Broadening the ARMC2 mutational phenotype: linking multiple morphological abnormalities of the Flagella to Pulmonary Manifestations in Primary Ciliary Dyskinesia

Abstract

Background

Severe asthenoteratozoospermia, a prevalent cause of male infertility, has increasingly been associated with ARMC2 variants that cause Multiple Morphological Abnormalities of the Sperm Flagella (MMAF). Although ARMC2 is also expressed in other ciliary structures, no studies have yet reported a link between ARMC2 gene variants and other symptoms of Primary Ciliary Dyskinesia (PCD).

Methods

Here, we performed whole-exome sequencing (WES) on Chinese subjects with MMAF to identify potential genetic variants. Sanger sequencing was used to validate the candidate variants. Sperm morphology was assessed using modified hematoxylin and eosin (H&E) staining, and transmission electron microscopy (TEM) was performed to observe the ultrastructural defects of the sperm flagella. Western blot analysis and immunofluorescence (IF) of spermatozoa were performed to evaluate variations in structural protein. Additionally, intracytoplasmic sperm injection (ICSI) was applied for assisted fertilization.

Results

We identified two compound heterozygous ARMC2 variants and one homozygous variant (P1: c.1030_1042del, p.T344fs/c.1331G > A, p.R444H; P2:c.1264C > T, p.R422X) in two unrelated individuals. Notably, in addition to MMAF, individual P2 exhibited classic symptoms of PCD in the lungs, including recurrent airway infections, bronchitis, and rhinosinusitis. Morphological and ultrastructural analyses of the spermatozoa obtained from the two individuals revealed dramatic disorganization in axonemal and peri-axonemal structures, as well as the absence of the axonemal central pair complex (CPC). Immunoblotting and immunofluorescence assays revealed the reduced expression of ARMC2 and the abnormality of various axonal structural proteins. Further assisted reproduction outcomes showed that one of the individuals conceived successfully after Intracytoplasmic Sperm Injection (ICSI).

Conclusions

Overall, this study significantly expanded the mutational phenotype of ARMC2, marking the first discovery of PCD-related pulmonary phenotypes outside of the reproductive system. This work establishes the association between ARMC2 and typical PCD and lays the groundwork for further investigation into the molecular mechanisms of ARMC2 in both flagellogenesis and ciliogenesis.

Background

Infertility is a concerning problem, affecting about 15% of couples of reproductive age worldwide [1], in which male factors account for about half of all cases of infertility [2]. Male infertility is a highly inherited condition and is usually classified into oligozoospermia, asthenozoospermia, teratozoospermia, and even azoospermia.

Male infertility can be attributed to a variety of factors, including disorders of the genitourinary system, environmental influences, and genetic components. Recently, the role of monogenic variants in male infertility has garnered increasing attention. It is believed that more than 4,000 genes are involved in the process of spermatogenesis [3]. Any defects in these pathogenic genes can hinder sperm production and result in male infertility. Recently, high-throughput sequencing technology has enabled researchers to identify an increasing number of genes necessary for sperm production, significantly advancing our understanding of the genetic basis of various forms of male infertility.

MMAF is a rare but serious subtype of asthenoteratozoospermia, characterized by multiple abnormalities on sperm flagella with a combination of short, absent, coiled, bent, and/or irregular-caliber flagella [4]. To date, genetic variants have been identified in more than 50 genes associated with MMAF, including AK7 (MIM: 615,364), AKAP4 (MIM: 300,185), ARMC2 (MIM: 618,424), CFAP43 (MIM: 617,558), CFAP44 (MIM: 617,559), CFAP65 (MIM: 614,270), CFAP69 (MIM: 617,949), DNAH1 (MIM: 603,332), FSIP2 (MIM: 618,153), QRICH2 (MIM: 618,304), SPEF2 (MIM: 610,172), TTC21A (MIM: 611,430), and WDR66 (also known as CFAP251, MIM: 618,146), DRC1(MIM:615,288) [5,6,7,8,9,10,11,12], et al. At present, the identified variants could account for about 60.0%−75.0% of infertile men diagnosed with MMAF, while the remaining cases (25.0%−40.0% of MMAF patients) have unknown genetic factors. As sperm flagella and motile cilia share similar “9 + 2” axonemal structures, malfunction of the axoneme in these organelles may lead to other ciliary abnormalities, causing phenotypes beyond the reproductive system, such as pulmonary phenotypes, associated with primary ciliary dyskinesia (PCD; MIM: 244,400) [13]. So far, approximately 40 disease-causing genes accounting for ~ 70% of PCD have been implicated in humans [14].

