Available Assays

Routine clinical testing for FMR1 mutations includes molecular assessment of both the trinucleotide repeat number and the FMR1 methylation status. Standard tech-

EcoRI

HindllL_

Methylation sensitive enzyme recognition sites:

Eagl

EcoRI Hindlll M

exon 1 —f PCR <Primers a exon 1 —f PCR <Primers a

Figure 6-1. Repeat expansion and methylation in FMR1. (a) Restriction enzyme map of FMR1, with locations of restriction enzyme sites, DNA probe, and PCR primers used in molecular testing. Top line indicates DNA fragments generated using EcoR I and Eag I as depicted in the Southern blot analysis in Figure 6-1b (2.8 kb and 5.2 kb fragments are detected by the DNA probe StB12.3). The vertical arrow indicates the location of the CGG repeat in exon 1.(b) Southern blot analysis of FMRI.Only the 2.8 kb fragment is detected in normal males (lane 1), while both the 2.8kb and 5.2 kb fragments are detected in normal females (lane 2) due to methylation associated with normal X inactivation. Completely methylated full mutations are depicted in lane 3 (affected male with full mutation) and lane 4 (affected female with full mutation contained on one of her X chromosomes; normal allele on her other X chromosome). Smeary signals occur due to variable repeat expansion within peripheral lymphocytes used for DNA isolation. Mosaic patterns are illustrated in lane 5 (male with partial methylation of full mutation), lane 6 (male with premutation/full mutation mosaicism), and lane 7 (female with premuta-tion/full mutation mosaicism). Lane 8 illustrates a transmitting male with a premutation and lane 9 illustrates a female with a premutation. Both premutations contain approximately 75 repeats. (c) PCR analysis of FMR1 repeats from five individuals separated on a 6% polyacrylamide gel. Lane 1 contains PCR products from a female with 20 and 30 repeats,respectively, contained within her two normal alleles. Lanes 2,3,and 5 are males with normal repeat alleles (40,30, and 20 repeats, respectively), while lane 4 illustrates a male with a 65-repeat premutation allele. Smeary signals result from DNA polymerase stuttering during the PCR amplification.

nical approaches include (1) double-digest Southern blot analysis using a methylation-sensitive restriction enzyme such as Eag I, BssH II, or Nru I along with a methylation-insensitive restriction enzyme such as EcoR I or Hind III4 and (2) polymerase chain reaction (PCR) assays specific for the CGG repeat segment of FMR1 (Figure 6-1).5 Specialized fragile X chromosome analysis, using special culture techniques to induce fragile sites, is no longer used for diagnosis of FXS due to low sensitivity. While only a very few FXS patients with point mutations in FMR1 have been identified, clinical molecular testing does not routinely investigate the gene for point mutations, deletions, insertions, or inversions downstream of the repeat segment.

In most laboratory settings PCR is used to size normal and premutation alleles with typical sensitivity up to 120 to 150 repeats. PCR product yield is inversely proportional to the number of trinucleotide repeats such that little or no product can be obtained when larger repeats are present. Some PCR-based testing protocols may have higher sensitivity regarding detection of larger repeats, yet few laboratories have adopted these practices due to technical difficulties. When used in conjunction with PCR, Southern blot analysis provides a more complete inspection of the gene by detecting multiple possible molecular events, including repeat expansion, DNA methylation, and the relatively rare FMR1 deletions around the trinucleotide repeat segment. Although it is not routinely performed in most clinical laboratory settings, a few laboratories utilize protein-based testing for FMRP. Since severity of the FXS phenotype appears to inversely correlate with FMRP expression, assessment of FMRP production in patients with methylation mosaicism may be a useful prognostic indicator of disease severity.6

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