Characterization of the split hand/split foot malformation locus SHFM1 at 7q21.3-q22.1 and analysis of a candidate gene for its expression during limb development
Characterization of the split hand/split foot malformation locus SHFM1 at 7q21.3-q22.1 and analysis of a candidate gene for its expression during limb developmentMichael A. Crackower1,2,+, Stephen W. Scherer2,+, Johanna M. Rommens1,2, Chi-Chung Hui1,3, Parvoneh Poorkaj4, Sylvia Soder2, Jan Maarten Cobben5, Louanne Hudgins6, James P. Evans7 and Lap-Chee Tsui1,2,*
1Department of Molecular and Medical Genetics, University of Toronto, Toronto, Canada, 2Department of Genetics and 3Department of Endocrinology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada, 4Department of Molecular Biotechnology, University of Washington, Seattle, Washington, USA, 5Department of Medical Genetics, University of Groningen, Groningen, The Netherlands, 6University School of Medicine and Children's Hospital and Medical Center, Seattle, Washington, USA and 7Division of Medical Genetics, Department of Medicine, University of North Carolina, Chapel Hill, USA
Received January 22, 1996; Revised and Accepted February 15, 1996
Split hand/split foot malformation (SHFM) is a heterogeneous limb developmental disorder, characterized by missing digits and fusion of remaining digits. An autosomal dominant form of this disorder (SHFM1) has been mapped to 7q21.3-q22.1 on the basis of SHFM-associated chromosomal rearrangements. Utilizing a YAC contig across this region, we have defined a critical interval of 1.5 Mb by the analysis of six interstitial deletion patients and mapped the translocation breakpoints of seven ectrodactyly patients within the interval. To delineate the basic molecular defect underlying SHFM, we have searched for candidate genes in a 500 kb region containing five of the translocation breakpoints. Three genes were identified, two genes of the Distal-less (dll) homeobox gene family, DLX5 and DLX6 and a novel gene, which we named DSS1. DSS1 is predicted to encode a highly acidic polypeptide with no significant similarity to any known proteins but 100% amino acid sequence identity with its murine homolog (Dss1). Using RNA in situ hybridization analysis, we detected a tissue-specific expression profile for Dss1 in limb bud, craniofacial primordia and skin. A deficiency in expression of DSS1, DLX5 and/or DLX6 during development may explain the SHFM phenotypes.
Split hand/split foot malformation (SHFM) is a form of ectrodactyly, characterized by deep median clefts, missing digits and a claw-like appearance of the distal extremities consistent with a developmental defect of the growth and patterning of the central digital rays (1 ). SHFM is clinically heterogeneous (2 ), presenting in both an isolated form and in combination with additional anomalies affecting the long bones (non-syndromic ectrodactyly) and/or other organ systems including the cranio- facial, genitourinary and ectodermal structures (syndromic ectrodactyly or SE) (3 ). The most notable example of syndromic ectrodactyly is the EEC (ectrodactyly, ectodermal dysplasia and cleft-lip/palate) syndrome (4 ).
Although most SHFM cases occur sporadically, familial forms have been observed with the majority showing autosomal dominant inheritance (3 ), while some autosomal recessive (5 ,6 ) and X-linked (7 ) patterns of inheritance have been reported. Peculiar patterns of transmission have been reported for >60% of SHFM pedigrees, in which about 30% of obligate carriers show no phenotypic abnormalities (1 ). In addition, variable expressivity is a hallmark of both syndromic and non-syndromic SHFM. Remarkably different phenotypes, ranging from mild abnormalities of a single limb to severe defects of all four limbs, may be observed. Moreover, segregation distortion with excessive transmission of SHFM from affected fathers to sons has been detected in some pedigrees (8 ,9 ).
A subset of SHFM and SE patients were found to have cytogenetically visible chromosome rearrangements. Molecular studies of these patients have led to the assignment of an autosomal dominant form of the disease to 7q21.3-q22.1 (10 ,11 ); the locus has been designated SHFM1 (McKusick #183600). A physical map consisting of overlapping yeast artificial chromosome (YAC) clones encompassing this entire region has been constructed (12 ). Together with the mapping of deletion breakpoints, a critical region of ~1.5 Mb could be established for the SHFM1 locus(12 ). Further, the breakpoints for six patients with translocation (or inversion) were localized within the critical region. Although these patients included both simple SHFM and complex SE, there was no obvious correlation between the sites of chromosomal disruption and clinical severity.
