scholarly journals The Role of RNA-Sequencing as a New Genetic Diagnosis Tool

2021 ◽  
Vol 9 (2) ◽  
pp. 13-21
Author(s):  
Philippa D. K. Curry ◽  
Krystyna L. Broda ◽  
Christopher J. Carroll
Keyword(s):  
Rna Sequencing ◽  
Rare Diseases ◽  
Diagnostic Yield ◽  
Neuromuscular Disorders ◽  
Genetic Diagnosis ◽  
Rna Seq ◽  
Novel Approach ◽  
Whole Exome ◽  
Diagnosis Tool

Abstract Purpose of Review Whole exome sequencing (WES) and whole-genome sequencing (WGS) are frontline approaches for the genetic diagnosis of rare diseases. However, WES/WGS fails in up to 75% of cases. Transcriptomics via RNA-sequencing (RNA-Seq) is a novel approach that aims to increase the diagnostic yield in rare diseases. Recent Findings Recent publications focus on the success of RNA-Seq for increasing diagnosis rates in WES/WGS-negative patients in up to 36% of cases, across a range of different diseases, sample sizes, and tissue types. Summary RNA-Seq is beneficial for aiding prioritisation of causative variants currently not detected or often overlooked by WES/WGS alone. An improvement in diagnostic yields has been demonstrated using multiple source tissues, with muscle and fibroblasts being the most representative, but the more accessible blood still demonstrating diagnostic success, particularly in neuromuscular disorders. The introduction of RNA-Seq to the genetic diagnosis toolbox promises to be a useful complementary tool to WES/WGS for improving genetic diagnosis in patients with rare disease.

scholarly journals How Machine Learning and Statistical Models Advance Molecular Diagnostics of Rare Disorders Via Analysis of RNA Sequencing Data

2021 ◽  
Vol 8 ◽  
Author(s):  
Lea D. Schlieben ◽  
Holger Prokisch ◽  
Vicente A. Yépez
Keyword(s):  
Machine Learning ◽  
Rna Sequencing ◽  
Rare Diseases ◽  
Statistical Models ◽  
Multiple Testing ◽  
Diagnostic Yield ◽  
Genetic Diagnosis ◽  
Sequencing Data ◽  
Rare Disorders ◽  
Aberrant Expression

Rare diseases, although individually rare, collectively affect approximately 350 million people worldwide. Currently, nearly 6,000 distinct rare disorders with a known molecular basis have been described, yet establishing a specific diagnosis based on the clinical phenotype is challenging. Increasing integration of whole exome sequencing into routine diagnostics of rare diseases is improving diagnostic rates. Nevertheless, about half of the patients do not receive a genetic diagnosis due to the challenges of variant detection and interpretation. During the last years, RNA sequencing is increasingly used as a complementary diagnostic tool providing functional data. Initially, arbitrary thresholds have been applied to call aberrant expression, aberrant splicing, and mono-allelic expression. With the application of RNA sequencing to search for the molecular diagnosis, the implementation of robust statistical models on normalized read counts allowed for the detection of significant outliers corrected for multiple testing. More recently, machine learning methods have been developed to improve the normalization of RNA sequencing read count data by taking confounders into account. Together the methods have increased the power and sensitivity of detection and interpretation of pathogenic variants, leading to diagnostic rates of 10–35% in rare diseases. In this review, we provide an overview of the methods used for RNA sequencing and illustrate how these can improve the diagnostic yield of rare diseases.

scholarly journals Exome sequencing in paediatric patients with movement disorders

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Anna Ka-Yee Kwong ◽  
Mandy Ho-Yin Tsang ◽  
Jasmine Lee-Fong Fung ◽  
Christopher Chun-Yu Mak ◽  
Kate Lok-San Chan ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Movement Disorders ◽  
Rare Variants ◽  
Diagnostic Yield ◽  
Neurological Diseases ◽  
Genetic Diagnosis ◽  
Potential Treatment ◽  
Paediatric Patients ◽  
Whole Exome

