Quy trình giải trình tự Sanger chẩn đoán các biến thể đa hình đơn Nucleotide trên gen CLDN-1 liên quan đến bệnh viêm da cơ địa

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Tác giả: Dương Hoàng Huy Lê, Đoàn Huỳnh Anh Phúc Nguyễn, Hữu Ngọc Tuấn Nguyễn

Ngôn ngữ: vie

Ký hiệu phân loại:

Thông tin xuất bản: Tạp chí Y Dược học Phạm Ngọc Thạch, 2023

Mô tả vật lý: tr.71

Bộ sưu tập: Metadata

ID: 339184

Thêm vào giỏ Liên kết toàn văn

- Giới thiệu: Tổn thương hàng rào bảo vệ da trong viêm da cơ địa (VDCĐ) có liên quan đến yếu tố di truyền, trong đó các biến thể đa hình đơn nucleotide (SNP) trên gen CLDN-1 mã hóa protein claudin-1 được mô tả có liên quan đến bệnh sinh của VDCĐ. Việc xác định chính xác các biến thể này góp phần thực hiện các nghiên cứu nhằm nâng cao hiệu quả quản lý bệnh nhân VDCĐ, trong đó kỹ thuật giải trình tự Sanger được xem là tiêu chuẩn vàng trong xác định các biến thể.- Mục tiêu: Xây dựng quy trình giải trình tự Sanger và khảo sát bốn biến thể rs17501010, rs9290927, rs9290929 và rs893051 trên gen CLDN-1 liên quan đến VDCĐ.- Đối tượng và phương pháp nghiên cứu: Thiết kế 4 cặp đoạn mồi đặc hiệu cho các biến thể, tối ưu hóa nhiệt độ bắt cặp của phản ứng PCR đa mồi chứa cả 4 biến thể và tối ưu hóa phản ứng giải trình tự Sanger trên hệ thống Applied Biosystems 3500 Series Genetic Analyzer của hãng Thermo Fishser. Áp dụng toàn bộ quy trình giải trình tự Sanger đã tối ưu lên 12 mẫu máu của bệnh nhân VDCĐ nhằm đánh giá thông số kỹ thuật và đặc điểm của các biến thể.- Kết quả: Xây dựng thành công quy trình giải trình tự Sanger bao gồm: thiết kế được bốn cặp đoạn mồi khuếch đại đặt hiệu bốn biến thể quan tâm, tối ưu hóa được nồng độ DNA, primer và chu trình nhiệt của phản ứng PCR đa mồi chứa bốn biến thể quan tâm, tối ưu hóa nồng độ DNA đầu vào của phản ứng giải trình tự. Khảo sát được 12/12 mẫu DNA từ bệnh nhân VDCĐ với tất cả kết quả giải trình tự có vị trí nucleotide nằm trong vùng QVB cao.- Kết luận: Quy trình có thể đưa vào ứng dụng để xác định biến thể đa hình đơn nucleotide. Đồng thời là các kết quả bước đầu, hứa hẹn ứng dụng kỹ thuật sinh học phân tử để khảo sát các đặc điểm di truyền ở bệnh viêm da cơ địa. Từ đó đưa ra các chiến lược phù hợp để tiếp cận và quản lý bệnh VDCĐ ở Việt Nam.Abstract- Introduction: Damage to the skin barrier in atopic dermatitis (AD) is related to genetic factors, in which a single nucleotide polymorphism (SNP) variant on the gene CLDN-1 encoding the claudin-1 protein has been described as having related to the pathogenesis of AD. Accurate identification of these variants contributes to the implementation of studies to improve the management of patients with AD, in which Sanger sequencing is considered the gold standard in identifying variants.- Objective: Develop Sanger sequencing and investigate variants rs17501010, rs9290927, rs893051 and rs9290929 on related CLDN-1 gene relatedatopic dermatitis.- Materials and methods: Design 4 pairs of primers specific for variants, optimize the pairing temperature of multiplex PCR containing all 4 variants, and optimize Sanger sequencing on Applied Biosystems 3500 Series Genetic Analyzer by Thermo Fishser. Applying the entire optimized Sanger sequencing procedure to 12 blood samples of AD patients to evaluate the specifications and characteristics of the variants.- Results: Successfully built Sanger sequencing included: designing four pairs of amplifying primers that signal the four variants of interest, optimizing the concentration of DNA, primer and thermal cycling of the multi - primed PCR reaction containing four variants of interest, optimizing the input DNA concentration of the sequencing reaction. Surveyed 12/12 DNA samples from AD patients with all sequencing results having nucleotide positions in the high QVB region.- Conclusion: He procedure is applicable to the identification of single nucleotide polymorphisms. At the same time, these are initial results, promising to apply molecular biology techniques to investigate genetic characteristics in atopic dermatitis. From there, provide appropriate strategies to approach and manage AD in Vietnam. DOI: 10.59715/pntjmp.2.1.9

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