A New ATTR Phe64Ile Mutation With Late-Onset Multiorgan Involvement

By Tarquini, Roberto Perfetto, Federico; Bergesio, Franco; Miliani, Anna; Pace, Stefano Del; Frusconi, Sabrina; Minuti, Barbara; Pelo, Elisabetta; Torricelli, Francesca

Keywords: Amyloidosis, transthyretin, cardiomyopathy, polyneuropathy Abbreviations: TTR= transthyretin; FAP=familial amyloid polyneuropathy; NYHA = New York Heart Association; NT- proBNP= N-terminal pro-brain natriuretic peptide; LVEF=left ventricular ejection fraction; IVRT=isovolumetric relaxation time

Abstract

We describe a novel transthyretin mutation in which phenylalanine is replaced with isoleucine in exon 3 at codon 64: Phe64Ile. The mutation was found in an isolated patient and it was not possible to perform a family study. The phenotype included heart and peripheral nerve involvement associated with a possible gastrointestinal and renal involvement.

Introduction

Transthyretin-related amyloidoses (ATTR) are systemic amyloidoses caused by mutated forms of transthyretin [1]. Gene coding for TTR (MIM# 176300) comprises 4 exons. To date, about 90 different amyloidogenic TTR point mutations have been described and most of them are associated with neuropathy and cardiomyopathy (so called familial amyloid polyneuropathy, FAP) [2-4]. During the past 20 years a number of FAP families have been found in non-endemic areas and now it is recognized that FAP exists in many nations worldwide, including Italy [5]. Kidney involvement is not frequent and it has been described in patients with some TTR mutations (S52P, S77Y, G17E, F33C, V30L) [6-9]. In this paper we describe a novel mutation which replaces a phenylalanine with an isoleucine in exon 3 at codon 64: Phe64Ile. The patient’s phenotype is characterized by cardiac and neurological deposits and probably by a gastrointestinal involvement.

Case report

In May 2004, a 78-year-old Caucasian man was admitted to the Internal Medicine Unit because of exertional fatigue and shortness of breath, associated leg edema, ascites and diarrhea. An electrocardiogram (ECG) showed atrial fibrillation (AF), while an echocardiogram revealed symmetric left ventricular hypertrophy with a sparkling appearance of the myocardium. The left ventricle ejection fraction (LVEF) was estimated to be 69%. A colonoscopy performed because of diarrhea was negative. Serum protein electrophoresis did not show an M component and a 24-h specimen of urine showed a mild proteinuria (200 mg) without Bence-Jones protein. The biopsy of subcutaneous abdominal fat, after Congo red staining, was positive for amyloid showing typical apple-green birefringence under polarized light. The bone marrow biopsy excluded plasma cell dyscrasia. The patient was discharged with the diagnosis of congestive heart failure secondary to cardiac amyloidosis and chronic atrial fibrillation. Five months later, he was admitted to the Internal Medicine Unit for fatigue, dyspnea at rest (NYHA IV), diarrhea, edema and hypesthesia of the legs. Family history revealed that his mother had died aged 81 years due to breast cancer and his father had died aged 102. He had no siblings or children. Physical examination showed an enlarged tender liver, pitting edema of the legs, irregular heart sounds due to atrial fibrillation and a 2/6 Levine grade systolic murmur over the precordium and at the apex. Tendon reflexes were absent, and there was a moderate sensory loss with “sock” distribution. Laboratory tests confirmed the absence of an M component in the serum and urine electrophoresis. Circulating free lambda and kappa light chains were in the normal range. Serum N- terminal pro-brain natriuretic peptide (NT-proBNP) level was increased (840 pg/ml, normal value <227). The complete blood count was normal and urinalysis showed mild proteinuria (150 mg/24 h). An ECG showed AF, right bundle branch block and left anterior fascicular block. The cardiac ultrasonographic examination revealed the presence of incomplete mitral valve closure with moderate mitral regurgitation. The left atrium was dilated (48 x 53 mm) with evidence of elevated left atrial pressure. There was a mild to moderate tricuspid insufficiency. The right ventricular systolic pressure was estimated to be 21 mmHg. The LV size was normal with increased thickness of both the interventricular septum (16 mm) and the posterior wall of the left ventricle (16 mm) indicating symmetric hypertrophy with a sparkling appearance of the myocardium. The left ventricular ejection fraction (LVEF) was estimated to be 65%. The E wave deceleration time was 72 ms, while isovolumetric relaxation time (TVRT) was 128 ms, suggesting LV diastolic dysfunction. A mild pericardial effusion was demonstrated. The electromyography of upper and lower limbs was consistent with severe axonal neuropathy. Abdominal ultrasonography revealed a mild hepatomegaly without splenomegaly and ascites. Orthostatic hypotension was present, but the test for autonomic dysfunction was not reliable because of the presence of AF. The absence either of serum and urinary M component or of chronic inflammatory or infectious diseases prompted us to perform the molecular analysis to rule out genetic forms of amyloidosis. The molecular analysis showed the hitherto undescribed Phe64Ile TTR mutation. The patient was submitted to the following therapy: furosemide (60 mg/day), spironolactone (100 mg/ day), carvedilol (up to 3.125 mg twice a day for 4 weeks, then 6.25 mg twice a day) and loperamide. This therapeutic regimen was associated with a rapid improvement in symptoms. Four months after discharge the patient showed an increased exercise capacity, reduction of dyspnea and the disappearance of ascites and pitting edema of the limbs. Diarrhea, however, was sometimes present. After the following 4 months the cardiac ultrasonographic examination showed dilation of both left and right atrium and increased LV thickness: the interventricular septum thickness was 22 mm and the posterior wall thickness was 22 mm with granular sparkling (Figure 1). The LVEF was 48% and diastolic dysfunction was suggested by the E wave deceleration time of 110 ms and by the IVRT of 72 ms. The ratio E/E' was 19 (normal value

Figure 1. Apical four-chamber view of a two-dimensional echocardiogram, shows marked thickening of cardiac walls, especially of the interventricular septum (22 mm) with a granular sparkling of the right ventricular free wall and interatrial septum. Left and right ventricular size is small while the atria were enlarged, with a size of 54 mm. The mitral and tricuspidal valves also appear thickened.

