遗传性凝血因子Ⅶ缺陷症及Wiskott-Aldrich综合征的临床及分子致病机理研究
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摘要
近年,随着人类基因组序列图完成及分子生物学技术的发展,几乎所有凝血蛋白及血小板膜蛋白等基因已被克隆,这有助于深入了解探索其结构与功能的关系。目前已发现很多遗传性出血性疾病的基因异常,基因诊断是今后遗传性出血性疾病研究的发展方向。遗传性凝血因子Ⅶ缺陷为常染色体隐性出血性疾病,其发病率约为1/500 000,仅次于血友病A、B,而且临床表现迥异。Wiskott-Aldrich综合征是WASP基因突变所致的X连锁隐性遗传病,其临床特征为血小板减少伴小血小板,湿疹及免疫缺陷等。本研究对7例遗传性凝血因子Ⅶ及7例Wiskott-Aldrich综合征的家系成员进行基因诊断并对其致病机制进行研究。本研究分为两部分:
第一部分遗传性凝血因子Ⅶ缺陷症的分子机制研究
第一节:7例遗传性凝血因子Ⅶ缺陷症患者及家系成员的临床与F7基因突变的研究
用一期法及ELISA检测PT、凝血因子Ⅶ活性及抗原等凝血指标进行表型诊断;用DNA测序方法对先证者及家系成员F7基因的全部外显子、侧翼区及5′和3′非翻译区进行分析,寻找基因突变位点;应用PCR-RFLP对新发现地突变的先证者及其家系成员相应基因片段进行酶切分析,证实测序所发现地突变;利用长链PCR分析无突变患者的基因组DNA排除大片段缺失;并用剪接分析软件分析剪接位点突变后效应。
在7例遗传性凝血因子Ⅶ缺陷症患者中,PT明显延长,FⅦ活性水平均降低(<5%),FⅦ抗原水平改变不一致,有的明显下降,有的仅轻度降低,其他凝血指标均正常;7例遗传性FⅦ缺陷患者中发现7种基因突变和3种F7基因多态性,其中18094C→T(Gln426X)、16750 C→T(Ser250Phe)(以ProFⅦ的Met为+1位)为国际首次报道,His408Gln,5076-5077 Del CT两种纯合突变为国内首次报道,15975G→A(IVS6-1G→A)、16039 G→A(Arg212Gln),17908 G→A(Arg364Gln)国际均已见报道,IVS6-1G→A突变重复出现于4个无亲缘关系的家系,His408Gln在两个无血缘关系的家庭中。除两例纯合突变外,其余均为双重杂合性突变;1例新的基因多态性10081 C→T,其杂合率为4%。其中5076-5077 Del CT纯合子、Gln426X与IVS6-1G→A双杂合子患者临床出血表现较重;Ser250Phe与IVS6-1G→A、His408Gln与Arg212Gln双杂合子患者出血症状较轻;His408Gln纯合子、Arg364Gln与IVS6-1G→A双杂合子及仅发现IVS6-1G→A突变的患者及家系成员无明显临床出血表现。
从上述结果可得出以下结论,7例遗传性FⅦ缺陷患者中发现了7种基因突变,其中2种突变为国际首次报道;IVS6-1G→A可能为中国汉族人的热点突变;但F7基因突变、FⅦ活性及抗原水平及临床表现无明显相关性。
第二节新的错义突变Ser250Phe致遗传性凝血因子Ⅶ缺陷的分子机制
Ser250Phe发生于FⅦ的丝氨酸蛋白酶活性中心附近,影响蛋白构象,使其活性及分泌合成受到影响。利用正常肝组织提取的mRNA构建正常(野生)FⅦcDNA真核表达载体(pcDNA3.1),在此基础上利用PCR介导的质粒定点诱变技术,用一对反向互补的突变引物PCR扩增后,Dpn I消化模板,然后转化到感受态细胞(DH5α)中修复。用脂质体法(Lipofectamine 2000)分别转染HEK293和CHO细胞,通过体外表达FⅦ活性及抗原的测定、Western Blotting分析、高尔基/内质网定位的质粒及免疫荧光技术及分子模型分析等方法研究Ser250Phe致遗传性凝血因子Ⅶ缺陷症的分子机制。
研究结果显示突变体Ser250Phe转染细胞后,在细胞裂解液中能检测到与野生型一致的FⅦ抗原量,但细胞培养上清中仅有极微量的FⅦ活性及抗原;亚细胞定位分析中,突变体能与内质网及高尔体共定位;分子模型分析示Ser250被较大侧链疏水的Phe替代后,改变了氢键位置并增大空间位阻,可能破坏了FⅦ的空间构象。
从上述研究结果可推出以下结论:Ser250Phe突变体构象改变,在细胞内能正常合成,而且能从内质网转运至高尔基体,但分泌障碍而导致FⅦ的表达降低。
第二部分7例Wiskott-Aldrich综合征的临床特点和基因分析
