Method

Cytosine-guanine-guanine (CGG) triplet repeats in the 5' untranslated region (5' UTR) of the FMR1 gene are detected by triplet repeat-primed PCR (RP-PCR) with fluorescently labeled primers followed by capillary electrophoresis in genomic DNA obtained from the submitted sample. Reference ranges: Normal: <45 CGG repeats, intermediate: 45-54 CGG repeats, premutation: 55-200 CGG repeats, full mutation: >200 CGG repeats. For alleles with 55-90 triplet repeats, the region surrounding the FMR1 repeat is amplified by PCR. The PCR amplicons are then processed through PacBio SMRTBell library prep and sequenced using PacBio long read technology. The number of AGG interruptions within the 55-90 triplet repeat is read directly from the resulting DNA sequences. Testing is performed by Invitae Corporation (1400 16th Street, San Francisco, CA 94103, #05D2040778) a College of American Pathologists (CAP)-accredited and Clinical Laboratory Improvement Amendments (CLIA)-certified clinical diagnostic laboratory.

Limitations

FMR1 testing is limited to repeat expansion analysis only, and does not include coding region sequence, CNV analysis or FMR1 methylation. Sizing accuracy is expected to be +/-1 for CGG repeat alleles less than or equal to 90 repeat units and +/-3 for CGG repeat alleles greater than 90 repeat units. If the two CGG repeat counts listed are the same, it most likely indicates homozygosity; however, in very rare scenarios it could be the result of biological or technical reasons including, but not limited to, sex chromosome anomalies, allelic dropout, or sample submission errors. This test is not intended to diagnose sex chromosome aneuploidy, although evidence of such incidental findings may be present in the analysis and reported. The number of AGG interruptions is only determined for females ≥12 years of age with triplet repeat sizes of 55-90. Due to somatic mosaicism and/or repeat instability of expanded alleles, repeat size identified in DNA isolated from peripheral blood, buccal cells, or saliva may not reflect the repeat size in untested tissues (e.g. brain, gonads). In addition, a negative result does not definitively rule out the presence of an expansion in the mosaic state, as the current test is not validated to detect low-level mosaic variants. This report reflects the analysis of an extracted genomic DNA sample. While this test is intended to reflect the analysis of extracted genomic DNA from a referred patient, in very rare cases the analyzed DNA may not represent that individual's constitutional genome, such as in the case of a circulating hematolymphoid neoplasm, bone marrow transplant, blood transfusion, chimerism, culture artifact or maternal cell contamination.