An enhanced polymerase chain reaction assay to detect pre- and full mutation alleles of the fragile X mental retardation 1 gene

Abstract

Several diagnostic strategies have been applied to the detection of FMR1 gene repeat expansions in fragile X syndrome. Here, we report a novel polymerase chain reaction-based strategy using the Expand Long Template PCR System (Roche Diagnostics, Mannheim, Germany) and the osmolyte betaine. Repeat expansions up to approximately 330 CGGs in males and up to at least approximately 160 CGGs in carrier women could be easily visualized on ethidium bromide agarose gels. We also demonstrated that fluorescence analysis of polymerase chain reaction products was a reliable tool to verify the presence of premutation and full mutation alleles both in males and in females. This technique, primarily designed to detect premutation alleles, can be used as a routine first screen for expanded FMR1 alleles.

Figure 1

Effect of betaine concentration on the PCR amplification of the CGG repeat region of the FMR1 gene. Three male subjects, carrying 20, 83, and ∼200 CGG repeats, were tested with increasing concentrations of betaine, from 1.3 to 2.2 mol/L. M, 100-bp ladder molecular weight marker; three marker bands of 300, 500, and 1000 bp are indicated.

Figure 2

Detection of PCR products from the amplification of the CGG region of the FMR1 gene. Five microliters of PCR product was loaded per lane: a, samples male; b, female samples. CGG genotype is indicated below each lane. M1, 100-bp ladder molecular weight marker. M2, molecular weight marker Hi-Low DNA Marker (Bionexus, Oakland, CA).

Figure 3

Automated fluorescence analysis of the PCR products for premutation and full mutation subjects. PCR reactions were performed using 1.7 mol/L betaine. Panels 1 and 2, premutation (male) carriers with alleles of 83 and 90 CGG repeats, respectively. Panels 3 to 5, full mutation males; descending array of peaks visible from 240 to 500 bp. Panel 6, full mutation carrier female with 35/>400 CGG repeats; a main offscale peak corresponds to the normal 35-CGG allele, and a set of low-intensity stutter bands is generated by the full mutation. Panel 7, full mutation mosaic male; the profile is similar to that of a full mutation carrier female in panel 6.

Figure 4

PCR fragment length (abscissa) versus number of CGG repeats (ordinate). ▴, data from 18 sequenced male subjects and 10 clones (Table 2). ○, derived from the average size of the stutter bands generated in 17 premutation or full mutation male subjects.