用流式细胞术、免疫比浊法及血细胞分析仪分别检测各患者细胞免疫功能、体液免疫功能及血小板计数及平均血小板体积;用DNA测序方法分析各患者WASP基因的外显子、侧翼区与3′与5′非翻译区,寻找基因突变,并与人类基因突变数据库比较。
7例Wiskott-Aldrich综合征患者均有皮肤粘膜的出血表现,反复感染高热,湿疹及反复腹泻,均无自身免疫疾病及肿瘤,临床评分为3或4分;骨髓检查均提示免疫性血小板减少性骨髓象;血小板均减少伴体积偏小,均有贫血且提示小细胞低色素性贫血,白细胞偏高;绝大部分患者(6/7例)的CD3+细胞减少,CD4+/CD8+比值紊乱,B细胞及NK细胞的比例正常;绝大部分患者(6/7例)的IgG升高,而IgA全部升高。在7例Wiskott-Aldrich综合征患者中发现了6种基因突变,除6783 C→G(Tyr102stop)突变发生于外显子3;另外5种突变均发生在外显子10, 10250C→T (Gln440stop),10216-10221 Ins G与9964 Del T均导致框移,分别于aa494及aa440处提前终止,10192-10203 Del GCCTGCCGGGG,10052-10059 Del GCTACTG均为小片段的缺失致框移;前三种突变患者母亲均为携带者,后二者母亲均为正常人,属于散发的病例;有一例患者虽然有WAS的临床表现,但现行的方法未能找到突变位点。
从上述结果可以看出,Wiskott-Aldrich综合征患者的血小板均减少且伴体积减小,但免疫功能及骨髓象改变变化较大,不具有诊断及预后价值;在基因检测中,发现了5例国际首次报道的突变,而且有两例散发的病例,这些突变均为无义及小插入或缺失突变。这些基因突变类型及位点与临床表现有一定的关系。
Inherited coagulation factorⅦdeficiency is a rare autosomal recessive bleeding disease, and has an estimated incidence of 1 per 500 000 in the general population. The hemorrhagic diathesis in affected patients can be considerably variable, and does not necessarily correlate with plasma FⅦactivity and antigen levels. The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia with low mean platelet volume , eczema, increased susceptibility to infections, autoimmune diseases, and malignancies. Gene analysis is helpful for the diagnosis and genetic counseling in WAS and hereditary factorⅦdeficiency. In this study the gene mutations of F7 and Wiskott Aldrich syndrome protein (WASP)gene were identified in the probands and their family members in 7 hereditary coagulation factorⅦdeficiency pedigrees and 7 Wiskott-Aldrich syndrome pedigrees, respectively. And the molecular mechanism caused these disorders and clinical characterization were also investigated in those pedigrees. Therefore the study comprises two parts.
Part 1:Study on the moleculr mechanism of inherited coagulation factorⅦdeficiency
Section 1: identification of F7 gene mutations in the probands and the part of family members in 7 inherited FⅦdeficiency pedigrees.
