scholarly journals A novel synonymous ABCA3 variant identified in a Chinese family with lethal neonatal respiratory failure

2021 ◽  
Author(s):  
Weifeng Zhang ◽  
Zhiyong Liu ◽  
Yiming Lin ◽  
Ruiquan Wang ◽  
Jinglin Xu ◽  
...  
Keyword(s):  
Respiratory Failure ◽  
Splice Site ◽  
Quantitative Polymerase Chain Reaction ◽  
Donor Site ◽  
Splice Donor Site ◽  
Patient Specific ◽  
Chinese Family ◽  
Compound Heterozygous ◽  
Loss Of Function ◽  
Synonymous Variant

Abstract Background: Lethal respiratory failure is mostly caused by pulmonary surfactant (PS) deficiency and is the main cause of neonatal death. PS metabolism dysfunction caused by mutations in the ABCA3 gene is a rare disease with very poor prognosis. However, the underlying mechanism of genetic mutations causing PS metabolism dysfunction has not been fully elucidated yet. This study aimed to identify the genetic features in a family with lethal respiratory failure.Methods: We studied members of a two-generation Chinese family including a female proband, her parents, her identical twin sister, and her older sister. Whole exome sequencing (WES), Sanger sequencing, and real-time quantitative polymerase chain reaction （PCR） were used to identify and validate the ABCA3 mutation. The potential pathogenicity of the identified synonymous variant was predicted using splice site algorithms (dbscSNV11_AdaBoost, dbscSNV11_RandomForest, and HSF).Results: All patients showed severe respiratory distress, which could not be relieved by mechanical ventilation, supplementation of surfactants, or steroid therapy, and died at an early age. Molecular genetic analysis revealed that the patients had compound heterozygous ABCA3 variants, including a novel synonymous variant c.G873A (p.Lys291Lys) in exon 8 derived from the mother and a heterozygous deletion of exons 4-7 derived from the father. The synonymous variant was consistently predicted to be a cryptic splice donor site that may lead to aberrant splicing of the pre-mRNA by three different splice site algorithms. The deletion of exons 4-7 of the ABCA3 gene was determined to be a loss-of-function pathogenic variant.Conclusions: We identified a novel synonymous variant and a deletion in the ABCA3 gene that may be responsible for the pathogenesis in patients in this family. These results enrich the known mutational spectrum of the ABCA3 gene. Moreover, the study of ABCA3 mutations is helpful for the realization of patient-specific therapies.

scholarly journals A novel synonymous ABCA3 variant identified in a Chinese family with lethal neonatal respiratory failure

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Weifeng Zhang ◽  
Zhiyong Liu ◽  
Yiming Lin ◽  
Ruiquan Wang ◽  
Jinglin Xu ◽  
...  
Keyword(s):  
Respiratory Failure ◽  
Splice Site ◽  
Pulmonary Surfactant ◽  
Sanger Sequencing ◽  
Monozygotic Twin ◽  
Splice Donor Site ◽  
Patient Specific ◽  
Chinese Family ◽  
Twin Sister ◽  
Synonymous Variant

