PREcision Diagnostics in Rare genetIC Diseases and Tumors - Long Read Sequencing

NCT ID: NCT06796751

Last Updated: 2025-01-31

Basic Information

• Recruitment Status: RECRUITING
• Total Enrollment: 30 participants
• Study Classification: OBSERVATIONAL
• Study Start Date: 2024-10-01
• Study Completion Date: 2026-09-30

Brief Summary

Using long-read sequencing (LRS) technology to achieve molecular diagnosis in patients with rare genetic diseases who have already been tested by state-of-the-art genetic analysis with ambiguous or negative results. This will lead to efficient and reliable identification and clinical interpretation of cryptic and complex structural genomic variants, which represent the central challenge for the coming decades in human genetics.

Detailed Description

The application of LRS to a diagnostic setting could have an impact on detection rate and diagnostic yield, leading to a better understanding of the etiology, prognosis and recurrence risk of rare genetic diseases (RGD), but also to a targeted treatment. One of the main benefits of LRS is the detection of balanced and unbalanced structural variants (SVs), including complex rearrangements, with high sensitivity and accuracy, through reliable alignment and precise breakpoint definition.

Among the main challenges of modern genetics, are identified 4 subgroups of patients that would benefit from the application of LRS to better characterize the genetic diagnosis and disease mechanisms.

• The first subgroup of patients are those with SVs of unknown significance or uncertain disease mechanism. In particular, clinical interpretation of duplications is challenging, due to the inability of Array Comparative Genomic Hybridization (aCGH) to detect whether they occur in tandem or are duplicated and inserted elsewhere in the genome, thus possibly disrupting genes involved in the duplication or altering their regulation.
• Other complex rearrangements can be studied with LRS to gain a better understanding of the molecular mechanisms of pathogenicity. A better definition of complex SVs, together with an accurate description of the phenotype, will allow the genotype-phenotype correlation to be determined.
• A third subgroup of patients eligible for the study are those with identified monoallelic alteration in autosomal recessive (AR) genes. The aim is to conduct a targeted analysis of the specific gene in order to look for a second mutation that wasn't detected by previous analysis (i.e. intronic variants, SVs, variants in difficult genomic regions).
• Lastly, patients with a phenotype that is strongly suggestive for a genetic condition, in whom several analyses, including Whole Exome-Sequencing (WES), retrieved negative results, could benefit from LRS. In this context, the aim is to apply a whole genome LRS approach to identify missed coding variants in difficult-to-sequence regions, variants in non-coding regions or SVs.

Conditions

Whole Exome Sequencing; Rare Diseases

Study Design

• Observational Model Type: COHORT
• Study Time Perspective: CROSS_SECTIONAL

Study Groups

Patients with negative results at WES analysis

Lastly, patients with a phenotype that is strongly suggestive for a genetic condition, in whom several analyses, including WES, retrieved negative results, could benefit from LRS.

DNA/RNA sequencing and bioinformatic data analysis

Intervention Type: GENETIC

DNA will be extracted from peripheral blood or from somatic tissues. In some cases a skin biopsy will be performed to obtain fibroblasts for further analysis (DNA/RNA extraction and preparation of cell culture for high-throughput genomic and epigenomic technique (Hi-C).

LRS will be performed on extracted DNA using Oxford Nanopore Technology by two different approaches:

• Target, in samples with monoallelic alterations in genes related to autosomal recessive disease;
• Genomic in other cases.

Sequencing data will be analyzed through a dedicated bioinformatics pipeline, to reconstruct the tridimensional structure of chromatin and the regions

Patients with CNVs of unknown significance

The first subgroup of patients are those with SVs of unknown significance or uncertain disease mechanism.

DNA/RNA sequencing and bioinformatic data analysis

Intervention Type: GENETIC

DNA will be extracted from peripheral blood or from somatic tissues. In some cases a skin biopsy will be performed to obtain fibroblasts for further analysis (DNA/RNA extraction and preparation of cell culture for high-throughput genomic and epigenomic technique (Hi-C).

LRS will be performed on extracted DNA using Oxford Nanopore Technology by two different approaches:

• Target, in samples with monoallelic alterations in genes related to autosomal recessive disease;
• Genomic in other cases.

