Unequivocal identification of the full composition of a gene is made difficult by the cryptic nature of regulatory elements. Regulatory elements are notoriously difficult to locate and may reside at considerable distances from the transcription units on which they operate and, moreover, may be incorporated into the structure of neighbouring genes. The importance of regulatory mutations as the basis of human abnormalities remains obscure. Here, we show that the chromosome 7q36 associated preaxial polydactyly, a frequently observed congenital limb malformation, results from point mutations in a Shh regulatory element. Shh, normally expressed in the ZPA posteriorly in the limb bud, is expressed in an additional ectopic site at the anterior margin in mouse models of PPD. Our investigations into the basis of the ectopic Shh expression identified the enhancer element that drives normal Shh expression in the ZPA. The regulator, designated ZRS, lies within intron 5 of the Lmbr1 gene 1 Mb from the target gene Shh. The ZRS drives the early spatio-temporal expression pattern in the limb of tetrapods. Despite the morphological differences between limbs and fins, an equivalent regulatory element is found in fish. The ZRS contains point mutations that segregate with polydactyly in four unrelated families with PPD and in the Hx mouse mutant. Thus point mutations residing in long-range regulatory elements are capable of causing congenital abnormalities, and possess the capacity to modify gene activity such that a novel gamut of abnormalities is detected.

/pdf/a-long-range-shh-enhancer-regulates-expression-in-the-1lf7f9g0ii.pdf

A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly

Cell signaling plays a key role in the development of all multicellular organisms. Numerous studies have established the importance of Hedgehog signaling in a wide variety of regulatory functions during the development of vertebrate and invertebrate organisms. Several reviews have discussed the signaling components in this pathway, their various interactions, and some of the general principles that govern Hedgehog signaling mechanisms. This review focuses on the developing systems themselves, providing a comprehensive survey of the role of Hedgehog signaling in each of these. We also discuss the increasing significance of Hedgehog signaling in the clinical setting.

Developmental roles and clinical significance of hedgehog signaling.

Preaxial polydactyly (PPD) is a common limb malformation in human. A number of polydactylous mouse mutants indicate that misexpression of Shh is a common requirement for generating extra digits. Here we identify a translocation breakpoint in a PPD patient and a transgenic insertion site in the polydactylous mouse mutant sasquatch (Ssq). The genetic lesions in both lie within the same respective intron of the LMBR1/Lmbr1 gene, which resides ≈1 Mb away from Shh. Genetic analysis of Ssq reveals that the Lmbr1 gene is incidental to the phenotype and that the mutation directly interrupts a cis-acting regulator of Shh. This regulator is most likely the target for generating PPD mutations in human.

https://www.pnas.org/content/pnas/99/11/7548.full.pdf

Disruption of a long-range cis-acting regulator for Shh causes preaxial polydactyly.

The limb bud is of paradigmatic value to understanding vertebrate organogenesis. Recent genetic analysis in mice has revealed the existence of a largely self-regulatory limb bud signalling system that involves many of the pathways that are known to regulate morphogenesis. These findings contrast with the prevailing view that the main limb bud axes develop largely independently of one another. In this Review, we discuss models of limb development and attempt to integrate the current knowledge of the signalling interactions that govern limb skeletal development into a systems model. The resulting integrative model provides insights into how the specification and proliferative expansion of the anteroposterior and proximodistal limb bud axes are coordinately controlled in time and space.

Vertebrate limb bud development : moving towards integrative analysis of organogenesis

Precise spatial and temporal control of developmental genes is crucial during embryogenesis. Regulatory mutations that cause the misexpression of key developmental genes may underlie a number of developmental abnormalities. The congenital abnormality preaxial polydactyly, extra digits, is an example of this novel class of mutations and is caused by ectopic expression of the signalling molecule Sonic Hedgehog (SHH) in the developing limb bud. Mutations in the long-distant, limb-specific cis-regulator for SHH ,c alled the ZRS, are responsible for the ectopic expression which underlies the abnormality. Here, we show that populations of domestic cats which manifest extra digits, including the celebrated polydactylous Hemingway’s cats, also contain mutations within the ZRS. The polydactylous cats add significantly to the number of mutations previously reported in mouse and human and to date, all are single nucleotide substitutions. A mouse transgenic assay shows that these single nucleotide substitutions operate as gain-of-function mutations that activate Shh expression at an ectopic embryonic site; and that the sequence context of the mutation is responsible for a variable regulatory output. The plasticity of the regulatory response correlates with both the phenotypic variability and with species differences. The polydactyly mutations define a new genetic mechanism that results in human congenital abnormalities and identifies a pathogenetic mechanism that may underlie other congenital diseases.

