Menkes病二家系遗传学分析

目的分析Menkes病基因变异特征。方法回顾分析2例Menkes病患儿的临床资料以及ATP7A基因检测结果。结果例1患儿为男性,2月龄,ATP7A基因第21外显子存在c.4077dupT的纯合变异。例2患儿为男性,4月龄,ATP7A基因第10外显子存在c.2354delC的纯合变异,2例患儿家系验证父母均为野生型。这2种变异在HGMD数据库中均未报道,根据ACMG指南划分为致病变异。结论对2例患儿家系的遗传学分析丰富了中国人ATP7A基因的变异谱,也为临床医师早期诊断和遗传咨询提供帮助。     

Menkes病患儿ATP7A基因突变分析

目的分析并确定2例Menkes病（MD）患儿的ATP7A基因突变及遗传特征。方法收集并分析2例Menkes病患儿的临床资料，提取其外周静脉血DNA，采用PCR法扩增ATPTA基因的23个外显子及其与内含子的连接区，并行DNA直接测序，异常者用DNA限制性内切酶酶切PCR扩增片段方法进行验证。结果发现2个半合子突变，1例在第12外显子检测到不连续的多处碱基缺失c．2589delTGAAGGA＋c．2598delTT＋c．2601delT＋c．2603delT＋c．2605delGTAGATGA（P．E864fsX883），导致移码突变。另1例在第15外显子检测到单个碱基缺失c．3045delT（p．T1016fsX1018），并发现移码突变。2例患儿的母亲均为相应突变但表型正常的携带者。结论发现2个国际上尚未见报道的新ATPTA基因突变（p．E864fsX883与p．T1016fsX1018），首次在国内明确了2例Menkes病患儿的基因诊断。     

Identification of three novel mutations in Japanese patients with Menkes disease and mutation screening by denaturing high performance liquid chromatography

BACKGROUND: Menkes disease is an X-linked recessive disorder resulting in a connective-tissue disturbance and profound neurodegeneration in early childhood. The gene for Menkes disease has been isolated and predicted to code for copper transporting ATPase. In this study, a mutation analysis in Japanese patients with Menkes disease was performed, as was a mutation screening by denaturing high performance liquid chromatography (DHPLC). METHODS: A mutation analysis on five Japanese patients with Menkes disease was performed using a direct sequencing method and DHPLC. RESULTS: Two nonsense mutations, two missense mutations and one splice donor site mutation were found. The DHPLC analysis showed differences in the peaks between the DNA fragments of wild type and heteroduplex (wild type and mutant). CONCLUSIONS: Three novel mutations (Asp1044Gly, Pro1279Leu and IVS21+1 g to a) were detected. The Asp1044Gly mutation destroys the highly conserved phosphorylation domain in exon 16. The splice site abnormality leads to a skipping of exon 21 coding for part of the seventh transmembrane domain. These two mutations could cause a severe protein dysfunction. Another missense mutation, Pro1279Leu, in exon 20 was found in a patient with a mild type of Menkes disease. It is speculated that this mutation partially maintains the ATP7A function is. A DHPLC analysis could detect these mutations. It is concluded that the best way to make a molecular diagnosis for Menkes disease is to first screen DNA samples for all exons using DHPLC, and thereafter perform direct sequencing for exons which have an abnormal elution profile in order to rapidly detect such mutations.     

