Loss-of-function mutations in IQCN cause male infertility in humans and mice owing to total fertilization failure.

Fertilization failure is a significant manifestation of unexplained male infertility. Previous work has suggested a genetic origin. In this study, we report on a man with unexplained infertility from a large consanguineous marriage family. Whole-exome sequencing and Sanger sequencing identified a homozygous frameshift variation of the IQ motif containing N (IQCN; GenBank: NM_001145304.1; c.1061_1062delAT; p.Y354Sfs*13) in the proband and one of his two brothers, who also remained infertile. Analyses of spermatozoa by quantitative RT-PCR indicated that the level of IQCN mRNA was significantly reduced compared to fertile men and the protein could not be detected by western blotting and immunofluorescent staining in the proband. Immunofluorescent staining of spermatozoa from fertile men showed that IQCN was located in the acrosomal region and translocated to the equatorial segment after the acrosome reaction. The proband spermatozoa had abnormal morphology and function. Finally, the proband couple underwent IVF with donor sperm and a healthy baby was born. Furthermore, we developed an Iqcn-KO mouse model using the CRISPR/Cas9 technique. Sperm quality, except for sperm motility, and the fertility of male Iqcn-/- mice were consistent with those of the proband. In conclusion, the findings in humans and mice demonstrate that the homozygous frameshift variant of IQCN causes male infertility owing to autosomal-recessive fertilization failure.

[Detection of pathogenic variants in four patients with globozoospermia].

OBJECTIVE: To explore the genetic basis for 4 patients with globozoospermia. METHODS: Semen and blood samples were collected from the patients for the determination of sperm concentration, viability, survival rate, morphology and acrosome antigen CD46. Meanwhile, DNA was extracted for whole exome sequencing (WES), and candidate variants were validated by Sanger sequencing. RESULTS: All of the four patients were found to harbor variants of the DPY19L2 gene. Patients 1 ~ 3 had homozygous deletions of the DPY19L2 gene. Sanger sequencing confirmed that the DPY19L2 gene in patient 3 was disrupted at a recombination breakpoint area BP2, resulting in nonallelic homologous recombination and complete deletion of the DPY19L2 gene. Patients 2 and 3 respectively harbored novel homozygous deletions of exons 2 ~ 22 and exons 14 ~ 15. Patient 4 harbored heterozygous deletion of the DPY19L2 gene, in addition with a rare homozygous deletion of the 3' UTR region. CONCLUSION: DPY19L2 gene variants probably underlay the globozoospermia in the four patients, which has fit an autosomal recessive pattern of inheritance and the characteristics of genomic diseases.

Cancer-testis antigen lactate dehydrogenase C4 as a novel biomarker of male infertility and cancer.

A unique lactate dehydrogenase (LDH) isoenzyme designated as lactate dehydrogenase C4 (LDH-C4) is found in mammalian mature testis and spermatozoa. Thus far, LDH-C4 has been well studied with regard to its gene and amino acid sequences, structure, biological properties, and peptide synthesis. Accumulating evidence has shown that LDH-C4 is closely related to spermatic energy metabolism and plays a critical role in sperm motility, capacitation, and fertilization. Defects in the catalytic activity of LDH-C4 are key to pathophysiological abnormalities underlying infertility. LDH-C4 was originally thought to be present only in mature testis and spermatozoa; however, recent studies have implicated LDH-C4 as a cancer-testis antigen (CTA), owing to its aberrant transcription in a broad spectrum of human neoplasms. This review highlights the recent findings on LDH-C4 with particular emphasis on its role in male infertility and tumors.

Premature termination codons in SMN1 leading to spinal muscular atrophy trigger nonsense-mediated mRNA decay.

