PMC5903162_01
Light microscopic and electron microscopic findings in the glomerulus on the first renal biopsy. a PAS staining: neither mesangial proliferation nor mesangial matrix expansion is identified. b Silver methenamine staining: spike formation is found in most of the glomeruli (arrows). c Electron microscopic findings (x2,500): highly electron dense deposits are found in subepithelial (asterisks), subendothelial (arrowhead), and mesangial areas (arrow). d Electron microscopic findings (x40,000): on larger magnification, highly electron dense deposits consist of microbubbles or microcysts.
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F1
Neither mesangial cell proliferation nor mesangial matrix expansion was marked (Fig. 1a). In silver methenamine-stained sections, "spike" formation as seen in MN was found in most of the glomeruli (Fig. 1b). EM study showed highly dense deposits in the subepithelial, subendothelial, and mesangial areas, which appeared like microbubbles or microcysts on higher magnification (Fig. 1c, d). The patient's sample showed the position of ApoE2/2 (Fig. 2a, lane 1). Genotype epsilon2/2 in the patient was identified by 91- and 83-bp fragments (Fig. 2b, lane 1).
Phenotype, genotype, and DNA sequence of ApoE in the patient. a ApoE phenotype analysis. The patient was identified as E2/2 (lane 1). Controls (lanes 2, 3, and 4) show E2/3, E3/3, and E3/4, respectively. b ApoE genotype analysis by RFLP using HhaI as a restriction enzyme. The patient was identified as epsilon2/2. Controls (lanes 2, 3, and 4) show epsilon2/2, epsilon3/4, and epsilon3/3, respectively. c RFLP for ApoE Toyonaka using Sacl as a restriction enzyme. Lanes M, 1, 2, and 3 show marker, PCR only, the patient (ApoE Toyonaka), and normal control, respectively. d Sequence analysis of APOE gene. A heterozygous missense mutation (c.644 C>G) in exon 4 leads to an amino acid substitution Cys (TGC, lower) for Ser (TCC, upper) at codon 197. ApoE, apolipoprotein E; RFLP, restriction fragment length polymorphism.
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F2
The patient's sample showed the position of ApoE2/2 (Fig. 2a, lane 1). Genotype epsilon2/2 in the patient was identified by 91- and 83-bp fragments (Fig. 2b, lane 1). The ApoE Toyonaka (Ser197Cys), as shown in Figure 2d, was also confirmed by restriction fragment length polymorphism analysis digested with SacI (Fig. 2c). The 335-bp fragment after cleavage by SacI suggested normal c.644C of ApoE in codon 197 (Fig. 2c, lane 3). The 358- and 335-bp fragments after cleavage by SacI showed a heterozygous novel mutation of ApoE Toyonaka (Fig. 2c, lane 2). ApoE DNA was performed as described previously. As a result, in our patient, we identified a homozygous polymorphism at codon 158 (epsilon2/2: c.526 C>T: Arg158Cys) (data not shown) and a heterozygous missense mutation (C to G) in exon 4 leading to an amino acid substitution Cys (TGC) for Ser (TCC) at codon 197 (c.644 C>G: p.S215C: Ser197Cys) (Fig. 2d).
Immunohistochemical staining for ApoE in the glomerulus. a Light microscopy specimen shows ApoE-positive deposits particularly in the subepithelial areas (arrows). b, c Electron microscopy shows black dots on higher magnification (c, arrowheads), which are positive signals for ApoE. ApoE, apolipoprotein E.
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F3
The 335-bp fragment after cleavage by SacI suggested normal c.644C of ApoE in codon 197 (Fig. 2c, lane 3). ApoE2/2, suggesting the involvement of a new form of ApoE-related disease. To clarify the accurate pathological changes, a second renal biopsy was conducted 9 years after the first one. Although the histological findings by LM and IF were similar to those of the first biopsy, the density of deposits on EM were more obvious, and ApoE was identified by immunohistochemical studies for LM and EM specimens using anti-ApoE antibody (Novus Biologicals, Littleton, CO, USA) (Fig. 3).
Summary of amino acid sequence coverage detected in the microdissected samples from the first and the second renal biopsy specimens, which showed high numbers of spectra matching ApoE protein with a probability greater than 95%. ApoE, apolipoprotein E.
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F4
As a result, the most abundant protein was ApoE in both samples (Fig. 4).