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Journal of Assisted Reproduction and... May 2015Recent studies have shown that genetic abnormalities may be responsible for most unknown cases of male infertility. Human Nsun7 gene, which is located on chromosome4,... (Comparative Study)
Comparative Study
PURPOSE
Recent studies have shown that genetic abnormalities may be responsible for most unknown cases of male infertility. Human Nsun7 gene, which is located on chromosome4, has a role in sperm motility by encoding the putative methyltransferase Nsun7 protein. The aim of the present study was to investigate the mutations of exon4 in the Nsun7 gene, which is associated with sperm motility defect.
METHODS
Semen samples including those of fertile normospermic (normal), infertile oligospermic (with normal sperm motility), and infertile asthenospermic (with reduced sperm motility) men were collected from the Omid and Fatemezahra IVF centres (Babol, Iran). These samples were then analysed on the basis of World Health Organization guidelines using the general phenol-chloroform DNA extraction method. Exon4 was amplified using Sun-F/Sun-R primers. Samples from asthenospermic men, which showed different patterns of movement on single-strand conformation polymorphism compared with normal and oligospermic samples, were identified and subjected to sequencing for further identification of possible mutations.
RESULTS
Analysis of extracted sperm proteins showed that the rate of Nsun7 decreased. Likewise, direct sequencing of PCR products, along with their analysis, confirmed the deletion mutation of adenine in location 11337 of the Nsun7 gene in asthenospermic men. Comparison of normal and mutant protein structures of Nsun7 indicated that the A11337-deletion of the exon4 resulted in the valine residues-157 with GTA-codon in normospermic replaced with TAG-early stop codon in asthenospermic samples, causing an abortive protein product with amino acid sequence shorter than normal. The secondary structure of the protein, the protein folding, and ligand binding sites were changed, indicating the impairment of the protein function.
CONCLUSIONS
Because the Nsun7 gene products have a role in sperm motility, it will lead to impairment in the activity of the protein and motility of sperm flagella as well as male infertility if a mutation occurs in this gene.
Topics: Adult; Amino Acid Sequence; Asthenozoospermia; Base Sequence; Case-Control Studies; DNA; Humans; Infertility, Male; Male; Methyltransferases; Molecular Sequence Data; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Prognosis; Proteolysis; Risk Factors; Semen; Sequence Deletion; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Sperm Motility; Young Adult
PubMed: 25702163
DOI: 10.1007/s10815-015-0443-0 -
Journal of Clinical Immunology May 2023Leukocyte adhesion deficiency-III (LAD-III) is an extremely rare autosomal recessive syndrome caused by mutations in FERMT3, the gene encoding kindlin-3. The genetic...
Leukocyte adhesion deficiency-III (LAD-III) is an extremely rare autosomal recessive syndrome caused by mutations in FERMT3, the gene encoding kindlin-3. The genetic alterations in this gene lead to abnormal expression or activity of kindlin-3 in leukocytes and platelets. Kindlin-3 acts as an important regulator of integrin activation. LAD-III has features of the bleeding syndrome of Glanzmann and also of leukocyte adhesion deficiency. In this study, we report on two families, one of Turkish and one of Syrian origin, with clinical features of LAD-III, loss of kindlin-3 protein expression, and a functional leukocyte defect. A novel, homozygous deletion in FERMT3 (c.921delC, p.Ser307Argfs*21) was found in the Turkish patient. The parents were carriers of the mutation, consistent with an autosomal recessive inheritance. A common c.1525C > T (p.Arg509*) mutation was found in the Syrian patient. In conclusion, beside the variant c.1525C > T in the FERMT3 gene, which was previously found in more than 15 patients in Anatolia, our study is the first to identify the novel homozygous variant c.921delC in the FERMT3 gene.
Topics: Humans; CD18 Antigens; Cell Adhesion; Homozygote; Leukocyte-Adhesion Deficiency Syndrome; Sequence Deletion; Turkey
PubMed: 36648575
DOI: 10.1007/s10875-022-01420-4 -
Scientific Reports Dec 2021A germline 29.5-kb deletion variant removes the 3' end of the APOBEC3A gene and a large part of APOBEC3B, creating a hybrid gene that has been linked to increased...
