Mutations in Frizzled 6 Cause Isolated Autosomal-Recessive Nail Dysplasia

15 March 2005

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2386 East Heritage Way, Suite B, Salt Lake City, Utah 84109 USA Phone +1-877-628-7300 • Email—[email protected] www.pachyonychia.org REPORT Mutations in Frizzled 6 Cause Isolated Autosomal-Recessive Nail Dysplasia

Anne-Sophie Fro¨jmark,1 Jens Schuster,1,6 Maria Sobol,1,2,6 Miriam Entesarian,1,5,6 Michaela B.C. Kilander,3 Dana Gabrikova,1,7 Sadia Nawaz,4 Shahid M. Baig,4 Gunnar Schulte,3 Joakim Klar,1 and Niklas Dahl1,*

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 char- acterized 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.

Congenital nail abnormalities are most often part of ecto- tions in the WNT-associated transcription factors dermal syndromes involving several epidermal append- LMX1B (MIM 602575) and MSX1 (MIM 142983), ages, whereas isolated and inherited nail dysplasias are involved in patterning and nail bed formation, cause very rare.1 Nail development is initiated at embryonic nail-patella (MIM 161200) and Witkop syndrome (MIM week 9 by mesenchymal condensation in the dorsal 189500), respectively.3,13,14 part of the distal digital tip. This is followed by the forma- To gain insight into molecular mechanisms regulating tion of a transverse nail fold while the underlying matrix nail development, we investigated two consanguineous primordium expands. The matrix induces the nail bed Pakistani families (F1 and F2) affected by autosomal-reces- and, subsequently, the formation of the nail plate.2 The sive isolated nail dysplasia (Figure 1A). Family F1 included formation of nails is initiated in the upper limb and four affected individuals, and family F2 included seven then proceeds to the hind limb, and the morphogenesis affected individuals. The affected individuals from family is similar in primates and rodents. The molecular mecha- F1 presented with a more severe nail dysplasia compared nisms underlying these processes are poorly understood, to affected individuals from family F2 (Figures 1B and but recent studies have shown that WNT-FZD signaling 1C). All affected individuals showed a variable degree of is important for the formation of ectodermal appendages, onychauxis (thick nails), hyponychia, and onycholysis of including nails.3–6 In humans, mutations in the WNT- ﬁngernails and toenails. Fingernails had a claw-like appear- signaling regulator PORCN (MIM 300651) are associated ance. No other disorders or anomalies of ectodermal tissues with focal dermal hypoplasia (FDH) (MIM 305600),7 (i.e., hair, teeth, sweat glands, or skin) were noted and indi- and mutations in the FZD agonists RSPO4 (MIM viduals with dysplastic nails had normal hearing, normal 610573) and RSPO1 (MIM 609595) are identiﬁed in psychomotor development and reported normal sweating both isolated anonychia (MIM 206800)8 and palmoplan- as well as normal hair growth. Four affected individuals tar hyperkeratosis with sex reversal,9 respectively. Further- from each family were available for clinical examinations. more, WNT10A (MIM 6062689) mutations are associated Available parents to individuals with nail dysplasia pre- with odontoonychodermal dysplasia (OODD) (MIM sented with normal nail morphology. Informed consent 257980) and ectodermal syndromes,10–12 whereas muta- was obtained from all individuals who participated in

1Department of Immunology, Genetics and Pathology, The Rudbeck Laboratory and Science for Life Laboratory, Uppsala University, 751 85 Uppsala, Sweden; 2Department of General and Molecular Genetics, National Taras Shevchenko University of Kyiv, Kiev 03680, Ukraine; 3Department of Physiology and Pharmacology, Section for Receptor Biology and Signaling, Karolinska Institutet, 171 76 Stockholm, Sweden; 4Human Molecular Genetics Laboratory, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad 38000, Pakistan; 5Current address: Department of Women’s and Children’s Health Center for Molecular Medicine, Karolinska University Hospital, 171 76 Stockholm, Sweden 6These authors contributed equally to this work 7Current address: Department of Biology, Faculty of Natural Sciences, University of Presov, Presov 08001, Slovakia *Correspondence: [email protected] DOI 10.1016/j.ajhg.2011.05.013. Ó2011 by The American Society of Human Genetics. All rights reserved.

