Small Exons Research Articles

Conserved role for spliceosomal component PRPF40A in microexon splicing.

Microexons (exons ≤30 nts) are important features of neuronal transcriptomes, but pose mechanistic challenges to the splicing machinery. We previously showed that PRP-40, a component of the U1 spliceosome, is globally required for microexon splicing in C. elegans. Here we show that the homologous PRPF40A is also globally required for microexon splicing in mouse neuroblastoma cells. We find that PRPF40A co-regulates microexons along with SRRM4, a neuron-specific regulator of microexon splicing. The relationship between exon size and dependence on PRPF40A/SRRM4 is distinct, with SRRM4-dependence exhibiting a size threshold (~30 nts) and PRPF40A-dependence exhibiting a graded decrease as exon size increases. Finally, we show that PRPF40A knockdown causes an increase in productive splicing of its spliceosomal binding partner Luc7l by skipping of a small poison exon. Similar homeostatic cross-regulation is often observed across paralogous RNA binding proteins. Here we find this concept likewise applies across evolutionarily unrelated but functionally and physically coupled spliceosomal components.

Emerging concepts of myosin 18A isoform mechanobiology in organismal and immune system physiology, development, and function.

Alternative and combinatorial splicing of myosin 18A (MYO18A) gene transcripts results in expression of MYO18A protein isoforms and isoform variants with different membrane and subcellular localizations, and functional properties. MYO18A proteins are members of the myosin superfamily consisting of a myosin-like motor domain, an IQ motif, and a coiled-coil domain. MYO18A isoforms, however, lack the ability to hydrolyze ATP and do not perform ATP-dependent motor activity. MYO18A isoforms are distinguished by different amino- and carboxy-terminal extensions and domains. The domain organization and functions of MYO18Aα, MYO18Aβ, and MYO18Aγ have been studied experimentally. MYO18Aα and MYO18Aβ have a common carboxy-terminal extension but differ by the presence or absence of an amino-terminal KE repeat and PDZ domain, respectively. The amino- and carboxy-terminal extensions of MYO18Aγ contain unique proline and serine-rich domains. Computationally predicted MYO18Aε and MYO18Aδ isoforms contain the carboxy-terminal serine-rich extension but differ by the presence or absence of the amino-terminal KE/PDZ extension. Additional isoform variants within each category arise by alternative utilization or inclusion/exclusion of small exons. MYO18Aα variants are expressed in somatic cells and mature immune cells, whereas MYO18Aβ variants occur mainly in myeloid and natural killer cells. MYO18Aγ expression is selective to cardiac and skeletal muscle. In the present review perspective, we discuss current and emerging concepts of the functional specialization of MYO18A proteins in membrane and cytoskeletal dynamics, cellular communication and signaling, endocytic and exocytic organelle movement, viral infection, and as the SP-R210 receptor for surfactant protein A.

Rbm45 Phylogenetics, Protein Domain Conservation, and Gene Architecture in Clade Metazoa

Mammalian Rbm45 is predominately expressed in neuronal tissue and is integral in brain development and neuronal differentiation under physiological conditions. Dysregulation of Rbm45 has been strongly associated with neurodegenerative disorders in humans and can drive hepatocellular carcinoma through reprogramming lipid metabolism. Intriguingly, Rbm45 is an ancient protein, evolutionarily conserved throughout metazoans, including in sponges which lack a nervous system. Curiously, the evolution of Rbm45 gene structure and protein domain conservation across kingdom Animalia is largely unknown. We performed phylogenetic analysis of Rbm45 nucleotide and amino acid sequences from 36 species representing 9 phyla: Porifera, Cnidaria, Priapulida, Mollusca, Brachiopoda, Arthropoda, Echinodermata, Hemichordata, and Chordata. While the tree from Rbm45 nucleotide sequence data resulted in clades Protostomia and Deuterostomia showing paraphyly, the phylogeny derived from Rbm45 amino acid sequence largely recapitulated known monophyletic relationships among metazoans. Human RBM45 protein structure includes three RNA-binding domains (RBD), a homo-oligomerization association (HOA) domain, a nuclear localization sequence (NLS), and a nuclear export sequence (NES). Multiple sequence alignment across the same 36 taxa used for phylogenetic analysis revealed conservation of all three RBDs, the HOA, and NLS; in contrast the NES was only detected in clade Craniata and not in clades Ambulacraria and Protostomia. Rbm45 gene structure analysis revealed increasing gene complexity concomitant with increasing evolutionary complexity. Rbm45 from non-bilaterian taxa had from 2 to 4 large exons, while bilaterian taxa had between 6 to17 small exons. These findings demonstrate that Rbm45 is an ancient, highly conserved gene among metazoans suggesting a function in a breadth of neural/sensory systems.

