Congenital Limb Malformations Research Articles

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

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.

Pathogenesis of split-hand/split-foot malformation.

Split-hand/split-foot malformation (SHFM), also known as ectrodactyly, is a congenital limb malformation, characterized by a deep median cleft of the hand and/or foot due to the absence of the central rays. SHFM may occur as an isolated entity or as part of a syndrome. Both forms are frequently found in association with chromosomal rearrangements such as deletions or translocations. Detailed studies of a number of mouse models for ectrodactyly have revealed that a failure to maintain median apical ectodermal ridge (AER) signalling is the main pathogenic mechanism. A number of factors complicate the identification of the genetic defects underlying human ectrodactyly: the limited number of families linked to each SHFM locus, the large number of morphogens involved in limb development, the complex interactions between these morphogens, the involvement of modifier genes, and the presumed involvement of multiple genes or long-range regulatory elements in some cases of ectrodactyly. So far, the only mutations known to underlie SHFM in humans have been found in the TP63 gene. The identification of novel human and mouse mutations for ectrodactyly will enhance our understanding of AER functions and the pathogenesis of ectrodactyly.

Why study human limb malformations?

Congenital limb malformations occur in 1 in 500 to 1 in 1000 human live births and include both gross reduction defects and more subtle alterations in the number, length and anatomy of the digits. The major causes of limb malformations are abnormal genetic programming and intra-uterine disruption to development. The identification of causative gene mutations is important for genetic counselling and also provides insights into the mechanisms controlling limb development. This article illustrates some of the lessons learnt from the study of human limb malformation, organized into seven categories. These are: (1) identification of novel genes, (2) allelic mutation series, (3) pleiotropy, (4) qualitative or (5) quantitative differences between mouse and human development, (6) physical and teratogenic disruption, and (7) unusual biological phenomena.

Current advances in Holt-Oram syndrome.

Holt-Oram syndrome is an autosomal-dominant condition characterized by congenital cardiac and forelimb anomalies. It is caused by mutations of the TBX5 gene, a member of the T-box family that encodes a transcription factor. Molecular studies have demonstrated that mutations predicted to create null alleles cause substantial abnormalities in both the limbs and heart, and that missense mutations of TBX5 can produce distinct phenotypes. One class of missense mutations causes significant cardiac malformations but only minor skeletal abnormalities; others might cause extensive upper limb malformations but less significant cardiac abnormalities. Intrafamilial variations of the malformations strongly suggest that genetic background or modifier genes play an important role in the phenotypic expression of HOS. Efforts to understand the intracellular pathway of TBX5 would provide a unique window onto the molecular basis of common congenital heart diseases and limb malformations.

A novel stable polyalanine [poly(A)] expansion in the HOXA13 gene associated with hand-foot-genital syndrome: proper function of poly(A)-harbouring transcription factors depends on a critical repeat length?

Hand-foot-genital syndrome (HFGS) is a dominantly inherited congenital malformation affecting the distal limbs and genitourinary tract. Here, we describe the phenotype and its molecular basis in a family that presented with HFGS. Genetic analysis revealed that the condition is caused by an 18-bp in-frame duplication within a cryptic trinucleotide repeat sequence encoding an 18-residue polyalanine tract in the homeoboxgene ( HOX) A13. This mutation expands the stretch with six extra alanine residues. Similar types of mutation (plus eight alanines) have recently been found in another HFGS family and also in the human HOXD13 gene (plus seven up to plus 14 residues) where it leads to synpolydactyly (SPD), a further congenital limb malformation rarely associated with genital abnormalities. As observed in our family, all the expanded tracts encoding polyalanine, either reported for HOXA13 or HOXD13, are quite stable when transmitted within affected families. Unlike disorders with unstable expansions of perfect trinucleotide repeats the molecular mechanism underlying these polyalanine expansions should be unequal crossing-over rather than replication slippage. The alanine tract elongation may prevent protein-protein interactions of the mutant HOXA13, thereby inducing a localized heterochrony in the sequence of distal limb and genitourinary development.