852 The American Journal of Human Genetics 88, 852–860, June 10, 2011 A F1 F2 I I

II II

III III 1 2 1 2 3 4 5 D8S521 3434 D8S521 23 31 2331 31 D8S1046 2121 D8S1046 11 11 1111 11 D8S1834 5452 D8S1834 13 33 1333 33 D8S385 1213 D8S385 41 12 3112 12 D8S1784 2223 D8S1784 11 12 1112 11

IV IV 3 3 5 5 6 1 2 3 4 1 2 3 4 5 D8S521 43 33 3213 D8S521 43 4313 12 33 D8S1046 12 22 2432 D8S1046 21 2111 11 11 D8S1834 25 55 5315 D8S1834 23 2333 31 33 D8S385 31 11 1121 D8S385 21 2111 23 11 D8S1784 32 22 2132 D8S1784 31 3111 21 11

V V 3 1 2 3 4 5 1 2 D8S521 33123333 32 D8S521 2333 D8S1046 22342222 34 D8S1046 1111 D8S1834 55135555 13 D8S1834 1333 D8S385 11211111 21 D8S385 4111 D8S1784 22312222 31 D8S1784 1111

Figure 1. 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. Finger- nails 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. this study, and the protocol was approved by the local the long arm of chromosome 8 (1570 SNPs). Within this ethical board at NIBGE, Faisalabad, Pakistan. region, affected individuals of family F2 were homozygous Blood samples were collected for DNA extraction from 22 over 800 kb (66 SNPs) (Figure S1A, available online). We per- available members of families F1 and F2, and we initially formed genetic linkage analysis by using polymorphic mi- genotyped DNA samples from the four affected family crosatellite markers16,17 on the long arm of chromosome members in family F1 by using the GeneChip Human 8 and obtained a maximum cumulative two-point LOD Mapping 250K SNP Array. Autozygosity mapping15 re- score of 3.87 (Q ¼ 0). The analysis confirmed homozygosity vealed one large homozygous region spanning 17 Mb on for the region in affected family members, and each family

