视紫红质基因在显性视网膜色素变性家系的突变筛查

目的：探讨视紫红质基因在视网膜色素变性疾病中的作用。方法：通过聚合酶链反应（PCR）对4个显性视网膜色素变性家系先证者视紫红质基因的5个外显子进行扩增，扩增产物进行直接测序。结果：视紫红质基因全部编码序列在4个家系先证者中均未发现碱基改变，TJE14及TJE15先证者第1外显子上游非编码区中第70个碱基出现MG套峰．在TJE15先证者第5外显子第1185碱基出现MC套峰；而在TJE16先证者中第1外显子上游非编码区中第45个碱基为纯合子GG，第70个碱基为纯合子AA；TJE17先证者第1外显子上游非编码区中第45个碱基为纯合子AA．第70个碱基为纯合子GG。结论：视紫红质基因在中国人群中突变率不如欧美人高，它可能不是导致中国人显性视网膜色素变性的主要致病基因。     

适当调整筛查切值降低先天性甲状腺功能低下症的漏诊率

目的 了解佛山市初生人口中先天性甲状腺功能低下症(congenital hypothyroidiSm，CH)的发生率，探讨适当调整初筛实验中召回复查的筛查切值(阳性切割点)，以降低先天性甲状腺功能低下症的漏诊率。方法 对81439例新生儿于出生72h后采集足跟血滤纸标本，荧光酶免疫分析法检测血中促甲状腺素(TSH)浓度，TSH初筛结果≥8mIU／L即召回复查；确诊以电化学发光法测定血清总三碘甲状腺原氨酸(TT3)、甲状腺素(TT4)、血清游离T3(FT3)、血清游离T4(FT4)及TSH浓度，并结合骨龄检查及甲状腺核素扫描以确诊。结果 以8mIU／L为筛查切值诊断CH，灵敏度达100％，、特异度91．24％；检出52例CH，发生率63．85／10万(1：1566)。结论 本组筛查结果显示佛山市初生人口中先天性甲状腺功能低下症的发生率高于国内主要城市报道。原因考虑与适当调整筛查切值，提高检测的敏感性，发现异常及时复查，充分利用本市新生儿疾病筛查工作三级管理网络对初筛结果异常者召回复查率达100％，减少漏诊有关。     

老年性甲状腺功能减退症伴精神障碍1例

患者，女．66岁。于2002年始出现颜面部浮肿、怕冷、反应迟钝、嗜睡，在当地医院诊断为原发性甲状腺功能减退症（甲减），但口服甲状腺素片不规则，也未定期监测甲状腺功能。入我院前2d出现颜面部轻度浮肿、嗜睡，间歇性发生胡言乱语、行为不能自控、怀疑被人迫害、拒绝饮食等精神行为异常，既往无心肝肾病史。查体：P60次／min，BP120／80mmHg，意识朦胧，面色蜡黄，颜面部轻度浮肿，甲状腺不大．心肺腹部无异常。双下肢无水肿，生理反射存在．病理反射未引出。人院后查血常规、尿常规、肝肾功能正常，     

Two novel mutations in the BCHE gene in patients with prolonged duration of action of mivacurium or succinylcholine during anaesthesia

BACKGROUND: Butyrylcholinesterase (BChE) hydrolyses the neuromuscular blocking agents, succinylcholine and mivacurium used during general anaesthesia. Hereditary low BChE activity may result in an extensively prolonged duration of action of these drugs, especially in patients who are homozygous for the atypical or silent variants. We present three novel mutations in the butyrylcholinesterase gene (BCHE) identified in three families in which a member had experienced severely prolonged duration of action of succinylcholine. METHODS: As the phenotypes of the three probands could not be established with certainty using conventional biochemical tests, DNA samples were collected from two of the probands and four relatives. Genotypes were determined using complete nucleotide sequencing. RESULTS: Three novel mutations were identified: BCHE*FS126, BCHE*I3E4-14C and BCHE*328D. The proband in family 1 was genotyped as BCHE*115D*I3E4-14C/BCHE*FS126, whereas the proband in family 3 was compound heterozygous for BCHE*328D and BCHE*142M. In both patients, BChE activity was below detection limit, and they experienced an extensively prolonged duration of action of succinylcholine. The proband in family 2 was not sequenced, but a relative was heterozygous for BCHE*FS126. BCHE*I3E4-14C was in linkage with a known silent variant. CONCLUSIONS: Two novel variants of BCHE are silencing the enzyme function. BCHE*FS126 results in a truncated protein lacking the active site and is therefore inactive. The second variant is BCHE*328D, also resulting in an inactive protein, as this change in amino acid is radical and furthermore situated in the gorge harbouring the active site. These variants result in extensively prolonged duration of action of succinylcholine.     

