Ann Lab Med.  2021 Jul;41(4):394-400. 10.3343/alm.2021.41.4.394.

Accuracy and Performance Evaluation of Triplet Repeat Primed PCR as an Alternative to Conventional Diagnostic Methods for Fragile X Syndrome

• Gu H
• Kim MJ
• Yang D
• Song JY
• Cho SI
• Park SS
• Seong MW

• KMID: 2512719
• DOI: http://doi.org/10.3343/alm.2021.41.4.394

Abstract

Background
Conventional diagnosis of fragile X syndrome (FXS) is based on a combination of fragment analysis (FA) and Southern blotting (SB); however, this diagnostic approach is time- and labor-intensive and has pitfalls such as the possibility of missing large number alleles. Triplet repeat primed PCR (TP-PCR) is a current alternative used to overcome these limitations. We evaluated the diagnostic usefulness of TP-PCR compared with the conventional diagnostic protocol consisting of FA and/or SB in terms of allele categorization, repeat number correlation, and zygosity concordance in female genetic carriers.
Methods
From November 2013 to March 2018, 458 patients (326 males, 132 females) were simultaneously examined using FA and/or SB and TP-PCR by detecting CGG repeat numbers in FMR gene and diagnosed as per American College of Medical Genetics guidelines.
Results
The TP-PCR results showed high concordance with the FA and/or SB results for all three aspects (allele categorization, repeat number correlation, and zygosity concordance in female genetic carriers). TP-PCR detected CGG expansions ≥ 200 in all full mutation (FM) allele cases in male patients, as well as both the normal allele (NL) and FM allele in female carriers. In premutation (PM) allele carriers, the TP-PCR results were consistent with the FA and/or SB results. In terms of zygosity concordance in female genetic carriers, 12 NL cases detected by TP-PCR showed a merged peak consisting of two close heterozygous peaks; however, this issue was resolved using a 10-fold dilution.
Conclusions
TP-PCR may serve as a reliable alternative method for FXS diagnosis.

Keyword

Fragile X syndrome; Fragment analysis; Southern blotting; Triplet repeat primed PCR; CGG expansion; Allele categorization; Repeat number; Zygosity

Figure

• 

  Fig. 1 Repeat number correlation based on repeat number range. The auxiliary line (y=x) in each graph is adapted for demonstrating their correlation. (A) A very strong correlation (P<0.001, r=0.987, N=352) of CGG repeat numbers was observed between FA and TP-PCR in the range from 0 to 80 of repeat numbers. (B) A moderate (P=0.078, r=0.614, N=9) correlation was observed between SB and TP-PCR in the range from 81 to 200 of repeat numbers., as obtaining an accurate CGG repeat value using SB is difficult compared with TP-PCR. Abbreviations: FA, fragment analysis; SB, Southern blotting; TP-PCR, triplet repeat primed PCR.

• 

  Fig. 2 Zygosity discrepancy between FA and TP-PCR and peak separation using a diluted DNA sample for TP-PCR. (A, B) One patient had 29 or 30 repeats when using FA but only 30 repeats using TP-PCR. (C) Allele separation was achieved, and two separate peaks were obtained using TP-PCR with a 10-fold dilution of the DNA sample. Abbreviations: FA, fragment analysis; TP-PCR, triplet repeat primed PCR.

• 

  Fig. 3 Mosaicism cases in fragile X syndrome. (A) SB results. Lanes 1 and 7 are the results of patient P4 and show mosaicism with NL, PM, and unmethylated FM. Smeared, continuous bands are observed in the boxed areas, indicating an unmethylated FM allele. Lanes 2 and 6 show FM in male carriers; Lanes 3, 4, and 5 show NL in females. (B) TP-PCR result of patient P4, with three very clear peaks in the areas of NL, PM, and FM. Abbreviations: SB, Southern blotting; TP-PCR, triplet repeat primed PCR; NL, normal allele; PM, premutation (allele); FM, full mutation (allele).

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