Molecular basis for the recently described hereditary

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1995 86: 4050-4053
Molecular basis for the recently described hereditary
hyperferritinemia- cataract syndrome: a mutation in the
iron-responsive element of ferritin L-subunit gene (the "Verona
mutation") [see comments]
D Girelli, R Corrocher, L Bisceglia, O Olivieri, L De Franceschi, L Zelante and P Gasparini
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RAPID COMMUNICATION
Molecular Basis for the Recently Described Hereditary HyperferritinemiaCataract Syndrome: A Mutation in the Iron-Responsive Element of Ferritin
L-Subunit Gene (the “Verona Mutation”)
By Domenico Girelli, Roberto Corrocher, Luigi Bisceglia, Oliviero Olivieri, Lucia De Franceschi, Leopoldo Zelante,
and Paolo Gasparini
Recently, we described anew genetic disorder(the “hereditary hyperferritinemia-cataract syndrome”) clinically characterized by the combination of elevated serum ferritin and
congenital bilateral nuclear cataract, both cotransmitted as
an autosomal dominant trait. In affected subjects, hyperferritinemia (ranging from 950 to 2,259 pg/L) is typically not
related to iron overload. Differently from subjects with hereditary hemochromatosis, they have normal to low levels
of serum iron and percent of transferrin saturation and absence of iron overload in parenchymal organs. When unnecessary phlebotomies are performed, they rapidly develop
iron-deficient anemia, with persistently elevated levels of
serum ferritin. By RNA-single-strand conformation polymorphism screeningof the L-subunitferritin geneon chromosome
19, we were able to identify in affected subjects a mutation
in the 5’ untranslated region. This mutation involvesthe five
[CAGUGI of the iron-responsive element
nucleotides sequence
(IRE), which is critical for the posttranscriptional regulation of
ferritin synthesis bymeans of IRE-bindingprotein(IRE-BP).
Thus. it is very likely to provide the molecular basis for the
iron-insensitive upregulation of ferritin synthesis in affected
subjects.
0 1995 by The American Societyof Hematology.
F
body iron status. Hereditary hemochromatosis, a parenchymaliron overload caused by excess iron absorption, has
long been considered the only genetic disorder with elevated
serum femtin.’
Recently, we have described a new genetic disorder characterized by a combination of high serum ferritin not related
to iron overload and congenital bilateral nuclear cataract,
which is transmitted as an autosomal dominant trait.8 Since
serum ferritin was determined in our patients using antibodies against the L-subunit, we suspected that the L-subunit
gene on chromosome 19 (in the region 19q13.3
19qter)
might be involved. Moreover, the recent assignment to chromosome 19q13.4 of MP19, one of the most abundant proteins of lens fiber cell which is likely tobe involved in
cataractogenesis,’ prompted us to actively search in this region of the human genome the gene responsible for the
“hyperferritinemia-cataract syndrome.” We searched for
mutations on genomic DNA using the RNA-single-strand
conformation polymorphism (RNA-SSCP) technique,”.” focusing our attention first on ferritin L gene.
In this report we describe a mutation in the 5’ UTR of the
gene for the ferritin L-subunit on chromosome 19q, which is
likely to provide the molecular basis for the upregulation of
ferritin synthesis in subjects affected by the “hyperferritinemia-cataract syndrome.”
