Molecular characterization of an Italian patient with plasminogen deficiency and ligneous conjunctivitis

Plasminogen deficiency is a rare disease characterized by ligneous conjunctivitis and infections. We observed a 3-year-old Italian boy presenting ligneous conjunctivitis and low plasma levels of plasminogen. Twenty-three different mutations on the PLG gene have been reported to date, but mutation analysis had been troublesome for the presence of highly homologous genes. The aim of the study was to identify the underlying mutation avoiding coamplification of unwanted genetic materials using a long polymerase chain reaction strategy, instead of the previously reported subcloning methods. By this simple strategy the complete sequence analysis of PLG gene was performed, and a previously reported missense homozygous mutation (K19E) was identified

Salvage therapy with high dose Intravenous Immunoglobulins in acquired Von Willebrand Syndrome and unresponsive severe intestinal bleeding

A 91-year-old woman affected with acquired Von Willebrand (VW) syndrome and intestinal angiodysplasias presented with severe gastrointestinal bleeding (hemoglobin 5\ua0g/dl). Despite replacement therapy with VW factor/factor VIII concentrate qid, bleeding did not stop (eleven packed red blood cell units were transfused over three days). High circulating levels of anti-VW factor immunoglobulin M were documented immunoenzimatically. Heart ultrasound showed abnormalities of the mitral and aortic valves with severe flow alterations. When intravenous immunoglobulins were added to therapy, prompt clinical and laboratory responses occurred: complete cessation of bleeding, raise in hemoglobin, VW factor antigen, VW ristocetin cofactor and factor VIII levels as well as progressive reduction of the anti-VWF autoantibody levels

Health-related quality of life and psychological well-being in elderly patients with haemophilia

Many persons with severe haemophilia reach seniority thanks to effective treatment. There is no information on health-related quality of life (HRQoL) of these patients, who had lived for many years when regular replacement therapy was unavailable. Italian patients with severe haemophilia aged 6565 years born in the 1940s or earlier were compared with men without bleeding disorders matched for age and geography. HRQoL was assessed via generic and disease-specific questionnaires. Potential associations with concomitant illnesses, orthopaedic status, physical functioning, cognitive status and depression were evaluated. In addition, the newly adapted HRQoL questionnaire specific for elderly persons with haemophilia (Haem-A-QoLEldlery) was psychometrically tested and validated. Thirty-nine patients, aged 65\u201378 years, were investigated, 33 with haemophilia A and six with haemophilia B, and compared to 43 controls, aged 65\u201379 years. Chronic blood borne viral infections, hypertension and arthropathy were more frequent in patients, whereas hypercholesterolemia and cardiovascular diseases were more frequent in controls. Psychometric characteristics of Haem-A-QoLElderly showed good to excellent values for reliability and validity. HRQoL was worse in patients at EQ-VAS, WHOQOL-BREF and WHOQOL-Old. The highest impairments were found in patients by means of the haemophilia-specific Haem-A-QoLElderly in such dimensions as \u2018physical activity & leisure\u2019, \u2018physical health\u2019 and \u2018view\u2019. A poor orthopaedic status was negatively associated with HRQoL. Compared to age-matched controls elderly patients with haemophilia had an impaired HRQoL in association with their health status. The newly developed Haem-A-QoLElderly proved to be a reliable and valid instrument for HRQoL assessment in elderly haemophilia patient

Molecular characterization in an Italian patient with plasminogen deficiency and ligneous conjunctivitis

