Routine screening for classical azoospermia factor deletions of the Y chromosome in azoospermic patients with Klinefelter syndrome

Aim： To evaluate the occurrence of classical azoospermia factor （AZF） deletions of the Y chromosome as a routine examination in azoospermic subjects with Klinefelter syndrome （KS）. Methods： Blood samples were collected from 95 azoospermic subjects with KS （91 subjects had a 47,XXY karyotype and four subjects had a mosaic 47,XXY/46, XY karyotype） and a control group of 93 fertile men. The values of testosterone, follicle stimulating hormone （FSH） and luteinizing hormone （LH） were measured. To determine the presence of Y chromosome microdeletions, polymerase chain reaction （PCR） of five sequence-tagged site primers （sY84, sY 129, sY 134, sY254, sY255） spanning the AZF region, was performed on isolated genomic DNA. Results： Y chromosome microdeletions were not found in any of the 95 azoosperrnic subjects with KS. In addition, using similar conditions of PCR, no microdeletions were observed in the 93 fertile men evaluated. The level of FSH in KS subjects was higher than that in fertile men （38.2 ± 10.3 mIU/mL vs. 5.4 ±2.9 mIU/mL, P 〈 0.001） and the testosterone level was lower than that in the control group （1.7 ±0.3 ng/mL vs. 4.3 ± 1.3 ng/mL, P 〈 0.001）. Conclusion： Our data and review of the published literature suggest that classical AZF deletions might not play a role in predisposing genetic background for the phenotype of azoospermic KS subjects with a 47,XXY karyotype. In addition, routine screening for the classical AZF deletions might not be required for these subjects. Further studies including partial AZFc deletions （e.g. gr/gr or b2/b3） are necessary to establish other mechanism underlying severe spermatogenesis impairment in KS.

The CRISPR/Cas9 induces large genomic fragment deletions of MSTN and phenotypic changes in sheep

The CRISPR/Cas9 system has been extensively used to engineer genetic loci for the generation of knockouts, insertions, and point mutations in animal models. However, many mutations that have been reported in animals are small insertions or deletions. This study used the CRISPR/Cas9 system to induce large DNA fragment deletions in MSTN via three guide RNAs in sheep. This successfully achieved the precise gene editing of the ovine MSTN gene by injecting both Cas9 m RNA and sg RNAs into embryos at the one-cell stage. Of 10 edited animals, 3 animals(30%) exhibited large genomic fragment deletions(~5 kb). Furthermore, the body weights of these 3 animals were significantly different(P0<0.0001, P15=0.001, P30=0.005, P60=0.027) between lambs with large deletions and wildtype lambs. In addition, the edited lambs were also significantly different(P0<0.0001, P15<0.0001, P30=0.002, P60=0.011) compared with wildtype. These results suggest that the generated MSTN knockout sheep is a reliable and effective animal model for further study. Furthermore, this method is time-and labor-saving, and efficient for the creation of animal models for agriculture, biology, and medicine.

Novel approach to identifying the hepatitis B virus pre-S deletions associated with hepatocellular carcinoma