3 例Menkes病患儿的临床与ATP7A基因分析及1例产前诊断研究

Menkes 病是一种罕见的X 连锁隐性遗传病,由于ATP7A 基因突变导致铜吸收障碍,铜相关酶功能缺陷,引起多系统功能障碍。该文拟通过对3 例Menkes 病患儿的临床经过和ATP7A 基因突变分析对该症进行研究,并对1 例再孕母亲进行产前诊断研究。3 例男婴于8~9 个月时来院就诊,均为婴儿期起病,主要表现为抽搐和智力运动落后,抗癫癎治疗无效,面色苍白,毛发稀疏、卷曲,小头,MRI 扫描显示脑萎缩、白质异常、基底节损害和脑血管形态改变,血浆铜蓝蛋白均显著降低,分别为70.2、73.5、81.0 mg/L（参考值210~530 mg/L）,符合经典型Menkes 病临床表型。例1 和2 的ATP7A 基因存在c.3914A>G（p. D1305G）突变,例3 为c.3265G>T（p.G1089X）突变,均为新生突变。c.3914A>G（p. D1305G）为已知突变,c.3265G>T（p.G1089X）为新突变,均为我国首次报道。例1 患儿的母亲再孕,于妊娠20 周时抽取羊水细胞,通过胎儿ATP7A 基因突变分析,进行产前诊断。羊水细胞ATP7A 基因未见c.3914A>G,提示胎儿未患与先证者相同的疾病。胎儿出生后发育正常。     

Menkes病临床及ATP7A基因突变和拷贝数改变分析

目的通过ATP7A基因突变及拷贝数改变（CNV）分析,了解13例临床诊断为经典型Menkes病患儿的临床表型以及ATP7A基因型特征,探讨其基因型、表型的相关性。方法收集并分析13例Menkes病患者的临床资料,采用DNA直接测序进行ATP7A基因突变检测,发现突变后用DNA限制性内切酶酶切进行验证;应用多重连接依赖的探针扩增技术（MLPA）对未发现突变的患儿行CNV分析,阳性患者用长片段PCR进行验证,并进行基因型、表型相关性分析。结果临床特点:（1）13例均为男性,有典型的毛发改变、癫发作、肌张力减低、智力运动发育迟缓和结缔组织异常,3例有阳性家族史;（2）起病年龄早,均在出生7个月内（3 d～7个月）,除1例患儿以智力运动发育落后起病外均以癫起病,伴严重发育停滞和倒退;（3）均呈进行性加重,随访5例患儿,均于14个月内死亡;（4）12/12例患儿（100%）铜蓝蛋白降低;6/8例患儿（75%）血清铜降低;4/4例患儿（100%）脑血管成像见血管走行紊乱,呈螺丝锥样改变,1例为47XXY/46XY嵌合体。基因突变确诊9例:（1）DNA直接测序检测到6种突变,包括2种缺失突变,2种错义突变及2种剪切位点突变,其中3种为国际上未报道过的新突变,均位于高度保守区,100例健康男性对照中相应位点均未发现上述突变;（2）MLPA检测发现1例患儿为第10外显子缺失突变,长片段PCR验证其断点为c.2173-346₂407-346del,共1 783 bp,导致p.F725_K802del,其母亲为表型正常的携带者。基因型-表型关系:同为c.2179G>A（p.G727R）突变患儿,47XXY/46XY嵌合体起病年龄早于核型正常者,且临床症状较重。结论 Menkes病ATP7A基因大片段缺失或重复突变检测中ML-PA方便、快捷,Menkes病的致病机制可能与ATP7A基因剂量关系密切。     

Molecular analysis and diagnosis in Japanese patients with Wilson's disease

BACKGROUND: Wilson's disease is characterized by the toxic accumulation of copper in the liver, brain, cornea and other organs. It is caused by both impaired excretion via the bile and impaired incorporation of copper into ceruloplasmin in the liver. The Wilson's disease gene (ATP7B) has been cloned as a putative copper-transporting P-type ATPase gene. We therefore analysed mutations of ATP7B in Japanese patients with Wilson's disease. METHODS: Twenty-three Japanese patients with Wilson's disease were investigated. In all patients, the ATP7B coding sequence, including exon-intron junctions, was analysed by restriction endonuclease digestion, mutation detected enhancement gel electrophoresis and/or direct sequencing analysis of amplified fragments. RESULTS: Thirteen mutations were identified, including seven missense mutations, four detections, one insertion and one exon skipping in the coding region. The most common mutations were 2874deletion(del)C in exon 13 and arginine (Arg)778 leucine (Leu) in exon 8. DISCUSSION: None of the observed mutations, except for 2302insertion(ins)C, have been previously detected in either European or North American patients. We conclude that the mutation spectrum of Wilson's disease may thus indicate a population-dependent pattern. Based on the population-dependent manner of the occurrence of ATP7B gene mutations, it may be possible to establish a molecular diagnosis system. A molecular diagnosis system is considered to be very effective for making a definitive diagnosis in very young patients and for also detecting carriers.     

