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. 2010 Mar;56(3):399-408.
doi: 10.1373/clinchem.2009.136101. Epub 2010 Jan 7.

A novel FMR1 PCR method for the routine detection of low abundance expanded alleles and full mutations in fragile X syndrome

Stela Filipovic-Sadic  1 , Sachin Sah, Liangjing Chen, Julie Krosting, Edward Sekinger, Wenting Zhang, Paul J Hagerman, Timothy T Stenzel, Andrew G Hadd, Gary J Latham, Flora Tassone
Affiliations

A novel FMR1 PCR method for the routine detection of low abundance expanded alleles and full mutations in fragile X syndrome

Stela Filipovic-Sadic et al. Clin Chem. 2010 Mar.

Abstract

Background: Fragile X syndrome (FXS) is a trinucleotide-repeat disease caused by the expansion of CGG sequences in the 5' untranslated region of the FMR1 (fragile X mental retardation 1) gene. Molecular diagnoses of FXS and other emerging FMR1 disorders typically rely on 2 tests, PCR and Southern blotting; however, performance or throughput limitations of these methods currently constrain routine testing.

Methods: We evaluated a novel FMR1 gene-specific PCR technology with DNA templates from 20 cell lines and 146 blinded clinical samples. The CGG repeat number was determined by fragment sizing of PCR amplicons with capillary electrophoresis, and results were compared with those for FMR1 Southern blotting analyses with the same samples.

Results: The FMR1 PCR accurately detected full-mutation alleles up to at least 1300 CGG repeats and consisting of >99% GC character. All categories of alleles detected by Southern blotting, including 66 samples with full mutations, were also identified by the FMR1 PCR for each of the 146 clinical samples. Because all full mutation alleles in samples from heterozygous females were detected by the PCR, allele zygosity was reconciled in every case. The PCR reagents also detected a 1% mass fraction of a 940-CGG allele in a background of 99% 23-CGG allele-a roughly 5- fold greater sensitivity than obtained with Southern blotting.

Conclusions: The novel PCR technology can accurately categorize the spectrum of FMR1 alleles, including alleles previously considered too large to amplify; reproducibly detect low abundance full mutation alleles; and correctly infer homozygosity in female samples, thus greatly reducing the need for sample reflexing to Southern blotting.

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Figures

Figure 1
Figure 1. Workflow for Amplification and Detection of FMR1 amplicons using Novel Fragile X PCR Reagents
Input genomic DNA is amplified by two gene-specific primers (Fwd and Rev) in a single tube. After amplification, the products, one for each allele present in the reaction, including mosaic alleles, are resolved by CE. The resulting electropherogram is interpreted relative to a sizing ladder to determine the number of CGG repeats for each amplicon. Alternatively, the amplicons can be resolved by agarose gel electrophoresis.
Figure 2
Figure 2. Gene-specific FMR1 PCR Reagents Detect the Full Range of CGG Repeat Lengths in Both Male and Female Cell Line Genomic DNA templates
A) PCR products from male gDNA templates. CCR catalog numbers for each template is given at the top with the CCR-provided CGG repeat number below. In cases where repeat quantification was indefinite, estimates from the data were provided by AGE (in parenthesis), as referenced to the MW sizing ladder. The white triangle marks the >1,000 CGG mosaic allele in NA07862. B) PCR products from female gDNA templates. Note that the full mutation band for NA05847 was estimated to be ~420 CGG, rather than 650 CGG.
Figure 3
Figure 3. FMR1 Southern Blotting and Gene-specific FMR1 PCR Provide Consistent Representations in both the Size and Distribution of Normal and Expanded Alleles
Top, FMR1 Southern blot results for a set of 17 clinical specimens. Regions of the blot that report unmethylated and methylated alleles are indicated. The white dashed line demarcates the size threshold for >200 CGG alleles that are also methylated. Asterisks in both the Southern blot and AGE gel below the blot denote methylated full mutation alleles mirrored in the PCR results below. The triangle marks indicate 1,300 CGG (sample #54) and 1200 CGG (sample #68) alleles, as sized by both Southern blot and AGE. Bottom, corresponding FMR1 PCR results as resolved by AGE. The colored bars on the sides of the gel image indicate the size of FMR1 amplicons by allele category. Note that the methylation state of some alleles as revealed by Southern blot explains differences in band mobility compared to AGE (particularly #56, 58, and 67).
Figure 4
Figure 4. Representative AGE and CE Profiles of Normal, Intermediate, Premutation, and Full Mutation FMR1 PCR Amplicons from Male and Female Clinical Specimens
A) Comparisons of AGE and CE data across all categories of FMR1 alleles. The black vertical line indicates the threshold between a normal, intermediate, or premutation amplicon (left of line) and full mutation amplicon (right of line). “Int,” Intermediate. B) CE electropherogram of a PCR Process Control comprised of 4 cell line gDNA templates of 20, 29, 31, 54, 119, and 199 CGG repeats.
Figure 4
Figure 4. Representative AGE and CE Profiles of Normal, Intermediate, Premutation, and Full Mutation FMR1 PCR Amplicons from Male and Female Clinical Specimens
A) Comparisons of AGE and CE data across all categories of FMR1 alleles. The black vertical line indicates the threshold between a normal, intermediate, or premutation amplicon (left of line) and full mutation amplicon (right of line). “Int,” Intermediate. B) CE electropherogram of a PCR Process Control comprised of 4 cell line gDNA templates of 20, 29, 31, 54, 119, and 199 CGG repeats.
Figure 5
Figure 5. Gene-specific FMR1 PCR Enables Detection of Low Abundance Expanded Alleles
A) FMR1 Southern blot data for 5 clinical specimens. The threshold for full mutation (FM, >200 CGG) unmethylated and methylated alleles is designated by the black dotted line. Note that the expanded alleles represented in samples #118 and #125 were very faint, but these were identified as full mutations. B) Corresponding FMR1 PCR data, as visualized by AGE. The threshold for a full mutation (FM) is indicated by the white dotted line. C) Corresponding FMR1 PCR data, as visualized by CE. The threshold for a full mutation (FM) is marked with the black vertical line. To facilitate data interpretation, the Y-axis scale was set to best suit the allele representations for each specimen: #22 (150 RFU), #101 (200 RFU), #63 (1000 RFU), #118 (1100 RFU), and #125 (1100 RFU). Note that the full mutations in #118 and #125 are readily discerned.
Figure 6
Figure 6. Gene-specific FMR1 PCR is 5-fold More Sensitive than Southern Blotting for the Detection of a Defined Full Mutation Allele
A total of 7 ug of gDNA was input into the Southern blot, and 40 ng gDNA into PCR. The limit of detection for both methods, expressed as percent 940 CGG full mutation allele in a background of excess 23 CGG allele and as revealed in the original autoradiograph or gel image, is marked with the asterisk.
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