Agnieszka Malcher

More than 70% of non-obstructive azoospermia (NOA) cases remain idiopathic, and the underlying genetic causes need to be investigated. In this study, we aimed to identify genes and variants associated with the etiology of NOA. Two NOA patients from consanguineous families, with normal karyotypes and no Y-chromosome microdeletions, were selected for whole-genome sequencing (WGS). Candidate variants were validated by Sanger sequencing. Structural protein modeling and assessment of mutation-induced changes in molecular interactions were performed using SWISS-MODEL and AlphaFold. We identified a candidate homozygous missense variant (c.899C>T, p.(Ser300Phe); rs375931805) in SOX30 in one patient; this transcription factor is highly expressed in the testis. In the second patient, a homozygous missense variant (c.1472C>T, p.(Thr491Met); rs746902232) in RNF220 and a hemizygous missense variant (c.2354A>G, p.(Gln785Arg) in the X-linked AKAP4 gene were simultaneously detected. Previous studies have shown that SOX30 knockout in mice leads to meiosis I arrest and impaired spermiogenesis. AKAP4 is exclusively expressed in the testis, and missense variants have been reported in different forms of male infertility. The co-occurrence of variants in AKAP4 and RNF220 may suggest an oligogenic etiology of NOA and contribute to the phenotypic variability associated with AKAP4 variants.

Reduced sperm count, impaired motility, and abnormal morphology are characteristics of oligoasthenoteratozoospermia (OAT). Genetic factors play an important role in spermatogenic failure, but the molecular basis of OAT is still not fully understood. Whole-genome sequencing (WGS) was performed in 17 infertile men with normal karyotypes and no Y-chromosome microdeletions, including 10 patients with azoospermia and 7 with OAT. Among the OAT group, three patients carrying prioritized rare variants with potential relevance to spermatogenesis were selected for detailed analysis and presentation in the current study. Variants were called using DeepVariant, and they were annotated by Ensembl Variant Effect Predictor (VEP). Variants were prioritized based on low allele frequency (< 1% in gnomAD), predicted functional impact, relevance to spermatogenesis, and consistency with a recessive inheritance model. Candidate variants were validated by Sanger sequencing and inspection of BAM files. Structural protein modeling and characterization of mutation-induced changes in molecular interactions were also performed using SWISS-MODEL and AlphaFold. We identified a hemizygous splice donor variant (c.*4 + 1G > T) in SSX1 and a missense variant (c.1190 A > G) in CPNE1 in one patient with OAT. SSX1 is a testis-expressed gene, while CPNE1 encodes a calcium-dependent phospholipid-binding protein. In another patient, a stop-gained variant (c.790 C > T) in SPTBN5 was identified, which encodes a spectrin beta chain protein. This study identifies rare candidate variants in SSX1 , CPNE1 , and SPTBN5 that may potentially be associated with the molecular basis of OAT. These findings provide preliminary evidence supporting further investigation of these genes in OAT and support the application of WGS in unresolved male infertility.

DDX53 (DEAD-box helicase 53, known also as CAGE) is an intronless gene on the X chromosome, which expression shows strong testis specificity. It belongs to the group of cancer-testis (CT) antigens, with most studies to date focusing on its role in cancer, but the precise biological function of DDX53 remains unclear. Previous reports identifying rare DDX53 variants in infertile men provided the rationale for investigating the role of DDX53 in the context of human spermatogenesis. By using the human seminoma cell line (TCam-2) as an in vitro male germline model, we aimed to investigate the function and molecular targets of DDX53 protein. Our eCLIP and RNA-seq data show that DDX53 protein directly interacts with numerous RNA molecules, drives transcriptome changes in human cells, and is involved in alternative splicing of RNA. Moreover, we identified potential DDX53 protein interactors using Co-IP-MS approach. Subcellular localization analysis by confocal microscopy indicated a predominantly cytoplasmic distribution of DDX53, with partial nuclear presence in TCam-2 cells. We also identified DDX53-positive structures that may correspond to germ granule-like assemblies, although their precise nature remains to be determined. Additionally, we confirmed DDX53 presence in human testis using a specific, commercially available anti-DDX53 antibody. Our data indicate that DDX53 protein acts as a regulator of RNA metabolism in human cells. Collectively, we show that DDX53 participates in transcriptome regulation (including splicing) in male germ cells and exhibits transcriptomewide RNA interactions, but its wider biological role remains to be clarified.

