Pathogenic gene variants in CCDC39, CCDC40, RSPH1, RSPH9, HYDIN, and SPEF2 cause defects of sperm flagella composition and male infertility

Abstract

Primary Ciliary Dyskinesia (PCD) is a rare genetic disorder affecting the function of motile cilia in several organ systems. In PCD, male infertility is caused by defective sperm flagella composition or deficient motile cilia function in the efferent ducts of the male reproductive system. Different PCD-associated genes encoding axonemal components involved in the regulation of ciliary and flagellar beating are also reported to cause infertility due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we performed genetic testing by next generation sequencing techniques, PCD diagnostics including immunofluorescence-, transmission electron-, and high-speed video microscopy on sperm flagella and andrological work up including semen analyses. We identified ten infertile male individuals with pathogenic variants in CCDC39 (one) and CCDC40 (two) encoding ruler proteins, RSPH1 (two) and RSPH9 (one) encoding radial spoke head proteins, and HYDIN (two) and SPEF2 (two) encoding CP-associated proteins, respectively. We demonstrate for the first time that pathogenic variants in RSPH1 and RSPH9 cause male infertility due to sperm cell dysmotility and abnormal flagellar RSPH1 and RSPH9 composition. We also provide novel evidence for MMAF in HYDIN- and RSPH1-mutant individuals. We show absence or severe reduction of CCDC39 and SPEF2 in sperm flagella of CCDC39- and CCDC40-mutant individuals and HYDIN- and SPEF2-mutant individuals, respectively. Thereby, we reveal interactions between CCDC39 and CCDC40 as well as HYDIN and SPEF2 in sperm flagella. Our findings demonstrate that immunofluorescence microscopy in sperm cells is a valuable tool to identify flagellar defects related to the axonemal ruler, radial spoke head and the central pair apparatus, thus aiding the diagnosis of male infertility. This is of particular importance to classify the pathogenicity of genetic defects, especially in cases of missense variants of unknown significance, or to interpret HYDIN variants that are confounded by the presence of the almost identical pseudogene HYDIN2.

Genetic Causes of Qualitative Sperm Defects: A Narrative Review of Clinical Evidence

“…Taken altogether, DNAI1 and DNAH5 abnormalities account for about 30% of PCD cases; however, mutations in the other thirty genes might lead to various ciliary ultrastructural defects and might explain 70% of the genetic causes of PCD [118]. As known and well-evident from our data collection, MMAF pathogenic genes are also often reported to result in PCD or PCD-like symptoms [71,111]. Those genes include genes encoding the axonemal ruler proteins CCDC39 and CCDC30 or genes encoding the proteins SPEF2 [111].…”

“…As known and well-evident from our data collection, MMAF pathogenic genes are also often reported to result in PCD or PCD-like symptoms [71,111]. Those genes include genes encoding the axonemal ruler proteins CCDC39 and CCDC30 or genes encoding the proteins SPEF2 [111]. Therefore, dividing genes causing only MMAF and genes causing PCD is not always easy and immediate, and it is possible to define MMAF and PCD as a "phenotypic continuum" [113].…”

Integrated miRNA and mRNA Sequencing Reveals the Sterility Mechanism in Hybrid Yellow Catfish Resulting from Pelteobagrus fulvidraco (♀) × Pelteobagrus vachelli (♂)

“…The genes involved in downregulation were identified as CFAP70 , TTC29 , CCDC40 , DNAAF4 , SYCE3 , STK36 , SPI1 , and EMX2 . These genes were found to be enriched in various pathways related to sex development, sperm growth, and sperm motility, such as organelle assembly, cilia movement, the development of the urogenital system, sperm production, and sperm deformity [ 47 , 48 ]. Significantly, various studies have shown that these genes contribute to reproductive development.…”

Molecular defects in primary ciliary dyskinesia are associated with agenesis of the frontal and sphenoid paranasal sinuses and chronic rhinosinusitis

“…Genetic diagnoses were established using regular gene testing including Sanger sequencing of PCD genes. In most cases, targeted PCD gene panels were used as previously described ( Raidt et al, 2022 ; Aprea et al, 2023 ). In a few cases, whole-exome sequencing was performed, and data were analyzed only for DNA variants in previously published PCD genes.…”

“…All DNA variants were evaluated according to the guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) ( Richards et al, 2015 ), and only pathogenic (class 5)/likely pathogenic variants (class 4) were included. The pathogenicity of genetic variants was determined as previously described using in silico calculation programs (e.g., Varsome) ( Raidt et al, 2022 ; Aprea et al, 2023 ). Gene nomenclature was used according to the current approved HGNC [human genome organization (HUGO); ( https://www.genenames.org/ )] ( Bruford et al, 2020 ).…”