High throughput screening for expanded CTG repeats in myotonic dystrophy type 1 using melt curve analysis.
DMPK gene
melt curve analysis
myotonic dystrophy
population screening
triplet-repeat expansion
Journal
Molecular genetics & genomic medicine
ISSN: 2324-9269
Titre abrégé: Mol Genet Genomic Med
Pays: United States
ID NLM: 101603758
Informations de publication
Date de publication:
04 2021
04 2021
Historique:
revised:
23
12
2020
received:
12
10
2020
accepted:
29
01
2021
pubmed:
25
2
2021
medline:
15
12
2021
entrez:
24
2
2021
Statut:
ppublish
Résumé
Myotonic dystrophy type 1 (DM1) is caused by CTG repeat expansions in the DMPK gene and is the most common form of muscular dystrophy. Patients can have long delays from onset to diagnosis, since clinical signs and symptoms are often nonspecific and overlapping with other disorders. Clinical genetic testing by Southern blot or triplet-primed PCR (TP-PCR) is technically challenging and cost prohibitive for population surveys. Here, we present a high throughput, low-cost screening tool for CTG repeat expansions using TP-PCR followed by high resolution melt curve analysis with saturating concentrations of SYBR GreenER dye. We determined that multimodal melt profiles from the TP-PCR assay are a proxy for amplicon length stoichiometry. In a screen of 10,097 newborn blood spots, melt profile analysis accurately reflected the tri-modal distribution of common alleles from 5 to 35 CTG repeats, and identified the premutation and full expansion alleles. We demonstrate that robust detection of expanded CTG repeats in a single tube can be achieved from samples derived from specimens with minimal template DNA such as dried blood spots (DBS). This technique is readily adaptable to large-scale testing programs such as population studies and newborn screening programs.
Sections du résumé
BACKGROUND
Myotonic dystrophy type 1 (DM1) is caused by CTG repeat expansions in the DMPK gene and is the most common form of muscular dystrophy. Patients can have long delays from onset to diagnosis, since clinical signs and symptoms are often nonspecific and overlapping with other disorders. Clinical genetic testing by Southern blot or triplet-primed PCR (TP-PCR) is technically challenging and cost prohibitive for population surveys.
METHODS
Here, we present a high throughput, low-cost screening tool for CTG repeat expansions using TP-PCR followed by high resolution melt curve analysis with saturating concentrations of SYBR GreenER dye.
RESULTS
We determined that multimodal melt profiles from the TP-PCR assay are a proxy for amplicon length stoichiometry. In a screen of 10,097 newborn blood spots, melt profile analysis accurately reflected the tri-modal distribution of common alleles from 5 to 35 CTG repeats, and identified the premutation and full expansion alleles.
CONCLUSION
We demonstrate that robust detection of expanded CTG repeats in a single tube can be achieved from samples derived from specimens with minimal template DNA such as dried blood spots (DBS). This technique is readily adaptable to large-scale testing programs such as population studies and newborn screening programs.
Identifiants
pubmed: 33624941
doi: 10.1002/mgg3.1619
pmc: PMC8123750
doi:
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
e1619Subventions
Organisme : NINDS NIH HHS
ID : K08 NS097631
Pays : United States
Organisme : NINDS NIH HHS
ID : K23 NS091511
Pays : United States
Organisme : NINDS NIH HHS
ID : R21 NS100040
Pays : United States
Organisme : NCATS NIH HHS
ID : R21 TR003184
Pays : United States
Organisme : NINDS NIH HHS
ID : R01 NS104010
Pays : United States
Organisme : NCBDD CDC HHS
ID : U01 DD001242
Pays : United States
Organisme : FDA HHS
ID : R01 FD006071
Pays : United States
Informations de copyright
© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.
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