Whole-exome sequencing identified a novel mutation of AURKC in a Chinese family with macrozoospermia

The proband was a 27-year-old treated for infertility at Anhui Provincial Hospital. The results of the patient’s semen tests are described in Table 1. The results revealed that the patient suffered from macrozoospermia (close to 100% large-headed spermatozoa with a sperm count of 1 M/ml) (Table 1). The parents of the patient were first-degree cousins (Fig. 1). The patient exhibited normal erection and ejaculation, and reported having regular sexual intercourse 2–3 times/week; however, his wife had been unable to get pregnant since they were married in 2015. The patient had no history of unhealthy activity or contact with adverse chemicals. Physical examination revealed normally developed male external genitalia, normal bilateral testicular size, and no abnormality in the bilateral spermatic veins upon palpation. The proband did not have primary microcephaly or respiratory disease. The chromosomal karyotype of the patient was normal (46; XY), and no deletion was found in the Y chromosome. The patient hormone levels were normal.

This project was approved by the ethics committee of Anhui Medical University. All study members gave their written informed consent before sample collection.

The genomic DNA from the patient and his parents was extracted from peripheral blood samples using a QIAamp DNA blood midi kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.

Genomic DNA from the proband and his parents was subjected to WES. WES was performed by the WuXi NextCODE in Shanghai on a HiSeq2000 sequencing platform (Illumina, San Diego, CA, USA). After the adaptors were removed, the WES raw reads were aligned to the reference genome Hg19 using the Burrows-Wheeler aligner, followed by removal of PCR duplicates. Variants including single-nucleotide polymorphisms and indels were identified using SAMtools and annotated by ANNOVAR software. A candidate gene was considered a variant that fulfilled the following criteria: (i) missense, nonsense, frame shift, and splice site variants, (ii) absent or rare (frequency below 1%) in the two databases (dbSNP, 1000G), and (iii) homozygous variants in the patient and heterozygous variants in his parents.

The mutation in AURKC in the proband and his parents was validated using Sanger sequencing. We amplified the PCR products of exon 3 of the AURKC gene using specific primes (the forward primer is 5′-AACCAGGATTCGAGTGTCTG-3′, and the reverse primer is 5′-CAATCTCCAGGTAGACGATGGAG-3′). Then, the PCR products were sequenced on an ABI 3730XL automated sequencer (Applied Biosystems, Forster City, CA, USA).

RNA extraction was carried out on the whole blood using TRI REAGENT®BD (Molecular Research Center) using the manufacturer’s protocol. Reverse transcription was performed in patient and his parents with 5 μl of extracted RNA. Hybridization of the oligo-dT was performed by incubating for 30 min at 42 °C and quenching on ice with the following mix: 5 μl of RNA, 10 μl of 2Xsupermix (10 mM, Pharmacia), 1 μl of gDNA (0.5 mM, Roche diagnostics), and 4 μl of H₂O. Then 2 μl of the obtained cDNA mix was used for the quantitative PCR (Q-PCR) using a StepOne-PlusTM Real-Time PCR System (Life Technologies) with Power SYBR Green PCR Master Mix (Life Technologies) according to the manufacturer’s protocol. Quantification of the fold change in gene expression was determined by the relative quantification method (2^−ΔΔCT) using the gapdh gene as a reference. Data are shown as the average fold increase standard error of the mean. Primers are described in supplementary file 1.

The patient and his wife underwent intracytoplasmic sperm injection (ICSI) at Anhui Provincial Hospital. Briefly, embryo culture was performed using Vitrolife G-SERIES™ culture media (Vitrolife, Goteborg, Sweden) according to the manufacturer’s instructions. The patient’s wife had undergone one ICSI cycle. Detailed results are described in the “Results” section.

Sequence alignment of the AURKC protein in different species was conducted using ClustalX2.1. The number of each species was as follows: Homo sapiens (NP_001015878.1), Mus musculus (AAI00338.1), Bos Taurus (NP_001180124.1), Desmodus rotundus (XP_024425801.1), Pan paniscus (XP_003816716.1), Pongo abelii (XP_002829903.1), and Rattus norvegicus (NP_001295465.1).

To identify the genetic cause of the macrozoospermia, we performed WES using peripheral blood genomic DNA from the patient and his parents to identify the putative pathogenic mutation. Considering the family history of consanguinity, we focused on homozygous mutations. After of the exclusions of frequent variants and application of technical and biological filters (see “Materials and methods” section and Fig. 2A), a limited list of homozygous mutations was established (Supplementary Table 1). To determine whether any of these 36 genes may be related to macrozoospermia, we first examined the tissue expression patterns for each gene. Among these homozygous variant genes, only two genes exhibited testis-enriched expression (Fig. S1). One of the identified genes was AURKC, and a new mutation (c.269G>A, GenBank accession number, NM_001015878) in exon 3 was identified in the patient by WES. Accordingly, the change in amino acids was determined to be Arg90Gln. Another identified gene was gametogenetin (GGN), and a mutation (c.148T>C, GenBank accession number, NM_152657.3) in exon 3 was identified in the patient by WES. Accordingly, the change in amino acids was determined to be Trp50Arg.

We next performed Sanger sequencing to verify the homozygous variant in the AURKC and GGN genes in both the patient and his parents. Exon 3 of the AURKC gene was amplified from genomic DNA of the patient and his parents using PCR. The homozygous missense variant in AURKC was validated in the patient, and his parents had heterozygous alleles (Fig. 2B).

Unfortunately, we could not get access to testicular biopsy samples from the affected patient and his parents; therefore, we set up Q-PCR to study the effect of the variation. Q-PCR analysis demonstrated that mRNA levels of AURKC were down-regulated significantly in the patient (Fig. 2C).

Bioinformatics analysis of the c.269G>A mutation in the AURKC gene by three online pathogenicity prediction tools (Polyphen-2, SIFT, Mutation taster) suggested that this mutation is most likely a disease-causing mutation (Supplementary Table 2). This variant is a novel mutation that was absent in the ExAC, 1000G, and gnomAD databases. The mutation site at R90 was highly conserved from human to zebrafish, indicating an important role of this site for the function of AURKC protein (Fig. 2D).

After centrifugation on density gradients and careful examination, a few “normal-looking” spermatozoa that fit into an ICSI micropipette were selected. One ICSI cycle was attempted for the patient at Anhui Provincial Hospital. For the ICSI cycles, we collected 10 eggs and 7 MII oocytes; none of the embryos developed to the blastocyst stage.