Scale representation of the segregating sites of human <i>CYP21</i> genes.

<p>Boxes symbolize the exons, red indicates the coding region, pink shows the untranslated regions. Segregating sites are denoted by their position, numbered from the start of the <i>CYP21A2</i> coding region in the sequence NT_007592.15: 31945792-31949720. The segregating site of the <i>CYP21A2</i> gene can be seen above the depicted gene. The segregating site of the <i>CYP21A1P</i> gene derived from an external dataset can be found below the depicted gene.</p

Scaled representation of the organization of human RCCX copy number variation (CNV) depicted by mono-, bi- and trimodular RCCX structure variants.

<p>The schematic abbreviations of RCCX structures are indicated on the left side; a module (a repeat) is abbreviated with two letters, the first represents the alleles of HERV-K CNV (L – the long allele or S – short allele [The abbreviation of these alleles comes from the traditional usage of long and short <i>C4</i> genes.]), and the second symbolizes the types of <i>C4</i> gene (A or B). The duplication of these two letters indicates bimodular RCCX structure, while the triplicate of the two letters means trimodular RCCX structures. Dotted lines indicate the module boundaries, and the directions of the ends of chromosome 6 are indicated by arrows under the scale bar. The variable region of bimodular RCCX contains two pairs of full-length genes, complement component 4 (<i>C4A</i> and <i>C4B</i>), steroid 21-hydroxylase (<i>CYP21A1P</i> and <i>CYP21A2</i>), and two pairs of a functional gene and a truncated pseudogene, serine/threonine kinase 19 (<i>STK19</i> and <i>STK19P</i>) and tenascin-X (<i>TNXA</i> and <i>TNXB</i>). The illustrated region spans from the telomeric end of exon 4 of <i>STK19</i> to the centromeric end of exon 28 of <i>TNXB</i>.</p

Spatial distributions of phylogenetic signals derived from the different orthologous and paralogous pairs of the human and chimpanzee full-length <i>CYP21</i> sequences.

<p>Blue line indicates the phlyogenetic signal of human paralogues (hA2-hA1P), red dashed line indicates <i>CYP21A2</i> orthologues (hA2-cA2), orange line indicates chimpanzee paralogues (cA2-cA1P) and green dashed line indicates <i>CYP21A1P</i> orthologues (hA1P-cA1P). The likelihood of closely related sequences to resemble each other more than random sequences of the same phylogenetic tree is expressed by ‘% of permutated trees’ in y axis. Schematic <i>CYP21</i> genes are indicated below the plot, high white boxes symbolize the exons, low white boxes represent the untranslated regions, and black lines indicate the introns and flanking regions.</p

Rooted maximum likelihood phylogenetic trees constructed from selected full-length sequences, intron 2 sequences and <i>CYP21</i> gene sequences without intron 2 of the great ape <i>CYP21</i> sequence dataset.

<p>The names of <i>CYP21</i> sequences of HLA-homozygous cell lines available from public databases are represented at the human sequences. Bootstrap values are shown next to the corresponding nodes, but the values within human clades are not presented for clarity. The scale bar indicates genetic distance. (A) Rooted ML tree of great ape <i>CYP21</i> full-length gene. (B) Rooted ML tree of great ape <i>CYP21</i> intron 2. (C) Rooted ML tree of great ape <i>CYP21</i> genes without intron 2.</p

Data_Sheet_1_Concentration and Subclass Distribution of Anti-ADAMTS13 IgG Autoantibodies in Different Stages of Acquired Idiopathic Thrombotic Thrombocytopenic Purpura.PDF

