Thomas Karaouzène’s research while affiliated with Grenoble Alpes University and other places

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Publications (49)


Fig. 2 Transmission electron microscopy analysis of sperm cells from CFAP43 and CFAP44 patients shows a severe axonemal disorganization. (Left panel) Longitudinal sections (scale bar = 500 nm) and cross-sections (scale bars, 100 nm) of sperm flagellum from control. (Central panel) Longitudinal sections (scale bars, 500 nm) and cross-sections (scale bars, 100 nm) of sperm flagellum from CFAP43-mutated patient (P 43-8). We can notice a short short tail corresponding to a cytoplasmic mass containing the different components of the flagellum, all unorganized. In CFAP43 upper cross-section, the CPC is not aligned with DMTs 3 and 8 (red line) and is rotated by 90°. We can observe the absence of central pair of microtubules in other cross-sections. (Right panel) Ultrastructure of CFAP44-mutated sperm (P 44-3) longitudinal sections (scale bars = 500 nm) show similar ultrastructure of short tail (cytoplasmic mass). In CFAP44 upper cross-section, the central pair is disassembled and displaced (red arrow). We can observe the absence of central pair of microtubules in other cross-sections. Scales bars for cross-sections = 100 nm
Coutton et al. - 2018 - Mutations in CFAP43 and CFAP44 cause male infertil
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October 2018

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Alexandra S. Vargas

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Figure 2 Location of FSIP2 mutations in the intron-exon structure and in the protein representation. Electropherograms of Sanger sequencing for all patients with FSIP2 mutations compared to the reference sequence are reported. Orange squares show the FSIP2_C domains (fibrous sheath-interacting protein 2, C-terminal, IPR031554) as predicted by InterPro server (https://www.ebi.ac.uk/interpro/). Mutations are annotated in accordance to the Human Genome Variation Society's recommendations. According to the InterPro server, three homologous domains called FSIP2_C (C-terminus of the FSIP2 protein) are present in the C terminal part of the protein. FSIP2_C domains are present in other proteins and have been found to be repeated up to 10 times but their function is not yet known (Finn et al., 2017).
Figure 3 Transmission electron microscopy analyses of sperm cells from a control subject (A, C, D) and patient FSIP2 _4 (B, E-H). (A) Longitudinal sections of sperm flagellum from control. In the midpiece (MP), outer dense fibers (ODF) are found longitudinally between the mitochondrial sheath (MS) and the axoneme (Ax). The fibrous sheath (FS) is found in the principal piece (PP). N, nucleus. (B) Longitudinal sections of sperm flagellum from patient FSIP2 _4. We notice an abnormal flagellum with the FS which appears severly disorganized and extends up to the sperm neck. ODFs remains present whereas the MS is totally absent from the midpiece likely due to an abnormal migration of the annulus. The central axoneme is not clearly visible. (C) Cross-sections of the MP in a control. The axoneme is surrounded by nine ODFs and the MS. The axoneme is composed of nine peripheral microtubules doublets (DMTs) and a central pair (CP) of singlet microtubules. (D) Cross-sections of the PP from a fertile control. The axoneme is surrounded by seven ODFs and by the FS composed of two longitudinal columns (LC) connected by circumferential ribs (CR). DA, dynein arms. (E-H) Cross-sections of sperm flagellum from patient FSIP2 _4. The FS is dysplastic, thickened and dramatically disorganized with randomly oriented fibers (E-G). When the CR seem present, we observed only one nascent and abnormal LC adjacent to doublets 3 or 8 (H). Various axonemal anomalies can be observed including the lack of the CP (E, F, H) or complete axonemal desorganization (G). Alterations of ODFs is also seen with surpernumeray (E), missing (F) or fully disorganized ODFs (G). DA are rarely visible (E, H).
Whole-exome sequencing identifies mutations in FSIP2 as a recurrent cause of multiple morphological abnormalities of the sperm flagella

