterogeneous conditions. We identified two consanguineous pedigrees in which some family members were affected by isolated nail dysplasia that suggested an autosomal-recessive inheritance pattern and was characterized by claw-shaped nails, onychauxis, and onycholysis. Genome-wide SNP array analysis of affected individuals from both families showed an overlapping and homozygous region of 800 kb on the long arm of chromosome 8. The candidate region spans eight genes, and DNA sequence analysis revealed homozygous nonsense and missense mutations in FZD6, the gene encoding Frizzled 6. FZD6 belongs to a family of highly conserved membrane-bound WNT receptors involved in developmental processes and differentiation through several signaling pathways. We expressed the FZD6 missense mutation and observed a quantitative shift in subcellular distribution from the plasma membrane to the lysosomes, where the receptor is inaccessible for signaling and presumably degraded. Analysis of human fibroblasts homozygous for the nonsense mutation showed an aberrant response to both WNT-3A and WNT-5A stimulation; this response was consistent with an effect on both canonical and noncanonical WNT-FZD signaling. A detailed analysis of the Fzd6−/− mice, previously shown to have an altered hair pattern, showed malformed claws predominantly of the hind limbs. Furthermore, a transient Fdz6 mRNA expression was observed in the epidermis of the digital tips at embryonic day 16.5 during early claw morphogenesis. Thus, our combined results show that FZD6 mutations can result in severe defects in nail and claw formation through reduced or abolished membranous FZD6 levels and several nonfunctional WNT-FZD pathways.

Main Text

Congenital nail abnormalities are most often part of ectodermal syndromes involving several epidermal appendages, whereas isolated and inherited nail dysplasias are very rare.1 Nail development is initiated at embryonic week 9 by mesenchymal condensation in the dorsal part of the distal digital tip. This is followed by the formation of a transverse nail fold while the underlying matrix primordium expands. The matrix induces the nail bed and, subsequently, the formation of the nail plate.2 The formation of nails is initiated in the upper limb and then proceeds to the hind limb, and the morphogenesis is similar in primates and rodents. The molecular mechanisms underlying these processes are poorly understood, but recent studies have shown that WNT-FZD signaling is important for the formation of ectodermal appendages, including nails.3–6 In humans, mutations in the WNT-signaling regulator PORCN (MIM 300651) are associated with focal dermal hypoplasia (FDH) (MIM 305600),7 and mutations in the FZD agonists RSPO4 (MIM 610573) and RSPO1 (MIM 609595) are identified in both isolated anonychia (MIM 206800)8 and palmoplantar hyperkeratosis with sex reversal,9 respectively. Furthermore, WNT10A (MIM 6062689) mutations are associated with odontoonychodermal dysplasia (OODD) (MIM 257980) and ectodermal syndromes,10–12 whereas mutations in the WNT-associated transcription factors LMX1B (MIM 602575) and MSX1 (MIM 142983), involved in patterning and nail bed formation, cause nail-patella (MIM 161200) and Witkop syndrome (MIM 189500), respectively.3,13,14

To gain insight into molecular mechanisms regulating nail development, we investigated two consanguineous Pakistani families (F1 and F2) affected by autosomal-recessive isolated nail dysplasia (Figure 1A). Family F1 included four affected individuals, and family F2 included seven affected individuals. The affected individuals from family F1 presented with a more severe nail dysplasia compared to affected individuals from family F2 (Figures 1B and 1C). All affected individuals showed a variable degree of onychauxis (thick nails), hyponychia, and onycholysis of fingernails and toenails. Fingernails had a claw-like appearance. No other disorders or anomalies of ectodermal tissues (i.e., hair, teeth, sweat glands, or skin) were noted and individuals with dysplastic nails had normal hearing, normal psychomotor development and reported normal sweating as well as normal hair growth. Four affected individuals from each family were available for clinical examinations. Available parents to individuals with nail dysplasia presented with normal nail morphology. Informed consent was obtained from all individuals who participated in this study, and the protocol was approved by the local ethical board at NIBGE, Faisalabad, Pakistan.

Figure 1.

Figure 1

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Pedigrees and Phenotypes of Families Affected by Nail Dysplasia

(A) Both families are consanguineous Pakistani pedigrees, and individuals who were examined and sampled for genetic analysis are indicated with numbers. Affected individuals are shown as filled symbols. Marker haplotypes on chromosome 8 spanning FZD6 locus are shown below the symbols and were generated as described.16,17

(B) Hands and feet of individual V:1 in family F1 showing onychauxis, hyponychia, and onycholysis of both finger- and toenails. Fingernails are claw-shaped.

(C) Hands and feet of individual III:5 in family F2. Onychauxis and onycholysis of both fingernails and toenails are less severe in individual III:5 than in individuals from family F1.