AIM: To develop a novel non-sequencing method for the detection of hepatitis B virus(HBV) pre-S deletion mutants in HBV carriers.METHODS: The entire region of HBV pre-S1 and pre-S2 was amplified by polymerase chain reaction(PCR). The size of PCR products was subsequently determined by capillary gel electrophoresis(CGE). CGE were carried out in a PACE-MDQ instrument equipped with a UV detector set at 254 nm. The samples were separated in 50 μm ID eCAP Neutral Coated Capillaries using a voltage of 6 kV for 30 min. Data acquisition and analysis were performed using the 32 Karat Software. A total of114 DNA clones containing different sizes of the HBV pre-S gene were used to determine the accuracy of the CGE method. One hundred and fifty seven hepatocellular carcinoma(HCC) and 160 non-HCC patients were recruited into the study to assess the association between HBV pre-S deletion and HCC by using the newly-established CGE method. Nine HCC cases with HBV pre-S deletion at the diagnosis year were selected to conduct a longitudinal observation using serial serum samples collected 2-9 years prior to HCC diagnosis.RESULTS: CGE allowed the separation of PCR products differing in size > 3 bp and was able to identify 10% of the deleted DNA in a background of wild-type DNA. The accuracy rate of CGE-based analysis was 99.1% compared with the clone sequencing results. Using this assay, pre-S deletion was more frequently found in HCC patients than in non-HCC controls(47.1% vs 28.1%, P < 0.001). Interestingly, the increased risk of HCC was mainly contributed by the short deletion of pre-S. While the deletion ≤ 99 bp was associated with a 2.971-fold increased risk of HCC(95%CI: 1.723-5.122, P < 0.001), large deletion(> 99 bp) did not show any association with HCC(P = 0.918, OR = 0.966, 95%CI: 0.501-1.863). Of the 9 patients who carried pre-S deletions at the stage of HCC, 88.9%(8/9) had deletions 2-5 years prior to HCC, while only 44.4%4(4/9) contained such deletions 6-9 years prior to HCC.CONCLUSION: CGE is a sensitive approach for HBV pre-S deletion analysis. Pre-S deletion, especially for short DNA fragment deletion, is a useful predictive marker for HCC.

PCR analysis of Yq microdeletions in infertile males, a study from South India

AIM: To estimate the frequency of microdeletions in the long arm of Y-chromosome of 20 infertile males from South India. METHODS: Polymerase chain reaction (PCR) amplification using Y-specific STS of azoospermia factor (AZF) regions i.e., SY 84 for AZFa, SY 127 for AZFb and SY 254 for AZFc. RESULTS: Of the 20 infertile subjects 3 (15 %), one azoospermic and two oligozoospermic, showed microdeletions in the AZF region of Y-chromosome. CONCLUSION: The frequency of deletions involving AZF region of the Y-chromosome is 15 % in azoospermic and severely oligozoospermic infertile men. PCR amplification of AZF locus is useful for the diagnosis of microdeletions in the Y-chromosome.

Influence of high-molecular-weight glutenin subunit deletions at the Glu-A1 and Glu-D1 loci on protein body development, protein components and dough properties of wheat(Triticum aestivum L.)

High-molecular-weight glutenin subunits(HMW-GSs) play a critical role in determining the viscoelastic properties of wheat. As the organelle where proteins are stored, the development of protein bodies(PBs) reflects the status of protein synthesis and also affects grain quality to a great extent. In this study, with special materials of four near-isogenic lines in a Yangmai 18 background we created, the effects of Glu-A1 and Glu-D1 loci deletions on the development and morphological properties of the protein body, protein components and dough properties were investigated. The results showed that the deletion of the HMW-GS subunit delayed the development process of the PBs, and slowed the increases of volume and area of PBs from 10 days after anthesis(DAA) onwards. In contrast, the areas of PBs at 25 DAA, the middle or late stage of endosperm development, showed no distinguishable differences among the four lines. Compared to the wild type and single null type in Glu-A1, the ratios of HMW-GSs to low-molecular-weight glutenin subunits(LMW-GSs), glutenin macropolymer(GMP) content, mixograph parameters as well as extension parameters decreased in the single null type in Glu-D1 and double null type in Glu-A1 and Glu-D1, while the ratios of gliadins(Gli)/glutenins(Glu) in those types increased. The absence of Glu-D1 subunits decreased both dough strength and extensibility significantly compared to the Glu-A1 deletion type. These results provide a detailed description of the effect of HMW-GS deletion on PBs, protein traits and dough properties, and contribute to the utilization of Glu-D1 deletion germplasm in weak gluten wheat improvement for use in cookies, cakes and southern steamed bread in China and liquor processing.

