The molecular genetics of human appendicular skeleton.

Disorders that result from de-arrangement of growth, development and/or differentiation of the appendages (limbs and digit) are collectively called as inherited abnormalities of human appendicular skeleton. The bones of appendicular skeleton have central role in locomotion and movement. The different types of appendicular skeletal abnormalities are well described in the report of "Nosology and Classification of Genetic skeletal disorders: 2019 Revision". In the current article, we intend to present the embryology, developmental pathways, disorders and the molecular genetics of the appendicular skeletal malformations. We mainly focused on the polydactyly, syndactyly, brachydactyly, split-hand-foot malformation and clubfoot disorders. To our knowledge, only nine genes of polydactyly, five genes of split-hand-foot malformation, nine genes for syndactyly, eight genes for brachydactyly and only single gene for clubfoot have been identified to be involved in disease pathophysiology. The current molecular genetic data will help life sciences researchers working on the rare skeletal disorders. Moreover, the aim of present systematic review is to gather the published knowledge on molecular genetics of appendicular skeleton, which would help in genetic counseling and molecular diagnosis.

Confirming the involvement of PIEZO2 in the etiology of Marden-Walker syndrome.

Pathogenic heterozygous variants in PIEZO2 typically cause distal arthrogryposis type 5 (DA5) and the closely related Gordon syndrome (GS). Only one case of PIEZO2-related Marden-Walker syndrome (MWS) has been reported to date. We report the phenotypic features of a Saudi female patient with features consistent with MWS in whom we identified a novel de novo likely pathogenic variant in PIEZO2. Our case lends support to the link between PIEZO2 and MWS.

Clubfoot Versus Positional Foot Deformities on Prenatal Ultrasound Imaging.

Clubfoot and positional foot deformities (eg, pes spinatus) may have the same aspects on prenatal ultrasound (US) imaging. Nevertheless, differentiating these entities is essential because their prognoses are different. This pictorial review illustrates the US findings of clubfoot and positional foot deformities. On the basis of clinical postnatal images, we describe a prenatal US technique that could give an accurate diagnosis. In this essay, we demonstrate that when a foot malposition is suspected, a systematic analysis with 3 rigorous planes could help differentiate positional foot deformities from malformations and define their types.

The developmental and genetic basis of 'clubfoot' in the peroneal muscular atrophy mutant mouse.

Genetic factors underlying the human limb abnormality congenital talipes equinovarus ('clubfoot') remain incompletely understood. The spontaneous autosomal recessive mouse 'peroneal muscular atrophy' mutant (PMA) is a faithful morphological model of human clubfoot. In PMA mice, the dorsal (peroneal) branches of the sciatic nerves are absent. In this study, the primary developmental defect was identified as a reduced growth of sciatic nerve lateral motor column (LMC) neurons leading to failure to project to dorsal (peroneal) lower limb muscle blocks. The pma mutation was mapped and a candidate gene encoding LIM-domain kinase 1 (Limk1) identified, which is upregulated in mutant lateral LMC motor neurons. Genetic and molecular analyses showed that the mutation acts in the EphA4-Limk1-Cfl1/cofilin-actin pathway to modulate growth cone extension/collapse. In the chicken, both experimental upregulation of Limk1 by electroporation and pharmacological inhibition of actin turnover led to defects in hindlimb spinal motor neuron growth and pathfinding, and mimicked the clubfoot phenotype. The data support a neuromuscular aetiology for clubfoot and provide a mechanistic framework to understand clubfoot in humans.

Foetal axillary lymphangioma with ipsilateral pes equinovarus: pitfalls in sonographic differential diagnosis (axillary lymphangioma & pes equinovarus).

Lymphangiomas are rare congenital malformations of the lymphatic system. Despite the benign histology, they are likely to grow rapidly and invade the surrounding tissues. In contrast to the cystic hygromas, lymphangiomas at the axillary region tend to have normal karyotype. However, associated hydrops makes the prognosis poor. Due to isolated few cases in the literature, the true incidence of foetal axillary lymphangiomas is not known. We present here a pre-natal ultrasonographic diagnosis of a 15-week foetus with rapidly growing axillary lymphangioma with ipsilateral foot abnormality which had normal karyotype.

