Novel Type Of Mutation Research Articles

CRISPR/Cas9-Mediated Targeted Mutagenesis of GmUGT Enhanced Soybean Resistance Against Leaf-Chewing Insects Through Flavonoids Biosynthesis.

Leaf-chewing insects are important pests that cause yield loss and reduce seed quality in soybeans (Glycine max). Breeding soybean varieties that are resistant to leaf-chewing insects can minimize the need for insecticide use and reduce yield loss. The marker gene for QTL-M, Glyma.07g110300 (LOC100775351) that encodes a UDP-glycosyltransferase (UGT) is the major determinant of resistance against leaf-chewing insects in soybean; it exhibits a loss of function in insect-resistant soybean germplasms. In this study, Agrobacterium-mediated transformation introduced the CRISPR/Cas9 expression vector into the soybean cultivar Tianlong No. 1 to generate Glyma.07g110300-gene mutants. We obtained two novel types of mutations, a 33-bp deletion and a single-bp insertion in the GmUGT coding region, which resulted in an enhanced resistance to Helicoverpa armigera and Spodoptera litura. Additionally, overexpressing GmUGT produced soybean varieties that were more sensitive to H. armigera and S. litura. Both mutant and overexpressing lines exhibited no obvious phenotypic changes. The difference in metabolites and gene expression suggested that GmUGT is involved in imparting resistance to leaf-chewing insects by altering the flavonoid content and expression patterns of genes related to flavonoid biosynthesis and defense. Furthermore, ectopic expression of the GmUGT gene in the ugt72b1 mutant of Arabidopsis substantially rescued the phenotype of H. armigera resistance in the atugt72b1 mutant. Our study presents a strategy for increasing resistance against leaf-chewing insects in soybean through CRISPR/Cas9-mediated targeted mutagenesis of the UGT genes.

Creation of Early Flowering Germplasm of Soybean by CRISPR/Cas9 Technology.

Soybean is an important economic crop and a typical short-day crop, sensitive to photoperiod, and has narrow geographical adaptative region, which limit the creation of transgenic materials and reduce the breeding efficiency of new varieties. In addition, the genetic transformation efficiency of soybean is lower than that of many other crops, and the available receptor genotypes are limited. In this study, Agrobacterium-mediated transformation were used to introduce the CRISPR/Cas9 expression vector into soybean cultivar Jack and generated targeted mutants of E1 gene controlling soybean flowering. We obtained two novel types of mutations, 11 bp and 40 bp deletion at E1 coding region, respectively, and frameshift mutations produced premature translation termination codons and truncated E1 proteins, causing obvious early flowering under long day condition. In addition, no off-target effects were observed by predicting and analyzing the potential off-target sites of E1 targets. Significant decreased E1 gene expression of two novel mutants showed that the truncated E1 protein disinhibited GmFT2a/5a and increasing GmFT2a/5a gene expressions resulted obvious early flowering. Homozygous trans-clean mutants without T-DNA elements were also obtained and showed early flowering under long day condition. The photo-insensitive soybean transformation receptor we created laid a foundation for breeding excellent transgenic receptors suitable for high latitudes.

Genetics of Osteopetrosis

The term osteopetrosis refers to a group of rare skeletal diseases sharing the hallmark of a generalized increase in bone density owing to a defect in bone resorption. Osteopetrosis is clinically and genetically heterogeneous, and a precise molecular classification is relevant for prognosis and treatment. Here, we review recent data on the pathogenesis of this disorder. Novel mutations in known genes as well as defects in new genes have been recently reported, further expanding the spectrum of molecular defects leading to osteopetrosis. Exploitation of next-generation sequencing tools is ever spreading, facilitating differential diagnosis. Some complex phenotypes in which osteopetrosis is accompanied by additional clinical features have received a molecular classification, also involving new genes. Moreover, novel types of mutations have been recognized, which for their nature or genomic location are at high risk being neglected. Yet, the causative mutation is unknown in some patients, indicating that the genetics of osteopetrosis still deserves intense research efforts.