Haploinsufficiency may be considered the cause of SHFM1 (12 ). Deletion, translocation or inversion involving the critical region may affect the activity of one or a series of genes, through either direct interruption of the gene(s) or their regulatory elements. To gain further insight into the molecular basis underlying SHFM1, we have initiated a systematic search for candidate genes located in the critical region. Our emphasis has been concentrated on a 500 kb region that spans five of the seven known translocation breakpoints. In this report, we describe the identification of three genes in this region. Two of them belong to the Drosophiladistal-less (dll) homeobox gene family, DLX5 and DLX6 (13 ), which have been previously implicated in limb development (13 -15 ). Here we provide a detailed characterization of the third gene which has not been previously described. We also present RNA in situ hybridization data to show its expression profile during mouse limb, craniofacial and skin development. All available data suggest that this gene is a strong candidate that should be considered when delineating the etiology of SHFM and SE.
In an attempt to delimit the critical region of the SHFM1 locus, we characterized four additional patients, namely, D6, D7, T6 and T8, with chromosome 7 abnormalities by FISH, somatic cell hybrid, densitometry, or microsatellite analysis, using the physical mapping reagents shown in Figure 1 . The clinical and cytogenetic description of all patients except D7 has been described (D6 in ref. 16 ; T6 in refs 12 and 17 ; T8 in ref. 18 ). D7 corresponded to an EEC patient with an interstitial deletion of 7q21.3 detectable by molecular analysis only. The deletion breakpoints of this patient were found to lie just outside of the region shown in Figure 1 , which confirmed the critical genetic region for SHFM1 but failed to narrow the position for the disease locus. The proximal deletion breakpoint of patient D6 has, however, allowed us to refine the critical interval to about 1000 kb. In addition, all seven translocation breakpoints could be mapped within a 700 kb region and five of them (T1, T2.2, T4, T5 and T6) within 500 kb of each other (see Fig. 1 ). As the latter breakpoint cluster was found to be encompassed by two overlapping YAC clones (HSC7E571 and HSC7E1131), it was reasonable to focus our initial gene identification study on these clones.
Direct cDNA selection, detection of evolutionarily conserved sequences and exon amplification were used to isolate putative transcribed sequences from genomic clones covering the critical region. In this paper we present the results from this search for the region encompassed by both HSC7E571 and HSC7E1131 (Fig. 1 ) (the clones isolated from HSC7E1170 and HSC7E1301 will be described elsewhere). A total of 97 cDNA fragments, 20 conserved DNA segments and 15 putative exons were characterized. These clones were sequenced, grouped and aligned, analyzed by northern blot with RNA from various tissues, examined for their correspondence to genomic DNA segments that showed sequence conservation with other species and used as probes to screen cDNA libraries. In addition, the cDNA sequences were used to search all available public databases for identity and homology and for coding and splicing potential. From this exhaustive analysis, so far, only three `bona fide' transcription units could be discerned (Fig. 1 ). The transcription orientation of these genes were determined by fine restriction enzyme mapping and blot hybridization analysis with different cDNA segments as probes (data not shown).
Two of the genes, DLX5 and DLX6, were reported previously (12 ,13 ). The 5' end of DLX6 could not be determined because it was not represented in any of the selected cDNA clones nor recovered from cDNA libraries made in the conventional manner. The third gene was not previously described and was thus assigned the gene symbol DSS1 (for deleted in the split hand/split foot SHFM1 region). As shown in Figure 1 , DSS1 was mapped towards the proximal end of YAC clone HSC7E571 and the gene appeared to be surrounded but not directly interrupted by any of the four rearrangement breakpoints mapped nearby. The closest breakpoint, T6, was localized within 10 kb of the 3'-end of the gene while three others, T4, T2.2 and T5, are on either side, within 100 kb (Fig. 1 ). It should be noted that T2.2 belonged to a simple SHFM patient and that the other three, to complex SE patients.
Based on the combined sequences of the various overlapping clones and two apparent full-length clones, a consensus cDNA of 494 bp could be established for DSS1 (Fig. 2 ). A putative open reading frame encoding for 70 amino acids was identified with the first available methionine codon being the initiation codon, which appeared to conform well to the Kozak consensus sequence (19 ). A polyadenylation signal (AATAAA) was found 11 bp upstream of the poly(A) tail. The deduced DSS1 polypeptide appeared to be highly acidic with 27 residues being either aspartic or glutamic acids (~40%). Database searches failed to detect any significant identity with known proteins or protein motifs, but a portion of the DSS1 DNA sequence was found in the dbEST database.
