scholarly journals A biallelic variant in CLRN2 causes non-syndromic hearing loss in humans

2021 ◽  
Author(s):  
Barbara Vona ◽  
Neda Mazaheri ◽  
Sheng-Jia Lin ◽  
Lucy A. Dunbar ◽  
Reza Maroofian ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Amino Acid ◽  
Rna Splicing ◽  
Stop Codon ◽  
Missense Variant ◽  
Homozygosity Mapping ◽  
Deafness Gene ◽  
Expression Studies ◽  
Hearing Function

AbstractDeafness, the most frequent sensory deficit in humans, is extremely heterogeneous with hundreds of genes involved. Clinical and genetic analyses of an extended consanguineous family with pre-lingual, moderate-to-profound autosomal recessive sensorineural hearing loss, allowed us to identify CLRN2, encoding a tetraspan protein, as a new deafness gene. Homozygosity mapping followed by exome sequencing identified a 14.96 Mb locus on chromosome 4p15.32p15.1 containing a likely pathogenic missense variant in CLRN2 (c.494C > A, NM_001079827.2) segregating with the disease. Using in vitro RNA splicing analysis, we show that the CLRN2 c.494C > A variant leads to two events: (1) the substitution of a highly conserved threonine (uncharged amino acid) to lysine (charged amino acid) at position 165, p.(Thr165Lys), and (2) aberrant splicing, with the retention of intron 2 resulting in a stop codon after 26 additional amino acids, p.(Gly146Lysfs*26). Expression studies and phenotyping of newly produced zebrafish and mouse models deficient for clarin 2 further confirm that clarin 2, expressed in the inner ear hair cells, is essential for normal organization and maintenance of the auditory hair bundles, and for hearing function. Together, our findings identify CLRN2 as a new deafness gene, which will impact future diagnosis and treatment for deaf patients.

scholarly journals Biallelic mutation of CLRN2 causes non-syndromic hearing loss in humans

2020 ◽  
Author(s):  
Barbara Vona ◽  
Neda Mazaheri ◽  
Sheng-Jia Lin ◽  
Lucy A Dunbar ◽  
Reza Maroofian ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Amino Acid ◽  
Rna Splicing ◽  
Stop Codon ◽  
Homozygosity Mapping ◽  
Biallelic Mutation ◽  
Deafness Gene ◽  
Expression Studies ◽  
Hearing Function

AbstractDeafness, the most frequent sensory deficit in humans, is extremely heterogenous with hundreds of genes probably involved. Clinical and genetic analyses of an extended consanguineous family with pre-lingual, moderate-to-profound autosomal recessive sensorineural hearing loss, allowed us to identify CLRN2, encoding a tetraspan protein as a new deafness gene. Homozygosity mapping followed by exome sequencing identified a 15.2 Mb locus on chromosome 4p15.32p15.1 containing a missense pathogenic variant in CLRN2 (c.494C>A, NM_001079827.2) segregating with the disease. Using in vitro RNA splicing analysis, we show that the CLRN2 c.494C>A mutation leads to two events: 1) the substitution of a highly conserved threonine (uncharged amino acid) to lysine (charged amino acid) at position 165, p.(Thr165Lys), and 2) aberrant splicing, with the retention of intron 2 resulting in a stop codon after 26 additional amino acids, p.(Gly146Lysfs*26). Expression studies and phenotyping of newly produced zebrafish and mouse models deficient for clarin 2 further confirm that clarin 2, expressed in the inner ear hair cells, is essential for normal organization and maintenance of the auditory hair bundles, and for hearing function. Together, our findings identify CLRN2 as a new deafness gene, which will impact future diagnosis and treatment for deaf patients.

