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Human Molecular Genetics, 2003, Vol. 12, No. 16 1959-1971
DOI: 10.1093/hmg/ddg212
© 2003 Oxford University Press

A genomic rearrangement resulting in a tandem duplication is associated with split hand–split foot malformation 3 (SHFM3) at 10q24

Xavier J. de Mollerat1,2, Fiorella Gurrieri3, Chad T. Morgan1, Eugenio Sangiorgi3, David B. Everman1,2, Paola Gaspari3, Jeanne Amiel4, Michael J. Bamshad5, Robert Lyle6, Jean-Louis Blouin6, Judith E. Allanson7, Bernard Le Marec8, Melba Wilson9, Nancy E. Braverman10, Uppala Radhakrishna6, Celia Delozier-Blanchet6, Albert Abbott2, Vincent Elghouzzi4, Stylianos Antonarakis6, Roger E. Stevenson1,2, Arnold Munnich4, Giovanni Neri3 and Charles E. Schwartz1,2,*

1Center for Molecular Studies, J.C. Self Research Institute of Human Genetics, Greenwood Genetic Center, Greenwood, SC, USA, 2Department of Genetics and Biochemistry, Clemson University, Clemson, SC, USA, 3Istituto di Genetica Medica, Università Cattolica, Roma, Italy, 4Département de Génétique and Unité INSERM U-393 Hôpital Necker-Enfants Malades, Paris, France, 5Department of Genetics, School of Medicine, University of Utah, Salt Lake City, UT, USA, 6Division of Medical Genetics, University of Geneva Medical School, Geneva, Switzerland, 7Department of Genetics, Children's Hospital of Eastern Ontario Research Institute, Ottawa, Ontario, Canada, 8Faculté de Médecine de Rennes I, Service de Pediatrie-Génétique Medicale, Rennes, France, 9Center for Human Genetics, Bar Harbor, ME, USA and 10John Hopkins University, School of Medicine, Baltimore, MD, USA

Received March 24, 2003; Accepted June 17, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Split hand–split foot malformation (SHFM) is characterized by hypoplasia/aplasia of the central digits with fusion of the remaining digits. SHFM is usually an autosomal dominant condition and at least five loci have been identified in humans. Mutation analysis of the DACTYLIN gene, suspected to be responsible for SHFM3 in chromosome 10q24, was conducted in seven SHFM patients. We screened the coding region of DACTYLIN by single-strand conformation polymorphism and sequencing, and found no point mutations. However, Southern, pulsed field gel electrophoresis and dosage analyses demonstrated a complex rearrangement associated with a ~0.5 Mb tandem duplication in all the patients. The distal and proximal breakpoints were within an 80 and 130 kb region, respectively. This duplicated region contained a disrupted extra copy of the DACTYLIN gene and the entire LBX1 and ß-TRCP genes, known to be involved in limb development. The possible role of these genes in the SHFM3 phenotype is discussed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ectrodactyly or split hand–split foot malformation (SHFM) is a human limb malformation characterized by hypoplasia/aplasia of the central digital rays and variable fusion of the remaining digits (MIM 183600). The condition occurs in 1 in 8500–25 000 newborns and accounts for 8–17% of all limb reduction defects (1–5). SHFM is clinically heterogeneous, presenting in both non-syndromic and syndromic forms. The non-syndromic form can be isolated (type I) or associated with long bone deficiency (type II—also called SHFLD; MIM 119100) (6). Among the several syndromes in which SHFM occurs, the most common is the EEC (ectrodactyly, ectodermal dysplasia, cleft lip/palate) syndrome (MIM 129900) (7).

When familial, SHFM is usually inherited in an autosomal dominant manner with incomplete penetrance, variable expressivity and segregation distortion with excessive transmission from affected males to sons (6,8–10). An X-linked form has been reported in a single family, and autosomal recessive inheritance has also been described (11–14). To date, five loci for non-syndromic and syndromic SHFM have been identified based upon linkage mapping and/or cytogenetic abnormalities: SHFM1 on chromosome 7q21.3–q22.1 (15,16), SHFM2 on Xq26 (11,13), SHFM3 on 10q24 (17,18), SHFM4 on 3q27 (19) and SHFM5 on 2q31 (20,21). P63, the gene responsible for SHFM4 on 3q27, is the only gene thus far identified in human SHFM (19,22). Interestingly, P63 mutations and abnormalities at the SHFM1 locus cause both syndromic and non-syndromic SHFM (22,23). In contrast, only non-syndromic SHFM has been mapped to the SHFM3 locus (17,18,24).

Studies of the naturally occurring Dactylaplasia (Dac) mouse, which has an SHFM-like phenotype, have been critical to investigation of the SHFM3 locus. The mouse phenotype bears striking similarity to typical human SHFM, with absence of central digits, underdevelopment or absence of metacarpal/metatarsal bones and syndactyly (25). The Dac phenotype is inherited as an autosomal semidominant trait, with the more severe phenotype (e.g. monodactyly) occurring in homozygotes (26,27). The Dac locus was mapped to chromosome 19 in a region syntenic to chromosome 10q24 in human (26). Therefore, the Dac mouse is considered to be the mouse model for SHFM3 in humans.

The Dac phenotype results from two different alleles, Dac1J and Dac2J (27). Both alleles arise from a disruption of the dactylin gene, which is a novel member of the F-box WD-40 protein family. F-box WD-40 proteins recruit specific target protein(s) through their WD-40 protein-protein binding domains for ubiquitin mediated degradation. The dactylin protein appears to function in maintaining the apical ectodermal ridge (AER) of the developing limb bud (27–29).

In humans, DACTYLIN has been mapped to the SHFM3 critical region (30) and is 87% identical to mouse dactylin at the nucleotide level. DACTYLIN is considered to be the best candidate gene for SHFM3 (27,30,31). To date, however, mutations in DACTYLIN have not been reported in sporadic cases of SHFM or familial cases mapping to the SHFM3 locus.

In this study, single-strand conformation polymorphism (SSCP) and sequencing analysis of the entire DACTYLIN coding region and flanking intronic sequences failed to detect mutations in affected individuals from six families linked to the SHFM3 locus. However, Southern analysis, pulsed field gel electrophoresis (PFGE) analysis and dosage analysis demonstrated complex rearrangements associated with large duplications of 10q24 in all six families and in an additional SHFM family in whom linkage analysis was not possible. These rearrangements create an extra copy of a portion of DACTYLIN, an extra copy of the complete ß-TRCP, POLL and LBX1 genes, and an extra copy of a sequence located ~400–550 kb centromeric to DACTYLIN and constitute the first evidence of genomic abnormalities within the SHFM3 critical region.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DACTYLIN analysis by SSCP and sequencing
No mutations were detected by either SSCP or sequencing in an affected individual from each of the six SHFM3-linked families. In patient LS, a g1134 G->A (p378T->T) change was identified in DACTYLIN exon 9. However, this neutral alteration was also found in a control individual.

Southern analysis
Southern analysis, using a variety of cDNA, exon-specific and intron-specific DACTYLIN probes, (Fig. 1A) was performed on an affected member of each linked family to screen for alterations or rearrangements that would have been missed by the SSCP and sequencing approaches. Altered bands were identified in three patients (DF, TU and LS; Fig. 1B).



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  		Figure 1. Southern analysis of SHFM patients DF, TU and LS. (A) Restriction map of the DACTYLIN gene (only the relevant enzyme sites are represented). E=EcoRI, P=PstI and H=HpaI. Scoring for presence (+) or absence (-) of the altered band using cDNA, exon-specific, and intron-specific DACTYLIN probes on an EcoRI filter localized the breakpoint (BP) region for each patient. (B) Southern hybridization of patients DF, TU and LS with probes DAC cDNA exon 4, DAC int 5 and DAC cDNA exons 1–4 respectively. The presence of an extra band in all three patients is indicated by the arrow. For patient DF, both the extra and control bands are present in the mouse–human monochromosomal hybrid cell line containing the affected chromosome (chromosome 10B), while only the normal band is present in the cell line containing the unaffected chromosome (chromosome 10A). The 10.2 kb band comes from mouse DNA. (C) Pedigree of DF's family and Southern hybridization with DAC exons 2–4 probe of an EcoRI filter. The 9 kb band is present only in the affected members of the family, which confirms segregation of the rearrangement within the family: solid symbols, affected individual; open symbols, normal.

