Uniparental disomy causes deficiencies of vitamin K-dependent proteins.

Essentials Vitamin K-dependent coagulant factor deficiency (VKCFD) is a rare autosomal recessive disorder. We describe a case of inherited VKCFD due to uniparental disomy. The homozygous mutation caused the absence of GGCX isoform 1 and overexpression of Δ2GGCX. Hepatic and non-hepatic vitamin K-dependent proteins must be assayed to monitor VKCFD treatment. SUMMARY: Background Inherited deficiency of all vitamin K-dependent coagulant factors (VKCFD) is a rare autosomal recessive disorder caused by mutations in the γ-glutamyl carboxylase gene (GGCX) or the vitamin K epoxide reductase gene (VKORC1), with great heterogeneity in terms of both clinical presentation and response to treatment. Objective To characterize the molecular basis of VKCFD in a Spanish family. Methods and Results Sequencing of candidate genes, comparative genomic hybridization and massive sequencing identified a new mechanism causing VKCFD in the proband. Uniparental disomy (UPD) of chromosome 2 caused homozygosity of a mutation (c.44-1G>A) resulting in aberrant GGCX splicing. This change contributed to absent expression of the mRNA coding for the full-length protein, and to four-fold overexpression of the smaller mRNA isoform lacking exon 2 (Δ2GGCX). Δ2GGCX might be responsible for two unexpected clinical observations in the patient: (i) increased plasma osteocalcin levels following vitamin K1 supplementation; and (ii) a mild non-bleeding phenotype. Conclusions Our study identifies a new autosomal disease, VKCFD1, caused by UPD. These data suggest that the Δ2GGCX isoform may retain enzymatic activity, and strongly encourage the evaluation of both hepatic and non-hepatic vitamin K-dependent proteins to assess differing responses to vitamin K supplementation in VKCFD patients.

Protocolo de estudio y tratamiento de la trombocitopenia inmune primaria (PTI-2010) / Protocol for the study and treatment of Immune Thrombocytopenic Purpura (ITP). ITP-2010

La trombocitopenia inmune primaria, anteriormente conocida como púrpura trombocitopénica inmune, es una enfermedad cuyo manejo diagnóstico y terapéutico ha sido siempre controvertido. La Sociedad Española de Hematología y Oncología Pediátricas, a través del grupo de trabajo de la PTI, ha actualizado el documento con las recomendaciones protocolizadas para el diagnóstico y tratamiento de esta enfermedad, basándose en las guías clínicas disponibles actualmente, revisiones bibliográficas, ensayos clínicos y el consenso de sus miembros. El objetivo principal es disminuir la variabilidad clínica en los procedimientos diagnósticos y terapéuticos con el fin de obtener los mejores resultados clínicos, con menor incidencia en la calidad de vida y los mínimos efectos adversos (AU)

Primary immune thrombocytopenia (ITP), formerly known as immune thrombocytopenic purpura, is a disease in which clinical and therapeutic management has always been controversial. The ITP working group of the Spanish Society of Paediatric Haematology and Oncology has updated its guidelines for diagnosis and treatment of ITP in children based on current guidelines, literature review, clinical trials and member consensus. The primary objective was to lessen clinical variability in diagnostic and therapeutic procedures in order to obtain best clinical results with minimal adverse events and good quality of life (AU)

[Protocol for the study and treatment of immune thrombocytopenic purpura (ITP). ITP-2010]. / Protocolo de estudio y tratamiento de la trombocitopenia inmune primaria (PTI-2010).

Primary immune thrombocytopenia (ITP), formerly known as immune thrombocytopenic purpura, is a disease in which clinical and therapeutic management has always been controversial. The ITP working group of the Spanish Society of Paediatric Haematology and Oncology has updated its guidelines for diagnosis and treatment of ITP in children based on current guidelines, literature review, clinical trials and member consensus. The primary objective was to lessen clinical variability in diagnostic and therapeutic procedures in order to obtain best clinical results with minimal adverse events and good quality of life.

