Clinical Features of Familial Amyloidotic Polyneuropathy With Transthyretin p.Ala117Ser Mutation in Mainland China

Objective: Our study aimed to report the clinical features of ATTR-PN with TTR p.Ala117Ser mutation in mainland China. Methods: Thirteen patients from 13 different families diagnosed with p.Ala117Ser ATTR-PN were identied from three centres. Clinical and laboratory data were retrospectively retrieved for analysis. Results: The male/female ratio was 11:2. All patients showed late onset, with the age of onset at 57.8 ± 5.8 years. The initial symptom was numbness of the lower or upper extremities in 9 patients (69.2%). Paraesthesia was present in all patients. Eleven patients (84.6%) had autonomic dysfunction. Cardiac, renal, hepatic, and ocular dysfunctions were noted in 8 (61.5%), 1 (7.7%), 2 (15.4%), and 3 (23.1%) patients, respectively. Nerve conduction studies have shown axonal-type sensorimotor polyneuropathy. The decline in sensory nerve action potentials was more noticeable than in compound muscle action potentials. The nerve damage present in the lower limbs was more severe than that in the upper limbs. Nerve biopsy revealed positive Congo red staining in 7/10 patients (70%). Conclusion: Our study is the largest population report on this rare p.Ala117Ser mutation in mainland China.


Introduction
Hereditary transthyretin amyloidosis with polyneuropathy (ATTR-PN) is an autosomal dominant genetic disorder, caused by point mutations of the TTR gene and is characterised by amyloid deposition in the peripheral nervous system and autonomic nervous system [1]. It is a systemic disease that involves various organs (such as the heart, eyes, kidney), and usually presents as progressive peripheral neuropathy with adult-onset. In 1952, Andrade rst described familial amyloid polyneuropathy (FAP) in northern Portugal [2]. It was subsequently reported in Japan (1968) [3] and Sweden (1976) [4]. ATTR-PN has been reported in 29 countries, including Korea [5], the United states of America [6], China [7], and many European countries [8].
The TTR gene is located on chromosome 18 and comprises four exons [9]. Currently, more than 130 mutations have been identi ed associated with this gene [1]. The p.Val30Met variant of TTR is most commonly identi ed among patients living in small clusters and scattered families worldwide and was rst described as the cause of FAP in 1984 [10]. Moreover, certain speci c mutations are associated with small clusters of families in particular areas. For example, the TTR p.Ala97Ser (now p.Ala117Ser) mutation is common among Chinese kindreds from Taiwan [11][12][13][14]. Subsequently, the rst FAP family with a proven missense mutation c.349G>T (p.Ala117Ser) in mainland China was reported in 2018 [15]. This study aimed to determine the clinical features of FAP with TTR p.Ala117Ser by studying 13 Chinese families from mainland China. To date, our study is the largest population report on this rare p.Ala117Ser mutation in mainland China.

Patients
Thirteen patients from 13 different families diagnosed with p.Ala117Ser ATTR-PN were identi ed from the Department of Neurology of three centers: Southern Medical University Nanfang Hospital, Peking University First Hospital, Central South University Third Xiangya Hospital, from September 2013 to September 2020. All patients conformed to the clinical and electrophysiologic criteria of idiopathic adult-onset axonal polyneuropathy: 1) length-dependent neuropathy with clinical evidence of limb weakness and sensory symptoms and 2) electrophysiologic evidence of axonal polyneuropathy on nerve conduction studies (NCS). All patients were con rmed of the mutation of p.Ala117Ser. Furthermore, the clinical and laboratory data were retrieved for analysis. Procedures of the tests (NCS, UCG, CSF examination, routine blood and urine examinations, genetic analysis, and nerve biopsies, etc) followed established protocols.

TTR gene analysis
Peripheral venous blood samples were obtained for DNA analysis from the patients. Genomic DNA was isolated from the blood samples following a standard protocol. Brie y, the 4 exons of the entire human TTR gene (NCBI Reference Sequence: NG_009490.1, NM_000371.3) were ampli ed by polymerase chain reaction (PCR). The PCR products were puri ed and sequenced directly by Sanger sequencing.