 
In patient DF, the cDNA probe containing DACTYLIN exons 1–4 detected an altered band with eight different restriction enzymes (EcoRI, PstI, BamHI, BglI, BglII, HindIII, XbaI and BclI). Using different probes containing exon 4 on an EcoRI filter, an extra 9 kb band was present in DF (Fig. 1B) and all affected members of the family, but was absent in the unaffected members and in 46 control individuals (Fig. 1C and data not shown). The EcoRI restriction map of the DACTYLIN gene identified a 6.4 kb fragment containing exons 2, 3 and 4 (Fig. 1A). This fragment alone was detected with a probe containing DACTYLIN exon 2, while the additional 9 kb band was detected with probes containing either exon 3 or exon 4 (Fig. 1A and B). Together, these results suggested that affected members of this family had a 10q24 genomic alteration in the 2.6 kb region between DACTYLIN exons 2 and 3 (Fig. 1A).

In patient TU, the DACTYLIN intron 5 probe detected an altered band with two different enzymes (EcoRI and PstI). On a PstI filter, this probe identified an extra 4.2 kb band (Fig. 1B) that segregated with the SHFM phenotype (data not shown). The PstI restriction map of DACTYLIN identified a 5.4 kb fragment containing exon 5 and the region corresponding to the intron 5 probe (Fig. 1A). Only the normal band was detected with an exon 5 probe, thus localizing a genomic alteration in this family to the 2 kb region between the exon 5 and intron 5 probes (Fig. 1A).

In patient LS, the probe containing DACTYLIN exon 1 detected an altered band with three different enzymes (HpaI, EcoRI and BclI). On an HpaI filter, this probe identifed an extra 10 kb band (Fig. 1B) that was not detected in 46 control individuals (data not shown). The HpaI restriction map of DACTYLIN identified a 14.9 kb fragment containing exon 1 (Fig. 1A). A probe located 10 kb upstream of exon 1, between the 5' HpaI site and exon 1, did not hybridize to the 10 kb band, thus localizing a genomic alteration to the 11 kb region between the DAC 5' probe and exon 1 (Fig. 1A).

Southern analysis of patients DH, VB and AC did not reveal any altered bands using cDNA probes containing exons 1–4 or exons 4–8 on EcoRI, BamHI, XbaI, HindIII, NcoI and NdeI filters.

Subgenomic library construction and characterization of a shifted band in patient DF
A subgenomic library for patient DF was constructed by cloning EcoRI genomic fragments, ranging from 8 to 10 kb, into a lambda vector in order to further characterize the 9 kb EcoRI fragment. Screening of this library with the DACTYLIN exon 4 probe identified a clone containing a 9.2 kb insert. Sequencing of this clone revealed an extra 5.2 kb sequence adjacent to 4 kb of DACTYLIN sequence which contained exons 3 and 4, intron 3 and adjacent portions of introns 2 and 4 (Fig. 2A). The breakpoint in DACTYLIN was located at position 871 125 bp on contig NT_033229, 1620 bp 5' of exon 3 (Fig. 2B). To confirm the rearrangement, PCR primers were designed in the new sequence and DACTYLIN intron 2 (Fig. 2A) and successfully amplified the expected 5.4 kb product in patient DF but not in a control individual (Fig. 2C). As additional confirmation, a probe to the extra 5.2 kb sequence (Cent1) hybridized to the 9 kb band previously detected with the DAC exon 4 probe in patient DF (Fig. 2D). Together, these data demonstrated the presence of an extra sequence disrupting the DACTYLIN gene between exons 2 and 3.



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  		Figure 2. Characterization of the 9 kb altered band detected on an EcoRI filter in patient DF. (A) Schematic of the insert of the clone containing the 9.2 kb altered band (estimated to be 9 kb by Southern). An extra sequence of 5.2 kb, located ~570 kb centromeric of the DACTYLIN gene, was adjacent to DACTYLIN intron 2. (B) Sequence of the breakpoint junction with the base pair position of the parental strand on the NT_033229 contig sequence. Sequence in italics represents the centromeric region located in database (AL357395). (C) PCR analysis using primer A (in the DACTYLIN gene) and B (in the centromeric sequence) from (A) revealed a PCR product of 5.4 kb only in patient DF but not in a control individual. (D) Southern blot analysis of EcoRI digested DNA from a control and patient DF using probe DAC exon 4 (left) or probe Cent1 located within the centromeric sequence (right). Both probes hybridize to the 9.2 kb band.

 
A BLASTN analysis (www.ncbi.nlm.nih.gov/blast) of this extra 5.2 kb sequence identified the BAC clone RP11-31L23 (GenBank no. AL357395), which is part of contig NT_033229 that also contains DACTYLIN. This extra sequence is ~570 kb centromeric to DACTYLIN, and the centromeric breakpoint in patient DF was located at position 300 773 bp on contig NT_033229 (Figs. 2B and 3A) in what we now refer to as the centromeric region.



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  		Figure 3. Computer-based sequencing of the SHFM3 critical region and breakpoint locations. (A) From top to bottom: the position of the STS markers, the BAC clones with their accession number assembled into the NT_033229 contig. Only the SHFM3 possible candidate genes are represented. PAX2, HUG1, HOX11, ß-TRCP and NFkB2 are in a centromeric to telomeric orientation. LBX1, DACTYLIN, FGF8, POLL and LDB1 are in telomeric to centromeric orientation. (B) location of the common duplicated region with the breakpoint (BP) regions (shaded boxed). The fine mapping of the breakpoint location for each patient is also represented (shaded arrows). Based upon the band sizes observed in the presence and absence of 5-azaC hypomethylating agent (data not shown), it was evident that the MluI site located between DACTYLIN and ß-TRCP and the 2 NruI sites 5' of DACTYLIN are methylated (asterisk).

 
PFGE analysis
The rearrangements detected in patients DF, TU and LS were further characterized by PFGE analysis. In addition, the other SHFM patients were included in this analysis to complete the mutational screening of DACTYLIN.

On a NruI filter, DACTYLIN cDNA probe DAC 4–8 (exons 4–8) detected extra fragments in patients DF, TU and LS, as anticipated from the Southern analysis, as well as in patients VB, DH and AC (Fig. 4A). For patient RH, a DACTYLIN exon 6 probe detected an extra fragment on an NruI filter which was also present in his affected mother but absent in his unaffected father (data not shown). The altered fragments present in all seven SHFM patients were similar in size ranging from ~550 to 610 kb (Fig. 4A) and none of these was detected in nine control individuals. In patients DF and TU, probe Cent1, located ~550 kb centromeric to DACTYLIN, recognized the altered band previously detected with probe DAC 4–8 (Fig. 4A). In all seven SHFM patients, probe Cent2, located between DACTYLIN and Cent1, hybridized to the extra fragments previously detected with DACTYLIN probes (Fig. 4A and B). Together these data suggested that the seven SHFM patients have a similar rearrangement involving the DACTYLIN gene and a sequence located ~460 kb (Cent2)–570 kb (Cent1) centromeric to this gene.



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  		Figure 4. PFGE analysis of samples from seven SHFM patients, digested with NruI. (A) Analysis of 6 SHFM patients (DF, TU, LS, VB, DH and AC) with probes DAC cDNA exons 4–8, Cent 1, Cent 2 and ß-TRCP exon1 were used. The presence of two bands (a and b) around 800 kb is due to the presence of a methylated NruI site located 5' of the FGF8 gene. A polymorphic band (c) is present in both control individual 2 and patient AC. Patient AC's altered band (d) is lower than the polymorphic band (c), resulting in the presence of a doublet (detected with DAC 4–8 and ß-TRCP probes). For patients LS and VB, the distinction between the altered band (detected with Cent 2 or ß-TRCP probes) and the polymorphic band could not be performed when using DACTYLIN specific probes. (B) Pedigree of RH's family and PFGE hybridization with Cent2 of a NruI filter. The 600 kb band is only present in the affected mother and the proband, which confirms segregation of the rearrangement within the family.