Zero incidence of inhibitor development in previously treated haemophilia A, HIV-negative patients upon exposure to a plasma-derived high-purity and double viral inactivated factor VIII concentrate.

Thirty-six haemophilia A, HIV-negative, previously treated patients were changed therapy to a highpurity and double-inactivated (solvent/detergent and dry-heating) previously unused factor VIII concentrate. The mean age of these patients was 27 years at the time of the change. Twenty-three patients were severe haemophiliacs (FVIII:C < 0.02 IU mL-1), seven moderate (FVIII:C between 0.02 and 0.05 IU mL-1) and six mild (FVIII:C > 0.05 IU mL-1). The mean follow-up with this single product was 16 months, with 82 accumulated exposure days and the mean consumption was 117,300 IU of FVIII corresponding to a mean of six batches per patient. No patient developed FVIII inhibitors (upper limit of the CI95: 7.98%), resulting in an incidence rate of 0/48 patient-years (upper limit of the CI95: 77/1000 patient-years). The change in therapy to this new factor VIII concentrate was not associated with the appearance of inhibitors.

Development of a factor VIII inhibitor in a newborn haemophiliac.

A major problem in the treatment of haemophilia A is the development of inhibitors (antibodies) against factor VIII. We report the case of a newborn male with no family history of haemophilia who developed an intracerebral haemorrhage. On day 10 post-delivery severe haemophilia A was diagnosed and treatment with recombinant FVIII (rFVIII) concentrate was started. Seventy-two hours later the presence of inhibitors was suspected because high doses of rFVIII were required to maintain therapeutic FVIII plasma levels. Days after, the inhibitor was detected. The quick detection of the inhibitor in this newborn haemophiliac allowed us to start the immunotolerance early, without interruption in the administration of rFVIII.

Simultaneous detection and typing of plum pox potyvirus (PPV) isolates by heminested-PCR and PCR-ELISA.

Two techniques for simultaneous detection and typing of plum pox potyvirus (PPV) isolates belonging to the D or M serotypes, heminested PCR (H-PCR) and PCR-ELISA, have been developed. Ten PPV isolates typed using PPV-D and PPV-M specific monoclonal antibodies by ELISA-DASI were used to validate these two methods. The results obtained show a complete coincidence of the nucleic acid-based techniques with the serological data. When serial dilutions of infected plant extracts were assayed, H-PCR and PCR-ELISA were found to be 100 times more sensitive than the more conventional immunocapture-PCR (IC-PCR) assay. Testing of 228 PPV-infected fruit tree samples coming from different hosts and locations indicated that so far only PPV type D appears to be present in Spain and in Chile. Coupled with print-capture sample preparation (Olmos et al., Nucl. Acids Res. 24, 2192-2193, 1996) the increased sensitivity provided by heminested-PCR allowed the detection of PPV targets of D and M types, in wingless individuals of the aphid vector Aphis gossypii.

Subtyping of Legionella pneumophila isolates by arbitrarily primed polymerase chain reaction.

Arbitrarily primed polymerase chain reaction (AP-PCR) was used to differentiate strains of Legionella pneumophila isolated from different water sources in a resort hotel in Benidorm, Alicante, Spain, where an outbreak of Legionnaires' disease occurred among a group of tourists between 65 and 80 years of age. All isolates were L. pneumophila serogroup 1, subtype Pontiac (Knoxville 1). Five different patterns (P1 to P5) were obtained by AP-PCR. The number of bands per pattern varied between 4 and 11. Patterns P1 and P2 represented 60 and 20% of L. pneumophila isolates, respectively. Since different subpopulations of L. pneumophila coexisted (up to three different AP-PCR patterns were identified in a single room), it was not possible to link an individual L. pneumophila strain to the occurrence of this outbreak.

DNA amplification fingerprinting for subtyping Neisseria gonorrhoeae strains.