/pdf/point-mutations-in-a-distant-sonic-hedgehog-cis-regulator-2kd45ilvrv.pdf

Point mutations in a distant sonic hedgehog cis-regulator generate a variable regulatory output responsible for preaxial polydactyly

Polydactyly is the most frequently observed congenital hand malformation with a prevalence between 5 and 19 per 10 000 live births. It can occur as an isolated disorder, in association with other hand/foot malformations, or as a part of a syndrome, and is usually inherited as an autosomal dominant trait. According to its anatomical location, polydactyly can be generally subdivided into pre- and postaxial forms. Recently, a gene responsible for preaxial polydactyly types II and III, as well as complex polysyndactyly, has been localised to chromosome 7q36.
In order to facilitate the search for the underlying genetic defect, we ascertained 12 additional families of different ethnic origin affected with preaxial polydactyly. Eleven of the kindreds investigated could be linked to chromosome 7q36, enabling us to refine the critical region for the preaxial polydactyly gene to a region of 1.9 cM. Our findings also indicate that radial and tibial dysplasia/aplasia can be associated with preaxial polydactyly on chromosome 7q36.
Combining our results with other studies suggests that all non-syndromic preaxial polydactylies associated with triphalangism of the thumb are caused by a single genetic locus, but that there is genetic heterogeneity for preaxial polydactyly associated with duplications of biphalangeal thumbs. Comparison of the phenotypic and genetic findings of different forms of preaxial polydactyly is an important step in analysing and understanding the aetiology and pathogenesis of these limb malformations.


Keywords: preaxial polydactyly; chromosome 7q36; localisation

Clinical and genetic studies on 12 preaxial polydactyly families and refinement of the localisation of the gene responsible to a 1.9 cM region on chromosome 7q36

A physical and transcriptional map of the preaxial polydactyly locus on chromosome 7q36.

The vertebrate limb bud develops along three different axes: proximodistal, anteroposterior, and dorsoventral. Several genetic factors responsible for control of each of the three limb axes have been identified. The genes involved interact in complex feedback loops to achieve proper arrangement and differentiation of tissues. Most of the available information on limb development and patterning has come from studies carried out in the lower vertebrates. In recent years, an increasing number of studies have been unraveling the genetic basis of human hand malformation phenotypes. At present, genes responsible for preaxial polydactyly, split hand/split foot malformation, and brachydactyly type C have been localized, and the gene responsible for synpolydactyly has been identified. In this paper, we present an overview of the genetic factors involved in limb development, followed by summarized discoveries in the genetics of human congenital hand malformations.

