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1.
JCI Insight ; 3(8)2018 04 19.
Article in English | MEDLINE | ID: mdl-29669941

ABSTRACT

Mutations in the gene encoding the protein kinase A (PKA) catalytic subunit α have been found to be responsible for cortisol-producing adenomas (CPAs). In this study, we identified by whole-exome sequencing the somatic mutation p.S54L in the PRKACB gene, encoding the catalytic subunit ß (Cß) of PKA, in a CPA from a patient with severe Cushing syndrome. Bioluminescence resonance energy transfer and surface plasmon resonance assays revealed that the mutation hampers formation of type I holoenzymes and that these holoenzymes were highly sensitive to cAMP. PKA activity, measured both in cell lysates and with recombinant proteins, based on phosphorylation of a synthetic substrate, was higher under basal conditions for the mutant enzyme compared with the WT, while maximal activity was lower. These data suggest that at baseline the PRKACB p.S54L mutant drove the adenoma cells to higher cAMP signaling activity, probably contributing to their autonomous growth. Although the role of PRKACB in tumorigenesis has been suggested, we demonstrated for the first time to our knowledge that a PRKACB mutation can lead to an adrenal tumor. Moreover, this observation describes another mechanism of PKA pathway activation in CPAs and highlights the particular role of residue Ser54 for the function of PKA.


Subject(s)
Adenoma/enzymology , Cushing Syndrome/diagnostic imaging , Cyclic AMP-Dependent Protein Kinases/genetics , Hydrocortisone/metabolism , Adenoma/metabolism , Adrenal Gland Neoplasms/diagnostic imaging , Adrenal Gland Neoplasms/pathology , Adrenal Gland Neoplasms/surgery , Adrenal Insufficiency/etiology , Adrenalectomy/methods , Adult , Catalytic Domain/genetics , Cushing Syndrome/pathology , Cushing Syndrome/surgery , Cyclic AMP Receptor Protein/metabolism , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits , Female , Holoenzymes/metabolism , Humans , Mutation , Treatment Outcome , Exome Sequencing/methods
2.
J Mol Diagn ; 19(5): 776-787, 2017 09.
Article in English | MEDLINE | ID: mdl-28826610

ABSTRACT

Pangenomic studies identified distinct molecular classes for many cancers, with major clinical applications. However, routine use requires cost-effective assays. We assessed whether targeted next-generation sequencing (NGS) could call chromosomal alterations and DNA methylation status. A training set of 77 tumors and a validation set of 449 (43 tumor types) were analyzed by targeted NGS and single-nucleotide polymorphism (SNP) arrays. Thirty-two tumors were analyzed by NGS after bisulfite conversion, and compared to methylation array or methylation-specific multiplex ligation-dependent probe amplification. Considering allelic ratios, correlation was strong between targeted NGS and SNP arrays (r = 0.88). In contrast, considering DNA copy number, for variations of one DNA copy, correlation was weaker between read counts and SNP array (r = 0.49). Thus, we generated TARGOMICs, optimized for detecting chromosome alterations by combining allelic ratios and read counts generated by targeted NGS. Sensitivity for calling normal, lost, and gained chromosomes was 89%, 72%, and 31%, respectively. Specificity was 81%, 93%, and 98%, respectively. These results were confirmed in the validation set. Finally, TARGOMICs could efficiently align and compute proportions of methylated cytosines from bisulfite-converted DNA from targeted NGS. In conclusion, beyond calling mutations, targeted NGS efficiently calls chromosome alterations and methylation status in tumors. A single run and minor design/protocol adaptations are sufficient. Optimizing targeted NGS should expand translation of genomics to clinical routine.


Subject(s)
Biomarkers, Tumor , Chromosome Aberrations , DNA Methylation , High-Throughput Nucleotide Sequencing , Mutation , Neoplasms/diagnosis , Neoplasms/genetics , Alleles , Computational Biology/methods , CpG Islands , DNA Copy Number Variations , Diagnostic Tests, Routine/methods , Gene Frequency , Genomics/methods , Genotype , High-Throughput Nucleotide Sequencing/methods , Humans , Polymorphism, Single Nucleotide , Sequence Analysis, DNA
3.
Br J Cancer ; 115(12): 1575-1583, 2016 Dec 06.
Article in English | MEDLINE | ID: mdl-27875525

ABSTRACT

BACKGROUND: Cervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality worldwide. CC pathogenesis is triggered when human papillomavirus (HPV) inserts into the genome, resulting in tumour suppressor gene inactivation and oncogene activation. Collecting tumour and blood samples is critical for identifying these genetic alterations. METHODS: BIO-RAIDs is the first prospective molecular profiling clinical study to include a substantial biobanking effort that used uniform high-quality standards and control of samples. In this European Union (EU)-funded study, we identified the challenges that were impeding the effective implementation of such a systematic and comprehensive biobanking effort. RESULTS: The challenges included a lack of uniform international legal and ethical standards, complexities in clinical and molecular data management, and difficulties in determining the best technical platforms and data analysis techniques. Some difficulties were encountered by all investigators, while others affected only certain institutions, regions, or countries. CONCLUSIONS: The results of the BIO-RAIDs programme highlight the need to facilitate and standardise regulatory procedures, and we feel that there is also a need for international working groups that make recommendations to regulatory bodies, governmental funding agencies, and academic institutions to achieve a proficient biobanking programme throughout EU countries. This represents the first step in precision medicine.


Subject(s)
Biological Specimen Banks , Uterine Cervical Neoplasms/pathology , Female , Humans
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