Double-gradient denaturing gradient gel electrophoresis assay for identification of L-ferritin iron-responsive element mutations responsible for hereditary hyperferritinemia-cataract syndrome: Identification of the new mutation Cl4G

L. Cremonesi, A. Fumagalli, N. Soriani, M. Ferrari, S. Levi, S. Belloli, G. Ruggeri, P. Arosio

Research output: Contribution to journalArticle

Abstract

Background: Hereditary hyperferritinemia-cataract syndrome is an autosomic dominant disorder caused by heterogeneous mutations on the iron-responsive element (IRE) of ferritin L-chain mRNA. The mutations described to date were identified by direct sequencing of DNA from probands with hyperferritinemia often associated to bilateral cataracts. A direct genetic approach on a large population is useful to recognize polymorphisms in the DNA region and the prevalence of mutations associated with minor increases in serum ferritin and subclinical cataracts. We developed a rapid DNA scanning technique to detect mutations in a single electrophoretic analysis. Methods: The double-gradient denaturing gradient gel electrophoresis (DG-DGGE) method consisted of PCR amplification of the target genomic DNA with GC-clamped oligonucleotides. The sequence encoded the 5′ untranslated flanking region of ferritin L-chain mRNA, which includes an IRE stem-loop structure. The product was subjected to DG-DGGE (8.5-15% polyacrylamide and 50-95% denaturant) to separate the homo- and heteroduplexes. Results: The method clearly identified all eight accessible mutations, including C-G transversions, which are the most difficult to detect. The method was applied to scan DNA samples from 50 healthy subjects and from 230 subjects with serum ferritin >400 μg/L. The new mutation G14C was identified. Conclusions: The DG-DGGE method detects all the mutations in the L-ferritin IRE sequence, is rapid and economical, and can be applied to scan large populations. The first population study indicated that the mutations are rare and may involve regions of the IRE structure not yet characterized.

Original languageEnglish
Pages (from-to)491-497
Number of pages7
JournalClinical Chemistry
Volume47
Issue number3
Publication statusPublished - 2001

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Apoferritins
Denaturing Gradient Gel Electrophoresis
Electrophoresis
Assays
Iron
Gels
Mutation
DNA
Ferritins
Messenger RNA
Cataract
Polymorphism
Oligonucleotides
Amplification
Population
Hyperferritinemia, hereditary, with congenital cataracts
5' Flanking Region
5' Untranslated Regions
Scanning
Serum

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

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title = "Double-gradient denaturing gradient gel electrophoresis assay for identification of L-ferritin iron-responsive element mutations responsible for hereditary hyperferritinemia-cataract syndrome: Identification of the new mutation Cl4G",
abstract = "Background: Hereditary hyperferritinemia-cataract syndrome is an autosomic dominant disorder caused by heterogeneous mutations on the iron-responsive element (IRE) of ferritin L-chain mRNA. The mutations described to date were identified by direct sequencing of DNA from probands with hyperferritinemia often associated to bilateral cataracts. A direct genetic approach on a large population is useful to recognize polymorphisms in the DNA region and the prevalence of mutations associated with minor increases in serum ferritin and subclinical cataracts. We developed a rapid DNA scanning technique to detect mutations in a single electrophoretic analysis. Methods: The double-gradient denaturing gradient gel electrophoresis (DG-DGGE) method consisted of PCR amplification of the target genomic DNA with GC-clamped oligonucleotides. The sequence encoded the 5′ untranslated flanking region of ferritin L-chain mRNA, which includes an IRE stem-loop structure. The product was subjected to DG-DGGE (8.5-15{\%} polyacrylamide and 50-95{\%} denaturant) to separate the homo- and heteroduplexes. Results: The method clearly identified all eight accessible mutations, including C-G transversions, which are the most difficult to detect. The method was applied to scan DNA samples from 50 healthy subjects and from 230 subjects with serum ferritin >400 μg/L. The new mutation G14C was identified. Conclusions: The DG-DGGE method detects all the mutations in the L-ferritin IRE sequence, is rapid and economical, and can be applied to scan large populations. The first population study indicated that the mutations are rare and may involve regions of the IRE structure not yet characterized.",
author = "L. Cremonesi and A. Fumagalli and N. Soriani and M. Ferrari and S. Levi and S. Belloli and G. Ruggeri and P. Arosio",
year = "2001",
language = "English",
volume = "47",
pages = "491--497",
journal = "Clinical Chemistry",
issn = "0009-9147",
publisher = "American Association for Clinical Chemistry Inc.",
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TY - JOUR

