Simple and Rapid Detection of the Newly Described Mutations in the HLA-H Gene

To the Editor:

A candidate gene for hereditary hemochromatosis has been recently described1 (Feder et al, 1996). Two different nucleotide substitutions have been found on the cDNA sequence of that gene in a sample of patients with hereditary hemochromatosis. The most important sequence variation is a G-to-A transition at nucleotide 845 of the open reading frame that is present in 83% of the 178 tested patients. It results in a cysteine-to-tyrosine substitution at amino acid 282 (mutation C282Y). The second substitution is found with a higher rate in the group of patients heterozygous for the C282Y substitution than in control individuals. It is responsible for a histidine to acid aspartic change at position 63 (mutation H63D).

It now seems important to test for these two mutations in various samples of patients with hereditary hemochromatosis (HH) as well as in control individuals to determine their frequency in different populations and their actual linkage with the disease. To investigate both substitutions we designed a simple and reliable assay based on modification of natural restriction sites.2 

Twenty unrelated patients with HH and 50 control individuals were examined. The patients were all diagnosed with hemochromatosis according to the following criteria: absence of any secondary cause of iron overload, evidence of a major iron overload removed by regular phlebotomies, and, in most cases, more than one affected individual in the pedigree. The control sample was made of 50 white individuals.

Blood samples were obtained after informed and written consent. DNA was extracted by using a simple salting out procedure.3 For each individual, we systematically amplified two polymerase chain reaction (PCR) fragments surrounding both mutations, using the pairs of primers previously described by Feder et al.1 The PCR mixtures contained 20 pmol of both primers, 200 μmol of each deoxy nucleotide triphosphate, 250 ng of genomic DNA, 10× buffer, and 0.75 U of Taq polymerase (Perkin Elmer Cetus Instruments, Norwalk, CT) in a final volume of 25 μL. Amplification was done using 5-minute denaturation at 94°C followed by 30 cycles of 30-second denaturation at 94°C, 30-second annealing at 56°C, and 1-minute extension at 72°C. A final extension cycle of 10 minutes at 72°C was performed. Ten microliters of the amplified products was subjected to digestion with Rsa I or SnabI for the C282Y mutation, or with Bcl I for the H63D substitution, according to the manufacturer's recommendations. The digested products were then run on a 2% agarose gel during 1 hour and photographed under UV light.

Genomic DNA of two HH patients, as well as a control sample, was sequenced by automatic DNA sequencing using the same PCR primers. One patient was homozygous for the Y282 allele while the second was homozygous for the D63 allele as determined by restriction site analysis. The sequence analysis confirmed the presence of the expected mutation in both cases (data not shown).

Using the restriction site method, we have tested until now 20 patients with HH. Of them, 13 (65%) were homozygous for the C282Y mutation, while 3 (15%) were heterozygous. Thus, a total of 29 of 40 chromosomes (72.5%) bore the major mutation. One patient presenting with a typical clinical and biologic picture of HH and subjected to periodic phlebotomy for 6 years was homozygous for the H63D substitution. Three other HH patients (15%) were heterozygous for this second variant, two of them being at the same time heterozygous for the C282Y mutant. Only two patients (10%) of this series had none of the two mutants. Among the 50 controls, 3 (6%) were found to be heterozygous for the C282Y mutation, 5 (10%) were heterozygous for the H63D substitution, and 1 subject was a compound heterozygote for both mutations. Two control individuals (4%) were found to be homozygous for the D63 allele. A total of 39 controls (78%) had none of both mutations. As shown in Fig 1, the diagnosis between normal, heterozygotes, and homozygotes for both mutations is easy using the chosen restriction enzymes.

Both substitutions can be identified with a number of restriction endonucleases, the mutation either creating or abolishing the recognition site. We have chosen the enzymes according to their cost and to their ability to give rise to fragments easily identifiable on a simple agarose gel. The method is rapid, inexpensive, and can be performed on large samples. It can be semi-automated as the PCR reaction, as well as the digestion, can take place in 96-well plates. The electrophoretic migration can be run in large agarose gels allowing to test as much as 40 patients in a same experiment. By contrast, it is also easy to test with this method a single or a few samples of patients, if necessary, with a similar cost and ease. This is not true when using methods based on hybridization with oligonuclucleotides such as the OLA assay4 recommended in the report of Feder et al.1 Moreover, those hybridization methods need specific equipment that are not available in each diagnosis laboratory.

The finding of mutations that can be considered as causative for HH is of utmost scientific and clinical importance. It gives rise to the possibility of a presymptomatic screening for the disease. Therefore, more data are needed to clarify the part played by these molecular abnormalities. The use of a simple and reliable test can help develop future testing for the disease possibly in large scale screening programs.