ARMC2 (MIM: 618,424) is located on chromosome 6 and contains 18 exons, encoding an armadillo protein with 867 amino acids, composed of 12 armadillo repeats (ARM-repeat) flanked by unique C-terminal and N-terminal domains (NCBI: NP_115507.4; UniProtKB: Q8NEN0), which is specifically expressed in human testes and spermatozoa. Coutton C et al. first established the pivotal role of ARMC2 in MMAF using whole-exome sequencing (WES) in a cohort comprising 168 individuals with infertility in 2019 [15]. Although ARMC2 has been observed to be abundantly expressed in epithelial cells with cilia and sperm flagella, initial findings indicated that ARMC2 defects were specific to male infertility. To date, no studies have confirmed that biallelic variants in ARMC2 lead to ciliary defects in other systems. However, it is noteworthy that genome-wide interaction studies have associated ARMC2 variants with diminished pulmonary function [16, 17], highlighting their potentially extensive involvement in ciliary biology that extends beyond their influence on fertility.

In this study, we have identified biallelic variants in the ARMC2 gene in two unrelated patients with MMAF. Notably, one patient exhibited additional classical PCD-related pulmonary symptoms, including recurrent airway infections, bronchitis, and rhinosinusitis, in addition to MMAF. Further analysis revealed impaired flagellar assembly in ARMC2 mutant individuals. Immunoblotting and immunofluorescence assays of the ARMC2 variant also resulted in significant reductions in ARMC2 protein levels and led to the loss of function of many axonemal structural proteins. Our findings expanded the genetic spectrum of the ARMC2 gene and provided further understanding of the links between ARMC2 gene variants and PCD.

Material and methods

Study participants

Subjects diagnosed with MMAF from the First Affiliated Hospital of Anhui Medical University (Hefei, China) were included in this study. The diagnosis and evaluation of subjects were conducted in strict accordance with the guidelines outlined in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen. Both participants had a normal 46, XY karyotype and no Y chromosome microdeletions. Potential causes of infertility, such as iatrogenic injury, reproductive tract infections, testicular inflammation, and drug exposure, were carefully excluded. Physical examinations revealed normal findings, verifying typical parameters for height, weight, hair distribution, mental state, testis size, and external genital organs.

Ethical approval for the genetic study was granted by the corresponding ethics committees of the First Affiliated Hospital of Anhui Medical University (PJ2023-04–19). Both individuals provided written informed consent prior to participation in the study.

Semen parameters and sperm morphological analysis

According to the guidelines of the World Health Organization (WHO, 2010) [18], subjects collected semen samples by masturbation after 2–7 days of abstinence. The samples were then analyzed in the source laboratory during routine biological examination. We used hematoxylin and eosin (H&E) staining to evaluate sperm morphology. For each subject, we examined at least 200 spermatozoa to evaluate the percentage of sperm with abnormal morphology.

Whole-Exome Sequencing (WES) and bioinformatic analysis

Genomic DNA was extracted from peripheral blood samples of asthenoteratozoospermic individuals for Whole-Exome Sequencing (WES). DNA was sheared into fragments, enriched using a Sure Select XT Human All Exon Kit, and sequenced using an Illumina HiSeq X-TEN platform. Sequenced reads were mapped to the human reference GRCh38/hg19 genome using Burrows-Wheeler Aligner (BWA) software [19]. Annotation of the variants was performed using ANNOVAR [20], incorporating data from several public databases, including the 1000 Genomes Project and gnomAD. Potentially deleterious variants were evaluated using SIFT, MutationTaster, PolyPhen-2, and the Combined Annotation-Dependent Depletion (CADD) score. Common variants with an allele frequency of > 0.05 were excluded. Our focus was on loss-of-function variants, including splicing variants (< 2 bp), stop gains, stop losses, frameshift indels and deleterious missense variants. Missense variants predicted to be deleterious by at least three of the four software tools- SIFT, PolyPhen-2, MutationTaster, and CADD (score > 20)- were defined as deleterious. We determined conservation among species by performing an alignment of the amino acid sequences of ARMC2 proteins whose data were obtained from the GenBank database. ARMC2 variants identified through WES were further validated by Sanger sequencing, with the primers used for validation provided in Table S1.