August 2018

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1,033 Reads

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112 Citations

Human Reproduction

Study question: Can whole-exome sequencing (WES) of infertile patients identify new genes responsible for multiple morphological abnormalities of the sperm flagella (MMAF)? Summary answer: WES analysis of 78 infertile men with a MMAF phenotype permitted the identification of four homozygous mutations in the fibrous sheath (FS) interacting protein 2 (FSIP2) gene in four unrelated individuals. What is known already: The use of high-throughput sequencing techniques revealed that mutations in the dynein axonemal heavy chain 1 (DNAH1) gene, and in the cilia and flagella associated protein 43 (CFAP43) and 44 (CFAP44) genes account for approximately one-third of MMAF cases thus indicating that other relevant genes await identification. Study design, size, duration: This was a retrospective genetics study of 78 patients presenting a MMAF phenotype who were recruited in three fertility clinics between 2008 and 2015. Control sperm samples were obtained from normospermic donors. Allelic frequency for control subjects was derived from large public databases. Participants/materials, setting, methods: WES was performed for all 78 subjects. All identified variants were confirmed by Sanger sequencing. Relative mRNA expression levels for the selected candidate gene (FSIP2) was assessed by quantitative RT-PCR in a panel of normal human and mouse tissues. To characterize the structural and ultrastructural anomalies present in patients' sperm, immunofluorescence (IF) was performed on sperm samples from two subjects with a mutation and one control and transmission electron microscopy (TEM) analyses was performed on sperm samples from one subject with a mutation and one control. Main results and the role of chance: We identified four unrelated patients (4/78, 5.1%) with homozygous loss of function mutations in the FSIP2 gene, which encodes a protein of the sperm FS and is specifically expressed in human and mouse testis. None of these mutations were reported in control sequence databases. TEM analyses showed a complete disorganization of the FS associated with axonemal defects. IF analyses confirmed that the central-pair microtubules and the inner and outer dynein arms of the axoneme were abnormal in all four patients carrying FSIP2 mutations. Importantly, and in contrast to what was observed in patients with MMAF and mutations in other MMAF-related genes (DNAH1, CFAP43 and CFAP44), mutations in FSIP2 led to the absence of A-kinase anchoring protein 4 (AKAP4). Limitations, reasons for caution: The low number of biological samples and the absence of a reliable anti-FSIP2 antibody prevented the formal demonstration that the FSIP2 protein was absent in sperm from subjects with a FSIP2 mutation. Wider implications of the findings: Our findings indicate that FSIP2 is one of the main genes involved in MMAF syndrome. In humans, genes previously associated with a MMAF phenotype encoded axonemal-associated proteins (DNAH1, CFAP43 and CFAP44). We show here that FSIP2, a protein of the sperm FS, is also logically associated with MMAF syndrome as we showed that it is necessary for FS assembly and for the overall axonemal and flagellar biogenesis. As was suggested before in mouse and man, our results also suggest that defects in AKAP4, one of the main proteins interacting with FSIP2, would induce a MMAF phenotype. Finally, this work reinforces the demonstration that WES sequencing is a good strategy to reach a genetic diagnosis for patients with severe male infertility phenotypes. Study funding/competing interest(s): This work was supported by the following grants: the 'MAS-Flagella' project financed by the French ANR and the DGOS for the program PRTS 2014 (14-CE15) and the 'Whole genome sequencing of patients with Flagellar Growth Defects (FGD)' project financed by the Fondation Maladies Rares for the program Séquençage à haut débit 2012. The authors have no conflict of interest.




