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\begin{document}

\title{Concern the pathogenicity of heterozygous mutation
c.346-1G\textgreater{}A in SOHLH1 gene for NOA}



\author[1]{LIU Mohan}%
\author[1]{Ying Shen}%
\author[2]{Su-Ren  Chen}%
\author[1]{Yihong Yang}%
\affil[1]{Sichuan University}%
\affil[2]{Beijing Normal University}%


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  \date{May 26, 2024}


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\textbf{Concern the pathogenicity ofheterozygous mutation
c.346-1G\textgreater{}A in \emph{SOHLH1} gene for NOA}

Mohan Liu\textsuperscript{a,b\#}, Ying Shen\textsuperscript{b\#}, SuRen
Chen\textsuperscript{c}*, Yihong Yang\textsuperscript{d}*

\textsuperscript{a} State Key Laboratory of Biotherapy and Cancer
Center, West China Hospital, Sichuan

University and Collaborative Innovation Center, Chengdu 610041, China.

\textsuperscript{b} Department of Obstetrics/Gynecology, Joint
Laboratory of Reproductive Medicine (SCU-CUHK), Key Laboratory~of
Obstetric, Gynecologic and Pediatric Diseases and~Birth
Defects~of~Ministry of Education, West China Second University Hospital,
Sichuan University, Chengdu 610041, China

\textsuperscript{c} Education Key Laboratory of Cell Proliferation \&
Regulation Biology, College of Life Sciences, Beijing Normal University,
Beijing 100875, China

\textsuperscript{d} Reproduction Medical Center of West China Second
University Hospital, Key

Laboratory of Obstetric, Gynecologic and Pediatric Diseases and Birth
Defects of

Ministry of Education, Sichuan University, Chengdu 610041, China

\textsuperscript{\#} These authors contributed equally to this work

*Correspondence should be addressed to: SuRen Chen: chensr@bnu.edu.cn;
Yihong Yang: zxgo88@163.com

To the Editor:

We have noticed that Choi Y et al. reported the heterozygous mutation in
\emph{SOHLH1} gene (NG\_033784.1: c.346-1G\textgreater{}A) caused the
original splicing site disruption and partial deletion within exon 4,
thus generated a truncated bHLH domain and resulted in nonobstructive
azoospermia (NOA) in two Korean patients (Choi et al., 2010). The
authors also performed hematoxylin-eosin staining (H\&E) to exam
patient's testis and the result showed a severe dysfunction in
spermatogenesis characterized by no spermatogonia, spermatocyte,
spermatids, or mature spermatozoa. Therefore, the author assumed this
heterozygous mutation in\emph{SOHLH1} resulted in NOA due to the absence
of normal spermatogenesis.

We would like to bring to your attention that we found this heterozygous
mutation in a Chinese patient A by whole-exome sequencing (WES) (Figure
1a). This patient did not suffer from NOA, whose sperm concentration and
sperm count were normal but had almost no motility (Table 1).
Particularly, most of the sperm from patient had amorphous head and the
anomies in flagella compared to the normal control by Papanicolaou
staining (Figure 1b). Moreover, scanning electron microscopy (SEM)
further confirmed that the sperm of patient had aberrant head including
round-head, double-head, tapered-head, pyriform head and/or irregular
head with short, bent, coiled and flagella (Figure 1c). Additionally, we
analyzed the ultrastructure of the patient's spermatozoa as well as the
normal control by transmission electron microscopy (TEM). TEM analysis
screened normal spermatozoa had proper ratio of head's length to width,
and the 9+2 structure (nine peripheral microtubule doublets and two
central microtubules, surrounded by outer dense fiber and fibrous
sheath) was integrated and regularly arranged. For patients, we observed
that sperm plasma membrane was swollen and damaged, cell nucleus
contained vacuoles, and mitochondria was empty foamed, combined with an
atypical 9+0 arrangement of axonemal microtubules in the sperm flagella
(Figure 1d). According to these findings, we speculated that patient A
suffered from teratozoospermia but not NOA.

Remarkably, we detected a homozygous mutation of c.346-1G\textgreater{}A
in \emph{SOHLH1} gene in another Chinese patient B (Figure 1a). For this
patient, his sperm count was extremely low. In addition, we found his
sperm morphology was also abnormal. Though Papanicolaou staining, we
observed deformities of head and flagella (Figure 1b). SEM and TEM
further identified abnormality of sperm morphology and defective sperm
ultrastructure (Figure 1c and d). Thus, patient B was affected to severe
oligozoospermia. Although c.346-1G\textgreater{}A in \emph{SOHIL1} gene
is a deleterious mutation which disrupted splice site, we have provided
evidence that heterozygous c.346-1G\textgreater{}A in \emph{SOHIL1} gene
was not responsible for NOA, and homozygous mutation led to severe
oligozoospermia.

