High-resolution methylation polymerase chain reaction for fragile X analysis: Evidence for novel FMR1 methylation patterns undetected in Southern blot analyses

Liangjing Chen, Andrew G. Hadd, Sachin Sah, Jeffrey F. Houghton, Stela Filipovic-Sadic, Wenting Zhang, Paul J Hagerman, Flora Tassone, Gary J. Latham

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

Purpose: Fragile X syndrome is associated with the expansion of CGG trinucleotide repeats and subsequent methylation of the FMR1 gene. Molecular diagnosis of fragile X currently requires Southern blot analysis to assess methylation. This study describes the evaluation of a polymerase chain reaction-only workflow for the determination of methylation status across a broad range of FMR1 genotypes in male and female specimens. Methods: We evaluated a novel method that combines allele-specific methylation polymerase chain reaction and capillary electrophoresis with eight cell line and 80 clinical samples, including 39 full mutations. Methylation status was determined using a three-step workflow: (1) differential treatment of genomic DNA using a methylation-sensitive restriction enzyme; (2) polymerase chain reaction with two sets of dye-tagged primers; and (3) amplicon sizing by capillary electrophoresis. All samples were analyzed by both methylation polymerase chain reaction and Southern blot analysis. Results: FMR1 methylation status and CGG repeat sizing were accurately and reproducibly determined in a set of methylation controls and genomic DNA samples representing a spectrum of CGG repeat lengths and methylation states. Moreover, methylation polymerase chain reaction revealed allele-specific methylation patterns in premutation alleles that were unobtainable using Southern blot analysis. Conclusions: Methylation polymerase chain reaction enabled high throughput, high resolution, and semiquantitative methylation assessments of FMR1 alleles, as well as determinations of CGG repeat length. Results for all samples were concordant with corresponding Southern blot analyses. As a result, this study presents a polymerase chain reaction-based method for comprehensive FMR1 analysis. In addition, the identification of novel methylation mosaic patterns revealed after polymerase chain reaction and capillary electrophoresis may be relevant to several FMR1 disorders.

Original languageEnglish (US)
Pages (from-to)528-538
Number of pages11
JournalGenetics in Medicine
Volume13
Issue number6
DOIs
StatePublished - Jun 2011

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Southern Blotting
Methylation
Polymerase Chain Reaction
Capillary Electrophoresis
Alleles
Workflow
Trinucleotide Repeat Expansion
Fragile X Syndrome
DNA
Coloring Agents

Keywords

  • autism
  • FMR1 gene
  • fragile X
  • methylation
  • PCR

ASJC Scopus subject areas

  • Genetics(clinical)

Cite this

High-resolution methylation polymerase chain reaction for fragile X analysis : Evidence for novel FMR1 methylation patterns undetected in Southern blot analyses. / Chen, Liangjing; Hadd, Andrew G.; Sah, Sachin; Houghton, Jeffrey F.; Filipovic-Sadic, Stela; Zhang, Wenting; Hagerman, Paul J; Tassone, Flora; Latham, Gary J.

In: Genetics in Medicine, Vol. 13, No. 6, 06.2011, p. 528-538.

Research output: Contribution to journalArticle

Chen, Liangjing ; Hadd, Andrew G. ; Sah, Sachin ; Houghton, Jeffrey F. ; Filipovic-Sadic, Stela ; Zhang, Wenting ; Hagerman, Paul J ; Tassone, Flora ; Latham, Gary J. / High-resolution methylation polymerase chain reaction for fragile X analysis : Evidence for novel FMR1 methylation patterns undetected in Southern blot analyses. In: Genetics in Medicine. 2011 ; Vol. 13, No. 6. pp. 528-538.
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AU - Houghton, Jeffrey F.

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