Detection of circulating tumour cells in peripheral blood with an automated scanning fluorescence microscope.

We have developed an automated, highly sensitive and specific method for identifying and enumerating circulating tumour cells (CTCs) in the blood. Blood samples from 10 prostate, 25 colorectal and 4 ovarian cancer patients were analysed. Eleven healthy donors and seven men with elevated serum prostate-specific antigen (PSA) levels but no evidence of malignancy served as controls. Spiking experiments with cancer cell lines were performed to estimate recovery yield. Isolation was performed either by density gradient centrifugation or by filtration, and the CTCs were labelled with monoclonal antibodies against cytokeratins 7/8 and either AUA1 (against EpCam) or anti-PSA. The slides were analysed with the Ikoniscope robotic fluorescence microscope imaging system. Spiking experiments showed that less than one epithelial cell per millilitre of blood could be detected, and that fluorescence in situ hybridisation (FISH) could identify chromosomal abnormalities in these cells. No positive cells were detected in the 11 healthy control samples. Circulating tumour cells were detected in 23 out of 25 colorectal, 10 out of 10 prostate and 4 out of 4 ovarian cancer patients. Five samples (three colorectal and two ovarian) were analysed by FISH for chromosomes 7 and 8 combined and all had significantly more than four dots per cell. We have demonstrated an Ikoniscope based relatively simple and rapid procedure for the clear-cut identification of CTCs. The method has considerable promise for screening, early detection of recurrence and evaluation of treatment response for a wide variety of carcinomas.

Enhanced intracellular availability and survival of hammerhead ribozymes increases target ablation in a cellular model of osteogenesis imperfecta.

Antisense hammerhead ribozymes have the capability to cleave complementary RNA in a sequence-dependent manner. In osteogenesis imperfecta, a genetic disorder of connective tissue, mutant collagen type I has been shown to participate in but not sustain formation of the triple helix. Selective ablation of mutant collagen gene transcript could potentially remove the mutant gene product and reverse the dominant-negative effect exerted by the abnormal protein. In earlier studies we showed that the hammerhead ribozyme Col1A1Rz547 selectively cleaved a mutant Col1A1 gene transcript in a murine calvarial osteoblast cell line. In order to test the possible therapeutic efficacy of this approach, a dramatic downregulation of the mutant transcript must be achieved, a function directly related to high steady-state level of intracellular ribozyme. We report significantly enhanced expression of Col1A1Rz547 by vaccinia T7 polymerase following infection with an attenuated T7-pol vaccinia virus as shown both by the intracellular level of the ribozyme and the cleavage of the mutant Col1A1 gene transcript. We also describe the engineering of a multimeric ribozyme construct comprising eight subunits, which can self-cleave to monomers. These studies suggest the potential use of multimeric ribozymes expressed by a vaccinia-based system in the therapy of a variety of disorders.

Split hand foot malformation is associated with a reduced level of Dactylin gene expression.

Split hand foot malformation (SHFM) is a congenital limb malformation presenting with a median cleft of the hand and/or foot, syndactyly and polydactyly. SHFM is genetically heterogeneous with four loci mapped to date. Murine Dactylaplasia (Dac) is phenotypically similar, and it has been mapped to a syntenic region of 10q24, where SHFM3 has been localized. Structural alterations of the gene-encoding dactylin, a constituent of the ubiquitinization pathway, leading to reduced levels of transcript have been identified in Dac. Here, we report a significant decrease of Dactylin transcript in several individuals affected by SHFM. This observation supports a central role for dactylin in the pathogenesis of SHFM.

Distal limb malformations: underlying mechanisms and clinical associations.

Congenital malformations of the extremities are conspicuous and have been described through the ages. Over the past decade, a wealth of knowledge has been generated regarding the genetic regulation of limb development and the underlying molecular mechanisms. Recent studies have identified several of the signaling molecules, growth factors, and transcriptional regulators involved in the initiation and maintenance of the apical ectodermal ridge (AER) as well as the molecular markers defining the three axes of the developing limb. Studies of abnormal murine phenotypes have uncovered the role played by genes such as p63 and Dactylin in the maintenance of AER activity. These phenotypes resemble human malformations and in this review we describe the underlying mechanisms and clinical associations of split hand/foot malformation and ectrodactyly-ectodermal dysplasia-cleft lip/palate syndrome, which have both been associated with mutations in the p63 gene.

