Neurological Sciences (2024) 45:1675–1684

[Link]

ORIGINAL ARTICLE

Early detection of nerve involvement in presymptomatic TTR​mutation

carriers: exploring potential markers of disease onset
Angela Romano1 · Valeria Guglielmino2 · Giulia Bisogni3 · Andrea Di Paolantonio4 · Andrea Truini5 ·
Angelo Maria Minnella2,6 · Maria Ausilia Sciarrone2 · Francesca Vitali2 · Martina Maceroni2,6 · Eleonora Galosi5 ·
Mario Sabatelli2,3 · Marco Luigetti2,1

Received: 31 August 2023 / Accepted: 30 October 2023 / Published online: 8 November 2023
© The Author(s) 2023

Abstract
Background Hereditary transthyretin (ATTRv) amyloidosis is a heterogeneous, progressive, multisystemic disease with a
life-threatening course if left untreated. Given the current availability of effective therapies, close follow-up of presymp-
tomatic TTR​mutation carriers is essential to recognize disease onset at the earliest sign. In addition to routine techniques,
in recent years several novel tools have been proposed, although a consensus on their use has not been reached yet. In this
paper, we aimed to evaluate possible markers of neuropathic disease onset intended to discriminate clinically asymptomatic
carriers from early symptomatic patients, thus allowing timely treatment initiation.
Methods Thirty-eight presymptomatic carriers were enrolled. Clinical and electrophysiological findings at first evaluation
and follow-up were collected. All carriers underwent an extensive clinical and instrumental evaluation according to the
standard clinical practice. One or more non-routine investigations, whose use in this field is not yet validated (henceforth
“unconventional”), were additionally assessed in a subgroup of individuals.
Results Based on the exclusive use of routine investigations, it was possible to define disease onset in 4/38 carriers dur-
ing the follow-up. Employing additionally one or more “unconventional” tests, abnormal findings, indicative of a possible
“conversion” to symptomatic disease, were detected in further 12 cases. More than half of our study cohort showed findings
suggestive of small nerve fiber (SF) involvement at either invasive or non-invasive tests.
Conclusions A close, multidisciplinary monitoring of presymptomatic TTR​mutation carriers is fundamental, and diagnostic
workup should include both routine and “unconventional” tests. Assessment of SF involvement is important also in non-
endemic countries.

Keywords Hereditary transthyretin amyloidosis · ATTRv-PN · Presymptomatic carriers · Disease onset biomarkers · Early
diagnosis

* Marco Luigetti
mluigetti@[Link] Introduction
1
UOC Neurologia, Fondazione Policlinico Universitario Hereditary transthyretin (ATTRv, v for “variant”) amyloi-
Agostino Gemelli IRCCS, Largo Agostino Gemelli, 8, dosis is a clinically and genetically heterogeneous, adult-
00168 Rome, Italy
onset, autosomal-dominant disease with variable penetrance,
2
Dipartimento Di Neuroscienze, Università Cattolica del caused by mutations in the gene encoding transthyretin (TTR​
Sacro Cuore, Rome, Italy
). This severe disease results from the multisystemic extra-
3
Centro Clinico NeMO Adulti, Fondazione Serena cellular deposition of misfolded variant TTR as insoluble
Onlus-Fondazione Policlinico Universitario Agostino
Gemelli IRCCS, Rome, Italy amyloid fibrils, causing progressive, irreversible organ dys-
4 function, with a poor prognosis if untreated [1–3].
UO Neurologia, Fondazione Poliambulanza, Brescia, Italy
Nowadays, the advent of new drugs acting at distinct
5
Department of Human Neuroscience, Sapienza University, stages of the amyloid cascade is dramatically changing dis-
Rome, Italy
ease course and clinical outcome [4].
6
UOC Oftalmologia, Fondazione Policlinico Universitario
Agostino Gemelli IRCCS, Rome, Italy

