Bi-allelic mutations in MOS cause female infertility characterized by preimplantation embryonic arrest.

STUDY QUESTION: Are mutations in MOS (MOS proto-oncogene, serine/threonine kinase) involved in early embryonic arrest in infertile women? SUMMARY ANSWER: We identified mutations in MOS that may cause human female infertility characterized by preimplantation embryonic arrest (PREMBA), and the effects of the mutations in human embryonic kidney 293T (HEK293T cells) and mouse oocytes provided evidence for a causal relation between MOS and female infertility. WHAT IS KNOWN ALREADY: MOS, an activator of mitogen-activated protein kinase, mediates germinal vesicle breakdown and metaphase II arrest. Female MOS knockout mice are viable but sterile. Thus, MOS seems to be an important part of the mammalian cell cycle mechanism that regulates female meiosis. STUDY DESIGN, SIZE, DURATION: Whole-exome sequencing, bioinformatics filtering analysis and genetic analysis were performed to identify two different biallelic mutations in MOS in two independent families. The infertile patients presenting with early embryonic arrest were recruited from October 2018 to June 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS: The female patients diagnosed with primary infertility were recruited from the reproduction centres of local hospitals. Genomic DNA from the affected individuals, their family members and healthy controls was extracted from peripheral blood. We performed whole-exome sequencing in patients diagnosed with PREMBA. Functional effects of the mutations were investigated in HEK293T cells by western blotting and in mouse oocytes by microinjection and immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE: We identified the homozygous missense mutation c.285C>A (p.(Asn95Lys)) and the compound heterozygous mutations c.467delG (p.(Gly156Alafs*18)) and c.956G>A (p.(Arg319His)) in MOS in two independent patients. The mutations c.285C>A (p.(Asn95Lys)) and c.467delG (p.(Gly156Alafs*18)) reduced the protein level of MOS, and all mutations reduced the ability of MOS to phosphorylate its downstream target, extracellular signal-regulated kinase1/2. In addition, the identified mutations reduced the capacity of exogenous human MOS to rescue the metaphase II exit phenotype, and the F-actin cytoskeleton of mouse oocytes was affected by the patient-derived mutations. LIMITATIONS, REASONS FOR CAUTION: Owing to the lack of in vivo data from patient oocytes, the exact molecular mechanism affected by MOS mutations and leading to PREMBA is still unknown and should be further investigated using knock-out or knock-in mice. WIDER IMPLICATIONS OF THE FINDINGS: We identified recessive mutations in MOS in two independent patients with the PREMBA phenotype. Our findings reveal the important role of MOS during human oocyte meiosis and embryonic development and suggest that mutations in MOS may be precise diagnostic markers for clinical genetic counselling. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (81725006, 81822019, 81771581, 81971450, 81971382,82001538 and 82071642), the project supported by the Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), the Project of the Shanghai Municipal Science and Technology Commission (19JC1411001), the Natural Science Foundation of Shanghai (19ZR1444500 and 21ZR1404800), the Shuguang Program of the Shanghai Education Development Foundation and the Shanghai Municipal Education Commission (18SG03), the Foundation of the Shanghai Health and Family Planning Commission (20154Y0162), the Capacity Building Planning Program for Shanghai Women and Children's Health Service and the collaborative innovation centre project construction for Shanghai Women and Children's Health. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

A novel homozygous missense variant in BTG4 causes zygotic cleavage failure and female infertility.

PURPOSE: "Zygotic cleavage failure" is an embryonic phenotype that causes female infertility and failure of in vitro fertilization and/or intracytoplasmic sperm injection. We aimed to identify pathogenic variants in a female infertility patient from a consanguineous family with the "zygotic cleavage failure" phenotype. METHODS: Whole-exome sequencing was performed in the affected patient; Sanger sequencing was used to confirm the identified variant. The functional effect of the identified variant was further investigated in HeLa cells. RESULTS: We identified a novel homozygous missense mutation in BTG4 (c.285G > C, p.W95C) in the affected individual. Co-immunoprecipitation in HeLa cells showed the complete loss of the interaction between the p.W95C BTG4 variant protein and CNOT7. CONCLUSION: This study confirms our previous research and expands the mutational spectrum of BTG4. Our findings complement the diagnostic genetic basis for "zygotic cleavage failure" patients and suggest that BTG4 may be a good target for future therapeutic strategies.