A germline 29.5-kb deletion variant removes the 3' end of the APOBEC3A gene and a large part of APOBEC3B, creating a hybrid gene that has been linked to increased APOBEC3 activity and DNA damage in human cancers. We genotyped the APOBEC3A/B deletion in hospital-based samples of 1398 Norwegian epithelial ovarian cancer patients without detected BRCA1/2 germline mutations and compared to 1,918 healthy female controls, to assess the potential cancer risk associated with the deletion. We observed an association between APOBEC3A/B status and reduced risk for ovarian cancer (OR = 0.75; CI = 0.61-0.91; p = 0.003) applying the dominant model. Similar results were found in other models. The association was observed both in non-serous and serous cases (dominant model: OR = 0.69; CI = 0.50-0.95; p = 0.018 and OR = 0.77; CI = 0.62-0.96; p = 0.019, respectively) as well as within high-grade serous cases (dominant model: OR = 0.79; CI = 0.59-1.05). For validation purposes, we mined an available large multinational GWAS-based data set of > 18,000 cases and > 26,000 controls for SNP rs12628403, known to be in linkage disequilibrium with the APOBEC3A/B deletion. We found a non-significant trend for SNP rs12628403 being linked to reduced risk of ovarian cancer in general and similar trends for all subtypes. For clear cell cancers, the risk reduction reached significance (OR = 0.85; CI = 0.69-1.00).
Topics: Aged; Biomarkers, Tumor; Case-Control Studies; Cytidine Deaminase; Female; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Genotype; Germ-Line Mutation; Humans; Middle Aged; Minor Histocompatibility Antigens; Ovarian Neoplasms; Polymorphism, Genetic; Proteins; Sequence Deletion
PubMed: 34873230
DOI: 10.1038/s41598-021-02820-z -
Human Genetics Nov 2022Cerebral cavernous malformations (CCM) are vascular malformations consisting of collections of enlarged capillaries occurring in the brain or spinal cord. These vascular...
Cerebral cavernous malformations (CCM) are vascular malformations consisting of collections of enlarged capillaries occurring in the brain or spinal cord. These vascular malformations can occur sporadically or susceptibility to develop these can be inherited as an autosomal dominant trait due to mutation in one of three genes. Over a decade ago, we described a 77.6 Kb germline deletion spanning exons 2-10 in the CCM2 gene found in multiple affected individuals from seemingly unrelated families. Segregation analysis using linked, microsatellite markers indicated that this deletion may have arisen at least twice independently. In the ensuing decades, many more CCM patients have been identified with this deletion. In this present study we examined 27 reportedly unrelated affected individuals with this deletion. To investigate the origin of the deletion at base pair level resolution, we sequenced approximately 10 Kb upstream and downstream from the recombination junction on the deleted allele. All patients showed the identical SNP haplotype across this combined 20 Kb interval. In parallel, genealogical records have traced 11 of these individuals to five separate pedigrees dating as far back as the 1600-1700s. These haplotype and genealogical data suggest that these families and the remaining "unrelated" samples converge on a common ancestor due to a founder mutation occurring centuries ago on the North American continent. We also note that another gene, NACAD, is included in this deletion. Although patient self-reporting does not indicate an apparent phenotypic consequence for heterozygous deletion of NACAD, further investigation is warranted for these patients.
Topics: Carrier Proteins; Hemangioma, Cavernous, Central Nervous System; Humans; Intellectual Disability; Micrognathism; Mutation; Pedigree; Proto-Oncogene Proteins; Ribs; Sequence Deletion
PubMed: 35488064
DOI: 10.1007/s00439-022-02458-5 -
Cancer Research and Treatment Oct 2016, the most frequently mutated gene in breast cancer, is more frequently altered in HER2-enriched and basal-like breast cancer. However, no studies have clarified the...
