Abstract
Familial amyloid polyneuropathy (FAP) is a dominantly inherited disorder. This study aims to explore the genetic features of a Han Chinese family with FAP, characterized by bloating, alternating diarrhea and constipation, and weakness in his feet. Amyloid presented histologically in the vessel walls of hepatic portal area and nerves of the surgically excised liver specimens from the proband by hematoxylin and eosin staining. Amyloid deposition was further confirmed with Congo red treatment. A c.349G>T transversion (p.Ala117Ser) in TTR gene exon 4 was identified in the proband with typical autonomic neuropathy and peripheral motor neuropathy, as well as in his asymptomatic son. The variant was not detected in 200 normal ethnically matched controls. These findings provide new insights into FAP cause and diagnosis and have implications for genetic counseling.
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Introduction
Familial amyloid polyneuropathy (FAP, OMIM 105210) is a rare, autosomal-dominant disease, characterized by amyloid accumulation in the peripheral nerves and other organs, including the heart, kidneys, and eyes [1]. FAP was first described in Portugal in 1952 and was originally thought to be endemic in only a few countries including Portugal, Japan, and Sweden. It was later reported in other locations [2, 3]. FAP can be caused by the following four genes: the transthyretin gene (TTR, OMIM 176300), the apolipoprotein A1 gene (APOA1, OMIM 107680), the gelsolin gene (GSN, OMIM 137350), and the beta-2-microglobulin gene (B2M, OMIM 109700) [4]. Of these, the TTR amyloidosis is the most common form [5]. The TTR p.Val50Met mutation was firstly described as the cause of FAP in 1984 [6]. Currently, more than 125 TTR mutations have been identified, of which 13 TTR mutations seem to be non-amyloidogenic. All are missense point mutations except for one microdeletion p.Val142del (http://www.amyloidosismutations.com/main_menu.html). The p.Val50Met mutation is the most common TTR mutation reported in 85% of the FAP patients from the Familial Amyloidotic Polyneuropathy World Transplantation Registry (FAPWTR) [5, 7].
Nerve length-dependent, sensory-motor, and autonomic polyneuropathy beginning in the feet is the neurological feature of TTR-FAP [1, 5]. The phenotypes vary dramatically between kindreds with different variants. It is difficult to establish a firm genotype-phenotype correlation in FAP. Clinical phenotype variability exists even among the same family and individuals with the same point mutation [3, 8, 9]. Though patients with TTR p.Ile127Val mutation were reported to have severe FAP with shorter median survival [10], individuals with compound heterozygous p.Val50Met and p.Thr139Met or p.Arg124His variants present with a mild form of FAP [8], indicating that the phenotype modifiers may be involved. TTR-FAP prevalence varies in different populations. There is a relatively high prevalence, 0.09% (1/1108) in northern Portugal, which is lower in the rest of Europe and USA (approximately 1/100,000) [3, 11]. TTR-FAP may present as sporadic cases in other non-endemic regions [12]. Sex ratio varied in different regions. Male-to-female ratios were significantly higher (10.7:1) in late-onset FAP TTR p.Val50Met Japanese patients, but much lower (0.9:1) in FAP TTR p.Val50Met Portuguese patients [10, 13]. TTR-FAP onset age ranges widely from 16 to 80 years old [12,13,14]. Disorder duration ranges from 2 to 21 years [14]. Age-dependent and geography-based penetrance has been described in the literature [14,15,16]. The penetrance was also significantly higher with maternally inherited TTR mutations [15, 17, 18].
In this article, we describe a Han Chinese family with c.349G>T (p.Ala117Ser) variant in the TTR gene. Bioinformation analysis along with the absence of the variant in 200 ethnically matched normal controls suggests that it may be a pathogenic variant.