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This has raised the need for reliable disease biomarkers screening. Our primary aim was to evaluate the percent-
aimed at monitoring disease progression and objectively age of carriers who converted to symptomatic ATTRv-PN
evaluating the response to pharmacological treatment. during the follow-up according to Consensus Criteria and
Similarly, the availability of effective therapies has pointed recommended routine tests. Moreover, we tried to assess
out the importance of the genetic counseling of at-risk the possible application of some still unvalidated tests in a
relatives of ATTRv patients and the regular monitoring of subgroup of carriers with normal or no definitive findings on
identified carriers of pathogenic TTR​variants [5]. Since the traditional investigations. This was done to evaluate if some
clinical benefit of all disease-modifying treatments (DMTs) of these “unconventional” tests could be more reliable for
is expected to be higher the sooner they are started, strict an early diagnosis, thus expanding the proposed diagnostic
monitoring of presymptomatic carriers, especially of those tools and prompting earlier initiation of DMTs.
close to the predicted age of disease onset (PADO), is indeed The study was conducted at the Institute of Neurology
essential to maximize the effectiveness of treatment. of the “Fondazione Policlinico Universitario Agostino
In 2018, a group of experts met to reach a consensus on Gemelli IRCCS” in Rome, Italy. The local Ethics Commit-
minimum criteria for defining disease onset and the best tee approved the study (Prot. ID 4108), and all participants
approach for the targeted monitoring of presymptomatic car- signed a written informed consent. All procedures were
riers [6]. Such recommendations suggest annual follow-up conducted in compliance with the ethical standards of the
of presymptomatic TTR​mutation carriers starting 10 years Declaration of Helsinki.
before the PADO, suggesting a list of investigations useful For the present study, we consecutively recruited subjects
for this purpose. Given the great clinical variability of the aged > 18 years with a confirmed pathogenic TTR​ variant
disease, a multidisciplinary approach is mandatory, and the and neither signs nor symptoms of multisystemic involve-
type of tests to be used should be tailored according to the ment definitely related to ATTRv amyloidosis at baseline
expected phenotypic presentation for that specific genotype evaluation, in regular follow-up at our Institute.
in each geographic region. The definition of “disease onset” For each subject, the following data were collected: TTR​
is based on a combination of signs/symptoms and abnormal variant, sex, age both at baseline evaluation and at the time
test findings [6]. of conversion (if applicable) or last follow-up, age at onset in
However, to date, there are no reliable biomarkers able other relatives, and the sex of the transmitting parent where
to identify the conversion from an asymptomatic status to available. PADO was estimated according to the literature
an overt illness. [6], using the age of onset of the index case (or the youngest
In the last few years, in addition to the recommended affected relative if many).
standard tests, further exams have emerged and have been We recorded any concomitant known cause or risk factor
gaining attention as potential biomarkers of both disease for neuropathy, such as diabetes, paraproteinemia, alcohol
onset and progression, such as skin biopsy [7, 8], quantita- abuse, vitamin B12 or B9 deficiencies, thyroid or chronic
tive sensory testing (QST) [9, 10], nerve ultrasound [11, kidney diseases, autoimmune disorders, or chronic viral
12], magnetic resonance (MRI) neurography [13], muscle infections (including HCV and HIV).
MRI [14, 15], serum or plasma levels of neurofilament light All enrolled subjects were evaluated roughly yearly
chain (NfL) [16–20], and extensive ophthalmological evalu- through an extensive clinical and instrumental neurological
ation [21]. evaluation according to the standard clinical practice [6]. A
In the present paper, we aimed to evaluate the potential subgroup of individuals with normal or no definitive find-
usefulness and reliability of some still unvalidated (hence- ings on routine tests was also evaluated using one or more
forth, “unconventional”) investigations, in addition to the ancillary tests aimed at exploring large or small nerve fiber
standard tests proposed by the 2018 Consensus, in the early involvement.
diagnosis of symptomatic ATTRv amyloidosis–related poly- Presymptomatic carriers, especially those with a TTR​var-
neuropathy (ATTRv-PN) in order to permit timely treatment iant usually associated with a mixed or cardiac phenotype,
initiation. were also referred to a cardiologist with expertise in amyloid
cardiomyopathies for baseline assessment and follow-up.
However, for the present paper, we chose to focus only on
Patients and methods the polyneuropathy (PN) assessment.

