Hereditary transthyretin amyloidosis in mainland China: a unicentric retrospective study

Abstract Objective This study aims to report the genotypes and phenotypes of hereditary transthyretin amyloidosis (ATTR) in a large Chinese cohort, yet the clinical and genetic profiles of ATTR remain elusive in mainland China. Methods Fifty‐four patients with molecularly confirmed ATTR from 39 unrelated families were identified by sequencing the TTR gene. Sural nerve biopsies were performed in 40 of these cases. The clinical and electrophysiological data were retrospectively collected and analyzed. Results The male/female ratio was 42:12. The average age of patients at the onset of the disease was 47.8 ± 13.0 years. The late‐onset type occurred in 29 cases (53.7%). Twenty‐two probands (56.4%) had a family history with ATTR. The initial symptoms were limb paresthesia in 33 cases (61.1%), autonomic dysfunction in 15 cases (27.8%), and blurred vision in 6 cases (11.1%). A total of 22 different TTR mutations were identified, including Val30Met (25.6%) in 10 families in North China and Ala97Ser in 4 families (10.3%) in South China. Electrophysiological studies revealed general sensorimotor axonal polyneuropathy in 33/44 cases (75.0%), mixed neuropathy with axonal and demyelinating impairment features in 9/44 cases (20.5%) and isolated carpal tunnel syndrome in two cases. Sural nerve biopsies revealed positive Congo red staining in 16/40 cases (40.0%). Conclusion Chinese patients with ATTR exhibited heterogeneous TTR genotypes and clinical phenotypes. Val30Met remains the most common mutation type in mainland China.


Introduction
Hereditary transthyretin amyloidosis (ATTR) is a progressive and life-threatening disease caused by TTR gene mutation with an autosomal dominant inheritance pattern. The disease is characterized by TTR amyloid fibrils deposition in multiple organs, leading to sensory-motor neuropathy, autonomic dysfunction, and cardiomyopathy, accompanied by vitreous opacities and renal insufficiency. 1 According to the age of onset, ATTR is classified as early-onset before 50 years old and late-onset after 50 years old. Slowly progressive polyneuropathy and cardiomyopathy are more common in the late-onset patients, while rapidly progressive polyneuropathy with prominent autonomic dysfunction is common in the early-onset ones. 2 Electrophysiological studies are generally compatible with sensory-motor axonal polyneuropathy, but sometimes with demyelinating features. 3 Pathologically, the loss of unmyelinated fibers occurs early, reduced density of small and then larger myelinated fibers is observed, and blood vessels are frequently invaded and destroyed by TTR accumulation. 4 Diagnosis of ATTR mainly depends on amyloid deposits in the tissues and TTR gene testing. So far, more than 150 mutations have been described in the TTR gene. 5 The prevalence of different mutations varies according to ethnicity and geographic locations. TTR Val30Met mutation is the most common pathogenic variant overall. 4 ATTR is a phenotypically and geographically variable disease. 6 There is a great phenotypic variability between patients, but length-dependent axonal, sensory-motor and autonomic polyneuropathies with varying degrees of other organ involvement are the distinct features of ATTR. 7,8 In Taiwan, TTR Ala97Ser is the most common mutation. 9,10 However, a limited number of cases of ATTR have been reported in the form of case reports in mainland China.  The clinical and genetic profiles of ATTR in mainland China remain elusive. Here, we report the phenotypes and genotypes of 54 patients in 39 unrelated Chinese families with ATTR.

Subjects and clinical data
The study was approved by the local ethics committee, and written informed consent was obtained from all patients participating in the study. We conducted a retrospective study of our cohort of 54 patients with ATTR from 17 provinces in mainland China who had been referred to our Department of Neurology, Peking University First Hospital, between January 2007 and September 2020. Clinical data and laboratory findings were collected. The severity of disability was evaluated according to the Coutinho stages of ATTR (stage 0, no symptoms; stage I, unimpaired ambulation, mostly with mild sensory, motor, and autonomic neuropathy in the lower limbs; stage II, assistance for ambulation required, mostly with a moderate motor, sensory, and autonomic impairment of the four limbs; stage III, wheelchair-bound or bedridden status with severe sensory, motor, and autonomic involvement of all limbs).

