Biomedical subjects
S Mundlos
Publications and source records attributed to S Mundlos.
Polytopic anomalies with agenesis of the lower vertebral column.
We describe clinical, pathological and radiological findings in 15 cases of sporadic and familial lower spine agenesis with additional anomalies of the axial skeleton and internal organs and speculate about the cause and pathogenesis of this malformation complex. We show that all of these findings are defects of blastogenesis, originate in the primary developmental field and/or the progenitor fields, thus representing polytopic field defects. This concept appears applicable in our cases and makes such terms such as "caudal regression syndrome" or "axial mesodermal dysplasia spectrum" redundant.
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.
Mutation analysis of core binding factor A1 in patients with cleidocranial dysplasia.
Cleidocranial dysplasia (CCD) is a dominantly inherited disorder characterized by patent fontanelles, wide cranial sutures, hypoplasia of clavicles, short stature, supernumerary teeth, and other skeletal anomalies. We recently demonstrated that mutations in the transcription factor CBFA1, on chromosome 6p21, are associated with CCD. We have now analyzed the CBFA1 gene in 42 unrelated patients with CCD. In 18 patients, mutations were detected in the coding region of the CBFA1 gene, including 8 frameshift, 2 nonsense, and 9 missense mutations, as well as 2 novel polymorphisms. A cluster of missense mutations at arginine 225 (R225) identifies this residue as crucial for CBFA1 function. In vitro green fluorescent protein fusion studies show that R225 mutations interfere with nuclear accumulation of CBFA1 protein. There is no phenotypic difference between patients with deletions or frameshifts and those with other intragenic mutations, suggesting that CCD is generally caused by haploinsufficiency. However, we were able to extend the CCD phenotypic spectrum. A missense mutation identified in one family with supernumerary teeth and a radiologically normal skeleton indicates that mutations in CBFA1 can be associated exclusively with a dental phenotype. In addition, one patient with severe CCD and a frameshift mutation in codon 402 had osteoporosis leading to recurrent bone fractures and scoliosis, providing first evidence that CBFA1 may help maintain adult bone, in addition to its function in bone development.
Cleidocranial dysplasia: clinical and molecular genetics.
Cleidocranial dysplasia (CCD) (MIM 119600) is an autosomal dominant skeletal dysplasia characterised by abnormal clavicles, patent sutures and fontanelles, supernumerary teeth, short stature, and a variety of other skeletal changes. The disease gene has been mapped to chromosome 6p21 within a region containing CBFA1, a member of the runt family of transcription factors. Mutations in the CBFA1 gene that presumably lead to synthesis of an inactive gene product were identified in patients with CCD. The function of CBFA1 during skeletal development was further elucidated by the generation of mutated mice in which the Cbfa1 gene locus was targeted. Loss of one Cbfa1 allele (+/-) leads to a phenotype very similar to human CCD, featuring hypoplasia of the clavicles and patent fontanelles. Loss of both alleles (-/-) leads to a complete absence of bone owing to a lack of osteoblast differentiation. These studies show that haploinsufficiency of CBFA1 causes the CCD phenotype. CBFA1 controls differentiation of precursor cells into osteoblasts and is thus essential for membranous as well as endochondral bone formation.
Characterization of a human homologue of the Saccharomyces cerevisiae transcription factor spt3 (SUPT3H).
Spt3 is a Saccharomyces cerevisiae transcription factor that is required in vivo for the transcription of a number of RNA polymerase II-transcribed genes. We report the cloning of the gene encoding the human homologue of Spt3, SUPT3H, and its initial functional analysis. The human and yeast Spt3 homologues share an overall identity of 30% that defines three conserved regions, suggesting possible functional domains. To determine whether SUPT3H is a true functional Spt3 homologue, we tested for complementation of an spt3Delta mutation in yeast. While expression of the full-length SUPT3H is unable to complement an spt3Delta mutation, expression of a human-yeast chimeric gene that contains 42% human sequences can partially complement an spt3Delta mutation. These data suggest that the transcriptional control carried out by Spt3 has been conserved from yeast to human.