Blood samples were collected for DNA extraction from 22 available members of families F1 and F2, and we initially genotyped DNA samples from the four affected family members in family F1 by using the GeneChip Human Mapping 250K SNP Array. Autozygosity mapping15 revealed one large homozygous region spanning 17 Mb on the long arm of chromosome 8 (1570 SNPs). Within this region, affected individuals of family F2 were homozygous over 800 kb (66 SNPs) (Figure S1A, available online). We performed genetic linkage analysis by using polymorphic microsatellite markers16,17 on the long arm of chromosome 8 and obtained a maximum cumulative two-point LOD score of 3.87 (Θ = 0). The analysis confirmed homozygosity for the region in affected family members, and each family had a distinct haplotype. The 800 kb region spans eight genes, including FZD6 (accession number NM_003506). We considered FZD6 a good candidate on the basis of previous findings showing involvement of WNT-FZD signaling in inherited anonychia as well as the role of FZD6 in hair patterning in mice.8,18 Sequencing of all seven exons of FZD6 revealed homozygosity for a nonsense mutation (c.1750G>T [p.Glu584X]; NM_003506.3) in the four affected members of family F1. In family F2 we identified a missense mutation (c.1531C>T [p.Arg511Cys]; NM_003506.3) homozygous in the four available and affected members (Figures 2A and 2B). The potential functional importance of the missense mutation is supported by the fact that the arginine residue is conserved in several species, including dogs, cattle and mice (Figure 2C). In each family, available parents of affected individuals were heterozygous for the respective mutation and unaffected siblings were either heterozygous for the mutation or homozygous for the wild-type (WT) sequence. The mutations were not present in chromosomes from 94 healthy unrelated Pakistani subjects or in chromosomes from 110 Swedish blood donors.

Figure 2.

Figure 2

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FZD6 Mutations Associated with Autosomal-Recessive Nail Dysplasia in Families F1 and F2

(A) The seven transmembrane FZD6 proteins showing positions of the two predicted mutations p.Arg511C and p.Glu584X in the intracellular domain (top). Boxes illustrate sequence chromatograms flanking the mutations (indicated with asterisks) from controls, carriers, and affected individuals in F1 (right box) and F2 (left box). The corresponding amino acid sequences are shown for WT FZD6 (above boxes) and mutated FZD6 (below boxes). The missense mutation is located 7 amino acids toward C-terminal of the conserved PDZ-binding motif (KTxxxW). Bidirectional sequence analysis of FZD6 included the seven exons, 5′ and 3′ UTRs, as well as the exon-intron boundaries on genomic DNA from individuals in families F1 and F2. The two identified mutations were not observed in 94 Pakistani and 110 Swedish control individuals. Primer sequences are available upon request.

(B) Multiple species alignment of orthologous FZD6 sequences from seven vertebrate species. The diagram shows residues 487–533 in human FZD6 (top) spanning the missense mutation p.Arg511C (gray area) and the corresponding amino acid sequence of orthologs. The following abbreviations are used: H.s., Homo sapiens; P.t., Pan troglodytes; C.f., Canis lupus familiaris, B.t., Bos taurus; M.m., Mus musculus; R.n., Rattus norvegicus; D.r., Danio rerio. The PDZ-binding motif is boxed.

FZD6 has previously shown to be important for the morphogenesis of hair follicles in both Drosophila and mice.18 Fzd6−/− mice present with abnormal macroscopic hair whorls, and Fzd3−/−; Fzd6−/− double-mutant mice have a disturbed pattern of inner-ear sensory hair cells specifying a role for FZD6 in planar-cell polarity.18,19 We reinvestigated the mouse model and found that about 50% of male, but not female, Fzd6−/− mice displayed abnormal claw morphology or absent claws when compared to WT mice (Figure 3A). At the age of 2 to 3 months, the claws disappeared or became rudimentary on the hind limbs. To further link expression of Fzd6 to early nail development, we investigated Fzd6 expression in mouse embryos at embryonic day (E) 14.5, 15.5, 16.5, and 17.5 at the time of early nail morphogenesis. Besides a general Fzd6 mRNA expression in murine skin,18 we observed a transient Fzd6 mRNA expression at the tip of the digits around E16.5, (Figure 3B) supporting a role for FZD6 in nail development. Microscopy revealed that the transient Fzd6 mRNA expression is mainly confined to the epidermis of the digital tips in a region corresponding to the developing nail bed and the ventral part of the digit (Figure 3C).

Figure 3.

Figure 3

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Hind-Limb Examination of the Fzd6−/− Male Mice

(A) Hind paws (left panel) and single digits (middle and right panel) of adult WT mice (upper panel) and adult Fzd6−/− mice (lower panel). Fzd6−/− mice display claw defects illustrated by absent or rudimentary claws (indicated by arrows). The boundaries of claw and claw rudiments, respectively, are marked (rightmost panel).

