Transthyretin Valine-94-Alanine, a Novel Variant Associated With Late-Onset Systemic Amyloidosis With Cardiac Involvement

By Kristen, Arnt V Ehlermann, Philipp; Helmke, Burkhard; Hund, Ernst; Haberkorn, Uwe; Linke, Reinhold P; Katus, Hugo A; Winter, Pia; Altland, Klaus; Dengler, Thomas J

Keywords: Dilated cardiomyopathy, cardiac amyloidosis, transthyretin Abbreviations: cTnT= cardiac troponin T; FAP= familial amyloid polyneuropathy; HIEF= hybrid isoelectric focusing; LV= left ventricular; NT-proBNP= N-terminal pro-natriuretic peptide; NYHA = New York Heart Association heart failure class; peakVO^sub 2^ =peak oxygen uptake

Abstract

A 63-year-old Caucasian male, diagnosed with dilated cardiomyopathy in 1993, remained clinically stable for several years. In 2003, a marked increase of N-terminal pro-natriuretic peptide serum level (611 ng/ml to 4926 ng/ml) was observed; left ventricular (LV) septum thickness was 10 mm. In addition, sensorimotor polyneuropathy and autonomic dysfunction occurred. Further progression of heart failure occurred despite unchanged systolic LV function. Endomyocardial biopsy in 2006 revealed transthyretin amyloidosis by Congo red and immunohistochemical staining, as well as Val94Ala substitution by transthyretin gene analysis. Cardiac amyloid deposition was quantified by technetium- 99m-3,3-diphosphono-1,2-propanodicarboxylic acid (^sup 99m^Tc-DPD) scintigraphy. Mutational search of the relatives (n=1) was unremarkable. The transthyretin Val94Ala mutation is characterized by sensorimotor polyneuropathy, autonomic dysfunction, and gastrointestinal and cardiac involvement with amyloid. This mutation is an addition to the growing spectrum of transthyretin mutations with late onset of clinical symptoms, but noteworthy because of progressive, finally disabling disease course. Final clinical assessment of severity of cardiac involvement in the present patient is rendered complex by possible concomitant or preceding idiopathic dilated cardiomyopathy.

Introduction

Amyloidosis is a generic term describing the extracellular deposition of beta-fibrils in targeted organs with a characteristic ultrastructure consisting of linear, non-branching fibrils and an affinity for Congo red dye [1]. Classification is usually determined by the major protein constituent found in the amyloid fibril deposits. Currently more than 25 different proteins have been identified in amyloid deposits [2]. Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant disease. Most FAP cases have been linked to single point mutations in the plasma protein transthyretin, synthesized in the liver, the choroid plexus, and the retina of the eyes. It plays a major role in the transport of thyroxine and retinol-loaded retinol-binding protein. Today about 100 transthyretin mutations are known; the vast majority are associated with a clinical phenotype, predominantly polyneuropathy and/or cardiomyopathy [3].

Here, we report for the first time a full clinical characterization of a point mutation that substitutes a valine for an alanine amino acid at position 94 of the 127 amino acid protein transthyretin, resulting in a sensorimotor polyneuropathy, severe autonomic dysfunction, and cardiomyopathy.