Analysis of DMD gene variants in a single center

Objective: To investigate the DMD genetic variants of the Chinese population with Duchenne (DMD) and Becker muscular dystrophies (BMD). Methods: A cross-sectional study was conducted on 2 690 unrelated patients with DMD and BMD aged 0-18 who visited the Genetic and Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University from January 2005 to February 2022. The clinical data, such as gender, age, clinical manifestations, and address, were collected. Multiplex ligation-dependent probe amplification, next generation sequencing panel, Sanger sequencing, and PCR amplification were used to detect the variants of the DMD gene in the patients, whose clinical information and gene detection results were descriptively analyzed. Results: The 2 690 patients included 2 648 males and 42 females, with an age of 6.0 (4.0, 9.0) years. The serum creatine kinase increased in all patients. Pathogenic DMD gene variants were detected in the 2 618 patients, including 1 875 cases (71.6%) large deletions, 231 cases (8.8%) duplications, and 512 cases (19.6%) small variants. Among the deletion variants, the deletion of 3 exons was the most common, accounting for 15.4% (288/1 875); and hotspot deletion involved exons 45 to 50, accounting for 6.3% (119/1 875). Exon 2 was the most common type duplication region, accounting for 13.0% (30/231). Small variants were distributed in all 79 exons of the DMD gene, with no hotspots. In addition, the 46 small variants were previously unreported. Conclusion: Exon deletion is the most common type of DMD gene variant, followed by small variants and exon duplication.

Regulation of micro- and small-exon retention and other splicing processes by GRP20 for flower development.

Pre-mRNA splicing is crucial for gene expression and depends on the spliceosome and splicing factors. Plant exons have an average size of ~180 nucleotides and typically contain motifs for interactions with spliceosome and splicing factors. Micro exons (<51 nucleotides) are found widely in eukaryotes and in genes for plant development and environmental responses. However, little is known about transcript-specific regulation of splicing in plants and about the regulators for micro exon splicing. Here we report that glycine-rich protein 20 (GRP20) is an RNA-binding protein and required for splicing of ~2,100 genes including those functioning in flower development and/or environmental responses. Specifically, GRP20 is required for micro-exon retention in transcripts of floral homeotic genes; these micro exons are conserved across angiosperms. GRP20 is also important for small-exon (51-100 nucleotides) splicing. In addition, GRP20 is required for flower development. Furthermore, GRP20 binds to poly-purine motifs in micro and small exons and a spliceosome component; both RNA binding and spliceosome interaction are important for flower development and micro-exon retention. Our results provide new insights into the mechanisms of micro-exon retention in flower development.

Identification and functional prediction of lncRNAs associated with intramuscular lipid deposition in Guangling donkeys.

