Ligase IV syndrome can present with microcephaly and radial ray anomalies similar to Fanconi anaemia plus fatal kidney malformations.

Ligase IV (LIG4) syndrome is a rare disorder of DNA damage repair caused by biallelic, pathogenic variants in LIG4. This is a phenotypically heterogeneous condition with clinical presentation varying from lymphoreticular malignancies in developmentally normal individuals to significant microcephaly, primordial dwarfism, radiation hypersensitivity, severe combined immunodeficiency and early mortality. Renal defects have only rarely been described as part of the ligase IV disease spectrum. We identified a consanguineous family where three siblings presenting with antenatal growth retardation, microcephaly, severe renal anomalies and skeletal abnormalities, including radial ray defects. Autozygosity mapping and exome sequencing identified a novel homozygous frameshift variant in LIG4, c.597_600delTCAG, p.(Gln200LysfsTer33), which segregated in the family. LIG4 is encoded by a single exon and so this frameshift variant is predicted to result in a protein truncated by 678 amino acids. This is the shortest predicted LIG4 protein product reported and correlates with the most severe clinical phenotype described to date. We note the clinical overlap with Fanconi anemia and suggest that LIG4 syndrome is considered in the differential diagnosis of this severe developmental disorder.

Upper limb phocomelia: A prenatal case of thrombocytopenia-absent radius (TAR) syndrome illustrating the importance of chromosomal microarray in limb reduction defects.

OBJECTIVE: To describe the ultrasonographic, pathologic and molecular findings in a fetus with TAR syndrome, and to illustrate the contribution of chromosomal microarray analysis (CMA) to the etiological investigation of fetal upper limb reduction defects. CASE REPORT: A 35-year-old woman was referred for Genetic Counseling after pregnancy termination for severe upper limb bilateral phocomelia detected in the second trimester. Fetal autopsy showed severe shortening of the arms and forearms. The fetal skeletal survey confirmed the absence of the radii, ulnae and humeri. CMA revealed an interstitial deletion in 1q21 including the RBM8A gene. Subsequent Sanger sequencing of this gene identified a hypomorphic mutant allele, c.-21G > A, confirming the diagnosis of TAR syndrome. CONCLUSION: The differential diagnosis of upper limb defects is broad. Identification of their cause is essential for adequate genetic counseling including prognosis and recurrence risk estimation. CMA should be considered in fetuses with upper limb reduction defects, especially when the thumbs are present.

Quantitative anatomy of the primary ossification center of the radial shaft in human fetuses.

PURPOSE: The medical literature still lacks studies on the size of the radial shaft primary ossification center, thus preventing us from potentially relevant data in diagnosing skeletal dysplasias, i.e., TAR syndrome, VATER syndrome, Holt-Oram syndrome, Fanconi anemia and Edwards syndrome, frequently characterized by disrupted or retarded fetal growth. MATERIALS AND METHODS: The size of the radial shaft primary ossification center in 47 (25 males and 22 females) spontaneously aborted human fetuses aged 17-30 weeks was studied by means of CT, digital image analysis and statistics. RESULTS: With neither sex nor laterality differences, the best-fit growth dynamics for the radial shaft primary ossification center was modeled by the following functions: y = - 10.988 + 1.565 × age ± 0.018 for its length, y = - 2.969 + 0.266 × age ± 0.01 for its proximal transverse diameter, y = - 0.702 + 0.109 × age ± 0.018 for its middle transverse diameter, y = - 2.358 + 0.203 × age ± 0.018 for its distal transverse diameter, y = -189.992 + 11.788 × (age)2 ± 0.018 for its projection surface area, and y = - 798.174 + 51.152 × age ± 0.018 for its volume. CONCLUSIONS: The morphometric characteristics of the radial shaft primary ossification center show neither sex nor bilateral differences. The radial shaft primary ossification center grows proportionately in length, transverse dimensions and volume, and quadratically in its projection surface area. The obtained numerical findings of the radial shaft ossification center are considered age-specific reference of relevance in both the estimation of fetal ages and the diagnostic process of congenital defects.

Prenatal diagnosis of radial ray defects by ultrasound: A report of 6 cases.

OBJECTIVE: All of the medical records of fetuses with the sonographic finding of radial ray defects (RRDs) between 2008 and 2015 were retrieved. The associated sonographic findings, cytogenetic results, and necropsy findings were correlated. CASE REPORT: There were 6 cases of RRD. Three cases were bilateral and the other 3 cases were unilateral. The gestational ages at diagnosis were between 12 and 24 weeks gestation. All women carrying fetuses with RRDs opted to terminate the pregnancy. There were 2 cases of trisomy 18, one case of thrombocytopenia-absent radius syndrome, and 2 cases of isolated RRD. Both cases of trisomy 18 had other sonographic abnormalities. CONCLUSION: RRD should be considered if a short radius and abnormal angulation of the wrist or thumb is noted. The use of 3-D ultrasound facilitates the diagnosis of RRD, even at early gestation, by providing a better surface appearance, panoramic views, and spatial orientation.

