Novel Compound Heterozygous Variants in CCDC40 Associated with Primary Ciliary Dyskinesia and Multiple Morphological Abnormalities of the Sperm Flagella

Xu, Yingjie; Yang, Binyi; Cheng, Lei; Yang, Danhui; Ding, Shuizi; Lu, Chenyang; Wang, Lin; Guo, Tao; Wang, Rongchun; Luo, Hong

doi:10.2147/pgpm.s359821

Cited by 9 publications

(12 citation statements)

References 36 publications

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“…They can severely reduce male fertility, but have received less attention in previous PCD studies ( Dávila Garza and Patrizio, 2013 ; Sironen et al, 2020 ). Although >50 PCD-associated pathogenic genes have been reported, a few have been reported to be associated with asthenoteratozoospermia, such as DNAH9 , DNAH1 , ARMC4 , BRWD1 , CCDC39 , CCDC40 , DRC1 , CFAP74 , and SPEF2 ( Neesen et al, 2001 ; Sha et al, 2020 ; Tu et al, 2020 ; Chen et al, 2021 ; Gao et al, 2021 ; Guo et al, 2021 ; Lei et al, 2022 ; Xu et al, 2022 ). In most other cases, researchers have not analyzed the semen of male patients.…”

Section: Discussionmentioning

confidence: 99%

Case Report: DNAAF4 Variants Cause Primary Ciliary Dyskinesia and Infertility in Two Han Chinese Families

Guo

Yang

et al. 2022

Front. Genet.

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Background: Primary ciliary dyskinesia (PCD) is a rare genetic disorder, predominantly autosomal recessive. The dynein axonemal assembly factor 4 (DNAAF4) is mainly involved in the preassembly of multisubunit dynein protein, which is fundamental to the proper functioning of cilia and flagella. There are few reports of PCD-related pathogenic variants of DNAAF4, and almost no DNAAF4-related articles focused on sperm phenotype. Moreover, the association between DNAAF4 and scoliosis has never been reported, to the best of our knowledge.Materials and Methods: We recruited two patients with a clinical diagnosis of PCD. One came from a consanguineous and another from a non-consanguineous family. Clinical data, laboratory test results, and imaging data were analyzed. Through whole exome sequencing, immunofluorescence, electron microscopy, high-speed video microscopy analysis, and hematoxylin–eosin (HE) staining, we identified the disease-associated variants and validated the pathogenicity.Results: Proband 1 (P1, F1: II-1), a 19-year-old man, comes from a non-consanguineous family-I, and proband 2 (P2, F2: II-1), a 37-year-old woman, comes from a consanguineous family-II. Both had sinusitis, bronchiectasis, situs inversus, and scoliosis. P1 also had asthenoteratozoospermia, and P2 had an immature uterus. Two homozygous pathogenic variants in DNAAF4 (NM_130810.4), c.988C > T, p.(Arg330Trp), and DNAAF4 (NM_130810.4), c.733 C > T, p.(Arg245*), were identified through whole exome sequencing. High-speed microscopy analysis showed that most of the cilia were static in P1, with complete static of the respiratory cilia in P2. Immunofluorescence showed that the outer dynein arms (ODA) and inner dynein arms (IDA) were absent in the respiratory cilia of both probands, as well as in the sperm flagellum of P1. Transmission electron microscopy revealed the absence of ODA and IDA of respiratory cilia of P2, and HE staining showed irregular, short, absent, coiled, and bent flagella.Conclusion: Our study identified a novel variant c.733C > T, which expanded the spectrum of DNAAF4 variants. Furthermore, we linked DNAAF4 to asthenoteratozoospermia and likely scoliosis in patients with PCD. This study will contribute to a better understanding of PCD.

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Section: Discussionmentioning

confidence: 99%

Case Report: DNAAF4 Variants Cause Primary Ciliary Dyskinesia and Infertility in Two Han Chinese Families

Guo

Yang

et al. 2022

Front. Genet.

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“…A recent study found that DRC1 variants could lead to PCD and MMAF, which is the same diagnosis as the patient we reported in this study ( Lei et al, 2022 ). So far, only several genes have been covered to be related to PCD with MMAF, including SPEF2 , CFAP74 , BRWD1 , CCDC39 , CCDC40 , ARMC4 , and DRC1 ( Sha et al, 2020 ; Tu et al, 2020 ; Chen et al, 2021 ; Gao et al, 2021 ; Guo et al, 2021 ; Lei et al, 2022 ; Xu et al, 2022 ). Since the axonemal ultrastructure of respiratory cilia and sperm flagella is highly consistent, it is necessary to consider their sperm motility and morphology when diagnosing a patient with PCD.…”

Section: Discussionmentioning

confidence: 99%

“…Nasal brush biopsy samples were imaged using an upright Olympus BX53 microscope (Olympus, Tokyo, Japan) and recorded using a scientific complementary metal oxide semiconductor camera (Prime BSI, Teledyne Photometrics Inc., United States) as previously described ( Xu et al, 2022 ).…”

Section: Methodsmentioning

confidence: 99%

“…Nasal epithelial tissues and sperm were fixed in 4% paraformaldehyde. Immunofluorescence on the slides was performed as described previously ( Xu et al, 2022 ). Briefly, the slides were incubated overnight at 4°C with the primary antibodies DRC4 (HPA041311, 1:50, Sigma-Aldrich, Missouri, United States), and anti-acetylated tubulin (T7451, 1:500, Sigma-Aldrich, Missouri, United States).…”