The Mouse Hoxd13spdh Mutation, a Polyalanine Expansion Similar to Human Type II Synpolydactyly (SPD), Disrupts the Function but Not the Expression of Other Hoxd Genes

Polyalanine expansion in the human HOXD13 gene induces synpolydactyly (SPD), an inherited congenital limb malformation. A mouse model was isolated, which showed a spontaneous alanine expansion due to a 21-bp duplication at the corresponding place in the mouse gene. This mutation (synpolydactyly homolog, spdh), when homozygous, causes malformations in mice similar to those seen in affected human patients. We have studied the genetics of this condition, by using several engineered Hoxd alleles, as well as by looking at the expression of Hox and other marker genes. We show that the mutated SPDH protein induces a gain-of-function phenotype, likely by behaving as a dominant negative over other Hox genes. The mutation, however, seems to act independently from Hoxa13 and doesn't appear to affect Hox gene expression, except for a slight reduction of the HOXD13 protein itself. Developmental studies indicate that the morphological effect is mostly due to a severe retardation in the growth and ossification of the bony elements, in agreement with a general impairment in the function of posterior Hoxd genes.

Deletions in HOXD13 Segregate with an Identical, Novel Foot Malformation in Two Unrelated Families

Synpolydactyly (SPD) is a dominantly inherited congenital limb malformation consisting of 3/4 syndactyly in the hands and 4/5 syndactyly in the feet, with digit duplication in the syndactylous web. The condition recently has been found to result from different-sized expansions of an amino-terminal polyalanine tract in HOXD13. We report a novel type of mutation in HOXD13, associated in some cases with features of classic SPD and in all cases with a novel foot phenotype. In two unrelated families, each with a different intragenic deletion in HOXD13, all mutation carriers have a rudimentary extra digit between the first and second metatarsals and often between the fourth and fifth metatarsals as well. This phenotype has not been reported in any mice with genetic modifications of the HoxD gene cluster. The two different deletions affect the first exon and the homeobox, respectively, in each case producing frameshifts followed by a long stretch of novel sequence and a premature stop codon. Although the affected genes may encode proteins that exert a dominant negative or novel effect, they are most likely to act as null alleles. Either possibility has interesting implications for the role of HOXD13 in human autopod development.

A new spontaneous mouse mutation of Hoxd13 with a polyalanine expansion and phenotype similar to human synpolydactyly

Human synpolydactyly (SPD) is an inherited congenital limb malformation caused by mutations in the HOXD13 gene. Heterozygotes are typically characterized by 3/4 finger and 4/5 toe syndactyly with associated duplicated digits; hands and feet of homozygotes are very small because of a shortening of the phalanges, metacarpal and metatarsal bones. Here we describe the phenotype and molecular basis of a spontaneous mutation of Hoxd13 in mice that provides a phenotypically and molecularly accurate model for human SPD. The new mutation, named synpolydactyly homolog (spdh), is a 21 bp in-frame duplication within a polyalanine-encoding region at the 5'-end of the Hoxd13 coding sequence. The duplication expands the stretch of alanines from 15 to 22; the same type of expansion occurs in human SPD mutations. spdh/spdh homozygotes exhibit severe malformations of all four feet, including polydactyly, syndactyly and brachydactylia. The phenotype of spdh is much more severe than that exhibited by mice with a genetically engineered, presumably null, disruption of Hoxd13. Thus spdh probably acts in a dominant-negative manner and will be valuable for examining interactions with other Hox genes and their protein products during limb development. Homozygous mice of both sexes also lack preputial glands and males do not breed; therefore, spdh/spdh mice may also be valuable in studies of reproductive physiology and behavior.

