Ignite Creation Date:
2024-10-25 @ 7:56 PM
Last Modification Date:
2024-10-26 @ 3:41 PM
Study NCT ID:
NCT06621732
Status:
NOT_YET_RECRUITING
Last Update Posted:
None
First Post:
2024-09-27
Brief Title:
Combating the Diagnostic Impasse in Mitochondrial Diseases a Transcriptomic Approach in Fibroblasts and Blood Cells
Sponsor:
None
Organization:
None
Study Overview
Official Title:
Combating the Diagnostic Impasse in Mitochondrial Diseases a Transcriptomic Approach in Fibroblasts and Blood Cells
Status:
NOT_YET_RECRUITING
Status Verified Date:
2024-09
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
No
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
RNAseq
Brief Summary:
Next-generation sequencing NGS and in particular whole exome sequencing WES or genome sequencing WGS has enabled a significant technical advance that has considerably improved genetic diagnostics However around 50 of patients still remain undiagnosed and are in diagnostic limbo One of the causes of this is pathogenic variants that modify transcript expression andor RNA splicing These variants may be located in deep intronic or intergenic regions or in the coding sequence synonymous or missense variants also having pathogenic consequences on splicing or gene expression It is very often difficult to interpret the pathogenicity of these variants which often remain variants of uncertain significance VSI The usefulness of transcriptome sequencing RNA-seq in the genetic diagnosis of MM has been demonstrated in recent years by several teams with diagnostic yields of 10 to 35 Cummings et al 2017 Kremer et al 2017 Alston et al 2018 These studies are ideally performed using muscle tissue as MMs are most often expressed in tissues with high energy metabolism such as muscle heart brain or liver Cummings et al 2017 However as biopsies of these tissues are difficult to obtain most transcript studies are performed using fibroblasts obtained from skin biopsies Kremer et al 2017 Saadi et al 2022 Indeed extreme regulatory defects such as loss of expression or aberrant splicing can be detected in fibroblasts even though the physiological consequence on fibroblasts may be negligible However some patients also refuse these biopsies and may remain in diagnostic limbo in the absence of functional analysis to confirm the pathogenicity of the variants identified RNA studies can also be performed using RNA extracted from blood cells on PAXGene tubes Frésard et al 2019 The quantity of RNA extracted is lower than that extracted from fibroblasts but this type of analysis i avoids a more invasive procedure ii saves technical time by avoiding the manips associated with cell culture and iii saves time for the patient by enabling immediate extraction from the blood tube without waiting for cell culture Frésard et al showed in patients with 16 different Mendelian pathologies that RNA-seq on blood cells identified a diagnosis in 75 of patients tested Frésard et al 2019 Their approach revealed both expression variations and splicing anomalies
We therefore propose to carry out a transcript study using high-throughput RNA sequencing RNA-Seq in parallel on RNA extracted from fibroblasts and on RNA extracted from blood cells on 10 patients with suspected mitochondrial disease in whom variants of uncertain significance in candidate genes VSI have been identified
We chose to target our study on patients with VSI previously identified by NGS to facilitate interpretation of the RNA-Seq data within the framework of a pilot study In these patients who carry variants in candidate genes we will focus our bioinformatics analysis on these genes For the interpretation of VSI a targeted approach using Sanger sequencing based on RT-PCR or quantification of gene expression using quantitative PCR is also feasible but requires custom development for each variant which is very time-consuming and not insignificantly expensive The advantage of an RNA-seq approach is that it homogenizes the diagnostic strategy for patients saves analysis time and therefore reduces the time spent in diagnostic wandering Finally the drastic reduction in the cost of NGS sequencing means that this technique could be used routinely as a complement to exomegenome sequencing It could therefore eventually be applied not only to patients with VSI but also in the absence of evidence of potentially pathogenic variants as an aid to filtering variants identified by WESWGS
We therefore propose to carry out a transcript study using high-throughput RNA sequencing RNA-Seq in parallel on RNA extracted from fibroblasts and on RNA extracted from blood cells on 10 patients with suspected mitochondrial disease in whom variants of uncertain significance in candidate genes VSI have been identified
We chose to target our study on patients with VSI previously identified by NGS to facilitate interpretation of the RNA-Seq data within the framework of a pilot study In these patients who carry variants in candidate genes we will focus our bioinformatics analysis on these genes For the interpretation of VSI a targeted approach using Sanger sequencing based on RT-PCR or quantification of gene expression using quantitative PCR is also feasible but requires custom development for each variant which is very time-consuming and not insignificantly expensive The advantage of an RNA-seq approach is that it homogenizes the diagnostic strategy for patients saves analysis time and therefore reduces the time spent in diagnostic wandering Finally the drastic reduction in the cost of NGS sequencing means that this technique could be used routinely as a complement to exomegenome sequencing It could therefore eventually be applied not only to patients with VSI but also in the absence of evidence of potentially pathogenic variants as an aid to filtering variants identified by WESWGS
Detailed Description:
None
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None