 
Multiple hybridizations with probes located throughout the centromeric region and DACTYLIN were used to refine the breakpoint regions in each patient. Scoring of the presence or absence of the altered band narrowed the breakpoint locations in the centromeric and the DACTYLIN regions. A polymorphic band (c) of ~550 kb was found in two controls and in patient AC using DACTYLIN probes and probes located 5' of DACTYLIN (Fig. 4A). In patient AC, the altered band could be distinguished from this polymorphic band because of a significant difference in size resulting in a doublet (c and d) (Fig. 4A). However, in patients LS and VB the presence of this polymorphic band interfered with the scoring for presence/absence of the altered band derived from the rearrangement and previously detected with the Cent2 probe.

Identification of the breakpoint junction in patient TU
Additional probing of an MluI filter identified the centromeric breakpoint in patient TU within a 24 kb region between the probe Cent13 (289 044–289 478 bp of contig NT_033229) and the next MluI site at position 264 876 bp (data not shown). Restriction map analysis of the 24 kb centromeric breakpoint region revealed 2 EcoRI and 2 PstI sites that were in the middle of the region. Southern analysis in patient TU demonstrated the presence of an altered band on an EcoRI filter (2.5 kb) and a PstI filter (4.2 kb) using the probe DAC int5 (Fig. 1B and data not shown). These findings suggested that the centromeric breakpoint is located in the vicinity of these EcoRI and PstI sites. PCR analysis was then performed using a primer located within the centromeric region containing the EcoRI and PstI sites and another primer located in intron 5 of DACTYLIN. A 2.1 kb band was amplified only from patient TU genomic DNA but not in DNA from two control individuals (data not shown). The sequence of this 2.1 kb band identified the breakpoint junction between DACTYLIN and the centromeric sequence (data not shown). The DACTYLIN breakpoint was located at position 863 909 bp of contig NT_033229, 2 kb downstream of exon 5. The centromeric breakpoint was located at position 282 974 bp, 18 kb from the centromeric breakpoint in patient DF (Fig. 3B).

Based upon PFGE results in the other five patients, the DACTYLIN breakpoints are within a 78.7 kb region between the probe DAC 5' (position 899 507–900 982 bp) and DACTYLIN exon 6 (position 820 652–820 913 bp; Fig. 3B). The centromeric breakpoints are within a 129.5 kb region between probe Cent6 (position 412 476–413 463 bp) and position 282 974 bp, the breakpoint identified in patient TU (Fig. 3B).

Southern analysis of human–mouse hybrid cell lines
Dosage analysis of an EcoRI filter revealed that the intensity of the normal DACTYLIN band was two to three times greater than the intensities of either altered band detected in DF and TU (data not shown). This finding suggested that DACTYLIN was present in more than two copies in patients DF and TU. This possibility was further tested using hybrid cell lines generated from these two patients. For patient DF, all four hybrid cell lines carried only one chromosome 10: two carried the chromosome 10 arbitrarily called ‘A’ and two carried the chromosome 10 called ‘B’. Chromosome 10B was considered the affected chromosome since it contained DNA marker alleles segregating with the phenotype (data not shown). Southern analysis was performed with EcoRI and BamHI using genomic DNA from a control individual and patient DF and DNA from two hybrids and a mouse fibroblast cell line (E2). On the EcoRI filter, a DACTYLIN exon 4 probe detected the extra 9 kb band and the normal 6.4 kb band in patient DF and cell line DF-Chr10B1, while only the normal band was seen in the control individual and cell line DF-Chr10A1 (Fig. 1B). An extra 10.2 kb band was seen only in the hybrid cell lines and in the mouse control because of cross hybridization with the dactylin mouse gene (Fig. 1B). On the BamHI filter, the same probe detected the extra 15 kb band and the normal 30 kb band in patient DF and cell line DF-Chr10B1, while only the normal band was seen in a control individual and cell line DF-Chr10A1 (data not shown). The presence of both the normal and altered bands in DF-Chr10B1 corresponding to the affected chromosome 10 suggested that at least a portion of DACTYLIN is duplicated on the affected chromosome in patient DF.

Interestingly, on both EcoRI and BamHI filters, the Cent1 probe detected 2 bands (the normal band and an extra band) in patient DF and DF Chr10B1, while only the normal band was detected in the control individual and DF-Chr10A1 (data not shown). The presence of both bands in DF-Chr10B1 suggested that the centromeric sequence is also duplicated on the affected chromosome in patient DF.

For patient TU, similar hybrids were created and Southern analysis was performed using DACTYLIN probes on PstI and EcoRI filters. Both normal and shifted bands were detected in the hybrids containing the affected chromosome, while only the normal band was detected in the hybrids containing the unaffected chromosome (data not shown). As in patient DF, the results were consistent with the presence of a partial DACTYLIN duplication on the affected chromosome.

Dosage analysis by slot-blot
Although the hybrid cell line studies demonstrated the presence of an extra copy of a portion of DACTYLIN on the affected chromosome of patients DF and TU, the exact DACTYLIN copy number in these patients could not be determined by dosage analysis of the Southern blots. Dosage analysis using a slot-blot method (32–34) was undertaken to determine if a duplication of DACTYLIN existed independent of the rearrangement or if the extra copy of DACTYLIN resulted from the rearrangement.

Hybridization of a filter containing genomic DNA from DF and two control individuals with DAC exon 4 probe revealed a statistically significant increase of the slope in patient DF, suggestive of an extra copy of the DACTYLIN exon 4 region in this patient (Fig. 5B and data not shown). A 3 : 2 ratio was observed between the slope observed in patient DF and in a control individual. This indicated that three copies of this portion of DACTYLIN are present in patient DF and two copies are present in the control individual and a trisomy 21 control. A DACTYLIN intron 2 probe, located on the other side of the breakpoint, is not duplicated in patient DF since no significant differences between the slopes were detected (Fig. 5C and data not shown). Furthermore, probe Cent1 is also duplicated while probe Cent12, localized on the other side of the centromeric breakpoint, is not (Fig. 5D and E and data not shown). Further hybridization with a ß-TRCP cDNA probe, located between DACTYLIN and Cent1, was performed and an increase of the slope was observed in patient DF (data not shown). Together, these data indicated that in patient DF the entire region between the DACTYLIN and the centromeric breakpoints is duplicated on the affected chromosome. This 570 kb duplicated region contains a portion of the DACTYLIN gene (exon 3 to the 3'-UTR), the complete ß-TRCP and POLL genes as well as LBX1, HUG1 and HOX11 genes which are centromeric to DACTYLIN.



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  		Figure 5. Graphical analysis of the densitometric values of test probes (y axis) vs the reference probe D4S12. In (A), the test probe is APP, a chromosome 21 specific probe; DAC exon 4 in (B); DAC intron 2 in (C), Cent1 in (D) and Cent12 in (E). (Diamond) Control; (Triangle) trisomy 21 individual; (Square) patient DF. In (A) the slope for the trisomy 21 patient is shifted, demonstrating duplication for the APP probe region in this patient. In (B) and (D) the slope for patient DF is shifted for probes DAC exon 4 and Cent1, respectively, but not for probes DAC intron 2 and Cent 12 in (C) and (E), respectively. The latter two probes are located on the other side of the DACTYLIN and centromeric breakpoints, respectively.

 
Slot blot analysis was then performed on the other six patients. In patients TU, LS, AC, DH and RH the region located between the DACTYLIN and centromeric breakpoints in each patient was also found to be present in three copies (data not shown). The dosage data were also used to narrow the DACTYLIN breakpoint region in patient VB that could not be determined by PFGE analysis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
A common genomic rearrangement was demonstrated to be associated with the SHFM phenotype in seven patients by using Southern, PFGE and dosage analyses as well as human–mouse hybrid cell lines containing a single chromosome 10 for two patients (DF and TU). The sequencing of the breakpoint junction in the same two patients revealed an extra sequence that is adjacent to DACTYLIN. This extra sequence is normally located ~570 kb centromeric to DACTYLIN. In the other five SHFM patients, the juxtaposition of both centromeric and DACTYLIN sequences was demonstrated by PFGE analysis. Additionally, Southern analysis of the single chromosome 10 hybrid cell lines of patients DF and TU and slot blot analysis of all seven patients demonstrated that the region located between the centromeric and DACTYLIN breakpoints is duplicated on one chromosome 10 in all the patients. Based on these findings, a model of an unequal recombination between the centromeric and DACTYLIN regions resulting in a tandem duplication is proposed to explain the rearrangement seen in these patients (Fig. 6). This model is consistent with the orientation of the sequences at the breakpoint junction, the size of the bands observed by PFGE and the duplication findings. The tandem duplication is likely the disease causing event for several reasons: (1) it segregates with the SHFM phenotype in the three families tested (DF, TU and RH); (2) the rearrangements detected in DF and LS were not detected in 46 control individuals; (3) the duplication is located on the affected chromosome, as demonstrated by the use of single chromosome 10 hybrid cell lines in patients DF and TU; and (4) none of the rearrangements detected by PFGE analysis in the seven SHFM patients was found in nine control individuals. Patient DH has the smallest duplicated region (440 kb) which defines the minimal duplicated region common to all patients. This narrows the number of genes included in the duplicated region to LBX1, ß-TRCP, POLL and a portion of the DACTYLIN gene (exon 9 to exon 6) involving a region of ~0.5 Mb (Fig. 6).



