Forkhead-associated phosphopeptide binding domain 1 (FHAD1) deficiency impaired murine sperm motility.

Background: Genetic knockout-based studies conducted in mice provide a powerful means of assessing the significance of a gene for fertility. Forkhead-associated phosphopeptide binding domain 1 (FHAD1) contains a conserved FHA domain, that is present in many proteins with phospho-threonine reader activity. How FHAD1 functions in male fertility, however, remains uncertain. Methods: Fhad1-/- mice were generated by CRISPR/Cas9-mediated knockout, after which qPCR was used to evaluate changes in gene expression, with subsequent analyses of spermatogenesis and fertility. The testis phenotypes were also examined using immunofluorescence and histological staining, while sperm concentrations and motility were quantified via computer-aided sperm analysis. Cellular apoptosis was assessed using a TUNEL staining assay. Results: The Fhad1-/-mice did not exhibit any abnormal changes in fertility or testicular morphology compared to wild-type littermates. Histological analyses confirmed that the testicular morphology of both Fhad1-/-and Fhad1+/+ mice was normal, with both exhibiting intact seminiferous tubules. Relative to Fhad1+/+ mice, however, Fhad1-/-did exhibit reductions in the total and progressive motility of epididymal sperm. Analyses of meiotic division in Fhad1-/-mice also revealed higher levels of apoptotic death during the first wave of spermatogenesis. Discussion: The findings suggest that FHAD1 is involved in both meiosis and the modulation of sperm motility.

A statistical investigation of parameters associated with low cell-free fetal DNA fraction in maternal plasma for noninvasive prenatal testing.

BACKGROUND: Noninvasive prenatal testing (NIPT) is the most common method for prenatal aneuploidy screening. Low fetal fraction (LFF) is the primary reason for NIPT failure. Consequently, factors associated with LFF should be elucidated for optimal clinical implementation of NIPT. METHODS: In this study, NIPT data from January 2019 to December 2022 from the laboratory records and obstetrical and neonatal data from the electronic medical records were collected and analyzed. Subjects with FF >3.50% were assigned to the control group, subjects with FF <3.50% once were assigned to the LFF group, and subjects with FF <3.50% twice were assigned to the repetitive low fetal fraction (RLFF) group. Factors, including body mass index (BMI), gestational age, maternal age, twin pregnancy, and in vitro fertilization (IVF) known to be associated with LFF were assessed by Kruskal-Wallis H test and logistic regression. Clinical data on first trimester pregnancy-associated plasma protein-A (PAPP-A), beta-human chorionic gonadotropin (ß-hCG), gestational age at delivery, birth weight at delivery, and maternal diseases were obtained from the hospital's prenatal and neonatal screening systems (twin pregnancy was not included in the data on gestational age at delivery and the control group did not include data on maternal diseases.), and were analyzed using Kruskal-Wallis H test and Chi-square test. RESULTS: Among the total of 63,883 subjects, 63,605 subjects were assigned to the control group, 197 subjects were assigned to the LFF group, and 81 subjects were assigned to the RLFF group. The median of BMI in the three groups was 22.43 kg/m2 (control), 25.71 kg/m2 (LFF), and 24.54 kg/m2 (RLFF). The median gestational age in the three groups was 130 days (control), 126 days (LFF), and 122/133 days (RLFF). The median maternal age in the three groups was 29 (control), 29 (LFF), and 33-years-old (RLFF). The proportion of twin pregnancies in the three groups was 3.3% (control), 10.7% (LFF), and 11.7% (RLFF). The proportion of IVF in the three groups was 4.7% (control), 11.7% (LFF), and 21.3% (RLFF). The factors significantly associated with LFF included BMI [2.18, (1.94, 2.45), p < 0.0001], gestational age [0.76, (0.67, 0.87), p < 0.0001], twin pregnancy [1.62, (1.02, 2.52), p = 0.0353], and IVF [2.68, (1.82, 3.86), p < 0.0001]. The factors associated with RLFF included maternal age [1.54, (1.17, 2.05), p = 0.0023] and IVF [2.55, (1.19, 5.54), p = 0.016]. Multiples of the median (MOM) value of ß-hCG and pregnant persons' gestational age at delivery were significantly decreased in the LFF and RLFF groups compared to the control group. CONCLUSION: According to our findings based on the OR value, factors associated strongly with LFF include a high BMI and the use of IVF. Factors associated less strongly with LFF include early gestational age and twin pregnancy, while advanced maternal age and IVF were independent risk factors for a second LFF result.

