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Title: MDE heteroduplex analysis of PCR products spanning each exon of the fibrillin (FBN1) gene greatly increases the efficiency of mutation detection in the Marfan syndrome

Defects in fibrillin (FNB1) cause the Marfan syndrome (MFS). Classic Marfan phenotype cosegregates with intragenic and/or flanking marker alleles in all families tested and a significant number of FBN1 mutations have been identified in affected individuals. Using a standard method of mutation detection, SSCP analysis of overlapping RT-PCR amplimers that span the entire coding sequence, the general experience has been a low yield of identifiable mutations, ranging from 10-20%. Possible explanations included low sensitivity of mutation screening procedures, under-representation of mutant transcript in patient samples either due to deletions or mutant alleles containing premature termination codons, clustering of mutations in yet uncharacterized regions of the gene, including regulatory elements, or genetic heterogeneity. In order to compensate for a potential reduced mutant transcript stability, we have devised a method to screen directly from genomic DNA. The intronic boundaries flanking each of the 65 FBN1 exons were characterized and primer pairs were fashioned such that all splice junctions would be included in the resultant amplimers. The entire gene was screened for a panel of 9 probands with classic Marfan syndrome using mutation detection enhancement (MDE) gel heteroduplex analysis. A mutation was identified in 5/9 (55%) of patient samples. All were either missensemore » mutations involving a cysteine residue or small deletions that did not create a frame shift. In addition, 10 novel polymorphisms were found. We conclude that the majority of mutations causing Marfan syndrome reside in the FBN1 gene and that mutations creating premature termination codons are not the predominant cause of inefficient mutation detection using RT-PCR. We are currently modifying screening methods to increase sensitivity and targeting putative FBN1 gene promoter sequences for study.« less
Authors:
;  [1] ; ;  [2]
  1. Johns Hopkins Univ. School of Med., Baltimore, MD (United States)
  2. Mount Sinai School of Med., New York, NY (United States)
Publication Date:
OSTI Identifier:
134268
Report Number(s):
CONF-941009--
Journal ID: AJHGAG; ISSN 0002-9297; TRN: 95:005313-1004
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; TRANSCRIPTION; SPLICING; DETECTION; SCREENING; HEREDITARY DISEASES; PATIENTS; PHENOTYPE; POLYMERASE CHAIN REACTION; EFFICIENCY; SENSITIVITY; STABILITY; CONNECTIVE TISSUE; EXONS; BIOLOGICAL MARKERS; DNA SEQUENCING; CODONS; INTRONS; GENE REPRESSORS