Case report

A 63-year-old Caucasian male was diagnosed in 1993 with dilated cardiomyopathy and markedly impaired left ventricular (LV) function (ejection fraction 20%), enlarged LV, and concomitant exclusion of coronary artery disease. Septum thickness was 10 mm; cardiac biomarkers were slightly increased (Table I), renal function was not impaired (creatinine 0.90 mg/dl, urea 36 mg/dl). Right ventricular endomyocardial biopsy revealed discordant hypertrophy and vacuolization of cardiomyocytes indicating dilated cardiomyopathy; amyloid staining was not performed. The patient was largely asymptomatic (NYHA II). He was on standard heart failure medication (beta-blocker, ACE-inhibitor, diuretics, digoxin, phenprocoumon; later spironolactone, and atorvastatin) and had stable course of the disease for several years. In 2003, a marked increase of N-terminal pro-natriuretic peptide (NT-proBNP) serum concentration was noted (Table I), while renal function remained unaltered. Endomyocardial biopsy did not show signs of ongoing inflammation or persistent viral genomes; amyloid staining was not performed at that time. In the following months the patient reported loss of appetite, diarrhea (3-5 x per day), fatigue, and muscular weakness resulting in a loss of weight (15 kg/9 months). Gastroscopy and colonoscopy revealed no intestinal amyloid deposition. Within the next year a decrease of exercise capacity (NYHA III, peak VO^sub 2^ 9.6 ml/kg/min) was observed. Cardiac biomarkers and LV wall further increased (Table I). Recurrent syncope presumably due to ventricular tachycardia and orthostatic deregulation had occurred; therefore an automated cardioverter-defibrillator was implanted (St. Jude Medical Epic + VR, Eschborn, Germany). Furthermore, he developed severe tetraparesis with predominant wasting of the proximal thigh and upper arm. Vibratory and deep sensibilities of the toe tip were markedly impaired. Motor nerve conduction studies of the peroneal (34.0 m/s; distal motor latency (dmL) 6.3 ms, amplitude 0.3 mV) and medianus (42.0 m/s; dmL 5.2 ms, amplitude 1.4 mV) revealed a fiber- length-dependent sensorimotor neuropathy. Due to progressive heart failure, despite unchanged systolic LV function (Table I), another endomyocardial biopsy was performed in 2006 revealing indications of dilated cardiomyopathy and pronounced subendocardial as well as peri- and endomysial Congo red staining indicating amyloid deposition. Immunohistochemical studies on paraffin-embedded tissue sections confirmed amyloidogenic transthyretin (ATTR) deposition by the use of the indirect peroxidase method and a panel of specific antibodies directed against amyloid fibril proteins as described previously [4].

Table I. Summary of the clinical and cardiac data.

Transthyretin DNA analysis was performed from peripheral blood. The coding exons for mature transthyretin [5,6] were amplified by polymerase chain reaction, purified, and sequenced in both directions as described previously [7], revealing the substitution of valine for alanine at position 94 of the transthyretin gene (Figure 1C). The microheterogeneity of transthyretin monomers was studied by double-one-dimensional electrophoretic procedure with vertical PAGE followed by horizontal hybrid isoelectric focusing in urea gradients as described previously [8] (Figure 1A, B).

Cardiac amyloid deposition was quantified by technetium-99m-3,3- diphosphono-1,2-propanodicarboxylic acid (^sup 99m^Tc-DPD) scintigraphy [9] (Figure 2). The myocardial tracer retention was 4.6% of the whole body activity at 3 h after injection. The ratio of the heart uptake to the contralateral reference region was 1.8.

Retrospective analysis of endomyocardial biopsy specimens (2003) as well as intestinal biopsy (2005) revealed amyloid deposition. The endomyocardial biopsy taken in 1993 was not available. Currently, the patient is on conservative heart failure treatment; due to intractable MRSA colonization and the advanced non-cardiac involvement the patient was deemed ineligible for heart and/or liver transplantation. The family history indicated that the proband’s father died at the age of 81 years from heart failure; he had Alzheimer’s disease. The mother died at the age of 71 from acute myocardial infarction. The index patient’s 67-year-old sister was asymptomatic for amyloidosis and mutational search was negative for the Val94Ala mutation.

Figure 1. (A) Urea titration curves under reducing conditions of transthyretin from the heterozygote carrier of normal transthyretin and the amyloidogenic variant ATTK Val94Ala. (B) Double one- dimensional electrophoretic pattern from vertical PAGE followed by horizontal hybrid isoelectric focusing in the presence of 2.7 M urea of normal plasma ATTK and ATTK Val94Ala sample from the index patient (arrow) to distinguish the mutant ATTK monomer from normal. (C) Direct nucleotide sequencing of the transthyretin exon 4. The arrow indicates the T/C ambiguity which results from the GTA to GCA change at codon 94 causing the novel substitution of valine for alanine at position 94 of the transthyretin gene.

Discussion

In this report we present a 63-year-old Caucasian male with dilated cardiomyopathy accelerated by an ATTK variant that has previously not been characterized in full clinical detail [10]. ATTR Val94Ala is associated with sensorimotor peripheral polyneuropathy, severe autonomic dysfunction, and amyloid cardiomyopathy with late onset of symptoms. Diagnosis was confirmed by endomyocardial biopsy with immunohistochemical studies and analysis of the transthyretin gene. ATTK-derived amyloid deposition in the affected tissues and the well-documented link between other transthyretin mutations, FAP and cardiomyopathy support the conclusion that the ATTK Val94Ala mutation causes FAP in this patient and at least contributes to the cardiomyopathy. As the only available family member was not affected, familial transmission versus spontaneous mutation cannot formally be discerned, but given the dominant autosomal genetics of other transthyretin mutations familial transmission is likely.