Transmission electron microscopy (TEM)

The patient's sperm and the control group's sperm were washed three times with 1 × phosphate-buffered saline (PBS) at 2500 rpm at 25 °C, and then fixed with 2.5% glutaraldehyde (pH 6.9) for more than 2 h at 4 °C. Immobilized sperm were fixed with 1% osmium tetroxide for 2 h at 4 °C, stained with 2% uranium acetate for 2 h, and dehydrated in a graded series of ethanol (50%, 70%, 90% and 100%) and 100% acetone. Then, the fixed spermatozoa were embedded in EPON 812 epoxy resin. Finally, it was cut into 100-nm sections by using an EM UC7 microtome (Leica, Wezlar, Germany), stained with lead citrate, and observed under a TEM (Thermo Fisher Scientific, Waltham, MA, USA) by using Talos L120C G2.

Western blot (WB) analysis

Protein was isolated from patients' sperm samples and control sperm samples by RIPA lysis buffer (Beyotime Biotechnology) for western blot analysis. Lysates were separated on 10% polyacrylamide gel by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to PDVF membrane. To prevent non-specific binding to the membrane, the blot was blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 at 25 °C for 1 h. The primary antibody diluted with TBST was then incubated overnight at 4 °C by shaking. Rabbit polyclonal anti-TOMM20 (1:1000), rabbit polyclonal anti-SPAG6 (1:1000), rabbit polyclonal anti-IFT88 (1:1000), rabbit polyclonal anti-ARMC2 (1:1000), and mouse polyclonal anti-β-Actin were used. Subsequently, these blots were washed in TBST and incubated at 25 °C for 1 h with 1:5000 dilutions of HRP-conjugated secondary antibody. Enhanced chemiluminescence (Biosharp, BL520A) was used to visualize the target protein, and β-Actin was used as a loading control.

Immunofluorescence analysis

Immunofluorescence analysis was performed with sperm cells from healthy controls and patients carrying ARMC2 variants. Sperm cells were washed in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 30 min at room temperature, and coated on a glass slide precoated with 0.1% poly-L-lysozyme (Thermo Scientific). Then, the slides were washed in PBS, blocked in 10% donkey serum, and incubated overnight at 4 °C with the following main antibodies: rabbit polyclonal anti-ARMC2 (1:100), rabbit polyclonal anti-TOMM20 (1:200), rabbit polyclonal anti-SPAG6 (Sigma-Aldrich, HPA038440, 1:200), rabbit polyclonal anti-IFT88 (Proteintech, 13,967–1-AP, 1:200), as well as mouse monoclonal anti-acetylated α-tubulin (Sigma-Aldrich, T6793, 1:200). After washing with PBS, slides were incubated for 1 h at 37 °C with highly cross-adsorbed secondary antibodies: anti-mouse Alexa Fluor 488 (Yeasen Biotechnology, USA, 34106ES60, 1:500) and anti-rabbit Alexa Fluor 594 antibodies (Jackson ImmunoResearch, USA, 111–585–003, 1:500). DNA was stained using Hoechst 33,342 (Thermo Fisher Scientific, USA, 62, 249, 1:1000). The images were captured with the LSM 800 confocal microscope (Carl Zeiss AG).

Intracytoplasmic sperm injection procedure

Among the individuals with the ARMC2 variant, one patient received ICSI treatment. The patient’s wife underwent a long protocol to induce ovulation. To enrich sperm vitality, semen samples were centrifuged with a discontinuous density gradient. After egg retrieval, mature oocytes and motile sperm were selected for ICSI. All embryos formed after standard embryo culture are preserved by vitrification for the next freeze–thaw cycle. Two months later, one or two viable blastocysts were thawed and transferred to the uterus of the female partner. Serum β-HCG level was measured on the 14th day after embryo transfer to confirm biochemical pregnancy, and clinical pregnancy was confirmed by B-ultrasound on the 30th day after embryo transfer.

