Browsing by Subject "KCNQ1"

Mutations in the KCNQ1 gene have been implicated in the onset of hypopituitarism. Regulating KCNQ1 expression would therefore enable future clinical research on the mechanism of the disease. CRISPR offers a flexible toolset for controlling genetic expression via knockout, knock-in, knockdown, and gene activation. Of these approaches, CRISPR activation (CRISPRa) is distinguished by its ability to induce gene overexpression in a cell’s native context, making it a valuable tool in the interrogation of genetic disorder pathogenesis. This thesis therefore tested the efficacy of a CRISPRa subsystem in increasing KCNQ1 expression. The CRISPRa subsystem, VPR, was chosen because of its high activation efficiency and the ease of controlling the activation system of its doxycycline-inducible mode of action. The cell line used for the experiment, HEK293, was similarly chosen because of its ease of culture and transfection. To validate the proper functioning of the activation system, expression rates of the related genes ASCL1 and GHRH were measured as positive controls. The activation system successfully upregulated the expression rates of the two genes. As the dCas9-VPR system is dependent on the Tet-ON operator for inducing activation in a controllable manner, a test for dCas9 leakage was conducted. RT-qPCR analysis showed the upregulation of ASCL1 expression in the uninduced state of the system, confirming the presence of dCas9-VPR leakage. The dCas9-VPR system finally aimed to test the expression rate of KCNQ1. Although one novel guide RNA successfully upregulated KCNQ1 expression, it did so inefficiently and its success was not shared by any of the other tested guide RNAs. Altogether, the dCas9-VPR system was successfully established in HEK293 cells, and the leakage of the inducible system was confirmed, however, KCNQ1 activation by CRISPRa requires further optimization.

Kv7.1 is a potassium ion channel comprised of the KCNQ1 protein, which can coassemble with distinct β-subunits modulating the channel functions in different tissues. In 2017, Raivio’s group (from the University of Helsinki) found two missense mutations in the KCNQ1 gene, p.(Arg116Leu) and p.(Pro369Leu), responsible for causing pituitary hormone deficiency and maternally inherited gingival fibromatosis. The facial features and bone structure pointed to a cranial neural crest (CNC)-derived phenotype caused by an alteration in the potassium channel balance, given that these cells form the bone and cartilage of the cranial zone. To understand the implication of the CNC in the KCNQ1 syndrome, I attempted to replicate the CNC differentiation protocol of Suga and Furue (2019) with the aim of obtaining cranial neural crest cells (CNCCs). This would enable future generation of a KCNQ1-related disease model. The differentiation process was carried out thrice, and two BMP4 concentrations (10 and 100 ng/ml) were assayed. The differentiated cells exhibited a CNC-like morphology as well as upregulation of the marker genes (TFAP2A, SOX10, DLX1, MSX1, and DLX2) associated to this cell lineage. However, the gene expression was low according to the qRT-PCR Ct values, which were in most cases higher than 30. Additionally, no differences were found between the two BMP4 treatments. Furthermore, the cells did not express KCNQ1, and thus the impact of the two KCNQ1 mutations was not investigated under this protocol. In conclusion, the protocol had a low efficiency in the generation of CNCCs that was not improved by increasing the BMP4 concentration. Further optimization of the protocol, such as the BMP4 concentration or the cell density of the culture, will be needed to improve its efficiency and obtain an adequate disease model.

Synnynnäinen ionikanavasairaus pitkä QT -oireyhtymä (long QT syndrome, LQTS) on perinnöllinen hengen-vaarallisia rytmihäiriöitä aiheuttava sairaus. LQTS johtuu sydänlihassolujen ionikanavien rakenteita koodaa-vien geenien mutaatioista. Yleisimmät mutaatiot ovat KCNQ1-geenissä, ja ne aiheuttavat sairauden alamuo-don LQT1. KCNQ1 sijaitsee kromosomin 11p15.5 leimautuneella alueella, ja se koodittaa jänniteriippuvaista kaliumkanavaa, Kv7.1:a. Kaksi KCNQ1:n aktivoivaa mutaatiota aiheuttavat autosomaalisesti dominantisti periytyvän kasvuhormonin vajauksen ja äidiltä perittynä ienfibromatoosin. Tutkimuksen tarkoituksena oli analysoida LQTS -potilaiden, joilla on toiminnan hävittävä mutaatio (loss-of-function mutaatio) KCNQ1-geenissä, kasvua ja endokriinisia ominaisuuksia. Keskityimme erityisesti varhaisen kasvun ja parent-of-origin -mutaation suhteeseen. Tutkimuksessa analysoitiin LQT1-potilaiden (n=104) syntymäpituutta ja -painoa, syntymän jälkeistä kasvua ensimmäisen vuoden osalta sekä potilaiden endokriinisia ominaisuuksia. Tutkimuksessa havaittiin, että poti-laat, jotka olivat perineet KCNQ1-mutaation äidiltään, olivat syntymässä lyhyempiä kuin potilaat, jotka olivat perineet mutaation isältään. Jatkoanalyysit osoittivat, että vain potilaat, joiden äidit olivat saaneet beetasal-paajaa raskaana ollessaan, olivat lyhyempiä ja kevyempiä kuin ne potilaat, jotka olivat perineet mutaation isältään. Äidin beetasalpaajan käyttö raskauden aikana oli myös yhteydessä matalampiin napa-TSH-pitoisuuksiin sekä merkittävään saavutuskasvuun ensimmäisen elinvuoden aikana. Myöhemmin eroa ei ha-vaittu. Tutkimuksemme mukaan KCNQ1:n loss-of-function -mutaatiot eivät ole yhteydessä epänormaaliin kasvuun. Sen sijaan analyysiemme mukaan äidin raskauden aikainen beetasalpaajan käyttö näyttää rajoittavan ras-kaudenaikaista LQT1-potilaiden kasvua, mitä seuraa nopea saavutuskasvu ensimmäisen elinvuoden aikaan.