ERRITIN is an ubiquitous iron storage protein present
in every cell of nearly all organisms. It is a multimer
shell composed of 24 heavy (H, Mr 21,000) and light (L,
Mr 19,000) subunits, surrounding a cavity that can accommodateup to 4,500 iron atoms in a readily available but
nontoxic form.’ The human genes for the H and L femtin
subunits have been assigned to chromosome 1 1 and 19, respectively.’ Iron availability finely regulates ferritin synthesis at the translational level by means of the so-called ironresponsive element binding protein (IRE-BP).3 In scarcity
of iron, this cytosolic 90,000 Mr protein binds to an IRE
situated on the S’-untranslatedregion (S’ UTR) of the ferritin
mRNA, thus inhibiting the initiation of the translation process. Alternatively, an excess of iron lowers the affinity of
the IRE-BP for the IRE stem-loop in ferritin mRNA, enabling efficient translation (Fig 1). In absence of conditions
such as cancer and inflammation, which can also modulate
femtin expression at transcriptional level by iron-independent, cytokine-mediated
the above-mentioned
mechanismis prominent, and represents perhaps the best
understood example of posttranscriptional gene regulation
in complex eukaryotes.?Ferritin is also present in thecirculation, representing a by-product of intracellular ferritin synthesis.‘ Serum ferritin levels generally reflect the size of iron
stores, providing a reliable marker for clinical evaluation of
From the Institute of Medical Pathology, Chairof Internal Medicine, University of Verona, and Service of Medical Genetics, CSS
Hospital, San Giovanni Rotondo, Foggia, Ituly.
Submitted July 31, 1995; accepted September 20, 1995.
Supported by grants from
the Ministry of the University and Technological Research 60% (to R.C.) and, in part, by the Ministry of
Health (to P.C.).
Address reprint requests to Roberto Corrocher, MD, Institute of
Medical Pathology, Chair of Internal Medicine, Policlinico Borgo
Roma, 37134 Verona, Italy.
The publication costsof this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/9.5/86/ 1-0046$3.00/0
4050
-
SUBJECTS AND METHODS
Subjects. A detailed description of subjects affected by the
“hyperfemtinemia-cataract syndrome” has been previously reported.RBriefly, they have congenital bilateral nuclear cataract and
serum ferritin ranging from 950 to 2,259 pg/L. Differently from
hereditary hemochromatosis patients, they have normal to low serum
iron and transferrin saturation, and no evidence of parenchymal iron
overload, as assessed by liver and bone marrowbiopsy. When unnecessary phlebotomies are performed, they rapidly develop iron-deficient anemia (reversed by adequate iron therapy), with persistently
elevated levels of serum ferritin. Further differences with respect
to hereditary hemochromatosis are represented by the autosomal
dominant transmission and the lack of relation with HLA. One of
the two families previously described was immediately available for
the genetic study. It was composed of 6 individuals, 3 affected
Blood, Vol 86, No 1 1 (December l ) , 1995:pp 4050-4053
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THE VERONA MUTATION
IRE-BP
IRE-BP
CL"
bind
bind
mRNA
mRNA
slcm-loop
Tramlation inhibited
Translalion pmccedr
M
N
ND
Fig 1. Iron-mediated regulation of ferritin at translational level.
(father and 2 children). and 3 not affected (mother and the 2 other
children).
A m / d i f i c d o u Nf ,gcwonric DNA. Genomic DNA was isolated
from peripheral hlood lymphocytes according to stantlard protocols.
DNA amplitication by polymerase chain reaction (PCR) was performed according to standard protocols byincorporating the T7
phage promoter sequence into one o f the PCR primers. In this manner.the amplified product canheprocessed by RNA-SSCP technique. Five different pairs o f primers were designed on the genomic
structure offerritin L-subunit gene (HSAFLI 2 and HSAFL34 EMRL
sequences). The sequences of the primers. their location. and the
size of the amplified products are reported i n Table I .
Scwrch , f i ~ rnew m t t r d o n s . The search for new mutation was
performed by RNA-SSCP technology, according to protocols previously described."'.'' After PCR reaction. transcription was performed with I O U of T7 RNA polymerase in a tinal volunw o f I O
pL containing I O mmol/L dithiothreitol ( D I T ) . 40 mmol/L Tris p H
7.5. 6 mmol/L MgCI?. 2 mmol/L spermidine. I O mmol/L NaCI. 5
nmol/L o f each ribonucleoside. I O U o f Rnasin. and 0 . 2 mL o f S35
UTP. Two microliters o f transcribed RNA was mixed with 48 pL
o f 95% formamide. 20 mmollL EDTA. 0.05% hromophenol blue.
and 0.05% xylene cyanol. The mixture was heated at95°C for 6
minutes and then chilled on ice for I O minutes. An aliquot o f 4.4
pL was then loaded onto a 6.5% nondenaturing polyacrylamide gel.