The American Journal of Human Genetics 88, 852–860, June 10, 2011 853 had a distinct haplotype. The 800 kb region spans eight A -NH3 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 8,18 FZD6 in hair patterning in mice. Sequencing of all seven KTxxxW exons of FZD6 revealed homozygosity for a nonsense mutation (c.1750G>T [p.Glu584X]; NM_003506.3) in the four C affected members of family F1. In family F2 we identified a missense mutation (c.1531C>T [p.Arg511Cys]; HOOC X NM_003506.3) homozygous in the four available and FZ6 508 509 510 511 512 513 581 582 583 584 585 586 affected members (Figures 2A and 2B). The potential func- wt Lys Arg Asn Arg Lys Asp Leu GlnGly Glu Thr Leu tional importance of the missense mutation is supported Control c.1531 C/C Control c.1750 G/G by the fact that the arginine residue is conserved in several species, including dogs, cattle and mice (Figure 2C). In each Carrier c.1531 C/T Carrier c.1750 G/T family, available parents of affected individuals were heterozygous for the respective mutation and unaffected Affected c.1531 T/T Affected c.1750 T/T * siblings were either heterozygous for the mutation or * homozygous for the wild-type (WT) sequence. The muta- FZ6 Lys Arg Asn Cys Lys Asp Leu GlnGly Stop Thr Leu tions were not present in chromosomes from 94 healthy mutant 508 509 510 511 512 513 581 582 583 584 585 586 unrelated Pakistani subjects or in chromosomes from 110 Swedish blood donors. B KTxxxW p.R511C H.s. VGISAVFWVGSKKTCTEWAGFFKRNRKRDPISESRRVLQESCEFFLKH FZD6 has previously shown to be important for the P.t...... morphogenesis of hair follicles in both Drosophila and C.f...... B.t...... K...... mice.18 Fzd6 / mice present with abnormal macroscopic M.m...... / / R.n...... C...... hair whorls, and Fzd3 ; Fzd6 double-mutant mice D.r. ...P.....S.R...SR..S.CR.AH.K.AV...... have a disturbed pattern of inner-ear sensory hair cells 18,19 specifying a role for FZD6 in planar-cell polarity. We re- Figure 2. FZD6 Mutations Associated with Autosomal-Recessive investigated the mouse model and found that about 50% Nail Dysplasia in Families F1 and F2 / of male, but not female, Fzd6 mice displayed abnormal (A) The seven transmembrane FZD6 proteins showing positions of the two predicted mutations p.Arg511C and p.Glu584X in the claw morphology or absent claws when compared to WT intracellular domain (top). Boxes illustrate sequence chromato- mice (Figure 3A). At the age of 2 to 3 months, the claws dis- grams flanking the mutations (indicated with asterisks) from appeared or became rudimentary on the hind limbs. To controls, carriers, and affected individuals in F1 (right box) and further link expression of Fzd6 to early nail development, F2 (left box). The corresponding amino acid sequences are shown for WT FZD (above boxes) and mutated FZD (below boxes). The we investigated Fzd expression in mouse embryos at 6 6 6 missense mutation is located 7 amino acids toward C-terminal of embryonic day (E) 14.5, 15.5, 16.5, and 17.5 at the time the conserved PDZ-binding motif (KTxxxW). Bidirectional of early nail morphogenesis. Besides a general Fzd6 sequence analysis of FZD6 included the seven exons, 50 and 30 mRNA expression in murine skin,18 we observed a transient UTRs, as well as the exon-intron boundaries on genomic DNA from individuals in families F1 and F2. The two identified muta- Fzd6 mRNA expression at the tip of the digits around tions were not observed in 94 Pakistani and 110 Swedish control E16.5, (Figure 3B) supporting a role for FZD6 in nail devel- individuals. Primer sequences are available upon request. opment. Microscopy revealed that the transient Fzd6 (B) Multiple species alignment of orthologous FZD6 sequences mRNA expression is mainly confined to the epidermis of from seven vertebrate species. The diagram shows residues 487–533 in human FZD (top) spanning the missense mutation the digital tips in a region corresponding to the developing 6 p.Arg511C (gray area) and the corresponding amino acid sequence nail bed and the ventral part of the digit (Figure 3C). of orthologs. The following abbreviations are used: H.s., Homo To determine the functional effects of the mutations, we sapiens; P.t., Pan troglodytes; C.f., Canis lupus familiaris, B.t., Bos obtained primary fibroblasts from one individual homozy- taurus; M.m., Mus musculus; R.n., Rattus norvegicus; D.r., Danio rerio. The PDZ-binding motif is boxed. gous for the FZD6 nonsense mutation. The mutant fibroblasts expressed 13% of FZD6 mRNA levels in primary control fibroblasts supporting nonsense-mediated mRNA missense mutation has no or little effect on total FZD6 decay (Figure S1B). We were unable to obtain primary levels. No expression was detected from FZD6(nonsense)- fibroblasts homozygous for the c.1531C>T missense muta- GFP (data not shown). tion. We next designed and expressed the WT as well as the FZD6 belongs to the heptahelical class of FZD receptors two mutant variants of FZD6 (WT; [FZD6(wt)], p.Glu584X with an internal PDZ-interacting motif necessary for the

[FZD6(nonsense)] and p.Arg511Cys [FZD6(missense)]) recruitment of the phosphoproteins Dishevelled (DVL) fused to green ﬂuorescent protein (GFP) in HEK293T cells. 1–3 (MIM 601365; 602151; 601368) and other signaling Similar GFP levels were observed in cells expressing the factors as well as for trafﬁcking of the receptor.20,21 Both

FZD6(wt) or FZD6(missense) variants, suggesting that the FZD6 mutations result in alterations of the intracellular

854 The American Journal of Human Genetics 88, 852–860, June 10, 2011 / Figure 3. 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 þ (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 a-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 proce- dures. Slides were scanned with the Mirax MIDI scanner (Zeiss) and analyzed with the MIRAX viewer 1.11 software.

tail, and we hypothesized that FZD6 with the missense plasma membrane, plasma membrane/vesicular, or pre- mutation might show (1) an abnormal turnover rate (2) dominantly vesicular patterns. Quantiﬁcation revealed an improper integration into the cell membrane, and/or that 71% of FZD6(wt)-GFP-expressing cells had a membra-

