Identification of a Novel TTR Gly67Glu Mutant and the First Case Series of Familial Transthyretin Amyloidosis in Hong Kong Chinese
By Mak, Chloe M Kwong, Yok-Lam; Lam, Ching-Wan; Chan, See-Ching; Lo, Chung-Mau; Fan, Sheung-Tak; Chang, Chee-My; Lau, Yuk-Kwong; U, Lok- Sun; Tam, Sidney
Keywords: Familial transthyretin amyloidosis, Hong Kong Chinese, TTR gene Abbreviations: ATTR = Familial transthyretin amyloidosis
Abstract
Familial transthyretin amyloidosis (ATTR), caused by mutant transthyretin deposition, is mainly characterized by peripheral neuropathy, autonomic dysfunction, and cardiomyopathy. There are few reports among the Chinese population. We previously described the TTR mutation (Val30Ala) in the first Hong Kong Chinese family with ATTR. In this study, we report the progress of this family and describe another three unrelated Chinese kinships newly diagnosed with ATTR. The second proband presented mainly with peripheral neuropathy, and genetic analysis of the TTR gene showed alanine-to- serine substitution at amino acid 97. The third proband complained of autonomic dysfunction, and a novel missense mutation of glycine- to-glutamate substitution at amino acid 67 was found. The fourth patient presented with peripheral neuropathy and diastolic cardiomyopathy with the mutation threonine-to-lysine at codon 59. Diagnosis was delayed for more than 2 years. We performed DNA analysis in 46 subjects and detected a total of 21 patients, including the four probands, affected with ATTR, 15 of whom were still at a symptom-free stage at the time of writing. We conclude that ATTR remains largely underdiagnosed in the Chinese population. A high clinical suspicion is crucial for a timely diagnosis and can thus lead to a significant decrease in morbidity and mortality.
Introduction
Familial transthyretin amyloidosis (ATTR) is a relentless and lethal disease, characterized by lateonset progressive polyneuropathy, autonomic dysfunction, gastrointestinal tract disorder, cardiomyopathy and sometimes fatal arrhythmias [1]. ATTR exhibits marked genetic anticipation in some families, with progressively earlier age of onset and increasing severity in successive generations. The transthyretin (TTR) gene, encoding for 127 amino acids, spans 6.9 kb on chromosome 18q12.1 and consists of four exons [2]. To date, more than 90 TTR mutations have been reported [3]. ATTR is considered a rare disease with an annual incidence about 1 in 125,000 [4]. Nonetheless, to our knowledge, there are few reports in the Chinese population [5-10]. We have previously characterized the genotype of the first Hong Kong Chinese family with ATTR [8]. In this study, we report the follow-up of this family and also describe the clinical and molecular characteristics of another three unrelated Hong Kong Chinese kinships recently diagnosed with ATTR. We believe that the disease remains largely under-diagnosed in the Chinese population. A high clinical suspicion is crucial for a correct and timely diagnosis and can lead to a significant decrease in morbidity and mortality.
Case A
The clinical history of this family has been described in our previous paper [8] (Figure 1A). Four patients had received liver transplantations with one domino (AIV-3 in 2004), one living- related (AIH-8 in 2006) and two cadaveric (AIV-15 in 2000 and AIV- 16 in 2005). Unfortunately, AIV-3 died of severe sepsis 3 months after the operation. AIV-15 continued to suffer from aggravating residual polyneuropathy and she had a sudden onset of left-sided sensorineural deafness. She also had an intracardiac pacemaker implantation in 2004 because of progressive amyloidotic cardiomyopathy with attacks of ventricular fibrillation. AIV-16 received a liver transplantation at a symptom-free stage, although the biopsy of gastric mucosa showed amyloid deposition. She enjoyed a good recovery with no signs of polyneuropathy. Intriguingly, AII- 4, who was a Chinese herbalist, died at age 78 years without any symptoms of ATTR; he had treated himself with multiple unknown traditional Chinese herbal medicines. Unfortunately, this patient refused any investigation.
Figure 1. Family pedigrees of (A) case A, (B) case B, and (C) case C are shown. The pedigrees may not be exhaustive. Age at symptom onset in affected subjects and age at death in deceased subjects are indicated in parentheses. Dotted: asymptomatic subjects with TTR mutant found; N, wild-type TTR confirmed by genetic test; ?, unknown status.
