Limb Phenotype Research Articles

A new mutation in the gene ROR2 causes brachydactyly type B1

Brachydactyly type B, an autosomal dominant disorder that is characterized by hypoplasia of the distal phalanges and nails, can be divided into brachydactyly type B1 (BDB1) and brachydactyly type B2 (BDB2). BDB1 is caused by mutations in the receptor tyrosine kinase gene ROR2, which maps to chromosome 9q22, whereas BDB2 is caused by point mutations in the bone morphogenetic protein antagonist NOGGIN. Here, we report a three-generation Chinese family with dominant inheritance of the BDB1 limb phenotype. Sequence analysis identified a novel heterozygous base deletion (c.1396-1398delAA) in the gene ROR2 in all affected family members. This new deletion is expected to produce a truncated Ror2 protein with a new polypeptide of 57 amino acids at the C-terminal.

Cellular basis of differential limb growth in postnatal gray short‐tailed opossums (Monodelphis domestica)

While growth has been studied extensively in invertebrates, the mechanisms by which it is controlled in vertebrates, particularly in mammals, remain poorly understood. In this study, we investigate the cellular basis of differential limb growth in postnatal Monodelphis domestica, the gray short-tailed opossum, to gain insights into the mechanisms regulating mammalian growth. Opossums are an ideal model for the study of growth because they are born with relatively large, well-developed forelimbs and small hind limbs that must "catch up" to the forelimb before the animal reaches adulthood. Postnatal Days 1-17 were identified as a key period of growth for the hind limbs, during which they undergo accelerated development and nearly quadruple in length. Histology performed on fore- and hind limbs from this period indicates a higher rate of cellular differentiation in the long bones of the hind limbs. Immunohistochemical assays indicate that cellular proliferation is also occurring at a significantly greater rate in the long bones of the hind limb at 6 days after birth. Taken together, these results suggest that a faster rate of cellular proliferation and differentiation in the long bones of the hind limb relative to those of the forelimb generates a period of accelerated growth through which the adult limb phenotype of M. domestica is achieved. Assays for gene expression suggest that the molecular basis of this differential growth differs from that previously identified for differential pre-natal growth in opossum fore- and hind limbs.

A novel mutation (g.106737G>T) in zone of polarizing activity regulatory sequence (ZRS) causes variable limb phenotypes in Werner mesomelia

Werner mesomelia is characterized by a sequence variation in the specific region (position 404) of the enhancer ZRS of SHH. The phenotype comprises variable mesomelia, abnormalities of the thumb and great toe and supernumerary digits. We describe extensive variation in limb phenotype in a large family and report on a novel sequence variation NG_009240.1: g.106737G>T (traditional nomenclature: ZRS404G>T) in the ZRS within the LMBR1 gene. The newly recognized clinical features in this family include small thenar eminence, sandal gap, broad first metatarsals, mesoaxial polydactyly, and postaxial polydactyly. We provide information on 12 affected family members. We review the literature on how a sequence variation in ZRS may cause such diverse phenotypes.

Dystrophic changes in extraocular muscles after gamma irradiation in mdx:utrophin(+/-) mice.

Extraocular muscles (EOM) have a strikingly different disease profile than limb skeletal muscles. It has long been known that they are spared in Duchenne (DMD) and other forms of muscular dystrophy. Despite many studies, the cause for this sparing is not understood. We have proposed that differences in myogenic precursor cell properties in EOM maintain normal morphology over the lifetime of individuals with DMD due to either greater proliferative potential or greater resistance to injury. This hypothesis was tested by exposing wild type and mdx:utrophin+/− (het) mouse EOM and limb skeletal muscles to 18 Gy gamma irradiation, a dose known to inhibit satellite cell proliferation in limb muscles. As expected, over time het limb skeletal muscles displayed reduced central nucleation mirrored by a reduction in Pax7-positive cells, demonstrating a significant loss in regenerative potential. In contrast, in the first month post-irradiation in the het EOM, myofiber cross-sectional areas first decreased, then increased, but ultimately returned to normal compared to non-irradiated het EOM. Central nucleation significantly increased in the first post-irradiation month, resembling the dystrophic limb phenotype. This correlated with decreased EECD34 stem cells and a concomitant increase and subsequent return to normalcy of both Pax7 and Pitx2-positive cell density. By two months, normal het EOM morphology returned. It appears that irradiation disrupts the normal method of EOM remodeling, which react paradoxically to produce increased numbers of myogenic precursor cells. This suggests that the EOM contain myogenic precursor cell types resistant to 18 Gy gamma irradiation, allowing return to normal morphology 2 months post-irradiation. This supports our hypothesis that ongoing proliferation of specialized regenerative populations in the het EOM actively maintains normal EOM morphology in DMD. Ongoing studies are working to define the differences in the myogenic precursor cells in EOM as well as the cellular milieu in which they reside.

