A point mutation in the iron-responsive element of the L-ferritin gene in a family with hereditary hyperferritinemia cataract syndrome

1Department of Medicine; 2Department of Pathology; 3Department of Medicine, Division of Gastroenterology, University of Western Ontario, London, Ontario Correspondence: Dr Paul Adams, Department of Medicine, London Health Sciences Centre, University Campus, 339 Windermere Road, PO Box 5339, London, Ontario N6A 5A5. Telephone 519-858-5125, fax 519-858-5114, e-mail padams@uwo.ca Received for publication August 17, 2004. Accepted January 19, 2005 K Wong, Y Barbin, S Chakrabarti, P Adams. A point mutation in the iron-responsive element of the L-ferritin gene in a family with hereditary hyperferritinemia cataract syndrome. Can J Gastroenterol 2005;19(4):253-255.

H ereditary hyperferritinemia cataract syndrome (HHCS) is an autosomal dominant disorder that was first described in 1995 independently by Bonneau et al (1) and Girelli et al (2).It is characterized by early-onset bilateral cataracts and high serum ferritin levels in the absence of iron overload.Iron studies are otherwise normal, with normal to low serum iron and transferrin saturation (3).Because of the assumption of iron overload, these patients are often mistakenly treated with repeat phlebotomies, resulting in iron deficiency anemia.The cataracts in this syndrome have a distinct morphology typified by slowly progressive cystic flecks with crystalline deposits in the lens cortex and nucleus that may be pathognomonic for HHCS (4).The prevalence of this syndrome is not known, but a research group in Australia estimated that there is a prevalence of at least one in 200,000 in southeastern Australia (4).Mutations in the iron-responsive element (IRE) of the L-ferritin gene located on chromosome 19q13.33-13.4 is the molecular basis of this genetic disorder.The IRE is a stem-loop structure located in the 5' noncoding region of the L-ferritin messenger RNA (mRNA).The role of the IRE is to interact with the cytoplasmic iron-responsive proteins (IRP) to regulate ferritin synthesis in response to cytoplasmic iron pools.When iron pools are adequate, the binding of the IRP to the IRE prevents the recruitment of the small ribosomal subunit to the mRNA, thereby, inhibiting the initiation of translation ( 5).The IRE is not unique to L-ferritin but is also found in H-ferritin, the 3' region of the transferrin receptor mRNA ( 6) and the erythroid aminolevulinate synthase mRNA (7).Mutations in the IRE have been shown to decrease its affinity for the IRP, leading to the unregulated, constitutive synthesis of the L-ferritin mRNA, irrespective of the body's iron status.This change in affinity has been demonstrated in vivo with lymphoblastoid cell lines (8) and in vitro using thermodynamic analysis (9).Ferritin is normally expressed in the lens and is thought to function as an antioxidant (4).It is the overproduction of L-ferritin resulting in intracellular accumulation in the lens that is thought to lead to the formation of cataracts in HHCS (4).
Almost 20 different mutations have been described in the IRE of the L-ferritin gene.Heterozygous point mutations in the 5' region of the L-ferritin gene account for the majority of the mutations with the exception of three deletion mutations (from two-base pair to 28-base pair deletions).We set out to determine whether the family in the present study had a mutation in the IRE.

CASE PRESENTATION
The proband (III-5) (Figure 1) was a 22-year-old woman who was initially found to have an elevated ferritin level of 1296 µg/L on bloodwork drawn during an episode of infectious mononucleosis at 16 years of age.She was referred to the hepatology clinic for further assessment of this persistent elevated ferritin.Aside from hyperferritinemia, her iron studies were otherwise normal.Her serum iron was 35.6 µg/mL and transferrin saturation was 45%.Workup included genetic testing for hemochromatosis which was negative for both the C282Y and H63D mutations of the hemochromatosis gene.A liver biopsy did not show evidence of iron overload with no stainable iron and a normal liver iron concentration of 5.4 µmol/g (normal 0 µmol/g to 35 µmol/g dry weight).For six years, she was noted to have a serum ferritin greater than 1000 µg/L.At 22 years of age, the patient underwent weekly 500 mL phlebotomy treatments and dropped her hemoglobin level from 153 g/L to 84 g/L over one month.She subsequently developed symptomatic cataracts and, given a positive family history of cataracts, the diagnosis of HHCS was made and she underwent surgical treatment.

