Novel mutations in PLCZ1 cause male infertility due to fertilization failure or poor fertilization

2020 ◽  
Vol 35 (2) ◽  
pp. 472-481 ◽  
Author(s):  
Zheng Yan ◽  
Yong Fan ◽  
Fei Wang ◽  
Zhiguang Yan ◽  
Menghui Li ◽  
...  
Keyword(s):  
Male Infertility ◽  
Conflicts Of Interest ◽  
Missense Mutations ◽  
Oocyte Activation ◽  
Fertilization Failure ◽  
Novel Mutations ◽  
Primary Infertility ◽  
Human Fertilization ◽  
Poor Fertilization

Abstract STUDY QUESTION Do sperm-specific phospholipase C zeta (PLCZ1) mutations account for male infertility due to fertilization failure? SUMMARY ANSWER Six novel mutations and one reported mutation in PLCZ1 were identified in five of 14 independent families characterized by fertilization failure or poor fertilization, suggesting that these mutations may be responsible for fertilization failure in men exhibiting primary infertility. WHAT IS KNOWN ALREADY PLCZ1 is essential for the induction of intracellular calcium (Ca2+) oscillations and the initiation of oocyte activation during mammalian fertilization. However, genetic evidence linking PLCZ1 mutations with male infertility remains limited. STUDY DESIGN, SIZE, DURATION Fourteen unrelated primary infertility patients were recruited into this study from January 2016 to December 2018; the patients exhibited total fertilization failure or poor fertilization, as evidenced by ICSI and sperm-related oocyte activation deficiencies identified in mouse oocyte activation assays. PARTICIPANTS/MATERIALS, SETTING, METHODS Genomic DNA samples were extracted from the peripheral blood of patients. The whole exons of PLCZ1 were sequenced by Sanger sequencing. The PLCZ1 sequences were aligned by CodonCode software to identify rare variants. The ExAC database was used to search for the frequency of corresponding mutations. The pathogenicity of identified variants and their possible effects on the protein were assessed in silico. PLCZ1 protein levels in semen samples were evaluated by western blotting. Oocyte activation ability was assessed by the injection of wild-type and mutant PLCZ1 cRNAs into human mature metaphase II (MII) oocytes in vitro. MAIN RESULTS AND THE ROLE OF CHANCE We identified six novel mutations and one reported mutation in PLCZ1 among five affected individuals. In addition to four novel missense mutations, two new types of genetic variants were identified, including one in-frame deletion and one splicing mutation. Western blot analysis revealed that PLCZ1 protein expression was not observed in the semen samples from the five affected patients. Microinjection with the PLCZ1 cRNA variants was performed, and a significant decrease in the percentage of pronuclei was observed for four novel missense mutations and one novel in-frame deletion mutation, suggesting that these mutations have a deleterious influence on protein function. By artificial oocyte activation treatment, the fertilization failure phenotypes of four affected patients were successfully rescued and three healthy babies were delivered. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION We screened only the whole exons of PLCZ1. Additional possible mutations in the non-coding region of PLCZ1 should be further studied. WIDER IMPLICATIONS OF THE FINDINGS Our study not only further confirms the important role of PLCZ1 in human fertilization but also expands the mutational spectrum of PLCZ1 associated with male infertility, which provides a basis for assessing genetic variation in PLCZ1 as a potential diagnostic marker for infertile men suffering from fertilization failure. STUDY FUNDING/COMPETING INTEREST(S) This research was supported by the National Natural Foundation of China (81 571 486 and 81 771 649). All authors have no conflicts of interest to declare.

scholarly journals A novel homozygous mutation of phospholipase C zeta leading to defective human oocyte activation and fertilization failure

2020 ◽  
Vol 35 (4) ◽  
pp. 977-985 ◽  
Author(s):  
Peng Yuan ◽  
Cen Yang ◽  
Yixin Ren ◽  
Jie Yan ◽  
Yanli Nie ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Human Oocyte ◽  
Homozygous Mutation ◽  
Whole Genome ◽  
C2 Domain ◽  
Oocyte Activation ◽  
Fertilization Failure ◽  
Human Fertilization

