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9AMYLOIDOGENIC AND NON-AMYLOIDOGENIC TRANSTHYRETIN MUTATIONS IN PATIENTS WITH CHRONIC HEART FAILURE FROM NORTH-WEST RUSSIA
K.V. Solovyovab, A.Ya. Goodkovabc, E.N. Sememinb,c, N.A. Grudininaa,c, A.N. Krutikovf, A.A. Poliakovab,c, E V. Shlyakhtobc,M.M. Shavlovskyab*
a Department of Molecular Genetics, Federal State Budgetary Scientific Institution "Institute of Experimental Medicine",
St. Petersburg, 197376 Russia
b I.P. Pavlov First Saint Petersburg State Medical University, Institute of Cardiovascular Diseases,
St. Petersburg, 197022 Russia
c V.A. Almazov Federal North-West Medical Research Centre, Ministry of Healthcare of the Russian Federaion,
St. Petersburg, 197341 Russia
ABSTRACT
The results of clinical and genetic study of patients with chronic heart failure (CHF) and heart remodeling by cardiomyopathy phenotype are presented. Amyloidogenic mutation V30M in the transthyretin gene (TTR-gene) was detected in four patients suffering from polyneuropathy with cardiac involvement. The possible role of other TTR-gene mutations detected in our cohort of patients, such as H90N, N98N, G6S and deletion (del9) of nine nucleotides in the noncoding region in exon 4, in the development of refractory CHF is discussed.
Keywords: transthyretin; cardiomyopathy; polyneuropathy; familial; amyloidosis.
Abbreviations: transthyretin (TTR); chronic heart failure (CHF); transthyretin amyloidosis (ATTR amyloidosis); familial amyloid polyneuropathy (FAP); restrictive cardiomyopathy (RCM); hypertrophic cardiomyopathy (HCM); dilated cardiomyopathy (DCM); single-strand conformation polymorphism analysis (SSCP); electrocardiogram (ECG); echocardiography (ECHO-CG); ejection fraction (EF); left ventricle (LV); left ventricular hypertrophy (LVH); left ventricular ejection fraction (LVEF); left atrium (LA); right ventricle (RV); right atrium (RA); myocardial mass index (MMI); heart rhythm (HR); arterial hypertension (AH).
Chronic heart failure (CHF) is the main cause of morbidity and mortality in the working-age population in developed countries. In elderly patients the frequency of CHF is especially high and ranges from 2.5% to 10.0%. Russia is among the countries where the frequency of this disease is very high. For example, in USA CHF affects 2.5% of population [1, 24-32], in Russia - up to 9.7%. Raise of number of new CHF cases is partially caused by increased lifespan in developed countries. Every year CHF affects 1% of individuals aged 60 and over and 10% of those aged 75 and over [2, 19-27].
Increase of morbidity and mortality caused by CHF should not be considered separately from various infiltrative myocardial diseases, in particular that are resulted from systemic amyloidosis. Quite common cause of CHF is a cardiomyopathy - a severe disease that can be caused by a variety of reasons including amyloidosis. One of the amyloidogenic proteins is transthyretin (TTR). Normally this protein functions as a transporter of thyroid hormones and retinol-binding protein [3, 3095-3101]. The TTR gene is located on chromosome 18 (18q11.2-q12.1) and consists of 4 exons [4, 243-248]. More than 100 mutations in the TTR gene have been described (http:// amyloidosismutations.com/mut-attr.php). Most of them are amyloidogenic, some are specific for certain ethnic groups only [5, 160-184]. There is no information on the spectrum of the TTR gene mutations in Russia. The only work dedicated to the association of mutation in the TTR gene with the amyloidosis in Russia is the work of Strokov and co-authors who have described the case of familial amyloid neuropathy associated with the Tyr114Cys mutation in the TTR gene [6, 67-72]. Our studies are the first comprehensive research of clinical and genetic aspects of systemic TTR amyloidosis in Russia [7, 543549; 8, 337-349]. In this report we present the results of the TTR gene analysis and correlation of revealed mutations with
clinical features in patients with cardiomyopathy who received treatment in Cardiomyopathy Laboratory of the Institute of Cardiovascular Diseases of Pavlov First Saint Petersburg State Medical University.
PATIENTS AND METHODS
Patients
257 patients with CHF and heart remodeling by cardiomyopathy phenotype (restrictive (RCM), hypertrophic (HCM), dilated (DCM) and the combination of morpfofunctional phenotypes (HCM + RCM, HCM + DCM and unclassified forms) have been examined. Probands were subjected to full clinical examination. The age of the patients varied from 45 to 72 years. In some cases relatives of probands have been examined.