(B) The X-Gal histochemical staining of embryonic hind limbs at E14.5, 15.5, 16.5, and 17.5 in mice heterozygous for Fzd6-nlacZ (Fzd6+/−). Transient X-Gal expression (blue) is shown at the digital tip (indicated by arrow) at E16.5 during nail morphogenesis. The X-Gal staining was performed in standard X-Gal staining solution as described.18

(C) Immunohistochemical staining of sectioned mice hind limbs from (B) showing X-Gal expression (brown) confined mainly to the epidermis (indicated by red arrow) around the digital tip and with a few expressed foci in the dermis. Sections are stained with α-X-Gal antibody (Abcam) and counterstained with hematoxylin (blue). The green asterisk denotes the digital tip and red asterisk denotes the dorsal part. The X-gal stained hind legs were paraffin embedded, sectioned, and deparaffinized according to standard procedures. Slides were scanned with the Mirax MIDI scanner (Zeiss) and analyzed with the MIRAX viewer 1.11 software.

To determine the functional effects of the mutations, we obtained primary fibroblasts from one individual homozygous for the FZD6 nonsense mutation. The mutant fibroblasts expressed 13% of FZD6 mRNA levels in primary control fibroblasts supporting nonsense-mediated mRNA decay (Figure S1B). We were unable to obtain primary fibroblasts homozygous for the c.1531C>T missense mutation. We next designed and expressed the WT as well as the two mutant variants of FZD6 (WT; [FZD6(wt)], p.Glu584X [FZD6(nonsense)] and p.Arg511Cys [FZD6(missense)]) fused to green fluorescent protein (GFP) in HEK293T cells. Similar GFP levels were observed in cells expressing the FZD6(wt) or FZD6(missense) variants, suggesting that the missense mutation has no or little effect on total FZD6 levels. No expression was detected from FZD6(nonsense)-GFP (data not shown).

FZD6 belongs to the heptahelical class of FZD receptors with an internal PDZ-interacting motif necessary for the recruitment of the phosphoproteins Dishevelled (DVL) 1–3 (MIM 601365; 602151; 601368) and other signaling factors as well as for trafficking of the receptor.20,21 Both FZD6 mutations result in alterations of the intracellular tail, and we hypothesized that FZD6 with the missense mutation might show (1) an abnormal turnover rate (2) an improper integration into the cell membrane, and/or (3) reduced capability for DVL recruitment. To further clarify any effects of the FZD6 missense variant, we analyzed the subcellular localization of FZD6(wt)-GFP and the FZD6(missense)-GFP by confocal imaging. Expression of the two FZD6-GFP variants in HEK293T cells revealed a clear difference in subcellular distribution. The FZD6(wt)-GFP was localized predominantly in the plasma membrane, and a few cells showed intracellular vesicles, whereas the FZD6(missense)-GFP showed the opposite pattern, that is the majority of cells had FZD6(missense)-GFP confined to intracellular vesicles (Figure 4A). In order to quantify the observed difference, we counted cells with plasma membrane, plasma membrane/vesicular, or predominantly vesicular patterns. Quantification revealed that 71% of FZD6(wt)-GFP-expressing cells had a membranous pattern, whereas only 14% of the FZD6(missense)-GFP transfected cells expressed the receptor in the membrane (Figure 4B). Conversely, 14% of the cells transfected with the FZD6(wt)-GFP showed a predominantly vesicular distribution; whereas 63% of cells expressing the mutant receptor showed a vesicular distribution. This suggested a quantitative defect of the FZD6 missense variant because of defects in intracellular transportation, impaired integration into the plasma membrane, accelerated internalization, and/or an increased degradation.

Figure 4.

Figure 4

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FZD6-GFP Missense Localizes Predominantly to Intracellular Vesicles of Lysosomal Character

(A) Overexpression of FZD6(wt)-GFP and FZD6(missense)-GFP in HEK293 cells results in plasmamembrane, plasmamembrane/vesicular, and vesicular receptor localization as assessed by confocal imaging.

(B) Quantification of cell categories according to receptor localization shows that FZD6(wt)-GFP is predominantly localized to the plasma membrane, whereas FZD6(missense)-GFP is localized to intracellular vesicles. Quantification was performed on 200 cells per transfection in three independent experiments. The mean values ± standard deviation (SD) were 70.7 ± 2.8 (FZD6[wt]membr); 14.3 ± 2.4 (FZD6[missense]membr), 15.5 ± 1.3 (FZD6[wt]membr to vesic), 22.5 ± 2.6 (FZD6[missense]membr to vesic), 13.8 ± 1.5 (FZD6[wt]vesic),and 63.2 ± 1.4 (FZD6[missense]vesic). Error bars denote ± SD.

