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PLoS Genetics Mar 2019The simplicity and the versatility of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR-Cas) systems have enabled the genetic...
The simplicity and the versatility of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR-Cas) systems have enabled the genetic modification of virtually every organism and offer immense therapeutic potential for the treatment of human disease. Although these systems may function efficiently within eukaryotic cells, there remain concerns about the accuracy of Cas endonuclease effectors and their use for precise gene editing. Recently, two independent reports investigating the editing accuracy of the CRISPR-Cas9 system were published by separate groups at the Wellcome Sanger Institute; our study-Iyer and colleagues [1]-defined the landscape of off-target mutations, whereas the other by Kosicki and colleagues [2] detailed the existence of on-target, potentially deleterious deletions. Although both studies found evidence of large on-target CRISPR-induced deletions, they reached seemingly very different conclusions.
Topics: Animals; CRISPR-Cas Systems; Cell Cycle; Cell Division; Gene Editing; Genetic Therapy; Genome; Genomics; Genotype; Humans; Mammals; Mutation Rate; Sequence Deletion; Zygote
PubMed: 30870431
DOI: 10.1371/journal.pgen.1007994 -
Journal of Cellular and Molecular... Feb 2022Non-obstructive azoospermia (NOA) is a common cause of male infertility, and genetic problems, such as chromosomal abnormalities and gene mutations, are important causes...
Non-obstructive azoospermia (NOA) is a common cause of male infertility, and genetic problems, such as chromosomal abnormalities and gene mutations, are important causes of NOA. Our centre received a case of NOA, in which no mature sperm was found during microdissection testicular sperm extraction. A postoperative pathological examination revealed that testicular spermatogenesis was blocked. Target region capture combined with high-throughput sequencing was used to screen for male infertility-related gene mutations. Sanger sequencing further confirmed that the SYCE1 gene, a central component of the synaptonemal complex (SC) during meiosis, had a homozygous deletion mutation in the tenth exon (c.689_690del; p.F230fs). Through molecular biological studies, we discovered altered expression and nuclear localization of the endogenous mutant SYCE1. To verify the effects in vitro, wild- and mutated-type SYCE1 vectors were constructed and transfected into a human cell line. The results showed that the expression and molecular weight were decreased for SYCE1 containing c.689_690del. In addition, mutated SYCE1 was abnormally located in the cytoplasm rather than in the nucleus. In summary, our research suggests that the novel homozygous mutation (c.689_690del; p.F230fs) altered the SYCE1 expression pattern and may have disturbed SC assembly, leading to male infertility and to a barrier to gamete formation. We reported for the first time that a frameshift mutation occurred in the exon region of SYCE1 in an NOA patient. This study is beneficial for accurate NOA diagnosis and the development of corresponding gene therapy strategies.
Topics: Azoospermia; DNA-Binding Proteins; Exons; Homozygote; Humans; Male; Mutation; Sequence Deletion; Testis
PubMed: 35023261
DOI: 10.1111/jcmm.17180 -
PloS One 2016The aim of this study is to identify the molecular basis of autosomal recessive congenital cataracts (arCC) in a large consanguineous pedigree.
PURPOSE
The aim of this study is to identify the molecular basis of autosomal recessive congenital cataracts (arCC) in a large consanguineous pedigree.
METHODS
All participating individuals underwent a detailed ophthalmic examination. Each patient's medical history, particularly of cataracts and other ocular abnormalities, was compiled from available medical records and interviews with family elders. Blood samples were donated by all participating family members and used to extract genomic DNA. Genetic analysis was performed to rule out linkage to known arCC loci and genes. Whole-exome sequencing libraries were prepared and paired-end sequenced. A large deletion was found that segregated with arCC in the family, and chromosome walking was conducted to estimate the proximal and distal boundaries of the deletion mutation.
RESULTS
Exclusion and linkage analysis suggested linkage to a region of chromosome 6p24 harboring GCNT2 (glucosaminyl (N-acetyl) transferase 2) with a two-point logarithm of odds score of 5.78. PCR amplifications of the coding exons of GCNT2 failed in individuals with arCC, and whole-exome data analysis revealed a large deletion on chromosome 6p in the region harboring GCNT2. Chromosomal walking using multiple primer pairs delineated the extent of the deletion to approximately 190 kb. Interestingly, a failure to amplify a junctional fragment of the deletion break strongly suggests an insertion in addition to the large deletion.
CONCLUSION
Here, we report a novel insertion/deletion mutation at the GCNT2 locus that is responsible for congenital cataracts in a large consanguineous family.