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  		Figure 6. Model of the tandem duplication detected in the seven SHFM patients created by an unequal recombination between sequences of the centromeric region and sequences of the DACTYLIN gene region. (A) Misalignment of the DACTYLIN and the centromeric sequences. (B) Result of an unequal recombination between the DACTYLIN and the centromeric sequences. The expected sizes of the NruI bands are indicated and the observed NruI band sizes are indicated in brackets.The asterisks define the methylated sites. The double-dagger symbol defines the expected and observed extra NruI fragment that is detected only in the SHFM patients.

 
Several other disorders have been reported to be associated with a submicroscopic duplication, deletion or inversion (35–37). These disorders are categorized as being genomic disorders in contrast to classic mendelian diseases (35). Regions of high similarity (>90% at the nucleotide level), known as low-copy repeats (LCRs), flank the genomic regions involved in the rearrangements (36,37). Indeed, misalignment and reciprocal recombination of these LCRs give rise to duplication, deletion or inversion of the regions flanked by these repeats (36–38). LCRs vary in size from 1 to 400 kb and can contain genes, pseudogenes, gene clusters or retroviral sequences. The tandem duplication detected in the SHFM patients suggests a similar mechanism for the rearrangements created by unequal crossing over events between highly similar regions of >1 kb and >90% identity. To look for LCRs in this region of chromosome 10, the 1.2 Mb of genomic sequence encompassing ~300 kb on either side of both breakpoint regions was analyzed using the DNA sequence comparisons program Miropeats (39). However, no evidence for the existence of such LCR sequences was identified, making it unlikely that LCRs mediate the rearrangements detected in the SHFM patients.

Several genomic rearrangements involving tandem duplications have been attributed to homologous recombination between Alu elements (40–45). Alu elements are the most abundant class of interspersed repeat sequences and represent 5–10% of the human genome. However, some regions that are prone to chromosomal rearrangement, like the chromosome 22q11 region, have more than 20% Alu elements. Using repeatmasker software (http://ftp.genome.washington.edu/cgi-bin/RepeatMasker), the repeat content of 100 kb of genomic sequence containing each breakpoint region was analyzed for interspersed repeat elements, and a high density of Alu elements (~20%) was observed in both regions. This finding suggests that the rearrangement detected in the SHFM patients may be mediated by an unequal homologous recombination between Alu elements located in the centromeric and DACTYLIN regions. However, analysis of the sequence of the breakpoint junction in patients DF and TU did not detect any residual Alu elements common to both parental strands or within 50 bp of the junctions. This would suggest instead a non-homologous recombination that may still be mediated by the repetitive elements. The mechanism of non-homologous recombination is poorly understood. It is believed to be a two-step process with chromatid cleavage in the first step and rejoining in the second (46). This process would explain why no recombination hot-spots (<2 kb) were found in the seven SHFM patients in contrast with other disorders involving homologous recombination (47,48). However, the breakpoints were still relatively clustered within over a region of ~100 kb. It is likely that the high density of repetitive elements in the region is responsible for bringing together the centromeric and DACTYLIN regions that are >400 kb apart, consequently favoring non-homologous recombination.

How the duplication leads to the SHFM phenotype is unknown. DACTYLIN is the best candidate gene for SHFM3 because of its role in the Dactylaplasia mice phenotype (27), which is presumed to be the animal model for SHFM3 (26). The Dac phenotype results from one of two different alleles, Dac1J and Dac2J and both heterozygous mice for Dac1J and Dac2J have an identical phenotype (27). The evidence suggests that the Dac2J allele causes the Dac phenotype by abolishing the normal dactylin transcript and/or creating a novel transcript. However, it is unclear if the Etn transposon insertion in the Dac1J allele is the disease-causing mutation, since homozygote Dac1J mice expressed normal levels of the dactylin transcript and no aberrant transcript was detected (27). In conjunction with this observation, our findings further question the role of dactylin in the Dac phenotype and DACTYLIN in human SHFM3, since they demonstrate partial DACTYLIN duplications in all patients tested. In five patients, extra DACTYLIN material is disrupted between exons 2 and 6 and in two patients the breakpoint is located no more than 8 kb upstream of the first exon. If DACTYLIN itself causes SHFM, it is possible that the rearrangements alter limb development by creating an aberrant DACTYLIN transcript that acts through a dominant negative mechanism. However, such a transcript has yet to be detected in our patients (data not shown) or in Dac1J mice (27).

Alternatively, the presence of a tandem duplication in the SHFM patients suggests that overexpression of another candidate gene may be responsible for the SHFM3 phenotype and possibly the Dac phenotype if dactylin itself is an innocent bystander in the Dac2J mouse. LBX1 and ß-TRCP are two potential candidate genes located in the duplicated region. LBX1 is a homeobox gene and plays an important function in developmental processes (49). However, this gene is mainly expressed in the central nervous system and its expression in the limb bud is restricted to the early myogenic cells, likely ruling out this gene as responsible for SHFM3 (49–51).

ß-TRCP is an F-Box-WD40 protein, like DACTYLIN, that is involved in the NFkB signaling transduction pathway. Several reports demonstrated a direct role of the NFkB pathway in limb development (52–56). Interestingly, three genes of the NFkB pathway (IKKa, NFkB2 and ß-TRCP) are within the critical region of SHFM3 defined by linkage mapping studies. IKKa knockout mice display severe limb and skin abnormalities. The NFkB pathway affects limb development by stimulating the expression of SHH and repressing the expression of BMP4 in the underlying mesenchyme (52). Therefore, NFkB activity is likely critical for the maintenance of the apical ectodermal ridge (AER) by controlling the expression of SHH and BMP4. The Dactylaplasia mouse phenotype, and probably the SHFM3 phenotype in human, is caused by a defect in the maintenance of the AER (29). Overexpression of ß-TRCP may disrupt NFkB activity giving rise to the SHFM3 phenotype. If this is the case, patients with trisomy of the 10q24 region, containing the ß-TRCP gene should display an SHFM-like phenotype. One patient with a de novo tandem duplication 10q24–q26 most likely including the ß-TRCP gene had mental retardation and characteristic facial features of the duplication 10q syndrome and minor malformation of the hands and feet (57). This patient had proximally implanted thumbs and toes, camptodactyly, a wide space between the first and second toes and syndactyly of the second and third toes (57). However, characteristic features of SHFM such as absence of the central digits and wide clefts in the hands/feet were not reported. Therefore, this patient does not seem to have a typical SHFM phenotype that would argue for a causative role of a dosage effect of ß-TRCP in the SHFM3 phenotype. However, the existence of a modifier locus for SHFM3 (25) may complicate the phenotypical analysis of this patient; indeed this patient may not display a SHFM phenotype because of his genotype at the modifier locus.

A cis-acting positional effect may be another mechanism that would cause the SHFM3 phenotype by disrupting the expression of the genes flanking the breakpoints of the duplication. Interestingly, the 400 kb proximal and distal flanking regions harbor numerous candidate genes with known or putative roles in limb development (Fig. 3A). FGF8 maps to the critical region of SHFM3 as defined by linkage mapping and its role in initiation, growth and patterning of the limb is extensively documented (58–60). However, direct sequencing and Southern analysis in SHFM3 patients did not reveal any mutations, ruling out this gene as the major cause of SHFM3 (61,62, Gurrieri and Schwartz, unpublished data). Suppressor of Fused (SUFU) is also located within the critical region and acts as negative regulator of the SHH pathway. However, SUFU mutations were recently found in patients with medulloblastoma (63) and no mutations were found among SHFM3 patients (62,64). HOX11 is mainly expressed in T-cells and disruption of this gene caused acute lymphoblastic leukemia in a patient with a chromosomal translocation t(10;14) (65). More importantly, the HOX11 murine homolog, tlx1, is not expressed in the limb bud and tlx1 knockout mice did not display any limb phenotype, arguing against a causative role of HOX11 (particularly through a loss of function mechanism) in SHFM3 (66).