(C) Colocalization of FZD6(wt)-GFP and FZD6(missense)-GFP transiently expressed in HEK293 cells with organelle markers (clathrin: clathrin-coated pits and early endosomes; transferrin: plasmamembrane and recycling endosomes; caveolin-1: caveolae; RAB5: early endosomes; TGN46: trans-Golgi network; lysotracker: lysosomes). Arrows indicate apparent colocalization. The scale bar indicates 10 μm. The human FZD6 construct was generated from the cDNA clone (IRAKp961D01133Q, imaGenes) and introduced into the vectors pAcGFP-N1 (FZD6). The FZD6 missense mutation (c.1531C>T) was introduced by in vitro mutagenesis (QuickChange II Site-Directed Mutagenesis Kit, Stratagene) into the pFZD6(wt)-GFP clone according to manufacturer's instructions. Transfection of HEK293T cells was performed with the calcium phosphate method and analyzed for GFP and expression by fluorescence microscopy on an LSM710 confocal microscope (Zeiss) 2 days after transfection. The following antibodies and markers were used for intracellular organelles: rabbit anti-clathrin (Abcam, ab14408), rabbit anti-caveolin-1 (Abcam, ab2910), rabbit anti-RAB5 (Abcam, ab13252), mouse anti-TGN46 (Abcam, ab2809), lysotracker DN-99, and Alexa555-coupled transferrin (Invitrogen).

The high proportion of cells expressing FZD6(missense)-GFP localized to intracellular vesicles made us hypothesize that the vesicles could belong to either the endosomal, caveosomal, or lysosomal pathway. In order to clarify the nature of the FZD6-GFP-positive intracellular vesicles, we performed colocalization experiments by using transferrin and antibodies against clathrin, caveolin-1, RAB5, the trans-Golgi network marker TGN46, and a lysosomal marker, lysotracker, in combination with the FZD6(wt)-GFP and FZD6(missense)-GFP, respectively. We could not detect any overlap with clathrin-, caveolin-1-, or RAB5-labeling endosomal compartments (Figure 4C). Furthermore, the trans-Golgi network marker TGN46 did not colocalize with GFP, indicating that the vesicular structures do not represent compartments in the excocytotic pathway. A partial colocalization was observed for GFP and transferrin, which is bound to the plasma membrane and some endosomal compartments. In contrast, we observed a clear colocalization for both FZD6(wt)-GFP and FZD6(missense)-GFP and lysotracker. This shows that the intracellular vesicles, observed predominantly in FZD6(missense)-GFP-expressing cells, are lysosomes. Thus, our observations indicate a quantitative shift of FZD6(missense)-GFP from the plasma membrane to lysosomes consistent with increased lysosomal degradation.22

FZDs contain a highly conserved KTxxxW sequence in the C terminus, just upstream of the mutated residue in missense FZD6.21 This domain is crucial for intracellular recruitment and interaction with Dishevelled (DVL) proteins, and we asked whether the recruitment of DVL is affected by the missense mutation. FZD6(wt)-GFP and FZD6(missense)-GFP were coexpressed with DVL1-FLAG in HEK293 cells. In the absence of FZD6 expression, DVL1-FLAG-positive cells showed characteristic aggregates in the cytosol in addition to a smaller percentage of cells with even distribution of DVL1-FLAG (Figure 5).23 Coexpression with FZD6(wt)-GFP induced a radical membrane recruitment of DVL1-FLAG. Interestingly, the membrane recruitment was also observed in the presence of FZD6(missense)-GFP, arguing that the mutated receptor is capable to interact with DVL. This is supported by immunoprecipitation showing that myc-FZD6(missense) binds to DVL2-3 (Figure S2). Moreover, intracellular FZD6(missense)-GFP within the lysosomal vesicles did not attract DVL1-FLAG (Figure 5), supporting the notion that FZD6 internalized into lysosomes is inactivated. In addition, employing indirect immunocytochemistry with an FLAG antibody and a Cy3-conjugated secondary antibody, we investigated the Förster resonance energy transfer (FRET) between FZD6-GFP and DVL1-FLAG in fixed cells after photoacceptor bleaching. FRET between FZD6(missense)-GFP and DVL1-FLAG was reduced in comparison to FZD6(wt)-GFP coexpressed with DVL1-FLAG (Figure S3). The combined results indicate that the missense mutation leads to increased internalization of the receptor and subsequent lysosomal degradation and thus a net reduction of FZD6 receptors in the plasma membrane.

Figure 5.

Figure 5

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FZD6-GFP-Mediated Recruitment of DVL1-FLAG

Overexpression of DVL1-FLAG in the absence of FZD6 in HEK293 results in punctuate DVL1-FLAG localization as shown in confocal microphotographs (upper panel). Coexpression of DVL1-FLAG with either FZD6(wt)-GFP or FZD6(missense)-GFP induces a redistribution and a recruitment of DVL1-FLAG to the plasma membrane (mid- and lower right). DVL1-FLAG is N-terminally tagged (from Madelon Maurice, Utrecht, The Netherlands). The scale bar indicates 10 μm.