Body mass index, gestational age, maternal age, twin pregnancy, and in vitro fertilization are associated with fetal fraction. We added the repetitive low fetal fraction population and used a large normal population as a control to identify the main factors associated with low fetal fraction.

[Analysis of clinical features and variants of NF1 gene in 12 patients with Neurofibromatosis type 1].

OBJECTIVE: To explore the types of NF1 gene variants and clinical characteristics among patients with Neurofibromatosis type I (NF1). METHODS: Clinical data of 12 patients diagnosed at Ningbo Women and Children's Hospital between December 2019 and May 2022 were retrospectively analyzed. The probands and their family members were subjected to high-throughput sequencing, and candidate variants were verified by Sanger sequencing and chromosome microarray analysis. RESULTS: The 12 patients had ranged from 4 months to 27 years old, with a male-to-female ratio of 2 : 1. Cafè-au-lait spots were found in all patients. 83.3% of them also had axillary and/or inguinal freckling, 58.3% had neurofibromas, and 16.7% had congenital pseudarthrosis of the tibia. Five types of NF1 gene variants were identified in the patients, including 5 nonsense variants, 4 frameshift variants, 1 missense variant, 1 splice variant, 1 large deletion involving the whole gene. Six patients were found to harbor de novo variants, 2 had inherited the variants from their parents, and 4 were not verified for their parental origin. The c.3379del (p.Thr1127Glnfs*15) and c.6628_6629del (p.Glu2210Thrfs*10) variants were unreported in literature and databases. CONCLUSION: Most NF1 patients may present with Cafè-au-lait spots initially and are due to pathogenic variant of the NF1 gene. High-throughput sequencing can efficiently identify such variants among the patients and enable the definite diagnosis.

Combination of trio-based whole exome sequencing and optical genome mapping reveals a cryptic balanced translocation that causes unbalanced chromosomal rearrangements in a family with multiple anomalies.

Background: Balanced translocation (BT) carriers can produce imbalanced gametes and experience recurrent spontaneous abortions (RSAs) and even give birth to a child with complex chromosomal disorders. Here, we report a cryptic BT, t(5; 6) (p15.31; p25.1), in the proband's grandmother, which caused unbalanced chromosomal rearrangements and various anomalies in the two subsequent generations. We also provide a thorough overview of the application of optical genome mapping (OGM) to identify chromosomal structural variants (SVs). Methods: Trio-based whole exome sequencing (Trio-WES) was conducted to explore the genetic basis of the phenotype of the proband and her mother. High-resolution karyotype analysis and OGM detection were performed on the proband's grandparents to trace the origin of the unbalanced rearrangements between chromosomes 5 and 6. A PubMed search was conducted with the following keywords: "OGM" and "SVs." Then, relevant studies were collected and systematically reviewed. Results: The proband and her mother presented with various anomalies, whereas the grandmother was healthy but had a history of four abnormal pregnancies. Trio-WES revealed a heterozygous duplication on the terminal region of chromosome 5p and a heterozygous deletion on the proximal end of chromosome 6p in the proband and her mother. High-resolution karyotype analysis revealed no aberrant karyotypes in either grandparent, whereas OGM detection revealed a cryptic BT, t(5; 6)(p15.31; p25.1), in the proband's grandmother. An overwhelming majority of research publications have verified the clinical utility of OGM in detecting SVs. Conclusion: The results of this study revealed that the unbalanced chromosomal rearrangements and many anomalies observed in multiple members of the family were attributable to the cryptic BT carried by the proband's grandmother. This study supports that OGM has a unique advantage for detecting cryptic BTs, and can be used as a first-tier genetic test for the etiological diagnosis of infertility, RSAs, and other complex genetic disorders.

