Lack of association of rs3798220 with small apolipoprotein(a) isoforms and high lipoprotein(a) levels in East and Southeast Asians

OBJECTIVE : The variant allele of rs3798220 in the apolipoprotein(a) gene (LPA) is used to assess the risk for coronary artery disease (CAD) in Europeans, where it is associated with short alleles of the Kringle IV-2 (KIV-2) copy number variation (CNV) and high lipoprotein(a) (Lp(a)) concentrations. No association of rs3798220 with CAD was detected in a GWAS of East Asians. Our study investigated the association of rs3798220 with Lp(a) concentrations and KIV-2 CNV size in non-European populations to explain the missing association of the variant with CAD in Asians. METHODS : We screened three populations from Africa and seven from Asia by TaqMan Assay for rs3798220 and determined KIV-2 CNV sizes of LPA alleles by pulsed-field gel electrophoresis (PFGE). Additionally, CAD cases from India were analysed. To investigate the phylogenetic origin of rs3798220, 40 LPA alleles from Chinese individuals were separated by PFGE and haplotyped for further SNPs. RESULTS : The variant was not found in Africans. Allele frequencies in East and Southeast Asians ranged from 2.9% to 11.6%, and were very low (0.15%) in CAD cases and controls from India. The variant was neither associated with short KIV-2 CNV alleles nor elevated Lp(a) concentrations in Asians. CONCLUSION : Our study shows that rs3798220 is no marker for short KIV-2 CNV alleles and high Lp(a) in East and Southeast Asians, although the haplotype background is shared with Europeans. It appears unlikely that this SNP confers atherogenic potential on its own. Furthermore, this SNP does not explain Lp(a) attributed risk for CAD in Asian Indians.http://www.elsevier.com/locate/atherosclerosis2016-10-31hb2016Chemical Patholog

Comprehensive characterization of the prostate tumor microenvironment identifies CXCR4/CXCL12 crosstalk as a novel antiangiogenic therapeutic target in prostate cancer

Background: Crosstalk between neoplastic and stromal cells fosters prostate cancer (PCa) progression and dissemination. Insight in cell-to-cell communication networks provides new therapeutic avenues to mold processes that contribute to PCa tumor microenvironment (TME) alterations. Here we performed a detailed characterization of PCa tumor endothelial cells (TEC) to delineate intercellular crosstalk between TEC and the PCa TME. Methods: TEC isolated from 67 fresh radical prostatectomy (RP) specimens underwent multi-omic ex vivo characterization as well as orthogonal validation of both TEC functions and key markers by immunohistochemistry (IHC) and immunofluorescence (IF). To identify cell-cell interaction targets in TEC, we performed single-cell RNA sequencing (scRNA-seq) in four PCa patients who underwent a RP to catalogue cellular TME composition. Targets were cross-validated using IHC, publicly available datasets, cell culture expriments as well as a PCa xenograft mouse model. Results: Compared to adjacent normal endothelial cells (NEC) bulk RNA-seq analysis revealed upregulation of genes associated with tumor vasculature, collagen modification and extracellular matrix remodeling in TEC. PTGIR, PLAC9, CXCL12 and VDR were identified as TEC markers and confirmed by IF and IHC in an independent patient cohort. By scRNA-seq we identified 27 cell (sub)types, including endothelial cells (EC) with arterial, venous and immature signatures, as well as angiogenic tip EC. A focused molecular analysis revealed that arterial TEC displayed highest CXCL12 mRNA expression levels when compared to all other TME cell (sub)populations and showed a negative prognostic role. Receptor-ligand interaction analysis predicted interactions between arterial TEC derived CXCL12 and its cognate receptor CXCR4 on angiogenic tip EC. CXCL12 was in vitro and in vivo validated as actionable TEC target by highlighting the vessel number- and density- reducing activity of the CXCR4-inhibitor AMD3100 in murine PCa as well as by inhibition of TEC proliferation and migration in vitro. Conclusions: Overall, our comprehensive analysis identified novel PCa TEC targets and highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis in PCa