Given the organization of the SHFM3 critical region, which contains multiple genes involved in the NFkB pathway and in limb development, it is conceivable that more than one gene in the region may be involved in the same pathway leading to SHFM. Hence, the Dac phenotype may be caused by two different mechanisms. For example, the Dac2J allele may cause limb malformation through loss of dactylin function and/or production of an aberrant transcript, while the Dac1J allele may cause increased dosage of a neighboring gene that normally downregulates dactylin or is downregulated by dactylin. In the families that we have studied, SHFM3 could result from either mechanism.

An important consequence of this study is the possible use of PFGE analysis with either DACTYLIN or centromeric probes for SHFM3 diagnosis. The novel NruI fragment observed by PFGE analysis with the probe Cent2 may be the best approach for SHFM3 diagnosis. Indeed, probe Cent2 detected an altered fragment in all the SHFM patients and the distinction between the normal and altered bands was fairly easy to ascertain. However, PFGE analysis requires the use of lymphoblastoid cell lines and these are not always available. An alternative approach would be to perform dosage analysis of the region using slot blot analysis or real time quantitative PCR. These approaches were recently initiated in three other families linked to chromosome 10q24. In two of these, slot blot analysis using probes ß-TRCP and DACTYLIN exon 6 demonstrated evidence of a duplicated fragment. In the third family, PFGE analysis using DACTYLIN exon 6 detected an extra band in an affected individual. Although further molecular characterization needs to be performed on these families, these preliminary data suggest a similar rearrangement with the other seven SHFM3 families.

This study raises several questions for future studies on SHFM3: (1) what is the mechanism of this rearrangement; (2) how common is this rearrangement among SHFM patients; and most importantly, (3) how does this rearrangement cause the SHFM3 phenotype? In a more general sense, this study reinforces the importance of Southern and PFGE screening to detect genomic rearrangements that can be missed by PCR-based approaches. These techniques will be essential for the identification of the rapidly growing family of genomic disorders.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
SHFM patients
All patients were ascertained for study based upon the presence of SHFM in one or more limbs. Informed consent was obtained prior to participation, and these studies were approved by the Institutional Review Committee of Self Regional Healthcare (Greenwood, SC, USA). Clinical details, pedigrees and linkage analysis of families of six of the seven patients (DF, AC, TU, LS, DH and VB) were previously described (18,24,62,67) and are summarized in Table 1. In all families, the phenotype was consistent with non-syndromic SHFM type I (6). Patient TU and some of his family members have thumb defects (duplicated and/or triphalangeal) with or without split feet. Patient RH has SHFM associated with medulloblastoma.


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  		Table 1. Clinical features of the seven SHFM patients
 
Analysis of SSCP
The sequence data from the full-length DACTYLIN cDNA (GenBank accession no. AF0281859) and genomic sequences from BAC clones RP11-190J1, RP11-148E14 and RP11-573E23 (GenBank accession nos AC010789, AL627144 and AL627424) were compared using the program Seqman 5.00© (DNASTAR Inc.) and Blast 2 sequence software (www.ncbi.nlm.nih.gov/Blast/). Divergence between the two sequences was taken to be indicative of the presence of an intron. BLAST analysis also identified a DACTYLIN pseudogene on chromosome 22q11. From the genomic structure, primers were designed to amplify the coding region of DACTYLIN for SSCP and sequence analysis. Primer sequences of the nine exons of the human DACTYLIN gene were designed using the program Primerselect 5.00© (DNASTAR Inc.). Individual exons, along with flanking intronic sequences, were amplified using 50 ng of genomic DNA in a total volume of 10 µl, containing 50 µM of each dNTPs, 1 µCi {alpha}32dCTP, 1 µM of each primer and 0.5 units of Taq DNA polymerase (Sigma, St Louis, MO, USA). The PCR conditions were as follows: one cycle at 95°C for 4 min; 20 cycles at 95°C for 30 s, annealing at 65–55°C (decrement of 0.5°C/cycle) for 30 s and elongation at 72°C for 30 s, then 15 cycles at 95°C for 30 s, 55°C for 30 s, 72°C for 30 s; one cycle at 72°C for 5 min. Five microliters of the radiolabeled PCR product were denatured with 5 µl of stop solution (95% formamide, 10 mM NaOH, 0.25% bromophenol blue, 0.25% xylene cyanol) at 94°C for 2 min before cooling on ice. Three microliters of the reaction mixture were loaded on a 0.5xMDETM Acrylamide gel (FMC, Rockland, ME, USA) prepared following the manufacturer's procedure. Electrophoresis was performed at a constant power of 8 W for 19 h at room temperature. The gel was attached onto 3MM Whatman filter paper, and dried before exposure to X-ray Biomax film, (Kodak, Rochester, NY, USA) using standard techniques.

Sequencing
PCR products were either directly purified or gel-purified using the QIAquick PCR purification and gel extraction kit (QIAGEN, Valencia, CA, USA), and sequenced using the Thermosequenase CyTM5.5 Dye Terminator kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and the forward or reverse exon-specific primer. A final purification was performed on Autoseq columns (Amersham Pharmacia Biotech, Piscataway, NJ, USA). All the samples were run on the automated laser fluorescence (ALF) DNA sequencer (Pharmacia Biotech, Piscataway, NJ, USA).

Southern analysis
Five micrograms of each patient's genomic DNA were singly digested overnight at 37°C using EcoRI, PstI, DraI, HindIII, XbaI, BclI, BglI, BglII, HpaI, BamHI and PvuII enzymes (New England Biolabs® Inc., Beverly, MA, USA). After digestion, samples were electrophoresed on a 0.8% Seakem® LE agarose gel (BMA, Rockland, ME, USA) and transferred to a Nytran membrane (Schleicher and Schuell, Keene, NH, USA) using an alkaline downward transfer procedure (68). The filters were pre-hybridized for 2–3 h at 65°C in hybridization solution (4xSSPE, 2xDenhardt's solution, 0.5% SDS, 6% polyethyleneglycol, 40 µg/ml denatured sonicated salmon sperm DNA) before adding the denatured probe. After 12–18 h of hybridization, filters were washed at 65°C, using a final wash of 0.1xSSC, 0.1% SDS. The filters were exposed to X-ray Biomax film (Kodak, Rochester, NY, USA), using standard techniques.

Subgenomic library construction
One-hundred and fifty micrograms of patient DNA was digested and electrophoresed using the same conditions as for the Southern analysis. The region containing the band of interest was cut out from the gel and the DNA was electroeluted and purified using Centricon® columns (Millipore Corporation, Bedford, MA, USA). The amount of DNA was quantified by UV (260 nm) detection and 0.2 µg were used in the ligation reaction containing 1 µg of the lambda zap express® vector (Stratagene, La Jolla, CA, USA). After packaging, the library was amplified and ~5x105 to 1x106 plaques were plated, blotted and probed following the manufacturer's protocol (Stratagene, La Jolla, CA, USA).

Lymphoblastoid, human-mouse hybrid cell lines and DNA extraction
Epstein–Barr virus-transformed lymphoblastoid cell lines were established from controls and patients following a standard procedure (69). Human-mouse hybrid cell lines from patients DF and TU were generated using Conversion technology (GMP genetics Inc., Waltham, MA, USA). Genomic DNA was isolated from peripheral blood or from the cell lines by high salt precipitation (70). Purified DNA was diluted to a concentration of 105 ng/µl.

PFGE analysis
High molecular weight DNA was isolated in agarose plugs as previously described (71) using lymphoblastoid cell lines. The plugs were rinsed twice in 50 ml of TE buffer and then equilibrated for 30 min in 300 µl of the appropriate endonuclease restriction buffer. Excess buffer was removed and the plugs were incubated at the appropriate temperature for 16 h with 50 U of endonuclease enzyme per reaction (71). Single digestion of the DNA plugs was performed using NotI, MluI, and NruI enzymes. Digested fragments in the range 50–500 kb were separated by electrophoresis on a 1% Seakem® Gold agarose gel (BMA, Rockland, ME, USA) at 200 V for 20 h, at 9°C in 0.5xTBE running buffer, using a 25 s pulse time on a Gene Navigator® apparatus (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The separation of fragments 400–900 kb was accomplished by electrophoresis at 170 V for 24 h, at 9°C in 0.5xTBE buffer, using a 100 s pulse time.

