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Novel mutations in the ferritin-L iron-responsive element that only mildly impair IRP binding cause hereditary hyperferritinaemia cataract syndrome.

Luscieti S, Tolle G, Aranda J, Campos CB, Risse F, Morán É, Muckenthaler MU, Sánchez M - Orphanet J Rare Dis (2013)

Bottom Line: Both mutations conferred reduced binding affinity on recombinant Iron Regulatory Proteins (IPRs) in EMSA experiments.Interestingly, the Badalona +36C > U mutation was found not only in heterozygosity, as expected for an autosomal dominant disease, but also in the homozygous state in some affected subjects.The Badalona +36C > U and Heidelberg +52 G > C mutations within the L-ferritin IRE only mildly alter the binding capacity of the Iron Regulatory Proteins but are still causative for the disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Predictive and Personalized Medicine of Cancer IMPPC, Ctra. de Can Ruti, Camí de les Escoles s/n, 08916, Badalona, Barcelona, Spain.

ABSTRACT

Background: Hereditary Hyperferritinaemia Cataract Syndrome (HHCS) is a rare autosomal dominant disease characterized by increased serum ferritin levels and early onset of bilateral cataract. The disease is caused by mutations in the Iron-Responsive Element (IRE) located in the 5' untranslated region of L-Ferritin (FTL) mRNA, which post-transcriptionally regulates ferritin expression.

Methods: We describe two families presenting high serum ferritin levels and juvenile cataract with novel mutations in the L-ferritin IRE. The mutations were further characterized by in vitro functional studies.

Results: We have identified two novel mutations in the IRE of L-Ferritin causing HHCS: the Badalona +36C > U and the Heidelberg +52 G > C mutation. Both mutations conferred reduced binding affinity on recombinant Iron Regulatory Proteins (IPRs) in EMSA experiments. Interestingly, the Badalona +36C > U mutation was found not only in heterozygosity, as expected for an autosomal dominant disease, but also in the homozygous state in some affected subjects. Additionally we report an update of all mutations identified so far to cause HHCS.

Conclusions: The Badalona +36C > U and Heidelberg +52 G > C mutations within the L-ferritin IRE only mildly alter the binding capacity of the Iron Regulatory Proteins but are still causative for the disease.

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(A, B) Direct electrophoretic mobility shift assays. Fluorescent labeled probes corresponding to FTL IRE wild type (lanes 1–2), the mutations +39ΔC (lane 3), Badalona +36C > U (lane 4), Milano +36C > G (lane 5), Heidelberg +52 G > C (lane 6) and Torino +29C > G (lane 7) were incubated with either rIRP1 (panel A) or rIRP2 (panel B) and the IRP-IRE complex resolved on acrylamide gels. One representative gel is shown. (C, D) Quantification of the signals in direct EMSAs compared to the wild type FTL IRE signal that was set to 100%. Means ± SD (standard deviation) of at least three independent experiments are shown. *** p < 0.001.
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Figure 3: (A, B) Direct electrophoretic mobility shift assays. Fluorescent labeled probes corresponding to FTL IRE wild type (lanes 1–2), the mutations +39ΔC (lane 3), Badalona +36C > U (lane 4), Milano +36C > G (lane 5), Heidelberg +52 G > C (lane 6) and Torino +29C > G (lane 7) were incubated with either rIRP1 (panel A) or rIRP2 (panel B) and the IRP-IRE complex resolved on acrylamide gels. One representative gel is shown. (C, D) Quantification of the signals in direct EMSAs compared to the wild type FTL IRE signal that was set to 100%. Means ± SD (standard deviation) of at least three independent experiments are shown. *** p < 0.001.

Mentions: For both mutations we next examined the ability of the mutated IRE to bind recombinant IRP1 or IRP2 by electrophoretic mobility shift assays (EMSAs). As expected little IRP binding occurs to the non-functional IRE structure (+39ΔC, Figure 3A and B, lanes 3), which was used as a positive control. Importantly, the Badalona +36C > U and Heidelberg +52 G > C mutations show a reduction of up to 30-40% in the binding to both IRPs (Figure 3A and B, lanes 4 and 6); a similar level of reduction was observed for the Torino +29C > G mutation ([11], Figure 3A and B, lanes 7), while the Milano +36C > G mutation [10] reduces its binding to both IRPs more drastically (Figure 3A and B, lanes 5). Next, we checked the Badalona +36C > U and Heidelberg +52 G > C changes by a more stringent assay, a competitive EMSA. The Badalona +36C > U mutation shows a mild but significant reduction in the efficiency of competition when compared to wild type unlabeled competitor (Figure 4A and C, lanes 17–22 compared to 3–8), while its corresponding control, the Milano +36C > G mutation, is inefficient in displacing the wild type probe; for this mutation a small degree of competition is only appreciable at 20x and 40x molar excess of competitor (Figure 4A and C, lanes 27–28). The Heidelberg +52 G > C mutation also shows a reduced capacity to compete with the FTL WT probe and behaves similarly to its corresponding control, the Torino +29C > G mutation (Figure 4A and C, lanes 31–36 and 37–42). Results obtained for IRP2 were comparable and consistent with previous data showing that IRP1 and IRP2 bind to the L-ferritin IRE with similar affinity (Figures 3 and 4).


