Detection of C282Y and H63D Mutations in the Hemochromatosis Gene Using SSCP and Sequencing

Ira W. Daly


Hereditary Hemochromatosis (HH) is a recessive disorder characterized by increased iron absorption that leads to irreversible tissue damage. The identification of mutations in the hemo-chromatosis gene, HFE, that cause HH has made it possible to test potential carriers of those mutations before the onset of the disease in adulthood. Conventional genetic testing for the C282Y and H63D mutations has relied on restriction fragment length polymorphisms (RFLP). This study utilizes single-strand conformation polymorphism (SSCP) analysis to detect point mutations in the regions containing codons 282 and 63 of HFE. Dideoxy sequencing of these regions confirmed SSCP detected mutations.

Hereditary Hemochromatosis (HH) is an autosomal recessive disorder characterized by increased iron absorption that leads to iron overload of parenchymal cells in many organs. Increased iron accumulation may lead to cirrhosis of the liver, hepatocellular carcinoma, diabetes, heart failure, arthritis, and hypogonadism. HFE, the gene imp-licated in HH, was cloned by Feder et al. in 1996 (1, 2). The HFE gene encodes a protein structurally similar to MHC class I-type molecules that interacts with the transferrin receptor and regulates the absorption of iron (3).
Hemochromatosis is one of the most common autosomal recessive disorders in Caucasians but is often overlooked in clinical diagnosis. Traditional diagnosis of the disease is based upon a combination of several parameters for measuring iron content (2). Onset of HH occurs in early adulthood and the first symptom is often the development of painful arthritis in the hands. The traditional methods of diagnosis are not useful in the presymptomatic stage of the disease unless there is a known familial history of HH. Phlebotomy is a highly effective means of preventing tissue damage, but is most often prescribed after the occurrence of damage (4).
Clinical studies have revealed multiple mutations, 10 or more (4, 5, 6), of the HFE gene in different ethnic groups. Caucasian subjects of North-ern European, and especially Celtic, descent are at high risk to be carriers, maybe as high as 1:8 (5), of one of two mutations implicated thus far in HH. The first of these mutations is H63D, in which histidine is replaced with aspartic acid in codon 63 caused by a C to G transition at position 187. A G to A transition at position 845 causes the more common C282Y mutation, in which the cysteine at codon 282 is replaced with tyrosine (7). The majority of patients with clinical HH are carriers of at least one allele with the C282Y mutation. Clinical manifestation of HH has been correlated to homozygosity for either of these mutations or compound heterozygosity, in which one allele of each mutation is present. It is important to note that compound heterozygotes of these and other alleles may develop clinical HH because other mutations or factors not associated with HFE may lead to the development of HH (7).
Recent studies have provided numerous methods for the detection of the C282Y and H63D mutations. Among these are PCR with sequence specific primers (8), PCR-single stranded con-formation polymorphism (SSCP)(9), and PCR-Restriction Fragment Length Polymorphism (1, 7). Here I describe an SSCP method for detection of point mutations in the HFE gene supported by sequencing of the HFE gene regions containing those mutations.
SSCP is routinely used to detect sequence changes for genetic testing. In SSCP analysis, PCR products of approximately 200 bp are denatured by heating, and cooled quickly to induce the formation of intrastrand secondary structures. The conformation of single strands is believed to be highly dependent upon sequence. In nondenaturing polyacrylamide gels, the electrophoretic mobility of single-stranded nucleic acid depends not only on size but also on sequence (10). Therefore, even single base changes can be detected. Alteration of the conditions for this procedure may be necessary for the highest fidelity (11). To minimize the possibility of misdiagnosis due to inherent errors in SSCP data, I employed dideoxy sequencing of the PCR product.


Figure 1-PCR of codon 282 (left) and codon 63 (right) regions of HFE, subjects 1-5 and negative control.

Figure 2-PCR-SSCP of the codon 282 region of HFE 50 ng of genomic DNA from subjects 1-4 was amplified by PCR in the presence of [32P] dGTP. The amplification products were denatured, quickly cooled, and electrophoresed in a polyacrylamide gel at 4C. Lane (-) is a negative control with no template DNA. Lane N is nondenatured product from subject 1. Exposure for autoradiography was overnight with an intensifying screen.