BACKGROUND AND AIMS: Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder caused by SMN1 gene mutations. About 40% of SMN1 subtle mutations produced premature termination codons (PTC). This study aims to determine the capacity of these PTCs to trigger nonsense-mediated mRNA decay (NMD) pathway. METHODS: Three nonsense mutations in SMN1, including c.43C > T, c.683T > A and c.844C > T, were investigated by using a minigene system and in vivo splicing assays. Two strategies were supplied: administration of cycloheximide (NMD inhibitor) and knockdown of UPF1 (a key NMD factor) in the cells carrying different minigenes. RESULTS: The wild-type minigene exclusively produced correctly spliced transcripts (FL-SMN1). Both the 683T > A and 844C > T expressed remarkably lower FL-SMN1 than the wild-type cells. After cycloheximide treatment, the FL-SMN1 levels in both the 683T > A and 844C > T were increased significantly compared with that of untreated cells. UPF1 knockdown in both the mutant 683T > A and 844C > T caused a dramatically augmentation of FL-SMN1 as compared to that in the cells treated with non-specific control siRNAs. CONCLUSION: Our data provide evidence that c.683T > A and c.844C > T, but not c.43C > T, in SMN1 leading to SMA trigger NMD using a minigene system. Therefore, NMD should be taken into consideration when exploring the pathogenetic mechanisms for these mutations.

Genetic mutation analysis of 22 patients with congenital absence of vas deferens: a single-center study†.

Congenital absence of the vas deferens (CAVD), a congenital malformation of the male reproductive system, causes obstructive azoospermia and male infertility. Currently, the cystic fibrosis transmembrane conductance regulator (CFTR) has been recognized as the main pathogenic gene in CAVD, with some other genes, such as adhesion G-protein-coupled receptor G2 (ADGRG2), solute carrier family 9 isoform 3 (SLC9A3), sodium channel epithelial 1 subunit beta (SCNN1B), and carbonic anhydrase 12 (CA12), being candidate genes in the pathogenesis of CAVD. However, the frequency and spectrum of these mutations, as well as the pathogenic mechanisms of CAVD, have not been fully investigated. Here, we sequenced all genes with potentially pathogenic mutations using next-generation sequencing and verified all identified variants by Sanger sequencing. Further bioinformatic analysis was performed to predict the pathogenicity of mutations. We described the distribution of the p.V470M, poly-T, and TG-repeat CFTR polymorphisms and identified novel missense mutations in the CFTR and SLC9A3 genes, respectively. Taken together, we identified mutations in the CFTR, ADGRG2, SLC9A3, SCNN1B, and CA12 genes in 22 patients with CAVD, thus broadening the genetic spectrum of Chinese patients with CAVD.

Pathogenesis of acephalic spermatozoa syndrome caused by SUN5 variant.

Acephalic spermatozoa syndrome (ASS) is a rare teratozoospermia that leads to male infertility. Previous work suggested a genetic origin. Variants of Sad1 and UNC84 domain containing 5 (SUN5) are the main genetic cause of ASS; however, its pathogenesis remains unclear. Here, we performed whole-exome sequencing in 10 unrelated ASS and identified 2 homozygous variants, c.381delA[p.V128Sfs7*] and c.675C>A[p.Y225X], and 1 compound variant, c.88 C > T[p.R30X] and c.381 delA [p.V128Sfs7*], in SUN5 in 4 patients. The c.381delA variant had been identified as pathogenic in previous reports, while c.675C>A and c.88 C > T were two novel variants which could lead to a premature termination codon (PTC) and resulted in loss of SUN5, and may also be pathogenic. SUN5 mRNA and protein were present at very low levels in ASS patients with SUN5 nonsense mutation. Furthermore, the distribution of outer dense fiber protein 1 (ODF1) and Nesprin3 was altered in sperm of ASS patients with SUN5 variants. The co-immunoprecipitation analysis indicated that SUN5 and ODF1, SUN5 and Nesprin3, and ODF1 and Nesprin3 interacted with each other in transfected HEK293T cells. Thus, we propose that SUN5, Nesprin3, and ODF1 may form a 'triplet' structure through interactions at neck of sperm. When gene variants resulted in a loss of SUN5, the 'triplet' structure disappears and then the head-tail junction becomes fragile, leading to the occurrence of ASS.

Further identification of a 140bp sequence from amid intron 9 of human FMR1 gene as a new exon.