Report of new variants in PPIL1 underlying type 14 pontocerebellar hypoplasia and their associated phenotypic manifestations in two fetuses.

Mutations in the PPIL1 gene have been linked to type 14 pontocerebellar hypoplasia (PCH14); however, prenatal clinical characteristics of PCH14 caused by mutations in the PPIL1 gene have not been reported. This study reports the first prenatal case of PCH14 diagnosed by whole-exome sequencing (WES). Two fetuses with severe microcephaly and cerebral dysplasia, along with their parents, underwent WES. The effects of the discovered PPIL1 variants on PPIL1 protein function were investigated using bioinformatics tools. WES revealed two compound heterozygous missense mutations in PPIL1, c.376C > G (p.His126Asp) and c.392G > T (p.Arg131Leu), inherited from the mother and father, respectively. The co-segregation of PPIL1 mutations in this family was confirmed using Sanger sequencing, identifying two PCH14-affected fetuses. Bioinformatics analysis revealed that these mutations could disrupt the formation of hydrogen bonds, altering the structural stability of the PPIL1 protein. This study is the first to define the clinical characteristics of PCH14 during pregnancy and reports a novel heterozygous missense variant, expanding the PCH14-related mutational spectrum of PPIL1.

[Prenatal diagnosis and genetic analysis for two Chinese pedigrees carrying large fragment deletions of 13q21].

OBJECTIVE: To explore the strategies of prenatal diagnosis and genetic counseling for fetuses of two families with large deletions of 13q21. METHODS: Two singleton fetuses who were diagnosed with chromosome 13 microdeletions by non-invasive prenatal testing (NIPT) at Ningbo Women and Children's Hospital in March 2021 and December 2021 respectively were selected as the study subjects. Chromosomal karyotyping and chromosomal microarray analysis (CMA) were carried on amniotic samples. Peripheral blood samples were collected from the two couples for CMA assay to determine the origin of abnormal chromosomes identified in the fetuses. RESULTS: The karyotypes of the two fetuses were both normal. CMA revealed that they have respectively harbored heterozygous deletions spanning 11.935 Mb at 13q21.1q21.33 and 10.995 Mb at 13q14.3q21.32, which were respectively inherited from their mother and father. Both deletions had low gene density and lacked haploinsufficient genes, and were predicted to be likely benign variants based on database and literature search. Both couples had opted to continue with the pregnancy. CONCLUSION: The deletions of the 13q21 region in both families may be of benign variants. As the follow-up time was short, there was no sufficient evidence for the determination of pathogenicity, though our finding may still provide a basis for the prenatal diagnosis and genetic counseling.

[A large-scale retrospective analysis of copy number variations in single center using ACMG-ClinGen latest guidelines].

OBJECTIVE: Through a retrospective large sample analysis of copy number variants in single center, we explored the technical standards for the interpretation and reporting of constitutional copy-number variants (CNVs) jointly proposed by the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen) in 2019, analyzing its impact on CNVs ratings and the improvement in the consistency of the classification of CNVs in clinical laboratories. METHODS: 236 CNVs that assessed as pathogenic, uncertain significant (including likely pathogenic, uncertain and likely benign) by the 2011 ACMG guidelines between August 2018 and December 2019 in our center were re-analyzed. Four working group members of the center reclassified and evaluated 235 CNVs according to 2019 ACMG guidelines. RESULTS: The consistency of clinical significance classification of CNVs was 91% and the α test coefficient was 0.98 among four working group members. Compared with the 2011 and 2019 ACMG technical standards for the CNVs classification, evaluation of pathogenicity and uncertain significant is basically consistent. 90% (45/50) of likely pathogenic and likely benign CNVs were Re-evaluated as variants of uncertain significance, and the difference is significant. CONCLUSION: The new version ACMG/ClinGen guidelines for the evaluation of CNVs developed semi-quantitative point-based scoring system and help to improve the consistency in clinical classifications. It can also make the interpretation of CNVs more standardized and transparent.