Identification of Two Novel Mitochondrial DNA Deletions Induced by Ionizing Radiation

Abstract Objective We identify ionizing radiation-induced mitochondrial DNA （mtDNA） deletions in human lymphocytes and their distribution in normal populations. Methods Long-range polymerase chain reactions （PCR） using two pairs of primers specific for the human mitochondrial genome were used to analyze the lymphoblastoid cell line following exposure to 10 Gy 6~Co y-rays. Limited-condition PCR, cloning and sequencing techniques were applied to verify the mtDNA deletions detected with long-range PCR. Human peripheral blood samples were irradiated with 0, 2 and 6 Gy ^60Co y-rays, and real-time PCR analysis was performed to validate the mtDNA deletions. In order to know the distribution of mtDNA deletions in normal population, 222 healthy Chinese adults were also investigated. Results Two mtDNA deletions, a 7455-bp deletion （nt475-nt7929 in heavy strand） and a 9225-bp deletion （nt7714 -nt369 in heavy strand）, occurring between two 8-bp direct repeats, were identified in lymphoblastoid cells using long-range PCR, limited-condition PCR and sequencing. These results were also observed for ^60Co y-rays irradiated human peripheral blood cells. Conclusion Two novel mtDNA deletions, a 7455-bp deletion and a 9225-bp deletion, were induced by ionizing radiation. The rate of the mtDNA deletions within a normal population was related to the donors＇ age, but was independent of gender.

Characterization of genetically engineered mouse models carrying Col2a1-cre-induced deletions of Lrp5 and/or Lrp6

Mice carrying Collagen2a1-cre-mediated deletions of Lrp5 and/or Lrp6 were created and characterized.Mice lacking either gene alone were viable and fertile with normal knee morphology.Mice in which both Lrp5 and Lrp6 were conditionally ablated via Collagen2al-cre-mediated deletion displayed severe defects in skeletal development during embryogenesis.In addition,adult mice carrying Collagen2al-cre-mediated deletions of Lrp5 and/or Lrp6 displayed low bone mass suggesting that the Collagen2a1-cre transgene was active in cells that subsequently differentiated into osteoblasts.In both embryonic skeletal development and establishment of adult bone mass,Lrp5 and Lrp6 carry out redundant functions.

Gonadotropin and Testosterone hormone’s serum levels and partial deletions in the AZFc region in Iranian oligozoospermia infertile males

To investigate the relation of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and Testosterone serum levels with partial deletions in the AZFc region in Iranian oligozoospermia males. Material and methods: thirty infertile oligozoospermia and 52 Iranian fertile men included. The hormonal assays were measured by the Radioimmunoassay (RIA). Multiplex polymerase chain reaction (M-PCR) using eight sequence-tagged site (STS) markers were measured on the Yq11 chromosome. Results: The mean of FSH and LH levels in all oligozoospermia males were higher than fertile men (p < 0.001) and testosterone was lower significantly (p < 0.001). Five patients showed partial deletions in AZFc region (four had gr/gr and one had b2/b3 deletions). Six fertile men showed partial deletions (five gr/gr and one b2/b3) with higher level of FSH, LH in their group (p < 0.05). Conclusion: According to high incidence of partial deletions in the AZFc region among Iranian oligozoospermia males, hormonal assay and molecular screening should be advised before considering for ART treatments.

Large deletions within the spinal muscular atrophy gene region in a patient with spinal muscular atrophy type 3

Spinal muscular atrophy （SMA） is an autosomal recessive neuromuscular disorder characterized by degeneration and loss of anterior horn cells in the spinal cord and brain stem nuclei, leading to progressive limb and trunk paralysis and muscular atrophy. Depending on the age of onset and maximum muscular function achieved, SMA is recognized as SMA1, SMA2, SMA3 or SMA4, and most patients have a deletion or truncation of the survival motor neuron 1 （SMN1） gene. In this report, we present a patient with a mild SMA phenotype, SMA3, and define his genetic abnormality. Tetra-primer amplification refractory mutation system PCR combined with restriction fragment length polymorphism analysis and array comparative genomic hybridization were used to determine the genetic variations in this patient. A 500 kb deletion in chromosome 5q13.2, including homozygous deletion of neuronal apoptosis inhibitory protein, and heterozygous deletion of occludin and B-double prime 1 was identified. This SMA region deletion did not involve SMN, indicating that SMN was likely to function normally. The phenotype was dependent of the large deletion and neuronal apoptosis inhibitory protein, occludin and B-double prime 1 may be candidate genes for SMA3.