Mutation screening
Genetic testing was performed and TTR genes were analyzed. We drew 5 mL of peripheral blood and extracted genomic DNA using the method of salt precipitation. Polymerase chain reaction (PCR) primers for exons 1-4 of the TTR gene were designed using Primer 3 software. Standard protocols were applied for PCR. The experimental products were purified and then sequenced using the ABI 3730XL automatic sequencing machine (Applied Biosystems, USA). Detected variants were confirmed in public databases including the Single Nucleotide Polymorphism database (dbSNP) (http://www.ncbi.nlm.nih.gov/ SNP/) and Mutations in Hereditary Amyloidosis (http:// amyloidosismutations.com/cdna-attr.html). Amino acid changes in TTR were numbered according to the beginning of the mature protein, as described historically, rather than including the 20 amino acid signal sequences.

Histopathology
Histopathological analysis was conducted to obtain the direct evidence for amyloid deposits through biopsy on the tissues. Sural nerve biopsies were performed in 40 patients. Nerve specimens were split into 2 sections. The first section was fixed in 4% formaldehyde, paraffin embedded, 8µm sections, and stained with hematoxylin and eosin and Congo red. Immunohistochemistry was used to confirm that amyloid was formed by TTR (DAKO, Denmark).
The second section was fixed in 3% glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated through serial alcohol baths, and embedded in Epon 812 (Electron Microscopy Sciences, USA). Semithin sections of sural nerve samples for light microscopy were stained with toluidine blue. Ultrathin sections of the nerve for electron microscopy were contrasted with uranyl acetate and lead citrate.

Reported cases in mainland China
We searched for cases in mainland China in English and Chinese that had been reported between January 2000 and September 2020. Key search terms included 'transthyretin familial amyloid polyneuropathy', 'familial amyloid polyneuropathy', 'transthyretin amyloidosis', 'ATTRamyloidosis', 'ATTR-PN', 'ATTR'.

Statistical analysis
The demographic data of the patients was included for descriptive statistics. Variables were presented as mean AE standard deviation. Continuous variables between two defined groups were compared using Student's t-test, while categorical variables were compared using Chi-square test or Fisher's exact test where appropriate. A level of significance of p < 0.05 was considered.

Clinical presentation
A total of 54 patients (12 female, 42 male) were collected from 39 unrelated families. There were 1 case without peripheral neuropathy in Coutinho stage 0, 33 cases (61.1%) in Coutinho stage I, 10 cases (18.5%) in Coutinho stage II and III, respectively.
The average age of patients at onset (AO) was 47.8 AE 13.0 years (range 23-68 years). The late-onset type occurred in 29 cases (53.7%) and early-onset type in 25 cases (46.3%). The positive family history was in 22 probands (56.4%). The mean course from initial symptoms to the last visit was 4.5 AE 3.4 years (Tables 1 and  2). Three patients had a long duration of 10-20 years, with alternating diarrhea and constipation as initial symptoms.
The initial symptoms were distal paresthesia in 33 cases (61.1%), including 5 patients with paresthesia of upper At the time of diagnosis, only 9 patients (16.7%) had symptoms of heart failure, but 40 patients (90.9%) showed cardiac hypertrophy from the echocardiography of 44 patients. Central nervous system involvement occurred in two cases, including transient ischemic attack-like episodes, and cerebral lobe hemorrhage. Interestingly, lower cranial nerve involvement including dysarthria or tongue fasciculation occurred in three cases, all of which were Ala97Ser mutation. Other rare symptoms included one case of renal involvement and one case of macroglossia, respectively (Tables 1-3).
The differences in clinical manifestations between earlyonset and late-onset probands from all the families were compared. A lower percentage of positive family histories and cases of Coutinho stage I, and a higher percentage of deep sensory impairment of upper limbs for late-onset probands suggested the likelihood of more common delay of diagnosis (Table 4).
Forty-four of these patients underwent electrophysiological studies at presentation. Most motor and sensory nerves showed generally axonal impairment in 33 cases including reduction of motor conduction velocity in the median nerve (4 cases), ulnar nerve (2 cases), and common peroneal nerve (3 cases), and motor distal latency prolongation in the ulnar nerve (1 case), common peroneal nerve (2 cases) and tibial nerve (4 cases). Because of CTS, two cases had isolated median neuropathy. Totally 46 patients were followed up. Three patients received liver transplantation, five patients were treated with tafamidis, and 16 patients were treated with diflunisal. Ten patients died either from respiratory failure after being bedridden or suddenly. The median interval between onset and death was 10 years (ranging from 4 to 25). Patient 20, 36 and 37, who received liver transplantation, reported slower progression of peripheral neuropathies than before. Six years after operation, patient 20 died suddenly. The diflunisal seemed unsatisfactory to the patients, due to the continuous progression of peripheral neuropathies despite its safety. Tafamidis has just been approved in China, so we still need to follow up the efficacy and safety of this drug.     (Fig. 1). Interestingly, all the families with Val30Met came from North China, while those with Ala97Ser came from South China. As the most common mutations in our cohort, a comparison of characteristics of cases with Val30Met and Ala97Ser was summarized in Table 3.