Table 1 . Semen Parameters of the Seven MMAF Subjects
Figure 2. Confirmation of the Presence of the WDR66 Deletion in Affected Individuals and Validation of Its Effect on mRNA and Protein (A) Compared with a control individual (C1), affected individuals P1 and P2 show a complete absence of sequence coverage of exons 20 and 21, as illustrated by IGV. (B) In humans, WDR66 encodes two transcripts: T1 (Ensembl: ENST00000288912.8; GenBank: NM_144668), in which 22 exons code for a 1,149 amino acid protein, and T2 (Ensembl: ENST00000397454.2; GenBank: NM_001178003), in which 18 exons code for a 941 amino acid protein. Only T1 is affected by the identified deletion (black double arrow). (C) Relative expression (2 ÀDDCt ) of both transcripts (T1 and T2) was performed on a human panel of cDNAs from two tissues expressing 9 þ 2 secondary motile axonemes (testis and lung) and two tissues expressing 9 þ 0 primary immotile axonemes (kidney and lung). GAPDH was used as a reference gene, and the T2 expression level in the brain was used as a reference sample. Data are expressed as mean 5 SD. (D) Representation of WDR66, encoded by T1. We note the presence of nine WD40repeat domains and one calcium-binding EF hand (a) in the C-terminal extremity. The first deleted amino acid is number 1,003. Deletion of exons 20 and 21 induces a frameshift. Translation of exon 22 is then incorrect for the first 25 amino acids (orange line) before a stop codon is induced 26 nucleotides after the first abnormal amino acid. The protein encoded by the deleted T1 is therefore expected to stop at amino acid 1,028. (E) Double immunofluorescence labeling of WDR66 and a-tubulin in human sperm cells. In control sperm, the yellow stain resulting from merged signals indicates the co-localization of both proteins within the flagellar axoneme. Typical sperm from affected subjects P1-P3 displayed abnormally shaped flagella with weak and punctuated WDR66 staining. The images shown are representative of many cells examined in two independent experiments. Scale bar, 10 mm.
Figure 4. TbWDR66 Is an Axoneme Protein, and Its C-Terminal Domain Is Required for Function in BSF Trypanosome (A) Control BSF cells (WT) or cells expressing TbWDR66 TY1 or TbWDR66-DCter TY1 were immunolabeled with anti-PFR (white) and anti-TY1 (red); anti-TY1 decorates the tagged proteins throughout the full length of the flagellum. Induction of RNAi for 48 hr (þRNAi) showed a decrease in fluorescence intensity of TbWDR66 TY1 labeling but not of TbWDR66-DCter TY1 labeling. Nuclei and kinetoplasts (mitochondrial genomes) were labeled with DAPI (blue). Scale bar, 5 mm. (B) Western-blot analysis of the presence of TbWDR66 TY1 and TbWDR66-DCter TY1 without (NI) and with 24, 48, and 72 hr of RNAi induction shows a decreased amount of TbWDR66 TY1 (indicated by the labeling percentage relative to the non-induced condition), whereas the amount of translated TbWDR66-DCter TY1 was not affected. Tubulin labeling was used as loading control. (C) TbWDR66 RNAi affects cell proliferation. Shown are growth curves for WT cells, and RNAi-non-induced or-induced cells expressing TbWDR66 TY1 or TbWDR66-DCter TY1. Cells were counted every 24 hr. The graph represents the cumulative number of cells per milliliter. Error bars represent the standard error from three independent experiments. (D) Electron microscopy images of thin sections of a WT cell (a) and a cell expressing TbWDR66 TY1 (b) or TbWDR66-DCter TY1 (c) and induced with RNAi for 48 hr show that the decreased amount of TbWDR66 induces severe disorganization of the axoneme and of the paraxonemal structure, which is not rescued by the presence of TbWDR66-DCter TY1. Scale bars, 50 nm.
A Homozygous Ancestral SVA-Insertion-Mediated Deletion in WDR66 Induces Multiple Morphological Abnormalities of the Sperm Flagellum and Male Infertility

August 2018

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229 Reads

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97 Citations

The American Journal of Human Genetics

Multiple morphological abnormalities of the sperm flagellum (MMAF) is a severe form of male infertility defined by the presence of a mosaic of anomalies, including short, bent, curled, thick, or absent flagella, resulting from a severe disorganization of the axoneme and of the peri-axonemal structures. Mutations in DNAH1, CFAP43, and CFAP44, three genes encoding axoneme-related proteins, have been described to account for approximately 30% of the MMAF cases reported so far. Here, we searched for pathological copy-number variants in whole-exome sequencing data from a cohort of 78 MMAF-affected subjects to identify additional genes associated with MMAF. In 7 of 78 affected individuals, we identified a homozygous deletion that removes the two penultimate exons of WDR66 (also named CFAP251), a gene coding for an axonemal protein preferentially localized in the testis and described to localize to the calmodulin- and spoke-associated complex at the base of radial spoke 3. Sequence analysis of the breakpoint region revealed in all deleted subjects the presence of a single chimeric SVA (SINE-VNTR-Alu) at the breakpoint site, suggesting that the initial deletion event was potentially mediated by an SVA insertion-recombination mechanism. Study of Trypanosoma WDR66's ortholog (TbWDR66) highlighted high sequence and structural analogy with the human protein and confirmed axonemal localization of the protein. Reproduction of the human deletion in TbWDR66 impaired flagellar movement, thus confirming WDR66 as a gene associated with the MMAF phenotype and highlighting the importance of the WDR66 C-terminal region.