Sequence variation in the human LPA gene

LPA is the major locus controlling Lp(a) plasma levels which vary extensively within and across populations and are associated with coronary heart disease. Identified causal genetic polymorphisms, including the KIV-2 CNV and other sequence variants, explain only a fraction of the variation in Lp(a) levels attributable to the LPA locus. Therefore additional sequence variation in LPA is expected to explain more of the observed variation in Lp(a) levels. One region of LPA not yet fully screened for such sequence variants is the KIV-2 CNV. Some variants in this region have been reported by the large re-sequencing projects but for those variants any data on the Lp(a) levels and the KIV-2 CNV size are missing. In one projects of this thesis, coding regions of the KIV 2 CNV were screened in individuals from six different populations (Gabonese Bantu, South African Bantu, Khoi San, Egyptians, Hong Kong Chinese and Austrians). LPA alleles of each individual were separated by PFGE and used for batchwise amplification of PCR fragments specific for the two exons of KIV-2. Despite a restricted sample size (in total 90 alleles) and limited sensitivity of the screening method used, several synonymous and non-synonymous variants as well as two previously unreported splice site variants were identified. Most of these variants were detected in African alleles. Among the variants found, an African specific acceptor splice site variant (K422 6T>G) associated with small KIV-2 CNV size was present in all African populations at high population frequencies (10% to 40%). This variant appears to be associated with low Lp(a) levels than expected considering the KIV-2 CNV size of the alleles carrying it. In contrast to the frequent African specific acceptor splice site variant, a rare donor splice site variant (K421 +1G>A) was found which was shared among Africans and Europeans and associated with non-detectable Lp(a). In Asians, only the synonymous variants that define the KIV 2 type B and type C were found at very high frequencies (70%) and observed with a broad range of different KIV 2 CNV sizes. These variants were rare in Europeans and Africans. A strong bottleneck suggested to have occurred during the migration of modern humans to East Asia might explain the enrichment of these variants in Asians. A key observation was that the variants which were frequent were shared among the KIV-2 copies of the alleles carrying them. The acceptor splice site variant as well as type B/C defining variants were observed at high intra allelic frequencies (relative proportion of the variant vs wild type carrying KIV-2 copies), i.e. they are shared among KIV-2 copies of an allele. Similar intra allelic frequencies were observed for the same variants carried on different sized KIV-2 allele. Given the association between elevated Lp(a) and risk for coronary heart disease (CHD), an accurate and cost-effective screening of individuals at high risk for CHD has become a target of research. Assessment of apo(a) isoform size, which is an important factor for risk evaluation, is laborious and costly. rs3798220 which is a non-synonymous SNP in the protease like domain of LPA has been shown to tag high risk LPA alleles in Europeans. This led to the proposition of its use as a cost-efficient risk marker. Whether it could serve the purpose to identify high risk LPA alleles in other populations appears doubtful given a negative result from a recent GWAS in Japanese. In a second project, the association of rs3798220 with Lp(a) levels and KIV-2 CNV size was investigated in different non-European populations from Africa (Khoi San, Gabonese Bantu and Egyptians), South Asia (North Indians and South Indians), and Southeast Asia (Hong Kong Chinese, Japanese, Indonesians, Thai and Trobriand Islanders), and a possible contribution to the Lp(a) attributed risk in CAD patients from North and South India was assessed. The variant was found to be very rare in Asian Indians, and could not explain the Lp(a) attributed risk for CAD in that population. In accordance with 1000G data, the variant was absent in autochthonous Africans but it was frequent in East and Southeast Asians with MAFs ranging from 2.9% to 11.6%. However, no association with either higher Lp(a) or short apo(a) isoforms/ KIV-2 CNV size was found. This is in contrast to the findings in Europeans where it was observed with short apo(a) isoforms and with high Lp(a). This finding suggests that rs379220 itself is not causal and its association with high Lp(a) could most likely be explained by the LD with short isoform size which come with very high Lp(a) levels in Europeans. The background haplotype of the variant carrying alleles was shared among Europeans and Asians. The findings reported in this dissertation suggest that the LPA locus harbors more sequence variation in the KIV-2 region, which might explain an additional fraction of variation in Lp(a) levels across ethnicities. Population specific differences exist in the association of SNPs with the KIV-2 CNV size, and the associated Lp(a) levels could be influenced by the differences in the LD patterns across populations. The SNP markers associated with Lp(a) levels or predicting the risk for CHD in one population may not be applicable to other populations. On the other hand, population specific SNPs in LD with a narrow range of KIV-2 CNV sizes can be causally associated with Lp(a) levels. One example could be the frequent African specific acceptor splice variant site, detected in the screening of the KIV-2 region, which is associated with short KIV-2 alleles and comparatively low Lp(a). This variant has potential clinical relevance for investigating the association between CHD risk and apo(a) isoform size and might provide some explanation for the missing association of the short apo(a) size with CHD in individuals of African descent. The nature of LD between SNPs and KIV-2 CNV size across populations can depend on the mode and the rate of size changing mutations at the CNV. The rs3798220 variant allele is observed with vastly different KIV-2 CNV sizes between Europeans and non-Europeans. The shared haplotype background of the variant carrying alleles in the two continental groups argues against it originating from a recurrent point mutation, but rather indicates that the CNV size has changed since the split of the populations. It remains to be determined whether CNV size changes can also explain the observation that the same variants are found on multiple KIV-2 copies on the same allele. Here, inter-locus gene conversion might also play an essential role. The same applies for the observation that similar intra allelic frequencies of specific KIV-2 variants are kept even between alleles of different KIV-2 CNV sizes. More extensive SNP haplotype data within the KIV-2 CNV may provide further insights into the mechanism of KIV-2 CNV size changes and their role in the spreading of variants across the KIV-2 copies.LPA is the major locus controlling Lp(a) plasma levels which vary extensively within and across populations and are associated with coronary heart disease. Identified causal genetic polymorphisms, including the KIV-2 CNV and other sequence variants, explain only a fraction of the variation in Lp(a) levels attributable to the LPA locus. Therefore additional sequence variation in LPA is expected to explain more of the observed variation in Lp(a) levels. One region of LPA not yet fully screened for such sequence variants is the KIV-2 CNV. Some variants in this region have been reported by the large re-sequencing projects but for those variants any data on the Lp(a) levels and the KIV-2 CNV size are missing. In one projects of this thesis, coding regions of the KIV 2 CNV were screened in individuals from six different populations (Gabonese Bantu, South African Bantu, Khoi San, Egyptians, Hong Kong Chinese and Austrians). LPA alleles of each individual were separated by PFGE and used for batchwise amplification of PCR fragments specific for the two exons of KIV-2. Despite a restricted sample size (in total 90 alleles) and limited sensitivity of the screening method used, several synonymous and non-synonymous variants as well as two previously unreported splice site variants were identified. Most of these variants were detected in African alleles. Among the variants found, an African specific acceptor splice site variant (K422 6T>G) associated with small KIV-2 CNV size was present in all African populations at high population frequencies (10% to 40%). This variant appears to be associated with low Lp(a) levels than expected considering the KIV-2 CNV size of the alleles carrying it. In contrast to the frequent African specific acceptor splice site variant, a rare donor splice site variant (K421 +1G>A) was foundArbeit an der Bibliothek noch nicht eingelangt - Daten nicht geprüftAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersInnsbruck, Med. Univ., Diss., 2015(VLID)81396

