1 Introduction
Left–right (LR) asymmetry is a widespread phenomenon in living organisms. Abnormal asymmetry is rare and can result from genetic mutations or environmental factors. The normal arrangement of visceral organs is known as situs solitus, and the inverted arrangement is referred to as situs inversus. Heterotaxia is a term used when subsets of organs show normal or aberrant positioning or morphology. Situs inversus and heterotaxia are collectively termed laterality defects, and they are often accompanied with congenital heart diseases, bronchiectasis, reproductive dysfunction, and other disorders [
1]. Advancements in genomics and sequencing technologies have revealed that gene-encoding ciliary components and planar cell polarity (PCP) proteins play a role in these conditions, leading to their classification as ciliopathies [
2].
The actin cytoskeleton is closely associated with ciliopathies because it regulates ciliogenesis and PCP [
3,
4]. Unconventional myosin-Id (MYO1D) functions as an actin-based motor protein and cooperates with the core PCP gene
Vangl2 in the formation of the animal LR axis [
5]. Previous research that used various model organisms has established that MYO1D deficiency can cause laterality defects [
5–
8]. However, evidence on its pathogenicity in humans is lacking.
In this study, we identified a novel biallelic variant of
MYO1D (NM_015194.2:exon12:c.1531G>A:p.D511N) in a proband from a consanguineous family, who presented with complex congenital heart disease, laterality defects, and asthenoteratozoospermia. This
MYO1D variant and another
MYO1D variant (NM_015194.2:c.2293C>T:p.P765S) found in an Arab family [
9] suggested that
MYO1D variants are responsible for laterality defects and congenital heart diseases and may play a role in sperm development.
2 Case report
This study was approved by the Ethics Committee of the Second Xiangya Hospital of Central South University, China, and written informed consent was obtained from subjects.
The proband, a 30-year-old man, presented with a complex set of medical conditions, including double outlet right ventricle defect, ventricular septal defect, patent foramen ovale, pulmonic stenosis, mirror image dextrocardia, and heterotaxia (Fig.1–1D).
After a genetic analysis, a novel variant in the MYO1D gene was identified as a potential candidate. Considering MYO1D’s role in ciliogenesis, we assessed the patient’s nasal mucociliary and spermatozoal functions. The nasal nitric oxide (nNO) concentration was measured to be 295 ppb, and the nNO production value was 117 nL/min, which exceeded the diagnostic cutoff value of 77 nL/min for primary ciliary dyskinesia (PCD). High-speed video microscopy revealed normal ciliary movement. Computer-aided sperm analysis was employed to assess the sperm function. In accordance with the WHO Laboratory Manual for the Examination and Processing of Human Semen (5th edition), sperm motility, progressive motility, and the normal morphology rate were calculated and found to be lower than the reference values, indicating asthenoteratozoospermia (Tab.1). In conclusion, aside from heterotaxia and complex congenital heart diseases, the patient also suffered from asthenoteratozoospermia. However, PCD was ruled out based on the evaluation of the nasal mucociliary function and the genetic analysis.
The patient was born into a consanguineous family, where his parents are cousins and have divorced (Fig.2). Whole exome sequencing was performed on the patient, and after data filtration [
10], seven variants were retained (Tab.2). Among these variants, a novel biallelic variant in the
MYO1D gene (NM_015194.2:exon12:c.1531G>A:p.D511N) was identified as the causative factor and validated through Sanger sequencing. The patient’s parents were found to carry the same heterozygous variant (Fig.2). The amino acid sequence of MYO1D around position 511 showed high conservation across multiple species, indicating its potential functional importance (Fig.2).
Bioinformatic analysis (MutationTaster, PolyPhen-2, SIFT, CADD, etc.) indicated that this variant was deleterious (Tab.2). The allele frequency of D511N was significantly less than 0.001, and no homozygotes were found in genetic databases. Furthermore, the variant was not detected in 200 unrelated ethnically matched healthy controls or in a cohort of 115 heterotaxia patients.
The two MYO1D variants identified in humans were mapped to the protein domain’s structure illustration. The D511N variant was located in the myosin motor domain, just before the actin binding domain, and P765S was situated in the nondomain region between IQ2 and TH1 domains (Fig.3).
The protein 3D model was downloaded from the AlphaFold Protein Structure Database, and the positions of 511, 765, and the actin-binding domain of MYO1D received high confidence scores. Protein models with the specific variants were generated using PyMOL. Similar to Alsafwani et al.’study, we observed minor structural changes when the 765th residue position was substituted by serine. However, the substitution of aspartic acid with asparagine at the 511th residue position did not result in any significant differences from the native structure (Fig.3). Electrostatic potential maps showed that both variants altered the surface potential (Fig.3 and 3D). No changes were observed in the actin-binding domain during the structural simulation process.
