scholarly journals A Novel Splice-Site Mutation in VEGFC Is Associated with Congenital Primary Lymphoedema of Gordon

2018 ◽  
Vol 19 (8) ◽  
pp. 2259 ◽  
Author(s):  
Noeline Nadarajah ◽  
Dörte Schulte ◽  
Vivienne McConnell ◽  
Silvia Martin-Almedina ◽  
Christina Karapouliou ◽  
...  
Keyword(s):  
Splice Site ◽  
Current Knowledge ◽  
Stop Codon ◽  
Novel Mutation ◽  
Premature Stop Codon ◽  
Lymph Vessel ◽  
Site Mutation ◽  
Exon 2 ◽  
Primary Lymphedema ◽  
Splice Site Variant

Lymphedema is characterized by chronic swelling of any body part caused by malfunctioning or obstruction in the lymphatic system. Primary lymphedema is often considered genetic in origin. VEGFC, which is a gene encoding the ligand for the vascular endothelial growth factor receptor 3 (VEGFR3/FLT4) and important for lymph vessel development during lymphangiogenesis, has been associated with a specific subtype of primary lymphedema. Through Sanger sequencing of a proband with bilateral congenital pedal edema resembling Milroy disease, we identified a novel mutation (NM_005429.2; c.361+5G>A) in VEGFC. The mutation induced skipping of exon 2 of VEGFC resulting in a frameshift and the introduction of a premature stop codon (p.Ala50ValfsTer18). The mutation leads to a loss of the entire VEGF-homology domain and the C-terminus. Expression of this Vegfc variant in the zebrafish floorplate showed that the splice-site variant significantly reduces the biological activity of the protein. Our findings confirm that the splice-site variant, c.361+5G>A, causes the primary lymphedema phenotype in the proband. We examine the mutations and clinical phenotypes of the previously reported cases to review the current knowledge in this area.

scholarly journals A rare large duplication of MLH1 identified in Lynch syndrome

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Abhishek Kumar ◽  
Nagarajan Paramasivam ◽  
Obul Reddy Bandapalli ◽  
Matthias Schlesner ◽  
Tianhui Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Lynch Syndrome ◽  
Splice Site ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Splice Donor Site ◽  
Laboratory Diagnostics ◽  
Mmr Genes ◽  
Base Pair Deletion ◽  
Splice Site Variant

Abstract Background The most frequently identified strong cancer predisposition mutations for colorectal cancer (CRC) are those in the mismatch repair (MMR) genes in Lynch syndrome. Laboratory diagnostics include testing tumors for immunohistochemical staining (IHC) of the Lynch syndrome-associated DNA MMR proteins and/or for microsatellite instability (MSI) followed by sequencing or other techniques, such as denaturing high performance liquid chromatography (DHPLC), to identify the mutation. Methods In an ongoing project focusing on finding Mendelian cancer syndromes we applied whole-exome/whole-genome sequencing (WES/WGS) to 19 CRC families. Results Three families were identified with a pathogenic/likely pathogenic germline variant in a MMR gene that had previously tested negative in DHPLC gene variant screening. All families had a history of CRC in several family members across multiple generations. Tumor analysis showed loss of the MMR protein IHC staining corresponding to the mutated genes, as well as MSI. In family A, a structural variant, a duplication of exons 4 to 13, was identified in MLH1. The duplication was predicted to lead to a frameshift at amino acid 520 and a premature stop codon at amino acid 539. In family B, a 1 base pair deletion was found in MLH1, resulting in a frameshift and a stop codon at amino acid 491. In family C, we identified a splice site variant in MSH2, which was predicted to lead loss of a splice donor site. Conclusions We identified altogether three pathogenic/likely pathogenic variants in the MMR genes in three of the 19 sequenced families. The MLH1 variants, a duplication of exons 4 to 13 and a frameshift variant, were novel, based on the InSiGHT and ClinVar databases; the MSH2 splice site variant was reported by a single submitter in ClinVar. As a variant class, duplications have rarely been reported in the MMR gene literature, particularly those covering several exons.

Hybrid minigene splicing assay verified the pathogenicity of a novel splice site variant in the dystrophin gene of a Chinese patient with typical Duchenne muscular dystrophy phenotype

2016 ◽  
Vol 54 (9) ◽  
pp. 1435-1440 ◽  
Author(s):  
Zhihong Wang ◽  
Yanhong Lin ◽  
Liping Qiu ◽  
Dezhu Zheng ◽  
Aizhen Yan ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Splice Site ◽  
Sanger Sequencing ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Splice Site Mutation ◽  
Dystrophin Gene ◽  
Site Mutation ◽  
Aberrant Splicing

