Abstract

Split-Hand/Foot Malformation (SHFM) is a complex limb malformation affecting the central rays of the autopod. We studied a large consanguineous kindred afflicted with autosomal recessive SHFM. Twelve affected members had central feet reductions with or without hand involvement while the remaining one had the mildest phenotype and atypical SHFM. We identified by homozygosity mapping a novel SHFM locus at 12q13.11–q13 with a maximum multipoint lod score of 5.47 and by subsequent candidate gene approach a homozygous missense WNT10b mutation (p.R332W) in all affected individuals but the atypical case plus in an asymptomatic female. We propose that either a second locus contributes to the manifestation of SHFM phenotype or a suppressor locus prevented trait manifestation in the non-penetrant female. We also investigated linkage to the five known SHFM loci. Four of the loci were excluded, while in TP63 [tumor protein p63 (SHFM4)], the only known gene responsible for SHFM, we detected in most affected subjects a rare insertion variant (rs34201045) at the alternate promoter used for transcription of the N-terminal-truncated p63 isotype. This is the first reported WNT10b mutation on the pathogenesis of limb development and recessive mutation in SHFM.

INTRODUCTION

Split-Hand/Foot Malformation (SHFM) is a complex limb malformation affecting the central rays of the hands and/or feet (MIM 183600, 313350, 600095, 605289 and 606708). The severity of SHFM is highly variable, grading from mild pre- or postaxial involvement as simple syndactyly to severe central clefting of the autopods (1,2). The condition occurs as either isolated or part of other anomalies. Familial SHFM is generally inherited as an autosomal dominant trait with reduced penetrance and variable expressivity both among and within affected families and even among the autopods of a single individual.

SHFM is genetically heterogenous, and four autosomal loci have been mapped, all with dominant inheritance (3–6): 7q21–22 (SHFM1), 10q24 (SHFM3), 3q28 (SHFM4) and 2q31 (SHFM5). Also, on chromosome Xq26, SHFM2 with semi-recessive effect has been described (7). The gene responsible has been identified only in SHFM4, as TP63 (tumor protein p63) (5), encoding a homolog of the tumor suppressor p53. Additionally, genomic rearrangements characterized in SHFM1, SHFM3 and SHFM5 cases have led to the identification of candidate genes (8).

Vertebrate limb develops from an embryonic limb bud that consists of a mesodermal core surrounded by ectodermal cells. Proper patterning of the limb bud is coordinated by both spatial and temporal expression of a variety of signaling molecules and transcription factors including fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), wingless-type MMTV integration site family member (WNT) and hedgehog signaling molecules, and homeobox proteins (9). At the apex of the limb bud, ectodermal cells differentiate into apical ectodermal ridge (AER) by inductive signals originating from the underlying mesenchyme. AER secretes FGFs to the sub-adjacent mesenchyme—the progress zone (PZ)—in order to keep those cells undifferentiated and proliferating. Interplay between AER and PZ determines the proximadistal polarity of the limb. FGF signaling from AER also maintains sonic hedgehog expression from zone of polarizing activity at the posterior mesenchyme, which controls anteroposterior polarity (10). Dorsoventral polarity is maintained by the non-AER ectoderm signaling: BMPs and Engrailed 1 from the ventral ectoderm and WNT7a from the dorsal ectoderm. Pathogenesis of SHFM has been attributed to defects in maintenance of AER activity that cause loss of central rays (11). TP63 was shown to be highly expressed in AER (12,13).

WNT signaling plays a role in vertebrate limb development (14). WNTs are secreted glycoproteins that are implicated in cell fate determination and cell growth, acting as short-range ligands in a variety of signaling pathways. Among those pathways, canonical Wnt/β-catenin is the best defined. Upon WNT binding to cell surface receptor Frizzled and co-receptor LRP5/6 (low density lipoprotein receptor-related protein), cytoplasmic β-catenin is stabilized, which then enters the nucleus and forms a transcriptional complex with TCF/LEF DNA binding proteins in order to induce the expression of target genes (15).

A definite autosomal recessive inheritance for isolated SHFM has been reported for one family only (16). Here we describe the clinical findings in that family and the genetic analyses comprising genome search, fine mapping and subsequent candidate gene approach. This is the first WNT10b mutation reported on the pathogenesis of limb development; the homozygous mutation was necessary but not sufficient for SHFM phenotype manifestation. We designated this novel locus as SHFM6.