Description of a novel mutation in the atpC gene in optochin-resistant Streptococcus pneumoniae strains isolates from Tunisia

Identification of Streptococcus pneumoniae among other α-haemolytic streptococci is based on phenotypic or genotypic characteristics such as colony morphology, bile solubility and optochin susceptibility. This study reports three optochin-resistant S. pneumoniae strains isolated from immunocompromised patients in Tunisia. The three isolates were positive for the bile solubility test. Biochemical identification with API® 20 Strep was not discriminatory for two strains. The three strains had different serotypes (6C, 19F and 23F) and three different sequence types (ST386, ST320 and ST326). Sequencing of the atpA and atpC genes for each strain showed only modification in atpC. The mutations Met13→Val or Val48→Ile were observed in two strains. However, in the third strain a novel type of mutation (Val15→Ile) was identified.

Genetic landscape remodelling in spinocerebellar ataxias: the influence of next-generation sequencing.

Hereditary cerebellar ataxias (HCAs) are clinically and genetically heterogeneous neurodegenerative disorders, characterised by a cerebellar syndrome and other neurological or non-neurological signs. So far, more than 20 genes have been described in autosomal dominant HCA; in autosomal recessive HCA, even more genes are involved, in often more complex phenotypes. Because of that complexity, the genetic diagnosis of these diseases is often based on the next-generation sequencing techniques. In this review paper, we discuss the major contributions that they have made to the genetic landscape of HCAs. Numerous novel genes have been identified; still more have recently been implicated in HCAs in addition to being responsible for other diseases. The phenotypic spectrum associated with a single gene constantly gains in complexity. Novel types of mutations or transmissions in known genes are regularly being identified. All these factors make genotype-phenotype correlations particularly difficult. Some but not all of this variability can be explained by different pathophysiological consequences (loss of function, gain of function, variable levels of haploinsufficiency). This also raises the question of modifier genes. Finally, we highlight some functional pathways that increasingly appear important in HCAs.

A novel homozygous Fas ligand mutation leads to early protein truncation, abrogation of death receptor and reverse signaling and a severe form of the autoimmune lymphoproliferative syndrome

We report a novel type of mutation in the death ligand FasL that was associated with a severe phenotype of the autoimmune lymphoproliferative syndrome in two patients. A frameshift mutation in the intracellular domain led to complete loss of FasL expression. Cell death signaling via its receptor and reverse signaling via its intracellular domain were completely abrogated. In vitro lymphocyte proliferation induced by weak T cell receptor stimulation could be blocked and cell death was induced by engagement of FasL in T cells derived from healthy individuals and a heterozygous carrier, but not in FasL-deficient patient derived cells. Expression of genes implicated in lymphocyte proliferation and activation (CCND1, NFATc1, NF-κB1) was increased in FasL-deficient T cells and could not be downregulated by FasL engagement as in healthy cells. Our data thus suggest, that deficiency in FasL reverse signaling may contribute to the clinical lymphoproliferative phenotype of ALPS.

Phenotypic and Molecular Characterization of Optochin-Resistant Streptococcus pneumoniae Isolates from Brazil, with Description of Five Novel Mutations in the atpC Gene

