Impact of the 14 bp InDel on the luciferase reporter gene expression when both construction types (1Fter-4R and 1Fter-2R primers) were considered in each cell line independently or in the four cell lines in combination.

<p>Impact of the 14 bp InDel on the luciferase reporter gene expression when both construction types (1Fter-4R and 1Fter-2R primers) were considered in each cell line independently or in the four cell lines in combination.</p

Significant differences in the modulation of the expression of the luciferase reporter gene considering either the cell type or the status of HLA-G expression, with 1Fter-2R and 1Fter-4R constructions and haplotypes combined.

<p>Points indicate the mean and vertical bars indicate the 95% confidence interval. The number of experiments considering each cell line or the status of HLA-G expression is indicated between parentheses. MW: Mann-whitney test; KW: Kruskal-Wallis test. (A) Modulation of luciferase gene expression in each cell line separately. The decrease in the luciferase gene activity in U251MG (HLA-G-) and FON cells (HLA-G+) was not statistically different. (B) Modulation of luciferase gene expression in the HLA-G- (M8 + U251MG) cells <i>vs</i> HLA-G + (FON + JEG-3) cells combined.</p

Site-directed mutagenesis replacing a T with a C at position +3035 in UTR-5 and UTR-7 (1Fter-2R constructions) does not significantly influence the levels of luciferase responses.

<p>The two-tailed Mann-Whitney test was used to compare the impact of wild type UTR-5 and UTR-7 to the impact of mutated UTR-5 and UTR-7 respectively in JEG-3 and U251MG cells. Data is presented as mean <b>+/-</b> SEM.</p

Modulation of the expression of the reporter gene luciferase by 3’UTR haplotypes in four cell lines expressing HLA-G or not.

<p>Modulation of the expression of the reporter gene luciferase by 3’UTR haplotypes in four cell lines expressing HLA-G or not.</p

Modulation of luciferase reporter gene expression in FON+, JEG-3, M8 and U251MG cells considering the constructions 1Fter-2R <i>vs</i> 1Fter-4R, is not statistically different, regardless of the haplotypes.

<p>Points indicate the means and vertical bars indicate the 95% confidence interval. The number of experiments considering each 1Fter construction is indicated between parentheses. MW: Mann-whitney test; KW: Kruskal-Wallis test. (A) Modulation of luciferase gene expression in the four cell lines combined. (B) Modulation of luciferase gene expression in each cell line independently. P values for Dunn’s multiple comparison test are indicated above and under the horizontal bar for the 1Fter-2R and 1Fter-4R constructions respectively (** p value < 0.01; *** p value < 0.001).</p

Significant differences in the modulation of the expression of the luciferase reporter gene in FON+, JEG-3, M8 and U251MG cells considering each 3’UTR haplotype independently, with the 1Fter-2R and 1Fter-4R constructions combined.

<p>Points indicate the means and vertical bars indicate the 95% confidence interval. The number of experiments considering each 3’UTR haplotype is indicated between parentheses. KW: Kruskal-Wallis test; * p value <0.05; ** p value < 0.01; *** p value < 0.001. (A) Modulation of luciferase expression in the four cell lines combined. (B) Modulation of luciferase expression in the HLA-G+ (FON + JEG-3) cell lines combined. (C) Modulation of luciferase expression in the HLA-G- (M8 + U251MG) cell lines combined. In (A) and (B) luciferase expression is significantly more intensely downregulated by UTR-5 and UTR-7 than by other haplotypes.</p

<i>HLA-G</i> 3’UTR polymorphisms.

<p>(A) Variations in <i>HLA-G</i> mRNA (red) along the 3’UTR. The less frequent variants are positioned below the most frequent one in the nucleotide sequence. Polymorphic sites are framed in bold <u>when the frequency of the</u> minor allele worldwide is higher than 1%. Purple single arrows point to the three predicted polyadenylation signals. Vertical lines with horizontal arrows indicate the 5’ end of the <i>HLA-G</i> nucleotide sequences for 1Fter, 2R and 4R primers used <u>in</u> pMIR-REPORT^™ constructions. (B) Sequence comparison of the most frequent 3’UTR haplotypes (21 worldwide populations; 2n = 3870) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169032#pone.0169032.ref025" target="_blank">25</a>] analyzed in the present study. Frequencies are indicated on the right part. UTR-1 and UTR-2 differ at 5 positions (pink rectangles). UTR-6 and UTR-18 (analyzed in the present work) only differ at one position and depending on the UTR typing strategy that was used (i.e., targeting or not +3227 SNP), UTR-18 could be confused with UTR-6 in several studies.</p

Demographic, clinical and laboratory findings of neuromyelitis optica (NMO) and multiple sclerosis (MS) Brazilian patients.

<p>Demographic, clinical and laboratory findings of neuromyelitis optica (NMO) and multiple sclerosis (MS) Brazilian patients.</p

Information of ancestry informative markers (AIMs) set for ancestral populations, multiple sclerosis (MS) and neuromyelitis optica (NMO) patients.

<p>(A) The panel of 12 ancestry informative markers (AIMs) for Africans (green), Europeans (red) and Amerindians (blue) were sufficient for an adequate discrimination among ancestral populations. (B) Principal components analysis (PCA) for NMO [Southeastern: Ribeirão Preto (NMO-RP), São Paulo (NMO-SP) and Belo Horizonte (NMO-BH); Central:-Goiânia (NMO-GO), and Northeastern: (Recife-Pernambuco (NMO-PE)] and MS patients from Ribeirão Preto (MS-RP) and control individuals from Ribeirão Preto (CTRL-RP) together with ancestral populations [Africans (green), Europeans (red) and Amerindians (blue)], showing that they clustered closer to Europeans than to Africans and Amerindians.</p

African ancestry indexes (AAI) distribution for ancestral populations, multiple sclerosis (MS) and neuromyelitis optica (NMO) patients.

<p>Distribution of African ancestry Index (AAI) for Africans (AFR-green), Amerindians (AMZ-blue) and Europeans (EUR-red) in NMO patients from several Brazilian regions [Southeastern: Ribeirão Preto (NMO-RP), São Paulo (NMO-SP) and Belo Horizonte (NMO-BH); Central:-Goiânia (NMO-GO), and Northeastern: (Recife-Pernambuco (NMO-PE)]; in MS patients from Ribeirão Preto (MS-RP); and in healthy controls from Ribeirão Preto (CTRL-RP).</p