Topics: Animals; Cataract; Child; Child, Preschool; Consanguinity; Female; Genetic Linkage; Genetic Loci; Humans; Infant; Male; Mice; Microsatellite Repeats; N-Acetylglucosaminyltransferases; N-Acetylhexosaminyltransferases; Pedigree; Sequence Deletion
PubMed: 27936067
DOI: 10.1371/journal.pone.0167562 -
Phytopathology Jan 2022' Liberibacter asiaticus' (Las) is the prominent species of associated with huanglongbing, a devastating disease of citrus worldwide. In this study, we report the...
' Liberibacter asiaticus' (Las) is the prominent species of associated with huanglongbing, a devastating disease of citrus worldwide. In this study, we report the identification of an ∼8.3-kb DNA region of the Las genome containing eight putative open reading frames flanked by two inverted repeats, which was not present in the Las str. psy62 genome. Comparisons with other genome sequences established this region as a unique genetic element associated with genome plasticity/instability. Primers specific for both the presence (Las wild type) and absence (Las mutant) of this region were designed to study the population dynamics and host adaptation of the two strains. Las populations with and/or without the wild-type strain were detected and differentiated in >2,300 samples that included psyllids, periwinkle, and several species of citrus. In psyllids, although a mixed population of the wild type and mutant was observed in most samples (88%), the wild-type Las was detected alone at a rate of 11%. In contrast, none of the infected citrus plants were positive for the wild type alone, which harbored either the mutant strain alone (8%) or a mixed population of the mutant and wild type (92%). Furthermore, the dynamics of these two major Las populations varied with different citrus hosts, whereas an in-depth study on grapefruit that did not rapidly succumb to disease revealed that the population of mutant alone increased with time, indicating that the absence of this genetic element is associated with the fitness of Las in planta under the selection pressure of its host.
Topics: Citrus; Liberibacter; Plant Diseases; Rhizobiaceae; Sequence Deletion
PubMed: 34645320
DOI: 10.1094/PHYTO-08-21-0325-FI -
The New Phytologist Nov 2019Distyly is an intriguing floral adaptation that increases pollen transfer precision and restricts inbreeding. It has been a model system in evolutionary biology since...
Distyly is an intriguing floral adaptation that increases pollen transfer precision and restricts inbreeding. It has been a model system in evolutionary biology since Darwin. Although the S-locus determines the long- and short-styled morphs, the genes were unknown in Turnera. We have now identified these genes. We used deletion mapping to identify, and then sequence, BAC clones and genome scaffolds to construct S/s haplotypes. We investigated candidate gene expression, hemizygosity, and used mutants, to explore gene function. The s-haplotype possessed 21 genes collinear with a region of chromosome 7 of grape. The S-haplotype possessed three additional genes and two inversions. TsSPH1 was expressed in filaments and anthers, TsYUC6 in anthers and TsBAHD in pistils. Long-homostyle mutants did not possess TsBAHD and a short-homostyle mutant did not express TsSPH1. Three hemizygous genes appear to determine S-morph characteristics in T. subulata. Hemizygosity is common to all distylous species investigated, yet the genes differ. The pistil candidate gene, TsBAHD, differs from that of Primula, but both may inactivate brassinosteroids causing short styles. TsYUC6 is involved in auxin synthesis and likely determines pollen characteristics. TsSPH1 is likely involved in filament elongation. We propose an incompatibility mechanism involving TsYUC6 and TsBAHD.
Topics: Amino Acid Sequence; Chromosomes, Artificial, Bacterial; Gene Expression Regulation, Plant; Genetic Loci; Genome, Plant; Genotype; Haplotypes; Mutation; Plant Proteins; Polymorphism, Genetic; Sequence Analysis, DNA; Sequence Deletion; Species Specificity; Turnera
PubMed: 31144315
DOI: 10.1111/nph.15970 -
Parkinsonism & Related Disorders Sep 2014Recently, it has been reported that carriers of a hemizygous chromosome 22q11.2 deletion may be at increased risk of early-onset Parkinson's disease. Herein, we propose... (Review)
Review
Recently, it has been reported that carriers of a hemizygous chromosome 22q11.2 deletion may be at increased risk of early-onset Parkinson's disease. Herein, we propose a hypothesis that it is not the microdeletion per se that is responsible for the phenotype but rather a complete loss of function of a gene within the region due to the combination of the deletion and another mutation on the alternate allele. Thus we propose the deletion may be highlighting a novel locus for a recessive form of early-onset Parkinson's disease.