Background<p>The acquired form of idiopathic thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease, in which the underlying deficiency of the ADAMTS13 protease is caused by autoantibodies, predominantly of the IgG isotype. Certain HLA-DR-DQ haplotypes were associated with the risk of developing TTP.</p>Objectives<p>To investigate the development of the ADAMTS13-specific antibody response during the course of the disease, we analyzed the concentration, subclass distribution, and inhibitory potential of anti-ADAMTS13 IgG autoantibodies in samples of TTP patients drawn during the first acute phase, in remission, and during relapse. Additionally, we compared the anti-ADAMTS13 IgG levels between patients carrying and not carrying risk and protective HLA-DR-DQ haplotypes.</p>Patients and Methods<p>We determined the anti-ADAMTS13 IgG concentration and subclass distribution in 101 antibody-positive samples of 81 acquired TTP patients by ELISA methods. The presence and semi-quantitative amount of anti-ADAMTS13 inhibitors were determined in 97 of 100 deficient samples, and the specific inhibitory potential of anti-ADAMTS13 autoantibodies was determined in 49 selected samples, by mixing ADAMTS13-activity assays. HLA-DR-DQ typing and haplotype prediction were performed in 70 of the above patients.</p>Results<p>We found that IgG1 and IgG4 were the predominant subclasses, present in almost all samples. While IgG1 was the dominant subclass in almost half of the samples taken during the first acute episode, IgG4 was dominant in all samples taken during or following a relapse. The inhibitory potential of the samples correlated with levels of the IgG4 subclass. Anti-ADAMTS13 antibodies of IgG4-dominant samples had higher specific inhibitory potentials than IgG1-dominant samples, independently of disease stage. Interestingly, we found that patients carrying the protective DR7-DQ2 and DR13-DQ6 haplotypes had higher anti-ADAMTS13 IgG levels.</p>Conclusion<p>Our results indicate that IgG4 becomes the dominant subtype at some point of the disease course, apparently before the first relapse, parallel to the increase in inhibitory potential of the anti-ADAMTS13 autoantibodies. Furthermore, we found an association between the genetic background and the antibody response in TTP.</p

Characteristics of Hungarian subjects with non-functioning adrenal incidentaloma before genetic exclusions.

<p>BMI – body mass index, ACTH – adrenocorticotrophic hormone, DHEA-S – dehydroepiandrosterone sulfate, LDDST – low dose dexamethasone test, DM 2– type 2 diabetes mellitus, CAH – congenital adrenal hyperplasia and <i>CYP21A2</i>– human steroid 21-hydroxlylase gene. Values are medians except for percentage values, and interquartile ranges are in parentheses.</p>a<p>There was no significant difference in prevalence (Fisher’s exact test: p = 1.0000) compared to healthy Hungarian control subjects.</p>b<p>There was no significant difference in prevalence (Fisher’s exact test: p = 0.5516) compared to healthy Hungarian control subjects.</p><p>Characteristics of Hungarian subjects with non-functioning adrenal incidentaloma before genetic exclusions.</p

Relationship between hormone levels in blood and <i>CYP21A2</i> haplotype carrier groups in subjects with non-functioning adrenal incidentaloma.

<p>c5/o and c8/0 indicate the carriers (heterozygotes) of <i>CYP21A2</i> haplotypes of cluster ih5 and cluster ih8, respectively, o/o abbreviates the genotypes of other haplotypes, and N indicates the number of subjects. K-S – Kolmogorov-Smirnov test, ANOVA – analysis of variance, K-W – Kruskal-Wallis test, M-W – Mann-Whitney test. The normalities of hormone datasets were checked by the K-S test; the datasets passing the normality test were investigated by ANOVA and t-test; the datasets not passing were examined by M-W and K-W tests. Median values are represented, interquartile ranges are shown in parentheses below the median values, ns means non-significant result, and significant values (p<0.05) with high power (power >0.8) are highlighted in bold characters. ACTH – adrenocorticotrophic hormone.</p>a<p>p values of the Newman-Keuls post-hoc test were 0.0263 between c5/o and o/o, 0.0411 between c8/o and o/o and 0.0002 between c5/o and c8/o.</p>b<p>Power was calculated by simulation.</p>c<p>p values of the Newman-Keuls post-hoc test were 0.0814 between c5/o and o/o, 0.7173 between c8/o and o/o and 0.0729 between c5/o and c8/o.</p>d<p>p values of the Newman-Keuls post-hoc test were 0.0958 between c5/o and o/o, 0.1610 between c8/o and o/o and 0.8720 between c5/o and c8/o.</p><p>Relationship between hormone levels in blood and <i>CYP21A2</i> haplotype carrier groups in subjects with non-functioning adrenal incidentaloma.</p