Acromesomelic dysplasia Maroteaux type maps to human chromosome 9.
Acromesomelic dysplasias are skeletal disorders that disproportionately affect the middle and distal segments of the appendicular skeleton. We report genetic mapping studies in four families with acromesomelic dysplasia Maroteaux type (AMDM), an autosomal recessive osteochondrodysplasia. A peak LOD score of 5.1 at recombination fraction 0 was obtained with fully informative markers on human chromosome 9. In three of the four families, the affected offspring are products of consanguineous marriages; if it is assumed that these affected offspring are homozygous by descent for the region containing the AMDM locus, a 6.9-cM AMDM candidate interval can be defined by markers D9S1853 and D9S1874. The mapping of the AMDM locus to human chromosome 9 indicates that AMDM is genetically distinct from the two other mapped acromesomelic dysplasias, Hunter-Thompson type and Grebe type, which are caused by mutations in CDMP1 on human chromosome 20.
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.
Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development.
We have generated Cbfa1-deficient mice. Homozygous mutants die of respiratory failure shortly after birth. Analysis of their skeletons revealed an absence of osteoblasts and bone. Heterozygous mice showed specific skeletal abnormalities that are characteristic of the human heritable skeletal disorder, cleidocranial dysplasia (CCD). These defects are also observed in a mouse Ccd mutant for this disease. The Cbfa1 gene was shown to be deleted in the Ccd mutation. Analysis of embryonic Cbfa1 expression using a lacZ reporter gene revealed strong expression at sites of bone formation prior to the earliest stages of ossification. Thus, the Cbfa1 gene is essential for osteoblast differentiation and bone formation, and the Cbfa1 heterozygous mouse is a paradigm for a human skeletal disorder.
Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia.
Cleidocranial dysplasia (CCD) is an autosomal-dominant condition characterized by hypoplasia/aplasia of clavicles, patent fontanelles, supernumerary teeth, short stature, and other changes in skeletal patterning and growth. In some families, the phenotype segregates with deletions resulting in heterozygous loss of CBFA1, a member of the runt family of transcription factors. In other families, insertion, deletion, and missense mutations lead to translational stop codons in the DNA binding domain or in the C-terminal transactivating region. In-frame expansion of a polyalanine stretch segregates in an affected family with brachydactyly and minor clinical findings of CCD. We conclude that CBFA1 mutations cause CCD and that heterozygous loss of function is sufficient to produce the disorder.
Mouse clavicular development: analysis of wild-type and cleidocranial dysplasia mutant mice.
Cleidocranial dysplasia (CCD) is an autosomal dominant disease characterized by hypoplasia or aplasia of clavicles, open fontanelles, and other skeletal anomalies. A mouse mutant, shown by clinical and radiographic analysis to be strikingly similar to the human disorder and designated Ccd, was used as a model for the human disorder. Since malformation of the clavicle is the hallmark of CCD, we studied clavicular development in wild-type and Ccd mice. Histology and in situ hybridization experiments were performed to compare the temporal and spatial expression of several genes in wild-type and Ccd mutant mouse embryos. Bone and cartilage specific markers--type I, II, and X collagens, Sox9, aggrecan, and osteopontin were used as probes. The analyses covered the development of the clavicle from the initial mesenchymal condensation at embryonic day 13 (E13) to the late mineralization stage at embryonic day 15.5. At day 13.5, cells in the center of the condensation differentiate into characteristic precursor cells that were not observed in other bone anlagen. In the medial part of the anlage these cells express markers of the early cartilage lineage (type II collagen and Sox9), whereas cells of the lateral part express markers of the osteoblast lineage (type I collagen). With further development the medial cells differentiate into chondrocytes and start to express chondrocyte-specific markers such as aggrecan. Cells of the lateral part differentiate into osteoblasts as indicated by the production of bone matrix and the expression of osteopontin. At day 14.5 a regular growth plate has developed between the two parts where type X collagen expression can be demonstrated in hypertrophic chondrocytes. The data indicate that the medial part of the clavicle develops by endochondral bone formation while the lateral part ossifies as a membranous bone. The clavicle of Ccd mice showed a smaller band of mesenchymal cell condensation than in wild-type mice. Cells of the condensation failed to express type I and type II collagen at E13.5. In the lateral part of the clavicle type I collagen expression was not detected until E14.5 and osteopontin expression only appeared at E15.5. At E15.5, a small ossification center appears in the lateral part which is, in contrast to the wild-type clavicular bone, solid and without primary spongiosa as well as bone marrow. In the medial portion, type II collagen expression and endochondral ossification never occurs in Ccd mice; this portion of the clavicle is therefore missing in Ccd.
