Background
Hereditary transthyretin (TTR) amyloidosis (ATTRv amyloidosis), which is caused by mutations in the
TTR gene, is an inherited systemic disorder characterized by extracellular amyloid deposits. Patients with ATTRv amyloidosis develop systemic symptoms such as sensorimotor neuropathy, autonomic dysfunction, cardiomyopathy, gastrointestinal dysfunction, renal failure, and ocular disorders [
1]. To date, more than 140 different mutations in the
TTR gene have been reported, most of which have been associated with ATTRv amyloidosis. Of the pathogenic TTR mutations, Val30Met is most frequently found worldwide [
2,
3].
TTR is a plasma protein, which is mainly synthesized in the liver, and acts as a transporter of thyroxine and retinol-binding protein with vitamin A. In the bloodstream, TTR forms a homotetramer with a dimer-of-dimers configuration. TTR mutations cause destabilization of TTR tetramers, which is believed to be a crucial step in formation of TTR amyloid [
4]. Regarding this mutation, late-onset patients from non-endemic areas show distinctive clinical features from early-onset patients from conventional endemic foci [
5]. Patients with ATTRv amyloidosis, particularly late-onset cases, tend to be initially misdiagnosed as having other diseases [
6].
Mass spectrometry (MS) is a powerful tool that can detect small molecular changes in proteins. Because the molecular masses of variant TTRs with amino acid exchanges differ from that of wild-type (WT) TTR, we developed diagnostic methods to detect variant TTRs in serum samples by means of several different mass spectrometric analyses such as matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) MS with immunoprecipitated (IP) serum TTR (IP-MALDI) [
7‐
9], electrospray ionization (ESI) MS with IP serum TTR (IP-ESI) [
10‐
12], and surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) MS ProteinChip system [
13,
14]. Although those methods are valuable for screening ATTRv amyloidosis and double-checking TTR variants in addition to genetic testing of the
TTR gene, they require significant time to analyze variant TTRs, and they also sometimes fail to detect TTRs because of technical difficulties related to the cumbersome immunoprecipitation with anti-TTR antibodies.
Here, we developed a new simple and reliable mass spectrometric method for ATTRv amyloidosis screening in which we can directly detect variant TTRs in serum samples without pre-purification by using MALDI-TOF MS (direct MALDI).
Discussion
Using this direct MALDI method, we rapidly detected variant TTRs in serum samples obtained from patients with ATTRv amyloidosis. We found a highly significant correlation between measured and theoretical mass shifts from WT TTR in serum samples from these patients.
Development of disease-modifying therapies for ATTRv amyloidosis has made dramatic advances. Liver transplantation (LT) has been performed to halt the progression of ATTRv amyloidosis. LT can lead to mutant TTR synthesized by a diseased liver being replaced with WT TTR produced by the healthy liver graft [
16,
17]. In addition, TTR tetramer stabilizers such as tafamidis and diflunisal were developed to prevent dissociation of the TTR tetramer to monomers and to inhibit progression of this disease [
18,
19]. Clinical studies have also revealed that gene-silencing therapies, such as the use of small interfering RNA and antisense oligonucleotides that target the
TTR gene, dramatically reduced disease-causing TTR expression by the liver and significantly improved symptoms of patients with ATTRv amyloidosis [
20,
21]. Early diagnosis of ATTRv amyloidosis is becoming more important so that these new disease-modifying therapies may be utilized earlier.
To detect variant TTR in serum samples from patients in a one-step procedure, we developed a new direct MALDI method, which did not require pre-purification such as immunoprecipitation with anti-TTR antibodies. This method required only 30 min to obtain results. Other mass spectrometric methods, such as IP-MALDI, IP-ESI, and SELDI, needed significant time to detect variant TTRs in serum samples because of cumbersome pre-purification such as immunoprecipitation with anti-TTR antibodies or use of the ProteinChip system (Table
2) [
7‐
14]. Direct MALDI is thus a simple means of detecting TTR variants for clinical screening compared with other methods.
Table 2
Comparison of different mass spectrometric methods to detect variant TTRs in serum samples
Immunoprecipitation | + | + | – | – |
ProteinChip system | – | – | + | – |
Running cost | Low | Low | High | Low |
Detection time | 2 days | 2 days | 3 h | 30 min |
Minimum mass difference that can be identified in each system | 14 Da | 13 Da | 15 Da | 12 Da |
Our direct MALDI method demonstrated a high sensitivity (91%) for detection of variant TTRs in our patients with various TTR mutations, although it failed to detect the rare TTR variants Glu61Lys and Glu89Gln, whose mass differences from WT TTR were 0.94 and 0.99
m/z, respectively. We should therefore note that direct MALDI could not distinguish mass differences of rare variant TTRs with small mass shifts, that is, TTR variants whose mass differences from WT TTR were less than 12 Da (Table
1). Direct MALDI succeeded, however, in detecting most variant TTRs, including the common Val30Met TTR. Direct MALDI may thus be useful for the screening of ATTRv amyloidosis.
Genetic testing is the most reliable tool for diagnosing inherited diseases. However, genetic testing usually takes a long time and is not infallible because of human errors [
22]. Therefore, to avoid misdiagnosis of ATTRv amyloidosis, we need an accurate system for double-checking results. Direct MALDI therefore promises to be a valuable tool for double-checking the diagnosis of ATTRv amyloidosis.
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