Genetics of limb development and congenital hand malformations

The mutations underlying the mouse Hemimelic extra toes(Hx) phenotype, characterized by preaxial polydactyly and shortening of the tibia, and theHammer toe(Hm) phenotype, characterized by syndactyly, have been localized very close to each other on mouse Chromosome (Chr) 5p (Green 1989; Mouse Genome Database 1999). Only a single recombination event between the Hm andHx mutations was detected in 3.664 offspring (Sweet 1982), and thus the two mutations might affect the same gene. TheHx mutant is characterized by preaxial polydactyly on all four feet, with the hind limbs more severely affected than the fore limbs. TheHx mutation arose in the B10.D2/nSn strain, but variable expression of the phenotype has been reported, possibly caused by the outbred genetic background used (Knudsen and Kochhar 1981). The heterozygous phenotype typically includes shortening of the radius, tibia, and talus with extra preaxial metacarpals, metatarsals, and digits. The fibula and ulna are normal in size but often bent. The humerus, femur, and limb girdles are normal, and no other skeletal defects are present (Green 1989). It has been assumed that the homozygous Hx condition is embryolethal at an early stage of development (Knudsen and Kochhar 1981). The Hm mutant (C3HeB/FeJLe-a/a-Ca J Sl Hm) displays a semi-dominant phenotype of soft-tissue syndactyly between digits 2, 3, 4, and 5 on all four feet, while digits 1 and 2 are separated normally. The hind feet are always more severely affected than the fore feet. Heterozygous mutants show partial syndactyly that extends to the base of the most distal phalanges. Syndactyly in the homozygous mutant is complete and extends to the top of the most distal phalanges. Both heterozygous and homozygous mutants show incomplete syndactyly of the digits on the fore feet that is gradually decreasing towards the preaxial side (Green 1989). Two human congenital hand malformations, preaxial polydactyly (PPD) and complex polysyndactyly (CPS), have been localized to human Chr 7q36 (Heutink et al. 1994; Tsukurov et al. 1994). This region is syntenic to the region of mouse Chr 5 where the Hx andHm phenotypes have been localized (Mouse Genome Database 1999). PPD is characterized by a triphalangeal thumb and index finger duplication. Tibial and radial abnormalities have been observed (Zguricas et al. 1999). CPS is characterized by preand postaxial limb anomalies combined with syndactyly of the fingers. The striking similarity between human and mouse phenotypes and the syntenic chromosomal location suggest that the mouseHmandHx mutations are equivalent to the human CPS and PPD mutations. To see whether the variable phenotype of the heterozygous Hx mutant stabilizes in an inbred genetic background, and to test whether theHx andHm phenotypes are variant expressions of the same genetic defect, we crossed the Hx mutation into the C3HeB/ FeJLe-a/a-Ca J background of theHmmouse. After ten generations the phenotype was stable, with all heterozygous Hx mutant mice showing preaxial polydactyly and normal tibia. This finding indicates that the variation in the phenotype of the original Hx mutant is most likely caused by the presence of genetic modifiers elsewhere on the genome that influence the expression of the Hx mutant phenotype. Because the Hx phenotype is still present in the Hm background, theHm andHx phenotypes are not the result of variant expression of a single mutation caused by differences in genetic background, but represent two distinct mutations. We then tested whether homozygous Hx mice are viable in this C3HeB/FeJLe-a/a-Ca J genetic background. Polymorphic marker D5mit387 lies in the proximity of theHx/Hm locus (Mouse Genome Database 1999) and was used to distinguish the mutant Hx from wildtype (+) alleles. In aHx mutant backcross we observed three recombinations in 161 offspring, correlating with a genetic distance betweenD5mit387 and Hx of 1.8 cM. Offspring from heterozygousHx crosses were tested for the presence of homozygosity for theD5mit387allele associated with the Hx mutation. In 93 offspring we found 28 mice homozygous for this allele (see Table 1). This finding is in agreement with Mendelian inheritance, assuming that the homozygous Hx condition is fully viable ( P < 0.05, chi square test). The entire offspring from a backcross of homozygousHx mutants withwt mates showed theHx mutant phenotype, confirming homozygosity of the parent Hx mice. HomozygousHx mutant mice could phenotypically be distinguished from heterozygousHx mutant mice; all four feet showed preaxial polydactyly with six to eight digits, but unlike the heterozygous Hx mutant mice, the bones of the extra preaxial digits tended to be fused. All homozygousHx mutant mice showed shortening of the tibia, which was not observed in the heterozygous Hx mutant, and in a few cases shortening of the radius was observed. Bone staining of neonates and adult animals revealed no other skeletal abnormalities. Intercrossing homozygous Hx mice yielded fully viable and fertile offspring that were born with normal embryo resorption rates and litter sizes (data not shown). We then used the homozygous Hx C3HeB/FeJLe-a/a-Ca J mutants (the same genetic background as the Hm mutant), to test whether theHx and Hm mutant phenotypes are caused by two independent mutations or whether they show genetic interaction. We therefore crossed homozygous Hm mutant mice with homo-

Henk C. Heus

Papers

Clinical and genetic studies on 12 preaxial polydactyly families and refinement of the localisation of the gene responsible to a 1.9 cM region on chromosome 7q36

A physical and transcriptional map of the preaxial polydactyly locus on chromosome 7q36.

Genetics of limb development and congenital hand malformations

Hemimelic extra toes and Hammer toe are distinct mutations that show a genetic interaction.