wiskott - aldrich syndrome ( was ) is an x - linked primary immunodeficiency characterized by a clinical triad of immunodeficiency , thrombocytopenia , and eczema ( 1 , 2 ) . was is caused by mutations in the gene encoding wiskott - aldrich syndrome protein ( wasp ) that affect all hematopoietic stem cells including lymphocytes , monocytes , neutrophils and platelets ( 3 ) . various wasp gene mutations have been reported in patients with was and x - linked thrombocytopenia , a clinically mild allelic variant ( 4 - 9 ) . in severe cases of was , the immunodeficiency limits the life expectancy of the patient , and immune reconstitution is the treatment of choice . ( 10 ) demonstrated correlations between the clinical phenotype , the extent of the mutation , and the presence or absence of wasp and also recommended hematopoietic stem cell transplantation ( hsct ) especially for the patients with wasp - negative was . recently , there have been reports of successful treatment of was by unrelated hsct with cord blood ( cb ) as well as bone marrow transplant when a matched sibling donor was unavailable ( 11 - 16 ) . although phenotypic corrections for immunologic and hematologic parameters have been reported , genotypic corrections for was with cord blood stem cell transplantation ( cbsct ) have not been demonstrated . in this report , we demonstrate 2 cases of was which were phenotypically and genotypically corrected with unrelated cbsct . patient 1 ( upn 1 ) is a male who was diagnosed with was at the age of 5 months . he presented with incidentally detected thrombocytopenia ( 23,000/l ) with skin eczema and severe , recurrent otitis media and diarrhea on admission . the second male patient ( upn 2 ) presented with neonatal thrombocytopenia at birth and received intermittent intravenous immunoglobulin ( ivig ) . thereafter he experienced skin eczema and recurrent infections such as cellulitis and pneumonia , until he visited our hospital at 3 months old . flow cytometric analysis of peripheral blood mononuclear cells ( pbmc ) for these 2 patients revealed a defect in wasp , leading to the diagnosis of was . subsequently , the nonsense mutations , arg211stop and arg13stop , were confirmed by genomic analysis ( 17 ) . before transplantation , these patients were treated with monthly infusions of ivig as well as supportive treatment but there was no clinical improvement . the pre - transplantation conditioning regimen for the 2 patients was 1 mg / kg of busulfan intravenously every 6 hr on days -9 through -6 . this was followed by 50 mg / kg of intravenous cyclophosphamide on days -5 through -3 and 30 mg / kg of intravenous antithymocyte globulin ( atg ) on days -3 through -1 . prophylaxis for acute graft versus host disease ( gvhd ) included continuous infusion of cyclosporine a beginning on day -1 , targeting whole blood levels to be 200 to 400 ng / ml , and 1 mg / kg / dose of methylprednisone every 12 hr on days 5 through 19 , and then a taper . the degree of human leukocyte antigen ( hla ) match confirmed by high resolution dna typing between the infused cb and the patients was 4/6 for upn 1 and 5/6 for upn 2 . infused cell doses of tnc and cd34 + cells for upn1 and upn2 were 6.2410/kg and 5.0810/kg for tnc , respectively , and 1.3310/kg and 4.810/kg for cd34 + cells , respectively . t- , nk- , and b - cell enumeration and quantitative immunoglobulin studies ( for immunoglobulin [ ig ] g , a , m , d , and e ) were performed . cytofluorographic analyses of lymphocyte subpopulations were performed with murine monoclonal antibodies conjugated to either fluorescein ( fitc ) or phycoerythrin ( pe ) and then analyzed by flow cytometry ( facscan ; becton dickinson , san jose , ca , u.s.a . ) . heparinized venous blood samples from patients and family members were fractionated on a ficoll - hypaque gradient to isolate pbmcs . for mutational analysis , genomic dna was extracted from the peripheral lymphocytes , and 12 wasp gene exons were amplified by polymerase chain reaction ( pcr ) followed by direct sequencing according to the protocol of sasahara et al . hematopoietic reconstitution following cbsct was uneventful , with an absolute neutrophil count ( anc ) of more than 500/l on days 31 and 13 and a platelet count of more than 20,000/l on days 58 and 50 , for upn 1 and upn 2 , respectively . molecular chimerism studies using the vntr method showed a complete donor cell type for these patients ( data not shown ) . acute gvhd did not occur , even after the infusion of hla 1 or 2 antigen mismatched cb . upn 1 experienced 1 episode of sepsis with b. cepacia during the pre - engraftment period without any complications . upn 2 experienced fever and respiratory distress with hypoxemia due to pulmonary edema as well as skin rashes on the trunk in the pre - engraftment period ( from days 8 to days 10 ) , which mimics engraftment syndrome . he recovered with oxygen supply via endotracheal tube and fluid restriction as well as methylprednisone therapy . both patients were clinically well without eczematous skin and recurrent infections at 60 months ( upn 1 ) and 55 months ( upn 2 ) post cbsct . clinical data regarding cbsct are shown in table 1 . the elevated total eosinophil counts and serum ige levels have been normalized since 7 months post - transplantation in both children ( table 2 ) . other immunologic parameters including igg , iga , igm , igd and lymphocyte subsets are summarized at table 2 . to determine whether the genotype was corrected by cbsct upn 1 had a single base substitution ( c665 t ) in exon 7 that results in an amino acid change in codon 211 ( arg211stop ) before cbsct . following cbsct , upn1 had a normal sequence at the mutation site in exon 7 of the wasp gene ( fig . the gene responsible for was ( wasp ) consists of 12 exons with 1,823 bp . wasp encodes a 502 amino acid protein that is expressed selectively in hematopoietic stem cell - derived lineages ( 4 ) . to date , approximately 100 mutations in the wasp gene have been described . these mutations consist of frameshift mutations , missense mutations , or splice - site mutations , which all give rise to aberrant transcription ( 4 - 9 ) . a correlation between clinical phenotype and genotype ( 10 ) observed that patients with missense mutations were wasp - positive , but patients with nonsense mutations , large deletions , small deletions , and small insertions were wasp - negative . lack of wasp expression was associated with susceptibility to bacterial , viral , fungal , and pneumocystis jiroveci infections and with severe eczema , intestinal hemorrhage , death from intracranial bleeding , and malignancies . they also revealed that the rates for overall survival and event - free survival were significantly lower in wasp - negative patients . conclusively , they recommended hsct to improve prognosis , especially for wasp - negative patients . recently , hsct has been the principal modality for correction of immune deficiencies such as was . since cb has been successfully transplanted to reconstitute patients with was , as first reported in 1994 ( 11 - 16 ) , cb appears to be an alternative donor source compared with matched unrelated bone marrow with successful engraftment associated with no to mild acute gvhd and without development of chronic gvhd . in this study , we identified 2 was patients who had nonsense mutations in wasp and we successfully treated them by unrelated cbsct with engraftment with no associated gvhd . in cbsct , the immunologic reconstitution resulted in consistent and stable t - cell , b - cell , and natural killer - cell development . the kinetics of recovery of phenotypic expression and function of the t cells occurred between 60 and 100 days and that of natural killer cells at approximately 180 days ( 19 ) . the immunologic parameters , especially cd8 of our patients also demonstrated normal values after cbsct . furthermore , following cbsct , we found that upn1 had a normal sequence at the mutation site in the exon 7 of wasp gene . while cbsct has been successfully performed for phenotypic correction including clinical features and immunological parameters in was , our study emphasizes the evidence of genetic correction as well as phenotypic correction by cbsct in was . to our knowledge , this is the first report documenting a genetic correction by cbsct in was . collectively , our data demonstrate that cbs could be an important source of stem cells for the phenotypic as well as genotypic correction of genetic diseases such as was .
we present two cases of wiskott - aldrich syndrome ( was ) , in which nonsense mutations in the wasp gene were corrected phenotypically as well as genotypically by unrelated cord blood stem cell transplantation ( cbsct ) . 
 two male patients were diagnosed with was at the age of 5-month and 3-month and each received unrelated cbsct at 16-month and 20-month of age , respectively . 
 the infused cord blood ( cb ) units had 4/6 and 5/6 hla matches and the infusion doses of total nucleated cells ( tnc ) and cd34 + cells were 6.24107/kg and 5.08107/kg for tnc and 1.33105/kg and 4.8105/kg for cd34 + cells , for upn1 and upn2 , respectively . 
 complete donor cell chimerism was documented by variable number tandem repeat ( vntr ) with neutrophil engraftment on days 31 and 13 and platelets on days 58 and 50 , respectively . 
 immunologic reconstitution demonstrated that cbsct resulted in consistent and stable t- , b- , and nk - cell development . 
 flow cytometric analysis for immunologic markers and sequence analysis of the wasp gene mutation revealed a normal pattern after cbsct . 
 these cases demonstrate that cbs can be an important source of stem cells for the phenotypical and genotypical correction of genetic diseases such as was .