Syndromic Ectrodactyly with Severe Limb, Ectodermal, Urogenital, and Palatal Defects Maps to Chromosome 19

Congenital limb malformations rank behind only congenital heart disease as the most common birth defects observed in infants. Finding genes that cause defects in human limb patterning should be straightforward but has been limited, in part, by the bewildering spectrum of phenotypes, which are difficult to separate into etiologically distinct disorders. One approach to the identification of relevant genes is to take advantage of unique extended kindreds in which a defect in limb patterning is segregating. Recently, a large Dutch family with ectrodactyly, ectodermal dysplasia, cleft palate, and urogenital defects (EEC) was described by Maas et al. We have studied this kindred and localized a gene causing EEC to a locus on chromosome 19, in a region defined by D19S894 and D19S416. A second extended kindred with EEC does not map to this locus, indicating that EEC is a genetically heterogeneous disorder. Growth and patterning of the limbs, teeth, hair, and genitourinary system are mediated in part by epithelial-mesenchyme inductive interactions. The identification of both the gene causing EEC and its mutation may further elucidate the general signals mediating inductive mechanisms.

Synpolydactyly phenotypes correlate with size of expansions in HOXD13 polyalanine tract.

Synpolydactyly (SPD) is a dominantly inherited congenital limb malformation. Typical cases have 3/4 finger and 4/5 toe syndactyly, with a duplicated digit in the syndactylous web, but incomplete penetrance and variable expressivity are common. The condition has recently been shown to be caused by expansions of an imperfect trinucleotide repeat sequence encoding a 15-residue polyalanine tract in HOXD13. We have studied 16 new and 4 previously published SPD families, with between 7 and 14 extra residues in the tract, to analyze the molecular basis for the observed variation in phenotype. Although there is no evidence of change in expansion size within families, even over six generations, there is a highly significant increase in the penetrance and severity of phenotype with increasing expansion size, affecting both hands (P = 0.012) and feet (P < 0. 00005). Affected individuals from a family with a 14-alanine expansion, the largest so far reported, all have a strikingly similar and unusually severe limb phenotype, involving the first digits and distal carpals. Affected males from this family also have hypospadias, not previously described in SPD, but consistent with HOXD13 expression in the developing genital tubercle. The remarkable correlation between phenotype and expansion size suggests that expansion of the tract leads to a specific gain of function in the mutant HOXD13 protein, and has interesting implications for the role of polyalanine tracts in the control of transcription.

Retinoic acid receptor alpha function in vertebrate limb skeletogenesis: a modulator of chondrogenesis.

Retinoic acid is a signaling molecule involved in the regulation of growth and morphogenesis during development. There are three types of nuclear receptors for all-trans retinoic acid in mammals, RAR alpha, RAR beta, and RAR gamma, which transduce the retinoic acid signal by inducing or repressing the transcription of target genes (Leid, M., P. Kastner, and P. Chambon. 1992. Trends Biochem. Sci. 17:427-433). While RAR alpha, RAR beta, and RAR gamma are expressed in distinct but overlapping patterns in the developing mouse limb, their exact role in limb development remains unclear. To better understand the role of retinoic acid receptors in mammalian limb development, we have ectopically expressed a modified RAR alpha with constitutive activity (Balkan, W., G.K. Klintworth, C.B. Bock, and E. Linney. 1992. Dev. Biol. 151:622-625) in the limbs of transgenic mice. Overexpression of the transgene was associated with marked pre- and postaxial limb defects, particularly in the hind limb, where expression of the transgene was consistently seen across the whole anteroposterior axis. The defects displayed in these mice recapitulate, to a large degree, many of the congenital limb malformations observed in the fetuses of dams administered high doses of retinoic acid (Kochhar, D.M. 1973. Teratology. 7:289-295). Further analysis of these transgenic animals showed that the defect in skeletogenesis resided at the level of chondrogenesis. Comparison of the expression of the transgene relative to that of endogenous RAR alpha revealed that downregulation of RAR alpha is important in allowing the chondrogenic phenotype to be expressed. These results demonstrate a specific function for RARalpha in limb development and the regulation of chondroblast differentiation.

