Study On The Clinical Features And Molecular Mechanisms Of Inherited Factor â…¦ Deficiency And Wiskott-Aldrich Syndrome

Inherited coagulation factorâ…¦deficiency 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 Fâ…¦activity 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 factorâ…¦deficiency. 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 factorâ…¦deficiency 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 factorâ…¦deficiencySection 1: identification of F7 gene mutations in the probands and the part of family members in 7 inherited Fâ…¦deficiency pedigrees.The phenotype diagnosis was established according to the coagulation parameters. The prothrombin time (PT) and the factorâ…¦activity were measured by one stage method, and the factorâ…¦antigen 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 factorâ…¦activity(<5%) and various levels of factorâ…¦antigen. 7 mutations and 3 gene polymorphisms in F7 were identified in the 7 pedigrees respectively. The two mutations: 18094Câ†'T(Gln426X)and 16750 Câ†'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 factorâ…¦. The patients carrying the homozygous 5076-5077 Del CT and compound heterozygous Gln426X and IVS6-1Gâ†'A presented with severe hemorrhage. However, the patients carrying the homozygous His408Gln and the compound heterozygous Arg364Gln and IVS6-1Gâ†'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 factorâ…¦deficiency, two of which have not been reported previously. Moreover, poor correlations have been found among F7 gene mutations,the levels of Fâ…¦in plasma and clinical manifestations. Section 2: a novel missense mutation close to the charge stabilizing system in a patient with congenital factorâ…¦deficiencyThe substitution of phenylalanine for serine at aa250 might change the conformation of factorâ…¦and impair the secretion and synthesis of Fâ…¦. The expression vector (pcDNA3.1) of Fâ…¦was 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 Fâ…¦in culture medium was detected by PT based on one stage method, and the antigen of Fâ…¦in 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 Fâ…¦molecules 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 Fâ…¦constructs 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 Fâ…¦were 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 Fâ…¦were 172.45±2.25% and 130.51±2.32 ng/ml, respectively. In the subcellular localization experiments, the fluorescent signals (green) for mutant Fâ…¦were colocalized with the perinuclear signals (red) from endoplasmic reticulum and Golgi apparatus respectively, which was the same as in the cells expressing wild type Fâ…¦. 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 Fâ…¦molecule 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 Fâ…¦deficiency.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.Ë‹immunothrombocytopenia purpuraËŠ 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.