WNT-FZD signaling constitutes a network of autocrine and paracrine pathways regulated via several feedback loops.21,24,25 The best known signaling pathway downstream of FZD is the WNT-β-catenin pathway leading to the stabilization of β-catenin and TCF/LEF-dependent transcription.26 However, it has been shown that FZD6 has the ability to interact with both WNT-3A (MIM 606359) and WNT-5A (MIM 164975), previously shown to recruit β-catenin-dependent as well as β-catenin-independent pathways, respectively.27 To further investigate the effect of mutated FZD6 on downstream signaling, we stimulated primary WT fibroblasts and patient fibroblasts homozygous for the FZD6 nonsense mutation with either WNT-3A or WNT-5A. WNT-3A stimulation resulted in an upregulation of β-catenin levels in control fibroblasts but not in patient fibroblasts (Figures 6A). This supports that FZD6 mediates β-catenin-dependent signaling and that FZD6-null mutant cells have lost their capability to respond to WNT-3A stimulation. Next, we analyzed levels of the WNT-signaling antagonist DKK1 in control and FZD6-mutated fibroblasts stimulated with WNT-3A or WNT-5A. After WNT-5A activation we observed upregulated levels of DKK1 mRNA in control fibroblasts but not in FZD6-null fibroblasts (Figure 6B). This suggests a perturbed response also to β-catenin-independent WNT-5A stimulation in the absence of FZD6. DKK1 and several other WNT-signaling inhibitors were recently shown to be induced by the transcription factor MSX1.28 In agreement with the results from DKK1 mRNA analysis, we observed increased MSX1 mRNA levels in WNT-5A-stimulated control fibroblasts but not in patient fibroblasts (Figure 6C). In combination, these results suggest that patient fibroblasts homozygous for the FZD6 nonsense mutation fail to respond properly to both WNT-3A and WNT-5A activation.

Figure 6.

Figure 6

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Effect of WNT-3A and WNT-5A Stimulation On Primary Fibroblast Cultures from an Affected Individual (V:1 form Family F1) and a Healthy Control

(A) β-catenin levels in control fibroblasts and in fibroblasts homozygous for the nonsense mutation p.Glu584X without stimulation (−) and after WNT-3A or WNT-5A stimulation (+). The graphs represent the relative amounts of β-catenin to β-actin as determined by immunoblot analysis (bottom). β-catenin accumulates upon WNT-3A stimulation in control cells but not in FZD6-null mutant cells (∗p < 0.05).

(B) Effect of WNT-5A stimulation on DKK1 expression. Quantitative RT/PCR analysis from total RNA isolated from control and mutated fibroblasts stimulated as in (A) shows that DKK1 is upregulated in control cells upon stimulation with WNT-5A but not WNT-3A (∗p < 0.05). Mutated fibroblasts do not show increased DKK1 transcript levels in response to WNT-5A.

(C) Effect of WNT-5A stimulation on MSX1 expression analyzed as in (B). MSX1 is upregulated in control fibroblasts but not in patient derived cells (∗p < 0.05). Error bars (in A–C) denote means ± SD.

Fibroblasts were cultured in either WNT-3A- or WNT-5A-conditioned RPMI medium, respectively, at 90% confluence and harvested after 24 hr and 48 hr, respectively. Levels of DKK1 and MSX1 transcripts were analyzed with the Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen) following manufacturers recommendations. For analysis of β-catenin expression, cells were lyzed in RIPA buffer (50 mM Tris/Cl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 1% Sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS]). Protein samples were separated on a 10% SDS-PAGE (NuPage, Invitrogen) and transferred to PVDF membranes (iBLOT transfer system, Invitrogen). Proteins were detected with primary α-β-catenin (Santa Cruz Biotechnology) and α-β-actin antibodies (Abcam), respectively. Proteins were visualized and quantified with the Odyssey infrared imaging system as previously described.36 Data obtained from independent tissue cultures were pooled and analyzed with student's two-tailed t test.

During normal nail formation, the nail plate must be properly attached to the nail bed. This process is presumably dependent on tight interaction and signaling between mesenchymal (matrix) cells forming the nail plate and the epidermal (nail bed) cells. Affected individuals in the F1 and F2 families have a retained regenerative capacity of the nail plate; this capacity suggests that the nail matrix is intact. The primary defect associated with FZD6 mutations seems to be related to perturbed formation and attachment of the nail plate shown as onycholysis. This is also consistent with the observations in the Fzd6−/− mice with claws that are easily lost with age and mechanical stress. The reason for the restriction of the claw phenotype to male mice is unclear, but one explanation is a more aggressive behavior. However, it cannot be excluded that altered WNT signaling caused by disrupted Fzd6 has a gender-restricted effect as shown for human RSPO1 mutations.9