Cross-sectional study of fetal long-bone length in an Iranian population at 17-25 weeks of gestation.

OBJECTIVE: To construct improved reference charts for fetal long bones in an Iranian setting and to compare them with previous studies. METHODS: The present prospective cross-sectional study included singleton fetuses assessed by ultrasonography at 17-25 weeks of gestation at the Comprehensive Medical Genetics Center, Shahid Soltani, Shiraz, Iran between May 1, 2012, and February 28, 2014. Exclusion criteria included conditions that could affect fetal growth. Fetal long bones (femur, humerus, tibia, fibula, ulna, and radius) were measured with ultrasonography and biometric charts were produced. Regression models were fitted to estimate bone lengths. The models produced were compared with those from previous studies in other populations. RESULTS: There were 660 singleton fetuses included and 660 femur, 633 humerus, 512 tibia, 498 fibula, 505 ulna, and 498 radius biometric measurements were recorded. The models generated to predict the length of the tibia, fibula, ulna, and radius from the length of the femur and humerus demonstrated a high goodness of fit when the predicted lengths were plotted against the actual lengths. Comparisons of mean lengths with previous studies suggested that long-bone length was affected by maternal ethnicity. CONCLUSION: The equations generated could be used to predict long-bone length in an Iranian population and ethnicity should be considered when using fetal long-bone length as a prenatal diagnostic tool.

Leptin administration affects growth and skeletal development in a rat intrauterine growth restriction model: preliminary study.

OBJECTIVE: Skeletal abnormalities are one of the hallmarks of growth delay during gestation. The aim of this study was to determine changes induced by leptin in skeletal growth and development in a rat model of intrauterine growth retardation (IUGR) and to elucidate the possible underlying mechanisms. METHODS: Intrauterine growth retardation was induced prepartum and the effects of leptin to mothers prenatally or to offspring postnatally were studied. Radii were harvested and tested mechanically and structurally. Tibias were evaluated for growth-plate morphometry. RESULTS: On day 40 postpartum, total bone length and mineral density and tibial growth-plate width and numbers of cells within its zones of offspring treated with leptin were significantly greater than in the control group. CONCLUSION: Postnatal leptin administration in an IUGR model improves the structural properties and elongation rate of bone. These findings could pave the way to preventing some phenotypic presentations of IUGR.

Are distributions of secondary osteon variants useful for interpreting load history in mammalian bones?

BACKGROUND/AIMS: In cortical bone, basic multicellular units (BMUs) produce secondary osteons that mediate adaptations, including variations in their population densities and cross-sectional areas. Additional important BMU-related adaptations might include atypical secondary osteon morphologies (zoned, connected, drifting, elongated, multiple canal). These variants often reflect osteonal branching that enhances toughness by increasing interfacial (cement line) complexity. If these characteristics correlate with strain mode/magnitude-related parameters of habitual loading, then BMUs might produce adaptive differences in unexpected ways. METHODS: We carried out examinations in bones loaded in habitual torsion (horse metacarpals) or bending: sheep, deer, elk, and horse calcanei, and horse radii. Atypical osteons were quantified in backscattered images from anterior, posterior, medial, and lateral cortices. Correlations were determined between atypical osteon densities, densities of all secondary osteons, and associations with habitual strain mode/magnitude or transcortical location. RESULTS: Osteon variants were not consistently associated with 'tension', 'compression', or neutral axis ('shear') regions, even when considering densities or all secondary osteons, or only osteon variants associated with relatively increased interfacial complexity. Similarly, marrow- and strain-magnitude-related associations were not consistent. CONCLUSION: These data do not support the hypothesis that spatial variations in these osteon variants are useful for inferring a habitual bending or torsional load strain history.

Teratogenic mechanisms of longitudinal deficiency and cleft hand.

