Association of CFAP65 rs56411706 with male infertility in 393 Vietnamese individuals

Abstract

Approximately two thousand genes have been found to be involved in spermatogenesis and their mutations have been reportedly associated with male infertility. Recent studies have shown that CFAP65 was crucial for spermatogenesis, and several mutations in this gene could result in male infertility. However, the association of polymorphisms in CFAP65 with male infertility remains unknown. In this study, the relationship between CFAP65 rs56411706 and male infertility was assessed in a Vietnamese population by 171 male infertility patients who had been diagnosed with non-obstructive azoospermia (NOA), oligozoospermia, or asthenozoospermia while 222 healthy controls were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Statistical analysis demonstrated that the allele frequencies of CFAP65 rs56411706 followed Hardy-Weinberg equilibrium (HWE) (p>0.05). The Chi-square test revealed no correlation between the polymorphism and male infertility in this study (p>0.05). This is the first study on the association between a single nucleotide polymorphism in the CFAP65 gene and male infertility in a Vietnamese population. The results of this study would help enrich the knowledge about the effects of CFAP65 polymorphisms on male infertility in the Vietnamese population.

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Introduction
Qualitative spermatogenesis defects are one of four
major etiological categories of male infertility [1]. It
is characterised by sperm motility, morphology, and
functional parameters such as DNA and chromatin integrity
[2]. Various clinical classications for qualitative defects
of spermatogenesis include “teratozoospermia” (reduced
percentage of sperm with normal morphology) [3],
“oligozoospermia” (reduced sperm count) [4], and
“asthenozoospermia” (reduced sperm motility) [2].
However, to describe more than one abnormality
within the semen parameters, other terms such as
oligoteratozoospermia, oligoasthenozoospermia, and
asthenoteratozoospermia are also used [2].
Asthenozoospermia, a type of asthenoteratozoospermia,
is characterised by the presence of sperm agellar
anomalies that occur at the end of spermatogenesis
and concurrently with head compaction and reshaping
[5]. Over 80% of male infertility cases exhibit some
alterations of sperm motility. The multiple morphological
abnormalities of asthenozoospermia phenotypes include
(1) morphological abnormalities such as short-coiled,
absent, bent, and/or irregular-calibre agella associated
with dramatically declined motility or (2) ultrastructural
agellar defects, e.g., absent central pair (CP),
disorganised double microtubules (DMT), dysplasia
of brous sheath (FS), or absence of dynein arms [2].
Several genes have so far been identied as having an
association with multiple morphological abnormalities of
sperm agella phenotypes [2, 6] leading to an absence
of inner dynein arm (IDA), disorganized 9+2, absent
CP, disorganised FS, or DMT [2]. Abnormalities of
CFAP genes, which have been found to be involved in
spermatogenesis, could cause fertility impairment in
males [7-11]. Notably, CFAP65 has been recognised
Association of CFAP65 rs56411706 with male
infertility in 393 Vietnamese individuals
Thuy Duong Nguyen1, 2*, Thi Khanh Ly Nguyen1, Thi Thu Ha Duong 1
1Institute of Genome Research, Vietnam Academy of Science and Technology
2Graduate University Science and Technology, Vietnam Academy of Science and Technology
Received 12 October 2021; accepted 1 December 2021
*Correspanding author: Email: tdnguyen@igr.ac.vn
Abstract:
Approximately two thousand genes have been found to be involved in spermatogenesis and their mutations
have been reportedly associated with male infertility. Recent studies have shown that CFAP65 was crucial for
spermatogenesis, and several mutations in this gene could result in male infertility. However, the association
of polymorphisms in CFAP65 with male infertility remains unknown. In this study, the relationship between
CFAP65 rs56411706 and male infertility was assessed in a Vietnamese population by 171 male infertility patients
who had been diagnosed with non-obstructive azoospermia (NOA), oligozoospermia, or asthenozoospermia
while 222 healthy controls were genotyped using polymerase chain reaction-restriction fragment length
polymorphism (PCR-RFLP). Statistical analysis demonstrated that the allele frequencies of CFAP65 rs56411706
followed Hardy-Weinberg equilibrium (HWE) (p>0.05). The Chi-square test revealed no correlation between
the polymorphism and male infertility in this study (p>0.05). This is the rst study on the association between
a single nucleotide polymorphism in the CFAP65 gene and male infertility in a Vietnamese population. The
results of this study would help enrich the knowledge about the eects of CFAP65 polymorphisms on male
infertility in the Vietnamese population.
Keywords: CFAP65, male infertility, PCR-RFLP, rs5611706, spermatogenic qualitative defects.
Classication number: 3.2
DOI: 10.31276/VJSTE.64(2).65-68

