抽象的
In this European study, the phenotype in 68 patients, homozygous or compound heterozygous for the G85E mutation, was investigated.
Each index case was compared with two cystic fibrosis (CF) patients from the same clinic, matched for age and sex: one with pancreatic sufficiency (PS) and one with pancreatic insufficiency (PI).
当比较31 G85E/F508DEL和F508DEL/F508DEL患者时,诊断时年龄中位年龄没有差异,平均汗水氯化值,最新的高度,最新的强迫呼气量,一秒钟,一秒钟,预测的一秒钟,慢性病。铜绿假单胞菌定植和典型的CF并发症。但是,PI在G85E/F508DEL组中的频率较低。与PS对照(n = 44)的55例G85E患者(已知第二个突变和未分类为轻度)的比较表明,G85E患者的汗水氯化物的含量明显更高,更常见于诊断,更高的PI患病率,更糟,更糟当前重量以实现高度,慢性较高的患病率P. aeruginosacolonisation and liver cirrhosis. Pulse-chase experiments revealed that G85E cystic fibrosis transmembrane conductance regulator failed to mature on a M470 as well as on a V470 background. Therefore, G85E is a class II mutation.
Although there is variability in its clinical presentation, G85E mutation results in a severe phenotype.
囊性纤维化(CF)是白人种群中最常见的致命性,先天性疾病。它是由编码囊性纤维化跨膜电导调节剂(CFTR)蛋白的两个基因的突变引起的。该基因在7号染色体的长臂上横跨约250 kb,并编码由环状腺苷单磷酸调节的氯化物通道。CFTR蛋白的缺陷负责氯化物的异常转运在外分泌细胞的顶膜上1,,,,2。Severe phenotypes are usually associated with high concentrations of sweat chloride, early onset of pancreatic insufficiency (PI) and severe lung disease. Mild phenotypes are associated with lower sweat chloride concentrations, pancreatic sufficiency (PS), variable lung disease and no history of meconium ileus. In addition, patients with atypical disease and normal sweat chloride values, as well as patients with disease manifestations limited to one organ, have been described. Whether these individuals can be considered affected by CF or not has not yet been fully elucidated2。在经典CF和汗水> 60 meq·L的患者中-1,可以将患者的胰腺状态视为疾病严重程度的标志:PS患者的汗水氯化物明显降低2,,,,slower progression of lung disease3,,,,4and usually carry at least one mild mutation5。
迄今为止,囊性纤维化遗传分析联盟已经报道了CFTR基因中> 1,000个不同的突变6。基因型 - 表型相关性因许多罕见突变而闻名。在所有突变报告中,全球G85E突变的频率约为0.2%。它在地中海地区更为普遍(西班牙1%,意大利1.7%)7,,,,8。G85E突变是一个错义突变:在外显子3中,在核苷酸位置386处,鸟嘌呤被腺苷代替,导致在第一个跨膜跨膜域中通过负电荷的谷氨酸取代甘氨酸9。Earlier reports about G85E concern a small number of patients. It is not clear whether the mutation correlates with a mild, a severe or a variable phenotype8-11。
本研究的目的是在一个大型研究组中确定纯合或复合杂合的CF患者的临床结果。此外,在评估了G85E突变构建体对CFTR蛋白表达的影响in vitrocell lines.
材料和方法
在CF主题网络中合作的医师12通过电子邮件联系。他们被要求报告有关G85E突变的纯合或复合杂合的患者的临床数据,并在两名对照CF患者上报告。对照患者需要对CF进行牢固的诊断,包括氯化物> 60 meq·L-1。They were matched for sex and age, their birth date being as close as possible to the index case. One control patient needed to have PI and one control patient needed to have PS. Exocrine pancreatic insufficiency was defined as >7 g of faecal fat loss per day and/or fat absorption <93% on 3‐day faecal fat balance.
