scholarly journals A Novel WT1 Mutation Identified in a 46,XX Testicular/Ovotesticular DSD Patient Results in the Retention of Intron 9

Biology ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1248
Author(s):  
Dmytro Sirokha ◽  
Olexandra Gorodna ◽  
Yakov Vitrenko ◽  
Nataliya Zelinska ◽  
Rafal Ploski ◽  
...  
Keyword(s):  
Splice Site ◽  
Rna Binding ◽  
De Novo ◽  
Zinc Fingers ◽  
Point Mutations ◽  
Whole Exome ◽  
Alternative Donor ◽  
Wt1 Mutation ◽  
Wt1 Protein ◽  
Genetic Rearrangements

The 46,XX testicular DSD (disorder/difference of sexual development) and 46,XX ovotesticular DSD (46,XX TDSD and 46,XX OTDSD) phenotypes are caused by genetic rearrangements or point mutations resulting in imbalance between components of the two antagonistic, pro-testicular and pro-ovarian pathways; however, the genetic causes of 46,XX TDSD/OTDSD are not fully understood, and molecular diagnosis for many patients with the conditions is unavailable. Only recently few mutations in the WT1 (WT1 transcription factor; 11p13) gene were described in a group of 46,XX TDSD and 46,XX OTDSD individuals. The WT1 protein contains a DNA/RNA binding domain consisting of four zinc fingers (ZnF) and a three-amino acid (KTS) motif that is present or absent, as a result of alternative splicing, between ZnF3 and ZnF4 (±KTS isoforms). Here, we present a patient with 46,XX TDSD/OTDSD in whom whole exome sequencing revealed a heterozygous de novo WT1 c.1437A>G mutation within an alternative donor splice site which is used for −KTS WT1 isoform formation. So far, no mutation in this splice site has been identified in any patient group. We demonstrated that the mutation results in the retention of intron 9 in the mature mRNA of the 46,XX TDSD/OTDSD patient. In cases when the erroneous mRNA is translated, exclusively the expression of a truncated WT1 +KTS protein lacking ZnF4 and no −KTS protein occurs from the mutated allele of the patient. We discuss potential mechanisms and pathways which can be disturbed upon two conditions: Absence of Zn4F and altered +KTS/−KTS ratio.

scholarly journals A Deep Learning approach predicts the impact of point mutations in intronic flanking regions on micro-exon splicing definition

2019 ◽  
Author(s):  
Lucas F. DaSilva ◽  
Ana C. Tahira ◽  
Vinicius Mesel ◽  
Sergio Verjovski-Almeida
Keyword(s):  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Point Mutations ◽  
Base Point ◽  
Validation Dataset ◽  
Systematic Assessment ◽  
Exon Splicing ◽  
Flanking Sequences ◽  
The Impact

AbstractWhile mammalian exons are on average 140-nt-long, thousands of human genes harbor micro-exons (≤ 39 nt). Large numbers of micro-exons have their splicing altered in diseases such as autism and cancer, and yet there is no systematic assessment of the impact of point mutations in intronic flanking-sequences on the splicing of a neighboring micro-exon. Here, we constructed a model using the Convolutional Neural Network (CNN) to predict the impact of point mutations in intronic-flanking-sequences on the splicing of a neighboring micro-exon. The prediction model was based on both the sequence contents and conservation among species of the two 100-nt intronic regions (5’ and 3’) that flank all human micro-exons and a set with the same number of randomly selected long exons. After training our CNN model, the micro-exon splicing event prediction accuracy, using an independent validation dataset, was 0.71 with an area under the ROC curve of 0.76, showing that our model had identified sequence patterns that have been conserved in evolution in the introns that flank micro-exons. Next, we introduced in silico point mutations at each of the 200 nucleotides in the introns that flank a micro-exon and used the trained CNN algorithm to predict splicing for every mutated intronic sequence version. This analysis identified thousands of point mutations in the flanking introns that significantly decreased the power of the CNN model to correctly predict a neighboring micro-exon splicing event, thus pointing to predictive bases in intronic regions important for micro-exon splicing signaling. We found these predictive bases to locate within conserved RNA-binding-motifs for RNA-binding-proteins (RBPs) known to relate to micro-exon splicing. Experimental data of minigene splicing reporter changes upon intron-base point-mutation confirmed the effect predicted by the CNN model for some of the micro-exon splicing events. The model can be used for validating novel micro-exons de novo assembled from RNA-seq data, and for an unbiased screening of introns, identifying genomic bases that have high micro-exon-splicing predictive power, possibly revealing critical point mutations that would be related in a yet unknown manner to a given disease.