Results

Bi-allelic ARMC2 variants were identified in two unrelated individuals with MMAF

In this study, we identified two compound heterozygous ARMC2 variants and one homozygous variant (P1: c.1030_1042del, p.T344fs/c.1331G > A, p.R444H; P2:c.1264C > T, p.R422X) in two unrelated individuals. Sanger sequencing confirmed that their parents were heterozygous carriers in the two unrelated families, which was consistent with an autosomal recessive mode of inheritance (Fig. 1A). The prevalence analysis from the 1000 Genomes and gnomAD databases revealed that these variants were either at low allele frequencies or unavailable in the general population, suggesting their rarity (Table S2). In addition, the frameshift and nonsense variants (M1, M3) identified in this study were assumed to induce premature termination codons and truncated proteins. The remaining missense variant, M2, caused the substitution of arginine (R) with histidine (H) and was highly conserved among species (Fig. 1B). It was also predicted to be deleterious by bioinformatic tools such as SIFT, PolyPhen-2, Mutation Taster, and CADD (Table S2).

Fig. 1

Identification of Bi-allelic variants in ARMC2. A Pedigrees of two families carrying ARMC2 variants. All the affected individuals have bi-allelic variants with a recessive inheritance mode. P1 carries compound heterozygous variants of ARMC2 derived from his parental heterozygous carriers. P2 has homozygous variants in ARMC2. Sanger sequencing results are shown below the pedigrees. The deleted region in the Sanger sequencing chromatogram for M1 is highlighted with a red box, and the position of each variant for M2 and M3 is indicated by a red arrow. B Schematic representation of the functional domains of ARMC2 and the locations of ARMC2 variants identified in this study. Arrows (red) and dotted lines (blue, red and black) show the positions of novel ARMC2 variants identified in the present study. Sequence alignments show that the amino acid affected by the M2 mutation is highly conserved across different species. The blue boxes indicate 12 ARM repeat domains as described by the Uniprot server. Abbreviations: M1, mutation 1; M2, mutation 2; M3, mutation 3; WT, wild type

Clinical features of two subjects with bi-allelic ARMC2 variants

Clinical examination revealed normal male testicular size, external genitalia, hormone levels, and secondary sexual characteristics. Chromosomal karyotype analysis showed no abnormalities. Semen analysis and sperm morphology assessment indicated asthenoteratozoospermia and typical MMAF. The P1 individual exhibited no other symptoms associated with PCD; however, the P2 individual exhibited not only typical MMAF but also characteristic PCD-related pulmonary phenotypes, including recurrent airway infections, bronchitis, and rhinosinusitis (Table 1). A chest CT scan of P2 showed bilateral lower lung bronchiectasis and typical imaging manifestations like signet ring sign (Fig. 2C). It was shown that semen volume and sperm concentration were within normal ranges in both individuals carrying bi-allelic ARMC2 variants, while a significant reduction was observed in sperm motility and progressive motility. Morphological analysis of the patients' sperm revealed a typical phenotype of MMAF, characterized by absent, coiled, short, bent, and irregular caliber flagella. Among these abnormalities, the most frequently observed defects were short and curly flagella in the sperm of both patients (Table 1 and Fig. 2A).

