Case Information
The patient, a 4-year-old spayed female cat, presented to the authors' (MT and TA) veterinary clinic with the chief complaint of vomiting, loss of energy, and anorexia. The patient had previously received the feline immunodeficiency virus/feline leukemia virus test at another veterinary hospital was negative (test kit unknown). The patient had numerous ulcers in the oral cavity and a mass on the lower part of the tongue, which was treated with antibiotics and anti-inflammatory drugs. The mass in the hypoglossal area was removed under general anesthesia and submitted to a commercial pathology lab, which was histopathologically diagnosed as lymphoplasmacytic stomatitis with granulation tissue.
About a year later, at age 5, in addition to the symptoms of gingivostomatitis, the patient began to exhibit polyuria and polydipsia, chronic renal insufficiency, and non-regenerative anemia. Therefore, she was treated with hematopoietic agents, oral medications for renal protection, and subcutaneous fluid replacement. Later, however, she showed gastrointestinal symptoms, then dehydration, further deterioration of renal function, hypothermia, loss of energy, anorexia, and severe non-regenerative anemia. Ultrasonography revealed mixed hypo- and hyperechoic nodules in the enlarged liver, so a fine needle aspiration of the liver was performed. Fine needle aspiration samples were submitted to a commercial pathology lab and cytologically diagnosed as hepatic amyloidosis.
Despite various symptomatic treatments, including two blood transfusions and hematopoietic agents for severe anemia, antibiotics, anti-inflammatory drugs, and hepatoprotective drugs, the patient died 14 days after the cytological diagnosis of amyloidosis.
Postmortem necropsy was performed promptly, and the liver and kidneys were sampled for histological examination. The liver was multinodularly enlarged, and blood clots were observed on the surface (Supplementary Fig. 1a). The kidney was slightly faded and had an uneven surface (Supplementary Fig. 1b).
Histopathology
Histologically, severe amyloid deposition was observed in the kidneys and liver (Fig. 1a). In the kidney, amyloid deposits were widely observed in the cortical glomeruli and medullary interstitium, with particularly severe deposits in the outer medulla (Figs. 1b-1d).
SAA genes in the case
Reverse transcription-polymerase chain reaction (RT-PCR) and sequencing revealed eight heterozygous single nucleotide substitutions (c.111G > C, c.140A > T, c.183T > T, c.183T > C, c.188A > G, c.231T > C, c.252A > G, c.272G > A, c.282T > C) in the SAA gene resulting in three amino acid substitutions (K29I, Q45R, S75N). Cloning of PCR products revealed that the amino acid substitution combinations are Ile29-Arg45-Asn75 (SAAIRN) and Lys29-Gln45-Ser75 (SAAKQS; Fig. 2a). BLAST analysis using the UniProt database showed that SAAKQS and SAAIRN were identical to entries M3WHE0 and A0A337SKP2, respectively.
Distribution of heterozygote-derived amyloids
To determine the respective renal distribution of amyloid derived from SAAIRN and SAAKQS, immunohistochemistry was performed using anti-SAAQ45 antibody, which specifically recognizes only SAA with Gln45, and anti-Pan-SAA antibody, which recognizes both SAAs with Gln45 or Arg45. All the renal amyloid deposits were positive for the anti-Pan-SAA antibody, indicating AA amyloidosis (Fig. 1e). Immunohistochemistry using anti-SAAQ45 antibody was positive for amyloid deposits in the glomeruli, but mostly negative for the medullary deposits except in the papillae (Fig. 1f). There were no noticeable distribution differences between the two antibodies in hepatic amyloid deposits (Supplementary Fig. 2).
Mass spectrometry demonstrated that SAA is the major component of amyloid deposits in glomeruli, outer medulla, and papilla (Supplementary Table 1). Analysis of SAA-derived peptides detected by mass spectrometry revealed that N-terminal Gln1-Arg72-derived peptides were frequently detected in all three regions, while C-terminal peptides were rare (Figs. 2b-2d). In all three regions, peptides with Ile29 were detected, but no peptides with Lys29 were detected. Focusing on the 45th amino acid residue, only Glu45 was detected in the glomeruli (Fig. 2b), only Arg45 in the outer medulla (Fig. 2c), and both Glu45 and Arg45 were in the papilla (Fig. 2d). The partial overlap of the mass spectrometric results of amyloid collected from the papilla with those of the outer medulla is expected because the two were mixed during microdissection. Therefore, the following discussion will primarily focus on the glomerular and outer medullary results.