晨尿铜/锌比值诊断儿童肝豆状核变性的敏感度和特异度研究

目的探讨晨尿铜/锌比值用于诊断儿童肝豆状核变性（WD）的可行性。方法收集复旦大学附属儿科医院2005年7月至2007年6月以肝病收住院 ≥3岁患儿的临床资料,并采集晨尿和24 h尿液标本。依据WD诊断标准和随访结果分为WD组和排除WD组。铜、锌浓度采用电感耦合等离子体质谱 仪测定。WD组和排除WD组晨尿铜/锌比值与24 h尿铜等指标行相关性检验。对WD组晨尿铜/锌比值、24 h尿铜及24 h尿铜/锌比值进行受试者工作 特征（ROC）曲线分析,并计算ROC曲线下面积和最佳诊断界值。结果研究期间WD组和排除WD组分别纳入24例和64例。晨尿铜/锌比值和24 h尿铜 的相关性较好（r=0.840,P=0.000）。WD组的24 h尿铜、晨尿铜/锌比值和24 h尿铜/锌比值中位数分别为87.1 μg、0.370和0.394,排除WD组 上述指标中位数分别为24.2 μg、0.051和0.061,两组差异有统计学意义（P均＝0.000）。两组24 h尿锌差异无统计学意义。24 h尿铜、晨尿 铜/锌比值和24 h尿铜/锌比值ROC曲线下面积分别为0.934、0.953和0.911,其最佳诊断界值分别为50.8、0.17 μg和0.12,取该界值时诊断WD 的敏感度和特异度分别为90.9%、93.7%,86.4%、92.2%和86.4%、85.9%。晨尿铜/锌比值取0.2时诊断WD的敏感度和特异度分别为81.8％和95.3 ％。结论晨尿铜/锌比值与24 h尿铜相关性好,诊断WD患儿的敏感度和特异度较高,有望成为WD的诊断指标。     

儿童肝豆状核变性临床表型及ATP7B基因突变关联性分析

目的 了解汉族儿童肝豆状核变性(WD) 患儿的临床表型与ATP7B基因突变的相关性。方法 以2005年7月至2012年12月就诊于复旦大学附属儿科医院确诊WD患儿为研究对象,按起病部位分为肝病型和神经型,以临床表现分为临床型和亚临床型。采用PCR技术扩增ATP7B基因全部外显子并直接测序。提取临床表型和基因型的信息;分析两者之间的相关性。结果 52个无亲缘关系家庭的53例WD患儿进行分析。肝脏损害52例（98.5%）。肝病型41例,神经型12例;临床型19例,亚临床型34例。①肝病型ALT升高明显;神经型24 h尿铜水平、胆汁酸水平和K-F环阳性率均显著高于肝病型;亚临床型起病年龄、ALT或AST异常率显著高于临床型;24 h尿铜水平、胆汁酸升高比例和K-F环阳性率显著低于临床型。②53例WD患儿ATP7B基因外显子序列分析发现致病性突变等位基因97个,突变频率为91.5%。纯合突变8例,复合杂合36例,杂合突变9例。错义突变23种,插入/缺失突变8种,无义突变2种,剪接突变3种。③错义突变与非错义突变,纯合突变与杂合突变患儿在起病年龄、K-F环阳性率、铜蓝蛋白水平、24 h 尿铜水平和ALT、AST异常率等方面差异无统计学意义。④3种高发突变p.Arg778Leu（35.0%,34/97）、p.Pro992Leu (15/97,15.5%)和p.Ala874Val/Pro(5/97,5.2%)在临床型和亚临床型、肝病型和神经型间的分布差异无统计学意义。⑤纯合、错义和错义+剪接突变在临床型和亚临床型、肝病型和神经型间的分布差异无统计学意义。结论 WD患儿几乎均有肝脏受累,多以肝病表现起病。ATP7B常见突变为p.Arg778Leu、p.Pro992Leu和p.Ala874Val,常见基因型和临床表型间未发现显著相关性。p.Ala874Val/Pro突变患儿起病年龄较低,剪接突变对血清铜蓝蛋白影响较小。     

