Investigation of a nonsense mutation located in the complex KIV-2 copy number variation region of apolipoprotein(a) in 10,910 individuals

Background The concentrations of the highly atherogenic lipoprotein(a) [Lp(a)] are mainly genetically determined by the LPA gene locus. However, up to 70% of the coding sequence is located in the complex so-called kringle IV type 2 (KIV-2) copy number variation, a region hardly accessible by common genotyping and sequencing technologies. Despite its size, little is known about genetic variants in this complex region. The R21X variant is a functional variant located in this region, but it has never been analyzed in large cohorts. Methods We typed R21X in 10,910 individuals from three European populations using a newly developed high-throughput allele-specific qPCR assay. R21X allelic location was determined by separating the LPA alleles using pulsed-field gel electrophoresis (PFGE) and typing them separately. Using GWAS data, we identified a proxy SNP located outside of the KIV-2. Linkage disequilibrium was determined both statistically and by long-range haplotyping using PFGE. Worldwide frequencies were determined by reanalyzing the sequencing data of the 1000 Genomes Project with a dedicated pipeline. Results R21X carriers (frequency 0.016–0.021) showed significantly lower mean Lp(a) concentrations (− 11.7 mg/dL [− 15.5; − 7.82], p = 3.39e−32). The variant is located mostly on medium-sized LPA alleles. In the 1000 Genome data, R21X mostly occurs in Europeans and South Asians, is absent in Africans, and shows varying frequencies in South American populations (0 to 0.022). Of note, the best proxy SNP was another LPA null mutation (rs41272114, D′ = 0.958, R2 = 0.281). D′ was very high in all 1000G populations (0.986–0.996), although rs41272114 frequency varies considerably (0–0.182). Co-localization of both null mutations on the same allele was confirmed by PFGE-based long-range haplotyping. Conclusions We performed the largest epidemiological study on an LPA KIV-2 variant so far, showing that it is possible to assess LPA KIV-2 mutations on a large scale. Surprisingly, in all analyzed populations, R21X was located on the same haplotype as the splice mutation rs41272114, creating “double-null” LPA alleles. Despite being a nonsense variant, the R21X status does not provide additional information beyond the rs41272114 genotype. This has important implications for studies using LPA loss-of-function mutations as genetic instruments and emphasizes the complexity of LPA genetics

Genetic characterization of the calcium-sensing receptor (CASR) in colorectal cancer cells and stable transfection of a colorectal cancer cell line with CASR