Pathology
Sural nerve biopsies were performed in 40 patients, all of whom presented with mild to severe axonal loss of both large and small myelinated fibers that were observed under a light microscope. Usually, clusters of regenerating myelinated fibers, degeneration of myelinated fibers, and occasionally, thin myelinated fibers and onion bulb formations were observed. Loss of nerve fibers of different diameters, especially the small myelinated and unmyelinated fibers, was further confirmed under an electron microscope. The positive rate of Congo red staining was 40.0% in sural nerves (16/40). The positive site also showed positive TTR staining. Under the electron microscope, massive filamentous amyloid deposits were observed (Fig. 2). Interestingly, the positive rate of Congo red staining was 55.6% (10/18) in early-onset cases, and 27.3% (6/22) in late-onset ones. It seemed that early-onset cases had higher positive rate, though no significant difference was found (p = 0.069).

Summary of reported cases of ATTR in mainland China
We reviewed 74 probands with ATTR. There were 32 kinds of mutations reported, among which Gly83Arg mutation was the dominating genotype (n = 11), with typical ocular involvement in all cases. Gly47Arg (n = 7), Val30Met (n = 6), and Val30Ala mutation (n = 6) were also frequently mentioned in previous reports, with prominent symptoms of peripheral neuropathy, autonomic dysfunction and cardiac involvement, followed by Ala97Ser mutation reported in 3 kindreds. Almost all the cases reported had prominent polyneuropathy and autonomic neuropathy, except some special cases that were closely related to a special genotype. Patients with Gly83Arg, Lys35Thr or Arg34Gly mutations mainly manifested themselves as ocular symptoms, and these cases were usually reported by ophthalmologists for presenting with blurred vision initially. Other special symptoms included chronic dry cough in Val30Ala and Ala97Ser, prominent psychiatric symptoms in Asp18Gly and Leu12-Pro, prominent cardiac symptoms in His88Arg, and renal involvement in Leu55Pro ( Table 5). The AO ranged from 17 to 69 years old.