Topics: Age of Onset; Animals; Chromosomes, Human, Pair 22; Genetic Predisposition to Disease; Humans; Mutation; Parkinson Disease; Sequence Deletion
PubMed: 25001314
DOI: 10.1016/j.parkreldis.2014.06.020 -
Journal of Infection and Chemotherapy :... Jun 2022Trichophyton rubrum is an anthropophilic dermatophyte that is most frequently isolated from onychomycosis (tinea unguium) worldwide. T. rubrum strains showing resistance...
INTRODUCTION
Trichophyton rubrum is an anthropophilic dermatophyte that is most frequently isolated from onychomycosis (tinea unguium) worldwide. T. rubrum strains showing resistance to the anti-fungal drug terbinafine (TRF) have also been isolated from human patients worldwide.
METHODS
In this study, we isolated a TRF-resistant strain (N99-2) of T. rubrum from a patient with recurrent tinea unguium. In vitro susceptibility of the clinical isolate to TRF, itraconazole (ITZ), ravuconazole (RVZ), and luliconazole (LCZ) was investigated using the Clinical & Laboratory Standards Institute M38-A2 test. To identify mutations, we compared the gene sequence of N99-2 to that of a TRF-susceptible strain of T. rubrum. Results; In N99-2, the minimum inhibitory concentrations were 32 mg/L for TRF, <0.03 mg/L for ITZ, <0.03 mg/L for RVZ, and <0.03 mg/L for LCZ. The squalene epoxidase (SQLE) gene sequence in N99-2 was determined to be 1467 bp in length, and it encoded a protein of 488 amino acids, beginning with a putative initiating methionine (ATG). The following mutations were identified from the SQLE of N99-2: L393F and Y394del.
CONCLUSIONS
This is the first report of the detection of a deletion mutation in SQLE in a TRF-resistant strain. The protein of SQLE is the target of TRF, and it is essential for cell membrane synthesis in dermatophytes. However, dermatophyte cells were found to undergo gene mutations to escape the effects of antifungal agents.
Topics: Amino Acid Sequence; Antifungal Agents; Drug Resistance, Fungal; Humans; Itraconazole; Microbial Sensitivity Tests; Onychomycosis; Sequence Deletion; Squalene Monooxygenase; Terbinafine; Trichophyton
PubMed: 35219578
DOI: 10.1016/j.jiac.2022.02.010 -
Journal of Molecular Neuroscience : MN Jan 2017Cerebral cavernous malformation (CCM) is a congenital vascular anomaly predominantly located within the central nervous system. Its familial forms (familial cerebral...
Identification of a Novel Deletion Mutation (c.1780delG) and a Novel Splice-Site Mutation (c.1412-1G>A) in the CCM1/KRIT1 Gene Associated with Familial Cerebral Cavernous Malformation in the Chinese Population.
Cerebral cavernous malformation (CCM) is a congenital vascular anomaly predominantly located within the central nervous system. Its familial forms (familial cerebral cavernous malformation (FCCM)), inherited in an autosomal dominant manner with incomplete penetrance, are attributed to mutations in CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10 genes. To date, little is known about the genetic alterations leading to FCCM in the Chinese population. We aimed to investigate the genetic defect of FCCM by DNA sequencing in Chinese families. This study enrolled five Chinese families with FCCM. All index cases underwent surgical treatment and were diagnosed with CCM by pathology; their relatives were diagnosed based on radiological and/or pathological evidence. Genomic DNA was extracted from peripheral blood and amplified using polymerase chain reaction (PCR) for DNA sequencing. The five families comprised a total of 21 affected individuals: 12 of these were symptomatic, and 9 were asymptomatic. Sequence analyses in the index patients disclosed three heterozygous loss-of-function mutations in the CCM1/KRIT1 gene in three families, respectively: a novel deletion mutation (c.1780delG; p.Ala594HisfsX67) in exon 16, a novel splice-site mutation (c.1412-1G>A) in the splice acceptor site in intron 13, and a previously described 4-bp deletion (c.1197_1200delCAAA; p.Gln401ThrfsX10) in exon 12. All of these mutations are predicted to cause a premature termination codon to generate a truncated Krev interaction trapped 1 (Krit1) protein. These mutations segregated in affected relatives. Our findings provided new CCM1 gene mutation profiles, which help to elucidate the pathogenesis of FCCM and will be of great significance in genetic counseling.
