Developing a Pipeline to Employ RNA-Seq as a Complementary Diagnostic Tool in Rare Diseases (ANTHEM)
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ClinicalTrials.gov Identifier: NCT05996731 |
Recruitment Status :
Recruiting
First Posted : August 18, 2023
Last Update Posted : March 15, 2024
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This project aims to identify, through RNA-Seq technology, the genetic alterations underlying undiagnosed rare diseases in pediatric and adult patients with early onset and with negative WES.
- Objective 1: Set up and validate techniques. Set-up and validation of the transcriptome analysis protocol in healthy subjects and in patients with known splicing alterations and/or altered RNA expression.
- Objective 2: Diagnostic phase. Study of splicing alterations and RNA levels in cultured fibroblasts obtained from skin biopsies of patients with rare genetic diseases and negative exome.
Exploratory goals
- Compare the RNA expression profile obtained from skin biopsy-derived fibroblasts with the RNA expression profile from blood. The most relevant results will be validated in qRT-PCR.
- To analyze the transcriptional and protein profile heterogeneity in skin-derived fibroblasts in enrolled subjects.
To explore the effects of genetic (from WES) and transcriptional (from RNA-seq) alterations in participants' plasma and serum.
Healthy controls Five healthy subjects will be recruited from the staff of the Mario Negri Institute for Pharmacological Research. The coded samples will be used to set up the method of isolation and culture of skin fibroblasts and RNA-Seq.
Validation group For the set-up and validation of the skin fibroblast isolation and RNA-Seq procedure, ten adult patients with known diagnosis and with alterations in RNA levels and/or splicing will be recruited as positive controls.
Patients who meet the requirements described above will be contacted by the doctors of the Daccò Center for an interview explaining the project. Those who agree to participate in the study will be asked to sign the informed consent before proceeding with the experimental part.
"Discovery/Exploration" group The exploration cohort will be composed of 30 symptomatic undiagnosed patients with suspected genetic disease (children and adults with infantile onset) belonging to the Clinical Center of the Mario Negri Institute for Pharmacological Research and for whom WES investigations did not reveal causative genetic alterations.
Condition or disease | Intervention/treatment | Phase |
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Atypical Hemolytic Uremic Syndrome Membranoproliferative Glomerulonephritis Autosomal Dominant Polycystic Kidney Healthy | Procedure: Skin biopsy | Not Applicable |
Study Type : | Interventional (Clinical Trial) |
Estimated Enrollment : | 105 participants |
Allocation: | Non-Randomized |
Intervention Model: | Parallel Assignment |
Masking: | None (Open Label) |
Primary Purpose: | Diagnostic |
Official Title: | Developing a Pipeline to Employ RNA-Seq as a Complementary Diagnostic Tool in Rare Diseases |
Actual Study Start Date : | February 21, 2024 |
Estimated Primary Completion Date : | September 2025 |
Estimated Study Completion Date : | September 2025 |
Arm | Intervention/treatment |
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Active Comparator: Healthy subjects
Five healthy donors will be asked to participate in the study to set up the condition for isolation and culture of skin-derived fibroblasts and to establish the RNA-Seq conditions and profile.
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Procedure: Skin biopsy
A punch biopsy is a 15-minute low-risk procedure performed through a sterile circular blade (usually 3-4 mm) under local anesthesia. Briefly, the instrument is rotated down through the forearm epidermis and dermis perpendicular to its physiological lines of relaxation and into the subcutaneous fat, by producing a cylindrical core. Punch biopsy site can be closed with a single suture and generally produce only a minimal scar. |
Experimental: Validation cohort
To develop a diagnostic pipeline for isolation and sequencing of mRNA from cultured skin fibroblasts, 10 adult patients with known genetic defects affecting RNA levels and/or splicing will be enrolled, as positive controls.
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Procedure: Skin biopsy
A punch biopsy is a 15-minute low-risk procedure performed through a sterile circular blade (usually 3-4 mm) under local anesthesia. Briefly, the instrument is rotated down through the forearm epidermis and dermis perpendicular to its physiological lines of relaxation and into the subcutaneous fat, by producing a cylindrical core. Punch biopsy site can be closed with a single suture and generally produce only a minimal scar. |
Experimental: Discovery cohort
The second group, the discovery cohort, will be composed of 30 undiagnosed symptomatic patients with clinical suspicion of a genetic disease (both children and adults with onset in infancy or early adulthood) referred to the Clinical Research Center for Rare Diseases "Aldo e Cele Daccò", and for which WES analyses did not reveal any causative genetic alteration. To this end, the investigators plan to recruit around 60 patients, their available parents and/or their available informative relatives who will undergo WES, if not previously done. On the basis of literature and their experience, the investigators expect that WES will be resolutive in 40-50% of cases. Consequently, investigators hypothesize to identify 30 patients with a negative WES who will enter the discovery RNA-Seq cohort.