MYO1D plasmids carrying the D511N and P765S point mutations were constructed and transfected into 293T cells to evaluate the effect of the variants. Western blot analysis revealed that the expression level of the MYO1D protein in the cells transfected with the D511N plasmid was significantly higher than that in the cells transfected with the normal (wild-type) plasmid, indicating that the D511N variant probably led to an overexpression of MYO1D, whereas the P765S variant slightly decreased its expression (Fig.3). The interaction of MYO1D with actin fibers is essential for its function [
6]. Co-immunoprecipitation experiments were conducted to assess this interaction [
11], and they revealed that the D511N variant did not affect the interaction of MYO1D with β-actin, but the P765S variant significantly enhanced the binding to β-actin (Fig.3). These findings suggest that the D511N and P765S variants exhibited a gain-of-function characteristic. However, given the recessive inheritance pattern of
MYO1D variants, our hypothesis leans toward a loss-of-function pathogenic mechanism. The two variants potentially affect MYO1D proteins by reducing their motor functionality, suppressing ATPase activity, disrupting their interaction with calmodulin chains, and other effects. The increase in expression and the intensified interactions might be compensatory reactions.
The spermatozoal length of the patient was generally 10 μm shorter than that of the control group, which is in accordance with the cilia phenotype observed in
myo1d knockout zebrafish [
5] (Fig.4–4C). Spermatozoa with a length larger than 50 μm were rarely found in the patient’s semen (Fig.4). Furthermore, functional analysis of sperm motility in the patient revealed a considerable reduction in progressive motility compared with the control group.
Sperm-associated antigen 6 (SPAG6), a causative gene of severe asthenoteratozoospermia, interacts with MYO1D and facilitates its translocation to the plasma membrane [
12,
13]. The ability of these variants to bind SPAG6 is consistent with their ability to bind β-actin. The D511N variant demonstrated a similar binding capacity as the native protein. The P765S variant showed a stronger binding capacity than the native proteins (Fig.4).
These findings establish a connection between sperm defects and MYO1D variants, thus providing valuable new clues for exploring infertility and reproductive health concerns.
3 Discussion
MYO1D deficiency causes laterality defects in zebrafish, frog, and
Drosophila models [
5–
8]. In humans, the first clinical evidence was from a heterotaxy patient in an Arab consanguineous family, where genetic analysis identified a homozygous variant (c.2293C>T) in the
MYO1D gene [
9]. This patient, similar to ours, suffered from complex congenital heart disorders and heterotaxy. These findings strongly suggest a conservative role of MYO1D in breaking LR symmetry [
6].
Clinically, cardiac diseases are prevalent in patients with heterotaxy [
12]. Large-scale forward genetic screening in mice has unveiled the central role of cilia in congenital heart disease and established a correlation between LR asymmetry and cardiac development [
13]. Studies on mice further revealed that the sonic hedgehog (SHH) signaling transduced by cilia coordinates LR patterning, heart looping, and differentiation of the heart tube and regulates subsequent events of heart development, including outflow tract septation and formation of the atrioventricular septum [
12,
14]. In zebrafish and
Xenopus, knockdown of
myo1d leads to short cilia in the LR organizer (LRO), and although the movement of short cilia may be normal, the LRO flow is altered [
5,
8]. These changes in cilia length can result in aberrant SHH signaling transduction, which may underlie defective cardiac jogging in zebrafish and congenital heart defects in humans [
5,
12,
15].
Male infertility is also a primary phenotype of ciliopathy [
16]. In addition to the structural components in cilia, the actin cytoskeleton contributes to spermatogenesis by cell polarity [
17]. Studies have shown strict spermatid apico-basal polarity and Sertoli cell polarity during spermatogenesis, and altered polarity can cause asthenoteratozoospermia and multiple morphological abnormalities of the sperm flagella (MMAF) [
17]. For instance, SPAG6 deficiency results in planar cell polarity (PCP) defects and hearing loss in mice, and in humans, homozygous
SPAG6 variants can induce nonsyndromic asthenoteratozoospermia with severe MMAF [
18,
19]. In
Drosophila larvae, Myo1d overexpression induces polarized reorganization of epithelial cells toward the dextral orientation [
6]. Furthermore, in zebrafish, Myo1d can functionally interact with the core PCP component Vangl2 to shape a productive LRO flow [
5]. These results illustrate that MYO1D regulates PCP, and MYO1D deficiency is a potential cause of male infertility. Notably, the patient’s sperm was dramatically shorter than the reference and normal sperm of the control subjects, a result that agrees with the findings of the model research and provides strong evidence that MYO1D supports spermatogenesis.
Airway cilia are the main “9+2” motile cilia, and they are frequently disordered and accompanied with heterotaxia and sperm defects. In this study, the patient exhibited reduced sperm motility, but the motility of the airway cilia remained normal. This discrepancy might be due to MYO1D’s role as an extra-ciliary component, unlike cilia structural genes, such as DNAH10 [
20,
21]. MYO1D deficiency does not directly affect the symmetric side-to-side beating of airway cilia; instead, it disrupts the rotational movement of sperm [
22]. Furthermore, a shortened sperm length can potentially interfere with the development of the sperm tail, a critical factor for mobility [
23].
4 Conclusions
In conclusion, this study demonstrated that biallelic variants in MYO1D are associated with laterality defects, congenital heart defects, and sperm defects in humans.
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