类风湿关节炎患者血清转甲状腺素蛋白化学修饰的蛋白质组学分析

目的通过检测类风湿关节炎（RA）患者血清转甲状腺素蛋白（TTR）的化学修饰类型和含量,探讨其在RA早期诊断中的潜在作用。方法采用酶联免疫吸附试验（ELISA）测定39名RA患者[早期（〈1年）16例,中晚期（〉2年）23例]、20名骨性关节炎（OA）患者及20名健康体检者血清TTR含量;采用Western blot测定血清TTR分子聚合体比例;采用基质辅助激光解吸离子化飞行时间质谱（MALDI-TOF-MS）技术鉴定血清中TTR蛋白的化学修饰类型和比例。结果 RA早期患者血清TTR含量为（502.46±109.25）mg/L,明显高于正常对照组[（424.98±118.52）mg/L]（P〈0.05）;RA中晚期患者[（440.67±94.15）mg/L]也有增高趋势,但与正常对照组比较差异无统计学意义（P=0.733）;OA组TTR含量为（363.91±106.41）mg/L,与正常对照组比较差异无统计学意义（P=0.072）。血清TTR化学修饰有4种蛋白峰：质荷比（m/z）13 749.86±1.48、m/z 13 829.63±2.76、m/z 13 870.70±2.70、m/z 13 927±5.77,分别对应野生型TTR（native TTR）、磺化TTR（sul-TTR）、半胱氨酰化TTR（cys-TTR）和半胱氨酰甘氨酰化TTR（cysgly-TTR）,且TTR修饰比例随病程发生变化。结论 RA患者血清TTR质谱分析发现4个TTR蛋白峰,且修饰比例随病程不同而发生改变;早期RA患者血清TTR含量增加,血清TTR水平有可能作为RA早期诊断的潜在指标。     

探讨鉴别诊断肺癌与肺感染患者的生物标志物

目的:探讨肺癌与肺感染患者之间生化成分含量以及蛋白组学变化,用于筛选诊断恶性胸腔积液的生物标志物。方法:(1)分别测定肺癌和肺感染患者血清和胸腔积液总蛋白、白蛋白、甘油三酯等10项生化成分含量,进一步比较两组之间各项指标的胸腔积液与血清的比值。(2)基质辅助激光电离飞行时间质谱(MALDI-TOF-MS)分析TTR蛋白化学修饰。结果:(1)肺癌患者血清中CHO、ApoA-1、TTR蛋白含量明显高于肺感染患者;而肺感染患者胸腔积液中ADA的活性高于肺癌患者。(2)计算个体胸腔积液与血清生化成分含量比值,肺癌患者TTR蛋白比例显著高于肺感染患者,ADA减低,与血清变化趋势相同,但更加明显。(3)肺癌患者及肺感染患者血清TTR蛋白均出现3种修饰类型,而肺癌患者胸腔积液中cysgly-TTR明显增高。结论:联合分析胸腔积液与血清TTR蛋白的比值和化学修饰,可能有助于鉴别诊断肺癌与肺感染。     

In vivo stabilization of mutant human transthyretin in transgenic mice

Transthyretin (TTR) is a 55 kD homotetrameric serum protein transporter of retinol binding protein charged with retinol and thyroxine (T4). The highly amyloidogenic human TTR variant in which leucine at position 55 is replaced by proline (L55P TTR) is responsible for aggressive fatal amyloidosis with peripheral and autonomic neuropathy, cardiomyopathy and nephropathy. Mice bearing one or two copies of a 19.2 kB human genomic fragment containing the entire coding sequence and the known control regions of the L55P TTR transgene, failed to develop TTR amyloidosis even though their sera contained mutant human TTR. The frequency of TTR tissue deposition was increased when the L55P TTR transgene was bred onto a murine TTR-null background. Denaturation of sera from the transgenic animals and murine TTR-knockouts expressing the human L55P TTR transgene revealed that the TTR tetramer was much more stable in the presence of the murine protein because the TTR circulates as hybrid human/murine heterotetramers. Intraperitoneal administration of diflunisal, a non-steroidal anti-inflammatory drug that binds to TTR in its T4-binding site and inhibits fibril formation in vitro, to human L55P TTR transgenic animals in which the murine TTR gene had been silenced, also stabilizes the circulating mutant protein to in vitro urea denaturation.     

Progression of cardiac amyloid deposition in hereditary transthyretin amyloidosis patients after liver transplantation

It has been hypothesized that transthyretin (TTR) amyloidosis may progress after orthotopic liver transplantation (OLT) as a result of continued amyloid fibril synthesis and deposition from normal TTR. To test this hypothesis amyloid fibrils were isolated from cardiac tissues of three patients who died 1½ to 5½ years after OLT: two with Val30Met and one with Thr60Ala TTR. The ratio of variant to normal TTR in each case was determined and compared with the ratio of variant to normal in cardiac tissues from seven patients who died with TTR amyloidosis but who had not had liver transplantation. Tissues from patients with TTR amyloidosis without OLT included three with Val30Met, two with Thr60Ala, one with ΔVal122, and one with Val122Ile. All tissues from patients without OLT had greater amounts of variant TTR than normal TTR except for the Val122Ile in which the ratio was 50:50. The overall median variant to normal ratio was 60:40 with a range of 50–70% variant. In contrast, the mean percentage of variant TTR in the three tissues from patients after OLT was 25% (range 20–35). These data are consistent with the continued deposition of normal TTR in cardiac tissue after liver transplantation.     

