Sensitivity of conformation sensitive gel electrophoresis in detecting mutations in Marfan syndrome and related conditions.

OBJECTIVE: It has been firmly established that mutations in the gene for fibrillin 1, FBN1, cause Marfan syndrome (MFS). FBN1 mutations can also cause other phenotypes, such as ectopia lentis (EL) and familial isolated thoracic aortic aneurysm and dissection (FAA). When the clinical presentation is typical, diagnosis of MFS is usually easy to make. However, there can be a marked phenotypic variation between affected subjects even in one family, and making the diagnosis can be challenging, especially in childhood. The objective of this study was to test the sensitivity of conformation sensitive gel electrophoresis (CSGE) for detecting mutations in FBN1 in MFS and related phenotypes. DESIGN: Setting up CSGE analysis for the FBN1 gene and testing the method first by screening coded samples from 17 MFS patients with previously detected FBN1 mutations. We then used a test set consisting of 46 coded samples representing MFS, related phenotypes, and controls. RESULTS: Sixteen of the 17 known mutations were detected. Altogether 23 mutations were detected in a test set consisting of 46 coded samples representing MFS, related phenotypes, and controls. Nineteen of the mutations were novel. The mutation was detected in 18 of the 20 MFS patients and in one patient with familial EL, but not in a patient with sporadic MASS syndrome, any of the five sporadic annuloaortic ectasia (AAE) patients, or any of the 15 controls. A FBN1 mutation was detected in four members of a multigeneration family with AAE, however. CONCLUSIONS: These results indicate that CSGE is highly sensitive for the detection of mutations in FBN1, and that molecular diagnostics is a useful means of confirming clinical diagnoses of MFS and related disorders. Further careful investigations are needed, however, in order to correlate the interfamilial and intrafamilial clinical variabilities of fibrillinopathies and mutations in FBN1.

Phenotypic expressions of a Gly 154Arg mutation in type II collagen in two unrelated patients with spondyloepimetaphyseal dysplasia (SEMD).

Type II collagenopathies consist of chondrodysplasias ranging from lethal to mild in severity. A large number of mutations has been found in the COL2A1 gene. Glycine substitutions have been the most common types of mutation. Genotype-phenotype correlations in type II collagenopathies have not been established, partly because of insufficient clinical and radiographic description of the patients. We found a glycine-to-arginine substitution at position 154 in type II collagen in two unrelated isolated propositi with spondyloepimetaphyseal dysplasia and provide a comparative clinical and radiographic analysis from birth to young adulthood for this condition. The clinical phenotype was disproportionate short stature with varus/valgus deformities of the lower limbs requiring corrective osteotomies, and lumbar lordosis. The skeletal radiographs showed an evolution from short tubular bones, delayed epiphyseal development, and mild vertebral involvement to severe metaphyseal dysplasia with dappling irregularities, and hip "dysplasia." The metaphyseal abnormalities disappeared by adulthood.

Double heterozygosity for pseudoachondroplasia and spondyloepiphyseal dysplasia congenita.

Pseudoachondroplasia (PSACH) and spondyloepiphyseal dysplasia congenita (SEDC) are autosomal dominant forms of short-limb short stature caused by mutations in genes that encode structural components of the cartilage extracellular matrix. PSACH results from mutations in the cartilage oligomeric matrix protein (COMP) gene, while SEDC is caused by mutations in the gene for type II procollagen (COL2A1). We report a child with a distinct skeletal dysplasia due to the combined phenotypes of PSACH and SEDC. The proband's mother had PSACH and his father had SEDC. The child was suspected of having both phenotypes on the basis of the severity of his clinical and radiographic findings, and this was confirmed by molecular analysis. The COMP gene mutation (C348R), while not previously published, is typical of those in PSACH patients, whereas the COL2A1 mutation (T1370M) is somewhat atypical, as it predicts an amino acid change within the carboxyl-terminal region of the protein. Both mutations segregated with their respective phenotypes within this family. The description and natural history of the double heterozygote phenotype may be useful in counseling families regarding risk and prognosis.

Widely distributed mutations in the COL2A1 gene produce achondrogenesis type II/hypochondrogenesis.

The COL2A1 gene was assayed for mutations in genomic DNA from 12 patients with achondrogenesis type II/hypochondrogenesis. The exons and flanking sequences of the 54 exons in the COL2A1 gene were amplified by a series of specific primers using PCR. The PCR products were scanned for mutations by conformation sensitive gel electrophoresis, and PCR products that generated heteroduplex bands were then sequenced. Mutations in the COL2A1 gene were found in all 12 patients. Ten of the mutations were single base substitutions that converted a codon for an obligate glycine to a codon for an amino acid with a bulkier side chain. One of the mutations was a change in a consensus RNA splice site. Another was an 18-base pair deletion of coding sequences. The results confirmed previous indications that conformation sensitive gel electrophoresis is highly sensitive for detection of mutations in large and complex genes. They also demonstrate that most, if not all, patients with achondrogenesis type II/hypochondrogenesis have mutations in the COL2A1 gene.

Novel COL1A1 mutation (G559C) [correction of G599C] associated with mild osteogenesis imperfecta and dentinogenesis imperfecta.

