Two Novel Missense Mutations and a 5bp Deletion in the Erythroid-Specific Promoter of the PKLR Gene in Two Unrelated Patients With Pyruvate Kinase Deficient Transfusion-Dependent Chronic Nonspherocytic Hemolytic Anemia.

We report two children with severe chronic hemolytic anemia, the cause of which was difficult to establish because of transfusion dependency. Reduced erythrocyte pyruvate kinase activity in their asymptomatic parents provided the diagnostic clues for mutation screening of the PKLR gene and revealed that one child was a compound heterozygote of a novel paternally derived 5-bp deletion in the promoter region (c.-88_-84delTCTCT) and a maternally derived missense mutation in exon nine (c.1174G>A; p.Ala392Thr). The second child was a compound heterozygote of two novel missense mutations, namely a paternally derived exon ten c.1381G>A (p.Glu461Lys) and a maternally derived exon seven c.907-908delCC (p.Pro303GlyfsX12) variant.

Risk for early pregnancy loss by factor XIII Val34Leu: the impact of fibrinogen concentration.

BACKGROUND: We have already described a significantly elevated overall risk for recurrent pregnancy loss (RPL) in women carrying the coagulation factor XIII (FXIII) Val34Leu and/or the plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism assuming that these polymorphisms contribute synergistically to RPL because of impaired hypofibrinolysis. Recent studies on FXIII indicate that the impact of the FXIII 34Leu genotype on fibrin structure and fibrinolysis is affected by fibrinogen concentration. Therefore, we reinvestigated the association between fibrinogen concentrations and FXIII Val34Leu with early RPL. MATERIALS AND METHODS: In this case-control study, we enrolled 49 women with a history of two consecutive or three to six nonconsecutive pregnancy losses between the 8th and 12th week of gestation and 48 healthy controls. The risk for RPL in carriers of FXIII 34Leu at fibrinogen levels above or below the median and first tertile of controls was evaluated. RESULTS: In carriers of the 34Leu allele, fibrinogen levels below the median (i.e., ≤ 300 mg/dl) and the first tertile (i.e., ≤ 284 mg/dl) of controls were associated with an increased risk for RPL [(2.9 (1.1-7.7), 3.9(1.0-15.0)]. CONCLUSIONS: The FXIII Val34Leu polymorphism may be associated with the development of early RPL in association with fibrinogen concentrations. At fibrinogen levels in the low normal range, FXIII 34Leu may modify fibrin structure toward an increased resistance to fibrinolysis.

Epidermal growth factor: the driving force in initiation of RPE cell proliferation.

BACKGROUND: To analyze whether epidermal growth factor (EGF) exerts regulatory effects on proliferation and differentiation in ARPE19 cells after different incubation periods (24 vs. 48 h) for obtaining ideal conditions for feasible rejuvenation and autologous transplantation of retinal pigment epithelial cells (RPE cells). METHODS: To evaluate gene expression patterns of RPE-specific differentiation and proliferation markers as well as transcriptional and translational changes of beta-catenin (ß-catenin)-signaling markers by fluorescence activated cell sorting (FACS) and reverse transcription - polymerase chain reaction (RT-PCR) after 24 h of EGF treatment. RESULTS: After 24 h of EGF treatment, a significant decrease of retinal pigment epithelium-specific protein 65 (RPE 65), cellular retinaldehyde-binding protein (CRALBP) and cytokeratin 18 in ARPE-19 cells was scaled. In addition, an increase of cyclin D1 expression and a significant decrease of glycogen synthase kinase-3beta (GSK-3ß) and beta-catenin (ß-catenin) were equally observed after 24 and 48 h of EGF treatment. Cell-cycle studies revealed an increase of ARPE cells in S-G2/M phase after 24 h of EGF treatment. CONCLUSIONS: Our data demonstrate the induction of proliferation and upregulation of the ß-catenin signaling pathway by EGF even after 24 h of incubation. As ideal cell culture conditions are essential for maintaining RPE-specific phenotypes, short incubation times enhance RPE cell quality for feasible rejuvenation and subsequent autologous transplantation of RPE cells.