scholarly journals Genotypes and Phenotypes of MEF2C Haploinsufficiency Syndrome: New Cases and Novel Point Mutations

2021 ◽  
Vol 9 ◽  
Author(s):  
Lin Wan ◽  
Xinting Liu ◽  
Linyan Hu ◽  
Huimin Chen ◽  
Yulin Sun ◽  
...  
Keyword(s):  
De Novo ◽  
Neurodevelopmental Disorder ◽  
Point Mutations ◽  
Clinical Manifestations ◽  
Poor Performance ◽  
Genetic Alterations ◽  
Mendelian Inheritance ◽  
Whole Exome ◽  
Patients With Epilepsy ◽  
Free Status

Aim: MEF2C haploinsufficiency syndrome (MCHS) is a severe neurodevelopmental disorder. We describe the clinical phenotypes and genotypes of seven patients with MCHS to enhance the understanding of clinical manifestations and genetic alterations associated with MCHS.Method: Seven patients (6 females and 1 male, aged between 2 years 5 months and 6 years) who had MEF2C mutations, and their parents underwent trio-based whole-exome sequencing; subsequently, their clinical features were assessed. A literature review of patients with MCHS was performed by searching the PubMed and Online Mendelian Inheritance in Man databases.Results: Seven mutations were identified, of which six were unreported in the past; of the reported cases, five patients had de novo mutations but two had an undefined inheritance pattern. All patients presented delays in developmental milestones, severe intellectual disabilities and lack of speech. Six patients exhibited infantile hypotonia, five patients experienced stereotypic movements and were unable to walk, four patients exhibited poor eye contact indicative of autism and two showed poor performance. While six patients experienced seizure, five among them became seizure free after receiving anti-seizure medicine. Three patients showed a regression in their development, whereas the mothers of two patients exhibited mosaicism but were healthy without any abovementioned symptoms.Interpretation: Regression was not a common phenomenon but occurred in MCHS. The prognosis of MCHS patients with epilepsy was good, but most patients can achieve a seizure-free status. Healthy people may have low-level mosaicism and carry a pathogenic MEF2C mutation.

scholarly journals Molecular basis for type 1 antithrombin deficiency: identification of two novel point mutations and evidence for a de novo splice site mutation

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3742-3748 ◽  
Author(s):  
K Jochmans ◽  
W Lissens ◽  
T Yin ◽  
JJ Michiels ◽  
L van der Luit ◽  
...  
Keyword(s):  
Splice Site ◽  
Molecular Basis ◽  
De Novo ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Protein Translation ◽  
Base Substitution ◽  
Nucleotide Position ◽  
At Deficiency

Inherited type 1 antithrombin (AT) deficiency is characterized by a reduction in both immunologically and functionally detectable protein. The disorder is associated with a high risk of thromboembolic disease. We have investigated the molecular basis of type 1 AT deficiency in three unrelated families. We have used the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis, followed by direct sequencing of the seven exons and the intron-exon junctions of the AT gene. Two novel point mutations were identified. A T to C single-base substitution was found in codon 421 in exon 6 (nucleotide position 13380), leading to an AT 421 isoleucine to threonine substitution. In another kindred, one of three Cs at nucleotide (nt) positions 5448 to 5450 in exon 3A (codon 151 or 152) was deleted, resulting in a frameshift mutation and predicting premature termination of protein translation at codon 251. In a third family, a previously reported G to A substitution, at nt position 9788 in intron 4, 14 bp in front of exon 5, was found. We have demonstrated the creation of a de novo exon 5 splice site by ectopic transcript analysis of lymphocyte mRNA. In all cases, the affected individuals were heterozygous for the mutation and no variant AT protein was detected.