[Analysis of TUBB2B gene variant in a fetus with complex cortical dysplasia with other brain malformations-7].

OBJECTIVE: To explore the genetic basis for a fetus with dysgenesis of corpus callosum and other brain malformations. METHODS: Whole exome sequencing was carried out for the fetus and its parents. Suspected pathogenic variants were verified by Sanger sequencing. RESULTS: A novel de novo missense variant c.758T>A (p.L253Q) of the TUBB2B gene was identified, which was unreported previously. Based on the guidelines from the American College of Medical Genetics, the c.758T>A variant was predicted to be likely pathogenic. Bioinformatics analysis predicted that the leucine at position 253 was highly conserved among various species, and the c.758T>A variant may impact the formation of hydrogen bonds between Leu253 and Asp249 and Met257 residues, which in turn may affect the combination of GTP/GDP and function of the TUBB2B protein. CONCLUSION: The c.758T>A variant of the TUBB2B gene probably underlay the fetal malformations in this Chinese family. Above discovery has enriched the spectrum of TUBB2B gene variants and provided a basis for genetic counseling and prenatal diagnosis.

[Variation analysis of EPG5 gene in a Vici syndrome family].

OBJECTIVE: To explore the genetic etiology of Vici syndrome in a Chinese family. METHODS: Whole exome sequencing (WES) technology was used to detect gene variants in a fetus of abnormal ultrasonic structure without abnormalities in routine chromosome karyotype analysis and SNP-array. Sanger sequencing and bioinformatics prediction were performed for the suspected variants of the fetus and parents. RESULTS: The fetus and the elder sister have carried c. 2427delC (p.T809fs) and c.1886A>T (p.E629V) compound heterozygous variants of the EPG5 gene, which were respectively inherited from their mother and father. Neither variant was reported previously. According to ACMG guidelines, the c.2427delC variant was predicted as pathogenic, while the c.1886A>T variant was of uncertain significance. PolyPhen-2 and PROVEAN software indicated that c.1886A>T variant was probably damaging. CONCLUSION: The c.2427delC and c.1886A>T variants of the EPG5 gene probably underlie the pathogenesis of the Vici syndrome in this family. Above finding has enriched the variational spectrum of EPG5 gene and provided a basis for genetic counseling and prenatal diagnosis for the family.

22q11.2 recurrent copy number variation-related syndrome: a retrospective analysis of our own microarray cohort and a systematic clinical overview of ClinGen curation.

BACKGROUND: Chromosomal 22q11.2 dosage changes in the recurrent region can lead to a series of clinically variable pediatric syndromes. This study conducted a retrospective analysis of microarray tested cases with 22q11.2 recurrent copy number variations (CNVs) at our laboratory from September 2018 to August 2021, and provides a systematical clinical overview of ClinGen curation. METHODS: The data of 34 microarray tested cases with 22q11.2 recurrent CNVs at our laboratory from September 2018 to August 2021 were retrospectively analyzed, and the variant types, abnormal chromosome regions, clinical phenotypes, and follow-up information were evaluated and summarized. A ClinGen Dosage Sensitivity Map was retrieved for "22q11.2". The information of each 22q11.2 recurrent region was collected and systematically classified. RESULTS: We reported 34 cases (including 18 22q11.2 microdeletion cases and 16 microduplication cases) from 8,465 microarrays. Of the 22q11.2 recurrent CNV-carried samples, 74% (25/34) comprised prenatal amniotic fluid or villus, and up to 50% (17/34) of the cases contained the proximal A-D interval. Across these 22q11.2 microdeletion samples, the congenital cardiovascular defect, which mainly included the tetralogy of fallot, ventricular septal defect, and patent foramen ovale, was identified as the most common feature (13/18, 72%). However, 22q11.2 microduplication cases exhibited a broad range of highly variable phenotypes, spanning from severe abnormality to mild characteristics and even the completely normal phenotype. This study also systematically reviewed the ClinGen dosage sensitivity curation on 22q11.2 recurrent regions, and found that A-D/A-B haploinsufficiency score reached "3", responsible for DiGeorge syndrome (DGS)/velocardiofacial syndrome (VCFS). Also, A-D/A-B triplosensitivity score "3" could further account for multiple variable phenotypes. CONCLUSIONS: Taken together, this study provides clinical overview of the ClinGen curation and data support for the American College of Medical Genetics and Genomics (ACMG) evaluation in the pathogenicity of each interval involved in 22q11.2 recurrent deletion and duplication. Certainly, more evidences on the genotype-phenotype contributions of different 22q11.2 recurrent CNVs need to be gathered.