Study design and population Clinical evaluation and conventional investigations

We carried out a single-center, observational, longitudinal, All subjects underwent a complete general and neurologi-
prospective study on a group of presymptomatic TTR​muta- cal examination (including the assessment of the Neu-
tion carriers identified in the context of familiar genetic ropathy Impairment Score, or NIS) [22]. Any change in

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blood pressure within 3 min of standing from a supine “Unconventional tests” evaluating small nerve
position was recorded to look for orthostatic hypoten- fibers
sion [23].
Clinical questionnaires, commonly used to assess the Cutaneous silent period (CSP)
quality of life and detect sensorimotor and/or autonomic
symptoms in ATTRv amyloidosis, were administered to all The CSP was obtained by stimulating the third and fourth
participants. These included the Norfolk Quality of Life- digits with ring electrodes and recording from the abductor
Diabetic Neuropathy (QOL-DN), Questionnaire Douleur brevis pollicis (ABP) and the abductor digiti minimi (ADM)
neuropathique 4 (DN4), and the Compound Autonomic muscles of the non-dominant hand, respectively, using sur-
Dysfunction Test (CADT) [24–26]. face electrodes, in compliance with standard protocols [32,
Conventional nerve conduction studies (NCSs) were 33]. The onset latency was measured at the beginning of the
performed employing a Dantec™ Keypoint® EMG/NCS abrupt EMG suppression, and the end latency at the recovery
equipment. NCSs of bilateral sural and tibial nerves and of EMG activity in each trace. The arithmetic difference
unilateral (non-dominant hand) sensory and motor ulnar between the 2 latencies defined CSP duration.
and median nerves were done according to standard pro-
tocols, using surface stimulating and recording electrodes Skin biopsy
[27]. The sural sensory nerve action potential (SNAP)
was recorded antidromically, whereas upper limb’ SNAPs Skin biopsy was performed and processed at Sapienza
were recorded using orthodromic techniques. Age- and University in a subgroup of carriers. Skin specimens were
sex-adjusted normative values of our laboratory were used obtained using a sterile 3-mm punch from a distal (calf) and
[28, 29]. a proximal (thigh) site, and processed according to protocols
The sudomotor function was assessed by Sudoscan™ described elsewhere [7].
(Impeto Medical, Paris, France), a non-invasive, rapid Intraepidermal nerve fiber density (IENFD) was meas-
test measuring electrochemical skin conductance (ESC) ured as the number of fibers/mm. Age-/sex-adjusted inter-
of feet and hands in response to a low voltage electri- national accepted normative values for both distal and proxi-
cal stimulus. Mean ESC values for the feet and the hands mal IENFD were used [34]. The ratio between distal and
were recorded. For interpreting the results, we referred to proximal IENFD (leg/thigh ratio) was also calculated to
the manufacturer’s reference intervals, obtained on a large evaluate whether small nerve fiber loss was consistent with
adult population [30]. We considered definitely abnormal a length-dependent pattern (cut-off value 0.48) [34].
those values in the “red/lower” range (feet ESC≤50 μS,
hands ESC≤40 μS, suggestive of severe sudomotor dys- In vivo corneal confocal microscopy (CCM)
function). Conversely, for values in the “yellow/interme-
diate” range (feet ESC 51–69 μS, hands ESC 41–59 μS, CCM was performed in a subgroup of carriers with the
indicative of mild/borderline sudomotor dysfunction), we Heidelberg Retinal Tomograph Rostock Corneal Module
considered these findings as suggestive of an early small (HRT-RCM) confocal laser scanning microscope (Heidel-
nerve fiber involvement only if at least another additional berg Engineering, GmBH, Dossenheim, Germany) [21].
test was abnormal as well. A qualitative evaluation was performed by an expert oph-
thalmologist who analyzed the subepithelial nervous plexus
(extension and density), the morphology of corneal nerves
“Unconventional tests” evaluating large nerve fibers (looking for nerve segmentation and/or fragmentation), and
the corneal nerve branch density.
Dorsal sural nerve (DSN)
Serum levels of NfL (sNfL)
NCSs of the DSN were performed in a subgroup of
enrolled subjects having normal findings on conventional Serum aliquots for determination of NfL levels were avail-
NCSs. Bilateral DSN was recorded antidromically, accord- able for a subgroup of carriers.
ing to standard protocols [31]. Blood samples were collected into serum separator tubes,
Age- and sex-related normative values of our laboratory clot for 30 min at room temperature, and then centrifuged at
were used. The ratio between the SNAP amplitudes of 2500 rpm for 15 min. Serum aliquots were stored at − 80 °C
the sural nerve and the DSN was also calculated, using a until analysis. Serum NfL concentration was measured using
cut-off value of 4.17 [31]. We considered definitely patho- the Simple Plex™ cartridge-based Assay on Ella™ platform
logical only those cases where both the age-/sex-adjusted (ProteinSimple, San Jose, CA, USA), according to the manu-
DSN SNAP and the sural/DSN SNAP ratio were abnormal. facturer’s instructions.