Abstract Background Movement disorders are a group of heterogeneous neurological diseases including hyperkinetic disorders with unwanted excess movements and hypokinetic disorders with reduction in the degree of movements. The objective of our study is to investigate the genetic etiology of a cohort of paediatric patients with movement disorders by whole exome sequencing and to review the potential treatment implications after a genetic diagnosis. Results We studied a cohort of 31 patients who have paediatric-onset movement disorders with unrevealing etiologies. Whole exome sequencing was performed and rare variants were interrogated for pathogenicity. Genetic diagnoses have been confirmed in 10 patients with disease-causing variants in CTNNB1, SPAST, ATP1A3, PURA, SLC2A1, KMT2B, ACTB, GNAO1 and SPG11. 80% (8/10) of patients with genetic diagnosis have potential treatment implications and treatments have been offered to them. One patient with KMT2B dystonia showed clinical improvement with decrease in dystonia after receiving globus pallidus interna deep brain stimulation. Conclusions A diagnostic yield of 32% (10/31) was reported in our cohort and this allows a better prediction of prognosis and contributes to a more effective clinical management. The study highlights the potential of implementing precision medicine in the patients.

scholarly journals MBRS-17. EXAMINING THE ROLE OF LHX9 IN GROUP 3 MEDULLOBLASTOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii401-iii401
Author(s):  
Sarah Injac ◽  
L Frank Huang ◽  
Stephen Mack ◽  
Frank Braun ◽  
Yuchen Du ◽  
...  
Keyword(s):  
Drug Repositioning ◽  
Single Agent ◽  
Xenograft Model ◽  
Cell Killing ◽  
Rna Seq ◽  
Loss Of Function ◽  
Novel Treatments ◽  
Novel Approach ◽  
Group 3

Abstract Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Despite major advances in our understanding of the biology of MB, novel treatments remain urgently needed. Using a chemical-genomics driven drug repositioning strategy, we identified the cardiac glycoside family of compounds as potential treatments for Group 3 MB. We subsequently demonstrated that single-agent treatment with digoxin prolongs survival in a patient-derived xenograft model (PDOX) of Group 3 MB to a degree comparable to radiation therapy, a mainstay in the treatment of MB. Finally, we examined the mechanism of digoxin-mediated cell killing using RNA-seq. This work identified LHX9, a member of the LIM homeobox family of transcription factors, as the gene most significantly down-regulated following treatment (Huang and Injac et al, Sci Trans Medicine, 2018). Homologs of LHX9 play key roles in cerebellar development via spatially and temporally restricted expression and LHX9 has been proposed as a core transcription factor (TF) in the regulatory circuitry of Group 3 tumors. Loss of function of other core TFs has been shown to impact MB growth. The role of LHX9 in MB, however, has not been previously experimentally evaluated. We now report that knockdown of LHX9 in MB-derived cell lines results in marked growth inhibition raising the possibility that loss of LHX9 plays a major role in digoxin-mediated cell killing and that LHX9 represents a key dependency required for the growth of Group 3 MB. Clinical targeting of core TFs would represent a novel approach to targeting this devastating disease.

scholarly journals RNA-Sequencing Analyses of Small Bacterial RNAs and their Emergence as Virulence Factors in Host-Pathogen Interactions

2020 ◽  
Vol 21 (5) ◽  
pp. 1627 ◽  
Author(s):  
Idrissa Diallo ◽  
Patrick Provost
Keyword(s):  
Rna Sequencing ◽  
Virulence Factors ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Rna Seq ◽  
Host Pathogen Interactions ◽  
Bioinformatics Analyses ◽  
Additional Layer ◽  
Host Pathogen

Proteins have long been considered to be the most prominent factors regulating so-called invasive genes involved in host-pathogen interactions. The possible role of small non-coding RNAs (sRNAs), either intracellular, secreted or packaged in outer membrane vesicles (OMVs), remained unclear until recently. The advent of high-throughput RNA-sequencing (RNA-seq) techniques has accelerated sRNA discovery. RNA-seq radically changed the paradigm on bacterial virulence and pathogenicity to the point that sRNAs are emerging as an important, distinct class of virulence factors in both gram-positive and gram-negative bacteria. The potential of OMVs, as protectors and carriers of these functional, gene regulatory sRNAs between cells, has also provided an additional layer of complexity to the dynamic host-pathogen relationship. Using a non-exhaustive approach and through examples, this review aims to discuss the involvement of sRNAs, either free or loaded in OMVs, in the mechanisms of virulence and pathogenicity during bacterial infection. We provide a brief overview of sRNA origin and importance and describe the classical and more recent methods of identification that have enabled their discovery, with an emphasis on the theoretical lower limit of RNA sizes considered for RNA sequencing and bioinformatics analyses.

scholarly journals Diagnostic yield of panel-based genetic testing in syndromic inherited retinal disease

