A novel homozygous mutation in ACTL7A leads to male infertility.

Male infertility, a global public health problem, exhibits complex pathogenic causes and genetic factors deserve further discovery and study. We identified a novel homozygous missense mutation c.224A > C (p.D75A) in ACTL7A gene in two infertile brothers with teratozoospermia by whole-exome sequencing (WES). In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) showed fertilization failure of the two affected couples. The three-dimensional (3D) models showed that a small section of α-helix transformed into random coil in the mutant ACTL7A protein and mutant amino acid lacked a hydrogen bond with Ser170 amino acid. Immunofluorescence revealed that ACTL7A protein was degraded in sperms of patients. Transmission electron microscopy (TEM) analysis of sperms from the infertile patients showed that the irregular perinuclear theca (PT) and acrosomal ultrastructural defects. Furthermore, ACTL7A mutation caused abnormal localization and reduced the expression of PLCZ1 in sperms of the patients, which may be the key reasons for the fertilization failure after ICSI. Our findings expand the spectrum of ACTL7A mutations and provide novel theoretical basis for genetic counseling.

Actl7a deficiency in mice leads to male infertility and fertilization failure.

Mutations in the Actl7a gene have been reported to lead to male infertility; however, the detailed mechanism of this phenomenon remains unknown. In this study, we constructed Actl7a gene knockout (KO) mice and found that Actl7a deficiency led to malformed formation of sperm acrosomes, male infertility, fertilization failure during in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), and reduced sperm-zona pellucida (ZP) binding ability. Moreover, we found that the localization of the zona pellucida binding protein (ZPBP) was altered in the sperm of Actl7a homozygous KO male mice, which may affect the sperm-zona pellucida binding ability. ACTL7A and ZPBP could form complex, which may be involved in acrosomal formation. Further studies found that localization and expression of the PLCZ1 protein were abnormal in misshapen sperm, leading to reduced calcium oscillations in oocytes. Herein, we provide more detailed mechanisms underlining Actl7a deficiency and male infertility.

Two novel STAT1 mutations cause Mendelian susceptibility to mycobacterial disease.

Mendelian susceptibility to mycobacterial disease (MSMD) is a rare monogenetic disease, which is characterized by susceptibility to some weakly virulent mycobacteria. Here, we explored the pathogenic genes and molecular mechanisms of MSMD patients. We recruited three patients diagnosed with MSMD from two families. Two novel mutations (c.1228A > G, p.K410E and c.2071A > G, p.M691V) in STAT1 gene were identified from two families. The translocation of K410E mutant STAT1 protein into nucleus was not affected. The binding ability between gamma-activating sequence (GAS) and K410E mutant STAT1 protein was significantly reduced, which will reduce the interaction between STAT1 protein with the promoters of target genes. The M691V mutant STAT1 protein cannot translocate into the nucleus after IFN-γ stimulation, which will affect the STAT1 protein form gamma-activating factors (GAF) and bind the GAS in the promoter region of downstream target genes. Taken together, our results showed that the mutation of K410E led to impaired binding of STAT1 to target DNA, and the mutation of M691V prevented the transport of STAT1 into the nucleus, which led to MSMD. Together, we identified two novel mutations (c.1228A > G, p.K410E and c.2071A > G, p.M691V) in STAT1 gene in MSMD patients, and deciphered the molecular mechanism of MSMD caused by STAT1 mutations.

A novel microdeletion upstream of HOXD13 in a Chinese family with synpolydactyly.

Synpolydactyly (SPD) is a digital malformation with the typical clinical phenotype of the webbing of 3/4 fingers and/or 4/5 toes, and combined with polydactyly. In this study, we investigated a Chinese family with SPD and genetic analysis found that all of the affected individuals in the family carry a heterozygous 11,451 bp microdeletion at chr2:176933872-176945322 (GRCh37), which is located upstream of HOXD13 gene, the known disease gene for SPD1. All the affected individuals in the family carry the heterozygous deletion variant, and the variant co-segregated with SPD in the family. Thus, we speculate that the 11,451 bp microdeletion is the disease-causing variant in the family. To date, the microdeletion associating with SPD1 which we identified is the smallest deletion upstream of the HOXD13 gene and not altering the sequence of the HOXD13 gene.

Novel mutations in ZP2 and ZP3 cause female infertility in three patients.