Background: RBMXL3 is a primate-specific gene localized on the X chromosome, which expression is detectable mainly in the male gonad. So far, very little is known about the RBMXL3 protein function and its molecular interactions. However, recent reports mention the RBMXL3 gene in the context of human spermatogenesis, cancer, and a breathing disorder that affects newborns. In this study, we investigate the RBMXL3’s molecular network on a genome-wide scale using the human seminoma cell line (TCam-2) as a male germline in vitro model. Methods: By using transcriptomic (RNA sequencing (RNA-seq) and enhanced crosslinking and immunoprecipitation (eCLIP)) and proteomic (Co-immunoprecipitation coupled with Mass Spectrometry, (Co-IP-MS)) approaches we show RBMXL3 importance in RNA metabolism. Additionally, Western Blot, qRT-PCR, immunostaining, and confocal imaging were used in order to investigate the function of RBMXL3. Finally, we used a plasmid-based L1 retrotransposition assay to demonstrate the suppressive effect of RBMXL3 on human Long Interspersed Nuclear Element-1 (LINE-1, L1) retrotransposition. Results: Our RNA-seq data show that RBMXL3 expression drives gene expression changes and influence alternative splicing in human cells. Moreover, by performing eCLIP we provide a proof that RBMXL3 binds to a wide range of RNA transcripts. Additionally, we confirmed the nuclear localization of RBMXL3 in TCam-2 cells and its presence in spermatogonia and spermatocytes within the human testis. Finally, we report for the first time that RBMXL3 restricts human LINE-1 retrotransposition. Conclusions: Our findings for the first time identify primate-specific RBMXL3 protein as a new upstream regulator of RNA metabolism, characterized by broad RNA-binding activity in human TCam-2 cells. Finally, we show that RBMXL3 expression heavily reduces LINE-1 retrotransposition in human cells, underlying RBMXL3 importance in maintaining genome integrity. Our data suggest that RBMXL3 may contribute to the regulation of transcriptome dynamics in male germ cells, while its broader functional implications remain to be determined. Graphical Created with BioRender.com

Duchenne Muscular Dystrophy (DMD) is a genetic disorder characterized by disruptions in the dystrophin gene. This study aims to investigate potential a therapeutic approach using genetically modified human iPS-derived mesoangioblast-like cells (HIDEMs) in mdx mouse model. This study utilizes patient-specific myoblasts reprogrammed to human induced pluripotent stem cells (iPSCs) and then differentiated into HIDEMs. Lentiviral vectors carrying microdystrophin sequences have been employed to deliver the genetic construct to express a shortened, functional dystrophin protein in HIDEMs. The study indicated significant changes within redox potential between healthy and pathological HIDEM cells derived from DMD patients studied by catalase and superoxide dismutase activities. Microdystrophin expressing HIDEMs also improved expression of genes involved in STARS (striated muscle activator of Rho signaling) pathway albeit in selective DMD patients (with mild phenotype). Although in vivo observations did not bring progress in the mobility of mdx mice with HIDEMs, microdystrophin interventions this may argue against “treadmill test” as suitable for assessment of mdx mice recovery. Low-level signaling of the Rho pathway and inflammation-related factors in DMD myogenic cells can also contribute to the lack of success in a functional study. Overall, this research contributes to the understanding of DMD pathogenesis and provides insights into potential novel therapeutic strategy, highlighting the importance of personalized gene therapy.