The role of sonic hedgehog in vertebrate development.
Members of the hedgehog family are important signalling molecules during embryonic development. One member, Sonic hedgehog, is expressed in embryonic structures such as the zone of polarizing activity in the posterior limb bud, the notochord, and the floor plate of the neural tube, where it plays a role in patterning of the embryo. Sonic hedgehog is synthesized as an inactive precursor which must be proteolytically cleaved and modified by the addition of a cholesterol moiety to become active as a signalling molecule. In this processing, the C-terminal region of Sonic hedgehog serves as both the endoprotease and a cholesterol transferase. The importance of cholesterol for Sonic hedgehog function may explain many of the profound developmental defects caused by perturbations of cholesterol metabolism. The receptor for Sonic hedgehog is Patched, a multi-pass transmembrane protein which forms a complex with Smoothened Mutations in Patched are associated with basal cell naevus syndrome, while mutations in Sonic hedgehog cause holoprosencephaly. Downstream targets of Sonic hedgehog signalling are transcription factors like Gli3, responsible for Greigs polycephalosyndactyly in humans and Hoxd13, responsible for polysyndactyly.
Heritable diseases of the skeleton. Part I: Molecular insights into skeletal development-transcription factors and signaling pathways.
The recent identification of the genetic basis of hereditary skeletal disorders is providing important insights into the intricate processes of skeletal formation, growth, and homeostasis. These processes include patterning events during condensation and differentiation of mesenchymal cells to form cartilage precursors of the future bones, the replacement of cartilage by bones through endochondral ossification, the growth of long bones through proliferation and differentiation of chondrocytes in growth plates, and bone formation through differentiation of osteoblasts from mesenchymal cells in areas of intramembranous ossification. Defects in any of these processes can give rise to skeletal abnormalities. Mutations in transcription factors such as HOX and PAX and members of the transforming growth factor-beta superfamily cause disorders associated with abnormal mesenchymal condensation, whereas defects in the transcription factor SOX-9 lead to abnormalities in chondrocyte differentiation. Abnormal growth plate function, resulting in dwarfism, is the consequence of mutations in receptors for fibroblast growth factors and parathyroid hormone-related peptide. Premature closure of cranial sutures in intramembranous ossification is a feature of syndromes due to mutations in fibroblast growth factor receptors.
Heritable diseases of the skeleton. Part II: Molecular insights into skeletal development-matrix components and their homeostasis.
A range of osteochondrodysplasias is caused by mutations in components of the extracellular matrix in cartilage and bone and in molecules that are important for posttranslational processing of such components. Mutations in the genes encoding the two polypeptide subunits of collagen I cause defects in the structure of bone matrix while mutations in genes encoding cartilage-specific collagens are responsible for several chondrodysplasias. Abnormalities in cartilage structure and function can also be due to mutations in structural noncollagenous components such as aggrecan and cartilage oligomeric matrix protein. Finally, several cartilage and bone disorders are due to abnormalities in sulfate transport and regulation of bone matrix homeostasis.