RESULTS

A large kindred from Eastern Turkey with 13 members afflicted with SHFM was investigated in this study (Fig. 1). All affected individuals were born to consanguineous parents. Clinical findings of nine of the cases have been described (16). Clinical evaluations for all cases are compiled in Table 1, and examples are presented in Figure 2. All affected members except individual 407 had central feet reductions with or without hand involvement. Individual 407 can best be described as atypical SHFM (17), having just unilateral hand syndactyly with no foot involvement. In general, females were less severely affected than the males, and hands were affected much less than the feet. The trait exhibited a great degree of variable expressivity among the affected subjects.

Figure 1.

Pedigree diagram for the SHFM kindred. The first three generations are partial. DNA available for the genome scan is marked with a plus sign, and DNA available later is marked with an asterisk. Symbols for affected individuals are shown in four quadrants according to upper and lower extremity involvement. Upper left quadrant represents the left hand and so forth.

Figure 1.

Pedigree diagram for the SHFM kindred. The first three generations are partial. DNA available for the genome scan is marked with a plus sign, and DNA available later is marked with an asterisk. Symbols for affected individuals are shown in four quadrants according to upper and lower extremity involvement. Upper left quadrant represents the left hand and so forth.

Figure 2.

Examples for autopod malformations in the kindred. (A) Foot phenotypes are demonstrated roughly in increasing severity: (1) postaxial syndactyly of toes 3–4, (2) preaxial syndactyly of toes 1–2, (3) conditions (1) and (2) together, (4) (3) together with loss of toe 2 and no clefting, (5) (3) together with loss of toe 2 and clefting, (6) loss of toe 3 with syndactyly of remaining toes in both pre-and postaxial direction with clefting, (7) (6) together with loss of toe 2 and no clefting, (8) (6) together with loss of toe 2 with clefting, (9) classical cleft foot. (B) Hand malformations in three males.

Figure 2.

Examples for autopod malformations in the kindred. (A) Foot phenotypes are demonstrated roughly in increasing severity: (1) postaxial syndactyly of toes 3–4, (2) preaxial syndactyly of toes 1–2, (3) conditions (1) and (2) together, (4) (3) together with loss of toe 2 and no clefting, (5) (3) together with loss of toe 2 and clefting, (6) loss of toe 3 with syndactyly of remaining toes in both pre-and postaxial direction with clefting, (7) (6) together with loss of toe 2 and no clefting, (8) (6) together with loss of toe 2 with clefting, (9) classical cleft foot. (B) Hand malformations in three males.

Table 1.

Review of the autopod malformations in the kindred. The degree of severity in foot malformations are numbered from 1 to 9 according to Figure 2

ID Casea Sex Autopod malformations
 
Photographs Radiographs 
   Left hand Right hand Left foot Right foot   
407 None Syndactyly type I (operated) None None − 
409 None Postaxial partial syndactyly (fingers 3–4) 
414 Postaxial partial syndactyly (fingers 3–4) None − − 
415 Postaxial syndactyly (fingers 3–4) with bone deformity, extra rudimentary bone, hypoplastic finger 2 Postaxial syndactyly (fingers 3–4) with bone deformity, flexion deformity of finger 2 
501 Postaxial syndactyly (fingers 3–4) with bone deformity Postaxial syndactyly (fingers 3–4) with almost fused nail beds − 
504 None None None − 
507 – None None None None 
508 None None None − 
509 None None 6, but toe 3 rudimentary − − 
510 – None None None 
511 Finger 5 clinodactyly Finger 5 clinodactyly − 
514 – None Postaxial partial syndactyly (fingers 3–4) − 
603 – Postaxial syndactyly (fingers 3–4) with bone deformity Postaxial syndactyly (fingers 3–4) with bone deformity, preaxial polydactyly type 1 − 
604 – None None − 
ID Casea Sex Autopod malformations
 