Methods
A dominant Han Chinese family (Taiwanese originally from Fujian) with FAP was enrolled in the Third Xiangya Hospital, Central South University, China (Fig. 1). The proband (Fig. 1, III-1) received a liver transplantation. Blood samples were collected from two members of the family and 200 unrelated, ethnically matched mainland Chinese, normal controls (age 40–70 years old). Informed consent was obtained from the individuals. The study received approval from the Ethics Committee of the Third Xiangya Hospital, Central South University, Changsha, Hunan, China.
Clinical Data
The proband presented with complaints of bloating, alternating diarrhea and constipation, and muscular weakness in his feet, over a year (Table 1). Neurological examination revealed muscle weakness in the lower extremities. Kidney function and ophthalmological examinations were normal. Cardiac ultrasound showed suspicious amyloid deposition. The proband’s son (IV-1) did not complain of any sensory-motor problem or similar gastrointestinal symptoms. The proband’s maternal grandfather (I-1), mother (II-2), and uncle (II-3) were unable to walk in their later years.
Light Microscope and Electron Microscopy Analysis
The hepatic specimens from the proband were fixed in 10% formalin and embedded in paraffin. The tissues were sectioned into 3 μm and stained with hematoxylin and eosin (HE) and Congo red. Electron microscopy samples were fixed in a 2.5% glutaraldehyde buffer for 2 h, then with osmium acid, dehydrated in acetone, and embedded with epoxy resin. The sections were observed under an electron microscope and photographed.
Gene Analysis
Genomic DNA was isolated from lymphocytes using the standard method [19]. Polymerase chain reaction (PCR) amplified the TTR gene (NCBI Reference Sequence: NG_009490.1, NM_000371.3) using a 9700 Thermal Cycler System (Applied Biosystems Inc., Foster City, USA), and PCR conditions were 95 °C for 3 min, followed by 35 cycles of 95 °C for 40 s, 58 °C for 35 s, 72 °C for 45 s, and a final extension step at 72 °C for 5 min. The primers used for PCR amplification cover all TTR gene coding regions and exon/intron boundaries, which were synthesized by Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China (Table 2). PCR products of 8.5 μl were digested by 0.8 U shrimp alkaline phosphatase and 8 U exonuclease I (Fermentas Inc., Burlington, Canada) in a 10-μl reaction volume. They were then sequenced directionally using an 8-capillary 3500 genetic analyzer (Applied Biosystems Inc., Foster City, USA). Three online tools, MutationTaster prediction (http://www.mutationtaster.org/), Sorting Intolerant from Tolerant (SIFT) prediction (http://sift.jcvi.org/), and HumVar-trained PolyPhen-2 (Polymorphism Phenotyping v2, http://genetics.bwh.harvard.edu/pph2/), were performed to estimate whether a variant affected protein structure or function [20, 21]. The structural and functional importance of the amino acid at the variant position was further assessed by National Center for Biotechnology Information-Basic Local Alignment Search Tool (NCBI-BLAST) in different species.
Results
Histopathologic evaluation of the excised liver specimens from the proband revealed that amyloid deposits were present in the perineurium and arteries of hepatic portal area by HE staining, further confirmed by Congo red treatment. There was no amyloid deposition in the hepatocytes. Electron microscopy revealed amyloid fibrils, which were crossed, or parallel-arranged, in bundles. Surrounding tissues were clear (Fig. 2).
A known heterozygous missense variant, c.349G>T (p.Ala117Ser), in the TTR gene, was identified in the proband (Fig. 1). Extended analysis of the family identified the identical c.349G>T variant in his asymptomatic son. This variant was absent in the 200 normal control subjects. This c.349G>T (p.Ala117Ser) variant was predicted to be disease causing, damaging, and probably damaging by MutationTaster, SIFT, and PolyPhen-2, respectively. The alanine at the mutated position (p.Ala117) is highly conserved in different species, suggesting its structural and functional importance (Fig. 3). Cartoon representation of the protein structure is shown in Fig. 4 created by PyMOL 1.7 based on the CPHmodels-3.3 [22]. Though recorded in the single nucleotide polymorphism database (rs267607161), there is no frequency data of this variant. The variant was absent in over 60,000 individuals in the Exome Aggregation Consortium (http://exac.broadinstitute.org/). According to the American College of Medical Genetics and Genomics guidelines [23], the c.349G>T (p.Ala117Ser) variant was classified as a “likely pathogenic” variant.