PURPOSE: The aim of this study was to identify the disease-causing mutations found in three infertile female patients who were diagnosed with abnormal zona pellucida (ZP) and empty follicle syndrome (EFS). METHODS: We performed whole-exome sequencing and Sanger sequencing to identify and verify the disease-causing mutations. Additionally, we performed Western blotting and mini-gene splicing assay to assess the effects of the mutations. RESULTS: We identified two novel compound heterozygous mutations in the ZP2 gene, a patient with an abnormal ZP carrying a novel compound heterozygous mutation (c.1695-2A>G and c.1831G>T, p.V611F) and a patient with EFS carrying a novel compound heterozygous mutation (c.1695-2A>G and c.1924 C>T, p.R642*). Furthermore, we identified a patient with typical abnormal ZP carrying a novel heterozygous mutation (c.400G>T, p.A134S) in the ZP3 gene. The splice site mutation (c.1695-2A>G) can cause abnormal pre-mRNA splicing that inserts an extra sequence of 61 bp in the mRNA of ZP2, and the missense mutation (c.1831G>T) can cause a decrease of ZP2 protein in HEK293 cells. CONCLUSION: We identified three novel mutations in the ZP2 gene and the ZP3 gene in three Chinese female patients with infertility. Our study expands the spectrum of ZP gene mutations and phenotypes and thus is beneficial in the genetic diagnosis of infertility in females.

A novel mutation in ZP3 causes empty follicle syndrome and abnormal zona pellucida formation.

PURPOSE: To identify disease-causing genes involved in female infertility. METHODS: Whole-exome sequencing and Sanger DNA sequencing were used to identify the mutations in disease-causing genes. We performed subcellular protein localization, western immunoblotting analysis, and co-immunoprecipitation analysis to evaluate the effects of the mutation. RESULTS: We investigated 17 families with female infertility. Whole-exome and Sanger DNA sequencing were used to characterize the disease gene in the patients, and we identified a novel heterozygous mutation (p.Ser173Cys, c.518C > G) in the ZP3 gene in a patient with empty follicle syndrome. When we performed co-immunoprecipitation analysis, we found that the S173C mutation affected interactions between ZP3 and ZP2. CONCLUSIONS: We identified a novel mutation in the ZP3 gene in a Chinese family with female infertility. Our findings thus expand the mutational and phenotypical spectrum of the ZP3 gene, and they will be helpful in precisely diagnosing this aspect of female infertility.

Deficiency of SCAMP5 leads to pediatric epilepsy and dysregulation of neurotransmitter release in the brain.

Secretory carrier membrane proteins (SCAMPs) play an important role in exocytosis in animals, but the precise function of SCAMPs in human disease is unknown. In this study, we identified a homozygous mutation, SCAMP5 R91W, in a Chinese consanguineous family with pediatric epilepsy and juvenile Parkinson's disease. Scamp5 R91W mutant knock-in mice showed typical early-onset epilepsy similar to that in humans. Single-neuron electrophysiological recordings showed that the R91W mutation significantly increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) at a resting state and also increased the amplitude of evoked EPSCs. The R91W mutation affected the interaction between SCAMP5 and synaptotagmin 1 and may affect the function of the SNARE complex, the machinery required for vesicular trafficking and neurotransmitter release. Our work shows that dysfunction of SCAMP5 shifted the excitation/inhibition balance of the neuronal network in the brain, and the deficiency of SCAMP5 leads to pediatric epilepsy.

Novel compound heterozygous mutations in WEE2 causes female infertility and fertilization failure.

PURPOSE: To identify the disease-causing gene in a family with female infertility and fertilization failure. METHODS: Whole-exome sequencing and Sanger sequencing were used to identify the disease-causing gene in a female with infertility and fertilization failure. Subcellular localization and western blot analysis were used to check the effect of mutations. RESULTS: We identified novel compound heterozygous mutations c.598C>T (p.Arg200Ter) and c.1319G>C (p.Trp440Ser) in WEE2 gene in a female with infertility and fertilization failure. The p.Arg200Ter mutant WEE2 gene produce truncated protein and mainly located in the nucleus, the same as the wild protein, while the p.Trp440Ser mutant WEE2 proteins are located in the nucleus and cytoplasm and the expression level of p.Trp440Ser mutant WEE2 protein is reduced significantly compared with that of wild-type WEE2. CONCLUSIONS: We discovered novel compound heterozygous mutations c.598C>T (p.Arg200Ter) and c.1319G>C (p.Trp440Ser) in WEE2 gene in a female whose oocytes could not form pronucleus after intracytoplasmic sperm injection (ICSI). Moreover, mutations in WEE2 gene affect the normal function of WEE2 proteins and cause fertilization failure.