Citations (15)


... In the present study, nineteen novel variants of FSIP2 and SPEF2 were identified in eleven patients with MMAF. Previous studies have reported that the frequency of biallelic FSIP2 mutations to be 2.9% (3/105) in patients with oligoasthenozoospermia [17], 5% (2/40) in Chinese men with MMAF [18], and 5.1% (4/78) in North African patients exhibiting the typical MMAF phenotype [19]. Our findings revealed a higher prevalence of FSIP2 variants (8.5%), with loss-of-function (LOF) variants accounting for 3.8% (4/106) and missense variants representing 4.7% (5/106) of cases. ...

Reference:

Novel variants of FSIP2 and SPEF2 cause varying degrees of spermatozoa damage in MMAF patients and favorable ART outcomes
Whole-exome sequencing identifies mutations in FSIP2 as a recurrent cause of multiple morphological abnormalities of the sperm flagella

Human Reproduction

... WDR12 homozygous mutation in men led to tapered-head spermatozoa [15]. In addition, the cilia-and flagella-associated protein 43 (CFAP43, also known as WDR96), CFAP44 (WDR52), CFAP251 (WDR66) and WDR63 proteins are thought to be related to the formation of sperm flagella, and deficiency of these genes in humans and mice can cause multiple morphological abnormalities of sperm flagella (MMAF) and male infertility [16][17][18][19][20]. Recently, our group has explored the function of WDR proteins in spermatogenesis. ...

A Homozygous Ancestral SVA-Insertion-Mediated Deletion in WDR66 Induces Multiple Morphological Abnormalities of the Sperm Flagellum and Male Infertility

The American Journal of Human Genetics

... Notably, PATL2 encodes an RNA-binding protein that was initially identified as a translational repressor in Xenopus oocytes [29,30], and the overexpression of PATL2 ortholog (labeled P100) represses mRNA translation and blocks oocyte meiosis I progression [30]. PATL2 loss-of-function mutations in humans lead to GV and MI arrest [22], but PATL2 knockout mice exhibit mild OMD phenotypes, mainly exhibiting early embryo arrest phenotype [31,32]. The Patl2-null mouse model revealed that PATL2 mainly functions as an adaptor protein to recruit EIF4E and CPEB1 to maintain the mRNA stability and maternal transcripts dosage in growing oocytes [31]. ...

PATL2 is a key actor of oocyte maturation whose invalidation causes infertility in women and mice

EMBO Molecular Medicine

... The CA plays a critical role in the development and regulation of motile cilia. Mutations in at least 11 CA-associated genes have been reported to cause motile ciliopathies, such as multiple morphological abnormalities of the sperm flagella (MMAF) and primary ciliary dyskinesia (PCD) [4][5][6][7][8][9][10][11][12][13][14]. PCD is a genetically and clinically heterogeneous disease, with an estimated global prevalence of approximately 1:10,000 [15], presenting with various combinations of symptoms including neonatal respiratory distress, persistent wet cough, recurrent and chronic upper and lower respiratory tract infections, bronchiectasis, otitis media with effusion, situs inversus or heterotaxy, hydrocephalus, and infertility [16]. ...

Absence of CFAP69 Causes Male Infertility due to Multiple Morphological Abnormalities of the Flagella in Human and Mouse

The American Journal of Human Genetics

... The flagellated protist Trypanosoma brucei is well known for causing sleeping sickness in Africa. In recent years, it has also emerged as a potent model organism to study cilia and flagella bringing useful insights to the comprehension of several ciliopathies [7][8][9] . Trypanosomes are fascinating organisms to study since they contain a mixture of universal features but also of unique characteristics, presumably linked to their divergent position in the eukaryotic phylogeny. ...