The criteria for patient inclusion in the study were: 1) the presence of clinical symptoms, 2) instrumental and morphological confirmation of cardiomyopathy diagnosis in accordance to the existing classification of cardiomyopathies [9, 55-62]. All patients were in New York Heart Association (NYHA) functional class II-IV. Also patients were diagnosed with diastolic dysfunction of the left and/or right ventricles (LV/RV) of the heart documented by echocardiography (ECHO-CG) and/or magnetic resonance tomography.
Immunochemical assays
The presence of amyloid deposits was tested by Congo red staining. All but one patient's biopsy specimens of the myocardial tissue and/or buccal mucosa were Congo red positive and exhibited bright green birefringence under polarized light microscopy. In some cases immunochemical staining using antisera to TTR and immunoglobulin kappa/ lambda light chains was performed.
Genetic analysis
Genomic DNA was isolated from patients' peripheral blood
samples using a standard method [10, 1245-1249]. Four pairs of primers [7, 543-549] for amplification of each of the TTR exons were designed using Fast-PCR free software (http:// fast-pcr.software.informer.com). Screening for mutations was carried out by means of single-strand conformation polymorphism analysis of DNA fragments (SSCP-analysis). For a better separation of single-stranded DNA conformers during SSCP-analysis Tris-glycine buffers and polyacrylamide gels of different concentrations were used. For detection of heterozygous deletions in DNA PCR products were cloned into the Taklon® (Medigen) vector system (Russia). Automatic
Mutations i
sequencing method was used for identification of the nucleotide substitutions. This procedure was carried out in Evrogen (Russia).
In all cases written informed consent was obtained from patients for using their genomic DNA for the TTR gene analysis. The study was approved by The Research Ethical Committee of Pavlov First Saint Petersburg State Medical University. RESULTS
We have revealed five different variants of the TTR gene which are summarized in Table 1.
Table 1.
the TTR gene
Mutation (name of protein variant) Sequence Variant Codon Change Location Patients and their relatives Review of the Literature
V30M c.142G > A GTG > ATG Exon 2 4 nonrelated patients reported
H90N c.328 C > A CAT > AAT Exon 3 1 patient and in his mother reported
N98N c.384 C > T AAC > AAT Exon 4 1 patient never reported before
del9 A GACTTCTCC noncoding region Exon 4 1 patient and in 2 his daughters reported
G6S c.76 G > A GGT > AGT Exon 2 1 patient reported
Mutation V30M (c.142G> A) in exon 2 of the TTR gene
Four patients were diagnosed with systemic transthyretin amyloidosis (ATTR amyloidosis) caused by V30M mutation using current international guidelines for the diagnosis of ATTR amyloidosis. All 4 patients with ATTRV30M amyloidosis had a similar clinical presentation.
Neurologic manifestations of patients with ATTRV30M amyloidosis
All our patients with ATTRV30M amyloidosis had decreased temperature and tactile sensitivity of the foot. Dysesthesia was frequently observed. Syndrome of mechanical allodynia or thermal allodynia was typical for patients. Hypoalgesia, hyperalgesia, hyperesthesia and hypoesthesia were registered. Later in the course of the disease the surface sensitivity decreased. Pareses appeared in the distal lower limb along with deep sensibility disorders. Sensitive ataxia appeared. Neuropathic pain resembled a burning sensation and became stronger at night. Later on, the sensory-motor disorders extended to the upper limbs. Autonomic nervous system disorders included anhidrosis, impotence, gastrointestinal violations (diarrhoea, constipation, nausea, vomiting), orthostatic hypotension.
Cardiac manifestations of patients with ATTRV30M amyloidosis
The results of ECHO-CG studies for patients with V30M mutation are shown in Tables 2 and 3. These patients demonstrated diastolic dysfunction and severe myocardial hypertrophy. At admission in 3 patients systolic function was preserved, in one patient it was reduced to 41.0% on the background of recurrent thromboembolism of the pulmonary artery small branches. Myocardial mass index (MMI) ranged from 178 (g/m2) to 274 (g/m2). In three patients the symmetric left ventricular hypertrophy (LVH) was detected, and one patient was diagnosed with asymmetric LVH (hypertrophy of the interventricular septum). In all cases there was a combination of morphofunctional phenotypes (HCM + DCM) indicating an advanced stage of the disease. The volume of the left atrium (LA) ranged from 83.5 to 112 ml and the right atrium (RA) - from 70 to 86 ml, respectively. In these patients thickening of the mitral and tricuspid valves were found. The severity of mitral and/or tricuspid regurgitation corresponded to I-II degree. All patients with V30M mutation had fluid in the pericardial cavity in small to moderate amount.