To investigate the function of DSS1 and its relationship with SHFM and SE, we examined the expression pattern of the gene using RNA in situ hybridization with whole mounts and sections of developing mouse embryos and newborn animals. For these studies, the murine cDNA clone, including the 3'-untranslated region, was used to generate sense and anti-sense riboprobes. As revealed by the results of whole mount studies shown in Figure 4 , expression of Dss1 could be detected at various stages of embryonic development. At embryonic day 9.5 (E9.5), Dss1 was found to express strongly in the mesenchyme of the first branchial arch and the frontonasal prominence (Fig. 4 a). As facial development progressed, the expression was laterally restricted in the maxillary and mandibulary prominence of the first and second branchial arch (data not shown), consistent with a role in the development of the bones of the lower face and jaw. By E12.0 the expression appeared to be restricted to the region of the prospective tooth buds (data not shown). In addition to the facial primordium, expression of Dss1 was detected in the early genital tubercle, but it was not followed after E12.5 (data not shown).
The results of embryo and newborn section analysis also revealed that the expression of Dss1 was widespread at low levels (data not shown), thus in good agreement with the northern blot results. In addition, Dss1 was found to be strongly expressed in the dermis of new born mice (Fig. 4 b-d). The latter expression pattern seemed relevant to the ectodermal dysplasia phenotype in the syndromic ectrodactyly patients.
The spatial and temporal pattern of expression of Dss1 was examined in the developing mouse limb bud by using whole mount RNA in situ hybridization (Fig. 5 ). At E9.5, when the first signs of limb bud outgrowth were evident (23 ), Dss1 expression could be detected ubiquitously throughout the mesenchyme of the structure but not in the ectoderm (data not shown). As the limb bud elongated, Dss1 was no longer expressed in the proximal mesenchyme. At E11.0, in the fore limb bud, expression was only apparent in the distal mesenchyme and the peripheral regions of the medial mesenchyme (Fig. 3 a). It was presumed that, at this stage, the mesenchymal cells of the central core of the limb bud were condensing to form the stylopodial or zeugopodial elements (23 ). In E12.5 embryos, Dss1 expression in the fore limb was found to be further restricted as the mesenchymal condensations occurred in the initial stages of digit formation (Fig. 3 b). At this stage, the Dss1 gene appeared active in the interdigital mesenchyme, but not in the condensing mesenchyme.
Through the molecular characterization of the genomic rearrangements found in a group of patients with SHFM and SE, we have identified a novel gene that appears to play an important part in limb, craniofacial, skin and genitourinary development. The gene, DSS1, which encodes a highly conserved and acidic protein, maps within the critical region and is closely surrounded, but not directly interrupted, by the chromosomal translocation breakpoints found in the patients. Based on RNA in situ hybridization analysis with the murine homolog, Dss1, the gene appears to express predominantly in the limb and facial primordia during days 9.5-13.5 of mouse development. It is also expressed strongly in the dermis of newborn mice, the early genital bud and possibly the tooth primordium. Thus, a reduction in the expression of this gene during human embryogenesis may not only explain the phenotype observed in SHFM1 patients, but also some forms of syndromic ectrodactyly including EEC.
A number of previous studies in mouse (13 ), rat (14 ) and chick (15 ) indicate that Dlx5 and Dlx6 are expressed during development in a unique spatial and temporal pattern. The expression pattern of the mouse Dlx5 and Dlx6 genes are almost identical (13 ). Both show strong signals in facial and branchial arch mesenchyme, otic vesicles and frontal ectoderm around olfactory placodes at E8.5-9 and in the developing forebrain in primordia of the ganglionic eminence and ventral diencephalic regions at E10. At midgestation (E12.5), they are expressed in almost every developing skeletal element and in the forebrain. Expression of Dlx5 can also be detected in regions of ossification, as well as ear ossicles and primordia of teeth. In the rat, the expression pattern has only been studied with Dlx5 and the pattern is essentially the same as that of the mouse, but showing an additional site in epidermis of the skin and the apical ectodermal ridge (AER) of limb buds (14 ). The study of Dlx5 in chick is primarily focused on limb development and the description of its expression pattern is more refined (15 ). In addition to AER, the expression of the chick Dlx5 gene is found in the mesoderm at the anterior margin of the limb bud and in a discrete group of mesodermal cells at the mid-proximal posterior margin that correspond to the posterior necrotic zone.
