Characterization of the morphological defects in the sperm of an infertile patient
In this study, we examined an infertile man from a consanguineous family (Fig. 1A). Routine sperm analysis indicated that the patient suffered from teratozoospermia. In particular, the Papanicolaou staining revealed that 83.8% of the spermatozoa presented a tapered-head sperm phenotype (Table 1 & Fig. 1B). To further characterize the spermatozoa defects of the patient, transmission electron microscopy (TEM) was used to analyze ultrastructure in sperm cells of the patient as well as the control. Compared with normal control, most sperm cells from the patient exhibited smaller and less condensed nuclei, while acrosomes of the spermatozoa of patient were either absent or morphologically defective (Fig. 1C). Thus, we speculate that the tapered-headed sperm might be the cause of the infertility for this patient.
Identification of a mutation in WDR12 in a patient with tapered-head sperm by WES analysis
To identify a possible genetic cause of the infertility in this patient, we analyzed the peripheral blood genomic DNA obtained from the patient and his parents using WES. WES data was analyzed and described in Figure 2. Briefly, variants with MAF >0.01 in human genetic variation databases (1000 Genomes, ESP6500, and ExAC) were excluded. Because of consanguinity loop in the family, we hypothesized that homozygous mutations in the patient may be responsible for the infertility. Further, we assumed a recessive mode of inheritance, which resulted in 13 variants in 12 genes (Fig. 2A). To investigate whether any of these 12 genes may be related to male infertility, we first excluded genes with no function in spermatogenesis based on data mining of the related literatures and genes which have no expression level in testis. Next, we excluded variants which predicted to be non-deleterious by >30% prediction tools (SIFT, Polyphen-2, CADD, Mutation assessor, Mutation taster, REVEL, MetalR, LRT, MetaSVM, FATHMM) (Fig. 2A &Table S2). These filters resulted in only two mutations (NM_005245.3, p.Asp1113Asn/c.3337G>A of FAT1 and NM_018256.3, p.Ser162Ala/c.484T>G of WDR12). Given that the patient did not display facial dysmorphism, colobomatous microphthalmia, ptosis, or syndactyly defects, which are associated with mutations in FAT1. we focused on the homozygous variant (p.Ser162Ala/c.484T>G ) of WDR12.
Validation by Sanger sequencing
Sanger sequencing revealed that a patient affected by teratozoospermia was homozygous for this WDR12 variant, whereas his parents carried the variant in a heterozygous state (Fig. 2B).
Detrimental effects of the identified WDR12 variant
To investigate the effect of the homozygous WDR12 (p.Ser162Ala/c.484T>G) variant at the molecular level, we analyzed WDR12 protein expression levels. Western blot analysis on spermatozoa protein extracts revealed WDR12 protein expression to be significantly decreased in the patient’s spermatozoa (Fig. 3A&B). Furthermore, we assessed WDR12 expression from WT and MT WDR12 expression plasmids in 293T cells respectively by western blot. The result showed that WDR12 protein level was highly expressed from the WT plasmid than the MT plasmid (Fig. 3C), suggesting that this missense variant affects WDR12 expression.
In silico analysis of the WDR12 mutation
Bioinformatic analysis utilizing online pathogenicity prediction tools (Polyphen-2, Sift, Mutation Assessor, CADD, Mutation taster) predicted that the c.484T>G mutation in WDR12 is probably a damaging mutation, suggesting that WDR12 (p.Ser162Ala/c.484T>G) should be a disease-causing mutation (Table. 2). The conservation of this mutation site was predicted by computational analysis and the result showed that Ser residue at the 162 site is highly conserved (Fig.3D). These results suggest the Ser residue at the 162 site had an important role.
WDR12 expression and localization in mouse testis
To explore the biological function of Wdr12 in the mouse, we detected tissue- and developmental stage-specific expression of WDR12. First, high expression of Wdr12 mRNA was observed in the adult mouse testis (Fig. 4 A). Further, Q-PCR and western blot analysis revealed that the expression of WDR12 was very weak in 7dpp mouse testis, but increased over time, peaking at 21dpp, which is coincident with the appearance of round spermatids. Then, WDR12 expression level was reduced in testes of adult mice (Fig. 4B, C). Immunohistochemical staining further showed that WDR12 is predominantly expressed in late pachytene spermatocytes and round spermatids (Fig. 4D). Collectively, the expression and localization patterns of WDR12 during spermatogenesis suggests its potential role in round spermatid development.
ICSI using the patient’s sperm and pregnancy outcome
After centrifugation on density gradients and careful examination, a few ‘normal-looking’ spermatozoa that fit into an ICSI micropipette were selected. ICSI cycle was attempted for the patient at the first affiliated hospital of USTC. Nine eggs were used for the ICSI cycle, and 4 eggs form viable embryos, then two embryos were transferred, and his wife became pregnant. These results indicate that the infertility of WDR12 homozygous mutation could be overcome by ICSI.