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Procedure: Skin biopsy
A punch biopsy is a 15-minute low-risk procedure performed through a sterile circular blade (usually 3-4 mm) under local anesthesia. Briefly, the instrument is rotated down through the forearm epidermis and dermis perpendicular to its physiological lines of relaxation and into the subcutaneous fat, by producing a cylindrical core. Punch biopsy site can be closed with a single suture and generally produce only a minimal scar. |
- Set up and validate the transcriptome analysis procedure in healthy controls and in patients diagnosed with known genetic diseases and with known splice/expression altering variants [ Time Frame: At day 0 ]WES analyses for DNA isolation
- Analyse alterations of mRNA levels and splicing in cultured fibroblasts derived from patients with rare diseases and an inconclusive WES. To investigate the heterogeneity of the transcriptomic and proteomic profile [ Time Frame: At day 0 ]Skin-derived fibroblasts
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Ages Eligible for Study: | Child, Adult, Older Adult |
Sexes Eligible for Study: | All |
Accepts Healthy Volunteers: | Yes |
Healthy subjects.
Inclusion Criteria:
- Male and female adults
- Written informed consent
Exclusion Criteria:
- Inability to understand the potential risk and benefits of the study
- Legal incapacity
Validation cohort.
Inclusion criteria:
- Male and female adults
- Genetic diseases affecting RNA levels (frameshifts, stop, large deletions, alteration of canonical splicing sites)
- Written informed consent
Exclusion criteria:
- Underage patients
- Inability to understand the potential risk and benefits of the study
- Legal incapacity
Discovery cohort.
Inclusion criteria:
- Male and female patients (children and adults with onset in infancy or early adulthood) with rare genetic undiagnosed diseases
- Patients with no strong candidates based on previous genetic analysis such as WES, but with clinically suspicion of a genetic rare disease
- Written informed consent
Exclusion criteria:
- Inability to understand the potential risk and benefits of the study
- Legal incapacity
To learn more about this study, you or your doctor may contact the study research staff using the contact information provided by the sponsor.
Please refer to this study by its ClinicalTrials.gov identifier (NCT number): NCT05996731
Contact: Marina Noris, PhD | +3903545351 | marina.noris@marionegri.it | |
Contact: Elena Bresin | +3903545351 | elena.bresin@marionegri.it |
Italy | |
Centro di Ricerche Cliniche per le Malattie Rare "Aldo e Cele Daccò" | Recruiting |
Ranica, BG, Italy, 24020 | |
Contact: Elena Bresin 0039 035 45351 elena.bresin@marionegri.it |
Principal Investigator: | Marina Noris, PhD | Istituto di Ricerche Farmacologiche Mario Negri IRCCS |
Responsible Party: | Mario Negri Institute for Pharmacological Research |
ClinicalTrials.gov Identifier: | NCT05996731 |
Other Study ID Numbers: |
ANTHEM-RNA-Seq |
First Posted: | August 18, 2023 Key Record Dates |
Last Update Posted: | March 15, 2024 |
Last Verified: | March 2024 |
Individual Participant Data (IPD) Sharing Statement: | |
Plan to Share IPD: | No |
Studies a U.S. FDA-regulated Drug Product: | No |
Studies a U.S. FDA-regulated Device Product: | No |
Undiagnosed genetic rare diseases Whole-exome sequencing RNA-Sequencing Molecular diagnosis Skin-derived fibroblasts |
Polycystic Kidney Diseases Glomerulonephritis Polycystic Kidney, Autosomal Dominant Hemolytic-Uremic Syndrome Atypical Hemolytic Uremic Syndrome Glomerulonephritis, Membranoproliferative Rare Diseases Disease Attributes Pathologic Processes Kidney Diseases, Cystic Kidney Diseases Urologic Diseases Female Urogenital Diseases Female Urogenital Diseases and Pregnancy Complications Urogenital Diseases |
Male Urogenital Diseases Abnormalities, Multiple Congenital Abnormalities Ciliopathies Genetic Diseases, Inborn Nephritis Uremia Anemia, Hemolytic Anemia Hematologic Diseases Thrombotic Microangiopathies Thrombocytopenia Blood Platelet Disorders Cytopenia Immune System Diseases |