PEDIATRICS Vol. 105 No. 2 February 2000, pp. 426-428

EXPERIENCE AND REASON:
Hereditary Hemochromatosis in Siblings: Diagnosis by Genotyping

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron metabolism, with a worldwide prevalence in whites of approximately 3 to 5 per 1000 population.^1-3 Unrecognized HH can lead to cirrhosis, diabetes mellitus, hyperpigmentation, and impotence in untreated individuals.³ Although usually not symptomatic until the ages of 50 to 70, HH has been described in children. Screening first-degree relatives of a person with HH has identified many children although symptomatic HH remains uncommon in children. However, with such a high prevalence of HH it is safe to say that all pediatricians have seen an affected child, but in the asymptomatic period. One of the principle tasks of the pediatrician is prevention; HH is a condition that, identified in the presymptomatic period, can be treated, avoiding potentially life-threatening complications. Recently a genetic defect has been described on the short arm of chromosome 6 that is found in nearly 90% of patients studied with HH.^3,4 Commercial testing for HH via mutational analysis is now possible. Once the general public becomes aware of the availability of HH testing, the pediatrician will be called on to screen potential affected children. In this article we describe a pair of siblings with HH identified by mutational analysis. Further, we review HH in children and discuss the rationale for screening children for this common inherited disorder.

	CASE REPORT

A 131/2-year-old white female was referred to pediatric gastroenterology for evaluation of increased serum iron levels. The patient was relatively healthy but had complained of decreased energy and fatigue for 3 to 4 months preceding the evaluation by her primary care physician. There was no history of weight loss, melena, hematochezia, or chronic emesis. She was menstruating. The past medical history was significant for benign Rolandic seizures (for which the patient received carbamazepine) and attention deficit disorder. The patient had no history of previous surgery or transfusion with blood products. Family history was negative for hemoglobinopathies, Wilson's disease, chronic hepatitis, and hemochromatosis.

As part of the laboratory evaluation for fatigue, serum iron studies were obtained. The results were as follows: serum iron: 186 µg/dL (nL: 40-155 µg/dL); total iron-binding capacity (TIBC): 212 µg/dL (nL: 240-400 µg/dL); ferritin: 267 ng/mL (nL: 10-291 ng/mL); and transferrin saturation (TS): 88% (nL: 20%-50%). The patient was referred to pediatric gastroenterology for further evaluation of the abnormal iron studies.

Physical examination at presentation revealed a slightly obese white female. There was no abnormal skin pigmentation, organomegaly, spider angiomata, or palmar erythema. Repeat laboratory studies obtained fasting revealed persistent elevation of TS ranging from 53% to 57%. A liver profile showed normal levels of alanine and aspartate aminotransferase, alkaline phosphatase, -glutamyl transpeptidase, bilirubin, and albumin. Prothrombin time and partial thromboplastin time were also normal. A percutaneous liver biopsy revealed early fibrous expansion of the portal areas and increased iron stores in periportal hepatocytes. No significant iron deposition was noted in Kupffer cells. The hepatic iron concentration (HIC) was 3750 µg/g dry weight (nL: 100-1600 µg/g) and the calculated hepatic iron index (HII) was 5.0. The patient was tested for the presence of mutational abnormalities consistent with HH. Briefly, DNA was isolated from blood and amplified by polymerase chain reaction (PCR) with specific primers. The PCR products were then subjected to restriction enzyme digestion followed by electrophoresis.³ Mutants are differentiated from normal based on specific patterns of bands on electrophoresis. The patient was homozygous for the C282Y mutation. Phlebotomy therapy was initiated. Ten units of blood were removed over 6 months with improvement in all parameters of iron homeostasis: serum iron: 73 µg/dL; TIBC: 275 µg/dL; ferritin: 54 ng/mL; and TS: 27%. The patient's symptoms of fatigue resolved with normalization of serum iron status.

The patient had a younger male sibling, 9 years old, who was also screened for evidence of hemochromatosis. His iron status was as follows: serum iron: 72-103 µg/dL; TIBC: 290-295 µg/dL; and TS: 24%-36%. He was also homozygous for the C282Y mutation. He has not yet begun phlebotomy therapy.

