Differential Association of Transthyretin Stability with Variant and Wild-Type Transthyretin Amyloid Cardiomyopathy

Transthyretin amyloid cardiomyopathy (ATTR-CM) is subclassified into wild-type (ATTRwt-CM) and variant (ATTRv-CM) based on the transthyretin (TTR) gene sequence. ATTRwt-CM occurs with a normal gene sequence, whereas ATTRv-CM is characterized by single nucleotide variations of the TTR gene, including the common Val122Ile variant.^1,2 The rate-limiting step for amyloidogenesis is TTR dissociation, which leads to protein misfolding, aggregation, and amyloid fibril deposition that results in cardiac dysfunction.² Critical aspects of ATTRwt-CM pathogenesis remain unknown.² A subunit exchange assay to assess TTR dissociation was developed to evaluate the response to TTR stabilizers.^3,4 Herein, we used this assay to determine differences in TTR stability among Val122Ile variant carriers, ATTRv-CM, ATTRwt-CM, and nonamyloid heart failure (HF) controls.

This is a prespecified substudy from the SCAN-MP (Screening of Cardiac Amyloidosis with Nuclear Imaging in Minority Populations study.⁵ SCAN-MP is approved by the Western Institutional Review Board and all subjects provided written informed consent. We included participants (n = 89) with an available measurement of TTR stability from May 2019 to January 2022. Participants were classified into 4 groups based on the presence of ATTR-CM (defined by Tc-99m-pyrophosphate [PYP] imaging) and gene testing for a TTR variant as: 1) nonamyloid HF controls (negative PYP and normal gene testing); 2) ATTRwt-CM [positive PYP and normal gene testing]; 3) Val122Ile carriers (negative PYP and abnormal genetic testing for variant Val122Ile); and 4) ATTRv-CM (positive PYP and abnormal genetic testing for variant Val122Ile). TTR kinetic stability was assessed by measuring the subunit exchange rate (K_ex), with higher rates indicating more unstable TTR.³ Recombinant dual-FLAG-tagged-C10A TTR (FT2-C10A-TTR)4 reporter TTR homotetramers were added to participant plasma samples at a concentration equal to the endogenous tetramers to measure the rate at which endogenous TTR subunits in plasma and the added FT 2recombinant subunits exchanged over 3 days.^3,4 Samples were analyzed at The Scripps Research Institute. Participants with ATTR-CM were not taking TTR stabilizers at the time of assessment.

Differences in characteristics and K_ex between groups were assessed with one-way ANOVA with the post hoc Tukey-Kramer test for parametric data or Kruskal-Wallis for nonparametric data. Categorical variables were assessed with the chi-square test or Fisher exact test where appropriate. Groups were compared using pairwise comparison analysis. TTR instability was classified as low, normal, and high using the mean ± 2 SD value from controls, which were normally distributed. Univariate linear regression analyses exploring the association between K_ex and: 1) TTR; and 2) holo-retinol-binding protein 4 (RBP4) concentrations were performed. A two-sided value of P < 0.05 was considered statistically significant. SAS Studio 3.81 (Enterprise Edition) was used for analyses.

Among the 89 participants, 67.4% were non-amyloid HF controls, 12.4% were Val122Ile carriers, 12.4% were ATTRwt-CM patients, and 7.9% were patients with Val122Ile ATTRv-CM. Most were male (60.7%) and self-identified as Black (79.8%). The mean serum TTR concentration was lower in ATTRv-CM than in controls (P < 0.0001) and ATTRwt-CM (P = 0.002) (Table 1).

TABLE 1.

Characteristics and Transthyretin Stability of Patients Classified by Amyloidosis Type and TTR Variant Carrier Status

		Study Groups According to TTR Gene Testing and PYP Result
	Overall (N = 89)	Nonamyloid HF (n = 60)	ATTRwt-CM (n = 11)	Val122Ile Carriers (n = 11)	Val122Ile ATTRv-CM (n = 7)	P Valuea
Baseline characteristics
Age, y	77 (67–85)	77 (66.5–83.5)	83 (79–91)	66 (64–77)	82 (74–85)	0.007 b
Male	54 (60.7)	37 (61.7)	7 (63.6)	5 (45.5)	5 (71.4)	0.703
Race						0.502
White	1 (1.1)	1 (1.7)	0 (0)	0 (0)	0 (0)
Black	71 (79.8)	43 (71.7)	11 (100)	10 (90.9)	7 (100)
Other	2 (2.3)	2 (3.3)	0 (0)	0 (0)	0 (0)
Unknown	15 (16.9)	14 (23.3)	0 (0)	1 (9.1)	0 (0)
Hispanic ethnicity	21 (23.6)	18 (30)	1 (9.1)	1 (9.1)	1 (14.3)	0.321
EF, %	60 (52–65)	60 (53–65)	61 (50–65)	61 (55–69)	52 (34–56)	0.076
eGFR, mL/min/1.73 m2	49 (35.1–66.1)	48.6 (34–64.9)	47.3 (36.4–51.6)	64.9 (46.6–78.2)	45.1 (24.9–71.4)	0.325
TTR, mg/dL						<0.0001 c
Mean ± SD	23 ± 7.1	25 ± 6.5	23 ± 5	19.4 ± 5.9	12 ± 5.2
Median (IQR)	24 (18–27)	25 (21–28.5)	24 (20–25)	20 (17–22)	12 (8–17)
TTR stability
TTR Kex, h−1						<0.0001 d
Mean ± SD	0.017 ± 0.005	0.015 ± 0.004	0.016 ± 0.003	0.025 ± 0.006	0.021 ± 0.005
Median (IQR)	0.016 (0.014–0.02)	0.015 (0.012–0.018)	0.016 (0.014–0.016)	0.024 (0.022–0.027)	0.021 (0.017–0.026)
Stability groupse
Low	1 (1.1)	1 (1.7)	0 (0)	0 (0)	0 (0)
Normal	76 (85.4)	57 (95)	11 (100)	4 (36.4)	4 (57.1)
High	12 (13.5)	2 (3.3)	0 (0)	7 (63.6)	3 (42.9)

