Allelic heterogeneity of alkaptonuria in Central Europe.

Defects of the homogentisate 1,2 dioxygenase (HGO; E.C. No. 1.13.11.5) have been identified as the molecular cause of alkaptonuria in humans (AKU) and the aku mouse. Here, we report on the genetic basis of 30 AKU patients from Central Europe. In addition to five mutations described previously, we have detected five novel HGO mutations. Recombinant expression of mutated HGO enzymes in E. coli demonstrates the inactivating effect of three of these mutations. A genetic epidemiologic study in Slovakia, the country with the highest incidence of alkaptonuria, demonstrates that two recurrent mutations (c.183-1G > A and Glyl61Arg) are found on more than 50% of AKU chromosomes. An analysis of the allelic association with intragenic DNA markers and of the geographic origins of the AKU chromosomes suggests that several independent founders have contributed to the gene pool, and that subsequent genetic isolation is likely to be responsible for the high prevalence of alkaptonuria in Slovakia.

Congenital deficiency of vitamin K dependent coagulation factors in two families presents as a genetic defect of the vitamin K-epoxide-reductase-complex.

Hereditary combined deficiency of the vitamin K dependent coagulation factors is a rare bleeding disorder. To date, only eleven families have been reported in the literature. The phenotype varies considerably with respect to bleeding tendency, response to vitamin K substitution and the presence of skeletal abnormalities, suggesting genetic heterogeneity. In only two of the reported families the cause of the disease has been elucidated as either a defect in the gamma-carboxylase enzyme (1) or in a protein of the vitamin K 2,3-epoxide reductase (VKOR) complex (2). Here we present a detailed phenotypic description of two new families with an autosomal recessive deficiency of all vitamin K dependent coagulation factors. In both families offspring had experienced severe or even fatal perinatal intracerebral haemorrhage. The affected children exhibit a mild deficiency of the vitamin K dependent coagulation factors that could be completely corrected by oral substitution of vitamin K. Sequencing and haplotype analysis excluded a defect within the gamma-carboxylase gene. The finding of highly increased amounts of vitamin K epoxide in all affected members of both families indicated a defect in a protein of the VKOR-multienzyme-complex. Further genetic analysis of such families will provide the basis for a more detailed understanding of the structure-function relation of the enzymes involved in vitamin K metabolism.

Evaluation of DHPLC in the analysis of hemophilia A.

The manifestation of hemophilia A, a common hereditary bleeding disorder in humans, is caused by abnormalities in the factor VIII (FVIII) gene. A wide range of different mutations has been identified and provides the genetic basis for the extensive variability observed in the clinical phenotype. The knowledge of a specific mutation is of great interest as this may facilitate genetic counseling and prediction of the risk of anti-FVIII antibody development, the most serious complication in hemophilia A treatment to date. Due to its considerable size (7.2 kb of the coding sequence, represented by 26 exons), mutation detection in this gene represents a challenge that is only partially met by conventional screening methods such as denaturing gradient gel electrophoresis (DGGE) or single stranded conformational polymorphism (SSCP). These techniques are time consuming, require specific expertise and are limited to detection rates of 70-85%. In contrast, the recently introduced denaturing high performance liquid chromatography (dHPLC) offers a promising new method for a fast and sensitive analysis of PCR-amplified DNA fragments. To test the applicability of dHPLC in the molecular diagnosis of hemophilia A, we first assessed a cohort of 156 patients with previously identified mutations in the FVIII gene. Applying empirically determined exon-specific melting profiles, a total of 150 mutations (96.2%) were readily detected. Five mutations (3.2%) could be identified after temperatures were optimized for the specific nucleotide change. One mutation (0.6%) failed to produce a detectable heteroduplex signal. In a second series, we analyzed 27 hemophiliacs in whom the mutation was not identified after extensive DGGE and chemical mismatch cleavage (CMC) analysis. In 19 of these patients (70.4%), dHPLC facilitated the detection of the disease-associated nucleotide alterations. From these findings we conclude that the dHPLC technology is a highly sensitive method well suited to the molecular analysis of hemophilia A.

A new cell culture-based assay quantifies vitamin K 2,3-epoxide reductase complex subunit 1 function and reveals warfarin resistance phenotypes not shown by the dithiothreitol-driven VKOR assay.