The human DLX5 and DLX6 were previously localized to the 7q22 region by fluorescence in situ hybridization (13 ). Their possible involvement in SHFM was also suggested (13 ). Our previous physical mapping studies placed these two genes in the SHFM1 critical interval, thus providing further support to this assumption. Based on the RNA in situ hybridization data, DLX5 and DLX6 are equally strong candidates as DSS1 in explaining the SHFM and SE phenotypes. It is of importance to note, however, that no mutation could be detected in these genes in sporadic SHFM patients (25 ). Moreover, although DLX5 and DLX6 are located within the critical interval defined by the deletion breakpoints, they are not interrupted by the translocation breakpoints. If reduced expression of the two genes is part of the etiology of SHFM and SE, they must be regulated by distantly located control elements which become separated in the translocations (see below). The same argument would apply to DSS1.
We have also designed oligonucleotide primers flanking the exons of DSS1 (Fig. 2 ) to facilitate the search for mutations in SHFM patients. All three exons have been scanned in 60 sporadic SHFM1 patients but, so far, no mutation could be detected (J. Evans and P. Charlton, unpublished data). In fact, previous studies showed that polymorphic markers located within the SHFM1-critical region failed to demonstrate linkage to the phenotype in several large SHFM families (26 -29 ). At least one additional adSHFM locus (named SHFM3) has been recently mapped on chromosome 10q (29 ). Genetic heterogeneity has also been suggested on the basis of different clinical subtypes of non-syndromic SHFM (30 ). It is probable that the SHFM and SE phenotypes are caused only by deletion or translocation affecting the SHFM1 locus. Therefore, searching for point mutations in DSS1, DLX5 and DLX6 may not be an effective strategy to demonstrate the role of these genes in limb development.
To explain how deletions and translocations at the SHFM1 locus could cause the SHFM phenotype, we have offered three different possibilities (12 ). We have first suggested there could be a large gene that was disrupted by all the translocations found in the patients. This explanation requires the presence of a gene of at least 700 kb in size, spanning most, if not all, of the translocation breakpoints. Despite exhaustive searching, we have not identified such a gene in the region. We have also suggested the presence of a number of genes in this region essential for limb development and that disruption of expression of any of them could lead to the observed phenotype. While this hypothesis is supported by the identification of three genes that share very similar expression patterns, none of them are directly interrupted by any of the translocation breakpoints nor have point mutations been detected in any of the sporadic patients. The third hypothesis that appears attractive is based on the notion of gene regulation by distant cis-acting regulatory elements, or long range position effects. Removal of these elements from DSS1, DLX5, DLX6, or other genes yet to be identified in this region may result in aberrant gene expression leading to the abnormal development.
The expression of DSS1 may be particularly susceptible to the proposed position effect because the gene is closely surrounded by the translocation breakpoints which do not interrupt any other known genes in the region. Other examples of genes for which a position effect has been proposed include SOX9 for campomelic dysplasia (31 ), PAX6 for aniridia (32 ), GLI3 for Greig's cephalopolysyndactyly (GCPS) (33 ), the Steel-locus for ovarian follicle development (34 ), the agouti coat color gene (35 ), POU3F4 for familial X-linked deafness (36 ) and myosin VI for Snell's waltzer deafness (37 ). For some of these genes, expression is affected by rearrangements 50-400 kb away. Therefore, the expression of DSS1, DLX5 and DLX6 may all be affected by the deletions as well as the translocations described for the SHFM and SE patients. The similar spatial and temporal expression pattern detected for these genes during development is in good agreement with this assumption. As there is no correlation between the extent of deletion or the location of translocation and the clinical phenotypes in SHFM and SE, the observed variable expressivity and penetrance are probably due to other genetic factors. All these hypotheses may be tested by the generation of mice with specific gene disruptions through homologous recombination (38 ).