Identification of Novel Point Mutations in Splicing Sites by the Integration of Exome and RNA Sequencing Data in Myeloproliferative Diseases

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2462-2462
Author(s):  
Roberta Spinelli ◽  
Rocco Piazza ◽  
Hima Raman ◽  
Alessandra Pirola ◽  
Simona Valletta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Exome Sequencing ◽  
Splice Site ◽  
Point Mutations ◽  
Rna Seq ◽  
Sequencing Data ◽  
Whole Exome ◽  
Splicing Variants ◽  
Sanger Method ◽  
Splicing Site

Abstract Abstract 2462 Point mutations in intronic regions near mRNA splice junctions can affect mRNA splicing, altering the resulting RNA sequence. The molecular characterization of in-frame or out-of-frame splicing variants in cancer samples can potentially assist in the molecular characterization of tumors. The aim of this study was to identify mutations located in the 5' or 3' exon-intron borders that affect RNA splicing using whole-exome sequencing analysis, a technique that targets coding sequences but also include the nearby intronic regions. In order to identify novel (in-frame and out-of-frame) splicing variants in myeloproliferative disorders we developed a bioinformatics procedure ‘Splice-Site Prediction Procedure to analyze Next Generation Sequencing data’ (SSPP-NGS). The SSPP-NGS bioinformatics method is an integration of two functional annotation tools for high-throughput sequencing data, ANNOVAR and MutationTaster and two canonical splice-site analysis tools, NetGene2 and Neural Network Promoter Prediction Tool (NNPPT). In addition, to assess the phenotypic effects of intronic mutations on mRNA splicing we combined DNA mutational screening analysis with RNA-Seq mediated gene expression profiling. Whole genome expression analysis was performed by using TopHat and Cufflinks: the first one is a splice junction mapper for RNA-Seq experiments able to mapp the reads against the junction to confirm them; the second one estimates gene expression, isoform-level expression, transcript abundance, differential gene expression and splicing. We used ANNOVAR and MutationTaster based on statistical Naive Bayes classifier to predict the non-coding mutations that affected physiological splicing. We then confirmed the results by queering NetGene2 and NNPPT using default parameters. Only the predictions found in all three programs were accepted as putative splicing variants and sequenced by Sanger method. We applied the entire procedure to whole exome sequencing data from 1 Ph+ leukemic patient sample (>80% myeloid cells) matched to autologous normal lymphocytes: on average, 70 million of paired-end reads and 5.2 gigabases (Gb) of sequences were generated per sample. A total of 177 candidate somatic point mutations (with minimum read depth of 20, minimum percent of substitution equal to 25% and minimum average Phred quality score of 30, corresponding to an accuracy of 99.9%, confirmed by at least 6 individual sequences) were found: 82/177 annotated in coding regions and 95/177 in non-coding regions. In particular 5/95 were located within 10-bp from a splicing junction. SSPS-NGS prediction analysis suggested the presence of 1/5 potential splicing site (predicting a loss of physiologic donor splicing site), while 4/5 were annotated as polymorphisms. The hypothetical splicing variant was located near the 5' donor splice site at position +1 in the intron between exons 5 and 6 of the GNAQ gene (IVS5+1C->T); it was present with a frequency of mutation of 35%, corresponding to its heterozygous presence in 88% of cells. The presence of this heterozygous mutation was confirmed by Sanger method. SSPS-NGS allowed us to focus on transcriptional analysis of this gene. RNA-seq analysis showed that 73% of GNAQ mRNA effectively skipped the upstream exon 5, resulting in a 4 to 6 frameshift fusion, which likely destroys the GTPase activity of GNAQ. No evidence of GNAQ exon 5 deleted RNA was found in additional 7 patients analyzed who lacked the intronic mutation. We extended the SSPN-NGS analysis to 7 myeloproliferative patients analyzed by exome sequencing. Three novel heterozygous splicing variants were identified, affecting the HOOK1, SMAD9 and DNAH9 genes. All mutations were confirmed by Sanger method. SSPS-NSG analysis predicted 1 loss of donor site in-frame (DNAH9) and 2 loss of acceptor splice site out of frame (HOOK1 and SMAD9), in one case with an activation of a new cryptic splicing site (HOOK1). RNA-seq analysis is in progress. In conclusion, the work presented here showed the applicability of SSPPs-NGS to whole-exome sequencing data as a tool to complement exome analysis, in order to identify novel splicing variants. Disclosures: No relevant conflicts of interest to declare.

scholarly journals RISK FACTORS, PREVALENCE AND DIAGNOSIS OF HUTCHISON GILFORD SYNDROME WITH SPECIAL REFERENCE TO CASE REPORTS

2017 ◽  
Vol 9 (5) ◽  
pp. 1
Author(s):  
Bhawana Sharma ◽  
Priyanka Sharma ◽  
S. C. Joshi
Keyword(s):  
Splice Site ◽  
De Novo ◽  
Case Reports ◽  
Genetic Disorder ◽  
Subcutaneous Fat ◽  
Point Mutations ◽  
Lamin A ◽  
Laboratory Findings ◽  
Progeria Syndrome ◽  
Hutchinson Gilford Progeria Syndrome

