Noninvasive prenatal diagnosis of facioscapulohumeral muscular dystrophy using SNP-based amplicon sequencing
Prenatal Genetics
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Primary Categories:
- Clinical Genetics
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Secondary Categories:
- Clinical Genetics
Introduction:
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant hereditary neuromuscular disorder. There is no effective treatment for FSHD, and due to the high recurrence risk of autosomal dominant inheritance, timely prenatal diagnosis and intervention are crucial. The Karyomap gene chip uses family-based SNP linkage analysis to provide the prenatal diagnosis of FSHD with good detection efficacy. However, it is limited by the risk of invasive sampling and high cost. Compared to preimplantation and invasive genetic diagnosis, noninvasive prenatal diagnosis (NIPD) for FSHD has more advantages but less research. This study aims to establish an NIPD for FSHD to develop a simple, rapid, low-cost diagnostic method that can be used in early pregnancy and explore its feasibility and clinical value.
Methods:
Thirteen high-risk FSHD1 families were prospectively collected. All pregnancies were single and no less than 7+0 weeks. Multiplex PCR probes, including 402 amplicons, were designed to cover high-frequency SNP sites (MAF >0.2) within 2 Mb upstream of the DUX4 gene. Genomic DNA from high-risk family members and maternal cell-free DNA were extracted, followed by library construction and sequencing using multiplex PCR probes. Haplotype inheritance of the fetus was determined using relative haplotype dosage analysis and the Bayes factor algorithm. The NIPD results were compared with invasive prenatal diagnostic results using Karyomap gene chips and Bionano single-molecule optical mapping for dual verification to assess diagnostic efficacy accurately.
Results:
Thirteen high-risk FSHD1 families were prospectively included, of which nine pregnant women were FSHD1 patients, and four pregnant women had spouses who were FSHD1 patients. All family samples passed amplicon sequencing and NIPD analysis on the first attempt, successfully identifying eight FSHD1-affected and five normal fetuses. The average gestational age of the thirteen pregnant women was 9+0(range: 7+0-11+2), and the fetal DNA concentration was 7.07%(range: 3.2%-18.6%). No chromosomal recombination events were detected. For maternal informative SNPs, the average numbers for Type 1 and Type 2 were 36(range: 12-74)and 36(range: 12-74); and for paternal informative SNPs, the average numbers for Type 3 and Type 4 were 21(range: 7-34) and 23(range: 16-36), respectively. All reports were given within seven days, and all NIPD results were consistent with the Karyomap gene chip and Bionano single-molecule optical mapping verification results, demonstrating 100% diagnostic accuracy.
Conclusion:
NIPD achieved 100% diagnostic accuracy and demonstrated excellent accuracy and reliability in the early detection of FSHD1. Meanwhile, the earliest gestational age for NIPD was 7+0 weeks, and the reports can be completed within 7 days, significantly shortening the clinical turnaround time compared to traditional invasive methods and effectively reducing maternal anxiety. In conclusion, This technology offers significant advantages in terms of testing cycle, diagnostic efficacy, cost, and safety. It is expected to become one of the preferred prenatal diagnostic methods for high-risk FSHD families, providing a safe, fast, and early diagnostic option for clinical practice.
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant hereditary neuromuscular disorder. There is no effective treatment for FSHD, and due to the high recurrence risk of autosomal dominant inheritance, timely prenatal diagnosis and intervention are crucial. The Karyomap gene chip uses family-based SNP linkage analysis to provide the prenatal diagnosis of FSHD with good detection efficacy. However, it is limited by the risk of invasive sampling and high cost. Compared to preimplantation and invasive genetic diagnosis, noninvasive prenatal diagnosis (NIPD) for FSHD has more advantages but less research. This study aims to establish an NIPD for FSHD to develop a simple, rapid, low-cost diagnostic method that can be used in early pregnancy and explore its feasibility and clinical value.
Methods:
Thirteen high-risk FSHD1 families were prospectively collected. All pregnancies were single and no less than 7+0 weeks. Multiplex PCR probes, including 402 amplicons, were designed to cover high-frequency SNP sites (MAF >0.2) within 2 Mb upstream of the DUX4 gene. Genomic DNA from high-risk family members and maternal cell-free DNA were extracted, followed by library construction and sequencing using multiplex PCR probes. Haplotype inheritance of the fetus was determined using relative haplotype dosage analysis and the Bayes factor algorithm. The NIPD results were compared with invasive prenatal diagnostic results using Karyomap gene chips and Bionano single-molecule optical mapping for dual verification to assess diagnostic efficacy accurately.
Results:
Thirteen high-risk FSHD1 families were prospectively included, of which nine pregnant women were FSHD1 patients, and four pregnant women had spouses who were FSHD1 patients. All family samples passed amplicon sequencing and NIPD analysis on the first attempt, successfully identifying eight FSHD1-affected and five normal fetuses. The average gestational age of the thirteen pregnant women was 9+0(range: 7+0-11+2), and the fetal DNA concentration was 7.07%(range: 3.2%-18.6%). No chromosomal recombination events were detected. For maternal informative SNPs, the average numbers for Type 1 and Type 2 were 36(range: 12-74)and 36(range: 12-74); and for paternal informative SNPs, the average numbers for Type 3 and Type 4 were 21(range: 7-34) and 23(range: 16-36), respectively. All reports were given within seven days, and all NIPD results were consistent with the Karyomap gene chip and Bionano single-molecule optical mapping verification results, demonstrating 100% diagnostic accuracy.
Conclusion:
NIPD achieved 100% diagnostic accuracy and demonstrated excellent accuracy and reliability in the early detection of FSHD1. Meanwhile, the earliest gestational age for NIPD was 7+0 weeks, and the reports can be completed within 7 days, significantly shortening the clinical turnaround time compared to traditional invasive methods and effectively reducing maternal anxiety. In conclusion, This technology offers significant advantages in terms of testing cycle, diagnostic efficacy, cost, and safety. It is expected to become one of the preferred prenatal diagnostic methods for high-risk FSHD families, providing a safe, fast, and early diagnostic option for clinical practice.