A spontaneous mouse mutant, dactylaplasia (Dac), with a phenotype closely resembling that of SHFM in humans has been described (39 ). The expression of the abnormal Dac phenotype is dependent on homozygosity for a recessive allele of another unlinked gene named mdac. Dac and mdac represent the only characterized two-locus system for a naturally occurring congenital malformation (40 ). Dac maps near Pax2 on mouse chromosome 19 (40 ) and based on syntenic relationships this is the same general region in human (10q23-q25) to which SHFM3 has been mapped (29 ). The mdac locus maps to mouse chromosome 13 which has regions of synteny on human chromosome 5q and 9q (41 ). While Dac may be a model for SHFM3, neither it or mdac share synteny to human chromosome 7q, so it is unlikely that they are involved in SHFM1. A two-locus model similar to Dac-mdac in humans might, however, explain some of the unusual features of inheritance observed in SHFM including the irregular forms of transmission, reduced penetrance and disturbed segregation ratios.
Finally, while DLX5 and DLX6, as members of the Distal-less family, may serve as transcription regulators that control the expression of other genes that are required for limb, craniofacial and skin development, it is difficult to predict the biological function for DSS1. However, the expression pattern of Dss1 during limb development is similar to that of Gli3 which may provide insight into its function. Gli3 encodes a zinc finger protein whose reduced expression is known to cause extra-toes inmouse (42 ) and GCPS in humans (33 ). GLI3 is thought to play a part in programmed cell death as its disruption results in an increase in the number of digits and the lack of digital separation. Similarly, the SHFM1 phenotype may be due to a loss of the overall control of programmed cell death in which an increase could result in the loss of digits and a decrease could result in the fusion of digits. Alternatively, it may be proposed that DSS1 is required to maintain cells in an undifferentiated state or that it promotes the proliferation of these cells. In support of this assumption, DSS1 expression is present in regions of rapid cell growth (limb bud, branchial arch, genital bud, skin) and excluded from regions of cell differentiation (e.g. digital condensations). In addition, DSS1 expression is widespread early in the developing embryo (not shown) and as development progresses and the fate of cell lineages become determined Dss1 expression dissipates and is found in late developing tissues that remain in an undifferentiated state. Although further studies are required to delineate the biological function(s) of DSS1 and those of DLX5 and DLX6, they are excellent candidates for study of limb development.
The precise breakpoints of rearrangements determined by FISH were localized within the cloned genomic DNA segments on the basis of altered signal intensity (as in the case of chromosome deletion) and splitting signals (for translocation), as described (12 ).
Direct cDNA selection was performed with YAC clones HSC7E571 (12 ) and HSC7E1131 (12 ) according to described methodology (43 ). Briefly, the YAC DNA was purified away from the endogenous yeast chromosomes by pulsed field gel electrophoresis, transferred to nylon membrane and used as substrate for exhaustive hybridization with PCR-amplified cDNA pools made from RNA isolated from 10 tissues (43 ). Exon amplification was conducted on `tiling path' phage and cosmid clones shown in Figure 1 using the pSPL3 vector and the protocols described by the supplier (Gibco/BRL). To identify evolutionarily conserved DNA sequences genomic subclones from the phage, cosmid and YACs were hybridized systematically against DNA from seven different species (shown in Fig. 2 ). The 132 DNA segments retrieved in these experiments that mapped back to the expected genomic clones were analyzed by DNA sequencing, northern blot analysis with RNA from various tissues as indicated and Southern blot analysis with genomic DNA (cloned and uncloned) according to standard protocols (44 ). The DNA sequences were grouped and aligned, if overlapping, examined for potential open-reading frames and used to screen GenBank, dbEST and other databases for sequence identity and possible motifs.
FC3 was a 1.5 kb full-length DLX5 cDNA isolated from a human frontal cortex library (Stratagene) with one of the selected clones. Similarly, one DLX6-specific clone (FB325) was isolated from a human fetal brain cDNA library (Stratagene). The latter cDNA (1.4 kb) was determined to be less than full length on the basis of the size of the mRNA (2.5 kb) and the result of DNA sequencing. The full-length DSS1 clones FC4C and FB41A were isolated from cDNA libraries of the frontal cortex and fetal brain, respectively. Five full-length Dss1 mouse clones were isolated from an oligo dT-primed cDNA library constructed in the [lambda]ZAP vector (Stratagene) with mRNA pools of the limb buds from E9.5 to E13.0 mouse embryos.