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A sNfL cut-off of 37.10 pg/mL was used, as recently pub- Table 1  Demographic data of our study cohort. Qualitative variables
lished [35]. are described with absolute frequencies and percentages (%). Quanti-
tative variables are summarized as mean ± standard deviation; median
[IQR, interquartile range]
Statistical analysis
Presymptomatic TTR​
mutation carriers
The sample was described in its clinical and demographic (n = 38)
characteristics using descriptive statistics techniques. Quali-
tative variables were described with absolute frequencies Male sex 20 (52.60%)
and percentages (%). Quantitative variables were summa- TTR​genotype
rized as mean ± standard deviation (SD), and/or as median - Val30Met 21 (55.3%)
and interquartile range [IQR]. - Phe64Leu 12 (31.6%)
For categorical variables, comparisons were performed - Glu89Gln 2 (5.3%)
by applying the chi-square test, or the Fisher exact test, as - Ala120Ser 1 (2.6%)
appropriate. For quantitative variables, the comparison of - Val122Ile 1 (2.6%)
means between two independent groups was performed with - Ile68Leu 1 (2.6%)
the independent samples t-test or the Mann–Whitney test, Inheritance
as appropriate. The normal distribution of quantitative data - Paternal line 19 (50.0%)
and homogeneity of variance were assessed with the Shap- - Maternal line 11 (28.9%)
iro–Wilk test and Levene’s test, respectively. - ­Unknowna 8 (21.1%)
Statistical analyses were performed using IBM® SPSS® Age at PST (years) 51.21 ± 12.85;
49.00 [40.00–63.00]
Statistics version 25.0. The significance level was set at
Age at first evaluation (years) 52.61 ± 12.14;
p < 0.05.
50.00 [42.75–63.25]
PADO (years) 60.00 ± 8.50;
60.50 [54.00–63.50]
Results Time-to-PADO (years) − 5.26 ± 11.90;
− 7.00 [− 14.00 to 5.75]
Study population Follow-up (months) 29.66 ± 30.67;
17.00 [3.75–55.00]
Thirty-eight presymptomatic carriers were enrolled. Twenty a
This subgroup of carriers had undergone a PST as siblings of an
of them (52.60%) were male, vs. eighteen females (47.40%), affected patient, without a known or confirmed diagnosis in their
with a male-to-female ratio of 1.11. All the enrolled carriers ancestors
were Caucasian (Italian), and all of them harbored a late- Abbreviations: TTR​, transthyretin; PST, presymptomatic genetic test;
onset TTR​variant, except for one female (F#21) carrying a PADO, predicted age of disease onset
V30M variant and having a PADO of 35 years. V30M and
F64L were the most common TTR​variants, being identified length-dependent sensory polyneuropathy as the first disease
in 21 (55.3%) and 12 (31.6%) individuals, respectively. manifestation, at 58 and 51 years old, respectively (Supple-
The demographic characteristics of the study cohort are mentary Table 1), and they could start a DMT according to
summarized in Table 1. minimum Consensus Criteria.
Conversely, in the monitoring, Sudoscan showed abnor-
Routine clinical and instrumental evaluation mal findings (either in the “red/lower” or “yellow/intermedi-
ate” range) in 13/38 carriers (34.21%, Table 2 and Supple-
Enrolled carriers were evaluated yearly for a mean follow-up mentary Table 1), most commonly after their PADO (10/13
of 29.7 (± 30.7) months. vs 3/13 before PADO, p = 0.001).
During the monitoring, 14/38 carriers (36.8%) com- Namely, two carriers (F#28, and M#20), both carry-
plained of symptoms possibly related to ATTRv (mainly ing a V30M variant (a 71-year-old woman with a familiar
distal limb dysesthesia and/or paresthesia, except for one age at onset of 63 years in the oldest affected brother and a
case with gastrointestinal symptoms). 54-year-old man with a paternal inheritance and a PADO of
Clinical examination and Sudoscan at baseline were unre- 60 years), showed definitely abnormal findings on Sudoscan
markable in all enrolled subjects. Conventional NCSs at t0 (i.e., in the “red/lower” range, suggestive of severe sudomo-
excluded a polyneuropathy in the whole cohort. tor dysfunction) as the first sign of neurological impairment;
During the follow-up, 2 siblings carrying a F64L vari- conventional NCSs were normal in both.
ant [one female (F#13) and one male (M#15), with a Additionally, Sudoscan revealed borderline/mild abnor-
paternal inheritance and a PADO of 76 years] developed mal ESC values (i.e., in the “yellow/intermediate” range)