Photographs Radiographs 
   Left hand Right hand Left foot Right foot   
407 None Syndactyly type I (operated) None None − 
409 None Postaxial partial syndactyly (fingers 3–4) 
414 Postaxial partial syndactyly (fingers 3–4) None − − 
415 Postaxial syndactyly (fingers 3–4) with bone deformity, extra rudimentary bone, hypoplastic finger 2 Postaxial syndactyly (fingers 3–4) with bone deformity, flexion deformity of finger 2 
501 Postaxial syndactyly (fingers 3–4) with bone deformity Postaxial syndactyly (fingers 3–4) with almost fused nail beds − 
504 None None None − 
507 – None None None None 
508 None None None − 
509 None None 6, but toe 3 rudimentary − − 
510 – None None None 
511 Finger 5 clinodactyly Finger 5 clinodactyly − 
514 – None Postaxial partial syndactyly (fingers 3–4) − 
603 – Postaxial syndactyly (fingers 3–4) with bone deformity Postaxial syndactyly (fingers 3–4) with bone deformity, preaxial polydactyly type 1 − 
604 – None None − 

Photographs and/or radiographs for individuals with a plus sign are available.

aCase numbering (16).

Linkage and mutation analysis

Data from the genome scan was subjected to two-point lod score analysis, assuming recessive inheritance with reduced penetrance (80%). Lod scores >1.5 were obtained for single markers on chromosomes 1p34, 3q13, 4p15 and 7p22, and for two consecutive markers on chromosome 12q12–13 (Fig. 3). Among those five loci, only for GATA91H06 at chromosome 12 the highest score was at θ = 0, and at this locus all affected individuals were homozygous for a haplotype apparently identical by descent (IBD). To further analyze the haplotypes in the homozygosity region, 13 additional known microsatellite markers (D12S1053, GATA167C12, D12S2194, D12S1296, D12S1687, D12S85, D12S1701, D12S1661, D12S1590, D12S1627, D12S1620, D12S1635 and D12S398) plus two identified in this study (D12SAAGGx18 and D12SAAAGx47) were employed. An ancestral recombination event between D12S1661 and D12S1590 together with a recent one between D12SAAGGx18 and D12SAAAGx47 in individual 511 delineated the gene region to a 1.71 Mb (mega base) interval (Fig. 4). The cumulative genotyping data assuming again 80% penetrance yielded maximum two-point and multipoint lod scores of 3.87 and 5.47, respectively (Table 2 and Fig. 5). However, a reportedly unaffected individual (507) was also homozygous for the haplotype. Nevertheless, candidate gene analysis was carried out in this region, as SHFM is generally well known for its incomplete penetrance. In the evaluation of the candidate genes, individual 407 who was homozygous only for a part of the haplotype was disregarded, since he displayed atypical SHFM.

Figure 3.

Two-point lod scores of the total data set generated by the genome scan (SuperLink) in a recessive model with reduced penetrance (80%). Maximum lod scores for each marker are plotted in the order of chromosomal position. Top five scores are given above the graph together with their loci and chromosomal positions.

Figure 3.

Two-point lod scores of the total data set generated by the genome scan (SuperLink) in a recessive model with reduced penetrance (80%). Maximum lod scores for each marker are plotted in the order of chromosomal position. Top five scores are given above the graph together with their loci and chromosomal positions.

Figure 4.

Partial pedigree diagram and haplotype analysis at 12p11.23–q13.13 for SHFM kindred. Phenotypes in generations 1–3 are not known, except for 307, 308 and 309. Alleles deduced from haplotype data are in italics.

Figure 4.

Partial pedigree diagram and haplotype analysis at 12p11.23–q13.13 for SHFM kindred. Phenotypes in generations 1–3 are not known, except for 307, 308 and 309. Alleles deduced from haplotype data are in italics.

Figure 5.

Multipoint linkage analysis of the 17.66 cM region at 12p11.23–q13.13 (SimWalk). Autosomal recessive inheritance and reduced penetrance (80%) were assumed.

Figure 5.

Multipoint linkage analysis of the 17.66 cM region at 12p11.23–q13.13 (SimWalk). Autosomal recessive inheritance and reduced penetrance (80%) were assumed.

Table 2.