Many studies have shown that long non-coding RNAs (lncRNAs) play key regulatory roles in various biological processes. However, the importance and molecular regulatory mechanisms of lncRNAs in donkey intramuscular fat deposition remain to be further investigated. In this study, we used published transcriptomic data from the longissimus dorsi muscle of Guangling donkeys to identify lncRNAs and obtained 196 novel lncRNAs. Compared with the coding genes, the novel lncRNAs and the known lncRNAs exhibited some typical features, such as shorter transcript length and smaller exons. A total of 272 coding genes and 52 lncRNAs were differentially expressed between the longissimus dorsi muscles of the low-fat and high-fat groups. The differentially expressed genes were found to be involved in various biological processes related to lipid metabolism. The potential target genes of differentially expressed lncRNAs were predicted by cis and trans. Functional analysis of lncRNA targets showed that some lncRNAs may act on potential target genes involved in lipid metabolism processes and regulate lipid deposition in the longissimus dorsi muscle. This study provides valuable information for further investigation of the molecular mechanisms of lipid deposition traits in donkeys, which may improve meat traits and facilitate the selection process of donkeys in future breeding.

Differential Myosin 5a splice variants in innervation of pelvic organs.

Introduction: Myosin proteins interact with filamentous actin and translate the chemical energy generated by ATP hydrolysis into a wide variety of mechanical functions in all cell types. The classic function of conventional myosins is mediation of muscle contraction, but myosins also participate in processes as diverse as exocytosis/endocytosis, membrane remodeling, and cytokinesis. Myosin 5a (Myo5a) is an unconventional motor protein well-suited to the processive transport of diverse molecular cargo within cells and interactions with multiprotein membrane complexes that facilitate exocytosis. Myo5a includes a region consisting of six small alternative exons which can undergo differential splicing. Neurons and skin melanocytes express characteristic splice variants of Myo5a, which are specialized for transport processes unique to those cell types. But less is known about the expression of Myo5a splice variants in other tissues, their cargos and interactive partners, and their regulation. Methods: In visceral organs, neurotransmission-induced contraction or relaxation of smooth muscle is mediated by Myo5a. Axons within urogenital organs and distal colon of rodents arise from cell bodies located in the major pelvic ganglion (MPG). However, in contrast to urogenital organs, the distal colon also contains soma of the enteric nervous system. Therefore, the rodent pelvic organs provide an opportunity to compare the expression of Myo5a splice variants, not only in different tissues innervated by the pelvic nerves, but also in different subcellular compartments of those nerves. This study examines the expression and distribution of Myo5a splice variants in the MPG, compared to the bladder, corpus cavernosum of the penis (CCP) and distal colon using immunohistochemistry and mRNA analyses. Results/discussion: We report detection of characteristic Myo5a variants in these tissues, with bladder and CCP displaying a similar variant pattern but one which differed from that of distal colon. In all three organs, Myo5a variants were distinct compared to the MPG, implying segregation of one variant within nerve soma and its exclusion from axons. The expression of distinct Myo5a variant arrays is likely to be adaptive, and to underlie specific functions fulfilled by Myo5a in those particular locations.

Identification and quantification of small exon-containing isoforms in long-read RNA sequencing data.

Small exons are pervasive in transcriptomes across organisms, and their quantification in RNA isoforms is crucial for understanding gene functions. Although long-read RNA-seq based on Oxford Nanopore Technologies (ONT) offers the advantage of covering transcripts in full length, its lower base accuracy poses challenges for identifying individual exons, particularly microexons (≤30 nucleotides). Here, we systematically assess small exons quantification in synthetic and human ONT RNA-seq datasets. We demonstrate that reads containing small exons are often not properly aligned, affecting the quantification of relevant transcripts. Thus, we develop a local-realignment method for misaligned exons (MisER), which remaps reads with misaligned exons to the transcript references. Using synthetic and simulated datasets, we demonstrate the high sensitivity and specificity of MisER for the quantification of transcripts containing small exons. Moreover, MisER enabled us to identify small exons with a higher percent spliced-in index (PSI) in neural, particularly neural-regulated microexons, when comparing 14 neural to 16 non-neural tissues in humans. Our work introduces an improved quantification method for long-read RNA-seq and especially facilitates studies using ONT long-reads to elucidate the regulation of genes involving small exons.

