Expansion of a CGG triplet repeat leading to DNA methylation and silencing of the FMR1 gene is the most frequent cause of fragile X syndrome. However, other mutations within the FMR1 gene have also been identified that cause fragile X syndrome. These include deletions, point mutations that disrupt RNA splicing and a missense mutation. EGL Genetics offers full gene sequencing to detect mutations other than CGG expansion as a cause of fragile X syndrome.
Sequencing of the FMR1 gene will only be done if the patient first tests negative for expansion of the CGG tract and FMR1 DNA methylation. The FMR1 gene consists of 17 exons. These coding exons, as well as the immediate flanking regions, are PCR amplified and sequenced in both forward and reverse strands. In addition, the entire FMR1 promoter, including the four known transcription factor binding sites and the transcription initiation site, are assessed by DNA sequencing. This analysis will therefore detect coding sequence changes, splice donor and acceptor site mutations and changes in the promoter sequence. In addition, both small and large deletions will be detected in males. Small deletions will also be detected in females, although larger deletions of the entire gene potentially could escape detection.
It is important to note that testing for expansion of the CGG tract and FMR1 DNA methylation alone does not rule out a diagnosis of fragile X syndrome or involvement of FMR1 in the patient's phenotype. Specialized consultation is available with Dr. Stephen Warren, an authority on FMR1, on the interpretation of missense mutations.
For patients with mutations not identified by full gene sequencing, a separate deletion/duplication assay is available using a targeted CGH array KQ.
Sequencing of the FMR1 gene will only be done if the patient first tests negative for expansion of the CGG tract and FMR1 DNA methylation. The FMR1 gene consists of 17 exons. These coding exons, as well as the immediate flanking regions, are PCR amplified and sequenced in both forward and reverse strands. In addition, the entire FMR1 promoter, including the four known transcription factor binding sites and the transcription initiation site, are assessed by DNA sequencing.
This test uses sequence analysis of the coding region of the FMR1 gene which is estimated to identify 90-95% of mutations. Mutations in the promoter region, some mutations in the introns, and other regulatory elements cannot be detected by this analysis. Large deletion and insertion mutations will not be detected by this assay. It is possible that some patients with a typical presentation may not carry a mutation detected by this analysis. This analysis may detect novel variants of unclear effect, which may require further studies.
Infants and Young Children (<2 years of age): 2-3 ml
Children > 2 years of age to 10 years old: 3-5 ml
Older Children & Adults: 5-10 ml
Autopsy: 2-3 ml unclotted cord or cardiac blood
Orangene™ Saliva Collection Kit used according to manufacturer instructions. Please contact EGL for a Saliva Collection Kit for patients that cannot provide a blood sample.
Isolation using the Perkin Elmer™Chemagen™ Chemagen™ Automated Extraction method or Qiagen™ Puregene kit for DNA extraction is recommended.
Submit copies of diagnostic biochemical test results with the sample. Sequence analysis is required before deletion/duplication analysis by targeted CGH array. If sequencing is performed outside EGL Genetics, please submit a copy of the sequencing report with the test requisition. Contact the laboratory if further information is needed.
- For Fragile X testing, CGG repeat analysis is the recommended first tier test. Sequencing and deletion/duplication analysis are also available and should follow CGG repeat analysis.
- FMR1 full gene sequencing is not recommended as a single-gene test due to the unlikelihood of a positive result. It is recommended that healthcare providers consider testing for FMR1 point mutations as part of a larger panel, such as an autism spectrum disorders or premature ovarian failure panel, depending on clinical indication. EGL offers panel testing for FMR1 sequencing and CNV analysis.