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. 2010 Sep;12(5):589-600.
doi: 10.2353/jmoldx.2010.090227. Epub 2010 Jul 8.

An information-rich CGG repeat primed PCR that detects the full range of fragile X expanded alleles and minimizes the need for southern blot analysis

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

An information-rich CGG repeat primed PCR that detects the full range of fragile X expanded alleles and minimizes the need for southern blot analysis

Liangjing Chen et al. J Mol Diagn. 2010 Sep.

Abstract

(CGG)(n) repeat expansion in the FMR1 gene is associated with fragile X syndrome and other disorders. Current methods for FMR1 molecular testing rely on Southern blot analysis to detect expanded alleles too large to be PCR-amplified and to identify female homozygous alleles that often confound interpretations of PCR data. A novel, single-tube CGG repeat primed FMR1 PCR technology was designed with two gene-specific primers that flank the triplet repeat region, as well as a third primer that is complementary to the (CGG)(n) repeat. This PCR was evaluated with 171 unique DNA samples, including a blinded set of 146 clinical specimens. The method detected all alleles reported by Southern blot analysis, including full mutations in 66 clinical samples and comprised up to 1300 CGG. Furthermore, a blinded cohort of 42 female homozygous and heterozygous specimens, including 21 with full mutation alleles, was resolved with 100% accuracy. Last, AGG interrupter sequences, which may influence the risk of (CGG)(n) expansion in the children of some carriers, were each correctly identified in 14 male and female clinical samples as referenced to DNA sequencing. As a result, this PCR provides robust detection of expanded alleles and resolves allele zygosity, thus minimizing the number of samples that require Southern blot analysis and producing more comprehensive FMR1 genotyping data than other methods.

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Figures

Figure 1
Figure 1
Workflow for amplification and detection of FMR1 amplicons using a three-primer FMR1 PCR. Input gDNA is amplified by two gene-specific primers (forward [Fwd] and reverse [Rev]) and a CGG repeat primer in a single tube. After amplification, the products, which include the full-length amplicon that completely encompasses the triplet repeat region and a multiplicity of CGG repeat primed products, are resolved by CE. The resulting electropherogram supports quantification of the number of CGG repeats, determination of the allele zygosity, and the sequence context of any AGG spacer elements.
Figure 2
Figure 2
CGG repeat primed PCR produces both full-length gene-specific FMR1 amplicons as well as triplet repeat-specific products that support absolute repeat quantification. A: Comparison of gene-specific PCR (agarose gel image) and repeat primed PCR (CE) of male cell line gDNA templates. Gene-specific PCR was performed as described previously.B: Comparison of gene-specific PCR (agarose gel image) and repeat primed PCR (CE) of female cell line gDNA templates. Inset: graphs offer a higher resolution view of the underlying data (see arrows). Determination of the number of CGG repeats was provided through absolute quantification, and compared with fragment sizing of the full-length amplicon (Table 1). The Coriell Cell Repositories catalog number for each template is provided on each electropherogram. The CGG number indicated with the corresponding arrow is the repeat number determined by counting the triplet repeat product peaks, whereas the number placed by the prominent gene-specific peak is the repeat number computed from fragment sizing of the full-length amplicon. PM or FM, gene-specific amplicons corresponding to the amplification of premutation or full mutation alleles, respectively.
Figure 3
Figure 3
Heterozygous female alleles produce an uninterrupted series of triplet repeat products that indicate the presence of a longer FMR1 allele and thus are readily distinguished from homozygous alleles. Note that PCR/CE of homozygous alleles (top) lack the characteristic and reproducible repeat primed signature (inset) of heterozygous alleles (bottom). FM, gene-specific PCR products that result from amplification of full mutation alleles.
Figure 4
Figure 4
The three-primer repeat primed PCR reports the presence of full mutation alleles even in the absence of a full-length amplicon. Fwd, forward; Rev, reverse.
Figure 5
Figure 5
The CGG RP PCR product profile can reveal both the number and sequence context of AGG elements that interrupt the CGG repeat segment. Top: Schematic representation of the impact of defined AGG sequences on the CGG RP PCR product profile for a male 30 CGG allele. Interrupting AGG elements are associated with a loss of product accumulation (and associated signal intensity) for those amplicons whose corresponding CGG primer contacts a templated AGG. Bottom: Annotation of an capillary electropherogram of a male 30 CGG allele with both CGG repeats, and the two interfering AGG sequences.
Figure 6
Figure 6
Identification of AGG interspersions in both male and female FMR1 templates. A: Capillary electropherogram and corresponding sequence context of AGG elements in male FMR1 alleles. The CGG repeat number determined from fragment sizing of the full-length, gene-specific peak is indicated for each gDNA template. B: Electropherogram and corresponding sequence context of AGG elements in female FMR1 alleles. The arrows mark the last CGG repeat associated with each of the two FMR1 alleles for each set of female samples. If the interpretation of the AGG sequence is ambiguous, more than one sequence possibility is given.
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