Alzheimer CSF biomarkers in routine clinical setting.

OBJECTIVES: Our work was aimed to evaluate Alzheimer's disease diagnosis improvement using cerebrospinal fluid biomarkers (CSF) in neurological daily practice. MATERIALS AND METHODS: For this purpose, 150 patients clinically and neurochemically classified as having AD or cognitive impairment with or without other dementia type were included in the study. The following CSF peptides were studied, blindly to the clinical diagnosis: beta-amyloid(1-42) peptide (Aß(1-42)), Tau (T-tau), threonine-181 hyperphosphorylated tau protein (P-tau(181)), and beta-amyloid(1-40) peptide (Aß(1-40)). From these measurements, Innotest® Amyloid Tau Index (IATI) was calculated for each patient. RESULTS: This assessment allowed to separate 83 biochemical profiles of AD and 67 non-Alzheimer's disease (non-AD), both AD and non-AD categories match with clinical data amounting to 73% and 90%, respectively. Among mild cognitive impairment (MCI) patients, CSF biomarkers led to discriminate those who are likely to be AD. We devoted a special section to Aß(1-40) which is not a routine parameter but can help to confirm a pathological amyloid process as Aß(1-42)/Aß(1-40) ratio underlining the real decline of the Aß(1-42). CONCLUSIONS: The interest of biomarkers and their ability to solve awkward cases were carefully noticed all the more when a discrepancy between clinical and CSF biological data was involved. The final proposed algorithm allowed to identify pathogenic forms of AD according to the prevailing role of hyperphosphorylated tau or amyloid beta peptide.

A new assay based on thrombin generation inhibition to detect both protein C and protein S deficiencies in plasma.

Activated protein C reduces thrombin generation by inactivating factors V and VIII in the presence of protein S. This prompted us to develop an assay which would allow specific exploration of this reaction. The total amount of thrombin formed in plasma after activation by tissue factor and phospholipids was reduced by adding thrombomodulin. This addition allowed protein C to be activated by endogenous thrombin. The inhibition of thrombin generation (ITG) due to protein C activation could be measured by comparing thrombin formation in the presence and in the absence of thrombomodulin. ITG increased with both protein C and protein S concentrations. Normal values of ITG expressed as a percentage were between 40 and 65% and were not influenced by age or sex. ITG increased in patients under heparin therapy, decreased in patients under oral anticoagulant therapy and was decreased in women using oral contraceptives. This method could be used for screening patients for protein C and protein S deficiencies.

Detection of a molecular defect in 40 of 44 patients with haemophilia B by PCR and denaturing gradient gel electrophoresis.

Oligonucleotides were computer designed to amplify by the polymerase chain reaction (PCR) the coding region, splice junctions, 112 bp of the 5' flanking region and 279 bp surrounding the polyadenylation site of the factor IX gene for analysis by denaturing gradient gel electrophoresis (DGGE). Forty-four unselected haemophilia B patients were studied of whom 24 had severe haemophilia and 20 had a mild to moderate form of the disease. Potential mutations were identified in 40 (91%) of the 44 cases. A defect could not be detected in three severe and one mild haemophiliac by DGGE analysis and direct sequencing of all the PCR fragments from these patients revealed no nucleotide alteration supporting the DGGE results. A total of 37 point mutations, two complete gene deletions and a duplication of 26 bp were found. The 37 point mutations included 35 single nucleotide substitutions, a deletion and an insertion of one nucleotide. The 35 single nucleotide substitutions included 26 missense mutations, seven nonsense mutations, a G (-6) to A transition in the promoter region and a G (30154) to A transition within the donor splice site of the last intron. Fifteen of these nucleotide substitutions involved CpG dinucleotides. Fifteen point mutations were found at codons where nucleotide substitutions had not been detected before. An insertion of a single nucleotide T at position 6370 and deletion of a G at nucleotide 30845 resulted in frameshift mutations creating stop codons at amino acid positions -2 and 250, respectively. A duplication of 26 bp (17747-17772) in exon V was found in a severe haemophilia patient resulting in a termination codon in exon VI. The detection of the mutation by the combined use of PCR, DGGE and direct sequencing was important for carrier diagnosis of 20 families with no prior history of haemophilia B.

Nucleotide substitutions at the -6 position in the promoter region of the factor IX gene result in different severity of hemophilia B Leyden: consequences for genetic counseling.

Mutations in the promoter region of the factor IX gene result in hemophilia B Leyden, which is characterized by considerable improvement in the disease after puberty. We have found that distinct nucleotide substitutions at the -6 position in the Leyden-specific (LS) region are associated with a different severity of hemophilia B. The proband (aged 2) from one family is a severe hemophiliac with factor IX activity (F.IXC) and antigen (F.IXAg) levels less than 1.0 U/dl. F.IXC and F.IXAg levels in two affected uncles are approximately 30% of normal levels. The LS region was targeted for analysis because the phenotypes suggested the inheritance of a factor IX Leyden gene. An abnormal TaqI digestion pattern was found in amplified DNA from the proband, and sequencing showed a G(-6) to C transversion that was linked to the disease in the family. In another family, two brothers (aged 8 and 9) suffer from mild hemophilia with F.IXC ranging from 7 to 10 U/dl and F.IXAg from 3 to 4 U/dl. They are the only documented members of the family with a bleeding tendency. Denaturing gradient gel electrophoresis on amplified fragments from one of the patient's genomic DNA corresponding to the 8 exons and flanking sequences of the factor IX gene suggested a defect only in a segment from the 5' region. This segment showed an altered TaqI digestion pattern, and sequencing demonstrated a G(-6) to A transition that was traced to the patient's mother and a grandmother. The different phenotypes associated with the G(-6) to A purine nucleotide transition compared with a G(-6) to C transversion provide evidence that this area is directly involved in the regulation of the human factor IX gene expression in vivo by binding of regulatory factors. The ability to predict that the conditions of a hemophilia B patient will improve with age has important implications for genetic counseling of the family. Therefore, the LS region should always be included when scanning the factor IX gene for mutations.