2019 ◽  
Vol 28 (5) ◽  
pp. 576-586 ◽  
Author(s):  
Omamah A. Jiman ◽  
◽  
Rachel L. Taylor ◽  
Eva Lenassi ◽  
Jill Clayton Smith ◽  
...  
Keyword(s):  
Clinical Diagnosis ◽  
Diagnostic Yield ◽  
Effective Means ◽  
Genetic Diagnosis ◽  
Retinal Disease ◽  
Human Phenotype ◽  
Clinical Notes ◽  
Inherited Retinal Disease ◽  
Genome Wide ◽  
Whole Exome

AbstractThirty percent of all inherited retinal disease (IRD) is accounted for by conditions with extra-ocular features. This study aimed to establish the genetic diagnostic pick-up rate for IRD patients with one or more extra-ocular features undergoing panel-based screening in a clinical setting. One hundred and six participants, tested on a gene panel which contained both isolated and syndromic IRD genes, were retrospectively ascertained from the Manchester Genomic Diagnostics Laboratory database spanning 6 years (2012–2017). Phenotypic features were extracted from the clinical notes and classified according to Human Phenotype Ontology; all identified genetic variants were interpreted in accordance to the American College of Medical Genetics and Genomics guidelines. Overall, 49% (n = 52) of patients received a probable genetic diagnosis. A further 6% (n = 6) had a single disease-associated variant in an autosomal recessive disease-relevant gene. Fifty-two percent (n = 55) of patients had a clinical diagnosis at the time of testing. Of these, 71% (n = 39) received a probable genetic diagnosis. By contrast, for those without a provisional clinical diagnosis (n = 51), only 25% (n = 13) received a probable genetic diagnosis. The clinical diagnosis of Usher (n = 33) and Bardet–Biedl syndrome (n = 10) was confirmed in 67% (n = 22) and 80% (n = 8), respectively. The testing diagnostic rate in patients with clinically diagnosed multisystemic IRD conditions was significantly higher than those without one (71% versus 25%; p value < 0.001). The lower pick-up rate in patients without a clinical diagnosis suggests that panel-based approaches are unlikely to be the most effective means of achieving a molecular diagnosis for this group. Here, we suggest that genome-wide approaches (whole exome or genome) are more appropriate.

scholarly journals Delineation of molecular findings by whole exome sequencing for suspected cases of paediatric-onset mitochondrial diseases in the Southern Chinese population

2020 ◽  
Author(s):  
Mandy Ho-Yin Tsang ◽  
Anna Ka-Yee Kwong ◽  
Kate Lok-San Chan ◽  
Jasmine Lee-Fong Fung ◽  
Mullin Ho-Chung Yu ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Chinese Population ◽  
Founder Mutation ◽  
Diagnostic Yield ◽  
Genetic Diagnosis ◽  
Mitochondrial Diseases ◽  
Chinese Patients ◽  
Southern Chinese ◽  
Whole Exome

Abstract BackgroundMitochondrial diseases (MDs) are a group of clinically and genetically heterogeneous disorders characterized by defects in oxidative phosphorylation. Since clinical phenotypes of MDs may be non-specific, genetic diagnosis is crucial for guiding disease management. In the current study, whole-exome sequencing (WES) was performed for our paediatric-onset MD cohort of a Southern Chinese origin, with the aim of identifying key disease-causing variants in the Chinese patients with MDs.MethodsWe recruited Chinese patients who had paediatric-onset MDs and a minimum mitochondrial disease criteria (MDC) score of 3. Patients with positive target gene or mitochondrial DNA sequencing results were excluded. WES was performed, variants with population frequency ≤1% were analysed for pathogenicity on the basis of the American College of Medical Genetics and Genomics guidelines.ResultsSixty-six patients with pre-biopsy MDC scores of 3–8 were recruited. The overall diagnostic yield was 35% (23/66). Eleven patients (17%) were found to have mutations in MD-related genes, with COQ4 having the highest mutation rate owing to the Chinese-specific founder mutation (4/66, 6%). Twelve patients (12/66, 18%) had mutations in non-MD-related genes: ATP1A3 (n=3, two were siblings), ALDH5A1 , ARX , FA2H , KCNT1 , LDHD , NEFL , NKX2-2 , TBCK , and WAC.ConclusionsWe confirmed that the COQ4 :c.370G>A, p.(Gly124Ser) variant was a recurrent founder mutation among the Southern Chinese population. Screening for this mutation should therefore be considered while diagnosing Chinese patients suspected to have MDs. Furthermore, WES has proven to be useful in detecting variants in patients suspected to have MDs because it helps to obtain an unbiased and precise genetic diagnosis for these diseases, which are genetically heterogeneous.