PURPOSE
, the most frequently mutated gene in breast cancer, is more frequently altered in HER2-enriched and basal-like breast cancer. However, no studies have clarified the role of status as a prognostic and predictive marker of triple-negative breast cancer (TNBC).
MATERIALS AND METHODS
We performed p53 immunohistochemistry (IHC), nCounter mRNA expression assay, and DNA sequencing to determine the relationship between alteration and clinical outcomes of TNBC patients.
RESULTS
Seventy-seven of 174 TNBC patients were found to harbor a mutation. Patients with missense mutations showed high protein expression in contrast to patients with deletion mutations (positivity of IHC: wild type vs. missense vs. deletion mutation, 53.6% vs. 89.8% vs. 25.0%, respectively; p < 0.001). mRNA expression was influenced by mutation status (mRNA expression [median]: wild type vs. missense vs. deletion mutation, 207.36± 132.73 vs. 339.61±143.21 vs. 99.53±99.57, respectively; p < 0.001). According to survival analysis, neither class of mutation nor protein or mRNA expression status had any impact on patient prognosis. In subgroup analysis, low mRNA expression was associated with poor prognosis in patients with a missense mutation (5-year distant recurrence-free survival [5Y DRFS]: low vs. high, 50.0% vs. 87.8%; p=0.009), while high mRNA expression with a deletion mutation indicated poor prognosis (5Y DRFS: low vs. high, 91.7% vs. 75.0%; p=0.316).
CONCLUSION
Association between mutation and expression indicates a potential prognostic marker of TNBC; hence both DNA sequencing and mRNA expression analysis may be required to predict the prognosis of TNBC patients.
Topics: Adult; Aged; Biomarkers, Tumor; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; High-Throughput Nucleotide Sequencing; Humans; Middle Aged; Mutation, Missense; Neoplasm Staging; Prognosis; RNA, Messenger; Sequence Deletion; Triple Negative Breast Neoplasms; Tumor Suppressor Protein p53
PubMed: 26910472
DOI: 10.4143/crt.2015.430 -
Molecular Neurodegeneration Sep 2016Christianson Syndrome, a recently identified X-linked neurodevelopmental disorder, is caused by mutations in the human gene SLC9A6 encoding the recycling endosomal...
BACKGROUND
Christianson Syndrome, a recently identified X-linked neurodevelopmental disorder, is caused by mutations in the human gene SLC9A6 encoding the recycling endosomal alkali cation/proton exchanger NHE6. The patients have pronounced limitations in cognitive ability, motor skills and adaptive behaviour. However, the mechanistic basis for this disorder is poorly understood as few of the more than 20 mutations identified thus far have been studied in detail.
METHODS
Here, we examined the molecular and cellular consequences of a 6 base-pair deletion of amino acids Glu(287) and Ser(288) (∆ES) in the predicted seventh transmembrane helix of human NHE6 expressed in established cell lines (CHO/AP-1, HeLa and neuroblastoma SH-SY5Y) and primary cultures of mouse hippocampal neurons by measuring levels of protein expression, stability, membrane trafficking, endosomal function and cell viability.
RESULTS
In the cell lines, immunoblot analyses showed that the nascent mutant protein was properly synthesized and assembled as a homodimer, but its oligosaccharide maturation and half-life were markedly reduced compared to wild-type (WT) and correlated with enhanced ubiquitination leading to both proteasomal and lysosomal degradation. Despite this instability, a measurable fraction of the transporter was correctly sorted to the plasma membrane. However, the rates of clathrin-mediated endocytosis of the ∆ES mutant as well as uptake of companion vesicular cargo, such as the ligand-bound transferrin receptor, were significantly reduced and correlated with excessive endosomal acidification. Notably, ectopic expression of ∆ES but not WT induced apoptosis when examined in AP-1 cells. Similarly, in transfected primary cultures of mouse hippocampal neurons, membrane trafficking of the ∆ES mutant was impaired and elicited marked reductions in total dendritic length, area and arborization, and triggered apoptotic cell death.
CONCLUSIONS
These results suggest that loss-of-function mutations in NHE6 disrupt recycling endosomal function and trafficking of cargo which ultimately leads to neuronal degeneration and cell death in Christianson Syndrome.