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66 june 2022 • Volume 64 number 2
as a new gene candidate for fertility impairment due to
its functions regarding sperm agellar morphology and
paraagellar rod synthesis [12, 13].
The CFAP65 encodes cilia- and agella-associated
protein 65, which is highly expressed in testis during
spermatogenesis. Several mutations on CFAP65 have
been reported to cause male infertility [8, 12, 14].
Consequently, the association between single nucleotide
polymorphisms on this gene and the risk of male fertility
is expected to be a good consideration. The polymorphism
of rs56411706 occurring in the coding region of the
CFAP65 gene was analysed in silico using SIFT. This
software predicts the eect of an amino acid substitution
on protein function based on sequence homology among
dierent species and physical properties. The obtained
score of 0.09 suggested that rs56411706 is a promising
candidate for an association study. Therefore, in this study,
we conducted the PCR-RFLP experiment to investigate
the relationship between CFAP65 rs56411706 and the
risk of male infertility in a Vietnamese population.
Materials and methods
Subjects
Our research subjects consisted of 171 men
diagnosed with NOA caused by either oligo/azoospermia
or asthenozoospermia and 222 healthy men with
normal semen who had conceived at least one child.
Unfertilised men with normal male reproductive organs
and hormone balances were screened for semen and
checked for microdeletion of the AZF region or other
major abnormalities in the Y chromosome. The study
was approved by the Ethical Review Committee of the
Institute of Genomics Research (No: 9-2019/NCHG-
HĐĐĐ). Prior to the commencement of the study, all
participating individuals gave written informed consent.
Genotype determination
The total DNA of 393 individuals was extracted from
peripheral blood samples using the GeneJet Whole Blood
Genomic DNA Purication (Thermo Fisher). Quantity of
genomic DNA was analysed by electrophoresis on 1%
agarose, and DNA was quantied using NanoDropTM
One spectrometer, Thermo Fisher. The gene fragment
containing polymorphism CFAP65 rs56411706 was
amplied by PCR with a specic pair of primers
sequenced F: 5’-CATTCTGCAAGGCGGTGATT-3’
and R: 5’-AGGCTAAATTTTCCC TGGGGC-3’. The
10-μl PCR reaction mixture contained 1 μl of 10X
PCR buer; 100 μM dNTPs; 0.15 μM of forward and
reverse PCR primer each; 0.1 U Ta q DNA polymerase
(Thermo Fisher); 10 ng genomic DNA; and H2O. The
PCR protocol consisted of denaturation at 95oC for 5 min
followed by 35 cycles of 95oC for 30 s, 60oC for 30 s, and
72oC for 30 s, and the nal extension for 7 min at 72oC.
Electrophoresis on a 1% agarose gel was performed using
3 μl of the PCR product for each sample. Products of PCR
reaction were subjected to 5 h digestion by HhaI at 37oC.
Digested products were separated by electrophoresis on
2% agarose gel. The number and size of DNA fragments
used to determine the genotype of CFAP65 rs56411706
are presented in Table 1.
Table 1. The number and size of DNA fragments of CFAP65
rs56411706 genotypes.
Genotype DNA fragments DNA length (bp)
CC 2 215, 112
CA 3 215, 112, 327
AA 1 327
Statistical analysis
Statistical analyses were conducted with the statistical
software R version 3.6.1 [15]. HWE equilibrium was
tested using HWE exact [16, 17]. Logistic regression
was used to estimate a 95% (CI) and odds ratio (OR)
for binary variables, with a p-value less than 0.05
considered signicant. A total of three genetic patterns
were investigated: dominant, recessive, and additive.
The allele composition, genotype of polymorphism
CFAP65 rs56411706, and the association between
single nucleotide polymorphism and infertility risk
were examined. SNPassoc [18] was used to perform
association analyses.
Results
Genotyping CFAP65 rs56411706
The specic PCR products were processed with the
restricted enzyme HhaI. The genotypes of CFAP65
rs56411706 were determined based on the size and number
of DNA bands of the restricted product (Table 1, Fig. 1).

