Background
Amyloidosis is a group of diseases characterised by deposition of insoluble fibrils derived from various proteins [
1]. Immunoglobulin related amyloidosis (Ig-amyloidosis) is one of the most prevalent renal amyloidosis and is classified into immunoglobulin light-chain (AL) amyloidosis, immunoglobulin heavy-chain (AH) amyloidosis, and, the recently established disease entity, immunoglobulin heavy-and-light-chain (AHL) amyloidosis [
1]. Most of the renal Ig-amyloidosis is AL amyloidosis, whereas AH and AHL amyloidosis are rare entities [
2,
3].
The classification of renal Ig-amyloidosis is generally performed by immunostaining of immunoglobulin light-chains and heavy-chains following positive Congo red stain [
1]. Recently, laser microdissection (LMD) of glomerular amyloid depositions combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has enabled more accurate classification [
1]. In large-scale studies classifying renal amyloidosis by immunostaining and LMD-LC-MS/MS [
2,
3], the amyloidosis with monotypic immunoglobulin light-chain and heavy-chain deposition were described as AHL amyloidosis. Therefore, at present, amyloidosis with monotypic immunoglobulin light-chain and heavy-chain deposition, with greater than or equal heavy-chain amount, is diagnosed as AHL amyloidosis [
3]. However, immunostaining and LMD-LC-MS/MS do not biochemically differentiate immunoglobulins that formed amyloid fibrils from non-amyloid, just deposited, immunoglobulins [
4,
5]. At the same time, the intact immunoglobulins could co-deposit with amyloid fibrils [
4‐
6] and be identified by immunostaining [
4,
6] and LMD-LC-MS/MS [
4,
5].
We herein report a case of renal Ig-amyloidosis due to lymphoplasmacytic lymphoma, with immunostaining and LMD-LC-MS/MS results consistent with previously reported AHL amyloidosis [
2,
3]. We attempted to confirm that both the immunoglobulin light-chain and heavy-chain were forming amyloid fibrils by LC-MS/MS of renal amyloid fibrils isolated by traditional amyloid purification method [
7]. The additional LC-MS/MS only identified immunoglobulin light-chains as the component of amyloid fibrils. This case demonstrated “AL amyloidosis with non-amyloid forming monoclonal immunoglobulin deposition” that might be diagnosed as AHL amyloidosis by immunostaining and LMD-LC-MS/MS.
Discussion
AHL amyloidosis is a disease entity where both the immunoglobulin light-chain and heavy-chain are composing amyloid fibrils, namely the concurrence of AL and AH amyloidosis [
1,
4].
In AL amyloidosis, excess production of amyloidogenic light-chain lead to amyloid fibril formation [
8]. In the present case, LMD-LC-MS/MS identified the peptide sequences of the kappa light-chain variable and constant domain (Fig.
2a), and the LC-MS/MS of isolated amyloid fibrils confirmed that variable domain were the component of the amyloid fibrils (Fig.
2b).
In contrast to AL amyloidosis, little is known about heavy-chain derived amyloid fibril formation. When reviewing the reported cases of AH amyloidosis (Table
1, [
2,
3,
9‐
17]), it was presumable that amyloid fibrils are composed of the variable domains with or without truncated small constant domains of immunoglobulin heavy-chain [
9,
10,
14‐
17]. In the present case, LMD-LC-MS/MS identified peptide sequences that were broadly distributed among the constant domain (CH1, CH2, CH3, and CH4 domains) of mu heavy-chain (Fig.
2a) that do not match the previously reported AH amyloidosis cases. Therefore, the LMD-LC-MS/MS results were suggestive that the identified mu heavy-chain was not composing amyloid fibrils, and the LC-MS/MS of isolated amyloid fibrils confirmed the speculation (Fig.
2b).
