that carriers may have increased levels of transthyretin. In the largest study to date, we show that T119M heterozygotes have a 17% (26 μg/mL) increase in transthyretin levels compared with noncarriers. These findings are in contrast to the more unstable, pathogenic TTR-variants, where carriers typically display lower transthyretin levels than normal.12 Therefore, the higher transthyretin levels indicate a protein less likely to dissociate and aggregate into fibrils.

=0.00540 50 60 70 80 90Age (years)Log-rank: P30h) BSBASIp Je|NOSeACIGeJeO JO eoUEpIOU! eAe|nWND

It has been shown both by extensive physicochemical stud- ies in vitro and by ex vivo studies of sera from T119M het- erozygotes14,19,20 that increased binding affinity for thyroxine reflects the stabilizing effect of T119M on the transthyretin tetramer. Furthermore, adding thyroxine, a natural ligand for transthyretin, has been shown to stabilize wild-type transthyre- tin and inhibit fibril formation in vitro.28 Therefore, increased plasma level of total thyroxine in heterozygotes is also a marker for the increased transthyretin tetramer stability attributable to T119M. We found that carriers had a 20% (19 nmol/L) increase in total thyroxine levels compared with noncarriers, but normal thyroid function, indicating that the increased thyroxine level was attributable to increased binding to transthyretin. Thus, our study demonstrates that T119M is functional and stabilizes transthyretin in the general population in vivo.

Figure 4. Cumulative incidence of cerebrovascular disease as a function of age in T119M heterozygotes (green) vs noncarriers (red) from the general population. Cerebrovascular disease was ischemic cerebrovascular disease (transitory ischemic attacks, amaurosis fugax, or ischemic stroke) or hemorrhagic stroke.

derived from normal wild-type transthyretin.6,9 Postmortem studies have shown that senile systemic amyloidosis affects ≈25% of individuals >80 years; however, the impact of this condition in the general population is not known.9,10 Although cardiomyopathy may be the most severe manifestation of senile systemic amyloidosis, this condition has been associated with an increased risk of myocardial infarction in autopsy studies,9,10 and possibly an increased risk of cardioembolic stroke.29,30 Furthermore, vascular deposits of transthyretin amyloid have been found systemically in cases with senile systemic amyloidosis,7,8 indicating that the vascular integrity may be compromised in other organs as well, including the brain. Therefore, we propose that the protective effect on vascular disease associated with the stabilizing variant T119M may be attributable to a reduced deposition of wild-type transthyretin in the wall of small arteries. In addition, in the present study, heterozygotes had increased life expectancy overall and

studies, but may suggest that drugs, such as tafamidis,4,5,25 which kinetically stabilize transthyretin, may have similar beneficial effects in the general population.

The stabilizing effect of the T119M variant in vitro has been convincingly shown by subunit exchange, where an increasing number of T119M subunits in the tetrameric structure associated strongly with decreased fibril formation and increased tetramer stability.16,17 The stabilizing property of the T119M variant is suggested to explain why compound heterozygotes for T119M and disease-causing mutations in TTR are protected from disease.14,15 However, the frequency and impact of T119M in the general population has not been determined previously. Thus far, only a few studies with a limited number of carriers have examined the effect of heterozygosity for T119M on plasma levels on transthyretin20,21,26,27; most suggest

Age at death (years)1009080P=0.04 P=0.01 P=0.03 P=0.61 P=0.002 H a __| 1 am _| i am _| i am _|) i mm _| iCC CT CC CT cc CT cc CT cc CT2 © 2 N Ke) Vv © ) Vv “Ay wv er Se & > e y LeP < Nw Ss S »” s p? << < < <All Vascular Cerebro- Non-vascular After diagnosiscauses disease vascular disease disease of vascular disease

Figure 5. Age at death from all causes, vascular disease, cerebrovascular disease, nonvascular disease, and death after diagnosis of cular disease in T119M heterozygotes vs noncarriers from the general population. Values are median and upper interquartile range. CC indicates noncarriers; and CT, T119M heterozygotes.

general population. Indirectly, these data, therefore, also sug- gest that amyloidosis as a cause of cerebrovascular disease may be relatively common in the general population. These results have potential clinical implications, because they sug- gest that current drugs, which inhibit the amyloid cascade by mimicking the stabilizing effect of T119M on the transthyre- tin tetramer,4,5,25 may slow the progression of vascular deposits leading to cerebrovascular disease in the general population.

among those who died from vascular and cerebrovascular disease, increased life expectancy after diagnosis of vascular disease and increased age at diagnosis of vascular disease compared with noncarriers. These results support that T119M may act through an effect on the vascular wall.

The sporadic form of cerebral amyloid angiopathy with deposition of Aβ, the protein also implicated in Alzheimer dis- ease, is a common condition affecting ≈40% to 50% of indi- viduals aged ≥60 years,13,31 and leading to an increased risk of both ischemic and hemorrhagic cerebral events. Several studies have shown that transthyretin can interact with Aβ and possibly inhibit Aβ amyloid deposition.32–34 Thus, another potential explanation for the apparent protective effect of T119M on cerebrovascular disease could be that T119M sta- bilizes transthyretin in the cerebrospinal fluid and, by binding Aβ, protects against sporadic cerebral amyloid angiopathy.