Dual functions for the ssDNA-binding protein RPA in meiotic recombination.

Meiotic recombination permits exchange of genetic material between homologous chromosomes. The replication protein A (RPA) complex, the predominant ssDNA-binding complex, is required for nearly all aspects of DNA metabolism, but its role in mammalian meiotic recombination remains unknown due to the embryonic lethality of RPA mutant mice. RPA is a heterotrimer of RPA1, RPA2, and RPA3. We find that loss of RPA1, the largest subunit, leads to disappearance of RPA2 and RPA3, resulting in the absence of the RPA complex. Using an inducible germline-specific inactivation strategy, we find that loss of RPA completely abrogates loading of RAD51/DMC1 recombinases to programmed meiotic DNA double strand breaks, thus blocking strand invasion required for chromosome pairing and synapsis. Surprisingly, loading of MEIOB, SPATA22, and ATR to DNA double strand breaks is RPA-independent and does not promote RAD51/DMC1 recruitment in the absence of RPA. Finally, inactivation of RPA reduces crossover formation. Our results demonstrate that RPA plays two distinct roles in meiotic recombination: an essential role in recombinase recruitment at early stages and an important role in promoting crossover formation at later stages.

The long intergenic non-protein coding RNA 707 promotes proliferation and metastasis of gastric cancer by interacting with mRNA stabilizing protein HuR.

Multiple studies have revealed that long non-coding RNAs (lncRNAs) extensively participate in human cancer malignant progression. The long intergenic non-protein coding RNA 707 (LINC00707), 3087 bp in length, was recently reported to be an essential oncogene in promoting lung adenocarcinoma cell proliferation and metastasis. However, its role in gastric cancer (GC) remains unclear. In this study, we identified that LINC00707 was excessively expressed in GC tissues and correlated with advanced stage, larger tumor size, lymph node metastasis and poorer prognosis in GC patients. In vitro and in vivo assays showed that LINC00707 promote GC cell proliferation and metastasis. Mechanistically, LINC00707 could abundantly interact with mRNA stabilizing protein HuR; "LINC00707-HuR" coalition ulteriorly combined with VAV3/F11R mRNAs and increased their stability. Taken together, our findings prove that LINC00707 may act as an oncogene in GC by regulating mRNA stability and serve as a potential target for GC diagnosis and prognosis.

HOXA11-AS: a novel regulator in human cancer proliferation and metastasis.

Multiple studies have demonstrated that lncRNAs extensively participate in human cancer proliferation and metastasis. Epigenetic modification, transcriptional and posttranscriptional regulatory mechanisms are involved in lncRNA-led tumorigenesis and transfer. Recently, a novel identified homeobox (HOX) A11 antisense lncRNA, HOXA11-AS, 1,628 bp in length, has been excessively highlighted to be an essential initiator and facilitator in the process of malignant tumor proliferation and metastasis. As found in many reports, HOXA11-AS can not only act as a molecular scaffold of PRC2, LSD1 and DNMT1 to epigenetically modify chromosomes in the nucleus but also occur as ceRNA competitively sponging miRNAs in the cytoplasm. Furthermore, HOXA11-AS may function as a potential biomarker for cancer diagnosis and prognosis. In this review, we summarize the evolvement and mechanisms of HOXA11-AS in proliferation and metastasis of various human cancers.

Human oocyte maturation arrest caused by a novel missense mutation in TUBB8.