(3) reduced capability for DVL recruitment. To further nous pattern, whereas only 14% of the FZD6(missense)- clarify any effects of the FZD6 missense variant, we GFP transfected cells expressed the receptor in the analyzed the subcellular localization of FZD6(wt)-GFP membrane (Figure 4B). Conversely, 14% of the cells trans- and the FZD6(missense)-GFP by confocal imaging. Expres- fected with the FZD6(wt)-GFP showed a predominantly sion of the two FZD6-GFP variants in HEK293T cells re- vesicular distribution; whereas 63% of cells expressing vealed a clear difference in subcellular distribution. The the mutant receptor showed a vesicular distribution. This

FZD6(wt)-GFP was localized predominantly in the plasma suggested a quantitative defect of the FZD6 missense membrane, and a few cells showed intracellular vesicles, variant because of defects in intracellular transportation, whereas the FZD6(missense)-GFP showed the opposite impaired integration into the plasma membrane, acceler- pattern, that is the majority of cells had FZD6(missense)- ated internalization, and/or an increased degradation.

GFP conﬁned to intracellular vesicles (Figure 4A). In order The high proportion of cells expressing FZD6(missense)- to quantify the observed difference, we counted cells with GFP localized to intracellular vesicles made us hypothesize

The American Journal of Human Genetics 88, 852–860, June 10, 2011 855 Figure 4. 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 5 standard deviation (SD) were 70.7 5 2.8 (FZD6[wt]membr); 14.3 5 2.4 (FZD6 5 5 5 [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 5 1.4 (FZD6[missense]vesic). Error bars denote 5 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 mm. 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). that the vesicles could belong to either the endosomal, cav- more, the trans-Golgi network marker TGN46 did not co- eosomal, or lysosomal pathway. In order to clarify the localize with GFP, indicating that the vesicular structures nature of the FZD6-GFP-positive intracellular vesicles, we do not represent compartments in the excocytotic performed colocalization experiments by using transferrin pathway. A partial colocalization was observed for GFP and antibodies against clathrin, caveolin-1, RAB5, the and transferrin, which is bound to the plasma membrane trans-Golgi network marker TGN46, and a lysosomal and some endosomal compartments. In contrast, we marker, lysotracker, in combination with the FZD6(wt)- observed a clear colocalization for both FZD6(wt)-GFP

GFP and FZD6(missense)-GFP, respectively. We could not and FZD6(missense)-GFP and lysotracker. This shows detect any overlap with clathrin-, caveolin-1-, or RAB5- that the intracellular vesicles, observed predominantly labeling endosomal compartments (Figure 4C). Further- in FZD6(missense)-GFP-expressing cells, are lysosomes.

856 The American Journal of Human Genetics 88, 852–860, June 10, 2011 DVL1-FLAG GFP merge Figure 5. 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 micro- photographs (upper panel). Coexpression of

no FZD 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 mm. [wt]-GFP 6 coexpressed with DVL1-FLAG (Figure S3). FZD 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. [miss]-GFP

6 WNT-FZD signaling constitutes a network of autocrine and paracrine pathways regu- 21,24,25 FZD lated via several feedback loops. The best known signaling pathway downstream of FZD is the WNT-b-catenin pathway leading to the stabilization of b-catenin Thus, our observations indicate a quantitative shift of and TCF/LEF-dependent transcription.26 However, it has