Plasminogen plays an important role in fibrinolysis and wound healing. Plasminogen deficiency is a rare disease associated with ligneous conjunctivitis. Since today 23 different mutations on PLG gene have been reported. We report a case of 3 year old Italian boy presenting ligneous conjunctivitis and low plasma levels of plasminogen (26%). Clinical history is unremarkable, and both parents are clinically healthy. Previously researches reported an extraordinary sequence homology between PLG genes, apo(A) genes and PRGs genes responsible of co-amplification problems of DNA fragments during PCR with a possible solutions based on amplification of PLG exons followed by subcloning and sequencing. We tried to carry out a faster strategy using Long PCR to analyse the gene portion between 5\u2019UTR and intron 5. The PCR product subsequently were extracted from the gel and used to perform a series of nested PCR on amplification of every single exons for further sequencing analysis. Exons 11, 12, 16 and 17 were amplified using the forward primer of the upstream exon and the reverse primer of the downstream exon; the product was gel extracted and sequenced too. All remaining exons without any co-amplification problem, were amplified and sequenced. This strategy revealed a missense homozygous mutation in exon 2, an A to G substitution leading to an amino acid exchange (Lys to Glu) at codon 19 (K19E). The same mutation in heterozygous state was present in both parents. This mutation has been previously reported in German, Italian and Scottish patients. Since the K19E mutation was also reported in homozygous state in a healthy Scottish blood donor with low levels of PLG and no clinical manifestations, its clinical significance and allele distribution in Europe populations could be the matter of further investigations

Long-term prophylaxis in severe factor VII deficiency

The spectrum of bleeding problems in FVII deficiency is highly variable and FVII levels and causative genetic mutations correlate poorly with the bleeding risk. Long-term prophylaxis is generally initiated in order to prevent subsequent CNS bleeding after a first event or in patients with other major/ life threatening/ frequent bleeding symptoms as gastrointestinal bleeding or hemarthrosis. However few data are available in the literature regarding FVII prophylaxis and clinical decisions cannot be based on evidence

Low-rate of complications after a long-term use of ateriovenous fistula (AVF) in hemophilic children

Background: Hemophilic children, undergoing regular prophylaxis or ITI, need a long-lasting uncomplicated venous access. Methods: Children lacking a venous access suitable for frequent infusions were eligible for AVF creation. AVF were accessed at home by parents. Doppler ultrasound of the limb and echocardiography were regularly performed. Results: Between 1999 and 2007, 43 AVF were created in 38 patients (FVIII/FIX <2%; median age 2.6 years, range: 0.9\u201311.9; 23 with inhibitors). AVF did not mature in 6 children (16%) and 5 of them underwent a second procedure that was successful in 4. Overall successful maturation was obtained in 36 AVF (84%) that were first accessed after a median of 56 days (21\u2013135) and used for a median of 5.1 years (0.7\u20137.9) for ITI (20), prophylaxis (11) and on-demand treatment (5). Complications not preventing AVF use were: thrombosis of a venous branch (1. 3%) and transient distal ischaemia (4. 11%). Other complications were: aneurysmatic dilatation (4. 11%) observed after a median of 5.4 years (3.5\u20137.7), limb hypertrophy (1. 3%) after 5.4 years and AVF overflow (1. 3%) after 6.9 years. These complications were reason for surgical dismantlement and transition to peripheral veins after a median of 6.6 years (range: 3.5\u20137.1). Uncomplicated AVF were dismantled after 4\u20137.4 years in 2 children who developed adequate peripheral veins. Conclusions: AVF were satisfactorily safe in hemophilic children, allowing long-term home treatment in 36/38 (95%). Regular follow-up allows early remedial intervention in case of complications; however, transition to peripheral veins should be implemented as soon as possibl

CYP2C9 genotypes and dose requirements during the induction phase of oral anticoagulant therapy