Table 2.
Structural changes in the myocardium and intracardiac hemodynamics in patients with V30M.
Sex/age/ mutation/ phenotype IVSd (mm) LVIDd (mm) LVPWd (mm) LVMI (g/m2) PAPs (mm Hg) RA volume (ml) LA volume (ml) Diameter LA (mm) Valves
f/58/ V30M/ HCM/ RCM 17.3 43.4 16.8 178.0 30.0 86.0 106.0 57.8 Valves are thickened, MR1,TR1,
f/63/ V30M/ HCM/ RCM 15.3 47.0 14.5 192.0 51.0 80.0 83.5 54.0 Valves are thickened, MR2,TR2,AR1,
f/72/ V30M/ HCM/ RCM 18.0 39.0 19.0 220.0 42.0 70.0 112.0 48.0 Valves are thickened, MR2, TR 1
m/66/ V30M/ HCM/ RCM 26.1 38.0 20.4 274.0 80.0 85.0 105.0 49.0 Valves are thickened, MR1, TR1
interventricular septal thickness (IVSd); left ventricular (LVMI); pulmonary artery systolic pressure (PAPs); mitral diastole internal dimension (LVIDd); left ventricular posterior regurgitation (MR); tricuspid regurgitation (TR); aortal wall diastole thickness (LVPWd); left ventricular mass index regurgitation (AR)
Table 3.
Systolic and diastolic myocardial functions in patients with V30M.
Sex/age/mutation/ phenotype LVEF(%) Type of diastolic dysfunction Pericardial fluid
f/58/V30M/HCM/RCM 66.0 Abnormal relaxation A moderate amount of fluid
f/63/V30M/HCM/ RCM 41.0 Pseudo-normalization A small amount of fluid
f/72/V30M/HCM/RCM 62.0 Restriction A small amount of fluid
m/66/V 30M/HCM/RCM 54.0 Restriction A small amount of fluid
The main clinical symptoms of studied patients with ATTRV30M amyloidosis were heart failure with preserved ejection fraction (EF) and peripheral and autonomic polyneuropathy of varying severity. All patients with confirmed ATTRV30M amyloidosis were in NYHA functional class III-IV. CHF (right ventricular or biventricular) manifested by peripheral edema, ascites, hydrothorax and hydropericardium. CHF was refractory to the applied therapy. Cardiac conduction and heart rhythm (HR) disturbances in all these patients were
presented by high grade supraventricular and ventricular arrhythmias. In three patients the pacemaker was implanted because of arrhythmias combined with atrioventricular and sinoatrial blockes (II-III degree) together with paroxysmal atrial fibrillation. In two patients with ATTRV30M amyloidosis low-voltage electrocardiogram (ECG) was observed. The ECG of one female patient with ATTRV30M amyloidosis is presented in Figure 1.
Figure 1. ECG of 63-years-old female with ATTRV30M amyloidosis showing low QRS voltage in limb leads, a pseudo-infarct pattern in the anteroseptal leads (a QS pattern, ST-segment elevation in leads V2-V4 and negative T waves in leads V4-V6), first-degree atrioventricular block and poor R wave progression in leads V1-V3.
In all patients amyloidosis was diagnosed at a late stage of the disease. Sudden death was pronounced in two cases. Increasing of the heart size was revealed in all patients by means of chest X-ray. In all patients with ATTRV30M amyloidosis there were no clinically significant renal disorders.
Illustrative case
A female patient since the age of 72 has been repeatedly hospitalized in different city hospitals due to the progressive biventricular (predominantly RV) CHF.
Admission complaints
Cardiovascular system
Dyspnea at rest or on minimal exertion, swelling of feet, legs, hips and abdomen were present. Presyncopes and syncopes, which predominantly occurred during the transition from the horizontal to the vertical position, were observed for 10-20 times per day.
Peripheral nervous system
Pin sensations in the limbs, spontaneous moderate pain of the skin all over the body, predominantly in the distal lower limbs were present. The pain was caused by mechanical irritation of the skin and intensified at night.