A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2501-2505 ◽  
Author(s):  
Maurizio Margaglione ◽  
Rosa Santacroce ◽  
Donatella Colaizzo ◽  
Davide Seripa ◽  
Gennaro Vecchione ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Splicing ◽  
Messenger Rna ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Autosomal Recessive Disorder ◽  
Chain Gene ◽  
Hemorrhagic Diathesis ◽  
Reading Frame ◽  
Congenital Afibrinogenemia

Abstract Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by a hemorrhagic diathesis of variable severity. Although more than 100 families with this disorder have been described, genetic defects have been characterized in few cases. An investigation of a young propositus, offspring of a consanguineous marriage, with undetectable levels of functional and quantitative fibrinogen, was conducted. Sequence analysis of the fibrinogen genes showed a homozygous G-to-A mutation at the fifth nucleotide (nt 2395) of the third intervening sequence (IVS) of the γ-chain gene. Her first-degree relatives, who had approximately half the normal fibrinogen values and showed concordance between functional and immunologic levels, were heterozygtes. The G-to-A change predicts the disappearance of a donor splice site. After transfection with a construct, containing either the wild-type or the mutated sequence, cells with the mutant construct showed an aberrant messenger RNA (mRNA), consistent with skipping of exon 3, but not the expected mRNA. Sequencing of the abnormal mRNA showed the complete absence of exon 3. Skipping of exon 3 predicts the deletion of amino acid sequence from residue 16 to residue 75 and shifting of reading frame at amino acid 76 with a premature stop codon within exon 4 at position 77. Thus, the truncated γ-chain gene product would not interact with other chains to form the mature fibrinogen molecule. The current findings show that mutations within highly conserved IVS regions of fibrinogen genes could affect the efficiency of normal splicing, giving rise to congenital afibrinogenemia.

scholarly journals Comparative functional characterization of novel non-syndromicGJB2gene variant p.Gly45Arg and lethal syndromic variant p.Gly45Glu

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2494 ◽  
Author(s):  
Juan Rodriguez-Paris ◽  
Jörg Waldhaus ◽  
Jeenal A. Gordhandas ◽  
Lynn Pique ◽  
Iris Schrijver
Keyword(s):  
Hearing Loss ◽  
Amino Acid ◽  
Gap Junctions ◽  
Functional Characterization ◽  
Missense Variant ◽  
Amino Acid Position ◽  
Cutaneous Infections ◽  
Transfected Cells ◽  
Novel Variant ◽  
And Function

We characterized a novelGJB2missense variant, c.133G>A, p.Gly45Arg, and compared it with the only other variant at the same amino acid position of the connexin 26 protein (Cx26) reported to date: c.134G>A, p.Gly45Glu. Whereas both variants are associated with hearing loss and are dominantly inherited, p.Gly45Glu has been implicated in the rare fatal keratitis-ichthyosis-deafness (KID) syndrome, which results in cutaneous infections and septicemia with premature demise in the first year of life. In contrast, p.Gly45Arg appears to be non-syndromic. Subcellular localization experiments in transiently co-transfected HeLa cells demonstrated that Cx26-WT (wild-type) and p.Gly45Arg form gap junctions, whereas Cx26-WT with p.Gly45Glu protein does not. The substitution of a nonpolar amino acid glycine in wildtype Cx26 at position 45 with a negatively charged glutamic acid (acidic) has previously been shown to interfere with Ca2+regulation of hemichannel gating and to inhibit the formation of gap junctions, resulting in cell death. The novel variant p.Gly45Arg, however, changes this glycine to a positively charged arginine (basic), resulting in the formation of dysfunctional gap junctions that selectively affect the permeation of negatively charged inositol 1,4,5-trisphosphate (IP3) and contribute to hearing loss. Cx26 p.Gly45Arg transfected cells, unlike cells transfected with p.Gly45Glu, thrived at physiologic Ca2+concentrations, suggesting that Ca2+regulation of hemichannel gating is unaffected in Cx26 p.Gly45Arg transfected cells. Thus, the two oppositely charged amino acids that replace the highly conserved uncharged glycine in p.Gly45Glu and p.Gly45Arg, respectively, produce strikingly different effects on the structure and function of the Cx26 protein.

scholarly journals Alteration in H-bond strength affects the stability of codon-anticodon interaction at in-frame UAG stop codon during in vitro translation

2021 ◽  
Author(s):  
Purnima Mala ◽  
Ishu Saraogi
Keyword(s):  
Amino Acid ◽  
Stop Codon ◽  
Protein Translation ◽  
In Vitro Translation ◽  
Natural Amino Acid ◽  
Additional Hydrogen Bond ◽  
Amino Acid Mutagenesis ◽  
The Stability ◽  
Decoding Ability

