Proteomic analysis of seminal plasma from asthenozoospermia patients reveals proteins that affect oxidative stress responses and semen quality

Asthenozoospermia （AS） is a common cause of human male infertility. In one study, more than 80% of the samples from infertile men had reduced sperm motility. Seminal plasma is a mixture of secretions from the testis, epididymis and several male accessory glands, including the prostate, seminal vesicles and Cowper＇s gland. Studies have shown that seminal plasma contains proteins that are important for sperm motility. To further explore the pathophysiological character of AS, we separated the seminal plasma proteins from AS patients and healthy donors using sodium dodecyl sulfate polyacrylamide gel electrophoresis and in-gel digestion, and then subjected the proteins to liquid chromatography-mass spectrometry （LC-MS/MS） analysis. A total of 741 proteins were identified in the seminal plasma, with a false discovery rate of 3.3%. Using spectral counting, we found that 45 proteins were threefold upregulated and 56 proteins were threefold downregulated in the AS group when compared with the control. Most of these proteins originated from the epididymis and prostate. This study identified a rich source of biomarker candidates for male infertility and indicates that functional abnormalities of the epididymis and prostate can contribute to AS. We identified D J-1--a protein that has been shown elsewhere to be involved in the control of oxidative stress （OS）-as a downregulated protein in AS seminal plasma. The levels of D J-1 in AS seminal plasma were about half of those in the control samples. In addition, the levels of reactive oxygen species were 3.3-fold higher in the AS samples than in the controls. Taken together, these data suggest that downregulation of DJ-1 is involved in OS in semen, and therefore affects the quality of the semen.

The role of Dby mRNA in early development of male mouse zygotes

Ejaculated mammalian spermatozoa contain a complex yet specific population of mRNA. However, the possible roles that mRNA has in early zygotic and embryonic development remain unclear. We found that Dby mRNA is selectively retained in capacitated mouse spermatozoa, and is transferred into the oocyte during fertilization by reverse transcription-polymerase chain reaction even though no DBY protein expression is detected. The cellular location ofDby mRNA is seen in the post-acrosome region, and it comprises nearly half of the mouse spermatozoa in in situ hybridization. In contrast, transcripts of the control gene, Smcy, are not detected in capacitated mouse spermatozoa, although the H-Y antigen encoded by Smcy is expressed on the surface of the spermatozoa. In our microinjection experiment, the zygotic development rate of the as-Dby male pronucleus injection group was significantly lower than that of the as-Smcy male pronucleus injection group （35.9% vs. 95%, P = 0.001） and the as-Dby female pronucleus injection group （35.9% vs. 93.8%, P = 0.001）. The rate of male-developed zygotes was also lower than that of the as-Smcy male pronucleus injection group （17.4% vs. 57.9%, P = 0.002） and the as-Dby female pronucleus injection group （17.4% vs. 54.1%, P = 0.002）. Thus, we conclude that Dby mRNA is selectively retained in capaci- tated mouse spermatozoa, and it has an important role in the early zygotic development of male mouse zygotes. This might imply that spermatozoa mRNA is involved in early zygotic and embryonic stages of reproduction.

Long-Term Impact of Maternal Protein Malnutrition on Learning and Memory Abilities and DNA Methylating Profiles of the Nervous System in Offspring Rats

Objective: To determine the mechanisms by which protein deficiency during pregnancy can lead to long-term alterations in learning and memory abilities of the offspring in rats. Study design: Fourty-two pregnant rats were fed control (n = 23) or low protein (n = 19) diets ad libitum until parturition. On the 8th week of post-natal life which represented early adulthood, eighty-four offsprings (control group: n = 52, LP group: n = 32) were determined their learning & memory ability by using the Morris water maze test. Six offprings’ brain tissue (control group: n = 3, LP group: n = 3) was also analysed for DNA methylating profiles, the GO and KEGG pathways, methylation status and twelve for protein expression (control group: n = 6, LP group: n = 6). Results: The offsprings of the protein-deficient-diet fed rats learnt faster initially then lagged behind those of the control rats, especially in female rats (p = 0.035). There were a series of genes methylated in the CpG island and pormoter area. Quantitative Mass Array data showed methylation differences in Grin2b and Grin2b_3CpG 3, 4, & 5 might be the target sites as shown by dual-luciferase assay. A decreased level of protein expression of NMDAR2B was observed. Conclusion: Differential methylation status in Grin2b and changes in expression of NMDAR2B may partially explain the long-term impact of maternal protein deficiency on the cognitive and learning capabilities of offsprings.