BACKGROUND AND OBJECTIVES: DNA amplification fingerprinting is used in most epidemiologic studies as a substitute for conventional typing methods. DNA amplification fingerprinting and conventional typing methods were compared in this epidemiologic study of Neisseria gonorrhoeae. GOAL OF THIS STUDY: To differentiate 70 Neisseria gonorrhoeae isolates from untreated patients with urogenital gonococcal infection. STUDY DESIGN: Gonococcal strains were characterized by auxotyping, serotyping, plasmid profile, antibiotic sensitivity, and DNA amplification fingerprinting. The method of unweighted pair-group average linkage was used for cluster analysis. Discriminatory power was calculated applying Simpson's index. RESULTS: Amplification of Neisseria gonorrhoeae DNA with primers OPA-03 and OPA-13 produced well-resolved patterns of 15 and 22 DNA fragments, respectively, with a discriminatory power (0.978 with OPA-13 and 0.967 with OPA-03) comparable to that obtained with auxotyping/serotyping combination (D:0.968) or with auxotype/serotype/plasmid profile combination (D:0.983). Correlation between DNA amplification fingerprinting pattern and auxotype/serotype class was not always uniform. Some strains with the same auxotype/serotype/plasmid profile were subdivided by DNA amplification fingerprinting, and vice versa. CONCLUSION: Although auxotype/serotype class and DNA amplification fingerprinting can be used in the epidemiologic characterization of strains, DNA amplification fingerprinting offers a better discriminatory index than the separate serotyping. It is especially useful for differentiating serologically identical strains and nontypable strains. A combination of serotyping and DNA amplification fingerprinting seems to be the best way to differentiate Neisseria gonorrhoeae strains in epidemiologic studies, bringing together the most simple techniques and the best discriminatory power among isolates.

Nested polymerase chain reaction for detection of Legionella pneumophila in water.

A highly sensitive nested polymerase chain reaction (PCR) method was evaluated for detection of Legionella pneumophila in water. Two sets of primers homologous to the coding region of the L. pneumophila macrophage infectivity potentiator (mip) gene were used. Even when starting from minute amounts of L. pneumophila DNA, the double PCR products were readily detected by direct visualization in ethidium-bromide-stained agarose gels. The method was tested on 34 potable water samples from a hospital building and compared with standard culture isolation. L. pneumophila was isolated in only twelve samples, whereas, by nested PCR, 19 samples were positive, 12 of them coincidental with culturing. These results indicate that nested PCR permits detection of L. pneumophila in samples where culturing fails, with the advantage of a rapid turnaround time, simplicity and the ability to detect non-culturable cells, fulfilling the requirements of sensitivity and specificity for routine use in an environmental laboratory.

[Progression to AIDS among HIV-seropositive hemophiliacs]. / Progresión a SIDA en hemofílicos seropositivos frente al VIH.

PURPOSE: To assess the incidence of AIDS, along with its origin and mortality rate, in a group of sero-positive haemophiliacs with long follow-up. MATERIAL AND METHODS: The progression into AIDS in 94 HIV-seropositive haemophiliacs (88 with haemophilia A and 6 with haemophilia B) followed since 1986 has been analyzed. The mean age was 19 years and 64% of the patients had severe haemophilia. All of them had been previously treated with non virus-inactivated factor concentrates. Was detected HIV antigen in 13 of them and 32 were treated with zidovudine. Actuarial analysis started from seroconversion date or from the first positive result and the average observation time was 90 months (5-97). RESULTS: Sixteen patients developed AIDS, resulting in an eight-years accumulative actuarial indice of about 24%. AIDS incidence was lower (p less than 0.0001) in haemophilia A (21%) than in haemophilia B (67%). The eight-years progression rate to AIDS showed a significant dependence on patient age, it being higher in patients aged greater than 30 (77%) than in those aged 15-30 (30%) and less than 15 (9%). Patients who presented CD4+ counts lower than 0.5 x 10(9)/L or CD4/CD8 ratios lower than 0.5 at the beginning of the observation period had a greater 8-year AIDS incidence (63% and 57% respectively) than the remaining patients (16% and 12%). At AIDS diagnosis, all patients had CD4+ lymphocyte counts lower than 0.2 x 10(9)/L, two had detectable HIV antigenaemia and six had been previously treated with zidovudine. The cause of AIDS was an opportunistic infection in all the cases namely, P. carinii pneumonia and candidiasis, but no secondary neoplasia was registered. Nineteen patients died during the follow-up, 12 because of AIDS and 7 because of other reasons unrelated to HIV-infection. CONCLUSION: Our results suggest that 25% of the haemophiliacs will develop AIDS eight years after seroconversion, and a decreasing incidence is not observed. Lower progression rate to AIDS in children than in adults is confirmed.