BACKGROUND: Warfarin directly inhibits the vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) enzyme to effect anticoagulation. VKORC1 function has historically been assessed in vitro using a dithiothreitol (DTT)-driven vitamin K 2,3-epoxide reductase (VKOR) assay. Warfarin inhibits wild-type VKORC1 function by the DTT-VKOR assay. However, VKORC1 variants with warfarin resistance-associated missense mutations often show low VKOR activities and warfarin sensitivity instead of resistance. OBJECTIVES: A cell culture-based, indirect VKOR assay was developed and characterized that accurately reports warfarin sensitivity or resistance for wild-type and variant VKORC1 proteins. METHODS: Human coagulation factor (F)IX and VKORC1 variants were coexpressed in HEK 293T cells under standardized conditions at various warfarin concentrations. Secreted FIX activity served as surrogate marker to report wild-type and variant VKORC1 inhibition by warfarin. RESULTS AND CONCLUSIONS: Warfarin dose-response curves fit to the secreted FIX activity data for coexpressed hVKORC1 wild-type, Val29Leu, Val45Ala and Leu128Arg variants. The corresponding calculated IC50 values were 24.7, 136.4, 152.0 and 1226.4 nm, respectively. Basal activities in the absence of warfarin for all VKORC1 variants were similar to that of wild-type VKORC1. Ranked IC50 values from the cell culture-based assay accurately reflect elevated warfarin dosages for patients with VKORC1 missense mutation-associated warfarin resistance.

Somatic mosaicism in hemophilia A: a fairly common event.

Mutations in the large gene of clotting factor VIII (FVIII) are the most common events leading to severe human bleeding disorder. The high proportion of de novo mutations observed in this gene raises the possibility that a significant proportion of such mutations does not derive from a single germ cell but instead should be attributed to a germline or somatic mosaic originating from a mutation during early embryogenesis. The present study explores this hypothesis by using allele-specific PCR to analyze 61 families that included members who had sporadic severe hemophilia A and known FVIII gene defects. The presence of somatic mosaicisms of varying degrees (0.2%-25%) could be shown in 8 (13%) of the 61 families and has been confirmed by a mutation-enrichment procedure. All mosaics were found in families with point mutations (8 [25%] of 32 families). In the subgroup of 8 families with CpG transitions, the percentage with mosaicism increased to 50% (4 of 8 families). In contrast, no mosaics were observed in 13 families with small deletions/insertions or in 16 families with intron 22 inversions. Our data suggest that mosaicism may represent a fairly common event in hemophilia A. As a consequence, risk assessment in genetic counseling should include consideration of the possibility of somatic mosaicism in families with apparently de novo mutations, especially families with the subtype of point mutations.

Missense mutations at ALA-10 in the factor IX propeptide: an insignificant variant in normal life but a decisive cause of bleeding during oral anticoagulant therapy.

Bleeding complications are the most common and unwanted side-effect of oral anticoagulant therapy. We report three patients in whom mutations in the factor IX (FIX) propeptide were found to cause severe bleeding during coumarin therapy. Strikingly, the bleeding occurred within the therapeutic ranges of the prothrombin time (PT) and international normalized ratio (INR). In all three patients coumarin therapy caused an unusually selective decrease of FIX activity (FIX:C) to levels below 1-3%. Upon withdrawal of coumarin, FIX:C increased to subnormal or normal values of 55%, 85% and 125%, respectively. Analysis of the FIX gene revealed two different missense mutations affecting the Ala-10 residue in the propeptide coding region: Ala[GCC] to Val[GTC] in two patients and Ala[GCC] to Thr[ACC] in one patient. No further mutation was detected by screening 195 random blood donors for mutations at Ala-10, thus excluding a frequent polymorphism at this position. The mutation in the FIX propeptide at a position which is essential for the carboxylase recognition site causes a reduced affinity of the carboxylase enzyme to the propeptide. This effect leads to an impaired carboxylase epoxidase reaction which is decisively triggered by the vitamin K concentration. Determination of FIX and APTT in addition to PT and INR is therefore recommended in coumarin-treated patients with an uncommon bleeding pattern.