\emph{SOHLH1} is a transcription factor which specifically expresses in
germ cells, and plays an important role in the development of
spermatogenic cells through transcriptional regulation of downstream
genes such as \emph{SYCP1} (Ballow D et al., 2006; Li et al., 2019).
Some researchers have focused on the relationship between
\emph{SOHLH1}and male infertility\emph{.} \emph{Sohlh1} knockout male
mice (\emph{Sohlh1\textsuperscript{-/-}} ) had a large number of
apoptotic spermatocytes in the meiotic stage, resulting in failure of
spermatogenesis and led to male infertility (Li et al., 2019).
Similarly, our patient B carrying homozygous\emph{SOHLH1} mutation
showed the severe reduction in total sperm number. Considering the low
homology of \emph{SOHLH1} gene in humans and mice
(https://www.ncbi.nlm.nih.gov/homologene/), it is reasonable that there
are some differences in phenotypes between humans and mice with
homozygous variation of \emph{SOHLH1} , but both due to a decrease in
the number of spermatozoa. However, Li et al. didn't mention any sperm
parameter about heterozygous male mice, and we speculate that
heterozygous male mice had normal fertility thus could produce the
homozygous mice. Moreover, our patient A with the heterozygous mutation
showed the normal sperm number. All the observations might be explained
that another normal allele of \emph{SOHLH1} could totally translated
into the intact protein and further performed normal biology functions,
while the homozygous mutation led to the complete absence of SOHLH1
expression. What's more, Song B et al. performed single nucleotide
polymorphism (SNP) linkage analysis of \emph{SOHLH1} , and discovered
that there was no relationship between the rs558113 in \emph{SOHLH1}gene
and susceptibility to NOA in Chinese population, suggesting that
heterozygous mutation in \emph{SOHLH1} may not associated to NOA (Song
et al., 2015). Moreover, no evidence suggests the \emph{SOHLH1} gene has
haploinsufficiency (https://clinicalgenome.org/). Remarkably, patient A
in our study followed ICSI, and his wife became pregnant and had
successful delivery. This outcome strongly suggested patient A could
select normal sperm to fertilization, and heterozygous mutation of
c.346-1G\textgreater{}A in \emph{SOHIL1} gene didn't result in NOA.
Actually, we didn't deny that heterozygous mutation of \emph{SOHLH1} has
a certain degree of contribution to male infertility, and it may combine
with other gene mutations or factors to drive the occurrence and
development of NOA, or epigenetic modifications affect the gene
transcriptional expression level.

According to our evidences, the heterozygous mutation of
c.346-1G\textgreater{}A in \emph{SOHIL1} was not the directly genetic
cause for NOA. Due to the complexity of the spermatogenesis process, and
the etiological factors of male infertility are mysterious, we should
cautious about mutations which discovered in patients and performed more
functional experiments to constitute the relationship between genotype
and phenotype, so that we can provide more strong and accurate evidence
for clinical diagnose.

\textbf{Figure Legend}

Figure 1 The morphology and ultrastructure of spermatozoa from two
patients. (a) Sequence chromatograms of two patients. Sanger sequencing
confirmed the \emph{SOHLH1} mutation in the patients. The red arrow
denotes the mutation site (NG\_033784.1: c.346-1G\textgreater{}A). (b)
Papanicolaou staining of patients' sperm, most of which were amorphous
head and irregular flagella. (c) The abnormal sperm phenotypes were
observed in the patients by SEM. (d) TEM showed the abnormal
ultrastructures of the head and flagella from the patients' spermatozoa.

\textbf{ACKNOWLEDGEMENTS}

We thank the study participants who provided the sperm samples and
clinical information. This work was supported by the Key research
project of Science \& Technology Department of Sichuan Province
{[}2020YJ0291{]}.

\textbf{CONFLICT OF INTEREST}

The authors declare no conflict of interests.

\textbf{DATA AVAILABILITY STATEMENT}

The data that support the findings of this study are available from the
corresponding author upon reasonable request.

\textbf{WEB RESOURCE}

NCBI:\emph{https://www.ncbi.nlm.nih.gov/homologene}

ClinGen:\emph{https://clinicalgenome.org}

\textbf{REFERENCE}

Ballow D, Meistrich ML, Matzuk M, Rajkovic A. (2006). Sohlh1 is
essential for spermatogonial differentiation. Dev. Biol, 294, 161-167.
doi:10.1016/j.ydbio.2006.02.027

Choi Y, Jeon S, Choi M, Lee MH, Park M, Lee DR, Jun KY, Kwon Y, Lee OH,
Song SH, Kim JY, Lee KA, Yoon TK, Rajkovic A, Shim SH. (2010). Mutations
in SOHLH1 gene associate with nonobstructive azoospermia. Hum. Mutat,
31, 788-793. doi:10.1002/humu.21264

Li Y, Qi W, Liu G, Du B, Sun Q, Zhang X, Jin M, Dong W, Liu J, Zheng Z
(2019). Sohlh1 is required for synaptonemal complex formation by
transcriptionally regulating meiotic genes during spermatogenesis in
mice. Mol. Reprod. Dev, 86, 252-264. doi:10.1002/mrd.23100 Song B, Zhang
Y, He XJ, Du WD, Ruan J, Zhou FS, Wu H, Zha X, Xie XS, Ye L, Wei ZL,
Zhou P, Cao YX. (2015). Association of genetic variants in SOHLH1 and
SOHLH2 with non-obstructive azoospermia risk in the Chinese population.
Eur. J. Obstet. Gynecol. Reprod. Biol., 184, 48-52.
doi:10.1016/j.ejogrb.2014.11.003

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