A novel mutation in the MITF may be digenic with GJB2 mutations in a large Chinese family of Waardenburg syndrome type Ⅱ

Waardenburg syndrome typeⅡ（WS2） is associated with syndromic deafness.A subset of WS2,WS2A,accounting for approximately 15%of patients,is attributed to mutations in the microphthalmia-associated transcription factor（MITF） gene.We examined the genetic basis of WS2 in a large Chinese family.All 9 exons of the MITF gene,the single coding exon（exon 2） of the most common hereditary deafness gene GJB2 and the mitochondrial DNA（mtDNA） 12S rRNA were sequenced.A novel heterozygous mutation c.[742_743delAAinsT;746_747delCA]in exon 8 of the MITF gene co-segregates with WS2 in the family.The MITF mutation results in a premature termination codon and a truncated MITF protein with only 247 of the 419 wild type amino acids.The deaf proband had this MITF gene heterozygous mutation as well as a c.[109G〉A]＋ [235delC]compound heterozygous pathogenic mutation in the GJB2 gene.No pathogenic mutation was found in mtDNA 12S rRNA in this family.Thus,a novel compound heterozygous mutation,c.[742_743delAAinsT;746_747delCA]in MITF exon 8 was the key genetic reason for WS2 in this family,and a digenic effect of MITF and GJB2 genes may contribute to deafness of the proband.

Changes in IncRNAs and related genes in β-thalassemia minor and β-thalassemia major

β-thalassemia is caused by β-globin gene mutations. However, heterogeneous phenotypes were found in individuals with same genotype, and still undescribed mechanism underlies such variation. We collected blood samples from 30 β-thalassemia major, 30 β-thalassemia minor patients, and 30 matched normal controls. Human lncRNA Array v2.0 （8 × 60 K, Arraystar） was used to detect changes in long non-coding RNAs （lncRNAs） and mRNAs in three samples each from β-thalassemia major, β-thalassemia minor, and control groups. Compared with normal controls, 1424 and 2045 lncRNAs were up- and downregulated, respectively, in β-thalassemia major patients, whereas 623 and 349 lncRNAs were up- and downregulated, respectively, in β-thalassemia minor patients. Compared with β-thalassemia minor group, 1367 and 2356 lncRNAs were up- and downregulated, respectively, in β-thalassemia major group. We selected five lncRNAs that displayed altered expressions （DQ583499, X-inactive specific transcript （Xist）, IincRNA-TPM1, MRFS16P, and lincRNA-RUNX2-2） and confirmed their expression levels in all samples using real-time polymerase chain reaction. Based on coding-non-coding gene co-expression network and gene ontology biological process analyses, several signaling pathways were associated with three common organ systems exhibiting β-thalassemia phenotypes： hematologic, skeletal, and hepatic systems. This study implicates that abnormal expression levels of lncRNAs and mRNA in β-thalassemia cases may be correlated with its various clinical phenotypes.

Chronic exposure to ethanol in male mice may be associated with hearing loss in offspring

Although paternal ethanol （EtOH） abuse has been shown to affect the growth and behavior of offspring, the exact molecular and mechanistic basis remains largely unclear. Methylation alterations in imprinted genes may be related to well-documented teratogenic effects of ethanol. Here we show that chronic paternal ethanol exposure increases the susceptibility to abnormal behavior in offspring through male game epigenetic alteration. In our study, different doses of ethanol （0, 1,1, 3.3 g kg^-1） were administered intra-gastrically to male mice and decreased sperm motility was found in the highest ethanol-exposed group compared with the controls. Data also showed a dose-dependent increase in deaf mice of the paternally ethanol-exposed groups. The methylation of H19, Peg3, Ndn and Snrpn was assessed in paternal spermatozoa and in the cerebral cortices of deaf mice. EtOH affected methylation of Peg3 （CpG 3, 7 and 9） in paternal spermatozoa and in the cerebral cortices of deaf mice, but the level of mRNA expression did not change, suggesting that other gene regulation may be involved in these processes. Overall, chronic paternal ethanol exposure could alter the methylation of imprinted genes in sire spermatozoa that could also be passed on to offspring, giving rise to developmental disorders. Our results provide possible epigenetic evidence for a paternal ethanol exposure contribution to Fetal Alcohol Syndrome （FAS）.

