Exp Eye Res. 2011 Dec 3;
Karring H, Runager K, Thøgersen IB, Klintworth GK, Højrup P, Enghild JJ

Different types of granular corneal dystrophy (GCD) and lattice corneal dystrophy (LCD) are associated with mutations in the transforming growth factor beta induced gene (TGFBI). These dystrophies are characterized by the formation of non-amyloid granular deposits (GCDs) and amyloid (LCD type 1 and its variants) in the cornea. Typical corneal non-amyloid deposits from GCD type 2 (R124H), amyloid from a variant of LCD type 1 (V624M) and disease-free tissue controls were procured by laser capture microdissection and analyzed by tandem mass spectrometry. Label-free quantitative comparisons of deposits and controls suggested that the non-amyloid sample (R124H) specifically accumulated transforming growth factor beta induced protein (TGFBIp/keratoepithelin/βig-h3), serum amyloid P-component, clusterin, type III collagen, keratin 3, and histone H3-like protein. The amyloid (V624M) similarly accumulated serum amyloid P-component and clusterin but also a C-terminal fragment of TGFBIp containing residues Y571-R588 derived from the fourth fasciclin-1 domain (FAS1-4), apolipoprotein E and apolipoprotein A-IV. Significantly, analyses of the amyloid sample also revealed the presence of the serine protease Htr (High-temperature requirement) A1 and a number of proteolytic cleavage sites in the FAS1-4 domain of TGFBIp. These cleavage sites were consistent with the ligand binding and proteolytic activity of HtrA1 suggesting that it plays a role in the proteolytic processing of the amyloidogenic FAS1-4 domain. Taken together, the data suggest that the amyloidogenic-prone region of the fourth FAS1 domain of TGFBIp encompasses the Y571-R588 peptide and that HtrA1 is involved in the proteolytic processing of TGFBIp-derived amyloid in vivo.

Jpn J Ophthalmol. 2012 Jan; 56(1): 26-30
Morita Y, Chikama T, Yamada N, Morishige N, Sonoda KH, Nishida T

The R124C mutation of the TGFBI gene gives rise to lattice corneal dystrophy type I, which is characterized by irregularity, turbulence, and opacity of the corneal epithelium. We investigated the efficacy of corneal epithelial debridement followed by application of autologous fibronectin eye drops in the treatment of patients with this mutation.Four patients (6 eyes; age range 25-57 years) treated between April 2006 and March 2008 were enrolled in the study. All patients had impaired visual acuity and recurrent corneal erosion. Corneal epithelial debridement was performed with a spatula at the pupillary zone, after which eye drops containing fibronectin purified from autologous serum were administered four times daily for 2 weeks to promote epithelial migration. Topical levofloxacin and betamethasone were also applied four times daily for 1 month.All corneas achieved resurfacing within a few days of debridement. The best corrected visual acuity of all eyes improved from an average logMAR of 0.80 (range 1.40 logMAR-0.52 logMAR) before treatment to a logMAR of 0.10 (range 0.30 logMAR-0.046 logMAR) by 2-4 months after debridement.Despite the limitation of this study setting, corneal epithelial debridement followed by administration of fibronectin eye drops seems to be effective in improving visual acuity in cases of lattice corneal dystrophy type I caused by the R124C mutation of the TGFBI gene. This treatment may be one option for temporarily improving visual acuity in lattice corneal dystrophy type I and thus may delay the requirement for phototherapeutic keratoplasty or keratoplasty.

Invest Ophthalmol Vis Sci. 2011 Oct; 52(11): 8401-6
Han YP, Sim AJ, Vora SC, Huang AJ

Specific components of transforming growth factor-beta-induced protein (TGFBIp) responsible for amyloid deposits in lattice corneal dystrophy (LCD) have not been delineated. LCD has been associated with various TGFBIp mutations such as R124C, L518P, and L527R. Using recombinant TGFBIp, this study was undertaken to identify TGFBIp components potentially contributing to the protein deposits in LCD.Recombinant wild-type (WT) TGFBIp and four mutants (R124C, R124H, L518P, and L527R) were generated in HEK293FT cells. WT and mutant TGFBIp were collected from crude cell lysates or purified from culture media. Immunoblot analyses were performed with four different anti-TGFBIp antibodies raised against various regions of TGFBIp.Consistent with the authors' previous findings, purified recombinant proteins are more prone to polymerize than crude cell lysates. As expected, all monomers and polymers of TGFBIp WT and mutants were detected by these antibodies. However, the authors noted WT and TGFBIp mutants showed differential reactivities with these antibodies. A 47-kDa band was detected in purified 2-tag proteins of L518P by all four antibodies. A unique 43-kDa band was detected in both 1-tag cell lysates and purified proteins of R124C by the authors' custom-made antibody (KE50) and a commercial anti-TGFBIp.Based on its universal reactivity with various antibodies, the authors surmise that the 47-kDa protein is a ubiquitous TGFBIp fragment derived from the N-terminus of the L518P mutant. The fact that the 43-kDa protein fragment was present primarily in R124C and R124H but not in WT implicates its potential role in the protein deposits of LCD.