Objective: Variant alleles of the CYP2C9 gene encoding the cytochrome P450 (CYP) enzyme (2C9*2 [Arg144Cys] and 2C9*3 [Ile359Leu]) are known to increase the anticoagulant effect of warfarin and decrease the mean daily dose required to maintain the international normalized ratio (INR) of the prothrombin time within the target therapeutic range. However, little information is available on the effect of CYP2C9 polymorphisms; on dose requirements during the most critical step of anticoagulant therapy, the induction phase. Methods. This retrospective study evaluated the dosages given to 125 patients who started therapy with warfarin in a clinical center where physicians used the same approach for dosing and frequency of monitoring. CYP2C9 allelic variants were evaluated by polymerase chain reaction followed by restriction enzyme analysis. Results. From the time of the first INR estimate (day 4) until the end of the induction phase (arbitrarily established at day 24), patients with 2C9*2 or 2C9*3 variant alleles required lower mean daily doses than patients carrying only wild-type alleles 2C9*1 (-17% and -40%, P < .0001). They also more frequently had INR values above the upper limit of the target range (3.0) (65% for 2C9*2/- and 66% for 2C9*3/- versus 33% for 2C9*1/*1; P = .006 and .012, respectively). Conclusions: The requirement of smaller doses of warfarin in relation to CYP2C9 polymorphisms is already manifest on the fourth day of treatment, at the time of the first INR estimate. CYP2C9 genotyping is as yet not warranted, but frequent INR monitoring with appropriate dose adjustments is recommended during the first 3 weeks of treatment to avoid overanticoagulation and the inherent risk of bleeding in carriers of variant alleles

Non-invasive tool for foetal sex determination in early gestational age

Free foetal DNA in maternal blood during early pregnancy is an ideal source of foetal genetic material for non-invasive prenatal diagnosis. The aim of this study was to evaluate the use of free foetal DNA analysis at early gestational age as pretest for the detection of specific Y-chromosome sequences in maternal plasma of women who are carriers of X-linked disorders, such as haemophilia. Real-time quantitative PCR analysis of maternal plasma was performed for the detection of the SRY or DYS14 sequence. A group of 208 pregnant women, at different gestational periods from 4 to 12 weeks, were tested to identify the optimal period to obtain an adequate amount of foetal DNA for prenatal diagnosis. Foetal gender was determined in 181 pregnant women sampled throughout pregnancy. Pregnancy outcome and foetal gender were confirmed using karyotyping, ultrasonography or after birth. The sensitivity, which was low between 4th and 7th week (mean 73%), increased significantly after 7+1th weeks of gestation (mean 94%). The latter sensitivity after 7+1th week of gestation is associated to a high specificity (100%), with an overall accuracy of 96% for foetal gender determination. This analysis demonstrates that foetal gender determination in maternal plasma is reliable after the 9th week of gestation and it can be used, in association with ultrasonography, for screening to determine the need for chorionic villus sampling for prenatal diagnosis of X-linked disorders, such as haemophili

Baseline factor VIII plasma levels and age at first bleeding in patients with severe forms of von Willebrand disease

Introduction: von Willebrand disease (VWD) is the most common inherited bleeding disorder. The age of bleeding onset is highly variable, also in patients with similar degree of severity. Aim: The primary aim of this study was to evaluate whether baseline factor VIII (FVIII) plasma levels correlate with age at first bleeding in patients with extremely low levels of VWF:RCo (<6 IU dL 121). Methods: One hundred and three patients with VWF:RCo <6 IU dL 121 (6 VWD1, 73 VWD2 and 24 VWD3) undergoing a medical examination between September 2010 and September 2013 were included. The relationship between baseline FVIII levels and age at first bleeding was tested in a multivariable linear regression model, adjusting for sex. Results: The median age at first bleeding was lower in patients with VWD3 than in those with severe forms of VWD1 or VWD2 (1 year vs. 7 and 8 years, respectively, P < 0.0001). A positive non-linear relationship between FVIII levels and age at first bleeding was found, the latter increasing by 5 years for every 10 IU dL 121 increase of FVIII (\u3b2 = 4.95 [95% CI: 2.02\u20137.87]) until levels of 30 IU dL 121, after which the age increased slowly. This relationship was not found in VWD 2A and 2B. In 65 patients (63%) there was a more than 6-month delay between bleeding onset and VWD diagnosis, with no difference over decades. Conclusions: Baseline FVIII plasma levels influence the age at bleeding onset in VWD patients with extremely low levels of VWF:RCo, except in those with types 2A and 2B