Solving Challenging Diagnoses Through Ultra-long Read Sequencing

Not Applicable

Active, not recruiting

• Conditions: Variant Nucleotide

• Registration Number: NCT06775613
• Lead Sponsor: IRCCS Azienda Ospedaliero-Universitaria di Bologna

Brief Summary

If the study is able to demonstrate that the LRS concretely overcomes the technical limitations of diagnostic methods routinely used in laboratories, its clinical application may make possible a more accurate analysis of repeated genomic regions and offer greater sensitivity for identifying SVs (Structural Variants)

Detailed Description

The diagnostic performance of LRS, by applying Oxford Nanopore Technology (ONT), will be evaluated through a real-time targeted approach and generation of ultra-long reads, to the identification of pathogenetic variants in genetic disorders with a currently challenging diagnosis due to the presence of pseudogenes (1) and to the precise mapping of SV breakpoints identified by aCGH for the definition of their clinical significance (2).

1. Patients with an established molecular diagnosis will be collected for each of the following disorders: 10 with autosomal dominant polycystic kidney disease (ADPKD) and 10 with CYP21 deficiency. Cases with ambiguous results to test possible ONT improvements in terms of time to results and precise molecular characterization will be included. The entire range of mutations affecting each causative gene (PKD1 and CYP21A2, respectively), which globally cover all the possible types of alterations (missense, nonsense, indels, exonic and gene deletions) will be included.

2. Collection of other 10 patients carrying copy number variants (CNVs) with aCGH-defined breakpoints mapping close to disease-genes possibly responsible for the observed clinical picture.

The main aim of this proposal is to evaluate ONT efficacy in resolving diagnostic challenges faced in the clinical genomics routine, in situations when a precise molecular diagnosis is often impossible or difficult and extremely time-consuming with current genetic tests. Sanger sequencing, NGS panels and MLPA are routinely used to identify pathogenic variants and CNVs responsible for many monogenic disorders and for the analysis through aCGH patients with isolated or syndromic intellectual disability without a specific clinical suspect. These analyses are long, technically laborious and often not individually conclusive because technical limitations may lead to ambiguous results and prevent definite diagnosis. The intent is to demonstrate that ONT, through a real-time targeted approach to increase coverage in clinically relevant regions during sequencing, outperforms current diagnostic tools in terms of rapidity and sensitivity, considerably improving the efficiency of the diagnostic process. The success of the proposed target ONT approach will provide the proof of principle to implement this strategy for the diagnosis of a wider spectrum of disorders already studied in our laboratory

Study Timeline

• Study Start: 2023-03-10
• Primary Completion: 2024-03-20
• Status Verified: 2024-10
• Study First Submitted: 2024-11-28
• Study First Submitted QC: 2025-01-09
• Last Update Submitted: 2025-01-09
• Study First Posted: 2025-01-15
• Last Update Posted: 2025-01-15
• Study Completion: 2025-12-31

Recruitment & Eligibility

• Status: ACTIVE_NOT_RECRUITING
• Sex: All
• Target Recruitment: 15

Inclusion Criteria

Not provided

Exclusion Criteria

Not provided

Study & Design

• Study Type: INTERVENTIONAL
• Study Design: PARALLEL

Primary Outcome Measures

Name	Time	Method
Genetic anomalies	24 months	* To assess the sensitivity of the LRS for identifying genetic variants in genomic regions that are difficult to analyse using the technologies routinely employed in diagnostic laboratories (NGS, Sanger sequencing, MLPA).; * Assess the ability of the LRS to precisely map unbalanced genomic alterations identified through aCGH, in order to define their clinical significance.

Secondary Outcome Measures

Name	Time	Method
Mapping the breaking points of CNVs	24 months	To verify whether the new sequencing technology leads to a reduction in analysis and reporting times compared to traditional methods.

Trial Locations

Locations (1)

IRCCS Azienda Ospedaliero-Universitaria di Bologna; 🇮🇹 Bologna, Italy