Variable clinical expression of an identical mutation in the ATP7A gene for Menkes disease/occipital horn syndrome in three affected males in a single family

Two maternal half-brothers presented with huge cephalic hematoma, fatal in one. Skin morphology disclosed lack of elastic fibres. Their maternal uncle is moderately mentally handicapped and has extensive connective tissue disorders. In all these patients, an identical missense mutation in the ATP7A gene was found and confirmed Menkes' disease.     

锌指蛋白A20对树突状细胞的免疫调控及其在炎症性肠病发病机制中的作用

泛素化修饰对蛋白质降解及多种细胞功能都具有重要的调控作用,如DNA损伤修复、细胞周期调控、细胞生长、免疫系统功能等。泛素修饰酶锌指蛋白A20被认为是调控机体免疫和炎症反应的重要门户,为NF-κB信号通路中的关键负性调控因子。树突状细胞(DC)是专职的抗原提呈细胞,可以通过多种受体识别炎性反应或病原微生物,是维持机体免疫稳态的关键调节者。近年来,有研究表明,A20在DC功能的调控中发挥着重要作用,可能参与了炎症性肠病的发生和发展。文章基于最新的研究进展,总结A20对DC的免疫调控及其在炎症性肠病发病机制中的作用。     

血小板-白细胞聚集体及其在川崎病发病中的研究进展北大核心CSCD

活化的血小板会与单核细胞、中性粒细胞、树突状细胞和淋巴细胞等各类白细胞相互作用,触发细胞间信号转导,从而导致血栓形成和炎症介质的大量合成。已在多种血栓性疾病和炎症性疾病中发现血液中血小板-白细胞聚集体水平升高,该文综述了最新文献,探讨血小板-白细胞聚集体形成机制、作用、检测方法及其在川崎病发病中的相关作用,为川崎病发病机制的研究提供了新思路。     

Epidemiology of neonatal group B streptococcal disease in the Netherlands before and after introduction of guidelines for prevention

OBJECTIVES: (1) To describe the epidemiology of neonatal group B streptococcal (GBS) disease over five years (1997-2001) in the Netherlands, stratified for proven and probable sepsis and for very early (<12 h), late early (12 h - <7 days) and late (7-90 days) onset sepsis. (2) To evaluate the effect of the introduction in January 1999 of guidelines for prevention of early onset GBS disease based on risk factors. METHODS: Data on cases were collected in collaboration with the Dutch Paediatric Surveillance Unit and corrected for under-reporting by the capture-recapture technique. RESULTS: Total incidence of proven very early onset, late early onset and late onset GBS sepsis was 0.32, 0.11 and 0.14 per 1000 live births, respectively, and of probable very early onset, late early onset and late onset GBS sepsis was 1.10, 0.18 and 0.02 per 1000 live births, respectively. Maternal risk factors were absent in 46% of the proven early onset cases. Considerably more infants with proven GBS sepsis were boys. 64% of the infants with proven very early onset GBS sepsis were first born compared with 47% in the general population. After the introduction of guidelines the incidence of proven early onset sepsis decreased considerably from 0.54 per 1000 live births in 1997-8 to 0.36 per 1000 live births in 1999-2001. However, there was no decrease in the incidence of meningitis and the case fatality rate in the first week of life. The incidence of late onset sepsis also remained unchanged. CONCLUSION: After the introduction prevention guidelines based on risk factors there has been a limited decrease in the incidence of proven early onset GBS sepsis in the Netherlands. This study therefore recommends changing the Dutch GBS prevention guidelines.     