In order to better understand the teratogenic mechanisms of congenital defects of the digits, we analyzed clinical cases and induced similar types of congenital hand anomalies in rat fetuses by oral administration of busulfan. In clinical cases, radial and ulnar deficiencies had common characteristic features. We induced radial and ulnar deficiencies in rat fetuses with the same drug. Radial and ulnar deficiencies induced in rats have similar clinical manifestations and these anomalies might be caused by the same teratogenic mechanism. Then, the formation of the digital rays was examined histologically. The results of histological examination suggested that these deficiencies were not caused by localized damage of the limb bud. They also suggested that the cause of missing digits in longitudinal deficiency is closely related to a deficit of mesenchymal cells in the limb bud. Cleft hand is considered to be one of the types of longitudinal deficiency. However, several investigators have suggested that the abnormal induction of finger rays in the process of formation of fingers induced central polydactyly, osseous syndactyly and also cleft hand. X-rays of the clinical cases and skeletal changes of the anomalies induced in rats appear to demonstrate that cleft hand formation proceeds from osseous syndactyly and central polydactyly. The results of our experimental study show that the critical periods of central polydactyly, osseous syndactyly and cleft hand are the same. They also suggest that central polydactyly, syndactyly and cleft hand might be induced when the same teratogenic factor acts on embryos at the same developmental stage in the human being. Because they have a similar causation, cleft hand, syndactyly and central polydactyly should be classified into the same entity, that is, abnormal induction of digital rays. Based on these clinical and experimental studies, we modified the Swanson classification. In our modified classification, typical cleft hand, syndactyly and polydactyly are included in the same category of abnormal induction of digital rays as the fourth new category.

Multiple roles of Hoxa11 and Hoxd11 in the formation of the mammalian forelimb zeugopod.

Mutations in the 5' or posterior murine Hox genes (paralogous groups 9-13) markedly affect the formation of the stylopod, zeugopod and autopod of both forelimbs and hindlimbs. Targeted disruption of Hoxa11 and Hoxd11 or Hoxa10, Hoxc10 and Hoxd10 result in gross mispatterning of the radius and ulna or the femur, respectively. Similarly, in mice with disruptions of both Hoxa13 and Hoxd13, development of the forelimb and hindlimb autopod is severely curtailed. Although these examples clearly illustrate the major roles played by the posterior Hox genes, little is known regarding the stage or stages at which Hox transcription factors intersect with the limb development program to ensure proper patterning of the principle elements of the limb. Moreover, the cellular and/or molecular bases for the developmental defects observed in these mutant mice have not been described. In this study, we show that malformation of the forelimb zeugopod in Hoxa11/Hoxd11 double mutants is a consequence of interruption at multiple steps during the formation of the radius and ulna. In particular, reductions in the levels of Fgf8 and Fgf10 expression may be related to the observed delay in forelimb bud outgrowth that, in turn, leads to the formation of smaller mesenchymal condensations. However, the most significant defect appears to be the failure to form normal growth plates at the proximal and distal ends of the zeugopod bones. As a consequence, growth and maturation of these bones is highly disorganized, resulting in the creation of amorphous bony elements, rather than a normal radius and ulna.

Which morphologies of synovial folds result from degeneration and/or aging of the radiohumeral joint: an anatomic study with cadavers and embryos.

The synovial folds of the radiohumeral joints in cadaveric elbows from 179 elderly subjects and 40 embryos were investigated macroscopically and histologically to determine any morphologic changes caused by aging or degeneration. The anterior and posterior folds found in the elderly population shared characteristics of folds seen in embryos, with some modifications, and were thought to originate from the primitive septum. Proportionally, the length, width, and thickness of these folds were consistent between adults and embryos. However, the embryonic folds showed a homogenous morphology. In contrast, in the adult the anterior fold was characterized by a shorter and narrower villous pattern, and the posterior fold tended to be wider. Lateral extension of the anterior or posterior folds was also observed. Moreover, the lateral fold, never seen in embryos, was present and characterized by a hard plicate pattern in the adult. These derived or specific morphologies in adults probably result from alterations in the movement of the radial head caused by aging.

IL-1- and TNF-induced bone resorption is mediated by p38 mitogen activated protein kinase.

We have previously shown that p38 mitogen-activated protein kinase (MAPK) inhibitors, which block the production and action of inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), are effective in models of bone and cartilage degradation. To further investigate the role of p38 MAPK, we have studied its activation in osteoblasts and chondrocytes, following treatment with a panel of proinflammatory and osteotropic agents. In osteoblasts, significant activation of p38 MAPK was observed following treatment with IL-1 and TNF, but not parathyroid hormone, transforming growth factor-beta (TGF-beta), 1,25(OH)(2)D(3), insulin-like growth factor-1 (IGF-1), or IGF-II. Similar results were obtained using primary bovine chondrocytes and an SV40-immortalized human chondrocyte cell line, T/C28A4. SB 203580, a selective inhibitor of p38 MAPK, inhibited IL-1 and TNF-induced p38 MAPK activity and IL-6 production (IC(50)s 0.3--0.5 microM) in osteoblasts and chondrocytes. In addition, IL-1 and TNF also activated p38 MAPK in fetal rat long bones and p38 MAPK inhibitors inhibited IL-1- and TNF-stimulated bone resorption in vitro in a dose-dependent manner (IC(50)s 0.3--1 microM). These data support the contention that p38 MAPK plays a central role in regulating the production of, and responsiveness to, proinflammatory cytokines in bone and cartilage. Furthermore, the strong correlation between inhibition of kinase activity and IL-1 and TNF-stimulated biological responses indicates that selective inhibition of the p38 MAPK pathway may have therapeutic utility in joint diseases such as rheumatoid arthritis (RA).