Novel biallelic mutations in MEI1: expanding the phenotypic spectrum to human embryonic arrest and recurrent implantation failure.

STUDY QUESTION: Are any novel mutations and corresponding new phenotypes, other than recurrent hydatidiform moles, seen in patients with MEI1 mutations? SUMMARY ANSWER: We identified several novel mutations in MEI1 causing new phenotypes of early embryonic arrest and recurrent implantation failure. WHAT IS KNOWN ALREADY: It has been reported that biallelic mutations in MEI1, encoding meiotic double-stranded break formation protein 1, cause azoospermia in men and recurrent hydatidiform moles in women. STUDY DESIGN, SIZE, DURATION: We first focused on a pedigree in which two sisters were diagnosed with recurrent hydatidiform moles in December 2018. After genetic analysis, two novel mutations in MEI1 were identified. We then expanded the mutational screening to patients with the phenotype of embryonic arrest, recurrent implantation failure, and recurrent pregnancy loss, and found another three novel MEI1 mutations in seven new patients from six families recruited from December 2018 to May 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS: Nine primary infertility patients were recruited from the reproduction centers in local hospitals. Genomic DNA from the affected individuals, their family members, and healthy controls was extracted from peripheral blood. The MEI1 mutations were screened using whole-exome sequencing and were confirmed by the Sanger sequencing. In silico analysis of mutations was performed with Sorting Intolerant From Tolerant (SIFT) and Protein Variation Effect Analyzer (PROVEAN). The influence of the MEI1 mutations was determined by western blotting and minigene analysis in vitro. MAIN RESULTS AND THE ROLE OF CHANCE: In this study, we identified five novel mutations in MEI1 in nine patients from seven independent families. Apart from recurrent hydatidiform moles, biallelic mutations in MEI1 were also associated with early embryonic arrest and recurrent implantation failure. In addition, we demonstrated that protein-truncating and missense mutations reduced the protein level of MEI1, while the splicing mutations caused abnormal alternative splicing of MEI1. LIMITATIONS, REASONS FOR CAUTION: Owing to the lack of in vivo data from the oocytes of the patients, the exact molecular mechanism(s) involved in the phenotypes remains unknown and should be further investigated using knock-out or knock-in mice. WIDER IMPLICATIONS OF THE FINDINGS: Our results not only reveal the important role of MEI1 in human oocyte meiosis and early embryonic development, but also extend the phenotypic and mutational spectrum of MEI1 and provide new diagnostic markers for genetic counseling of clinical patients. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research and Development Program of China (2018YFC1003800, 2017YFC1001500, and 2016YFC1000600), the National Natural Science Foundation of China (81725006, 81822019, 81771581, 81971450, and 81971382), the project supported by the Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), the Project of the Shanghai Municipal Science and Technology Commission (19JC1411001), the Natural Science Foundation of Shanghai (19ZR1444500), the Shuguang Program of the Shanghai Education Development Foundation and the Shanghai Municipal Education Commission (18SG03), the Shanghai Health and Family Planning Commission Foundation (20154Y0162), the Strategic Collaborative Research Program of the Ferring Institute of Reproductive Medicine, Ferring Pharmaceuticals and the Chinese Academy of Sciences (FIRMC200507) and the Chongqing Key Laboratory of Human Embryo Engineering (2020KFKT008). No competing interests are declared. TRIAL REGISTRATION NUMBER: N/A.

Novel mutations in LHCGR (luteinizing hormone/choriogonadotropin receptor): expanding the spectrum of mutations responsible for human empty follicle syndrome.

PURPOSE: To screen novel mutations in LHCGR responsible for empty follicle syndrome and explore the pathological mechanism of mutations. METHODS: Four affected individuals diagnosed with infertility-associated anovulation or oligo-ovulation from three independent families were recruited. Sanger sequencing was used to identify the LHCGR mutations in affected individuals. Western blot was performed to evaluate the effects of mutations on LHCGR protein levels. Immunofluorescence was done to explore the effects of mutations on LHCGR subcellular localization. The ATP levels were measured to infer the functional effects of the mutations on LHCGR. RESULTS: In the present study, three novel biallelic mutations in LHCGR were identified in four affected individuals from three independent families with empty follicle syndrome or oligo-ovulation. All biallelic mutations were inherited from the proband of their parents. The western blot showed that the identified mutations decreased LHCGR protein level and altered the glycosylation pattern. The immunofluorescence showed an ectopic subcellular localization of LHCGR in cultured HeLa cells. Besides, the mutations in LHCGR also reduced the cellular ATP consumption. CONCLUSION: These findings confirm previous studies and expand the mutational spectrum of LHCGR, which will provide genetic diagnostic marker for patients with empty follicle syndrome.