DNA hybridization probes
DACTYLIN cDNA probes, containing exons 1–4 (DAC 1–4), exons 2–4 (DAC 2–4) and exons 4–8 (DAC 4–8) were generated by RT–PCR amplification of RNA extracted from a lymphoblastoid cell line from a control individual. Hybridization of individual exons was performed using PCR products generated with SSCP primers. The genomic sequence corresponding to the DACTYLIN gene region (contig NT_033229.1) was submitted to the repeatmasker software available at http://ftp.genome.washington.edu/cgi-bin/RepeatMasker. Intronic probes were then developed using primers residing in non-repetitive sequences of the NT_033229.1 contig. Each intronic probe was amplified using 10 ng of BAC DNA when available or 100 ng of total genomic DNA extracted from a control individual. For intronic probes larger than 3 kb, long-range PCR was performed using Taq polymerase with buffer 1 from the Expand Long Template PCR system (Roche, Indianapolis, IN, USA) following the manufacturer's procedures. Prior to hybridization, intronic probes were preassociated with sonicated human placental DNA (Sigma, St Louis, MO, USA) in 50 000-fold excess, at 65°C for 90 min. All the PCR products used as probes were gel-purified with the QIAquick gel extraction kit (QIAGEN, Valencia, CA, USA) and sequenced. PCR products were labeled using the RadprimeTM labeling system (Gibco-Invitrogen, Carlsbad, CA, USA) in the presence of 50 µCi of {alpha}32dCTP (NEN, Boston, MA, USA).

Quantification of chromosome 10 sequences by slot-blot analysis
The slot-blot technique was used, with modifications, for the quantification of chromosome 10 sequences (32,33). In summary, DNA was quantified by a fluorescence-based assay using the dye PicoGreen® (Molecular Probes, Eugene, OR, USA). The DNA concentration was then adjusted to 50 ng/µl by dilution in 10 mM Tris–HCl buffer, pH 7.5, 1 mM EDTA and in 0.2 M of NaOH. The samples were incubated at 37°C for 20 min and 12 serial dilutions (0.2–1.5 µg) in 0.15 M NaOH and 0.1xSSC were blotted onto GeneScreen Plus® (NEN®, Life Science Products, Boston, MA, USA) using a slot blot apparatus (Minifold II; Schleicher & Schuell, Keene, NH, USA). After the DNA solution went through, each slot was washed with 2xSSC and the whole membrane was then rinsed in 2xSSC for 5 min and baked at 80°C for 20 min. Blots were probed with a reference probe, D4S12, prior to the hybridization with the test probes from chromosomes 21 and 10. All the probes were sequenced and tested by Southern hybridization to confirm their specificity and lack of hybridization to repetitive sequences. Band intensities were quantified using the Phospho-imager FUJIX BAS 1000 (Fuji Medical Systems, Stamford, CT, USA). The chromosome 10 signal ( y axis) was then plotted against the reference probe signal (x axis) and the slopes were statistically analyzed and their comparison was assessed by t-test for significance as previously described (32,33).

To test the efficacy of the slot-blot method, DNAs from a control individual, patient DF and a trisomic 21 individual were used. Successive probing with a reference probe, D4S12 and a chromosome 21 probe, corresponding to the APP gene, revealed an extra copy of the APP gene only in the trisomic 21 patient. This was characterized by a statistically significant (P<0.001) increase of the slope representative of the correlation between signals from the reference probe and the chromosome 21 probe (Fig. 6A).


   ACKNOWLEDGEMENTS
 
We thank the patients and families for their participation. Sequencing analysis was performed by Susan Daniels and sample coordination was provided by Cindy Skinner of the Center for Molecular Studies (CMS). Tonya Moss was responsible for maintaining the patients' cell lines. Special thanks are due to Dr William Marcotte Jr, at Clemson University, for his support and scientific advice. We also thank Dr Julianne Collins for her help with the statistical analysis of the data. This work was supported in part by a grant from the South Carolina Department of Disabilities and Special Needs and NIH grant R24MH57840 (R.E.S.). This paper is dedicated to the memory of Ethan Francis Schwartz, 1996–1998.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Center for Molecular Studies, J.C. Self Research Institute, One Gregor Mendel Circle, Greenwood, SC 29646, USA. Tel: +1 8649418140; Fax: +1 8643881707; Email: schwartz{at}ggc.org Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Calzolari, E., Manservigi, D., Garani, G.P., Cocchi, G., Magnani, C. and Milan, M. (1990) Limb reduction defects in Emilia Romagna, Italy: epidemiological and genetic study in 173,109 consecutive births. J. Med. Genet., 27, 353–357.[Abstract/Free Full Text]

  2. Nelson, K. and Holmes, L.B. (1989) Malformations due to presumed spontaneous mutations in newborn infants. New Engl. J. Med., 320, 19–23.[Abstract]

  3. Froster-Iskenius, U.G. and Baird, P.A. (1989) Limb reduction defects in over one million consecutive live births. Teratology, 39, 127–135.[CrossRef][Web of Science][Medline]

  4. Bod, M., Czeizel, A. and Lenz, W. (1983) Incidence at birth of different types of limb reduction abnormalities in Hungary 1975–1977. Hum. Genet., 65, 27–33.[CrossRef][Web of Science][Medline]

  5. Riano, G., I, Fernandez, T.J., Garcia, L.E., Moro, B.C., Mosquera, T.C., Rodriguez, F.A., Suarez, M.E., Ariza, H.F. and Franganillo, F.A. (2000) [Limb reduction defects in Asturias (1986–1997): prevalence and clinical presentation]. An. Esp. Pediatr., 52, 362–368.[Medline]

  6. Zlotogora, J. (1994) On the inheritance of the split hand/split foot malformation. Am. J. Med. Genet., 53, 29–32.[CrossRef][Web of Science][Medline]

  7. Roelfsema, N.M. and Cobben, J.M. (1996) The EEC syndrome: a literature study. Clin. Dysmorphol., 5, 115–127.[CrossRef][Web of Science][Medline]

  8. Temtamy, S.A. and McKusick, V.A. (1978) The genetics of hand malformations. Birth Defects Orig. Artic. Ser., 14, i-619.

  9. Spranger, M. and Schapera, J. (1988) Anomalous inheritance in a kindred with split hand, split foot malformation. Eur. J. Pediatr., 147, 202–205.[CrossRef][Web of Science][Medline]

  10. Jarvik, G.P., Patton, M.A., Homfray, T. and Evans, J.P. (1994) Non-Mendelian transmission in a human developmental disorder: split hand/split foot. Am. J. Hum. Genet., 55, 710–713.[Web of Science][Medline]

  11. Ahmad, M., Abbas, H., Haque, S. and Flatz, G. (1987) X-chromosomally inherited split-hand/split-foot anomaly in a Pakistani kindred. Hum. Genet., 75, 169–173.[CrossRef][Web of Science][Medline]

  12. Zlotogora, J. and Nubani, N. (1989) Is there an autosomal recessive form of the split hand and split foot malformation? J. Med. Genet., 26, 138–140.[Abstract/Free Full Text]

  13. Faiyaz ul, H.M., Uhlhaas, S., Knapp, M., Schuler, H., Friedl, W., Ahmad, M. and Propping, P. (1993) Mapping of the gene for X-chromosomal split-hand/split-foot anomaly to Xq26–q26.1. Hum. Genet., 91, 17–19.[Web of Science][Medline]

  14. Gul, D. and Oktenli, C. (2002) Evidence for autosomal recessive inheritance of split hand/split foot malformation: a report of nine cases. Clin. Dysmorphol., 11, 183–186.[CrossRef][Web of Science][Medline]

  15. Scherer, S.W., Poorkaj, P., Allen, T., Kim, J., Geshuri, D., Nunes, M., Soder, S., Stephens, K., Pagon, R.A. and Patton, M.A. (1994) Fine mapping of the autosomal dominant split hand/split foot locus on chromosome 7, band q21.3–q22.1. Am. J. Hum. Genet., 55, 12–20.[Web of Science][Medline]

