Clinical Severity and Thermodynamic Effects of Iron-responsive Element Mutations in Hereditary Hyperferritinemia-Cataract Syndrome

Abstract

Hereditary hyperferritinemia-cataract syndrome (HHCS) is a novel genetic disorder characterized by elevated serum ferritin and early onset cataract formation. The excessive ferritin production in HHCS patients arises from aberrant regulation of L-ferritin translation caused by mutations within the ironresponsive element (IRE) of the L-ferritin transcript. IREs serve as binding sites for iron regulatory proteins (IRPs), iron-sensing proteins that regulate ferritin translation. Previous observations suggested that each unique HHCS mutation conferred a characteristic degree of hyperferritinemia and cataract severity in affected individuals. Here we have measured the in vitro affinity of the IRPs for the mutant IREs and correlated decreases in binding affinity with clinical severity. Thermodynamic analysis of these IREs has also revealed that although some HHCS mutations lead to changes in the stability and secondary structure of the IRE, others appear to disrupt IRP-IRE recognition with minimal effect on IRE stability. HHCS is a noteworthy example of a human genetic disorder that arises from mutations within a protein-binding site of an mRNA cis-acting element. Analysis of the effects of these mutations on the energetics of the RNA-protein interaction explains the phenotypic variabilities of the disease state. Several reports have described a new autosomal dominant disorder called hereditary hyperferritinemia-cataract syndrome (HHCS)1 (1-12). This condition is characterized by a combination of elevated levels of serum ferritin and congenital nuclear cataract. Human ferritin is composed of two different types of subunit (H-ferritin and L-ferritin), although the relative proportion of each subunit in the ferritin copolymer varies among tissues (13). Through the use of monoclonal antibodies specific for either H-or L-ferritin, the excess serum ferritin in HHCS patients has been shown to be predominantly L-ferritin (3, 7). A close relationship has also been established between monocyte L-ferritin content and serum ferritin concentration (7), suggesting that the excess production of L-ferritin in cells is directly responsible for the hyperferritinemia. Although serum ferritin is thought to reflect body iron stores, individuals with HHCS do not appear to suffer from iron overload (1, 2).The expression of both H-ferritin and L-ferritin is normally regulated according to intracellular iron concentration by means of iron-responsive elements (IREs) that are found within the 5Ј-untranslated regions of their transcripts (14, 15). These regulatory elements interact with the iron regulatory proteins IRP1 and IRP2, which are both capable of sensing cellular iron status. Under conditions of intracellular iron depletion, both IRP1 and IRP2 bind to IREs with high affinity. The binding of an IRP to the ferritin IRE prevents the binding of the 43 S translation initiation complex to the transcript (16, 17) and thus blocks translation of the ferritin protein. When intracellular iron concentrations rise, IRP1 acquires a [4Fe-4S] cluster ...

Clinical features and molecular analysis of seven British kindreds with hereditary hyperferritinaemia cataract syndrome

“…6,14,16,21,22,26,27 Mutagenesis studies of (mRNA)FTL IREs in vitro suggest that the primary nucleotide sequence in the apical loop of the motif is an important determinant of IRP binding. 15,28 Our findings support this hypothesis since two of the observed nucleotide substitutions lay within the apical loop of the IRE motif. We have also shown that substitutions in the upper stem of the IRE are predicted to change the secondary structures of the IRE apical loops.…”

“…13,14 Nucleotide substitutions that disrupt the secondary structure of IRE stem-loop motifs reduce the affinity of IRP binding in vitro. 15 It has therefore been proposed that HHCS arises through disruption of the L-ferritin RP-IRE interaction in vivo leading to failure of suppression of constitutive translation of (mRNA)FTL.…”

Multiple determinants within iron-responsive elements dictate iron regulatory protein binding and regulatory hierarchy

“…2A). From these data, the apparent K d of IRP1 was determined to be 17.5 pM for L-ferritin IRE, which is in general agreement with values others have reported using cytosol (Haile et al 1989;Barton et al 1990) or purified protein (Alam et al 1989;Allerson et al 1999). The ferritin IRE binding isotherm reached saturation and was easily modeled with a Hill coefficient of 1, indicating a lack of cooperativity in binding.…”

“…These mutations cause the disease hereditary hyperferritinemia cataract syndrome (HHCS), and the effect on IRP binding affinity correlates with the clinical severity of the disease (Girelli et al 1995;Allerson et al 1999). Therefore, we analyzed IRP1 affinity for L-ferritin IREs containing mutations associated with HHCS in order to evaluate the relationship between IRE binding affinity and regulation in the context of the binding hierarchy for natural IREs.…”

Molecular cloning, overexpression, purification, and sequence analysis of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide

“…Over recent years, conservation measures have been put in place and scientists have realized tremendous success in areas such as breeding, ecology, and propagation, among others. Meanwhile, giant panda research and conservation has been increasingly focusing on the level of gene functionality, which is currently drawing much attention in this field (Allerson et al, 1999;Liao et al, 2003;Hou et al, 2012;Hou et al, 2013;Wang et al, 2015;Wang et al, 2016). In this study, we designed primers based on the available FTL gene sequences of several mammalian species, including Homo sapiens, Cavia porcellus, Equus caballus, and Felis catus, and used reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify FTL complementary DNA (cDNA) from total RNA isolated from giant panda skeletal muscle.…”