NovoRNABreak: local assembly for novel splice junction and fusion transcript detection from RNA-seq data

We present novoRNABreak, a unified framework for cancer specific novel splice junction and fusion transcript detection in RNA-seq data obtained from human cancer samples. novoRNABreak is based on a local assembly model, which offers a tradeoff between the alignment-based and de novo whole transcriptome assembly (WTA) methods. This approach is accurate and sensitive in assembling novel junctions that are difficult to directly align or have multiple alignments. Additionally, it is more efficient due to the strategy that focuses on junctions rather than full length transcripts. The performance of novoRNABreak is demonstrated by a comprehensive set of experiments using synthetic data generated based on genome reference, as well as real RNA-seq data from breast cancer and prostate cancer samples. The results show that our tool has a better performance by fully utilizing unmapped reads and precisely identifying the junctions where short reads or small exons have multiple alignments. novoRNABreak is a fully-fledged program available on GitHub (https:// github.com/KChen-lab/novoRNABreak).

LB968 COL7A1 frame restoration by CRISPR/Cas9-facilitated, NHEJ-mediated removal of mutant exons as a reliable broad-range therapeutic strategy for RDEB

Bi-allelic frameshift mutations leading to PTC in COL7A1 are frequent causal mutations of recessive dystrophic epidermolysis bullosa (RDEB). Several studies including ours showed that COL7A1 exons encoding the collagenous domain of type VII collagen (C7) could be therapeutically skippable without apparent functional consequences for protein function. However, the long-term in vivo phenotypic consequences of targeted exon deletion and concomitant COL7A1 reframing have been assessed only for the small exon 80 (36 bp) but are not yet clear for other larger exons.

Dominant Distal Myopathy 3 (MPD3) Caused by a Deletion in the HNRNPA1 Gene.

Background and ObjectivesTo determine the genetic cause of the disease in the previously reported family with adult-onset autosomal dominant distal myopathy (myopathy, distal, 3; MPD3).MethodsContinued clinical evaluation including muscle MRI and muscle pathology. A linkage analysis with single nucleotide polymorphism arrays and genome sequencing were used to identify the genetic defect, which was verified by Sanger sequencing. RNA sequencing was used to investigate the transcriptional effects of the identified genetic defect.ResultsSmall hand muscles (intrinsic, thenar, and hypothenar) were first involved with spread to the lower legs and later proximal muscles. Dystrophic changes with rimmed vacuoles and cytoplasmic inclusions were observed in muscle biopsies at advanced stage. A single nucleotide polymorphism array confirmed the previous microsatellite-based linkage to 8p22-q11 and 12q13-q22. Genome sequencing of three affected family members combined with structural variant calling revealed a small heterozygous deletion of 160 base pairs spanning the second last exon 10 of the heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) gene, which is in the linked region on chromosome 12. Segregation of the mutation with the disease was confirmed by Sanger sequencing. RNA sequencing showed that the mutant allele produces a shorter mutant mRNA transcript compared with the wild-type allele. Immunofluorescence studies on muscle biopsies revealed small p62 and larger TDP-43 inclusions.DiscussionA small exon 10 deletion in the gene HNRNPA1 was identified as the cause of MPD3 in this family. The new HNRNPA1-related phenotype, upper limb presenting distal myopathy, was thus confirmed, and the family displays the complexities of gene identification.

