Pitfalls in the Genetic Diagnosis of Hereditary Hemochromatosis

Abstract

The widespread use of the genotype assay that identifies the common C282Y mutation in the HFE gene has allowed an earlier diagnosis to be made in many subjects. A significant number of these patients may have no evidence of phenotypic disease and have a normal serum ferritin level. This phenomenon is more common when the genotype assay is used to screen populations rather than higher-risk groups such as family members of a proband with hereditary hemochromatosis. Moreover, patients with significant iron overload may be wild type for the C282Y mutation and have no other demonstrable mutation of the HFE gene. The HFE genotype assay has recently been found to give a false-positive C282Y homozygous result in half of the subjects in one population screening study due to the presence of a single nucleotide polymorphism (SNP) that interfered with primer binding in the PCR assay. The problem may be overcome by using alternate primers. A number of other groups have confirmed the finding but in a much smaller number of subjects, whereas others found that their assays were not affected by the SNP. The use of the HFE genotype assay as the sole diagnostic criterion for hereditary hemochromatosis is not recommended. The genotype assay should be used as an adjunct to the established methods of demonstrating iron overload and be viewed as a predictor of either the presence of iron overload or the subsequent development of iron overload during an individual's lifetime.

Filaggrin Genotyping

“…But with 2282del positive samples (i.e., 2282del4 heterozygotes), the reduced wildtype signal results in a reduced "wildtype signal to mutation signal ratio" that falls outside of both the wildtype and the heterozygote range. A similar fi nding has also been seen in other genotyping assays like the HFE gene mutations H63D, and S65C [ 166 ].…”

Risk of Misdiagnosis Due to Allele Dropout and False-Positive PCR Artifacts in Molecular Diagnostics

“…18, 29 Our results show that i) these events can occur with sufficient frequency in the context of moderate-to-high volume routine testing over periods of months to years, resulting in a potentially important number of erroneous clinical diagnoses; ii) most of them are because of unpredictable sequence-independent factors rather than sequence variants and thus may not be prevented even by careful primer design, iii) error rates vary substantially among different amplification target loci; and iv) the use of two independent genotyping assays based on different sets of primers is both a feasible and efficient way of detecting and hence minimizing these artifacts. For the remaining unresolved cases, the addition of sequencing information (again using two different sets of primers to generate the amplicons before sequencing) will ensure maximum accuracy of the diagnostic information.…”