The phenotype diagnosis was established according to the coagulation parameters. The prothrombin time (PT) and the factorⅦactivity were measured by one stage method, and the factorⅦantigen was assayed by enzyme linked immunosorbent assay(ELISA). The polymerase chain reaction (PCR) and sequencing were performed to identify the mutations in F7 gene (including all the exons and exon-intron boundaries and 3′, 5′untranslation region) of 7 probands and their family members. The novel mutations were confirmed by PCR-restrict fragment length polymorphism assay. The long chain PCR was performed to exclude the large deletion of F7 gene, and spliceosome mutations were analyzed by the computational method.
The probands had normal activated partial thromboplastin time and thrombin time, prolonged prothrombin time, significantly low level of factorⅦactivity(<5%) and various levels of factorⅦantigen. 7 mutations and 3 gene polymorphisms in F7 were identified in the 7 pedigrees respectively. The two mutations: 18094C→T(Gln426X)and 16750 C→T(Ser250Phe)(the initial methionine numbered as +1 for amino acid) have not previously been reported, and the homozygous mutations of His408Gln,5076-5077 Del CT were first reported in China and other mutations have been described previously. In addition, the spiceosome mutation IVS6-1G>A was identified in the four unrelated pedigrees, and the missense mutation His408Gln was identified in two unrelated pedigrees. two mutations (His408Gln,5076-5077 Del CT) of them were homozygous, and other mutations were compound heterozygous. The clinical presentations of the affected patients were highly diverse, which showed poor correlations with the gene mutations and the levels of factorⅦ. The patients carrying the homozygous 5076-5077 Del CT and compound heterozygous Gln426X and IVS6-1G→A presented with severe hemorrhage. However, the patients carrying the homozygous His408Gln and the compound heterozygous Arg364Gln and IVS6-1G→A had no clinical manifestation. other patients showed mild bleeding tendency.
This study shows 7 mutations identified in F7 of 7 unrelated patients with inherited coagulation factorⅦdeficiency, two of which have not been reported previously. Moreover, poor correlations have been found among F7 gene mutations,the levels of FⅦin plasma and clinical manifestations.
Section 2: a novel missense mutation close to the charge stabilizing system in a patient with congenital factorⅦdeficiency
The substitution of phenylalanine for serine at aa250 might change the conformation of factorⅦand impair the secretion and synthesis of FⅦ. The expression vector (pcDNA3.1) of FⅦwas constructed with mRNA extracted from normal liver tissue. To introduce the Ser250Phe into the wild type vector, the PCR mediated site direct mutagenesis of plasmid by Dpn I digestion was performed with a high fidelity polymerase and reverse complementary mutagenic primers. The variant constructs were transformed into DH5αcompetent cells and the sequence of variant was confirmed using the direct sequencing with general primer. The wild type and variant constructs were transiently transfected into human embryonic kidney 293 cells using lipofectamine 2000 reagents. The activity of FⅦin culture medium was detected by PT based on one stage method, and the antigen of FⅦin culture medium and cell lysate was measured by ELISA and Western blotting, respectively. The subcellular localization of mutants were performed in Chinese hamster ovary cells with fluorescent antibody and red fluorescent protein vector which can visualize the endoplasm reticulum and Golgi apparatus.
Western blotting analysis of cell lysate samples of the wild type and variant constructs revealed that the intracellular recombinant FⅦmolecules had a same band of approximately 50 kDa. However, only wild type constructs molecules in culture medium had a band of approximately 50 kDa. In addition, when activity level of the wild type FⅦconstructs in culture medium was taken as 100%, the level of activity of mutant in culture medium was 4.12±0.61%. The levels of antigen in culture medium of wild type and mutant FⅦwere 37.77±2.30 ng/ml and 4.02±0.52 ng/ml, respectively, whereas the levels of antigen in cell lysate of the wild type and mutant FⅦwere 172.45±2.25% and 130.51±2.32 ng/ml, respectively. In the subcellular localization experiments, the fluorescent signals (green) for mutant FⅦwere colocalized with the perinuclear signals (red) from endoplasmic reticulum and Golgi apparatus respectively, which was the same as in the cells expressing wild type FⅦ. In the crystallographic study, Ser250 is located at the third residues prior to the catalytic actve site, His253. Its substitution by phenylalanine (having bulkier side chain) might result in a severe conformational change of FⅦmolecule due to disrupting the normal hydrogen network responsible for maintaining tertiary structure.