AbstractBackground:Duchenne muscular dystrophy (DMD) is typically caused by disrupting the reading frame of the dystrophin gene: approximately 70%–80% of mutational events are represented by deletions or duplications of one or more exons in the dystrophin gene, and the remaining cases by subtle mutations, including point mutations, small indels, small inversions, and complex small rearrangements. The dystrophin gene is the largest known gene with one of the highest known rates of new mutations.Methods:Deletions and duplications were detected in theDMDgene of the proband by using multiple ligation-dependent probe amplification (MLPA). Targeted next-generation sequencing (NGS) was used in the subtle mutation detection, followed by Sanger sequencing confirmation. The effect of the mutation on the splicing of theDMDgene was assessed by bioinformatics prediction and hybrid minigene splicing assay (HMSA).Results:Neither duplication nor deletion was found in theDMDgene of the proband. While a novel splice site mutation c.6762+1G>C was identified in the proband by NGS and Sanger sequencing, and his mother was heterozygous at the same site. Bioinformatics predicted that the 5′ donor splice site of intron 46 disappeared because of the mutation, which would lead to aberrant splicing and introduce premature stop codon. The HMSA results were in agreement with the prediction.Conclusions:The novel splice site mutation caused DMD in the proband by aberrant splicing. We suggested that combined applications of MLPA, NGS, HMSA and bioinformatics are comprehensive and effective methods for diagnosis and aberrant splicing study of DMD.

scholarly journals Peripheral blood mononuclear cells express mutated NCCT mRNA in Gitelman's syndrome: evidence for abnormal thiazide-sensitive NaCl cotransport.

1998 ◽  
Vol 9 (5) ◽  
pp. 819-826
Author(s):  
N Abuladze ◽  
N Yanagawa ◽  
I Lee ◽  
O D Jo ◽  
D Newman ◽  
...  
Keyword(s):  
Splice Site ◽  
Mononuclear Cells ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Splice Site Mutation ◽  
Paternal Allele ◽  
Site Mutation ◽  
Peripheral Blood Mononuclear ◽  
22Na Uptake ◽  
Nacl Cotransport

Genetic analysis has demonstrated complete linkage between the human thiazide-sensitive sodium chloride cotransporter gene (NCCT or TSC) and Gitelman's syndrome (GS). Several genomic NCCT mutations have been reported. This study was performed to determine whether peripheral blood mononuclear cells (PBMC) express NCCT mRNA and whether defective PBMC NaCl cotransport could be demonstrated in GS. PBMC were isolated from two brothers with GS, their parents, and healthy control subjects. Northern analysis revealed that NCCT mRNA is expressed in PBMC. The sequence of full-length NCCT cDNA amplified from normal PBMC was identical to human renal NCCT cDNA. Two different mutations were detected in the patients' NCCT cDNA (compound heterozygote). In cDNA derived from the patient's maternal allele, exon 24 was deleted, resulting in a premature stop codon (after amino acid 920). cDNA derived from the patient's paternal allele had an additional 119-bp insertion between exons 3 and 4, generating a premature stop codon (after amino acid 187). The patient's genomic DNA had a previously described 5' splice site mutation in intron 24, GGT --> GTT (maternal allele), and a new 3' splice site mutation in intron 3, CAG --> CAA (paternal allele), which resulted in the activation of a nearby cryptic splice site in intron 3. The latter mutation was not present in 300 normal chromosomes. To determine the functional significance of these findings, chlorothiazide-inhibitable 22Na uptake was measured in PBMC from control subjects, the parents, and the patients with GS in the presence of bumetanide. In control PBMC, chlorothiazide inhibited 22Na uptake by approximately 9%. PBMC from the two patients with GS failed to respond to chlorothiazide. These results demonstrate that PBMC can be used for mutational analysis of NCCT mRNA in patients with GS. Furthermore, functional evidence is provided that the underlying cause of GS is defective NCCT NaCl cotransport.

Ectopic Transcript Analysis Indicates that Allelic Exclusion is an Important Cause of Type I Protein C Deficiency in Patients with Nonsense and Frameshift Mutations in the PROC Gene

1996 ◽  
Vol 75 (06) ◽  
pp. 870-876 ◽  
Author(s):  
José Manuel Soria ◽  
Lutz-Peter Berg ◽  
Jordi Fontcuberta ◽  
Vijay V Kakkar ◽  
Xavier Estivill ◽  
...  
Keyword(s):  
Splice Site ◽  
Protein C ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Allelic Exclusion ◽  
Polymorphism Analysis ◽  
Type I ◽  
Protein C Deficiency ◽  
Nonsense Mutations ◽  
Stop Codons