Homozygous Mutations in BTG4 Cause Zygotic Cleavage Failure and Female Infertility.

Zygotic cleavage failure (ZCF) is a unique early embryonic phenotype resulting in female infertility and recurrent failure of in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI). With this phenotype, morphologically normal oocytes can be retrieved and successfully fertilized, but they fail to undergo cleavage. Until now, whether this phenotype has a Mendelian inheritance pattern and which underlying genetic factors play a role in its development remained to be elucidated. B cell translocation gene 4 (BTG4) is a key adaptor of the CCR4-NOT deadenylase complex, which is involved in maternal mRNA decay in mice, but no human diseases caused by mutations in BTG4 have previously been reported. Here, we identified four homozygous mutations in BTG4 (GenBank: NM_017589.4) that are responsible for the phenotype of ZCF, and we found they followed a recessive inheritance pattern. Three of them-c.73C>T (p.Gln25Ter), c.1A>G (p.?), and c.475_478del (p.Ile159LeufsTer15)-resulted in complete loss of full-length BTG4 protein. For c.166G>A (p.Ala56Thr), although the protein level and distribution of mutant BTG4 was not altered in zygotes from affected individuals or in HeLa cells, the interaction between BTG4 and CNOT7 was abolished. In vivo studies further demonstrated that the process of maternal mRNA decay was disrupted in the zygotes of the affected individuals, which provides a mechanistic explanation for the phenotype of ZCF. Thus, we provide evidence that ZCF is a Mendelian phenotype resulting from mutations in BTG4. These findings contribute to our understanding of the role of BTG4 in human early embryonic development and provide a genetic marker for female infertility.

Bi-allelic Missense Pathogenic Variants in TRIP13 Cause Female Infertility Characterized by Oocyte Maturation Arrest.

Normal oocyte meiosis is a prerequisite for successful human reproduction, and abnormalities in the process will result in infertility. In 2016, we identified mutations in TUBB8 as responsible for human oocyte meiotic arrest. However, the underlying genetic factors for most affected individuals remain unknown. TRIP13, encoding an AAA-ATPase, is a key component of the spindle assembly checkpoint, and recurrent homozygous nonsense variants and a splicing variant in TRIP13 are reported to cause Wilms tumors in children. In this study, we identified homozygous and compound heterozygous missense pathogenic variants in TRIP13 responsible for female infertility mainly characterized by oocyte meiotic arrest in five individuals from four independent families. Individuals from three families suffered from oocyte maturation arrest, whereas the individual from the fourth family had abnormal zygote cleavage. All displayed only the infertility phenotype without Wilms tumors or any other abnormalities. In vitro and in vivo studies showed that the identified variants reduced the protein abundance of TRIP13 and caused its downstream molecule, HORMAD2, to accumulate in HeLa cells and in proband-derived lymphoblastoid cells. The chromosome mis-segregation assay showed that variants did not have any effects on mitosis. Injecting TRIP13 cRNA into oocytes from one affected individual was able to rescue the phenotype, which has implications for future therapeutic treatments. This study reports pathogenic variants in TRIP13 responsible for oocyte meiotic arrest, and it highlights the pivotal but different roles of TRIP13 in meiosis and mitosis. These findings also indicate that different dosage effects of mutant TRIP13 might result in two distinct human diseases.

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

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.

A homozygous mutation in CMAS causes autosomal recessive intellectual disability in a Kazakh family.