It can be inferred from these results of experiments that recombinant mutant can normally be synthesized in cell and transported from the endoplasmic reticulum to Golgi apparatus, but secreted inefficiently. And the compound heterozygous mutation (16750C>T and 15975G>A) are responsible for the FⅦdeficiency.
Part 2: the analysis of the clinical phenotype and gene mutation in 7 patients with Wiskott-Aldrich syndrome.
The T lymphocyte subtypes were measured by flow cytometer using fluorecent antibody against the CD3, CD4, CD8, CD19 and CD16+56. The routine blood tests including platelet count and the mean platelet volume were performed by complete blood analyzer Sysmex XE2100. The immunoturbidimetry was performed to measure the serum immunoglobulin of patients with Wiskott-Aldrich syndrome. The polymerase chain reaction and sequencing were performed to identify the mutations in WASP gene (including all the exons and exon-intron boundaries and 3′, 5′untranslation region) of 7 patients with Wiskott-Aldrich syndrome and their family members.
The clinical scores of 7 patients with Wiskott-Aldrich syndrome were 3 or 4. Those patients had petechiae, easy bruises, eczema, bloody diarrhea, recurrent infection and fever. The routine blood tests revealed that they had reduced platelet counts with low mean platelet volume , decreased hemoglobin and increased leukocyte count.ˋimmunothrombocytopenia purpuraˊ were revealed in the bone marrow examination. The distribution of T lymphoctye subgroups in the probands was abnormal. The percent of CD3+ T cells were decreased, but the fractions of B and NK cells were normal.The majority of patients with WAS had increased levels of IgG and IgA in serum. 6 mutations have been identified in 7 patients with WAS. The 6783 C>G in exon 3 resulted in premature stopping at Tyr102, and the 10216-10203 Del G and 9964 Ins G in exon 10 resulted in frame shift and stopping at aa494 and aa440, respectively. These mutations were transmitted from their mothers. Two small deletions 10192-10203 Del GCCTGCCGGGG,10052-10059 Del GCTACTG in exon 10 also resulted in frame shift, and their mothers were not carrier. In addition, one patient had classical clinical manifestations of WAS, but no mutation could be identified using the current PCR and sequencing.
In the present study, the patients with WAS had reduced platelet count with small platelet. immunologic test results were considerately variable and microscope examination of bone marrow was not useful for the diagnosis and prognosis of WAS. 5 novel mutations have been reported and two small deletion mutations have identified in two patients with no family history.
第一部分遗传性凝血因子Ⅶ缺陷症的分子机制研究
第一节:7例遗传性凝血因子Ⅶ缺陷症患者及家系成员的临床与F7基因突变的研究
用一期法及ELISA检测PT、凝血因子Ⅶ活性及抗原等凝血指标进行表型诊断;用DNA测序方法对先证者及家系成员F7基因的全部外显子、侧翼区及5′和3′非翻译区进行分析,寻找基因突变位点;应用PCR-RFLP对新发现地突变的先证者及其家系成员相应基因片段进行酶切分析,证实测序所发现地突变;利用长链PCR分析无突变患者的基因组DNA排除大片段缺失;并用剪接分析软件分析剪接位点突变后效应。
