Functional Tests to Resolve Unsolved Rare Diseases. Rares.

Study Overview
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Brief Summary
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Rares diseases are a heterogeneous group of conditions which need important tools for diagnosis. The use of high-throughput sequencing is able to diagnose half of the patients. For the other part it is impossible to conclude due to the presence of variants of unknown significance (VOUS). Functional analysis are needed to bring strong argument to reclassify variants as pathogenic or benign. The main objective is to evaluate the diagnosis yield of this strategy.

Detailed Description
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The main objective is the improvement of the diagnosis of rare genetic diseases. The investigator lab is expert for diagnosis of some rare diseases such as neurodevelopmental disorder, albinism, cystic fibrosis and congenital heart defect. Actually with implementation of high-throughput sequencing for diagnosis, a high number of genetic variants are found and need to be interpretated. The ACMG classification is used to classify variants with argument of variant frequency, predicted effect on protein and in-silico prediction. Functional evidence is a strong argument to help classify VOUS. The investigators propose the use of RNA-Seq, minigene and luciferase assay for study of VOUS to bring argument to classify them as benign or pathogenic.

Official Title
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Resolving Unsolved Rare Diseases : Functional Tests and New Diagnosis Strategy to Study Genetic Variants From High-throughput Sequencing (RID)

Conditions
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Albinism, Rubinstein-Taybi Syndrome, Cystic Fibrosis, Congenital Heart Defect, Periventricular Nodular Heterotopia, Neurodegeneration With Brain Iron Accumulation (NBIA), Intellectual Disability

Intervention / Treatment
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* Genetic: Ex-vivo approach concerning 25 patients
* Genetic: In-vitro approach concerning 25 patients


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Minor and adult patient. Registered for the social security system. Informed consent signed by patient or parent of a minor patient. Patient affected by one of the rare diseases studied (albinism, congenital heart defect, cystic fibrosis, neurodevelopmental disease) Patient bearing variants of unknown significance (VOUS) Exclusion Criteria: Refusal to participate in research protocol. Patient under administrative supervision Pregnant or nursing women
Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Diagnostic
Intervention Model: Single Group Assignment
Interventional Model Description: Ex-vivo approach concerning 25 patients with blood sample in PAXgene tubes or skin biopsy and RNA-Seq analysis. In-vitro approach concerning 25 patients without specific samples needed for analysis in minigene or luciferase assay.
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Other: Ex-vivo and In-vitro approach<br>Ex-vivo approach concerning 25 patients with blood sample in PAXgene tubes or skin biopsy and RNA-Seq analysis. In-vitro approach concerning 25 patients without specific samples needed for analysis in minigene or luciferase assay | Genetic: Ex-vivo approach concerning 25 patients<br>* Ex-vivo approach concerning 25 patients with blood sample in PAXgene tubes or skin biopsy and RNA-Seq analysis<br>Genetic: In-vitro approach concerning 25 patients<br>* In-vitro approach concerning 25 patients without specific samples needed for analysis in minigene or luciferase assay<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Proportion of VOUS reclassified as pathogenic (class 5) or benign (class 1) | It's the proportion of VOUS that could be definitively reclassified as pathogenic (class 5) or benign (class 1) according to the ACMG classification (Richards et al., 2015 and Appendix 1). Indeed currently only variants considered as pathogenic or probably pathogenic make it possible to confirm a diagnosis and to propose genetic offer genetic counseling to families and perform a prenatal diagnosis. This is an evaluation that will be carried out at the end of the analyses carried out | Inclusion visit | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Pre-analysis process : Time of sample transport to the laboratory | Time of transport to the laboratory. To calculate this time, the time of collection and the time of receipt by the and the time of reception by the molecular genetics technician will be recorded | Inclusion visit | 
| Pre-analysis process : Quality of RNA extraction (RNA Integrity Number, RIN) | RNA quality measurement by RIN (RNA integrity number): very good >7, good >/=5, poor <5. Only RNA with RIN >5 will be retained. | Inclusion visit | 
| Praticability :Characteristics and number of CPU (Central Processing Unit) | Evaluation of bioinformatic ressources by measure of number of CPU needed and turnaround time for processing data | Inclusion visit | 
| Praticability : Training time of Biologists for interpretation | Evaluation of training time needed to interpret the data | Inclusion visit | 
| Global cost | Evaluation of cost of global analyse and each test | Inclusion visit | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Variant of unknown significance, Functional genetics, Translational research, RNAseq, Gene expression regulation