Intellectual disability (ID) describes a wide range of serious human diseases caused by defects in central nervous system development and function. Some mutant genes have been found to be associated with these diseases, but not all cases can be explained, thus suggesting that other disease-causing genes have not yet been discovered. Sialic acid is involved in a number of key biological processes, including embryo formation, nerve cell growth, and cancer cell metastasis, and very recently it has been suggested that N-acetylneuraminic acid synthase-mediated synthesis of sialic acid is required for brain and skeletal development. CMP-sialic acid synthetase (CMAS) is one of four enzymes involved in NeuNAc metabolism, as it catalyzes the formation of CMP-NeuNAc. Before the present study, no links between mutations in CMAS and incidences of human ID had been reported. In the current study, we recruited a recessive nonsyndromic ID pedigree with consanguineous marriage in which all patients have typical clinical manifestations of ID. We identified the NM_018686.3:c.563G > A (p.Arg188His) substitution in CMAS as being responsible for the disease in this family. Conservation analysis, structural prediction, and enzyme activity experiments demonstrated that (p.Arg188His) influences protein dimerization and alters CMAS enzyme activity. Our results offer a new orientation for future research and clinical diagnosis.

Pregnancy and Live Birth In Women With Pathogenic LHCGR Variants Using Their Own Oocytes.

CONTEXT: The LH/chorionic gonadotropin receptor (LHCGR) is mainly expressed in gonads and plays important roles in estradiol production, ovulation, and luteal formation. Women with pathogenic LHCGR variants suffer from infertility, and successful fertility treatments for such women have never been reported. OBJECTIVE: The purpose of this study was to determine whether women with pathogenic LHCGR variants can achieve successful pregnancies through in vitro fertilization. DESIGN: Three women with LH resistance and infertility and their parents underwent exome sequencing. The biochemical characteristics and functional effects of LHCGR mutation were assessed in transfected human embryonic kidney -293T cells and primary granulosa cells. RESULTS: All affected women harbored pathogenic LHCGR variants. The LHCGR variants lacked cell surface localization and signal transduction abilities in vitro and in vivo. After dual triggering and prolonging the interval between triggering and oocyte pick-up, all three patients achieved oocytes and high-quality embryos. After frozen embryo transfer, one woman successfully birthed twins, and one woman successfully birthed a live boy. Apart from difficulties in oocyte retrieval, no obvious abnormalities in fertilization or during embryo development and pregnancy were identified in these patients. CONCLUSIONS: This study is, to our knowledge, the first to report successful assisted reproductive treatment of women with pathogenic LHCGR variants using their own oocytes. Our results supported that defects in LHCGR disrupted ovulation but had no effect on fertilization and embryo development.

Identification of a novel homozygous mutation in the MYO15A gene in a Kazakh family with non-syndromic hearing loss.

BACKGROUND: Millions of people around the world are plagued by hearing loss. More than 50% of congenital or pre-lingual deafness is associated with genetic factors and has highly genetic heterogeneity. To date, although hundreds of genes have been found to be implicated in non-syndromic deafness, there are still lots of genes or loci that we need to verify. METHODS: In this study, we performed target sequencing and Sanger sequencing in a Kazakh consanguineous family with autosomal recessive non-syndromic hearing loss. Following that, functional and structural studies predicted the pathogenic effect of novel mutations by use of the online tools. RESULTS: We identified a novel homozygous mutation p.R3191C in MYO15A gene causing deafness in this family. The mutation p.R3191C co-segregated with the disease phenotype in this family and was not present in any public databases. Automatic tools predict that the novel mutation makes a great impact on the function and structure of MYO15A protein. CONCLUSIONS: This is a novel mutation of MYO15A causing deafness and also the first report of MYO15A mutations causing deafness in the Kazakh families. This finding expanded the spectrum of MYO15A mutations, making it more precise for future genetic diagnosis in patients with deafness.

A pannexin 1 channelopathy causes human oocyte death.

Connexins and pannexins are two protein families that play an important role in cellular communication. Pannexin 1 (PANX1), one of the members of pannexin family, is a channel protein. It is glycosylated and forms three species, GLY0, GLY1, and GLY2. Here, we describe four independent families in which mutations in PANX1 cause familial or sporadic female infertility via a phenotype that we term "oocyte death." The mutations, which are associated with oocyte death, alter the PANX1 glycosylation pattern, influence the subcellular localization of PANX1 in cultured cells, and result in aberrant PANX1 channel activity, ATP release in oocytes, and mutant PANX1 GLY1. Overexpression of a patient-derived mutation in mice causes infertility, recapitulating the human oocyte death phenotype. Our findings demonstrate the critical role of PANX1 in human oocyte development, provide a genetic explanation for a subtype of infertility, and suggest a potential target for therapeutic intervention for this disease.

Novel mutations and structural deletions in TUBB8: expanding mutational and phenotypic spectrum of patients with arrest in oocyte maturation, fertilization or early embryonic development.