Digit evolution in gymnophthalmid lizards.

The tetrapod limb is a highly diverse structure, and reduction or loss of this structure accounts for many of the limb phenotypes observed within species. Squamate reptiles are one of the many tetrapod lineages in which the limbs have been greatly modified from the pentadactyl generalized pattern, including different degrees of reduction in the number of limb elements to complete limblessness. Even though limb reduction is widespread, the evolutionary and developmental mechanisms involved in the formation of reduced limb morphologies remains unclear. In this study, we present an overview of limb morphology within the microteiid lizard group Gymnophthalmidae, focusing on digit arrangement. We show that there are two major groups of limb-reduced gymnophthalmids. The first group is formed by lizard-like (and frequently pentadactyl) species, in which minor reductions (such as the loss of 1-2 phalanges mainly in digits I and V) are the rule; these morphologies generally correspond to those seen in other squamates. The second group is formed by species showing more drastic losses, which can include the absence of an externally distinct limb in adults. We also present the expression patterns of Sonic Hedgehog (Shh) in the greatly reduced fore and hindlimb of a serpentiform gymnophthalmid. Our discussion focuses on identifying shared patterns of limb reduction among tetrapods, and explaining these patterns and the morphological variation within the gymnophthalmids based on current knowledge of the molecular signaling pathways that coordinate limb development.

A Trans-Acting Protein Effect Causes Severe Eye Malformation in the Mp Mouse

Mp is an irradiation-induced mouse mutation associated with microphthalmia, micropinna and hind limb syndactyly. We show that Mp is caused by a 660 kb balanced inversion on chromosome 18 producing reciprocal 3-prime gene fusion events involving Fbn2 and Isoc1. The Isoc1-Fbn2 fusion gene (Isoc1Mp) mRNA has a frameshift and early stop codon resulting in nonsense mediated decay. Homozygous deletions of Isoc1 do not support a significant developmental role for this gene. The Fbn2-Isoc1 fusion gene (Fbn2 Mp) predicted protein consists of the N-terminal Fibrillin-2 (amino acids 1–2646, exons 1–62) lacking the C-terminal furin-cleavage site with a short out-of-frame extension encoded by the final exon of Isoc1. The Mp limb phenotype is consistent with that reported in Fbn2 null embryos. However, severe eye malformations, a defining feature of Mp, are not seen in Fbn2 null animals. Fibrillin-2Mp forms large fibrillar structures within the rough endoplasmic reticulum (rER) associated with an unfolded protein response and quantitative mass spectrometry shows a generalised defect in protein secretion in conditioned media from mutant cells. In the embryonic eye Fbn2 is expressed within the peripheral ciliary margin (CM). Mp embryos show reduced canonical Wnt-signalling in the CM – known to be essential for ciliary body development - and show subsequent aplasia of CM-derived structures. We propose that the Mp “worse-than-null” eye phenotype plausibly results from a failure in normal trafficking of proteins that are co-expressed with Fbn2 within the CM. The prediction of similar trans-acting protein effects will be an important challenge in the medical interpretation of human mutations from whole exome sequencing.