Responsible genes in children with primary vesicoureteral reflux: findings from the Chinese Children Genetic Kidney Disease Database

Background Primary vesicoureteral reflux(VUR)is a common congenital anomaly of the kidney and urinary tract(CAKUT)in childhood.The present study identified the possible genetic contributions to primary VUR in children.Methods Patients with primary VUR were enrolled and analysed based on a national multi-center registration network(Chinese Children Genetic Kidney Disease Database,CCGKDD)that covered 23 different provinces/regions in China from 2014 to 2019.Genetic causes were sought using whole-exome sequencing(WES)or targeted-exome sequencing.Results A total of 379 unrelated patients(male:female 219:160)with primary VUR were recruited.Sixty-four(16.9%)children had extrarenal manifestations,and 165(43.5%)patients showed the coexistence of other CAKUT phenotypes.Eighty-eight patient(23.2%)exhibited impaired renal function at their last visit,and 18 of them(20.5%)developed ESRD at the median age of 7.0(IQR 0.9–11.4)years.A monogenic cause was identified in 28 patients(7.39%).These genes included PAX2(n=4),TNXB(n=3),GATA3(n=3),SLIT2(n=3),ROBO2(n=2),TBX18(n=2),and the other 11 genes(one gene for each patient).There was a significant difference in the rate of gene mutations between patients with or without extrarenal complications(14.1%vs.6%,P=0.035).The frequency of genetic abnormality was not statistically significant based on the coexistence of another CAKUT(9.6%vs.5.6%,P=0.139,Chi-square test)and the grade of reflux(9.4%vs.6.7%,P=0.429).Kaplan–Meier survival curve showed that the presence of genetic mutations did affect renal survival(Log-rank test,P=0.01).PAX2 mutation carriers(HR 5.1,95%CI 1.3–20.0;P=0.02)and TNXB mutation carriers(HR 20.3,95%CI 2.4–168.7;P=0.01)were associated with increased risk of progression to ESRD.Conclusions PAX2,TNXB,GATA3 and SLIT2 were the main underlying monogenic causes and accounted for up to 46.4%of monogenic VUR.Extrarenal complications and renal function were significantly related to the findings of genetic factors in children with primary VUR.Like other types of CAKUT,several genes may be responsible for isolated VUR.

Research progress in pathogenic genes of hereditary non-syndromic mid-frequency deafness

Hearing impairment is considered as the most prevalent impairment worldwide. Almost 600 million people in the world suffer from mild or moderate hearing impairment, an estimated 10% of the human population. Genetic factors play an important role in the pathogenesis of this disorder. Hereditary hearing loss is divided into syndromic hearing loss （associated with other anomalies） and non-syndromic hearing loss （not associated with other anomalies）. Approximately 80% of genetic deafness is non-syndromic. On the basis of the frequency of hearing loss, hereditary non-syndromic hearing loss can be divided into high-, mid-, low-, and total-frequency hearing loss. An audiometric finding of mid-frequency sensorineural hearing loss, or a ＂bowl-shaped＂ audiogram, is uncommon. Up to now, merely 7 loci have been linked to mid-frequency hearing loss. Only four genetic mid- frequency deafness genes, namely, DFNA10 （EYA4）, DFNA8/12 （TECTA）, DFNA13 （COLIIA2）, DFNA44 （CCDC50）, have been reported to date. This review summarizes the research progress of the four genes to draw attention to mid-frequency deafness genes.

Expression of Cx43-related microRNAs in patients with tetralogy of Fallot

Background:Abnormal expression of connexin 43(Cx43)has been reported to play an important role in the development of conotrunccal anomalies.However,less is known about the underlying reason for its abnormal expression.MicroRNAs(miRNAs),as an important part of gene expression regulation,have been implicated in some cardiac diseases.This study aimed to investigate the expression of Cx43 and its related miRNAs in patients with tetralogy of Fallot(TOF),and illustrate the potential role of abnormal miRNAs regulation to Cx43 expression in the pathology of TOF.Methods:Real-time polymerase chain reaction was used to detect the expression of Cx43 and 10 Cx43-related miRNAs in the myocardium from 30 TOF patients and 10 normal controls.Immunohistochemistry was used to detect Cx43 protein expression.Putative miRNA binding sites in the 3'UTR of Cx43 were examined in 200 TOF patients and 200 healthy individuals,using Sanger sequencing,to exclude sequence variations resulting in binding diffi culties of miRNAs.Results:Cx43 mRNA and protein expression in the myocardium tissue was significantly increased in TOF patients.The expression of MiR-1 and 206 was significantly decreased in the TOF patients as compared with the controls(P<0.05).No obvious difference was observed in the expression of the other 7 miRNAs between the TOF patients and controls(P>0.05).No meaningful sequence variation was detected in the putative miR1/206 binding sites in the 3'UTR of Cx43.Conclusions:This study indicated that miR-1 and 206 is down-regulated in TOF patients,which may cause an up-regulation of Cx43 protein's synthesis.It provided a clue that miR-1 and 206 might be involved in the pathogenesis of TOF,additional experiments are needed to determine if in fact,miR-1 and 206 contribute substantially to TOF.