在7例遗传性凝血因子Ⅶ缺陷症患者中,PT明显延长,FⅦ活性水平均降低(<5%),FⅦ抗原水平改变不一致,有的明显下降,有的仅轻度降低,其他凝血指标均正常;7例遗传性FⅦ缺陷患者中发现7种基因突变和3种F7基因多态性,其中18094C→T(Gln426X)、16750 C→T(Ser250Phe)(以ProFⅦ的Met为+1位)为国际首次报道,His408Gln,5076-5077 Del CT两种纯合突变为国内首次报道,15975G→A(IVS6-1G→A)、16039 G→A(Arg212Gln),17908 G→A(Arg364Gln)国际均已见报道,IVS6-1G→A突变重复出现于4个无亲缘关系的家系,His408Gln在两个无血缘关系的家庭中。除两例纯合突变外,其余均为双重杂合性突变;1例新的基因多态性10081 C→T,其杂合率为4%。其中5076-5077 Del CT纯合子、Gln426X与IVS6-1G→A双杂合子患者临床出血表现较重;Ser250Phe与IVS6-1G→A、His408Gln与Arg212Gln双杂合子患者出血症状较轻;His408Gln纯合子、Arg364Gln与IVS6-1G→A双杂合子及仅发现IVS6-1G→A突变的患者及家系成员无明显临床出血表现。
从上述结果可得出以下结论,7例遗传性FⅦ缺陷患者中发现了7种基因突变,其中2种突变为国际首次报道;IVS6-1G→A可能为中国汉族人的热点突变;但F7基因突变、FⅦ活性及抗原水平及临床表现无明显相关性。
第二节新的错义突变Ser250Phe致遗传性凝血因子Ⅶ缺陷的分子机制
Ser250Phe发生于FⅦ的丝氨酸蛋白酶活性中心附近,影响蛋白构象,使其活性及分泌合成受到影响。利用正常肝组织提取的mRNA构建正常(野生)FⅦcDNA真核表达载体(pcDNA3.1),在此基础上利用PCR介导的质粒定点诱变技术,用一对反向互补的突变引物PCR扩增后,Dpn I消化模板,然后转化到感受态细胞(DH5α)中修复。用脂质体法(Lipofectamine 2000)分别转染HEK293和CHO细胞,通过体外表达FⅦ活性及抗原的测定、Western Blotting分析、高尔基/内质网定位的质粒及免疫荧光技术及分子模型分析等方法研究Ser250Phe致遗传性凝血因子Ⅶ缺陷症的分子机制。
研究结果显示突变体Ser250Phe转染细胞后,在细胞裂解液中能检测到与野生型一致的FⅦ抗原量,但细胞培养上清中仅有极微量的FⅦ活性及抗原;亚细胞定位分析中,突变体能与内质网及高尔体共定位;分子模型分析示Ser250被较大侧链疏水的Phe替代后,改变了氢键位置并增大空间位阻,可能破坏了FⅦ的空间构象。
从上述研究结果可推出以下结论:Ser250Phe突变体构象改变,在细胞内能正常合成,而且能从内质网转运至高尔基体,但分泌障碍而导致FⅦ的表达降低。
第二部分7例Wiskott-Aldrich综合征的临床特点和基因分析
用流式细胞术、免疫比浊法及血细胞分析仪分别检测各患者细胞免疫功能、体液免疫功能及血小板计数及平均血小板体积;用DNA测序方法分析各患者WASP基因的外显子、侧翼区与3′与5′非翻译区,寻找基因突变,并与人类基因突变数据库比较。
7例Wiskott-Aldrich综合征患者均有皮肤粘膜的出血表现,反复感染高热,湿疹及反复腹泻,均无自身免疫疾病及肿瘤,临床评分为3或4分;骨髓检查均提示免疫性血小板减少性骨髓象;血小板均减少伴体积偏小,均有贫血且提示小细胞低色素性贫血,白细胞偏高;绝大部分患者(6/7例)的CD3+细胞减少,CD4+/CD8+比值紊乱,B细胞及NK细胞的比例正常;绝大部分患者(6/7例)的IgG升高,而IgA全部升高。在7例Wiskott-Aldrich综合征患者中发现了6种基因突变,除6783 C→G(Tyr102stop)突变发生于外显子3;另外5种突变均发生在外显子10, 10250C→T (Gln440stop),10216-10221 Ins G与9964 Del T均导致框移,分别于aa494及aa440处提前终止,10192-10203 Del GCCTGCCGGGG,10052-10059 Del GCTACTG均为小片段的缺失致框移;前三种突变患者母亲均为携带者,后二者母亲均为正常人,属于散发的病例;有一例患者虽然有WAS的临床表现,但现行的方法未能找到突变位点。
从上述结果可以看出,Wiskott-Aldrich综合征患者的血小板均减少且伴体积减小,但免疫功能及骨髓象改变变化较大,不具有诊断及预后价值;在基因检测中,发现了5例国际首次报道的突变,而且有两例散发的病例,这些突变均为无义及小插入或缺失突变。这些基因突变类型及位点与临床表现有一定的关系。
Inherited coagulation factorⅦdeficiency is a rare autosomal recessive bleeding disease, and has an estimated incidence of 1 per 500 000 in the general population. The hemorrhagic diathesis in affected patients can be considerably variable, and does not necessarily correlate with plasma FⅦactivity and antigen levels. The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia with low mean platelet volume , eczema, increased susceptibility to infections, autoimmune diseases, and malignancies. Gene analysis is helpful for the diagnosis and genetic counseling in WAS and hereditary factorⅦdeficiency. In this study the gene mutations of F7 and Wiskott Aldrich syndrome protein (WASP)gene were identified in the probands and their family members in 7 hereditary coagulation factorⅦdeficiency pedigrees and 7 Wiskott-Aldrich syndrome pedigrees, respectively. And the molecular mechanism caused these disorders and clinical characterization were also investigated in those pedigrees. Therefore the study comprises two parts.
