Hereditary Transthyretin Amyloidosis- Clinical and Genetic Characteristics of a Multiracial South-East Asian Cohort in Singapore

ABBREVIATIONS

AAO

age at onset

ACMG

American College of Medical Genetics

ATTRv

amyloidosis, hereditary transthyretin amyloidosis

ECG

electrocardiography

VUS

variants of unknown significance

INTRODUCTION

Hereditary transthyretin amyloidosis (ATTRv), an autosomal dominant disorder caused by missense mutations of the transthyretin (TTR) gene, leads to multisystemic extracellular deposition of transthyretin amyloid fibrils. Genotypic and phenotypic heterogeneity between populations have been described(1). The disease onset ranges from the 20 s to the 80 s [2, 3], and may typically be classified as early-onset (age < 50 years) and late-onset disease (age≥50 years)(3).

To date, cases of ATTRv amyloidosis have mainly been documented from endemic regions including Portugal, Japan, Majorca, Cyprus and Sweden (3–8). The ATTR-V30 M (p.Val50Met) variant is the most common reported variant associated with ATTRv amyloidosis in endemic areas. Consequently, it is also the best studied genotype. Studies looking into genotype-phenotype correlation involving early and late-onset disease, as well as disease anticipation, have largely been reported on cohorts of ATTR-V30 M patients [3, 9].

Apart from ATTR-V30M, there are currently approximately 130 other known pathogenic variants of the TTR gene. The ATTR-A97S (p.Ala117Ser) variant is the most common variant reported amongst ethnic Chinese Malaysian patients with ATTRv amyloidosis [10, 11]. This variant has also been frequently observed among ethnic Chinese patients in Taiwan (12–14). ATTRv amyloidosis in other racial groups within South-East Asia is however, not well studied, and genotype-phenotype correlations in ATTRv amyloidosis amongst South-East Asian patients is currently underreported.

Although treatment options for ATTRv amyloidosis were limited previously, recent development of disease modifying therapies have shown significant efficacy in slowing clinical progression of the disease [15]. In light of this, the need to diagnose this treatable genetic disease within different populations worldwide has gained importance. Our study aims to describe the phenotypic and genotypic profile of patients with ATTRv amyloidosis in Singapore, a multi-racial nation-state in South-East Asia.

METHODS

We identified patients with genetically proven ATTRv amyloidosis, diagnosed at the National Neuroscience Institute (Tan Tock Seng Hospital and Singapore General Hospital) between January 2007 and June 2020. All patients were evaluated by neuromuscular specialists.

The diagnosis of ATTRv amyloidosis was based on the detection of a TTR gene mutation via Sanger sequencing of the TTR gene(16). Retrospective chart review was performed for all patients. Collated data included the following: gender, age at onset of disease, family history, clinical characteristics, TTR-FAP clinical staging [17], biopsy studies, electrophysiological tests, genetic test for the TTR gene, electrocardiography (ECG), transthoracic echocardiography, cardiac magnetic resonance imaging (MRI), and treatment received. The age at onset (AAO) of disease was defined as the age at which the patient first developed neurological or systemic symptoms suggestive of ATTRv amyloidosis. Patients were grouped into (a) early onset–AAO of symptoms < 50 years, and (b) late-onset–AAO≥50 years. A positive family history was defined as the presence of either an affected family member with a reported history of somatic neuropathic symptoms or with genetically confirmed ATTRv amyloidosis. Time to diagnosis was defined as time from onset of first symptom to time of definitive diagnosis of ATTRv amyloidosis. Delayed diagnosis was defined as time from onset of first symptom to time of diagnosis of more than 24 months. Clinical staging of disease was done based on the ATTRv amyloidosis clinical staging scale as follows: stage 0 –asymptomatic, stage 1 - ambulatory without walking aids, stage 2 -ambulatory with walking aid, and stage 3 - wheelchair/bedridden [17]. Severity of cardiac symptoms at the time of diagnosis was graded based on the New York Heart Association (NYHA) functional classification [18].