Abstract STUDY QUESTION Is a novel homozygous phospholipase C zeta (PLCζ), c.1658 G>C; p. R553P mutation in the C2 domain associated with the outcomes of recurrent fertilization failure after ICSI? SUMMARY ANSWER PLCζ, c.1658 G>C led to defective human oocyte activation and fertilization failure, while this mutation in the C2 domain of PLCζ did not compromise concentration, motility and chromosome ploidy of sperm. WHAT IS KNOWN ALREADY Sperm-specific PLCζ is now widely considered to be the physiological stimulus that evokes intracellular calcium (Ca2+) oscillations, which are essential for egg activation during mammalian fertilization. Thus far, few genetic studies have shown that different point mutations in the PLCζ gene are associated with male infertility. STUDY DESIGN, SIZE, DURATION This was a basic medical research to assess pathogenicity for novel mutation in the C2 domain of PLCζ during human fertilization. PARTICIPANTS/MATERIALS, SETTING, METHODS Single-cell omics were applied to analyze the DNA methylation state of the fertilization failure oocytes and the ploidy of the patient’s sperm. Whole genome sequencing data for the patient were analyzed for mutations in PLCζ. Sanger sequencing confirmed the presence of a rare variant, and then the mutant and wild-type PLCζ mRNA were injected to observe oocyte activation. MAIN RESULTS AND THE ROLE OF CHANCE The fertilization failure oocytes (n = 4) were triploid and lacking proper DNA demethylation. The whole genome sequencing analysis revealed a novel missense homozygous mutation in PLCζ, c.1658 G>C; p. R553P, which leads to the conversion of arginine 553 to proline. This point mutation does not affect the production of the corresponding protein in sperm. However, microinjection of the mRNA transcribed from the PLCζ R553P mutation gene failed to trigger oocyte activation and the subsequent embryo development. LIMITATIONS, REASONS FOR CAUTION Only one patient with PLCζ mutations was available because of its rare incidence. WIDER IMPLICATIONS OF THE FINDINGS Notably, we discovered a novel homozygous mutation in PLCζ, which results in an abnormal conformation at the C2 domain of the PLCζ protein. Our findings indicate an essential role of PLCζ in human fertilization and the requirement of a normal structure of C2 domain in PLCζ-mediated physiological function. STUDY FUNDING/COMPETING INTEREST(S) This project is funded by the National Natural Science Foundation of China (31571544, 31871482, 31871447) and National Key Research and Development Program (2018YFC1004000, 2017YFA0103801). All authors declared no competing interests. TRIAL REGISTRATION NUMBER Not applicable.

scholarly journals The identification of novel mutations in PLCZ1 responsible for human fertilization failure and a therapeutic intervention by artificial oocyte activation

2020 ◽  
Vol 26 (2) ◽  
pp. 80-87 ◽  
Author(s):  
Jian Mu ◽  
Zhihua Zhang ◽  
Ling Wu ◽  
Jing Fu ◽  
Biaobang Chen ◽  
...  
Keyword(s):  
Human Reproduction ◽  
Missense Mutations ◽  
Oocyte Activation ◽  
Fertilization Failure ◽  
Gene Encoding ◽  
Human Fertilization ◽  
Molecular Events ◽  
Whole Exome ◽  
Artificial Oocyte Activation ◽  
Biallelic Mutations

Abstract Fertilization involves a series of molecular events immediately following egg–sperm fusion; Ca2+ oscillations are the earliest signaling event, and they initiate the downstream reactions including pronucleus formation. Successful human reproduction requires normal fertilization. In clinical IVF or ICSI attempts, some infertile couples suffer from recurrent fertilization failure. However, the genetic reasons for fertilization failure are largely unknown. Here, we recruited several couples diagnosed with fertilization failure even though their gametes are morphologically normal. Through whole-exome sequencing and Sanger sequencing, we identified biallelic mutations in gene-encoding phospholipase C zeta 1 (PLCZ1) in four independent males in couples diagnosed with fertilization failure. Western blotting showed that missense mutations decreased the level of PLCZ1 and that nonsense or frameshift mutations resulted in undetectable or truncated proteins. Expression of these mutations in mice significantly reduced the levels of oocyte activation. Artificial oocyte activation in patient oocytes could rescue the phenotype of fertilization failure and help establish pregnancy and lead to live birth. Our findings expand the spectrum of PLCZ1 mutations that are responsible for human fertilization failure and provide a potentially feasible therapeutic treatment for these patients.

