Clinical And Molecular Study On Fechtner Syndrome

Fechtner syndrome is a rare autosomal dominant disorder characterized by the triad of thrombocytopenia,giant platelet and inclusion bodies in neutrophils and renal lesion, deafness and ocular lesion.It results from mutations in the human MYH9 gene,which encodes nonmuscle myosin-ⅡA heavy chain.Differences in the heavy chains produce three isoforms of classⅡnon-muscle myosins(A,B and C),which are widely distributed in most tissues and cells.They participate in mobility,cytokinesis,phagocytosis, maintenance of cell shape Nearly all forms are expressed in kidney,cochlea and lens. However,megakaryocytic and granulocytic lineage express onlyⅡ-A.Little is known about the pathogenesis of MYH9 syndrome and specific functional role of nonmuscle myosin.Objective:To identify molecular mechanism underlying Fechtner syndrome in a Chinese family by exploring clinic and genetic defect,the pathogenesis of renal lesion and functional study of non-muscle myosin-ⅡA.Methods:1.The characteristic morphological features of platelets and leukocytes are examined on blood smears with Wright's-Giemsa staining.Genomic DNA was isolated from peripheral blood of the proband and 9 members of his family.All the exons and exon-intron boundaries of the MYH9 gene were amplified by PCR followed by direct sequencing.2.Ultrastructure of platelet and leukocyte are investigated under electron microscope.3.Indirect immunofluorescence with DAPI staining technology was used to observe the location of nonmuscle myosinⅡA in patients' peripheral blood smear.4.Pathological features of glomerulonephritis were explored by HE staining,immunochemistry,immunofluorescence and electron microscopy of kidney tissues.5.HEK-293 cells are adopted to perform co-immunoprecipitation study.Results:1.Patients present characteristic clinical features including macrothrombocytopenia, leukocyte inclusions and/or hereditary nephritis.A heterozygous C to T mutation was found in the proband and three members of his family at nucleotide 5981 in exon 40 of MYH9 gene,resulting in a nonsense mutation which encoded truncated protein owing to its premature termination at the Arg 1933 codon.2.Although the platelet is large,the relative numbers of granules,mitochondria and dense bodies is not unusual.Microtubles and elements of the dense tubular system of channels are present.General features of neutrophil morphology are normal.However,a small inclusion is present in the cytoplasm and its area is clear of granules and other organdelles and contains ribosomes.3.There appears abnormal nonmuscle myosinⅡA aggregates in cytoplasm of neutuophils which is similar to the Wright's-Giemsa staining in location and size of inclusions.4.Immunochemistry analysis shows thatⅡA is expressed in the podocyte of glomeruli and distal convoluted ubular,and faintly in the brush berder in proximal tubular.Histological examination observes that glome-rulosclerosis and decreased expression ofⅡA in podocyte.By standard immunofluorescence,the expression ofⅡA in podocyte is higher than normal podocytes and loss of interpodocyte slit diaphragm.5.The calculatedⅡA/β-actin ratio for Fechtner syndrome granulocytes was 39%of control conditions(median:range 33%-44%).The expression ofⅡA andⅡB are identified in HEK-293 cells by RT-PCR.The interaction ofⅡA andⅡB is confirmed by co-immunoprecipation in HEK-293 cells.Conclusions:1.We first report a Chinese family with Fechtner syndrome.2.The patient with R1933X mutaion of MYH9 gene presents inherited glomerulonephritis,which,to our knowledge,has not been reported in the literature.3.Indirect immunofluorescence with DAPI staining technology is a simple and sensitive method for the diagnosis of MYH9 syndrome.4.ⅡA has probably an effect on SD molecule or the alteration of podocyte cytoskeleton,which leads to the appearance of glomerulonephritis.5.As far as granulocytes are concerned,a dominant-negative effect of mutant allele is involved in the formation of inclusion bodies.ⅡB compensate for the isoform A defect derived from MYH9 mutations,and delay or prevent the development of clinically relevant abnormalities.