Somatic neuropathy was defined as any peripheral neuropathy with involvement of sensory and/or motor nerves and can be subdivided into either a small fibre neuropathy or large fibre polyneuropathy. Small fibre neuropathy was defined as the presence of sensory symptoms or signs, with predominant involvement of pain and temperature modalities, in the presence of a normal nerve conduction study. Large fibre polyneuropathy was defined as the presence of peripheral neuropathy in the presence of an abnormal nerve conduction study.

Carpal tunnel syndrome was classified as a connective tissue manifestation of ATTRv amyloidosis. It was defined based on standard electrophysiological criteria, with/ without associated clinical symptoms and signs [19]. Autonomic dysfunction was defined based on presence of the following symptoms/ signs in isolation or combination: recurrent orthostatic hypotension, syncope, diarrhea or constipation, urinary retention or incontinence, hypohidrosis or hyperhidrosis, erectile dysfunction, and/or involuntary loss of weight. Orthostatic hypotension was defined as a sustained reduction of systolic blood pressure of≥20 mmHg or diastolic blood pressure of≥10 mmHg within 3 min of standing from a supine position [20].

Established electrophysiological criteria were used to define axonal neuropathy and demyelinating neuropathy [21]. Where performed, the autonomic function test consisted of the following assessments: sympathetic skin response for evaluation of sudomotor function; heart rate response to posture change and deep breathing and Valsalva ratio for evaluation of cardiovagal function; blood pressure change from supine to standing and on 60 degrees tilt for evaluation of sympathetic adrenergic function. Cardiac MRIs were reported by a cardiovascular radiologist. The following radiological features were used to identify cardiac amyloid: 1. diffuse sub-endocardial or transmural late gadolinium enhancement, 2. increased myocardial native T1 signal, 3. increased extracellular volume fraction, and 4. inability to suppress myocardial signal with phase sensitive inversion recovery late gadolinium enhancement imaging [22].

For patients who had not undergone prior genetic testing of the TTR gene, Sanger sequencing of exons 2 to 4 of the TTR gene of DNA isolated from frozen sural nerve biopsy specimens was performed. DNA was first extracted from frozen nerve tissue using Reliaprep gDNA tissue Miniprep system (Promega) following manufacturer’s guidelines. The TTR gene (NG_009490.1) exons 2 to 4 were amplified by polymerase chain reaction using exTEN 2X PCR Master Mix (1st BASE) and primers (see supplementary Table 1) at an annealing temperature of 58°C. After amplification, PCR products were treated with FastAP Alkaline Phosphatase (Thermo Scientific) and Exonuclease I (Thermo Scientific) and subjected to direct sequencing using BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) on an ABI 3130xl Genetic Analyzer (Applied Biosystems).The identified TTR variants were referenced against public databases including ClinVar (www.ncbi.nlm.nih.gov/clinvar/) and Varsome (varsome.com), and their pathogenicity was classified based on the American College of Medical Genetics (ACMG) criteria [23]. These variants were annotated based on the amino acid position of the mature protein [24] as well as nomenclature recommended by the Human Genome Variation Society [25]. For patients harboring TTR variants of unknown significance (VUS), liquid chromatography tandem mass spectrometry (LC MS/MS) was performed on sural nerve biopsy specimens for molecular confirmation of transthyretin protein deposits (Mayo clinic laboratories), to confirm pathogenicity.

Statistical analyses were performed with IBM SPSS Statistics, version 23, IBM. Demographic, clinical, and investigative parameters were compared between patients with early and late onset disease, and patients of different genotypes. A comparative analysis of median time from symptom onset to diagnosis between patients with and without positive family history was performed using the Mann-Whitney U test. Two-tailed p values of < 0.05 were considered statistically significant.

This study was approved by the Singapore Health Services Centralized Institutional Review Board (CIRB 2018-2341). Written informed consent was obtained from all patients.

RESULTS

We identified 29 patients from 19 unrelated families. The demographics and clinical features are summarized in Table 1. The individual patients’ clinical and genotype data are summarized in supplemental Table 2. Seventeen patients (58.6%) were male. The median age of onset was 57 (mean 56, range 30–70) years. Twenty-four (82.8%) patients were Singaporean, four were from Vietnam, and one from Indonesia. Of the 24 Singaporeans, 21 were Chinese, one Malay, and two were of Burmese origin.