Multiexon deletions in the type I collagen COL1A2 gene in osteogenesis imperfecta type IB. Molecules containing the shortened alpha2(I) chains show differential incorporation into the bone and skin extracellular matrix.
Osteogenesis imperfecta (OI) type IB is a rare subset of the mildest form of OI, clinically characterized by moderate bone fragility, blue sclera, and dentinogenesis imperfecta. Cultured skin fibroblasts from two unrelated individuals (OI-197 and OI-165) with the typical features of OI type IB produced shortened alpha2(I) chains. Reverse transcription-polymerase chain reaction of the alpha2(I)-cDNA revealed deletions in the triple helical domain of 5 exons (exons 7-11) in OI-197, and 8 exons (exons 10-17) in OI-165. This exon skipping was caused by genomic deletions in one allele of COL1A2 with the breakpoints located in introns 6 and 11 in OI-197, and introns 9 and 17 in OI-165. The secretion and deposition of the mutant collagen into the matrix was measured in vitro in cultures of skin fibroblasts and bone osteoblasts, grown in the presence of ascorbic acid to induce collagen matrix formation and maturation, as well as in collagen extracts from skin and bone. The secretion of mutant collagen was impaired and long term cultures of fibroblasts showed that the mutant collagen was not incorporated into the mature collagenous matrix produced in vitro by skin fibroblasts from both patients. Likewise, the shortened alpha2(I) chain was not demonstrable in skin extracts. In contrast, bone extracts from OI-197 showed the presence of the mutant collagen. This incorporation of the abnormal collagen into the mature matrix was also demonstrated in long term cultures of the patient's osteoblasts. The deposition of the mutant collagen by bone osteoblasts but not by skin fibroblasts demonstrates a tissue specificity in the incorporation of mutant collagen into the matrix which may explain the primary involvement of bone and not skin in these patients.
Cleidocranial dysplasia in mice.
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An alpha 1(II) Gly913 to Cys substitution prevents the matrix incorporation of type II collagen which is replaced with type I and III collagens in cartilage from a patient with hypochondrogenesis.
A heterozygous mutation in the COL2A1 gene was identified in a patient with hypochondrogenesis. The mutation was a single nucleotide transition of G3285T that resulted in an amino acid substitution of Cys for Gly913 in the alpha 1(II) chain of type II collagen. This amino acid change disrupted the obligatory Gly-X-Y triplet motif required for the normal formation of a stable collagen triple helix and prevented the deposition of type II collagen into the proposita's cartilage, which contained predominantly type I and III collagens and minor amounts of type XI collagen. Biosynthetic analysis of collagens produced and secreted by the patient's chondrocytes cultured in alginate beads was consistent with the in vivo matrix composition, demonstrating that the main products were type I and III collagens, along with type XI collagen. The synthesis of the cartilage-specific type XI collagen at similar levels to controls indicated that the isolated cartilage cells had re-differentiated to the chondrocyte phenotype. The chondrocytes also produced small amounts of type II collagen, but this was post-translationally overmodified and not secreted. These data further delineate the biochemical and phenotypic consequences of mutations in the COL2A1 gene and suggest that cartilage formation and bone development can take place in the absence of type II collagen.
An unknown spondylo-meta-epiphyseal dysplasia in sibs with extreme short stature.
In three sibs of Jordanian descent a unique type of severe spondylo-meta-epiphyseal dysplasia results in extreme disproportionate dwarfism. They have a distinct facial appearance with hypotelorism, prognathia, and hypodontia. The limbs are short and the hands and feet stubby. Radiologically, the irregular end plates of the vertebral bodies, the very small and late appearing epiphyseal ossification centres, and the hypoplastic acetabular roofs are most impressive. Histopathologic studies of the growth plate demonstrate characteristic findings with fingerprint-like inclusion bodies in the hypertrophic chondrocytes. This seems to be a distinct, autosomal recessive skeletal dysplasia.