Topics: Animals; Ataxia; Brain; Cell Death; Cell Line, Tumor; Endocytosis; Endosomes; Epilepsy; Genetic Diseases, X-Linked; Humans; Intellectual Disability; Mice; Microcephaly; Neurons; Ocular Motility Disorders; Protein Transport; Sequence Deletion; Sodium-Hydrogen Exchangers
PubMed: 27590723
DOI: 10.1186/s13024-016-0129-9 -
Journal of Veterinary Internal Medicine 2014Pituitary dwarfism in German Shepherd Dogs is associated with autosomal recessive inheritance and a mutation in LHX3, resulting in combined pituitary hormone deficiency....
BACKGROUND
Pituitary dwarfism in German Shepherd Dogs is associated with autosomal recessive inheritance and a mutation in LHX3, resulting in combined pituitary hormone deficiency. Congenital dwarfism also is encountered in breeds related to German Shepherd Dogs, such as Saarloos and Czechoslovakian wolfdogs.
OBJECTIVES
To investigate whether Saarloos and Czechoslovakian wolfdog dwarfs have the same LHX3 mutation as do Germans Shepherd Dog dwarfs. A specific aim was to determine the carrier frequency among Saarloos and Czechoslovakian wolfdogs used for breeding.
ANIMALS
Two client-owned Saarloos wolfdogs and 4 client-owned Czechoslovakian wolfdogs with pituitary dwarfism, 239 clinically healthy client-owned Saarloos wolfdogs, and 200 client-owned clinically healthy Czechoslovakian wolfdogs.
METHODS
Genomic DNA was amplified using polymerase chain reaction (PCR). In the Saarloos and Czechoslovakian wolfdog dwarfs, PCR products were analyzed by sequencing. DNA fragment length analysis was performed on the samples from the clinically healthy dogs.
RESULTS
Saarloos and Czechoslovakian wolfdog dwarfs have the same 7 bp deletion in intron 5 of LHX3 as do German Shepherd Dog dwarfs. The frequency of carriers of this mutation among clinically healthy Saarloos and Czechoslovakian wolfdogs used for breeding was 31% and 21%, respectively.
CONCLUSIONS AND CLINICAL IMPORTANCE
An LHX3 mutation is associated with pituitary dwarfism in Saarloos and Czechoslovakian wolfdogs. The rather high frequency of carriers of the mutated gene in the 2 breeds emphasizes the need for screening before breeding. If all breeding animals were genetically tested for the presence of the LHX3 mutation and a correct breeding policy would be implemented, this disease could be eradicated completely.
Topics: Animals; Dog Diseases; Dogs; Dwarfism, Pituitary; Female; Genetic Association Studies; Growth Hormone; Heterozygote; Insulin-Like Growth Factor I; LIM-Homeodomain Proteins; Male; Sequence Deletion; Transcription Factors
PubMed: 25273400
DOI: 10.1111/jvim.12448 -
G3 (Bethesda, Md.) May 2018The CRISPR/Cas9 system, which relies on RNA-guided DNA cleavage to induce site-specific DNA double-strand breaks, is a powerful tool for genome editing. This system has...
The CRISPR/Cas9 system, which relies on RNA-guided DNA cleavage to induce site-specific DNA double-strand breaks, is a powerful tool for genome editing. This system has been successfully adapted for the fission yeast by expressing Cas9 and the single-guide RNA (sgRNA) from a plasmid. In the procedures published to date, the cloning step that introduces a specific sgRNA target sequence into the plasmid is the most tedious and time-consuming. To increase the efficiency of applying the CRISPR/Cas9 system in fission yeast, we here developed a cloning-free procedure that uses gap repair in fission yeast cells to assemble two linear DNA fragments, a gapped Cas9-encoding plasmid and a PCR-amplified sgRNA insert, into a circular plasmid. Both fragments contain only a portion of the or marker so that only the correctly assembled plasmid can confer uracil prototrophy or blasticidin resistance. We show that this gap-repair-based and cloning-free CRISPR/Cas9 procedure permits rapid and efficient point mutation knock-in, endogenous N-terminal tagging, and genomic sequence deletion in fission yeast.