Progeria also known Hutchinson–Gilford progeria syndrome (HGPS), is an extremely rare genetic disorder. The prevalence of HGPS is 1 in 4-8 million newborns. Progeria causes premature, rapid aging shortly after birth present within the first year of life. Recently, de novo point mutations in the Lmna gene at position 1824 of the coding sequence have been found in persons with HGPS. Lmna encodes lamin A and C, the A-type lamins, which are an important structural component of the nuclear envelope and play a role in protein processing. The most common HGPS mutation is located at codon 608 (G608G). This mutation responsible for creating a cryptic splice site within exon 11, which deletes a proteolytic cleavage site within the expressed mutant lamin A. In Progeria, gene mutation results in the deletion of a Zmpste24/FACE1 splice site in prelamin A, preventing end terminal cleavage. The result of this point mutation can be observed by the main clinical and radiological features include alopecia, thin skin hypoplasia of nails, loss of subcutaneous fat, and osteolysis. The common symptoms of HGPS is a loss of eyebrows and eyelashes which can observed in early childhood and due to receding hairline and blading can also observed. Generally, this patient has facial character include microganthia (small jaw), craniofacial disproportion, prominent eyes, scalp veins and alopecia (loss of hair), restricted joint mobility and severe premature atherosclerosis. Laboratory findings are unremarkable, with the exception of an increased urinary excretion of hyaluronic acid. There is presently no effective therapy is available for Hutchinson-Gilford progeria syndrome (HGPS) but, it is essential to monitor carefully cardiovascular and cerebrovascular disease So, Treatment usually includes low dose aspirin which helps prevent the atherothrombotic events, stroke and heart attacks by hindering platelet aggregation

scholarly journals The zinc fingers of the SR-like protein ZRANB2 are single-stranded RNA-binding domains that recognize 5' splice site-like sequences

2009 ◽  
Vol 106 (14) ◽  
pp. 5581-5586 ◽  
Author(s):  
F. E. Loughlin ◽  
R. E. Mansfield ◽  
P. M. Vaz ◽  
A. P. McGrath ◽  
S. Setiyaputra ◽  
...  
Keyword(s):  
Splice Site ◽  
Rna Binding ◽  
Zinc Fingers ◽  
Binding Domains ◽  
Rna Binding Domains

scholarly journals Comparison of the frequencies of ENU-induced point mutations in male germ cells and inherited germline mutations in their offspring

2021 ◽  
Vol 43 (1) ◽  
Author(s):  
Kenichi Masumura ◽  
Tomoko Ando ◽  
Naomi Toyoda-Hokaiwado ◽  
Akiko Ukai ◽  
Takehiko Nohmi ◽  
...  
Keyword(s):  
Germ Cells ◽  
De Novo ◽  
Genetic Diseases ◽  
Point Mutations ◽  
Gene Mutations ◽  
Next Generation ◽  
Male Germ Cells ◽  
Gene Coding ◽  
Whole Exome ◽  
And Control

Abstract Background Gene mutations induced in germ cells may be transmitted to the next generation and cause adverse effects such as genetic diseases. Certain mutations may result in infertility or death in early development. Thus, the mutations may not be inheritable. However, the extent to which point mutations in male germ cells are transmitted to the next generation or eliminated during transmission is largely unknown. This study compared mutation frequencies (MFs) in sperm of N-ethyl-N-nitrosourea (ENU)-treated gpt delta mice and de novo MFs in the whole exome/genome of their offspring. Results Male gpt delta mice were treated with 10, 30, and 85 mg/kg of ENU (i.p., weekly × 2) and mated with untreated females to generate offspring. We previously reported a dose-dependent increase in de novo MFs in the offspring estimated by whole exome sequencing (WES) (Mutat. Res., 810, 30–39, 2016). In this study, gpt MFs in the sperm of ENU-treated mice were estimated, and the MFs per reporter gene were converted to MFs per base pair. The inherited de novo MFs in the offspring (9, 26 and 133 × 10− 8/bp for 10, 30, and 85 mg/kg ENU-treated groups, respectively) were comparable to those of the converted gpt MFs in the sperm of ENU-treated fathers (6, 16, and 69 × 10− 8/bp). It indicated that the gpt MFs in the ENU-treated father’s sperm were comparable to the inherited de novo MFs in the offspring as estimated by WES. In addition, de novo MFs in the offspring of 10 mg/kg ENU-treated and control fathers were estimated by whole genome sequencing (WGS), because WES was not sufficiently sensitive to detect low background MF. The de novo MF in the offspring of the ENU-treated fathers was 6 × 10− 8/bp and significantly higher than that of the control (2 × 10− 8/bp). There were no significant differences in de novo MFs between gene-coding and non-coding regions. WGS analysis was able to detect ENU-induced characteristic de novo base substitutions at a low dose group. Conclusions Despite a difference between exome/genome and exogenous reporter genes, the results indicated that ENU-induced point mutations in male germ cells could be transmitted to the next generation without severe selection.