We have studied the decoding ability of a non-standard nucleobase modified tRNA for non-natural amino acid mutagenesis. The insertion of 2, 6-diaminopurine (D) base at the 3rd position of a tRNA anticodon enabled us to evaluate the effect of an additional hydrogen bond during translation. The presence of D at the tRNA anticodon led to stabilization of the codon-anticodon interaction due to an additional H-bond between the N2-exocyclic amine of D and the C2 carbonyl group of uracil during protein translation. While decoding UAG codons using stop codon suppression methodology, the enhanced codon-anticodon interaction improved codon readthrough and synthesis of modified protein with a non-natural amino acid at multiple sites. Our findings imply that the number of hydrogen bonds at the tRNA-mRNA duplex interface is an important criterion during mRNA decoding and improves protein translation at multiple UAG stop sites. This work provides valuable inputs towards improved non-natural amino acid mutagenesis for creating functional proteins.

scholarly journals Missense variant in LOXHD1 is associated with canine nonsyndromic hearing loss

2021 ◽  
Author(s):  
Marjo K. Hytönen ◽  
Julia E. Niskanen ◽  
Meharji Arumilli ◽  
Casey A. Brookhart-Knox ◽  
Jonas Donner ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Cell Function ◽  
Autosomal Recessive Inheritance ◽  
Missense Variant ◽  
Homozygosity Mapping ◽  
Large Animal Model ◽  
Large Animal ◽  
Hearing Disorder ◽  
Nonsyndromic Hearing Loss ◽  
Cochlear Hair Cell

AbstractHearing loss is a common sensory deficit in both humans and dogs. In canines, the genetic basis is largely unknown, as genetic variants have only been identified for a syndromic form of hearing impairment. We observed a congenital or early-onset sensorineural hearing loss in a Rottweiler litter. Assuming an autosomal recessive inheritance, we used a combined approach of homozygosity mapping and genome sequencing to dissect the genetic background of the disorder. We identified a fully segregating missense variant in LOXHD1, a gene that is known to be essential for cochlear hair cell function and associated with nonsyndromic hearing loss in humans and mice. The canine LOXHD1 variant was specific to the Rottweiler breed in our study cohorts of pure-bred dogs. However, it also was present in some mixed-breed dogs, of which the majority showed Rottweiler ancestry. Low allele frequencies in these populations, 2.6% and 0.04%, indicate a rare variant. To summarize, our study describes the first genetic variant for canine nonsyndromic hearing loss, which is clinically and genetically similar to human LOXHD1-related hearing disorder, and therefore, provides a new large animal model for hearing loss. Equally important, the affected breed will benefit from a genetic test to eradicate this LOXHD1-related hearing disorder from the population.

A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2501-2505
Author(s):  
Maurizio Margaglione ◽  
Rosa Santacroce ◽  
Donatella Colaizzo ◽  
Davide Seripa ◽  
Gennaro Vecchione ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Splicing ◽  
Messenger Rna ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Autosomal Recessive Disorder ◽  
Chain Gene ◽  
Hemorrhagic Diathesis ◽  
Reading Frame ◽  
Congenital Afibrinogenemia

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by a hemorrhagic diathesis of variable severity. Although more than 100 families with this disorder have been described, genetic defects have been characterized in few cases. An investigation of a young propositus, offspring of a consanguineous marriage, with undetectable levels of functional and quantitative fibrinogen, was conducted. Sequence analysis of the fibrinogen genes showed a homozygous G-to-A mutation at the fifth nucleotide (nt 2395) of the third intervening sequence (IVS) of the γ-chain gene. Her first-degree relatives, who had approximately half the normal fibrinogen values and showed concordance between functional and immunologic levels, were heterozygtes. The G-to-A change predicts the disappearance of a donor splice site. After transfection with a construct, containing either the wild-type or the mutated sequence, cells with the mutant construct showed an aberrant messenger RNA (mRNA), consistent with skipping of exon 3, but not the expected mRNA. Sequencing of the abnormal mRNA showed the complete absence of exon 3. Skipping of exon 3 predicts the deletion of amino acid sequence from residue 16 to residue 75 and shifting of reading frame at amino acid 76 with a premature stop codon within exon 4 at position 77. Thus, the truncated γ-chain gene product would not interact with other chains to form the mature fibrinogen molecule. The current findings show that mutations within highly conserved IVS regions of fibrinogen genes could affect the efficiency of normal splicing, giving rise to congenital afibrinogenemia.