表面活性蛋白B、甲状腺转录因子在新生儿肺透明膜病中的表达及意义

目的探讨表面活性蛋白B（SP-B）与甲状腺转录因子（TTF-1）在新生儿肺透明膜病中的表达及其意义。方法采用SP法进行免疫组织化学染色,对肺透明膜病组（HMD）22例、对照组18例检测SP-B、TTF-1在肺泡和呼吸性支气管的表达与分布。结果HMD组中SP-B表达水平显著降低（P〈0．01）,其中有1例未见SP-B表达;TTF-1表达减少（P〈0．05）。结论SP-B、TTF-1的表达与新生儿肺透明膜病密切相关,检测SP-B表达水平对了解新生儿肺透明膜病的发生具有重要意义。     

手足口病的临床特征及中枢神经系统并发症高危因素分析

目的 探讨不同肠道病毒所致手足口病的临床特征及发生中枢神经系统并发症(CNSC)的高危因素.方法 分析272例手足口病患儿,其中肠道病毒71型(EV71)感染92例、柯萨奇病毒组16型(CA16)感染31例、非EV71非CA16感染149例.有CNSC 42例,无CNSC 230例.比较分析不同肠道病毒感染患儿的临床特征,同时对伴有CNSC者进行多因素Logistic分析.结果 3组不同肠道病毒感染患儿的年龄、外周血自细胞计数(WBCC)、C-反应蛋白(CRP)、血糖(BG)水平差异无显著性(P＞0.05);而体温、热程、中性粒细胞百分比(NP)、肢体抖动(MJOL)及CNSC发生率3组间差异有显著性(P＜0.05).多因素Logistic分析发现,MJOL、WBCC＞1.2×109/L、EV71感染是CNSC的高危因素,而且MJOL具有最大相对危险度(OR=242.848,P=0.000).Logistic线性预测因子Z=-12.825+5.492(MJOL)+2.624(WBCC＞1.2×109/L)+1.900(FV71感染).结论 (1)EV71感染者比其他肠道病毒感染者有更高体温、NP、CNSC及MJOL发生率,热程比CA16感染者更长.(2)FV71感染、MJOL、WBCC＞1.2 x109/L是手足口病发生CNSC的高危因素,尤其MJOL对预测CNSC有重要意义.     

Gender-related effects of prenatal administration of estrogen and progesterone receptor antagonists on VEGF and surfactant-proteins and on alveolarisation in the developing piglet lung

Background

Vascular endothelial growth factor (VEGF) is essential for embryonic lung development and has been shown to be regulated by estradiol (E2) and progesterone (P).

Aim

To investigate the effects of prenatal E2 and P withdrawal by specific receptor antagonists on the mRNA expression of VEGF, surfactant proteins (SP-B and SP-C) and on alveolarisation in lung tissue of male and female pig fetuses.

Methods

Fetuses from 10 sows were randomized to receive either both an intramuscular injection of the E2 receptor blocker ICI 182.780 and the P receptor blocker RTI 3021-022 (ICI + RTI, n = 5) or a placebo injection (n = 5) at 90 days of gestation (DOG, 115 = term). After delivery by cesarean section on 114 DOG, tissue of the left lingula of the piglet's lung (28 placebo, 26 ICI + RTI) was obtained to determine the mRNA expression of VEGF, SP-B and SP-C. Lungs from 15 placebo and 14 ICI + RTI group piglets were removed and alveolar counts performed.

Results

The ICI + RTI group showed significantly lower SP-C mRNA expression and alveolar counts compared to the placebo group (p = 0.04 and 0.03, respectively). Diminished alveolarisation in the ICI + RTI group was mainly due to the reduction of alveolar counts in male piglets (p = 0.02). Within the placebo group VEGF and SP-B mRNA expression in male piglets were significantly lower compared to female piglets (p = 0.01 and 0.004, respectively). ICI + RTI treatment abolished this gender-related difference.

Conclusion

Estradiol and P antagonism affected gender-related differences of key proteins for pulmonary function and development and especially in males was associated with diminished alveolarisation.