A Novel Intergenic ETnII-β Insertion Mutation Causes Multiple Malformations in Polypodia Mice

Mouse early transposon insertions are responsible for ∼10% of spontaneous mutant phenotypes. We previously reported the phenotypes and genetic mapping of Polypodia, (Ppd), a spontaneous, X-linked dominant mutation with profound effects on body plan morphogenesis. Our new data shows that mutant mice are not born in expected Mendelian ratios secondary to loss after E9.5. In addition, we refined the Ppd genetic interval and discovered a novel ETnII-β early transposon insertion between the genes for Dusp9 and Pnck. The ETn inserted 1.6 kb downstream and antisense to Dusp9 and does not disrupt polyadenylation or splicing of either gene. Knock-in mice engineered to carry the ETn display Ppd characteristic ectopic caudal limb phenotypes, showing that the ETn insertion is the Ppd molecular lesion. Early transposons are actively expressed in the early blastocyst. To explore the consequences of the ETn on the genomic landscape at an early stage of development, we compared interval gene expression between wild-type and mutant ES cells. Mutant ES cell expression analysis revealed marked upregulation of Dusp9 mRNA and protein expression. Evaluation of the 5′ LTR CpG methylation state in adult mice revealed no correlation with the occurrence or severity of Ppd phenotypes at birth. Thus, the broad range of phenotypes observed in this mutant is secondary to a novel intergenic ETn insertion whose effects include dysregulation of nearby interval gene expression at early stages of development.

Application of synthetic photostable retinoids induces novel limb and facial phenotypes during chick embryogenesisin vivo

We have recently developed a range of synthetic retinoid analogues which include the compounds EC23 and EC19. They are stable on exposure to light and are predicted to be resistant to the normal metabolic processes involved in the inactivation of retinoids in vivo. Based on the position of the terminal carboxylic acid groups in the compounds we suggest that EC23 is a structural analogue of all-trans retinoic acid (ATRA), and EC19 is an analogue of 13-cis retinoic acid. Their effects on the differentiation of pluripotent stem cells has been previously described in vitro and are consistent with this hypothesis. We present herein the first description of the effects of these molecules in vivo. Retinoids were applied to the anterior limb buds of chicken embryos in ovo via ion-exchange beads. We found that retinoid EC23 produces effects on the wing digits similar to ATRA, but does so at two orders of magnitude lower concentration. When larger quantities of EC23 are applied, a novel phenotype is obtained involving production of multiple digit 1s on the anterior limb. This corresponds to differential effects of ATRA and EC23 on sonic hedgehog (shh) expression in the developing limb bud. With EC23 application we also find digit 1 phenotypes similar to thumb duplications described in the clinical literature. EC23 and ATRA are shown to have effects on the entire proximal–distal axis of the limb, including hitherto undescribed effects on the scapula. This includes suppression of expression of the scapula marker Pax1. EC23 also produces effects similar to those of ATRA on the developing face, producing reductions of the upper beak at concentrations two orders of magnitude lower than ATRA. In contrast, EC19, which is structurally very similar to EC23, has novel, less severe effects on the face and rarely alters limb development. EC19 and ATRA are effective at similar concentrations. These results further demonstrate the ability of retinoids to influence embryonic development. Moreover, EC23 represents a useful new tool to investigate developmental processes and probe the mechanisms underlying congenital abnormalities in vertebrates including man.

Embryonic mechanical and soluble cues regulate tendon progenitor cell gene expression as a function of developmental stage and anatomical origin

Stem cell-based engineering strategies for tendons have yet to yield a normal functional tissue, due in part to a need for tenogenic factors. Additionally, the ability to evaluate differentiation has been challenged by a lack of markers for differentiation. We propose to inform tendon regeneration with developmental cues involved in normal tissue formation and with phenotypic markers that are characteristic of differentiating tendon progenitor cells (TPCs). Mechanical forces, fibroblast growth factor (FGF)-4 and transforming growth factor (TGF)-β2 are implicated in embryonic tendon development, yet the isolated effects of these factors on differentiating TPCs are unknown. Additionally, developmental mechanisms vary between limb and axial tendons, suggesting the respective cell types are programmed to respond uniquely to exogenous factors. To characterize developmental cues and benchmarks for differentiation toward limb vs. axial phenotypes, we dynamically loaded and treated TPCs with growth factors and assessed gene expression profiles as a function of developmental stage and anatomical origin. Based on scleraxis expression, TGFβ2 was tenogenic for TPCs at all stages, while loading was for late-stage cells only, and FGF4 had no effect despite regulation of other genes. When factors were combined, TGFβ2 continued to be tenogenic, while FGF4 appeared anti-tenogenic. Various treatments elicited distinct responses by axial vs. limb TPCs of specific stages. These results identified tenogenic factors, suggest tendon engineering strategies should be customized for tissues by anatomical origin, and provide stage-specific gene expression profiles of limb and axial TPCs as benchmarks with which to monitor tenogenic differentiation of stem cells.

