Basic Research in
Cardiology
Basic Res Cardio186:567-571 (1991)
TransthyTetin Leu 68 in a form of cardiac amyloidosis M. R. Almeidal'2), A. Hesse3), A. Steinmetz3), B. Maisch3), K. Altland4), R. P. LinkeS), M. A. Gawinowicz6), and M. J. M. Saraiva 1'2) 1) Centro de Estudos de Paramiloidose 2) Instituto de Ci~ncias Biom6dicas, Universidade do Porto, 4300 Porto, Portugal 3) Zentrum Innere Medizin, Philips-Universit~it, D-3550 Marburg, Germany 4) Institut ftir Humangenetik Justus-Liebig-Universit~it, D-6300 Giegen, Germany 5) Max-Planck-Institut fttr Biochemie, D-8033 Martinsried, Germany 6) Department of Medicine, Columbia University, New York, USA Summary: A form of transthyretin (TTR)-related cardiac amyloidosis was previously described in a German patient. Electrophoretic analysis of plasma TI'R showed the presence of an electrically neutral variant. We have now characterized the variant transthyretin by comparative peptide mapping, aminoacid and DNA sequencing procedures. A new mutation in TTR with a substitution of leucine for isoleucine at position 68 of the monomer is described. Key words: Amyloidosis; cardiac '_amyloidosis;cardiomyopathy; _transthyretin variant Introduction
Several forms of familial amyloidosis have been associated with different transthyretin (TTR) variants. All the known variants arise from single amino acid substitutions due to single point mutations in the coding region of the ~ITR gene. Most of these variants are associated with neuropathies and show variable degrees of cardiac involvement. The TTR variants which were described to solely involve cardiomyopathy are TTR Thr 45 (9), TTR Met 111 (6) and T ~ R Ile 122 (3, 8). The variants TTR Ala 49 (1), TTR Ala 60 (12), and TTR Asn 90 (11) are associated with cardiomyopathy and neuropathy. A form of TTR-related cardiac amyloidosis, was previously described in a German patient (4). This individual was reported to have primarily a cardiomyopathy and signs of polyneuropathy. Amyloid material in a cardiac biopsy stained positive for TTR antibodies. Electrophoretic analysis of his plasma TTR by double one-dimensional electrophoresis with polyacrylamide gels followed by isoelectric focusing (as described by Altland et al. (2) with modifications designed for the study of folding-unfolding reactions of TTR in the presence of urea) showed that this was a electrically neutral variant. Electrophoretic analysis of the patient's family members showed the patient's son to also be a carrier of the variant. We have characterized this TTR variant by protein and D N A analysis and found a substitution of leucine for isoleucine at position 68 of the TTR monomer. Material and Methods
Subject
Blood was obtained from a 63-year-old male German patient with cardiac amyloidosis (who is a carrier of variant TTR, reported previously (4)), 700
568
Basic Research in Cardiology, Vol. 86, No. 6 (1991)
Protein analysis Comparative tryptic peptide mapping of plasma TFR from the patient and of normal TFR was performed by high-performance liquid chromatography (HPLC). TFR was isolated from plasma by procedures described previously (7), including ion-exchange and affinity chromatography on BlueSepharose. Approximately 1 mg of each TTR sample was digested with trypsin, and the resulting peptides were separated by HPLC on a reverse-phase C8 column with a gradient of 0-30 % of acetonitrile in 0.1% trifluoroacetic acid, for 85 rain (8). Some of the peptides were collected and subjected to sequence analysis on an Applied Biosystems model 470A gas-phase sequencer equipped with a 120A PTH analyzer.
D N A sequencing Exon 3 of the TTR gene was amplified from DNA isolated from leucocytes of the patient using conditions previously described (1), except for performing an asymmetric amplification with 5 pmol of biotinylated primer A (5' TCCTCCATGCGTAACTFAAT 3') and 100 pmol of primer B (5' A C T G T G C A T I T C C T G G A A T G 3'). The amplified DNA was linked to avidin-agarose (5). The slurry was applied to a Sephadex G-50 column and the amplified strand (antisense) was eluted with NaOH 0.2 M; after neutralization with ammonium acetate 5M, the single strand DNA was precipitated with ethanol and diluted with 20 ~1 of TE. 7 ~1 of the single strand DNA was sequenced with 10 pmol of primer A using Sequenase version 2.0 (USB) with c~-32p-dATP. The sequencing gel was exposed to xray film for 24 h.