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Table 2  Tests employed to assess large and small nerve fibers The mean age at conversion in this subgroup was
involvement in our study cohort, and count/percentage of abnormal 58.50 ± 8.81 years (median 56.00, IQR 51.75–67.75).
findings. Both routine and “unconventional” tests (in italics) are
reported
The mean PADO was 68.75 ± 8.46 years (median
69.50, IQR 60.75–76.00), with a mean time-to-PADO
Examined cases Abnormal findings of − 10.25 ± 14.48 years (median − 12.00. IQR − 23.25 to
[count (% in relation to [count (% in relation
the whole study cohort)] to the examined 4.50). In 3 out of 4 cases (75%), conversion to symptomatic
cases)] disease was detected before the individual’s PADO (up to
25 years before).
Investigations aimed at exploring large nerve fibers
Routine NCSs 38 (100%) 2 (5.26%)
NCSs of DSN 30 (78.95%) 10 (33.33%)a “Unconventional” tests
Investigations aimed at exploring small nerve fibers
Sudoscan 38 (100%) 13 (34.21%)b DSN
CSP 30 (78.95%) 2 (6.67%)c
Skin biopsy 19 (50.0%) 11 (57.89%) Evaluation of DSN was available in 30/38 enrolled subjects,
CCM 7 (18.42%) 7 (100%) all of which with normal conventional NCSs. Ten of the
examined subjects (33.33%) had both abnormal “corrected”
a
Only cases where both the age-/sex-adjusted DSN SNAP and the SNAP amplitude and sural/DSN SNAP ratio (mean ratio
sural/DSN SNAP ratio were abnormal were considered definitely
pathological
4.57 ± 1.47, median 4.68, IQR 3.14–5.30).
b
Both cases indicative of severe and mild/borderline sudomotor dys-
function are included CSP
c
Given the absence of normative data for CSP, we considered defi-
nitely abnormal only those cases for which it was absent CSP was obtained in 30 carriers, and it was absent from both
Abbreviations: NCSs, nerve conduction studies; DSN, dorsal sural ABP and ADM in 2 of them (Table 2 and Supplementary
nerve; CSP, cutaneous silent period; CCM, in vivo corneal confocal
microscopy Table 1). Both cases showed abnormal findings on other
tests as well.
Namely, skin biopsy proved signs of skin denervation in
at lower and/or upper limbs in further 11 cases (feet ESC: one case (a 60-year-old man carrying a Val122Ile variant,
mean 63.55 ± 7.38 μS; hands ESC: mean 61.09 ± 10.71 μS). with a PADO of 72 years, complaining of distal limb par-
Among this latter group, 8 agreed to undergo skin biopsy, esthesia; M#35). Additionally, he showed abnormal NCSs
which confirmed abnormal findings in 6 of them. Overall, of DSN [36].
7/11 individuals with “intermediate” ESC values had at The other carrier (a 45-year-old man harboring a
least another abnormal test (namely, skin biopsy in 6 cases, Glu89Gln variant, with a PADO of 56 years; M#34) com-
NCSs of DSN in 3 cases, CCM in 2 cases). As concerns the plained of severe gastrointestinal symptoms (namely daily
remaining 4 carriers (F#3, M#4, M#8, M#27), showing nor- diarrhea), suggesting a possible autonomic dysfunction. The
mal findings on all other neurological investigations besides hypothesis of a prevalent small nerve fiber involvement was
Sudoscan, none of them complained of any symptoms, and further supported by the evidence of abnormal findings on
none of them was then considered “converted” to sympto- CCM. A skin biopsy has been then recommended but the
matic disease. patient refused.
Interestingly, in 2/11 cases (M#26, M#27), abnormal As concerns the remaining 28 carriers, given the absence
Sudoscan findings were suggestive of a non-length-depend- of normative values, we cannot draw any conclusion. How-
ent pattern, but both carriers refused to undergo skin biopsy. ever, they showed a higher onset latency and a tendency
towards a shorter duration as compared to a control group,
Overall conventional diagnostic tests as already published [37].

