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Stickler syndrome type I (STL1) is a phenotypically heterogeneous disorder characterized by ocular and extraocular features. It is caused by null-allele mutations in the COL2A1 gene that codes for procollagen II. COL2A1 precursor mRNA undergoes alternative splicing, resulting in two isoforms, a long form including exon 2 (type IIA isoform) and a short form excluding exon 2 (type IIB isoform). The short form is predominantly expressed by differentiated chondrocytes in adult cartilage, and the long form in chondroprogenitor cells during early development and in the vitreous of the eye, which is the only adult tissue containing procollagen IIA. Recent evidence indicates that due to the tissue-specific expression of these two isoforms, premature termination codon mutations in exon 2 cause Stickler syndrome with minimal or no extraocular manifestations. We describe here two mutations in exon 2 of COL2A1 in three patients with predominantly ocular Stickler syndrome: Cys64Stop in two patients, and a novel structural mutation, Cys57Tyr, in one patient. RT-PCR of total lymphoblast RNA from one patient with the Cys64Stop mutation revealed that only the normal allele of the IIA form was present, indicating that the mutation resulted either in complete loss of the allele by nonsense-mediated mRNA decay or by skipping of exon 2 via nonsense-mediated altered splicing, resulting in production of the type IIB isoform. The results of COL2A1 minigene expression studies suggest that both Cys64Stop and Cys57Tyr alter positive cis regulatory elements for splicing, resulting in a lower IIA:IIB ratio., (2007 Wiley-Liss, Inc.)

The importance of the genetic component in high myopia has been well established in population and family studies, but only a few candidate genes have been explored to date. The extracellular matrix small leucine-rich repeat proteins/proteoglycans (SLRPs) regulate collagen fibril diameter and spacing. Given their role in extracellular matrix assembly and expression in the eye, they are likely to regulate its shape and size. Analysis of 85 English and 40 Finnish subjects with high myopia (refractive error of -6 diopters [D] or greater) resulted in 23 sequence variations in four SLRP genes, LUM, FMOD, PRELP, and OPTC. We observed higher number of variations in OPTC in English patients than in controls (p=0.042), and a possibly protective variation in LUM (c.893-105G>A) with p-value of 0.0043. Two intronic variations, six nonsynonymous and one synonymous amino acid changes, were not found in any of the nonmyopic controls. Five changes were detected in opticin, Thr177Arg, Arg229His, Arg325Trp, Gly329Ser, and Arg330His, and all but one (Arg229His) were shown to cosegregate with high myopia in families with incomplete penetrance. A homology model for opticin revealed that Arg229His and Arg325Trp are likely to disrupt the protein structure, and PolyPhen analysis suggested that Thr177Arg, Arg325Trp, and Gly329Ser changes may be damaging. A Leu199Pro change in lumican and Gly147Asp and Arg324Thr variations in fibromodulin are located in the highly conserved leucine-rich repeat (LRR) domains. This study provides new insight into the genetics of high myopia, suggesting that sequence variations in the SLRP genes expressed in the eye may be among the genetic risk factors underlying the pathogenesis of high myopia., (Copyright 2006 Wiley-Liss, Inc.)

Osteogenesis imperfecta (OI) is caused by mutations in COL1A1 and COL1A2 that code for the alpha1 and alpha2 chains of type I collagen. Phenotypes correlate with the mutation types in that COL1A1 null mutations lead to OI type I, and structural mutations in alpha1(I) or alpha2(I) lead to more severe OI types (II-IV). However, correlative analysis between mutation types and OI associated hearing loss has not been previously performed. A total of 54 Finnish OI patients with previously diagnosed hearing loss or age 35 or more years were analyzed here for mutations in COL1A1 or COL1A2. Altogether 49 mutations were identified, of which 41 were novel. The 49 mutations represented the molecular genetic background of 41.1% of the Finnish OI population. A total of 38 mutations were in COL1A1 and 11 were in COL1A2. Of these, 16 were glycine substitutions and 16 were splicing mutations in alpha1(I) or alpha2(I). In addition, 17 null allele mutations were detected in COL1A1. A total of 32 patients (65.3%) with a mutation had hearing loss. That is slightly more than in our previous population study on Finnish adults with OI (57.9%). The association between the mutation types and OI type was statistically evident. Patients with COL1A1 mutations more frequently had blue scleras than those with COL1A2 mutations. In addition, patients with COL1A2 mutations tended to be shorter than those with COL1A1 mutations. However, no correlation was found between the mutated gene or mutation type and hearing pattern. These results suggest that the basis of hearing loss in OI is complex, and it is a result of multifactorial, still unknown genetic effects., (Copyright 2004 Wiley-Liss, Inc.)

