In this study, we identified sequence variations in the EGR4 gene of patients with idiopathic non-obstructive spermatogenetic impairment. Spermatogenesis is followed by the differentiation of primordial germ cells into motile spermatozoa. This process is controlled by numerous factors [23, 24], and disruption of these factors may affect the quality and quantity of spermatozoa production and fertility in males. Many genes have been associated with spermatogenesis, but the pathophysiological mechanisms of these genes have not been elucidated. EGR4 is expressed in germ cells and has a crucial role in spermatogenesis by regulating germ cell maturation during pachytene. In addition, the functional loss of EGR4 blocks spermatogenesis, thereby reducing spermatozoa production [8].

We identified eight variants in the EGR4 gene of Korean males with impaired spermatogenesis. However, these variants were all listed in the NCBI Single Nucleotide Polymorphisms (SNP) database [25], and the allele frequencies of the variants alleles were not significantly different between patients and controls. Therefore, the significance of these variations in spermatogenesis is not clear. The allele frequency of each variant was very low (less than 0.01), and variants not found in the controls were only identified in one patient. We were also unable to elucidate the biochemical and physiological significance of each variant. Instead, we evaluated the pathological significance of the variants using 7 computational prediction algorithms: PolyPhen-2, SIFT, Mutation Taster, fathmm, Mutation assessor, SNPs &GO and SNAP².

Four variants were not identified in the controls, and three were nonsynonymous. The c.65_66InsG (p. Cys23Leufs*37) variant was detected in only one patient with oligozoospermia. This insertion shifted the reading frame and generated a premature stop codon. It was predicted as “damaging” by the Mutation Taster and SNAP2 database. Haploinsufficiency is a well-known mechanism of many genetic diseases [26–28]. In null mouse models, hemizygotes (+/−) may show a normal phenotype because a deleted allele may not affect its phenotype. However, a gene product of a variant allele may interfere with a normal gene product. The allele frequency of the insertion was very rare and may have been responsible for the reduced sperm count in the patient.

The c.236C > T (p. Pro79Leu) variant was detected in only one patient. The variant was also identified by the 1000 genomes project with a MAF of 0.0003. Proline and leucine are nonpolar amino acids, so the mutability of this variant should be negligible. However, this type of substitution could affect protein function. Computational analyses predicted the variant as both “damaging” (SIFT) and “benign” (PolyPhen-2); therefore, its pathological significance is unclear. The nonsynonymous variant c.1294G > T (p. Val432Leu) was also only identified in one patient and had already been identified by the 1000 genomes project with a MAF of 0.0016. Valine and leucine are structurally similar; therefore, nonpolar amino acids of this variant should be insignificant. The synonymous c.1230G > A (p. Thr410) variant was also only found in one patient with impaired spermatogenesis and had also previously been identified by the 1000 genomes project with a MAF of 0.0008.

The remaining four variants, including one nonsynonymous variant, were identified in both patients and controls. The c.214C > A (p. Arg72Ser) variant had a MAF of 0.009, and the c.867C > G (p. Leu289) variant had a MAF of 0.023 in our Korean population. These frequencies were much higher than those reported in the NCBI SNP database. The c.867C > G (p. Leu289) variant was predicted to be “disease causing” by Mutation Taster. The substitution may affect a splice site [16, 17]. However, this variant occurred more frequently in controls and is considered to be a normal variation in the Korean population. Two of our identified variants (c.1488C > T and c2373 + 52C > T) are considered common variants; c.1488C > T has been reported in European (MAF: 0.0636) and African (MAF: 0.4554) populations and was also identified by the 1000 genomes project (MAF: 0.2845). Here, we found that the MAF for this variant in a Korean population is similar to that previously reported in an African population. The c.2373 + 52C > T variant was located in the 3′-UTR and was tightly linked to the c.1448C > T variant in our Korean population; the C allele of the c1448C > T variant was coupled to the C allele of the c.2373 + 52C > T variant. The haplotype frequencies did not differ between the two groups.

SNPs and other structural variants have been associated with impaired spermatogenesis in different populations, but the same variants have not yet been reported in more than one population [29–32]. Chuncheng et al. performed the largest genetic association study in patients with non-obstructive azoospermia and identified SNPs associated with five potentially related genes. They suggested that these SNPs act as cofactors rather than directly affecting spermatogenesis because they were also present at a lower frequency in fertile men. These variants may cause mild impairment of spermatogenesis, but this could be worsened in the presence of other cofactors [33]. Tourtellotte et al. suggested that the EGR4 gene can compensate for the function of EGR1 in regulating LH during steroidogenesis [34]. This supports the idea that EGR1 has a dominant role in maintaining male fertility, while EGR4 can compensate for the functional loss of EGR1 in germ cells.