Abstract Background Lethal respiratory failure is primarily caused by a deficiency of pulmonary surfactant, and is the main cause of neonatal death among preterm infants. Pulmonary surfactant metabolism dysfunction caused by variants in the ABCA3 gene is a rare disease with very poor prognosis. Currently, the mechanisms associated with some ABCA3 variants have been determined, including protein mistrafficking and impaired phospholipid transport. However, some novel variants and their underlying pathogenesis has not been fully elucidated yet. In this study we aimed to identify the genetic features in a family with lethal respiratory failure. Methods We studied members of two generations of a Chinese family, including a female proband, her parents, her monozygotic twin sister, and her older sister. Trio whole exome sequencing (WES) were used on the proband and her parents to identify the ABCA3 variants. Sanger sequencing and real-time quantitative polymerase chain reaction (PCR) were used on the monozygotic twin sister of proband to validate the ABCA3 synonymous variant and exon deletion, respectively. The potential pathogenicity of the identified synonymous variant was predicted using the splice site algorithms dbscSNV11_AdaBoost, dbscSNV11_RandomForest, and Human Splicing Finder (HSF). Results All patients showed severe respiratory distress, which could not be relieved by mechanical ventilation, supplementation of surfactant, or steroid therapy, and died at an early age. WES analysis revealed that the proband had compound heterozygous ABCA3 variants, including a novel synonymous variant c.G873A (p.Lys291Lys) in exon 8 inherited from the mother, and a heterozygous deletion of exons 4–7 inherited from the father. The synonymous variant was consistently predicted to be a cryptic splice donor site that may lead to aberrant splicing of the pre-mRNA by three different splice site algorithms. The deletion of exons 4–7 of the ABCA3 gene was determined to be a likely pathogenic variant. The variants were confirmed in the monozygotic twin sister of proband by Sanger sequencing and qPCR respectively. The older sister of proband was not available to determine if she also carried both ABCA3 variants, but it is highly likely based on her clinical course. Conclusions We identified a novel synonymous variant and a deletion in the ABCA3 gene that may be responsible for the pathogenesis in patients in this family. These results add to the known mutational spectrum of the ABCA3 gene. The study of ABCA3 variants may be helpful for the implementation of patient-specific therapies.

scholarly journals Novel Loss-of-Function Variants in CDC14A are Associated with Recessive Sensorineural Hearing Loss in Iranian and Pakistani Patients

2020 ◽  
Vol 21 (1) ◽  
pp. 311 ◽  
Author(s):  
Julia Doll ◽  
Susanne Kolb ◽  
Linda Schnapp ◽  
Aboulfazl Rad ◽  
Franz Rüschendorf ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Sensorineural Hearing Loss ◽  
Hearing Impairment ◽  
Splice Site ◽  
Donor Site ◽  
Sensorineural Hearing ◽  
Splice Donor Site ◽  
Loss Of Function ◽  
Altered Expression ◽  
Initial Symptoms

CDC14A encodes the Cell Division Cycle 14A protein and has been associated with autosomal recessive non-syndromic hearing loss (DFNB32), as well as hearing impairment and infertile male syndrome (HIIMS) since 2016. To date, only nine variants have been associated in patients whose initial symptoms included moderate-to-profound hearing impairment. Exome analysis of Iranian and Pakistani probands who both showed bilateral, sensorineural hearing loss revealed a novel splice site variant (c.1421+2T>C, p.?) that disrupts the splice donor site and a novel frameshift variant (c.1041dup, p.Ser348Glnfs*2) in the gene CDC14A, respectively. To evaluate the pathogenicity of both loss-of-function variants, we analyzed the effects of both variants on the RNA-level. The splice variant was characterized using a minigene assay. Altered expression levels due to the c.1041dup variant were assessed using RT-qPCR. In summary, cDNA analysis confirmed that the c.1421+2T>C variant activates a cryptic splice site, resulting in a truncated transcript (c.1414_1421del, p.Val472Leufs*20) and the c.1041dup variant results in a defective transcript that is likely degraded by nonsense-mediated mRNA decay. The present study functionally characterizes two variants and provides further confirmatory evidence that CDC14A is associated with a rare form of hereditary hearing loss.

scholarly journals Novel compound heterozygous CLCNKB gene mutations (c.1755A>G/c.848_850delTCT) cause classic Bartter syndrome

2018 ◽  
Vol 315 (4) ◽  
pp. F844-F851 ◽  
Author(s):  
Chunli Wang ◽  
Ying Chen ◽  
Bixia Zheng ◽  
Mengshu Zhu ◽  
Jia Fan ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Donor Site ◽  
Gene Mutations ◽  
Bartter Syndrome ◽  
Splice Donor Site ◽  
Chinese Family ◽  
Compound Heterozygous ◽  
Nucleotide Mutation ◽  
Clcnkb Gene