Visual loss in AIDS patients

<h3>ABSTRACT</h3> Split-Hand/Foot Malformation type 3 (SHFM3) is a congenital limb malformation associated with tandem duplications at the <i>LBX1</i>/<i>FGF8</i> locus. Yet, the disease patho-mechanism remains unsolved. Here we investigated the functional consequences of SHFM3-associated rearrangements on chromatin conformation and gene expression <i>in vivo</i> in transgenic mice. We show that the <i>Lbx1</i>/<i>Fgf8</i> locus consists of two separate, but interacting, regulatory domains. Re-engineering of a SHFM3-associated duplication and a newly reported inversion in mice resulted in restructuring of the chromatin architecture. This led to an ectopic activation of the <i>Lbx1</i> and <i>Btrc</i> genes in the apical ectodermal ridge (AER) in an <i>Fgf8-</i>like pattern. Artificial repositioning of the AER-specific enhancers of <i>Fgf8</i> was sufficient to induce misexpression of <i>Lbx1</i> and <i>Btrc</i>. We provide evidence that the SHFM3 phenotype is the result of a combinatorial effect on gene misexpression and dosage in the developing limb. Our results reveal new insights into the molecular mechanism underlying SHFM3 and provide novel conceptual framework for how genomic rearrangements can cause gene misexpression and disease.

Correlation of expression of Wnt-1 in developing limbs with abnormalities in growth and skeletal patterning.

The Wnt genes are members of a family of vertebrate genes related to the Drosophila gene wingless (wg). They encode secreted molecules that are thought to be important in patterning and growth control during ontogenesis. Several such genes are transcribed in localized domains during limb budding and morphogenesis. We report here a congenital limb malformation in a mouse transgenic line that ectopically expresses Wnt-1 in the developing limbs. The hemizygote phenotype, which is inherited as an autosomal dominant trait, presents extensive distal truncations of skeletal elements, skeletal fusions and interdigital webbing. The data shown here demonstrate that abnormal Wnt-1 expression is correlated with retarded mesenchymal condensations replaced by highly proliferative cells in the limb bud. This seems to lead to an inability of the affected cells to participate in normal skeletal development leading to the adult defects.

A cytogenetic study on congenital limb malformations in the Japanese monkey (Macaca fuscata)

A cytogenetic investigation was performed on 88 Japanese monkeys (Macaca fuscata) with abnormal limbs from 11 free-ranging provisioned troops including nine individuals with abnormalities indistinguishable as to whether they were congenital or injurious. All of the monkeys with abnormal limbs including the nine questionable individuals had the same karyotypes as those of normal individuals. The chromosome number was 42, consisting of 20 bi-arm autosome pairs and a submetacentric X-chromosome and Y-chromosome. The ninth chromosome pair, which was the only chromosome pair with remarkable secondary constriction, displayed length polymorphism of the centromeric C-band and secondary constriction in both deformed and normal monkeys. These kinds of variants have also been commonly found in other monkey species, which have almost the same karyotype as the Japanese monkey and have not been reported to show frequent occurrence of limb malformation. We concluded therefore that chromosomal abnormalities could be excluded from the main causal factors for limb malformations of the Japanese monkey.

エラータ : ニホンザルの四肢奇形と有機塩素系農薬の関連について

エラータ : ニホンザルの四肢奇形と有機塩素系農薬の関連について

Relation of the Congenital Limb-malformation in the Japanese Monkeys and Organochlorin Pesticides

It has been often found that the provisionized Japanese monkeys bore limb-malformed babies since 1955 in Japan. We reported that some organochlorin pesticide residues might have related to the cause of the malformation of limbs. It was found that the tissues of the dead Japanese monkeys were contaminated with Heptachlor epoxide, Dieldrin, BHC, DDT, and Chlordane isomers. The concentrations of Heptachlor epoxide in the malformed monkeys were significantly higher (kidney, p<0.05) than those in the normal monkeys. The levels of Heptachlor epoxide (kidney) and Dieldrin (liver) in the adult female monkeys who had borne malformed babies were also significantly higher (p<0.05) than those in the adult females who had not.

Behavior of infant Japanese monkeys (Macaca fuscata) with congenital limb malformations during their first three months.