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Cardiology
Basic Res Cardio186:567-571 (1991)
TransthyTetin Leu 68 in a form of cardiac amyloidosis M. R. Almeidal'2), A. Hesse3), A. Steinmetz3), B. Maisch3), K. Altland4), R. P. LinkeS), M. A. Gawinowicz6), and M. J. M. Saraiva 1'2) 1) Centro de Estudos de Paramiloidose 2) Instituto de Ci~ncias Biom6dicas, Universidade do Porto, 4300 Porto, Portugal 3) Zentrum Innere Medizin, Philips-Universit~it, D-3550 Marburg, Germany 4) Institut ftir Humangenetik Justus-Liebig-Universit~it, D-6300 Giegen, Germany 5) Max-Planck-Institut fttr Biochemie, D-8033 Martinsried, Germany 6) Department of Medicine, Columbia University, New York, USA Summary: A form of transthyretin (TTR)-related cardiac amyloidosis was previously described in a German patient. Electrophoretic analysis of plasma TI'R showed the presence of an electrically neutral variant. We have now characterized the variant transthyretin by comparative peptide mapping, aminoacid and DNA sequencing procedures. A new mutation in TTR with a substitution of leucine for isoleucine at position 68 of the monomer is described. Key words: Amyloidosis; cardiac '_amyloidosis;cardiomyopathy; _transthyretin variant Introduction
Several forms of familial amyloidosis have been associated with different transthyretin (TTR) variants. All the known variants arise from single amino acid substitutions due to single point mutations in the coding region of the ~ITR gene. Most of these variants are associated with neuropathies and show variable degrees of cardiac involvement. The TTR variants which were described to solely involve cardiomyopathy are TTR Thr 45 (9), TTR Met 111 (6) and T ~ R Ile 122 (3, 8). The variants TTR Ala 49 (1), TTR Ala 60 (12), and TTR Asn 90 (11) are associated with cardiomyopathy and neuropathy. A form of TTR-related cardiac amyloidosis, was previously described in a German patient (4). This individual was reported to have primarily a cardiomyopathy and signs of polyneuropathy. Amyloid material in a cardiac biopsy stained positive for TTR antibodies. Electrophoretic analysis of his plasma TTR by double one-dimensional electrophoresis with polyacrylamide gels followed by isoelectric focusing (as described by Altland et al. (2) with modifications designed for the study of folding-unfolding reactions of TTR in the presence of urea) showed that this was a electrically neutral variant. Electrophoretic analysis of the patient's family members showed the patient's son to also be a carrier of the variant. We have characterized this TTR variant by protein and D N A analysis and found a substitution of leucine for isoleucine at position 68 of the TTR monomer. Material and Methods
Subject
Blood was obtained from a 63-year-old male German patient with cardiac amyloidosis (who is a carrier of variant TTR, reported previously (4)), 700
568
Basic Research in Cardiology, Vol. 86, No. 6 (1991)
Protein analysis Comparative tryptic peptide mapping of plasma TFR from the patient and of normal TFR was performed by high-performance liquid chromatography (HPLC). TFR was isolated from plasma by procedures described previously (7), including ion-exchange and affinity chromatography on BlueSepharose. Approximately 1 mg of each TTR sample was digested with trypsin, and the resulting peptides were separated by HPLC on a reverse-phase C8 column with a gradient of 0-30 % of acetonitrile in 0.1% trifluoroacetic acid, for 85 rain (8). Some of the peptides were collected and subjected to sequence analysis on an Applied Biosystems model 470A gas-phase sequencer equipped with a 120A PTH analyzer.
D N A sequencing Exon 3 of the TTR gene was amplified from DNA isolated from leucocytes of the patient using conditions previously described (1), except for performing an asymmetric amplification with 5 pmol of biotinylated primer A (5' TCCTCCATGCGTAACTFAAT 3') and 100 pmol of primer B (5' A C T G T G C A T I T C C T G G A A T G 3'). The amplified DNA was linked to avidin-agarose (5). The slurry was applied to a Sephadex G-50 column and the amplified strand (antisense) was eluted with NaOH 0.2 M; after neutralization with ammonium acetate 5M, the single strand DNA was precipitated with ethanol and diluted with 20 ~1 of TE. 7 ~1 of the single strand DNA was sequenced with 10 pmol of primer A using Sequenase version 2.0 (USB) with c~-32p-dATP. The sequencing gel was exposed to xray film for 24 h.
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