Molecular analysis

After receiving written informed consent, genomic DNA was isolated from 200 [mu]l of peripheral blood by the QIAamp DNA Blood kit (Qiagen GmbH, Germany). The TTR gene was amplified and polymerase chain reaction (PCR) products were sequenced by the ABI PRISM BigDye Terminators cycle sequencing kit and electrophoresed on the 3100 Genetic Analyzer (Applied Biosystems, Foster City, KA, USA). Data analysis revealed that the proband was heterozygous for the TTT[arrow right]ATT substitution at codon 64 of exon 3, which replaced a phenylalanine with an isoleucine (Phe64Ile) (Figure 2). Mutation was validated on a second independent PCR product.

Data obtained from the SeqScape v2.1.1 Software were aligned with the wild-type TTR gene sequence by the SMART web-based tool (http:// smart.embl-heidelberg.de/) to analyze conserved motifs of TTR.

Figure 2. Identification of the Phe64Ile substitution (T to A nucleotide change) in exon 3 by DNA direct sequencing. Top, the sequence of the wild-type sample; bottom, the patient’s sequence. The arrow indicates the mutation. Please see manuscript online for colour image.

Discussion

In this report we identify a previously undescribed mutation: Phe64Ile. The major clinical characteristics of this new TTR variant seem to be the late-onset sensorimotor polyneuropathy of the lower limbs, severe cardiomyopathy and probable gastrointestinal involvement. Despite the fact that amyloid deposition within tissues other than periumbilical fat tissue was lacking, clinical and laboratory findings are typical for amyloid involvement of peripheral nervous system and heart. Gastrointestinal symptoms, in particular chronic diarrhea, might have been die consequence either of autonomic involvement and/or of direct amyloid deposition although a gastrointestinal biopsy was not performed. An immunohistochemical or biochemical characterization of amyloid in the tissue is lacking; however, the absence of a monoclonal component and a negative history of chronic illness stands against a different origin of the amyloid deposit. The Phe64Ile mutation is located at a highly conserved residue of the TTR-related proteins. Furthermore at the same position other mutations have been described [10-13]. Li et al. [10], in a proband of Italian origin affected by polyneuropathy, described the Phe64Leu mutation without a specific correlation genotype-phenotype. Also Ferlini et al. [11], described two families of Italian origin with the Phe64Leu mutation. One family comprised six heterozygous patients in two generations affected by both polyneuropathy and/or cardiomyopathy, with the onset of the disease occurring in the 7th decade of life. The other study included only the proband since he was adopted; this patient had a homozygous genotype and his symptoms started when he was aged 49. In another Italian family, Uemichi et al. [13] also identified the Phe64Ser substitution. This mutation segregates with a phenotype that differs from those of the Phe64Leu mutation and is characterized by an oculoleptomeningeal amyloidosis. Unfortunately, as regards the novel mutation we describe, we lack any information about the patient’s kindred and possible phenotypic differences among generations. The clinical manifestations of this mutation seem to occur late, involving first the peripheral nervous system and men the heart. Clinical follow-up suggests that this type of TTR variant is not particularly aggressive to the peripheral nervous system but, despite the late onset, the cardiac involvement becomes massive inducing rapid and severe congestive heart failure. As reported by Hattori et al. [14], patients with FAP related to non-Val30Met ATTRs frequently showed a late onset of symptoms, but a more pronounced cardiac dysfunction, including intractable heart failure, in addition to peripheral somatic and/or autonomic neuropathy. These authors suggest that aging may play an important pathogenetic role in the development of cardiac involvement. Because of the late onset of the ATTRs, a misdiagnosis of senile systemic amyloidosis may have critical implications for the patients and their relatives, emphasizing the importance of molecular studies to screen for hereditary mutations in these elderly patients. Several factors, other than the TTR mutation may be related to the expression of the disease. The deposition of amyloid fibrils in tissues and organs could be influenced by: the presence of modifier genes or polymorphisms; the functional interactions among different gene products; and/ or various physiological and environmental factors [15-19]. References

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ROBERTO TARQULNI1, FEDERICO PERFETTO1, FRANCO BERGESIO2, ANNA MILLANI1, STEFANO DEL PACE3, SABRLNA FRUSCONI4, BARBARA MLNUTI4, EUSABETTA PELO4, & FRANCESCA TORRICELLI4

1 Dipartimento di Medicina Interna, Azienda Universitaria Ospedaliera Careggi, Florence, Italy, 2 Unitd di Nefrologia, Azienda Universitaria Ospedaliera Careggi, Florence, Italy, 3 Dipartimento Area Critica Medico Chirurgica, University of Florence, Italy, and 4 SOD Diagnostica Genetica, Azienda Universitaria Ospedaliera Careggi, Florence, Italy

Correspondence: Roberto Tarquini, Azienda Universitaria Ospedaliera Careggi, Department of Internal Medicine, Viale Morgagni, 85 50143 Florence, Italy. Tel: +39-055-7947845. Fax: +39- 055-417121. E-mail: rtarquini@unifi.it

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