Dependency of phenprocoumon dosage on polymorphisms in the VKORC1 and CYP2C9 genes.

OBJECTIVES: Polymorphisms in the vitamin K epoxide-reductase-complex-1 (VKORC1) and the cytochrome-P450-isozyme (CYP2C9) genes account for therapeutic responses to vitamin K antagonists (VKA). This study aimed to investigate the prevalence of VKORC1 and CYP2C9 polymorphisms among patients under phenprocoumon and its influence on the VKA dosage. METHODS: Patients under phenprocoumon were screened for the polymorphisms -1639G > A and 3730G > A in the VKORC1 gene and 430C > T and 1075A > C in the CYP2C9 gene by means of a StripAssay. Baseline clinical and laboratory parameters were registered. RESULTS: Among 53 patients (28 females, mean age 72.5 years), VKORC1 polymorphisms were found in 34 [-1639G > A: homozygote (n = 11), heterozygote (n = 23)] and 28 [3730G > A: homozygote (n = 7), heterozygote (n = 21)] patients. Thirteen patients were compound heterozygote. CYP2C9 polymorphisms were found in 12 [430G > T: homozygote (n = 1), heterozygote (n = 11)] and 7 [1075A > C: homozygote (n = 0), heterozygote (n = 7)] patients. Seventeen patients had at least one VKORC1 and one CYP2C9 polymorphism. Mean phenprocoumon dosage per week to achieve therapeutic anticoagulation was lower (higher) in patients with than without the VKORC1 polymorphism -1639G > A (3730G > A) or the CYP2C9 polymorphisms. Despite the presence of VKORC1 or CYP2C9 polymorphisms, mean International Normalized Ratio was not significantly different between patients with and without polymorphisms. CONCLUSIONS: Though VKORC1 and CYP2C9 polymorphisms influence the phenprocoumon dosage necessary to achieve therapeutic anticoagulation, anticoagulation is therapeutic if carefully monitored.

Hydrogen breath testing versus LCT genotyping for the diagnosis of lactose intolerance: a matter of age?

BACKGROUND: Two single nucleotide polymorphisms (-13910 C/T and -22018 G/A) upstream of the lactase gene (LCT) have been found to be associated with lactose tolerance in Europeans. METHODS: In one hundred and twenty Austrian outpatients, who visited the physician's office for symptoms of irritable bowel syndrome (IBS), hydrogen breath testing (HBT) and LCT genotyping by polymerase chain reaction and reverse-hybridisation were performed in parallel. RESULTS: The coincidence between a genotype suggesting lactase non-persistence (lactose intolerance) and a positive HBT result was almost perfect (97.4% for LCT-13910 C/T and 100% for LCT-22018 G/A). Between a genotype indicating lactase persistence (lactose tolerance) and a negative HBT result the coincidence was lower (72% and 71.4%, respectively). Among heterozygotes, there was a statistically significant increase in the proportion of positive HBT results with age. Both SNPs were in accordance in 117/120 (97.5%) patients. CONCLUSION: Genetic analysis of LCT-13910 C/T and LCT-22018 G/A is a good indicator for the presence of lactose intolerance. Because age, as well as a number of secondary causes (e.g. celiac disease), can influence HBT results, it is useful to combine HBT and genetic analysis in the diagnostic assessment of IBS.

Coagulation factor XI: a database of mutations and polymorphisms associated with factor XI deficiency.