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Sural nerve (or sensory branch of the super cial peroneal nerve) biopsy was performed under local skin and tissue anaesthesia, excluding the nerve. Nerve specimens were processed for routine stains (haematoxylin-eosin for overview and nerve morphology; Congo red for amyloid) on frozen sections and semi-thin sections (azure-methylene blue). Serial consecutive sections were assessed. Electron microscopy samples were xed in a 2.5% glutaraldehyde buffer for 2h, then with osmium acid, dehydrated in acetone, and embedded with epoxy resin. The sections were observed under an electron microscope and photographed.

Statistical analyses
We used IBM SPSS Version 26 and Microsoft O ce Excel. Descriptive statistics are mainly used in this study. Numeric variables are here described as means ± SD, numbers (n) with percentages (%) or median values, etc. Results were considered signi cant at p<0.05.

Clinical features
The basic information of the probands (11 males and 2 females) in the 13 families with p.Ala117Ser ATTR-PN is summarised in Table 1. All probands were from South China (see Figure 1). The age of onset was 57.8 ± 5.8 years (range 47-66). The time-period from the onset to the nal diagnosis was 5.1 ± 3.8 years (range 1-13), and neurological assessments were performed at the time of diagnosis. Initial symptoms were numbness of the lower or upper extremities (9 patients), weakness of the lower limbs (1 patient), diarrhoea and constipation (1 patient), erectile dysfunction (1 patient), and chest tightness (1 patient). No cranial nerve defects, such as dysphagia, dysarthria, or atrophy of the tongue, were observed in any of the patients. Other symptoms included oedema (7 patients), dry cough (5 patients), and haemorrhagic rash (2 patients). The various system dysfunction scores of the probands are shown in Figure 2. Case history Total Neurophysiological manifestations NCS showed axonal-type sensorimotor polyneuropathy (detailed in Table 3). Most patients had long distal latency of the upper or lower extremities, but it was much smaller than the demyelinating lesion. All patients had reductions in the amplitudes of compound muscle action potentials (CMAPs) and sensory nerve action potentials (SNAPs) from mild to severe. It is worth noting that the decline in SNAP was more noticeable than that in CMAP. The nerve damage of the lower limbs was more severe than that of the upper limbs. The SNAP of most of the lower extremities was not observed. Additionally, a decrease in the F wave was observed in nearly all tested nerves. Histopathological ndings Ten patients underwent a sural or peroneal nerve biopsy. The main pathological changes in peripheral nerves were moderately to severely decreased myelinated and unmyelinated nerve bres, accompanied by degenerative or regenerative changes in the axons and myelin sheath of myelinated nerve bres, which is in accordance with the pathological characteristics of chronic active mixed peripheral neuropathy. Nerve biopsy revealed positive Congo red staining in 7/10 patients (70%). Congo red staining showed multiple red-stained amyloid deposits and bright apple green coloration under a polarising microscope. Axonal degeneration of green substances was found in some large myelinated bres by MGT staining. The histopathologic ndings of patient no. 10 are showed in Figure 3.

