1/2. Abnormal propeptide processing resulting in the presence of two abnormal species of protein c in plasma: characterization of the dysfunctional protein c Padua3 (protein c(R-1L/propeptide)).A heterozygous G-->T transversion at position 1388 of the protein c (PC) gene which predicted the substitution of Arg(-1) to a Leu (PC(R-1L)) was identified in a thrombophilic patient. The PC(R-1L) was purified from the patient's plasma by immunoaffinity chromatography using Ca -independent and Ca -dependent monoclonal antibodies. NH2-terminal sequencing of the light chain of PC(R-1L) revealed two amino acid sequences: one was identical to the complete propeptide sequence of PC, while the other matched the normal PC light chain sequence elongated by one amino acid (leucine at position 1). Activated PC(R-1L/propeptide) exhibited normal amidolytic and impaired anticoagulant activity. Thus, the substitution of a Leu for an Arg at position -1 of PC shifts the propeptidase cleavage site by one amino acid. In addition, in PC(R-1L/propeptide) the propeptide cleavage at Lys(-2) is less efficient since approximately 60% of PC variant molecules present in patient's plasma retained the entire propeptide. Our findings suggest that depending on the specific amino acid substitution at position-1, PC can be secreted in plasma containing the entire propeptide attached to the light chain. Impaired interaction of elongated APC molecules with a membrane-surface and/or factor va which is the physiological substrate for APC, is manifested in vivo by thrombophilia.- - - - - - - - - - ranking = 1keywords = chromatography (Clic here for more details about this article) |
2/2. A novel TPM1 mutation in a family with hypertrophic cardiomyopathy and sudden cardiac death in childhood.We sought to define the pathogenic mutation in a family with hypertrophic cardiomyopathy (HC) and a markedly arrhythmogenic phenotype. The proband was an 8-year-old female with a sentinel event of sudden death. Screening echocardiograms revealed HC in 2 of her 3 siblings and her father. Her youngest male sibling was diagnosed with HC at age 2 years and died suddenly at age 6 years from ventricular fibrillation despite an implanted cardioverter defibrillator. Using dna extracted from peripheral lymphocytes, linkage exclusion was performed by haplotype analysis of polymorphic markers for the HC genes. genes not excluded by linkage were analyzed for mutations using denaturing high-performance liquid chromatography (DHPLC) and direct dna sequencing. Using this strategy, a 610 T>G nucleotide substitution in the alpha-tropomyosin gene (TPM1) was identified resulting in a novel L185R (leucine [L] to arginine [R]) missense mutation. This mutation was a spontaneous germ-line mutation originating in the proband's father. L185R-TPM1 cosegregated with family members having clinical evidence of HC, including the proband as confirmed by molecular autopsy. The mutation was not present in 400 reference alleles. Thus, a novel missense mutation in TPM1 was discovered in a family with HC and sudden death in childhood. Unlike previously defined mutations that may disrupt the interactions between alpha-tropomyosin monomers, the L185R mutation may affect troponin-T binding. Defining the pathogenic mutation enabled definitive molecular diagnosis of 2 surviving children.- - - - - - - - - - ranking = 1keywords = chromatography (Clic here for more details about this article) |