Hereditary factor XI deficiency is a rare bleeding disorder that is found worldwide. Rapidly increasing numbers of mutations and polymorphisms in various populations have been reported. However, the number of identified mutations given in recent literature and available databases is named to be not more than 35. We assumed that this is clearly too low and that to date no comprehensive survey of mutations associated with factor XI deficiency is available. To provide a complete database of mutations and polymorphisms associated with factor XI deficiency we collected all available data on hereditary factor XI deficiency from main biological and medical databases [http://ncbi.nlm.nih.gov/pubmed and http://ncbi.nlm.nih.gov/omim (OMIM reference 264900) and the Human Gene Mutation Database for F11 mutations http://uwcmml1s.uwcm.ac.uk/uwcm/mg/search/119891.html] as well as from contributions to international congresses. As of 8 June 2004 the number of reported causative mutations is 81, of which 12 have been described in unrelated individuals by more than one study group. For three frequently observed mutations [type II and type III mutations (Gln116Stop and Phe283Leu) and Cys38Arg] common founders have been described. Furthermore, 20 polymorphisms have been described in association with factor XI deficiency, three of which have been reported by two independent study groups. For the majority, allele frequencies have been published for in the Caucasian and/or Black population.

Elevated coagulation factor VIII and the risk for recurrent early pregnancy loss.

Inherited and acquired thrombophilia are associated with recurrent pregnancy loss. Recently, an increased risk for thromboembolic disease was described for patients with elevated coagulation factor VIII, but it is unknown whether there is also an association to early pregnancy loss. We therefore evaluated the relation between recurrent early pregnancy loss and levels of coagulation factor VIII. We enrolled 49 unrelated Caucasian women with a history of 2-6 early pregnancy losses and 48 healthy controls, who had delivered at least one term infant and had never experienced pregnancy loss. We determined factor V Leiden-, G20210A prothrombin-, MTHFR C677T- and A1298C-gene mutations, levels of antithrombin, protein C, protein S, factor VIII, C-reactive protein and antiphospholipid antibodies. There was a significantly higher rate of pregnancy losses in women with Antiphospholipid Syndrome (p = 0.043). Furthermore, plasma levels of coagulation factor VIII were significantly higher in cases than in controls (130.5 IU/dl +/- 25.4 vs 119.5 IU/dl +/- 24.1; p = 0.032) and appeared independent of C-reactive protein (R = 0.146, p = 0.323 in cases; R = -0.028, p = 0.850 in controls). The relative risk for recurrent pregnancy loss in women with factor VIII levels above 151 IU/dl (90(th) percentile of controls) was 2.5 (0.7 - 8.9, 95 percent confidence interval), for levels above 156 IU/dl (95(th) percentile of controls) 3.9 (0.8 - 20.0, 95 percent confidence interval). Elevated maternal plasma levels of coagulation factor VIII tend to be associated with an increased risk for recurrent early pregnancy loss.

Effects of ultra-marathon on circulating DNA and mRNA expression of pro- and anti-apoptotic genes in mononuclear cells.

We investigated the effects of an ultra-marathon on cell-free plasma DNA as well as on mRNA expression of pro-apoptotic (Bax, Bad), anti-apoptotic (Bcl-2) and cell-protective (Hsp70, Hsp27 and Hsp32) genes in mononuclear blood cells (MNCs). Blood samples were drawn from 14 athletes before and immediately after 6-h run. In addition, blood samples were also collected and analyzed 2 and 24 h after the end of the run. Levels of plasma DNA were significantly increased immediately after the marathon (P < 0.001) and were still higher 2 h later (P < 0.005), but significantly lower than those immediately after the race (P < 0.05). Cell-free plasma DNA returned to pre-race levels 24 h after the run. mRNA expressions of Hsp70, Hsp32 and Bax significantly increased in MNCs after the race, whereas Hsp27 and Bad mRNA expression levels showed no significant changes. Bcl-2 expressions decreased immediately after the race (P < 0.001), but increased in the 24 h later (P < 0.05). We conclude that apoptotic ladders of cell-free DNA following exhaustive exercise originate from apoptotic cells and that not only skeletal muscle cells but also leukocytes contribute to this phenomenon.