P–568 Homozygous Pathogenic Variants in ACTL9 Cause Fertilization Failure and Male Infertility in Human and Mouse

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
J Dai ◽  
T Zhang ◽  
J Guo ◽  
Q Zhou ◽  
Y Gu ◽  
...  
Keyword(s):  
Mouse Model ◽  
Male Infertility ◽  
Therapeutic Option ◽  
Calcium Ionophore ◽  
Calcium Oscillations ◽  
Oocyte Activation ◽  
Fertilization Failure ◽  
Pathogenic Variants ◽  
Male Factors ◽  
Poor Fertilization

Abstract Study question What are the other male factors that cause total fertilization failure (TFF) excepting for variants in PLCZ1? Summary answer Homozygous variants in ACTL9 (actin like 9) cause abnormal localization of PLCζ in a loosened perinuclear theca (PT) structure and leads to TFF. What is known already In previous studies, investigators have reported that the female factors in TFF after intracytoplasmic sperm injection (ICSI) include pathogenic variants in WEE2, TLE6, and TUBB8, whereas for male factors, pathogenic variants in PLCZ1 were reported to be the primary cause of TFF, which account for approximately 30% of couples with male factors in TFF excluding globozoospermia. Most recently, it was reported that pathogenic variants in ACTL7A led to reduced expression and abnormal localization of PLCζ, thereby identifying this genetic variant as a potential cause of TFF. Study design, size, duration Fifty-four infertile couples with TFF or poor fertilization (fertilization rate of < 20%) at the Reproductive and Genetic Hospital of CITIC-Xiangya during January 2014 to June 2020 were recruited into this study. Participants/materials, setting, methods Male factors were identified in (MOAT). WES analysis was used to analyze the genetic factors of individuals with male factors. Sperm morphological study was conducted by H&E staining and TEM. Immunostaining of PLCζ was used to analyze the status of sperm-borne activation factor. A knock-in mouse model was generated by CRISPER-Cas9 technology. Sperm from homozygous Actl9 variant mice were analyzed by TEM and ICSI. ICSI with AOA was performed in couples with ACTL9 variants. Main results and the role of chance A total of 54 couples with TFF or poor fertilization were screened, with 21 couples determined to have a male infertility factor by MOAT. Whole-exome sequencing of these 21 male individuals identified three homozygous pathogenic variants in ACTL9 in three individuals. ACTL9 variations led to abnormal ultrastructure of the PT, with PLCζ absent in the head and present in the neck of the mutant sperm, which contributed to failed normal calcium oscillations in oocytes and subsequent TFF. The key roles of ACTL9 in the PT structure and TFF after ICSI were further confirmed in Actl9-mutated mouse model. Furthermore, assisted oocyte activation by calcium ionophore exposure successfully overcame TFF and achieved live births in a couple with an ACTL9 variant. Limitations, reasons for caution The mechanism of how ACTL9 regulate PLCζ remains unknown. Wider implications of the findings: It provided a genetic marker and a therapeutic option for individuals who have undergone ICSI without successful fertilization. Trial registration number not applioable

scholarly journals Detection and Analysis of Gene Mutations in Patients with Glanzmann's Thrombasthenia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2373-2373
Author(s):  
Boyang Sun ◽  
Donglei Zhang ◽  
Huiyuan Li ◽  
Xueqing Dou ◽  
Renchi Yang
Keyword(s):  
Clinical Laboratory ◽  
Conflicts Of Interest ◽  
Gene Mutations ◽  
Adenosine Diphosphate ◽  
Severe Bleeding ◽  
Missense Mutations ◽  
Novel Mutations ◽  
Genetic Characteristics ◽  
Pathogenic Variants ◽  
Standards And Guidelines

Background: Glanzmann thrombasthenia (GT) is a rare inherited disorder of bleeding, and it is characterized by the impaired or absent platelet aggregation to multiple physiologic agonists such as collagen, adenosine diphosphate (ADP), arachidonic acid(AA), but normal reaction to ristocetin. There is qualitative or quantitative defect in platelet integrin αIIbβ3(GPIIb/IIIa). Pathogenic variants of either αIIb or β3 unit could cause GT. The database of gene mutations is continuously updated on the Internet (http://www.hgmd.org); it totally lists 236 variants of ITGA2B gene and 170 variants of ITGB3 gene. Aim: To characterize the clinical manifestation and molecular basis of GT patients in China, and update the pathogenic variants database. Method: Clinical features are evaluated in 104 patients with GT. New generation sequencing was performed with a custom designed panel for the bleeding and platelet disease involving 76 genes, while ITGA2B and ITGB3 were enrolled. Result: The initial bleeding occurred before 1 age in most patients. Incidence of consanguinity is 12.5%. Symptoms lessened with age in about 30% patients. Female patients suffered more severe bleeding than male patients. Fifty different mutations were detected, among which 15 were novel. Most patients were compound heterozygotes and most mutations detected were missense mutations. Among 15 novel mutations, there were 7 missense mutations, 2 nonsense mutations, 2 splicing mutations, 4 frameshift mutations. Pathogenicity of all novel mutations were evaluated according to the standards and guidelines of ACMG. All variants detected were pathogenic or likely pathogenic. Furthermore, c.1750C>T [p.R584X] and c.2333A>C [p.Q778P] in ITGA2B were detected in 10 and 16 unrelated families, strongly suggesting a founder effect. Conclusion: Our study reports the largest cohort of GT in China, describing the clinical, laboratory and genetic characteristics of 104 patients. We found 15 novel pathogenic mutations in ITGA2B and ITGB3 causing GT. Theses novel findings expand the GT mutation spectrum. Disclosures No relevant conflicts of interest to declare.