Discussion
This study detected the presence of amyloid deposits in the perineurium and arteries of the proband’s hepatic portal area using HE staining. Amyloid deposition exhibited affinity for Congo red. A heterozygous missense variant c.349G>T (p.Ala117Ser), previously reported as Ala97Ser by other studies [24,25,26,27,28,29,30], was identified in the proband’s and his asymptomatic son’s TTR gene. Amyloid deposition in tissue and a proven amyloidogenic variant in the TTR gene confirmed this patient’s diagnosis of TTR-FAP. Patients with p.Ala117Ser TTR-FAP usually had a late age at onset, different from those with TTR p.Val50Met mutation [25, 26]. Almost all TTR p.Ala117Ser patients have motor and sensory symptoms. Autonomic symptoms, such as gastrointestinal symptoms and orthostatic hypotension, are common (Table 3). The patient in our study showed gastrointestinal symptoms, autonomic nerve function damage, and lower limb weakness, which are typical manifestations in the TTR-FAP cases. He presented no significant sensory symptom. Disease progression is usually described as having three stages according to the patients’ signs and symptoms. Stage I patients are ambulatory. Stage II patients are ambulatory but require assistance. Stage III patients are either bedridden or wheelchair-bound [3]. The proband in this study is in the early stages of the disease and suffers mild motor impairment of the lower extremities, moderate autonomic manifestations, and full ambulation.
The human TTR gene, located on 18q12.1, includes 4 exons spanning over 7 kb and encodes 147 amino acids. The TTR protein is a 56 kDa homotetrameric protein formed by the 127-residue polypeptides. It is a soluble protein circulating in peripheral blood and cerebrospinal fluid [5]. Half of the residues in each monomer are composed of two β-sheets, each of which is composed of four strands. The remaining residues loop attaches to the β-strands [31]. TTR, as a plasma-transport protein for thyroxin (T4) and vitamin A, is primarily synthesized in the liver [5, 7]. The remainder is in choroid plexus cells and retinal cells [5]. Energetic studies of a large number of recombinant TTR variants suggested that amyloidogenic mutations destabilize the native quaternary and tertiary structures of TTR, thereby inducing conformational changes [32, 33]. When the TTR gene mutates, TTR tetramer dissociates into monomers as the initial step which allows subsequent partial misfolding and misassembly. This leads to the formation of TTR amyloid fibrils and several aggregate morphologies [32, 34]. Dissociation of TTR tetramer into monomers depends on pH. Under acidic conditions, tetrameric TTR mutant dissociates into monomers to a much greater extent than that of wild-type TTR [33, 35]. The mutated alanine p.Ala117 located on the carboxy terminus, the F-strand of the TTR molecule, is part of the hydrophobic core [26, 36]. A misTTR antibody and a peptide inhibitor that selectively target TTR residues in the F-strand can inhibit fibrillogenesis or protein aggregation [37, 38], which supports the importance of F-strand for TTR protein aggregation. The substitution of alanine p.Ala117 with the less hydrophobic serine might destabilize the structure and cause the dissociation of the TTR tetramer.
Transgenic Drosophila melanogasters, with TTR Leu55Pro or engineered TTR Val14Asn/Val16Glu, showed peripheral toxicity, accompanied by premature death and locomoter behavioral alterations [39]. In transgenic mice carrying human TTR mutants, amyloid deposition was detected in the gastrointestinal tract and other organs and tissues, which became more remarkable with aging [40, 41].
