【作者】 张志勇；

【导师】 杨锡强；

【作者基本信息】 重庆医科大学 ， 儿科学， 2010， 博士

【摘要】 第一部分24例Wiskott-Aldrich综合征临床特征与分子特点分析目的:探讨来自中国23个不同家系的24例Wiskott-Aldrich综合征患儿的临床特征及分子特点。方法:2007年4月～2009年7月收集在重庆医科大学附属儿童医院治疗的24例疑诊Wiskott-Aldrich综合征患儿外周静脉血,采用流式细胞术(FCM)检测患儿外周血单个核细胞(PBMC)中WASP表达。扫描电镜观察患儿外周血淋巴细胞形态。PCR扩增WASP基因序列并直接双向测序分析24例患儿及亲属基因突变情况。总结确诊WAS患儿的临床资料。结果:24例患儿均为男性,具有典型WAS的临床特征。其中2例发生自身免疫性溶血性贫血(AIHA),1例患视网膜母细胞瘤(RB)。5例淋巴细胞扫描电镜(SEM)检查可见典型微绒毛异常。21例患儿采用FCM检测外周血PBMC的WASP表达,18例患儿WASP不表达,3例WASP部分表达。WASP基因分析在23例患儿中发现20种不同突变,包括错义突变5例,无义突变4例,缺失突变4例,插入突变3例,拼接位点突变6例,复合突变1例。其中新型突变7例。突变分布于WASP基因7个外显子和4个内含子。1例WAS患儿在WASP基因编码区未发现基因突变。3例WASP部分表达患儿WASP基因分析发现1例WASP基因第二位点突变。22例明确基因诊断的患儿的母亲及相关亲属进行WASP基因检测,在20个家系中发现23例WASP基因突变携带者。5例WAS患儿接受造血干细胞移植,移植后5例均正常表达WASP,但1例死于爆发性巨细胞病毒感染。结论:通过对24例WAS患儿基因分析,发现7个新型WASP基因突变位点。在中国人群中报道1例WASP基因第二位点突变。FCM检测和WASP基因分析能确诊并检出携带者,有利于WAS患儿的及时治疗和相关的遗传咨询。第二部分21例先天性无丙种球蛋白血症的临床特征和分子特点目的:分析和探讨21例先天性无丙种球蛋白血症患儿的临床特征及分子特点。方法:2008年3月～2010年2月收集在重庆医科大学附属儿童医院就诊的21例先天性无丙种球蛋白血症患儿及亲属外周静脉血,采用RT-PCR扩增BTK基因序列并直接双向测序分析患儿及亲属基因突变情况。对未发现BTK基因突变的患儿进一步采用PCR扩增常染色体隐性遗传无丙种球蛋白血症的相关基因,包括μHc,λ5,Igα和Igβ。收集21例先天性无丙种球蛋白血症患儿的临床资料。结果:21例先天性无丙种球蛋白血症患儿起病年龄0.9±0.5岁,诊断年龄6.3±3.0岁。呼吸道感染是最常见的临床表现(n=20, 95.2%),其它依次为关节炎(n=8, 38.1%),中耳炎(n=8, 38.1%),腹泻(n=6, 28.6%),皮肤感染(n=6, 28.6%)和脑膜脑炎(n=1, 4.8%)。1例患儿发生前B细胞白血病,2例患儿因反复肺炎进展为慢性肺疾病。BTK基因分析在18例患儿中发现16种不同突变,其中包括7种新型突变(del 373-441, 504 del G, 537 del C, 851 del A, 1637 G>A, 1879 T>C, del 1482-1882)。突变类型包括缺失突变6例,错义突变4例,无义突变3例,拼接位点突变5例。3例患儿未见BTK基因突变。18例BTK基因突变中10例位于酪氨酸激酶区,4例位于血小板-白细胞C激酶底物同源区,3例位于Src同源区3,1例跨越酪氨酸激酶同源区、Src同源区3、Src同源区2和酪氨酸激酶区。对BTK基因未见突变的3例患儿进行常染色体隐性遗传无丙种球蛋白血症的相关基因筛选,发现1个μHC基因的复合突变(1956 G>A, 170-175 insert C)。结论:通过对21例中国先天性无丙种球蛋白血症患儿分子特点分析,发现7个BTK基因的新型突变。在中国人群中首次报道μHC基因突变的临床特征。基因分析有助于先天性无丙种球蛋白血症患儿的明确诊断,有利于发现携带者和进行遗传咨询。第三部分IL-7Rα基因缺陷导致严重联合免疫缺陷病和Omenn综合征的临床特征和分子特点目的:探讨IL-7Rα基因缺陷导致严重联合免疫缺陷病和RAG1基因突变导致Omenn综合征的临床特征和分子特点。方法: 2008年2月～11月在重庆医科大学附属儿童医院收治3例男性患儿,均在生后早期出现反复、严重的感染,抗生素治疗效果较差。其中2例患儿全身反复出现大片红色斑丘疹、脱屑及肝脾肿大。采用PCR方法扩增患儿及父母IL-7Rα基因和RAG1/RAG2基因,PCR产物直接进行双向序列测定。采用25个TCRVβ亚家族的特异性正向引物和1个共同的Cβ反向引物扩增TCRVβ进行克隆谱型分析。采用STR分析除外母源性T细胞植入。结果:患儿1的免疫球蛋白IgG 686.7mg/dL,IgA 24.9 mg/dL,IgM 20.6 mg/dL, IgE 2.3IU/ml。淋巴细胞分类T淋巴细胞(CD3+) 0,B淋巴细胞(CD19+)58%,NK细胞(CD16+CD56+)42%。基因分析患儿为IL-7Rα基因的复合杂合突变(638 C>T;IVS4(+1)G>A),父母均为携带者。第4内含子剪接位点突变(IVS4(+1)G>A)为首次报道的突变类型。RT-PCR检测发现患儿IL-7RαmRNA表达明显降低。患儿IL-7RαcDNA经巢式PCR扩增并进行T-A克隆,测序发现外显子4出现64个核苷酸缺失(496-559 del, K158fsX160)。患儿2的免疫球蛋白IgG 1150mg/dL,IgA 80mg/dL,IgM 180mg/dL,IgE 5.4IU/ml。淋巴细胞分类T淋巴细胞(CD3+) 69%,B淋巴细胞(CD19+)3%,NK细胞(CD16+CD56+)27%。患儿3免疫球蛋白IgG 4847mg/dL(IVIG后),IgA 46mg/dL,IgM 129 mg/dL,IgE 1.2IU/ml。淋巴细胞分类T淋巴细胞(CD3+)21%,B淋巴细胞(CD19+)1%,NK细胞(CD16+CD56+)69%。PHA刺激后患儿2和3淋巴细胞增殖均极度低下。STR分析除外患儿2和3母源性T细胞植入可能。RAG1基因组DNA测序发现患儿2为RAG1基因的复合突变(G1983A, R624H; C2444T, R778W)。患儿3为RAG1基因的纯合缺失突变(del2302, I729X)。TCRVβ克隆谱型分析显示患儿2的25个TCRVβ亚家族均为单克隆或寡克隆,患儿3的14个TCRVβ亚家族为单克隆或寡克隆,11个TCRVβ亚家族极弱或缺失。结论:在中国人群中首次报道了2例明确基因诊断的Omenn综合征和1例IL-7Rα基因缺陷导致严重联合免疫缺陷病的临床特点和基因突变类型,发现1个RAG1基因和1个IL-7Rα基因的新型突变。更多还原