	DISCUSSION

Top Introduction Discussion Conclusion References

HH is an autosomal recessive disorder of inappropriately high iron absorption and progressive iron deposition in various organs, particularly the liver and pancreas. It is a common disease in whites, with a prevalence of a heterozygous state of 1 in 10 and a homozygous state of 3 to 5 per 1000 general population.^1-3 The prevalence of HH in children with 1 parent with homozygous HH is approximately 1 in 20.²

Although common, HH typically remains asymptomatic until the ages of 50 to 70, making it a relatively rare "disease' in children. The majority of children reported to date have been identified during screening of family members of a patient with HH. Symptomatic disease in children, although rare, presents with some of the features observed in adults including fatigue, abnormal skin pigmentation, abnormal liver tests, cirrhosis, cardiomyopathy, hypogonadism, diabetes mellitus, and recurrent abdominal pain.^5-10 Similar to adults with HH, symptomatic HH in children is more common in males. In one report of children with HH, the male to female ratio was 1:2 for asymptomatic disease and 3:1 for symptomatic disease.¹⁰ In children, HH should be distinguished from 2 other rare iron overload syndromes, neonatal hemochromatosis (more appropriately termed neonatal iron storage disease) and juvenile hemochromatosis. Neonatal iron storage disease presents within hours of birth with severe hepatic dysfunction. The mechanism for iron overload in this condition remains unresolved. Juvenile hemochromatosis shares features with HH but the clinical course is more severe and the onset of symptoms is earlier. Recently, juvenile hemochromatosis has been shown to be genetically distinct from HH.¹¹

In HH the importance of diagnosis in the "presymptomatic' period can not be overemphasized. Subjects with HH without cirrhosis have a normal life expectancy when treated with phlebotomy.^3,12 Conversely, a decreased life expectancy is observed in HH with cirrhosis, diabetes mellitus, and inability to deplete iron by phlebotomy in 18 months.¹³ Attempts have been made to identify a reliable screening test to identify affected individuals before symptoms. Measurement of various serum markers of iron overload have been used, including serum iron, serum ferritin, and TS. The sensitivity in diagnosing HH with any single marker varies from 68% to 85%. The highest sensitivity (94%) is reached using the combination of serum ferritin and TS.¹⁴ Elevated serum TS is the most specific marker for HH.¹⁴ False-positive testing may occur if blood is obtained after meals, thus an elevated TS must be verified by repeat testing with fasting blood levels. False-negative testing may occur in the young, particularly females.¹ This reflects the limited time for iron accumulation and the loss of iron during menstruation. In children a TS >50% and/or a ferritin >90% of normal, age-specific levels warrants further evaluation.¹⁰

Although serum iron studies can be used for screening, definitive diagnosis requires further testing. Presently, the "gold standard" is documentation of an elevated HIC, which requires a liver biopsy. However, an elevated HIC is not diagnostic of HH. The challenge for the physician is to differentiate HH from the various causes of secondary iron overload. The HII, which is the HIC (in micromoles of iron per gram of dry weight) divided by the patient's age (in years), is useful in making this differentiation. Typically, the HIC increases with age in homozygous HH but not in patients with secondary iron overload or in patients who are heterozygous for HH.¹⁵ It has been estimated that in homozygous HH, serum ferritin increases by approximately 65 ng/mL/y, which is equivalent to the absorption of approximately 500 mg of iron/y.¹⁴ A HII >1.9 has been considered diagnostic of HH; conversely, a HII <1.9 could be associated with secondary iron overload or heterozygosity.¹⁶ However, recent studies have shown that approximately 10% to 15% of homozygous HH patients have a HII <1.9.^3,17 The histologic appearance on the biopsy may also be useful. In HH, iron is found in hepatocytes, beginning in zone 1 and with progression of the disease extending, to zone 3. There is minimal, if any, Kupffer cell iron deposition until late in the disease.¹⁰ In secondary iron overload, the majority of the iron is found in Kupffer cells.

The discovery of a gene for HH, referred to as HFE, was first reported in 1996.⁴ Two mutations have been identified in HFE; one results in a change in cysteine at position 282 to tyrosine (C282Y), the second results in a change in histidine at position 63 to aspartate (H63D).⁴ At least 4 studies have determined the presence of these genetic abnormalities in patients with typical phenotypic hemochromatosis. Homozygosity for the C282Y mutation was found in 82% to 100%,^418-20, while compound heterozygosity, 1 allele for C282Y and 1 allele for H63D, was found in 4% to 5%.^4,18,19 These studies suggest that H63D may play a role in the development of clinically significant iron overload when found in conjunction with C282Y.²¹ Approximately 8% to 10% of patients with phenotypic hemochromatosis do not have evidence of this genetic abnormality, suggesting another syndrome of iron overload in these individuals.²¹

The use of mutation analysis for the determination of HH has revolutionized the diagnosis and subsequent treatment of HH. As the genetic abnormality is present from birth, it will obviously be positive before the development of abnormalities in serum iron markers. The male sibling we presented, where the C282Y mutation was present but serum iron markers were normal, provides evidence of this. Mutational analysis could supplant liver biopsy as an essential diagnostic test, although liver biopsy will remain useful in determining the extent of hepatic injury in patients with symptomatic HH. As stated above, 8% to 10% of patients with typical phenotypic hemochromatosis do not have the described genetic abnormality.²² In these patients and their family members, diagnosis will continue to depend on serum iron markers and liver biopsy. The use of mutation analysis would avoid a potentially dangerous procedure, as well as lead to significant cost-saving.

