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* These authors contributed equally
An accurate and robust polymerase chain reaction-based assay for quantifying cytosine-guanine-guanine trinucleotide repeats in the Fragile X mental retardation-1 gene facilitates molecular diagnosis and screening of Fragile X syndrome and Fragile X-related disorders with shorter turn-around time and investment in equipment.
Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5’ untranslated region (UTR) of the Fragile X mental retardation-1 (FMR1) gene promoter. Conventionally, capillary electrophoresis fragment analysis on a genetic analyzer is used for the sizing of the CGG repeats of FMR1, but additional Southern blot analysis is required for exact measurement when the repeat number is higher than 200. Here, we present an accurate and robust polymerase chain reaction (PCR)-based method for quantification of the CGG repeats of FMR1. The first step of this test is PCR amplification of the repeat sequences in the 5’UTR of the FMR1 promoter using a Fragile X PCR kit, followed by purification of the PCR products and fragment sizing on a microfluidic capillary electrophoresis instrument, and subsequent interpretation of the number of CGG repeats by referencing standards with known repeats using the analysis software. This PCR-based assay is reproducible and capable of identifying the full range of CGG repeats of FMR1 promoters, including those with a repeat number of more than 200 (classified as full mutation), 55 to 200 (premutation), 46 to 54 (intermediate), and 10 to 45 (normal). It is a cost-effective method that facilitates classification of the FXS and Fragile X-associated disorders with robustness and rapid reporting time.
Fragile X syndrome (FXS) and Fragile X associated disorders, e.g., tremor and ataxia syndrome (FX-TAS), and primary ovarian insufficiency (FX-POI) are mainly caused by cytosine-guanine-guanine (CGG) trinucleotide repeat expansion in the 5’ untranslated region (UTR) of the Fragile X mental retardation-1 (FMR1) gene on Xq27.31,2. The FMR1 encoded protein (FMRP) is a polyribosome-associated RNA-binding protein that functions in neuronal development and synaptic plasticity by regulating alternative splicing, stability, and dendritic transport of mRNA or modulating synthesis of partial postsynaptic proteins3,4,5,6,7.
The dynamic variation with a CGG repeat size of >200 is described as full mutation, which induces the aberrant hypermethylation and subsequent transcriptional silencing of the FMR1 promoter8. The resulting absence or lack of the FMRP protein disrupts normal neuronal development and causes FXS9, characterized by various clinical symptoms, including moderate to severe intellectual disability, developmental delay, hyperactive behaviors, poor contacts and autistic manifestations10,11,12. The presentation in female FXS patients is generally milder than that in males. The CGG repeat size ranging from 55 to 200 and 45 to 54 are classified as premutation and intermediate status, respectively. Due to the high degree of instability, the CGG repeat size in a premutation or intermediate allele presumably expands when transmitted from parents to offspring13,14. Thus, carriers with premutation alleles are at high risk of having children affected with FXS because of the repeat expansion, and in some cases, intermediate alleles can expand their repeat size to the full mutation range over two generations15,16. Furthermore, males with premutation also convey increased risk of developing late-onset FX-TAS17,18,19, while premutation females are predisposed for both FX-TAS and FX-POI20,21,22. Recently, it has been reported that autistic spectrum disorders with developmental delay and problems in social behaviors are presented in children with premutation FMR1 alleles23,24.
To determine the exact CGG repeat size is of great significance for classification and prediction of the FXS and Fragile X-associated disorders25,26. Historically, the CGG repeat region-specific polymerase chain reaction (PCR) with fragment sizing plus Southern blot analysis have been the gold standard for molecular profiling of the FMR1 CGG repeat27. However, traditional specific PCR is less sensitive to large premutations with more than 100 to 130 repeats and is incapable of amplifying full mutations27,28. Furthermore, capillary electrophoresis on a traditional genetic analyzer for repeat sizing fails to detect FMR1 PCR products with more than 200 CGG repeats. The Southern blot analysis enables differentiation of a wider range of repeat size, from normal to full mutation repeat numbers, and has been widely used for confirming full mutations (in males) and differentiating heterozygous alleles with a full mutation from apparently homozygous alleles with normal repeat sizes (in females). However, the resolution for quantifying the repeats is limited. More importantly, this step-by-step testing strategy is labor-intensive, time-consuming, and cost-ineffective.