Objective findings
Nervous system
Predomination of polyneuropathy syndrome with peripheral tetraparesis (predominantly in the distal parts of lower limbs) was observed. Superficial and deep sensory modalities were seriously affected. Syndrome of mechanical allodynia was present. Decreased sensation in the inner surface of the feet, atrophy of muscles of shoulder girdle, hypotrophy of hand muscles and a reduction of strength of the distal hand were found.
Cardiovascular system
Skin pallor, jugular venous distention and acrocyanosis were observed. The pulse was 76 beats per minute. Blood pressure (BP) on lying was 105/60 mm Hg, BP on sitting - 95/50 mm Hg, BP on standing at the first minute - 85/40 mm Hg, BP on standing at the third minute - 70/40 mm Hg. Drop in BP was
accompanied by blackout, somnolentia and chest discomfort.
Auscultation
Heart tones were muffled, the first heart sound was weakened on the apex, accent of the second heart sound was on the pulmonary artery. The breathing was harsh with decreased breath sounds in the lower lung fields. Also during examination of lungs, fine moist rales were heard and hypostatic phenomena were revealed.
The abdomen was distended with ascites. Peripheral edema was observed.
ECG
ECG showed a normally functioning pacemaker. HR was 80 beats per minute. Block of the anterior superior division of the left bundle branch was registered. ECG showed a pseudo-infarct pattern in the anteroseptal leads (a QS pattern, ST-segment elevation and positive/negative T waves in leads V1-V5). No hemodynamically significant stenosis was shown by coronarography. The ECG Holter monitoring showed supraventricular arrhythmias atypical for healthy individuals, including a paroxysm of supraventricular tachycardia (HR from 113 to 120 beats per minute). Polymorphic polytopic ventricular extrasystoles (Lown's grade III-IV) were registered.
ECHO-CG
Severe left atrial (LA) dilation, increased thickness of the interatrial septum, severe symmetric concentric LVH (MMI 220g/m2), RV hypertrophy (anterior wall thickness was 0.7 cm) (Figures 2, 3 and 4). The ventricles were not dilated. The ultrasound density of the ventricular myocardium was significantly increased. Normal LV global contractility (EF 62%), increased pressure in pulmonary artery (42 mm Hg), pseudonormal LV filling, RV diastolic dysfunction, reduced systolic myocardial velocities of all LV segments (S' 0,3-0,4 cm/s for fibrous ring of the mitral valve) were detected. RV contractility, assessed by tissue Doppler imaging was on the lower limit of normal (S' 11 cm/s for fibrous ring of the tricuspid valve).
Figure 2. Echocardiogram of 72-years-old female with ATTRV30M amyloidosis showing severe LVH. A) Parasternal long-axis image. B) Parasternal short-axis image. C) LV M-mode measurements.
Figure 3. Two-dimensional speckle-tracking echocardiography image with the automatically derived curves of segmental longitudinal strain of 72-years-old female with ATTRV30M amyloidosis. Global longitudinal strain in two-chamber LV view is reduced to -6.5%.
Figure 4. Two-dimensional speckle-tracking echocardiographic image with the automatically derived curves of segmental longitudinal strain of 72-years-old female with ATTRV30M amyloidosis. Global longitudinal strain in four-chamber LV view is reduced to -5.8%.
Biopsy findings
Deposits of amyloid were detected in the adipose tissue and in buccal mucosa.
Diagnosis
Systemic amyloidosis with cardiac and peripheral nervous involvement was diagnosed.
Genetic research
The PCR product of exon 2 of the patient's TTR gene showed an abnormal SSCP pattern. Sequencing of this PCR product revealed a heterozygous substitution c.142G>A, resulting in the amino acid change V30M.
Other mutations in the TTR gene
In four individuals from the selected cohort of patients DNA analysis disclosed four other defects in the TTR gene
(H90N, del9, G6S and N98N) (summarized in Table 1). No clinical manifestations of polyneuropathy were revealed in any patient with CHF and non-V30M mutations. ECG results in patients with non-V30M mutation are presented in Tables 4 and 5. All these patients had a pronounced LVH. MMI ranged from 157 to 193 g/m2. Diastolic dysfunction was found in three patients. At admission in two patients systolic function was preserved and in two other patients it was reduced to 37.0% and to 47.3% on the background of recurrent thromboembolism of the pulmonary artery small branches. All patients had different cardiomyopathy phenotypes. The combination of morphofunctional phenotypes (HCM + RCM) was found in one patient. HCM, DCM and unclassified variant were found in three other patients. In patients with non-V30M
mutations thickening of the mitral and tricuspid valves and no clinically significant renal disorders. Amyloid deposits were a small amount of fluid in the pericardial cavity were found found in biopsy specimens of the myocardial tissue, buccal (Table 5). In all patients with non-V30M mutations there were mucosa and skin of 2 patients.