The median time from symptom onset to diagnosis was 26 months (range 6–110 months); no significant difference was noted between patients with and without family history [median 22 (range 6–110) vs 34 (range 12–66) months, p = 0.26]. Delayed diagnosis occurred in 16/29 (55.2%) patients, ten of whom had a positive family history (reasons for delayed diagnosis are detailed in supplemental Table 2).

At initial presentation, 10 (34.5%) had somatic neuropathy, nine (31.0%) had carpal tunnel syndrome, eight (27.6%) had autonomic dysfunction, while two (6.9%) had cardiac symptoms. At diagnosis, 22 (75.9%) were in stage 1, five (17.2%) stage 2, and two (6.9%) stage 3. At the most recent clinical follow-up [time from symptom onset to last follow up: 62 (range 6–161) months], 26 (89.7%) patients had somatic neuropathy, 23 (79.3%) had autonomic dysfunction, 17 (58.7%) had carpal tunnel syndrome while nine (31.0%) had cardiac dysfunction.

Salient investigations and treatment are summarized in Table 2. Twenty-eight (96.6%) patients had abnormal NCS results. Of the 12 patients who underwent autonomic function evaluation, 11 (91.7%) had features of autonomic dysfunction. 24/24 (100.0%) had abnormal echocardiographic findings, while 11/11 (100.0%) had cardiac MRI findings demonstrating features of cardiac amyloidosis. Twenty-three patients underwent histological evaluation; 16/17 (94.1%) had amyloid deposits in sural nerve tissues while 6/6 (100.0%) had amyloid deposits in extraneural tissues.

Five patients had early-onset disease (Table 3; Supplementary Table 2). Median age at onset was 42 (mean 39, range 30–48) years. All five had a positive family history. The following variants were identified in this group: ATTR-V30M (p.Val50Met), ATTR-G47A (p.Gly67Ala), ATTR-S50I (p.Ser70Ile), ATTR-A97S (p.Ala117Ser). Two female early-onset patients had mothers who were in the late-onset group: patient 23 [AAO 30; mother (patient 2): AAO 53; ATTR-S50I], and patient 27 [AAO 46; mother (patient 7): AAO 59; ATTR-A97S]. Patient 1 (AAO 42), a female, had an affected father (AAO 50; ATTR-G47A), while a male sibling pair, patients 5 (AAO 30) and 25 (AAO 48), had an affected mother (AAO 70; ATTR-V30M). One young-onset patient with ATTR-A97S presented with isolated, unilateral carpal tunnel syndrome; histological examination of the transverse carpal ligament of the patient revealed the presence of amyloid deposits on congo-red staining.

There were 24 patients in our cohort with late-onset disease (Table 3; Supplementary Table 2). Median age at onset was 61 (mean 60, range 51–70) years. In this group, 17 (70.8%) had a positive family history, of which nine (37.5%) had a positive parental history. We identified the following variants in this group: ATTR-V30M (p.Val50Met), ATTR-R34T (p.Arg54Thr), ATTR-S50I (p.Ser70Ile), ATTR-E66D (p.Glu86Asp), ATTR-A81V (p.Ala101Val), ATTR-H88R (p.His108Arg) and ATTR-A97S (p.Ala117Ser). The genotype frequencies are summarized in Table 4. 8/24 (33.3%) had carpal tunnel syndrome, 8/24 (33.3%) had somatic neuropathy, 6/24 (25.0%) had autonomic dysfunction and 2/24 (8.3%) had cardiac dysfunction at onset.

We identified two late-onset disease patients (patient 24 and 6) with variants previously classified as variants of unknown significance, namely ATTR-E66D (p.Glu86Asp, ClinVar variant ID: 935886) and ATTR-A81V (p.Ala101Val, ClinVar variant ID: 495842). Clinical features of these patients are described below:

Patient 24, a Chinese female with ATTR-E66D variant (c.258A>T, p.Glu86Asp, Table 4), presented at 64 years of age with painless weakness and numbness sequentially affecting the lower limbs followed by upper limbs. She had no positive family history. She subsequently developed bilateral carpal tunnel syndrome at age 69, followed by congestive cardiac failure, and gastrointestinal symptoms [diarrhea, gastroparesis] at 72 years. Sural nerve biopsy revealed the presence of amyloid deposits in epineural blood vessels and connective tissue (Supplementary Figure 1a). LC MS/MS performed on paraffin embedded sural nerve biopsy specimen detected a peptide profile consistent with transthyretin amyloid deposition. This variant has not been reported in control databases, including GnomAD and ExAC; it has been predicted through multiple lines of in-silico tools to be pathogenic. It affects a highly conserved amino acid position (Genomic Evolutionary Rate Profiling (GERP) score: 5.5399) within a hot- spot region in exon 3 of the TTR gene, with several other pathogenic variants in the close vicinity. Based on the above evidence, the variant is reclassified as pathogenic based on the ACMG guideline(23).

ATTR-A97S was the most common variant (Table 4) in our cohort, identified in 14 patients across seven ethnic Chinese families. This variant constituted 66.7%of ethnic Chinese patients and 48.3%of the entire cohort. All except one (92.9%) patient had late-onset disease. Six (42.9%) presented with carpal tunnel syndrome and four (28.6%) had somatic polyneuropathy (all large fibre polyneuropathy). Thirteen (92.9%) patients developed both large fibre polyneuropathy and autonomic dysfunction over the course of follow up [median 62 (range 26–118) months]. The TTR-V30M variant accounted for only four (13.8%) patients in our cohort.

Of the 23 patients diagnosed after 2012, 16 (69.6%) received disease modifying therapy (Table 2). Fifteen patients were treated with diflunisal (one patient also underwent liver transplantation), while one was treated with tafamidis. Ten patients died during the course of follow-up [median time from symptom onset to death: 90 (range 38 –161) months] (Table 1). Six patients (60.0%) died from cardiac causes and two (20.0%) from pneumonia; cause of death was uncertain in two patients.

DISCUSSION

We present the phenotypic and genotypic characteristics of a cohort of South-East Asian patients with ATTRv amyloidosis. Our cohort includes the first description of ATTRv amyloidosis in an ethnic Malay patient, as well as patients from Indonesia, Vietnam and Myanmar, suggesting that ATTRv amyloidosis may be under-diagnosed in the South-East Asian region. We also report two novel pathogenic ATTRv amyloidosis variants.

While carpal tunnel syndrome is classified as a non-specific early connective tissue manifestation ATTRv amyloidosis [30], histological examination of transverse carpal ligament for the presence of transthyretin amyloid can serve as evidence to support the presence of systemic amyloid deposition [31] and hence guide the early initiation of disease modifying therapy, as illustrated in our patient. Echoing prior reports stating that carpal tunnel syndrome can be the first symptom of ATTRv amyloidosis and can precede the onset of other symptoms by up to ten years [32], we observe that carpal tunnel syndrome is also a common first symptom within our cohort, accounting for 9 (31.0%) of patients. Further studies are required to better identify characteristics of carpal tunnel syndrome associated with ATTRv amyloidosis in non-endemic populations in order to guide early diagnosis.

Anticipation has previously been reported in patients with the ATTR-V30 M variant, most pronounced in mother-to-son inheritance, but also noted in mother-to-daughter and father-to-son inheritance [9, 27]. Our study revealed anticipation in two mother-daughter pairs with ATTR-S50I and ATTR-A97 S variants. This suggests that anticipation with mother-to-daughter inheritance may occur in genotypes other than the ATTR-V30 M variant. Further verification is important to guide genetic counselling in families of affected patients.

Delayed diagnosis of sporadic late-onset individuals, a phenomenon encountered in non-endemic populations [28], is a significant problem, since studies have shown that many disease modifying therapies are not effective in late stage ATTRv amyloidosis [29]. In our cohort, delayed diagnosis occurred in 16/29 (55.2%) patients. Intriguingly, there was no significant difference in median time from symptom onset to diagnosis in patients with and without positive family history. Several possible causes may contribute to delayed diagnosis, including local cultural norms of not openly discussing medical disorders even amongst family members. Measures to promote early and open discussion regarding genetic testing for family members of affected individuals may facilitate early diagnosis. Interventions aimed at increasing physician awareness of the condition, as well as availability of non-invasive diagnostic tests for patients with neuropathy of uncertain etiology should be explored to facilitate early diagnosis of ATTRv amyloidosis.

