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Case Reports
. 2016 Jul 7;99(1):174-87.
doi: 10.1016/j.ajhg.2016.05.028.

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

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Case Reports

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

Nikhita Ajit Bolar et al. Am J Hum Genet. .
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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.

Figures

Figure 1
Figure 1
Pedigrees, Schematic Protein Representation, and Conservation of SEC61A1 (A) Circles indicate women, squares indicate males; filled symbols indicate affected individuals; plus sign indicates presence of SEC61A1 variation; minus sign indicates absence of SEC61A1 variation. (B) Position of the p.Val67Gly change in the plug region and the p.Thr185Ala change in the fifth transmembrane region of the translocase-transmembrane domain of SEC61A1. (C) Conservation of the Val67 and Thr185 amino acids throughout evolution.
Figure 2
Figure 2
Kidney Biopsy from Individual 1-II:3 with the p.Thr185Ala Substitution (A) Clusters of atrophic tubules surrounding glomeruli with collapsed or rudimentary capillary tufts (marked by asterisks). A part of affected tubules shows thickening of tubular basement membranes; the remaining are endocrine-type atrophic tubules with narrowed lumina and simplified epithelium. PAS staining. (B) Prominent cystic dilatation of Bowmann’s spaces (marked by asterisks), which are sometimes filled with finely granular proteinaceous material. PAS staining. (C and D) SEC61A protein was detected immunohistochemically in proximal tubules (PT) and distal tubules (DT) in kidney tissue in both affected individual (C) and a control subject (D). Compare coarsely granular intracytoplasmic staining in the affected individual with finely granular and less intensive pattern in the control subject. (E) Renin was undetectable immunohistochemically in juxtaglomerular apparatus (marked by arrows) in an affected individual but weak finely granular staining was found in the cytoplasm of renal tubules. (F) Comparison with strong renin positivity in juxtaglomerular apparatus and negativity in tubular epithelium in a control subject. Scale bars represent 100 μm in (A) and (B), 30 μm in (C) and (D), and 50 μm in (E) and (F).
Figure 3
Figure 3
Structural Topology of the Mutations in Sec61 The cryo-EM based models of translocons translating hydrophilic peptide with opened pore (PDB: 4CG5) and translating peptide inserted to membrane possessing a pore sealed by a plug domain (PDB: 4CG6) were used for illustration. Regions containing mutations are depicted with cartoon representation, namely plug domain (p.Val67Gly) in magenta and TM5 (p.Thr185Val) in orange. Mutated residues are highlighted as dots and/or sticks. (A) Overall structure of Sec61 and location of the mutated residues. (B) Orientation of the mutated residues in the Sec61 structure with opened translocation pore (left) and with pore sealed by a plug domain (right). (C) Left: constriction ring formed by apolar residues represented by green (TM2, TM7, and TM10) and orange (positioned at TM5 near Thr185 residue) spheres; mutated residues are shown as red spheres. Right: detail of TM5 illustrating hydrogen bonding of hydroxyl Thr185 with carbonyls of Leu181 and Phe182.
Figure 4
Figure 4
Transient Expression and Intracellular Localization of SEC61A1_FLAG Variants in Human Embryonic Kidney 293 Cells (A) Western blot detection of transiently expressed wild-type and mutated SEC61A1_FLAG proteins and endogenously expressed SEC61A, SEC61B, and tubulin at 36 hr post-transfection. (B) Quantitative image analysis of wild-type and mutated SEC61A1_FLAG proteins demonstrating decreased amounts of mutated proteins compared to the wild-type. Results represent means of fold change ± SD of the relative signal intensities of mutated proteins to the wild-type protein from three biological replicates. Signal intensities of SEC61A1_FLAG proteins were normalized to that of α-tubulin. Statistical significance was assessed using Student’s t test; p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (C) Quantitative image analysis of the endogenously expressed SEC61A and SEC61B demonstrating that overexpression of neither protein significantly affected relative amounts of the endogeneously expressed SEC61A1 and SEC61B transient expression of SEC61A1. Results represent means of fold change ± SD from three biological replicates of the relative signal intensities of SE61A over SEC61B in cells expressing individual mutated SEC61A1_FLAG protein and empty vector compare to that of cells expressing the wild-type protein. Signal intensities of SEC61A and SEC61B were normalized to that of α-tubulin. Statistical significance was assessed using Student’s t test. (D) Immunofluorescence analysis showing that the wild-type SEC61A1_FLAG is present in a finely granular (subpanel A) or coarsely granular (subpanel D) structures. Co-staining of wt-SEC61A1_FLAG with Golgi matrix protein GM130 (subpanel B) and with Protein disulphide isomerase (PDI) (subpanel E), a marker of endoplasmic reticulum (ER), demonstrating absence of the wt-SEC61A1_FLAG in the Golgi (subpanel C) but presence in the ER (subpanel F). p.Val67Gly (subpanels G and J) and p.Thr185Ala (subpanels M and P) variants of SEC61A1_FLAG are present in a form of intracellular clumps that are more pronounced in the latter. Co-staining with GM130 (subpanels H and N) and PDI (subpanels K and Q) demonstrating localization of both mutant proteins in the Golgi (subpanels I and O) as well as in ER (subpanels L and R). The degree of SEC6A1A_FLAG colocalization with selected markers is demonstrated by the fluorescent signal overlap coefficient values that range from 0 to 1. The resulting overlap coefficient values are presented as the pseudo color which scale is shown in corresponding lookup table.
Figure 5
Figure 5
Intracellular Localization of SEC61A1 in Affected Kidney (A and D) In affected kidney, SEC61A1 is present in coarsely granular structures. (B, C, E, and F) Co-staining of SEC61A1 with Protein disulphide isomerase (PDI) (B), a marker of endoplasmic reticulum (ER), and with 58k Golgi-protein (E) demonstrate localization of the SEC61A1 in the ER (C) and in the Golgi (F). (G and J) In control kidney, SEC6A1A is present in finely granular structures. (H, I, K, and L) Co-staining with (H) PDI and (K) GM130 demonstrating localization of SEC61A1 in ER (I) but not in the Golgi (L). The degree of SEC6A1A colocalization with selected markers is demonstrated by the fluorescent signal overlap coefficient values that ranging from 0 to 1. The resulting overlap coefficient values are presented as the pseudo color which scale is shown in corresponding lookup table.
Figure 6
Figure 6
Suppression of SEC61A1 Leads to Pronephric Tubular Atrophy in Zebrafish (A–C) Whole-mount immunostaining of 4 days post-fertilization (dpf) zebrafish larvae with anti-Na+/K+-ATPase alpha subunit monoclonal antibody (α6F) shows the overall anatomy of the pronephric ducts (pd) and pronephric tubules (pt), which become progressively convoluted in control larvae. Three levels of convolution were assessed: convoluted (normal) (A and A′), V-shaped (B and B′), and straight pronephric tubule (C and C′). The pronephric ducts are normal. (D) Qualitative scoring of the tubular atrophy was performed in larvae batches injected with sec61al2 MO alone; MO and mutant (p.Thr185Ala) RNA or (p.Val67Gly) RNA; MO and wild-type (WT) capped-RNA; WT and mutant RNAs alone; and control. (E) Results of Fisher’s exact test conducted between pairs of conditions. The significant p values (< 0.05) are highlighted in red.

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