Qualitative assessment of FMR1 (CGG)n triplet repeat status in normal, intermediate, premutation, full mutation, and mosaic carriers in both sexes: implications for fragile X syndrome carrier and newborn screening.
OBJECTIVE
Fragile X syndrome is caused by expansion and subsequent methylation of a CGG trinucleotide repeat in the FMR1 5'-untranslated region. Southern blot analysis is typically required to determine expansion size for triplet repeat lengths >200. We describe a triplet-primed polymerase chain reaction-based method using automated capillary electrophoresis detection for qualitative assessment of expanded CGG repeats.


METHODS
The assay uses triplet-primed polymerase chain reaction in combination with GC-melting reagents and substitution of 7-deaza-2-deoxyGTP for dGTP. Amplicons are resolved by capillary electrophoresis.


RESULTS
A distinctive pattern of tapering or "stutter" polymerase chain reaction amplification was evident on capillary electrophoresis in male and female patients harboring all expanded allele lengths examined (up to 2000 CGG repeats) and could be used to differentiate normal, intermediate, premutation, and full mutation alleles. Full mutation alleles exhibited an additional late-migrating amplicon on capillary electrophoresis. Mixing experiments demonstrated sensitivity as low as 1% for detection of the full mutation allele. In a 1275-sample concordance study against our existing polymerase chain reaction platform (with Southern blot analysis for repeat lengths ≥55), the triplet-primed polymerase chain reaction method exhibited 100% concordance for normal, intermediate, expanded, and full mutation alleles. This method also detected the full mutation alleles in DNA isolated from blood spots.


CONCLUSIONS
This assay provides an accurate assessment of FMR1 repeat status and holds promise for use in carrier and newborn screening. The method distinguishes normal homozygous females from full mutation carrying females. Although the method is not useful for accurate sizing, it supplements the classic polymerase chain reaction method and results in significant reduction in the number of Southern blot analyses required to be performed in the laboratory to accurately assess the FMR1 genotype in all individuals.
Hantash
Feras M
FM
Department of Molecular Genetics, Nichols Institute, Quest Diagnostics, San Juan Capistrano, California 92690, USA. Feras.X.Hantash@questdiagnostics.com
Goos
Dana G
DG
Tsao
David
D
Quan
Franklin
F
Buller-Burckle
Arlene
A
Peng
Mei
M
Jarvis
Michael
M
Sun
Weimin
W
Strom
Charles M
CM
D020121:5' Untranslated Regions / Q000235:genetics; D015139:Blotting, Southern; D004247:DNA / Q000235:genetics; D019075:Electrophoresis, Capillary; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D005600:Fragile X Syndrome / Q000175:diagnosis* / Q000235:genetics*; D005820:Genetic Testing; D006579:Heterozygote; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D009030:Mosaicism; D009154:Mutation; D015997:Neonatal Screening; D016133:Polymerase Chain Reaction; D013673:Technology Assessment, Biomedical; D018911:Trinucleotide Repeats
D020121:5' Untranslated Regions; D004247:DNA; D051860:Fragile X Messenger Ribonucleoprotein 1; C497683:FMR1 protein, human