Two-point lod scores for 17 markers at the 23.94 Mb region on chromosome 12p11.23–q13.13 (SuperLink). Autosomal recessive inheritance with reduced penetrance was assumed

Marker Position
 
ZmaxθMLE0.00 0.05 0.10 0.20 0.30 0.40 
 Mb cMa         
D12S1042 27.54 52.09 0.62 0.15 −8.03 −0.10 0.45 0.61 0.36 0.09 
D12S1053 29.22 53.79 0.91 0.00 0.91 0.86 0.77 0.54 0.31 0.13 
GATA167C12 37.48 58.19 0.22 0.15 −0.83 0.05 0.19 0.20 0.11 0.03 
D12S2194 38.74 59.12 3.61 0.00 3.61 3.40 3.12 2.42 1.61 0.76 
D12S1296 40.45 (59.7) 2.20 0.00 2.20 2.02 1.79 1.26 0.74 0.30 
D12S1301 42.35 61.09 1.62 0.00 1.62 1.41 1.20 0.80 0.45 0.17 
D12S1687 43.01 61.09 2.94 0.00 2.94 2.77 2.52 1.93 1.25 0.56 
D12S85 45.62 63.46 2.94 0.00 2.94 2.69 2.41 1.80 1.14 0.49 
D12S1701 46.21 64.12 3.50 0.00 3.50 3.30 3.04 2.37 1.58 0.74 
D12S1661 46.89 65.12 1.32 0.00 1.32 1.17 1.01 0.67 0.36 0.13 
D12S1590 47.82 65.54 1.65 0.00 1.65 1.44 1.24 0.84 0.48 0.19 
D12S1627 48.07 65.71 3.57 0.00 3.57 3.26 2.90 2.10 1.24 0.47 
D12SAAGGx18 48.46 (66.0) 3.87 0.00 3.87 3.54 3.15 2.29 1.38 0.55 
D12SAAAGx47 48.60 (66.2) 1.20 0.10 −2.35 1.16 1.20 0.94 0.59 0.26 
D12S1620 48.89 66.22 0.65 0.10 −2.31 0.55 0.65 0.54 0.33 0.14 
D12S1635 49.32 66.22 2.58 0.05 −0.99 2.58 2.52 2.05 1.39 0.66 
D12S297 50.90 68.44 1.61 0.10 −4.38 1.49 1.61 1.23 0.60 0.05 
Marker Position
 
ZmaxθMLE0.00 0.05 0.10 0.20 0.30 0.40 
 Mb cMa         
D12S1042 27.54 52.09 0.62 0.15 −8.03 −0.10 0.45 0.61 0.36 0.09 
D12S1053 29.22 53.79 0.91 0.00 0.91 0.86 0.77 0.54 0.31 0.13 
GATA167C12 37.48 58.19 0.22 0.15 −0.83 0.05 0.19 0.20 0.11 0.03 
D12S2194 38.74 59.12 3.61 0.00 3.61 3.40 3.12 2.42 1.61 0.76 
D12S1296 40.45 (59.7) 2.20 0.00 2.20 2.02 1.79 1.26 0.74 0.30 
D12S1301 42.35 61.09 1.62 0.00 1.62 1.41 1.20 0.80 0.45 0.17 
D12S1687 43.01 61.09 2.94 0.00 2.94 2.77 2.52 1.93 1.25 0.56 
D12S85 45.62 63.46 2.94 0.00 2.94 2.69 2.41 1.80 1.14 0.49 
D12S1701 46.21 64.12 3.50 0.00 3.50 3.30 3.04 2.37 1.58 0.74 
D12S1661 46.89 65.12 1.32 0.00 1.32 1.17 1.01 0.67 0.36 0.13 
D12S1590 47.82 65.54 1.65 0.00 1.65 1.44 1.24 0.84 0.48 0.19 
D12S1627 48.07 65.71 3.57 0.00 3.57 3.26 2.90 2.10 1.24 0.47 
D12SAAGGx18 48.46 (66.0) 3.87 0.00 3.87 3.54 3.15 2.29 1.38 0.55 
D12SAAAGx47 48.60 (66.2) 1.20 0.10 −2.35 1.16 1.20 0.94 0.59 0.26 
D12S1620 48.89 66.22 0.65 0.10 −2.31 0.55 0.65 0.54 0.33 0.14 
D12S1635 49.32 66.22 2.58 0.05 −0.99 2.58 2.52 2.05 1.39 0.66 
D12S297 50.90 68.44 1.61 0.10 −4.38 1.49 1.61 1.23 0.60 0.05 

aEstimated cM distances are given in parenthesis.

bMaximum two-point lod score.

c Maximum likelihood estimate (MLE) for recombination fraction (θ).