Human genomic DNA fragments hybridizing to the cDNA clones were isolated and cloned into pBluescripttm (Stratagene) for sequencing analysis. The exon-intron boundaries were identified by sequence alignment. The transcription initiation sites for DSS1 were determined by the use of the 5'-RACE (rapid amplification of cDNA ends) kit (Life Technologies). Caco-2 and fibroblast total cellular RNA was reverse transcribed with an anti-sense oligonucleotide primer (5'-TATGAAGTCTCCATCTTAT-3'). The first round PCR was performed in a total volume of 50 [mu]l for 35 cycles with the primer annealing at 50oC, followed by a `nested-PCR' with gene specific primer (5'-TCTCTAGTTCAGCTCGTAAC-3'). PCR products were subcloned into the pCRIItm TA cloning vector (Invitrogen).
Mouse (CD1 strain) embryos were used in this study. Staging of the developing embryos was determined in reference to the first midday when vaginal plug was detected as E0.5. Whole-mount in situ hybridization using a digoxigenin-labeled RNA probe and an alkaline phosphatase-coupled anti-digoxigenin antibody was performed as described (45 ). Paraffin sections of developmentally staged embryos were hybridized with 35S- labeled sense and anti-sense probes as described previously (42 ). Adjacent sections were examined to allow accurate localization of expression patterns.
The authors acknowledge Jacques Michaud, Hai Shienne Chen and Eddy Wong for technical assistance and Jean Weissenbach for unpublished data on genetic markers. The work is supported by grants from the Canadian Genetic Disease Network, the Howard Hughes Medical Institute (International Scholar program) and the Canadian Genome Analysis and Technology Program to L-C.T and the N.I.H. (HD31153) to J.P.E. M.A.C. is supported by an M.R.C. studentship.
1 Temtamy, S.A. and McKusick, V.A. (1978) In The Genetics of Hand Malformations. Alan R. Liss, Inc., New York, pp. 53-71.
2 Thompson, M.W., McInnes, R.R. and Willard, H.F. (1991) Genetics in Medicine, 5th edn. W.B. Saunders, Toronto, pp. 83-85.
3 McKusick, V.A. (1992) In Mendelian Inheritance in Man. Catalogs of Autosomal Dominant, Autosomal Recessive and X-linked Phenotypes. Johns Hopkins University Press, Baltimore, 10th edn, pp. 874-875.
4 Rodini, E.S.O. and Richieri-Costa, A. (1990) EEC syndrome: report on 20 new patients, clinical and genetic considerations. Am. J. Med. Genet., 37, 42-53.
5 Freire-Maia, A. (1971) A recessive form of ectrodactyly and its implications in genetic couselling. J. Hered., 62, 53.MEDLINE Abstract
6 Verma, I., Joseph, R., Bhargava, S. and Mehta, S. (1976) Split-hand/split-foot deformity inherited as an autosomal recessive trait. Clin. Genet., 9, 8-14.MEDLINE Abstract
7 Faiyaz-ul-Haque, 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.MEDLINE Abstract
8 Stevenson, A.C. and Jennings, L.M. (1960) Ectrodactyly-evidence in favour of a disturbed segregation in the offspring of affected males. Ann. Hum. Genet., 24, 89-96.
9 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.MEDLINE Abstract
10 Scherer, S.W., Poorkaj, P., Allen, T., Kim, J., Geshuri, D., Nunes, M., Soder, S., Stephens, K., Pagon, R.A., Patton, M.A., Berg, M.A., Donlon, T., Rivera, H., Pfeiffer, R.A., Naritomi, K., Hughes, H., Genuardi, M., Gurrieri, F., Neri, G., Lovrein, E, Magenis, E., Tsui, L-C. and Evans, J.P. (1994) Fine mapping of the autosomal dominant split hand split foot malformation on chromosome 7 band q21.3-q22.1. Am. J. Hum. Genet., 55, 12-20. MEDLINE Abstract
11 Marinoni, J.C., Stevenson, R.E., Evans, J.P., Geshuri, D., Phelan, M.C. and Schwartz, C.E. (1994) Split foot and developmental retardation associated with a deletion of three microsatellite markers in the 7q21.2-q22.1. Clin. Genet., 47, 90-95.