Inactivated variants in CLCNKB gene encoding the basolateral chloride channel ClC-Kb cause classic Bartter syndrome characterized by hypokalemic metabolic alkalosis and hyperreninemic hyperaldosteronism. Here, we identified two cBS siblings presenting hypokalemia in a Chinese family due to novel compound heterozygous CLCNKB mutations (c.848_850delTCT/c.1755A>G). Compound heterozygosity was confirmed by amplifying and sequencing the patientʼs genomic DNA. The synonymous mutation c.1755A>G (Thr585Thr) was located at +2 bp from the 5′ splice donor site in exon 15. Further transcript analysis demonstrated that this single nucleotide mutation causes exclusion of exon 15 in the cDNA from the proband and his mother. Furthermore, we investigated the expression and protein trafficking change of c.848_850delTCT (ΔTCT) and exon 15 deletion (ΔE15) mutation in vitro. The ΔE15 mutation markedly decreased the expression of ClC-Kb and resulted in a low-molecular-weight band (~55 kDa) trapping in the endoplasmic reticulum, while the ΔTCT mutant only decreased the total and plasma membrane ClC-Kb protein expression but did not affect the subcellular localization. Finally, we studied the physiological functions of mutations by using whole cell patch-clamp and found that the ΔE15 or ΔTCT mutation decreased the current of the ClC-Kb/barttin channel. These results suggested that the compound defective mutations of the CLCNKB gene are the molecular mechanism of the two cBS siblings.

scholarly journals A 5′ splice site mutation affecting the pre-mRNA splicing of two upstream exons in the collagen COL1A1 gene. Exon 8 skipping and altered definition of exon 7 generates truncated proα1(I) chains with a non-collagenous insertion destabilizing the triple helix

1994 ◽  
Vol 302 (3) ◽  
pp. 729-735 ◽  
Author(s):  
J F Bateman ◽  
D Chan ◽  
I Moeller ◽  
M Hannagan ◽  
W G Cole
Keyword(s):  
Splice Site ◽  
De Novo ◽  
Donor Site ◽  
Mrna Splicing ◽  
Splice Donor Site ◽  
Sequence Motif ◽  
Type I ◽  
Splice Donor ◽  
Site Mutation ◽  
Definition Of

A heterozygous de novo G to A point mutation in intron 8 at the +5 position of the splice donor site of the gene for the pro alpha 1(I) chain of type I procollagen, COL1A1, was defined in a patient with type IV osteogenesis imperfecta. The splice donor site mutation resulted not only in the skipping of the upstream exon 8 but also unexpectedly had the secondary effect of activating a cryptic splice site in the next upstream intron, intron 7, leading to re-definition of the 3′ limit of exon 7. These pre-mRNA splicing aberrations cause the deletion of exon 8 sequences from the mature mRNA and the inclusion of 96 bp of intron 7 sequence. Since the mis-splicing of the mutant allele product resulted in the maintenance of the correct codon reading frame, the resultant pro alpha 1(I) chain contained a short non-collagenous 32-amino-acid sequence insertion within the repetitive Gly-Xaa-Yaa collagen sequence motif. At the protein level, the mutant alpha 1(I) chain was revealed by digestion with pepsin, which cleaved the mutant procollagen within the protease-sensitive non-collagenous insertion, producing a truncated alpha 1(I). This protease sensitivity demonstrated the structural distortion to the helical structure caused by the insertion. In long-term culture with ascorbic acid, which stimulates the formation of a mature crosslinked collagen matrix, and in tissues, there was no evidence of the mutant chain, suggesting that during matrix formation the mutant chain was unable to stably incorporated into the matrix and was degraded proteolytically.

scholarly journals A Deep Exon Cryptic Splice Site Promotes Aberrant Intron Retention in a Von Willebrand Disease Patient

2021 ◽  
Vol 22 (24) ◽  
pp. 13248
Author(s):  
John G. Conboy
Keyword(s):  
Secondary Structure ◽  
Splice Site ◽  
Intron Retention ◽  
Donor Site ◽  
Von Willebrand Disease ◽  
Splice Donor Site ◽  
Cryptic Splice Site ◽  
Splice Donor ◽  
Nucleotide Mutation ◽  
Von Willebrand