为了描述疾病的严重程度,使用了以下临床变量:诊断时的年龄;使用毛果果离子噬菌体的汗液氯化物浓度13;表现症状(幼虫,不壮成长,脂肪肝,呼吸道疾病);高度的最新体重;最新的强迫呼气量在一秒钟内(FEV1) % 预料到的;存在慢性铜绿假单胞菌殖民化;并发生并发症,例如肝肝硬化,囊性纤维化相关糖尿病(CF-RD),远端肠梗阻综合征(DIOS)和胰腺炎。
使用Prader的标准计算重量的高度百分位数et al.14。ChronicP. aeruginosacolonisation was defined as stated in the European consensus report: presence ofP. aeruginosain the bronchial tree for 6 months, based on at least three positive sputum cultures at 1‐month intervals15。Liver cirrhosis was reported when a firm (left) liver (lobe) was felt and ultrasound confirmed signs of cirrhosis or when there was a large palpable spleen. CF‐RD refers to disturbed glucose metabolism treated with insulin. DIOS was defined as intermittent abdominal pain and a palpable faecal mass in the right abdominal quadrant, leading to hospital admission for medical or surgical treatment. Pancreatitis was considered as any acute episode of abdominal pain associated with serum amylase levels ≥1.5 times above the upper limit of normal for the individual laboratories.
For evaluation of lung function, FEV1values were used at their most recent assessment and they were determined as recommended by the ATS consensus16。All values were expressed as percent predicted value by Quanjer and Porsboom17。
In order to have a clear-cut evaluation of the influence of the G85E mutation, a primary analysis for G85E/F508del patientsversus进行F508DEL/F508DEL PI患者。还将G85E患者与PI和PS的组进行了比较。轻度突变以主要方式赋予PS表型5。From the group of G85E patients, thirteen with unknown second mutation or with a second mutation known to be associated with PS were eliminated.
对于连续的,正态分布的数据,结果表示为平均值±SEM,并通过配对t检验进行比较。偏斜的数据表示为中位数(四分位间范围)。对于连续变量,使用了Wilcoxon签名的级别测试。为了分析二进制变量,使用了Fisher的精确测试。
Apart from studying the phenotype in patients carrying the G85E mutation, the G85E mutation was expressed in cell lines, and the influence of the mutation on CFTR protein biosynthesis and expressionin vitrowas studied. This technique helps to determine which mutation class G85E belongs to, at least in the chosen expression system18。G85E‐CFTR cDNA/pcDNA3 expression vectors, either on a M470‐CFTR or V470‐CFTR background, were made by means of the TransformerTM值根据先前描述的方案,使用诱变底漆5' -gga gat tta ttct ttct atg aaa tct ttt -3',使用诱变的诱变试剂盒(Clontech,Clontech,Palo Alto,CA,USA)。19。The complete CFTR‐coding region of these constructs was verified by sequencing. Expression vectors were transiently transfected in COS‐1 cells. After metabolic labelling of transfected cells, pulse-chase experiments were performed over different time periods according to previously described protocols19,,,,except for immunoprecipitation of CFTR with a C‐terminal CFTR antibody (R&D systems, Minneapolis, MN, USA).
结果
Twenty-one centres in seven European countries contributed data. Data were collected on the phenotype of 68 patients homozygous or compound heterozygous for the G85E mutation. Some centres were unable to supply data on matched PS patients.
Genotypes
表1列出了所有患者的基因型⇓。
在G85E组的68例患者中,有34例携带G85E/F508DEL突变。在26例患者中,第二个突变是已知的,但不是F508DEL:其中21例患者已知第二个突变与严重疾病有关。在这些患者的两个患者中,第二突变被认为是轻度突变。在八名患者中,第二个突变尚不清楚。在三名患者中,第二突变的表型尚不清楚。一个指数病是G85E突变的纯合子,是PS。G85E组的两名患者除了两名患者都是高加索人:一名是高加索/非洲(Antilles),一名是非洲人。
PI组的所有患者均携带至少一个F508DEL突变。68例PI患者中有58名是F508DEL纯合子。在八个中,第二个突变是已知的。
在PS组中,只有一名患者是F508DEL纯合子。33例患者是F508DEL杂合子,但在其中15例中,第二个突变尚不清楚。十二例患者在两个CFTR基因上都有已知但非F508DEL突变。在九名患者中,既不知道突变。至少有13名患者携带了已知赋予轻度疾病的突变18,,,,20。
G85E/F508DEL与F508DEL/F508DEL相比
This analysis concerns 31 patients and is presented in table 2⇓:three of the 34 patients with G85E/F508del genotype in whom the PI control patient did not have a F508del/F508del genotype were excluded from analysis. Mean current age, median age at diagnosis, mean sweat chloride value, most recent weight for height, most recent FEV1% pred, per cent of patients with chronicP. aeruginosa表2报道了定植和典型的CF并发症⇓,,,,and did not differ between the two subgroups. However, PI was less frequent in the G85E/F508del group. In addition, at time of diagnosis, steatorrhea and failure to thrive were less frequent in G85E/F508del patients. Chronic or recurrent respiratory symptoms occurred equally frequently in both groups.
