DataSheet_1_A Mendelian randomization study to assess the genetic liability of type 1 diabetes mellitus for IgA nephropathy.docx

BackgroundThe prevalence of immunoglobulin A nephropathy (IgAN) seems to be higher in patients with type 1 diabetes mellitus (T1DM) than that in the general population. However, whether there exists a causal relationship between T1DM and IgAN remains unknown.MethodsThis study conducted a standard two-sample Mendelian randomization (MR) analysis to assess the causal inference by four MR methods, and the inverse variance-weighted (IVW) approach was selected as the primary method. To further test the independent causal effect of T1DM on IgAN, multivariable MR (MVMR) analysis was undertaken. Sensitivity analyses incorporating multiple complementary MR methods were applied to evaluate how strong the association was and identify potential pleiotropy.ResultsMR analyses utilized 81 single-nucleotide polymorphisms (SNPs) for T1DM. The evidence supports a significant causal relationship between T1DM and increased risk of IgAN [odds ratio (OR): 1.39, 95% confidence interval (CI): 1.10–1.74 for IVW, p 0.05).ConclusionsThis MR study provided evidence that T1DM may be a risk factor for the onset of IgAN, which might be driven by LDL-c. Lipid-lowering strategies targeting LDL-c should be enhanced in patients with T1DM to prevent IgAN.</p

MALDI-TOF-MS analysis of peptide mass fingerprint of native 1Aiy1 subunit.

<p>Peptide mass is available at <a href="http://www.expasy.org/tools/peptide-mass.html" target="_blank">http://www.expasy.org/tools/peptide-mass.html</a>.</p

Properties of the primary structure of the HMW-GS from <i>Ag. intermedium</i> in comparison with those of the representatives of wheat HMW-GS.

a<p>The nucleotide number of Open Reading Frame;</p>b<p>The number of Cys.</p

SDS-PAGE (A) and Western blotting (B) analysis of HMW-GSs of <i>Ag. intermedium</i>.

<p>Lane 1 shows the named HMW-GSs from common wheat variety Chinese Spring as a control. Lanes 2∼7 show the HMW-GSs from six representative seeds of the <i>Ag. intermedium</i> line used in this study. The seven expressed HMW-GSs with distinct electrophoretic mobility comparing with Chinese Spring were detected by SDS-PAGE (A) and were confirmed using Western blotting experiment with polyclonal antibody specific for HMW-GSs (B). Among the seven HMW-GSs from <i>Ag. intermedium</i>, three subunits (marked with solid triangles in lane 2 of B) share comparable electrophoretic mobility with Chinese Spring, the other four subunits (marked with hollow triangles in lane 2 of B) moved faster than those HMW-GSs from Chinese Spring.</p

Illustration for the developmental mechanism of two hybrid HMW-GSs based on unequal double crossover hypothesis.

<p>The broken line box indicates the double crossover region. The xy and yx represent the hybrid subunit with 5′region of x-type and 3′region of y-type and the hybrid subunit with 5′region of y-type and 3′region of x-type, respectively.</p

Phylogenetic tree of <i>Thinopyrum intermedium</i> ( = <i>Ag. intermedium</i>) and some representative HMW-GSs from <i>Triticeae</i>.

<p>This phylogenetic tree was constructed with Maximum Likelihood Estimation method based on the nucleotide sequences encoding signal peptide and N-terminal conserved region of HMW-GSs plus the next three repeat units, one dodecapeptide, one undecapeptide and one hexapeptide repeat. D-hordein from barley was used as outgroup. The species names of HMW-GS genes in this figure are consistent with their accession names in GenBank, so here we replaced <i>Agropyron intermedium</i> with <i>Thinopyrum intermedium</i>.</p

Alignments and clustering analyses based on N-terminal (A), C-terminal (B) and the last 93 residues in the repetitive region (C) of the HMW-GSs from <i>Ag. intermedium</i> and several representative HMW-GSs from <i>Triticum</i> genus.

<p>Noticeably, the subunit 1Aix1 possesses a N-terminal clustered to x-type subunits (A) and a C-terminal and last part of repetitive region clustered to y-type subunits (B and C). Conversely, the subunit 1Aiy1 possesses a N-terminal more similar to y-type subunits (A) and a C-terminal and last part of repetitive region more similar to x-type subunits (B and C). The default parameters were used for full alignment and clustering analysis of sequences by aid of DNAMAN version 5. 2. 2.</p

SDS-PAGE (A) and Western blotting (B) analysis of HMW-GSs from <i>Ag. intermedium</i> and bacterial expression products.

<p>Lane 1 is HMW-GSs from common wheat variety Chinese Spring, the four expressed HMW-GSs are noted on left; Lane 2 is native HMW-GS from the seed of <i>Ag. intermedium</i> same as the lane 2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087477#pone-0087477-g001" target="_blank">Figure 1</a>, the seven expressed HMW-GSs are marked with triangles; Lane 3, 5, 7, 9, 11, 13 and 15 are total cell proteins from IPTG induced <i>E. coli</i> containing pET-<i>Glu-1Ai1</i>, pET-<i>Glu-1Ai2</i>, pET-<i>Glu-1Ai3</i>, pET-<i>Glu-1Ai4</i>, pET-<i>Glu-1Ai5</i> pET-<i>Glu-1Ai6</i> and pET-<i>Glu-1Ai7</i>, respectively, whereas the dextral lanes for each of them shows the total cell proteins from their bacterial cells without induction of IPTG. The seven expressed target proteins in <i>E. coli</i>, which were detected by SDS-PAGE (marked with arrows in A) and were confirmed by Western blotting (lanes 3, 5, 7, 9, 11, 13 and 15 in B), share comparable electrophoretic mobility with those native HMW-GSs from <i>Ag. intermedium</i> (lane 2).</p

Genomic locations of glucokinase genes in diverse mammalian genomes.

1<p>Bases spanning the N-terminus to C-terminus of the encoded protein.</p>2<p>Y, indicates that all 11 exons (2 tissue specific and 9 shared) could be predicted from the genomic sequence. N, indicates that at least one exon could not be predicted.</p>3<p>None indicates that a gene was not annotated in the genome assembly.</p>4<p>A liver specific 1^st exon was not found in the genomic sequence, but there is no gap in this sequence.</p

Alignment of glucokinase gene sequences from diverse mammals.

<p>A genomic sequence alignment was generated by MultiPipMaker <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045824#pone.0045824-Schwartz1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045824#pone.0045824-Schwartz2" target="_blank">[25]</a>. The sequence is numbered (in kilobases, k) from the 5′ end of the liver-specific transcript, with 5′ flanking sequence numbered backwards. Exons are represented as tall boxes, and are numbered from the 5′ end of the transcripts. The arrow, labeled GCK, represents the liver-specific glucokinase transcript. Beta refers to the 1^st exon of the beta-cell-specific GCK transcript, which is spliced to join exon 2. Tissue-specific first exons are labeled as 1B, for the pancreatic beta-cell-specific 1^st exon, and 1L, for the liver-cell-specific 1^st exon. Filled tall boxes are coding exon sequences, while shaded boxes are untranslated sequences. The percentage sequence identity (if above 50%) of the mouse, cow, horse, hyrax, and Tasmanian devil GCK genomic sequences to the human genomic sequence is shown for each species below the human genomic region schematic. Repetitive DNA elements, and sequence shown high GC content are also identified using the symbols shown in box at the lower right.</p