Loss of function variant in CIP2A associated with female infertility with early embryonic arrest and fragmentation.

BACKGROUND: Early embryonic arrest and fragmentation (EEAF) is a common cause of female infertility, but the genetic causes remain to be largely unknown. CIP2A encodes the cellular inhibitor of PP2A, playing a crucial role in mitosis and mouse oocyte meiosis. METHODS: Exome sequencing and Sanger sequencing were performed to identify candidate causative genes in patients with EEAF. The pathogenicity of the CIP2A variant was assessed and confirmed in cultured cell lines and human oocytes through Western blotting, semi-quantitative RT-PCR, TUNEL staining, and fluorescence localization analysis. FINDINGS: We identified CIP2A (c.1510C > T, p.L504F) as a novel disease-causing gene in human EEAF from a consanguineous family. L504 is highly conserved throughout evolution. The CIP2A variant (c.1510C > T, p.L504F) reduced the expression level of the mutant CIP2A protein, leading to the abnormal aggregation of mutant CIP2A protein and cell apoptosis. Abnormal aggregation of CIP2A protein and chromosomal dispersion occurred in the patient's oocytes and early embryos. We further replicated the patient phenotype by knockdown CIP2A in human oocytes. Additionally, CIP2A deficiency resulted in decreased levels of phosphorylated ERK1/2. INTERPRETATION: We first found that the CIP2A loss-of-function variant associate with female infertility characterized by EEAF. Our findings suggest the uniqueness and importance of CIP2A gene in human oocyte and early embryo development. FUNDING: This work was supported by National Key Research and Development Program of China (2023YFC2706302), the National Natural Science Foundation of China (81000079, 81170165, and 81870959), the HUST Academic Frontier Youth Team (2016QYTD02), and the Key Research of Huazhong University of Science and Technology, Tongji Hospital (2022A20).

A Novel Homozygous Nonsense Mutation in ZP1 Causes Female Infertility due to Empty Follicle Syndrome.

ZP1 is a critical glycoprotein in the formation of the zona pellucida. It plays an indispensable role in the maturation of oocytes. To identify the causative gene of empty follicle syndrome (EFS) in a patient from a consanguineous family, whole-exome sequencing was performed in the proband. We identified a novel homozygous nonsense mutation c.1260C > G (p. Tyr420X) in the ZP1 gene from two primary infertile patients. Western blot showed that Y420X mutation in ZP1 gene produced a truncated protein. However, the mutation had no significant effect on subcellular localization of the mutant protein. Our findings confirmed the important role of the ZP1 gene in human female reproduction, enriched the mutation spectrums of ZP1 gene, and expanded its applications in the clinical and molecular diagnoses of EFS.

Highly Dispersed Antimony-Bismuth Alloy Encapsulated in Carbon Nanofibers for Ultrastable K-Ion Batteries.

Antimony-based alloys have appealed to an ever-increasing interest for potassium ion storage due to their high theoretical capacity and safe voltage. However, sluggish kinetics and the large radius of K+ lead to limited rate performance and severe capacity fading. In this Letter, highly dispersed antimony-bismuth alloy nanoparticles confined in carbon fibers are fabricated through an electrospinning technology followed by heat treatment. The BiSb nanoparticles are uniformly confined into the carbon fibers, which facilitate rapid electron transport and inhibit the volume change during cycling owing to the synergistic effect of the BiSb alloy and carbon confinement engineering. Furthermore, the effect of a potassium bis(fluorosulfonyl)imide (KFSI) electrolyte with different concentrations has been investigated. Theoretical calculation demonstrates that the incorporation of Bi metal is favorable for potassium adsorption. The combination of delicate nanofiber morphology and electrolyte chemistry endows the fiber composite with an improved reversible capacity of 274.4 mAh g-1, promising rate capability, and cycling stability upon 500 cycles.

Novel Compound Heterozygous Mutation in FSIP2 Causes Multiple Morphological Abnormalities of the Sperm Flagella (MMAF) and Male Infertility.