Here, we present an accurate and robust PCR-based method for quantification of the CGG repeats of FMR1. The first step of this test is PCR amplification of the repeat sequences in the 5’UTR of the FMR1 promoter using Fragile X PCR kit. The PCR products are purified and fragment sizing is performed on a microfluidic capillary electrophoresis instrument, and subsequent interpretation of the number of CGG repeats using the analysis software by referencing standards with known repeats based on the rationale that PCR fragment length is directly proportional to the number of CGG repeats. The PCR system include reagents that facilitate the amplification of the highly GC-rich trinucleotide repeat region. This PCR-based assay is reproducible and capable of identifying all ranges of CGG repeats of FMR1 promoters. This is a cost-effective method that can find wide application in molecular diagnosis and screening of FXS and Fragile X-related disorders with less turn-around time and investment in equipment and thus, can be utilized in a broader spectrum of clinical laboratories.
Ethical approval was granted by the Joint Chinese University of Hong Kong―New Territories East Cluster Clinical Research Ethics Committee (Reference Number: 2013.055)
1. PCR amplification
2. Purification of the PCR Products
3. Fragment Sizing of PCR Products
4. Analyze the Fragment Sizing Results
NOTE: The reference samples should be amplified and analyzed by the same thermal cycler and bioanalyzer in the same batch with the unknown samples.
The sizing results of the premutation female reference sample (NA20240, repeat sizes of 30 and 80) and the full mutation female reference sample (NA20239, repeat sizes of 20 and 200) are shown in Figure 1A and Figure 1B, respectively. Typically, two marker peaks (lower marker 50 base pairs [bp] and upper marker 10,380 bp) are included in the fragment size profile. There is usually a primer complex peak with a size of nearly 95 bp. Through the reference sample, a...
FXS is the second most common cause of intellectual impairment after trisomy 21, accounting for nearly one-half of X-linked mental retardation30, which may affect approximately 1 in 4,000 males and 1 in 8,000 females. More importantly, nearly 1 in 250–1,000 females carry a premutation, and this frequency is 1 in 250–1,600 in males26,31,32,33. Since the risk o...
The authors have nothing to disclose.
This research was supported by grants from NSFC Emergency Management Project (Grant No. 81741004), the National Natural Science Foundation of China (Grant No. 81860272), the Major Research Plan of the Provincial Science and Technology Foundation of Guangxi (Grant No. AB16380219), the China Postdoctoral Science Foundation Grant (Grant No. 2018M630993), and the Guangxi Natural Science Foundation (Grant No. 2018GXNSFAA281067).