Electrophoresis was performed at 30 W constant power for 13 hours.
Table 1. Description of Primers Used in This Study
Fragment
Size
5'UTR
287
Exon 1
Exon 2
Exon 3
Exon 4
Primers
F-TCCTTGCCACCGCAGATTG
R-TTGGCAAGAAGGAGCTAACI
208
F-ATCTCCTGCTTCTGGGA
R-GCAGCTGGAGGAAATTAG*
221
F-CTCCCGCTAACCATTGT
R-CTGGGAGATGTAGTCCAT'
275
F-AGGTITAGTTCTATGTGCC
R-AGGTGTGAATGAGGCTCTG'
F-TTAATCTGCCAACTGGCTGC
319
R-AAGCTGCCTATTGGCTGGAl
The sequence of the primers are 5' to 3'. The asterisks indicate the
primers to whomthe l
7 tail bas been added to carry out RNA-SSCP
assays. Themodified primerused to test the presence of the mutation
creating an artificial Dde I restriction site was: CGGATGTGTTCGTCACTCA. This reverse primer was combined in the PCR reaction with the
F primer of the 5'UTR.
Abbreviations: F, forward; R, reverse.
Fig 2. (Top) DNA automatic sequence showing, in one affected
the presenceof an ambiguity l*) corresponding to a
patient (M),
nucleotidechange G to C; the sequence of onenormal control (NI is
also reported. (Bottom) Demonstration of the mutated sequence in
the affected patient by restriction-generationPCR using a modified
primer to create a Dde I artificial restriction site. In the presence of
the mutation, an additional band of 157 bp is present.
After electrophoresis, the pel was dried and suh.jected to autoradiography for I2 hours. Samples showing an electrophoretically altered
mobility were then sequenced o n an automatic sequencer (Applied
Biosystem 373A). according to manufacturer's protocols.
RESULTS
The S'UTR plus the four exons of ferritin L gene have
been screened for the presence of nucleotide alterations. An
electrophoretically abnormal bandwas detected in the affected members of the family analyzing the fragment corresponding to the S'UTR of the gene. Sequence analysis alC substitution at nucleotide
lowed us toidentify a G
position 147 of theferritin L gene sequence (EMBL sequence name HSAFL12) (Fig 2, top). The mutation could
be easily analyzed byPCRand agarose gel electrophoresis
using a modified primer which introduces two base substitutions adjacent to the mutation site and create an artificial
D& I restriction site," as shown on Fig 2 (bottom).
-
DISCUSSION
The main feature of therecentlydescribed"hereditary
hyperferritinemia-cataract syndrome" appears to bean excess of ferritin synthesis that is independent of the body iron
stores. For example. if a mistaken diagnosis of hereditary
hemochromatosis is made in affected subjects. they rapidly
develop iron deficiency anemia in response to phlebotomies,
whereas serum ferritin levels remain substantially elevated.x
This prompted us to focus our attention firstonferritin L
gene. RNA-SSCP technology is an accurate and sensitive
method able to detect almost a l l kinds of nucleotide alterations present in a given DNA fragment."'." It allowed us
to detect in affected subjects a nucleotide alteration in the
S'UTR of the ferritin L gene. This mutation (named the
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4052
GlRELLl ET AL
*C
@J
A-U
A-U
C-G
U-A
Fig 3. Particular of the IRE in
the S’UTR of ferritin mRNA. *,
Mutation detected in subjects
affected by the “hereditary
hyperferritinemia-cataract syndrome.” The G to C mutation
involves one of the five highly
conserved nucleotidesthat characterize the CAGUG loop saquence of IRE.