Table 1 Clinical features in Men Harboring ARMC2 Variants

Fig. 2

Sperm Morphology and Ultrastructure Analyses for Men Harboring ARMC2 Variants. A Light microscopy analysis of spermatozoa from the control and men harboring ARMC2 variants. Most spermatozoa from men harboring ARMC2 variants have flagella that are short, coiled, or of irregular caliber. The spermatozoa from subjects P1 and P2 are given as examples of typical MMAF phenotypes observed in men harboring ARMC2 variants. Scale bars: 10 μm. B TEM analyses of sperm cells from a fertile control and two men harboring ARMC2 variants. The control individual shows the typical ‘‘9 + 2’’ microtubule structure, including nine peripheral microtubule doublets paired with nine outer dense fibers and the central pair of microtubules, surrounded by the organized mitochondrial sheath or fibrous sheath. The ultrastructure observed in cross-sections of the flagella in men harboring ARMC2 variants indicates the misarranged outer dense fibers and disorganization of the peripheral microtubules with a lack of the central pair of microtubules. Scale bars: 500 nm. Abbreviations: MS, mitochondrial sheath (yellow arrows); CP, central pair of microtubules (orange arrows); MD, microtubules doublet (green arrows); ODF, outer dense fiber (blue arrows). C In the lung window, P2-I demonstrated bilateral lower lung bronchiectasis with characteristic imaging features, including the signet ring sign (red arrows). In the mediastinal window, P2-II displayed normal anatomical positioning of the liver (blue arrows), spleen (yellow arrows), and stomach (green arrows), with no evidence of situs inversus

To investigate the sperm flagellar ultrastructure of individuals harboring bi-allelic ARMC2 variants, we performed TEM analyses of spermatozoa obtained from two patients and healthy control individuals. More than 50 flagella cross-sections were randomly observed under TEM to evaluate the microtubule assembly in sperm flagella. In the control sperm, typical “9 + 2” microtubule structures were observed, consisting of a central pair of microtubules surrounded by nine peripheral microtubule doublets. These doublets were supported by nine radial spoke complexes and were encased by outer dense fibers (ODF). However, the sperm flagella of the two individuals harboring biallelic ARMC2 variants mainly presented a dramatic disorganization in axonemal or other peri-axonemal structures. The main defect observed in sperm flagella was the lack of a central pair of microtubules, resulting in an abnormal “9 + 0” configuration. In addition, the peripheral microtubule doublets, outer dense fibers, and mitochondrial sheath were also translocated and disordered (Fig. 2B).

Sperm flagellar component defects in patients with bi-allelic ARMC2 variants

To investigate the impact of ARMC2 biallelic variants on sperm flagellar composition, we first examined the expression and localization of ARMC2. WB and immunofluorescence analyses revealed that ARMC2 immunostaining was predominantly localized along the entire length of the flagella, with limited protein signals observed in the acrosomal region and neck of normal sperm. However, in the P2 individual carrying the ARMC2 homozygous variant, these signals were almost absent (Fig. 3A). Correspondingly, WB analysis demonstrated that ARMC2 was either absent or significantly downregulated in spermatozoa from the P2 individual (Fig. 3B). To comprehensively investigate the effects of biallelic variant in ARMC2 on the assembly or anchoring of axonemal proteins complexes, WB and immunofluorescence analyses were subsequently performed with antibodies targeting proteins located in various axonemal and peri-axonemal structures of the sperm flagella, including SPAG6 (a component of CP complex), TOMM20 (a mitochondrial outer membrane protein) and IFT-88 (a component of IFT-B subcomplex). WB assays performed on spermatozoa from subjects P1 and P2 revealed a dramatic reduction or complete absence of SPAG6 signals. Moreover, immunostaining assays performed specifically on spermatozoa from subject P2 also showed a complete absence of SPAG6 signals. In addition, WB assays revealed that the expression levels of TOMM20 and IFT-88 were significantly reduced in P1, whereas the reduction was not evident in P2. Immunofluorescence assays also showed similar results (Fig. 4A-D). This finding implied that ARMC2 deficiency primarily led to a significant reduction in SPAG6 levels, thereby impairing the assembly of the central pair complex (CPC).

