Completion of the Last Half of the Structure of the Human Gene for the Proα1 (I) Chain of Type I Procollagen (COL1A1)

Westerhausen, A; Constantinou, Constantinos D.; Packs, Michael; Peng, Minzhong; Hanning, Charles R.; Olsen, Anne S.; Prockop, Darwin J.

doi:10.1016/s0934-8832(11)80191-5

Cited by 17 publications

(9 citation statements)

References 12 publications

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“…3, panel B). The sequences of the patients were identical to the published sequence of human COL1A1 intron 36 (22) except that there were 3 additional nt, CCA, located 83 nt from the 5Ј-end of the intron. We also found 2 single nt changes, C3 A and T3 G at positions 81 and 100, respectively, of the intron.…”

Section: Resultsmentioning

confidence: 76%

Alternative Splicing in COL1A1 mRNA Leads to a Partial Null Allele and Two In-frame Forms with Structural Defects in Non-lethal Osteogenesis Imperfecta

Wang

Forlino

Marini

1996

Journal of Biological Chemistry

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We have identified a novel multiexon genomic deletion in one COL1A1 collagen allele that results in three alternative forms of mutant mRNA. This mutation occurs in a 9-year-old girl and her father, both affected with severe type III osteogenesis imperfecta (OI). We previously reported detection of a mismatch in their ␣1(I) amino acids 558 -861 region by RNA/RNA hybrid analysis (Grange, D. K., Gottesman, G. S., Lewis, M. B., and Marini, J. C. (1990) Nucleic Acids Res. 18, 4227-4236). Single Strand Conformational Polymorphism further localized the mRNA mutation to the amino acids 579 -679 coding region. At the gene level, polymerase chain reaction (PCR) amplification of patient leukocyte DNA from the exon 33-38 region yielded the normal 1004-base pair (bp) fragment and an additional 442-bp fragment. Sequencing of the shorter genomic PCR product confirmed the presence of a 562-bp deletion, extending from the last 3 nucleotides (nt) of exon 34 to 156 nt from the 3-end of intron 36. The genomic deletion was also detected in the clinically normal grandmother, who was confirmed to be a mosaic carrier.PCR amplification and RNase protection experiments were used to investigate the mRNA structure and occurrence of alternative splicing. One form of the mutant cDNA has a deletion with end points that are identical to the genomic deletion. This results in a combination deletion/insertion, with a deletion of amino acids 603-639 followed by an insertion of 156 nt from the 3-end of intron 36. In addition, we found two alternatively spliced forms. One form uses a cryptic donor site in exon 34 and the exon 37 acceptor. The second form uses the normal exon 32 splice donor and exon 37 acceptor. Use of the cryptic donor results in a coding sequence that is out-of-frame. Both the retained intron form and the use of the exon 32 donor site result in coding sequences that are in-frame. This is the first report of a collagen defect in OI with alternative splicing generating both in-frame and out-of-frame forms of mRNA. Although the in-frame forms constitute more than 60% of the mRNA from the mutant allele, no mutant protein chain was identified. Collagen produced by cultured OI osteoblasts showed a significant increase in the relative amount of type III collagen but no mutant ␣1(I) chain. Osteogenesis imperfecta (OI)1 is a heritable disorder of connective tissue; its most significant clinical feature is fragile bones that are susceptible to fracture from minimal trauma (1). The phenotypic spectrum of OI is described by the Sillence classification and ranges from perinatal lethal to minimal skeletal involvement. The full clinical spectrum of OI has been demonstrated to be caused by defects in the structure or synthesis of type I collagen, the most abundant protein of bone and skin (for reviews, see Refs. 2 and 3). In the majority of instances, the mild OI type I is caused by defects that effectively create a null ␣1(I) allele (4), such as a frameshift resulting in premature chain termination. In these cases, all the secreted collagen is s...