Effect of histidine-tag and R457H and E580Q mutations on catalytic activity of recombinant human cytochrome P450 oxidoreductase

Cytochrome P450 oxidoreductase (POR) transfers electrons from NADPH to several oxygenase enzymes including cytochrome P450 (CYP). Genetic mutations in the POR gene have recently been identified and associated with an autosomal recessive genetic disease. In this study, Vmax, Km, and Vmax/Km values of cytochrome c reduction and NADPH oxidation activities for R457H variant, histidine-tagged wild-type, and histidine-tagged E580Q were compared with those for wild-type. Vmax/Km values of cytochrome c reduction for the R457H variant and histidine-tagged wild-type were 8% and 26%, respectively, of wild-type, whereas Vmax/Km values of NADPH oxidation for the R457H variant and histidine-tagged wild-type were similar to those for wild-type. The kinetic parameters of the histidine-tagged E580Q variant were similar to those for histidine-tagged wild-type, suggesting that E580Q mutation may be of minor importance in interindividual variation in drug response. These results suggest that R457H but not E580Q is essential for the deficiency of POR activities and that the histidine-tagged system would be inappropriate for POR function.     

PCR-HRM分析筛查成骨不全一家系患儿基因突变

【摘要】目的 采用PCR-高分辨率熔解曲线（HRM）分析筛查成骨不全（OI）一家系患儿（先证者）COL1A1基因突变位点,探讨其基因型与临床表型的联系。方法对先证者进行家族史及临床资料的调查,采集先证者、家属及 50名正常对照者血液标本,应用PCR-HRM分析筛查先证者及正常对照者COL1A1基因突变,基因测序确证突变位点。结果先证者COL1A1基因17外显子筛查结果异常,其熔解温度（Tm）值比正常对照者Tm值低约0.4℃。先证者与正常对照者的标准熔解曲线及差异熔解曲线均有明显差异。测序结果为c.1138G>A,突变导致380位氨基酸由甘氨酸（Gly）变成丝氨酸（Ser）：p.（Gly380Ser）,为错义突变。先证者父亲、祖母均具有相同突变位点。先证者母亲及正常对照者基因测序结果无此突变。该突变在中国人群中未见报道。该家系遗传特征为常染色体显性遗传,先证者临床诊断为Ⅳ型OI,临床表型较严重。结论PCR-HRM分析是有效的OI基因筛查新方法。COL1A1基因 c.1138G>A突变在中国人群中为新发现的突变位点。α螺旋结构域Gly被替换可能导致较严重的临床表型。 COL1A1     

ABL激酶区突变的检测与伊马替尼耐药

目的研究慢性粒细胞白血病(chronic myeloid leukemia,CML)患者ABL酪氨酸激酶区突变及伊马替尼耐药。方法用巢式RT-PCR对35例CML患者骨髓液BCR-ABL mRNA内ABL激酶区序列进行逆转录、扩增,测序和同源性比较分析ABL激酶区突变,并分析其伊马替尼耐药。结果 35例患者检出突变16例,阳性率45.71%。CML慢性期、加速期、急变期患者突变率分别为47.83%(11/23)、50%(1/2)和40%(4/10),其差异无显著性(P=0.858);16例突变患者中,共检出26种点突变,包括Y253H、T315I、F317L、M351T、L387F、A380D、H396R、G398R、D455G、E459K和1例185 bp碱基缺失突变等。其中A269V、D274G、S286G、V299M、C305Y、R307W、G312R、I314V、L324Q、K356E、D381G、K403E、K419E、L471P、A474T和S485P突变暂未见文献报道,可能为新的突变。在这些新发现的突变中,D274G、S286G、C305Y和K356E等突变可能与伊马替尼(imatinib,IM)治疗抗性有关。结论约半数患者存在ABL激酶区突变,突变位点广泛,性质多样,既存在单一位点突变,也可多位点同时存在,突变的发生与年龄、性别无关,部分突变与伊马替尼耐药有关。ABL基因激酶区突变检测有助于酪氨酸激酶抑制剂疗效的评估、治疗方案的调整。     