ANO5Gene Analysis in a Large Cohort of Patients with Anoctaminopathy: Confirmation of Male Prevalence and High Occurrence of the Common Exon 5 Gene Mutation

Limb girdle muscular dystrophy type 2L or anoctaminopathy is a condition mainly characterized by adult onset proximal lower limb muscular weakness and raised CK values, due to recessive ANO5 gene mutations. An exon 5 founder mutation (c.191dupA) has been identified in most of the British and German LGMD2L patients so far reported. We aimed to further investigate the prevalence and spectrum of ANO5 gene mutations and related clinical phenotypes, by screening 205 undiagnosed patients referred to our molecular service with a clinical suspicion of anoctaminopathy. A total of 42 unrelated patients had two ANO5 mutations (21%), whereas 14 carried a single change. We identified 34 pathogenic changes, 15 of which are novel. The c.191dupA mutation represents 61% of mutated alleles and appears to be less prevalent in non-Northern European populations. Retrospective clinical analysis corroborates the prevalently proximal lower limb phenotype, the male predominance and absence of major cardiac or respiratory involvement. Identification of cases with isolated hyperCKaemia and very late symptomatic male and female subjects confirms the extension of the phenotypic spectrum of the disease. Anoctaminopathy appears to be one of the most common adult muscular dystrophies in Northern Europe, with a prevalence of about 20%-25% in unselected undiagnosed cases.

Mouse limbs expressing only the Gli3 repressor resemble those of Sonic hedgehog mutants

Anterioposterior vertebrate limb patterning is controlled by opposing action between Sonic Hedgehog (Shh) and the Gli3 transcriptional repressor. Unexpectedly, Gli3Δ699 mutant mice, which are thought to express only a Gli3 repressor and not the full-length activator, exhibit limb phenotypes inconsistent with those of Shh mutant mice. Therefore, it remains debatable whether Shh patterns the anterioposterior limb primarily by inhibiting generation of the Gli3 repressor. However, one caveat is that Gli3Δ699 may not be as potent as the natural form of Gli3 repressor because of the nature of the mutant allele. In the present study, we created a conditional Gli3 mutant allele that exclusively expresses Gli3 repressor in the presence of Cre recombinase. Using this mutant, we show that the phenotypes of mouse limbs expressing only the Gli3 repressor exhibit no or single digit, resembling those of Shh mutant limbs. Consistent with the limb phenotypes, the expression of genes dependent on Shh signaling is also inhibited in both mutants. This inhibition by the Gli3 repressor is independent of Shh. Thus, our study clarifies the current controversy and provides important genetic evidence to support the hypothesis that Shh patterns the anterioposterior limb primarily through the inhibition of Gli3 repressor formation.

Overdosage of Hand2 causes limb and heart defects in the human chromosomal disorder partial trisomy distal 4q

Partial trisomy distal 4q (denoted 4q+) is a human chromosomal disorder caused by duplication of the distal end of the long arm of chromosome 4 (Chr4). This disorder manifests typical phenotypes, including craniofacial, renal, heart and thumb developmental defects. Although these clinical features are likely caused by a dosage imbalance in the gene network involving the trisomic region, the causative gene or genes and the molecular bases are largely unknown. Here, we report mouse Recombination-induced mutation 4 (Rim4) as a model animal of 4q+. The Rim4 genome contains an insertion of a 6.5 Mb fragment from mouse chromosome 8 into chromosome 6. This insertion fragment contains 17 genes, including Hand2, that encode the basic helix-loop-helix transcription factor and is syntenic to the distal end of human Chr4, 4q32.3 to 4q34.1, which is responsible for 4q+. A comparison of phenotypes between patients with Rim4 and 4q+ revealed that Rim4 shows direct parallels with many phenotypes of 4q+ such as craniofacial, heart, cervical vertebra and limb deformities. Rebalancing the gene dosage by a genetic cross with Hand2 knockout mice ameliorated symptoms of the heart and limb deformities of Rim4. Conversely, an increase in copy number of Hand2 in wild-type mice recaptures the heart and limb deformities of Rim4. Our results collectively demonstrate that overdosage of Hand2 is a major cause for at least the limb and heart phenotypes of 4q+ and that mouse Rim4 provides a unique animal model for understanding the molecular bases underlying the complex phenotypes of 4q+.