The databases reported 51 genes in the 1.71 Mb gene region identified. WNT10b stood out as the best candidate, being highly expressed in AER (18) and limbs of developing mice (19). We identified a C→T transition in the homozygous state in exon 5 of the gene in all affected subjects except 407, as well as in individual 507. Several evidences indicated that this variant was a mutation and not a simple polymorphism. It was predicted to be damaging with high confidence by online tools predicting effect of amino acid substitutions on protein function, including PolyPhen (http://genetics.bwh.harvard.edu/pph/), SIFT (Sorting Intolerant From Tolerant, http://blocks.fhcrc.org/sift/SIFT.html) and SNPs3D (http://www.snps3d.org/). A control group of 200 individuals were screened for the mutation to achieve at least 95% power to distinguish a normal sequence variant (20), and the mutation was not found in any. At the protein level, mutation c.994C→T (GenBank accession no. NM_003394.2) leads to the substitution of an amino acid with a residue having completely different biochemical properties: positively charged arginine is replaced with a non-polar tryptophan: p.R332W (GenBank accession no. NP_003385.2). The residue has been strictly conserved in all paralogs and across species (Fig. 6).

Figure 6.

c.994C→T transition associated with SHFM phenotype. (A) 1.71 Mb gene region between markers D12S1661 and D12SAAAGx47. Genomic structure of WNT10b and schematic representation of WNT10b protein are also shown (modified from NCBI gene viewer). The sites of mutation c.994C→T and the corresponding p.R332W amino acid change are indicated. (B) Chromatograms showing the c.994C→T transition in a heterozygote and a homozygote and the wild-type allele in a control. (C) SSCP results for c.994C→T screening using primer pairs P2–P3 in the SHFM kindred and six control individuals (C1–C6) are shown and alleles are compiled in the table. The wild-type allele is labeled 1 and mutant allele 2. (D) Confirmation of c.994C→T transition by PCR-RFLP assay (P2–P3 primers and NlaIV digestion). (E) Evolutionary conservation of WNT10b p.332R amino acid.

Figure 6.

c.994C→T transition associated with SHFM phenotype. (A) 1.71 Mb gene region between markers D12S1661 and D12SAAAGx47. Genomic structure of WNT10b and schematic representation of WNT10b protein are also shown (modified from NCBI gene viewer). The sites of mutation c.994C→T and the corresponding p.R332W amino acid change are indicated. (B) Chromatograms showing the c.994C→T transition in a heterozygote and a homozygote and the wild-type allele in a control. (C) SSCP results for c.994C→T screening using primer pairs P2–P3 in the SHFM kindred and six control individuals (C1–C6) are shown and alleles are compiled in the table. The wild-type allele is labeled 1 and mutant allele 2. (D) Confirmation of c.994C→T transition by PCR-RFLP assay (P2–P3 primers and NlaIV digestion). (E) Evolutionary conservation of WNT10b p.332R amino acid.

To ascertain that the mutation was indeed non-penetrant in 507, repeated radiological investigations of the hands and feet were performed, and no evidence supportive of SHFM phenotype was found. Individual 407, having no foot involvement, no bone defect and no WNT10b mutation, was left out in the subsequent linkage analyses for SHFM.

Linkage to chromosomes 3q13, 4p15 and 7p22 was not supported by the results of the additional genotyping with a total of 13 markers and the haplotype segregation analyses at those loci. But at 1p34.3–p32.3, an ∼20 Mb haplotype justified with 17 additional markers and delineated by D1S496 and D1S200 was found in all affected subjects except for 510 in either heterozygous or homozygous state. This haplotype was not present in individual 507 either.

Haplotype analysis at five known SHFM loci

We investigated by haplotype segregation analysis whether any of the five known SHFM loci could possibly be contributing to the etiology of SHFM in our family. Only two loci, SHFM4 at 3q28 and SHFM2 at Xq26.2-27.1 seemed candidates. Due to a common haplotype observed in many of our SHFM subjects at 3q28 and to similar phenotypic similarity between members of our family and SHFM4 cases reported (5), we screened all of the exons of TP63 for mutations in individual 409, who was homozygous at the locus. No obvious disease causing mutation was found, but a rare insertion polymorphism (rs34201045) was detected at the alternate promoter used for the transcription of the N-terminal-truncated p63 isotype (ΔNp63). The frequency of the rare insertion variant (c.-71insAG from GenBank accession no. AF091627) in 100 control chromosomes was 0.073 and that of the frequent allele 0.69. We detected also a novel allele (c.-71insAGAG) at a frequency of 0.24. We did not detect any homozygotes for the rarest allele in the control samples. All SHFM subjects but 508 were either homozygous or heterozygous for the rarest allele.