Table 1
Reported cases of renal immunoglobulin heavy-chain amyloidosis and amyloid responsible region of immunoglobulin heavy-chain
65/F | WB/AASA | Amyloid purification | IgG-κ | ND | VH + Cγ3 | 22 kDa | VH directly contiguous with CH3 | 1990 | |
59/M | WB/AASA | Amyloid purification | IgG-κ | K(−), λ(−) | vH | 11 kDa | VH with truncated D segment | 1994 | |
64/M | Immunostaining | | IgA-κ | IgA(+), κ(−), λ(−) | NA | NA | NA | 2002 | |
56/M | Immunostaining | | IgM-λ | IgM(+), κ(−), λ(−) | NA | NA | NA | 2003 | |
53/F | Immunostaining | | IgG-λ | IgG(+), κ(−), λ(+) | NA | NA | NA | 2003 | |
72/F | AASA | Amyloid purification | IgG-λ | IgG(−), κ(−), λ(−) | vH | 11 kDa | truncated VH | 2004 | |
61/F | AASA | Amyloid purification | IgG-κ | IgG(−), κ(−), λ(−) | vH | 11 kDa | VH with truncated D segment | 2006 | |
67/M | LC-MS/MS | Amyloid purification | IgM-κ | K(−), λ(−) | vH | 11 kDa | truncated VH | 2008 | |
50/M | LC-MS/MS | LMD | IgG-λ | ND | VH + Cγ3 | NA | NA | 2010 | |
36/M | LC-MS/MS | LMD | IgG-κ | IgG(+), IgA(+), IgM(+), κ(+), λ(+) | VH + Cγ3 | NA | NA | 2010 | |
ND | LC-MS/MS | LMD | IgA-λ | IgA(+), λ(+) | VH + Cα1 | NA | NA | 2013 | |
ND | LC-MS/MS | LMD | IgG-λ | IgG(+), λ(+) | Cγ1 | NA | NA | 2013 | |
ND | LC-MS/MS | LMD | negative | IgG(+) | Cγ1, Cγ4 | NA | NA | 2013 | |
ND | Immunostaining | | IgG-λ | IgG1,(+) | NA | NA | NA | 2013 | |
70/F | LC-MS/MS | Amyloid purification | IgG-λ | IgG(−), κ(−), λ(−) | VH + Cγ1 | 11 kDa | VH with truncated CHI | 2015 | |
There are several hypotheses to explain the glomerular mu heavy-chain deposition. First, there are reported cases of AL amyloidosis combined with non-amyloid monoclonal immunoglobulin deposition diseases (MIDDs) [
18]. However, in the present case, there were no histological findings to support the concurrence of non-amyloid MIDD [
1]. Second, concurrence of AL amyloidosis and heavy-chain disease cannot be denied [
19]. However, plasma electrophoresis did not revealed free heavy-chain and smears and flow cytometry of the bone marrow aspirate were consistent with the lymphoplasmacytic lymphoma without detection of IgM positive light-chain negative lymphoplasmacytoid lymphocytes [
20], Therefore, we speculate that the identified mu heavy-chain was derived from monoclonal IgM-kappa that non-specifically co-deposited with amyloid fibrils [
5], while the exact origin remains unknown.
This case documented “AL amyloidosis with non-amyloid forming monoclonal immunoglobulin deposition” using immunostaining and two distinct LC-MS/MS analyses. In fact, the existence of the present condition has been speculated upon the initial description of AHL amyloidosis [
4]. The concern was in part because of the technical limitations of immunostaining and LMD-LC-MS/MS which do not differentiate amyloid fibrils from non-amyloid monoclonal immunoglobulin deposition [
4,
5]. Moreover, because of the extremely low prevalence of AH amyloidosis [
2‐
4,
21], it was questioned whether a single patient may develop both AL and AH amyloidosis [
4]. On the other hand, co-deposition of non-amyloid immunoglobulin with amyloid fibrils is a well-described phenomenon [
4‐
6,
22]. Therefore, we speculate that the present condition might not be so rare and might be more prevalent than “true” AHL amyloidosis.
Conclusion
We demonstrated “AL amyloidosis with non-amyloid forming monoclonal Ig deposition” disguised as AHL amyloidosis. The confident exclusion of the present condition should be required to diagnose AHL amyloidosis.
Concise method for the two LC-MS/MS analyses
LMD [
2,
3,
18]: Formalin-fixed paraffin-embedded sections were stained with congo-red dye, and the positive areas were extracted using an LMD system (LMD 7000; Leica Microsystems Inc., Tokyo, Japan). The extraction was solubilised in 10 mM Tris/1 mM EDTA/0.002% Zwittergent buffer and digested overnight with trypsin.
Amyloid purification [
7,
14‐
17]: Fresh frozen renal tissue was homogenised in Tris-buffered saline, after which it was centrifuged and the supernatant was decanted. The procedure was repeated twice. The resultant pellet was solubilised in 6 M guanidine/0.5 M Tris-buffered saline, after which it was centrifuged and the supernatant was dialysed against distilled water. The sample was solubilised in gel loading buffer containing 5% 2-mercaptoethanol and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The entire electrophoresed sample was excited and digested overnight with trypsin.
The samples were analysed by LC-MS/MS (Nano LC DiNa; KYA Technologies Co., Tokyo, Japan; and QExactive; Thermo Fisher Scientific Inc., Waltham, MA).
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