scholarly journals Missense Variant in LOXHD1 is Associated With Canine Nonsyndromic Hearing Loss

2021 ◽  
Author(s):  
Marjo K Hytönen ◽  
Julia E Niskanen ◽  
Meharji Arumilli ◽  
Casey A Knox ◽  
Jonas Donner ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Cell Function ◽  
Autosomal Recessive Inheritance ◽  
Missense Variant ◽  
Homozygosity Mapping ◽  
Large Animal Model ◽  
Large Animal ◽  
Hearing Disorder ◽  
Nonsyndromic Hearing Loss ◽  
Cochlear Hair Cell

Abstract Hearing loss is a common sensory deficit both in humans and dogs. In canines the genetic basis is largely unknown, as genetic variants have only been identified for a syndromic form of hearing impairment. We observed a congenital or early-onset sensorineural hearing loss in a Rottweiler litter. Assuming an autosomal recessive inheritance, we used a combined approach of homozygosity mapping and genome sequencing to dissect the genetic background of the disorder. We identified a fully segregating missense variant in LOXHD1, a gene that is known to be essential for cochlear hair cell function and associated with nonsyndromic hearing loss in humans and mice. The canine LOXHD1 variant was specific to the Rottweiler breed in our study cohorts of pure-bred dogs. However, it also was present in mixed-breed dogs, of which the majority showed Rottweiler ancestry. Low allele frequencies in these populations, 2.6 % and 0.04 %, respectively, indicate a rare variant. To summarize, our study describes the first genetic variant for canine nonsyndromic hearing loss, which is clinically and genetically similar to human LOXHD1-related hearing disorder, and therefore, provides a new large animal model for hearing loss. Equally important, the affected breed will benefit from a genetic test to eradicate the hearing disorder from the population.

scholarly journals Exon skipping via chimeric antisense U1 snRNAs to correct Retinitis Pigmentosa GTPase-Regulator (RPGR) splice defect.

2021 ◽  
Author(s):  
Giuseppina Covello ◽  
Gehan H. Ibrahim ◽  
Niccoló Bacchi ◽  
Simona Casarosa ◽  
Michela A Denti
Keyword(s):  
Retinitis Pigmentosa ◽  
Rna Splicing ◽  
Stop Codon ◽  
Genetic Defect ◽  
Exon Skipping ◽  
Model Systems ◽  
Preclinical Model ◽  
Inherited Retinal Dystrophies ◽  
Retinitis Pigmentosa Gtpase Regulator

Inherited retinal dystrophies are caused by mutations in more than 250 genes, each of them carrying several types of mutations that can lead to different clinical phenotypes. Mutations in Retinitis Pigmentosa GTPase-Regulator (RPGR) cause X-linked Retinitis pigmentosa (RP). A nucleotide substitution in intron 9 of RPGR causes the increase of an alternatively spliced isoform of the mature mRNA, bearing exon 9a (E9a). This introduces a stop codon, leading to truncation of the protein. Aiming to restore impaired gene expression, we developed an antisense RNA-based therapeutic approach for the skipping of RPGR E9a. We designed a set of specific U1 antisense snRNAs (U1x_asRNAs) and tested their efficacy in vitro, upon transient co-transfection with RPGR minigene reporter systems in HEK-293T and PC-12 cell lines. We thus identified three chimeric U1_asRNAs that efficiently mediate E9a skipping, correcting the genetic defect. Unexpectedly, the U1-5' antisense construct, which exhibited the highest exon-skipping efficiency in PC-12 cells, induced E9a inclusion in HEK-293T cells, indicating caution in the choice of preclinical model systems when testing RNA splicing-correcting therapies. Our data provide a proof of principle for the application of U1_snRNA exon skipping-based approach to correct splicing defects in RPGR.