Genotyping at Xq26.2–27.1 with four markers excluded linkage to SHFM2. However, we identified a common haplotype at Xq22.3–25 (between markers DXS6797 and ATCT003) in all affected males but 604.

Table 3 summarizes the genotyping results on chromosomes 12q13.2, 1p34.3–32.3, 3q28 (SHFM4) and Xq22.3–25.

Table 3.

Summary of haplotypes for all affected individuals plus individual 507 at four loci. A plus sign indicates that the relevant mutation, haplotype or polymorphism is present

Locus Model 407 409 414 415 501 504 507 508 509 510 511 514 603 604 
12q13.2 (WNT10b, c.994C→T) ARb −/− +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ 
1p34.3–32.3 ADc −/− +/+ +/+ +/+ +/− +/− −/− +/− +/− −/− +/− +/− +/− +/+ 
3q28a (SHFM4) AD 0/2 1/1 0/1 1/1 1/2 0/1 1/2 0/2 0/1 1/2 1/1 1/2 0/1 1/1 
Xq22.3–25 X-Rd +/− −/− +/− +/− +/− +/− +/− − 
Locus Model 407 409 414 415 501 504 507 508 509 510 511 514 603 604 
12q13.2 (WNT10b, c.994C→T) ARb −/− +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ 
1p34.3–32.3 ADc −/− +/+ +/+ +/+ +/− +/− −/− +/− +/− −/− +/− +/− +/− +/+ 
3q28a (SHFM4) AD 0/2 1/1 0/1 1/1 1/2 0/1 1/2 0/2 0/1 1/2 1/1 1/2 0/1 1/1 
Xq22.3–25 X-Rd +/− −/− +/− +/− +/− +/− +/− − 

aAlleles represent the number of AG dinucleotide insertion in the promoter region of TP63 encoding the ΔNp63 isotype (0: no insAG, 1: insAG, 2: insAGAG).

bAutosomal recessive.

cAutosomal dominant.

dX-linked recessive.

DISCUSSION

The family we analyzed in this study was the only one reported so far exhibiting autosomal recessive inheritance for SHFM (16). Incomplete penetrance reported for all SHFM with known locus except for the X-linked one (a single family) strongly implies the contribution of more than one locus to each autosomal SHFM. In addition, general variability of the trait among the autopods of a single individual further complicates the picture, indicating that the contributions of genes to the trait are not absolutely additive. Therefore, our results that revealed the complex inheritance for SHFM in the family studied were not unexpected. A homozygous mutation in WNT10b having a penetrance of 92.3% underlies the trait in subjects with foot involvement, while the atypical SHFM individual did not carry the mutation at all.

The finding that SHFM phenotype did not manifest in 507 who was homozygous for the mutation could be explained by two alternative models: Either the coinheritance of additional locus/loci was essential for SHFM manifestation, or a suppressor mutation prevented trait manifestation. The first model proposes oligogenic inheritance as explained earlier. As for the second model, the sample size was too small for a meaningful investigation, 507 being the only individual homozygous for p.R332W and not manifesting SHFM. Suppressor genes/loci for other human traits have been reported (21). Since sisters of 507 are only very mildly affected, it is possible that only minor genetic differences have led to a shift from the equilibrium for the normal phenotype in favor of the SHFM phenotype.