Table 4.
Structural changes in the myocardium and intracardiac hemodynamics in patients with non-V30M.
Sex/age/ mutation/ phenotype IVSd (mm) LVIDd (mm) LVPWd (mm) LVMI (g/m2) PAPs (mm Hg) RA volume (ml) LA volume (ml) Diameter LA (mm) Valves
m/45/ H90N/ HCM/ RCM 16.1 46.9 16.1 193.0 35.0 96.0 104.0 54.5 Valves are thickened, MR1, TR1
f/56/ N98N/ HCM 16.0 45.0 14.0 110.0 45.0 40.0 50.0 35.0 Valves are thickened, MR1, TR1, light valve AU
m/55/ del9/ DCM 10.9 84.2 8.4 168.0 42.0 85.0 120.0 62.0 Valves are thickened, MR3, TR3, PR3, AR1
m/51/ G6S 20.0 40.2 11.0 157.0 29.6 65.0 72.0 42.0 Valves are thickened, MR1, TR1
interventricular septal thickness (IVSd); left ventricular (LVMI); pulmonary artery systolic pressure (PAPs); mitral diastole internal dimension (LVIDd); left ventricular posterior regurgitation (MR); tricuspid regurgitation (TR); pulmonary wall diastole thickness (LVPWd); left ventricular mass index regurgitation (PR); aortal regurgitation (AR)
Table 5.
Systolic and diastolic myocardial functions in patients with non-V30M.
Sex/age/mutation/ phenotype LVEF(%) Type of diastolic dysfunction Pericardial fluid
m/45/H90N/HCM/RCM 47.3 Restriction A moderate amount of fluid
f/56/N98N/HCM 69.0 Normal A small amount of fluid
m/55/del9/DCM 37.0 Restriction A moderate amount of fluid
m/51/G6S 60.0 Restriction LV+RV A moderate amount of fluid
Mutation H90N (c.328 A> C) in exon 3 of the TTR gene A 45-year-old man was admitted to the hospital with congestive heart failure. The patient's ECG (Figure 5) showed HR of 97 beats per minute, frequent atrial extrasystoles, moderate sinus tachycardia, signs of RV and LV hypertrophy, signs of enlargement of the RA, slowing of atrioventricular conduction (PQ = 0,21), low QRS voltage in all leads, a QS
pattern in leads V1-V4 and ST-segment elevation in leads V1-V4. Coronary angiography revealed the absence of hemodynamically significant stenosis of coronary arteries. Sudden death occurred due to the Morgagni-Adams-Stokes attack caused by complete sinoatrial block and sinus node arrest with the escape rhythm arising from the atrioventricular junction.
Figure 5. ECG of 45-year-old man with mutation H90N in the TTR gene showing low QRS voltage in all leads, a QS pattern in leads V1-V4 and ST-segment elevation in leads V1- V4.
Autopsy findings
Deposits of amyloid were detected in the myocardium (perivascular and intramuscular). A slight coronary atherosclerosis was found. Immunohistochemical study revealed positive staining for TTR, immunoglobulin lambda light chains and serum amyloid P component.
Diagnosis
Systemic amyloidosis with cardiac, lung and mucous membranes involvement was diagnosed.
Genetic research
The PCR product of exon 3 of the patient's TTR gene showed an abnormal SSCP pattern. Sequencing of this PCR product revealed a heterozygous substitution c.328 C> A, resulting in the amino acid change H90N. This mutation was detected in the heterozygous state in the proband's 74-year-old mother and was not detected in his 21-year-old son [7, 543-549].
Deletion (del9) of nine nucleotides in exon 4 of the TTR gene
A 55-year-old man was diagnosed with DCM caused by viral myocarditis. The clinical course of the disease in this patient was indistinguishable from the disease course of other patients with DCM complicated with progressive CHF and high grade ventricular arrhythmias. Dilatation of all heart cavities was observed. Severe systolic dysfunction (LV ejection fraction varied from 13% to 37%) and restrictive type of diastolic dysfunction were present. Sudden death due to the ventricular fibrillation occurred at the age of 57 years.