BACKGROUND: The disease gene of fragile X syndrome, FMR1 gene, encodes fragile X mental retardation protein (FMRP). The alternative splicing (AS) of FMR1 can affect the structure and function of FMRP. However, the biological functions of alternatively spliced isoforms remain elusive. In a previous study, we identified a new 140bp exon from the intron 9 of human FMR1 gene. In this study, we further examined the biological functions of this new exon and its underlying signaling pathways. RESULTS: qRT-PCR results showed that this novel exon is commonly expressed in the peripheral blood of normal individuals. Comparative genomics showed that sequences paralogous to the 140 bp sequence only exist in the genomes of primates. To explore the biological functions of the new transcript, we constructed recombinant eukaryotic expression vectors and lentiviral overexpression vectors. Results showed that the spliced transcript encoded a truncated protein which was expressed mainly in the cell nucleus. Additionally, several genes, including the BEX1 gene involved in mGluR-LTP or mGluR-LTD signaling pathways were significantly influenced when the truncated FMRP was overexpressed. CONCLUSIONS: our work identified a new exon from amid intron 9 of human FMR1 gene with wide expression in normal healthy individuals, which emphasizes the notion that the AS of FMR1 gene is complex and may in a large part account for the multiple functions of FMRP.

Biallelic mutations in DCDC2 cause neonatal sclerosing cholangitis in a Chinese family.

BACKGROUND: Neonatal sclerosing cholangitis (NSC) is a severe cholestatic liver disease, which often develops into end-stage liver disease in childhood and requires liver transplantation. Mutations in CLDN1 and DCDC2 are confirmed to be the main pathogenic mechanism of NSC. METHODS: Whole exon sequencing (WES) was performed to find the possible disease-causing mutations of this family. The mutation was confirmed by Sanger sequencing, and large fragment copy number variation was confirmed by qPCR. RESULTS: We found novel biallelic mutations c.[705-2A>G];[923_1023del] in the DCDC2 gene of the proband. The proband's father had the heterozygous mutation c.705-2A>G, and his mother had a heterozygous c.923_1023del. The proband's younger brother, who had similar clinical manifestations, was found the same biallelic mutations with the proband. CONCLUSION: Novel biallelic mutations were identified in DCDC2 of this Chinese family, according to the American College of Medical Genetics and Genomics (ACMG) guidelines for interpretation of sequence variants, both mutations were classified as pathogenic, which might be the cause of NSC in this family.

Splicing of exon 9a in FMR1 transcripts results in a truncated FMRP with altered subcellular distribution.

FMRP is an RNA-binding protein, loss of which causes fragile X syndrome (FXS). FMRP has several isoforms resulted from alternative splicing (AS) of fragile X mental retardation 1 (FMR1) gene, but their biological functions are still poorly understood. In the analysis of alternatively spliced FMR1 transcripts in the blood cells from a patient with FXS-like phenotypes (normal CGG repeats and no mutation in coding sequence of FMR1), we identified three novel FMR1 transcripts that include a previously unidentified microexon (46 bp), terming the exon 9a. This microexon exists widely in unaffected individuals, inclusion of which introduces an in-frame termination codon. To address whether these exon 9a-containing transcripts could produce protein by evading nonsense-mediated decay (NMD), Western blot was used to analysis blood cell lysate from unaffected individuals and a 34 kDa protein that consistent in size with the molecular weight of the predicted truncated protein produced from mRNA with this microexon was found. Meanwhile, treatment of peripheral blood mononuclear cells with an inhibitor of NMD (Cycloheximide) did not result in significant increase in exon 9a-containing transcripts. Using confocal immunofluorescence, we found the truncated protein displayed both nuclear and cytoplasmic localization in HEK293T and HeLa cells due to lacking C-terminal domains including KH2, NES, and RGG, while the full-length FMRP protein mainly localized in the cytoplasm. Therefore, we hypothesize that the inclusion of this microexon to generate exon 9a-containing transcripts may regulate the normal functionality of FMRP, and the dysregulation of normal FMRP due to increased exon 9a-containing alternatively spliced transcripts in that patient may be associated with the manifestation of FXS phenotype.

[Prokaryotic expression and purification of mouse recombinant myelin proteolipid protein polypeptide].