Name | Company | Catalog Number | Comments |
Agilent 2100 Bioanalyzer instrument: 0.2 mL PCR tubes | Axygen | PCR-02D-C | |
Agilent 2100 Bioanalyzer instrument: 1X TE buffer, pH 8.0, Rnase-free | Ambion | AM9849 | |
Agilent 2100 Bioanalyzer instrument: 2100 Bioanalyzer instrument | Agilent | G2939AA | |
Agilent 2100 Bioanalyzer instrument: 96-well PCR Plate | Thermo Fisher | AB0800 | |
Agilent 2100 Bioanalyzer instrument: Electrode cartridge | Agilent | Supplies equipment of the 2100 Bioanayzer instrument | |
Agilent 2100 Bioanalyzer instrument: IKA vortex mixer | Agilent | Supplies equipment of the 2100 Bioanayzer instrument | |
Agilent 2100 Bioanalyzer instrument: Sizing software 2100 Expert software | Agilent | Supplies equipment of the 2100 Bioanayzer instrument | |
Agilent 2100 Bioanalyzer instrument: Test chips | Agilent | Supplies equipment of the 2100 Bioanayzer instrument | |
Agilent DNA 7500 kit | Agilent | 5067-1506 | For Fragment sizing |
Agilent DNA 7500 kit: DNA 7500 Ladder (yellow cap) | Agilent | In kit: Agilent DNA 7500 kit (catalog number: 5067-1506) | |
Agilent DNA 7500 kit: DNA 7500 Markers (green cap) | Agilent | In kit: Agilent DNA 7500 kit (catalog number: 5067-1506) | |
Agilent DNA 7500 kit: DNA chips | Agilent | In kit: Agilent DNA 7500 kit (catalog number: 5067-1506) | |
Agilent DNA 7500 kit: DNA Dye Concentrate (blue cap) | Agilent | In kit: Agilent DNA 7500 kit (catalog number: 5067-1506) | |
Agilent DNA 7500 kit: DNA Gel Matrix Vial (red cap) | Agilent | In kit: Agilent DNA 7500 kit (catalog number: 5067-1506) | |
Agilent DNA 7500 kit: Electrode Cleaner | Agilent | In kit: Agilent DNA 7500 kit (catalog number: 5067-1506) | |
Agilent DNA 7500 kit: Spin Filter | Agilent | Supplies of Agilent DNA 7500 kit (catalog number: 5067-1506) | |
Agilent DNA 7500 kit: Syringe | Agilent | Supplies of Agilent DNA 7500 kit (catalog number: 5067-1506) | |
Chip priming station | Agilent | 5065-4401 | Supplies equipment of the 2100 Bioanayzer instrument |
Cubee Mini-centrifuge | GeneReach | aqbd-i | |
Filter plate vacuum Manifold: MultiScreenHTS Vacuum Manifold | Merck Millipore | MSVMHTS00 | Vacuum instrument for Filter plate vacuum Manifold for PCR product purification |
Filter plate vacuum Manifold: Silicone stopper | Merck Millipore | XX2004718 | Filter plate vacuum Manifold |
Filter plate vacuum Manifold: Vacuum pump | Merck Millipore | WP6122050 | Filter plate vacuum Manifold |
Filter plate vacuum Manifold: Waste collection vessel | Merck Millipore | XX1004705 | Filter plate vacuum Manifold |
FragilEase Fragile X PCR kit | PerkinElmer | 3101-0010 | For PCR amplification |
FragilEase Fragile X PCR kit: Sample Diluent | PerkinElmer | In kit: FragilEase Fragile X PCR kit (catalog number: 3101-0010 ) | |
FragilEase PCR Buffer mix | PerkinElmer | In kit: FragilEase Fragile X PCR kit (catalog number: 3101-0010 ), containing primers. Primer sequences: TCAGGCGCTCAGCTCCGTTTCGGTTTCA (forward) FAM-AAGCGCCATTGGAGCCCCGCACTTCC (reverse) | |
FragilEase Polymerase | PerkinElmer | In kit: FragilEase Fragile X PCR kit (catalog number: 3101-0010 ) | |
FraXsoft analysis software | PerkinElmer | ||
NanoDrop ND-2000 Spectrophotometer | Thermo Fisher | ||
Paper towels | |||
PCR clean up plate: NucleoFast 96 PCR plate | MACHEREY-NAGEL | 743100 | |
reference DNA sample | Coriell | NA20240 & NA20239 | |
S1000 96-well Thermal Cycler | Bio-Rad | 1852196 | This can be replaced by other Thermal Cyclers (eg. Veriti™ 96-Well Thermal Cycler, Applied Biosystems, catalog number: 4375786) |
TriNEST Incubator/Shaker instrument | PerkinElmer | 1296-0050 | |
UltraPure DNase/RNase-Free Distilled Water | Life Technologies | 10977015 | For 2100 Bioanalyzer electrode cleaning |
Vortex-Genie 2 | Scientific Industries | SI-0256 (Model G560E) | Conventional vortex mixer |
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