Autopsy findings
Deposits of amyloid were detected in the skin, mucous membranes, myocardium and lungs. Immunohistochemical study revealed positive staining for TTR.
Diagnosis
Systemic amyloidosis with cardiac, skin, lung and mucous membranes involvement was diagnosed.
Genetic research and family history
The PCR product of exon 4 of the patient's TTR gene showed an abnormal SSCP pattern. Also heteroduplexes were observed. Cloning and sequencing revealed a nine nucleotide deletion (del9) (GACTTCTCC) at position 6776 (from start codon at position 98782 according to the reference sequence AC079096 (NCBI)) in the noncoding region of exon 4 in the mutant allele of patient's TTR gene.
The deletion was detected in the heterozygous state in two proband's daughters from different marriages and was not detected in his son [7, 543-549]. No other family members had undergone genetic testing.
Patient's daughters (24 and 13 years old) didn't have any clinical symptoms of cardiomyopathy. However, eldest daughter's biopsy specimen of buccal mucosa was positive for amyloid deposition. The medical records of the patient's father and of the patient's older brother were obtained and reviewed. Both of them had died suddenly at the same age of 57 with a similar clinical picture (DCM and congestive heart failure). Thus, the patient was diagnosed with familial form of DCM.
Synonymous substitution N98N (c.384 C> T) in exon 4 of the TTR gene
A 56-year-old woman was admitted to the hospital with the acute LV failure and increased BP (180/100 mm Hg). For the time of the survey the duration of arterial hypertension (AH)
did not exceed one year. The severe concentric, asymmetric LVH was revealed. The degree of LVH did not correlated with the time of the duration and the severity of the AH. Patient was diagnosed with symmetric concentric LVH. The biopsy of buccal mucosa was negative for amyloid deposition.
Diagnosis
AH stage II, risk IV was diagnosed.
Genetic research
The PCR product of exon 4 of the patient's TTR gene showed an abnormal SSCP pattern. Sequencing of this PCR product revealed a substitution c.328 C> A with no amino acid alteration N98N. The potential association of this mutation with the disease is an object for further discussion.
Mutation G6S (c.76 G> A) in exon 2 of the TTR gene
A 51-year-old man was admitted to the hospital on an emergency basis with suspicion of intestinal bleeding which was not confirmed later on. The patient had complaints of weakness, palpitations, dizziness and loss of consciousness. He had lost 20 kg in 3-4 months on the background of weakness, nausea, vomiting, lack of appetite, fainting when going from horizontal to vertical position, low-grade fever and increased stool frequency up to 12 times daily. Fibrogastroduodenoscopy was performed and biopsy of mucosa was taken. Acute surgical disease and oncopathology were excluded. Coronarography did not reveal hemodynamically significant stenoses.
ECG showed low QRS voltage, first-degree atrioventricular block, biatrial and RV enlargement. ECHO-CG showed interventricular septum thickening (in the basal segments -20,0 mm, in the middle third - 12,5 mm), LV posterior wall thickening (11,0 mm), signs of concentric LV remodeling, diastolic dysfunction and preserved left ventricular ejection fraction (LVEF). Mitral, aortic and tricuspid valve leaflets were thickened.
The examination also revealed a progressive intoxication, malabsorption syndrome, severe hypoproteinemia, hypoalbuminemia, dyselectrolytemia, anemia,
thrombocytopenia, orthostatic hypotonia and heart failure. Bence Jones protein was detected in the urine. The patient developed a sudden asystole, respiratory arrest occurred and the blood pressure could not be determined. Emergency resuscitation efforts were unsuccessful and the patient died.
Autopsy findings
Amyloid deposits were found in autopsy specimens.
Diagnosis
The combination of symptoms and the results of surveys aroused suspicion for systemic AL-amyloidosis with gastrointestinal and cardiac involvement.
Genetic research
The PCR product of exon 2 of the patient's TTR gene showed an abnormal SSCP pattern. Sequencing of this PCR product revealed a heterozygous substitution c.76 G> A, resulting in the amino acid change G6S.
DISCUSSION
The spectrum of TTR mutations varies significantly between different ethnic groups and geographical regions. In 257 patients with CHF from St. Petersburg (Russia) we have identified five different variants of the TTR gene (Table 1).