Long non-coding RNA SAP30-2:1 is downregulated in congenital heart disease and regulates cell proliferation by targeting HAND2

Congenital heart disease(CHD)is the most common birth defect worldwide.Long non-coding RNAs(lncRNAs)have been implicated in many diseases.However,their involvement in CHD is not well understood.This study aimed to investigate the role of dysregulated lncRNAs in CHD.We used Gene Expression Omnibus data mining,bioinformatics analysis,and analysis of clinical tissue samples and observed that the novel lncRNA SAP30-2:1 with unknown function was significantly downregulated in damaged cardiac tissues from patients with CHD.Knockdown of lncRNA SAP30-2:1 inhibited the proliferation of human embryonic kidney and AC16 cells and decreased the expression of heart and neural crest derivatives expressed 2(HAND2).Moreover,lncRNA SAP30-2:1 was associated with HAND2 by RNA immunoprecipitation.Overall,these results suggest that lncRNA SAP30-2:1 may be involved in heart development through affecting cell proliferation via targeting HAND2 and may thus represent a novel therapeutic target for CHD.

HIRA Gene is Lower Expressed in the Myocardium of Patients with Tetralogy of Fallot

Background：The most typical cardiac abnormality is conotruncal defects （CTDs） in patients with 22q11 deletion syndrome （22q11DS）.HIRA （histone cell cycle regulator） gene,as one of the candidate genes located at the critical region of 22q11DS,was reported as possibly relevant to CTD in animal models.This study aimed to analyze the level of expression of the HIRA gene in tetralogy of Fallot （TOF） patients and the potential DNA sequence variations in the promoter region.Methods：The messenger RNA （mRNA） expression was examined with quantitative real-time polymerase chain reaction in 39 myocardial tissues of the right ventricular outflow tract （RVOT） from TOF patients and 4 myocardial tissues of RVOT from noncardiac death children.The protein expression was detected using immunohistochemistry in 12 TOF patients and 4 controls.A total of 100 TOF cases and 200 healthy controls were recruited for DNA sequencing.Results：The mRNA and protein expressions of the HIRA gene in the myocardium of the TOF patients were both significantly lower as compared to the controls （P 〈 0.05）.Five single nucleotide polymorphisms （SNPs）,including g.4111A〉G （rs1128399）,g.4265C〉A （rs4585115）,g.4369T〉G （rs2277837）,g.4371C〉A （rs148516780）,and g.4543T〉C （rs111802956）,were found in the promoter region of the HIRA gene.There were no significant differences of frequencies in these SNPs between the TOF patients and the controls （P 〉 0.05）.Conclusion：The abnormal lower expression of the HIRA gene in the myocardium may participate in the pathogenesis of TOF.

Biological Functions and Research Methods of Long Noncoding RNAs

Long noncoding RNAs(lncRNAs)are functional RNA molecules which are longer than 200 nucleotides in length that do not encode proteins;instead,they regulate target gene expression at transcriptional,posttranscriptional,and epigenetic levels.LncRNAs play important roles in various biological processes such as dosage compensation,genomic imprinting,cell cycle regulation,and cell differentiation.Although their characterizations have been relatively straightforward with recent advances in modern biology,the functions of lncRNAs are largely unknown.Herein,we discuss the biological functions and research methods of lncRNAs.

A Novel Functional Missense Mutation p.T219A in Type Gaucher＇s Disease

Background： Gaucher＇s disease （GD） is an autosomal recessive disorder caused by a deficiency of acid β-glucosidase （glucocerebrosidase [GBA]） that results in the accumulation of glucocerebroside within macrophages. Many mutations have been reported to be associated with this disorder. This study aimed to discover more mutations and provide data for the genetic pattern of the gene, which will help the development of quick and accurate genetic diagnostic tools for this disease. Methods： Genomic DNA was obtained from peripheral blood leukocytes of the patient and Sanger sequencing is used to sequence GBA gene. Sequence alignments of mammalian β-GBA （GCase） and three-dimensional protein structure prediction of the mutation were made. A construct of this mutant and its compound heterozygous counterpart were used to measure GCase in vitro. Results： GCase is relatively conserved at p.T219A. This novel mutation differs from its wild-type in structure. Moreover, it also causes a reduction in GCase enzyme activity. Conclusion： This novel mutation （c.655A〉G, p.T219A） is a pathogenic missense mutation, which contributes to GD.