MLH1 R217C/BRAF V600E 突变型家族性甲状腺乳头状癌永生化细胞系的建立及鉴定

摘要： 目的 建立家族性甲状腺乳头状癌 （FPTC） 永生化细胞系, 为研究家族性非髓样甲状腺癌 （FNMTC） 提供新的途径。方法 选取 FPTC 患者的肿瘤标本, 采用消化法分离原代细胞。使用改良的 DMEM/F12 培养基, 添加促甲状腺激素 （TSH）、 三碘甲状腺原氨酸 （T3）、 表皮细胞生长因子 （EGF） 及氢化可的松等进行培养。在原代细胞早期分 2种方式导入外源基因 SV40T/TERT 用于诱导永生化。利用 RT-PCR 检测甲状腺过氧化物酶（TPO）、 甲状腺球蛋白（TG）、 促甲状腺激素受体 （TSHR）、 钠/碘共同转运体（NIS） 等基因的表达, 同时利用免疫荧光技术检测 TPO 及磷脂酰肌醇蛋白聚糖-3（GPC3）蛋白的表达。提取该患者外周血 DNA, 进行突变基因的检测。提取细胞基因组 PCR 扩增后测序, 进行突变基因的检测。结果 分离的 FPTC 原代细胞呈梭形或不规则多角形贴壁生长; 细胞生长 6 个月, 转染 SV40T 组（标记为 FPTC-S）传代至 p26, FPTC 细胞传代至 p23, 转染 SV40T+hTERT 组（标记为 FPTC-ST）传代至p19, 细胞增殖能力较好; FPTC-S 与 FPTC-ST 细胞均能够在基因水平稳定表达 TPO、 TG 与 TSHR; 患者外周血中存在 MLH1 R217C 胚系突变; 原代细胞存在 BRAF V600E 突变; 原代细胞及永生化的细胞系中均存在 MLH1 R217 基因突变。结论 本研究初步建立了 FPTC 永生化细胞系, 且该细胞系中存在 MLH1 R217C 及 BRAF V600E 突变, 该细胞系将为研究以上突变及其相互作用关系提供研究模型。     

CYP17基因的新的突变导致二例17α羟化酶与17,20裂链酶联合缺乏

目的：分析2例无血缘关系的17α羟化酶与17，20裂链酶联合缺乏征患者的CYP17基因序列。方法：外周血中提取基因组DNA。设计引物行PCR反应（包括8个外显子及内、外显子交界处和启动子）并对PCR产物进行序列测定。结果：在2例患者的CYP17基因中，共发现了3种基因突变，且均位于第6外显子。例1有2种突变：其一是1103C〉A．即第368编码子由CCT→CAT，编码的氨基酸由脯氨酸转变为组氨酸（Pro368His）；其二是插入缺失突变，985缺失TAC插入AA（985delTACinsAA）。例2是一纯合子突变：1135T〉C，即第379编码子由TCC转变为CCC，编码的氨基酸由丝氨酸转变为脯氨酸（Ser379Pro）。结论：发现了3种突变。其中985delTACinsAA是一种新的突变形式。3种突变均发生在第6外显子。提示该区域与P450C17酶活性密切相关。     

Characterization of the biochemical and structural phenotypes of four CYP1B1 mutations observed in individuals with primary congenital glaucoma

OBJECTIVE: The objective of this study was to examine the biochemical and physical properties of cytochrome P450 1B1 (CYP1B1) mutants, test our hypothesis that primary congenital glaucoma (PCG)-causing mutants have altered metabolic activity, and correlate these to structural changes in the molecule. METHODS: CYP1B1.1 cDNA was mutated to four forms found in individuals with the PCG phenotype, Y81N, E229K, A330F, and R368H. Expression and stability of the mutant hemoproteins and their ability to metabolize beta-estradiol, arachidonic acid, and retinoids, were determined. Alterations in mutant properties were related to structural changes by in silico examination, on the basis of the CYP1A2 crystal structure. RESULTS: CYP1B1 mutations strongly affected the stability, ease of heterologous expression, and enzymatic properties of the protein. These were related to the location of the amino acid substitutions in the CYP1B1 structure. Three of the mutations involve residues located on the surface of CYP1B1, Y81N, and E229K near the distal surface, and R368H near the proximal surface. The former two substitutions, Y81N and E229K, caused greatly reduced stability at 4 degrees C. Y81N severely inhibited all substrate turnover, but E229K only inhibited arachidonate turnover and exhibited minimal effect on efficiency of retinoid metabolism and estradiol metabolism. The R368H mutation is relatively conservative, affecting charge-pairing with the deeper-located D374, but it severely inhibited metabolism of all substrates tested, and, like Y81N, expression of the enzyme is less facile than CYP1B1wt. The A330F mutation replaces a small alanine by a bulky phenylalanine in the enzyme active site and had major impact on substrate binding, turnover, uncoupling, and metabolite pattern. CONCLUSION: Consistent with the hypothesis, these PCG-related mutations cause identifiable structural changes negatively impacting CYP1B1 biochemistry and stability.     

