Research On Gene And Molecular Mechanism In Two Pedigrees With Wiskott-Aldrich Syndrome

Part 1 Research on gene and molecular mechanism in one edigree with Wiskott-Aldrich SyndromeObjective: To investigate the relationship between the genotype and clinical phenotype of a family with Wiskott-Aldrich syndrome(WAS).Methods:1.Collected the family history of the patients of WAS.2.Collected the peripheral blood of the proband and his cousin used for laboratory examination.3.The expression of the WAS proteins(WASp)of proband and his mother was analyzed by Flow cytometry(FCM)and Western blot(WB).4.The next generation sequencing(NGS)technology was used for targeted sequencing proband and his cousin screening for disease-causing gene and Sanger sequencing technology was used to verify the gene analysis of the proband,his parents,his cousin and his cousin's patients.5.Bioinformatics software was used to predict the pathogenicity of the genetic results.Results: 1.The proband and his cousin were patients of WAS.The proband had repeated infections with severe eczema.Except for the proband,his cousin also had several clinical manifestations,the proband and his cousin with scores of 4 and 5;2.The proband and his cousin were mild to moderate anemia,and the blood routine count showed that white blood cells were increased and platelet count were severely decreased.The immunoglobulins Ig G,Ig A and Ig M of the proband were decreased,immunoglobulin Ig E was normal,while Ig G and Ig E of his cousin were increased,Ig A was decreased and Ig M was normal.The total T cells were increased,and CD19+/20+ was decreased in the lymphocytes' detection of the two patients.CD3+ and CD3-CD56+ were increased,CD3+CD4+ and CD3+CD4+/CD3+CD8+ were normal,and CD3+CD8+ was decreased in the the lymphocytes' detection of the proband.Cousin's CD3+CD8+ and CD3-CD56+ were normal,while CD3+CD4+ and CD3+CD4+/CD3+CD8+ were reduced;3.The WASp expression of the proband was 3.8%,his mother was 72.8%,and the WASp expression in the normal control was 96%.The WB result was consistent with the FCM result;4.The exon 2 c.158T>C mutation was founded in both boys and their mothers,causing the amino acid p.L53 P changed,two mothers were all heterozygous of the mutant gene.Meanwhile,his cousin had ivs2 273+14C>T of the WAS gene;5.The results of predicting the pathogenicity of the gene mutation showed that both two mutations may be pathogenic.Conclusions:1.The mutation c.158T>C of WAS gene may be the molecular basis of the amily of WAS.2.The reduced or even absent expression of WASp resulted in severe linical phenotype of the proband.3.Clinically,the use of NGS should be considered to help identify the tiology of WAS patients and is important for subsequent treatment.Part 2 Genetic and molecular pathogenesis analysis of a female pedigree with skewed X-chromosome inactivation of Wiskott-Aldrich syndromeObjective: To investigate the molecular genetic mechanism of a female patient with Wiskott-Aldrich syndrome(WAS).Methods: 1.Collected the family history of a patient clinically suspected as WAS.2.Collected the peripheral blood of proband and her mother used for examining detailed laboratory tests.3.The expression of WAS proteins(WASp)of the proband and her mother was analyzed by Flow cytometry(FCM)and Western blot(WB).4.The next generation sequencing(NGS)technology was used targeted sequencing for proband screening for disease-causing gene and Sanger sequencing technology was used to verify the gene analysis of the proband and her parents.5.The polymorphism of the CAG repeats of exon 1 of the androgen receptor(AR)gene was amplified by PCR(Polymerase chain reaction),and used to analyze the X-chromosome inactivation.Results: 1.The pedigree investigation revealed that her uncles had immune thrombocytopenia(ITP),and the female family members had no obvious syndromes,which was consistent with the recessive genetic characteristic of the X-chromosome;2.The proband was moderately anemic,small platelets with thrombocytopenia,and abnormal immune system.Her titer of Mycoplasma pneumoniae antibody was 1: 160.Her mother was associated with moderate anemia,and the immunoglobulin Ig M and Ig A were lower than the normal level,and there was no obvious abnormality in other examinations;3.The WASp expression of the proband was 20.6% and her mother was 50.4%,both significantly lower than which of the normal control;4.A heterozygous mutation c.173C>T in exon 2 of the WAS gene was found in the proband,which resulted in the change of the p.P58 L,and proline was replaced by leucine.Her mother was a carrier of the mutant gene and her father was normal;5.DNA methylation analysis found that the X-chromosome inactivation was existed in both the proband and his mother after digestion by methylation-sensitive endonuclease Hpa ?.The X-chromosome of the proband carrying the normal WAS gene was inactivated,and the X-chromosome where the mutant gene located remained active,causing that the heterozygous carriers showed corresponding clinical symptoms.Conclusion: The results alert us to realize the fact that female heterozygous carriers of X-chromosome recessive genetic disease also have the possibility to develop the disease,whose need to pay great attention.