Acta Neurol Taiwan. 2011 Jun; 20(2): 155-60
Hsieh ST

Familial amyloid polyneuropathy (FAP) is a major etiology in differential diagnosis of symmetric axonalform polyneuropathy, but had been considered an unusual disease in Taiwan. We have reviewed the pathology of nerve biopsies and sequenced the entire 4 exons of transthyretin (TTR), the most common genetic mutation of FAP. Our studies indicated that the mutation of TTR at Ala97Ser (TTR Ala97Ser) was a new mutation only reported in ethnic Taiwanese, and this mutation accounted for the most frequent etiology of adult-onset pan-modality (involving motor, sensory, and autonomic components of peripheral nerves) polyneuropathy with the pathology of axonal degeneration type. Over the past 10 years, there have been advancements in the management of FAP due to TTR mutations: (1) symptomatic treatments of dyaautonomia, especially orthostatic hypotension, and (2) therapies with liver transplantation and small molecules to reduce or stabilize TTR.

Hum Pathol. 2011 Sep; 42(9): 1259-64
Sueyoshi T, Ueda M, Jono H, Irie H, Sei A, Ide J, Ando Y, Mizuta H

Transthyretin-derived amyloid deposition is commonly found in intercarpal ligaments of patients with senile systemic amyloidosis. However, the frequency of transthyretin-derived amyloid deposits in ligaments of other tissues remains to be elucidated. This study aimed to determine the frequency of amyloid deposition and the precursor proteins of amyloid found in orthopedic disorders. We studied 111 specimens from patients with carpal tunnel syndrome (flexor tenosynovium specimens), rotator cuff tears (rotator cuff tendon specimens), and lumbar canal stenosis (yellow ligament specimens). To identify amyloid precursor proteins, we used immunohistochemical staining with antibodies that react with transthyretin, immunoglobulin light chain, amyloid A protein, and β(2)-microglobulin. By means of Congo red staining, we identified 47 (42.3%) amyloid-positive samples, 39 of which contained transthyretin-derived amyloid (18 flexor tenosynovium specimens, 5 rotator cuff tendon specimens, and 16 yellow ligament specimens). Genetic testing and/or clinical findings suggested that all patients with transthyretin amyloid deposits did not have familial amyloidotic polyneuropathy. The occurrence of amyloid deposition in those tissues depended on age. These results suggest that transthyretin-derived amyloid deposits may occur more frequently in various ligaments and tendons than originally expected. In the future, such amyloid deposits may aid determination of the pathogenesis of ligament and tendon disorders in older patients.

J Biol Chem. 2011 Mar 25; 286(12): 10856-63
Ozawa D, Kaji Y, Yagi H, Sakurai K, Kawakami T, Naiki H, Goto Y

Mutations in keratoepithelin are associated with blinding ocular diseases, including lattice corneal dystrophy type 1 and granular corneal dystrophy type 2. These diseases are characterized by deposits of amyloid fibrils and/or granular non-amyloid aggregates in the cornea. Removing the deposits in the cornea is important for treatment. Previously, we reported the destruction of amyloid fibrils of β(2)-microglobulin K3 fragments and amyloid β by laser irradiation coupled with the binding of an amyloid-specific thioflavin T. Here, we studied the effects of this combination on the amyloid fibrils of two 22-residue fragments of keratoepithelin. The direct observation of individual amyloid fibrils was performed in real time using total internal reflection fluorescence microscopy. Both types of amyloid fibrils were broken up by the laser irradiation, dependent on the laser power. The results suggest the laser-induced destruction of amyloid fibrils to be a useful strategy for the treatment of these corneal dystrophies.