BMP-Mediated Functional Cooperation between Dlx5;Dlx6 and Msx1;Msx2 during Mammalian Limb Development

The Dlx and Msx homeodomain transcription factors play important roles in the control of limb development. The combined disruption of Msx1 and Msx2, as well as that of Dlx5 and Dlx6, lead to limb patterning defects with anomalies in digit number and shape. Msx1;Msx2 double mutants are characterized by the loss of derivatives of the anterior limb mesoderm which is not observed in either of the simple mutants. Dlx5;Dlx6 double mutants exhibit hindlimb ectrodactyly. While the morphogenetic action of Msx genes seems to involve the BMP molecules, the mode of action of Dlx genes still remains elusive. Here, examining the limb phenotypes of combined Dlx and Msx mutants we reveal a new Dlx-Msx regulatory loop directly involving BMPs. In Msx1;Dlx5;Dlx6 triple mutant mice (TKO), beside the expected ectrodactyly, we also observe the hallmark morphological anomalies of Msx1;Msx2 double mutants suggesting an epistatic role of Dlx5 and Dlx6 over Msx2. In Msx2;Dlx5;Dlx6 TKO mice we only observe an aggravation of the ectrodactyly defect without changes in the number of the individual components of the limb. Using a combination of qPCR, ChIP and bioinformatic analyses, we identify two Dlx/Msx regulatory pathways: 1) in the anterior limb mesoderm a non-cell autonomous Msx-Dlx regulatory loop involves BMP molecules through the AER and 2) in AER cells and, at later stages, in the limb mesoderm the regulation of Msx2 by Dlx5 and Dlx6 occurs also cell autonomously. These data bring new elements to decipher the complex AER-mesoderm dialogue that takes place during limb development and provide clues to understanding the etiology of congenital limb malformations.

Sonic hedgehog signaling directly targets Hyaluronic Acid Synthase 2, an essential regulator of phalangeal joint patterning

Sonic hedgehog (Shh) signal, mediated by the Gli family of transcription factors, plays an essential role in the growth and patterning of the limb. Through analysis of the early limb bud transcriptome, we identified a posteriorly-enriched gene, Hyaluronic Acid Synthase 2 (Has2), which encodes a key enzyme for the synthesis of hyaluronan (HA), as a direct target of Gli transcriptional regulation during early mouse limb development. Has2 expression in the limb bud is lost in Shh null and expanded anteriorly in Gli3 mutants. We identified an ∼3kb Has2 promoter fragment that contains two strong Gli-binding consensus sequences, and mutation of either site abrogated the ability of Gli1 to activate Has2 promoter in a cell-based assay. Additionally, this promoter fragment is sufficient to direct expression of a reporter gene in the posterior limb mesenchyme. Chromatin immunoprecipitation of DNA-Gli3 protein complexes from limb buds indicated that Gli3 strongly binds to the Has2 promoter region, suggesting that Has2 is a direct transcriptional target of the Shh signaling pathway. We also showed that Has2 conditional mutant (Has2cko) hindlimbs display digit-specific patterning defects with longitudinally shifted phalangeal joints and impaired chondrogenesis. Has2cko limbs show less capacity for mesenchymal condensation with mislocalized distributions of chondroitin sulfate proteoglycans (CSPGs), aggrecan and link protein. Has2cko limb phenotype displays striking resemblance to mutants with defective chondroitin sulfation suggesting tight developmental control of HA on CSPG function. Together, our study identifies Has2 as a novel downstream target of Shh signaling required for joint patterning and chondrogenesis.