Exon Deletions of Parkin Gene in Patients with Parkinson Disease

Summary: Mutations in the parkin gene have recently been identified in familial and isolated patients with early-onset Parkinson disease (PD) and that subregions between exon 2 and 4 of the parkin gene are hot spots of deletive mutations. To study the distribution of deletions in the parkin gene among variant subset patients with PD in China, and to explore the role of parkin gene in the pathogenesis of PD, 63 patients were divided into early onset and later onset groups. Exons 1-12 were amplified by PCR, templated by the genomic DNA of patients, and then the deletion distribution detected by agarose electrophoresis. Four patients were found to be carrier of exon deletions in 63 patients with PD. The location of the deletion was on exon 2 (1 case), exon 3 (2 cases) and exon 4 (1 case). All patients were belong to the group of early onset PD. The results showed that parkin gene deletion on exon 2, exon 3 and exon 4 found in Chinese population contributes partly to early onset PD.

A VNTR ELEMENT ASSOCIATED WITH STEROID SULFATAES GENE DELETIONS STIMULATES RECOMBINATIONIN CULTURED CELLS

Steroid sulfatase deficiency is a common genetic disorder, with a prevalence of approximately one in every 3500 males world wide.About 90% of these patients have complete gene deletions, which appear to result from recombination between members of a low-copy repeat family (CR1-232 is the prototype) that flank the gene. RUI and RU2 are two VNTR elements found within each of these family members.The RU1 consists of 30bp repeating units and its length shows minimal variation among individuals. The RU2 element consists of repeating sequences which are highly asymmetric, with about 90% purines and no C's on one strand, and range from 0. 6kb to over 23kb among different individuals. We conducted a study to determine if the RU1 and RU2 elements can promote recombination in an in vivo test system.We inserted these elements adjacent to the neo gene in each of two pSV2neo derivatives. One of which has a deletion in the 5' portion of neo gene and the other having a deletion in the 3'portion. These plasmids were combined and used to transfect EJ human bladder tumor cells. Survival of cells in G418 indicates restoration of a functional neo gene by recombination between two deletion constructs. Thus counting G418 resistant colonies gives a quantitative measure of the enhancement of recombination by the inserted VNTR elements.The results showed no effect on recombination by the inserted RU1 element(compared to the insertion of a nonspecific sequence), while the RU2 element stimulated.recombination by 3. 5-fold (P< 0.01). A separate set of constructs placed RU1 or RU2 within the nitron of an exon trapping vector. Following transfection of cells, recombination events were monitored by a quantitative PCR assay that detected the approximation of primer banding sites (as a result of recombination).These studies showed that, as in the first set of experiments, the highly variable RU2 element is capable of stimulating somatic recombination in tnammalian cells.

Ratio quantification of gene dosage by Agilent 2100 Bioanalyzer for detection of somatic gene deletions

We applied the Agilent 2100 Bioanalyzer, a microfluidics-based electrophoresis instrument, and introduced an accurate and consistent parameter, the relative product yield ratio (ROY), to detect somatic gene deletions in tumor cells. Briefly for such purpose, the Agilent 2100 Bioanalyzer quantified the ROY of a target gene to an endogenous internal control, both of which were initially co-amplified by Robust-Dosage PCR (RD- PCR). Herein, we extensively validated this approach. We first tested the effect of well positions on the Agilent DNA chip. We found that no matter which wells the samples were loaded in, the ROY was consistent with coefficient of variation (CV) < 2%. Then we tested the effect of product concentrations that varied 8-fold, and the ROY was also consistent with CV < 3.5%. Furthermore, we applied this approach to identify six somatic KRAS deletions in non-small cell lung cancer patients, confirming our previous findings. Thus, the Agilent 2100 Bioanalyzer is simple, accurate, quick, and ultimately able to replace conventional gel electrophoresis for the detection of somatic gene deletions.