Detecting copy number variation in next generation sequencing data from diagnostic gene panels

BackgroundDetection of copy number variation (CNV) in genes associated with disease is important in genetic diagnostics, and next generation sequencing (NGS) technology provides data that can be used for CNV detection. However, CNV detection based on NGS data is in general not often used in diagnostic labs as the data analysis is challenging, especially with data from targeted gene panels. Wet lab methods like MLPA (MRC Holland) are widely used, but are expensive, time consuming and have gene-specific limitations. Our aim has been to develop a bioinformatic tool for CNV detection from NGS data in medical genetic diagnostic samples.ResultsOur computational pipeline for detection of CNVs in NGS data from targeted gene panels utilizes coverage depth of the captured regions and calculates a copy number ratio score for each region. This is computed by comparing the mean coverage of the sample with the mean coverage of the same region in other samples, defined as a pool. The pipeline selects pools for comparison dynamically from previously sequenced samples, using the pool with an average coverage depth that is nearest to the one of the samples. A sliding window-based approach is used to analyze each region, where length of sliding window and sliding distance can be chosen dynamically to increase or decrease the resolution. This helps in detecting CNVs in small or partial exons. With this pipeline we have correctly identified the CNVs in 36 positive control samples, with sensitivity of 100% and specificity of 91%. We have detected whole gene level deletion/duplication, single/multi exonic level deletion/duplication, partial exonic deletion and mosaic deletion. Since its implementation in mid-2018 it has proven its diagnostic value with more than 45 CNV findings in routine tests.ConclusionsWith this pipeline as part of our diagnostic practices it is now possible to detect partial, single or multi-exonic, and intragenic CNVs in all genes in our target panel. This has helped our diagnostic lab to expand the portfolio of genes where we offer CNV detection, which previously was limited by the availability of MLPA kits.

Alternative microexon splicing by RBFOX2 and PTBP1 is associated with metastasis in colorectal cancer.

The splicing of microexons (very small exons) is frequently dysregulated in the brain of individuals with autism spectrum disorder. However, little is known of the patterns, regulatory mechanisms and roles of microexon splicing in cancer. We here examined the transcriptome-wide profile of microexon splicing in matched colorectal cancer (CRC) and normal tissue specimens. Out of 1492 microexons comprising 3 to 15 nucleotides, 21 (1%) manifested differential splicing between CRC and normal tissue. The 21 genes harboring the differentially spliced microexons were enriched in gene ontology terms related to cell adhesion and migration. RNA interference-mediated knockdown experiments identified two splicing factors, RBFOX2 and PTBP1, as regulators of microexon splicing in CRC cells. RBFOX2 and PTBP1 were found to directly bind to microexon-containing pre-mRNAs and to control their splicing in such cells. Differential microexon splicing was shown to be due, at least in part, to altered expression of RBFOX2 and PTBP1 in CRC tissue compared to matched normal tissue. Finally, we found that changes in the pattern of microexon splicing were associated with CRC metastasis. Our data thus suggest that altered expression of RBFOX2 and PTBP1 might influence CRC metastasis through the regulation of microexon splicing.

Accurate spliced alignment of long RNA sequencing reads.

MotivationLong-read RNA sequencing technologies are establishing themselves as the primary techniques to detect novel isoforms, and many such analyses are dependent on read alignments. However, the error rate and sequencing length of the reads create new challenges for accurately aligning them, particularly around small exons.ResultsWe present an alignment method uLTRA for long RNA sequencing reads based on a novel two-pass collinear chaining algorithm. We show that uLTRA produces higher accuracy over state-of-the-art aligners with substantially higher accuracy for small exons on simulated and synthetic data. On simulated data, uLTRA achieves an accuracy of about 60% for exons of length 10 nucleotides or smaller and close to 90% accuracy for exons of length between 11 and 20 nucleotides. On biological data where true read location is unknown, we show several examples where uLTRA aligns to known and novel isoforms containing small exons that are not detected with other aligners. While uLTRA obtains its accuracy using annotations, it can also be used as a wrapper around minimap2 to align reads outside annotated regions.Availabilityand implementationuLTRA is available at https://github.com/ksahlin/ultra.Supplementary information Supplementary data are available at Bioinformatics online.

Identification of a unique endoplasmic retention motif in the Xenopus GIRK5 channel and its contribution to oocyte maturation.