The CDKL5 disorder is an independent clinical entity associated with early-onset encephalopathy

The clinical understanding of the CDKL5 disorder remains limited, with most information being derived from small patient groups seen at individual centres. This study uses a large international data collection to describe the clinical profile of the CDKL5 disorder and compare with Rett syndrome (RTT). Information on individuals with cyclin-dependent kinase-like 5 (CDKL5) mutations (n=86) and females with MECP2 mutations (n=920) was sourced from the InterRett database. Available photographs of CDKL5 patients were examined for dysmorphic features. The proportion of CDKL5 patients meeting the recent Neul criteria for atypical RTT was determined. Logistic regression and time-to-event analyses were used to compare the occurrence of Rett-like features in those with MECP2 and CDKL5 mutations. Most individuals with CDKL5 mutations had severe developmental delay from birth, seizure onset before the age of 3 months and similar non-dysmorphic features. Less than one-quarter met the criteria for early-onset seizure variant RTT. Seizures and sleep disturbances were more common than in those with MECP2 mutations whereas features of regression and spinal curvature were less common. The CDKL5 disorder presents with a distinct clinical profile and a subtle facial, limb and hand phenotype that may assist in differentiation from other early-onset encephalopathies. Although mutations in the CDKL5 gene have been described in association with the early-onset variant of RTT, in our study the majority did not meet these criteria. Therefore, the CDKL5 disorder should be considered separate to RTT, rather than another variant.

Phenotype profile of a genetic mouse model for Muenke syndrome.

The Muenke syndrome mutation (FGFR3 (P250R)), which was discovered 15 years ago, represents the single most common craniosynostosis mutation. Muenke syndrome is characterized by coronal suture synostosis, midface hypoplasia, subtle limb anomalies, and hearing loss. However, the spectrum of clinical presentation continues to expand. To better understand the pathophysiology of the Muenke syndrome, we present collective findings from several recent studies that have characterized a genetically equivalent mouse model for Muenke syndrome (FgfR3 (P244R)) and compare them with human phenotypes. FgfR3 (P244R) mutant mice show premature fusion of facial sutures, premaxillary and/or zygomatic sutures, but rarely the coronal suture. The mice also lack the typical limb phenotype. On the other hand, the mutant mice display maxillary retrusion in association with a shortening of the anterior cranial base and a premature closure of intersphenoidal and spheno-occipital synchondroses, resembling human midface hypoplasia. In addition, sensorineural hearing loss is detected in all FgfR3 (P244R) mutant mice as in the majority of Muenke syndrome patients. It is caused by a defect in the mechanism of cell fate determination in the organ of Corti. The mice also express phenotypes that have not been previously described in humans, such as reduced cortical bone thickness, hypoplastic trabecular bone, and defective temporomandibular joint structure. Therefore, the FgfR3 (P244R) mouse provides an excellent opportunity to study disease mechanisms of some classical phenotypes of Muenke syndrome and to test novel therapeutic strategies. The mouse model can also be further explored to discover previously unreported yet potentially significant phenotypes of Muenke syndrome.

Twist2 contributes to termination of limb bud outgrowth and patterning through direct regulation of Grem1

Twist1 has been demonstrated to play critical roles in the early development of neural crest and mesodermally derived tissues including the limb. Twist2 has been less well characterised but its relatively late onset of expression suggests specific roles in the development of a number of organs. Expression of Twist2 within the developing limbs begins after formation of the limb bud and persists within the peripheral mesenchyme until digital rays condense. We have used RCAS-mediated overexpression in chick to investigate the function of Twist2 in limb development. Viral misexpression following injection into the lateral plate mesoderm results in a spectrum of hypoplastic limb phenotypes. These include generalized shortening of the entire limb, fusion of the autopod skeletal elements, loss of individual digits or distal truncation resulting in complete loss of the autopod. These phenotypes appear to result from a premature termination of limb outgrowth and manifest as defective growth in both the proximal–distal and anterior–posterior axes. In situ hybridisation analysis demonstrates that many components of the Shh/Grem1/Fgf regulatory loop that controls early limb growth and patterning are downregulated by Twist2 overexpression. Grem1 has a complementary expression pattern to Twist2 within the limb primordia and co-expression of both Grem1 and Twist2 results in a rescue of the Twist2 overexpression phenotype. We demonstrate that Twist proteins directly repress Grem1 expression via a regulatory element downstream of the open reading frame. These data indicate that Twist2 regulates early limb morphogenesis through a role in terminating the Shh/Grem1/Fgf autoregulatory loop.