A genotype-phenotype analysis of a three-generation family segregating for an autosomal-dominant osteogenesis imperfecta (OI) variant is reported here. The family was ascertained through the presentation of a proband concerned about discoloration of her teeth, found to be dentinogenesis imperfecta (DGI). Examination of 36 family members identified 15 individuals with DGI. Linkage studies were performed for genetic markers from candidate intervals known to contain genes responsible for DGI on chromosomes 4q, 7q, and 17q. Conclusive evidence for linkage of DGI was obtained to genetic markers on chromosome 17q21-q22 (DLX-3, Z(max) = 5.34, theta = 0.00). All DGI-affected family members shared a common haplotype, which was not present in individuals without DGI. Haplotype analysis sublocalized the gene to a 5-cM genetic interval that contained the collagen 1 alpha 1 (COL1A1) gene. More than 150 different COL1A1 gene mutations have been associated with various forms of OI, and five of these have been associated with DGI and type IV OI. After excluding these five mutations, mutational analysis was performed on the remaining exons including intron--exon boundaries, which resulted in identification of a Gly559Cys mutation in exon 32, present in all DGI-affected family members. Clinical features segregating with this G559C mutation included hyperextensible joints, joint pain and an increased propensity for bone fractures with moderate trauma. This is the first report of joint pain associated with a COL1A1 mutation and DGI. The mild skeletal features and reduced penetrance of the non-dental findings illustrate the importance of genetic evaluations for families with a history of DGI.

MicroSAGE analysis of 2,353 expressed genes in a single cell-derived colony of undifferentiated human mesenchymal stem cells reveals mRNAs of multiple cell lineages.

Mesenchymal stem cells (MSCs) isolated from the bone marrow of adult organisms are capable of differentiating into adipocytes, chondrocytes, myoblasts, osteoblasts, and hematopoiesis-supporting stroma. We recently demonstrated that MSCs also adopt glial cell fates when transplanted into the developing central nervous system and hence can produce tissue elements derived from a separate embryonic layer. Despite these remarkable properties, it has been difficult to establish specific criteria to characterize MSCs. Using a modified protocol for micro-serial analysis of gene expression, we cataloged 2,353 unique genes expressed by a single cell-derived colony of undifferentiated human MSCs. This analysis revealed that the MSC colony simultaneously expressed transcripts characteristic of various mesenchymal cell lineages including chondrocytes, myoblasts, osteoblasts, and hematopoiesis-supporting stroma. Therefore, the profile of expressed transcripts reflects the developmental potential of the cells. Additionally, the MSC colony expressed mRNAs characteristic of endothelial, epithelial and neuronal cell lineages, a combination that provides a unique molecular signature for the cells. Other expressed transcripts included various products involved in wound repair as well as several neurotrophic factors. A total of 268 novel transcripts were also identified, one of which is the most abundantly expressed mRNA in MSCs. This study represents the first extensive gene expression analysis of MSCs and as such reveals new insight into the biology, ontogeny, and in vivo function of the cells.

Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: comparison with denaturing gradient gel electrophoresis and nucleotide sequencing.

Previously, an assay called conformation sensitive gel electrophoresis (CSGE) was developed for scanning PCR products for the presence of single-base and larger base mismatches in DNA. The assay was based on the assumption that mildly denaturing solvents in an appropriate buffer can accentuate the conformational changes produced by single-base mismatches in double-stranded DNA and thereby increase the differential migration in electrophoretic gels of heteroduplexes and homoduplexes. Here the sensitivity of assays by CSGE was improved by limiting the maximal size of the PCR products to 450 bp and making several changes in the conditions for PAGE. With the improved conditions, CSGE detected all 76 previously identified single-base changes in a large series of PCR products from collagen genes that contain multiple exons with highly repetitive and GC-rich sequences. In a survey of 736 alleles of collagen genes, CSGE detected 223 unique single-base mismatches that were confirmed by nucleotide sequencing. CSGE has the advantage over other methods for scanning PCR products in that it is simple, requires no special preparation of PCR products, has a large capacity, and does not use radioactivity.

Two new recurrent nucleotide mutations in the COL1A1 gene in four patients with osteogenesis imperfecta: about one-fifth are recurrent.

Previous observations on mutations causing osteogenesis imperfecta (OI) suggested that unrelated patients had private mutations. Here preliminary studies on two patients with type I OI indicated that some mutations in the COL1A1 gene for type I procollagen cannot be detected by analyses of cDNAs. Therefore, we developed a protocol whereby 43 exon and exon flanking sequences of the COL1A1 gene can be amplified by PCR and scanned for mutations by denaturing gradient gel electrophoresis. Two new recurrent nucleotide mutations in the gene were found in four apparently unrelated patients with OI. Analysis of previous publications indicated that up to one-fifth of the mutations causing OI are recurrent in the sense that they were identical in apparently unrelated probands. About 80% of these identical mutations were in CpG dinucleotide sequences.