Part 1:Study on the moleculr mechanism of inherited coagulation factorⅦdeficiency
Section 1: identification of F7 gene mutations in the probands and the part of family members in 7 inherited FⅦdeficiency pedigrees.
The phenotype diagnosis was established according to the coagulation parameters. The prothrombin time (PT) and the factorⅦactivity were measured by one stage method, and the factorⅦantigen was assayed by enzyme linked immunosorbent assay(ELISA). The polymerase chain reaction (PCR) and sequencing were performed to identify the mutations in F7 gene (including all the exons and exon-intron boundaries and 3′, 5′untranslation region) of 7 probands and their family members. The novel mutations were confirmed by PCR-restrict fragment length polymorphism assay. The long chain PCR was performed to exclude the large deletion of F7 gene, and spliceosome mutations were analyzed by the computational method.
The probands had normal activated partial thromboplastin time and thrombin time, prolonged prothrombin time, significantly low level of factorⅦactivity(<5%) and various levels of factorⅦantigen. 7 mutations and 3 gene polymorphisms in F7 were identified in the 7 pedigrees respectively. The two mutations: 18094C→T(Gln426X)and 16750 C→T(Ser250Phe)(the initial methionine numbered as +1 for amino acid) have not previously been reported, and the homozygous mutations of His408Gln,5076-5077 Del CT were first reported in China and other mutations have been described previously. In addition, the spiceosome mutation IVS6-1G>A was identified in the four unrelated pedigrees, and the missense mutation His408Gln was identified in two unrelated pedigrees. two mutations (His408Gln,5076-5077 Del CT) of them were homozygous, and other mutations were compound heterozygous. The clinical presentations of the affected patients were highly diverse, which showed poor correlations with the gene mutations and the levels of factorⅦ. The patients carrying the homozygous 5076-5077 Del CT and compound heterozygous Gln426X and IVS6-1G→A presented with severe hemorrhage. However, the patients carrying the homozygous His408Gln and the compound heterozygous Arg364Gln and IVS6-1G→A had no clinical manifestation. other patients showed mild bleeding tendency.
This study shows 7 mutations identified in F7 of 7 unrelated patients with inherited coagulation factorⅦdeficiency, two of which have not been reported previously. Moreover, poor correlations have been found among F7 gene mutations,the levels of FⅦin plasma and clinical manifestations.
Section 2: a novel missense mutation close to the charge stabilizing system in a patient with congenital factorⅦdeficiency
The substitution of phenylalanine for serine at aa250 might change the conformation of factorⅦand impair the secretion and synthesis of FⅦ. The expression vector (pcDNA3.1) of FⅦwas constructed with mRNA extracted from normal liver tissue. To introduce the Ser250Phe into the wild type vector, the PCR mediated site direct mutagenesis of plasmid by Dpn I digestion was performed with a high fidelity polymerase and reverse complementary mutagenic primers. The variant constructs were transformed into DH5αcompetent cells and the sequence of variant was confirmed using the direct sequencing with general primer. The wild type and variant constructs were transiently transfected into human embryonic kidney 293 cells using lipofectamine 2000 reagents. The activity of FⅦin culture medium was detected by PT based on one stage method, and the antigen of FⅦin culture medium and cell lysate was measured by ELISA and Western blotting, respectively. The subcellular localization of mutants were performed in Chinese hamster ovary cells with fluorescent antibody and red fluorescent protein vector which can visualize the endoplasm reticulum and Golgi apparatus.