Novel approach to the molecular diagnosis of Marfan syndrome: application to sporadic cases and in prenatal diagnosis.

Marfan syndrome is an autosomal dominant disorder affecting the skeletal, ocular, and cardiovascular systems. Defects in the gene that encodes fibrillin-1 (FBN1), the main structural component of the elastin-associated microfibrils, are responsible for the disorder. Molecular diagnosis in families with Marfan syndrome can be undertaken by using intragenic FBN1 gene markers to identify and track the disease allele. However, in sporadic cases, which constitute up to 30% of the total, DNA-based diagnosis cannot be performed using linked markers but rather requires the identification of the specific FBN1 gene mutation. Due to the size and complexity of the FBN1 gene, identification of a causative Marfan syndrome mutation is not a trivial undertaking. Herein, we describe a comprehensive approach to the molecular diagnosis of Marfan syndrome that relies on the direct analysis of the FBN1 gene at the cDNA level and detects both coding sequence mutations and those leading to exon-skipping, which are often missed by analysis at the genomic DNA level. The ability to consistently determine the specific FBN1 gene mutation responsible for a particular case of Marfan syndrome allows both prenatal and pre-implantation diagnosis, even in sporadic instances of the disease.

Acheiropodia is caused by a genomic deletion in C7orf2, the human orthologue of the Lmbr1 gene.

Acheiropodia is an autosomal recessive developmental disorder presenting with bilateral congenital amputations of the upper and lower extremities and aplasia of the hands and feet. This severely handicapping condition appears to affect only the extremities, with no other systemic manifestations reported. Recently, a locus for acheiropodia was mapped on chromosome 7q36. Herein we report the narrowing of the critical region for the acheiropodia gene and the subsequent identification of a common mutation in C7orf2-the human orthologue of the mouse Lmbr1 gene-that is responsible for the disease. Analysis of five families with acheiropodia, by means of 15 polymorphic markers, narrowed the critical region to 1.3 cM, on the basis of identity by descent, and to <0.5 Mb, on the basis of physical mapping. Analysis of C7orf2, the human orthologue of the mouse Lmbr1 gene, identified a deletion in all five families, thus identifying a common acheiropodia mutation. The deletion was identified at both the genomic-DNA and mRNA level. It leads to the production of a C7orf2 transcript lacking exon 4 and introduces a premature stop codon downstream of exon 3. Given the nature of the acheiropodia phenotype, it appears likely that the Lmbr1 gene plays an important role in limb development.

Delivery of a hammerhead ribozyme specifically downregulates mutant type I collagen mRNA in a murine model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a systemic heritable disorder of connective tissue, caused by a mutation in one of the genes for type I collagen, whose cardinal manifestation is bone fragility. Several studies have identified two molecular mechanisms of collagen type I defects. In chain exclusion, the mutant chain is not incorporated into the collagen triple helix, whereas in chain nonexclusion, it is. The dominant-negative effect of nonexcluded mutations must be taken into account in all strategies aimed at correcting the collagen defects in individuals affected with moderate or several OI. Herein, we describe the application of hammerhead ribozymes to selectively target the mutant minigene transcript expressed in a murine calvarial osteoblast cell line. Active and control inactive ribozymes were tested in vitro on both mutant and normal targets and in the minigene-expressing cell line. Active ribozyme cleaved its target with high efficiency and specificity in both a time-dependent and dose-dependent manner. After delivery of a ribozyme expression construct, intracellular ribozyme was detected, along with a relative reduction in mutant transcript level.

Orthopaedic manifestations of Ehlers-Danlos syndrome.