Fibrillin is the major component of extracellular microfibrils. Mutations in the fibrillin gene on chromosome 15 (FBN1) were first described in the heritable connective disorder, Marfan syndrome (MFS). FBN1 has also been shown to harbor mutations related to a spectrum of conditions phenotypically related to MFS, called "type-1 fibrillinopathies." In 1995, in an effort to standardize the information regarding these mutations and to facilitate their mutational analysis and identification of structure/function and phenotype/genotype relationships, we created a human FBN1 mutation database, UMD-FBN1. This database gives access to a software package that provides specific routines and optimized multicriteria research and sorting tools. For each mutation, information is provided at the gene, protein, and clinical levels. This tool is now a worldwide reference and is frequently used by teams working in the field; more than 220,000 interrogations have been made to it since January 1998. The database has recently been modified to follow the guidelines on mutation databases of the HUGO Mutation Database Initiative (MDI) and the Human Genome Variation Society (HGVS), including their approved mutation nomenclature. The current update shows 559 entries, of which 421 are novel. UMD-FBN1 is accessible at www.umd.be/. We have also recently developed a FBN1 polymorphism database in order to facilitate diagnostics., (Copyright 2003 Wiley-Liss, Inc.)

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.

Ehlers-Danlos syndrome type IV (EDS IV) is an autosomal dominant disorder characterized by fragile skin, blood vessels, and internal organs and associated with decreased production, secretion, or thermal stability of type III procollagen. Mutations in the gene for type III procollagen have been identified in patients exhibiting decreased secretion or thermal stability of the protein, but no defect has been elucidated to explain the decreased production of type III procollagen in some patients with EDS IV. We report on a patient with a moderate case of EDS IV who produced decreased amounts of type III procollagen despite normal levels of translatable type III procollagen mRNA. S1 nuclease analysis of the type III procollagen mRNA indicated a defect in the region encoding exon 27. Sequence analysis of cDNA clones and genomic fragments generated by polymerase chain reaction amplification revealed that sequences encoded by exon 27 were absent from 3 out of 5 cDNA clones and that a G at the +5 position of the splice donor site in intron 27 was changed to an A in one allele of the patient's type III procollagen gene. Using a cDNA-genomic DNA hybrid probe in S1 nuclease analysis, fragments consistent with mRNA species containing and lacking exon 27 were detected in a 1:1 ratio. Pulse label and chase experiments in the presence or absence of brefeldin A indicated that most of the type III procollagen molecules synthesized by the patient's fibroblasts were not secreted into the medium but were degraded in the endoplasmic reticulum-Golgi compartment by a nonlysosomal mechanism.

A search for mutations in the gene for type II procollagen (COL2A1) was carried out in a family with late-onset spondyloepiphyseal dysplasia resulting in short sature, restricted mobility and severe pain in joints, deforming arthritis in the hips, and claudication. Analysis of the HindIII and VNTR polymorphisms at the COL2A1 gene in the family raised the possibility that the gene cosegregated with the disease. Screening for mutations in the COL2A1 gene using PCR-denaturing gradient get electrophoresis suggested a sequence variation in exon 19 of one allele of the COL2A1 gene in the proband. Direct sequencing of the PCR products for exon 19 revealed a single base mutation that converted the codon of -GGT- for glycine at alpha 1-247 to -AGT-, a codon for serine. The mutant that converted the present in all affected family members, but absent in nonaffected members and in a group of 50 unrelated healthy individuals. It was also absent in 20 unrelated patients with chondrodysplasia and 30 unrelated patients with early-onset osteoarthritis.

The direct sequencing of the human type II procollagen (COL2A1) gene from polymerase chain reaction (PCR)-amplified genomic DNA is described. Thirty-two regions of the COL2A1 gene were asymmetrically amplified with intron primers which were specifically chosen to amplify a region spanning 500 to 800 bp of sequence encoding one or more exons and their accompanying intervening sequences. Primers for dideoxynucleotide sequencing of the PCR products were then designed to provide complete exon sequence information and to insure that intron:exon splice junction sequence data would be obtained. Amplification and sequencing reactions were performed on an automated workstation to facilitate the handling of multiple DNA templates. The procedure allowed efficient sequencing of over 25,000 bp of each allele of the COL2A1 gene per diploid genome. We used this method for the comparative analyses of COL2A1 sequences in DNA isolated from the blood of 42 unrelated individuals and we identified 21 neutral sequence variants in the gene. The sequence variations were confirmed by independent assays, including restriction enzyme digestion. The sequence variants described here will be important for identifying haplotypes of the type II procollagen gene that will be useful in defining a genetic etiology for diseases of cartilaginous tissues.