What genetic testing is recommended for a lap panel that includes Fragile X syndrome?

Genetic Testing for Fragile X Syndrome in a Laboratory Panel

For a laboratory panel that includes Fragile X syndrome testing, PCR-based methods combined with Southern blot analysis should be used as the standard approach, with newer long-read sequencing methods considered for comprehensive characterization when clinically indicated. 1

Standard Testing Methodology

First-tier Testing

• PCR-based methods:
  • Triplet repeat-primed PCR (TP-PCR) should be used as the initial screening test
  • Can accurately detect CGG repeats in normal range (5-44 repeats), intermediate range (45-54 repeats), and premutation range (55-200 repeats)
  • Can detect full mutations (>200 repeats) with sensitivity of 99%
  • Resolves female zygosity issues that often confound PCR interpretation 1, 2

Second-tier Testing

• Southern blot analysis:
  • Required for confirmation of full mutations
  • Necessary to determine methylation status
  • Uses restriction enzymes (typically EcoRI and EagI) with probes (commonly StB12.3)
  • Essential for detecting methylation mosaicism 1

Comprehensive FMR1 Analysis

For more detailed characterization, newer methods can be considered:

• Long-read sequencing approaches:
  • Detect CGG repeat expansions with higher sensitivity (0.5-1% in background of normal alleles)
  • Identify AGG interruptions within CGG repeats (important for risk assessment)
  • Detect rare intragenic variants and large deletions
  • Provide more comprehensive characterization of the FMR1 locus 3

Clinical Indications for Testing

According to ACMG and other professional guidelines:

• For autism spectrum disorder (ASD):

  • Fragile X testing is recommended as first-tier testing for males with unexplained ASD
  • Should be performed alongside chromosomal microarray (CMA) 1

• For global developmental delay/intellectual disability:

  • Fragile X testing should be first-line testing for all patients
  • Particularly important when there is:
    • Family history of Fragile X syndrome
    • Undiagnosed intellectual disability
    • Presence of dysmorphic features 1

Technical Considerations

• Sample requirements:

  • Peripheral blood is the preferred specimen
  • DNA extraction methods should preserve high molecular weight DNA for Southern blot

• Quality control:

  • Include positive controls representing different mutation categories
  • Use characterized reference materials with specific CGG repeat sizes 1

• Reporting considerations:

  • Report should classify results using defined categories:
    • Normal: 5-44 CGG repeats
    • Intermediate/gray zone: 45-54 CGG repeats
    • Premutation: 55-200 CGG repeats
    • Full mutation: >200 CGG repeats
  • Include recommendations for genetic counseling for positive results 1

Common Pitfalls to Avoid

1. Missing mosaic patterns:

   • Some patients have size mosaicism (premutation and full mutation)
   • Others have methylation mosaicism (methylated and unmethylated full mutations)
   • Both Southern blot and PCR are needed for complete characterization 1

2. Overlooking rare variants:

   • Approximately 1% of Fragile X cases are due to intragenic variants or deletions
   • Consider comprehensive sequencing when clinical suspicion is high but standard testing is negative 4

3. Misinterpreting female results:

   • Homozygous normal females can be misinterpreted without proper controls
   • TP-PCR or long-read sequencing can resolve zygosity issues 2

By implementing this comprehensive testing approach, laboratories can provide accurate diagnosis of Fragile X syndrome, which is essential for appropriate clinical management and genetic counseling.