  16. Scherer, S.W., Poorkaj, P., Massa, H., Soder, S., Allen, T., Nunes, M., Geshuri, D., Wong, E., Belloni, E. and Little, S. (1994) Physical mapping of the split hand/split foot locus on chromosome 7 and implication in syndromic ectrodactyly. Hum. Mol. Genet., 3, 1345–1354.[Abstract/Free Full Text]

  17. Nunes, M.E., Schutt, G., Kapur, R.P., Luthardt, F., Kukolich, M., Byers, P. and Evans, J.P. (1995) A second autosomal split hand/split foot locus maps to chromosome 10q24–q25. Hum. Mol. Genet., 4, 2165–2170.[Abstract/Free Full Text]

  18. Gurrieri, F., Prinos, P., Tackels, D., Kilpatrick, M.W., Allanson, J., Genuardi, M., Vuckov, A., Nanni, L., Sangiorgi, E., Garofalo, G. et al. (1996) A split hand-split foot (SHFM3) gene is located at 10q24->25. Am. J. Med. Genet., 62, 427–436.[CrossRef][Web of Science][Medline]

  19. Celli, J., Duijf, P., Hamel, B.C., Bamshad, M., Kramer, B., Smits, A.P., Newbury-Ecob, R., Hennekam, R.C., Van Buggenhout, G., van Haeringen, A. et al. (1999) Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome. Cell, 99, 143–153.[CrossRef][Web of Science][Medline]

  20. Boles, R.G., Pober, B.R., Gibson, L.H., Willis, C.R., McGrath, J., Roberts, D.J. and Yang-Feng, T.L. (1995) Deletion of chromosome 2q24–q31 causes characteristic digital anomalies: case report and review. Am. J. Med. Genet., 55, 155–160.[CrossRef][Web of Science][Medline]

  21. Goodman, F.R., Majewski, F., Collins, A.L. and Scambler, P.J. (2002) A 117-kb microdeletion removing HOXD9-HOXD13 and EVX2 causes synpolydactyly. Am. J. Hum. Genet., 70, 547–555.[CrossRef][Web of Science][Medline]

  22. van Bokhoven, H., Hamel, B.C., Bamshad, M., Sangiorgi, E., Gurrieri, F., Duijf, P.H., Vanmolkot, K.R., van Beusekom, E., van Beersum, S.E., Celli, J. et al. (2001) p63 Gene mutations in eec syndrome, limb-mammary syndrome, and isolated split hand–split foot malformation suggest a genotype–phenotype correlation. Am. J. Hum. Genet., 69, 481–492.[CrossRef][Web of Science][Medline]

  23. Ianakiev, P., Kilpatrick, M.W., Toudjarska, I., Basel, D., Beighton, P. and Tsipouras, P. (2000) Split-hand/split-foot malformation is caused by mutations in the p63 gene on 3q27. Am. J. Hum. Genet., 67, 59–66.[Medline]

  24. Raas-Rothschild, A., Manouvrier, S., Gonzales, M., Farriaux, J.P., Lyonnet, S. and Munnich, A. (1996) Refined mapping of a gene for split hand–split foot malformation (SHFM3) on chromosome 10q25. J. Med. Genet., 33, 996–1001.[Abstract/Free Full Text]

  25. Chai, C.K. (1981) Dactylaplasia in mice a two-locus model for development anomalies. J. Hered., 72, 234–237.[Abstract/Free Full Text]

  26. Johnson, K.R., Lane, P.W., Ward-Bailey, P. and Davisson, M.T. (1995) Mapping the mouse dactylaplasia mutation, Dac, and a gene that controls its expression, mdac. Genomics, 29, 457–464.[CrossRef][Web of Science][Medline]

  27. Sidow, A., Bulotsky, M.S., Kerrebrock, A.W., Birren, B.W., Altshuler, D., Jaenisch, R., Johnson, K.R. and Lander, E.S. (1999) A novel member of the F-box/WD40 gene family, encoding dactylin, is disrupted in the mouse dactylaplasia mutant. Nat. Genet., 23, 104–107.[CrossRef][Web of Science][Medline]

  28. Seto, M.L., Nunes, M.E., MacArthur, C.A. and Cunningham, M.L. (1997) Pathogenesis of ectrodactyly in the Dactylaplasia mouse: aberrant cell death of the apical ectodermal ridge. Teratology, 56, 262–270.[CrossRef][Web of Science][Medline]

  29. Crackower, M.A., Motoyama, J. and Tsui, L.C. (1998) Defect in the maintenance of the apical ectodermal ridge in the Dactylaplasia mouse. Dev. Biol., 201, 78–89.[CrossRef][Web of Science][Medline]

  30. Ianakiev, P., Kilpatrick, M.W., Dealy, C., Kosher, R., Korenberg, J.R., Chen, X.N. and Tsipouras, P. (1999) A novel human gene encoding an F-box/WD40 containing protein maps in the SHFM3 critical region on 10q24. Biochem. Biophys. Res. Commun., 261, 64–70.[CrossRef][Web of Science][Medline]

  31. Sifakis, S., Basel, D., Ianakiev, P., Kilpatrick, M. and Tsipouras, P. (2001) Distal limb malformations: underlying mechanisms and clinical associations. Clin. Genet., 60, 165–172.[CrossRef][Web of Science][Medline]

  32. Rahmani, Z., Blouin, J.L., Creau-Goldberg, N., Watkins, P.C., Mattei, J.F., Poissonnier, M., Prieur, M., Chettouh, Z., Nicole, A. and Aurias, A. (1989) Critical role of the D21S55 region on chromosome 21 in the pathogenesis of Down syndrome. Proc. Natl Acad. Sci. USA, 86, 5958–5962.[Abstract/Free Full Text]

  33. Blouin, J.L., Rahmani, Z., Chettouh, Z., Prieur, M., Fermanian, J., Poissonnier, M., Leonard, C., Nicole, A., Mattei, J.F. and Sinet, P.M. (1990) Slot blot method for the quantification of DNA sequences and mapping of chromosome rearrangements: application to chromosome 21. Am. J. Hum. Genet., 46, 518–526.[Web of Science][Medline]

  34. Delabar, J.M., Chettouh, Z., Rahmani, Z., Theophile, D., Blouin, J.L., Bono, R., Kraus, J., Barton, J., Patterson, D. and Sinet, P.M. (1992) Gene-dosage mapping of 30 DNA markers on chromosome 21. Genomics, 13, 887–889.[CrossRef][Web of Science][Medline]

  35. Lupski, J.R. (1998) Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits. Trends Genet., 14, 417–422.[CrossRef][Web of Science][Medline]

  36. Inoue, K. and Lupski, J.R. (2002) Molecular mechanisms for genomic disorders. A. Rev. Genom. Hum. Genet., 3, 199–242.

  37. Stankiewicz, P. and Lupski, J.R. (2002) Genome architecture, rearrangements and genomic disorders. Trends Genet., 18, 74–82.[CrossRef][Web of Science][Medline]

  38. Ji, Y., Eichler, E.E., Schwartz, S. and Nicholls, R.D. (2000) Structure of chromosomal duplicons and their role in mediating human genomic disorders. Genome Res., 10, 597–610.[Abstract/Free Full Text]

  39. Parsons, J.D. (1995) Miropeats: graphical DNA sequence comparisons. Comput. Appl. Biosci., 11, 615–619.[Abstract/Free Full Text]

  40. Stoppa-Lyonnet, D., Carter, P.E., Meo, T. and Tosi, M. (1990) Clusters of intragenic Alu repeats predispose the human C1 inhibitor locus to deleterious rearrangements. Proc. Natl Acad. Sci. USA, 87, 1551–1555.[Abstract/Free Full Text]

  41. Pousi, B., Hautala, T., Heikkinen, J., Pajunen, L., Kivirikko, K.I. and Myllyla, R. (1994) Alu-Alu recombination results in a duplication of seven exons in the lysyl hydroxylase gene in a patient with the type VI variant of Ehlers-Danlos syndrome. Am. J. Hum. Genet., 55, 899–906.[Web of Science][Medline]

  42. Rudiger, N.S., Gregersen, N. and Kielland-Brandt, M.C. (1995) One short well conserved region of Alu-sequences is involved in human gene rearrangements and has homology with prokaryotic chi. Nucl. Acids Res., 23, 256–260.[Abstract/Free Full Text]