WNT-FZD signaling is indispensable for numerous developmental processes such as tissue morphogenesis, differentiation, and regeneration in all animals.26 It has become evident that many WNT-FZD interactions and their downstream pathways are integrated in extensive cross-talks with shared components.21,29,30 Furthermore, FZDs have the ability to elicit different cellular responses in different environments.31 Previous studies have shown that FZD6 can act as a negative regulator of β-catenin signaling32 as well as a mediator of the planar cell polarity (PCP) pathway.18 We show here that FZD6-null mutant cells fail to respond to both WNT-3A and WNT-5A stimulation in vitro when compared to control cells. Hence, FZD6 can elicit both β-catenin-dependent and -independent signaling presumably determined by the local dosage of ligands, other FZDs, and coreceptors, as suggested from other studies.33–35 Furthermore, subcellular studies show that FZD6 containing the missense mutation is localized predominantly in lysosomes when compared to the WT FZD6. Thus, the FZD6 missense variant is presumably subject to increased internalization and subsequent degradation and a reduced capability to interpret WNT stimulation. In conclusion, our combined results show that isolated nail dysplasia can be caused by dysfunctional FZD6 or loss of FZD6 with a subsequent misregulation of several FZD6-mediated pathways required for proper formation and regeneration of nails throughout life.

Acknowledgments

We thank all family members who participated in this study. Mice targeted for Fzd6 were generously shared by Jeremy Nathans, Howard Hughes Medical Institute and Johns Hopkins University. Wnt producing 3T3 fibroblast lines were a gift from Peter Mark, Universitätklinikum Rostock. This work was supported by grants from the Swedish Research Council (K2010-66X-10829-17-3) and Swedish Links, Asia (348-2008-6069), the Indevelops fund, Thuréus fund, Uppsala University Hospital, Uppsala University, and the Science for Life Laboratory. M.B.C.K. was supported by the Karolinska Institutet's Keratitis, Ichthyosis, and Neurosensory Deafness Syndrome program; J.K. was supported by the Swedish Society for Medical Research, and M.S. was supported by the Swedish Institute. Work in the laboratory of G.S. was supported by the Swedish Research Council (K2008-68P-20810-01-4, K2008-68X-20805-01-4), Swedish Cancer Society (CAN 2008/539), and the Knut and Alice Wallenberg Foundation (KAW2008.0149).

Supplemental Data

Document S1. Three Figures
mmc1.pdf (262.8KB, pdf)

Web Resources

The URLs for data presented herein are as follows:

Accession Numbers

The GenBank accession numbers for human FZD6 and human FZD6 protein, human chromosome 8 clone containing FZD6, human FZD6 variant c.1531C>T, and human FZD6 variant c.1750G>T are NM_003506 and NP_003497, AC025370.12, NM_003506.3, and NM_003506.3, respectively.