【Abstract】 Part one Analysis of Clinical and Molecular Characteristics of Wiskott-Aldrich Syndrome in 24 Patients from 23 Unrelated Chinese FamiliesObjective: In this study we analyzed the clinical, immunological and molecular characteristics of 24 children with Wiskott-Aldrich syndrome (WAS) from 23 unrelated Chinese families, in an attempt to provide information for improving the diagnosis and treatment of WAS in China.Methods: Totally 24 male children from 23 unrelated Chinese families admitted to Chongqing Children’s Hospital during April 2007 and July 2009 were included in this study. WASP expression in PBMCs was detected by flow cytometry(FCM). PBMCs were also examined by scanning electron microscopy(SEM). WASP gene was amplified by polymerase chain reaction (PCR) and directly sequenced to analyze mutations of the WASP gene in patients and their female relatives. The clinic findings of the children with WAS were collected and analyzed.Results: Twenty-four children with WAS met the clinical diagnostic criteria of WAS. Two cases suffered from autoimmune hemolytic anemia (AIHA) and one case suffered from bilateral retinoblastoma (RB). Scanning electron microscopy (SEM) in five WAS patients demonstrated abnormal lymphocytes, including the presence of sparse, blunted, or disrupted microvilli. Of the 21 cases of children with WAS, 18 cases showed no WASP expression and three cases showed partial expression of WASP. WASP gene detection was performed in the 24 patients and 20 different WASP gene mutations were detected in 23 cases, including five cases of missense mutation, four cases of nonsense mutation, four cases of deletion mutation, three cases of insert mutation, six cases of splice site mutation and one case of complex mutation. The remaining one case showed no mutation in the coding regions of the WASP gene. Of the 20 WASP gene mutations, seven were novel mutations, including a complex mutation (168 C> A; 747-748 del T; 793-797 del C; 1185 insert C; Dup 1251-1267; 1277 insert A and 1266 C> G, 1267-1269 del C). WASP gene analysis revealed a second-site mutation occurred in one of the three patients with partial expression of WASP. These mutations were distributed in seven exons (exons 1, 2, 4, 7, 8, 10, 11) and four introns (introns 1, 3, 8, 9) of the WASP gene. Genetic study for carrier status was carried out in 22 families with definite genetic diagnosis and 23 carriers of WASP mutations were identified in 20 families. Five patients underwent hematopoietic stem cell transplantation (HSCT) before 5 years old. All the five patients exhibited normal expression of WASP two months after transplantation and one case died of cytomegalovirus infection-induced interstitial lung disease following transplantation.Conclusion: Seven novel mutations were identified in 24 children with WAS and a second-site mutation of WASP gene was reported in China. WASP expression detected by flow cytometry and WASP gene analysis can make a definite diagnosis of WAS and identify mutation carriers, beneficial for timely treatment and genetic counseling for children with WAS.Part two Clinical Characteristics and Molecular Analysis of 21 Chinese Children with Congenital AgammaglobulinemiaObjective: Congenital agammaglobulinemia is a humoral primary immunodeficiency and affected