Genomic fingerprinting of penicillinase-producing strains of Neisseria gonorrhoeae in Valencia, Spain.

OBJECTIVE: To compare the value of different markers and their combinations with the restriction enzyme technique in the differentiations of penicillinase-producing N. gonorrhoeae (PPNG) strains. MATERIALS AND METHODS: 17 PPNG strains isolated from symptomatic, untreated male patients with urethritis were characterised by antibiotic sensitivity testing, auxotyping, serotyping, plasmid profile, and restriction endonuclease fingerprinting (Hind III digestion). Cluster analysis with the method of unweighted pair-group average (UPGMA) linkage was used to calculate similarity or dissimilarity for PPNG strains. MAIN RESULTS: Either auxotyping or plasmid profile alone differentiated three groups of PPNG strains, whereas the combination auxotyping/serotyping identified 10. Although the combination auxotyping/serotyping/plasmid profile and the restriction enzyme technique showed a similar discrimination ability (differentiation of 11 PPNG strains), genomic fingerprinting gave highly specific restriction patterns on individual gonococcal isolates. CONCLUSIONS: The combination of different markers gave more epidemiological information than the use of only one. The sequence of discriminating ability for PPNG strains was: auxotyping/serotyping less than auxotyping/serotyping/plasmid profile less than restriction patterns of genomic DNA.

[Membrane proteins electrophoretic profiles study applied to Neisseria gonorrhoeae epidemiology]. / Aplicación del estudio de los perfiles electroforéticos de proteínas de membrana en la epidemiología de Neisseria gonorrhoeae.

The analysis of electrophoretic profiles of membrane proteins is one of the epidemiological methods of bacterial typing. The profiles of membrane proteins of 95 isolates were studied for valuing their usefulness in the epidemiology of N. gonorrhoeae. The results were compared with the obtained using other characterization methods (auxotyping, serotyping and antimicrobial sensibility). The proteins I and II (PI and PII) showed clear differences between isolates. Only protein-I (PI) with constant molecular weight for each isolate was valid to discriminate between strains. It was observed correlation between serovariety IA and molecular weight of PI 33.6-36 kD, and the serovariety IB with molecular weight 35.5-37 kD. Though it wasn't possible discriminated between the different serovarieties. It was proved a sensibility decrease to penicillin, tetracycline and cloramfenicol in those strains with molecular weight of PI greater than 35.5 kD (serogroup WII/WIII). In the 80% of the isolates considered multiple antibiotic-resistant it was observed a significant increase of the membrane protein dough of 52 kD. All the strains with this protein increased were multiple antibiotic-resistant.

[Human immunodeficiency virus infection in hemophiliacs: prevalence and current clinical situation]. / Infección por el virus de la inmunodeficiencia humana en hemofílicos: prevalencia y situación clínica actual.

The prevalence of HIV-antibody was studied in a cohort of 156 haemophiliacs controlled since before 1986. Ninety four patients (60%) were HIV-seropositive, and all of them had been previously treated with non-heated factor concentrates. Seroprevalence was 50% by 1983. Evidence for previous negative results was only available in 19 seropositive patients and all of them seroconverted before 1985. For the 148 patients exposed to non-heat treated factor concentrates, severe haemophiliacs and those with factor consumption greater than 100 x 10(3) had the maximal rate of seropositivity (81% and 75% respectively, p less than 0.005). Fifteen patients developed AIDS (10% of the HIV-positive), 12 of which have died. HIV infection has a high prevalence and it has become the most important cause of death in our haemophiliacs.