Globally, considering routine tests and minimum Consensus Skin biopsy

Criteria, 4 out of 38 subjects (10.53%) had a confirmed diag-
nosis of symptomatic ATTRv-PN and could start specific Skin biopsy was performed in 19/38 subjects (Table 2 and
anti-amyloid treatment. Supplementary Table 1). The mean age at skin biopsy was
Among these, 2 patients initially developed length- 54.74 ± 12.81 years. Considering age- and sex-adjusted
dependent sensory PN, whereas the remaining 2 patients normative values, a reduced IEFND was evident in 11/19
showed small nerve fiber involvement as the first sign of (57.89%), in 5 of whom before their PADO (vs 6 after
neurological impairment. PADO, p = 0.690). Actually, a 42-year-old man carrying a

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Phe64Leu variant (M#16) showed abnormal findings at a whereas CSP was absent in 2 cases (Table 2 and Supple-
time-to-PADO of approximately − 20 years. mentary Table 1). Interestingly, observed findings were sug-
The mean proximal (thigh) IEFND was 10.23 ± 2.07 fib- gestive of a non-length-dependent pattern in 11 cases (i.e.,
ers/mm (median 10.62, IQR 9.34–11.74). The mean distal 9 cases documented by skin biopsy, 2 cases by Sudoscan).
(leg) IEFND was 7.14 ± 1.55 fibers/mm (median 7.12, IQR
6.37–8.33). The mean leg/thigh IEFND ratio was 0.71 ± 0.24 sNfL
fibers/mm (median 0.68, IQR 0.56–0.86). Notably, in 9/11
carriers, skin denervation was not consistent with a length- Serum levels of NfL were available for 35 out of 38 car-
dependent pattern (leg/thigh ratio > 0.48). riers (all the 16 “converted” carriers vs. 19 out of 22 still-
presymptomatic carriers) (Supplementary Table 1).
CCM Values above the cut-off threshold of 37.10 pg/mL were
detected only in the subgroup of “converted” carriers (9/16).
Fourteen eyes of 7 carriers were analyzed using CCM. None Mean values of sNfL were significantly higher in
of them complained of ocular symptoms. All the examined this subgroup (44.91 ± 39.34 pg/mL, median 37.14,
eyes (100%) presented a rarefied subepithelial nervous IQR 11.42–74.78) than in still-presymptomatic carriers
plexus (for extension and density), nerve segmentation and/ (13.67 ± 10.56 pg/mL, median 11.70 pg/ml, IQR 4.12–21.20;
or fragmentation (increased beads), and a reduced branch mean difference 31.24 pg/mL; p = 0.005).
density (Table 2 and Supplementary Table 1).
Six out of 7 carriers underwent skin biopsy as well, prov- Comparisons between the whole subgroup
ing abnormal in 3 of them (50.0%). of “converted carriers” and the subgroup
of still‑presymptomatic carriers
Overall “unconventional” diagnostic tests
Considering the whole subgroup of “converted” carri-
Globally, considering “unconventional” diagnostic tests in ers (n = 16), these individuals were significantly older
addition to conventional exams, 16 individuals (42.10%) (61.19 ± 11.51 years) as compared to the subgroup of still-
could have been considered affected, as compared to 4 indi- presymptomatic carriers (n = 22; 50.05 ± 10.22 years) (mean
viduals (10.53%) considered “converted” based on only rou- difference 11.14 years; p = 0.003). However, there was not
tine tests proposed by the Consensus. Thus, a diagnosis of any statistically significant difference between these 2 sub-
disease onset could have been reached in further 12 cases groups in terms of time-to-PADO, although being shorter in
[in 5 before their PADO (up to 21 years before) vs 7 after the first group (− 2.06 ± 12.77 years vs. − 7.59 ± 10.94 years;
PADO, p = 0.163]. mean difference 5.53 years; p = 0.160).
The mean age at conversion in this further subgroup of
12 individuals was 62.08 ± 12.49 years (median 65.50, IQR
48.50–72.75). The mean PADO was 61.42 ± 6.36 years Discussion
(median 62.00, IQR 55.25–66.00), with a mean time-to-
PADO of 0.67 ± 11.52 years (median 4.50. IQR − 10.25 to In this study, we aimed to evaluate the potential use of sup-
11.00). plementary, not yet validated investigations, in addition to
standardized diagnostic tests, in the early diagnosis of symp-
Assessment of large and small nerve fiber tomatic ATTRv-PN amyloidosis. In our cohort, it was pos-
involvement sible to confirm disease onset using conventional instrumen-
tal tools in approximately 10% of individuals (4/38) during
Overall, large nerve fiber involvement was evident in 12/38 their periodic monitoring. Indeed, in further 12 cases (nearly
carriers (31.58%). In 2 of them, length-dependent polyneu- 32%), one or more “unconventional” tests resulted abnormal,
ropathy was detected by routine NCSs. Both reduced SNAP suggesting a possible “conversion” to overt disease also in
of DSN and abnormal sural/DSN SNAP ratio were evident those cases showing normal or not definitive findings on
in further 10 individuals with normal findings on standard clinical examination and routine investigations. Interestingly,
NCSs (Table 2 and Supplementary Table 1). in many cases, conversion occurred long before PADO (up
Twenty-one/38 carriers (55.26%) showed abnormal find- to 25 years earlier), supporting the importance of starting
ings in at least one test aimed at exploring small nerve fibers monitoring even before the 10-year cut-off.
(including both routine and “unconventional” exams). Glob- Defining disease onset in ATTRv amyloidosis is chal-
ally, Sudoscan revealed abnormal findings in 13 cases (also lenging, as there is significant variability in disease
including those cases with mild/borderline values), skin expression across individuals and geographic areas. More-
biopsy in 11 cases, and CCM in all the 7 examined cases, over, ATTRv amyloidosis is a multisystemic disease, and

