Aportación de la secuenciación masiva de nueva generación en el diagnóstico del síndrome de cáncer de mama y ovario hereditario mediante el uso de un panel de genes

Resum

The incorporation of next generation sequencing in Genomics Laboratory in Clinical Analysis Service of University Hospital Virgen de la Arrixaca (HCUVA) has allowed the study of Hereditary Breast and Ovarian Cancer (HBOC) through the performance of 26 genes panel, in which high and moderate penetrance genes were included. However, the increase of information in comparison with the genetic study of only BRCA1 and BRCA2 must be accompanied of a correct interpretation of this information. Therefore, it was necessary to evaluate the clinical utility of genetic studies through multigene pane testing in the studied population. Taking this general objective into account, also the following secondary objectives were addressed: establishing the diagnostic yield of multigene panel testing analysis in the studied population with a previous uninformative test for BRCA1 and BRCA2, evaluating the selection criteria, characterizing molecularly and clinically the variants encountered, evaluating the mutation rate of the studied genes, performing genotype-phenotype correlation study in those clinically relevant variants and describing the incidence of variants of uncertain significance variants (VUS) as well as prioritized VUS. In this thesis the study population were 138 families with a previous uninformative test for BRCA1 and BRCA2 from Genetic Counselling Units from Medical Oncology Services of HCUVA and University Hospital Morales Meseguer, which had been included for meeting additional criteria to those proposed by Spanish Society of Medical Oncology (SEOM). After the analysis through the multigene panel BRCA Hereditary MasterTM Plus (Agilent¿), the found variants were classified molecularly and clinically by using different bioinformatics tools, databases, bibliography and in silico studies. With this methodology an algorithm for prioritizing VUS was stablished. Subsequently, the verification of clinically variants (pathogenic and likely pathogenic) found and the extension of study to family members were performed by Sanger sequencing, so information of genotype and phenotype was obtained. Finally, to probe the founder effect of the variant c.8251_8254del in ATM a microsatellite analysis in families affected a control population was carried out. The use of the multigene panel described above increased the diagnostic yield by 8% in the studied population, as described in bibliography. To that end, the implementation of additional criteria of selection did not contribute significantly to increase the diagnostic yield in comparison with those proposed by SEOM. With regard to the molecular and clinical characterization of multigene panel generated a great number of missense variants which, in most cases, were classified clinically as variants with uncertain significance. However, most of pathogenic and probably pathogenic variants were nonsense or frameshift. Thanks to the incorporation of a VUS prioritizing algorithm, the percentage of families that carried this type of variants was reduced from 52% to 20%, so it is recommendable to use this tool in the analysis of genetic studies. This study also contributed to the identification of 14 previously non described variants (4 clinically relevant and 10 with unknown significance). Likewise, it is worth noting that prevalence of mutations in the ATM (4.35%) has been the highest described until now as well as the contribution to the detection rate of the variant c8251_8254del in ATM, found in two families of the study, and which has a common origin in Murcia. With respect to the genotype-phenotype correlation studies performed in carriers of pathogenic variants in non BRCA genes the most common cancer was unilateral breast cancer, followed by ovarian cancer. In respect of the histopathologic features of breast cancer cases, all of them were positive for hormonal receptors, except he carrier of a pathogenic variant in TP53. This result is similar to the found in other studies.