At present, routine population screening for HH by mutational analysis can not be suggested.²³ There are still questions that need to be answered. Does the genetic abnormality have complete penetrance? At what age do iron studies become abnormal? What is the ideal time for initiating phlebotomy? Until further research dictates differently, a reasonable approach to the diagnosis of HH in children is outlined below (Fig 1).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   An approach to the evaluation of the child at risk for HH. This flow diagram represents a reasonable approach to the evaluation of a child at risk for HH because of a parent (proband) known to have hemochromatosis. The first step is to determine if the parent has a mutational abnormality associated with HH. If the proband's mutational analysis is negative, then assessment of iron status in the child should be done around puberty. Iron studies can be repeated periodically until they indicate iron overload; at that point a liver biopsy should be performed to determine the HII. If the proband's mutational analysis is positive, the child should undergo mutational analysis. If the mutational analysis on the child is negative, no further evaluation is needed. If the mutational analysis on the child is positive, fasting serum iron studies should be performed. If the iron studies indicate iron overload, phlebotomy should be initiated. If the iron studies are normal they should be repeated periodically (every 1 to 2 years). # = mutational analysis C282Y/C282Y and C282Y/H63D; ## = mutational analysis wild type/C282Y, wild type/H63D, wild type/wild type, H63D/H63D; *fasting iron studies include TIBC, serum iron, and ferritin; **abnormal includes a TS >50% or a ferritin >90% of normal age-specific levels.

When an adult patient has HH and wants to determine if their child is also affected, mutation analysis for C282Y and H63D should be performed. This test is commercially available. If the proband is homozygous for C282Y, then all children of the proband should undergo mutation analysis. If mutational analysis is negative in the child, no further evaluation is needed. If mutational analysis is positive, fasting serum iron studies should be performed. If there is evidence of iron overload, (elevated TS, elevated ferritin) phlebotomy should be initiated, consisting of removal of 5 to 7 mL/kg of blood every week until the iron studies normalize (TS <50% and ferritin <50 ng/mL).²¹ Phlebotomy can then be performed every 3 to 4 months to keep the serum iron studies normal. Our patient who received phlebotomy treatment had normalization of iron studies after the removal of 10 units of blood. The number of units of blood that must be removed to normalize iron status depends on the degree of iron overload. Normalization may occur with as little as 8 to 11 units of blood.¹⁷

If the child is homozygous for the C282Y mutation but the initial serum iron studies are normal, the iron status should be rechecked periodically. Strict guidelines can not be suggested at this time, as it has not been determined at what age iron studies reflect iron overload in HH. A reasonable approach would be to retest at puberty and then every 1 to 2 years. Once iron overload is documented, phlebotomy should be initiated as outlined above.

If the proband does not have a mutation consistent with HH, the child should be screened with fasting serum iron markers. If normal, the iron studies should be repeated as outlined above. Once serum tests become abnormal a liver biopsy should be performed to document hepatic iron overload. If there is an elevated HIC, phlebotomy should be initiated.

In the rare child with symptoms suggestive of hemochromatosis, but without a family history, serum iron studies should be drawn. Further evaluation and treatment should be dictated by the results of the serum iron studies.

	CONCLUSION

Top Introduction Discussion Conclusion References

In summary, we report the use of mutation analysis in the diagnosis of HH in children. The importance of this new diagnostic test is the ability to document a potentially serious disease before symptoms develop, thus allowing timely screening and early intervention. Commercial testing is now available. The appropriate use of this test may lead to an earlier diagnosis of HH, making this a treatable disease of childhood, rather than a potentially serious disease of adulthood. At the present time routine population screening can not be suggested. The question of insurability not withstanding, presently there is no cost-effective means to attain this goal. Further research is needed to determine when iron studies indicate overload, thus allowing better, more economical, recommendations on the appropriate timing for screening.

Michael J. Nowicki, MD
Division of Pediatric Gastroenterology
University of Mississippi Medical Center
Jackson, MS 39216

Bruce R. Bacon, MD
Division of Gastroenterology and Hepatology
Saint Louis University School of Medicine
St Louis, MO 63110

	FOOTNOTES

Received for publication Jan 11, 1999; accepted May 26, 1999.

Address correspondence to Michael J. Nowicki, MD, Department of Pediatrics, Division of Pediatric Gastroenterology and Nutrition, University of Mississippi Medical Center, 2500 N State St, Jackson, MS 39216. E-mail: mnowicki{at}ped.umsmed.edu

	ABBREVIATIONS

HH, hereditary hemochromatosis; TIBC, total iron-binding capacity; TS, transferrin saturation; HIC, hepatic iron concentration; HII, hepatic iron index; PCR, polymerase chain reaction.

	REFERENCES

Top Introduction Discussion Conclusion References

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