Multiple morphological abnormalities of the sperm flagella (MMAF), characteristic with bent, short, coiled, absent, and abnormal caliber flagella, is an important basis of male infertility. Genetic factors account for a large proportion of patients with MMAF. The fibrous sheath interacting protein 2 (FSIP2) has a significant function in the spermatogenesis and flagellar motility. In our study, a novel compound heterozygous mutation (c.1494C > A, p.C498* and c.11020_11024del, p.Tyr3675Cysfs*3) in FSIP2 gene was identified in an infertile male patient with MMAF. H&E staining presented typical MMAF phenotype and thick neck, midpiece in the patient's sperm cells. Transmission electron microscopy observation showed abnormal mitochondrial arrangement and disorganization and dysplastic of the fibrous sheath (FS), which were verified again under light microscopy. Immunofluorescence (IF) analysis of FISP2 expression showed that FSIP2 was absent in the flagellum of the patient's sperm cells. Our findings will be helpful to the precise diagnosis of MMAF and male infertility and enrich the mutational spectrum of FSIP2 gene.

Novel Heterozygous Mutations in ZP2 Cause Abnormal Zona Pellucida and Female Infertility.

Zona pellucida (ZP) which is an extracellular matrix consisting of ZP1, ZP2, ZP3, and ZP4 plays a vital role in oocyte maturity, early embryonic development, and fertilization process. Any alterations of structure or function may lead to the abnormal formation of ZP and female infertility. Two novel heterozygous mutations c.1859G > A (p.Cys620Tyr) and c.1421 T > C (p.Leu474Pro) in ZP2 gene were recognized in three patients from two unrelated families with abnormal ZP and female infertility in this study. The expression constructs carrying wild-type ZP2 gene, c.1859G > A (p.Cys620Tyr) mutant ZP2 gene, and c.1421 T > C (p.Leu474Pro) mutant ZP2 gene were transfected into CHO cells respectively. There was a remarkable decrease in the expression of p.Cys620Tyr mutant protein with western blot. In addition, secretion of p.Leu474Pro mutant protein in the culture medium reduced markedly compared with that of wild-type ZP2 protein. Furthermore, co-immunoprecipitation showed that the p.Leu474Pro mutation affected the interaction between ZP2 and ZP3. Prediction of three-dimensional (3D) structure of the proteins showed that p.Cys620Tyr mutation altered the disulfide bond of ZP2 protein and may affect its function. These findings extend the ranges of mutations of ZP2 gene. Meanwhile, it will be helpful to the precise diagnosis of abnormal ZP.

Tsga10 is essential for arrangement of mitochondrial sheath and male fertility in mice.

BACKGROUND: Male infertility is a major issue in human reproduction health, yet known genetic factors are only responsible for a small fraction of cases. TSGA10 is a testis-specific protein that is highly conserved among different species. A previous study has reported a homozygous mutation in TSGA10 in a male infertile patient; however, function analysis of Tsga10 genes in knockout mice has not yet been undertaken. OBJECTIVES: The aim of the present work was to analyse the function of TSGA10 protein in the spermatogenesis of Tsga10+/- mice. MATERIALS AND METHODS: Tsga10+/- mice were generated by CRISPR/Cas9 technology, in vitro fertilization (IVF), western blot, co-immunoprecipitation and other methods were used to the function analysis. RESULTS: Heterozygous Tsga10 male mice created by CRISPR/Cas9 were infertile and presented significantly reduced sperm motility because of disordered mitochondrial sheath formation. Furthermore, TSGA10 can interact with GRP78 and NSUN2, which are associated with peri-implantation lethality and the gonadotropin-releasing hormone (GnRH) network. DISCUSSION AND CONCLUSION: We demonstrate that deficiency of Tsga10 gene can lead to male infertility in mice. TSGA10 is involved in the correct arrangement of mitochondrial sheath in spermatozoa. Future studies on TSGA10 include an in-depth exploration of the underlying mechanisms of TSGA10 in spermatogenesis, early embryonic development and GnRH network.

Three novel compound heterozygous IL12RB1 mutations in Chinese patients with Mendelian susceptibility to mycobacterial disease.

Mendelian Susceptibility to Mycobacterial Diseases (MSMD) is a primary immunodeficiency disease (PID) characterized by variable susceptibility to weakly virulent mycobacteria (Bacille Calmette-Guerin, BCG) and various intramacrophagic bacteria, fungi, parasites. Mycobacterial disease generally begins in childhood, more rarely during adolescence and adulthood. The pathogenesis of MSMD is the inherited impaired production of interferon gamma (IFN-γ) or inadequate response to it. Autosomal recessive IL12RB1 deficiency is the most common genetic etiology of MSMD. Here we identified three novel compound heterozygous mutations in IL12RB1 gene (c.635G>A, c.765delG; c.632G>C, c.847C>T; c.64G>A, c.1673insGAGCTTCCTGAG) in three Chinese families with MSMD.