Fig. 3

Expression Analysis and Location of ARMC2 Protein in Sperm Flagella. A ARMC2 immunostaining in human spermatozoa from a normal control and men harboring ARMC2 variants. Sperm cells were stained with anti-ARMC2 (green) and anti-Ac-Tubulin (red) antibodies. DNA was counterstained with Hoechst as a nuclei marker. ARMC2 immunostaining is localized along the entire flagella but is almost absent in the sperm flagella of men harboring ARMC2 variants. Scale bar: 10 μm. B Immunoblotting analysis of ARMC2 protein in sperm lysates from a normal control and men harboring ARMC2 variants (P1 and P2). WB assays suggested that the level of ARMC2 was reduced significantly in the sperm from men harboring ARMC2 variants when compared to the level in sperm from a normal control man. β-Actin was used as a loading control

Fig. 4

The localization and levels of flagella-associated proteins in ARMC2 patients and controls. (A-C) Immunofluorescence assays of flagella-associated proteins, including TOMM20 (mitochondrial outer membrane protein), SPAG6 (a component of the CP complex), and IFT 88 (a component of IFT-B subcomplex) in ARMC2 patients and controls. Anti-TOMM20 (red in A) localized at mitochondrial sheath, Anti-SPAG6 (red in B), Anti-IFT-88 (green in C) were normally localized along the sperm flagella in the control sperm. A complete absence of SPAG6 signals was observed in P2. Anti-Ac-Tubulin (green in A and B, red in C) marked the sperm flagella, and Hoechst (blue) marked the nucleus of spermatozoa. Scale bar: 10 μm. (D) WB assays analyzed the expression levels of TOMM20, SPAG6, and IFI-88 in sperm obtained from ARMC2 individuals and normal controls. The results of WB assays were consistent with those of immunofluorescence assays described above. β-Actin was used as a loading control

Favorable outcomes via ICSI in subjects with ARMC2 variants

The partners of the two individuals harboring bi-allelic ARMC2 variants had been unable to conceive spontaneously without contraception for over two years. Current studies have predominantly indicated that ICSI is an effective treatment for patients with MMAF. The partner of P2 had an irregular menstrual cycle. Seven oocytes were retrieved, all of which were at the metaphase II (MII) stage and subsequently microinjected. Successful fertilization occurred in seven oocytes, which then cleaved. On the first day post-fertilization, four embryos showed two pronuclei (2PN), and three showed no pronuclei (0PN). By the fifth and sixth days, six blastocysts were developed and subsequently cryopreserved. Following thawing and embryo transfer, a positive urinary hCG test was observed 14 days post-transfer. At 30 days post-transfer, a singleton intrauterine pregnancy was confirmed via ultrasound. Ultimately, a healthy baby boy was successfully delivered (refer to Table 2 for detailed data). Although we lack information on the assisted reproductive cycle of P1, the successful outcome in P2 suggested that ICSI could be a viable clinical treatment option for patients with ARMC2 variants.

Table 2 The clinical outcomes of ARMC2 mutated subject ICSI

Discussion

In this study, we identified bi-allelic variants of ARMC2 in two unrelated individuals with MMAF. Moreover, in one of these patients, we found for the first time that bi-allelic variants of ARMC2 not only lead to MMAF but may also be associated with PCD-related pulmonary phenotypes. This expands the phenotypic spectrum of ARMC2 variants and suggests that ARMC2 is a new candidate gene for PCD.

ARMC2 encodes a protein containing several armadillo (ARM) repeats [21], which are involved in a wide range of cellular functions, including cytoskeleton regulation, intracellular signal transduction, and protein degradation or folding [22]. Previous studies had shown that ARM-containing domain may have been a key domain in a protein family related to the structure and function of flagella/cilla. Many members of the ARMC protein family were closely related to spermatogenesis or ciliogenesis. For example, ARMC9 played an important role in ciliary development and was related to Joubert syndrome, a hereditary disease [23]. ARMCX4 was found to be involved in the differentiation of spermatogonial stem cells during spermatogenesis [24]. More interestingly, as a sperm mitochondrial outer membrane-related protein, ARMC12 interacted with mitochondrial proteins, such as VDAC2 and VDAC3, to participate in the formation of the sperm mitochondrial outer membrane and mitochondrial sheath [25]. Another protein related to the ARM-domain, CFAP69, was closely associated with the formation of central microtubules during sperm flagella development [8]. In our study, all MMAF-related variants in ARMC2 were located in the ARM domain, which may lead to damage in the ARM domain of the ARMC2 protein. IF and WB analyses also showed that the expression of ARMC2 in the sperm of subjects harboring ARMC2 variants was significantly reduced compared with normal controls. Therefore, it is speculated that the defect in the ARM domain of the ARMC2 protein may be an important factor contributing to the MMAF phenotype.