颅骨锁骨发育不全家系基因多态性与表型关系的研究

目的研究家族性颅锁骨发育不全家系基因型与临床表型的关系。方法提取临床收集的一个先天性颅锁骨发育不全家系中患者和健康成员外周血基因组DNA,PCR扩增RUNX2/CBFA1基因7个编码外显子及其侧翼内含子序列,分别进行正反向测序,对发现的突变位点行酶切分析验证。结果家系中两位患者（先证者及其父亲）显示cDNA 346T→A的杂合突变,使编码的色氨酸变成精氨酸（W 116R）,属错义突变。酶切结果进一步证实了该错义突变。测序还发现先证者父亲cDNA 198G→A的杂合突变,致第66位氨基酸的密码子GCG被GCA取代,但二者均编码丙氨酸,属同义突变。先证者及家系健康成员中未见此改变。结论 RUNX2/CBFA1基因346T→A杂合突变是该家系发病的分子基础。     

Mutational analysis of the highly conserved arginine within the Glu/Asp-Arg-Tyr motif of the alpha(1b)-adrenergic receptor: effects on receptor isomerization and activation

We have suggested previously that both the negatively and positively charged residues of the highly conserved Glu/Asp-Arg-Tyr (E/DRY) motif play an important role in the activation process of the alpha(1b)-adreneric receptor (AR). In this study, R143 of the E/DRY sequence in the alpha(1b)-AR was mutated into several amino acids (Lys, His, Glu, Asp, Ala, Asn, and Ile). The charge-conserving mutation of R143 into lysine not only preserved the maximal agonist-induced response of the alpha(1b)-AR, but it also conferred high degree of constitutive activity to the receptor. Both basal and agonist-induced phosphorylation levels were significantly increased for the R143K mutant compared with those of the wild-type receptor. Other substitutions of R143 resulted in receptor mutants with either a small increase in constitutive activity (R143H and R143D), impairment (R143H, R143D), or complete loss of receptor-mediated response (R143E, R143A, R143N, R143I). The R413E mutant displayed a small, but significant increase in basal phosphorylation despite being severely impaired in receptor-mediated response. Interestingly, all the arginine mutants displayed increased affinity for agonist binding compared with the wild-type alpha(1b)-AR. A correlation was found between the extent of the affinity shift and the intrinsic activity of the agonists. The analysis of the receptor mutants using the allosteric ternary complex model in conjunction with the results of molecular dynamics simulations on the receptor models support the hypothesis that mutations of R143 can drive the isomerization of the alpha(1b)-AR into different states, highlighting the crucial role of this residue in the activation process of the receptor.     

An open population screening study for HFE gene major mutations proves the low prevalence of C282Y mutation in Central Italy

BACKGROUND: The C282Y mutation in the HFE gene is responsible for most cases of hereditary haemochromatosis. AIM: To investigate the allele frequency of HFE mutations and the associations between mutations and cases of iron overload or liver diseases in an open population of Central Italy. METHODS: A total of 502 individuals over 8 years of age, comprising 203 males and 299 females, who were residents in Arsita (a small town in Central Italy), were assayed for: C282Y, H63D and S65C mutations of the HFE gene by TaqMan probes; body mass index, serum ferritin, transferrin saturation, transaminases, GGT, glucose, insulin, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, HBV and HCV serum markers. Information was obtained on alcohol intake. Liver ultrasound was performed in 334 (67%) subjects. RESULTS: The allele frequencies for C282Y, H63D and S65C were 2%, 15%, and 0.01%, respectively. C282Y/wt was found in 19 subjects (4%), H63D/wt in 127 (25%), H63D/H63D in 11 (2%) and S65C/wt in one (2.0 per thousand). No homozygosity for C282Y or compound mutation (C282Y/H63D) was found in the study population, but 27 subjects (5%) had TfSat >45% (including 10 subjects with high serum ferritin). Overall, 49 subjects (9.8%) were HCV-RNA-positive. Logistic regression analysis indicated that male gender (P = 0.000) and hepatic steatosis (P = 0.017) were independent variables correlating to a high serum ferritin. CONCLUSION: C282Y HFE mutation is less frequent in Central Italy than in Northern Italy.