Pathology of Helicases and Premature Aging: Study by Derivation of hiPS

Study Overview
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Brief Summary
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Topic of this work is the involvement of replicative helicases in human premature ageing syndrome. Replicative helicases are ubiquitous and essential during numerous reactions of the DNA metabolism. The family of replicative helicases (RecQL) is involved in the replication/repair of the DNA and in the telomere maintenance. There are 5 enzymes in human and 3 of them are involved in clinically recognizable syndromes: WRN for the Werner syndrome, BLM for the Bloom syndrome and RECQL4 for the Rothmund Thomson syndrome. All are responsive of a high cancer risk due to genomic instability. Molecular and cellular mechanisms involved in these diseases of ageing are unknown. Moreover, for all of them, there is not therapeutic or preventive solution.

Detailed Description
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For understanding the involved mechanisms we would like to model the 3 diseases with hiPS (human induced Pluripotent Stem cells) from somatic cells of patients. The patient recruitment was organized by the Montpellier and Nîmes public hospitals. The project is to generate a hiPS cell line for the 3 syndromes from fibroblasts and/or blood samples. Then, we could induce differentiation of hiPS to a target cell line of the diseases. Finally we could study the disease development following the genomic instability (karyotype, array-CGH) and the cellular ageing (senescence-associated heterochromatin foci, telomere length). For each mutated enzyme, we will perform a transcriptional profiling (splice, mRNA quantification) and protein studies (western blot). All results will be compared to wild type cells.

Official Title
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Pathology of Helicases and Premature Aging: Study by Derivation of hiPS

Conditions
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Age Problem

Intervention / Treatment
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* Other: taking of cutaneous cells


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Patinet with one of the 3 helicase-associated precoce aging desease Exclusion Criteria: Minor and /or mentally incapable patient

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 65 Years

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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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Taking of cutaneous cells by biopsy<br>Taking of cutaneous cells by biopsy and a sample of blood | Other: taking of cutaneous cells<br>* Taking of cutaneous cells by biopsy Sample of blood<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Genomic instability : analysis | Molecular analysis of hiPS cell derived from pathological tissue (karyotype, array-CGH) | 1 year | 
| Genomic instability : size of telomers | size of the telomers which will be quantified under microscope after fluorescent marking in situ of telomeric sequences (Q-FISH technique) | 1 year | 
| Genomic instability : Duplication of centrosomes | duplication of centrosomes which is often associated with chromosomal segregation errors and genomic instability. This analysis will be done by immunolabelling using antibodies specific to the 2 main components of centrosomes, pericentrin and -tubulin. | 1 year | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| cellular ageing : molecular analysis of hiPS cell derived from pathological tissue | Analysis of senescence-associated heterochromatin foci, telomere length (Q-FISH) | 2 years | 
| cellular ageing : IPS line with the criteria defined for morphological characterization | expression of specific surface markers (specifics markers : TRA-1-60, SSEA-4), ability to re-differentiate in the 3 embryonic layers (specifics markers : SMA, MAP2, FOXA2) | 2 years | 
| cellular ageing : molecular characterization | lengthening of telomeric sequence size (Q-FISH), re-expression of pluripotency genes (QRTPCR), transcriptional profile of iPS cell lines. | 2 years | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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helicase, induced pluripotent stem cells