Examples for the possible distribution of sequence variations in KIV-2 CNV alleles.

<p>Sequence variants affecting KIV-2 copies (shown as filled circles) can be present in single or several KIV-2 copies within alleles of different sizes. (A) Low and high intra-allelic frequencies of a variant on short (e.g. 5 KIV-2) alleles. (B) Low and high intra-allelic frequencies of a variant for longer (limited to 10 KIV-2 for graphic display) alleles. (C) The same number of KIV-2 copies harbors the variant on a short and a longer allele, i.e. the intra-allelic frequency of the variant is higher on the short allele. Thus detection of the variant would be more likely if present on the short allele. (D) Both alleles have the same intra-allelic frequency (20%), though the number of copies carrying the variant is different. Hence the probability of detection would be the same. (E) The order of variant carrying KIV-2 copies within the allele might vary. These different scenarios cannot be distinguished by present methods. (F) Different variants can be allocated in <i>cis</i>, shown for a genotype with one short and one longer allele or (G) in <i>trans</i>. While scenarios F and G cannot be distinguished in analyses based on diploid samples, this is possible in our analysis based on separated alleles.</p

Distribution of sequence variation within KIV-2 across KIV CNV alleles and populations.

<p>The frequency distribution of the KIV CNV alleles is given for all samples, and by population. Alleles carrying variants within the screened KIV-2 exon 1 and KIV-2 exon 2 domains are colored according to the variation found in the batchwise screening. Alleles harboring several variants are striped. Note that for the individual alleles, the number of KIV-2 copies carrying the depicted variants cannot be derived from this figure. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121582#pone.0121582.s011" target="_blank">S6 Table</a> for these intra-allelic frequencies. Inserts show the mean KIV CNV sizes (i.e. number of KIV-2 repeats plus the nine non-repetitive KIVs), and the mean allele-associated Lp(a) concentrations in mg/dl for the populations.</p

<i>LPA</i> gene structure.