Clinical Performance of Cell-Free Fetal DNA Testing for Fetal Aneuploidies and Subchromosomal Deletions/Duplications in a Cohort of 19,531 Pregnancies

Objective:We aim to assess the clinical performance of cell-free fetal DNA(cffDNA)testing for detecting common fetal aneuploidies as well as subchromosomal deletions/duplications and explore the pregnancy decisions in screen-positive cases.Methods:A cohort of 19,531 pregnant women was offered cffDNA testing for detection of trisomies 21,18,and 13(T21,T18,and T13);sex chromosome aneuploidies(SCAs);and subchromosomal deletions/duplications.Screen-positive cases were confirmed by karyotyping and single-nucleotide polymorphism array analysis.Results:A total of 47 cases failed the test.The overall screen-positive rate of chromosomal abnormalities was 1.07%(208/19,484),including 57 cases with T21,18 cases with T18,7 cases with T13,106 cases with SCAs,and 20 cases of subchromosomal deletions/duplications.Positive predictive values were 91.30%(42/46),38.46%(5/13),33.33%(2/6),41.33%(31/75),and 27.78%(5/18),respectively.There was no significant difference in the screening of fetal chromosomal aneuploidies in the high-risk group compared with the low-risk group(P>0.05).All of the pregnant women who had confirmed fetal T21,T18,or T13 terminated their pregnancies,except for a case of T13 mosaic,whereas 45.16%(14/31)of women with fetal SCAs continued their pregnancies.Furthermore,17 pregnant women with positive screens for T21,T18,or T13 without a subsequent diagnosis chose to terminate their pregnancy,whereas 29 of 31 women with SCAs chose to continue their pregnancies.Conclusions:CffDNA testing exhibited good screening accuracy for T21,T18,and T13 and also contributed to detecting fetal SCAs and subchromosomal deletions/duplications.Pregnant women with fetal 47,XXX or 47,XYY were more willing to terminate their pregnancy than those with fetal 45,X or 47,XXY.

Melatonin improves synapse development by PI3K/Akt signaling in a mouse model of autism spectrum disorder

Autism spectrum disorders are a group of neurodevelopmental disorders involving more than 1100 genes,including Ctnnd2 as a candidate gene.Ctnnd2knockout mice,serving as an animal model of autis m,have been demonstrated to exhibit decreased density of dendritic spines.The role of melatonin,as a neuro hormone capable of effectively alleviating social interaction deficits and regulating the development of dendritic spines,in Ctnnd2 deletion-induced nerve injury remains unclea r.In the present study,we discove red that the deletion of exon 2 of the Ctnnd2 gene was linked to social interaction deficits,spine loss,impaired inhibitory neurons,and suppressed phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt) signal pathway in the prefrontal cortex.Our findings demonstrated that the long-term oral administration of melatonin for 28 days effectively alleviated the aforementioned abnormalities in Ctnnd2 gene-knockout mice.Furthermore,the administration of melatonin in the prefro ntal cortex was found to improve synaptic function and activate the PI3K/Akt signal pathway in this region.The pharmacological blockade of the PI3K/Akt signal pathway with a PI3K/Akt inhibitor,wo rtmannin,and melatonin receptor antagonists,luzindole and 4-phenyl-2-propionamidotetralin,prevented the melatonin-induced enhancement of GABAergic synaptic function.These findings suggest that melatonin treatment can ameliorate GABAe rgic synaptic function by activating the PI3K/Akt signal pathway,which may contribute to the improvement of dendritic spine abnormalities in autism spectrum disorders.

