Although infertility due to hypogonadism is a clinical manifestation of primary or idiopathic hemochromatosis, this is the first report of the frequency of HFE mutations among infertile/subfertile men in comparison to fertile controls. The HFE gene was cloned in 1996 and the two mutations we studied were found in HH patients [6]. The majority of HH patients consists of C282Y homozygotes [6,19]. Although the role of the H63D mutation on the HFE gene was at first controversial [20], its deleterious nature was established when an excess of H63D alleles was demonstrated in HH patients [21,22], and compound heterozygotes for the C282Y and H63D mutations were found to express HH, even though with a penetrance of only 1.5% of that of C282Y homozygotes [7]. In vitro evidence for the functional consequences of the H63D mutation was also reported [23].
We did not find the C282Y mutation among the sub­fertile/infertile men or among fertile controls in this study. This is in accordance with the absence of the C282Y mutation among more than 400 individuals tested in the Republic of Macedonia (unpublished results).
Although the genotype and allele frequencies of the H63D mutation did not differ in the total group of infertile/subfertile men when compared to the fertile controls, a significantly higher frequency was found in the men manifesting severe oligozoospermia (sperm counts of £1.0 x 106/mL). This suggests a possible association of the H63D mutation with severe oligozoospermia. This allele may have a direct role on spermatogenesis, act as a modifier allele or may be a marker for a linked gene implicated in spermatogenesis.
Although the first mentioned role of the H63D mutation is unlikely, the possibility cannot be excluded. Namely, infertility can be an early symptom of primary hemochromatosis in men with severe oligozoospermia, who may also carry an unidentified HFE mutation. Most of the infertile/subfertile men we studied are less than 35 years old, whereas symptoms of primary hemochromatosis are usually diagnosed when the patients are more than 50 years old [24].
Other studies have suggested the existence of a gene, or genes, required for normal spermatogenesis on the short arm of chromosome 6, near the HFE gene. The HFE gene is located at the 6p21.3 region, within the major histo­compatibility complex (MHC) region, some 4.5 Mb from the human leukocyte antigen (HLA) A. Several studies have observed significant differences in HLA class I, and especially class II, allele frequency between males with impaired spermatogenesis and control subjects [25-27]. Using a digital differential display, a candidate gene for infertility (TET7) was identified, which maps to 6p21 and is a human homologue of mouse Tpx-1 [3]. In the rat, Tpx-1 is involved in the specific cell-cell interaction between Sertoli and spermatogenic cells [28]. In addition, three cases of chromosomal breakpoints involving 6p21 in infertile males have been reported [3].
The HFE gene is located within the histone gene cluster in the vicinity of the histone H1t gene. The testis specific linker histone H1t is expressed in tetraploid primary spermatocytes during spermatogenesis, and may function in establishing an open chromatin structure for the replacement of histones by transition proteins and protamines [29,30]. Histone H1t synthesis has been detected only in primary spermatocytes, the protein representing approximately 60% of the total H1 histone complement in these cells. Other linker histones present at a significant level in primary spermatocytes include the testis-enriched H1a and H1c variants [31]. Despite its abundance in male germ cells, inactivation of the H1t gene in mice showed that it is not required for spermatogenesis [32-34]. The ratio of total histone H1 to DNA does not drop in germinal cells in mice that are H1t-deficient, suggesting compensation by histones H1a and H1c for the missing H1t [33]. In enriched populations of the germinal cells that contain H1t (pachytene spermatocytes and round spermatids), failure to make H1t leave the chromatin of spermatids with only 75% of the total H1 normally present [34]. Also, homozygous H1t-null mice have normal fertility and show no obvious phenotypic consequence due to the lack of this histone [34]. Since the H1t gene is located only a few kilbobases (kb) from the HFE gene on chromosome 6, we can speculate that the association of the H63D allele in our subjects with severe oligozoospermia may be due to the involvement of the H1t gene in human spermato­genesis. Although mice without H1t genes are fertile [34], it cannot be excluded that this gene has a different and more important role in human spermatogenesis.
In conclusion, we have shown an association of the HFE H63D mutation with severe oligozoospermia. Further studies on men with severe oligozoospermia from different populations are warranted to confirm this association.