Similar to other non-endemic populations, the genetic epidemiology of our cohort is unique to this region. The most common genotype was ATTR-A97S; affecting 66.7%of ethnic Chinese patients and 48.3%of the entire cohort. This is similar to reports from Malaysian Chinese and Taiwanese cohorts [10, 13], likely explained by the migration of many ethnic Chinese from Southern China to Singapore, Malaysia and Taiwan previously. Clinical characteristics of these patients are similar to previously described cohorts with the ATTR-A97S variant [12, 13], with carpal tunnel syndrome being the most common presenting symptom, and subsequent progression to large fibre and autonomic neuropathies during the course of follow-up. Furthermore, we identified two ethnic Chinese patients with novel pathogenic variants of ATTRv amyloidosis, namely ATTR-E66D and ATTR-A81V. Both patients presented with large fibre polyneuropathy and later developed cardiac involvement. Our study further expands the repertoire of pathogenic variants known to cause ATTRv amyloidosis.

The notably low prevalence of the ATTR-V30 M variant (13.8%) is unlike that seen in endemic populations. Prior treatment trials of disease modifying therapy, such as tafamidis, have focused on Western cohorts with predominantly ATTR-V30 M [33] genotype, hence limiting generalizability of its therapeutic effect to other populations harboring different ATTRv amyloidosis genotypes. Similarly, while the role of liver transplantation in patients with ATTR-V30 M variant is established [34], its benefit in other genotypes is less clear. So far, only the treatment trial for patisiran (APOLLO) included a significant proportion of Asian patients with ATTRv amyloidosis [35]. Further studies of liver transplantation and disease modifying agents should preferably include Asian patients, with non ATTR-V30 M genotypes, to accurately ascertain the efficacy of these treatments in such patients.

Our study also demonstrates the limited use of disease modifying agents in our cohort. Following publication of the treatment trial on tafamidis [33] in 2012, only 66.7%of patients diagnosed after 2012 were initiated on disease modifying treatment. A majority of these patients were treated off-label with diflunisal, a drug which, while not licensed for use for the treatment of ATTRv amyloidosis in both Europe and the United States of America, is more affordable compared to the other disease modifying agents. One patient also underwent liver transplantation while another patient was treated with tafamidis. A significant proportion declined treatment due to concerns regarding medication costs as well as medication side effects. The low usage of other disease modifying agents apart from diflunisal is likely secondary to factors relating to the local health financing system. With increasing recognition of the disease burden associated with ATTRv amyloidosis and emphasis on precision medicine, collaborative efforts between referral centers to better capture regional epidemiology of the disease and develop cost-effective treatment strategies are needed in South-East Asia [36].

There are some limitations to our study. Firstly, this was a retrospective study with inherent biases of a retrospective chart-review. Clinical and laboratory evaluations were clinician-determined, and therefore not standardized. Measurements such as neuropathy impairment score and modified body mass index were not performed, limiting our ability to accurately quantify longitudinal disease progression. Secondly, our cohort was limited to patients presenting or referred to neurology services; hence, ATTRv amyloidosis patients with cardiac predominant disease and minimal or no neuropathic symptoms would have been missed, thus limiting precise phenotypic correlations. Lastly, we only identified five patients with early-onset disease, with a limited longitudinal follow-up period in two of these patients, again limiting genotype-phenotype correlation. Nonetheless, our study provides useful insights into the phenotypic characteristics and genotypic features of a South-East Asian cohort diagnosed with ATTRv amyloidosis.

In conclusion, our study of a multiracial, multinational South-East Asian cohort with ATTRv amyloidosis demonstrated mutations in ethnic Malay, Indonesian, Vietnamese and Burmese patients that were not previously described. The most common variant in our cohort is ATTR-A97S. We report two new variants in the TTR gene that were confirmed to be pathogenic. We also demonstrate the potential for disease anticipation in non ATTR-V30 M disease genotypes and an atypical clinical presentation in a patient with early-onset ATTR-A97S variant. Our study findings emphasize the need for collaborative studies to prospectively evaluate the phenotypic and genotypic features of non-endemic ATTRv amyloidosis cohorts worldwide so as to facilitate inclusivity in multi-center, disease modifying treatment clinical trials.