Development of bioinformatics tools for the characterization of B cell neoplasms
- Autores: Azahara Maria Fuentes Trillo
- Directores de la Tesis: Felipe Javier chaves Martínez (dir. tes.), María José Terol Castera (codir. tes.), Blanca Navarro Cubells (codir. tes.)
- Lectura: En la Universitat de València ( España ) en 2023
- Idioma: inglés
- Tribunal Calificador de la Tesis: María Luisa Mansego Talavera (presid.), Miguel Morard Pedrouzo (secret.), Davide Rossi (voc.)
- Programa de doctorado: Programa de Doctorado en Biomedicina y Biotecnología por la Universitat de València (Estudi General)
- Materias:
- Enlaces
- Tesis en acceso abierto en: TESEO
- Resumen
The adaptive immune system is orchestrated by a wide battery of antibodies, secreted by B lymphocytes, in charge of the recognition of antigenic molecules. In order to express the Immunoglobulin-type receptor transmembrane protein on their surface, B cells suffer recombination of the receptor Heavy and Light Chains genes. This occurs during their development and involving V(D)J gene segments (one of each of the possible V, D and J genes rearrange, conforming the IGH locus variable domain). Additional variability is conferred on the receptor after antigen exposure, when somatic mutations are introduced to augment the specificity of antigen-receptor recognition. The degree of mutation in the IGH locus is especially determinant as a prognostic factor in CLL, where patients classified as mutated tend to have more indolent disease course compared to patients with unmutated B cell receptors.
The main aim of this thesis work is the development of bioinformatics tools for clinical determinations of the IGH locus in CLL patients. For that purpose, a simple and cost-effective library preparation protocol was employed to sequence the VDJ region of the IGH locus, and a specific bioinformatics pipeline for analysis of B cell clones based on the construction of a consensus sequence was designed (BMyRepCLL). A specific cut-off method was implemented within this workflow to differentiate the clonal and subclonal rearrangements fraction among patients. A second pipeline (CLL Immcantation) was adapted from the Immcantation Framework; a suite which unites tools to analyze immune repertoires data. Both workflows have been used to analyze a dataset of 314 CLL patients previously diagnosed with the standard criteria and protocols, and to compare the assessment of mutational status. Moreover, the results from BMyRepCLL have been validated exhaustively against the gold-standard. Since both pipelines are based in different methodologies, CLL Immcantation was used for benchmarking the characterization of samples with multiple clones by BMyRepCLL, as well as to perform downstream analyses specific to immune repertoire data.
Results show agreement between BMyRepCLL and SSeq regarding IGHV and IGHJ genes annotation and CDR3 sequence extraction. The mutational status was characterized accordingly in 99% of the rearrangements. Clones were detected with a specificity and sensitivity of 97% and 100%, respectively. With the development of these methods we contribute to the standardization of NGS protocols for the determination of IGH locus in CLL, which will be highly advantageous for augmenting drastically the scope of SSeq and allowing to study in deep the clonal architecture in CLL.