Hum Pathol. 2011 Feb; 42(2): 236-43
Tsuchiya-Suzuki A, Yazaki M, Kametani F, Sekijima Y, Ikeda S

Wild-type transthyretin is inherently an amyloidogenic protein, but its contribution to the formation of amyloid fibrils remains unclear in familial amyloid polyneuropathy patients. Our aim in this study was to elucidate the ratio of wild-type transthyretin in amyloid deposits in familial amyloid polyneuropathy patients. Abdominal fat amyloid fibrils in 44 familial amyloid polyneuropathy patients with amyloidogenic transthyretin Val30Met who had not undergone liver transplantation were examined. The amyloid fibrils were extracted from abdominal fat tissues and the composition ratios of wild-type and variant transthyretin were analyzed with liquid chromatography tandem mass spectrometry. The amyloid fibrils in abdominal fat tissues were composed of not only variant but also wild-type transthyretin in most patients (mean ratio, 40.7% ± 27.5%). The composition ratios of wild-type transthyretin in patients older than 50 years were significantly higher than those in patients younger than 50 (50.7% ± 26.9% versus 30.7 ± 24.8%). Our results indicate that wild-type transthyretin significantly contributes to the formation of amyloid fibrils in familial amyloid polyneuropathy patients with amyloidogenic transthyretin Val30Met, and its contribution tends to increase in older patients, suggesting that aging may play an important role in wild-type transthyretin-derived amyloid fibril formation in familial amyloid polyneuropathy patients. This is the first report showing the relationship between wild-type transthyretin deposition and aging in familial amyloid polyneuropathy patients. In addition, wild-type transthyretin may be more strongly amyloidogenic than previously considered because it is detectable even in amyloid fibrils isolated from young familial amyloid polyneuropathy patients.

J Genet Couns. 2010 Dec; 19(6): 585-92
Santos PI, Figueiredo E, Gomes I, Sequeiros J

This study is an investigation of the impact of familial amyloid polyneuropathy type I (FAP I, ATTR V30M) on death anxiety and symbolic immortality. Templer and Drolet's scales were administered to 524 individuals: (1) 84 relatives at risk, (2) 92 relatives not at risk for FAP I; and (3) a control group (n = 348) with no known hereditary disease in their families. At-risk relatives had, on average, a higher score for death anxiety and a lower score for symbolic immortality, than either those not-at-risk or controls. There were no significant differences in scores on either measure for those not-at-risk versus controls. Being at risk increases death anxiety and threatens the sense of symbolic immortality and psychosocial wellbeing. This may be true for other serious hereditary disorders as well. Genetic counsellors should become familiar with these concepts, feel comfortable initiating discussions about death with their patients, and be able to identify and reinforce their patients' and family members' sense of symbolic immortality.

Mol Vis. 2010; 16: 1429-38
Paliwal P, Sharma A, Tandon R, Sharma N, Titiyal JS, Sen S, Kaur P, Dube D, Vajpayee RB

To screen a cohort of corneal dystrophy patients from North India for mutations in the transforming growth factor beta induced (TGFBI) gene, to correlate genotypes to phenotypes, to describe structural implications of various mutations on protein function, and to discuss the implications for diagnosis.Eighty affected individuals from 61 unrelated families, who were diagnosed with autosomal dominant granular and/or lattice corneal dystrophy, were recruited for the study. Detailed clinical evaluation was undertaken for these patients to establish their corneal phenotypes. Genomic DNA was isolated from peripheral blood samples and all exons of TGFBI were screened for mutations by polymerase chain reaction (PCR) and direct DNA sequencing. Protein molecular dynamics (MD) simulations were performed for the mutations detected to assess the changes in protein structure.The most common mutations seen were Arg555Trp and Arg124Cys. Two novel mutations, Ser516Arg (c.DNA1548C>G), with a phenotype similar to granular corneal dystrophy I (GCDI), and Leu559Val (c.DNA1675T>G), with an atypical phenotype closely resembling epithelial basement membrane dystrophy/map dot fingerprint dystrophy, were identified. Protein modeling studies involving wild type and mutant protein indicated that the Leu559Val is a destabilizing mutation and that Ser516Arg could adversely affect the specific binding of Fas1 domain 4 with other proteins. In addition, two single-nucleotide polymorphisms, rs4669 and rs11331170, were also identified. Mutations were not identified in 8 affected individuals, 6 of whom were diagnosed with bowman layer dystrophy and 2 with lattice corneal dystrophy.This is the first comprehensive report of TGFBI mutations covering a large part of North India. Identification of novel mutations, the presence of phenotypic variability, and the genetic heterogeneity seen in our cases stress the need for mandatory screening of TGFBI for precise diagnosis and classification of corneal dystrophies.