SummaryNonsense mutations, deletions and splice site mutations are a common cause of type I protein C deficiency. Either directly or indirectly by altering the reading frame, these' lesions generate or may generate premature stop codons and could therefore be expected to result in premature termination of translation. In this study, the possibility that such mutations could instead exert their pathological effects at an earlier stage in the expression pathway, through “allelic exclusion” at the RNA level, was investigated. Protein C (PROC) mRNA was analysed in seven Spanish type I protein C deficient patients heterozygous for two nonsense mutations, a 7bp deletion, a 2bp insertion and three splice site mutations. Ectopic RNA transcripts from patient and control lymphocytes were analysed by RT-PCR and direct sequencing of amplified PROC cDNA fragments. The nonsense mutations and the deletion were absent from the cDNAs indicating that only mRNA derived from the normal allele had been expressed. Similarly for the splice site mutations, only normal PROC cDNAs were obtained. In one case, exclusion of the mutated allele could be confirmed by polymorphism analysis. In contrast to these six mutations, the 2 bp insertion was not associated with loss of mRNA from the mutated allele. In this case, cDNA analysis revealed the absence of 19 bases from the PROC mRNA consistent with the generation and utilization of a cryptic splice site 3’ to the site of mutation, which would result in a frameshift and a premature stop codon. It is concluded that allelic exclusion is a common causative mechanism in those cases of type I protein C deficiency which result from mutations that introduce premature stop codons

Molecular and Cellular Characterization of a New α-Thalassemia Mutation (HBA2:c.94A>C) Generating an Alternative Splice Site and a Premature Stop Codon

Hemoglobin ◽  
2012 ◽  
Vol 36 (3) ◽  
pp. 244-252 ◽  
Author(s):  
Talal Qadah ◽  
Jill Finlayson ◽  
Christopher Newbound ◽  
Nicole Pell ◽  
Michelle Pascoe ◽  
...  
Keyword(s):  
Alternative Splice ◽  
Splice Site ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Alternative Splice Site ◽  
Thalassemia Mutation

scholarly journals CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary cells

2020 ◽  
Author(s):  
Mitchell G. Kluesner ◽  
Walker S. Lahr ◽  
Cara-Lin Lonetree ◽  
Branden A. Smeester ◽  
Patricia N. Claudio-Vázquez ◽  
...  
Keyword(s):  
Splice Site ◽  
Stop Codon ◽  
Cytidine Deaminase ◽  
Gene Knockout ◽  
Premature Stop Codon ◽  
Splice Sites ◽  
Immune Synapse ◽  
Base Editing ◽  
Human T Cells ◽  
Site Targeting

ABSTRACTBase editors allow for precise nucleotide editing without the need for genotoxic double-stranded breaks. Prior work has used base editors to knockout genes by introducing premature stop codons or by disrupting conserved splice-sites, but no direct comparison exists between these methods. Additionally, while base editor mediated disruption of splice sites has been used to shift the functional isoform pool, its utility for gene knockout requires further validation. To address these needs, we developed the program SpliceR (z.umn.edu/spliceR) to design cytidine-deaminase base editor (CBE) and adenosine-deaminase base editor (ABE) splice-site targeting guides. We compared the splice-site targeting and premature stop codon introduction in a knockout screen against the TCR-CD3 immune synapse in primary human T-cells. Our data suggests that 1) the CBE, BE4 is more reliable than the ABE, ABE7.10 for splice-site targeting knockout and 2) for both CBEs and ABEs, splice-donor targeting is the most reliable approach for base editing induced knockout.

scholarly journals A new mutation at exon 2 of hprt1 locus causing Lesch-Nyhan Syndrome.

2015 ◽  
Vol 3 (1) ◽  
pp. 18-21
Author(s):  
Adriana María Gil Zapata ◽  
Adriana Castillo Pico ◽  
Leonor Gusmão ◽  
António Amorim ◽  
Fernando Rodríguez Sanabria
Keyword(s):  
Genetic Counseling ◽  
Inborn Error ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Dna Fragments ◽  
Hprt Gene ◽  
New Mutation ◽  
Exon 2 ◽  
The Family ◽  
Hypoxanthine Guanine Phosphoribosyl Transferase

Introduction: Lesch-Nyhan síndrome (LNS) is an X-linked recessive inborn error of metabolism, due to deficiency of the enzyme Hypoxanthine-guanine-phosphoribosyl transferase (HGPRT; EC.2.4.2.8) resulting in hyperuricemia, neurological and behavioural disturbances. In the present work, we report the results of the study of a Colombian family, where LNS was previously clinically and biochemically diagnosed. Material and Methods: The full HPRT gene, including 9 exons and 8 introns, was amplified on eight separate DNA fragments. Both strands, forward and reverse, of the amplified DNA fragments were analyzed and the obtained sequences were compared with those deposited at National Center for Biotechnology Information. Results and conclusions: Sequence analysis allowed the detection of new LNS causing mutation, an adenine deletion in exon 2 of HPRT1 gene resulting in a frameshift which determines a premature stop codon. This study, besides adding a new mutation to the already large spectrum of disease causing variation at HPRT, allows therefore providing genetic counseling for the family as well as prenatal diagnosis.

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