Developmental morphological and histological studies on structures of the human fetal elbow joint.

In the present work, morphological changes in the developing human elbow joint were studied at different prenatal ages (8, 12, 16, 20, 29 and 40 weeks) and were compared with the same structures in the adult joint. The elbow joint had gone through its most important developmental changes during the 20th week of prenatal life, probably due to the direct dynamic effect of the newly developed fetal movement. During later prenatal development, the articular surfaces of the lower end of humerus and the upper ends of radius and ulna developed their characteristic congruencies, so that the highly curved convexities always articulate with the highly curved concavities. That process progressed postnatally and even till adult age. In full-term infants it was found that the lower end of humerus had acquired its adult shape, while the shape of the upper ends of radius and ulna were still not fully developed. They continued development in postnatal life even till adult age. In the present work, histological prenatal studies were done on longitudinal sections from the back of the capsule and synovial tissue, early (8 weeks) and late in full term, and the results were also compared with the same structures in adults. It was found that at all ages, the capsules were formed of cellular and fibrous elements, but at early prenatal age (8 weeks), this cellular condensation was more massive and prominent while in full-term infants, it became generally more fibrous, but was still different compared to adults. Basic cellular structures of the synovial tissue changed very little during the late prenatal developmental stage, as it did not become more fibrous than cellular during these periods, but differences in vascularity became more obvious. The cartilaginous content of the articular surface at 8 weeks was highly cellular with very little intercellular matrix. In contrast to that of full term, this cartilage became fully chondrogenous with a notable decrease in cellular density and massive increase in matrix content.

Development of the human elbow joint.

Many studies have been published on the development of the human elbow joint, but authors disagree on its morphogenetic timetable. Most discrepancies center on the cavitation of the elbow joint (including the humeroradial, humeroulnar, and superior radioulnar joints), and the organization of the tunnel of the ulnar nerve. We summarize our observations on the development of the elbow joint in 49 serially sectioned human embryonic (n = 28) and fetal (n = 21) upper limbs. During week 12, ossification begins in the epiphyses of the elements comprising the elbow joint. At the end of the embryonic period, the shallow groove between the posterior aspect of the medial epicondyle and the olecranon process, begins to be visible. The elbow joint cavity appears in O'Rahilly stage 21 (51 days) at the level of the humeroulnar and humeroradial interzones. Formation of the cavity begins at the medialmost portion of the humeroradial interzone and the lateralmost portion of the humeroulnar interzone. The annular ligament begins to develop in O'Rahilly stage 21 (51 days), and the superior radioulnar joint cavity appears between this ligament and the lateral aspect of the head of the radius during O'Rahilly stage 23 (56 days). We established the morphogenetic timetable of the human elbow joint.

1,25-dihydroxyvitamin D3 inhibits parathyroid hormone-related peptide mRNA expression in fetal rat long bones in culture.

When fetal rat long bones are incubated in the presence of 10(-8) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], steady-state parathyroid hormone-related peptide (PTHrP) mRNA levels are decreased. This decrease is temporary: it is observed as soon as after 3 h of exposure and reaches a nadir after 6 h. At that time, PTHrP mRNA levels are significantly lower in the experimental than in the control bones. However the inhibitory effect vanishes after 24 h, despite continuous exposure to 1,25(OH)2D3 for even 48 h. This is the first report showing that PTHrP mRNA expression can be regulated in rat fetal long bones in vitro by 1,25(OH)2D3.

Maturational disturbance of chondrocytes in Cbfa1-deficient mice.

