Frequency of COL4A3/COL4A4 mutations amongst families segregating glomerular microscopic hematuria and evidence for activation of the unfolded protein response. Focal and segmental glomerulosclerosis is a frequent development during ageing.

Familial glomerular hematuria(s) comprise a genetically heterogeneous group of conditions which include Alport Syndrome (AS) and thin basement membrane nephropathy (TBMN). Here we investigated 57 Greek-Cypriot families presenting glomerular microscopic hematuria (GMH), with or without proteinuria or chronic kidney function decline, but excluded classical AS. We specifically searched the COL4A3/A4 genes and identified 8 heterozygous mutations in 16 families (28,1%). Eight non-related families featured the founder mutation COL4A3-p.(G1334E). Renal biopsies from 8 patients showed TBMN and focal segmental glomerulosclerosis (FSGS). Ten patients (11.5%) reached end-stage kidney disease (ESKD) at ages ranging from 37-69-yo (mean 50,1-yo). Next generation sequencing of the patients who progressed to ESKD failed to reveal a second mutation in any of the COL4A3/A4/A5 genes, supporting that true heterozygosity for COL4A3/A4 mutations predisposes to CRF/ESKD. Although this could be viewed as a milder and late-onset form of autosomal dominant AS, we had no evidence of ultrastructural features or extrarenal manifestations that would justify this diagnosis. Functional studies in cultured podocytes transfected with wild type or mutant COL4A3 chains showed retention of mutant collagens and differential activation of the unfolded protein response (UPR) cascade. This signifies the potential role of the UPR cascade in modulating the final phenotype in patients with collagen IV nephropathies

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

Summary of pathogenic <i>COL4A3/A4</i> mutations found in Greek-Cypriot families studied here.

<p>One additional deletion mutation was detected in a Cypriot of Romanian origin.</p><p>Summary of pathogenic <i>COL4A3/A4</i> mutations found in Greek-Cypriot families studied here.</p

Mutations detected in <i>COL4A3</i> and <i>COL4A4</i> genes.

<p>*these mutations tested negative in an additional collection of 52 patients with chronic kidney disease.</p><p>**this mutation was detected only in a single patient during screening of 153 patient samples. It was subsequently detected in six of 120 Cypriot controls. It is a suspect founder mutation and is under further investigation.</p>+<p>these mutations tested negative in an additional collection of 40 patients with glomerulonephritis of unknown aetiology CY, Cypriot samples; RO, Romanian sample; ND: Not Done; NA: Not applicable.</p><p>Mutations detected in <i>COL4A3</i> and <i>COL4A4</i> genes.</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

Clinical, pathologic and mutational analysis results for the families that mutations were found in the present patient cohort.

<p>Clinical, pathologic and mutational analysis results for the families that mutations were found in the present patient cohort.</p