Haplotype tree of <i>CYP21A2</i> intron 2 and the 5′-part of exon 3, and the <i>CYP21A2</i> haplotype clusters with multiple significances found by tree scanning, and p-values of the haplotype clusters related to different steroid hormone levels in subjects with non-functioning adrenal incidentaloma.

<p>Circles represent the <i>CYP21A2</i> intron 2 and the 5′-part of exon 3 haplotypes, lines with or without small black circles indicate the allele differences (character-state changes) between haplotype variants and gray shapes encompass the haplotype clusters with multiple significances. The values without parentheses are uncorrected p-values, and the values with parentheses are the corrected step-down permutational p-value after enforcing monotonicity. ACTH – adrenocorticotrophic hormone.</p

Concentrations of hormones in blood decomposed by <i>CYP21A2</i> intron 2 haplotype carrier groups in subjects with non-functioning adrenal incidentaloma.

<p>c5/o and c8/o indicate the carriers (heterozygotes) of c5 and c8 haplotypes, respectively, and o/o abbreviates the genotypes of other haplotypes. Boxes indicate interquartile ranges, lines in boxes show the medians, whiskers represent the 5^th–95^th percentiles, asterisks above the boxes indicate the significant differences between the hormone levels of genotypes. One, two and three asterisks indicate p<0.05, p<0.01 and p<0.001 respectively (t-test or Mann-Whitney test). A) Serum cortisol level after adrenocorticotropic hormone (ACTH) stimulation. B) Serum aldosterone level after ACTH stimulation. C) Serum 17-OH-progesterone level after ACTH stimulation. D) Serum corticosterone level after ACTH stimulation. E) Serum 11-deoxycortisol level after metyrapone administration. F) Baseline ACTH level.</p

DataSheet_1_Complement C7 and clusterin form a complex in circulation.docx

IntroductionThe complement system is part of innate immunity and is comprised of an intricate network of proteins that are vital for host defense and host homeostasis. A distinct mechanism by which complement defends against invading pathogens is through the membrane attack complex (MAC), a lytic structure that forms on target surfaces. The MAC is made up of several complement components, and one indispensable component of the MAC is C7. The role of C7 in MAC assembly is well documented, however, inherent characteristics of C7 are yet to be investigated.MethodsTo shed light on the molecular characteristics of C7, we examined the properties of serum-purified C7 acquired using polyclonal and novel monoclonal antibodies. The properties of serum‑purified C7 were investigated through a series of proteolytic analyses, encompassing Western blot and mass spectrometry. The nature of C7 protein-protein interactions were further examined by a novel enzyme-linked immunosorbent assay (ELISA), as well as size‑exclusion chromatography. ResultsProtein analyses showcased an association between C7 and clusterin, an inhibitory complement regulator. The distinct association between C7 and clusterin was also demonstrated in serum-purified clusterin. Further assessment revealed that a complex between C7 and clusterin (C7-CLU) was detected. The C7-CLU complex was also identified in healthy serum and plasma donors, highlighting the presence of the complex in circulation. DiscussionClusterin is known to dissociate the MAC structure by binding to polymerized C9, nevertheless, here we show clusterin binding to the native form of a terminal complement protein in vivo. The presented data reveal that C7 exhibits characteristics beyond that of MAC assembly, instigating further investigation of the effector role that the C7-CLU complex plays in the complement cascade. </p