Mol Vis. 2010; 16: 224-30
Zhong X, Chen S, Huang W, Yang J, Chen X, Zhou Y, Zhou Q, Wang Y

To report novel transforming growth factor beta-induced (TGFBI) mutations responsible for lattice corneal dystrophy (LCD), the associated genotype-phenotype correlation, and structural changes in the mutant proteins in three Chinese families.Three unrelated Chinese families were diagnosed as Type I LCD. Mutations in TGFBI were detected by sequencing all of the 17 exons and splice sites of the gene. Phenotype, including corneal erosions, and opacification in the families were compared. Structural changes of the mutant proteins were modeled. One hundred healthy volunteers were recruited as controls for sequence analysis of TGFBI.Two novel mutations, c.(1702G>C and 1706T>A; p.Arg514Pro and Phe515Leu) in TGFBI were identified in Family 1. Two known hotspot mutations, c. 531C>T (p. Arg124Cys) and c.1876A>G (p.His572Arg), were revealed in Family 2 and Family 3, respectively. Sequence analysis in the 100 healthy control subjects, the unaffected members in Family 1, and evolutionary alignment showed that the novel mutations occurred in the conserved amino acids. Structural modeling revealed changes in the 2nd structure of the mutant proteins, but did not detect gross structural changes. Mutations c.(1702G>C and 1706T>A; p.Arg514Pro and Phe515Leu) and the c. 531C>T (p. Arg124Cys) were present in the corneas with sever opacification.The novel mutations c.(1702G>C and 1706T>A; p.Arg514Pro and Phe515Leu), c. 531C>T (p. Arg124Cys), c.1876A>G (p.His572Arg) in TGFBI were responsible for LCD in the 3 families. Mutations c.(1702G>C and 1706T>A) (p.Arg514Pro and Phe515Leu) and the c. 531C>T (p. Arg124Cys) were associated with more severe LCD phenotypes in the families. These results provide more data for molecular diagnosis and prognosis of the disease.

J Genet Couns. 2009 Oct; 18(5): 483-93
Paneque M, Lemos C, Sousa A, Velázquez L, Fleming M, Sequeiros J

To identify possible factors affecting the psychological impact of pre-symptomatic testing for spinocerebellar ataxia type 2 (SCA2) and familial amyloid polyneuropathy (FAP ATTRV30M), we studied (1) the effect of previous experience with the disease in the family, (2) kinship with the closest affected relative and (3) gender of affected parent, when adapting to test results; as well as (4) differences in the course of psychological wellbeing in 63 subjects ( 28 at-risk for FAP ATTRV30M, and 35 at risk for SCA2), who pursued predictive testing for these diseases, in Cuba and in Portugal. Our research shows that individuals with little or no experience with the disease in their family exhibited more anxiety; at-risk subjects for SCA2 or FAP ATTRV30M who had a first degree relative with the disease showed lower levels of anxiety and depression during pre-symptomatic testing. Also those with an affected mother had lower levels of depression, either immediately, or one year after receipt of test results. Adaptation to pre-symptomatic testing results differed for subjects at-risk for the two different conditions. Unlike the FAP ATTRV30M families, carriers for SCA2 reported pathological levels of depression immediately after-testing (3 weeks), although those levels had returned to normal levels at 6 months. Subjects at-risk for FAP ATTRV30M tended to have less anxiety than those tested for SCA2, at the one-year follow-up. Overall, depression levels improved over time, while anxiety remained more constant. A longer awareness of the disease in the family, closer kinship, and a transmitting mother all lessened the impact of pre-symptomatic testing, as expressed by the post-test levels of anxiety and depression.

Biochem Biophys Res Commun. 2009 Oct 16; 388(2): 256-60
Lee KW, Lee DH, Son H, Kim YS, Park JY, Roh GS, Kim HJ, Kang SS, Cho GJ, Choi WS

Transthyretin (TTR) is a human disease-associated amyloidogenic protein that has been implicated in senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). FAP typically results in severe and early-onset disease, and the only therapy established so far is liver transplantation; thus, developing new strategies for treating FAP is of paramount interest. Clusterin has recently been proposed to play a role as an extracellular molecular chaperone, affecting the fibril formation of amyloidogenic proteins. The ability of clusterin to influence amyloid fibril formation prompted us to investigate whether clusterin is capable of inhibiting TTR amyloidosis. Here, we report that clusterin strongly interacts with wild-type TTR and TTR variants V30M and L55P under acidic conditions, and blocks the amyloid fibril formation of TTR variants. In particular, the amyloid fibril formation of V30M TTR in the presence of clusterin is reduced to level similar to wild-type TTR. We also demonstrated that clusterin is an effective inhibitor of L55P TTR amyloidosis, the most aggressive form of TTR diseases. The mechanism by which clusterin inhibits TTR amyloidosis appears to be through stabilization of TTR tetrameric structure. These findings suggest the possibility of using clusterin as a therapeutic agent for TTR amyloidosis.