Mutation V30M
Mutation V30M was found in the heterozygous state in
four unrelated patients. This mutation is the most common and well-studied variant worldwide [11, 1057-1062]. The frequency and penetrance of V30M varies in different ethnicities. It was originally described in Portugal patients with familial amyloid polyneuropathy (FAP). Two other endemic regions are Japan and Sweden. The highest frequency of familial TTR amyloidosis caused by mutation V30M is estimated to be 1 in 538 in northern Portugal [12, 116-118]. The lowest frequency of ATTRV30M amyloidosis is in USA (1 in 100000 individuals) [13, 5345-5378]. V30M variant is also common in Greece, Italy and France [14, 1413-1417; 15, 143-153; 16, 524-526]. The age of the onset and the symptoms of the disease vary across populations, even in endemic regions the differences occur. So, in Portuguese and Japanese patients with TTR-FAP early-onset is described [17, 1001-1007; 18, 288-294], whereas to Swedish patients later age of onset is reported [19, 351-354]. In endemic regions cardiac involvement in patients with ATTRV30M amyloidosis is rare (occurs approximately in one of five patients). The clinical presentation is dominated by arrhythmias requiring pacemaker implantation. Development of congestive CHF is not typical for these patients. For patients from non-endemic regions, one of which is North-Western region of Russia, late onset (60 years old and over), severe cardiac involvement (predominantly presented by RCM) and congestive CHF are typical [12, 116-118; 20, 1951-1962].
The main clinical feature of ATTRV30M amyloidosis is a sensorimotor peripheral neuropathy with severe impairment of the autonomic nervous system. Nervous system impairment often does not have any specific features. Therefore, in some cases it is difficult to distinguish familial amyloid polyneuropathy from cases of inherited and acquired neuropathy, such as chronic inflammatory demyelinating polyradiculoneuropathy. Patients with ATTR amyloidosis are often mistakenly diagnosed with chronic inflammatory demyelinating polyradiculoneuropathy [4, 243-248; 21, 191-196; 22, 693-698]. In such cases as presented in our study, ATTR amyloidosis is diagnosed using the results of immunohistochemical study ofbiopsied tissue and genetic research. In a cohort of CHF patients the proportion of ATTRV30M amyloidosis was 1.56%.
All our patients with mutation V30M had late-onset disease (55-65 years and older). Other typical features of the disease included severe concentric symmetric LVH, left ventricular or biventricular diastolic dysfunction, biatrial dilation and congestive CHF. Disproportionate thickening of LV and RV walls in the absence of hypertension, low-voltage ECG, progressive sinoatrial block and atrioventricular block were typical. Life-threatening ventricular and supraventricular rhythm disturbances were found in all such patients.
Mutation H90N
Mutation was detected in one patient (42 years old) and in his mother (74 years old). Mutation H90N was firstly described in the patient with FAP and was thought to cause this disease [23, 1240-1246]. Although later on, it was shown that the presence of this mutation does not lead to the development of the amyloidosis [24, 1004-1008]. Currently mutation H90N is considered to be non-amyloidogenic [5, 160-184].
Clinical manifestations of the disease such as progressive CHF and recurrent thromboembolia of pulmonary artery branches were present in the patient, but were absent in his
mother. Cardiac involvement is common in patients with systemic amyloidosis. The key to the diagnosis of heart disease in systemic amyloidosis is the presence of combination of significant myocardial hypertrophy, low-voltage ECG and congestive predominantly RV CHF refractory to therapy. The presence of intraventricular blocks, sinoatrial blocks and pseudo-infarct pattern is also typical. All these symptoms were observed in our patient. The unusual feature in the clinical picture of the disease in this patient was the absence of signs of peripheral and autonomic neuropathies. The patient's sudden death occurred due to complete sinoatrial block. Immunohistochemical study of autopsy specimens revealed positive staining for two amyloidogenic proteins - TTR and immunoglobulin lambda light chains. Unfortunately the form of TTR in amyloid deposits is unknown. Final autopsy diagnosis was systemic amyloidosis with cardiac, lung and mucous membranes involvement. We assume that in this case the presence of two fibrillogenic proteins influenced the disease course and presumably led to the severe form of systemic amyloidosis which caused fast death of the patient. Thus, the problem concerning amyloidogenic properties of H90NTTR variant remains unsolved.
Deletion del9
The deletion del9 was detected in the heterozygous state in one patient (56 years old) and in his two daughters from different marriages (12 and 30 years). This DNA alteration was described earlier in a group of children with mental retardation from one of China's provinces [25, 230-235]. However, del9 also was found in the group of healthy persons from the same province. These data suggest that there is no association between the presence of del9 and the genesis of mental retardation.