Sequencing data will be analyzed through a dedicated bioinformatics pipeline, to reconstruct the tridimensional structure of chromatin and the regions

Patients with complex genomic rearrengements

Other complex rearrangements can be studied with LRS (i.e. ring chromosomes, isochromosome, chromosomal translocations, somatic SV found in tumors, etc.) to gain a better understanding of the molecular mechanisms of pathogenicity.

DNA/RNA sequencing and bioinformatic data analysis

Intervention Type: GENETIC

DNA will be extracted from peripheral blood or from somatic tissues. In some cases a skin biopsy will be performed to obtain fibroblasts for further analysis (DNA/RNA extraction and preparation of cell culture for high-throughput genomic and epigenomic technique (Hi-C).

LRS will be performed on extracted DNA using Oxford Nanopore Technology by two different approaches:

• Target, in samples with monoallelic alterations in genes related to autosomal recessive disease;
• Genomic in other cases.

Sequencing data will be analyzed through a dedicated bioinformatics pipeline, to reconstruct the tridimensional structure of chromatin and the regions

Patients with a monoallelic variant in an AR gene

A third subgroup of patients eligible for the study are those with identified monoallelic alteration in autosomal recessive (AR) genes.

DNA/RNA sequencing and bioinformatic data analysis

Intervention Type: GENETIC

DNA will be extracted from peripheral blood or from somatic tissues. In some cases a skin biopsy will be performed to obtain fibroblasts for further analysis (DNA/RNA extraction and preparation of cell culture for high-throughput genomic and epigenomic technique (Hi-C).

LRS will be performed on extracted DNA using Oxford Nanopore Technology by two different approaches:

• Target, in samples with monoallelic alterations in genes related to autosomal recessive disease;
• Genomic in other cases.

Sequencing data will be analyzed through a dedicated bioinformatics pipeline, to reconstruct the tridimensional structure of chromatin and the regions

Interventions

DNA/RNA sequencing and bioinformatic data analysis

DNA will be extracted from peripheral blood or from somatic tissues. In some cases a skin biopsy will be performed to obtain fibroblasts for further analysis (DNA/RNA extraction and preparation of cell culture for high-throughput genomic and epigenomic technique (Hi-C).

LRS will be performed on extracted DNA using Oxford Nanopore Technology by two different approaches:

• Target, in samples with monoallelic alterations in genes related to autosomal recessive disease;
• Genomic in other cases.

Sequencing data will be analyzed through a dedicated bioinformatics pipeline, to reconstruct the tridimensional structure of chromatin and the regions

Intervention Type: GENETIC

Eligibility Criteria

Inclusion Criteria

• patients/relatives of patients with Copy Number Variations (CNVs), previously detected by aCGH, with uncertain clinical significance;
• patients/relatives of patients with inconclusive WES and aCGH data (no pathogenic/likely pathogenic variant);
• patients/relatives of patients with a known single hit (a pathogenic or likely pathogenic variant) in an AR gene detected with WES or aCGH;
• patients/relatives of patients with a finding of complex structural variants whose molecular disease mechanism is to be elucidated.

Exclusion Criteria

• none

• Minimum Eligible Age: 28 Days
• Eligible Sex: ALL
• Accepts Healthy Volunteers: No

Sponsors

IRCCS Azienda Ospedaliero-Universitaria di Bologna

OTHER

Sponsor Role: lead

Responsible Party

Responsibility Role: SPONSOR

Principal Investigators

Tommaso Pippucci, Biologist

Role: PRINCIPAL_INVESTIGATOR

IRCCS Azienda Ospedaliero-Universitaria di Bologna

Locations

IRCCS Azienda Ospedaliero-Universitaria di Bologna

Bologna, Bologna, Italy

Site Status: RECRUITING

Countries

Italy

Central Contacts

Tommaso Pippucci, Biologist

Role: CONTACT

tommaso.pippucci@aosp.bo.it

0512142892

Facility Contacts

Tommaso Pippucci, Biologist

Role: primary

tommaso.pippucci@aosp.bo.it

0512142892

Other Identifiers

PNRR2022

Identifier Type: OTHER_GRANT

Identifier Source: secondary_id

PREDICT-LRS

Identifier Type: -

Identifier Source: org_study_id