Case B
A 57-year-old man (BII-3) presented with a dry cough and significant weight loss of 20 lbs for 1 year. He was treated for asthma. In the subsequent year, he complained of progressive bilateral limb numbness and proximal muscle weakness. Magnetic resonance imaging showed mild spinal stenosis at the cervical and lumbar regions. He was initially managed as prolapsed lumbar disc. He also suffered from alternate constipation and watery diarrhea with three bowel openings a day. Physical examination was initially unremarkable. Nerve conduction velocity (NCV) showed severe axonal sensorimotor polyneuropathy with prolonged medial nerve distal motor latency. Extensive investigations were unfruitful revealing normal liver and renal function, negative autoimmune markers, normal blood lead and mercury levels, and negative urinary porphyrins. Sural nerve biopsy was performed after 2 years of his initial presentation and showed eosinophilic deposits on the perineural vessels consistent with amyloidosis. The second elder brother (BII-2) had a history of ischemic heart disease with negative coronary angiogram findings at age 37. He had two episodes of minor stroke in his 50s. At age 58, he presented with acute abdominal distension and was diagnosed with congestive heart failure, with dilated cardiomyopathy, mild mitral and tricuspid valve regurgitation, and a left ventricular ejection fraction (LVEF) of 20%. He had no symptoms of autonomic dysfunction.
Case C
A 49-year-old male Macau resident (CII-10) who had previously enjoyed good health complained of a subtle change in bowel habit with mild diarrhea, passing loose stools two to four times a day with alternate constipation, for 2 years. Later, he had bilateral limb numbness and postural dizziness. Three years after the first presentation, he suffered from urinary and fecal incontinence and required intermittent self-catheterization and diapers. Physical examination revealed severe orthostatic hypotension, grade II systolic murmur at apex, and decreased lower limb muscle power. Echocardiogram showed mitral valve regurgitation, diastolic dysfunction and hypertrophic cardiomyopathy with an LVEF of 60%. NCV revealed length-dependent attenuation of action potential amplitudes with normal velocity parameters. Amyloid deposition was shown in the biopsy of abdominal fatty issue. Immunohistochemical staining for transthyretin was not available in the local hospitals. His father (CI-2), the eldest brother (CII-6) and five paternal relatives (CM, CII-1, CII2, CII-3 and CII-4) died in their mid-40s to 60s in Hong Kong.
Case D
A 48-year-old man (D-1) presented in 2003 with bilateral ankle swelling, and shortness of breath on exertion. He also complained of difficulty in rising from squatting, climbing up stairs, lower limb numbness, diarrhea and impotence in the subsequent 2 years. Echocardiogram showed markedly thickened ventricular myocardium and heart valves with granular coarse appearance, suggestive of amyloidotic diastolic cardiomyopathy. Sural nerve biopsy confirmed amyloidosis. His mother and his maternal uncle died at ages 48 and 45 years, respectively.
Method and materials
Genomic DNA was extracted from peripheral blood samples using a QIAamp Blood Kit (Qiagen, Hilden, Germany) after informed consent was obtained. All four coding exons and their flanking introns of the TTR gene were amplified for the four probands by polymerase chain reaction (PCR) and sequenced directly according to our previously published method [8]. Family screening was performed for adults older than 18 years after proper genetic counseling and informed consents were obtained for members of family A: AIII-4, AIII-5, AIII-8, AIV-1 to -9 and AIV-12; family B: BII-1 to -5, BHI- 1 to -11; and family C: CII-5, CIII-1, CIII-2, Cffl-3, CIII-5, Cin- 6, CIII-10 to -13, and IIIC-18). Blood samples of overseas relatives were collected by local doctors and sent to us for DNA analysis. In case C, restriction fragment length polymorphism was conducted using 10 [mu]l of the initial PCR products and incubated at 25[degrees]C overnight with two units of BspCNI. The restriction fragments were then separated by 4% agarose gel electrophoresis, under 100 V for 1 h.