Mutation in type II procollagen (COL2A1) that substitutes aspartate for glycine alpha 1-67 and that causes cataracts and retinal detachment: evidence for molecular heterogeneity in the Wagner syndrome and the Stickler syndrome (arthro-ophthalmopathy)

A search for mutations in the gene for type II procollagen (COL2A1) was carried out in affected members of a family with early-onset cataracts, lattice degeneration of the retina, and retinal detachment. They had no symptoms suggestive of involvement of nonocular tissues, as is typically found in the Stickler syndrome. The COL2A1 gene was amplified with PCR, and the products were analyzed by denaturing gradient gel electrophoresis. The results suggested a mutation in one allele for exon 10. Sequencing of the fragment demonstrated a single-base mutation that converted the codon for glycine at position alpha 1-67 to aspartate. The mutation was found in three affected members of the family available for study but not in unaffected members or 100 unrelated individuals. Comparison with previously reported mutations suggested that mutations introducing premature termination codons in the COL2A1 gene are a frequent cause of the Stickler syndrome, but mutations in the COL2A1 gene that replace glycine codons with codons for bulkier amino acid can produce a broad spectrum of disorders that range from lethal chondrodysplasias to a syndrome involving only ocular tissues, similar to the syndrome in the family originally described by Wagner in 1938.

Analysis of the COL1A1 and COL1A2 genes by PCR amplification and scanning by conformation-sensitive gel electrophoresis identifies only COL1A1 mutations in 15 patients with osteogenesis imperfecta type I: identification of common sequences of null-allele mutations.

Although >90% of patients with osteogenesis imperfecta (OI) have been estimated to have mutations in the COL1A1 and COL1A2 genes for type I procollagen, mutations have been difficult to detect in all patients with the mildest forms of the disease (i.e., type I). In this study, we first searched for mutations in type I procollagen by analyses of protein and mRNA in fibroblasts from 10 patients with mild OI; no evidence of a mutation was found in 2 of the patients by the protein analyses, and no evidence of a mutation was found in 5 of the patients by the RNA analyses. We then searched for mutations in the original 10 patients and in 5 additional patients with mild OI, by analysis of genomic DNA. To assay the genomic DNA, we established a consensus sequence for the first 12 kb of the COL1A1 gene and for 30 kb of new sequences of the 38-kb COL1A2 gene. The sequences were then used to develop primers for PCR for the 103 exons and exon boundaries of the two genes. The PCR products were first scanned for heteroduplexes by conformation-sensitive gel electrophoresis, and then products containing heteroduplexes were sequenced. The results detected disease-causing mutations in 13 of the 15 patients and detected two additional probable disease-causing mutations in the remaining 2 patients. Analysis of the data developed in this study and elsewhere revealed common sequences for mutations causing null alleles.

Identification of COL2A1 gene mutations in patients with chondrodysplasias and familial osteoarthritis.

OBJECTIVE: To use a recently developed procedure for analysis of blood leukocyte DNA to detect mutations in the gene for type II procollagen (COL2A1) in patients with cartilage diseases ranging from early-onset familial osteoarthritis (OA) to lethal chondrodysplasias. METHODS: The technique of denaturing gradient gel electrophoresis was used to scan polymerase chain reaction (PCR) products from 45 exons and exon-flanking sequences of the COL2A1 gene in more than 70 patients with cartilage diseases whose severity ranged from mild to lethal. PCR products with abnormal migrations were then sequenced. RESULTS: Among the 3 patients with lethal hypochondrogenesis who were analyzed, all 3 were found to have a mutation in the COL2A1 gene. Among 17 patients with spondyloepiphyseal or spondyloepimetaphyseal dysplasia, 2 well-defined and 2 probable mutations were found. Among 15 patients with the Wagner-Stickler syndrome, 2 well-defined and 2 probable mutations were found. Among 45 patients with early-onset familial OA, 1 probable mutation was found. CONCLUSION: Using the procedure developed for analysis of the COL2A1 gene, mutations were detected in > 20% of patients with chondrodysplasias and up to 2% of patients with early-onset familial OA. However, these percentages are only minimal estimates because all possible mutations in the gene cannot be detected with this procedure.

Splicing mutations of 54-bp exons in the COL11A1 gene cause Marshall syndrome, but other mutations cause overlapping Marshall/Stickler phenotypes.

Stickler and Marshall syndromes are dominantly inherited chondrodysplasias characterized by midfacial hypoplasia, high myopia, and sensorineural-hearing deficit. Since the characteristics of these syndromes overlap, it has been argued whether they are distinct entities or different manifestations of a single syndrome. Several mutations causing Stickler syndrome have been found in the COL2A1 gene, and one mutation causing Stickler syndrome and one causing Marshall syndrome have been detected in the COL11A1 gene. We characterize here the genomic structure of the COL11A1 gene. Screening of patients with Stickler, Stickler-like, or Marshall syndrome pointed to 23 novel mutations. Genotypic-phenotypic comparison revealed an association between the Marshall syndrome phenotype and splicing mutations of 54-bp exons in the C-terminal region of the COL11A1 gene. Null-allele mutations in the COL2A1 gene led to a typical phenotype of Stickler syndrome. Some patients, however, presented with phenotypes of both Marshall and Stickler syndromes.