The current reference treatment for TTR-FAP is liver transplantation [12, 32]. Liver transplantation is recommended to early onset TTR-FAP patients with p.Val50Met mutation before 50 years old except for women, aiming to remove the main source of systemic mutant TTR [12]. To our knowledge, this is the first liver transplantation case reported with a TTR p.Ala117Ser variant, and the patient had a liver transplantation at an early stage of TTR-FAP. By 6 months after surgery, patient’s gastrointestinal symptoms eased. There had been no further progression of the neuropathy though long-term effects need further follow-up observation.
Recently, some new therapeutic strategies intended to stabilize TTR have become available. Tafamidis, a specific TTR stabilizer, is the first TTR-FAP drug approved for use in Europe and some other countries (Japan, Mexico, and Argentina) [32, 42]. In the latest study of early treatment with tafamidis over a 5.5-year period, it resulted in delay in neurologic progression and long-term preservation of nutritional status [43]. Some other new therapeutic strategies for TTR amyloidosis including antibody [44, 45], TTR siRNA treatment [46], and tauroursodeoxycholic acid and curcumin [47] are currently being explored, which may shed a new light on the therapy of TTR-FAP.
Conclusions
The missense variant c.349G>T (p.Ala117Ser) of the TTR gene may be responsible for the Han Chinese family with FAP. Sanger sequencing of TTR gene provides a cost-effective approach to identify variant responsible for patients of FAP. These findings provide new insights into FAP cause and diagnosis and have implications for genetic counseling.
References
Conceição I (2012) Clinical features of TTR-FAP in Portugal. Amyloid 19(Suppl 1):71–72
Andrade C (1952) A peculiar form of peripheral neuropathy; familiar atypical generalized amyloidosis with special involvement of the peripheral nerves. Brain 75:408–427
Ando Y, Coelho T, Berk JL et al (2013) Guideline of transthyretin-related hereditary amyloidosis for clinicians. Orphanet J Rare Dis 8:31
Delpech M, Valleix S (2012) Amyloid polyneuropathies--biochemical and genetic aspects. Bull Acad Natl Med 196:1309–1318
Planté-Bordeneuve V, Said G (2011) Familial amyloid polyneuropathy. Lancet Neurol 10:1086–1097
Saraiva MJ, Birken S, Costa PP et al (1984) Family studies of the genetic abnormality in transthyretin (prealbumin) in Portuguese patients with familial amyloidotic polyneuropathy. Ann N Y Acad Sci 435:86–100
Wilczek HE, Larsson M, Ericzon BG et al (2011)Long-term data from the Familial Amyloidotic Polyneuropathy World Transplant Registry (FAPWTR). Amyloid 18(Suppl 1):193–195
Zeldenrust SR (2012)Genotype-phenotype correlation in FAP. Amyloid 19(Suppl 1):22–24
Ikeda S, Nakazato M, Ando Y et al (2002) Familial transthyretin-type amyloid polyneuropathy in Japan: Clinical and genetic heterogeneity. Neurology 58:1001–1007
Mariani LL, Lozeron P, Théaudin M et al (2015)Genotype-phenotype correlation and course of transthyretin familial amyloid polyneuropathies in France. Ann Neurol 78:901–916
Sousa A, Coelho T, Barros J et al (1995) Genetic epidemiology of familial amyloidotic polyneuropathy (FAP)-type I in Póvoa do Varzim and Vila do Conde (north of Portugal). Am J Med Genet 60:512–521
Adams D, Cauquil C, Labeyrie C et al (2016) TTR kinetic stabilizers and TTR gene silencing: A new era in therapy for familial amyloidotic polyneuropathies. Expert Opin Pharmacother 17:791–802
Misu Ki, Hattori N, Nagamatsu M et al (1999)Late-onset familial amyloid polyneuropathy type I (transthyretin Met30-associated familial amyloid polyneuropathy) unrelated to endemic focus in Japan. Clinicopathological and genetic features. Brain 122:1951–1962