  43. Whitman, S.P., Strout, M.P., Marcucci, G., Freud, A.G., Culley, L.L., Zeleznik, L., Mrozek, K., Theil, K.S., Kees, U.R., Bloomfield, C.D. and Caligiuri, M.A. (2001) The partial nontandem duplication of the MLL (ALL1) gene is a novel rearrangement that generates three distinct fusion transcripts in B-cell acute lymphoblastic leukemia. Cancer Res., 61, 59–63.[Abstract/Free Full Text]

  44. Kutsche, K., Ressler, B., Katzera, H.G., Orth, U., Gillessen-Kaesbach, G., Morlot, S., Schwinger, E. and Gal, A. (2002) Characterization of breakpoint sequences of five rearrangements in L1CAM and ABCD1 (ALD) genes. Hum. Mutat., 19, 526–535.[CrossRef][Web of Science][Medline]

  45. Kolomietz, E., Meyn, M.S., Pandita, A. and Squire, J.A. (2002) The role of Alu repeat clusters as mediators of recurrent chromosomal aberrations in tumors. Genes Chromosomes Cancer, 35, 97–112.[CrossRef][Web of Science][Medline]

  46. Hu, X.Y., Ray, P.N. and Worton, R.G. (1991) Mechanisms of tandem duplication in the Duchenne muscular dystrophy gene include both homologous and nonhomologous intrachromosomal recombination. EMBO J., 10, 2471–2477.[Web of Science][Medline]

  47. Pentao, L., Wise, C.A., Chinault, A.C., Patel, P.I. and Lupski, J.R. (1992) Charcot–Marie–Tooth type 1A duplication appears to arise from recombination at repeat sequences flanking the 1.5 Mb monomer unit. Nat. Genet., 2, 292–300.[CrossRef][Web of Science][Medline]

  48. Strout, M.P., Marcucci, G., Bloomfield, C.D., and Caligiuri, M.A. (1998) The partial tandem duplication of ALL1 (MLL) is consistently generated by Alu-mediated homologous recombination in acute myeloid leukemia. Proc. Natl Acad. Sci. USA, 95, 2390–2395.[Abstract/Free Full Text]

  49. Jagla, K., Dolle, P., Mattei, M.G., Jagla, T., Schuhbaur, B., Dretzen, G., Bellard, F. and Bellard, M. (1995) Mouse Lbx1 and human LBX1 define a novel mammalian homeobox gene family related to the Drosophila lady bird genes. Mech. Dev., 53, 345–356.[CrossRef][Web of Science][Medline]

  50. Brohmann, H., Jagla, K. and Birchmeier, C. (2000) The role of Lbx1 in migration of muscle precursor cells. Development, 127, 437–445.[Abstract]

  51. Gross, M.K., Moran-Rivard, L., Velasquez, T., Nakatsu, M.N., Jagla, K. and Goulding, M. (2000) Lbx1 is required for muscle precursor migration along a lateral pathway into the limb. Development, 127, 413–424.[Abstract]

  52. Bushdid, P.B., Brantley, D.M., Yull, F.E., Blaeuer, G.L., Hoffman, L.H., Niswander, L. and Kerr, L.D. (1998) Inhibition of NF-kappaB activity results in disruption of the apical ectodermal ridge and aberrant limb morphogenesis. Nature, 392, 615–618.[CrossRef][Medline]

  53. Hu, Y., Baud, V., Delhase, M., Zhang, P., Deerinck, T., Ellisman, M., Johnson, R. and Karin, M. (1999) Abnormal morphogenesis but intact IKK activation in mice lacking the IKKalpha subunit of IkappaB kinase. Science, 284, 316–320.[Abstract/Free Full Text]

  54. Takeda, K., Takeuchi, O., Tsujimura, T., Itami, S., Adachi, O., Kawai, T., Sanjo, H., Yoshikawa, K., Terada, N. and Akira, S. (1999) Limb and skin abnormalities in mice lacking IKKalpha. Science, 284, 313–316.[Abstract/Free Full Text]

  55. Li, Q., Lu, Q., Hwang, J.Y., Buscher, D., Lee, K.F., Izpisua-Belmonte, J.C. and Verma, I.M. (1999) IKK1-deficient mice exhibit abnormal development of skin and skeleton. Genes Dev., 13, 1322–1328.[Abstract/Free Full Text]

  56. Fisher, C. (2000) IKKalpha-/- mice share phenotype with pupoid fetus (pf/pf) and repeated epilation (Er/Er) mutant mice. Trends Genet., 16, 482–484.[CrossRef][Web of Science][Medline]

  57. Tomkins, D.J., Gitelman, B.J. and Roberts, M.H. (1983) Confirmation of a de novo duplication, dup(10) (q24 leads to q26), by GOT1 gene dosage studies. Hum. Genet., 63, 369–373.[CrossRef]

  58. Crossley, P.H., Minowada, G., MacArthur, C.A. and Martin, G.R. (1996) Roles for FGF8 in the induction, initiation, and maintenance of chick limb development. Cell, 84, 127–136.[CrossRef][Web of Science][Medline]

  59. Lewandoski, M., Sun, X. and Martin, G.R. (2000) Fgf8 signalling from the AER is essential for normal limb development. Nat. Genet., 26, 460–463.[CrossRef][Web of Science][Medline]

  60. Moon, A.M. and Capecchi, M.R. (2000) Fgf8 is required for outgrowth and patterning of the limbs. Nat. Genet., 26, 455–459.[CrossRef][Web of Science][Medline]

  61. Ozen, R.S., Baysal, B.E., Devlin, B., Farr, J.E., Gorry, M., Ehrlich, G.D. and Richard, C.W. (1999) Fine mapping of the split-hand/split-foot locus (SHFM3) at 10q24: evidence for anticipation and segregation distortion. Am. J. Hum. Genet., 64, 1646–1654.[CrossRef][Web of Science][Medline]

  62. Tackels, D. (1996) Mapping of the third locus for split hand–split foot malformation, SHFM3. Clemson University.

  63. Taylor, M.D., Liu, L., Raffel, C., Hui, C.C., Mainprize, T.G., Zhang, X., Agatep, R., Chiappa, S., Gao, L., Lowrance, A. et al. (2002) Mutations in SUFU predispose to medulloblastoma. Nat. Genet., 31, 306–310.[CrossRef][Web of Science][Medline]

  64. Grimm, T., Teglund, S., Tackels, D., Sangiorgi, E., Gurrieri, F., Schwartz, C. and Toftgard, R. (2001) Genomic organization and embryonic expression of Suppressor of Fused, a candidate gene for the split-hand/split-foot malformation type 3. FEBS Lett., 505, 13–17.[CrossRef][Web of Science][Medline]

  65. Hatano, M., Roberts, C.W., Minden, M., Crist, W.M. and Korsmeyer, S.J. (1991) Deregulation of a homeobox gene, HOX11, by the t(10;14) in T cell leukemia. Science, 253, 79–82.[Abstract/Free Full Text]

  66. Raju, K., Tang, S., Dube, I.D., Kamel-Reid, S., Bryce, D.M. and Breitman, M.L. (1993) Characterization and developmental expression of Tlx-1, the murine homolog of HOX11. Mech. Dev., 44, 51–64.[CrossRef][Web of Science][Medline]

  67. Le Marec, B., Odent, S. and Treguier, C. (1990) Triphalangeal thumb and split foot in the same family. Genet. Couns., 1, 251–258.[Medline]

  68. Chomczynski, P. (1992) One-hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal. Biochem., 201, 134–139.[CrossRef][Web of Science][Medline]

  69. Bolton, B.J. and Spurr, N.K. (1996) Culture of Immortalized Cells. Wiley-Liss, New York.

  70. Schwartz, C.E., Ulmer, J., Brown, A., Pancoast, I., Goodman, H.O. and Stevenson, R.E. (1990) Allan-Herndon syndrome. II. Linkage to DNA markers in Xq21. Am. J. Hum. Genet., 47, 454–458.[Web of Science][Medline]

  71. Herrmann, B.G., Barlow, D.P. and Lehrach, H. (1987) A large inverted duplication allows homologous recombination between chromosomes heterozygous for the proximal t complex inversion. Cell, 48, 813–825.[CrossRef][Web of Science][Medline]


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