Locomotion and mother-infant interactions of a severely malformed, a slightly malformed, and a normal infant were observed in a free-ranging group of Japanese monkeys (Macaca fuscata). The severely malformed infant had no feet and had hands consisting of 2 digits. The slightly malformed infant had normal feet and hands consisting of 2 digits. The severely malformed infant was more retarded in development of posture and locomotion than the other infants; however, it developed locomotor patterns appropriate to its defects. All infants spent approximately the same amount of time in contact with their mothers. High mother-infant contact of malformed infants was maintained because their mothers were very attentive and carried and held them. Mothers reared their handicapped infants even though the infants lacked the movement necessary for clinging.

A classification for congenital limb malformation

The classification for congenital limb malformations adopted by the American Society for Surgery of the Hand, the International Federation of Societies for Surgery of the Hand, and the International Society of Prosthetics and Orthotics is presented. This method groups similar patterns of deficiencies according to the parts that have been primarily affected by certain embryological failures, whether the insult involves a total part (skeletal and soft tissue) or only the dermomyofascial structures. The main categories of this classification are (I) failure of formation of parts, (II) failure of differentiation (separation) of parts, (III) duplication, (IV) overgrowth, (V) undergrowth, (VI) congenital constriction band syndrome, and (VII) generalized skeletal abnormalities. The rationale and method of use of the classification are discussed. This method has been tested and used in a variety of centers and has been found to be properly conceived and practical.

The role of morphogenetic cell death during abnormal limb-bud outgrowth in mice heterozygous for the dominant mutation Hemimelia-extra toe (Hm x )

ABSTRACT A dominant mutation in the mouse, Hemimelia-extra toe (Hmx), induces congenital limb malformations in heterozygotes. Typical expression includes axial shortening of the radius, tibia and talus (‘hemimelia’), with supernumerary metacarpals, metatarsals, and digits (‘polydactyly’). Pathogenesis was investigated during developmental stages 16 through 22 (11th through 15th days of gestation). Full expression was apparent during stage 20 when the limb pattern was comprised of pre-cartilaginous anlagen. Formation of a pre-axial protrusion on the autopod during stage 17 or 18 was the earliest gross abnormality, and foreshadowed the development of supernumerary digits. Microscopically, there was an alteration in the pattern of physiologic cellular degeneration (PCD) programmed to occur within the zeugopod and autopod. The ‘opaque patch’ (mesodermal necrotic zone normally occurring between tibial and fibular anlagen) was overextended pre-axially causing resorption of the tibial precartilage. Additionally, PCD normally occurring within the basal cell layer of the apical ectodermal ridge (AER) and the ‘foyer primaire préaxial’ was not expressed in the mutant autopod. This occurred in association with outgrowth of the protrusion. The pre-axial portion of the AER remained in an abnormally thickened, viable, proliferative state, and did not undergo scheduled degression. This may have been the basis for prolonged induction of pre-axial outgrowth. Paucity of mesenchymal cell filopodial processes extended along the basal lamina, as well as a rarefaction of the filamentous material normally associated with the mesodermal face of the basal lamina, was detected at the pre-axial AER-mesenchymal interface on stage 18. A potential involvement of epithelial-mesenchymal interactions in the induction of epithelial PCD is discussed.

Congenital central ray deficiency in the hand—A survey of 59 cases and subclassification

Eighty-nine hands were studied in 59 patients with central ray deficiency. A subclassification into two subgroups was established based on the clinical and radiological findings-subgroup I: typical type and subgroup II: (atypical type) with type a, syndactylous type, and type b, polydactylous type. In subgroup I, the sequential severity of deficiency ranged from a partial defect of phalanges of the middle finger to a monodigit hand. The central digital elements were fused to adjacent digital rays in subgroup II-type a. Supernumerary bony elements were seen in subgroup II-type b. The close relationship between central ray deficiency, syndactyly, and polydactyly was discussed from the standpoint of development of the hand. The classification of central ray deficiency into the longitudinal deficiency category of the International Classification of Congenital Limb Malformations was recommended.