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detecting the first involved tissue requires a multidisci- The cutaneous silent period and in vivo corneal confocal
plinary approach carried out by expert specialists from microscopy are further non-invasive, easy-to-perform tech-
across many fields. niques aimed at exploring SFs. Even if underused, they seem
From the neurological point of view, in the follow-up of to have an emerging role in the monitoring of disease onset
TTR​mutation carriers, an extensive neurological examina- and progression in ATTRv amyloidosis.
tion and traditional NCSs are essential to identify and evalu- CSP is a spinal inhibitory reflex whose afferent arc is
ate the extent of peripheral nerve involvement. NCSs are mediated almost exclusively by high-threshold, Aδ sensory
also essential as longitudinal assessment measures to detect nerve fibers [32, 45–47]. Despite the absence of normative
any change over time from the baseline in neurophysiologi- data, recent reports suggest an early alteration of CSP in
cal data [6, 38]. Such a change may indeed precede by as terms of onset latency and duration both in ATTRv patients
much as 2 years the onset of symptoms [38]. and presymptomatic carriers [37, 48].
The sural nerve is the sensory nerve most commonly CCM, providing in vivo imaging of corneal nerve fib-
assessed in the diagnostic workup of suspected length- ers, represents another promising sensitive tool in the early
dependent polyneuropathy. However, the segment routinely detection of nerve damage in peripheral neuropathies mainly
explored in daily practice is proximal to the sites initially affecting small fibers [49], including ATTRv-PN amyloi-
affected by distal polyneuropathies. The study of the sural dosis [21, 50, 51]. In patients with SFN, abnormalities on
lateral dorsal cutaneous branch could overcome this limita- CCM may sometimes even precede loss of intraepidermal
tion, therefore allowing early detection of peripheral nerve nerve fibers in skin biopsies [49], as supported by our find-
damage [27, 31, 39]. In our cohort, longitudinal conven- ings as well.
tional NCSs were able to detect sensory polyneuropathy as Nevertheless, although being available only in a few highly
the first disease sign only in 2 cases. Conversely, the study of specialized centers, skin biopsy still represents the gold stand-
the DSN proved abnormal in more than 30% of the examined ard for the diagnosis of SFN. Providing information about
carriers, supporting its potential role in early diagnosis of somatic and autonomic SFs, this minimally invasive tech-
ATTRv-PN amyloidosis. nique appears to be a promising tool in the assessment of
Moreover, routine electrophysiological studies are often presymptomatic TTR​ mutation carriers. Interestingly, skin
completely normal in those cases characterized by early, biopsy can disclose pathological changes even several years
exclusive involvement of small nerve fibers (SFs). Impor- before the onset of symptoms, supporting the role of IENFD
tantly, SF involvement is often the first manifestation in quantification as a possible biomarker for subclinical ATTRv
early-onset ATTRv amyloidosis, while it is usually consid- disease [7]. Moreover, this technique can help to shed light
ered less prominent in late-onset cases, especially in the very on pathophysiologic mechanisms underlying SFNs. The leg/
early stages. thigh IENFD ratio can be used indeed as a parameter to dis-
Several non-invasive techniques, such as sympathetic skin criminate between length-dependent small-fiber neuropathy
response (SSR), QST, and laser evoked potentials (LEPs), and small-fiber sensory ganglionopathy [34]. Curiously, in a
can be useful to investigate small-fiber neuropathy (SFN) recent paper by Leonardi et al., skin denervation was some-
[40, 41]. However, all of them are time-consuming and not times more pronounced in proximal than distal sites, suggest-
available anywhere. ing a non-length-dependent pattern in at least a subgroup of
Sudoscan is a simple, quick diagnostic test, whose role subjects [7]. In line with literature data, a reduced IEFND was
in ATTRv amyloidosis has already been assessed [42–44], evident in more than half of the examined cases of our cohort,
showing a high diagnostic accuracy, especially in the early- with most cases (9/11) showing findings suggestive of a non-
onset population. Hence, its application as a screening test length-dependent involvement.
in TTR​ mutation carriers has already been recommended Globally, more than half of our study cohort (21/38) showed
by the 2018 Consensus, mainly in endemic areas. Con- abnormal findings on one or more (either invasive or non-inva-
versely, in non-endemic countries (such as Italy), although sive) tests aimed at exploring SFs. Altogether, our data support
a demonstrated role in monitoring disease progression and the importance of exploring not only large nerve fibers but also
severity [43, 44], its utility for early diagnosis has always small nerve fibers in the monitoring of TTR​presymptomatic
been considered uncertain. However, in our cohort, 2 people carriers, independently from the geographic origin.
first developed confirmed SFN (documented by Sudoscan), Interestingly, in a subgroup of individuals, observed find-
with initial sparing of large-diameter myelinated nerve fib- ings were suggestive of a non-length-dependent pattern. As
ers. Slightly abnormal ESC values have been documented a matter of fact, even if progressive, length-dependent poly-
in further 11 individuals. All but 4 (7/11) presented at least neuropathy is the most common neurological presentation in
another abnormal test, confirming the value of this tool in non-endemic areas, other phenotypes, including multifocal
the early detection of small nerve fiber impairment also in neuropathy with onset in the upper limbs, have been reported
the presence of mild/borderline sudomotor dysfunction. as well [52].