Celsr3 is required in motor neurons to steer their axons in the hindlimb.

The cadherin Celsr3 regulates the directional growth and targeting of axons in the CNS, but whether it acts in collaboration with or in parallel to other guidance cues is unknown. Furthermore, the function of Celsr3 in the peripheral nervous system is still largely unexplored. Here we show that Celsr3 mediates pathfinding of motor axons innervating the hindlimb. In mice, Celsr3-deficient axons of the peroneal nerve segregate from those of the tibial nerve but fail to extend dorsally, and they stall near the branch point. Mutant axons respond to repulsive ephrinA-EphA forward signaling and glial cell-derived neurotrophic factor (GDNF). However, they are insensitive to attractive EphA-ephrinA reverse signaling. In transfected cells, Celsr3 immunoprecipitates with ephrinA2, ephrinA5, Ret, GDNF family receptor α1 (GFRα1) and Frizzled3 (Fzd3). The function of Celsr3 is Fzd3 dependent but Vangl2 independent. Our results provide evidence that the Celsr3-Fzd3 pathway interacts with EphA-ephrinA reverse signaling to guide motor axons in the hindlimb.

Prenatal ultrasound diagnosis of a 48,XXYY syndrome.

The 48,XXYY syndrome is a rare uncommon gonosome aneuploidy and its incidence is estimated to be 1:18,000-1:40,000. The phenotype associated with this syndrome, classically described as Klinefelter variant, is extremely variable but developmental abnormalities are always present. Ultrasound signs during pregnancy are inconsistent, and only three prenatal cases have been described in the literature. Here, we report a case of 48,XXYY syndrome identified in prenatal period because of the presence of polyhydramnios and bilateral clubfeet on second trimester ultrasound. This observation shows the importance of chromosomal prenatal diagnosis in cases with bilateral clubfeet on morphologic ultrasound. This diagnosis is essential for further characterization of the prenatal phenotype and to improving genetic counselling.

Copy number analysis of 413 isolated talipes equinovarus patients suggests role for transcriptional regulators of early limb development.

Talipes equinovarus is one of the most common congenital musculoskeletal anomalies and has a worldwide incidence of 1 in 1000 births. A genetic predisposition to talipes equinovarus is evidenced by the high concordance rate in twin studies and the increased risk to first-degree relatives. Despite the frequency of isolated talipes equinovarus and the strong evidence of a genetic basis for the disorder, few causative genes have been identified. To identify rare and/or recurrent copy number variants, we performed a genome-wide screen for deletions and duplications in 413 isolated talipes equinovarus patients using the Affymetrix 6.0 array. Segregation analysis within families and gene expression in mouse E12.5 limb buds were used to determine the significance of copy number variants. We identified 74 rare, gene-containing copy number variants that were present in talipes equinovarus probands and not present in 759 controls or in the Database of Genomic Variants. The overall frequency of copy number variants was similar between talipes equinovarus patients compared with controls. Twelve rare copy number variants segregate with talipes equinovarus in multiplex pedigrees, and contain the developmentally expressed transcription factors and transcriptional regulators PITX1, TBX4, HOXC13, UTX, CHD (chromodomain protein)1, and RIPPLY2. Although our results do not support a major role for recurrent copy number variations in the etiology of isolated talipes equinovarus, they do suggest a role for genes involved in early embryonic patterning in some families that can now be tested with large-scale sequencing methods.

Micro-magnetic resonance imaging and embryological analysis of wild-type and pma mutant mice with clubfoot.