scholarly journals Analysis of single nucleotide variants in CRISPR-Cas9 edited zebrafish embryos shows no evidence of off-target inflation

2019 ◽  
Author(s):  
Marie R Mooney ◽  
Erica E Davis ◽  
Nicholas Katsanis
Keyword(s):  
De Novo ◽  
Point Mutations ◽  
Enzyme Engineering ◽  
Single Nucleotide Variants ◽  
Guide Rna ◽  
Whole Exome ◽  
Two Generations ◽  
Sgrna Design ◽  
Favorable Conditions

AbstractTherapeutic applications of CRISPR-Cas9 gene editing have spurred innovation in Cas9 enzyme engineering and single guide RNA (sgRNA) design algorithms to minimize potential off-target events. While recent work in rodents outlines favorable conditions for specific editing and uses a trio design to control for the contribution of natural genome variation, the potential for CRISPR-Cas9 to induce de novo mutations in vivo remains a topic of interest. In zebrafish, we performed whole exome sequencing (WES) on two generations of offspring derived from the same founding pair: 54 exomes from control and CRISPR-Cas9 edited embryos in the first generation (F0), and 16 exomes from the progeny of inbred F0 pairs in the second generation (F1). We did not observe an increase in the number of transmissible variants in edited individuals in F1, nor in F0 edited mosaic individuals, arguing that in vivo editing does not precipitate an inflation of deleterious point mutations.

scholarly journals Whole-exome sequencing with targeted analysis and epilepsy after acute symptomatic neonatal seizures

2021 ◽  
Author(s):  
Adam L. Numis ◽  
Gilberto da Gente ◽  
Elliott H. Sherr ◽  
Hannah C. Glass
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
De Novo ◽  
List Type ◽  
Sequencing Analysis ◽  
Neonatal Seizures ◽  
Pathogenic Variants ◽  
Targeted Analysis ◽  
Whole Exome ◽  
Epilepsy Gene

Abstract Background The contribution of pathogenic gene variants with development of epilepsy after acute symptomatic neonatal seizures is not known. Methods Case–control study of 20 trios in children with a history of acute symptomatic neonatal seizures: 10 with and 10 without post-neonatal epilepsy. We performed whole-exome sequencing (WES) and identified pathogenic de novo, transmitted, and non-transmitted variants from established and candidate epilepsy association genes and correlated prevalence of these variants with epilepsy outcomes. We performed a sensitivity analysis with genes associated with coronary artery disease (CAD). We analyzed variants throughout the exome to evaluate for differential enrichment of functional properties using exploratory KEGG searches. Results Querying 200 established and candidate epilepsy genes, pathogenic variants were identified in 5 children with post-neonatal epilepsy yet in only 1 child without subsequent epilepsy. There was no difference in the number of trios with non-transmitted pathogenic variants in epilepsy or CAD genes. An exploratory KEGG analysis demonstrated a relative enrichment in cell death pathways in children without subsequent epilepsy. Conclusions In this pilot study, children with epilepsy after acute symptomatic neonatal seizures had a higher prevalence of coding variants with a targeted epilepsy gene sequencing analysis compared to those patients without subsequent epilepsy. Impact We performed whole-exome sequencing (WES) in 20 trios, including 10 children with epilepsy and 10 without epilepsy, both after acute symptomatic neonatal seizures. Children with post-neonatal epilepsy had a higher burden of pathogenic variants in epilepsy-associated genes compared to those without post-neonatal epilepsy. Future studies evaluating this association may lead to a better understanding of the risk of epilepsy after acute symptomatic neonatal seizures and elucidate molecular pathways that are dysregulated after brain injury and implicated in epileptogenesis.

Sign in / Sign up

Export Citation Format

Share Document