Copy Number Analysis of Complement C4A, C4B and C4A Silencing Mutation by Real-Time Quantitative Polymerase Chain Reaction

Peer reviewe

Diversity of Extended HLA-DRB1 Haplotypes in the Finnish Population

Peer reviewe

Primer sequences of <i>C4A</i>, <i>C4B, CTins</i> and <i>Beta-actin</i> qPCR runs.

<p>Abbreviations:</p><p>qPCR (real-time quantitative polymerase chain reaction),</p><p><i>CTins</i> (CT-insertion mutation of <i>C4A</i> leading to non-expression).</p>a<p>Specific bases are underlined.</p

Method validation with selected samples.

<p><b>A.</b> Immunophenotyping. The gel is skewed in the middle, leading to lower position of protein band levels on the left. All samples were analysed in a replicate run. <b>B.</b> Real-time quantitative PCR (qPCR) for copy number variation. The y-axis depicts the linear view of the fluorescence rate (from 0 to 0.6 in the full picture, from 0 to 0.06 in the magnification) and the x-axis the number of cycles (from 0 to 30 in the full picture and 15 to 21 in the magnification). Each curve represents the mean of two replicates of a sample. The lowest horizontal line represents non-template controls (sterile water), negative control and samples with zero copies of <i>C4B</i> having zero fluorescence due to undetectable amounts of DNA (TX-2144 and TX-2147). The curves from left to right depict samples with <i>C4B</i> CNV 3, 2 and 1 (TX-1586, TX-2170 and TX-2158, TX-2209 and TX-2284, respectively). The number of cycles at which the fluorescence curve cuts the threshold (the red horizontal line) is recorded; the greater amount of genes indicates the lower number of cycles surpassing the threshold. <i>C4A</i> and <i>CTins</i> qPCR runs resulted in similar output. <b>C.</b> Parallel results of C4 analyses. Functional <i>C4A</i> CNV was assessed by reducing the amount of <i>CTins</i> from <i>C4A</i> CNV.</p

Two-locus haplotypes of <i>HLA-DRB1</i> with <i>BTNL2</i> block (>1.0%)#.

<p>#Rare haplotypes were excluded.</p><p>f = frequency.</p

Two-locus haplotypes of HLA-DRB1 with C4 block (>1.0%)#.

#<p>Rare haplotypes were excluded.</p><p>Q0 = null allele.</p><p>InsCT = insertion in C4A gene.</p><p>f = frequency.</p

The Finnish <i>DRB1</i>∼<i>DQB1∼DPB1</i> haplotype frequencies (>1%) and comparison with other populations [41]–[46].

<p>f = frequency.</p><p>Total number of haplotypes in the population sample (Haplotypes/2n) shown in frequencies (f).</p

C4 blocks (C4_1– C4_6) and their frequencies (>1.0%).

<p>C4 blocks (C4_1– C4_6) and their frequencies (>1.0%).</p

<i>TNF</i> blocks (<i>TNF_1– TNF_9</i>) and their frequencies (>1.0%).<i>HLA-DRB1</i> Haplotypes with <i>TNF, BTNL2</i> and <i>C4</i> Blocks.

<p><i>TNF</i> blocks (<i>TNF_1– TNF_9</i>) and their frequencies (>1.0%).<i>HLA-DRB1</i> Haplotypes with <i>TNF, BTNL2</i> and <i>C4</i> Blocks.</p