Discussion
The occurrence of ATTR-PN is relatively rare in China. In the 1990s, a family with ATTR-PN was rst diagnosed at the Peking Union Medical College Hospital [16]. It is estimated that there are approximately 1997 cases in China (ranging from 435 to 10134) [17]. Over 40 case reports have been published in recent years, including reports of multiple ATTR-PN families. Studies describe a mutation in the TTR gene, which is different from that observed in Europe [18][19][20][21]. Shortly before, a unicentric retrospective study reported that TTR p.Val30Met remains the most common mutation type in mainland China [22]. However, TTR p.Ala117Ser mutations are relatively rare. Based on previous studies, the TTR p.Ala117Ser mutation was one of the most common variants in the Chinese population, especially in Chinese kindreds from Taiwan.
This mutation has never been reported in Caucasian populations. Detailed haplotype analyses demonstrated a shared haplotype in most patients with the p.Ala117Ser mutation in Taiwan, suggesting a founder effect [23]. Except for the rst report of the FAP family with a proven TTR p.Ala117Ser mutation from mainland China [15], our study reported another 13 pedigrees with the same mutation. Furthermore, all probands came from Southern China, including Hunan and Guangdong Province. We speculate that the TTR p.Ala117Ser mutation rst originated in mainland China despite the lack of genetic veri cation.
Our study demonstrated the characteristics of ATTR-PN with the TTR p.Ala117Ser mutation in mainland China. Similar to previous reports, there were signi cantly more men than women diagnosed with this mutation. It is unclear whether there are protective factors in women. The Ala117Ser patients showed late onset -almost all were over 50 years of age. It was different from Val30Met patients, which showed early or late onset [24]. The course from the onset to nal diagnosis ranged from 1 to 13 years, while there was no signi cant correlation between this and the severity of clinical manifestations. Numbness of the lower or upper extremities was one of the most common initial symptoms.
Because of its insidious onset, the diagnosis of this disease is usually delayed, rendering treatment di cult. Apart from peripheral nerve dysfunction, autonomic dysfunction was also particularly prominent. Constipation was more common than orthostatic hypotension, and it (constipation) often manifested as alternating between diarrhoea and constipation. Remarkably, almost all males had erectile dysfunction at an early stage, which is an important feature that differentiates them from female patients. Cardiac dysfunction was the most common involvement of other organs. One of the 13 patients died of a cardiac incident shortly after the diagnosis. A unique phenotype of ATTR-PN has been reported in a case report describing a distinctive chronic dry cough [15]. In our study, ve patients had similar symptoms. It occurred at different stages of the disease. Further research is needed to con rm this mechanism. Although nerve biopsy helped diagnose the disease, not all patients in our study underwent a nerve biopsy. Only 7/10 (70%) patients showed positive Congo red staining. Patients with ATTR-PN negative Congo red staining might get misdiagnosed. Neurophysiological examinations also helped with the diagnosis. The nal diagnosis was based on genetic and pathological examinations.
In addition, many factors, such as the rarity of the disease in the general population, physicians' lack of understanding of various clinical features, and limited diagnostic tools (i.e. histopathology and gene screening) lead to a high misdiagnosis rate and poor prognosis. ATTR-PN is often misdiagnosed as chronic idiopathic axonal polyneuropathy, chronic in ammatory demyelinating polyneuropathy, and lumbar spinal stenosis. Diabetes or chronic alcoholism may cause polyneuropathies such as ATTR-PN. It may also be misdiagnosed as Charcot-Marie-Tooth or motor neuron disease. Delayed diagnosis is the main obstacle to the optimal management of ATTR-PN in China. There is usually an interval of several years from the initial clinical symptoms of the disease to the nal diagnosis. Because of the signi cant unmet medical needs for this rare and fatal disease, there is an urgent need to raise disease awareness to facilitate early diagnosis and timely treatment.

Conclusion
In this study, we described the characteristics of ATTR-PN with TTR p.Ala117Ser mutation. To date, our study is the largest population report on this rare mutation in mainland China. However, a large-scale study is needed to explore a complete picture of the disease.

Declarations
Availability of data and materials The data sets generated during and/or analysed during the current study are available from the corresponding author on reasonable request..
All data generated and analyzed during this study are included in this article. shows the various system dysfunction scores of all patient based on Table 2. One point for each of the following symptoms: Paresthesia, Sensory dissociation, allodynia, weakness, amyotrophy, decreased re exs, carpal tunnel syndrome, diarrhea, constipation, weight reduction, orthostatic hypotension, hyperhidrosis, erectile dysfunction, urine retention, arrhythmia, cardiac hypertrophy, symptomatic heart failure, hepatic dysfunction, renal dysfunction, vision loss, vitreous opacity, cataract, glaucoma.