To date there are no diseases known to be associated with the presence of del9. Our data could not confirm the association of del9 in patient's TTR gene with the development of systemic amyloidosis. Although, it is necessary to mention that immunohistochemical study of autopsy specimens revealed positive staining with TTR-antibodies. We had no opportunity to establish which form of TTR (normal or mutated) was present there. TTR-related amyloidoses are phenotypically heterogeneous [15, 143-153] and therefore the clinical features of the disease of patient with del9, such as a severe biventricular heart failure, ventricular HR disorders (Lown's grade V) and heart remodeling by DCM phenotype, could be the symptoms of ATTR amyloidosis. Typically ATTR amyloidosis is a late-onset disease [13, 5345-5378]. At the moment of the examination clinical signs of the cardiomyopathy were absent in two patient's daughters, but it may be due to their young age. We assume that further studies are needed to establish the possible role of del9 in the genesis of the disease.
Mutation N98N
Mutation N98N (c.384 C> T) was detected in the heterozygous state in one patient. This patient had clinically detectable heart disturbances (AH, severe concentric asymmetric LVH), but his biopsy specimen of buccal mucosa was negative for amyloid deposition. This patient had undergone the genetic testing, before the morphological analysis of biopsy was performed. Mutation N98N was not previously described in the literature. It is very likely that this mutation is non-amyloidogenic. We assume that it is not involved in the development of the disease
in this patient.
We have found genetic alterations in the TTR gene in 8 from 257 patients with CHF. Also alterations were revealed in 3 relatives of probands. However, the presence of mutation in the TTR gene is not a sufficient condition for development of amyloidosis, because some mutations in the TTR gene are known to be non-amyloidogenic. Sporadic form of TTR-related amyloidosis could be caused by fibrillogenesis of wildtype TTR [26, 2843-2845; 27, 171-180]. Therefore, in order to exclude or to confirm ATTR amyloidosis in the patients without mutations in the TTR gene, it is necessary to carry out additional immunohisto chemical studies. Also cardiomyopathies in the surveyed patients can arise from deposits of other amyloid precursor proteins, for example such as apolipoprotein A1 [28, 75-84].
Often the ECG features of ATTR and AL amyloidosis are similar. In spite of difficulties it is critical to differentiate familial and senile amyloidosis from AL amyloidosis because of the differences in therapy. At present the only specific treatment for TTR-associated FAP is the orthotopic liver transplantation. This procedure can remove 95% of the production of mutant form of TTR and can slow the progression of the disease. Liver transplantation in TTR-FAP cases could be carried out with the assistance of Familial Amyloidotic Polyneuropathy World Transplant Registry (FAPWTR) [29, 1-18]. Many patients after liver transplantation can live a relatively normal life, although organs impairments that occurred before the transplantation do not reverse. According to the FAPWTR data a 10-year survival rate after liver transplantation is higher in V30M patients in comparison with non-V30M patients [30, 64-71]. It is essential to diagnose the amyloidosis at the early stage, before cardiac involvement occurs, because there are some evidences indicating that even after liver transplantation, normal TTR, produced by donor's liver, can deposit on existing amyloid [27, 171-180]. At present, it is recommended to carry out liver transplantation only in patients with early stage of the disease, because of difficulties connected with progressive amyloid cardiomyopathy or neuropathy [29, 1-18]. For patients with advanced amyloidosis the treatment strategy is focused on the symptomatic relief. Cardiac involvement is common in patients with ATTR amyloidosis, but neurologic manifestations of amyloidosis often lead to the early disability of patients and therefore can mask cardiac manifestations. We assume that identification of the most common mutations in the TTR gene in patients with early stage of CHF with different cardiomyopathy phenotypes from Saint-Petersburg and establishing of genotype-phenotype correlation typical for North-Western region together with revealing of familial cases will lead to the improvement of diagnostics and optimization of medical tactics for treating of amyloidosis.
CONCLUSION
From a selected cohort of patients from North-Western region of Russia four patients were diagnosed with ATTRV30M amyloidosis. In this regard, we assume that it is important to improve the diagnostics of TTR-related familial amyloidosis in Russia. First of all for the improvement of diagnostics it is necessary to carry out regular monitoring of the cardiovascular system in patients with neuropathic pains or progressive sensation disturbances of unknown etiology. Also it is
important to pay greater attention to familial history of patients with polyneuropathy followed by HCM, RCM or DCM.
DECLARATION OF INTEREST
This work was supported by RFBR grant #14-04-01912. The authors report no conflicts of interest in this work.
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