Long-Read Sequencing

Comprehensive Analysis of Fragile X Syndrome (CAFXS)

Addressing Factors Often Overlooked in Standard Screening

Closing the Gap in Screening: The Impact of AGG Analysis

Fragile X syndrome (FXS) is considered the most common inherited cause of intellectual disability and the second most prevalent cause after Down syndrome.¹ It is primarily caused by overexpanded CGG repeats in the FMR1 gene.

While standard testing detects these CGG repeats, it often misses a crucial stability factor - AGG interruptions. The presence of AGG interruptions can reduce the likelihood of full-mutation expansion. A population-based study found that incorporating AGG interruption analysis modified the risk assessment for 83.2% of premutation carriers.² This allows for much more accurate genetic counseling.

Rate of full mutation expansions sorted by repeat size and number of AGG interruption

Information derived from reference³

Road Ahead - The Next Fragile X Testing

Comprehensive Analysis of Fragile X Syndrome (CAFXS) offers an advanced solution for FXS analysis, addressing previously overlooked factors. It offers one-stop detection of CGG/AGG repeat with >99.9% accuracy, and FMR1 variants often missed by PCR-based assays. This helps to resolve borderline and cases challenging cases, and support better informed decision-making.

Comparison: CAFXS vs. Routine Methods

Items

TP-PCR + CE

Southern blot analysis

CAFXS

CGG Repeat

At risk of misinterpretation (high number)

Low resolution

Precise (Up to 940 repeats⁴)

Allele Zyosity

✔

AGG Interruptions

Limited

✔

Methylation

✔

Proven Performance

Offers 2–4 times greater sensitivity than TP-PCR, detecting mosaicism as low as 0.5%⁴
Identified more mosaic carriers (3/62) that tested negative via TP-PCR and Southern Blot⁴
Detects critical deletions in exon 1/upstream regions where TP-PCR primers fail to bind, as well as the loss of entire FMR1⁴

When considering our send-out sequencing services:

Consultation: Contact our team for the most current test specifications.
Sample Preparation: Check sample types and shipment requirements to ensure high-quality results. Please check your local export regulations and logistics partners.
Submission: Contact Xcelom when placing an order. Include the completed Test Request and Consent Form, along with any required documents.

Sample Requirements

Peripheral Blood: 2 mL in EDTA tube

Dried Blood Spot (DBS): 3 spots, ≥ 8mm diameter each

Long-fragment gDNA

'**For prenatal cases, please include maternal sample for STR to exclude maternal contamination.

Transport Conditions

2-8℃, arrive within 72 hours

Testing Scope

Basic Panel

Determines the number of CGG repeats in the 5' UTR region of the FMR1 gene, and AGG interruption numbers and positions

Comprehensive Panel

Determines the number of CGG repeats in the 5' UTR region of the FMR1 gene, AGG interruption numbers and positions, and exon 1 deletions

Turnaround Time (TAT)

15 working days

Xcelom Limited and Berry Genomics provide an end-to-end turnkey solution for labs wishing to conduct this test in-house.

Laboratory Setup: Full consultation
Workflow Integration: SOPs, reagents, and staff training
Bioinformatics: Complete software suite for sample management and automated report generation

Platform

PacBio Sequel IIe, Vega and Revio system

Sample Type

gDNA from dried blood spot, blood, buccal swab, and amniotic fluid

Test per Batch

1-384 Tests per SMRT Cell (Mixed Batching Supported)

Operation Time

~ 67/ 74.5 hours (Hand on time: ~10.5 hours)

Automation

Supported

Coverage

Targets CGG and AGG number/position in 5' UTR region, and exon 1 deletion in the FMR1 gene

Full-length FMR1 gene analysis available upon request

The provided time is based on the PacBio Sequel IIe system and may vary across different labs and systems.

Resources

CAFXS Brochure

References:

1. Saldarriaga W, Tassone F, González-Teshima LY, Forero-Forero JV, Ayala-Zapata S, Hagerman R. Fragile X syndrome. Colomb Med (Cali). 2014;45(4):190-198.

2. Westemeyer M, Saucier J, Wallace J, et al. Clinical experience with carrier screening in a general population: support for a comprehensive pan-ethnic approach .Genet Med. 2020;22(8):1320-1328.

3. Nolin SL, Glicksman A, Ersalesi N, et al. Fragile X full mutation expansions are inhibited by one or more AGG interruptions in premutation carriers. Genet Med. 2015;17(5):358-364.

4. Liang Q, Liu Y, Liu Y, et al. Comprehensive Analysis of Fragile X Syndrome: Full Characterization of the FMR1 Locus by Long-Read Sequencing. Clin Chem. 2022;68(12):1529-1540.