DBS Screening for Glycogen Storage Disease Type 1a: Detection of c.648G>T Mutation in G6PC by Combination of Modified Competitive Oligonucleotide Priming-PCR and Melting Curve Analysis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Patients and Controls
2.1.1. Informed Consents and Ethics Committee Approval
2.1.2. Patient Clinical Information
2.2. Detection of G6PC and CFTR
2.2.1. Preparation of DBS Samples from Controls and Patients
2.2.2. Construction of Carrier Status Model
2.2.3. Outline of PCR Detection System for c.648G>T in G6PC
- First-round PCR amplification of G6PC intron 4–exon 5 and CFTR exon 4–intron 4 using the outer primer sets was done by conventional PCR. A small, punched circle from the DBS was used directly for PCR, without any DNA extraction or purification procedures.
- Second-round PCR amplification of G6PC intron 4–exon 5 and CFTR exon 4 using the inner primer sets was done by real-time mCOP-PCR. The two alleles, c.648G (wild type) and c.648T (mutant), were specifically amplified in this step.
- Melting curve analysis of the amplification products was immediately started after the second-round PCR amplification ended. The G6PC peaks, c.648G and c.648T, were clearly separated from the CFTR peaks.
2.2.4. First-Round PCR: Multiplex Amplification of G6PC and CFTR Outer Fragments
2.2.5. Second-Round PCR: Multiplex Amplification of G6PC and CFTR Inner Fragments
2.2.6. Melting Curve Analysis
2.3. Sequencing Analysis
2.4. Statistical Analysis
3. Results
3.1. First-Round PCR Followed by Gel Electrophoresis
3.2. Second-Round PCR Followed by Gel Electrophoresis
3.3. Amplification Curve Analysis of the Second-Round PCR Products
3.4. Melting Curve Analysis of the Second-Round PCR Products
3.5. GSD1a Carrier Model Screening for GSDIa
3.6. Peak Height Ratio of G6PC vs. CFTR
4. Discussion
4.1. Necessity of GSD1a Screening in the Context of Gene Replacement Therapy
4.2. Nested mCOP-PCR as a New Screening System to Detect c.648G>T in G6PC
4.3. Screening Strategy for GSDIa in the Real World
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Samples | Ratios | Mean | SD |
---|---|---|---|
Control DBS (n = 50) | G-ratio (G6PC [G]/CFTR) | 1.20 | ± 0.30 |
T-ratio (G6PC [T]/CFTR) | 0.06 | ± 0.02 | |
Patient DBS (n = 4) | G-ratio (G6PC [G]/CFTR) | 0.08 | ± 0.02 |
T-ratio (G6PC [T]/CFTR) | 1.43 | ± 0.08 |
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Niba, E.T.E.; Wijaya, Y.O.S.; Awano, H.; Taniguchi, N.; Takeshima, Y.; Nishio, H.; Shinohara, M. DBS Screening for Glycogen Storage Disease Type 1a: Detection of c.648G>T Mutation in G6PC by Combination of Modified Competitive Oligonucleotide Priming-PCR and Melting Curve Analysis. Int. J. Neonatal Screen. 2021, 7, 79. https://doi.org/10.3390/ijns7040079
Niba ETE, Wijaya YOS, Awano H, Taniguchi N, Takeshima Y, Nishio H, Shinohara M. DBS Screening for Glycogen Storage Disease Type 1a: Detection of c.648G>T Mutation in G6PC by Combination of Modified Competitive Oligonucleotide Priming-PCR and Melting Curve Analysis. International Journal of Neonatal Screening. 2021; 7(4):79. https://doi.org/10.3390/ijns7040079
Chicago/Turabian StyleNiba, Emma Tabe Eko, Yogik Onky Silvana Wijaya, Hiroyuki Awano, Naoko Taniguchi, Yasuhiro Takeshima, Hisahide Nishio, and Masakazu Shinohara. 2021. "DBS Screening for Glycogen Storage Disease Type 1a: Detection of c.648G>T Mutation in G6PC by Combination of Modified Competitive Oligonucleotide Priming-PCR and Melting Curve Analysis" International Journal of Neonatal Screening 7, no. 4: 79. https://doi.org/10.3390/ijns7040079