As for the variability of the phenotype, the simplest model would suggest that combinations of various loci shared by various combinations of SHFM subjects act in concert with the WNT10b. An interesting locus example was 1p34.3–p32.3, where a haplotype was found in all SHFM subjects except the atypical case and 510, in either heterozygous or homozygous state. It is remarkable that the phenotype of 510 was unique in the family in the sense that she had postaxial involvement in the affected foot. Postaxial syndactyly in foot was not observed alone in any other member of the family, but in association with a preaxial defect. Another locus of interest was 3q27, harboring TP63, the gene for SHFM4. We analyzed all 16 exons of TP63 encoding the six p63 isotypes (22). Only a rare insertion polymorphism at the promoter for isotype ΔNp63 was identified in all SHFM subjects but 508 (Table 3). No functional study has been reported for the alleles. ΔNp63 lacks the transactivation domain for transcriptional activity and was suggested to have dominant-negative effects on p53 and p63 activities (22). Whether any of the loci found to be shared by most of the SHFM cases indeed contribute to phenotype severity cannot be assessed for sure before the responsible genes are identified. One factor that hinders such an evaluation is that a quantitative phenotypic classification was not possible in our SHFM subjects. Moreover, complex inheritance is generally complicated also by environmental factors, and nothing is known yet about the effect of environment on SHFM phenotype.

A general feature of SHFM is that males are more affected than females. No locus on the X-chromosome was shared by all male SHFM subjects in the family. Locus Xq22.3–25 was shared by all but 604. Although it is possible that the locus may have a modifier effect on the severity in males, acting as an X-linked recessive locus, it is more likely that in this family the trait severity is sex influenced rather than modified by an X-linked gene.

Homozygous WNT10b mutation was definitely non-penetrant in 507, since radiological investigations excluded SHFM. No malformation could be observed in hands and feet. However, two clinical findings in her feet are remarkable: unusually large navicular bones fused to accessory bones and reduced bone density as evidenced by the hollow appearance in calcaneus bones. Available radiograms allowed the investigation of those phenotypes in two SHFM cases as well: The navicular bones were bilaterally large but not fused in individual 409, who also had a weak calcaneus appearance. In the atypical SHFM case 407, who did not carry mutation p.R332W at all, the bone density was normal and the navicular bones were neither large nor fused. The somewhat lower bone density in mutation homozygotes is in agreement with the positive role of Wnt10b in osteoblastogenesis and regulation of bone density (23,24).

The other perplexing phenotype was that of individual 407, who did not carry the mutation. He exhibited the atypical SHFM phenotype: just a mild hand involvement; a photograph of the hand before operation has been previously presented (16). This together with the observation that seven of the remaining 12 SHFM cases had hand involvement (in addition to foot) could suggest that a particular set of genes underlie hand involvement. SHFM individuals 407, 409, 414 and 514 were afflicted with postaxial cutaneous hand syndactyly type I, while three males (501, 415 and 603) had severe hand malformations. We propose that yet unidentified locus/loci other than gene WNT10b could be responsible for the mild syndactyly phenotype without bone involvement as in 407, 409, 414 and 514. As for 501, 415 and 603, phenotype is perhaps too complex to distinguish a probable syndactyly component alone. Further evidence in support of the presence of a hand-involvement locus is the phenotype of brothers 603 and 604: both have severely affected feet as their father, but only 603 had hand involvement, and it was as severe as the father’s.

Oligogenic inheritance might be not very rare in bone morphogenesis. Recently SHFM with long bone deficiency was shown to be linked simultaneously to two dominant loci in an extended kindred (25), but still parental consanguinity raised the question whether a recessive gene could also be involved. The clinical variability was extremely high as in the family we studied.

This is the first report on the pathogenesis of a WNT10b mutation in limb development. WNT10b acts as a key signaling molecule promoting osteoblastogenesis and inhibiting adipogenesis. WNT genes were shown to be mutated in at least four other autosomal recessive developmental human disorders: WNT3 in tetra-amelia (26), WNT7a in Fuhmann syndrome and Al-Awardi/Raas-Rothschild syndrome (AARRS) (27), and WNT10a in Odonto-onycho-dermal dysplasia (28). It is remarkable that mutation p.R292C in WNT7a reported for AARRS is at the paralogous position to mutation p.R332W identified in this study. This position is perhaps important for regulation or receptor recognition by the WNT protein.