Der Calcium-sensing receptor (CaSR) ist ein G-Protein gekoppelter Rezeptor und ist primär in der Nebenschilddrüse exprimiert. Die Hauptaufgabe des Rezeptors ist die Regulierung des Calciumhaushalts. Weiters ist dieser jedoch auch in unterschiedliche physiologische als auch pathophysiologische Bedingungen wie Hormonsekretion, Proliferation und Differenzierung involviert. Ziel dieser Masterarbeit ist es das CASR Gen in Zelllinien des Colorectalcarcinoms genetisch zu charakterisieren und eine stabil transfizierte Zelllinie mit einer CaSR Überexpression zu erstellen. Die Hypothese hierfür ist, dass der CaSR ein Tumorsuppressor ist und Überexpression des CaSR in transfizierten Zellen zu verminderter Proliferation und erhöhter Differenzierung als auch Apoptose führt. Mittels DNA Sequenzierung wurden sieben colorectale Zelllinien auf Mutationen im CASR Gen überprüft und detektierte Varianten anhand der Datenbank Alamut® Visual analysiert, um Informationen über die möglichen Auswirkungen der Varianten auf die Aktivität des CaSR zu erhalten. Die Transfektion wurde mit zwei unterschiedlichen Plasmiden (cDNA3.1/kan(+)-FLAG-CaSR-EGFP und cDNA3.1/kan(+)-FLAG-EGFP) an den Zelllinien HT-29 und SW-480 mittels LipofectamineTM LTX 3000 durchgeführt. Zur weiterführenden Untersuchung der transfizierten Zellen wurde der MAPK Phosphorylation Antibody Array durchgeführt. Zehn Genvarianten wurden detektiert, davon sieben Polymoprhismen und drei Mutationen. Drei dieser Polymorphismen führen zu Missense-Substitutionen im codierenden Bereich und vier Polymorphismen liegen im nicht-codierenden Bereich des Gens. Mutationen wurden nur in der Zelllinie DLD-1 gefunden. Dabei geht die Anzahl der Mutationen mit der Passagennummer einher. Die stabile als auch die transiente Transfektion der Zelllinie HT-29 waren nicht erfolgreich. So wurde alternativ die Zelllinie SW-480 transient transfiziert Der MAPK Phsophorylation Antibody Array an transfizierten SW-480 Zellen zeigte eine geringe Auswirkung der Behandlung mit Kalzium Um die Bedeutung von Mutationen des CASR Gens in colorectalem Karzinom zu verstehen, sind noch weitere Untersuchungen notwendig. Als weiterführendes Experiment ist eine stabile Transfektion an der Zelllinie SW-480 inklusive in vitro und in vivo Experimenten geplant.The calcium sensing receptor (CaSR) is a G protein-coupled receptor and is mainly expressed in the parathyroid gland. Its central role is the regulation of calcium homeostasis but it also regulates other processes, as hormone secretion, proliferation, differentiation. In cancer it can act either as an oncogene or as a tumour suppressor depending on the tissue. The aim of this master thesis is to genetically characterise the CASR in CRC cell lines and to establish a stable transfected CRC cell line overexpressing CaSR. The hypothesis is that the CaSR is an oncogene and its overexpression leads to decreased proliferation and increased differentiation and apoptosis, by regulating phosphorylation of signalling molecules in the transfected cells. We analysed seven CRC cell lines by DNA sequencing using specific primers for the CASR. Detected CASR variants were analysed with the mutation software Alamut® Visual and compared to the literature to obtain information about the potential effect of the variant on the activity of the CaSR. The transfection was performed on HT-29 and SW-480 cells using LipofectamineTM LTX 3000 and two different plasmids: cDNA3.1/kan(+)-FLAG-CaSR-EGFP and cDNA3.1/kan(+)-FLAG-EGFP. As a functional assay the MAPK Phosphorylation Antibody Array was performed. Ten variants were detected: seven polymorphisms and three mutations. Three polymorphisms cause missense substitutions in the coding region and four polymorphisms were in the non-coding region of the gene. The variants leading to mutant proteins were found only in the DLD-1 cell line. Stable as well as transient transfection of HT-29 cells did not work. Alternatively SW-480 cells were transiently transfected. The MAPK Phosphorylation Antibody Array showed only a minor effect of the treatment with calcium. To understand the significance of CaSR mutants in colorectal tumorigenesis further investigations are needed. As a future experiment a stable transfection on SW-480 cells as well as in vitro and afterwards in vivo studies with those will be performed.vorgelegt von: Rebecca Maria GrüneisWien, FH Campus Wien, Masterarb., 2017(VLID)229341

Frequent LPA KIV-2 Variants Lower Lipoprotein(a) Concentrations and Protect Against Coronary Artery Disease

Background: Lipoprotein(a) (Lp(a)) concentrations are a major independent risk factor for coronary artery disease (CAD) and are mainly determined by variation in LPA. Up to 70% of the LPA coding sequence is located in the hypervariable kringle IV type 2 (KIV-2) region. It is hardly accessible by conventional technologies, but may contain functional variants. Objectives: This study sought to investigate the new, very frequent splicing variant KIV-2 4733G>A on Lp(a) and CAD. Methods: We genotyped 4733G>A in the GCKD (German Chronic Kidney Disease) study (n = 4,673) by allele-specific polymerase chain reaction, performed minigene assays, identified proxy single nucleotide polymorphisms and used them to characterize its effect on CAD by survival analysis in UK Biobank (n = 440,234). Frequencies in ethnic groups were assessed in the 1000 Genomes Project. Results: The 4733G>A variant (38.2% carrier frequency) was found in most isoform sizes. It reduces allelic expression without abolishing protein production, lowers Lp(a) by 13.6 mg/dL (95% CI: 12.5-14.7; P A and 4925G>A, another KIV-2 splicing mutation, reduces Lp(a) by 31.8 mg/dL and most importantly narrows the interquartile range by 9-fold (from 42.1 to 4.6 mg/dL) when compared to the wild type. In UK Biobank 4733G>A alone and compound heterozygosity with 4925G>A reduced HR for CAD by 9% (95% CI: 7%-11%) and 12% (95% CI: 7%-16%) (both P < 0.001). Frequencies in ethnicities differ notably. Conclusions: Functional variants in the previously inaccessible LPA KIV-2 region cooperate in determining Lp(a) variance and CAD risk. Even a moderate but lifelong genetic Lp(a) reduction translates to a noticeable CAD risk reduction