In the SPAG16
regions analyzed, both major and minor alleles were present in the study group for four known polymorphisms, and no previously unreported mutations were detected. At rs2042791 [29
], the minor allele replacement of an adenosine by a cytosine residue causes the replacement of a glutamine at position 361 [GenBank: NP_078808.3] with a histidine. Previous genotyping studies have reported a minor allele frequency of 0.39 [26
] in individuals of Caucasian ancestry for this SNP. Fisher’s exact test demonstrated no significant difference between the measured minor allele frequency of 0.408 in our sample population and that established in control populations (p
0.05). While the present study lacks sufficient power to detect small effects of these alleles on variation in observed parameters of sperm characterization, among patients carrying this SNP no single factor was uniformly deficient within the distribution of sub-normal semen analysis of the study participants. Full genotype and semen analysis results are included for reference (Additional file 2
: Table S2).
The lack of association between this SNP and sperm dysfunction is not surprising, given that the glutamine replacement by histidine is predicted to be tolerated in the protein structure [27
]. Further, the amino acid is not conserved in mammals – Mus musculus
[GenBank:AAI20669.1, NP_080004.1, NP_083436.2] and Rattus norvegicus
[GenBank:NP_001128200.1, AAI58603.1] reference sequences for the orthologous proteins in fact report a histidine at this position, consistent with the human minor allele. The same amino acid replacement is predicted in Pan troglodytes
[GenBank:XP_001148592.1, XP_526016.2, XP_001148393.1]. These data are consistent with the hypothesis that structural rather than sequence homology is essential to maintenance of axoneme function.
The rs2042792 minor allele was not found at a significantly different frequency than in the reference population (p
0.05), and did not correlate with assessed sperm phenotypes.
Rs2042791 exists in LD with rs2042792 in our population, but no phenotypic consequences were associated with any of the observed haplotypes.
Minor allele replacement of an adenosine with a cytosine in rs12623569 causes an amino acid replacement at position 425 [GenBank:NP_078808.3], with a threonine taking the place of a lysine. Previous genotyping studies have shown a minor allele frequency for this SNP in individuals of Caucasian ancestry of 0.30 [26
]. The present sample population exhibited a minor allele frequency of 0.333, which was not significantly different than that in control populations (p
0.05). While the lysine at position 425 encoded by the major allele is present in other mammals (Mus musculus
– GenBank:NP_080004.1, NP_083436.2; Rattus norvegicus
– GenBank:NP_001128200.1, AAI58603.1), replacement by threonine is predicted to be tolerated by the protein structure. Interestingly, minor allele carriage was found to be nominally associated with a higher percentage of normal accessory fibers of the axoneme. Although the statistical significance of this association was lost upon correction for multiple testing, further studies on a larger study population may be warranted to identify contributions of the SNP to sperm flagellar structure.
At rs16851495, minor allele replacement of guanine with an adenine residue does not affect protein translation directly, as the position lies outside the translated exon region. However, minor allele carriage was nominally associated with the reduced presence of normal axoneme structure in the study population. The statistical significance of this association did not withstand correction for multiple testing. However, the limited power of our study may have contributed to the absence of a robust association.
For all SNPs discussed, both heterozygous and homozygous individuals were present in the sample population, and statistical analysis demonstrated no difference in genotype frequency between the sample and control populations (p
0.05). While the study did not have sufficient power to analyze possible contributions to complex traits, the results negate the hypothesis that amino acid modification at the tested positions may result in a loss of protein function that would mimic the severe phenotypes observed in transgenic mouse studies.
Our results suggest that non-synonymous amino acid substitutions at residues 361 and 425 in the SPAG16 protein are not sufficient to explain a reduction of sperm motility and fertility index, the presence of axonemal/periaxonemal alterations, or an increased percentage of sperm pathologies in the assessed patient population. Based on the strong homology between members of the WD protein family, it is tempting to suspect that these resides may be non-essential in other WD repeat proteins as well.
The profound defects observed in studies of Spag16 gene effects in other species suggest that a functional mutation would significantly perturb sperm function, and would be observed even in a small sample population. Our observations do not, however, preclude the possibility that alternative variations in the SPAG16 gene cause an increase in sperm ultrastructural alterations, and a reduction in sperm motility. Indeed, our previous studies suggest that more significant SPAG16 mutations, such as a frame shift, can reduce sperm central apparatus stability. Further studies of SPAG16 gene variation are warranted to offer a comprehensive understanding of the gene’s contributions to male fertility. The sensitivity of the present study to detect variation with the sample is limited by the lack of similarly extensive sperm analysis data, in particular TEM ultrastructural analysis, from a control population. Future development of this area of research should include control samples from men with known fertility and/or sperm count, motility, and structure in the normal range.
Future studies in mammals are necessary as well to explore the nature of Spag16
gene evolution, and the possibility that male germ cell-specific isoforms exist in multiple species. The only identified rat SPAG16 protein is similar to SPAG16S in size, and is derived from mRNA similar to murine SPAG16S
, with a 5′
UTR from the region upstream of the first exon suggestive of independent transcription, rather than splice variation. A putative SPAG16S
promoter and 5′UTR have been identified in humans as well [GenBank: EF591776.1], raising the possibility that SPAG16S maybe be common to mammalian male germ cells. We have recently shown that murine SPAG16S is enriched within nuclear speckles of male germ cells [30
]. This unique localization, combined with phenotypic results from various Spag16
transgenic mice, strongly suggests a unique processing role for the SPAG16S protein, and thus a distinct role for the Spag16
gene apart from its structural role in the axoneme.