FZD6(missense)-GFP from the plasma membrane to lyso- been shown that FZD6 has the ability to interact with somes consistent with increased lysosomal degradation.22 both WNT-3A (MIM 606359) and WNT-5A (MIM FZDs contain a highly conserved KTxxxW sequence in 164975), previously shown to recruit b-catenin-dependent the C terminus, just upstream of the mutated residue in as well as b-catenin-independent pathways, respectively.27 21 missense FZD6. This domain is crucial for intracellular To further investigate the effect of mutated FZD6 on down- recruitment and interaction with Dishevelled (DVL) stream signaling, we stimulated primary WT fibroblasts proteins, and we asked whether the recruitment of DVL and patient fibroblasts homozygous for the FZD6 is affected by the missense mutation. FZD6(wt)-GFP and nonsense mutation with either WNT-3A or WNT-5A. b FZD6(missense)-GFP were coexpressed with DVL1-FLAG WNT-3A stimulation resulted in an upregulation of -cate- in HEK293 cells. In the absence of FZD6 expression, nin levels in control fibroblasts but not in patient DVL1-FLAG-positive cells showed characteristic aggregates fibroblasts (Figures 6A). This supports that FZD6 mediates in the cytosol in addition to a smaller percentage of cells b-catenin-dependent signaling and that FZD6-null mutant with even distribution of DVL1-FLAG (Figure 5).23 Coex- cells have lost their capability to respond to WNT-3A stim- pression with FZD6(wt)-GFP induced a radical membrane ulation. Next, we analyzed levels of the WNT-signaling recruitment of DVL1-FLAG. Interestingly, the membrane antagonist DKK1 in control and FZD6-mutated fibroblasts recruitment was also observed in the presence of FZD6(mis- stimulated with WNT-3A or WNT-5A. After WNT-5A acti- sense)-GFP, arguing that the mutated receptor is capable to vation we observed upregulated levels of DKK1 mRNA in interact with DVL. This is supported by immunoprecipita- control fibroblasts but not in FZD6-null fibroblasts tion showing that myc-FZD6(missense) binds to DVL2-3 (Figure 6B). This suggests a perturbed response also to b (Figure S2). Moreover, intracellular FZD6(missense)-GFP -catenin-independent WNT-5A stimulation in the within the lysosomal vesicles did not attract DVL1-FLAG absence of FZD6. DKK1 and several other WNT-signaling

(Figure 5), supporting the notion that FZD6 internalized inhibitors were recently shown to be induced by the tran- into lysosomes is inactivated. In addition, employing indi- scription factor MSX1.28 In agreement with the results rect immunocytochemistry with an FLAG antibody and from DKK1 mRNA analysis, we observed increased MSX1 a Cy3-conjugated secondary antibody, we investigated mRNA levels in WNT-5A-stimulated control ﬁbroblasts the Fo¨rster resonance energy transfer (FRET) between but not in patient ﬁbroblasts (Figure 6C). In combination,

FZD6-GFP and DVL1-FLAG in ﬁxed cells after photoaccep- these results suggest that patient ﬁbroblasts homozygous tor bleaching. FRET between FZD6(missense)-GFP and for the FZD6 nonsense mutation fail to respond properly

DVL1-FLAG was reduced in comparison to FZD6(wt)-GFP to both WNT-3A and WNT-5A activation.

The American Journal of Human Genetics 88, 852–860, June 10, 2011 857 0,6 Control nitca-β/ninetac-β Control Figure 6. Effect of WNT-3A and WNT-5A A β-catenin Patient 0,5 B nitc 0,02 * Patient DKK1 * Stimulation On Primary Fibroblast 0,4 a 0,015 - Cultures from an Affected Individual (V:1 0,3 β/ 0,01 1 form Family F1) and a Healthy Control

0,2 KKD 0,005 0,1 (A) b-catenin levels in control ﬁbroblasts 0 0 and in ﬁbroblasts homozygous for the Control FZD6[nonsense] Control FZD6[nonsense] WNT3A --++-- WNT3A --++-- nonsense mutation p.Glu584X without WNT5A ----++ WNT5A ----++ stimulation () and after WNT-3A or WNT-5A stimulation (þ). The graphs repre- β-catenin sent the relative amounts of b-catenin to b-actin as determined by immunoblot analysis (bottom). b-catenin accumulates upon β-actin WNT-3A stimulation in control cells but not in FZD6-null mutant cells (*p < 0.05). (B) Effect of WNT-5A stimulation on DKK1 C expression. Quantitative RT/PCR analysis 6E-05 from total RNA isolated from control and MSX1 WNT5a* Control

ni 5E-05 Patient mutated ﬁbroblasts stimulated as in (A) tc 4E-05 a-β/ shows that DKK1 is upregulated in control 3E-05