scholarly journals TPP2 mutation associated with sterile brain inflammation mimicking MS

2018 ◽  
Vol 4 (6) ◽  
pp. e285 ◽  
Author(s):  
Eva M. Reinthaler ◽  
Elisabeth Graf ◽  
Tobias Zrzavy ◽  
Thomas Wieland ◽  
Christoph Hotzy ◽  
...  
Keyword(s):  
Protein Expression ◽  
Missense Variant ◽  
Homozygosity Mapping ◽  
Brain Inflammation ◽  
Postmortem Brain ◽  
Loss Of Function ◽  
Expression Studies ◽  
Postmortem Brain Tissue ◽  
Tripeptidyl Peptidase ◽  
Tripeptidyl Peptidase Ii

ObjectiveTo ascertain the genetic cause of a consanguineous family from Syria suffering from a sterile brain inflammation mimicking a mild nonprogressive form of MS.MethodsWe used homozygosity mapping and next-generation sequencing to detect the disease-causing gene in the affected siblings. In addition, we performed RNA and protein expression studies, enzymatic activity assays, immunohistochemistry, and targeted sequencing of further MS cases from Austria, Germany, Canada and Jordan.ResultsIn this study, we describe the identification of a homozygous missense mutation (c.82T>G, p.Cys28Gly) in the tripeptidyl peptidase II (TPP2) gene in all 3 affected siblings of the family. Sequencing of all TPP2-coding exons in 826 MS cases identified one further homozygous missense variant (c.2027C>T, p.Thr676Ile) in a Jordanian MS patient. TPP2 protein expression in whole blood was reduced in the affected siblings. In contrast, TPP2 protein expression in postmortem brain tissue from MS patients without TPP2 mutations was highly upregulated.ConclusionsThe homozygous TPP2 mutation (p.Cys28Gly) is likely responsible for the inflammation phenotype in this family. TPP2 is an ubiquitously expressed serine peptidase that removes tripeptides from the N-terminal end of longer peptides. TPP2 is involved in various biological processes including the destruction of major histocompatibility complex Class I epitopes. Recessive loss-of-function mutations in TPP2 were described in patients with Evans syndrome, a rare autoimmune disease affecting the hematopoietic system. Based on the gene expression results in our MS autopsy brain samples, we further suggest that TPP2 may play a broader role in the inflammatory process in MS.

scholarly journals The effect of novel mutations on the structure and enzymatic activity of unconventional myosins associated with autosomal dominant non-syndromic hearing loss

Open Biology ◽  
2014 ◽  
Vol 4 (7) ◽  
pp. 140107 ◽  
Author(s):  
Tae-Jun Kwon ◽  
Se-Kyung Oh ◽  
Hong-Joon Park ◽  
Osamu Sato ◽  
Hanka Venselaar ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Protein Function ◽  
Autosomal Dominant ◽  
Missense Variant ◽  
Myosin Vi ◽  
Protein Modelling ◽  
Functional Studies ◽  
Genetic Hearing Loss ◽  
Modelling Studies

Mutations in five unconventional myosin genes have been associated with genetic hearing loss (HL). These genes encode the motor proteins myosin IA, IIIA, VI, VIIA and XVA. To date, most mutations in myosin genes have been found in the Caucasian population. In addition, only a few functional studies have been performed on the previously reported myosin mutations. We performed screening and functional studies for mutations in the MYO1A and MYO6 genes in Korean cases of autosomal dominant non-syndromic HL. We identified four novel heterozygous mutations in MYO6 . Three mutations (p.R825X, p.R991X and Q918fsX941) produce a premature truncation of the myosin VI protein. Another mutation, p.R205Q, was associated with diminished actin-activated ATPase activity and actin gliding velocity of myosin VI in an in vitro analysis. This finding is consistent with the results of protein modelling studies and corroborates the pathogenicity of this mutation in the MYO6 gene. One missense variant, p.R544W, was found in the MYO1A gene, and in silico analysis suggested that this variant has deleterious effects on protein function. This finding is consistent with the results of protein modelling studies and corroborates the pathogenic effect of this mutation in the MYO6 gene.

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