References

  • 1.Hamm H., Karl S., Bröcker E.B. Isolated congenital nail dysplasia: a new autosomal dominant condition. Arch. Dermatol. 2000;136:1239–1243. doi: 10.1001/archderm.136.10.1239. [DOI] [PubMed] [Google Scholar]
  • 2.Seitz C.S., Hamm H. Congenital brachydactyly and nail hypoplasia: clue to bone-dependent nail formation. Br. J. Dermatol. 2005;152:1339–1342. doi: 10.1111/j.1365-2133.2005.06509.x. [DOI] [PubMed] [Google Scholar]
  • 3.Duverger O., Morasso M.I. Role of homeobox genes in the patterning, specification, and differentiation of ectodermal appendages in mammals. J. Cell. Physiol. 2008;216:337–346. doi: 10.1002/jcp.21491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Hill T.P., Taketo M.M., Birchmeier W., Hartmann C. Multiple roles of mesenchymal beta-catenin during murine limb patterning. Development. 2006;133:1219–1229. doi: 10.1242/dev.02298. [DOI] [PubMed] [Google Scholar]
  • 5.Kawakami Y., Capdevila J., Büscher D., Itoh T., Rodríguez Esteban C., Izpisúa Belmonte J.C. WNT signals control FGF-dependent limb initiation and AER induction in the chick embryo. Cell. 2001;104:891–900. doi: 10.1016/s0092-8674(01)00285-9. [DOI] [PubMed] [Google Scholar]
  • 6.Soshnikova N., Zechner D., Huelsken J., Mishina Y., Behringer R.R., Taketo M.M., Crenshaw E.B., 3rd, Birchmeier W. Genetic interaction between Wnt/beta-catenin and BMP receptor signaling during formation of the AER and the dorsal-ventral axis in the limb. Genes Dev. 2003;17:1963–1968. doi: 10.1101/gad.263003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Grzeschik K.H., Bornholdt D., Oeffner F., König A., del Carmen Boente M., Enders H., Fritz B., Hertl M., Grasshoff U., Höfling K. Deficiency of PORCN, a regulator of Wnt signaling, is associated with focal dermal hypoplasia. Nat. Genet. 2007;39:833–835. doi: 10.1038/ng2052. [DOI] [PubMed] [Google Scholar]
  • 8.Blaydon D.C., Ishii Y., O'Toole E.A., Unsworth H.C., Teh M.T., Rüschendorf F., Sinclair C., Hopsu-Havu V.K., Tidman N., Moss C. The gene encoding R-spondin 4 (RSPO4), a secreted protein implicated in Wnt signaling, is mutated in inherited anonychia. Nat. Genet. 2006;38:1245–1247. doi: 10.1038/ng1883. [DOI] [PubMed] [Google Scholar]
  • 9.Parma P., Radi O., Vidal V., Chaboissier M.C., Dellambra E., Valentini S., Guerra L., Schedl A., Camerino G. R-spondin1 is essential in sex determination, skin differentiation and malignancy. Nat. Genet. 2006;38:1304–1309. doi: 10.1038/ng1907. [DOI] [PubMed] [Google Scholar]
  • 10.Adaimy L., Chouery E., Megarbane H., Mroueh S., Delague V., Nicolas E., Belguith H., de Mazancourt P., Megarbane A. Mutation in WNT10A is associated with an autosomal recessive ectodermal dysplasia: the odonto-onycho-dermal dysplasia. Am. J. Hum. Genet. 2007;81:821–828. doi: 10.1086/520064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Bohring A., Stamm T., Spaich C., Haase C., Spree K., Hehr U., Hoffmann M., Ledig S., Sel S., Wieacker P., Röpke A. WNT10A mutations are a frequent cause of a broad spectrum of ectodermal dysplasias with sex-biased manifestation pattern in heterozygotes. Am. J. Hum. Genet. 2009;85:97–105. doi: 10.1016/j.ajhg.2009.06.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Nawaz S., Klar J., Wajid M., Aslam M., Tariq M., Schuster J., Baig S.M., Dahl N. WNT10A missense mutation associated with a complete odonto-onycho-dermal dysplasia syndrome. Eur. J. Hum. Genet. 2009;17:1600–1605. doi: 10.1038/ejhg.2009.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Dreyer S.D., Zhou G., Baldini A., Winterpacht A., Zabel B., Cole W., Johnson R.L., Lee B. Mutations in LMX1B cause abnormal skeletal patterning and renal dysplasia in nail patella syndrome. Nat. Genet. 1998;19:47–50. doi: 10.1038/ng0598-47. [DOI] [PubMed] [Google Scholar]
  • 14.Jumlongras D., Bei M., Stimson J.M., Wang W.F., DePalma S.R., Seidman C.E., Felbor U., Maas R., Seidman J.G., Olsen B.R. A nonsense mutation in MSX1 causes Witkop syndrome. Am. J. Hum. Genet. 2001;69:67–74. doi: 10.1086/321271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Carr I.M., Flintoff K.J., Taylor G.R., Markham A.F., Bonthron D.T. Interactive visual analysis of SNP data for rapid autozygosity mapping in consanguineous families. Hum. Mutat. 2006;27:1041–1046. doi: 10.1002/humu.20383. [DOI] [PubMed] [Google Scholar]
  • 16.Entesarian M., Carlsson B., Mansouri M.R., Stattin E.L., Holmberg E., Golovleva I., Stefansson H., Klar J., Dahl N. A chromosome 10 variant with a 12 Mb inversion [inv(10)(q11.22q21.1)] identical by descent and frequent in the Swedish population. Am. J. Med. Genet. A. 2009;149A:380–386. doi: 10.1002/ajmg.a.32663. [DOI] [PubMed] [Google Scholar]
