Clinical And Molecular Analysis Of Primary Immunodeficiency Disease

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.