Optochin (Opt) susceptibility is used largely for the identification of Streptococcus pneumoniae in diagnostic laboratories. Opt-resistant (Opt(r)) S. pneumoniae isolates have been reported, however, indicating the potential for misidentification of this important pathogen. Point mutations in the atpC gene have been associated with the emergence of Opt(r) S. pneumoniae, but data on the characterization of such atypical variants of S. pneumoniae are still limited. The present report describes the results of a polyphasic approach to identifying and characterizing 26 Opt(r) S. pneumoniae isolates recovered from patients or carriers living in Brazil. Sixteen isolates consisted of heterogeneous populations, and 10 isolates were homogeneously Opt(r). The isolates had different serotypes and antimicrobial susceptibility profiles. They also presented diverse genetic characteristics, as indicated by pulsed-field gel electrophoresis (PFGE), multilocus variable-number tandem-repeat analysis (MLVA), and pspA gene typing. Except for Opt MICs (4- to 64-fold higher among Opt(r) variants), Opt(r) and Opt-susceptible (Opt(s)) subpopulations originating from the same culture had identical characteristics. Sequencing of the atpC gene of the Opt(r) variants revealed 13 different nucleotide changes distributed among eight different codons. Changes in codon 49 were the most frequent, suggesting that this might be a hot spot for optochin resistance-conferring mutations. On the other hand, five novel types of mutations in the atpC gene (Met13Ile, Gly18Ser, Gly20Ala, Ala31Val, and Ala49Gly) were identified. In silico prediction modeling indicated that the atpC gene mutations corresponded to alterations in the transmembrane region of the ATPase, leading to a higher hydrophobicity profile in α-helix 1 and to a lower hydrophobicity profile in α-helix 2.

Intragenic deletion as a novel type of mutation in Wolman disease

Two clinically distinct disorders, Wolman disease (WD) and cholesteryl ester storage disease (CESD), are allelic autosomal recessive disorders caused by different mutations in lysosomal acid lipase (LIPA) which encodes for an essential enzyme involved in the hydrolysis of intracellular cholesteryl esters and triglycerides. We describe a case of lysosomal acid lipase deficiency in an infant with WD and report on a novel mutation type, intragenic deletion.

Novel frameshift mutation in Troponin C (TNNC1) associated with hypertrophic cardiomyopathy and sudden death

PurposeHypertrophic cardiomyopathy is the most common cause of sudden death in young people, including trained athletes, and is caused by mutations in genes encoding proteins of the cardiac sarcomere. Mutations in the Troponin C gene (TNNC1) are a rare genetic cause of hypertrophic cardiomyopathy. We describe a novel type of mutation (c.363dupG) in Troponin C, a rare form of hypertrophic cardiomyopathy. A family in which a 19-year-old asymptomatic male died of sudden cardiac death due to hypertrophic cardiomyopathy was genetically studied by sequencing 17 genes associated with hypertrophic cardiomyopathy or its phenocopies. A c.363dupG mutation in Troponin C was identified, and tested across the family. We report the first frameshift mutation (c.363dupG or p.Gln122AlafsX30) in Troponin C causing hypertrophic cardiomyopathy (and sudden cardiac death) in a 19-year-old male, and have demonstrated that the mutation segregates with hypertrophic cardiomyopathy within the family.

Large genomic deletions: A novel cause of Ullrich congenital muscular dystrophy

Two mutational mechanisms are known to underlie Ullrich congenital muscular dystrophy (UCMD): heterozygous dominant negatively-acting mutations and recessively-acting loss-of-function mutations. We describe large genomic deletions on chromosome 21q22.3 as a novel type of mutation underlying recessively inherited UCMD in 2 families. Clinically unaffected parents carrying large genomic deletions of COL6A1and COL6A2also provide conclusive evidence that haploinsufficiency for COL6A1and COL6A2is not a disease mechanism for Bethlem myopathy. Our findings have important implications for the genetic evaluation of patients with collagen VI-related myopathies as well as for potential therapeutic interventions for this patient population.

P1.11 Large genomic deletions as a novel type of mutation in Ullrich CMD

have performed oral function and swallowing test (ST) with surface electrode EMG and videofluoroscopic examination of swallowing (VF) in two unrelated very mild FCMD patients, who were able to walk and step up-down stairs in their childhood. Genomic DNA analysis of these cases showed 3 kb-RT insertion and no mutation in coding region and exon–intron junction of this gene. However, the haplotype analysis of the FKTN gene with six microsatellite markers revealed these patients have different haplotype from other severe type of FCMD. The ST showed severe dysfunction of swallowing in contradiction to their facial appearance and behavioral state. These results indicate that there may be potential risk for dysphagia and the periodical examination of ST is important for keeping the quality of life even in the very mild case of FCMD.

