A rapid and accurate methylation‐sensitive high‐resolution melting analysis assay for the diagnosis of Prader Willi and Angelman patients

• Published in: Molecular genetics & genomic medicine Vol. 7; no. 6; pp. e637 - n/a
• Main Authors: Ribeiro Ferreira, Igor, Darleans dos Santos Cunha, Wilton, Henrique Ferreira Gomes, Leonardo, Azevedo Cintra, Hiago, Lopes Cabral Guimarães Fonseca, Letícia, Ferreira Bastos, Elenice, Clinton Llerena, Juan, Farias Meira de Vasconcelos, Zilton, Cunha Guida, Letícia
• Format: Journal Article
• Language: English
• Published: United States John Wiley & Sons, Inc 01-06-2019; John Wiley and Sons Inc; Wiley
• Subjects: Alleles; Angelman syndrome; Angelman Syndrome - blood; Angelman Syndrome - diagnosis; Angelman Syndrome - genetics; Bisulfite; Chromosome 15; Chromosomes; Chromosomes, Human, Pair 15 - genetics; Cytogenetics; Deoxyribonucleic acid; Diagnosis; Diagnostic systems; DNA; DNA methylation; DNA Methylation - genetics; DNA Primers - genetics; Electrophoresis; Epigenesis, Genetic - genetics; Female; Genetic analysis; Genetic disorders; Genomic imprinting; high‐resolution melting; Humans; Laboratories; Male; Melting; Method; Molecular biology; MS‐HRM; MS‐PCR; Mutation; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Polymerase chain reaction; Polymerase Chain Reaction - methods; Prader Willi syndrome; Prader-Willi Syndrome - blood; Prader-Willi Syndrome - diagnosis; Prader-Willi Syndrome - genetics; Primers; Sampling methods; snRNP Core Proteins - genetics; snRNP Core Proteins - metabolism; Uniparental disomy; MS-HRM; Angelman syndrome; MS-PCR; Prader Willi syndrome; high-resolution melting
• ISSN: 2324-9269; 2324-9269
• DOI: 10.1002/mgg3.637

Background
Prader Willi (PWS) and Angelman (AS) syndromes are rare genetic disorders characterized by deletions, uniparental disomy, and imprinting defects at chromosome 15. The loss of function of specific genes caused by genetic alterations in paternal allele causes PWS while the absence in maternal allele results AS. The laboratory diagnosis of PWS and AS is complex and demands molecular biology and cytogenetics techniques to identify the genetic mechanism related to the development of the disease. The DNA methylation analysis in chromosome 15 at the SNURF‐SNRPN locus through MS‐PCR confirms the diagnosis and distinguishes between PWS and AS. Our study aimed to establish the MS‐PCR technique associated with High‐Resolution Melting (MS‐HRM) in PWS and AS diagnostic with a single pair of primers.
Methods
We collected blood samples from 43 suspected patients to a cytogenetic and methylation analysis. The extracted DNA was treated with bisulfite to perform comparative methylation analysis.
Results
MS‐HRM and MS‐PCR agreed in 100% of cases, identifying 19(44%) PWS, 3(7%) AS, and 21(49%) Normal. FISH analysis detected four cases of PWS caused by deletions in chromosome 15.
Conclusion
The MS‐HRM showed good performance with a unique pair of primers, dispensing electrophoresis gel analysis, offering a quick and reproducible diagnostic.
Prader Willi (PWS) and Angelman (AS) syndromes are complex genetic diseases characterized by chromosomal abnormalities. The diagnostic of both syndromes demands cytogenetic and molecular techniques. This study aimed to improve the diagnosis of PWS and AS through High‐Resolution Melting (MS‐HRM) analysis. Our MS‐HRM approach reduces the time taken to an accurate and reproducible diagnostic.