12 Scherer, S.W., Poorkaj, P., Massa, H., Soder, S., Allen, T., Nunes, M., Geshuri, D., Wong, E., Belloni, E., Little, S., Zhou, L., Becker, D., Kere, J., Ignatius, J., Niikawa, N., Fukushima, Y., Hasegawa, T., Weissenbach, J., Boncinelli, E., Trask, B., Tsui, L-C. and Evans, J.P. (1994) Physical mapping of the split hand/split foot locus on chromosome 7 and implication in syndromic ectrodactyly. Hum. Mol. Genet., 3, 1345-1354.MEDLINE Abstract
13 Simeone, A., Acampora, D., Pannese, M., D'Esposito, M., Stornaiuolo, A., Gulisano, M., Mallamaci, A., Kastury, K., Druck, T., Huebner, K. and Boncinelli, E. (1994) Cloning and characterization of two new members of the vertebrate Dlx gene family. Proc. Natl Acad. Sci. USA, 91, 2250-2254.MEDLINE Abstract
14 Zhao, G-Q., Zhao, S., Zhou, X., Eberspaecher, H., Solursh, M. and de Crombrugghe, B. (1994) rDlx, a novel distal-less-like homeoprotein is expressed in developing cartilages and discrete neuronal tisues. Dev. Biol., 164, 37-51.MEDLINE Abstract
15 Ferrari, D., Sumoy, L., Gannon, J., Sun, H., Brown, A.M.C., Upholt, W.B. and Kosher, R.A. (1995) The expression pattern of the Distal-less homeobox gene Dlx-5 in the developing chick limb bud suggests its involvement in apical ectodermal ridge activity, pattern formation and cartilage differentiation. Mech. Dev., 52, 257-264.MEDLINE Abstract
16 Hudgins, L., Massa, H., Disteche, C., Scherer, S.W., Tsui, L-C., Trask, B. and Evans, J.P. (1994) Identification of a microdeletion at 7q21.3 with fluorescence in situ hybridization with split hand/split foot (ectrodactyly) (abstract). Am. J. Hum. Genet., 3, A107.
17 Ignatius, J., Knuutila, S., Scherer, S.W., Trask, B. and Kere, J. (1995) Split hand/split foot malformation, deafness and mental retardation with a complex cytogenetic rearrangement involving 7q21.3. Clin. Genet. in press.
18 Cobben, J.M., Verheij, J.B.G.M., Eisma, W.H., Robinson, P.H., Zwierstra, R.P., Leegte, B. and Castedo, S. (1995) Bilateral split hand/split foot malformation and inv(7)(p22q21.3). J. Med. Genet., 32, 375-378.MEDLINE Abstract
19 Kozak, M. (1987) An analysis of 5'-nonencoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res., 15, 8125-8148.MEDLINE Abstract
20 Frohman, M.A., Dush, M.K. and Martin, G.R. (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl Acad. Sci. USA, 85, 8998-9002. MEDLINE Abstract
21 Sharp, P.A. (1992) TATA-binding protein is a classless factor. Cell, 68, 819-821.MEDLINE Abstract
22 Mount, S.M. (1982) A catalogue of splice junction sequences. Nucleic Acids Res., 10, 459-472.MEDLINE Abstract
23 Wanek, N., Muneoka, K., Holler-Dinsmore, G., Burton, R. and Bryant, S.V. (1989) A staging system for mouse limb bud development. J. Exp. Zool., 249, 41-49.MEDLINE Abstract
24 Roberts, D.J. and Tabin, C. (1994) The genetics of human limb development. Am. J. Hum. Genet., 55, 1-6.MEDLINE Abstract
25 Genuardi, M., Gurrieri, F., Nanni, L., Boncinelli, E., Evans, J.P. and Neri, G. (1995) Mutational analysis of distal-less genes in split hand/split foot anomaly (abstract). Am. J. Hum. Genet., 57S, A133.
26 Palmer, S.E., Scherer, S.W., Kukolich, M., Wijsman, E.M.,Tsui, L-C., Stephens, K. and Evans, J.P. (1994) Evidence for locus heterogeneity in human autosomal dominant split hand/split foot malformation. Am. J. Hum. Genet., 55, 21-26.MEDLINE Abstract
27 Marinoni, J-C., Boyd, E., Sherman, S. and Schwartz, C. (1994) Familial split hand/split foot long bone deficiency does not segregate with markers linked to the SHFD1 locus in 7q21.3-q22.1. Hum. Mol. Genet., 3, 1355-1357.MEDLINE Abstract
28 Gurrieri, F., Genuardi, M., Chiurazzi, P., Gillessen-Kaesbach, G. and Neri, G. (1994) Exclusion of linkage between autosomal dominant split hand/split foot and markers from chromosome 7q: further evidence for genetic heterogeneity. Am. J. Hum. Genet., 55, 853-855.MEDLINE Abstract
29 Nunez, M.E., Schutt, G., Kukolich, M., Byers, P. and Evans, J.P. (1996) A second autosomal split hand/split foot locus maps to chromosome 10q25. Hum. Mol. Genet. in press.