Several genes causing MMAF have been identified, and some of these also result in other PCD-related phenotypes. For example, mutations in the DNAH1 gene, which encodes an inner arm heavy chain dynein, have been associated with MMAF, exhibiting no apparent PCD phenotypes. However, defects in DNAH1 have also been documented to elicit a PCD phenotype in a female of Arabian descent, as well as a PCD phenotype accompanied by male infertility in mice [26, 27]. Guo et al. reported a BRWD1 variant associated with MMAF and PCD-like symptoms in humans [28]. Additionally, mutations in CFAP47, a gene previously implicated in MMAF, also play a role in the respiratory defects observed in PCD patients [29]. This suggests that some individuals diagnosed with MMAF may exhibit a mild or atypical PCD phenotype. As for ARMC2, it is known as a gene linked to MMAF and is implicated in diminished lung function. However, there have been no previous reports of other typical PCD-related symptoms associated with ARMC2 variants. We have identified the initial case of a patient harboring a homozygous ARMC2 variant presenting with MMAF and typical PCD-related lung manifestations. Variants in the ARMC2 gene are hypothesized to trigger PCD via multiple mechanisms. Firstly, such variants might induce aberrations in the axonemal structure, compromising the functionality of dynein arms and nexin links that are crucial for the synchronized movement of cilia, thereby leading to the hallmark ciliary motility deficits in PCD. Secondly, given the potential role of ARMC2 in ciliary assembly or maintenance, variants could result in anomalies of ciliogenesis or ciliary structural integrity, further contributing to the complex pathophysiology of PCD. Future studies should include comprehensive functional analyses of ARMC2 to determine its role in cilia biology. Additionally, genetic screening in larger cohorts of PCD patients, coupled with advanced imaging techniques to assess ciliary structure and motility, will be instrumental in establishing a definitive link between ARMC2 variants and PCD.

Assisted reproductive technology (ART), such as IVF (In vitro fertilization) and ICSI, has become an important tool in treating infertile couples [30]. For MMAF, there is no empirical drug treatment to improve semen parameters; therefore, ICSI may be the only choice for MMAF-affected cases [31]. In previous reports, several variants of ARMC2 were identified in MMAF patients, and one patient achieved good clinical results after undergoing ICSI-assisted reproductive therapy. In this study, the fertilization rate, pregnancy rate, and delivery rate of the patient's partner were all 100%. Our study further confirmed that patients with bi-allelic variants in the ARMC2 gene can achieve favorable fertility outcomes after undergoing ICSI, even with PCD.

Although our study provides valuable insights into the association between biallelic ARMC2 mutations and phenotypes such as MMAF and PCD, several limitations should be noted. First, the small sample size of only two cases limits the generalizability of our conclusions, and larger cohort studies are needed to validate the relationship between biallelic ARMC2 mutations and these clinical phenotypes. Second, due to the unavailability of appropriate samples, we were unable to conduct immunofluorescence analysis for P1. Third, the lack of imaging scans for P1, along with the absence of nasal nitric oxide (NO) measurements and TEM analysis of respiratory cilia, restricted our ability to comprehensively evaluate the patient's condition. These limitations underscore the need for future studies with larger sample sizes and more thorough phenotypic assessments to confirm and expand upon our findings.

Conclusion

In conclusion, our study further confirmed the association between bi-allelic variants in ARMC2 and human MMAF. More importantly, we have established, for the first time, a relationship between bi-allelic variants in ARMC2 and the typical pulmonary phenotype associated with PCD, beyond reproductive phenotypes. Additionally, our results also indicated that male infertility and MMAF resulting from bi-allelic variants in ARMC2 can be overcome by ICSI, even in the presence of typical PCD. Overall, this research enhanced our understanding of the genetic underpinnings of both flagellar assembly and ciliary function. It offers valuable insights into the genetic causes of male infertility associated with PCD and establishes a foundation for further exploration into the molecular mechanisms of ARMC2 in flagellogenesis and ciliogenesis.