METHODS DNA analysis
Genomic DNA was extracted from peripheral blood leukocytes of eight individuals in the pedigree (Figure 1), four of whom were affected by cataracts.The 5' region of the L-ferritin gene was amplified by polymerase chain reaction (PCR) using flanking primers (forward 5'-TCCTTGCCACCGCAGATTG-3'; reverse 5'-TTGGCAAGAAGGAGCTAACC-3') (10).PCR reactions were made up to a total volume of 50 µL containing 25 pmol of each primer, 1 U Taq polymerase and 2.5 µmol magnesium chloride.PCR reactions were carried out under the following conditions using the PTC-100 Thermocycler: denaturation at 94°C for 30 s, annealing at 54°C for 30 s and extension at 72°C for 90 s.Nested PCR was performed using the same primers to yield a 287-base pair product.PCR products were gel purified and DNA extracted using QIAquick Gel Extraction Kit (QIAGEN Inc, Canada).DNA was sent for direct sequencing at the Robarts Research Institute (London, Ontario) sequencing laboratory.

RESULTS
Serum ferritin levels in affected individuals were elevated and ranged from 1307 µg/L to 1976 µg/L.The ferritin levels in the unaffected individuals ranged from 17 µg/L to 20 µg/L.Sequencing showed a heterozygous point mutation at position +32 (G→T) in the affected members (Figure 2).This mutation was located in the bulge of the IRE.

DISCUSSION
During the workup of an individual for hyperferritinemia, a family was identified with a clinical suspicion of HHCS.The affected individuals of this family were found to have high serum ferritin levels (1307 µg/L to 1976 µg/L) and early-onset cataracts requiring surgical intervention.DNA analysis identified a heterozygous mutation (G32T) in the bulge of the IRE in the affected members.This mutation has also been described in six individuals in France (11,12) and one family in Australia (4).The G32T mutation has previously been shown to lead to a decrease in binding affinity of IRE to the IRP by in vitro gel retardation assays performed by Martin et al (12).Mutations in the bulge of the IRE are the most common and account for approximately 50% of all mutations described in the IRE (11).
There is a spectrum in the clinical presentation of HHCS varying from mildly elevated ferritin levels and asymptomatic  cataracts, to ferritin levels greater than 1000 µg/L and cataracts requiring surgical intervention in childhood.There have been suggestions that each unique mutation may correspond to the severity of HHCS and that varying degrees of the effect on binding affinity may explain this phenotypic variability.This variation of binding affinity was demonstrated by Allerson et al (9) using thermodynamic analysis.Although there appears to be a correlation between each mutation and disease severity, the range of ferritin levels in individuals with the same mutation is wide and the age of onset of cataracts varies among families with the same mutation as well as among members of the same family.This suggests that there may be other determinants, such as environmental factors, that affect the penetrance of this genetic disorder or alter the phenotypic manifestation (13).Wide ranges in ferritin levels may or may not reflect coexisting inflammatory conditions.In the present study, the patient developed symptoms of cataracts in her early 20s; however, individual IV:4, who was six years of age, was already known to have cataracts on ophthalmological examination.Timely diagnosis of this disorder has been challenging because of the low awareness of this condition among physicians.HHCS should be added to the differential for hyperferritinemia; in particular, in the absence of iron overload.However, the finding of high ferritin levels is often incidental because testing is not routine in ophthalmology practice.Although Craig et al (4) have described the distinct morphology of these cataracts, suggesting that they may be pathognomonic, the lack of familiarity with this disease prevents an early ophthalmological diagnosis.The treatment for this disorder is surgical and there is currently no known therapy for preventing or slowing the formation of cataracts; however, accurate diagnosis of this disorder may prevent inappropriate phlebotomy treatments leading to iron deficiency anemia.