-
“Verona mutation”) is a G
Csubstitutioninthe
third
residue of the 5-base sequence (CAGUG) that characterizes
the loop structure of the IRE (Fig 3). The IRES are stemloop structures of approximately 28 nucleotides, which are
both necessary and sufficient for iron-mediatedregulation
offerritin biosynthesis’jand highly conservedduring the
evolution; in particular, the CAGUG sequence of the loop
is present in allthe ferritin mRNAsexamined inverteb r a t e ~ . ’IRE-RP
~
is the cytoplasmic proteinthatmaintain
cellular iron homeostasis by coordinating the translation of
ferritin and transferrin receptor mRNAs. There is strong evidence that IRE-BP recognizes the IRE as a sequencdstructure motif,15 leading to inhibition of ferritin translation, except when iron is abundant. Extensive in vitro studies using
synthetic IRE-analogue oligonucleotides have tested the effects of specific site-directed mutations either in the loop or
in the upper stem.’.’’ These mutational data suggested that
the integrity of the CAGUG sequence in the loop as well as
the basepairing along the upper stem are critical for the
IRWIRE-BP high-affinity interaction. Also, a single-basedeletion in the loop abolished the iron-mediated translational
regulationintransfectedcells.I3
More recently, 28 altered
IRES with single-basemutations (substitution or deletion)in
the loop or in the upper stem weretested for interaction
with the IRE-BP, all resulting in a substantially decreased
binding.“ It is noteworthy that this study includedthe evaluC single-base
ationofa synthetic IRE-RNA with aG
substitution in thethird residue of the CAGUG loop sequence,whichwas identical tothespontaneous mutation
detected by us. Competition experiments showed that this
IRE-mutant interacted with the IRE-BPwith a 28-fold lower
affinitythan didthenative RNA.16 This bulk of in vitro
observations strongly supportsfor the importance of the
spontaneous G
Cmutation we detectedinsubjects affected by the “hyperferritinemia-cataract syndrome.” By
altering the IRE/IRE-BP interaction, the Verona mutation is
expected to determinethe lack of attenuationofferritin
mRNA translation, leading ultimately to iron-insensitive, uncontrolled ferritin synthesis. In this way, the “hyperferritinemia-cataract syndrome”appearstobe
aunique human
-
-
model providing insight into the in vivo regulation of iron
homeostasis.
At present, we do not have aclear-cutexplanation
for
the presence of bilateral cataract in subjects affected by the
genetic disorder of the iron metabolism here described. Hereditary cataract is genotypically and phenotypically heterogeneous, and couldbe caused by either dysfunction of genes
coding for lens-specific proteins or by alteration of the environment of the lens.I7 Recently, a membrane protein (MP19)
of lens fiber cell hasbeen assigned to chromosome 19ql3.4,‘
near the L-ferritin gene. However, it is difficult to see how
a point mutation in the L-ferritin gene can lead to abnormal
expression of thenearby MP19 gene.Similarly, it seems
unlikely that the simultaneous presence of cataract and hyperferritinemia may be caused by a cosegregation of a mutated allele in each of the two genes lying in close vicinity.
On the other hand, it is of interest to note that several lines
of evidence argue in favour of a role of iron-related mechanisms in cataracts formation. First, iron-catalyzed reactions
have been implicated in causing lens oxidative damagesimilar to that seen in cataractogenesis,” and lensepithelial cells
have been shown capable of active ferritin synthesis, especially in response to oxidative damage.I9 Second, ocular siderosis caused by retention of an iron-containing intraocular
foreign body is frequently followed by cataract formation.’“
Third,cataracts havebeenreported
in patients with iron
overload, especially when treated with the iron chelator deferroxamine.”.*? Thus, it seems reasonable to speculate that
the inappropriate production of ferritin may be directly responsible for cataract formation in individuals affected by
this disorder of iron metabolism. Whether this hypothesis is
true and the molecular mechanism(s) by which the disorder
of ferritin synthesis may favor per se lens opacity remain to
be determined.
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