Ehlers-Danlos syndrome is the most prevalent heritable disorder of connective tissue. Musculoskeletal problems include joint pain, swelling and instability, and spinal deformity. This study was undertaken to assess functional orthopaedic problems of patients with Ehlers-Danlos syndrome. Sixty patients with genetically verified Ehlers-Danlos syndrome (range, 8-60 years; mean, 34 years) who attended a National Ehlers-Danlos Syndrome Foundation learning conference were evaluated by questionnaire, clinical examination, and when indicated, radiographs. A database of 250 items per patient was constructed and statistically assessed using analysis of variance. Because of rarity of Types VII and VIII, these two patients were dropped from the analysis. Fifty-eight patients had Ehlers-Danlos syndrome Types I, II, III, or IV and form the study cohort. Among these four types, there were no significant differences in history of joint dislocation, swelling, or types of orthopaedic surgical procedures experienced. Thirty patients with Type III Ehlers-Danlos syndrome reported joint pain more frequently than did patients with Types I, II, or IV. Ambulation was impaired significantly in patients with Type III disorder as a whole, as was functional hand strength and upper extremity function. Back or neck pain was a common (67.2%) report among patients with all types of disease but did not correlate with the presence or absence of spinal deformity. Contrary to most previous reports, the patients in this study showed that Type III Ehlers-Danlos syndrome was the most debilitating form with respect to musculoskeletal function.

Localization of an acromesomelic dysplasia on chromosome 9 by homozygosity mapping.

The acromesomelic dysplasias (AMDs) are a group of genetic disorders that primarily affect the middle and distal segments of the extremities. A form of AMD is present on the isolated island of St Helena in the South Atlantic, which has a population of approximately 5500 derived from a number of founder individuals. DNA from four affected individuals and 11 first-degree relatives in four related nuclear families segregating an AMD was collected for gene mapping studies. Six consecutive markers on chromosome 9, spanning an approximately 5 cM region, showed identical homozygosity in all affected individuals, thus identifying a region of homozygosity by descent. Multipoint analysis generated a maximum lod score of Z = 2.85. These data localize the gene for this dysplasia to the pericentromeric region of chromosome 9 where the gene for the Maroteaux form of AMD is situated. The identification of the gene responsible for this disorder may shed further light on the complex processes involved in limb morphogenesis.

Split-hand/split-foot malformation is caused by mutations in the p63 gene on 3q27.

Split-hand/split-foot malformation (SHFM), a limb malformation involving the central rays of the autopod and presenting with syndactyly, median clefts of the hands and feet, and aplasia and/or hypoplasia of the phalanges, metacarpals, and metatarsals, is phenotypically analogous to the naturally occurring murine Dactylaplasia mutant (Dac). Results of recent studies have shown that, in heterozygous Dac embryos, the central segment of the apical ectodermal ridge (AER) degenerates, leaving the anterior and posterior segments intact; this finding suggests that localized failure of ridge maintenance activity is the fundamental developmental defect in Dac and, by inference, in SHFM. Results of gene-targeting studies have demonstrated that p63, a homologue of the cell-cycle regulator TP53, plays a critically important role in regulation of the formation and differentiation of the AER. Two missense mutations, 724A-->G, which predicts amino acid substitution K194E, and 982T-->C, which predicts amino acid substitution R280C, were identified in exons 5 and 7, respectively, of the p63 gene in two families with SHFM. Two additional mutations (279R-->H and 304R-->Q) were identified in families with EEC (ectrodactyly, ectodermal dysplasia, and facial cleft) syndrome. All four mutations are found in exons that fall within the DNA-binding domain of p63. The two amino acids mutated in the families with SHFM appear to be primarily involved in maintenance of the overall structure of the domain, in contrast to the p63 mutations responsible for EEC syndrome, which reside in amino acid residues that directly interact with the DNA.

The genetic basis of aortic disease. Marfan syndrome and beyond.

The Marfan syndrome and related disorders are systemic disorders of connective tissue. Proximal aorta is usually dilated. The molecular basis of Marfan syndrome has been elucidated, thus allowing prenatal diagnosis. Life expectancy has markedly improved due to the widespread use of beta-adrenergic receptor inhibitors and improved surgical management of the aortic disease.

A novel human gene encoding an F-box/WD40 containing protein maps in the SHFM3 critical region on 10q24.