patients have extremely low levels of peripheral B cells and profound deficiency of all immunoglobulin isotypes. In this study, the phenotypes and genotypes of 21 male Chinese children with congenital agammaglobulinemia were investigated and analyzed to improve care plans.Methods: From March, 2008 to February, 2010, 21 Chinese children with congenital agammaglobulinemia from 21 unrelated families were enrolled into the present study. Amplification of BTK gene of the patients and relatives was carried out in four overlapping sections by RT-PCR. The candidate genes of autosomal-recessive agammaglobulinemia, includingμheavy chain,λ5, Igαand Igβwere also screened by PCR in 3 patients without mutations in the BTK gene. Clinical data of the children with congenital agammaglobulinemia were collected and analyzed.Results: The mean age of onset of the 21 patients was 0.9±0.5 years old and the mean age at diagnosis was 6.3±3.0 years old. Of the 21 children with congenital agammaglobulinemia showing recurrent infections, respiratory tract infection was the most common (n=20, 95.2%), followed by arthritis (n=8, 38.1%), otitis media (n=8, 38.1%), diarrhea (n=6, 28.6%), skin infection (n=6, 28.6%) and meningocephalitis (n=1, 4.8%). There were 2 cases in which chronic lung disease (CLD) was developed due to recurrent pneumonia up to the time of diagnosis and one child developed pre-B cell leukemia at 10 years old despite adequate IVIG treatment. Sixteen different mutations in the BTK gene were identified in 18 patients, including six cases of deletion mutation, four cases of missense mutation, three cases of nonsense mutation and five cases of splice site mutation. The remaining three cases had no mutation in the coding regions of the BTK gene. Of the 16 BTK gene mutations, seven novel mutations were also identified, including five deletion mutations and two missense mutations (del 373-441, 504 del G, 537 del C, 851 del A, 1637 G>A, 1879 T>C, del 1482-1882). Ten of eighteen mutations in the BTK gene were located in the TK domain, four in the PH domain, three in the SH3 domain and one spanned the TH, SH3, SH2 and TK domains. Candidate genes of autosomal-recessive agammaglobulinemia were also screened in three patients without mutations in the BTK gene. A compound heterozygosity mutation in theμHC gene was identified in one patient (1956 G>A, 170-175 insert C).Conclusion: Seven novel mutations of BTK gene were identified in 21 patients with congenital agammaglobulinemia. A compound heterozygosity mutation in theμHC gene with autosomal recessive inheritance was firstly reported from China. Molecular genetic test is an important tool for definitive and early diagnosis of congenital agammaglobulinemia and may contribute to accurate carrier detection and prenatal diagnosis.Part three Characterization of a Compound Heterozygosity Mutation of the Interleukin-7 ReceptorαGene in a Chinese Patient with Severe Combined Immunodeficiency and Recombinant Active Gene 1 Mutations in Two Patients with Omenn SyndromeObjective: In this study we analyzed the clinical and molecular characteristics