Developing a Pipeline to Employ RNA-Seq as a Complementary Diagnostic Tool in Rare Diseases

NCT05996731 | Recruiting

• 1 other identifier: ANTHEM-RNA-Seq
• Study Type: interventional
• Target: 105
• Locations: 1 country
• Sites: 1

Brief Summary

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.

Conditions

Atypical Hemolytic Uremic Syndrome; Membranoproliferative Glomerulonephritis; Autosomal Dominant Polycystic Kidney; Healthy

Keywords

Undiagnosed genetic rare diseases; Whole-exome sequencing; RNA-Sequencing; Molecular diagnosis; Skin-derived fibroblasts

Outcome Measures

Primary Outcomes (2)

• 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

  WES analyses for DNA isolation

  At day 0

• 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

  Skin-derived fibroblasts

  At day 0

Study Arms (3)

Healthy subjects

ACTIVE COMPARATOR

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.

Procedure: Skin biopsy

Validation cohort

EXPERIMENTAL

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.

Procedure: Skin biopsy

Discovery cohort

EXPERIMENTAL

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.

Procedure: Skin biopsy

Interventions

Skin biopsy PROCEDURE

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.

Discovery cohort; Healthy subjects; Validation cohort

Eligibility Criteria

• Sex: all
• Healthy Volunteers: Yes
• Age Groups: Child (0-17), Adult (18-64), Older Adult (65+)

You may qualify if:

• Male and female adults
• Written informed consent

You may not qualify if:

• Inability to understand the potential risk and benefits of the study
• Legal incapacity
• Validation cohort.
• Male and female adults
• Genetic diseases affecting RNA levels (frameshifts, stop, large deletions, alteration of canonical splicing sites)
• Written informed consent
• Underage patients
• Inability to understand the potential risk and benefits of the study
• Legal incapacity
• Discovery cohort.
• 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
• Inability to understand the potential risk and benefits of the study
• Legal incapacity

Sponsors & Collaborators

• Mario Negri Institute for Pharmacological Research: lead

Study Sites (1)

Centro di Ricerche Cliniche per le Malattie Rare "Aldo e Cele Daccò"

Ranica, BG, 24020, Italy

RECRUITING

Related Publications (3)

• Kremer LS, Bader DM, Mertes C, Kopajtich R, Pichler G, Iuso A, Haack TB, Graf E, Schwarzmayr T, Terrile C, Konarikova E, Repp B, Kastenmuller G, Adamski J, Lichtner P, Leonhardt C, Funalot B, Donati A, Tiranti V, Lombes A, Jardel C, Glaser D, Taylor RW, Ghezzi D, Mayr JA, Rotig A, Freisinger P, Distelmaier F, Strom TM, Meitinger T, Gagneur J, Prokisch H. Genetic diagnosis of Mendelian disorders via RNA sequencing. Nat Commun. 2017 Jun 12;8:15824. doi: 10.1038/ncomms15824.

  PMID: 28604674 BACKGROUND

• Lee H, Huang AY, Wang LK, Yoon AJ, Renteria G, Eskin A, Signer RH, Dorrani N, Nieves-Rodriguez S, Wan J, Douine ED, Woods JD, Dell'Angelica EC, Fogel BL, Martin MG, Butte MJ, Parker NH, Wang RT, Shieh PB, Wong DA, Gallant N, Singh KE, Tavyev Asher YJ, Sinsheimer JS, Krakow D, Loo SK, Allard P, Papp JC; Undiagnosed Diseases Network; Palmer CGS, Martinez-Agosto JA, Nelson SF. Diagnostic utility of transcriptome sequencing for rare Mendelian diseases. Genet Med. 2020 Mar;22(3):490-499. doi: 10.1038/s41436-019-0672-1. Epub 2019 Oct 14.

  PMID: 31607746 BACKGROUND

• Yepez VA, Mertes C, Muller MF, Klaproth-Andrade D, Wachutka L, Fresard L, Gusic M, Scheller IF, Goldberg PF, Prokisch H, Gagneur J. Detection of aberrant gene expression events in RNA sequencing data. Nat Protoc. 2021 Feb;16(2):1276-1296. doi: 10.1038/s41596-020-00462-5. Epub 2021 Jan 18.

  PMID: 33462443 BACKGROUND

MeSH Terms

Conditions

Atypical Hemolytic Uremic Syndrome; Glomerulonephritis, Membranoproliferative; Polycystic Kidney, Autosomal Dominant

Condition Hierarchy (Ancestors)

Hemolytic-Uremic Syndrome; Uremia; Kidney Diseases; Urologic Diseases; Female Urogenital Diseases; Female Urogenital Diseases and Pregnancy Complications; Urogenital Diseases; Male Urogenital Diseases; Anemia, Hemolytic; Anemia; Hematologic Diseases; Hemic and Lymphatic Diseases; Thrombotic Microangiopathies; Thrombocytopenia; Blood Platelet Disorders; Cytopenia; Glomerulonephritis; Nephritis; Immune System Diseases; Polycystic Kidney Diseases; Kidney Diseases, Cystic; Abnormalities, Multiple; Congenital Abnormalities; Congenital, Hereditary, and Neonatal Diseases and Abnormalities; Ciliopathies; Genetic Diseases, Inborn

Study Officials

• Marina Noris, PhD

  Istituto Di Ricerche Farmacologiche Mario Negri

  PRINCIPAL INVESTIGATOR

Central Study Contacts

Marina Noris, PhD

CONTACT

+3903545351; marina.noris@marionegri.it

Elena Bresin

CONTACT

+3903545351; elena.bresin@marionegri.it

Study Design

• Study Type: interventional
• Phase: not applicable
• Allocation: NON RANDOMIZED
• Masking: NONE
• Purpose: DIAGNOSTIC
• Intervention Model: PARALLEL
• Sponsor Type: OTHER
• Responsible Party: SPONSOR

Study Record Dates

• First Submitted: August 2, 2023
• First Posted: August 18, 2023
• Study Start: February 21, 2024
• Primary Completion (Estimated): June 1, 2026
• Study Completion (Estimated): June 1, 2026
• Last Updated: March 23, 2026

Record last verified: 2026-03

Data Sharing

• IPD Sharing: Will not share

Locations

IT (1)