T1 - Double-gradient denaturing gradient gel electrophoresis assay for identification of L-ferritin iron-responsive element mutations responsible for hereditary hyperferritinemia-cataract syndrome

T2 - Identification of the new mutation Cl4G

AU - Cremonesi, L.

AU - Fumagalli, A.

AU - Soriani, N.

AU - Ferrari, M.

AU - Levi, S.

AU - Belloli, S.

AU - Ruggeri, G.

AU - Arosio, P.

PY - 2001

Y1 - 2001

N2 - Background: Hereditary hyperferritinemia-cataract syndrome is an autosomic dominant disorder caused by heterogeneous mutations on the iron-responsive element (IRE) of ferritin L-chain mRNA. The mutations described to date were identified by direct sequencing of DNA from probands with hyperferritinemia often associated to bilateral cataracts. A direct genetic approach on a large population is useful to recognize polymorphisms in the DNA region and the prevalence of mutations associated with minor increases in serum ferritin and subclinical cataracts. We developed a rapid DNA scanning technique to detect mutations in a single electrophoretic analysis. Methods: The double-gradient denaturing gradient gel electrophoresis (DG-DGGE) method consisted of PCR amplification of the target genomic DNA with GC-clamped oligonucleotides. The sequence encoded the 5′ untranslated flanking region of ferritin L-chain mRNA, which includes an IRE stem-loop structure. The product was subjected to DG-DGGE (8.5-15% polyacrylamide and 50-95% denaturant) to separate the homo- and heteroduplexes. Results: The method clearly identified all eight accessible mutations, including C-G transversions, which are the most difficult to detect. The method was applied to scan DNA samples from 50 healthy subjects and from 230 subjects with serum ferritin >400 μg/L. The new mutation G14C was identified. Conclusions: The DG-DGGE method detects all the mutations in the L-ferritin IRE sequence, is rapid and economical, and can be applied to scan large populations. The first population study indicated that the mutations are rare and may involve regions of the IRE structure not yet characterized.

AB - Background: Hereditary hyperferritinemia-cataract syndrome is an autosomic dominant disorder caused by heterogeneous mutations on the iron-responsive element (IRE) of ferritin L-chain mRNA. The mutations described to date were identified by direct sequencing of DNA from probands with hyperferritinemia often associated to bilateral cataracts. A direct genetic approach on a large population is useful to recognize polymorphisms in the DNA region and the prevalence of mutations associated with minor increases in serum ferritin and subclinical cataracts. We developed a rapid DNA scanning technique to detect mutations in a single electrophoretic analysis. Methods: The double-gradient denaturing gradient gel electrophoresis (DG-DGGE) method consisted of PCR amplification of the target genomic DNA with GC-clamped oligonucleotides. The sequence encoded the 5′ untranslated flanking region of ferritin L-chain mRNA, which includes an IRE stem-loop structure. The product was subjected to DG-DGGE (8.5-15% polyacrylamide and 50-95% denaturant) to separate the homo- and heteroduplexes. Results: The method clearly identified all eight accessible mutations, including C-G transversions, which are the most difficult to detect. The method was applied to scan DNA samples from 50 healthy subjects and from 230 subjects with serum ferritin >400 μg/L. The new mutation G14C was identified. Conclusions: The DG-DGGE method detects all the mutations in the L-ferritin IRE sequence, is rapid and economical, and can be applied to scan large populations. The first population study indicated that the mutations are rare and may involve regions of the IRE structure not yet characterized.

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