Topics: Central Nervous System Vascular Malformations; Codon, Nonsense; Exons; Female; Gene Deletion; Humans; Introns; KRIT1 Protein; Male; Microtubule-Associated Proteins; Pedigree; Proto-Oncogene Proteins; RNA Splicing
PubMed: 27649701
DOI: 10.1007/s12031-016-0836-2 -
Molecular Medicine Reports Jan 2020Wilson disease (WD) is a rare autosomal recessive genetic disorder that causes abnormal copper metabolism, resulting in pathological accumulation of copper in the liver,...
A novel gross deletion and breakpoint junction sequence analysis of ATP7B in a Chinese family with Wilson disease using next‑generation sequencing and Sanger sequencing.
Wilson disease (WD) is a rare autosomal recessive genetic disorder that causes abnormal copper metabolism, resulting in pathological accumulation of copper in the liver, brain and other organs. Mutations in the ATPase copper transporter 7B (ATP7B) gene, which encodes a membrane P‑type adenosine triphosphatase, have been identified as being responsible for WD. The present study analyzed clinical data and collected DNA samples from a pediatric patient with WD and her healthy parents. Mutation screening for ATP7B was performed using direct sequencing, multiplex ligation‑dependent probe amplification(MLPA), next‑generation sequencing (NGS) and Sanger sequencing of the breakpoint junction sequence. The patient (age, 2.7 years) presented with early‑onset hepatic disease. The present study identified compound heterozygous mutations of ATP7B, including a heterozygous mutation (p.Arg1,041Trp) and a novel heterozygous gross deletion of a 57,771 bp fragment (chr13: 52490972‑52548742) (GRCh37) from partial exon2‑ exon21 to external ATP7B sequence (15.833bp) in the patient. Analysis of the family members of the patient showed that the missense mutation and the gross deletion mutation were inherited from her mother and father, respectively. Microhomology and inverted repeat sequences, which may mediate the deletion mutation, were identified through sequence analysis on both sides of the breakpoints of this deletion. The present study provided additional information on the genotypic spectrum of the ATP7B gene, particularly with regard to early onset hepatic disease, as observed in the present patient with WD. The identification of the precise breakpoint junction sequence warrants further investigation of DNA break and recombination mechanisms. In detecting precise deletions, the NGS associated with Sanger sequencing of breakpoint junction sequence have been found to have more advantages than MLPA.
Topics: Adult; Age of Onset; Child, Preschool; Copper; Copper-Transporting ATPases; Female; Genetic Predisposition to Disease; Hepatolenticular Degeneration; Heterozygote; High-Throughput Nucleotide Sequencing; Humans; Male; Mutation; Pedigree; Sequence Deletion
PubMed: 31746411
DOI: 10.3892/mmr.2019.10830 -
Genetics and Molecular Research : GMR Nov 2015The aim of this study is to investigate the ability to prenatally diagnose phenylketonuria (PKU) by using phenylalanine hydroxylase (PAH) gene mutation analysis combined...
The aim of this study is to investigate the ability to prenatally diagnose phenylketonuria (PKU) by using phenylalanine hydroxylase (PAH) gene mutation analysis combined with short tandem repeat (STR) linkage analysis in 118 fetuses from 112 Chinese families. Genomic DNA was extracted from the peripheral blood from members of 112 families and the exons and exon-intron boundaries of the PAH gene were amplified by PCR. PCR products were analyzed by bi-directional Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). The three variable number of tandem repeat (VNTR) markers PAH-1, PAH-26, PAH-32 were used in the prenatal diagnosis for the PKU families. We identified a spectrum of 63 different mutations, including 61 point mutations and indels, two large exon deletion mutations, and five novel mutations. A substantial proportion of mutant alleles were accounted for by p.R243Q (15.62%), EX6-96AG (9.82%), p.V399V (7.59%), p.Y356X (6.70%), and p.R413P (5.36%). The same mutations were identified in 31 prenatally genotyped fetuses. We identified 58 fetuses that carried only one mutant allele and 29 fetuses that carried no mutations of PAH and were presumed normal. PAH gene mutation analysis combined with STR linkage analysis can provide rapid and accurate prenatal diagnosis for PKU families.
Topics: Alleles; Asian People; Exons; Female; Genetic Linkage; Genotype; Humans; Introns; Microsatellite Repeats; Phenylalanine Hydroxylase; Phenylketonurias; Point Mutation; Pregnancy; Prenatal Diagnosis; Sequence Deletion
PubMed: 26600521
DOI: 10.4238/2015.November.18.25