Objective To explore the etiology of human oocyte maturation arrest in two infertile Chinese sisters. Methods Clinical examination and genetic testing of all available family members were conducted, and the findings were used to create a pedigree. Mutation screening using PCR amplification and DNA Sanger sequencing of the entire tubulin beta 8 class VIII gene ( TUBB8) including intron-exon boundaries was performed to identify mutations. Results A novel missense TUBB8 mutation (c.1054G > T, p.A352S) in the patient and her elder sister was detected and shown to be associated with oocyte maturation arrest. Conclusion Our findings expand the known mutation spectrum of TUBB8 and provide insights into the etiology of human oocyte maturation arrest.

MORC2B is essential for meiotic progression and fertility.

The microrchidia (MORC) family proteins are chromatin-remodelling factors and function in diverse biological processes such as DNA damage response and transposon silencing. Here, we report that mouse Morc2b encodes a functional germ cell-specific member of the MORC protein family. Morc2b arose specifically in the rodent lineage through retrotransposition of Morc2a during evolution. Inactivation of Morc2b leads to meiotic arrest and sterility in both sexes. Morc2b-deficient spermatocytes and oocytes exhibit failures in chromosomal synapsis, blockades in meiotic recombination, and increased apoptosis. Loss of MORC2B causes mis-regulated expression of meiosis-specific genes. Furthermore, we find that MORC2B interacts with MORC2A, its sequence paralogue. Our results demonstrate that Morc2b, a relatively recent gene, has evolved an essential role in meiosis and fertility.

Accelerated reproductive aging in females lacking a novel centromere protein SYCP2L.

Menopause results from loss of ovarian function and marks the end of a woman's reproductive life. Alleles of the human SYCP2L locus are associated with age at natural menopause (ANM). SYCP2L is a paralogue of the synaptonemal complex protein SYCP2 and is expressed exclusively in oocytes. Here we report that SYCP2L localizes to centromeres of dictyate stage oocytes, which represent the limited pool of primordial oocytes that are formed perinatally and remain arrested till ovulation. Centromere localization of SYCP2L requires its C-terminal portion, which is missing in truncated variants resulting from low-frequency nonsense mutations identified in humans. Female mice lacking SYCP2L undergo a significantly higher progressive loss of oocytes with age compared with wild-type females and are less fertile. Specifically, the pool of primordial oocytes becomes more rapidly depleted in SYCP2L-deficient than in wild-type females, such that with aging, fewer oocytes undergo maturation in developing follicles. We find that a human SYCP2L intronic single nucleotide polymorphism (SNP) rs2153157, which is associated with ANM, changes the splicing efficiency of U12-type minor introns and may therefore regulate the steady-state amount of SYCP2L transcript. Furthermore, the more efficiently spliced allele of this intronic SNP in SYCP2L is associated with increased ANM. Our results suggest that SYCP2L promotes the survival of primordial oocytes and thus provide functional evidence for its association with ANM in humans.

Gold nanoparticle incorporated inverse opal photonic crystal capillaries for optofluidic surface enhanced Raman spectroscopy.

Novel transducers are needed for point of care testing (POCT) devices which aim at facile, sensitive and quick acquisition of health related information. Recent advances in optofluidics offer tremendous opportunities for biological/chemical analysis using extremely small sample volumes. This paper demonstrates nanostructured capillary tubes for surface enhanced Raman spectroscopy (SERS) analysis in a flow-through fashion. The capillary tube integrates the SERS sensor and the nanofluidic structure to synergistically offer sample delivery and analysis functions. Inside the capillary tube, inverse opal photonic crystal (IO PhC) was fabricated using the co-assembly approach to form nanoscale liquid pathways. In the nano-voids of the IO PhC, gold nanoparticles were in situ synthesized and functioned as the SERS hotspots. The advantages of the flow-through SERS sensor are multifold. The capillary effect facilities the sample delivery process, the nanofluidic channels boosts the interaction of analyte and gold nanoparticles, and the PhC structure strengthens the optical field near the SERS hotspots and results in enhanced SERS signals from analytes. As an exemplary demonstration, the sensor was used to measure creatinein spiked in artificial urine samples with detection limit of 0.9 mg/dL.