Discussion
In our study, we reported the clinical and genetic profiles of ATTR in 54 patients from 39 families, which is currently the largest ATTR cohort in mainland China. A study from Taiwan investigated the clinical features and mutational spectrum of a cohort of 79 patients with 6 kinds of TTR mutations from 57 pedigrees, which is the largest ATTR cohort in Chinese populations so far. It was found that Ala97Ser mutation was the most common cause of ATTR in Taiwan, accounting for 91.2% of the pedigrees. 9 In our cohort, patients with 22 kinds of different TTR mutations came from 17 provinces across mainland China, reflecting the more real genotypes of Chinese populations.
In our cohort, TTR Val30Met mutation was the most common cause of ATTR in mainland China, especially in North China, accounting for 25.6% of the families. Only 30.0% of the probands had the positive family history, and late-onset type occurred in all the patients except one, who showed simple alternating diarrhea and constipation at the age of 43, more than 10 years earlier than other systems involvement. That is different from the endemic area in Portugal and Japan, where early-onset type and known family history are common. 52,53 The fact that very few TTR Val30Met mutation cases have been reported before in mainland China probably means that such cases might have been greatly underestimated. As in the Taiwan cohort, our patients with the TTR Ala97Ser mutation usually presented with a late-onset peripheral neuropathy and autonomic dysfunction. 9 Unlike the genetic situation in the north of China, TTR Ala97Ser mutation is the dominant mutation type in the south. According to previous studies, the most common mutation is Thr60Ala in the UK and US, Val30Met in Japanese non-endemic areas, and Asp38Ala in South Korea, indicating different genetic characteristics of ATTR in nonendemic areas of the world. [54][55][56][57] Additionally, another 20 kinds of TTR mutations have been found in our cohort, five of which have never been reported in mainland China before. It has to be acknowledged that our center is in Beijing, a northern city of China, which may lead to genotype bias, indicating the need for multicenter epidemiological investigations of ATTR in the future.
In the cohort, the most common clinical manifestations were peripheral neuropathy, in which, chronic sensory or sensorimotor polyneuropathy was the dominant type. Above 30% of the cases had neuropathic pain, which could occur as the initial symptoms, indicating early small nerve fiber involvement in ATTR. 9 Generally speaking, autonomic neuropathy was more common in early-onset patients in previous reports. 53 From our experience, autonomic neuropathy in late-onset patients was not few, but showed a mild degree, which tended to be neglected. That is an important cause of delayed diagnosis, if autonomic neuropathy is considered the distinctive feature of ATTR. 53 Cardiomyopathy, although frequently observed in this study, was not an onset symptom, which was consistent with the results of a previous study in Chinese populations. 14 Cardiac involvement was usually associated with symptoms of heart failure, and indicated the clinical course of the disease. 14, 52 We observed no more than nine patients with symptoms of heart failure during diagnosis, but over 90% of the patients had abnormal findings in echocardiography, underscoring the importance of cardiac evaluation of each patient with ATTR, even among those without heart symptoms. Ocular amyloidosis occurred in 25.9% of our patients, which was the case with Mayo Clinic patients with ATTR (24%). 58 The present study also indicated that hearing loss was not a rare symptom, suggesting amyloid deposition in the various anatomical structures of the inner and middle ear. 59 Renal involvement, including a nephritic syndrome and progressive renal failure, which occurred in about one in three patients in Portugal, 53 was rare in our cohort. Central nervous system involvement was observed in one case with the Tyr114Cys, who presented with transient ischemic attack-like episodes of dysarthria, hemihypoesthesia, hemiplegia and psychiatric symptoms, and in another case with Val30Ala as evidenced by cerebral lobe hemorrhage, thus highlighting the need for early investigations of central nervous system amyloidosis. 60 Interestingly, chronic cough was observed in 7 patients with TTR Val30Met, Ala97Ser, Phe33Val, Glu42Gly mutation. Nevertheless, the cause of chronic dry cough remains unknown, but could be attributed to deposition of amyloid in the vagal nerve or denervation hypersensitivity of the upper airways. 19 Three patients had lower cranial nerve involvement, including dysarthria or tongue fasciculation with Ala97Ser mutation, to our knowledge, which was not mentioned in previous studies with Ala97Ser mutation. 9,10 In our cohort, we confirmed that 75.0% of the cases had sensory-motor axonal neuropathies in the electrophysiological studies, while 20.5% of the cases showed mixed neuropathies with demyelinating patterns, which made it difficult to differentiate ATTR from some acquired demyelinating neuropathies. 61 Isolated CTS has been found in our cohort, which could be an early neuropathy pattern of ATTR. 4,5 Sural biopsy was less likely to detect amyloid deposition, but several other biopsy sites other than nerves may contribute more to the diagnosis of ATTR. 62 Interestingly, the trend that the positive rate of amyloid deposition of sural nerves was lower in late-onset patients was found, possibly because the blood-nerve barrier in the distal part of peripheral nerves may be preserved later in late-onset ATTR. 63 We found mild to severe loss of myelinated fibers of different diameters, especially the small-diameter and unmyelinated fibers, which started from the asymptomatic stage of ATTR. 64 Thin myelinated fibers and onion bulb formations were observed occasionally, which indicated involvement of myelin. Similar pathologic findings were observed in previous studies, which revealed severe axonal loss and occasional segmental demyelination-remyelination in nerve biopsy. 3 We have reported the largest ATTR cohort in mainland China. There are several limitations to our study. Firstly, our study is a retrospective study, in which recall bias may affect the results. Secondly, in our cohort, more cases come from North China, so the clinical and genetic characteristics may not reflect the real feathers of mainland China. Finally, all the probands were admitted to our Neurological Department first, so in our cohort, it was difficult to include ATTR cases without peripheral neuropathy. Therefore, future multicenter or multi-disciplinary studies are needed to identify ATTR phenotypes and genotypes in mainland China.

Conclusion
This study reported the clinical and genetic characteristics of ATTR in a single center in mainland China, and revealed the heterogeneity of ATTR phenotypes and genotypes in Chinese populations. Val30Met is the most common mutation type in mainland China, but it might be greatly underestimated because of the high proportion of sporadic and late-onset patients. There are genotypic differences between North and South China.