STUDY QUESTION: Are there any new type of mutations and novel phenotypes in patients with arrest in oocyte maturation, fertilization or early embryonic development having tubulin beta eight class VIII (TUBB8) mutations? SUMMARY ANSWER: We identified new types of mutations in TUBB8 associated with maturation, fertilization and developmental arrest. WHAT IS KNOWN ALREADY: We previously found heterozygous mutations and a homozygous frameshift/internal seven amino acid deletion in TUBB8 that are responsible for oocyte maturation arrest. STUDY DESIGN, SIZE, DURATION: We recruited 10 new primary infertility patients from 9 families from December 2015 to May 2016, most of which exhibited failures in oocyte maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ten primary infertility patients were recruited from the reproduction centers in local hospitals. Genomic DNA samples from the affected individuals, their family members and healthy controls were extracted from peripheral blood. TUBB8 in the DNA samples were sequenced by Sanger sequencing. TUBB8 sequence was then aligned by CodonCode software to identify rare variants. ExAC database was used to search frequency of corresponding mutations. In silico analysis of mutations was used by Polyphen and PROVEAN. Phenotypes of oocytes and embryos were evaluated by light microscopy, polarization microscopy or immunolabeling. MAIN RESULTS AND THE ROLE OF CHANCE: Besides several novel heterozygous missense mutations, we also identified other new types of genetic variants, including homozygous mutations and a de novo compound heterozygous mutation. We also found a patient with a homozygous deletion of the whole TUBB8 gene, which is the first reported case of a large structural variation in this gene. In addition, we found different mutations in TUBB8 that could result in variability in oocyte/embryo phenotypes, including oocyte maturation arrest, first polar body (PB1) oocytes that cannot be fertilized, and PB1 oocytes that can be fertilized but arrest at an early embryonic stage. LIMITATIONS, REASONS FOR CAUTION: The exact molecular mechanism has not been analyzed and should be further investigated in the future. In addition, immunostaining of more oocytes with mutations and checking spindle status of oocytes with mutations non-invasively by polarization microscopy needs to be done in order to determine exact stage of PB1 oocytes and the functional differences of these mutations. WIDER IMPLICATIONS OF THE FINDINGS: The results not only emphasize the important role of TUBB8 in oocyte maturation, fertilization and early embryonic development but they also provide a basis for determining the genetic variations in TUBB8 as a potential additional criterion for evaluating the quality of patients' functional PB1 oocytes. STUDY FUNDING/COMPETING INTERESTS: National Key R&D Program of China (2016YFC1000600); Basic Research Program of China (2015CB943300); National Natural Science Foundation of China (81270747 and 81571501). No competing interests declared.

Mutations in TUBB8 cause a multiplicity of phenotypes in human oocytes and early embryos.

BACKGROUND: TUBB8 is a primate-specific ß-tubulin isotype whose expression is confined to oocytes and the early embryo. We previously found that mutations in TUBB8 caused oocyte maturation arrest. The objective was to describe newly discovered mutations in TUBB8 and to characterise the accompanying spectrum of phenotypes and modes of inheritance. METHODS AND RESULTS: Patients with oocyte maturation arrest were sequenced with respect to TUBB8. We investigated the effects of identified mutations in vitro, in cultured cells and in mouse oocytes. Seven heterozygous missense and two homozygous mutations were identified. These mutations cause a range of folding defects in vitro, different degrees of microtubule disruption upon expression in cultured cells and interfere to varying extents in the proper assembly of the meiotic spindle in mouse oocytes. Several of the newly discovered TUBB8 mutations result in phenotypic variability. For example, oocytes harbouring any of three missense mutations (I210V, T238M and N348S) could extrude the first polar body. Moreover, they could be fertilised, although the ensuing embryos became developmentally arrested. Surprisingly, oocytes from patients harbouring homozygous TUBB8 mutations that in either case preclude the expression of a functional TUBB8 polypeptide nonetheless contained identifiable spindles. CONCLUSIONS: Our data substantially expand the range of dysfunctional oocyte phenotypes incurred by mutation in TUBB8, underscore the independent nature of human oocyte meiosis and differentiation, extend the class of genetic diseases known as the tubulinopathies and provide new criteria for the qualitative evaluation of meiosis II (MII) oocytes for in vitro fertilization (IVF).