Nevertheless, interpretation of cardiac disease in the present patient is complex because the initial biopsy specimen was not available and the early clinical picture was one of dilated cardiomyopathy characterized by markedly reduced systolic LV function and increased LV diameter in the absence of increased LV wall thickness. Retrospective analysis of the endomyocardial biopsy was positive for amyloid coinciding with the beginning of the accelerated course of cardiac symptoms (increase of NT-proBNP and LV wall). Thus, cardiomyopathy in this patient might be explained retrospectively by: (i) cardiac dysfunction exclusively due to ATTK, initially non-hypertrophic; (ii) pre-existing familial or idiopathic dilated cardiomyopathy accelerated by late onset of clinically relevant ATTK disease; and (iii) idiopathic dilatative cardiomyopathy with clinically irrelevant ATTK deposition representing an epiphenomenon. Figure 2. Cardiac amyloid deposition visualized and quantified by the bone scanning agent technetium-99m- 3,3-diphosphono-1,2propanodicarboxylic acid (^sup 99m^Tc-DPD) scintigraphy. RVL, right-ventral-left; LDR, left-dorsal-right.

In our interpretation, pre-existing dilated cardiomyopathy with acceleration due to late-onset amyloidosis is favored by the clinical course of cardiac amyloid disease especially as progression of cardiac symptoms and echocardiography parameters coincides with occurrence of FAP symptoms and the accelerated increase in LV wall thickness, a signature finding in patients with AL and ATTK cardiac disease [11]. Also, elevated serum levels of NT-proBNP that appeared disproportionate to the clinical symptoms have been reported with cardiac amyloid disease even in the absence of LV impairment [12,13]. Clinical validity of cardiac involvement by Val94Ala ATTK disease can therefore only be assessed incompletely, as the relative of the index patient does not show the transthyretin mutation. Sensorimotor polyneuropathy might also be explained by critical illness polyneuropathy; this, however, appears unlikely as FAP symptoms occurred more than 2 years prior to the patient’s first major infection.

We presented for the first time a full clinical description of the ATTK Val94Ala mutation characterized by sensorimotor polyneuropathy, autonomic dysfunction, and gastrointestinal and cardiac involvement of amyloidosis. This mutation is an addition to the growing spectrum of transthyretin mutations with late-onset of clinical symptoms, but noteworthy because of progressive, finally disabling disease course. Conclusive clinical assessment of severity of cardiac involvement in the present patient is rendered complex by possible simultaneous or preceding idiopathic dilated cardiomyopathy.

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ARNT V. KRISTEN1, PHILIPP EHLERMANN1, BURKHARD HELMKE2, ERNST HUND3, UWE HABERKORN4, REINHOLD P. LINKE5, HUGO A. KATUS1, PIA WINTER6, KLAUS ALTLAND6, & THOMAS J. DENGLER1

1 Department of Cardiology, Medical University of Heidelberg, Im Neuenheimer Feld 410, D-69120 Heidelberg, Germany, 2 Institute of Pathology, University of Heidelberg, Im Neuenheimer Feld 220/221, D- 69120 Heidelberg, Germany,3 Department of Neurology, and 4 Department of Nuclear Medicine, University of Heidelberg, Im Neuenheimer Feld 400, D-69120 Heidelberg, Germany, 5 amYmed, IZB, Am Khpferspitz 19, D-82152 Munchen, Germany, and 6 Institute of Human Genetics, Justus-Liebig-Universitat, Schlangenzahl 14, D-35392 Giessen, Germany

Correspondence: Amt V. Kristen, Department of Cardiology, Angiology and Respiratory Medicine, University Hospital Heidelberg, Im Neuenheimer Feld 410, D-69120 Heidelberg, Germany. Tel: +49-6221- 568611. Fax: +49-6221-565515. E-mail: Arnt_Kristen@med.uni- heidelberg.de

Copyright Taylor & Francis Ltd. Dec 2007

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