A translationally silent single nucleotide mutation in exon 44 (E44) of the von Willebrand factor (VWF) gene is associated with inefficient removal of intron 44 in a von Willebrand disease (VWD) patient. This intron retention (IR) event was previously attributed to reordered E44 secondary structure that sequesters the normal splice donor site. We propose an alternative mechanism: the mutation introduces a cryptic splice donor site that interferes with the function of the annotated site to favor IR. We evaluated both models using minigene splicing reporters engineered to vary in secondary structure and/or cryptic splice site content. Analysis of splicing efficiency in transfected K562 cells suggested that the mutation-generated cryptic splice site in E44 was sufficient to induce substantial IR. Mutations predicted to vary secondary structure at the annotated site also had modest effects on IR and shifted the balance of residual splicing between the cryptic site and annotated site, supporting competition among the sites. Further studies demonstrated that introduction of cryptic splice donor motifs at other positions in E44 did not promote IR, indicating that interference with the annotated site is context dependent. We conclude that mutant deep exon splice sites can interfere with proper splicing by inducing IR.

Efficacy of DNA versus RNA NGS-based Methods in MET Exon 14 skipping mutation detection.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 9036-9036
Author(s):  
Magdalena Jurkiewicz ◽  
Anjali Saqi ◽  
Mahesh M Mansukhani ◽  
Vaishali Hodel ◽  
Anamaria Krull ◽  
...  
Keyword(s):  
Splice Site ◽  
Donor Site ◽  
Driver Mutations ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Acceptor Site ◽  
Female Ratio ◽  
Lung Carcinomas ◽  
Rna Analysis ◽  
Lung Adenocarcinomas

9036 Background: Exon 14 skipping mutations in the mesenchymal-epithelial transition ( MET) gene are reported in 2-5% of lung adenocarcinomas and are mutually exclusive of other driver mutations. Small-molecule MET tyrosine kinase inhibitors, capmatinib and tepotinib, showed durable responses in previously treated and treatment-naïve patients harboring MET-exon-14 skipping mutations. Studies suggest that for detection of MET-ex14 mutations, DNA-based assays alone may be sub-optimal when compared to RNA-based NGS assays. We compared the performance of DNA and RNA-based assays for detection of MET-ex14 variants. Methods: We examined NGS-based profiling data of lung adenocarcinomas (or when this diagnosis could not be excluded) to identify MET-ex14 mutations missed by DNA but identified by RNA analysis. The carcinomas were profiled by a DNA-based NGS panel that targets MET exons 2, 14, 16, 18 and 19. Cases without driver mutations were reflexed to an NGS-based RNA fusion panel (Archer’s Anchored Multiplex PCR). Results: Over a 21-month period, MET-ex14 skipping events were detected in 16/644 (2.5%) lung carcinomas by DNA profiling. RNA analysis on driver-negative cases identified 9 additional MET-ex14 mutations. All 16 MET-ex14 DNA variants occurred at or around the intron 14 splice donor site, as the assay did not include the intron 13 splice acceptor site. Clinical characteristics of the MET positive cohort include a male to female ratio of 0.8:1.0, an average age of 76.5 years and 52% non-smoker status. All tumors were adenocarcinomas (including one with a < 10% spindle/pleomorphic component) with the exception of 3 adenosquamous carcinomas and 1 squamous cell carcinoma. Conclusions: DNA based NGS-panels can potentially miss MET-ex14 skipping events in lung carcinomas, when the primers do not target both 3′ splice site of intron 13, and the 5′ splice site of intron 14. A reflex work flow interrogating RNA fusions can potentially capture such events. The clinical and molecular characterization of the variants detected only by RNA NGS assays warrants further exploration.

scholarly journals Isoform-specific loss of dystonin causes hereditary motor and sensory neuropathy

2020 ◽  
Vol 6 (5) ◽  
pp. e496
Author(s):  
William W. Motley ◽  
Stephan Züchner ◽  
Steven S. Scherer
Keyword(s):  
Exome Sequencing ◽  
Messenger Rna ◽  
Dna Analysis ◽  
Transmembrane Domain ◽  
Donor Site ◽  
Sensory Neuropathy ◽  
Splice Donor Site ◽  
Compound Heterozygous ◽  
Human Phenotype ◽  
Charcot Marie Tooth

