A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene

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 males^26,31,32,33. Since the risk of CGG repeat expansion to full mutations when transmitting premutation alleles to offspring dramatically elevates, for example, from 4% when the maternal repeat size is 55–59 to 98% when the size is 100–200¹⁴, the determination of the CGG repeat size at a wider range could facilitate diagnosis of FXS and screening of premutation carriers for their reproductive planning. The PCR-based method introduced here is accurate, rapid and robust to amplify the repeat sequences in the 5’UTR of the FMR1 promoter and quantify the full spectrum of CGG repeat numbers on a microfluidic capillary electrophoresis instrument, and thus can boost its wide application in molecular diagnosis and screening of FXS and Fragile X-related disorders with less turn-around time and investment in equipment. The bioanalyzer instrument can be confidently utilized in low to moderate throughput test settings for repeat size measurement. The equipment is much smaller, less costly and simpler to maintain than other capillary electrophoresis instruments, such as ABI capillary genetic analyzers. For high volume screening, the MultiDX system containing up to 384-sample model provides an extensive flexibility and throughput for the tests.

The assay includes four main steps: PCR amplification of the repeat sequences in the 5’UTR of the FMR1 promoter (PCR set-up and PCR amplification takes nearly 3.5 h), purification of the PCR products (takes nearly 1 h), fragment sizing on a microfluidic capillary electrophoresis instrument (takes nearly 1 h), and interpretation of the number of CGG repeats using the analysis software by inferring from the reference standards with known repeats (takes nearly 0.5 h). In total, the turn-around time of this assay is approximately 6 h. For optimal performance, DNA samples should be purified to remove putative interfering substances such as proteins and high salt concentrations. Additionally, the recommended amount of input DNA is 50–100 ng per 20 µL of PCR reaction. DNA amounts greater than 150 ng per 20 µL of the PCR system have been shown to result in poor amplification of large repeat alleles. Furthermore, it is strongly recommended to set up at least two reference samples with well characterized repeat sizes in each PCR run for simultaneous analysis of repeat numbers as quality control and subsequent automated fragment sizing in the analysis software²⁹. Typically, the R² value of the linear regression curve derived from the reference samples should be greater than 0.98. In addition, the PCR products should be purified before microfluidic capillary electrophoresis to improve detection efficiency. As the PCR primer is labeled with FAM fluorescence²⁹, fragment sizing with an appropriate electrophoresis system (e.g., bioanalyzer and the MultiDX system) can be directly performed without additional labeling.

A variety of methodologies for the diagnosis and study of FXS and related disorders have been comprehensively reviewed by Bruce E. Hayward et al., including DNA-based assays and FMRP protein assays³⁴. As in most cases the molecular basis of FXS is dynamic mutation characterized by an expansion CGG repeat in the promoter region of FMRP1 gene, Southern blotting and amplification-based assays by using genomic DNA are the most commonly used assays for determination of the repeat number. It is well known that PCR-based assays overweigh Southern blotting in cost-effectiveness and minimum DNA amount requirement. However, amplification of the CGG-repeat is a main challenge since high GC content can affect the efficiency, and much effort has been made for the optimization of the PCR system over the years³⁴. The fragile X PCR kit has been optimized for accurate amplification of the entire CGG trinucleotide repeats, and a validation study on the performance of this PCR-based method has been previously reported by our group²⁹. A similar PCR-based method was reported by Mailick Seltzer et al. in 2012³⁵, but the ABI 3730xl instrument was used for repeat size determination and only individuals with repeat size less than 200 were identified. This may be due to the fact that their chosen platform was unable to detect the larger repeats with repeat size above 200. In contrast, the bioanalyzer instrument we use offers a more robust fragment sizing assay, as it can accurately and cost-effectively detect the full spectrum of the FMR1 CGG repeat including full mutations²⁹.

Except repeat number, several other factors also need to be ascertained for appropriate diagnosis of FXS and FXS-related disorders, including FMR1 mutation, the extent of methylation and mosaicism³⁴. The methylation status can be monitored by various methods such as incorporation of pre-digestion steps using methylation-sensitive restriction enzyme or bisulfite modification assay³⁴, however, our assay is incapable of determining the methylation status of the 5’UTR of the FMR1 promoter. Additionally, the expansion risk of a FMR1 allele also depends on the presence of AGG interruptions, which can stabilize the gene during transmission. PCR-based repeat assays have been modified to detect AGG-interruptions whereas digestion enzyme instability is one of the main limitations. Triplet-primed (TP) PCR by using a hybrid PCR primer binding in CGG repeats or AGG interruptions is another type of PCR assay which can detect CGG repeat size and AGG interruptions simultaneously. However, the FMR1 gene-specific PCR method we have described is unable to identify the AGG interruptions unless FMR1 gene sequencing after the amplification is performed. Recently, Hayward et al. reported the PCR methods used in their own laboratory for determination all the parameters necessary for a complete genetic workup or thorough laboratory study, including assays that can detect repeat number, AGG interruption status and methylation status³⁶. Unfortunately, neither assay is capable of comprehensively determining all these factors up to date.

It is worth noting that the assay is unable to detect deletions or single-nucleotide variants within the FMR1 gene, which accounts for approximately 1% of the FXS cases²⁷. Rarely, in individuals who have cellular mosaicism for the FMR1 repeat, PCR may give a false negative result because of failure in detecting mosaicism for larger premutation and full-mutation alleles^37,38. It has been demonstrated that the threshold of this PCR-based assay for mosaic full mutation male sample (341 repeats) is 2.5% when the peak detector threshold is set at three fluorescence units above baseline²⁹. Southern blot analysis is recommended in the cases if mosaic alleles are indicated. Furthermore, with regards to the electrophoresis platforms, previous study has indicated that compared with capillary electrophoresis systems (e.g., ABI 3130XL instrument), the bioanalyzer instrument is incapable of differentiating the normal homozygous female samples with individual fragment peaks from female samples with heterozygous repeat sizes that have repeat number differences of four or less²⁹. However, these single-peak samples could be classified as normal, because it has been validated that this PCR-based pipeline enables robust amplification and detection of full mutation or premutation alleles, which minimizes the possibility of false negative in this situation. Regardless of the above limitations, the method can be utilized as a first-tier pipeline for molecular identification of FXS and Fragile X-associated diseases with cost-effectiveness, robustness and rapid reporting time, supplemented by sequencing of the FMR1 gene and Southern blot analysis of methylation levels.