It was a challenge to work with such a large family with a large number of affected members having such a variable phenotype. Several frequent haplotypes were observed. It is difficult to speculate which of those haplotypes harbor additional genes contributing to the trait, not only because of the high consanguinity but also because several inheritance models could be proposed for the highly variable phenotype. The hands were either not affected at all or much less affected than the feet, indicating that separate but overlapping molecular mechanisms may exist for morphogenesis of hand and foot. Individual 407 lacked both WNT10b mutation and foot malformation. It is therefore justified to propose that mutations in that gene underlie typical recessive SHFM phenotype and work in concert with defects in several other genes to fully manifest the phenotype. In spite of the high parental consanguinity, the genetic background in the family cannot be expected to be as uniform as in inbred mice with dactylaplasia, the mouse model for SHFM. This may explain why a clear-cut phenotypic classification could not be drawn in humans and effects of locus combinations on the phenotype could not be established. Nevertheless, recessive genetic components have never been considered previously in SHFM families, and this report emphasizes the importance of studying such components and evaluates possible complex inheritance models, which are extensions of an underlying recessive gene defect.

MATERIALS AND METHODS

Subjects

Nine cases from the kindred investigated in this study have been described as afflicted by an autosomal recessive form of SHFM (16). Blood samples of 21 individuals were kindly supplied by Dr Davut Gül and processed thereafter for the initial genome scan. Blood samples of an additional 12 individuals were obtained by the authors and employed for mutation analysis and fine mapping studies. DNA was extracted from whole blood using standard methods. Recent photographs and/or radiographs of 12 subjects were taken during this study. Informed consent was obtained from/for all subjects. The study was approved by the Committee on Research with Human Participants at Boğaziçi University.

Molecular genetics

Genotyping and statistical analyses

A genome scan for 21 members of the family (Fig. 1) was performed at NHLBI (National Heart, Lung and Blood Institute) Mammalian Genotyping Service (Contract Number HV48141) with Marshfield Screen Set 13 (29). The set contained 405 polymorphic microsatellite markers that spanned autosomes and sex chromosomes with an average density of 10 centi Morgans (cM). The results of the genome scan were obtained in the LINKAGE file format and were initially analyzed under the model of recessive inheritance, reduced penetrance, and a disease gene frequency of one in 100 000. Linkage data of individual 407 was not introduced to the computer programs, but used only in the haplotype segregation analyses. The updated genetic and physical positions of the markers were obtained from the Rutgers second generation combined linkage and physical map [NCBI (National Centre for Biotechnology Information) Build 36] of the human genome (http://compgen.rutgers.edu/maps/). This map was also used to estimate genetic positions of markers whenever not known. Software package easyLINKAGE (30) was used for detecting genotyping errors, calculating two-point (SuperLink) and multipoint (SimWalk2) lod scores and constructing haplotypes (Genehunter, with split families).

Those loci yielding relatively high lod scores and exhibiting shared IBD haplotypes in most of the affected members as assessed by haplotype segregation analysis were analyzed in our laboratory using more densely spaced markers and also including other family members that later joined the study. Microsatellite markers reported in the databases were selected with priority for genotyping. For regions where no markers were reported, the DNA sequence was introduced to Tandem Repeats Finder program (http://tandem.bu.edu/trf/trf.html) and oligonucleotide primer pairs were designed to amplify the repeats found (Supplementary Material, Table S1).

Mutation analyses

All four coding exons of WNT10b and flanking intronic sequences were PCR amplified with intronic primers (Supplementary Material, Table S1) designed using the Primer3 software and checked for genome uniqueness by in silico PCR. PCR products were subjected to single strand conformational polymorphism (SSCP) analysis and subsequent DNA sequencing. Similarly, all 15 exons of TP63 were screened for mutations.

Screening the family members and the control group for the identified WNT10b mutation c.994C→T was performed either by SSCP analysis of fragments amplified with primers P3–P4 or by endonuclease NlaIV digestion of fragments amplified using P2–P3 (Supplementary Material, Table S1).

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG Online.

FUNDING

This work was supported by the State Planning Organization (97K120660). A.T. was partially supported by the Turkish Academy of Sciences. S.A.U. was a fellow of the Scientific and Technological Research Council of Turkey. GenBank Accession Numbers: NM_003394.2, NP_003385.2, NM_003722.3, NP_003713.3 and AF091627; EMBL Accession Number O00744.

ACKNOWLEDGEMENTS

We thank the family that participated in the study; Dr Davut Gül for the initial blood samples and Dr Ayşegül Bursali for her help in clinical evaluations. We gratefully acknowledge NHLBI Mammalian Genotyping Service (Contract Number HV48141) for the genome scan.

Conflict of Interest statement. None declared.

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