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

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

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

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

table_1_Secreted Phospholipases A2 in Hereditary Angioedema With C1-Inhibitor Deficiency.PDF

Background<p>Hereditary angioedema (HAE) caused by deficiency (type I) or dysfunction (type II) of the C1 inhibitor protein (C1-INH-HAE) is a disabling, potentially fatal condition characterized by recurrent episodes of swelling. We have recently found that patients with C1-INH-HAE have increased plasma levels of vascular endothelial growth factors and angiopoietins (Angs), which have been associated with vascular permeability in several diseases. Among these and other factors, blood endothelial cells and vascular permeability can be modulated by extracellular or secreted phospholipases A₂ (sPLA₂s).</p>Objective<p>We sought to investigate the enzymatic activity and biological functions of sPLA₂ in patients with C1-INH-HAE.</p>Methods<p>sPLA₂s enzymatic activity was evaluated in the plasma from 109 adult patients with C1-INH-HAE and 68 healthy donors in symptom-free period and attacks. Plasma level of group IIA sPLA₂ (hGIIA) protein was measured in selected samples. The effect of C1-INH-HAE plasma on endothelial permeability was examined in vitro using a vascular permeability assay. The role of hGIIA was determined using highly specific sPLA₂ indole inhibitors. The effect of recombinant hGIIA on C1-INH activity was examined in vitro by functional assay.</p>Results<p>Plasma sPLA₂ activity and hGIIA levels are increased in symptom-free C1-INH-HAE patients compared with controls. sPLA₂ activity negatively correlates with C1-INH protein level and function. C1-INH-HAE plasma increases endothelial permeability in vitro, and this effect is partially reverted by a specific hGIIA enzymatic inhibitor. Finally, recombinant hGIIA inhibits C1-INH activity in vitro.</p>Conclusion<p>sPLA₂ enzymatic activity (likely attributable to hGIIA), which is increased in C1-INH-HAE patients, can promote vascular permeability and impairs C1-INH activity. Our results may pave the way for investigating the functions of sPLA₂s (in particular, hGIIA) in the pathophysiology of C1-INH-HAE and may inform the development of new therapeutic targets.</p

image_1_Secreted Phospholipases A2 in Hereditary Angioedema With C1-Inhibitor Deficiency.PDF

Background<p>Hereditary angioedema (HAE) caused by deficiency (type I) or dysfunction (type II) of the C1 inhibitor protein (C1-INH-HAE) is a disabling, potentially fatal condition characterized by recurrent episodes of swelling. We have recently found that patients with C1-INH-HAE have increased plasma levels of vascular endothelial growth factors and angiopoietins (Angs), which have been associated with vascular permeability in several diseases. Among these and other factors, blood endothelial cells and vascular permeability can be modulated by extracellular or secreted phospholipases A₂ (sPLA₂s).</p>Objective<p>We sought to investigate the enzymatic activity and biological functions of sPLA₂ in patients with C1-INH-HAE.</p>Methods<p>sPLA₂s enzymatic activity was evaluated in the plasma from 109 adult patients with C1-INH-HAE and 68 healthy donors in symptom-free period and attacks. Plasma level of group IIA sPLA₂ (hGIIA) protein was measured in selected samples. The effect of C1-INH-HAE plasma on endothelial permeability was examined in vitro using a vascular permeability assay. The role of hGIIA was determined using highly specific sPLA₂ indole inhibitors. The effect of recombinant hGIIA on C1-INH activity was examined in vitro by functional assay.</p>Results<p>Plasma sPLA₂ activity and hGIIA levels are increased in symptom-free C1-INH-HAE patients compared with controls. sPLA₂ activity negatively correlates with C1-INH protein level and function. C1-INH-HAE plasma increases endothelial permeability in vitro, and this effect is partially reverted by a specific hGIIA enzymatic inhibitor. Finally, recombinant hGIIA inhibits C1-INH activity in vitro.</p>Conclusion<p>sPLA₂ enzymatic activity (likely attributable to hGIIA), which is increased in C1-INH-HAE patients, can promote vascular permeability and impairs C1-INH activity. Our results may pave the way for investigating the functions of sPLA₂s (in particular, hGIIA) in the pathophysiology of C1-INH-HAE and may inform the development of new therapeutic targets.</p