Background: Telomere shortening is linked to a range of different human diseases, hence reliable measurement methods are needed to uncover such associations. Among the plethora of telomere length measurement methods, qPCR is reported as easy to conduct and a cost-effective approach to study samples with low DNA amounts. Methods: Cancer cells' telomere length was evaluated by relative and absolute qPCR methods. Results: Robust and reproducible telomere length measurements were optimized taking into account a careful reference gene selection and by knowing the cancer cells ploidy. qPCR data were compared to "gold standard" measurement from terminal restriction fragment (TRF). Conclusions:Our study provides guidance and recommendations for accurate telomere length measurement by qPCR in cancer cells, taking advantage of our expertise in telomere homeostasis investigation in primary cutaneous T-cell lymphomas.Furthermore, our data emphasize the requirement of samples with both, high DNA quality and high tumor cells representation. K E Y W O R D S cancer, qPCR, southern blot, telomere length, tumor 3154 | ROPIO et al. (ERDF), COMPETE 2020 -Operacional Programme for Competitiveness and Internationalization (POCI) and by Portuguese funds through FCT -Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior.
Cutaneous T-cell lymphomas (CTCL) are telomerase-positive tumors expressing hTERT, although neither gene rearrangement/amplification nor promoter hotspot mutations could explain the hTERT re-expression. As the hTERT promoter is rich in CpG, we investigated the contribution of epigenetic mechanisms in its reexpression. We analyzed hTERT promoter methylation status in CTCL cells compared to healthy cells. Gene-specific methylation analyses revealed a common methylation pattern exclusively in tumor cells. This methylation pattern encompassed a hypermethylated distal region from-650bp to-150bp and a hypomethylated proximal region from-150bp to +150bp. Interestingly, the hypermethylated region matches with the recently named TERT Hypermethylated Oncogenic Region (THOR). THOR has been associated with telomerase reactivation in many cancers, but it has so far not been reported in cutaneous lymphomas. Additionally, we assessed the effect on THOR of two histone deacetylase inhibitors (HDACi), romidepsin and vorinostat, both approved for CTCL treatment as well as a DNA methyltransferase inhibitor (DNMTi) 5-azacytidine, unapproved for CTCL. Contrary to our expectations, the findings reported herein revealed that THOR methylation is relatively stable under these epigenetic drugs' pressure, whereas these drugs reduced the hTERT gene expression.
BackgroundCockayne Syndrome (CS) is a rare autosomal recessive disorder characterized by neurological and sensorial impairment, dwarfism, microcephaly and photosensitivity. CS is caused by mutations in ERCC6 (CSB) or ERCC8 (CSA) genes.MethodsThree patients with CS were referred to the Medical Genetics Unit of Saint Joseph University. Sanger sequencing of both ERCC8 and ERCC6 genes was performed: ERCC8 was tested in all patients while ERCC6 in one of them.ResultsSequencing led to the identification of three homozygous mutations, two in ERCC8 (p.Y322* and c.843 + 1G > C) and one in ERCC6 (p.R670W). All mutations were previously reported as pathogenic except for the c.843 + 1G > C splice site mutation in ERCC8 which is novel.ConclusionsMolecular diagnosis was established in all patients included in our study. A genotype-phenotype correlation is discussed and a link, between mutations and some specific religious communities in Lebanon, is suggested.Electronic supplementary materialThe online version of this article (10.1186/s12881-018-0677-7) contains supplementary material, which is available to authorized users.
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