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1682 Neurological Sciences (2024) 45:1675–1684

Conclusions Pfizer, and Csl Behring. G.B. received financial grants (honoraria and
speaking) from Alnylam, and travel grants from Pfizer, Kedrion, and
Grifols. A.D.P. received financial grants (honoraria and speaking) from
Overall, our data support the importance of the periodic Akacia, Alnylam, Sobi, and travel grants from Akcea. M.A.S. received
monitoring of TTR​ mutation carriers, suggesting the need travel grants to attend scientific meetings from Sobi. F.V. received
for broadening the neurological diagnostic workup in the travel grants to attend scientific meetings from Alnylam. M.S. received
financial grants (honoraria and speaking) from Akcea. M.L. received
context of a multidisciplinary evaluation. This is fundamen- financial grants (honoraria and speaking) from Akcea, Alnylam, and
tal to detect disease onset at the earliest convenience, since a Pfizer, and travel grants from Akcea, Alnylam, Sobi, Pfizer, Kedrion,
subclinical stage, to effectively modify the disease’s natural Csl Behring, and Grifols. All other Authors reported no disclosures.
history and prognosis.
However, we cannot certainly imagine the actual appli- Open Access This article is licensed under a Creative Commons Attri-
cation of all these techniques in daily clinical practice. It bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
should be thus necessary to define the most accurate tests as you give appropriate credit to the original author(s) and the source,
in terms of sensibility and specificity to avoid performing provide a link to the Creative Commons licence, and indicate if changes
unnecessary “overlapping” investigations, aimed at explor- were made. The images or other third party material in this article are
ing the same system. In addition to the investigations already included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
approved and recommended by the Consensus, from a neu- the article’s Creative Commons licence and your intended use is not
rological point of view, it would be worth applying the permitted by statutory regulation or exceeds the permitted use, you will
study of the dorsal sural nerve and the skin biopsy (or the need to obtain permission directly from the copyright holder. To view a
corneal confocal microscopy) in order to combine a neuro- copy of this licence, visit [Link]
physiological test aimed at exploring the large nerve fibers
(probably with a higher sensibility as compared to standard
NCSs) with a technique intended to assess small nerve fiber
involvement. References
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