与PI和PS匹配的对照组相比
Comparison of this larger G85E patient group with their PI controls (n=55; table 3⇓) showed the same results as the comparison between the G85E/F508del subgroupversusF508DEL/ F508DEL组。平均当前年龄,诊断中位年龄,汗水氯化值的中位年龄,当前体重,最新FEV没有差异1% pred, prevalence ofP. aeruginosa发现定殖或CF并发症。同样,患有PI和脂肪敏性的患者百分比较低,或者在诊断时未能蓬勃发展。
Comparison of the G85E group with PS controls (n=44; table 3⇑)表明,G85E患者的汗水氯化物明显更高(p <0.0018),更常见的是脂肪性症状和在诊断时未能繁殖的症状(p <0.0001),胰腺不足的患病率更高,当前体重较差(p <0.0001),较高对于高度(p <0.02),慢性的患病率较高P. aeruginosa定植(p <0.0083)和肝硬化(p <0.05)。最近的FEV1% pred was 69±4.8% in the G85E group and 85±6.6% in the PS group (p=0.08). Median age at diagnosis was 22 months in the G85E group and 44 months in the PS group (p=0.13). CF‐RD was more frequent (11versus2%, p=0.1).
如果在分析中考虑了所有G85E患者(n = 68),则这些最后的差异再次达到统计显着性(数据未显示)。G85E组的三名患者死亡:两名患者死于10岁,其中一名Burkholderia cepacia殖民化;一名患者在肺移植后26岁时死亡。这进一步记录了G85E患者的严重肺部疾病。总G85E组的胰腺功能不全的频率为57%。在总G85E组中,PI患者的年龄为23.0±1.7岁,PS 16.1±1.7岁的患者的年龄为23.0±1.7岁。PI患者比PS患者大得多(P = 0.0045)。
兄弟姐妹对
There were six sibling pairs in the G85E group. Two sib-pairs, all compound heterozygous for the G85E and F508del mutations, were discordant for pancreatic disease manifestation. Out of 23‐yr-old dizygotic twins, the boy suffered from PI, had a sweat chloride level of 106 mEq·L-1,,,,a weight for height percentile of 55 and liver cirrhosis. His twin sister was PS, had a sweat chloride value of 84 mEq·L-1and a weight for height percentile of 100. Both were chronically colonised withP. aeruginosa。胰腺功能的另一个同胞不一致为21岁和23岁。年轻的pi sib的汗水值为97 meq·l-1and a weight for height percentile of 90. The PS sib had a sweat chloride value of 116 mEq·L-1高度百分比为30。P. aeruginosa。
In vitro功能性质的G85E雌性生殖道
The degree of G85E‐CFTR maturation was investigated. It is known that polymorphic loci, such as M470V, can affect the properties of CFTR19。但是,G85E -CFTR基因的单倍型背景大多未知。G85E was, therefore, studied either on a M470 or V470 background. G85E‐V470‐, G85E‐M470‐ and wildtype‐V470‐CFTR were transiently expressed in COS cells. Pulse-chase experiments, lasting 30 min, 1 h 30 min and 3 h 30 min, showed maturation to the complex-glycosylated C‐form for wild type‐V470‐CFTR. In contrast, G85E‐V470‐ and G85E‐M470‐CFTR failed to mature to the complex-glycosylated C‐form at all time periods (fig. 1⇓).
Discussion
CF patients with the G85E mutation have a severe phenotype. When comparing G85E/F508del and F508del/F508del patients, there are no differences in age at diagnosis, sweat chloride value, parameters evaluating lung disease, most recent weight for height, nor CF complications. Conversely, comparing G85E patients with PS patients it has been shown here that the G85E group has more severe disease when considering the same parameters. Conversely, there are some differences with classical PI patients: 43% of the G85E patients are PS; and steatorrhea and failure to thrive are less frequent presenting symptoms. Disease variability for pancreatic function is present.