New Type of Mutations in Three Spanish Families with Choroideremia

Choroideremia (CHM) is an X-linked ophthalmic disease. The gene associated with CHM (REP-1) encodes a ubiquitously expressed protein that is indispensable for the posttranslational activation of retina-specific Rab protein. Different mutations, including large genomic rearrangements involving the REP-1 gene, are responsible for CHM, but they all cause the protein to be truncated or absent. The authors screened 20 Spanish families with clinical diagnoses of CHM to determine the molecular cause of the disease. First, the authors performed haplotype analyses to determine whether the disease is linked to the REP-1 gene. In families in whom the disease segregated with the CHM locus (n = 14), mutational screening of the REP-1 gene was performed. In 13 of the 14 families in which the phenotype segregated with the CHM locus, the authors identified the mutation associated with the disease. Eight different molecular defects that led to truncation and one that led to complete absence of the REP-1 protein were found in nine families and one family, respectively. Furthermore, the authors identified a novel type of mutation in the REP-1 gene in three families. This novel type of mutation did not result in a truncated or absent protein. Rather, these patients lost different parts of the REP-1 mRNA in-frame that in all the cases encode a conserved protein domain implicated in the interaction with Rab proteins. Based on the different mutations found, the authors propose a four-step protocol for the molecular diagnosis of CHM.

Selection for High-Level Telithromycin Resistance in Staphylococcus aureus Yields Mutants Resulting from an rplB -to- rplV Gene Conversion-Like Event

While most Staphylococcus aureus telithromycin-resistant mutants isolated in this study possessed duplications within rplV (encoding ribosomal protein L22), four isolates possessed insertions within rplV that were identical to a portion of the gene rplB (encoding ribosomal protein L2). This novel type of mutation is the result of an apparent gene conversion-like event.

Diversity of mutations in the atpC gene coding for the c Subunit of F0F1 ATPase in clinical isolates of optochin-resistant Streptococcus pneumoniae from Brazil.

We report the characteristics of four optochin-resistant (Opt(r)) Streptococcus pneumoniae isolates from Brazil. All four Opt(r) isolates presented mutations in the nucleotide sequence coding for the c subunit of F(0)F(1) ATPase. Two isolates showed mutations in codons 23 (leading to the deduced amino acid substitution isoleucine instead of alanine) and 49 (serine instead of alanine, a novel type of mutation detected at this position), respectively. Two additional novel mutations, both located in codon 45, were detected in the other two isolates, corresponding to leucine or valine (instead of phenylalanine). The data indicate that three previously unrecognized alterations were detected in the atpC gene of S. pneumoniae and that Opt resistance among Brazilian pneumococcal isolates is not related to a specific pneumococcal serotype, antimicrobial-resistance profile, or clonal group.

Novel Type of Hepatitis B Virus Mutation: Replacement Mutation Involving a Hepatocyte Nuclear Factor 1 Binding Site Tandem Repeat in Chronic Hepatitis B Virus Genotype E