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Section: Resultsmentioning

confidence: 76%

Alternative Splicing in COL1A1 mRNA Leads to a Partial Null Allele and Two In-frame Forms with Structural Defects in Non-lethal Osteogenesis Imperfecta

Wang

Forlino

Marini

1996

Journal of Biological Chemistry

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“…To define the consensus sequences, genomic DNAs from eight unrelated probands with OI were used as separate templates for PCR with primers based on published sequences of the COL1A1 gene (Chu et al 1985;D'Alessio et al 1988;Mä ä ttä et al 1991;Westerhausen et al 1991). The PCR reactions were performed with a commercial DNA polymerase (AmpliTaq᭨ Gold; Perkin-Elmer) in a 40-ml volume, with thermal cycling at 95ЊC for 10 min, for one cycle, followed by 95ЊC for 40 s, 60ЊC for 40 s, and 72ЊC for 50 s, for 35 cycles.…”

Section: Defining the Consensus Sequences Of The 5 Half Of The Col1a1mentioning

confidence: 99%

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

Körkkö

Ala‐Kokko

Paepe

et al. 1998

The American Journal of Human Genetics

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161

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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.

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“…Accordingly, a new assay was developed on the basis of reverse transcription of the mRNA and then PCR amplification. The sequences of the 3'-end of the human and mouse mRNAs for proal(1) chains are highly conserved but contain several differences (Mooslehner & Harbers, 1988;Bernard et al, 1983;Westerhausen et al, 1991). The primer for reverse transcription and the different primers for PCR were designed so as to be complementary to identical sequences in human and mouse proal(1) mRNAs ( Figure 2) and thereby ensure that the efficiency of both the reverse transcriptase reaction and PCR was the same for both templates.…”

Section: Resultsmentioning

confidence: 99%

Tissue- and development-specific expression in transgenic mice of a type I procollagen (COL1A1) minigene construct with 2.3 kb of the promoter region and 2 kb of the 3'-flanking region. Specificity is independent of the putative regulatory sequences in the first intron

Sokolov¹,

Mays²,

Khillan³

et al. 1993

Biochemistry

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Previous reports have provided inconsistent data as to the cis-regulatory elements that are essential for correct expression of the gene for the pro alpha 1 (I) chain of type I procollagen (COL1A1) in the many tissues in which the protein is synthesized. Here, two internally deleted minigene versions of the human COL1A1 gene were used to prepare transgenic mice. The constructs made it possible to test regulatory sequences in the normal context of the gene. Also, in contrast to the reporter genes used in previous experiments, the constructs made it possible to assay quantitatively expression of the exogenous genes relative to expression of the endogenous COL1A1 gene, both as mRNA and as protein. The average level of expression of the minigenes varied among three transgenic lines, but the ratio of expression of the minigenes to expression of the endogenous gene was the same in all transgenic mice of a given line. Within the same line, the ratio of expression was essentially the same in nine or more tissues in which expression of the endogenous gene varied widely. Also, the ratio of expression within a given line was the same in 15-day-old embryos and in mice ranging in age from 4 days to 4 months. In addition, the ratio remained constant during repair of a surgical wound. The results demonstrated, therefore, that the minigene constructs with about 2.3 kb of the promoter region and about 2 kb of the 3'-flanking region contained all of the sequences necessary for correct expression of the genes in a tissue-specific and development-specific manner.(ABSTRACT TRUNCATED AT 250 WORDS)

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Completion of the Last Half of the Structure of the Human Gene for the Proα1 (I) Chain of Type I Procollagen (COL1A1)

Cited by 17 publications

References 12 publications

Alternative Splicing in COL1A1 mRNA Leads to a Partial Null Allele and Two In-frame Forms with Structural Defects in Non-lethal Osteogenesis Imperfecta

Alternative Splicing in COL1A1 mRNA Leads to a Partial Null Allele and Two In-frame Forms with Structural Defects in Non-lethal Osteogenesis Imperfecta

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

Tissue- and development-specific expression in transgenic mice of a type I procollagen (COL1A1) minigene construct with 2.3 kb of the promoter region and 2 kb of the 3'-flanking region. Specificity is independent of the putative regulatory sequences in the first intron

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