Mutations in CFAP43 and CFAP44 cause male infertility and flagellum defects in Trypanosoma and human

... To investigate possible infertility-related genes at common breakpoints on each chromosome and the relationship between these breakpoints and the type of infertility (pregestational or gestational), the data obtained from patients' records were compared with the literature, the results of which are shown in Table 7 Kueng et al. 1997;Maruoka et al. 1998;Ou et al. 1999;Abusheikha et al. 2001;Crackower et al. 1979;Kirby et al. 2004;Reynolds et al. 2005;Galan et al. 2006;Choi et al. 2007;Dieterich et al. 2007;Perera et al. 2007;Sc et al. 2007;Lamarca et al. 2007;Singh et al. 2008Singh et al. , 2019Li et al. 2009Li et al. , 2011Li et al. , 2022Wyatt et al. 2010;Nagai et al. 2011;Saadi et al. 2011;Zheng et al. 2012Zheng et al. , 2020Buchold and Eddy 2012;Heaydaryan et al. 2013;Hodžić et al. 2013;de Mateo and Sassone-Corsi 2014;Purandhar et al. 2014;Devi et al. 2015;Guan et al. 2015;Kwon et al. 2015;Li et al. 2012;Mateusz Kurzawski et al. 2015;Mobasheri et al. 2015;Wang et al. 2015Wang et al. , 2023Tsatsanis et al. 2015;Chirn et al. 2015;Aydos et al. 2016;Marques et al. 2016;Cai et al. 2017;Ghasemi et al. 2017;Kherraf et al. 2017;Sha et al. 2017;Regions and Genes 2018;An et al. 2018;Beurois et al. 2019;Giebler et al. 2019;Qiu et al. 2019;Yoshitaka Fujiharaa et al. 2019;Musio 2020;Liudkovska and Dziembowski 2021;Pantos et al. 2021;Sabetian et al. 2021;Tu et al. 2021;Yuan et al. 2021;Du et al. 2021;Wu et al. 2021;zur Lage P, et al. 2021zur Lage P, et al. . 2021Dong et al. 2022;Jones and Naylor 2022;Woodhouse et al. 2022;Amaral 2022;Baker et al. 2022;Akram et al. 2023;Greither et al. 2023;Gusmano et al. 2023). ...

SPINK2 deficiency causes infertility by inducing sperm defects in heterozygotes and azoospermia in homozygotes

EMBO Molecular Medicine

... In a 2014 report by the FORGE (Finding of Rare Disease Genes) Canada Consortium, WES was only successful in identifying genes in 45% of sporadic cases and 43% of recessive diseases in non-consanguineous families. WGS, where both coding and non-coding regions are sequenced, has several advantages over WES, as it generates a higher level of resolution [105], information on copy number variants (CNVs) [106,107], and epigenomic information [108]. The limited number of recent male factor infertility studies utilizing WGS that have been published are primarily case reports or studies with small cohorts [109][110][111][112][113][114][115][116]. ...

Whole-exome sequencing of familial cases of multiple morphological abnormalities of the sperm flagella (MMAF) reveals new DNAH1 mutations

Human Reproduction

... It is clear that a sperm might have genetic defects that result in subsequent failure of the fertilization process, which could affect embryonic development. Over the years, studies on PLCζ have formed the basis by which we understand the correlation between oocyte activation and the spermatic factor PLCζ, coded by the PLCZ1 gene [40]. ...

Homozygous mutation of PLCZ1 leads to defective human oocyte activation and infertility that is not rescued by the WW binding protein PAWP

Human Molecular Genetics

... X is abundantly expressed in sperm acrosomes, is secreted from activated spermatozoa during acrosome reaction, and hydrolyzes sperm membrane phospholipids to release DHA and DPA as well as LPC.24,69 Pla2g10 −/− spermatozoa show impairment of the late phase of the progesterone-induced acrosome reaction,70 and the reduced ability of Pla2g10 −/− sperm to fertilize oocytes is rescued by supplementation with DPA or LPC during in vitro fertilization.24,69 Hereafter, we focus mainly on the established immune-related roles of sPLA 2 -X in specific tissues where this enzyme is substantially expressed, while the controversial functions reported in other tissues with little or no sPLA 2 -X expression are briefly summarized at the end of this section. ...

Progesterone-induced acrosome exocytosis requires sequential involvement of calcium-independent iPLA2[beta] and group X sPLA2

Journal of Biological Chemistry

... Кодируемый белок экспрессируется в ходе спермиогенеза и остается в зрелых сперматозоидах. В течение сперматогенеза белок SUN5 находится в составе ядерной оболочки, однако в зрелых сперматозоидах локализован в имплантационной ямке HTCA [37]. ...

Dynamics of Sun5 Localization during Spermatogenesis in Wild Type and Dpy19l2 Knock-Out Mice Indicates That Sun5 Is Not Involved in Acrosome Attachment to the Nuclear Envelope