of a patient with the interleukin-7 receptorαgene mutation and two patients with Omenn Syndrome from China.Methods: Three male patients admitted to Chongqing Children’s Hospital during February, 2008 to Novermber, 2008 were enrolled in the present study. They all suffered from recurrent fever, persistent cough and diarrhea soon after birth and infections could not be controlled by treatment of antibiotics. In addition, two patients had the characteristics of generalized erythematous skin rash and hepatosplenomegaly. IL-7Rαand RAG1/RAG2 were amplified by PCR from genomic DNA of the patients and their parents. TCRBV-specific PCR amplifications were performed using a panel of 25 BV-specific forward primers and a common BC-specific reverse primer. Sequencing was performed directly on the PCR products in forward and reverse. The PCR products of TCRBV were analyzed by the method of genescan. Analysis of short tandem repeat (STR) was performed to rule out the possibility of graft-versus-host disease (GVHD).Results: The serum immunoglobulin (Ig) profile of case 1 was IgG 686.7mg/dL, IgM 20.6 mg/dL, IgA 24.9 mg/dL and IgE 2.3IU/mL. There were no T-cells but increased percentage of B-cells (58%) and NK cells (42%) present in the peripheral blood. The patient had a compound heterozygosity mutation in the interleukin-7 receptorαgene (638 C>T; IVS4(+1)G>A) and both his parents were carriers. The splice-site mutation in intron 4 of IL-7Rαwas firstly reported. The IL-7RαmRNA expression of the patient was remarkably reduced whereas the parents had relatively normal IL-7RαmRNA expression. IL-7RαcDNA of the patient was amplified by nested PCR and a 64 bp deletion was found in exon 4 of IL-7Rα. The serum Ig profile of case 2 was IgG 1150mg/dL, IgM 180mg/dL, IgA 80mg/dL and IgE 5.4IU/mL. The peripheral blood lymphocyte subset in case 2 was T lymphocyte (CD3+) 69%, B lymphocyte (CD19+) 3% and NK cells(CD16+CD56+) 27%. The serum Ig profile of case 3 was IgG 4847 mg/dL, IgM 129 mg/dL, IgA 46 mg/dL and IgE 5.4IU/mL. (The patient was administered IVIG previously). The peripheral blood lymphocyte subset in case 3 was T lymphocyte (CD3+) 21%, B lymphocyte (CD19+) 1% and NK cells (CD16+CD56+) 69%. Lymphocyte proliferative responses were markedly reduced after PHA stimulation in cases 2 and 3. Gene analysis of RAG1 and RAG2 showed that case 2 had a compound heterozygosity mutation in the RAG1 gene (1983 G>A, R624H; 2444 C>T, R778W). In case 3, a homozygous deletion mutation with a premature stop codon was identified at residue 2302 of RAG1 gene (del2302, I729X) and both his parents were carriers. The deletion mutation in RAG1 gene was a novel mutation. In case 2, 25 TCRVβsubfamilies presented monoclonal or ologoclonal peaks. Only monoclonal or ologoclonal peaks of 14 TCRVβsubfamilies were identified in case 3 and another 11 TCRVβsubfamilies were very weak or absent . Conclusions: This is the first report about mutations in the interleukin-7 receptorαgene and Omenn syndrome with definite gene mutation in Chinese patients and two novel mutations of IL-7Rαgene and RAG1 were identified.更多还原