Western blotting analysis of cell lysate samples of the wild type and variant constructs revealed that the intracellular recombinant FⅦmolecules had a same band of approximately 50 kDa. However, only wild type constructs molecules in culture medium had a band of approximately 50 kDa. In addition, when activity level of the wild type FⅦconstructs in culture medium was taken as 100%, the level of activity of mutant in culture medium was 4.12±0.61%. The levels of antigen in culture medium of wild type and mutant FⅦwere 37.77±2.30 ng/ml and 4.02±0.52 ng/ml, respectively, whereas the levels of antigen in cell lysate of the wild type and mutant FⅦwere 172.45±2.25% and 130.51±2.32 ng/ml, respectively. In the subcellular localization experiments, the fluorescent signals (green) for mutant FⅦwere colocalized with the perinuclear signals (red) from endoplasmic reticulum and Golgi apparatus respectively, which was the same as in the cells expressing wild type FⅦ. In the crystallographic study, Ser250 is located at the third residues prior to the catalytic actve site, His253. Its substitution by phenylalanine (having bulkier side chain) might result in a severe conformational change of FⅦmolecule due to disrupting the normal hydrogen network responsible for maintaining tertiary structure.
It can be inferred from these results of experiments that recombinant mutant can normally be synthesized in cell and transported from the endoplasmic reticulum to Golgi apparatus, but secreted inefficiently. And the compound heterozygous mutation (16750C>T and 15975G>A) are responsible for the FⅦdeficiency.
Part 2: the analysis of the clinical phenotype and gene mutation in 7 patients with Wiskott-Aldrich syndrome.
The T lymphocyte subtypes were measured by flow cytometer using fluorecent antibody against the CD3, CD4, CD8, CD19 and CD16+56. The routine blood tests including platelet count and the mean platelet volume were performed by complete blood analyzer Sysmex XE2100. The immunoturbidimetry was performed to measure the serum immunoglobulin of patients with Wiskott-Aldrich syndrome. The polymerase chain reaction and sequencing were performed to identify the mutations in WASP gene (including all the exons and exon-intron boundaries and 3′, 5′untranslation region) of 7 patients with Wiskott-Aldrich syndrome and their family members.
The clinical scores of 7 patients with Wiskott-Aldrich syndrome were 3 or 4. Those patients had petechiae, easy bruises, eczema, bloody diarrhea, recurrent infection and fever. The routine blood tests revealed that they had reduced platelet counts with low mean platelet volume , decreased hemoglobin and increased leukocyte count.ˋimmunothrombocytopenia purpuraˊ were revealed in the bone marrow examination. The distribution of T lymphoctye subgroups in the probands was abnormal. The percent of CD3+ T cells were decreased, but the fractions of B and NK cells were normal.The majority of patients with WAS had increased levels of IgG and IgA in serum. 6 mutations have been identified in 7 patients with WAS. The 6783 C>G in exon 3 resulted in premature stopping at Tyr102, and the 10216-10203 Del G and 9964 Ins G in exon 10 resulted in frame shift and stopping at aa494 and aa440, respectively. These mutations were transmitted from their mothers. Two small deletions 10192-10203 Del GCCTGCCGGGG,10052-10059 Del GCTACTG in exon 10 also resulted in frame shift, and their mothers were not carrier. In addition, one patient had classical clinical manifestations of WAS, but no mutation could be identified using the current PCR and sequencing.
In the present study, the patients with WAS had reduced platelet count with small platelet. immunologic test results were considerately variable and microscope examination of bone marrow was not useful for the diagnosis and prognosis of WAS. 5 novel mutations have been reported and two small deletion mutations have identified in two patients with no family history.
引文
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