ObjectiveTo determine the genetic cause of axonal Charcot-Marie-Tooth disease in a small family with 2 affected siblings, one of whom had cerebellar features on examination.MethodsWhole-exome sequencing of genomic DNA and analysis for recessively inherited mutations; PCR-based messenger RNA/complementary DNA analysis of transcripts to characterize the effects of variants identified by exome sequencing.ResultsWe identified compound heterozygous mutations in dystonin (DST), which is alternatively spliced to create many plakin family linker proteins (named the bullous pemphigoid antigen 1 [BPAG1] proteins) that function to bridge cytoskeletal filament networks. One mutation (c.250C>T) is predicted to cause a nonsense mutation (p.R84X) that only affects isoform 2 variants, which have an N-terminal transmembrane domain; the other (c.8283+1G>A) mutates a consensus splice donor site and results in a 22 amino acid in-frame deletion in the spectrin repeat domain of all BPAG1a and BPAG1b isoforms.ConclusionsThese findings introduce a novel human phenotype, axonal Charcot-Marie-Tooth, of recessive DST mutations, and provide further evidence that BPAG1 plays an essential role in axonal health.

scholarly journals Comparison of in vitro and in vivo splice site selection in kappa-immunoglobulin precursor mRNA.

1988 ◽  
Vol 8 (6) ◽  
pp. 2610-2619 ◽  
Author(s):  
D E Lowery ◽  
B G Van Ness
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Donor Site ◽  
Splice Donor Site ◽  
Splice Sites ◽  
Splice Donor ◽  
Splice Site Selection ◽  
Nuclear Extracts

The processing of a number of kappa-immunoglobulin primary mRNA (pre-mRNA) constructs has been examined both in vitro and in vivo. When a kappa-immunoglobulin pre-mRNA containing multiple J segment splice sites is processed in vitro, the splice sites are used with equal frequency. The presence of signal exon, S-V intron, or variable (V) region has no effect on splice site selection in vitro. Nuclear extracts prepared from a lymphoid cell line do not restore correct splice site selection. Splice site selection in vitro can be altered by changing the position or sequence of J splice donor sites. These results differ from the processing of similar pre-mRNAs expressed in vivo by transient transfection. The 5'-most J splice donor site was exclusively selected in vivo, even in nonlymphoid cells, and even in transcripts where in vitro splicing favored a 3' J splice site. The in vitro results are consistent with a model proposing that splice site selection is influenced by splice site strength and proximity; however, our in vivo results demonstrate a number of discrepancies with such a model and suggest that splice site selection may be coupled to transcription or a higher-order nuclear structure.

scholarly journals Novel Splice Site Pathogenic Variant of EFTUD2 Is Associated with Mandibulofacial Dysostosis with Microcephaly and Extracranial Symptoms in Korea

Diagnostics ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 296
Author(s):  
So Young Kim ◽  
Da-hye Lee ◽  
Jin Hee Han ◽  
Byung Yoon Choi
Keyword(s):  
Splice Site ◽  
De Novo ◽  
Donor Site ◽  
Elongation Factor ◽  
Splice Donor Site ◽  
Functional Verification ◽  
Pathogenic Potential ◽  
Mandibulofacial Dysostosis ◽  
Minigene Assay

Elongation factor Tu guanosine-5’-triphosphate (GTP) binding domain containing 2 (EFTUD2) encodes a major component of the spliceosomal GTPase and, if mutated, causes mandibulofacial dysostosis with microcephaly (MFDM; MIM#610536). Despite the increasing number of potentially pathogenic variants reported in the literature, most previous studies have relied solely on in silico prediction of the pathogenic potential of EFTUD2 variants, which may result in misclassification of the variant’s pathogenicity. Given the importance of the functional verification of EFTUD2 variants, we identified a novel splice donor site variant, c.271+1G>A of EFTUD2, whose pathogenicity was clearly verified at the RNA level using a minigene assay. A child with MFDM, mixed hearing loss, microcephaly, and a congenital cardiac defect was identified with this variant, which arose in a de novo fashion. The minigene assay showed erroneous integration of the 118 bp IVS3 of EFTUD2 exclusively among the c.271+1G>A variant clone. We first applied the minigene assay to identify the splice function of a splice site variant of EFTUD2, thereby allowing for in vitro functional verification of splice site variants in EFTUD2.

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