A novel ZRS mutation in a Balochi tribal family with triphalangeal thumb, pre‐axial polydactyly, post‐axial polydactyly, and syndactyly

Limb malformations are one of the most common types of human congenital malformations. Mutations in the ZRS enhancer of Sonic Hedgehog are thought to be responsible for pre-axial polydactyly in multiple independent families. Here, we describe a large Balochi tribal family from Southern Punjab, Pakistan, with a variable set of limb malformations and a novel ZRS mutation. The family has a limb phenotype characterized by triphalangeal thumb, pre-axial polydactyly, and post-axial polydactyly. There is also a high degree of phenotypic heterogeneity with less common clinical findings in the affected family members that include osseous syndactyly of forth-fifth fingers, clinodactyly, hypoplasia of mesoaxial fingers, and bifid halluces. The presentation in most of the affected patients was bilateral and symmetrical. A heterozygous C>A mutation at position 287 of the ZRS enhancer (chr7:156,584,283; hg19) was detected in all affected subjects and is absent from four unaffected family members, 42 unrelated samples, and multiple databases of human variation. Combined, these results identify a novel ZRS287 C>A mutation which leads to a variable spectrum of limb phenotypes.

Disparate Igf1 Expression and Growth in the Fore‐ and Hind Limbs of a Marsupial Mammal (Monodelphis domestica)

Proper regulation of growth is essential to all stages of life, from development of the egg into an embryo to the maintenance of normal cell cycle progression in adults. However, despite growth's importance to basic biology and health, little is known about how mammalian growth is regulated. In this study, we investigated the molecular basis of the highly disparate growth of opossum fore- and hind limbs in utero. We first used a novel, opossum-specific microarray to identify several growth-related genes that are differentially expressed in opossum fore- and hind limbs of comparable developmental stages. These genes included Igf1. Given Igf1's role in the growth of other systems, we further investigated the role of Igf1 in opossum limb growth. Supporting the microarray results, RT-PCR indicated that Igf1 levels are approximately two times higher in opossum fore- than hind limbs. Consistent with this, while Igf1 transcripts were readily detectable in opossum forelimbs using whole-mount in situ hybridization, they were not detectable in opossum hind limbs. Furthermore, opossum limbs treated with exogenous Igf1 protein experienced significantly greater cellular proliferation and growth than control limbs in vitro. Taken together, results suggest that the differential expression of Igf1 in developing opossum limbs contributes to their divergent rate of growth, and the unique limb phenotype of opossum newborns. This study establishes the opossum limb as a new mammalian model system for study of organ growth.

A novel 13 base pair insertion in the sonic hedgehog ZRS limb enhancer (ZRS/LMBR1) causes preaxial polydactyly with triphalangeal thumb

Mutations in the Sonic hedgehog limb enhancer, the zone of polarizing activity regulatory sequence (ZRS, located within the gene LMBR1), commonly called the ZRS), cause limb malformations. In humans, three classes of mutations have been proposed based on the limb phenotype; single base changes throughout the region cause preaxial polydactyly (PPD), single base changes at one specific site cause Werner mesomelic syndrome, and large duplications cause polysyndactyly. This study presents a novel mutation-a small insertion. In a Swedish family with autosomal-dominant PPD, we found a 13 base pair insertion within the ZRS, NG_009240.1:g.106934_106935insTAAGGAAGTGATT (traditional nomenclature: ZRS603ins13). Computational transcription factor-binding site predictions suggest that this insertion creates new binding sites and a mouse enhancer assay shows that this insertion causes ectopic gene expression. This study is the first to discover a small insertion in an enhancer that causes a human limb malformation and suggests a potential mechanism that could explain the ectopic expression caused by this mutation.