Neonatal Screening for the Hemochromatosis Defect

To the Editor:

Hereditary hemochromatosis (HC) is an autosomal recessive disorder of iron metabolism that is characterized by inappropriate iron absorption and storage of excess iron in the parenchymal cells of major organs, primarily the liver, pancreas, heart, pituitary, and joints. High levels of iron stored in these organs can lead to cirrhosis, hepatocellular carcinoma, cardiac dysfunction, diabetes, arthritis, hypogonadism, and premature death.1 However, patients can have a normal life expectancy if the disorder is diagnosed in the early stages and phlebotomy therapy is undertaken to remove the excess iron.2 

A novel major histocompatibility complex (MHC) class I-like gene termed HLA-H was recently identified telomeric of the classical MHC complex on the short arm of chromosome 6 and proposed as a candidate for HC.3 In this initial report, 83% of HC patients were found to be homozygous for a single missense mutation, causing an amino acid substitution of cysteine to tyrosine at residue 282 (C282Y).3 Subsequent studies have found 92.4%⁴ and 100%⁵ of HC patients from well-characterized families homozygous for this mutation, providing very strong supporting evidence for the C282Y mutation in HLA-H as the genetic defect responsible for HC. This gene has now been named HFE in accordance with the accepted nomenclature for the HC locus.6,7 

HC is a common disorder in Caucasian populations but one that can be difficult to diagnose by biochemical analyses. Previous studies using biochemical expression of iron overload have indicated that the frequency of HC homozygotes in Caucasian populations is likely to be between 3 and 5 per 1,000.8,9 

The recent identification of HFE and the presence of a single causative mutation provide an opportunity for the population frequency of the mutant allele to be accurately determined. Comparison of the frequency of HC as determined by biochemical analyses with that determined by detection of the mutant allele will provide insight into the significance of environmental and other genetic factors in determining the phenotypic expression of this disorder.

The present study used a random sample of Australian neonates to determine the prevalence of the C282Y mutation in HLA-H. A total of 1,660 blood samples were obtained from consecutive neonatal screening cards collected by the Brisbane neonatal screening unit. The population of the region from which these samples were collected is predominantly Caucasian. Samples collected were anonymous and this study was approved by the Ethics Committees of The Royal Children's Hospital (Brisbane) and The Queensland Institute of Medical Research.

Three-millimeter squares were cut from dried blood spots using sterile scalpel blades. These squares were then fixed by soaking with absolute methanol and allowed to air dry (for approximately 30 minutes) at room temperature. Samples were then placed in 0.6-mL PCR tubes with 40 μL of sterile ddH₂O and incubated at 60°C for 30 minutes, followed by 96°C for 30 minutes in a Perkin Elmer 9600 thermal cycler (Perkin Elmer, Norwalk, CT) to elute the DNA. Twenty microliters of the resulting supernatant was used in subsequent polymerase chain reaction (PCR).

PCR amplification of the region containing the C282Y mutation was performed using the oligonucleotide primers previously described.3 PCR was performed in 25-μL volumes containing 200 ng of each primer, 400 μmol/L dNTPs, 1.5 mmol/L MgCl₂ , and 1.5 U of Taq polymerase (Cetus, Sunnyvale, CA) under the following conditions: 94°C for 5 minutes, followed by 35 cycles of 94°C for 30 seconds, 55°C for 50 seconds, and 72°C for 50 seconds and then a final step of 72°C for 5 minutes. PCR was performed in a Perkin Elmer 9600 thermal cycler.

The presence of the C282Y mutation creates a SnaBI restriction site in the amplified product enabling detection of the mutation by enzyme digest. Twelve microliters of the amplified product was digested with 2 U SnaBI (Promega, Madison, WI) for 2 hours at 37°C in a total volume of 20 μL. Samples were then analyzed on a 2% agarose gel. A control sample, known to be homozygous for C282Y, was included to confirm complete digestion of all samples.

Of the 1,660 samples analyzed, 8 (1 in 200 or 0.48%) were homozygous for the C282Y mutation and 186 (1 in 9 or 11.2%) were heterozygous. This gives a frequency for the HC allele of 0.061 in this predominantly Caucasian population. Previous estimates of the frequency of homozygotes for this disorder have been lower than this; however, all previous screening studies have relied on biochemical and/or pathological expression of HC for diagnosis.8,9 This indicates that phenotypic expression of HC may be prevented in approximately one third of individuals homozygous for the mutant allele, due to environmental factors such as dietary iron intake and physiological blood loss, as well as sex and age. In addition, a small percentage of patients heterozygous for the C282Y mutation have been shown to meet clinical diagnostic criteria for HC,10 further indicating the significance of environmental and possibly other genetic factors in expression.

The C282Y mutation can be easily and rapidly detected; thus, population screening is feasible. However, because many of those homozygous for this defect will not develop iron overload requiring treatment, the cost effectiveness of widespread population screening requires further evaluation. However, detection of the mutation is useful in confirming the diagnosis in those with increased iron indices.