CELLULAR NEUROCIENCE | NEURONAL SIGNALING | CARDIOVASCULAR RESEARCH | METABOLISM | LIPID SIGNALING AND HOMEOSTASIS | ENDOCRINOLOGY | CELLULAR PHYSIOLOGY | TARGETED GENE-EXPRESSION ANALYSIS | MEMBRANE BIOLOGY | TELOMERE RESEARCH
TARGETED GENE-EXPRESSION ANALYSES
Jakob Stenman, M.D., Dr.Med.Sci., Head
Kien Dang, M.D.
The aim of the research group is to develop novel diagnostic assays based on PCR and Next Generation Sequencing. Technical development is focused on applications where there are specific difficulties in utilizing conventional PCR, as well as on improvements in pre-sequencing amplification.
During the past year we have worked intensively on a novel technique for ultra-sensitive detection of expressed KRAS mutations in patients with colorectal cancer. This technique could provide a means not only for disease monitoring from plasma samples, but also for early detection of malignant transformation in pre-malignant conditions, such as Barrett’s esophagus and Primary Sclerosing Cholangitis, where there are specific difficulties with currently available diagnostic tests. Our first paper describing this technique has recently been published. We have applied this technique for studying the occurrence of expressed KRAS and BRAF mutations in pediatrics patients with inflammatory bowel disease
and patients treated for oesophageal atresia in their early childhood (manuscripts under preparation). In the coming year we are planning to study a large sample series consisting of 800 colon cancer FFPE (formalin-fixed, paraffin-embedded) samples.
Figure. Schematic of the Extendable Blocking Probe reverse transcription (ExBP-RT) method that enables ultra-sensitive detection of expressed mutation though allele-specific reverse transcription. The method was published in Nucleic Acids Research in January 2015.
We have continued development of a novel PCR amplification technology called Heat Pulse Extension PCR (HPE-PCR). Specifically, we have shown that difficulties in amplifying long GC rich and repetitive sequences can be overcome by using pulsatile temperature cycling during the extension step of each PCR cycle to destabilize secondary structures in the template. We have previously demonstrated the utility of this technique by amplifying repetitive expansions in Fragile X syndrome as well as in Type I Myotonic Dystrophy. Currently, we are focusing on improving the reaction efficiency further in order to enable amplification of longer repeat expan21 sions that are present in Amyotrophic Lateral Sclerosis (ALS). We have also achieved very promising results in utilizing HPE-PCR for reduction of GCbias in sequencing pre-amplification and thus improving the coverage of poorly amplifying regions. European and US patents were granted for the HPEPCR technology during 2014.
Jakob Stenman, M.D., Dr.Med.Sci., Head
Räsänen K, Dang KX, Mustonen H, Ho TH, Lintula S, Koistinen H, Stenman UH, Haglund C, Stenman J. MAPK inhibitors induce serine peptidase inhibitor Kazal type 1 (SPINK1) secretion in BRAF V600E mutant colorectal adenocarcinoma. Mol Oncol. 2018; 12:224-238.
Dang KX, Ho T, Sistonen S, Koivusalo A, Pakarinen M, Rintala R, Stenman UH, Orpana A, Stenman J. No tissue expression of KRAS or BRAF mutations in 61 adult patients treated for esophageal atresia in early childhood. Eur J Pediatr Surg. 2017; Sep 5. [Epub ahead of print]
Ho TH, Dang KX, Lintula S, Hotakainen K, Feng L, Olkkonen VM, Verschuren EW, Tenkanen T, Haglund C, Kolho KL, Stenman UH, Stenman J. Extendable blocking probe in reverse transcription for analysis of RNA variants with superior selectivity. Nucleic Acids Res. 2015; 43:e4.
Kakkola L, Denisova OV, Tynell J, Viiliäinen J, Ysenbaert T, Matos RC, Nagaraj A, Ohman T, Kuivanen S, Paavilainen H, Feng L, Yadav B, Julkunen I, Vapalahti O, Hukkanen V, Stenman J, Aittokallio T, Verschuren EW, Ojala PM, Nyman T, Saelens X, Dzeyk K, Kainov DE. Anticancer compound ABT- 263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice. Cell Death Dis 2013; 25;4:e742.
Orpana AK, Ho TH, Alagrund K, Ridanpää M, Aittomäki K, Stenman J. Novel heat pulse extension- PCR-based method for detection of large CTG-repeat expansions in myotonic dystrophy type 1. J Mol Diagn 2013; 15:110–5.
Koivuniemi R, Mäkelä J, Hokkanen ME, Bruelle C, Ho TH, Ola R, Korhonen L, Schröder J, Kataoka H, Lindholm D. Hepatocyte growth factor activator inhibitor-1 is induced by bone morphogenetic proteins and regulates proliferation and cell fate of neural progenitor cells. PLoS One 2013; 8:e56117.
Hyrskyluoto A, Pulli I, Törnqvist K, Huu Ho T, Korhonen L, Lindholm D. Sigma-1 receptor agonist PRE084 is protective against mutant huntingtin-induced cell degeneration: involvement of calpastatin and the NF-κB pathway. Cell Death Dis 2013; 23;4:e646.
Denisova OV, Kakkola L, Feng L, Stenman J, Nagaraj A, Lampe J, Yadav B, Aittokallio T, Kaukinen P, Ahola T, Kuivanen S, Vapalahti O, Kantele A, Tynell J, Julkunen I, Kallio-Kokko H, Paavilainen H, Hukkanen V, Elliott RM, De Brabander JK, Saelens X, and Kainov DE. Obatoclax, saliphenylhalamide and gemcitabine inhibit influenza A virus infection. J. Biol. Chem 2012; 287:35324-32.Feng L, Lintula S, Ho TH, Anastasina M, Paju A, Haglund C, Stenman U-H, Hotakainen K, Orpana A, Kainov D and Stenman J. Technique for strand-specific gene- expression analysis and monitoring of primer-independent cDNA synthesis in reverse transcription. Biotechniques 2012; 52:263-70.