Heterozygous Loss-of-Function SEC61A1 Mutations Cause Autosomal-Dominant Tubulo-Interstitial and Glomerulocystic Kidney Disease with Anemia

Abstract

Autosomal-dominant tubulo-interstitial kidney disease (ADTKD) encompasses a group of disorders characterized by renal tubular and interstitial abnormalities, leading to slow progressive loss of kidney function requiring dialysis and kidney transplantation. Mutations in UMOD, MUC1, and REN are responsible for many, but not all, cases of ADTKD. We report on two families with ADTKD and congenital anemia accompanied by either intrauterine growth retardation or neutropenia. Ultrasound and kidney biopsy revealed small dysplastic kidneys with cysts and tubular atrophy with secondary glomerular sclerosis, respectively. Exclusion of known ADTKD genes coupled with linkage analysis, whole-exome sequencing, and targeted re-sequencing identified heterozygous missense variants in SEC61A1-c.553A>G (p.Thr185Ala) and c.200T>G (p.Val67Gly)-both affecting functionally important and conserved residues in SEC61. Both transiently expressed SEC6A1A variants are delocalized to the Golgi, a finding confirmed in a renal biopsy from an affected individual. Suppression or CRISPR-mediated deletions of sec61al2 in zebrafish embryos induced convolution defects of the pronephric tubules but not the pronephric ducts, consistent with the tubular atrophy observed in the affected individuals. Human mRNA encoding either of the two pathogenic alleles failed to rescue this phenotype as opposed to a complete rescue by human wild-type mRNA. Taken together, these findings provide a mechanism by which mutations in SEC61A1 lead to an autosomal-dominant syndromic form of progressive chronic kidney disease. We highlight protein translocation defects across the endoplasmic reticulum membrane, the principal role of the SEC61 complex, as a contributory pathogenic mechanism for ADTKD.

Autosomal Dominant Tubulointerstitial Kidney Disease

“…35 Two families have been identified with this condition. In one family, intrauterine growth retardation, dysplastic kidneys and anemia were present.…”

De Novo Disruption of the Proteasome Regulatory Subunit PSMD12 Causes a Syndromic Neurodevelopmental Disorder

“…The CRISPR/ Cas9 efficiency was assessed as previously described. 49 In brief, DNA from each 2 day post fertilization (dpf) F0 embryo was extracted by proteinase K digestion (Life technologies, AM2548). The CRISPR-targeted region was amplified by PCR.…”

“…Standard whole-mount zebrafish immunostaining was performed on CRISPR experiments as previously described. 49,50 To visualize the axonal tracts in the brain, including the optic tecta and the cerebellum, we stained 3 dpf mutant and control embryos with an anti-acetylated tubulin primary antibody (T7451, mouse, Sigma-Aldrich; 1:1,000 dilution) and the Alexa Fluor goat anti-mouse IgG (A21207, Invitrogen; 1:500 dilution) as a secondary antibody. 50 We observed that the size of the optic tecta was significantly smaller in CRISPR F0 mutants than in the control larvae (p % 0.0001, n ¼ 30-50 larvae/batch, repeated; Figures 4A and 4D).…”

Mutations in TMEM260 Cause a Pediatric Neurodevelopmental, Cardiac, and Renal Syndrome

“…We and others have demonstrated previously the utility of zebrafish models in testing the candidacy of rare mutations in syndromic disorders; [14][15][16][17][18][19][20][21] this approach includes an experimental paradigm in which expression of mutations in the context of spliced isoforms can be used for testing the specific effect of such alleles. 14,22-24 For TMEM260, we utilized this approach to ask (1) whether the transient suppression or introduction of deletions at the locus can reproduce key aspects of the human pathology, (2) whether the two known isoforms of the human locus might have different abilities to rescue relevant phenotypes, and (3) whether the long TMEM260 isoform harboring individual 2-II-4's mutation (c.1698_1701del) has the ability to rescue relevant phenotypes.…”

“…In addition to the rescue experiments, we tested the specificity of the phenotype by generating deletions in the zebrafish locus with CRISPR/Cas9-mediated genome editing. We designed and validated three CRISPR guide RNAs (G2, G3, and G4; Table S4) targeting exons 2, 7, and 5 of D. rerio tmem260, respectively, according to previously described methods, 16 and we injected guide RNA and Cas9 into single-cell embryos (n ¼ 48-74 embryos per batch). We established the efficiency of each reagent by genomic DNA isolation, heteroduplex detection on 15% polyacrylamide gel electrophoresis (n ¼ 6 embryos tested per condition), 25 and subsequent cloning and sequencing of PCR amplicons to estimate mosaicism.…”