Within classical CF, the pancreatic status differentiates mild and severe CF disease3-5,,,,21。PS patients have a milder expression than PI patients, with regards to sweat chloride value, mean patient age and pulmonary evolution22,,,,23。Moreover, classical PS patients seem to be intermediate between PI and non-classical CF patients with regards to sweat chloride values and nasal potential difference measurements24。The pancreatic phenotype is thought to be more dependent on the CFTR genotype than the pulmonary phenotype, for which the environment and modifying genes play a larger role2,,,,5,,,,18,,,,20,,,,25,,,,26。在研究的总G85E组中,PI的频率为57%。在携带F508DEL,N1303K和W1282X等突变的患者中,与严重的表型相关,> 95%的患者具有PI21。In patients carrying mutations such as R117H, A455E, R334W and 3849+10 kb C>T, which are reported to correlate with a milder phenotype, 40–87% have PS21,,,,27。
Familial concordance of pancreatic function within a family is high26。Indeed, only two of six sibling pairs in this study were discordant for pancreatic status. Intra-familial discordance for pancreatic status has, however, been reported previously28。Conversely, patients may be PS at diagnosis and evolve towards PI22。In accordance with this report, the G85E PS patients in the present study are significantly younger than the G85E PI patients.
Modifying genes can alter the clinical presentation29。实际上,在第19q13q113.2区域已经鉴定出了幼虫的修饰基因。30。Modifying genes can, at present, not yet be studied, but they could explain the rather infrequent occurrence of meconium ileus in the larger group of G85E patients.
在先前的研究中,人们发现G85E雌性生殖道fails to mature and, therefore, is a class II mutation31。通常,II类突变导致严重的CF疾病18。因此,G85E -CF患者组疾病严重程度的异质性对于II类突变而言是显着的,除非该突变是由其他染色体突变赋予的。众所周知,多态基因座,例如Tn,TGm,M470V,影响CFTR的性质。但是,G85E -CFTR基因的单倍型背景大多未知。因此,G85E -CFTR在M470和V470背景下进行了研究,以研究G85E -CFTR特性是否受到M470V位置的氨基酸的影响,并且以这种方式可以解释观察到的疾病变异性。但是,G85E -M470-和G85E -V470 -CFTR都完全未能成熟。基于现在in vitro发现以及根据患者组的临床发现,G85E可以分类为与严重疾病相关的突变。
A variable such as the age at diagnosis may be biased by the introduction of screening programmes that may artificially lower the age at diagnosis in PS patients. Even so, the age at diagnosis was lower in G85E patients, supporting the idea of a more severe phenotype. Respiratory disease is the most important cause of morbidity and mortality in CF20。In the present study G85E patients have more severe lung disease as assessed both by more frequent chronicP. aeruginosainfection and worse most recent FEV1%pred。
In conclusion, G85E is associated with a severe phenotype. The much larger patient sample compared to previous reports makes a correct evaluation of the phenotype more likely. There is some variability in the phenotypic expression of pancreatic disease. The aetiology of this is uncertain but modifying genes and environmental factors could be responsible.
致谢
欧洲G85E调查贡献者:T。Bienvenu(法国巴黎HôpitalCochin);R. Cabanas(西班牙圣地亚哥医院诊所);T. Casals(西班牙巴塞罗那的Duran I Reynals医院);G. Castaldo(Federico II大学 - 意大利纳波利市CEINGE);C. Castellani(意大利维罗纳的Ospedale Civile Maggiore);J. Dapena(U.K. de Boeck(比利时鲁汶大学的加斯特堡大学医院);E. Dequeker(欧洲CF主题网络,人类遗传学系,鲁汶。比利时);G. Evans-Jones(英国切斯特的切斯特医院Chester Hospital Trust的伯爵夫人);D. Feldmann(法国巴黎HôpitalA。Trousseau); I. Galeva (Pediatric Clinic State University Hospital, Sofia, Bulgaria); A. Göçmen (Hacettepe Univ. Pediatric Chest Diseases, Ankara, Turkey); E. Gunn (Papworth Hospital, Cambridge, UK); J. Manzanares (Hospital Universitario “12 de Octubre”, Madrid, Spain); C. Olveira (C.H. Carlos Haya, Malaga, Spain); R. Padoan (Istituti Clinici di Perfezionamento, Milano, Italy); G. Picherot (Hôpital St. Nazaire, France); L. Romano (Istituto Giannina Gaslini, Geneva, Italy); J.L. Seculi (Hospital S. Joan de Deu, Barcelona, Spain); J. Sirvent (C.H. Juan Canalejo, Coruna, Spain); G. Taccetti, Centro Regionale Toscano Fibrosi Cistica, Firenze, Italy); B. Togores (Hospital Son Dureta, Palma Spain); C. Vazquez (Hopital de Cruces, Baracaldo, Spain).
- ReceivedFebruary 10, 2003.
- 公认2004年1月22日。
- © ERS Journals Ltd