<i>MYH9</i> rs11089788 statistical analysis for a replicate cohort (D) and for the sum of cohorts B and D, that gave statistical significance.

<p>Patients in cohort D belong to families that segregate microscopic hematuria but no known mutation has been found in any of the genes <i>COL4A3</i>, <i>COL4A4</i>, or <i>CFHR5</i>, so far.</p

Description of cohorts and patients under study.

<p>Please note that cohort B1 is the male only patients with CFHR5 nephropathy.</p><p>MH: Microscopic Hematuria, ESKD: End Stage Kidney Disease, XLAS: X-linked Alport syndrome.</p>1<p>“Mild” patients born before 01/1963. Gender difference (Mild vs Severe) is not significant (p = 0.141).</p>2<p>“Mild” patients born before 01/1975. Gender difference (Mild vs Severe) is significant (p = 0.001).</p>3<p>“Severe” patients: ESKD≤40 yo.</p>4<p>“Mild” patients born before 01/1979. Gender difference (Mild vs Severe) is significant (p = 0.001).</p

Genotype associations for the three <i>MYH9</i> variants genotyped in this study.

<p>P-values were calculated by Pearson Chi-Square test. The whole CFHR5 cohort (B) was genotyped only for <i>MYH9</i> rs11089788, the only SNP that gave p-value close to 0.1 for the male CFHR5 patients (B1).</p>*<p>P-values calculated by Fisher's Exact Test (2-sided) due to existence of genotypes values less than 10.</p>**<p>Odds ratio (OR) cannot be estimated due to zero genotypic values in the “Severe” category.</p

Genotype distribution of the studied <i>MYH9</i> variants, by cohort and by severity.

<p>Genotype distribution of the studied <i>MYH9</i> variants, by cohort and by severity.</p

Mutant <i>COL4A3</i> chains expressed in AB8/13 cultured podocytes demonstrate a trend for increased intracellular retention.

<p>(a) AB8/13 cells were transiently transfected with expression vectors containing wild-type <i>COL4A3</i>-WT or the mutant <i>COL4A3</i> (p.G1334E, p.G871C, p.G484R, p.A587G) cDNAs that included a HA epitope at C-terminus. Single chain expression was measured via Western blot analysis of the cell lysate, 48 h after transfection. No HA antigen was detected in AB8/13 cells transfected with a construct expressing the empty vectors. Shown is a representative Western blot of proteins in cell lysates. (b) All mutant chains show a trend towards increased intracellular retention as compared to the wild type chain, although not reaching significance at the 48 h time point. Shown is densitometry analysis data normalized to tubulin expression. Data are represented as means ± SEM of n≥3 independent experiments.</p

Τable 1. Information on mutations and reagents used for their identification.

<p>If no restriction enzyme is given, detection was performed by direct Sanger DNA sequencing.</p><p>Τable 1. Information on mutations and reagents used for their identification.</p

Synonymous and non-synonymous polymorphisms detected in <i>COL4A3</i> and <i>COL4A4</i> in this work.

<p>*Polymorphisms found by NGS.</p><p>Synonymous and non-synonymous polymorphisms detected in <i>COL4A3</i> and <i>COL4A4</i> in this work.</p

Overexpression of wild type or mutant <i>COL4A3</i> chains induces XBP1 splicing in AB8/13 cells.

<p>(a) Representative experiment of reverse transcription-PCR using XBP1 mRNA as template, from AB8/13 cells transiently expressing <i>COL4A3</i>-WT (A3/WT) or the mutant chains G1334E, G871C, G484R (<i>COL4A3</i>). PCR products were run on 3% agarose gel. It is apparent that over-expression of all chains induces XBP1 splicing, as evidenced by the appearance of the spliced band (s) when the PCR product is cut with the restriction enzyme Pst1. (h) hybrid, (u) unspliced (b) RT-PCR is quantified via densitometric analysis of the bands after PstI digestion as follows: ratio of the spliced band and the sum of the two PstI digest bands (s/(u1+u2), with PstI digestion. Hybrid band (h) was considered as equally contributing to unspliced and spliced bands. There is statistically significant XBP1 splicing when either WT or any of the mutant chains is overexpressed in AB8/13 cells. L19 was used as an internal PCR control. Data are means ± SEM of three independent experiments.</p

Chaperone BiP protein and PERK, a transmembrane protein kinase of the PEK family resident in the endoplasmic reticulum membrane, are deregulated in AB8/13 podocytes transfected with various <i>COL4A3</i> mutant chains.

<p><b><u>a, c</u>:</b> AB8/13 cells were transiently transfected with expression vectors containing either wild-type <i>COL4A3</i> chain or one of several mutant chains. Transfection with lipofectamine only (lipo) served as a negative control. Protein expression of the UPR markers was measured 48 hours after transfection via Western blotting. β-tubulin expression in the same samples was used as equal loading control. Shown are representative blots with differential expression levels of BiP and p-PERK for the various mutant proteins. <b><u>b, d</u>:</b> Western blotting as above, was quantified via densitometric analysis. BiP and p-PERK are up-regulated in cells over-expressing the mutant <i>COL4A3</i>-p.(G1334E), <i>COL4A3</i>-p.(G871C) and <i>COL4A3</i>-p.(G484R) while there is a trend for <i>COL4A3</i>-p.(A587G), as compared to cells expressing the wild type chain. Data are means ± SEM (n = 4 for BiP; n = 7 for p-PERK) *p<0.05; **p<0.01.</p

Electropherograms showing causative mutations found in <i>COL4A3</i> (a) and in <i>COL4A4</i> (b) genes during the course of this work.

<p>In (c) is shown a 52 bp deletion in <i>COL4A4</i> which encompasses 50 bp of exon 20 plus the two conserved <b><i>gt</i></b> bp at the donor site of intron 20. In addition to the translation frameshift introduced, it is expected that aberrant splicing will occur.</p