Lack of an Additive Effect between the Deletions of Klf5 and Nkx3-1 in Mouse Prostatic Tumorigenesis

Prostate cancer is one of the most common malignancies.The development and progression of prostate cancer are driven by a series of genetic and epigenetic events including gene amplification that activates oncogenes and chromosomal deletion that inactivates tumor suppressor genes.Whereas gene amplification occurs in human prostate cancer,gene deletion is more common,

Large chromosomal segment deletions by CRISPR/LbCpf1-mediated multiplex gene editing in soybean

The creation of new soybean varieties has been limited by genomic duplication and redundancy.Efficient multiplex gene editing and large chromosomal segment deletion through clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)systems are promising strategies for overcoming these obstacles.CRISPR/Cpf1 is a robust tool for multiplex gene editing.However,large chromosomal excision mediated by CRISPR/Cpf1 has been reported in only a few non-plant species.Here,we report on CRISPR/LbCpf1-induced large chromosomal segment deletions in soybean using multiplex gene targeting.The CRISPR/LbCpf1 system was optimized for direct repeat and guide RNA lengths in crispr RNA(crRNA)array.The editing efficiency was evaluated using LbCpf1 driven by the CaMV35S and soybean ubiquitin promoter.The optimized system exhibited editing efficiencies of up to 91.7%.Our results showed eight gene targets could be edited simultaneously in one step when a single eight-gRNA-target crRNA array was employed,with an efficiency of up to 17.1%.We successfully employed CRISPR/LbCpf1 to produce small fragments(<1 Kb)and large chromosomal segment deletions(10 Kb-1 Mb)involving four different gene clusters in soybean.Together,these data demonstrate the power of the CRISPR/LbCpf1 platform for multiplex gene editing and chromosomal segment deletion in soybean,supporting the use of this technology in both basic research and agricultural applications.

Chromosomal deletions mediated by CRISPR/Cas9 in Helicoverpa armigera

Helicoverpa armigera, cotton bollworm, is one of the most disastrous pests worldwide, threatening various food and economic crops. Functional genomic tools may provide efficient approaches for its management. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, dependent on a single guide RNA (sgRNA), has been used to induce indels for targeted mutagenesis in cotton bollworm. However, genomic deletions may be more desirable to disrupt the function of noncoding genes or regulatory sequences. By injecting two sgRNAs with Cas9 protein targeting different exons, we obtained predictable genomic deletions of several hundred bases. We achieved this type of modification with different combinations of sgRNA pairs, including HaCad and HaABCC2. Our finding indicated that CRISPR/Cas9 can be used as an efficient tool to engineer genomes with chromosomal deletion in H. armigera.

HBV surface antigen proteins with deletions in the preS region

Four pairs of HBV surface antigen genes, in which the preS region was partially deleted,were constructed by the polymerase chain reaction (PCR). The comparison of the levels of the expression in mammalian cells of these genes and the ones constructed before, and the properties of these gene products showed that the missing of a part of the preS region did not affect the overall spatial structure of the S region and the surface localization of the preS region. The removal of the preS1 retention sequence (a. a. 2-19) alleviated significantly the shelter of the major antigenic determinants in the S region by the preS sequence. It was found that the long preS region seriously impaired the secretion of the surface antigen proteins from mammalian cells. In addition to the previously reported preSl retention sequence, the preSl sequence (a.a. 48-65) may also inhibit the secretion of the surface antigen proteins, whereas the preS2 region exerts no major influence on the retention of the large surface antigen

Cre-recombinase systems for induction of neuronspecific knockout models:a guide for biomedical researchers

Gene deletion has been a valuable tool for unraveling the mysteries of molecular biology.Early approaches included gene trapping and gene targetting to disrupt or delete a gene randomly or at a specific location,respectively.Using these technologies in mouse embryos led to the generation of mouse knocko ut models and many scientific discoveries.The efficacy and specificity of these approaches have significantly increased with the advent of new technology such as cluste red regula rly inters paced short palindromic repeats for targetted gene deletion.However,several limitations including unwanted off-target gene deletion have hindered their widespread use in the field.Crerecombinase technology has provided additional capacity for cell-specific gene deletion.In this review,we provide a summary of currently available literature on the application of this system for targetted deletion of neuronal genes.This article has been constructed to provide some background info rmation for the new trainees on the mechanism and to provide necessary information for the design,and application of the Cre-recombinase system thro ugh reviewing the most f requent promoters that are currently available for genetic manipulation of neuro ns.We additionally will provide a summary of the latest technological developments that can be used for targeting neurons.This may also serve as a general guide for the selection of appropriate models for biomedical research.