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Title: Identification of five novel FBN1 mutations by non-radioactive single-strand conformation analysis

Abstract

Marfan syndrome (MFS), one of the most common genetic disorders of connective tissue, is characterized by variable manifestations in skeletal, cardiovascular and ocular systems. Mutations in the fibrillin gene on chromosome 15 (FBN1) have been shown to cause MFS. To examine the relationship between FBN1 gene mutations, fibrillin protein function and MFS phenotypes, we screened for alternations in the fibrillin coding sequence in fibroblast derived cDNA from MFS patients. To date, abnormally migrating bands in more than 20 unrelated MFS patients have been identified by using non-radioactive single-strand conformation analysis and silver staining. Five altered bands have been directly sequenced. Two missense mutations and three splice site mutations have been identified. Both missense mutations substitute another amino acid for a cysteine residue (C1402W and C1672R) in EGF-like motifs of the fibrillin polypeptide chain. The two splice site mutations are at nucleotide positions 6994+1 (G{yields}A), and 7205-2 (A{yields}G) and result in in-frame skipping of exon 56 and 58, respectively. Skipping of exon 56 occurs in 50% of mutant transcripts. Use of a cryptic splice site 51 bp upstream of the normal donor site results in half of the mutant transcripts containing part of exon 56. Both products contain in-frame deletions. Anothermore » splice site mutation, identified by exon screening from patient genomic DNA using intron primers, is at nucleotide position 2293+2 (T{yields}A), but the predicted exon skipping has not been detected at the RT-PCR level. This may be due to instability of the mutant transcript. Including the mutations reported here, a total of 8 out of 36 published FBN1 gene mutations involve exon skipping. It may be inferred that FBN1 exon skipping plays an important pathogenic role in MFS.« less

Authors:
; ; ;  [1]
  1. Stanford Univ. Medical Center, Stanford, CA (United States)
Publication Date:
OSTI Identifier:
134252
Report Number(s):
CONF-941009-
Journal ID: AJHGAG; ISSN 0002-9297; TRN: 95:005313-0988
Resource Type:
Journal Article
Resource Relation:
Journal Name: American Journal of Human Genetics; 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; GENETIC MAPPING; STRUCTURAL CHEMICAL ANALYSIS; SPLICING; TRANSCRIPTION; SCREENING; PATIENTS; SKELETAL DISEASES; SENSE ORGANS DISEASES; CARDIOVASCULAR DISEASES; PHENOTYPE; HUMAN CHROMOSOME 15; CONNECTIVE TISSUE; EXONS; DNA SEQUENCING; NUCLEOTIDES; AMINO ACIDS; INTRONS

Citation Formats

Liu, W., Qian, C., Comeau, K., and Francke, U. Identification of five novel FBN1 mutations by non-radioactive single-strand conformation analysis. United States: N. p., 1994. Web.
Liu, W., Qian, C., Comeau, K., & Francke, U. Identification of five novel FBN1 mutations by non-radioactive single-strand conformation analysis. United States.
Liu, W., Qian, C., Comeau, K., and Francke, U. 1994. "Identification of five novel FBN1 mutations by non-radioactive single-strand conformation analysis". United States. doi:.
@article{osti_134252,
title = {Identification of five novel FBN1 mutations by non-radioactive single-strand conformation analysis},
author = {Liu, W. and Qian, C. and Comeau, K. and Francke, U.},
abstractNote = {Marfan syndrome (MFS), one of the most common genetic disorders of connective tissue, is characterized by variable manifestations in skeletal, cardiovascular and ocular systems. Mutations in the fibrillin gene on chromosome 15 (FBN1) have been shown to cause MFS. To examine the relationship between FBN1 gene mutations, fibrillin protein function and MFS phenotypes, we screened for alternations in the fibrillin coding sequence in fibroblast derived cDNA from MFS patients. To date, abnormally migrating bands in more than 20 unrelated MFS patients have been identified by using non-radioactive single-strand conformation analysis and silver staining. Five altered bands have been directly sequenced. Two missense mutations and three splice site mutations have been identified. Both missense mutations substitute another amino acid for a cysteine residue (C1402W and C1672R) in EGF-like motifs of the fibrillin polypeptide chain. The two splice site mutations are at nucleotide positions 6994+1 (G{yields}A), and 7205-2 (A{yields}G) and result in in-frame skipping of exon 56 and 58, respectively. Skipping of exon 56 occurs in 50% of mutant transcripts. Use of a cryptic splice site 51 bp upstream of the normal donor site results in half of the mutant transcripts containing part of exon 56. Both products contain in-frame deletions. Another splice site mutation, identified by exon screening from patient genomic DNA using intron primers, is at nucleotide position 2293+2 (T{yields}A), but the predicted exon skipping has not been detected at the RT-PCR level. This may be due to instability of the mutant transcript. Including the mutations reported here, a total of 8 out of 36 published FBN1 gene mutations involve exon skipping. It may be inferred that FBN1 exon skipping plays an important pathogenic role in MFS.},
doi = {},
journal = {American Journal of Human Genetics},
number = Suppl.3,
volume = 55,
place = {United States},
year = 1994,
month = 9
}
  • Mutations in the p53 tumor-suppressor gene are commonly found in human cancers of diverse origin. Once of a number of methods developed to analyze large numbers of DNA samples for specific mutations is the single-strand conformation polymorphism (SSCP) analysis. This method is particularly well suited for analysis of tissues, such as brain tumors, with mixed cell populations. It takes advantage of the fact that, in a mixed cell population containing DNA with and without a mutation (e.g., the p53 gene mutation), both molecular species will be amplified by the PCR. A mutation within a PCR-amplified DNA fragment will alter themore » secondary structure of the amplified fragment and affect its electrophoretic mobility in a nondenaturing gel. The DNA fragments with the mutation are detected as an aberrantly migrating allele that can be seen concurrently with the wild-type allele. Although many studies have used this technique to screen for p53 mutations in tumors, with detection of a number of different mutations the limit of detection of point mutations in a background of wild-type DNA is not known. To test this, mixtures of mutant DNA from tumor D317 with a G-to-A point mutation in codon 272 of the p53 gene; or from tumor D263 (with a G-to-A point mutation in codon 175 of the p53 gene) and wild-type DNA from leukocytes, in ratios of 1:100, 5:95, 10:90, 15:85, 50:50, and 30:70, were prepared. The mixtures containing 100 ng of DNA were amplified using standard PCR technique. After the double-stranded DNAs were denatured, the DNA samples were loaded and electrophoresed on a nondenaturing acrylamide gel. The mutant allele was detectable even when the ratio of mutant to wild-type DNA was 5:95 in tumor D317. For tumor D263, the mutant allele was detectable when the ratio of mutant to wild-type DNA was 15:85, and it was not detectable at 10:90.« less
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