Cbfa1, a transcription factor that belongs to the runt-domain gene family, plays an essential role in osteogenesis. Cbfa1-deficient mice completely lacked both intramembranous and endochondral ossification, owing to the maturational arrest of osteoblasts, indicating that Cbfa1 has a fundamental role in osteoblast differentiation. However, Cbfa1 was also expressed in chondrocytes, and its expression was increased according to the maturation of chondrocytes. Terminal hypertrophic chondrocytes expressed Cbfa1 extensively. The significant expression of Cbfa1 in hypertrophic chondrocytes was first detected at embryonic day 13.5 (E13.5), and its expression in hypertrophic chondrocytes was most prominent at E14.5-16.5. In Cbfa1-deficient mice, whose entire skeleton was composed of cartilage, the chondrocyte differentiation was disturbed. Calcification of cartilage occurred in the restricted parts of skeletons, including tibia, fibula, radius, and ulna. Type X collagen, BMP6, and Indian hedgehog were expressed in their hypertrophic chondrocytes. However, osteopontin, bone sialoprotein, and collagenase 3 were not expressed at all, indicating that they are directly regulated by Cbfa1 in the terminal hypertrophic chondrocytes. Chondrocyte differentiation was severely disturbed in the rest of the skeleton. The expression of PTH/PTHrP receptor, Indian hedgehog, type X collagen, and BMP6 was not detected in humerus and femur, indicating that chondrocyte differentiation was blocked before prehypertrophic chondrocytes. These findings demonstrate that Cbfa1 is an important factor for chondrocyte differentiation.

Regulation of chondrocyte differentiation by Cbfa1.

Cbfa1, a developmentally expressed transcription factor of the runt family, was recently shown to be essential for osteoblast differentiation. We have investigated the role of Cbfa1 in endochondral bone formation using Cbfa1-deficient mice. Histology and in situ hybridization with probes for indian hedgehog (Ihh), collagen type X and osteopontin performed at E13.5, E14.5 and E17.5 demonstrated a lack of hypertrophic chondrocytes in the anlagen of the humerus and the phalanges and a delayed onset of hypertrophy in radius/ulna in Cbfa1-/- mice. Detailed analysis of Cbfa1 expression using whole mount in situ hybridization and a lacZ reporter gene reveled strong expression not only in osteoblasts but also in pre-hypertrophic and hypertrophic chondrocytes. Our studies identify Cbfa1 as a major positive regulator of chondrocyte differentiation.

Ossification and growth rates of the limb long bones during the prehatching period in the quail (Coturnix coturnix japonica).

The timing of ossification and the growth of six long bones of the prehatching period in the quail was studied. Ninety-nine quail eggs were incubated and in total nine fetuses were selected daily from the sixth to the sixteenth day of incubation. The fetuses were stained with alizarine and alcian blue double colouration. The fetuses were studied under the stereoscopic microscope and linear measurements were obtained from the humerus, ulna, radius, femur, tibia and fibula. The first appearance of the primary ossification centres in the diaphysis of the studied bones was found to occur between the sixth and the seventh day of incubation. Different growth patterns between the bones of the leg and of the wing were observed. Humerus and tibia showed the greatest growth rate while the radius and fibula showed the lowest.

Control of the limb bud outgrowth in quail-chick chimera.

It is well known that the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) play a major role in growth and patterning of the limb. But a mechanism underlying species-specific growth of the limb has not yet been fully elucidated. To investigate the role of AER and ZPA in limb size control, we constructed quail-chick limb chimeras. When we grafted a whole forelimb bud from one species to another, the size of the developed grafted limb was comparable to the limb of the donor species. Moreover, we demonstrated that neither the interspecific substitution of the posterior half region of the limb bud containing the ZPA nor the exchange of the ectodermal component of the limb involving the AER could alter the species-specific size of the limb. These results indicate that the factors affecting the size of the limb are already involved in the mesodermal component of the limb bud at stage 20 of chick embryo. Thus, the mesoderm dictates limb specificity including size.

[Congenital thumb hypoplasia. Clinical study of twenty patients]. / L'hypoplasie congénitale du pouce. Etude clinique à propos de 20 patients.

Congenital hypoplasia of the thumb is often seen in association with longitudinal failure of formation of the radius. A clinical study of 20 children, with 26 cases of hypoplasia of the thumb was conducted to evaluate the surgical indications according to the stage of the malformation. The results were evaluated for each patient of the study, with the position of the thumb, its spontaneous movements and its function. In grade II hypoplasia, treatment consisted of reanimation of the hypoplastic thumb by one or more tendinon transfers. In grade IV and V, pollicisation is the treatment of choice. In our experience, the surgical indications raise a problem essentially in grade III hypoplasia of Blauth's classification. In these cases, pollicisation gives the best results. Reanimation of the thumb with tendinon transfers can sometimes be used when parents refuse amputation of the thumb, when the first metacarpal is not excessively hypoplastic, and when the child is old enough to allow early physiotherapy.