scholarly journals Delineation of molecular findings by whole exome sequencing for suspected cases of paediatric-onset mitochondrial diseases in the Southern Chinese population

2020 ◽  
Author(s):  
Mandy Ho-Yin Tsang ◽  
Anna Ka-Yee Kwong ◽  
Kate Lok-San Chan ◽  
Jasmine Lee-Fong Fung ◽  
Mullin Ho-Chung Yu ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Chinese Population ◽  
Founder Mutation ◽  
Diagnostic Yield ◽  
Genetic Diagnosis ◽  
Mitochondrial Diseases ◽  
Chinese Patients ◽  
Southern Chinese ◽  
Whole Exome

Abstract Background Mitochondrial diseases (MDs) are a group of clinically and genetically heterogeneous disorders characterized by defects in oxidative phosphorylation. Since clinical phenotypes of MDs may be non-specific, genetic diagnosis is crucial for guiding disease management. In the current study, whole-exome sequencing (WES) was performed for our paediatric-onset MD cohort of a Southern Chinese origin, with the aim of identifying key disease-causing variants in the Chinese patients with MDs.Methods We recruited Chinese patients who had paediatric-onset MDs and a minimum mitochondrial disease criteria (MDC) score of 3. Patients with positive target gene or mitochondrial DNA sequencing results were excluded. WES was performed, variants with population frequency ≤1% were analysed for pathogenicity on the basis of the American College of Medical Genetics and Genomics guidelines.Results Sixty-six patients with pre-biopsy MDC scores of 3–8 were recruited. The overall diagnostic yield was 35% (23/66). Eleven patients (17%) were found to have mutations in MD-related genes, with COQ4 having the highest mutation rate owing to the Chinese-specific founder mutation (4/66, 6%). Twelve patients (12/66, 18%) had mutations in non-MD-related genes: ATP1A3 (n=3, two were siblings), ALDH5A1, ARX, FA2H, KCNT1, LDHD, NEFL, NKX2-2, TBCK, and WAC.Conclusions We confirmed that the COQ4:c.370G>A, p.(Gly124Ser) variant was a recurrent founder mutation among the Southern Chinese population. Screening for this mutation should therefore be considered while diagnosing Chinese patients suspected to have MDs. Furthermore, WES has proven to be useful in detecting variants in patients suspected to have MDs because it helps to obtain an unbiased and precise genetic diagnosis for these diseases, which are genetically heterogeneous.

The Efficacy of Whole Genome Sequencing and RNA-Seq in the Diagnosis of Whole Exome Sequencing Negative Patients with Complex Neurological Phenotypes

2021 ◽  
Author(s):  
Bianca Blake ◽  
Lauren I. Brady ◽  
Nicholas A. Rouse ◽  
Peter Nagy ◽  
Mark A. Tarnopolsky
Keyword(s):  
Whole Genome Sequencing ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Genome Sequencing ◽  
Diagnostic Yield ◽  
Whole Genome ◽  
Rna Seq ◽  
Complete Coverage ◽  
Whole Exome ◽  
Chromosome Microarray

AbstractWhole-genome sequencing (WGS) is being increasingly utilized for the diagnosis of neurological disease by sequencing both the exome and the remaining 98 to 99% of the genetic code. In addition to more complete coverage, WGS can detect structural variants (SVs) and intronic variants (SNVs) that cannot be identified by whole exome sequencing (WES) or chromosome microarray (CMA). Other multi-omics tools, such as RNA sequencing (RNA-Seq), can be used in conjunction with WGS to functionally validate certain variants by detecting changes in gene expression and splicing. The objective of this retrospective study was to measure the diagnostic yield of duo/trio-based WGS and RNA-Seq in a cohort of 22 patients (20 families) with pediatric onset neurological phenotypes and negative or inconclusive WES results in lieu of reanalysis. WGS with RNA-Seq resulted in a definite diagnosis of an additional 25% of cases. Sixty percent of these solved cases arose from the identification of variants that were missed by WES. Variants that could not be unequivocally proven to be causative of the patients' condition were identified in an additional 5% of cases.