We report the cloning and characterization of a new human gene, Dactylin, encoding a novel member of the F-box/WD40 protein family. The Dactylin gene comprises nine exons distributed in more than 85 kb of genomic DNA and encoding a protein with four WD40 repeats and an F-box motif. Northern blot analysis demonstrates a single 2.8 kb transcript in brain, kidney, lung and liver. FISH hybridization localized Dactylin to 10q24.3. Using an Msc I SNP identified in the first exon of the gene, we were able to assign Dactylin within the critical region for Split Hand Split Foot malformation (SHFM3) that has been mapped to 10q24. The SHFM3 phenotype includes absence or hypoplasia of the central digital rays, a deep median cleft and syndactyly of the remaining digits. Recent studies have demonstrated the importance of F-box/WD40 proteins in the regulation of developmental processes, by a mechanism of specific ubiquitinization and subsequent proteolysis of target proteins belonging to the Wnt, Hh and NF-kappaB signaling pathways. The chromosomal location of Dactylin and its putative function as an F-box/WD40 repeat protein, likely to be involved in key signaling pathways crucial for normal limb development, make it a promising candidate gene for SHFM3.

Hammerhead ribozymes targeted to the FBN1 mRNA can discriminate a single base mismatch between ribozyme and target.

Hammerhead ribozymes are catalytic RNA molecules that can act in trans, with ribozyme and substrate being two different oligoribonucleotides with regions of complementarity. Mutations in the gene for fibrillin-1 (FBN1) cause Marfan syndrome. The majority of mutations are single-base changes, many of which exert their effect via a dominant-negative mechanism. Previously we have shown that an antisense hammerhead ribozyme, targeted to the FBN1 mRNA can reduce deposition of fibrillin to the extracellular matrix of cultured fibroblasts, suggesting it may be possible to utilize ribozymes to down regulate the production of mutant protein and thus restore normal fibrillin function. This might be achieved by the mutation creating a ribozyme cleavage site that is not present in the normal allele, however this is likely to limit the number of mutations that could be targeted. Alternatively, it might be possible to target the mutant allele via the ribozyme binding arms. To determine the potential of ribozymes to preferentially target mutant FBN1 alleles via the latter approach, the effect of mismatches in helix I of a hammerhead ribozyme, on the cleavage of fibrillin (FBN1) mRNA was investigated. A single base mismatch significantly reduced ribozyme cleavage efficiency both in vitro and in vivo. The discrimination between fully-matched and mismatched ribozyme varied with the length of helix I, with the discrimination being more pronounced in ribozymes with a shorter helix. These data suggest that it should be possible to design hammerhead ribozymes that can discriminate between closely related (mutant and normal) target RNAs varying in as little as a single nucleotide, even if the mutation does not create a ribozyme cleavage site.

Ehlers-Danlos syndromes: revised nosology, Villefranche, 1997. Ehlers-Danlos National Foundation (USA) and Ehlers-Danlos Support Group (UK).

Categorization of the Ehlers-Danlos syndromes began in the late 1960s and was formalized in the Berlin nosology. Over time, it became apparent that the diagnostic criteria established and published in 1988 did not discriminate adequately between the different types of Ehlers-Danlos syndromes or between Ehlers-Danlos syndromes and other phenotypically related conditions. In addition, elucidation of the molecular basis of several Ehlers-Danlos syndromes has added a new dimension to the characterization of this group of disorders. We propose a revision of the classification of the Ehlers-Danlos syndromes based primarily on the cause of each type. Major and minor diagnostic criteria have been defined for each type and complemented whenever possible with laboratory findings. This simplified classification will facilitate an accurate diagnosis of the Ehlers-Danlos syndromes and contribute to the delineation of phenotypically related disorders.

Grebe syndrome: clinical and radiographic findings in affected individuals and heterozygous carriers.

