The Molecular Diagnosis And Pathogenetic Study Of Three Kinds Of Genetic Diseases

Antithrombin（AT） Leu99 mutations are common mutations which lead to the type II inherited AT deficiency. To figure out the pathogenetic mechanisms of AT Leu99 mutations, the function and structure of Leu99 mutant AT were studied in this paper. Besides, one Chinese pedigree of osteopetrosis and seven Chinese pedigrees of X linked adrenal hypoplasia congenita（XL-AHC） were enrolled in this paper. The clinical, biochemical and radiological manifestations of the pedigrees were studied. The TCIRG1 and CLCN7 genes were analysed in the pedigree of osteopetrosis, while the NR0B1 gene were detected in the pedigrees of XL-AHC. The effect of CLCN7 c.1798-1G>T was evaluated by transcriptional experiment.Inherited AT deficiency, a major risk factor for venous thromboembolic disease, is usually caused by the mutations of AT gene. AT Leu99 is one of the hot spots of mutations and have been reported in several pedigrees. In order to figure out the molecular mechanisms of inherited AT defiency caused by AT Leu99 mutations, various AT Leu99 mutants including Leu99 Phe, Leu99 Val, Leu99 Ala, Leu99 Ile and Leu99 Ser were constructed. AT mutants were purified from drosophila expression system. No transcriptional difference was detected in the transient transfection assay. Compared to the WT AT, AT Leu99 Phe and Leu99 Val showed decreased expression in the pulse-chase assay（36.9% and 13.8% respectively）. The binding of low molecular weight heparin sodium to AT Leu99 Val, Leu99 Ala, Leu99 Ile and Leu99 Ser were also decreased in the heparin binding assay [（44.8±3.6） %,（118.9±14.0） %,（15.2±8.8） % and（23.0±8.2） % respectively]. In the surface plasmon resonance（SPR） assay, the binding of FIIa to AT Leu99 Val, Leu99 Ala, Leu99 Ile and Leu99 Ser were 13%, 57%, 3% and 29%, while the binding of FXa to AT Leu99 Val, Leu99 Ala, Leu99 Ile and Leu99 Ser were 7%, 51%, 1% and 25%. The AT activity（AT:A） of WT, Leu99 Val, Leu99 Ala, Leu99 Ile and Leu99 Ser AT, which detected by chromogenic assay, were 146.5%, 21.4%, 120.9%, 10.8% and 39.0% respectively. Circular dichroism（CD） analyses showed reduced α-helix and increased β-sheet of Leu99 mutant AT. The decreased Leu99 mutant AT:Ag and the damaged binding between Leu99 mutant AT and heparin/FIIa/FXa resulted in decreased AT:A and inherited AT deficiency.Osteopetrosis is a heritable bone disorder that exhibits highly clinical and genetical heterogeneity, and is caused by defective osteoclastic resorption. In the present study, the clinical, biochemical and radiological manifestations were described in a patient with osteopetrosis. Sequence analysis identified the compound heterozygous mutations, c.909C>A（p.Tyr303X） and c.2008C>T（p.Arg670X）, in TCIRG1, and a heterozygous splicing mutation, c.1798-1G>T, in the chloride channel 7 gene（CLCN7）. Two aberrant forms of the CLCN7 transcripts, c.1798₁883（exon 20） deletion predicted to cause p.Leu601 Glyfs X13, and the c.1798₁821 deletion, the first 24 bp of the exon 20, predicted to cause p.Gly600_Gln607del, were detected by further analysis of the splicing patterns in the leukocytes. The patient was finally diagnosed with the autosomal recessive severe osteopetrosis on the basis of clinical and biochemical parameters, radiological changes and genetic defects. To the best of our knowledge, this is the first reported case of a patient with osteopetrosis who carried TCIRG1 and CLCN7 mutations. In addition, among the three mutations, TCIRG1 c.909C>A and CLCN7 c.1798-1G>T were novel.XL-AHC is a condition clinically featuring adrenal insufficiency and hypogonadotropic hypogonadism caused by mutations of NR0B1. Seven patients of XL-AHC were studied. Seven NR0B1 mutations including c.871T>G（p.Trp291Gly）, c.376 G > A（p.Val126Met）, c.195₂07delins TG（p.Cys66 Glyfs X2）, c.833 T > C（p.Leu278Pro）, c.543 del A（p.Gly183 Valfs X80）, c.1102 del T（p.Cys368 Alafs X3） and c.501 del A（p.Gly169 Alafs X94） were identified in the study. Among these mutations, three were novel, i.e. c.871T>G, c.195₂07delins TG and c.1102 del T. No significant correlation was found between the phenotypes and the NR0B1 mutations. NR0B1 detection is helpful for the diagnostic of XL-AHC.