  • 17.Klar J., Schweiger M., Zimmerman R., Zechner R., Li H., Törmä H., Vahlquist A., Bouadjar B., Dahl N., Fischer J. Mutations in the fatty acid transport protein 4 gene cause the ichthyosis prematurity syndrome. Am. J. Hum. Genet. 2009;85:248–253. doi: 10.1016/j.ajhg.2009.06.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Guo N., Hawkins C., Nathans J. Frizzled6 controls hair patterning in mice. Proc. Natl. Acad. Sci. USA. 2004;101:9277–9281. doi: 10.1073/pnas.0402802101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Wang Y., Guo N., Nathans J. The role of Frizzled3 and Frizzled6 in neural tube closure and in the planar polarity of inner-ear sensory hair cells. J. Neurosci. 2006;26:2147–2156. doi: 10.1523/JNEUROSCI.4698-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Schulte G., Bryja V. The Frizzled family of unconventional G-protein-coupled receptors. Trends Pharmacol. Sci. 2007;28:518–525. doi: 10.1016/j.tips.2007.09.001. [DOI] [PubMed] [Google Scholar]
  • 21.Schulte G. International Union of Basic and Clinical Pharmacology. LXXX. The class Frizzled receptors. Pharmacol. Rev. 2010;62:632–667. doi: 10.1124/pr.110.002931. [DOI] [PubMed] [Google Scholar]
  • 22.Bross P., Corydon T.J., Andresen B.S., Jørgensen M.M., Bolund L., Gregersen N. Protein misfolding and degradation in genetic diseases. Hum. Mutat. 1999;14:186–198. doi: 10.1002/(SICI)1098-1004(1999)14:3<186::AID-HUMU2>3.0.CO;2-J. [DOI] [PubMed] [Google Scholar]
  • 23.Bryja V., Schulte G., Rawal N., Grahn A., Arenas E. Wnt-5a induces Dishevelled phosphorylation and dopaminergic differentiation via a CK1-dependent mechanism. J. Cell Sci. 2007;120:586–595. doi: 10.1242/jcs.03368. [DOI] [PubMed] [Google Scholar]
  • 24.Macdonald B.T., Semenov M.V., He X. SnapShot: Wnt/beta-catenin signaling. Cell. 2007;131:1204. doi: 10.1016/j.cell.2007.11.036. [DOI] [PubMed] [Google Scholar]
  • 25.Semenov M.V., Habas R., Macdonald B.T., He X. SnapShot: Noncanonical Wnt Signaling Pathways. Cell. 2007;131:1378. doi: 10.1016/j.cell.2007.12.011. [DOI] [PubMed] [Google Scholar]
  • 26.Logan C.Y., Nusse R. The Wnt signaling pathway in development and disease. Annu. Rev. Cell Dev. Biol. 2004;20:781–810. doi: 10.1146/annurev.cellbio.20.010403.113126. [DOI] [PubMed] [Google Scholar]
  • 27.Yamamoto S., Nishimura O., Misaki K., Nishita M., Minami Y., Yonemura S., Tarui H., Sasaki H. Cthrc1 selectively activates the planar cell polarity pathway of Wnt signaling by stabilizing the Wnt-receptor complex. Dev. Cell. 2008;15:23–36. doi: 10.1016/j.devcel.2008.05.007. [DOI] [PubMed] [Google Scholar]
  • 28.Revet I., Huizenga G., Koster J., Volckmann R., van Sluis P., Versteeg R., Geerts D. MSX1 induces the Wnt pathway antagonist genes DKK1, DKK2, DKK3, and SFRP1 in neuroblastoma cells, but does not block Wnt3 and Wnt5A signalling to DVL3. Cancer Lett. 2010;289:195–207. doi: 10.1016/j.canlet.2009.08.019. [DOI] [PubMed] [Google Scholar]
  • 29.McNeill H., Woodgett J.R. When pathways collide: collaboration and connivance among signalling proteins in development. Nat. Rev. Mol. Cell Biol. 2010;11:404–413. doi: 10.1038/nrm2902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.van Amerongen R., Nusse R. Towards an integrated view of Wnt signaling in development. Development. 2009;136:3205–3214. doi: 10.1242/dev.033910. [DOI] [PubMed] [Google Scholar]
  • 31.Ye X., Wang Y., Cahill H., Yu M., Badea T.C., Smallwood P.M., Peachey N.S., Nathans J. Norrin, frizzled-4, and Lrp5 signaling in endothelial cells controls a genetic program for retinal vascularization. Cell. 2009;139:285–298. doi: 10.1016/j.cell.2009.07.047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Golan T., Yaniv A., Bafico A., Liu G., Gazit A. The human Frizzled 6 (HFz6) acts as a negative regulator of the canonical Wnt. beta-catenin signaling cascade. J. Biol. Chem. 2004;279:14879–14888. doi: 10.1074/jbc.M306421200. [DOI] [PubMed] [Google Scholar]
  • 33.Mikels A.J., Nusse R. Purified Wnt5a protein activates or inhibits beta-catenin-TCF signaling depending on receptor context. PLoS Biol. 2006;4:e115. doi: 10.1371/journal.pbio.0040115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Nikolova T., Wu M., Brumbarov K., Alt R., Opitz H., Boheler K.R., Cross M., Wobus A.M. WNT-conditioned media differentially affect the proliferation and differentiation of cord blood-derived CD133+ cells in vitro. Differentiation. 2007;75:100–111. doi: 10.1111/j.1432-0436.2006.00119.x. [DOI] [PubMed] [Google Scholar]
  • 35.Stoick-Cooper C.L., Moon R.T., Weidinger G. Advances in signaling in vertebrate regeneration as a prelude to regenerative medicine. Genes Dev. 2007;21:1292–1315. doi: 10.1101/gad.1540507. [DOI] [PubMed] [Google Scholar]
  • 36.Schuster J., Karlsson T., Karlström P.O., Poromaa I.S., Dahl N. Down-regulation of progesterone receptor membrane component 1 (PGRMC1) in peripheral nucleated blood cells associated with premature ovarian failure (POF) and polycystic ovary syndrome (PCOS) Reprod. Biol. Endocrinol. 2010;8:58. doi: 10.1186/1477-7827-8-58. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

Document S1. Three Figures
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