Planté-Bordeneuve V, Carayol J, Ferreira A et al (2003) Genetic study of transthyretin amyloid neuropathies: Carrier risks among French and Portuguese families. J Med Genet 40:e120
Hellman U, Alarcon F, Lundgren HE et al (2008) Heterogeneity of penetrance in familial amyloid polyneuropathy, ATTR Val30Met, in the Swedish population. Amyloid 15:181–186
Saporta MA, Zaros C, Cruz MW et al (2009) Penetrance estimation of TTR familial amyloid polyneuropathy (type I) in Brazilian families. Eur J Neurol 16:337–341
Bonaïti B, Olsson M, Hellman U et al (2010) TTR familial amyloid polyneuropathy: Does a mitochondrial polymorphism entirely explain the parent-of-origin difference in penetrance? Eur J Hum Genet 18:948–952
Bonaïti B, Alarcon F, Bonaïti-pellié C et al (2009)Parent-of-origin effect in transthyretin related amyloid polyneuropathy. Amyloid 16:149–150
Yuan L, Deng X, Song Z et al (2015) Genetic analysis of the RAB39B gene in Chinese Han patients with Parkinson's disease. Neurobiol Aging 36:2907.e11–2907.e12
Kumar P, Henikoff S, Ng PC (2009) Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 4:1073–1081
Adzhubei I, Jordan DM, Sunyaev SR (2013) Predicting functional effect of human missense mutations using PolyPhen-2. Curr Protoc Hum Genet Chapter 7:Unit7.20
Nielsen M, Lundegaard C, Lund O et al (2010) CPHmodels-3.0--remote homology modeling using structure-guided sequence profiles. Nucleic Acids Res 38:W576–W581
Richards S, Aziz N, Bale S et al (2015) Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 17:405–424
Hsieh ST (2011) Amyloid neuropathy with transthyretin mutations: overview and unique Ala97Ser in Taiwan. Acta Neurol Taiwan 20:155–160
Lai HJ, Chiang YW, Yang CC et al (2015) The temporal profiles of changes in nerve excitability indices in familial amyloid polyneuropathy. PLoS One 10:e0141935
Yang NC, Lee MJ, Chao CC et al (2010) Clinical presentations and skin denervation in amyloid neuropathy due to transthyretin Ala97Ser. Neurology 75:532–538
Liu YT, Lee YC, Yang CC et al (2008) Transthyretin Ala97Ser in Chinese-Taiwanese patients with familial amyloid polyneuropathy: Genetic studies and phenotype expression. J Neurol Sci 267:91–99
Chao CC, Huang CM, Chiang HH et al (2015) Sudomotor innervation in transthyretin amyloid neuropathy: pathology and functional correlates. Ann Neurol 78:272–283
Tachibana N, Tokuda T, Yoshida K et al (1999) Usefulness of MALDI/TOF mass spectrometry of immunoprecipitated serum variant transthyretin in the diagnosis of familial amyloid polyneuropathy. Amyloid 6:282–288
Klein CJ, Vrana JA, Theis JD et al (2011) Mass spectrometric-based proteomic analysis of amyloid neuropathy type in nerve tissue. Arch Neurol 68:195–199
Blake CC, Geisow MJ, Swan ID et al (1974) Structure of human plasma prealbumin at 2-5 A resolution. A preliminary report on the polypeptide chain conformation, quaternary structure and thyroxine binding. J Mol Biol 88:1–12
Sekijima Y (2015) Transthyretin (ATTR) amyloidosis: Clinical spectrum, molecular pathogenesis and disease-modifying treatments. J Neurol Neurosurg Psychiatry 86:1036–1043
Lashuel HA, Lai Z, Kelly JW (1998) Characterization of the transthyretin acid denaturation pathways by analytical ultracentrifugation: implications for wild-type, V30M, and L55P amyloid fibril formation. Biochemistry 37:17851–17864
Lai Z, Colón W, Kelly JW (1996) The acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can self-assemble into amyloid. Biochemistry 35:6470–6482