1XSM cells upon stimulation with WNT-5A but 2E-05 < 1E-05 not WNT-3A (*p 0.05). Mutated fibro- 0 blasts do not show increased DKK1 tran- WNT3A --++-- script levels in response to WNT-5A. WNT5A -- (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 5 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 b-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 a-b-catenin (Santa Cruz Biotechnology) and a-b-actin antibodies (Abcam), respectively. Proteins were visual- ized 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 (PCP) pathway.18 We show here that FZD6-null mutant properly attached to the nail bed. This process is presum- cells fail to respond to both WNT-3A and WNT-5A stimula- ably dependent on tight interaction and signaling between tion in vitro when compared to control cells. Hence, FZD6 mesenchymal (matrix) cells forming the nail plate and the can elicit both b-catenin-dependent and -independent epidermal (nail bed) cells. Affected individuals in the F1 signaling presumably determined by the local dosage of and F2 families have a retained regenerative capacity of ligands, other FZDs, and coreceptors, as suggested from the nail plate; this capacity suggests that the nail matrix is other studies.33–35 Furthermore, subcellular studies show intact. The primary defect associated with FZD6 mutations that FZD6 containing the missense mutation is localized seems to be related to perturbed formation and attachment predominantly in lysosomes when compared to the WT of the nail plate shown as onycholysis. This is also consis- FZD6. Thus, the FZD6 missense variant is presumably tent with the observations in the Fzd6 / mice with claws subject to increased internalization and subsequent that are easily lost with age and mechanical stress. The degradation and a reduced capability to interpret WNT reason for the restriction of the claw phenotype to male stimulation. In conclusion, our combined results show mice is unclear, but one explanation is a more aggressive that isolated nail dysplasia can be caused by dysfunctional behavior. However, it cannot be excluded that altered FZD6 or loss of FZD6 with a subsequent misregulation of

WNT signaling caused by disrupted Fzd6 has a gender- several FZD6-mediated pathways required for proper restricted effect as shown for human RSPO1 mutations.9 formation and regeneration of nails throughout life. WNT-FZD signaling is indispensable for numerous developmental processes such as tissue morphogenesis, differentiation, and regeneration in all animals.26 It has Supplemental Data become evident that many WNT-FZD interactions and Supplemental Data include three ﬁgures and can be found with their downstream pathways are integrated in extensive this article online at http://www.cell.com/AJHG/. 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 Acknowledgments b that FZD6 can act as a negative regulator of -catenin We thank all family members who participated in this study. Mice signaling32 as well as a mediator of the planar cell polarity targeted for Fzd6 were generously shared by Jeremy Nathans,

858 The American Journal of Human Genetics 88, 852–860, June 10, 2011 Howard Hughes Medical Institute and Johns Hopkins University. BMP receptor signaling during formation of the AER and the Wnt producing 3T3 fibroblast lines were a gift from Peter Mark, dorsal-ventral axis in the limb. Genes Dev. 17, 1963–1968. Universita¨tklinikum Rostock. This work was supported by grants 7. Grzeschik, K.H., Bornholdt, D., Oeffner, F., Ko¨nig, A., del from the Swedish Research Council (K2010-66X-10829-17-3) Carmen Boente, M., Enders, H., Fritz, B., Hertl, M., Grasshoff, and Swedish Links, Asia (348-2008-6069), the Indevelops fund, U., Ho¨fling, K., et al. (2007). Deficiency of PORCN, a regulator Thureús fund, Uppsala University Hospital, Uppsala University, of Wnt signaling, is associated with focal dermal hypoplasia. and the Science for Life Laboratory. M.B.C.K. was supported by Nat. Genet. 39, 833–835. the Karolinska Institutet’s Keratitis, Ichthyosis, and Neurosensory 8. Blaydon, D.C., Ishii, Y., O’Toole, E.A., Unsworth, H.C., Teh, Deafness Syndrome program; J.K. was supported by the Swedish M.T., Ru¨schendorf, F., Sinclair, C., Hopsu-Havu, V.K., Tidman, Society for Medical Research, and M.S. was supported by the N., Moss, C., et al. (2006). The gene encoding R-spondin 4 Swedish Institute. Work in the laboratory of G.S. was supported (RSPO4), a secreted protein implicated in Wnt signaling, is by the Swedish Research Council (K2008-68P-20810-01-4, K2008- mutated in inherited anonychia. Nat. Genet. 38, 1245–1247. 68X-20805-01-4), Swedish Cancer Society (CAN 2008/539), and 9. Parma, P., Radi, O., Vidal, V., Chaboissier, M.C., Dellambra, E., the Knut and Alice Wallenberg Foundation (KAW2008.0149). Valentini, S., Guerra, L., Schedl, A., and Camerino, G. (2006). R-spondin1 is essential in sex determination, skin differentia- Received: March 11, 2011 tion and malignancy. Nat. Genet. 38, 1304–1309. Revised: May 1, 2011 10. 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