scholarly journals A de novo paradigm for male infertility

2021 ◽  
Author(s):  
MS Oud ◽  
RM Smits ◽  
HE Smith ◽  
FK Mastrorosa ◽  
GS Holt ◽  
...  
Keyword(s):  
Male Infertility ◽  
De Novo ◽  
P Value ◽  
Missense Mutations ◽  
Loss Of Function ◽  
De Novo Mutations ◽  
Systematic Analysis ◽  
Significant Enrichment

IntroductionDe novo mutations (DNMs) are known to play a prominent role in sporadic disorders with reduced fitness1. We hypothesize that DNMs play an important role in male infertility and explain a significant fraction of the genetic causes of this understudied disorder. To test this hypothesis, we performed trio-based exome-sequencing in a unique cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare protein altering DNMs were classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of Loss-of-Function (LoF) DNMs in LoF-intolerant genes (p-value=1.00×10-5) as well as predicted pathogenic missense DNMs in missense-intolerant genes (p-value=5.01×10-4). One DNM gene identified, RBM5, is an essential regulator of male germ cell pre-mRNA splicing2. In a follow-up study, 5 rare pathogenic missense mutations affecting this gene were observed in a cohort of 2,279 infertile patients, with no such mutations found in a cohort of 5,784 fertile men (p-value=0.009). Our results provide the first evidence for the role of DNMs in severe male infertility and point to many new candidate genes affecting fertility.

scholarly journals Sperm-oocyte interplay: an overview of spermatozoon’s role in oocyte activation and current perspectives in diagnosis and fertility treatment

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mohammad Ishraq Zafar ◽  
Shi Lu ◽  
Honggang Li
Keyword(s):  
Diagnostic Tests ◽  
Scientific Information ◽  
Male Gamete ◽  
Fertility Treatment ◽  
Normal Embryo ◽  
Oocyte Activation ◽  
Fertilization Failure ◽  
Sperm Selection ◽  
Reproductive Techniques

AbstractThe fertilizing spermatozoon is a highly specialized cell that selects from millions along the female tract until the oocyte. The paternal components influence the oocyte activation during fertilization and are fundamental for normal embryo development; however, the sperm-oocyte interplay is in a continuous debate. This review aims to analyze the available scientific information related to the role of the male gamete in the oocyte activation during fertilization, the process of the interaction of sperm factors with oocyte machinery, and the implications of any alterations in this interplay, as well as the advances and limitations of the reproductive techniques and diagnostic tests. At present, both PLCζ and PAWP are the main candidates as oocyte activated factors during fertilization. While PLCζ mechanism is via IP3, how PAWP activates the oocyte still no clear, and these findings are important to study and treat fertilization failure due to oocyte activation, especially when one of the causes is the deficiency of PLCζ in the sperm. However, no diagnostic test has been developed to establish the amount of PLCζ, the protocol to treat this type of pathologies is broad, including treatment with ionophores, sperm selection improvement, and microinjection with PLCζ protein or RNA.

scholarly journals Molecular Biology of Spermatogenesis: Novel Targets of Apparently Idiopathic Male Infertility

2020 ◽  
Vol 21 (5) ◽  
pp. 1728 ◽  
Author(s):  
Rossella Cannarella ◽  
Rosita A. Condorelli ◽  
Laura M. Mongioì ◽  
Sandro La Vignera ◽  
Aldo E. Calogero
Keyword(s):  
Molecular Biology ◽  
Male Infertility ◽  
Current Evidence ◽  
Idiopathic Male Infertility ◽  
Oocyte Fertilization ◽  
Human Fertilization ◽  
Sperm Dna ◽  
Infertile Couples ◽  
Insight Into

Male infertility affects half of infertile couples and, currently, a relevant percentage of cases of male infertility is considered as idiopathic. Although the male contribution to human fertilization has traditionally been restricted to sperm DNA, current evidence suggest that a relevant number of sperm transcripts and proteins are involved in acrosome reactions, sperm‒oocyte fusion and, once released into the oocyte, embryo growth and development. The aim of this review is to provide updated and comprehensive insight into the molecular biology of spermatogenesis, including evidence on spermatogenetic failure and underlining the role of the sperm-carried molecular factors involved in oocyte fertilization and embryo growth. This represents the first step in the identification of new possible diagnostic and, possibly, therapeutic markers in the field of apparently idiopathic male infertility.