Novel mutations in the ferritin-L iron-responsive element that only mildly impair IRP binding cause hereditary hyperferritinaemia cataract syndrome.

Luscieti S, Tolle G, Aranda J, Campos CB, Risse F, Morán É, Muckenthaler MU, Sánchez M - Orphanet J Rare Dis (2013)

(A, B) Direct electrophoretic mobility shift assays. Fluorescent labeled probes corresponding to FTL IRE wild type (lanes 1–2), the mutations +39ΔC (lane 3), Badalona +36C > U (lane 4), Milano +36C > G (lane 5), Heidelberg +52 G > C (lane 6) and Torino +29C > G (lane 7) were incubated with either rIRP1 (panel A) or rIRP2 (panel B) and the IRP-IRE complex resolved on acrylamide gels. One representative gel is shown. (C, D) Quantification of the signals in direct EMSAs compared to the wild type FTL IRE signal that was set to 100%. Means ± SD (standard deviation) of at least three independent experiments are shown. *** p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: (A, B) Direct electrophoretic mobility shift assays. Fluorescent labeled probes corresponding to FTL IRE wild type (lanes 1–2), the mutations +39ΔC (lane 3), Badalona +36C > U (lane 4), Milano +36C > G (lane 5), Heidelberg +52 G > C (lane 6) and Torino +29C > G (lane 7) were incubated with either rIRP1 (panel A) or rIRP2 (panel B) and the IRP-IRE complex resolved on acrylamide gels. One representative gel is shown. (C, D) Quantification of the signals in direct EMSAs compared to the wild type FTL IRE signal that was set to 100%. Means ± SD (standard deviation) of at least three independent experiments are shown. *** p < 0.001.
Mentions: For both mutations we next examined the ability of the mutated IRE to bind recombinant IRP1 or IRP2 by electrophoretic mobility shift assays (EMSAs). As expected little IRP binding occurs to the non-functional IRE structure (+39ΔC, Figure 3A and B, lanes 3), which was used as a positive control. Importantly, the Badalona +36C > U and Heidelberg +52 G > C mutations show a reduction of up to 30-40% in the binding to both IRPs (Figure 3A and B, lanes 4 and 6); a similar level of reduction was observed for the Torino +29C > G mutation ([11], Figure 3A and B, lanes 7), while the Milano +36C > G mutation [10] reduces its binding to both IRPs more drastically (Figure 3A and B, lanes 5). Next, we checked the Badalona +36C > U and Heidelberg +52 G > C changes by a more stringent assay, a competitive EMSA. The Badalona +36C > U mutation shows a mild but significant reduction in the efficiency of competition when compared to wild type unlabeled competitor (Figure 4A and C, lanes 17–22 compared to 3–8), while its corresponding control, the Milano +36C > G mutation, is inefficient in displacing the wild type probe; for this mutation a small degree of competition is only appreciable at 20x and 40x molar excess of competitor (Figure 4A and C, lanes 27–28). The Heidelberg +52 G > C mutation also shows a reduced capacity to compete with the FTL WT probe and behaves similarly to its corresponding control, the Torino +29C > G mutation (Figure 4A and C, lanes 31–36 and 37–42). Results obtained for IRP2 were comparable and consistent with previous data showing that IRP1 and IRP2 bind to the L-ferritin IRE with similar affinity (Figures 3 and 4).

Bottom Line: Both mutations conferred reduced binding affinity on recombinant Iron Regulatory Proteins (IPRs) in EMSA experiments.Interestingly, the Badalona +36C > U mutation was found not only in heterozygosity, as expected for an autosomal dominant disease, but also in the homozygous state in some affected subjects.The Badalona +36C > U and Heidelberg +52 G > C mutations within the L-ferritin IRE only mildly alter the binding capacity of the Iron Regulatory Proteins but are still causative for the disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Predictive and Personalized Medicine of Cancer IMPPC, Ctra. de Can Ruti, Camí de les Escoles s/n, 08916, Badalona, Barcelona, Spain.

ABSTRACT

Background: Hereditary Hyperferritinaemia Cataract Syndrome (HHCS) is a rare autosomal dominant disease characterized by increased serum ferritin levels and early onset of bilateral cataract. The disease is caused by mutations in the Iron-Responsive Element (IRE) located in the 5' untranslated region of L-Ferritin (FTL) mRNA, which post-transcriptionally regulates ferritin expression.

Methods: We describe two families presenting high serum ferritin levels and juvenile cataract with novel mutations in the L-ferritin IRE. The mutations were further characterized by in vitro functional studies.

Results: We have identified two novel mutations in the IRE of L-Ferritin causing HHCS: the Badalona +36C > U and the Heidelberg +52 G > C mutation. Both mutations conferred reduced binding affinity on recombinant Iron Regulatory Proteins (IPRs) in EMSA experiments. Interestingly, the Badalona +36C > U mutation was found not only in heterozygosity, as expected for an autosomal dominant disease, but also in the homozygous state in some affected subjects. Additionally we report an update of all mutations identified so far to cause HHCS.

Conclusions: The Badalona +36C > U and Heidelberg +52 G > C mutations within the L-ferritin IRE only mildly alter the binding capacity of the Iron Regulatory Proteins but are still causative for the disease.

Show MeSH
Related in: MedlinePlus