The genetic diversity of hepatitis B virus (HBV) strains has evolved through mutations such as point mutations, deletions or insertions, and recombination. We identified and characterized a novel type of mutation which is a complex of external insertion, deletion, and internal duplication in sequences from one of six patients with chronic hepatitis B virus genotype E (HBV/E). We provisionally named this mutation a "replacement mutation"; the core promoter upstream regulatory sequence/basic core promoter was replaced with a part of the S1 promoter covering the hepatocyte nuclear factor 1 (HNF1) binding site, followed by a tandem repeat of the HNF1 site. A longitudinal analysis of the HBV population over 6 years showed the clonal change from wild-type HBV/E to replacement-mutant type, resulting in a lower hepatitis B (HB) e antigen titer, a high HBV DNA level in serum, and progression of liver fibrosis. In an in vitro study using a replication model, the replacement-mutant HBV showed higher replication levels than the wild-type HBV/E replicon, probably mediated by altered transcription factor binding. Additionally, this HNF1 site replacement mutation was associated with excessive HB nucleocapsid protein expression in hepatocytes, in both in vivo and in vitro studies. This novel mutation may be specific to HBV genotype E, and its prevalence requires further investigation.

Genomic rearrangements in the CFTR gene: extensive allelic heterogeneity and diverse mutational mechanisms.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR/ABCC7). Despite the extensive and enduring efforts of many CF researchers over the past 14 years, up to 30% of disease alleles still remain to be identified in some populations. It has long been suggested that gross genomic rearrangements could account for these unidentified alleles. To date, however, only a few large deletions have been found in the CFTR gene and only three have been fully characterized. Here, we report the first systematic screening of the 27 exons of the CFTR gene for large genomic rearrangements, by means of the quantitative multiplex PCR of short fluorescent fragments (QMPSF). A well-characterized cohort of 39 classical CF patients carrying at least one unidentified allele (after extensive and complete screening of the CFTR gene by both denaturing gradient gel electrophoresis and denaturing high-performance liquid chromatography) participated in this study. Using QMPSF, some 16% of the previously unidentified CF mutant alleles were identified and characterized, including five novel mutations (one large deletion and four indels). The breakpoints of these five mutations were precisely determined, enabling us to explore the underlying mechanisms of mutagenesis. Although non-homologous recombination may be invoked to explain all five complex lesions, each mutation appears to have arisen through a different mechanism. One of the indels was highly unusual in that it involved the insertion of a short 41 bp sequence with partial homology to a retrotranspositionally-competent LINE-1 element. The insertion of this ultra-short LINE-1 element (dubbed a "hyphen element") may constitute a novel type of mutation associated with human genetic disease.

A novel termination codon mutation of the WAS gene in a Thai family with Wiskott-Aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by microthrombocytopenia, eczema, immunodeficiency, and susceptibility to lymphoid malignancy. Loss-of-function mutations in WAS gene have been identified to cause disorders with platelet defects including WAS and X-lined thrombocytopenia. Mutations anticipated to yield truncated or no protein have been associated with the more severe presentations of WAS. Activating mutations in WAS gene result in an entirely different phenotype, an X-linked severe congenital neutropenia. Case report We describe a Thai family with classic WAS. The proband, a one-year-old boy presented with recurrent mucous bloody diarrhea, recurrent otitis media, chronic eczema, thrombocytopenia, and small platelet sizes, the patient's older brother who was also had persistent thrombocytopenia died at the age of seven months from severe pneumonia. Immunoblot analysis demonstrated that the proband's cells lacked WAS protein expression. Mutation analysis of the proband and his mother for the entire coding region of WAS identified a novel type of mutation, a termination codon mutation, X503R. The change is expected to result in an elongated mRNA that would code for a WASP of 581 amino acid residues instead of the normal 502 residues. Because of the absence of WASP expression, we speculated that the termination codon mutation causes reduced mRNA stability. Conclusion Our finding supported that WAS mutations resulted in no protein are associated with a severe phenotype of WAS.