Objective To establish and optimize the prokaryotic expression method for the recombinant mouse myelin proteolipid protein (PLP, 139-208 aa) which is a critical immunogenic polypeptide of PLP. Methods The sequence coding for PLP139-208 polypeptide was cloned into pET-32a(+) vector. Afterwards, the expression vector prepared in this research was transformed into E. coli BL21, and the recombinant PLP polypeptide was induced to express by isopropyl-ß-D-thiogalactoside (IPTG). Two key prokaryotic expression conditions, IPTG's induction length and temperature, were analyzed for further optimization. The recombinant PLP polypeptide was induced to express by the expression method under the optimal expression conditions, and then was purified by Ni-NTA agarose and amylose resin. Finally, the gain of PLP139-208 polypeptide was verified by Western blot analysis. Results The results in the combinatorial optimization revealed that the expression of PLP139-208 was obtained at a satisfactory level when it was incubated at 23DegreesCelsius for 20 hours with the IPTG concentration of 0.5 mmol/L. Conclusion The optimized prokaryotic expression method for the recombinant mouse PLP139-208 was successfully established and effectively performed. This will shed light on the further researches on the improved preparation for experimental autoimmune encephalitis (EAE, an animal model of multiple sclerosis) and the underlying mechanism underlying PLP-induced autoimmune demyelination.

Hybrid minigene splicing assay verifies the pathogenicity of a novel splice site variant in the COL1A1 gene of a chinese patient with osteogenesis imperfecta type I.

BACKGROUND: Osteogenesis imperfecta (OI) is a rare genetic bone disease associated with brittle bones and fractures. Among all known types, OI type I is the most common type and characterized by increased bone fragility, low bone mass, distinctly blue-gray sclera, and susceptibility to conductive hearing loss beginning in adolescence. Mutations in genes encoding type I collagen (COL1A1 and COL1A2) contribute to the main pathogenic mechanism of OI. METHODS: Subtle mutation of the COL1A1 gene in the proband was detected by targeted next-generation sequencing (NGS) and confirmed by Sanger sequencing. We then assessed the effect of the mutation on the splicing of the COL1A1 gene by bioinformatics prediction and hybrid minigene splicing assay (HMSA). RESULTS: A novel splice site mutation c.1821+1 G>C was discovered in the proband by NGS and further confirmed by Sanger sequencing, which was also simultaneously identified from the proband's mother and elder sister. Bioinformatics predicted that this mutation would result in a disappearance of the 5' donor splice site in intron 26, thereby leading to abnormal splicing and generation of premature stop codon. The follow-up experimental data generated by HMSA was consistent with this prediction. CONCLUSION: Our study identified a novel splice site mutation that caused OI type I in the proband by abnormal splicing and demonstrated that combined applications of NGS, bioinformatics and HMSA are comprehensive and effective methods for diagnosis and aberrant splicing study of OI.

[Genetic analysis of a pedigree affected with congenital high myopia caused by a novel splice site variant of COL11A1 gene].

OBJECTIVE: To analyze genetic variant in a pedigree affected with congenital high myopia. METHODS: Whole exome sequencing (WES) was carried out for the proband. Suspected variation was verified with Sanger sequencing. The pedigree was also subjected to co-segregation analysis. RESULTS: WES has identified a novel splice site heterozygous variant (c.2556+1G>A) in the COL11A1 gene in the proband. Co-segregation analysis of the pedigree showed that the affected mother and two daughters of the proband have carried the same variant(c.2556+1G>A), while his unaffected father and sister did not. Based on the ACMG Standards and Guidelines for the Interpretation of Sequence Variants, the variant was classified as "likely pathogenic" (PVS1+PM2). CONCLUSION: A novel splice variant (c.2556+1G>A) of the COL11A1 gene has been identified in a pedigree affected with congenital high myopia, which probably underlies the disease.

Small Supernumerary Ring Chromosome Derived from an Inverted Duplication of 13q11.2q14 in a Fetus with Coarctation of the Aorta.