Values are n (%) or median (IQR), unless otherwise indicated. Bold indicates statistical significance.

^aP values for comparisons between the 4 study groups, further pairwise comparisons are shown in superscripts.

^bPairwise comparison: ATTRwt-CM vs controls (P = 0.040), ATTRwt-CM vs carriers (P = 0.015).

^cPairwise comparison: carriers vs controls (P = 0.033), ATTRv-CM vs controls (P < 0.0001), ATTRv-CM vs ATTRwt-CM (P = 0.002).

^dPairwise comparison: carriers vs controls (P < 0.0001), ATTRv-CM vs controls (P = 0.006), ATTRwt-CM vs carriers (P < 0.0001), ATTRwt-CM vs ATTRv-CM (P = 0.051), carriers vs ATTRv-CM (P = 0.222), ATTRwt-CM vs controls (P = 0.995).

^eClassified as low (K_ex <0.007), normal (K_ex 0.007–0.023), and high (K_ex >0.023).

ATTR = transthyretin amyloidosis; ATTRv-CM = variant transthyretin amyloid cardiomyopathy; ATTRwt-CM = wild-type transthyretin amyloid cardiomyopathy; EF = ejection fraction; eGFR = estimated glomerular filtration rate; K_ex = subunit exchange rate; PYP = technetium pyrophosphate scintigraphy; TTR = transthyretin.

Overall, the K_ex was 0.017 ± 0.005 h⁻¹ with the highest rates identified in Val122Ile carriers and ATTRv-CM. Val122Ile carriers (0.025 ± 0.006 h⁻¹) and patients with ATTRv-CM (0.021 ± 0.005 h⁻¹) had higher K_ex than controls (0.015 ± 0.004 h⁻¹) (P < 0.0001 and P = 0.006, respectively). Val122Ile carriers, without and with ATTR-CM, had more unstable TTR than ATTRwt-CM (0.016 ± 0.003 h⁻¹) (P < 0.0001 and P = 0.051, respectively). No differences were seen between Val122Ile carriers and ATTRv-CM (P = 0.222) or between ATTRwt-CM and controls (P = 0.994). K_ex rates were categorized as low (<0.007), normal (0.007–0.023), or high (>0.023). Sixty-four percent of Val122Ile carriers and 43% of ATTRv-CM cases were classified as high, while all ATTRwt-CM were classified as normal (Table 1). No significant associations were found between K_ex and TTR or RBP4 concentrations.

We demonstrate greater TTR instability in a cohort of African American patients with the Val122Ile variant, irrespective of phenotypic ATTR-CM. In contrast, TTR stability in patients with ATTRwt-CM was indistinguishable from nonamyloid HF controls. This finding suggests that TTR instability might contribute differently to the pathogenesis of ATTR-CM in these 2 scenarios. Despite TTR dissociation being rate limiting for amyloidogenesis, our data showing indistinguishable stability suggests that other factors may contribute to ATTRwt-CM pathogenesis, such as alterations in protein homeostasis.² However, improved TTR stability is important in ATTRwt-CM; the stabilizing agents tafamidis and acoramidis have been proven effective in all ATTR-CM types.^1,4 The K_ex has been used to assess the efficacy of tafamidis in ATTRwt-CM.⁴ Our findings suggest that future investigations might be useful to assess the association of K_ex with prognosis and therapeutic response in ATTRv-CM. Finally, no differences were seen in TTR instability between Val122Ile carriers and ATTRv-CM, suggesting that this is not the sole factor contributing to clinical penetrance.

This study has limitations. It was underpowered to correlate K_ex with phenotypical characteristics because the data were available from a subset of cases with a low ATTR-CM frequency. The K_ex might be insensitive to identify a small fraction of unstable TTR in ATTRwt-CM that may be sufficient to result in clinical phenotype. Additionally, the K_ex might vary between subjects as reported previously.³ The interaction of TTR and RBP4 was explored as a factor affecting the K_ex; however, no association was identified. Last, other mechanisms potentially contributing to ATTR-CM pathogenesis were not measured.²

In conclusion, TTR instability was associated with the Val122Ile variant, irrespective of ATTRv-CM. Future investigations should assess the association of TTR instability with clinical penetrance and disease outcomes.

What is the clinical question being addressed?

Does transthyretin stability differ in inherited and noninherited ATTR-CM vs controls?

What is the main finding?

Transthyretin was more unstable in patients with variant Val122Ile than wild-type or controls. Measuring transthyretin stability might aid in assessing prognosis and response to stabilizer therapy in future studies