A novel termination codon mutation of the WAS gene in a Thai family with Wiskott-Aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by microthrombocytopenia, eczema, immunodeficiency, and susceptibility to lymphoid malignancy. Loss-of-function mutations in WAS gene have been identified to cause disorders with platelet defects including WAS and X-linked thrombocytopenia. Mutations anticipated to yield truncated or no protein have been associated with the more severe presentations of WAS. Activating mutations in WAS gene result in an entirely different phenotype, an X-linked severe congenital neutropenia. We describe a Thai family with classic WAS. The proband, a one-year-old boy presented with recurrent mucous bloody diarrhea, recurrent otitis media, chronic eczema, thrombocytopenia, and small platelet sizes. The patient's older brother who also had persistent thrombocytopenia died at the age of seven months from severe pneumonia. Immunoblot analysis demonstrated that the proband's cells lacked WAS protein expression. Mutation analysis of the proband and his mother for the entire coding region of WAS identified a novel type of mutation, a termination codon mutation, X503R. The change is expected to result in an elongated mRNA that would code for a WASP of 581 amino acid residues instead of the normal 502 residues. Because of the absence of WASP expression, we speculate that the termination codon mutation causes reduced mRNA stability. Our findings supported that WAS mutations resulted in no protein are associated with a severe phenotype of WAS.

The Functional Integrity of the Serpin Domain of C1-inhibitor Depends on the Unique N-terminal Domain, as Revealed by a Pathological Mutant

C1-inhibitor (C1-Inh) is a serine protease inhibitor (serpin) with a unique, non-conserved N-terminal domain of unknown function. Genetic deficiency of C1-Inh causes hereditary angioedema. A novel type of mutation (Delta 3) in exon 3 of the C1-Inh gene, resulting in deletion of Asp62-Thr116 in this unique domain, was encountered in a hereditary angioedema pedigree. Because the domain is supposedly not essential for inhibitory activity, the unexpected loss-of-function of this deletion mutant was further investigated. The Delta 3 mutant and three additional mutants starting at Pro76, Gly98, and Ser115, lacking increasing parts of the N-terminal domain, were produced recombinantly. C1-Inh76 and C1-Inh98 retained normal conformation and interaction kinetics with target proteases. In contrast, C1-Inh115 and Delta 3, which both lack the connection between the serpin and the non-serpin domain via two disulfide bridges, were completely non-functional because of a complex-like and multimeric conformation, as demonstrated by several criteria. The Delta 3 mutant also circulated in multimeric form in plasma from affected family members. The C1-Inh mutant reported here is unique in that deletion of an entire amino acid stretch from a domain not shared by other serpins leads to a loss-of-function. The deletion in the unique N-terminal domain results in a "multimerization phenotype" of C1-Inh, because of diminished stability of the central beta-sheet. This phenotype, as well as the location of the disulfide bridges between the serpin and the non-serpin domain of C1-Inh, suggests that the function of the N-terminal region may be similar to one of the effects of heparin in antithrombin III, maintenance of the metastable serpin conformation.

Chemotherapeutically induced deletion of expanded triplet repeats

The number of neurodegenerative disorders associated with the expansion of DNA repeats, currently about 18, continues to increase as additional diseases caused by this novel type of mutation are identified. Typically, expanded repeats are biased toward further expansion upon intergenerational transmission, and disease symptoms show an earlier age of onset and greater severity as the length of the triplet repeat tract increases. Most diseases exhibit progressive neurological and/or muscular degeneration that can lead to total disability and death. As yet, no treatment exists for the genetic basis of any repeat disease. Given that the severity of these diseases is related to repeat tract length, reducing repeat lengths might delay the onset and reduce disease severity. Here, we test the hypothesis that the introduction of damage into DNA, which results in subsequent repair events, can lead to an increased rate of repeat deletion. Applying a sensitive genetic assay in Escherichia coli [Mut. Res. 502 (2002) 25], we demonstrate that certain DNA damaging agents, including EMS, ENU, UV light, and anticancer agents mitomycin C, cisplatin, and X-rays increase the rate of deletion of (CTG)·(CAG) repeats in a length and orientation dependent fashion. In addition, oxidative damage to DNA also increases the deletion rate of repeats. These results suggest that a chemotherapeutic approach to the reduction in triplet repeat length may provide one possible rationale to slow, stop, or reverse the progression of these diseases.