Here, we report a molecular characterization of a small supernumerary marker chromosome (sSMC) derived from the most proximal region of 13q present in a fetus with coarctation of the aorta at ultrasound examination during prenatal diagnosis. Cultured umbilical cord blood cells showed a de novo extra ring-shaped sSMC in 76% of the cells using a standard banding technique. SNP array revealed a tetrasomy of about 28.4 Mb in the long arm of chromosome 13 from band 13q11 to 13q14.11 in the fetus's cells. Metaphase/interphase FISH using specific probes located at 13q11, 13q12.11, and 13q14.11, respectively, demonstrated that the supernumerary ring chromosome was derived from an inverted duplication of the region 13q11q14.11 with a conventional centromere. To the best of our knowledge, this is the first time that an inverted duplication of the most proximal region 13q11q14.11 in a ring chromosome is characterized. The findings we presented here deepen our understanding of the clinical consequences of tetrasomy in this region and may be of help for further studies of critical regions in chromosome 13.

The clinical application of preimplantation genetic diagnosis for X-linked retinitis pigmentosa.

OBJECTIVE: To investigate the usefulness of preimplantation genetic diagnosis (PGD) based on mutated allele revealed by sequencing with aneuploidy and linkage analyses (MARSALA) for a pedigree with X-linked retinitis pigmentosa (XLRP). METHODS: One pathogenic mutation (c.494G > A) of the retinitis pigmentosa GTPase regulator (RPGR) gene was identified in a pedigree affected by XLRP. Then, PGD was carried out for the couple, of which the wife was an XLRP carrier. Three blastocysts were biopsied and then MARSALA was performed by next-generation sequencing (NGS). Prenatal diagnosis was also carried out to confirm the PGD results. RESULTS: Three blastocysts were all unaffected. Then, one of the embryos was chosen randomly to be transferred, and the pregnancy was acquired successfully. The results of prenatal diagnosis were consistent with the PGD results. The fetus did not carry RPGR mutation (c.494G > A) and had normal chromosome karyotype. As a result, a healthy baby free of XLRP condition was born. CONCLUSION: The PGD method based on MARSALA was established and applied to a family with XLRP successfully. MARSALA will be a valid tool, not only for XLRP families but also for families affected with other monogenetic disorders, to prevent transmission of the genetic disease from parents to offspring.

3D-cultured adipose tissue-derived stem cells inhibit liver cancer cell migration and invasion through suppressing epithelial-mesenchymal transition.

Adipose tissue-derived stem cells (ADSCs) are considered promising candidates for stem cell therapy; however, the tumorigenicity of ADSCs remains controversial. The present study aimed to investigate the association between ADSCs and liver cancer cells, and to determine whether culture methods could influence the effects of ADSCs on liver cancer cell growth in vitro. Liver cancer cells were treated with ADSCs-conditioned medium (CM) that was collected using the two-dimensional (2D) culture method, sphere culture method, or three-dimensional (3D) culture method. After that, cell viability and apoptosis were measured using CCK-8 and Annexin V-FITC assay, respectively; the cell motility and adhesive capacity were analyzed by scratch wound healing and cell adhesion assay, respectively; the cell migration and invasion were examined by Transwell units; and the molecular mechanisms of ADSCs on effecting epithelial mesenchymal transition signaling pathway were further analyzed. The results demonstrated that ADSCs­CM was able to inhibit the growth of liver cancer cells by inhibiting cell proliferation and promoting cell apoptosis, as well as by suppressing cell motility, adhesive capacity, migration and invasion. In addition, ADSCs­CM was able to suppress cell growth via the downregulation of epithelial­mesenchymal transition signaling. Notably, the enhanced inhibitory effects of ADSCs on liver cancer cell growth could be achieved after cultu-ring using a 3D approach. These findings suggested that ADSCs may provide a novel promising therapeutic approach for the treatment of patients with liver cancer, and the 3D culture method may provide a novel approach to explore the association between ADSCs and cancer.

Associations of rs823128, rs1572931, and rs823156 polymorphisms with reduced Parkinson's disease risks.