30 Zlotogora, J. (1994) On the inheritance of the split hand/split foot malformation. Am. J. Med. Genet., 53, 29-32.MEDLINE Abstract
31 Wagner, T., Wirth, J., Meyer, J., Zabel, B., Held, M., Zimmer, J., Pasantes, J., Bricarelli, F.D., Keutel, J., Hustert, E., Wolf, U., Tommerup, N., Schempp, W. and Scherer, G. (1994) Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell, 79, 1111-1120.MEDLINE Abstract
32 Fantes, J., Redeker, B., Breen, M., Boyle, S., Brown, J., Fletcher, J., Jones, S., Bickmore, W., Fukushima, Y., Mannens, M., Danes, S., van Heyningen, V. and Hanson, I. (1995). Aniridia-associated cytogenetic rearrangements suggest that a position effect may cause the mutant phenotype. Hum. Mol. Genet.,4, 415-422.MEDLINE Abstract
33 Vortkamp, A., Gessler, M. and Grzeschik, K-H. (1991) GLI3 zinc-finger gene interrupted by translocations in Greig syndrome families. Nature, 352, 539-540.MEDLINE Abstract
34 Bedell, M.A., Brannan, C.I., Evans, E.P., Copeland, N.G., Jenkins, N.A. and Donovan, P.J. (1995). DNA rearrangements located over 100 kb 5' of the Steel (Sl)-coding region in Steel-panda and Steel-contrasted mice deregulate Sl expression and cause female sterility by disrupting ovarian follicle development. Genes Dev.,9, 455-470.MEDLINE Abstract
35 Duhl, D.M.J., Vrieling, H., Miller, K.A., Wolff, G. and Barsh, G.S. (1994) Neomorphic agouti mutations in obese yellow mice. Nature Genet., 8, 59-65.
36 de Kok, Y.J.M., Merkx, G.F.M., van der Maarel, S.M., Huber, I., Malcolm, S., Ropers, H-H. and Cremers, F.P.M. (1995) A duplication/paracentric inversion associated with familial X-linked deafness (DFN3) suggests the presence of a regulatory element more than 400 kb upstream of the POU3F4 gene. Hum. Mol. Genet., 4, 2145-2150.
37 Avraham, K.B., Hasson, T., Steel, K.P., Kingsley, D.M., Russell, L.B., Mooseker, M.S., Copeland, N.G. and Jenkins, N.A. (1995) The mouse Snell's waltzer deafness gene encodes an unconventional myosin required for structural integrity of inner ear hair cells. Nature Genet., 11, 369-375.MEDLINE Abstract
38 Thomas, K.R. and Capecchi, M.R. (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell, 51, 503-512.MEDLINE Abstract
39 Chai, C.K. (1981) Dactylaplasia in mice, a two-locus model for developmental anomalies. J. Hered., 72, 234-237.MEDLINE Abstract
40 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.MEDLINE Abstract
42 Hui, C-C. and Joyner, A.L. (1993) A mouse model of Greig cephalopolysyndactyly syndrome: the extra-toesJ mutation contains an intragenic deletion of the Gli3 gene. Nature Genet., 3, 241-246.MEDLINE Abstract
43 Rommens, J.M., Mar, L., McArthur, J., Tsui, L-C. and Scherer, S.W. (1994) In Hochgeschwender, U.and Gardiner, K. (eds), The Identification of Transcribed Sequences. Plenum Press, pp. 65-79.
44 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York.
45 Ang, S-L. and Rossant, J. (1993) Anterior mesendoderm induces mouse Engrailed genes in explant cultures. Development, 118, 139-149.MEDLINE Abstract
*To whom correspondence should be addressed
+These authors contributed equally to this work
This page is maintained by OUP admin. Last updated Thu Oct 31 15:23:49 GMT 1996. Part of the OUP Journals World Wide Web service.Copyright Oxford University Press, 1996