Association of endothelial protein C receptor haplotypes, factor V Leiden and recurrent first trimester pregnancy loss.

OBJECTIVES: We evaluated whether the endothelial protein C receptor (EPCR) haplotypes A1 and A3 exert effects on the development of recurrent pregnancy loss (RPL) in association with factor V Leiden. DESIGN AND METHODS: We determined the EPCR haplotypes A1 and A3 and factor V Leiden in 49 women with a history of RPL and 48 parous controls. RESULTS: In carriers of factor V Leiden the A1 haplotype decreased the relative risk for RPL from 2.2 to 1.0. CONCLUSIONS: The EPCR A1 haplotype tends to modulate the risk for RPL in carriers of factor V Leiden.

Association of the protein Z intron F G79A gene polymorphism with recurrent pregnancy loss.

OBJECTIVE: To investigate the association of the common protein Z (PZ) intron F G79A gene polymorphism with recurrent early pregnancy loss (RPL) and its gene-gene interaction with known thrombophilic risk factors for RPL. DESIGN: Case control study. SETTING: University clinic. PATIENT(S): We enrolled 49 women with a history of two consecutive or three to six nonconsecutive pregnancy losses between the 8th and 12th weeks of gestation and 48 age-matched parous controls without a history of pregnancy complications. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Allele frequencies of the PZ intron F G79A polymorphism and its gene-gene interaction with known risk factors for RPL. RESULT(S): Fourteen case subjects (28.6%) and 24 control subjects (50.0%) carried at least one A allele. This was associated with a significant reduction of the relative risk for recurrent pregnancy loss (odds ratio [OR] 0.4, 95% confidence interval [CI] 0.2-0.9; adjusted OR 0.3, 95% CI 0.1-0.8). Coexistence of any thrombophilic risk factor studied with the 79A allele resulted in a clear reduction of the primal relative risk for recurrent pregnancy loss. CONCLUSION(S): The isolated presence of the PZ intron F 79A allele as well as the combination with known thrombophilic risk factors was protective against RPL between the 8th and 12th weeks of gestation.

Coagulation factor XI gene analysis in three factor XI deficient Austrian patients.

OBJECTIVES: Hereditary factor XI deficiency is a rare bleeding disorder with worldwide distribution. In Austrian patients only one mutation leading to congenital factor XI deficiency has been reported. In the present study, we identified the molecular basis of factor XI deficiency in three Austrian patients. METHODS: Patients attended hospital for other reasons than bleeding disorders. Routine laboratory tests revealed prolonged APTTs due to decreased factor XI levels. We performed automated fluorescent sequencing of the promotor region, exons 1-15 and the flanking intronic regions of the factor XI gene. The mutations found were confirmed by restriction enzyme analysis or sequencing of the non-coding strand. RESULTS: Fluorescent sequencing revealed two novel mutations, the nonsense mutation Gln116X (443C>T) in exon 5 and a deletion of Ile197 and Asp198 (687_692delTCGACA) in exon 7. Furthermore, we detected a heterozygous A>G exchange at the third nucleotide of IVS6 (IVS 6 +3A>G), which had already been reported in a FXI deficient individual of French Basque origin. CONCLUSION: While the IVS 6 +3A>G decreases the calculated splice consensus score from 0.98 in the wild type to 0.56 in the altered sequence and therefore interferes with the consensus splice sequence, the complete loss of the two amino acids Ile197 and Asp198 is expected to interfere with the steric structure and hence the functions of the third apple domain. The Gln116X leads to a premature termination codon resulting in a lack of the light as well as parts of the heavy chain of the FXI protein, most likely resulting in rapid degradation of the truncated mRNA.

Detection of single nucleotide polymorphisms in coagulation factor XI deficient patients by multitemperature single-strand conformation polymorphism analysis.