Gross similarities between the external appearance of the hind limbs of the peroneal muscle atrophy (pma) mouse mutant and congenital talipes equinovarus (CTEV), a human disorder historically referred to as 'clubfoot', suggested that this mutant could be a useful model. We used micro-magnetic resonance imaging to visualize the detailed anatomy of the hind limb defect in mutant pma mice and performed 3D comparisons between mutant and wild-type hind limbs. We found that the pma foot demonstrates supination (i.e. adduction and inversion of the mid foot and fore foot together with plantar flexion of the ankle and toes) and that the tibiale and distal tarsals display 3D abnormalities in positioning. The size and shape of the tibia, fibula, tarsal and metatarsal bones are similar to the wild-type. Hypoplasia of the muscles in the antero-lateral (peroneal) compartment was also demonstrated. The resemblance of these features to those seen in CTEV suggests that the pma mouse is a possibly useful model for the human condition. To understand how the observed deformities in the pma mouse hind foot arise during embryonic development, we followed the process of foot rotation in both wild-type and pma mutant mice. Rotation of the hind foot in mouse embryos of wild-type strains (CD-1 and C57/Black) occurs from embryonic day 14.5 onwards with rotation in C57/Black taking longer. In embryos from both strains, rotation of the right hind foot more commonly precedes rotation of the left. In pma mutants, the initiation of rotation is often delayed and rotation is slower and does not reach completion. If the usefulness of the pma mutant as a model is confirmed, then these findings on pma mouse embryos, when extrapolated to humans, would support a long-standing hypothesis that CTEV is due to the failure of completion of the normal process of rotation and angulation, historically known as the 'arrested development hypothesis'.

Quantitative evaluation in vivo of the degree of differentiation of hindlimb cartilage in a rat clubfoot model.

The Limb Morphogenetic Differentiation Scoring system introduced by Neubert and Barrach in 1977 has been used in drug testing as a measure of the degree of cartilage growth inhibition especially for forelimb in vitro. There is no scoring system to quantify the degree of hindlimb bud cartilage differentiation in vivo. A total of 60 female Sprague-Dawley rats weighing 220-250 g were assigned at random to six control groups and six experimental groups on day 0 of pregnancy. The experimental groups were treated with all-trans-retinoic acid (ATRA). A new limb morphogenetic differentiation scoring system was developed and used to quantify the degree of development of the hindlimb buds from the fetuses at embryonic days E13 to E18. The differentiation of cartilages assessed by the new scoring system showed a statistically significant difference between the experimental group and the control group from E13 to E18 (T-test, p < 0.05). Cartilage growth (the proximodistal length) in the control group increased gradually from E14, reaching its peak at E17, but in the experimental group the growth at E13, E16, E17, and E18 was significantly shorter (p < 0.05). In conclusion, the new limb morphogenetic differentiation scoring system described here can be used to quantify the degree of inhibition of the hindlimb bud development by teratogenic drugs or materials, and morphogenetic differentiation in vivo.

Mechanical properties of human fetal talus.

Mechanical characterization of human cartilage anlagen is required to effectively model congenital musculoskeletal deformities. Such modeling can effectively explore the effect of treatment procedures and potentially suggest enhanced treatment methods. Using serial MRI, we have noted shape changes of the cartilaginous hindfoot anlagen in patients with clubfoot, suggesting they are soft and deformable. We therefore determined the stress relaxation behavior of cartilage plugs obtained from third-trimester stillborn fetuses in unconfined and confined compression geometries. The material parameters determined were the aggregate modulus H(A) = 0.15 +/- 0.07 MPa, Poisson's ratio nu = 0.4 +/- 0.06, Young's modulus E(s) = 0.06 +/- 0.03 MPa, and permeability coefficients k(0) = 2.01 +/- 0.8 x 10(-14) m(4) N(-1) s(-1) and M = 4.6 +/- 1.0. As compared with adult articular cartilage, stiffness was an order of magnitude lower than the values reported in the literature, suggesting the relative softness of the tissue, and the permeability was an order of magnitude higher, indicating relative ease of flow in the tissue. Poisson's ratio also was close to the higher end of the range reported in previous studies. Such material is expected to deform and relax to larger extents. These findings are consistent with the deformability of the cartilage anlagen during manipulation and casting for treatment of clubfoot.

[The functions of GLI3 and EN1 genes in idiopathic congenital talipes equinovarus].