Endogenous tissue factor pathway inhibitor in vascular smooth muscle cells inhibits arterial thrombosis

Tissue factor pathway inhibitor （TFPI） is the main inhibitor of tissue factor-mediated coagulation. TFPI is expressed by endothelial and smooth muscle cells in the vasculature. Endothefium-derived TFPI has been reported to play a regulatory role in arterial thrombosis. However, the role of endogenous TFPI in vascular smooth muscle cells （VSMCs） in thrombosis and vascular disease development has yet to be elucidated. In this TFPI^Flox mice crossbred with Sma-Cre mice were utilized to establish TFPI conditional knockout mice and to examine the effects of VSMC-directed TFPI deletion on development, hemostasis, and thrombosis. The mice with deleted TFPI in VSMCs （TFP^Sma） reproduced viable offspring. Plasma TFPI concentration was reduced 7.2% in the TFPIsma mice compared with TFPI^Flox littermate controls. Plasma TFPI concentration was also detected in the TFPI^Tle2 （mice deleted TFPI in endothefial ceils and cells of hematopoietic origin） mice. Plasma TFPI concentration of the TFPI^Tle2 mice was 80.4% lower （P 〈 0.001） than that of the TFPI^Flox mice. No difference in hemostatic measures （PT, APTT, and tail bleeding） was observed between TFPIsma and TFPI^Flox mice. However, TFP^Sma mice had increased ferric chloride-indueed arterial thrombosis compared with TFPI^Flox littermate controls. Taken together, these data indicated that endogenous TFPI from VSMCs inhibited ferric chloride-induced arterial thrombosis without causing hemostatic effects.

Ablation of Zfhx4 results in early postnatal lethality by disrupting the respiratory center in mice

Breathing is an integrated motor behavior that is driven and controlled by a network of brainstem neurons.Zfhx4 is a zinc finger transcription factor and our results showed that it was specifically expressed in several regions of the mouse brainstem.Mice lacking Zfhx4 died shortly after birth from an apparent inability to initiate respiration.We also found that the electrical rhythm of brainstem‒spinal cord preparations was significantly depressed in Zfhx4-null mice compared to wild-type mice.Immunofluorescence staining revealed that Zfhx4 was coexpressed with Phox2b and Math1 in the brainstem and that Zfhx4 ablation greatly decreased the expression of these proteins,especially in the retrotrapezoid nucleus.Combined ChIP‒seq and mRNA expression microarray analysis identified Phox2b as the direct downstream target gene of Zfhx4,and this finding was validated by ChIP‒qPCR.Previous studies have reported that both Phox2b and Math1 play key roles in the development of the respiratory center,and Phox2b and Math1 knockout mice are neonatal lethal due to severe central apnea.On top of this,our study revealed that Zfhx4 is a critical regulator of Phox2b expression and essential for perinatal breathing.

Genetic Architecture of Childhood Kidney and Urological Diseases in China

Kidney disease is manifested in a wide variety of phenotypes,many of which have an important hereditary component.To delineate the genotypic and phenotypic spectrum of pediatric nephropathy,a multicenter registration system is being imple-mented based on the Chinese Children Genetic Kidney Disease Database(CCGKDD).In this study,all the patients with kidney and urological diseases were recruited from 2014 to 2020.Genetic analysis was conducted using exome sequencing for families with multiple affected individuals with nephropathy or clinical suspicion of a genetic kidney disease owing to early-onset or extrarenal features.The genetic diagnosis was confirmed in 883 of 2256(39.1%)patients from 23 provinces in China.Phenotypic profiles showed that the primary diagnosis included steroid-resistant nephrotic syndrome(SRNS,23.5%),glomerulonephritis(GN,32.2%),congenital anomalies of the kidney and urinary tract(CAKUT,21.2%),cystic renal disease(3.9%),renal calcinosis/stone(3.6%),tubulopathy(9.7%),and chronic kidney disease of unknown etiology(CKDu,5.8%).The pathogenic variants of 105 monogenetic disorders were identified.Ten distinct genomic disorders were identified as pathogenic copy number variants(CNVs)in 11 patients.The diagnostic yield differed by subgroups,and was highest in those with cystic renal disease(66.3%),followed by tubulopathy(58.4%),GN(57.7%),CKDu(43.5%),SRNS(29.2%),renal calcinosis/stone(29.3%)and CAKUT(8.6%).Reverse phenotyping permitted correct identification in 40 cases with clinical reassessment and unexpected genetic conditions.We present the results of the largest cohort of children with kidney disease in China where diagnostic exome sequencing was performed.Our data demonstrate the utility of family-based exome sequencing,and indicate that the combined analysis of genotype and phenotype based on the national patient registry is pivotal to the genetic diagnosis of kidney disease.