Section: Methodsmentioning

confidence: 99%

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Case Report: Whole-Exome Sequencing-Based Copy Number Variation Analysis Identified a Novel DRC1 Homozygous Exon Deletion in a Patient With Primary Ciliary Dyskinesia

et al. 2022

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Objective: Whole-exome sequencing (WES) based copy number variation (CNV) analysis has been reported to improve the diagnostic rate in rare genetic diseases. In this study, we aim to find the disease-associated variants in a highly suspected primary ciliary dyskinesia (PCD) patient without a genetic diagnosis by routine WES analysis.Methods: We identified the CNVs using the “Exomedepth” package in an undiagnosed PCD patient with a negative result through routine WES analysis. RNA isolation, PCR amplification, and Sanger sequencing were used to confirm the variant. High-speed video microscopy analysis (HSVA) and immunofluorescence analysis were applied to detect the functional and structural deficiency of nasal cilia and sperm flagella. Papanicolaou staining was employed to characterize the morphology of sperm flagella.Results: NC_000002.11(NM_145038.5): g.26635488_26641606del, c.156-1724_244-2550del, r.156_243del, p. (Glu53Asnfs*13), a novel DRC1 homozygous CNV, was identified by WES-based CNV analysis rather than routine variants calling, in a patient from a non-consanguineous family. HSVA results showed no significant change in ciliary beating frequency but with reduced beating amplitude compared with normal control, and his spermatozoa were almost immotile. The diagnosis of multiple morphological abnormalities of the sperm flagella (MMAF) was established through sperm motility and morphology analysis. PCR amplification and Sanger sequencing confirmed the novel variant of DRC1. Immunofluorescence showed that both cilia and sperm flagella were deficient in protein expression related to the dynein regulatory complex.Conclusion: This report identifies a novel DRC1 disease-associated variant by WES-based CNV analysis from a highly suspected PCD patient with MMAF. Our findings not only expand the genetic spectrum of PCD with MMAF but suggest that in combination with CNV analysis might improve the efficiency of genetic tests.

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“…Genetic origin accounts for 35–60% of these MMAF cases. Several gene-families are identified as associated with flagella, including the AKAP family, DANI family, DNAH family, RSPH family, CCDC family, CFAP family, TTC family, and some single genes [ 1 , 9 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 ].…”

Section: Introductionmentioning

confidence: 99%

LRRC46 Accumulates at the Midpiece of Sperm Flagella and Is Essential for Spermiogenesis and Male Fertility in Mouse

Yin

et al. 2022

IJMS

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The sperm flagellum is essential for male fertility. Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. MMAF phenotypes are understood to result from pathogenic variants of genes from multiple families including AKAP, DANI, DNAH, RSPH, CCDC, CFAP, TTC, and LRRC, among others. The Leucine-rich repeat protein (LRRC) family includes two members reported to cause MMAF phenotypes: Lrrc6 and Lrrc50. Despite vigorous research towards understanding the pathogenesis of MMAF-related diseases, many genes remain unknown underlying the flagellum biogenesis. Here, we found that Leucine-rich repeat containing 46 (LRRC46) is specifically expressed in the testes of adult mice, and show that LRRC46 is essential for sperm flagellum biogenesis. Both scanning electron microscopy (SEM) and Papanicolaou staining (PS) presents that the knockout of Lrrc46 in mice resulted in typical MMAF phenotypes, including sperm with short, coiled, and irregular flagella. The male KO mice had reduced total sperm counts, impaired sperm motility, and were completely infertile. No reproductive phenotypes were detected in Lrrc46−/−female mice. Immunofluorescence (IF) assays showed that LRRC46 was present throughout the entire flagella of control sperm, albeit with evident concentration at the mid-piece. Transmission electron microscopy (TEM) demonstrated striking flagellar defects with axonemal and mitochondrial sheath malformations. About the important part of the Materials and Methods, SEM and PS were used to observe the typical MMAF-related irregular flagella morphological phenotypes, TEM was used to further inspect the sperm flagellum defects in ultrastructure, and IF was chosen to confirm the location of protein. Our study suggests that LRRC46 is an essential protein for sperm flagellum biogenesis, and its mutations might be associated with MMAF that causes male infertility. Thus, our study provides insights for understanding developmental processes underlying sperm flagellum formation and contribute to further observe the pathogenic genes that cause male infertility.

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Novel Compound Heterozygous Variants in CCDC40 Associated with Primary Ciliary Dyskinesia and Multiple Morphological Abnormalities of the Sperm Flagella

Cited by 9 publications

References 36 publications

Case Report: DNAAF4 Variants Cause Primary Ciliary Dyskinesia and Infertility in Two Han Chinese Families

Case Report: DNAAF4 Variants Cause Primary Ciliary Dyskinesia and Infertility in Two Han Chinese Families

Case Report: Whole-Exome Sequencing-Based Copy Number Variation Analysis Identified a Novel DRC1 Homozygous Exon Deletion in a Patient With Primary Ciliary Dyskinesia

LRRC46 Accumulates at the Midpiece of Sperm Flagella and Is Essential for Spermiogenesis and Male Fertility in Mouse

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