Miyata M, Sato T, Mizuguchi M et al (2010) Role of the glutamic acid 54 residue in transthyretin stability and thyroxine binding. Biochemistry 49:114–123
Yasuda T, Sobue G, Doyu M et al (1994) Familial amyloidotic polyneuropathy with late-onset and well-preserved autonomic function: A Japanese kindred with novel mutant transthyretin (Ala97 to Gly). J Neurol Sci 121:97–102
Galant NJ, Bugyei-Twum A, Rakhit R et al (2016) Substoichiometric inhibition of transthyretin misfolding by immune-targeting sparsely populated misfolding intermediates: A potential diagnostic and therapeutic for TTR amyloidoses. Sci Rep 6:25080
Saelices L, Johnson LM, Liang WY et al (2015) Uncovering the mechanism of aggregation of human transthyretin. J Biol Chem 290:28932–28943
Pokrzywa M, Dacklin I, Hultmark D et al (2007) Misfolded transthyretin causes behavioral changes in a drosophila model for transthyretin-associated amyloidosis. Eur J Neurosci 26:913–924
Shimada K, Maeda S, Murakami T et al (1989) Transgenic mouse model of familial amyloidotic polyneuropathy. Mol Biol Med 6:333–343
Yi S, Takahashi K, Naito M et al (1991) Systemic amyloidosis in transgenic mice carrying the human mutant transthyretin (Met30) gene. Pathologic similarity to human familial amyloidotic polyneuropathy, type I. Am J Pathol 138:403–412
Dubrey S, Ackermann E, Gillmore J (2015) The transthyretin amyloidoses: Advances in therapy. Postgrad Med J 91:439–448
Waddington Cruz M, Amass L, Keohane D et al (2016) Early intervention with tafamidis provides long-term (5.5-year) delay of neurologic progression in transthyretin hereditary amyloid polyneuropathy. Amyloid 23:178–183
Hosoi A, Su Y, Torikai M et al (2016) Novel antibody for the treatment of transthyretin amyloidosis. J Biol Chem 291:25096–25105
Richards DB, Cookson LM, Berges AC et al (2015) Therapeutic clearance of amyloid by antibodies to serum amyloid P component. N Engl J Med 373:1106–1114
Coelho T, Adams D, Silva A et al (2013) Safety and efficacy of RNAi therapy for transthyretin amyloidosis. N Engl J Med 369:819–829
Teixeira CA, Almeida Mdo R, Saraiva MJ (2016) Impairment of autophagy by TTR V30M aggregates: In vivo reversal by TUDCA and curcumin. Clin Sci (Lond) 130:1665–1675
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We thank the participating members and investigators for their cooperation and efforts in collecting clinical and genetic information and DNA specimens.
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Informed consent was obtained from the individuals. The study received approval from the Ethics Committee of the Third Xiangya Hospital, Central South University, Changsha, Hunan, China.
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This work was supported by National Key Research and Development Program of China (2016YFC1306604), National Basic Research Program of China (2014CB542400), National Natural Science Foundation of China (81670216), Natural Science Foundation of Hunan Province (2015JJ4088 and 2016JJ2166), Grant for the Foster Key Subject of the Third Xiangya Hospital Clinical Laboratory Diagnostics (H.D.), the New Xiangya Talent Project of the Third Xiangya Hospital of Central South University (20150301), China.
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Chen, Q., Yuan, L., Deng, X. et al. A Missense Variant p.Ala117Ser in the Transthyretin Gene of a Han Chinese Family with Familial Amyloid Polyneuropathy. Mol Neurobiol 55, 4911–4917 (2018). https://doi.org/10.1007/s12035-017-0694-0
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DOI: https://doi.org/10.1007/s12035-017-0694-0