G protein‐activated inward‐rectifying potassium (K+) channels (Kir3/GIRK) participate in cell excitability. The GIRK5 channel is present in Xenopus laevis oocytes. In an attempt to investigate the physiological role of GIRK5, we identified a noncanonical di‐arginine endoplasmic reticulum (ER) retention motif (KRXY). This retention motif is located at the N‐terminal region of GIRK5, coded by two small exons found only in X. laevis and X. tropicalis. These novel exons are expressed through use of an alternative transcription start site. Mutations in the sequence KRXY produced functional channels and induced progesterone‐independent oocyte meiotic progression. The chimeric proteins enhanced green fluorescent protein (EGFP)‐GIRK5‐WT and the EGFP‐GIRK5K13AR14A double mutant, were localized to the ER and the plasma membrane of the vegetal pole of the oocyte, respectively. Silencing of GIRK5 or blocking of this channel by external barium prevented progesterone‐induced meiotic progression. The endogenous level of GIRK5 protein decreased through oocyte stages in prophase I augmenting by progesterone. In conclusion, we have identified a unique mechanism by which the expression pattern of a K+ channel evolved to control Xenopus oocyte maturation.

Silencing of SRRM4 suppresses microexon inclusion and promotes tumor growth across cancers.

RNA splicing is widely dysregulated in cancer, frequently due to altered expression or activity of splicing factors (SFs). Microexons are extremely small exons (3-27 nucleotides long) that are highly evolutionarily conserved and play critical roles in promoting neuronal differentiation and development. Inclusion of microexons in mRNA transcripts is mediated by the SF Serine/Arginine Repetitive Matrix 4 (SRRM4), whose expression is largely restricted to neural tissues. However, microexons have been largely overlooked in prior analyses of splicing in cancer, as their small size necessitates specialized computational approaches for their detection. Here, we demonstrate that despite having low expression in normal nonneural tissues, SRRM4 is further silenced in tumors, resulting in the suppression of normal microexon inclusion. Remarkably, SRRM4 is the most consistently silenced SF across all tumor types analyzed, implying a general advantage of microexon down-regulation in cancer independent of its tissue of origin. We show that this silencing is favorable for tumor growth, as decreased SRRM4 expression in tumors is correlated with an increase in mitotic gene expression, and up-regulation of SRRM4 in cancer cell lines dose-dependently inhibits proliferation in vitro and in a mouse xenograft model. Further, this proliferation inhibition is accompanied by induction of neural-like expression and splicing patterns in cancer cells, suggesting that SRRM4 expression shifts the cell state away from proliferation and toward differentiation. We therefore conclude that SRRM4 acts as a proliferation brake, and tumors gain a selective advantage by cutting off this brake.

The complete chloroplast genome of Bupleurum marginatum var. stenophyllum (H. Wolff) Shan & Yin Li (Apiaceae), a new substitution for Chinese medicinal material, Bupleuri Radix (Chai hu)

The root of Bupleurum marginatum var. stenophyllum (H. Wolff) Shan & Yin Li (Apiaceae), a new substitution for the popular Chinese medicinal material, Bupleuri Radix (Chai hu), is not easily distinguishable via traditional methods. The complete chloroplast genome sequence of B. marginatum var. stenophyllum was characterized using next-generation sequencing and the de novo assembly method. The complete genome was 155,576 bp in length and contained two inverted repeat (IR) regions of 26,311 bp, a large single-copy (LSC) region of 85,351 bp, and a small single-copy (SSC) region of 17,603 bp. It encoded 113 unique genes consisting of 79 protein-coding genes (PCGs), 30 transfer RNA genes, and four ribosomal RNA genes. Importantly, three genes (petB, petD and rps16) with small exon, and one trans-splicing gene (rps12) were correctly annotated. The overall GC content of the B. marginatum var. stenophyllum chloroplast genome is 37.7%. The phylogenetic analyses indicated that B. marginatum var. stenophyllum was closely related to B. marginatum. Moreover, many genetic information sites were available for distinguishing B. marginatum var. stenophyllum from the official ‘Chai hu’ plant sources, B. scorzonerifolium Willd. and B. chinense DC.

