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Title: A method distinguishing expressed vs. null mutations of the Col1A1 gene in osteogenesis imperfecta

Abstract

Osteogenesis imperfecta (OI) is a heterogeneous group of heritable disorders of bone characterized by increased susceptibility to fracture. Most of the causative mutations were identified in patients with the lethal form of the disease. Attention is now shifting to the milder forms of OI where glycine substitutions and null producing mutations have been found. Single amino acid substitutions can be identified by RT/PCR of total cellular RNA, but this approach does not work well for null mutations since the defective transcript does not accumulate in the cytoplasm. We have altered our RNA extraction method to separate RNA from the nuclear and cytoplasmic compartments of cultured fibroblasts. Standard methods of mutation identification (RT/PCR followed by SSCP) is applied to each RNA fraction. DNA from an abnormal band on the SSCP gel is eluted and amplified by PCR for cloning and sequencing. Using this approach we have identified an Asp to Asn change in exon 50 (type II OI) and a Gly to Arg in exon 11 (type I OI) of the COL1A1 gene. These changes were found in both nuclear and cytoplasmic compartments. These putative mutations are currently being confirmed by protein studies. In contrast, three patients with mild OI associatedmore » with reduced {proportional_to}(I)mRNA, had distinguishing SSCP bands present in the nuclear but not the cytoplasmic compartment. In one case a frame shift mutation was observed, while the other two revealed polymorphisms. The compartmentalization of the mutant allele has directed us to look elsewhere in the transcript for the causative mutation. This approach to mutation identification is capable of distinguishing these fundamentally different types of mutations and allows for preferential cloning and sequencing of the abnormal allele.« less

Authors:
; ;  [1]
  1. and others
Publication Date:
OSTI Identifier:
134302
Report Number(s):
CONF-941009-
Journal ID: AJHGAG; ISSN 0002-9297; TRN: 95:005313-1035
Resource Type:
Journal Article
Journal Name:
American Journal of Human Genetics
Additional Journal Information:
Journal Volume: 55; Journal Issue: Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; GENES; GENE MUTATIONS; TRANSCRIPTION; DNA-CLONING; DNA SEQUENCING; DETECTION; SKELETAL DISEASES; PATIENTS; HEREDITARY DISEASES; EXONS; POLYMERASE CHAIN REACTION; BANDING TECHNIQUES; AMINO ACIDS; RNA; MESSENGER-RNA; PROTEINS

Citation Formats

Redford-Badwal, D A, Stover, M L, and McKinstry, M. A method distinguishing expressed vs. null mutations of the Col1A1 gene in osteogenesis imperfecta. United States: N. p., 1994. Web.
Redford-Badwal, D A, Stover, M L, & McKinstry, M. A method distinguishing expressed vs. null mutations of the Col1A1 gene in osteogenesis imperfecta. United States.
Redford-Badwal, D A, Stover, M L, and McKinstry, M. 1994. "A method distinguishing expressed vs. null mutations of the Col1A1 gene in osteogenesis imperfecta". United States.
@article{osti_134302,
title = {A method distinguishing expressed vs. null mutations of the Col1A1 gene in osteogenesis imperfecta},
author = {Redford-Badwal, D A and Stover, M L and McKinstry, M},
abstractNote = {Osteogenesis imperfecta (OI) is a heterogeneous group of heritable disorders of bone characterized by increased susceptibility to fracture. Most of the causative mutations were identified in patients with the lethal form of the disease. Attention is now shifting to the milder forms of OI where glycine substitutions and null producing mutations have been found. Single amino acid substitutions can be identified by RT/PCR of total cellular RNA, but this approach does not work well for null mutations since the defective transcript does not accumulate in the cytoplasm. We have altered our RNA extraction method to separate RNA from the nuclear and cytoplasmic compartments of cultured fibroblasts. Standard methods of mutation identification (RT/PCR followed by SSCP) is applied to each RNA fraction. DNA from an abnormal band on the SSCP gel is eluted and amplified by PCR for cloning and sequencing. Using this approach we have identified an Asp to Asn change in exon 50 (type II OI) and a Gly to Arg in exon 11 (type I OI) of the COL1A1 gene. These changes were found in both nuclear and cytoplasmic compartments. These putative mutations are currently being confirmed by protein studies. In contrast, three patients with mild OI associated with reduced {proportional_to}(I)mRNA, had distinguishing SSCP bands present in the nuclear but not the cytoplasmic compartment. In one case a frame shift mutation was observed, while the other two revealed polymorphisms. The compartmentalization of the mutant allele has directed us to look elsewhere in the transcript for the causative mutation. This approach to mutation identification is capable of distinguishing these fundamentally different types of mutations and allows for preferential cloning and sequencing of the abnormal allele.},
doi = {},
url = {https://www.osti.gov/biblio/134302}, journal = {American Journal of Human Genetics},
number = Suppl.3,
volume = 55,
place = {United States},
year = {Thu Sep 01 00:00:00 EDT 1994},
month = {Thu Sep 01 00:00:00 EDT 1994}
}