scholarly journals Delineation of molecular findings by whole-exome sequencing for suspected cases of paediatric-onset mitochondrial diseases in the Southern Chinese population

2020 ◽  
Vol 14 (1) ◽  
Author(s):  
Mandy H.Y. Tsang ◽  
Anna K.Y. Kwong ◽  
Kate L.S. Chan ◽  
Jasmine L.F. Fung ◽  
Mullin H.C. Yu ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Chinese Population ◽  
Founder Mutation ◽  
Diagnostic Yield ◽  
Genetic Diagnosis ◽  
Mitochondrial Diseases ◽  
Chinese Patients ◽  
Southern Chinese ◽  
Whole Exome

Abstract Background Mitochondrial diseases (MDs) are a group of clinically and genetically heterogeneous disorders characterized by defects in oxidative phosphorylation. Since clinical phenotypes of MDs may be non-specific, genetic diagnosis is crucial for guiding disease management. In the current study, whole-exome sequencing (WES) was performed for our paediatric-onset MD cohort of a Southern Chinese origin, with the aim of identifying key disease-causing variants in the Chinese patients with MDs. Methods We recruited Chinese patients who had paediatric-onset MDs and a minimum mitochondrial disease criteria (MDC) score of 3. Patients with positive target gene or mitochondrial DNA sequencing results were excluded. WES was performed, variants with population frequency ≤ 1% were analysed for pathogenicity on the basis of the American College of Medical Genetics and Genomics guidelines. Results Sixty-six patients with pre-biopsy MDC scores of 3–8 were recruited. The overall diagnostic yield was 35% (23/66). Eleven patients (17%) were found to have mutations in MD-related genes, with COQ4 having the highest mutation rate owing to the Chinese-specific founder mutation (4/66, 6%). Twelve patients (12/66, 18%) had mutations in non-MD-related genes: ATP1A3 (n = 3, two were siblings), ALDH5A1, ARX, FA2H, KCNT1, LDHD, NEFL, NKX2-2, TBCK, and WAC. Conclusions We confirmed that the COQ4:c.370G>A, p.(Gly124Ser) variant, was a founder mutation among the Southern Chinese population. Screening for this mutation should therefore be considered while diagnosing Chinese patients suspected to have MDs. Furthermore, WES has proven to be useful in detecting variants in patients suspected to have MDs because it helps to obtain an unbiased and precise genetic diagnosis for these diseases, which are genetically heterogeneous.

scholarly journals Comprehensive MicroRNA Expression Profile of the Mammary Gland in Lactating Dairy Cows With Extremely Different Milk Protein and Fat Percentages

2020 ◽  
Vol 11 ◽  
Author(s):  
Xiaogang Cui ◽  
Shengli Zhang ◽  
Qin Zhang ◽  
Xiangyu Guo ◽  
Changxin Wu ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Rna Sequencing ◽  
Differentially Expressed Genes ◽  
Small Rna ◽  
Milk Protein ◽  
Milk Composition ◽  
Differentially Expressed ◽  
Small Rna Sequencing ◽  
Rna Seq

A total of 31 differentially expressed genes in the mammary glands were identified in our previous study using RNA sequencing (RNA-Seq), for lactating cows with extremely high and low milk protein and fat percentages. To determine the regulation of milk composition traits, we herein investigated the expression profiles of microRNA (miRNA) using small RNA sequencing based on the same samples as in the previous RNA-Seq experiment. A total of 497 known miRNAs (miRBase, release 22.1) and 49 novel miRNAs among the reads were identified. Among these miRNAs, 71 were found differentially expressed between the high and low groups (p &lt; 0.05, q &lt; 0.05). Furthermore, 21 of the differentially expressed genes reported in our previous RNA-Seq study were predicted as target genes for some of the 71 miRNAs. Gene ontology and KEGG pathway analyses showed that these targets were enriched for functions such as metabolism of protein and fat, and development of mammary gland, which indicating the critical role of these miRNAs in regulating the formation of milk protein and fat. With dual luciferase report assay, we further validated the regulatory role of 7 differentially expressed miRNAs through interaction with the specific sequences in 3′UTR of the targets. In conclusion, the current study investigated the complexity of the mammary gland transcriptome in dairy cattle using small RNA-seq. Comprehensive analysis of differential miRNAs expression and the data from previous study RNA-seq provided the opportunity to identify the key candidate genes for milk composition traits.

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