To investigate the role of gene Gli3 in idiopathic congenital talipes equinovarus (ICTEV), we constructed the Gli3 luciferase reporter gene expression vectors to analyze the promoter activity of the rat gene Gli3. The regulatory element in the promoter region of the rat Gli3 was predicted using P-Match software and further verified by ChIP experiment. Meanwhile, the correlation between the rat En1 and ICTEV was evaluated by RT-PCR, immunohistochemistry, and Western blotting analyses. The result from P-Match software prediction showed that only one of the three possible En1 binding sites in Gli3 promoter region was interacted directly with En1 in vivo, which was confirmed by ChIP analysis. The results from RT-PCR, immunohistochemistry and Western blotting analyses suggested that En1 was down-regulated in ICTEV model rats compared to the controls. Our results indicated that En1 might be the negative regulatory element in the upstream of Gli3. The low expression level of EN1 in ICTEV could contribute to the up-regulation of GLI3, which led to the genesis of ICTEV.

A model for clubfoot based on micro-CT data.

The pathological anatomy of idiopathic clubfoot has been investigated for more than 180 years using anatomy, computed tomography (CT), histology and microscopy. Seven idiopathic clubfeet and two normal feet of aborted fetuses were dissected in the present study, with special emphasis on the shape of the cartilage and bones. A three-dimensional (3D) micro-CT system, which generates a series of X-ray attenuation measurements, was used to produce computed reconstructed 3D data sets of each of the separated bones. Based on the micro-CT data scans a high-definition 3D colour printing system was used to make a four times enlarged clubfoot model, precisely presenting all the bony malformations. This model reflects the complexity of the anatomy of this disease and is designed to be used in the workshops of orthopaedic surgeons and physiotherapists, for training in new surgical and manipulation techniques.

Additional muscle in idiopathic club foot.

During the dissection of seven club feet of foetuses, aborted between the 25th and 37th week of gestation, an additional muscle bundle was found in a right foot. This muscle arose from both heads of the gastrocnemius muscle, continued downwards onto the posterior surface of the soleus, crossed the calcaneal tendon and assumed a position on the lateral side of the calcaneal tendon. The muscle's tendon was fixed just laterally to the calcaneal tendon in the calcaneal tuberosity with some tiny fibres branching off into the superior fibular retinaculum.

Clinical, cytogenetic and molecular investigation in a fetus with Wolf-Hirschhorn syndrome with paternally derived 4p deletion. Case report and review of the literature.

Wolf-Hirschhorn (4p-) syndrome (WHS), caused by partial deletion of the short arm of chromosome 4, has been extensively described in children and young adults. Knowledge on fetuses with WHS is still limited due to the small number of published cases. We report on a fetus with prenatally diagnosed severe intrauterine growth retardation, reduced thoracal diameter, clubfeet deformity and midface hypoplasia including slight microretrognathia indicative for fetal karyotyping. Chromosome analysis after amniocentesis revealed a de novo terminal deletion of chromosome 4p [karyotype: 46,XX,del(4) (p16)] which was confirmed by FISH. Analyses of a set of polymorphic markers mapping in 4pter->4p15.3 showed absence of paternal haplotypes. These observations corroborate the preferential paternal origin of the de novo 4p deletion in WHS patients. Furthermore, the distal breakpoint could be narrowed to band 4p16.1. At autopsy, the fetus showed typical craniofacial dysmorphic signs of WHS, severe IUGR and delayed bone age. This report suggests the possibility of recognising the particular phenotype of WHS in utero by prenatal ultrasound and emphasises the importance of karyotyping fetuses with severe IUGR, especially when the amount of amniotic fluid is normal.

Congenital talipes equinovarus (clubfoot): a disorder of the foot but not the hand.

Idiopathic (non-syndromic) congenital talipes equinovarus, or clubfoot, is a poorly understood but common developmental disorder of the lower limb, which affects at least 2 per 1000 Scottish births (ISD data). It is defined as a fixation of the foot in a hand-like orientation--in adduction, supination and varus--with concomitant soft tissue abnormalities. Despite advances in treatment, disability often persists. The aetiology of the condition has been little studied and is poorly understood. Neurological, muscular, bony, connective tissue and vascular mechanisms have been proposed, but the only firm evidence is that the mildest cases appear to be associated with intra-uterine posture. There is evidence for a genetic contribution to congenital talipes equinovarus aetiology. Its incidence varies with ethnic group, and we found that a family history is present in 24-50% of cases, depending on the population studied. Complex segregation analysis suggests that the most likely inheritance pattern is a single gene of major effect operating against a polygenic background. Possible mechanisms for congenital talipes equinovarus are discussed.