<p>The <i>LPA</i> gene contains 27 non-repetitive exons including the 5’UTR (grey), one copy each of kringle (K) domains KIV-1, KIV-3 to KIV-10 (black), KV (green), all comprising two exons, followed by six exons encoding the protease-like domain (purple) and the 3’UTR (grey). The KIV-2 domain (red) varies in number from 1 to >40 copies, thereby forming the KIV-2 CNV. Each KIV-2 repeat is approximately 5.5 kb long, composed of the first exon (160 bp), a long intron (4 kb), the second exon (182 bp), and a short intron (1.2 kb). Depicted is the general exon/intron structure of the <i>LPA</i> gene, with one KIV-2 repeat, and an example containing six KIV-2 repeats and, hence, 15 KIV repeats (as is the case for <i>LPA</i> in the human reference sequence).</p

Sequence Variation within the KIV-2 Copy Number Polymorphism of the Human <i>LPA</i> Gene in African, Asian, and European Populations

<div><p>Amazingly little sequence variation is reported for the kringle IV 2 copy number variation (KIV 2 CNV) in the human <i>LPA</i> gene. Apart from whole genome sequencing projects, this region has only been analyzed in some detail in samples of European populations. We have performed a systematic resequencing study of the exonic and flanking intron regions within the KIV 2 CNV in 90 alleles from Asian, European, and four different African populations. Alleles have been separated according to their CNV length by pulsed field gel electrophoresis prior to unbiased specific PCR amplification of the target regions. These amplicons covered all KIV 2 copies of an individual allele simultaneously. In addition, cloned amplicons from genomic DNA of an African individual were sequenced. Our data suggest that sequence variation in this genomic region may be higher than previously appreciated. Detection probability of variants appeared to depend on the KIV 2 copy number of the analyzed DNA and on the proportion of copies carrying the variant. Asians had a high frequency of so-called KIV 2 type B and type C (together 70% of alleles), which differ by three or two synonymous substitutions respectively from the reference type A. This is most likely explained by the strong bottleneck suggested to have occurred when modern humans migrated to East Asia. A higher frequency of variable sites was detected in the Africans. In particular, two previously unreported splice site variants were found. One was associated with non-detectable Lp(a). The other was observed at high population frequencies (10% to 40%). Like the KIV 2 type B and C variants, this latter variant was also found in a high proportion of KIV 2 repeats in the affected alleles and in alleles differing in copy numbers. Our findings may have implications for the interpretation of SNP analyses in other repetitive loci of the human genome.</p></div

Positions of the PCR primers and regions amplified for cloning and batchwise screening.

<p>Below a stretch of KIV-2 domains showing the positions of the exons and the short and long introns in the KIV-2 CNV, the regions amplified by the different PCRs are depicted by horizontal lines with the names of the PCRs written above and the primers below. PCRs “421” and “422” are spanning the first and second exon of the KIV-2 respectively, and also contain the flanking intron sequences. These two PCRs were used in the batchwise screening and cloning. PCR “412to422” amplifies both exons and the long intron, while PCR “422to421” includes both exons and the short intron. The locations of the different primers used for sequencing are shown above the depicted stretch of KIV-2 CNV. The figure is drawn to scale (2.5 cm = 1 kb).</p

S1 File -

This study aimed to synthesize fluoride-doped bioactive glass (F-BG) based thermo-sensitive injectable hydrogel for endodontic applications. The structural and phase analyses were done with Fourier Transform Infrared spectroscopy and X-ray Diffraction, respectively. The setting time of prepared injectable was investigated at 21°C (in the presence and absence of an ultrasonic scalar) and at 37°C. Flowability was tested according to ISO-6876:2012 specifications, whereas injectability was checked by extrusion method using 21-, 22-, and 23-gauge needles. The in vitro bio-adhesion and push-out bond strength were studied on days 7 and 90 and compared with the commercially available TotalFill®. The ion release profile was analyzed for up to 30 days with Inductively Coupled Plasma Optical Emission Spectroscopy. The fluoride release analysis was conducted periodically for up to 21 days in deionized water and artificial saliva using an ion-selective electrode. The final setting time at 21°C, 21°C+ultrasonic scalar, and 37°C were 38.66±3.21, 29.12±1.23, and 32±3.46 min, respectively. The flowability was 25±3.94 mm, and the injectability coefficient was ≥70.3 for 22, 21, and 57% in a 23-gauge needle. Fluoride release in deionized water was found to be significantly higher than in artificial saliva and increased with time. A significant difference in bond strength was found between days 7 and 90, where the strength was increased, and a new apatite layer was formed on the tooth surface. A rapid release of calcium, phosphate, and silicon ions was seen initially, whereby the continuous release of these ions was observed for up to 30 days. The prepared F-BG injectable hydrogel has shown promising results and has the potential to be used as an endodontic sealer.</div