Detection of exon location in eukaryotic DNA using a fuzzy adaptive Gabor wavelet transform

The existing model-independent methods for the detection of exons in DNA could not prove to be ideal as commonly employed fixed window length strategy produces spectral leakage causing signal noise The Modified-Gabor-wavelet-transform exploits a multiscale strategy to deal with the issue to some extent. Yet, no rule regarding the occurrence of small and large exons has been specified. To overcome this randomness, scaling-factor of GWT has been adapted based on a fuzzy rule. Due to the nucleotides' genetic code and fuzzy behaviors in DNA configuration, this work could adopt the fuzzy approach. Two fuzzy membership functions (large and small) take care of the variation in the coding regions. The fuzzy-based learning parameter adaptively tunes the scale factor for fast and precise prediction of exons. The proposed approach has an immense plus point of being capable of isolating detailed sub-regions in each exon efficiently proving its efficacy comparing with existing techniques.

DeSALT: fast and accurate long transcriptomic read alignment with de Bruijn graph-based index

The alignment of long-read RNA sequencing reads is non-trivial due to high sequencing errors and complicated gene structures. We propose deSALT, a tailored two-pass alignment approach, which constructs graph-based alignment skeletons to infer exons and uses them to generate spliced reference sequences to produce refined alignments. deSALT addresses several difficult technical issues, such as small exons and sequencing errors, which break through bottlenecks of long RNA-seq read alignment. Benchmarks demonstrate that deSALT has a greater ability to produce accurate and homogeneous full-length alignments. deSALT is available at: https://github.com/hitbc/deSALT.

Detection of Complex Structural Variations of X-Linked Deafness-2 (DFNX2) by Single-Molecule Sequencing

Purpose: To investigate genetic causes of X-linked deafness-2 (DFNX2) and analyse the efficiencies and advantages/disadvantages of different sequencing methods. Methods: From 2016 to 2018, 12 unrelated patients with characteristic high-resolution computed tomographic images showing incomplete partition type III (IP-III) were enrolled in this study. Targeted next-generation sequencing (NGS) and Nanopore long-read sequencing were used to analyse the genetic etiology. Results: Six variants of POU3F4 were identified in this cohort by NGS. The six variants were all located upstream of the POU-specific domain and were considered pathogenic due to truncated protein. Among six patients with negative NGS results, three patients were volunteered to choose further Nanopore sequencing and were all found to have structural variations (SVs). We detected an 870 kb deletion in J0012, located at ChrX:81,140,313-82,011,100, approximately 750 kb upstream of the POU3F4 gene. Non-homologous end joining may be a deletion mechanism. An 8 Mb inversion was found in J0011 at ChrX:82,146,071-90,188,492. The sequences of the breakpoint analysis suggested non-allelic homologous recombination occurred within a single chromatid. A 6 kb deletion at ChrX:81,096,652-81,102,705 was found in J0007, which is a region with a high density of retrotransposons. Conclusions: The most common mutations in patients with IP-III were point mutations and small INDELs or exons that can be detected by Sanger sequencing and NGS. Single-molecule long-fragment sequencing technology provides a new technique to detect the pathogenic SVs in patients with negative NGS results. Thus, the DNA diagnostic strategy for IP-III and its efficiency have been greatly improved. At the same time, the sequence characteristics around the breakpoint and the possible mechanism of SV formation should be explored further. Funding Statement: This work was supported by National Key Research and Development Program of China (2016YFC1000700, 2016YFC1000704 and 2017YFC1001800), Shanghai Key Laboratory of Translational Medicine on Ear and Nose diseases (14DZ2260300). Declaration of Interests: The authors have declared that no competing interests exist. Ethics Approval Statement: This investigation was performed with the approval of the ethics committees of the Chinese PLA General Hospital. All of the subjects or their guardians signed the informed consent forms prior to blood sampling.