Topics: Base Sequence; CRISPR-Cas Systems; Cloning, Molecular; DNA Repair; Gene Editing; Gene Knock-In Techniques; Point Mutation; Schizosaccharomyces; Sequence Deletion; Temperature
PubMed: 29703785
DOI: 10.1534/g3.118.200164 -
Animal Genetics Oct 2023Cerebellar hypoplasia is a heterogeneous neurological condition in which the cerebellum is smaller than usual or not completely developed. The condition can have genetic...
Cerebellar hypoplasia is a heterogeneous neurological condition in which the cerebellum is smaller than usual or not completely developed. The condition can have genetic origins, with Mendelian-effect mutations described in several mammalian species. Here, we describe a genetic investigation of cerebellar hypoplasia in White Swiss Shepherd dogs, where two affected puppies were identified from a litter with a recent common ancestor on both sides of their pedigree. Whole genome sequencing was conducted for 10 dogs in this family, and filtering of these data based on a recessive transmission hypothesis highlighted five protein-altering candidate variants - including a frameshift-deletion of the Reelin (RELN) gene (p.Val947*). Given the status of RELN as a gene responsible for cerebellar hypoplasia in humans, sheep and mice, these data strongly suggest the loss-of-function variant as underlying these effects. This variant has not been found in other dog breeds nor in a cohort of European White Swiss Shepherds, suggesting a recent mutation event. This finding will support the genotyping of a more diverse sample of dogs, and should aid future management of the harmful allele through optimised mating schemes.
Topics: Animals; Dogs; Humans; Cerebellum; Dog Diseases; Frameshift Mutation; Mammals; Mutation; Sequence Deletion; Switzerland; Reelin Protein
PubMed: 37334487
DOI: 10.1111/age.13336 -
Bosnian Journal of Basic Medical... Feb 2020Presbycusis, or age-related hearing loss, is a prevalent disease that severely affects the physical and mental health of the elderly. Oxidative stress and mitochondrial...
Presbycusis, or age-related hearing loss, is a prevalent disease that severely affects the physical and mental health of the elderly. Oxidative stress and mitochondrial (mt)DNA deletion mutation are considered as major factors in the pathophysiology of age-related hearing loss. The 4977-bp deletion in human mtDNA (common deletion, corresponding to the 4834-bp mtDNA deletion in rats) is suggested to be closely associated with the pathogenesis of age-related hearing loss. Superoxide dismutase 2 (SOD2), an isoform of SOD that is exclusively expressed in the intracellular mitochondrial matrix, plays a crucial role in oxidative resistance against mitochondrial superoxide. Previous research has shown that methylation of the promoter region of the SOD2 gene decreased the expression of SOD2 in marginal cells (MCs) extracted from the inner ear of rats subjected to D-galactose-induced mtDNA4834 deletion. However, the relationship between SOD2 methylation and mtDNA4834 deletion under oxidative stress remains to be elucidated. Herein, an oxidative damage model was established in the extracted MCs using hydrogen peroxide (H2O2), which increased the methylation level of SOD2 and the copy number of mtDNA4834 mutation in MCs. Decreasing the methylation level of SOD2 using 5-azacytidine, a DNA methylation inhibitor, reduced oxidative stress and the copy number of mtDNA4834 mutation and inhibited H2O2-induced apoptosis. The present work demonstrates that decreasing the methylation of SOD2 suppresses the mtDNA4834 deletion in MCs under oxidative stress and provides potential insights to the intervention therapy of aging-related hearing loss.
Topics: Animals; Cell Culture Techniques; DNA, Mitochondrial; Disease Models, Animal; Ear, Inner; Hydrogen Peroxide; Methylation; Oxidative Stress; Presbycusis; Rats; Rats, Wistar; Sequence Deletion; Superoxide Dismutase
PubMed: 31465718
DOI: 10.17305/bjbms.2019.4353