scholarly journals Identification of New BMP6 Pro-Peptide Mutations in Patients with Unexplained Iron-Overload

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 264-264
Author(s):  
Chiara Piubelli ◽  
Annalisa Castagna ◽  
Giacomo Marchi ◽  
Monica Rizzi ◽  
Fabiana Busti ◽  
...  
Keyword(s):  
Iron Overload ◽  
Conflicts Of Interest ◽  
Late Onset ◽  
Hereditary Hemochromatosis ◽  
3D Structure ◽  
Potential Candidate ◽  
Missense Mutations ◽  
Tertiary Referral Centre ◽  
Bioinformatic Tools

Abstract Background: Hereditary Hemochromatosis (HH) is a genetically heterogeneous disorder caused by mutations in at least 5 different genes (HFE, HJV, TFR2, SLC40A1, and HAMP) involved in the production and function of the liver hormone hepcidin, a key regulator of iron metabolism. Nevertheless, patients with a HH-like phenotype that remains unexplained, despite extensive sequencing of the known genes, are not infrequently seen at referral centres, implicating the role of still unknown genetic factors. A compelling candidate is Bone Morphogenetic Protein 6 (BMP6), a member of TGFb superfamily, whose expression is stimulated by increased iron stores in the liver. BMP6 acts as a major activator of the BMP-SMAD signalling pathway, ultimately leading to the upregulation of hepcidin gene transcription. Indeed, early this year French Authors have described 3 heterozygous missense mutations in BMP6 (p.Pro95Ser, p.Leu96Pro, and p.Gln113Glu) in 6 unrelated patients with mild to moderate, late onset, unexplained iron overload (Daher R, Gastroenterology 2016). Methods: we recently updated our next generation sequencing (NGS)-based second level genetic test for the molecular diagnosis of non-HFE HH (Badar S, Am J Hematol 2016), by adding a number of novel potential candidate genes, including BMP6, to the panel of the 5 known HH genes. This test was applied to 38 patients evaluated at our tertiary referral centre for iron disorders, because of an unexplained iron overload phenotype. Results: we found 3 heterozygous missense mutations in BMP6 gene in 4 patients with unexplained, late-onset, iron overload, from 3 different families. Their relevant clinical data are summarized into Table 1. Of note, 1 mutation (p.Leu96Pro) was the same recently described by Daher et al. and proven to be functional. The other two mutations (p.Glu112Gln, p.Arg257His) were novel, predicted damaging by bioinformatic tools, and both located in the pro-peptide domain, known to be crucial for appropriate BMP6 processing and secretion. They were further studied by in silico modelling, based on the available 3D structure of the TGFb, which also resulted to be consistent with their pathogenetic role. Conclusions: to the best of our knowledge, our results provide the first independent confirmation of the likely causal role of BMP6 mutations in late onset, moderate iron overload phenotype, unrelated to mutations in the established 5 HH genes. Disclosures No relevant conflicts of interest to declare.

scholarly journals A de novo paradigm for male infertility

2021 ◽  
Author(s):  
Joris Veltman ◽  
Manon Oud ◽  
Roos Smits ◽  
Hannah Smith ◽  
Francesco Mastrorosa ◽  
...  
Keyword(s):  
Male Infertility ◽  
De Novo ◽  
P Value ◽  
Missense Mutations ◽  
Loss Of Function ◽  
De Novo Mutations ◽  
Systematic Analysis ◽  
Significant Enrichment

Abstract De novo mutations (DNMs) are known to play a prominent role in many sporadic disorders with reduced fitness. We hypothesize that DNMs play an important role in male infertility and explain a significant fraction of the genetic causes of this understudied disorder. We performed a trio-based exome-sequencing study in a unique cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare protein altering DNMs were classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of Loss-of-Function (LoF) DNMs in LoF-intolerant genes (p-value=1.00x10-5) as well as predicted pathogenic missense DNMs in missense-intolerant genes (p-value=5.01x10-4). One DNM gene identified, RBM5, is an essential regulator of male germ cell pre-mRNA splicing. In a follow-up study, 5 rare pathogenic missense mutations affecting this gene were observed in a cohort of 2,279 infertile patients, with no such mutations found in a cohort of 5,784 fertile men (p-value=0.009). Our results provide the first evidence for the role of DNMs in severe male infertility and point to many new candidate genes affecting fertility.

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