Grebe syndrome is a recessively inherited acromesomelic dysplasia. We studied, clinically and radiographically, 10 affected individuals, originating from Bahia, Brazil. The phenotype is characterized by a normal axial skeleton and severely shortened and deformed limbs, with a proximo-distal gradient of severity. The humeri and femora were relatively normal, the radii/ulnae and tibiae/fibulae were short and deformed, carpal and tarsal bones were fused, and several metacarpal and metatarsal bones were absent. The proximal and middle phalanges of the fingers and toes were invariably absent, while the distal phalanges were present. Postaxial polydactyly was found in several affected individuals. Several joints of the carpus, tarsus, hand, and foot were absent. Heterozygotes presented with a variety of skeletal manifestations including polydactyly, brachydactyly, hallux valgus, and metatarsus adductus. Grebe syndrome is caused by a missense mutation in the gene encoding cartilage-derived morphogenetic protein-1.

Disruption of human limb morphogenesis by a dominant negative mutation in CDMP1.

Chondrodysplasia Grebe type (CGT) is an autosomal recessive disorder characterized by severe limb shortening and dysmorphogenesis. We have identified a causative point mutation in the gene encoding the bone morphogenetic protein (BMP)-like molecule, cartilage-derived morphogenetic protein-1 (CDMP-1). The mutation substitutes a tyrosine for the first of seven highly conserved cysteine residues in the mature active domain of the protein. We demonstrate that the mutation results in a protein that is not secreted and is inactive in vitro. It produces a dominant negative effect by preventing the secretion of other, related BMP family members. We present evidence that this may occur through the formation of heterodimers. The mutation and its proposed mechanism of action provide the first human genetic indication that composite expression patterns of different BMPs dictate limb and digit morphogenesis.

Chronic pain is a manifestation of the Ehlers-Danlos syndrome.

The Ehlers-Danlos syndrome (EDS) is a group of heritable systemic disorders of connective tissue manifesting joint hypermobility, skin extensibility, and tissue fragility. Although the presence of pain has been documented in the various types of the EDS, its natural history, distribution, and management have not been defined. We conducted a structured interview in 51 individuals affected with different types of EDS. Affected individuals reported chronic pain of early onset involving most frequently the shoulders, hands, and knees. Pain was generally refractory to a variety of pharmacologic and physical interventions. Chronic pain is a common manifestation of EDS.

Delivery of a hammerhead ribozyme specifically down-regulates the production of fibrillin-1 by cultured dermal fibroblasts.

The hammerhead ribozyme is a small catalytic RNA molecule. Potential hammerhead ribozymes that possess a catalytic domain and flanking sequence complementary to a target mRNA can cleave in trans at a putative cleavage site within the target molecule. We have investigated the potential of hammerhead ribozymes to down-regulate the product of the fibrillin-1 gene (FBN1). Fibrillin is a 347 kDa glycoprotein that is a major constituent of the elastin-associated microfibrils. Mutations in the FBN1 gene are responsible for Marfan syndrome (MFS), a common systemic disorder of the connective tissue. Many FBN1 mutations responsible for MFS appear to act in a dominant-negative fashion, raising the possibility that reduction of the amount of product from the mutant FBN1 allele might be a valid therapeutic approach for MFS. A trans-acting hammerhead ribozyme (FBN1-RZ1) targeted to the 5' end of the human FBN1 mRNA has been designed and synthesized, and shown to cleave its target efficiently in vitro. FBN1-RZ1 cleavage is magnesium dependent and efficient at both 37 and 50 degrees C. Delivery of the FBN1-RZ1 ribozyme into cultured dermal fibroblasts, by receptor-mediated endocytosis of a ribozyme-transferrin-polylysine complex, specifically reduces both cellular FBN1 mRNA and the deposition of fibrillin in the extracellular matrix. These results suggest that the use of hammerhead ribozymes is a valid approach to the study of fibrillin gene expression and possibly to the development of a therapeutic approach to MFS.

Preimplantation genetic diagnosis in Marfan syndrome.

The in vitro fertilization technology coupled with the ability to amplify DNA from a single cell has been used for the preimplantation genetic diagnosis of Marfan syndrome. An intragenic FBN1 gene marker has been used to track the inheritance of this disorder in a family. Marker genotyping was established following two rounds of amplification. Whenever possible, two blastomeres were separately assayed per embryo. The transfer of five embryos resulted in a singleton pregnancy and the birth of a full-term male infant.