The PARK16 locus is considered to play a protective role in Parkinson's disease (PD). However, the epidemiological evidence on the relationships between PARK16 single-nucleotide polymorphisms (rs823128, rs1572931, and rs823156) and PD is inconsistent. Therefore, we carried out a meta-analysis to validate the relationships and performed a bioinformatic analysis to explore putative regulation mechanisms of the single-nucleotide polymorphisms in PD. Through meta-analysis, we confirmed that minor variants of rs823128A>G, rs1572931C>T, and rs823156A>G played protective roles in PD. Through bioinformatic analysis, we predicted that rs823128, rs1572931, and rs823156 as noncoding variants of NUCKS1, RAB29, and SLC41A1, respectively, might affect PD risk by altering the transcription factor-binding capability of the genes. These findings suggest new clues for PD research and potential targets for PD prevention and treatment.

[Phenotypic and genetic analysis of an inv dup(15) case with a BP3:BP3 rearrangement].

OBJECTIVE: To analyze a case of supernumerary marker chromosome (SMC) with combined genetic techniques and explore its correlation with the clinical phenotype. METHODS: The SMC was analyzed with G-banded karyotyping, multiplex ligation dependent probe amplification (MLPA), fluorescence in situ hybridization (FISH), and single nucleotide polymorphism array (SNP-array). RESULTS: G-banding analysis indicated that the patient has a karyotype of 47,XX,+mar. MLPA showed that there were duplications of proximal 15q. FISH assay using D15Z4 probes indicated that the SMC was a pseudodicentric chromosome derived from chromosome 15. And SNP-array revealed that there were two extra copies of 15q11-13 region spanning from locus 20 161 372 to 29 071 810. CONCLUSION: The duplication of Prader-Willi/Angelman syndrome critical region probably underlies the abnormal phenotype of the inv dup(15) case with a BP3:BP3 rearrangement.

A new bioinformatic insight into the associated proteins in psychiatric disorders.

BACKGROUND: Psychiatric diseases severely affect the quality of patients' lives and bring huge economic pressure to their families. Also, the great phenotypic variability among these patients makes it difficult to investigate the pathogenesis. Nowadays, bioinformatics is hopeful to be used as an effective tool for the diagnosis of psychiatric disorders, which can identify sensitive biomarkers and explore associated signaling pathways. METHODS: In this study, we performed an integrated bioinformatic analysis on 1945 mental-associated proteins including 91 secreted proteins and 593 membrane proteins, which were screened from the Universal Protein Resource (Uniport) database. Then the function and pathway enrichment analyses, ontological classification, and constructed PPI network were executed. RESULTS: Our present study revealed that the majority of mental proteins were closely related to metabolic processes and cellular processes. We also identified some significant molecular biomarkers in the progression of mental disorders, such as HRAS, ALS2, SLC6A1, SLC39A12, SIL1, IDUA, NEPH2 and XPO1. Furthermore, it was found that hub proteins, such as COMT, POMC, NPS and BDNF, might be the potential targets for mental disorders therapy. Finally, we demonstrated that psychiatric disorders may share the same signaling pathways with cancers, involving ESR1, BCL2 and MAPK3. CONCLUSION: Our data are expected to contribute to explaining the possible mechanisms of psychiatric diseases and providing a useful reference for the diagnosis and therapy of them.

[Construction and application of eukaryotic expression vector of fragile X mental retardation 1 (FMR1) gene].

Objective To construct a eukaryotic expression vector of human fragile X mental retardation 1 (FMR1) gene and establish stably transfected HeLa cells. Methods The full-length FMR1 cDNA fragment was amplified by PCR and inserted into eukaryotic expression vector pEGFP-N2 using restriction enzyme. The recombinant plasmid pEGFP-N2-FMR1, after identified by restriction digestion and DNA sequencing, was transfected into HeLa cells by lipofectamine 2000. The stably transfected cell line was obtained by screening with G418. The expression and subcellular distribution of FMR protein was identified by Western blotting and immunofluorescence staining combined with laser-scanning confocal microscopy. Results Restriction digestion and DNA sequencing revealed that the eukaryotic expression plasmid of pEGFP-N2-FMR1 was successfully constructed. Besides, Western blotting and immunofluorescence staining showed that GFP-FMR protein was expressed in HeLa cells, which mainly was localized in the cytoplasm. Conclusion The recombinant eukaryotic expression vector of pEGFP-N2-FMR1 has been constructed successfully and stably expressed FMR protein in HeLa cells.