Results
In case A, the mutation was an alanine-for-valine substitution at amino acid 30 [8]. More than 50% of the offspring at risk in the Val 30Ala ATTR kindred were affected. In case B, DNA sequencing showed a thymine for guanine substitution in the first base of codon 97, i.e. GCC[arrow right]TCC (Ala97Ser) in exon 4. This mutation was previously reported in a Taiwanese patient characterized predominantly by polyneuropathy and cardiomyopathy [11]. In case C, we identified a novel missense mutation of a glycine-toglutamate substitution at amino acid position 67 in exon 2, i.e. GGG[arrow right]GAG (Gly67Glu). This missense mutation is a non-conservative change and was not found in 50 normal healthy subjects. Restriction fragment length polymorphism using BspCNI was performed for Gly67Glu. In Gly67Glu, the mutant created one restriction site of BspCNI. The heterozygote mutant gave three fragments of 317 bp, 240 bp and 77 bp, whereas the wild-type gave only one fragment of 317 bp. Because the immunohistochemical staining for transthyretin was not available in our hospital, the diagnosis of ATTR relied on typical phenotype and genetic results. In case D, we found a missense mutation of threonine-to-lysine substitution in amino acid position 59, i.e. ACA[arrow right]AAA (Thr59Lys) in exon 3. Cardiac amyloidosis was the major presentation in the first reported Italian family [12] and also in our patient. Discussion
ATTR has been reported in different ethnic groups but infrequentiy among the Chinese population. In this paper, we describe four Hong Kong Chinese ATTR families with a total of 21 affected subjects confirmed by genetic analysis. All the four families were of Guangdong ethnicity with no foreign ancestors and were unrelated as they harbored four different mutations (Table I). Clinical presentations varied widely and were usually non-specific in the early stage, making diagnosis difficult. Observed lag time of accurate diagnosis was at least 2 years after initial onset of symptoms, hindered usually by its non-specific nature and unavailable or incomplete family history. Advances in DNA technology make the detection of asymptomatic carriers easier and faster. In the four families, we performed DNA analysis in 46 family members, including the four probands. We detected a total of 21 affected ATTR patients, 15 of whom were still at a symptom-free stage at the time of writing. We used the protocol published by Sales-Luis et al. to monitor these carriers with yearly investigations [13]. liver transplantation is the only curative treatment for symptomatic ATTR patients at this time because the liver is the major organ synthesizing the amyloidogenic transthyretin. Liver transplantation can purportedly abort disease progression. Nonetheless, it is difficult to decide the optimal time point for liver transplantation especially in the young asymptomatic carriers, given the comorbidity, risks of complications and the need for life-long immunosuppression [10]. Studies on drugs that stabilize the native tetrameric conformation of the mutant transthyretins and inhibit fibril formation have shown preliminary notable results [14-16]. One of the most promising candidate drugs is diflunisal which stabilizes transthyretin against dissociation required for amyloidogenesis [17,18]. Phase II of the clinical trial “the effect of diflunisal on familial amyloidosis” is currently being conducted by the National Institute of Neurological Disorders and Stroke and Food and Drug Administration. New therapeutic approaches for ATTR may be available in the near future to solve this dilemma [19,20]. In conclusion, we diagnosed four unrelated Hong Kong Chinese kindreds of transthyretin amyloidosis (ATTR) with a total of 21 genotype-confirmed patients. ATTR is largely under-reported among the Chinese population.
Table I. Symptomatic ATTR patients with available phenotypes and genotypes.
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CHLOE M. MAK1, YOK-LAM KWONG2, CHTNG-WAN LAM3, SEE-CHLNG CHAN4, CHUNG-MAU LO4, SHEUNG-TAK FAN4, CHEE-MY CHANG5, YUK-KWONG LAU5, LOK- SUN U6, & SIDNEY TAM1
1 Division of Clinical Biochemistry, Queen Mary Hospital, Hong Kong SAR, China, 2 Division of Haematology and Oncology, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong SAR, China, 3 Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong SAR, China, 4 Department of Surgery, The University of Hong Kong, Queen Mary Hospital, Hong Kong SAR, China, 5 Department of Medicine, Ruttonjee and Tang Shiu Kin Hospital, Hong Kong SAR, China, and 6 Unit of Internal Medicine, Centra Hospitalar Conde de San Januario, Macau, China
Correspondence: Dr Sidney Tarn, Division of Clinical Biochemistry, Queen Mary Hospital, Hong Kong SAR, China. Tel: (852)- 28553202. Fax: (852)-28559915. E-mail: sidneytam@hku.hk
Copyright Taylor & Francis Ltd. Dec 2007
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