Figure 3-Sequencing of the codon 282 containing region of HFE revealed point mutations in subjects 2 and 3. Subject 1 (3A) has a wild type G at position 845. Subject 2 has an A at position 845 (3B). Subject 3 is heterozygous, with both G and A at position 845 of HFE (3C). The G to A transition results in a tyrosine for cysteine substitution. Subject 2's sequencing film is shown (3D). Sequencing was performed by a modified dideoxy method.

Figure 4-Aligned sequences of codon 282 and codon 63 of HFE. Codons 282 and 63 are shown in bold.

PCR of the codon 62 and codon 282 regions of HFE from genomic DNA for all samples yielded amplification products between 100 and 200 base pairs. Control reactions without template DNA for both regions produced no reaction products. All PCR products were electrophoresed in a 0.8% agarose gel concurrent with a 100 bp DNA ladder. Upon staining with ethidium bromide bands consistent with the expected size of approximately 160 bp were observable (Fig 1).
SSCP of the codon 282 containing region of HFE's exon 4 revealed identical banding patterns for subjects 1 and 4. Subjects 2 and 3 exhibited banding patterns different from each other and those of subjects 1 and 4.
Sequencing of the codon 282 and codon 63 regions was performed by a modified dideoxy method. The 165 bp codon 282 containing region was sequenced for all five subjects (Figures 3 and 4). The 162 bp codon 63 containing region was sequenced for subjects 1 and 5 (Fig 4). Figure 3A-3C shows base transitions at codon 282 (base 845) for subjects 1, 2, and 3. Figure 4 gives sequence alignments with HFE regions.
Subjects 1 and 4 exhibited identical SSCP banding patterns. Sequencing of the codon 282 region showed that subject 1 and subject 4 have a wild type G (base 845) at codon 282. Subject 2, a known C282Y homozygous mutant, had a unique SSCP banding pattern that is distinctly different from that of the wild type. The G to A substitution expected for this subject was confirmed by sequencing. Subject 3's SSCP banding pattern can be described as a combination of the patterns seen for subjects 1 and 2, consistent with the banding pattern expected for a heterozygous individual. Sequencing of the codon 282 region for subject 3 indicated that this person is heterozygous for the C282Y mutation as predicted by SSCP. Lastly, subject 5's SSCP banding pattern was inconclusive (not shown). However, this individual proved to be wild type upon DNA sequencing.
Both subjects whose codon 63 regions were sequenced (2 and 5) exhibited the wild type sequence. SSCP analysis of the codon 63 region initially indicated that subject 2 possessed a heterozygous mutation within this region. This was not confirmed by sequencing of the region and time limitations on the project prevented further investigation.

SSCP has been used extensively to screen for inherited mutations (11, 15, 16, and 17). While SSCP is a powerful tool for detecting point mutations, the parameters of the assay require strict control. The balance between temperature fluctuations and weak local stabilizing forces such as intrastrand base pairing and or stacking presumably determines SSCP banding patterns (10). Varying temperature or additive concentrations may alter the electrophoretic mobility of SSCP bands (10). Altering the concentrations of additives such as glycerol or formamide, or varying the running temperature may increase the sensitivity of SSCP (13). Also, free nucleotides present in the PCR reaction can anneal to product strands and cause alterations in mobility (14). Because the variation of these many parameters may affect the sensitivity of SSCP, it is important to maintain strict control when utilizing SSCP in genetic testing.
This study has preliminarily shown that SSCP can be a useful tool for the detection of HFE point mutations. The different mobilities of wild type and mutant alleles are clearly shown. Additionally, the sensitivity of the assay is such that it can be used not only for genotyping diagnosed HH patients, but also to screen for carriers of the C282Y mutation. However, because the sensitivity of the assay may vary under mildly different conditions, it is critically important to run known controls when using SSCP for genetic screening. Additionally, when possible, dideoxy sequencing should be relied upon to clarify uncertainties and to confirm results from SSCP

*Please see full paper for references.


I am especially grateful to Mr. Rocco Coli, Ms. Sabrina Volpi, Ms. Amy Kozak, Dr. Sylvia Anderson and Dr. Berish Rubin for their countless hours of patient assistance with this project.

This document was last modified 01/31/2006.
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