Factor XI (FXI) deficiency is a rare inherited disorder which can cause bleeding complications especially in case of hemostatic challenge and/or in tissues with high fibrinolytic activity. A number of causative mutations have been described in FXI deficient individuals which have been detected by various screening methods. In this study, we present the application of the multitemperature single-strand conformation polymorphism analysis (MSSCP) on the FXI gene, a recently developed methodology for the detection of single nucleotide exchanges. We analyzed a total of 217 polymerase chain reaction (PCR) fragments from the promoter region as well as from exons 1-7 and 11-15 and compared the results to automatic fluorescent sequencing. A total of 29 PCR fragments showed single nucleotide exchanges in conventional fluorescent sequencing, representing 10 different mutations (nine missense mutations, one small deletion) and four frequent polymorphisms. With MSSCP electrophoresis at a standard temperature profile (gel temperature 35-20-10 degrees C) we were able to detect 13 of 14 (93%) different nucleotide exchanges in 25 of 29 PCR fragments (86%). Hence, the detection rate for genetic variations in the FXI gene was 86%. To evaluate the reproducibility, MSSCP was performed twice for 174 PCR fragments and the consistency between two electrophoretic runs was 99%. We conclude that the MSSCP is a sensitive, fast, and cost effective screening method for the detection of FXI gene mutations.

Risk-factor profile in severe, generalized, obliterating vascular disease.

A 74-year-old woman had a history over 25 years of endarterectomy of both renal arteries, iliac venous thrombosis, pulmonary embolism, left internal carotid artery endarterectomy, coronary angioplasty, aortocoronary bypass grafting, occlusion of the right axillary artery, lower-limb claudication due to common iliac artery aneurysm, external iliac artery stenosis, multiple femoral artery stenoses, bifurcational stent grafting, occlusion of the left brachial artery and the right external iliac artery, and stroke. Assessment of the risk-factor profile revealed an absence of classic risk factors but the presence of the factor V Leiden mutation, the methylenetetrahydrofolate reductase AI298C mutation, the HFE C282Y mutation, plasminogen activator inhibitor-1 gene mutation, the -455 G/A fibrinogen gene polymorphism, the epsilon3/epsilon4 apolipoprotein E -675 4G gene polymorphism, and hyperhomocysteinemia. This case shows that severe, generalized, occlusive vascular disease may be due to the combination of various genetic risk factors for atherosclerosis and venous thromboembolism.

Plasminogen activator inhibitor 1 4G/5G polymorphism and coagulation factor XIII Val34Leu polymorphism: impaired fibrinolysis and early pregnancy loss.

BACKGROUND: A successful outcome of pregnancy depends on proper placental formation. In the very beginning of this process, trophoblast invasion and fibrin deposition into the wall of the decidual veins play an important part. Two polymorphisms, coagulation factor XIII (FXIII) Val34Leu and plasminogen activator inhibitor 1 (PAI-1) 4G/5G, interfere with fibrin cross-linking and regulation of fibrinolysis and may therefore contribute to early pregnancy loss. METHODS: We enrolled 49 unrelated Caucasian women with a history of two consecutive or three to six nonconsecutive early pregnancy losses and 48 unrelated parous healthy controls without a history of pregnancy loss and evaluated them for the following genetic variants: the factor V Leiden and prothrombin G20210A gene mutations, the methylenetetrahydrofolate reductase C677T and A1298C polymorphisms, and the PAI-1 4G/5G and FXIII Val34Leu polymorphisms. RESULTS: For the isolated occurrence of PAI-1 4G/5G or FXIII Val34Leu, we found no statistically significant difference between cases and controls. For homozygosity of either or compound carrier status of both mutations, the overall relative risk for early pregnancy loss was significantly increased (odds ratio = 2.4; 95% confidence interval, 1.1-5.5; P = 0.032). We observed no statistically relevant association of any of the other tested mutations with early pregnancy loss. CONCLUSION: Homozygosity for PAI-1 4G or FXIII 34Leu polymorphisms as well as compound carrier status is associated with early pregnancy loss.