Heterogeneity of penetrance in familial amyloid polyneuropathy,ATTR Val30Met,in the Swedish population

Transthyretin (TTR) familial amyloid polyneuropathies (FAP) are autosomal dominant devastating afflictions. They were first described in Portugal, later in Japan and Sweden and are now recognized worldwide. The TTR Val30Met mutation is the most common, and depending on the geographic origin, a wide variation in age at onset of the disease is observed. In Europe, northern Sweden is the second most prevalent area of the disease, and a late age of onset of 56 years has been reported. The present study aims to estimate the penetrance in TTR Val30Met Swedish families. Genealogical investigations, clinical data and genotyping were obtained in 77 TTR-Val30Met Swedish families. The penetrance in Val30Met carriers and variation within the endemic area, according to gender and transmitting parents were calculated by a newly developed bias-free method. The penetrance estimates were low, i.e. 1.7% and 22% at age 30 and 60 years, respectively, and far from complete (69%) by age 90 years. Differences between Piteå and Skellefteå regions were observed. Moreover, penetrance was significantly higher when the mutation was inherited from the mother than from the father. The low penetrance observed in TTR FAP kindreds and its variations is important information for the genetic counseling and treatment of Swedish FAP patients and their families.     

Genetic testing improves identification of transthyretin amyloid (ATTR) subtype in cardiac amyloidosis

Amyloidosis is a group of conditions characterized by the accumulation of amyloid deposits in various tissues. Among these disorders, ATTR amyloidosis occurs either with or without a TTR pathogenic variant. Treatment for amyloidosis depends on the subtype, which is often identified through a tissue biopsy followed by liquid chromatography tandem mass spectrometry (LC–MS/MS). Genetic testing may be done to confirm these results for patients with ATTR amyloidosis; however, the necessity of genetic testing after LC–MS/MS has not been evaluated. A retrospective review identified 153 patients diagnosed with biopsy-proven ATTR amyloidosis, and 56 of these patients underwent both genetic testing and LC–MS/MS. LC–MS/MS and proteomics correctly reported the mutant peptide and heterozygosity in 47/56 (84%) cases. It failed to identify two individuals who were homozygous for the ATTRV122I mutation and failed to detect the following mutations in six other individuals: ATTRA19D, ATTRF44L, ATTRT60A, ATTRI68L and ATTRV122I. Therefore, LC–MS/MS is not sufficient to rule out a pathogenic mutation in cases of ATTR amyloid, and genetic testing should be performed in most cases of ATTR amyloidosis. Correct recognition of hereditary ATTR amyloidosis is important for estimating prognosis, proper familial counselling and guiding use of therapies, such as liver transplantation.     

Low Prevalence of Clinically Apparent Cardiac Amyloidosis Among Carriers of Transthyretin V122I Variant in a Large Electronic Medical Record

BackgroundTransthyretin (TTR) gene mutations are the most common cause of hereditary amyloidosis. Valine replaced by isoleucine in position 122 (V122I) variant is common, particularly in the black population. Carriers of V122I have increased risk for developing cardiac amyloidosis. Despite a relatively high prevalence, the penetrance of V122I is not firmly established. This study sought to determine the prevalence of clinically apparent cardiac amyloidosis among carriers of the TTR V122I variant.MethodsBioVU, a Vanderbilt University resource linking DNA samples and pre-existing genetic data to de-identified electronic medical records was used to identify TTR V122I mutation carriers. Automated billing code queries (International Classification of Diseases, 9th revision codes), problem list searches, and manual chart reviews were used to identify subjects with clinically diagnosed cardiac amyloidosis.ResultsAmong 28,429 subjects with available genotype data, 129 were V122I carriers. Carriers had a median age of 42 years (interquartile range 16-64). Noncarriers had a median age of 62 years, (interquartile range 41-77). The carrier rate was 3.7% in blacks and 0.02% in whites. Overall, the prevalence of clinically apparent cardiac amyloidosis was 0.8% in carriers and 0.04% in noncarriers (P = .05). Above age 60, the prevalence of cardiac amyloidosis was 2.6% in carriers and 0.06% in noncarriers (P = .03).ConclusionCarriers of the TTR V122I variant are at a higher risk for development of cardiac amyloidosis, particularly at age>60 years. However, clinically apparent cardiac amyloidosis in this population was uncommon. These results support that the penetrance of TTR V122I is age dependent and suggest it may be significantly lower than previously reported.     

Haplotype analysis of French,British and other European patients with familial amyloid polyneuropathy (met 30 and tyr 77)

Familial amyloid polyneuropathy (FAP) is an autosomal dominant disorder originally and most frequently described in Portugal. The usual constituent amyloid fibril protein is transthyretin (TTR) and the most frequent mutation in the TTR gene associated with FAP (including all Portuguese cases) is that at position 30 (met 30). Three different TTR haplotypes have been described in association with the met 30 mutation in European patients. We studied the haplotypes of 27 families (24 French, 2 British and 1 Greek) with FAP met 30 by analysing three polymorphisms in introns of the TTR gene. We also studied 6 families (2 British, 3 French and 1 Spanish) with FAP tyr 77. There were two main haplotypes in French patients with FAP met 30, one most commonly seen in the French families of Portuguese descent which was the same haplotype as previously described in Portuguese patients (haplotype I) and another haplotype (III) detected in most informative French families not of Portuguese origin. The age of onset of symptoms was consistently later in French than in Portuguese patients and in patients with haplotype III as the disease-associated haplotype rather than haplotype I. British and French patients with the tyr 77 mutation had different haplotypes. The most likely explanation of these findings is multiple founders of both mutations.