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Fig. 1. Restriction enzyme-digested PCR products on agarose gel
2%.
Note: M: marker 100 bp; 1: uncut PCR product; 2: homozygous AA (1
band of 327 bp); 3-5: wildtype with the homozygous CC (2 bands of 112
and 215 bp); 6, 7: heterozygous CA (3 bands of 112, 215, and 327 bp).
Statistical analyses showed no dierences in genotype
composition and allele frequency between the case
and control groups (Table 2). The distribution of this
polymorphism was examined across two groups and as
a whole by using the Chi-square test. The population
followed HWE equilibrium (p>0.05) with CC/CA/
AA genotype compositions of 0.72, 0.24, and 0.04,
respectively (Table 2).
Table 2. Genotype composition and allele frequency of
polymorphism CFAP65 rs56411706.
Group
Genotype Allele frequency
(%) p HWE
CC CA AA C A
Control (n=222) 123 (0.72) 42 (0.24) 6 (0.04) 0.84 0.16 0.99 +
Case (n=171) 160 (0.72) 55 (0.24) 7 (0.04) 0.84 0.16 0.70 +
Total (n=393) 283 (0.72) 97 (0.24) 13 (0.04) 0.84 0.16 0.99 +
Note: HWE: Hardy-Weinberg equilibrium; +: follow the Hardy-Weinberg
equilibrium.
Association analysis of CFAP65 rs56411706 with
risk of male infertility
Chi-square testing on all three dominant, recessive,
and additive models showed no association between
this polymorphism and the risk of male infertility with a
p-value greater than the signicance level of 0.05 (Table 3).
Discussion
The gene CFAP65 is evolutionarily conserved in
several species [8]. The putative homolog of CFAP65
is abundantly expressed in adult male mice, according
to the murine testicular transcriptome analysis [8, 19].
Similarly, in Chlamydomonas, CFAP65 is highly activated
during agellar regeneration [20]. The disruption of
sperm motility caused by CFAP65 was rst discovered
in Rose comb chickens [21]. A 2 bp frameshift mutation
later conrmed the pathogenicity of CFAP65 mutation in
male mice [8]. Remarkably, phenotypes of mutated mice
with CFAP65-frameshift-caring mutation were consistent
with human asthenozoospermia [8].
In humans, CFAP65, comprising 13 transcripts,
is found on 2q35. The cilia and agella-associated
protein 65, which is encoded by this gene, is highly and
preferentially expressed in the testis according to the
ENCODE [22], FANTOM [23], and GTEx databases
[24]. Furthermore, proteomic analyses have found
CFAP65 in the centrioles of human spermatozoa [19].
In a study by W. Wang, et al. (2019) [14], CFAP65 was
identied as the causative gene for completely immotile
spermatozoa. According to the proteomic analysis, in
the absence of CFAP65 during manchette organization,
both mitochondrial sheath (MS) assembly and acrosome
formation were unable to function properly [8].
Importantly, endogenous immunoprecipitation and
immunostaining experiments revealed that CFAP65
might form a cytoplasmic protein network with MNS1,
ZPBP1, RSPH1, TPPP2, and SPACA1 [13] in which
the perturbations to the CFAP65-centered proteostasis
network caused a series of defects in sperm head and
agella leading to severe asthenoteratospermia [13].
A homozygous nonsense mutation (p.Glu1781*) in
CFAP65 was identied in a consanguineous Chinese
family with an abnormal agella patient [19], and a
biallelic mutation in CFAP65 was also identied to cause
Table 3. Association between CFAP65 rs56411706 and male
infertility.
Model Control (n=222)Case (n=171) OR 95% CI p
Additive 0.296
CC 160(72.07%) 123 (71.93%) 1.00
CA 55 (24.77% ) 42 (24.56%) 1.006 0.631-1.609 0.977
AA 7 (3.16%) 6 (3.51%) 0.894 0.283-2.909 0.848
Dominant
CA+CC 215 (96.84%) 165 (96.49%) 1.00
AA 7 (3.16%) 6 (3.51%) 0.89 0.28-2.86 0.84
Recessive
AA+AC 160 (72.07%) 123 (71.93%) 1.00
CC 62 (27.93%) 48 (28.07%) 0.99 0.636-1.553 0.975
Note: n: number of individuals; OR: odds ratio; 95% CI: 95% confidence
interval; p: measured using Chi-square test.

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asthenozoospermia.
In CFAP65 rs56411706 polymorphism, a nucleotide
was substituted at NM_194302.4:c.1024G>T. This
substitution altered alanine to serine at position 342.
Although the CFAP65 rs56411706 polymorphism did
not show any association with male infertility in the
Vietnamese population, our ndings provide insights
into CFAP65 single nucleotide polymorphisms of male
infertility.
Conclusions
In this study, the CFAP65 rs56411706 polymorphism
was analysed among 393 studied subjects. Results
revealed the frequencies of genotypes CC/CA/CC to
be 0.72/0.24/0.04, respectively. Their distribution all
followed HWE. However, no relationship was established
between CFAP65 rs56411706 and male infertility
(p>0.05). To gain more insight into this association,
examinations of other polymorphisms in the CFAP65
need to be considered.
ACKNOWLEDGEMENTS
We thank all sample donors for contributing to this
research. This research was funded by the Ministry of
Science and Technology, Vietnam (60/19-ĐTDL.CN-
XNT).
COMPETING INTERESTS
The authors declare that there is no conict of interest
regarding the publication of this article.
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